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Pharmacognostical studies and anti inflammatory effect of kakamachi on albino rats – Gangoor, Department of Dravya Guna, Post Graduate Studies & Research Centre, D.G. MELMALAGI AYURVEDIC MEDICAL …

Pharmacognostical studies and anti inflammatory effect of kakamachi on albino rats – Gangoor, Department of Dravya Guna, Post Graduate Studies & Research Centre, D.G. MELMALAGI AYURVEDIC MEDICAL COLLEGE,GADAG

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  • 1. “PHARMACOGNOSTICAL STUDIES & ANTI-INFLAMMATORY EFFECT OF KAKAMACHI ON ALBINO RATS”. AN EXPERIMENTAL STUDY BY Dr. Shivakumar S. Gangoor. Dissertation submitted to the Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore. In partial fulfillment of Regulations for award of degree of DOCTOR OF MEDICINE (AYURVEDA VACHASPATI) IN DRAVYAGUNA Under the guidance of Under the co-guidance of DR. G. V. MULAGUND. DR. KUBER. SANKH. M.D. (AYU) M.D. (AYU) PROFESSOR & H.O.D, LECTURER DEPARTMENT OF DEPARTMENT OFP. G. A. R. Center DRAVYAGUNA. P. G. A. R. Center DRAVYAGUNA. POST GRADUATE DEPARTMENT OF DRAVYAGUNA D.G MELMALAGI AYURVEDIC MEDICAL COLLEGE AND RESEARCH CENTER, GADAG - 582103. 2006
  • 2. Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore. DECLARATION BY THE CANDITATE I hereby declare that this dissertation / thesis entitled“PHARMACOGNOSTICAL STUDIES AND ANTI-INFLAMMATORYEFFECT OF KAKAMACHI”- By an Experimental Study is a bonafideand genuine research work carried out by me under the guidance of Dr. G. V.MULAGUND. MD (Ayu),Professor and H.O.D, Post Graduate Department ofDravyaguna, Shri D.G.M.A.M.C and Research center Gadag.Date: Signature of the CandidatePlace: (Dr.Shivakumar. S. Gangoor)
  • 3. CERTIFICATE BY THE GUIDE This is to certify that the dissertation entitled “PHARMACOGNOSTICALSTUDIES AND ANTI-INFLAMMATORY EFFECT OF KAKAMACHI ONALBINO RATS ” – BY an Experimental Study is a bonafide research workdone by Dr. Shivakumar. S. Gangoor in partial fulfillment of the requirementfor the degree of Ayurveda Vachaspati in Drvyaguna. This work is applied, scientific and an original contribution in the field ofresearch in Ayurveda. I am fully satisfied with his original work and recommended the dissertation tobe put before the adjudication. Signature of the Guide Dr. G. V. MULAGUND. M.D (AYU) Professor and H.O.D Post Graduate Department of Dravyguna, D.G.M. Ayurvedic Medical College, Gadag.
  • 4. ENDORSEMENT BY THE HOD, PRINCIPAL HEAD OF THE INSTITUTION This is to certify that the dissertation entitled “PHARMACOGNOSTICALSTUDIES AND ANTI-INFLAMMATORY EFFECT OF KAKAMACHI ONALBINO RATS ”- By an Experimental Study is a bonafide research work done byDr.Shivakumar S. Gangoor under the guidance of Dr. G. V.MULAGUND. MD(Ayu) Professor and H.O.D, Post Graduate Department of Dravyaguna, ShriD.G.M.A.M.C and research center Gadag and contributed good values to theAyurvedic research. We here with forward this dissertation for the evaluation and adjudication. Seal & Signature of the HOD Seal & Signature of thePrincipal Dr. G. V. Mulagund. Dr. G. B.Patil.
  • 5. COPYRIGHT Declaration by the Candidate I here by declare that the Rajiv Gandhi University of Health Sciences,Karnataka shall have the rights to preserve, use and disseminate this dissertation /thesis in print or electronic format for academic / research purpose.Date: Signature of the CandidatePlace: Dr.Shivakumar S. Gangoor. © Rajiv Gandhi University of Health Sciences, Karnataka.
  • 6. CERTIFICATE BY THE CO-GUIDE This is to certify that the dissertation entitled “PHARMACOGNOSTICALSTUDIES AND ANTI-INFLAMMATORY EFFECT OF KAKAMACHI ONALBINO RATS ” – BY an Experimental Study is a bonafide research workdone by Dr. Shivakumar. S. Gangoor in partial fulfillment of the requirement forthe degree of Ayurveda Vachaspati in Drvyaguna. This work is applied, scientific and an original contribution in the field of researchin Ayurveda. I am fully satisfied with his original work and recommended the dissertation to beput before the adjudication. Signature of the Co- Guide Dr. KUBER.SANKH. M.D (AYU) Lecturer, Post Graduate Department of Dravyguna, D.G.M. Ayurvedic Medical College, Gadag.
  • 7. ABSTRACTBackground : Inflammation is fundamentally a protective response. These are exogenousand endogenous stimuli can cause cell injury. Due to cell injury there will bealterations in vascular caliber which leads to an increase in blood flow, changes inmicro vascular structure allow the plasma, proteins and leucocytes to leave thecirculation and get accumulated at the focus of injury leading to swelling. There aremultiple remedies available to reduce it with maximum side effects andcomplications. Inflammation is common pathological condition. It is livoited to any of the agegroup nor to any socio-economic class of society. It is having greater influence oftreatment factors and conditions. Since ancient times human societies have searched effective drugs of antiinflammatory actions. Kakamachi is also a best example for an anti-inflammatorydrug. The main objectives of the present protocol are1) Pharmacognostical Study: • Microscopic Evaluation • Standardization and validation • Extraction of phyto-chemical constituents. • TLC, UV and I R.2) Screening of fraction for anti-inflammatory activity.3) To evaluate the anti-inflammatory effect of Kakamachi on Carreginine induced albino rats in four different groups.
  • 8. Method:For thoroughly completion of present protocol needs fallowing essentialities such asa) Source of data: present study i) Literary source of data ii) Experimental source of data It includes phyto-chemical investigations of the extraction. i.e. fractionationand characterization etc..b) Method of Collection of Data: In the present protocol the dried powder of Solanum nigrum extracted withalcohol soxhlet apparatus is fractionated and is characterized. Fractionated principletested for anti-inflammatory activity by Carreginine induced Rat paw oedema.a) Measurement by Carreginine induced inflammation in the hind paw of albino rats.b) Inflammation is measured before and after administration of Carreginine.c) The Carreginine 0.01 ml is injected to the dorsum of the foot.d) After one hour and three hours the readings were taken with the help of plethismograph.Observation; In pharmacognostical study, morphologically colour of the leaves is greenwith bitter taste. Shape is of ovate with characteristics acceptable odour and physicalevaluations of these respective entities are established. In phytochemical observationsof extractive and different preliminary phytochemical tests are Observed andIdentification by TLC are done and focused U V and I R spectroscopy observation onthe experimental studies are done on the basis of selection of Animal sample size,preparation of the test drug.
  • 9. Interpretations and Conclusions: Different interoperations are done according to different points of view inconcern with the protocol and the study concern conclusions were drawn.Key words Kakamachi. Solanum nigrum. Pharmacognostical. Experimental. Anti-inflammatory.
  • 10. ABBREVIATIONSA.H. Ashtanga HridayaA.P.I Ayurvedic Pharmacopoeia of IndiaA.S Astang sangrahaB.P Bhava PrakashnighantuC.S. Charaka SamhitaChi. ChikitsasthanaD.N Dhanvantari NighantuGroup C Control GroupGroup S Standard GroupGroup T Trial GroupK.N Kaideva NighantuM.N. Madanpala NighantuM.E.S. Maximal Electro-ShockR.N. Raj NighantuS.N Sodal nighantuS.S. Susharutha samhitaSh. N Shaligram NighantuSu. SutrasthanaT.L.C Thin Layer ChromatographyP.N. Priya NighantuTemp TemperatureD.W. Distilled water
  • 11. ACKNOWLEDGEMENT I express my deep sense of gratitude to the Principal Dr. G. B. Patil andManagement Committee for giving me the opportunity to pursue my post graduationstudies in this institution. I am deeply inducted to my respected guide Dr. G. B. Mulgund M.D. (Ayu)Prof. & HOD Dept. of P.G. Studies in Dravya Guna for his inspiring paramountthoughts, zealous suggestions and guidance rendered for the successful completion ofthis research work. I also offer my deep felt gratitude to my co-guide Dr. Kuber Sankh M.D.(Ayu) for his invaluable support without which the study could not have materialized. I am thankful to associate guide Sri. B.S. Hunasi Lecturer Dept. ofPharmacognosy B.L.D.E. Pahrmacy College, Bijapur. and Botanist Sri. S.A. Kapalifor their timely guidance and help. The completion of this dissertation has a lot to dowith their constant support. I would like to express my profound gratitude to my department incharge Dr.G.S. Hiremath M.D. (Ayu), Dr. Shashidhar Nidagundi and Dr. Smt. Veena Kori fortheir guidance and inspiration given at various stages of my work. I express my sincere thanks to Sri. Nandakumar for his help in statisticalanalysis of results. I am highly indebted to Dr. A.I. Akki, Dr. S.V. Teggi, Dr. V. M. Sabarar, Dr.Wali, Dr. N.S. Shettar, Sri. Somashekahr T. for the able guidance and inspirationgiven at different stages of my work. I take this opportunity to thank the post graduate teaching staff of thisinstitution especially Dr. M.C. Patil, Dr. K.S.R. Prasad, Dr. Shivaramudu, Dr.
  • 12. Shashidhar Dodamani and other lecturers of our college for their help and suggestionsduring my post graduation studies. I sincerely thank to my beloved classmates Dr. Jagadeesh H., Dr. AshokBingi, Dr. Shivakumar Sajjan, Dr. Savita G., Dr. Anand, Dr. C.B. Inamdar and allclassmates of other post graduation branches for their constant co-operation and help. I wish to convey my thanks to beloved librarian Sri. V.M. Mundinmani andS.B. Sureban for providing essential references in the study. I wish to convey my thanks to beloved friends Mr. S.M. Teggi, Mr. B.A.Tenginkai, Mr. M.H. Vaijapur, Mr. R.M. Vaijapur for their co-operation. I remember with respect my revered parents for their inspiration andencouragement. I sincerely thanks to Smt & Sri. S.B. Gangoor for their profound and neverending love. I am grateful to my sisters Smt. Vijaylaxmi and Smt. Saraswati for theiraffection. I am highly indebted to my mother-in-law and father-in-law Smt & Sri.K.S. Anneppanavar for their affection and love. This is incomplete without remembering my beloved wife Smt. Sunita whohelped in all respects to complete this valuable dissertation work and lastly sonNiranjan & lovely daughter Akshata for their affection. I express my gratitude to all those who helped me directly or indirectly incompleting this work.Date :Place : Dr. S. S. Gangoor
  • 13. CONTENTSSl. No Particulars Page No i) Acknowledgement ii) Abbreviations iii) Abstract iv) List of Tables 1. Introduction 1-3 2. Objectives 4 3. Review of literature 5-54 Drug Review Disease Review 4. Methodology 55-70 Pharmacognostical study Phytochemical study Experimental study 5. Observations and Results 71-110 6. Discussion 111-119 7. Conclusion 120 8. Recommendation for further study 121 9. Summary 122-125 10. Bibliography 11. Annexure Photographs
  • 14. INTRODUCTION INTRODUCTION Ayurveda is a primordial system of medicine. The quest of the man is to livehappily, health is the elemental factor for happiness. The task of the medicine is topreserve and restore the health by relieving the sufferings. Understanding themedicine is essential to reach these both the goals. Pain is universally understood as asign of diseases. It is the most common symptom that brings a patient to physiciansattention. The knowledge of this condition, to the modern medical science is just twocentury old. This is well known to Ayurveda since 5000 years. The modern line oftreatment that provides a range of analgesic, physiotherapy and surgery but not a finalanswer and there is a common problem of reoccurrence. An Ayurvedic approach ishelpful to improve the quality of life in-patient suffering from shotha. Ayurvedic management measures seem to be more satisfactory because oftheir simplicity, applicability, and easy availability and cost effectively. It is a seriousburning problem of the society. It is our contractual obligation to provide propermanagement to the patients who are suffering from it and make them get through theproblem. In the drug review, the different references from Samhitas, Vedas, in modernbooks have been mentioned with different synonyms, having different importantvaluable properties. The useful part of Kakamachi has panchanga having with doses, churna 3 to 6gms., kasaya 50 to 100 gms, patra swarasa 25 ml, phala churna 3 to 6 gms and usesof Solanum nigrum according to external application and amayika prayoga. The whole work comprises different sections. First section deals with theReview of literature under various sub headings wherein literatures available from 1
  • 15. INTRODUCTIONdifferent references have been dealt. Material and Methods, Pharmacognostical study,Phytochemical and Experimental studies are made use in this work. Observations andResults, Discussions and Conclusion are given. It is hoped that the humble efforts in the form of this dissertation will help inunderstanding the effect of above mentioned measures as well as planning the futureresearch to find out the definite cure from Ayurvedic therapeutics. Inflammation is well known by all the human beings because each person ofthe world is certainly met with this condition at least once in their lifetime, whichaffects all the age groups and to both the sexes. It makes the human beings to feelfatigue, uneasiness, swelling, redness, and tenderness and disturb the day today life. Shotha is well known to mankind since the beginning, its references are foundin Vedas. Which are the older written documents of knowledge, mentioned thatinflammation is first disease to incarnate on the earth. In all Samhita’s, the shotha isexplained in detail. "Charaka" who is well known for his skills of chikitsa explained.It shows the redness, tenderness of the intolerable pain symptoms of shotha. Alsomany others like Brihathrayees, Laghutrayees and recent authors were resolvedsufficient literature to explain the Shotha (Inflammation). Inflammation is best defined in the teleogical terms, specifically it is a seriesof molecules and cellular responses acquired during evaluation, designed to eliminateforeign agents and promote repairs of damaged tissues. Unfortunately, these are notinfallible, pathogens have concurrently evolved inclusion to avoid elimination. Newpathogens occasionally emerge from the environment and under some circumstancesaberrant to inner inflammatory responses and damages the host. The complexity ofthe inner inflammatory system is a reflection of millions of years of mentalchallenges. It is becoming apparent that the system is not simple but rather a 2
  • 16. INTRODUCTIONcompletely opposite of responses that were assembled approximately and whereelicited may synergize autogenic each other. The cursory of inflammations are multitudinous. Almost anything that injuresliving tissues there occurs the inflammation. By fast the most frequent causes ofinflammation is trauma, the minor injuries that escape our attention as well, andinfection by Bacteria, Viruses, Fungi as Parasites, to these must be added a host ofothers cause exogenous and endogenous. Such as eradication, poisoning, metallicdisorders and derangements of the immune system. The body reacts to almost anykind of injury by inflammation and other kinds of responses may follow butinflammation is the first and most constant response. In the present Phytochemical study of Solanum nigrum extraction, thepreliminary phytochemical tests were conducted and observed the presence orabsence of Proteins, Alkaloids, Saponnins, Tannins, Triterpenoids and Carbohydrateswith their characterizational findings, also with UV and IR methods and appropriatetechniques on identification by TLC method were done. During experimental study total 24 albino Rats were taken and subjected themin four groups (Group I to IV). Group I was standard while Group II was trial group with minimum dose andwhile group III was trial group with maximum dose and where as Group IV wasVehicle group. Here Carreginine was induced to all the 24 Albino rats by injecting inhind paw technique method. First inflammation on hind paw is observed andmeasured. After induction of Carreginine to all the groups, immediately theinflammation was observed and measured. 3
  • 17. AIMS AND OBJECTIVES AIMS AND OBJECTIVES1. Pharmacognostical study- • Pharmacological Evaluation. • Microscopical Evaluation • Standardization and Validation • Extraction of Phyto chemical constituents • T.L.C, UV IR2. Screening of fraction for Anti-Inflammatory activity3. To evaluate the Anti-Inflammatory effect of Kakamachi on Carageenen induced albino rats in four different groups. 4
  • 18. DRUG REVIEW DRUG REVIEWBOTANICAL NAME: Solanum nigrum. i. KULA : KANTHAKARI. II. GANAS Charkokta ganas : Tikta skanda.BOTANICAL CLASSIFICATION: Classification according to Bentham and Hooker. Table No : 1 Kingdom Plantae Division Spermatophyta Class Dicotyledonae Sub-class Gamopetalae Order Polemoniales Series Bicarpellatae Family Solanaceae Genus Solanum Species nigrum According to Engler and Prantl. Table No : 2 Kingdom Plantae Division Embryophyta siphonogamy Class Dicotyledon Sub-class Sympetalae Order Tubiflorae Family Solanaceae Genus Solanum Species nigrum 5
  • 19. DRUG REVIEW According to Hutchinson Table No : 3 Phylum Angiospermae Sub-phylum Dicotyledones Division Herbaceae Order Solanales Family Solanaceae Genus Solanum Species nigrumDESCRIPTION OF FAMILY SOLANACEAE The family is spread over 90 genera and 2,000 species, Randle considered 85genera and 2,000 species, according to others the number of species included underthis is as much as 2,200. The genus Solanum alone has about 1,700 species. In Indiathe family is represented by approximately 21 genera and about 70 species.HABIT: Majority of the plants are annual herbs, some are shrubs, rarely soft woodtrees. Some of the plants are climbing or a few armed with spines. The genusSolanum with about 1,700 species shows diverse habits being herbs, shrubs, trees oreven creepers and woody climbers.VEGETATIVE CHARACTERISTICS: A branched taproot, the roots are thick and forked resembling human being inminiature, stem erect, climbing, spinous, tuberous, herbaceous, sometimes woody,branched, cylindrical, solid, sometimes hollow, hairy or glabrous. Leaves exstipulate, 6
  • 20. DRUG REVIEWpetiolate, rarely sessile, usually alternate, sometimes opposite, simple entire,sometimes pinnately divided, spinous, variegated, reticulate and unicostate.FLORAL CHARACTERISTICS: Inflorescence usually cymose, extra axillary and leaf opposed cymes, helicoidcymes, axillary umbellate cymess, rarely helicoid, sometimes the flowers are solitaryor terminal in the main or lateral axis. Flowers bracteate or ebracteate, hypogynous,bisexual, actinomorphic, complete sometimes zygomorphic, usually pentamerous,variously coloured, large and showy in many species, sepals 5, fused 5, partite orlobed –lobes sometimes 4-6, corolla tubular or companulate urceolate when mature,ovate or imbricate, persistent, in Physalis calyx persistent and develops a bladder likehusk round the fruit, petals 5,fused,rotae, companulate, infundibular, lobed with 5 ormore lobes or bilabiate, ovate, sometimes imbricate, hairy on the outside variouslycoloured, covered with stellate hairs. Stamens 5,sometimes 4, free, alternate with thepetals, epipetalous, filaments usually of unequal length, rarely the stamens are 4 andare didinamous or only two, anthers two celled usually coming close together to forma cone round the pistil, dehiscing by terminal pores or longitudinal slits, connective,sometimes becoming tetra or tri to penta locular due to the formation of a false septa,placentation axile, ovules many in each locule placed on a swollen and obliqueplacenta. Fruit a berry, seeds small, numerous, smooth or pitted, endospermic,endosperm fleshy, embryo curved.FLORAL FORMULA+, K(5), C(5), A 5, G(2) 7
  • 21. DRUG REVIEWFLORAL DIAGRAMFigure No 1.USEFUL PLANTS: S. tuberosum, S. xanthocarpum, S. melongena, S. nigrum.DESCRIPTION OF SOLANUM NIGRUMCOLLECTION: Kakamachi patra is collected in varsharutu.SYNONYMS TABLE NO : 4Bhavaprakash Nighantu Kakamachi,Dhwanksha, maachi, Kakahava, Vayasi.Nighantu Adarsha Kakashva, Vayasi.Dhanavantari Nighantu Kakhva, Vayasi, Katvi, Katuphala, Rasayanavara.Raja Nighantu Vayasi, Kakamaachi, katu.Kaideva Nighantu Kakasahava, kakamaachi, Kamata, Jaganephala, Sarvatikta, Bahuphala, Swadupakaphala, Kamachi, Kakini, Kushtaghni, Vayasi, Dhwanksha maachi, Gudaphala, Rasayanavara, Katu. 8
  • 22. DRUG REVIEWVERNACULAR NAMESArabic : Ambussalap, Enabeddir.Assam : PichakatiBengal : Gurkamai, Kakmachi, Mako, Tulidun.Bombay : Ghati, Kamuni, Mako.English : Black Nightshade, Garden Nightshade, Hounds Berry, Morelle, Petty morel.Gujarat : Piludi.Hindi : Gurukamai, Kabaiya, Makoi.Latin : Solanum nigrum.Malayalam : Trong parachichit.Marathi : Ghati, Kakmachi, Kamoni, Laghukavali, Meko.Persian : RubahtareekPortuguese : Herva moura, Solano.Punjab : Kakmach, Kambei, Kwansaf, Mako, Riaungi.Sanskrit : Bahuphala, Bahutikta, Dhvankshamachi, Ghanaghana, Gucchaphala, Jaghenephala, Kaka, Kakamachi, Kakamata, Kakini, Katuphala, Kushtaghni, Rasayani, Sarvatikta, Sundari, Svadupaka, Tiktika, Vayasavha, Vayasi.Sindha : KanperumSinhalese : Kalukanweriya, Kalukungwareiya, Tibbatu.Tamil : ManattakkaliTelugu : Gajuchettu, Kachi, Kakamachi, Kamachi, Kanchipundu, Kasaka.Urdu : Makoya. 9
  • 23. DRUG REVIEWTYPES:In Nighantu two types are mentioned. They are1. Shweta2. RaktaMORPHOLOGY OF SOLANUM NIGRUM Solanum nigrum is wild herb growing for most part of the year in wasteplaces, sometimes reaching a height of 90 cm. A variable annual, stem erect, glabrous or more less pubescent, muchdivaricately branched. Leaves numerous, 2.5-9 by 2.5 cm, Ovate-lanceolate, subacuteor acuminate, thin, entire sinuate toothed, tapering in to the petiole; petioles 2 cmlong. Flowers small, in extra axillary subumbellate 3-8 flowered cymes; peduncles 6-20 mm long, slender; pedicels 6-10 mm long, very slender. Calyx 3 mm long,glabrous or nearly so; lobes 5, oblong, obtuse, 1.25 mm long, not enlarged in fruit.Corolla 4-8 mm long, divided more than ½ way down into 5 oblong subacute lobes.Filaments short, flattened, hairy at the base ; anthers 2.5 mm long, yellow, oblong,obtuse, nothed at the apex. Ovary globose, glabrous; Style cylindric, hairy. Berry 6mm in diameter, globose, usually purplish black but sometimes red or yellow, smoothshining. Seeds discoid, 1.5 mm in diameter, minutely pitted, yellow. Calyx 5 sepals,gamosepalous, lobes oblong, acute, white, vivate or imbricate aestivation.Corolla- 5 petals, gamopetalous, rotate, lobes oblong, acute, white, twisted or ovateaestivation.Androecium- 5 stamens alternate to petals, polyandrous, epipetalous, filaments short,anthers oblong, connivent in a cone like structures, bicelled, introse, yellow, dehiscingby apical pores. 10
  • 24. DRUG REVIEW Gynoecium- 2 carpals (bicarpellary), syncarpous, ovary obliquely placed in the flower, ovary superior, globose, bilocular, axile placentation, many shining ovules present on placenta, style simple hairy at the base, stigma one. DISTRIBUTION: Throughout India, Ceylon and all temperate and tropical regions of the world. PROPERTIES Table No : 5Name of Text Rasa Guna Viryo Vipaka Karma DoshaghnataRaja Nighantu Tika, Laghu Ushna Katu Vajeekara Rasayana Katu Shotha hara Tridosha haraDhanavantari Tikta Laghu Ushna Katu Swarya, Sarakam,Nighantu Kushtanashaka, Vajeekara Rasayana Tridosh haraKaideva Tikta Snigdha Ushna Katu HridyaNighantuNighantu Laghu Shukra vardhaka, Tridosha Swaraya, nashaka SarakaBhavaprakash Tikta, Snigdha, Ushna Katu Swaraya, ShukraNighantu Katu Ushna janana, Rasayana, Tridosha Netraya, Shotha hara nashaka KushtaghnaPriya Nighantu Tikta Ushna Katu Balya, Rasayana Shotha hara, Mutrala YakritrogaAdarsha Katu Ushna Katu Kushtaghna,Nighantu Tikta Shothaghna Rasayana Tridosha haraSusruta Samhita Katu Naatius Mushika visha Tikta hna nashaka, Tilla roga Sheeta nasahaka Tridosha haraCharaka Katu Naatius Kushtaghnam Tridosha haraSamhita Tikta hna Rasayanam Sheeta 11
  • 25. DRUG REVIEWPARTS USED Pushpa, Phala, Panchaga, Beeja.MATRA (DOSE) : Table No : 6 1. Swarasa 10-20 ml 2. Phala Choorna 1-3 gm 3. Arka 20-50 mlTOXIC EFFECTS: Excessive intake of fruit of Solanum nigrum leads to chardi, Trishna,Udarashoola, Taraka Vispharana, Shirashool, Bhrama, Pralap, Akshepa, Sanyasa andat the severity may lead to Death.TREATMENT FOR TOXIC EFFECTS: Treatment is as similar to Dhatura visha chikitsa.USES OF SOLANUM NIGRUMExternal Uses : Shotha hara, Vrunashodhana, Vedana sthapana, Savarnikarana.Internal Uses :Digestive System: Deepana, Yakrut uttejaka, Pittasaraka and Rechaka.Circulatory System: Hridya, Rakta shodaka, Shothahara and reduces hypertension.Respiratory System : Kaphaghna, Hikkanigrahana and Shwasahara.Urinary System : Mootrala.Skin : Swedajanana and KushtaghnaFever : Jwaraghna. 12
  • 26. DRUG REVIEWTHERAPEUTIC USES:1. Useful in diseases of the heart , eyes, piles, leucoderma, itch, worms in the ear, dysentary, hiccough, vomitting, asthama, bronchitis, fever, urinary discharges, improves the voice, favour conception and facilitate delivary and rat bite.2. The root bark is laxative and useful in the diseases of the ear, eye, and the nose. Good for ulcers on the neck, burning of the throat, inflammation of the liver, chronic fever, gripping and is harmful to the pregnant women.3. The Leaves have a bad odour and taste and used for headache and the diseases of the nose.4. The fruit is useful in thirst due to fever and inflammation.5. The seeds are laxative and are useful in giddiness, gonorrhea, thirst and inflammation.6. In Bengal the berrys are employed in fever, diarrhoea, eye diseases and hydrophobia.7. The juice in doses of six to eight ounces is given in the treatment of chronic enlargement of the liver and considered as valuable alternative. It acts as a hydrogogue catharic and diuretic.8. The syrup acts as an expectorant and diaphoretic, used as a cooling drink in fevers.9. In North-western province the juice is used in blood spitting, piles, dysentary etc.10. A decoction of the leaves in the dosage of drachm thrice daily in the treatment of dropsical affectin by the mooden sheriff. Its action is diuretic and laxative.11. The young shoots are given in the chronic skin diseases and psoriasis in the konkan region. 13
  • 27. DRUG REVIEW12. The Chinese employ the juice of the leaves to alleviate the pain in inflammation of the kidneys , bladder and in virulent in gonorrhoea.13. The plant is given internally for cardialgia and externally in nephritic colic, corroding ulcer, suppurating chancre and in severe burnings and herpes.14. The decoction of the leaves is used as diuretic and depurative.15. The plant is used by Europians for convulsions. The herb is one of the native remedies for local application to anthrax pustules and a paste of the green berries is applied to ringworm. The Xosas use the plant for disinfecting anthrax infected meat.16. The Zulus administered as an enema to infants with abdominal upsets. The Sutos rub the burnt and powdered root in to the incisions on the back for relief of lumbgo.17. The fruit in combination with other drug is prescribed for snake bite and scorpion sting, but it is not an antidote to either snake venom or scorpion venom.18. The leaves are employed as poultice over rheumatic and gouty joints and as a remedy in skin diseases.19. Freshly prepared fluid extract from all the portions of the plant has been recommended in dropsy, in heart diseases, skin diseases, piles, gonorrhoea, inflammatory swellings and chronic cirrhosis of the liver and seen in doses of ½ to 2 drachms.20. A Syrup of the plant is useful as an cooling drink in fevers and promotes perspiration.21. Heated warm leaves are applied to the painful and swollen part of the testicles.22. Decoction of the berries and flowers are useful in cough. 14
  • 28. DRUG REVIEWviii) CHEMICAL COMPOSITION The analysis of the leaves gave the following values in 100 gm of ediblematerial.Moisture- 82.1Protein- 5.9Fat- 1.0Minerals- 2.1Carbohydrates- 8.9Calcium- 410 mgPhosphorus- 70 mgIron- 20.5Leaf is a rich source of Riboflavin. The values for various vitamins present in the leafare (in 100 mg edible material)Riboflavin- 0.59Nicotanic acid- 0.92Vitamin C- 11 mgThe higher values for the vitamin C (20-40 mg) have been reported.The fruits contain-Glucose-15-20%Vitamin –CB-Carotene Green unripe fruits, however contain glycolalkaloids, consumption of thesame leads to toxic hazards to human beings as well as to the livestocks. Ripe fruitcontains very little alkaloids and can be consumed without ill effects. 15
  • 29. DRUG REVIEW Seeds forming 9.5 percent of protein on dry weight basis. They yield agreenish yellow oil-21.5 % with the following physical and chemical constituents-Specific gravity-0.9198Acid value- 11.62Saponification value- 184.0Acet value- 25.7Iod value- 123.2Hehner value- 92.9R M value- 0.66Unsapon matter- 1.4 %The component fatty acids of the oil areLinolic- 46.63Oleic- 49.73Palmitic- 1.76Steraic- 1.88The unsaponifiable matter contains sitosterol. Immature green fruit of the plant contains four steroidal glycol alkaloids viz; • Solamargine • Solasonine • Solanigrine • Solanigrinine All these yeild Solasodine as the aglycone. It also contains a steroidal genin,tigogenin. (0.101 %) Solanigrine and (0.431 %) Solasonine are present in leavesrespectively. 16
  • 30. DRUG REVIEWECONOMIC IMPORTANCE : Economically the Solanaceae family is fairly important. It comprises severalplants of food value, medicinal value, vegetable and ornamental values. In whichSolanum nigrum posses medicinal properties. The fruits are given in fevers, diarrhoea,eye diseases and hydrophobia. The juice of plant is given in chronic enlargements ofthe liver, in bleeding piles, dysentery. The fruits are edible and very much liked by thechildren. 17
  • 31. DISEASE REVIEW REVIEW OF INFLAMMATION1. INTRODUCTION There are many features in the normal structure and physiologies of the body,which serve, protect it against injury. The continuity of skin and mucous surfacesprotects to a considerable extent against minor injuries. Normal or increased secretionfrom nose, eyes, urinary tract. Movements like that of eyelids or cilia of the trachealmucosa chemical constituents such as of gastric juice is other instances of naturaldefense mechanism against injury or bacterial invasion. Reflexes such as sneezing andcough are definite protective mechanisms, immune bodies present in the body fluids,which may be increased by the survival of natural disease and by the artificialprocesses, endow the organism with an important means of defense. Inflammation in addition to above means seems to be a better defensivereaction against an injury locally. It may be associated with general changes such asfires, leukocytosis and development of immune substances, but it itself operates onlyat the site of injury. The same exogenous and endogenous stimuli that causes cell injury also elicita complex reaction in vascularized connective tissues called inflammation. Reducedto its simplest terms, inflammation is a protective response intended to eliminate theinitial cause of cell injury as well as the necrotic cells and tissues resulting from theoriginal insult. Inflammation accomplishes its protective mission by dilating,destroying or otherwise neutralizing harmful agonists (e.g. microbes or toxins). It thussets into motion the events that eventually heal and reconstitute the sites of injury.Thus inflammation is also intimately interviews with repair processes wherebydamaged tissue is replaced by the regeneration of parenchymal cells, and loss byfilling of any residual defect fibrous scar tissues. 18
  • 32. DISEASE REVIEW Although inflammation helps to clear infections and repairs, makes woundhealing possible, both inflammation and repairs have considerable potential to causeharm. Thus inflammatory responses are the basis of life threatening anaphylaticreactions to insect bites or drugs, as well as to cause certain diseases such asrheumatoid arthritis and athero-sclerosis, other harmful examples includeinflammation in the peritoneum leading to fibrous bands that cause intestinalobstruction or pericardial inflammation resulting in lenses encasing scar that impairscardiac function. The inflammatory response has many roles; these include circulating cells andplasma proteins, vascular wall cells and extra cellular matrix of the surroundingconnective tissue. The circulating cells include bone marrow derived polymorpho-nucleus. Leukocytes (neutrophils), eosinophils, basophils, lymphocytes, monocytesand platelets, the circulating proteins include clotting factors; kininogens andcomplement components, largely synthesized by the vascular wall cells include theendothelial cells in direct contact with the blood as well as the underlying smoothmuscle cells that are important to the vessels. The connective tissue cells includesentinels, macrophages and lymphocytes, in addition to the fibroblasts that synthesizethe extra cellular matrix and can proliferate to fill in a wound, the extra cellular matrix(ECM) consist of fibrous structural proteins (e.g. collagen and elastin), get formingproteglycous and adhesive glycoprotein (e.g. fibronectin) that are the cells – FCM=ECM-ECM connectors, as we see, these all interact to resolve local injury andrestore normal tissue function. Inflammation is divided into two basic patterns. Acute inflammation is ofrelatively short duration, lasting from a few minutes to a few days and is characterized 19
  • 33. DISEASE REVIEWby fluid and plasma protein exudation, a predominantly neutrophilic leukocyteaccumulation. Chronic inflammation is of longer duration (day to years) and typicalby influx of lymphocytes and macrophages associated with vascular proliferation andscarring. However these basic forms of inflammation may have many factors andhistological appearances. The reaction of vascularized living tissue to local injury, invertebrates with novascular system, single celled organisms, and multicellular parasites have their ownresponses to local injury. These include phagocytosis of the injurious agents,endropment of the irritants by specialized cells haemocytes, which ingest it andneutralization of noxious stimuli by hypertrophy of the cell or one of its organelles.The inflammatory process in higher forms is the reaction of blood vessels leading tothe accumulation of fluid and blood cells. The inflammatory response is closely interwined with the process of repair, itserves to destroy, dilute the injurious agent, but sets into motion a complex series ofevents, heal and reconstitute the damaged tissue as far as possible.2. DEFINITION AND TERMINOLOGYDEFINITION: Inflammation is defined as the local response of living mammalian tissues toinjury due to any agent. It is a body defense reaction in order to eliminate or limit thespread of injurious agent as well as to remove the consequent necrosed cells andtissues.TERMINOLOGY: The majority of inflammatory serous are named by adding the suffix It is tothe anglicized form of the Greek hour of the tissue or organ involved. Thus we speakof dermatitis, gastritis, meningitis and so on. 20
  • 34. DISEASE REVIEW The suffix It is derived from the Greek for belonging to the names ending in Itis do not imply the cause of the inflammation. For example, dermatitis may be due tothe infection or chemical injury, radiation or immunologic reaction and so on. All thename implies is inflammation of the skin. Inflammatory diseases have been known since ancient times and like others ofancient lineage, they sometimes bear strange names derived from the past Erysipelas aspreading Streptococcal infection of the subcutaneous tissue of the face was named bythe great Hippocrates 300 years before christ pneumonia is another old term still incommon use, these terms will be defined when we come to the diseases of the organsaffected. Inflammation is best defined in teleological terms, specifically it is a series ofmolecules and cellular responses acquired during evaluation designed to eliminateforeign agents and promote repair of damaged tissues. Unfortunately, these responsesare not infallible. Pathogens have concurrently evolved mechanism to avoidelimination; new pathogens occasionally emerge from the environment and undersome circumstances aberrant immuno inflammatory response damages the host. Thecomplexity of the immuno inflammatory system is a reflection of millions of years ofenvironmental challenges. It is becoming apparent that the system is not simple butrather a complex composite of responses that were assembled opportunitically andwhen elicited may synergize or antagonize each other.3. HISTORICAL BACKGROUND For centuries, humans have identified inflammation fire, undoubtedly, as aresult of the experience of reduces heat and pain associated its occurrence.Interestingly scientific investigation of inflammation has extended this analogy. Atthe microscopic level, inflammation is described as an accumulation of leukocytes 21
  • 35. DISEASE REVIEWthat “spread” within tissues and then ultimately “burn out” and heal or lead“smoldering’ conditions. Similarly at the molecular level, leukocytes use an oxidativemechanism in essence a form of biologic fire that destroys micro-organisms anddamages tissues. Despite the essential truth of our intuitive sense of inflammation, theobjective under starting has come slowly. At least since the eighteenth century, inflammation has been considered asmanifestation of immunity, but the mechanisms governing inflammatory events haveremained enigmatic until recent decades. During the late nineteenth and much of thetwentieth centuries, important advances were made regard to manipulating gum oralimmunity as evidenced by the development of vaccines and the use of antiserum.These great advances tended to over shadow the role of phagocyte cells in resistanceto infection as promoted by the Zoologist Elie Metchnikoff like wisehistopathologists. Such as Rudolf Virechow and Julius Cohnheim speculated thatcellulus and vascular components of inflammation were critical elements, but theycould not ascertain full significance or relationship to humeral immunity. It is nowunderstood that inflammation is a dynamic interaction of humeral of cellularresponses. Recently studies of inflammation have focused on revealing the“molecules language” that dictates the observed events clearly. There is a complex eraof both humeral and cellular signals that determine the quality, intensity and durationof inflammation. The features of inflammation were described in an Egyptian paper around3000 BC. celsus, a Roman writer of the first century AD listed the four cardinal signsof inflammation, they were rubor (redness) tumor (swelling), calor (heat) and dolor(pain). These signs are more prominent in acute inflammation than in chronicinflammation. Later Virechow added the fifth sign of functiolaesa (loss of function). 22
  • 36. DISEASE REVIEW Inflammation is not a disease but a non-specific response that has a salutoneffect on its host, this obvious fact was noticed by the Scottish surgeon John Hunter in1793. Julius Cohnheim (1839-1884) who provided one of the first and bestmicroscopic description of inflammation and observed inflamed blood vessels in thinand transparent membranes such as in the mesentery and tongue of frog. Noting theinitial vasolidation and changes in blood flow, the subsequent oedema caused byincreased vascular permeability and the characteristic leukocyte emigration. Elie Metchnikoff, a Russian biologist in 1882 discovered the process ofphagocytosis by observing the ingestion of rose thorns by amebocytes of starfishlarvae and of bacteria by mammalian leukocytes and concluded that the purpose ofinflammation was to bring phagocytic cells to the injured area to engulf invadingbacteria. Metchnikoff contradicted the prevailing theory, that the purpose ofinflammation (humoral theory of immunity) was to bring in factors from the serum toneutrilize the infectious agents. It became clear that both cells (phagocytes) and serumfactors (antibodies) were critical for defense against micro-organisms, thisstrengthened by the discovery of the antitoxins by Behring and Kitasato (1890) On the basis of simple experimental studies of inflammatory response on skin.Sir Thomas Lewis established the concept that chemical substances such as histaminelocally induced by injury, mediate the vascular changes of inflammation, thisfundamental concept underlies the important discoveries of chemical mediators ofinflammation and the use of anti-inflammatory agents.4. CAUSES OF INFLAMMATION1. The outstanding cause is injury by living agents; these can induce progressive inflammation due to two basic procones, early inflammatory response and 23
  • 37. DISEASE REVIEW healing. These processes generally have protective role against injurious agents. Inflammation is distinct from infection, is being a protective response by the body and invasion into the body by harmful microbes and their ill effects by toxins. The causes may be due to physical agents i.e., heat, cold radiation, mechanical trauma.2. Chemical agents i.e., organic and inorganic poisons3. Infective agents like bacteria, viruses and their toxins4. Immunological agents like cell mediated and antigen antibody reactions. CAUSES OF INFLAMMATION CHART NO – 1 INJURIOUS AGENTS Living agents Non-living agents Physical agents Chemical agents I) Mechanical trauma I) Strong acids ii) Presence of foreign body ii) Strong alkalies iii) Under heat and cold iii) poisons iv) Pressure v) Ionising radiation5. SIGNS AND SYMPTOMS The Roman writer Celsus described four cardinal signs of inflammation as (a) Rubor (Redness): An acutely inflamed tissue appears red due to dilatation of small blood vessels within the damaged area. E.g., Skin affected by sunburn, cellulites by bacterial infection etc. 24
  • 38. DISEASE REVIEW (b) Calor (Heat): Increase in temperature is seen only in peripheral parts of the body such as skin. It is due to increased blood flow (hyperaemia) through the region resulting in vascular dilatation and delivery of warm blood to the area. E.g., Systemic fever. (c) Tumor (Swelling): Swelling results from oedema, the accumulation of fluid in the extra vascular space as part of the fluid exudates and to a much lesser extent, from the physical mass of the inflammatory cells migrating into the area. (d) Dolor (Pain): Pain is the best-known feature of acute inflammation. It results partly from stretching and distortion of tissues due to inflammatory oedema and in particular from pus under pressure in an abscess cavity. Chemical mediators like bradykinin, prostglandins and serotonin are known to induce pain. (e) Loss of function (Functio laesa): Movement of an inflamed area is consciously and reflexly inhibited by pain, while severe swelling may physically immobilize the tissues.6. CLASSIFICATION OF INFLAMMATION CHART NO-2 CLASSIFICATION OF INFLAMMATIONAcute inflammation Sub Acute inflammation Chronic inflammationi) Catarrhal 1) Diffusedii) Fibrinous or Serofibrinous 2) Suppurativeiii) Suppurative or purulent 3) Granulomatousiv) Haemorrhagic 4) Fibrinoidv) Pseudo membranousvi) Allergic 25
  • 39. DISEASE REVIEWI. ACUTE INFLAMMATION:a) CATARRHAL ACUTE INFLAMMATION: This is confined to mucous membrane, the mucous cells of the lining and ofthe mucous glands secrete large quantities of mucin, there is molecular desquamationof the surface lining, not so pronounced as to appear as ulceration. The discharge onthe surface or in the lumen consists of mucous plasma, leucocytes and desquamatedepithelial cells, it is mucoid in the early stages and becomes mucopurulent later.Acute catarrhal inflammation is mildest form of inflammation, but in some organs itcan progress to termination and can become chronic. E.g. Rhinitis of common cold,catarrhal bronchitis.b) FIBRINOUS OR SEROFIBRINOUS INFLAMMATION: This is seen in the serous membranes, the pericardium, pleura, peritoneum etc.It consists of plenty of fibrin in the exudates derived from the exudate plasma,irrespective of the nature of the irritant, they react in a stereotyped manner.Neutrophils and macrophages are seen in the fibrinous network.c) SUPPURATIVE OR PURULENT INFLAMMATION: This type is characterized by the formation of pus. In solid tissues and organssuppurative inflammation causes Abscesses, in contrast to the circumscribed affectionin abscess formation the process may become diffuse and spreading. Diffuse lesionsin the connective tissue are described as “Phlegmonous Inflammation or Cellulites”.d) ACUTE HAEMORRHAGIC INFLAMMATION: In certain cases, bacterial toxins produce intense injury to the capillary walland allow large number of Red Blood Corpuscles to escape, making the exudateshaemorrhagic. E.g. In Influenza Pneumonia, Acute Glomerulonephritis etc. 26
  • 40. DISEASE REVIEWe) PSEUDO MEMBRANOUS INFLAMMATION: This form of inflammatory reaction is characterized by the formation of amembrane usually made up of precipitated fibrin, necrotic epithelium andinflammatory white cells. The reaction is encountered only on mucosal surfacescommonly in the Pharynx, Larynx, Respiratory passage etc. The membrane formationresults from an acute inflammation response to a powerful necrotizing toxin, outpouring of exudates traps the necrotic and cellular debris producing a dirty gray-white, rubbery membrane on the eroded surfaces. E.g. In Diphtheria.f) ALLERGIC INFLAMMATION: In this, Inflammation is primarily and exclusively by antigen-antibodyreactions. The injury in this is due to the occurrence of antigen-antibody union onwalls of the tissue cells, hypersensitivity has much greater significance. The vascularchanges are associated with a cellular exudates consisting of neutrophils, eosinophils,plasma cells and macrophages. Eosinophils may be spectacular components, tendencyto necrosis and tissue decay due to accelerated reaction and increase in the Phagocyticpower of the Leucocytes with increase of eosinophils are distinct features in allergicinflammation.2. CHRONIC INFLAMMATION The acute inflammatory response is unable to remove or neutralize aninjurious agent; the response is modified to chronic. It is not usual for a chronicresponse to last many months but years. As the inflammatory process continues, fluidexudates diminishes and cellular response assumes dominance, the chronic responseis dominated by a massive build up of cells in the affected tissue. These cells areprimarily macrophages and lymphocytes. In chronic inflammation the agent and thehost are just capable of resisting each other. The agents involved are of low inevitable 27
  • 41. DISEASE REVIEWability. They are unable to penetrate deeply in to or spread throughout the body of thehost. Such agents may be bacteria, fungi, larger parasites. Foreign bodies which areinsoluble in body’s fluid can also elicit a chronic inflammatory response. Regardless of the specific nature of the inciting agent, its presence in the tissuepromotes a long term conflict with the phagocytic cells of the host; heavy infiltrationby the inflammatory cells progressively interferes with normal function. When theprocess continues over a month and years, the function detoriates as tissue isdestroyed, accumulating inflammatory cells replace functional tissues and scarringdevelops. This deterioration ultimately leads to somatic death.TYPES OF CHRONIC INFLAMMATIONSFollowing Four types are included in Chronic Inflammation: 1) Chronic Diffused Inflammation 2) Chronic Suppurative Inflammation 3) Chronic Granulomatous Inflammation and 4) Chronic Fibrinoid Inflammation.1) CHRONIC DIFFUSED INFLAMMATION: Exudates, which may be diffused or focal shows lymphocytes, plasma cellsand macrophages, under certain stimuli macrophages develop into epitheloid cells andmultinucleated giant cells. Fibroblasts are present, in older lesions fibrosisconspicuous. E.g. Chronic Ulcer.2) CHRONIC SUPPURATIVE INFLAMMATION: It is a non-specific inflammatory cell infilteration, in which infiltration bypolymorphs and abscess formation. E.g. Actinomycosis 28
  • 42. DISEASE REVIEW3) CHRONIC GRANULOMATOUS INFLAMMATION: It is characterized by the formation of Granulomas, a tiny lesion composed ofepithelial cells and lymphoid cells at the periphery, also granulomas may have giantcells, necrosis and fibrosis. It is seen in specific infective granulomas as inTuberculosis, syphilis. Leprosy etc.4) CHRONIC FIBRINOID INFLAMMATION: It is a degenerative phenomenon like rheumatoid arthritis, reheumatic feveretc. DIFFERENCES BETWEEN ACUTE INFLAMMATION & CHRONIC INFLAMMATION Table No 7Acute inflammation Chronic Inflammation1.Duration: Usually for days or weeks 1. Duration: For months or Years2. Cardinal Signs: Present 2. Cardinal Signs: Doubtful or not perceptible3.Vascular Changes: Present 3.Vascular Changes: Not Marked4. Exudation of Plasma: Present 4. Exudation of Plasma: Doubtful or Absent5. Cellular Exudates: 5.Cellular Exudates:Neutrophils initially lates Macrophages and Histocytes Plasma cells lymphocytesFibroblasts Stage of repair Lymphocytes Fibroblasts are present Neutrophilsare few. absent or very less in numbers. 29
  • 43. DISEASE REVIEW7. GENERAL MECHANISM OF INFLAMMATION The starting point of inflammation is the cell damage, living or inert, the cellswhich do not damage are not capable of producing inflammation. However once thedamage has occurred the reaction takes place inevitably and proceeds through adefinite series of events to its ultimate end, i.e. repair of damage and restoration offunction. CHART NO- 3GENERAL MECHANISM OF INFLAMMATION • Vascular phenomenon changes Vasodilation Active hyperemia Capillary dilation Static of blood Sludging of red cells in the capillaries Pavementation of leukocytes Exudation of fluid and out pouring of polymorphs • Cellular response Local Exudation Fluid of exudation Cells of the exudation General response Fever degeneration Leukocytosis • Repair of tissues 30
  • 44. DISEASE REVIEW Degenerative Albuminous degeneration a. Repuls degeneration b. Healing cloudy swelling c. Regeneration Fatty degeneration a. Suppuration b. Abusers c. Boil d. Carbuncle e. Cellulites Proliferative Phagocytes a. Attachment b. Engulfment (i) Azophil (ii) Special groused c. Killing and degeneration (i) Oxygen dependent mechanism (ii) Oxygen independent mechanismCells included in the inflammatory exudates are : • Neutrophil • Eosinophils • Mast cells • Lymphocytes 31
  • 45. DISEASE REVIEW • Plasma cells • MacrophagesMECHANISM OF INFLAMMATION -Management of inflammation and action of non-steroidal anti-inflammatory drugs(NSAID) Irrespective of the nature of causative factors or the type of inflammation, thetissue reaction in first few hours is stereotyped and similar. Its pathology can beunderstood in the fallowing steps. 1.Vascular phenomenon 2.Cellular phenomenon 3.Repair1.Vascular phenomenon:Following changes will occur in vascular phenomenon one by one i. Vasodilatation: Proceeded by transient vasoconstriction ii. Active hyperemia iii. Capillary dilatation iv. Stasis of Blood v. Sludging of red cells in the capillaries. vi. Pavementation of leukocytes. vii. Exudation of fluid and out pouring of polymorphs.There are number of cells present in the inflammatory exudates which performvarious functions. Those are a. Neutrophils b. Eosinophils 32
  • 46. DISEASE REVIEW c. Mast cells d. Lymphocytes e. Plasma cells f. MacrophagesA brief description and the role is as follows.a) Neutrophils: Neutrophils or Polymorphs are the cells along with Basophils and Eosinophils are known as granulocytes, due to the presence of granules in the cytoplasm, contains substances like proteases,myloperoxidase and alkaline phosphates. Neutrophils are actively motile, get collected arround blood vessels and passes through the tissue by active amoeboid movements, there from the first line of defense in bacterial infections.b) Eosinophils: These are larger than the Neutrophils and constitute 2-4% of the total blood leucocytes. They appear only at the sites of inflammatory exudates of diseases of immunological origin. They remain in the circulation for a short peroid and rapidly attracted in the tiisues by the raised concentration of released histamine. Eosinophils are phagocytic in nature. These may share many structural and functional similarities with neutrophils, like their production in the bone marrow, locomotion, lobed nucleus and presence of granules in the cytoplasm. Granules of eosinophils are rich in myeloperoxidase than neutrophils and lack lysozyme. High level of steroid harmone (eosinopenia) leads to fall in number, and even disappear from the blood. These may be increased in certain conditions like Allergy, Parasitic infestations, skin diseases and certain malignant lymphomas.c) Mast cells: These cells contain coarse basophilic granules in the cytoplasm and a polymorphous nucleus, These granules are laden with heparin and 33
  • 47. DISEASE REVIEW histamine. The functions of mast cells in normal Human being is not vivid. They are believed to produce the acid.d) Plasma cells: These are larger than the lymphocytes with more abundant cytoplasm and an eccentric nucleus. These cells are normally not seen in peripheral blood. They develop from lymphocytes and are rich in RNA and Gama-globulin in their cytoplasm. There is an inter relationship between Plasmocytosis and Hyperglobulinaemia. These cells are most active in antibody synthesis. The number will increase when prolonged infection with immunological responses. e.g. Syphilis, Rheumatoid Arthritis, Tuberculosis, Hypersensitivity states and multiple myeloma.e) Macrophages: These are large mono-nucleated cells play an important role in the stages of Acute and Chronic Inflammations. It is believed that many lymphocytes derived from the blood are converted in to microphages at the site of inflammation, by increase in cytoplasm and enlargement of the nucleus and are derived from Kuffer cells of the liver and histocytes. They form the scavenger cells of inflammation, combine to form giant cells and need phagocytic action. The giant cells are mainly of : i. Tumour Giant Cells a. Anaplastic Cancer Giant Cells b. Reed-Sternberg Giant Cells and c. Giant Cells of Tumour ii. Foreign Body Giant Cells.i. Tumour Giant Cells: It is of fallowing typesa. Anaplastic Cancer Giant Cells: 34
  • 48. DISEASE REVIEW These are larger and have numerous nuclei which are hyper chromatic andvary in size. These giant cells are not derived from macrophages but are formed fromdividing nuclei of the neoplastic cells.b. Reed-Sternberg Giant Cells: These are malignant tumour giant cells , which are binucleate and are seen invarious histologic types of Hodgkins lymphomas.c. Giant Cells of Tumour: These cells are usually found in the bones, uniform distribution andosteoclastic giant cells spread in the stroma.ii. Foreign Body Giant Cells:- These contain numerous nuclei, which are uniform in size and shape andresemble the nuclei of macrophages, these nuclei are scattered throughout thecytoplasm, these are usually seen in the chronic infective granulomas.2.CELLULAR RESPONSE: Cellular response can be explained in to a) Local response and b) Generalresponse. The local response starts with the pavementing and emigration ofleukocytes before the cells of the damaged tissue release some chemicals that bringabout all the changes. The Vasodilation and increased permeability is due to thesubstances like "leukotoxin" and "histamines" are produced by the damaged tissuecells. These stimuli activate both Hematogenic and Histogenic cells, which carry outvarious functions like vascular phenomenon, pavementation and emigration ofleukocytes and various other cellular responses. 35
  • 49. DISEASE REVIEW Following a local injury, there is a transient constriction in the vessels thatcause local Ischemia. This mommentary constriction is followed by an active phase ofhyperemia with dilation of vessels, this dilation chiefly occurs in arteries and venulesand at last in the capillary bed. The active hyperemia lasts for a few hours. Thecapillaries get changed and become prominent. The blood vessels keep on dilating and the active hyperemia is fallowed by thestagnation of blood, which is termed as stasis and the clotting takes place in thevessels. The blood supply to the tissue is lost and necrosis sets in the vasodilation thatfollows the transient vasoconstriction (due to neural reflex) It is recognized that asensory fibre has a vasodilator branch to an arteriole. When there is an injury, there isa reflex vasoconstriction and thus vasodilation sets in. The vasodilation occurs under the influence of chemical mediators also, it waspostulated by Lewis (1927) that "H" like substance cause vasodilation. FurtherMenklin postulated about "Leukotoxin". In addition, it increases the vascularpermeability and responsible for the emigration of leukocytes, the slowing of bloodflow in the vessels may be attributed to swelling of the endothelial lining of the bloodvessels, vasodilatation that decreases the pressure. Loss of blood fluid in interstitialspaces makes the blood viscous. There is sludging of red cells. The red cells becomesticky and adhere to one another in masses and to the walls of the vessels. In this slower blood stream, rearrangement of the corpuscles takes place, undernormal condition, the red and white cells flow intermingled in the central part of thevessels forming an axial stream, which is separated from the wall by a clear plasmaticzone free from cells. When there is some degree of injury, the leukocytes fall out ofthe axial stream and come to occupy the plasmatic zone adhere to the vessel wall andseem to drag themselves along with difficulty. In this way, the inner wall of the 36
  • 50. DISEASE REVIEWcapillary becomes paved by a broken line of leukocytes without the admixture of asingle red blood cell. This arrangement is called pavementing of leukocytes. Normally the endothelial cells of the blood vessels and blood cells repel oneanother due to change in the electrical potential, where these carry negative chargeswith them, hence the repulsion. The endothelium becomes positvely charged and theleukocytes are the first of the cellular elements to be attracted to and adhere to thelining of the vessel. The endothelial cells also become enlarged, proliferated and thus assumeround shape. The inter-endothelial space widens and through these spaces leukocytesemigrate.A) EXUDATION: After dilation of blood vessels, the solid and fluid contents of plasma as wellas of the blood cells pass through the vessel walls and constitute within the tissues.Thus, the fluid is rich in protein contents and the cells constitute the exudates.B) FLUID OF THE EXUDATE: Normally the walls of the blood vessels are permeable to the fluid, some ionicsalts and the molecules with molecular weight less than 10,000 Daltons, large part ofthe fluid get reabsorbed and the remaining is carried by lymph channels. But during inflammation, this exudate fluid, which originates from plasmadiffers from it in several aspects. Its solid contents are almost double than the contentsof the lymph. Its specific gravity is 1.020. The main reason is that serum albumin andserum globulin is present in exudate. The fluid molecules come out of vessels due toincreased permeability of vessels. 37
  • 51. DISEASE REVIEW Besides this protein, the exudate contains various extractions like Urea, fibrinforming elements, mucin ferments and immune bodies, oxidases, lipases and trysin.All type of immune bodies may be found including cytolysin, haemolysin,bacteriolysin, agglutinins, opsonins and complement fixing bodies. The formats andthe immune substances are distinctly higher in oedema than oedema produced due toother causes.FUNCTION OF FLUIDS OF EXUDATE :1. It dilutes the soluble poisons and irritates, thereby reduces their direct effect.2. It provides over runs of escape for the metabolites which are formed in excess.3. It maintains normal hydrogen concentration. The protolytic enzymes serve to complete the solution of the tissues whichhave been injured or killed, thus aid in their removal. Exudate is rich in fibrin formingproteins. The environment in which it is present favours fibrin formation, this servesas the limits of extent of the inflammatory process. The fibrin mesh in the lymphaticsserve as a filter and with solid materials especially bacteria, if it is not dissolving byprotolytic enzymes it provides a frame work on which connective tissue grows andhealing takes place.C) CELLS OF THE EXUDATES: After pavementing the leukocytes emigrate in extra vascular spaces, as theyemerge from the outer margin of endothelium, a new basement membrane formsbetween them and the endothelium disappears permitting release of the leukocytes into the extra vascular space without leaving any defect behind them, under theinfluence of various chemical mediators, this phenomenon is known as "Chemotaxis". 38
  • 52. DISEASE REVIEWD) PHAGOCYTES: Phagocytes can be resolved in to three distinct ways 1. Attachment of the Particle to the surface of the phagocyte. 2. Engulfment. 3. Killing and degeneration of the ingested microbe or particle.1. Attachment of the Particle to the surface of the phagocyte: These cells are recognized and attracted to bacteria by chemotactic factorsreleased by bacterial products as well as by tissue proteins, in order to establish abond between bacteria and the cell membrane of phagocytic cells, the microorganisms get coated with opsonins which are naturally occurring factors in theserum. The main opsonin present in the serum IgG opsonin, is the Fe fragment ofimmunoglobulin G, antibody in the serum that coats the bacteria. C3b opsonin is thefragment of completement, which is generated by activation of completementpathway. Lactins are carbohydrate-bonding proteins in the plasma, which bind tobacterial cell wall. Receptors mediated attachment of opsonized bacteria has been therecognization step of Phagocytosis.2. Engulfment: Leukocytes are able to respond to opsonins for that they display leukocytebinding sites, once an opsonin coated particle is bound to the surface receptors of thephagocyte, the particle is readily engulfed. Binding of a particle to the phagocyticmembrane elicits the engulfment, the phagocytic membrane flows around the particleto enclose it in a cytoplasmic phagosome. Granular appearing lysosomes that contain 39
  • 53. DISEASE REVIEWdestructive agents then merge with the phagosome membrane, thereby bringing theircontents in to contact with the particle. As lysosome merge with the phagosomes theirnumber within the phagocyte are reduced and the phagocyte is degenerated. If theparticle is too large to be easily engulfed i.e. a multicellular parasite. There may beregurgitation of the granule contents in to the tissue spaces. The leukocytes attemptingto engulf this large surface experiences frustrated phagocytosis and releases toxic anddegradative substances that damage the basement membrane and the surrounding cellsand the matrix. The neutrophil cytoplasm contains two types of granules Azusophil andSpecific granules. Lysosomes contain acid hydrolases, neutral proteases, cationicproteins, lysozyme.i) AZUSOPHIL: These lysosomes contain acid hydrolases, neutral proteases, cationic proteins,lysozyme.ii) SPECIFIC GRANULES: Which contain lysozyme and lectoferiin but no hydrolases or peroxides.Macrophages also contain azusophil granule. The process of degranulation pour in tothe phagosome powerful enzymes which kill the bacteria by different mechanisms.3. KILLING AND DEGENARATION: The micro-organisms after being killed by antibacterial substances aredegraded by hydrolytic enzymes, their mechanism fails to kill and degrade somebacteria.These are of mainly two types, Oxygen dependent and Oxygen Independent.1. Oxygen dependent: It is an important mechanism of microbicidal killing by the production ofreactive oxygen metabolites (O2 H2O2, OH, HOCL, HOL, HOBr) a phase of increased 40
  • 54. DISEASE REVIEWoxygen consumption by activated phagocytic leukocytes in presence of NADPHoxidase.The NADPH oxidase which is present in the cell membrane of phagosomereduces oxygen to superoxide ion (O2). 2O2 NADPH 2O2 (superoxide anion) 2O2 + 2H─ H2O2 Suproxide is subsequently converted in to Hydrogen peroxide (H2O2) hasbactericidal properties, this bactericidal activity is carried out either enzymemyeloperoxidase present in the granules of neutrophils and monocytes or the enzymemyeloperoxidse acts on Hydrogen peroxide in the presence of halides (Chlorides,Iodide, Bromides) to form hypophalous acid (HOCl ). Which is more potentantibacterial agent than hydrogen peroxide. Reactive oxygen metabolites are useful in eliminating microbial organismsthat grow within phagocytes.2. Oxygen Independent: This mechanism involves the release of substances that damage bacterial cellwalls, disrupt bacterial replication and produce a low pH within the phagosomesresulting from accelerated glycolysis, Which may be directly toxic and may indirectlyaid the function of other enzymes.b) GENERAL RESPONSE: The general celleular respons may be Fever and Leukocytosis.i. Fever: Fever is one of the most prominent systemic manifestation, particularly ininflammation associated with spread of organisms into the blood stream. The patientmay have high fever charecterised by dramatic swings in the temparatur. The origin ofthe fever may be uncertain, although it may be caused by the release of bacterial 41
  • 55. DISEASE REVIEWendotoxins. In addition interleukin – 1 (IL-), prevously described as endogeneouspyrogen released from the leukocytes is an important mediator of hyper pyrexia. Thismediator is taken up by lymphatics from the site of imflammation, which is circulatedin blood stream. It is believed that IL-1 initiates fever by inducing the synthesis ofPGE2 in the anterior hypothalamas and stimulating thermoregulatory centres.ii. Leukocytosis: Increase in the number of circulating white cell is another charecteristicsignificance of acute and chronic inflammation. When the inflammation is deep andthe local symptomatology is either not observes or impossible to demonstrate, thenleukocytosis is of assistance in determining the severity of the infection and thedegree of the resistance offered by the body. This requires not only an absolute count(i.e. the total number of white cells per mm) but also the relative number of each typeof leukocytes particularly the number of neutrophils. In making the differential count,the number of each kind of white cells in hundred is counted. • A high absolute count (T.L.C) with a high neutrophil percentage indicate severe infection and good body resisitance. • A high absolute count with a moderate neutrophil percentage indicates a moderate infection and good body resisitance. • A low absolute count with high neutrophil percentage indicates severe infection and weak resisitance of the body. • All inflammatory states do not evoke neutrophilic leukocytosis. Infectious mono nucleoses, whooping cough, mumps, rubella and undulent fever charecteristicaly produce lymphocytosis. 42
  • 56. DISEASE REVIEW Allergic inflammatory reaction like hay fever, bronchial asthama and parasiticinfection typically elicit eosinophilia. Some infections may cause leukopenia instead,like infections caused by viruses, rickettsiae, protozoa and salmonelloses.C. TISSUE CHANGES OR REPAIR: There are two types of tissue changes a. Degenerative b. Proliferative.a. Degenerative: The two most common degenerations are 1. Albuminous degeneration or cloudy swelling 2. Fatty degeneration1. Albuminous degeneration or cloudy swelling: It is closely related to hydrophic or vascular degeneration. Being manifestationof a disturbance of protein metabolism, is called as Albuminous degeneration. It maybe caused by the bacterial toxins, chemical poison, malnutrition and otherdisturbances. The principle organs showing cloudy swelling are Kidneys, Liver and Heartmuscles. The organ affected is slightly enlarged owing to swelling of the cells ofwhich it is composed. It is pale as blood vessels being compressed by the swollencells. The cut surface has rather cloudy appearance and slightly opaque.2. Fatty degeneration: Fatty degeneration or Fatty metamorphosis is a true sickness of cells causedby some injurious influences. It is best seen in Liver, Kidney and Myocardium. Thefat metabolism is interferred with fat accumulations in the cell. In some cases there ismerely unmarking of fat already present. The causes of fatty degenerations are 43
  • 57. DISEASE REVIEW • Poisons • Anoxia The organs look fatty. The liver and kidneys are pallor and softer than normal.The heart is soft and flabby. Ultimately the tissue becomes necrosed. If the irritant is intense, the effect is degenerative destruction. If it is mild, actsas stimulant and leads to proliferation. At the centre of the inflammatory area theaction of the irritant is severe, degeneration predominates at the periphery and theaction is mild. The tissue may be stimulated to proliferation. thus this part of theinflammation is known as repair or healing.i) SUPPURATION: If the dead tissue in an inflammed area undergoes softening and liquification isknown as the process of Suppuration, the fluid formed is called the Pus, by thismethod the dead material is removed from the body.There are three requisites forsuppuration. i. Necrosis ii. Presence of sufficient leukocytes iii. Digestion of the dead materials by protolytic fermentation. If any one of these are absent suppuration does not occurs.The digestiveenzymes are produced mainly by the leukocytes to a lesser extent by the necrosedtissue cells and the infecting bacteria. These are neutralized by anti enzymes presentin the serum. If there is less leukocytes or more serum liquification does not takeplace.ii. ABSCESS: It is an example of a localized suppuration. The inflammation is limited to thearea and as the irritant is pyogenic, pus is produced. The cells in the centre of the 44
  • 58. DISEASE REVIEWinflammatory area are killed and liquified by protolytic enzymes. In this way a cavityis produced which contains pus. The wall of the abscess cavity consists of damagedbut still living tissues. Hence the spread of infection is limited, this limiting zone iscrowded with polymorph nucleus leukocytes and macrophages filled with debris. Puscells are continuously dicharged from this, thus the abscess is chronic or if theinfection is dying out, the macrophages will greatly outnumber the polymorphsfurther out, the tissue become more and more normal. If the infection is continuously active more and more material is added to theabscess, So that the pressure within the abscess increases. Thus it points in thedirection of less resistance. If the abscess enters the muscle sheath, it may extentalong a considerable distance.iii BOIL: It is an abscess of a Sebaceous gland or a hair follicle caused by theStaphylococcus aureus, which has penetrated the opening of the duct. There is amarked fibroblastic proliferation, which with intercellular formation of fibrin, causesthe characteristic indusation. The tension becomes high and causes pain. There maybe a very little liquification of the necrosed tissue, So that the centre of the boil iscomposed of a solid "Core" instead of Pus.iv. CARBUNCLE: It is the infection spread to the subcutaneous tissue where it causes a morediffused lesion which discharges on the surface by a series of openings. The pusbecomes inspected and the dead tissue is converted into a mass of fatty debris inwhich lime salts may be deposited. 45
  • 59. DISEASE REVIEWv. CELLULITIS: When the suppuration spreads through the tissues, the condition is calledcellulitis. The fibrin that forms as a result of proliferation, inflammed part limit theinfection.3. REGENERATION: This implies to a complete renewal of the tissue as in liver. The process ofhealing if fundamentally the same in all the wounds. It consists of two parts. Removalof inflammatory material and necrotic debris, which may be more or little.Replacement or reconstruction of the original tissue to a greater degree as much aspossible. The repair involves the invasion and replacement of dying and dead tissue byimmature mesenchyma called Granulation tissue, which is a highly vascular tissue.On account of its cellularity a granulating surface has a remarkable power ofresistance against bacterial infection. The granulation tissue grows to maturity frombelow to up words. When the wound is aseptic the epithelium will grow in from theedges as a delicate blue pellicles, gradually it becomes thick and opaque. When the surface is covered by epithelium, the process of devascularizationbegins. The new vessels, being no more needed will gradually disappear. The scar thatis red becomes white and bloodless. 8. REVIEW OF ANTI-INFLAMMATION Drugs which can normally be used to almost every type of inflammatoryconditions are known as anti-inflammatory drugs These have two major groups. 1. Steroidal anti-inflammatory drugs in the form of gluco corticoids. 2. Non-Steroid anti-inflammatory drugs (NSAID)Mode of action of steroidal anti-inflammatory drugs 46
  • 60. DISEASE REVIEW ACTH and Glucocorticoids prevent the clinical features of inflammation i.e.local heat, redness pain and swelling. Their action is based on reducing the increasedpermeability of capillaries and maintenance of the integrity of the cell membraneeven in the presence of toxins. Stabilization of lysosomes release from thegranulocytes by inhibiting phagocytosis. Also acts on fats phase of inflammation andinhibits capillary proliferation deposition of collagen cicatrisation. But fibrous tissueonce formed is not dissolve by corticosteroids. Hence the anti-inflammatory effect of steroidal therapy is non specific. Thesteroid inhibit the phospholipase enzyme which is essential to activate the cellmembrane. This activation release the arachidonic acid, which metabolises to produceinflammation. But once the chain is initiated it cannot be stopped by the steroidaltherapy, moreover in large doses, it may interface with wound healing.9. CLASSIFICATION OF ANTI-INFLAMMATION These are broadly classified into two groups. Narcotic Analgesics or opoidanalgesics and Non-narcotic analgesics or non opoid analgesic or NSAID, nonsteroidal anti-inflammatory drugs.1. NARCOTIC ANALGESICS: These agents are capable of relieving severe degree of pain but are moderatelyor strongly addicting. This group includes opoids which binds to the opoid receptors.These, further classified in to Natural opium alkaloids ( e.g. Morphin)., Semi-synthetic opiums (e.g. Diacetyle morphin or acetyle morphin) and Synthetic opoid(e.g. Pethedine).2. NON-NARCOTIC ANALGESICS OR NON OPOID ANALGESIC orNSAID: 47
  • 61. DISEASE REVIEW In this the agents relieve mild to moderate degrees of pain and are consideredas non active. This group includes aspirin analgesic and other analgesics andantipyretic drugs and these have no affinity for the opoid receptors, but the site ofaction is only peripheral and further NSAID is divided in to Potent anti-inflammatoryand good analgesics, Potent anti-inflammatory and good analgesics, Moderate anti-inflammatory and moderate analgesics and Poor anti-inflammatory and goodanalgesics.A. POTENT ANTI-INFLAMMATORY AND GOOD ANALGESICS: i. Salcylates e.g. Aspirin 2. Oxy cum derivatives e.g. Paraoxicam.B. POTENT ANTI-INFLAMMATORY AND GOOD ANALGESICS: i. Pyrazolin derivatives e.g. Phenyl butazone. ii. In dol derivatives e.g. Indomethacin.3. MODERATE ANTI-INFLAMMATORY AND MODERATEANALGESICS: i. Propyonic acid derivatives e.g. Ibuprofen ii. Anthranlic derivatives e.g. Mefenamic acid. iii. Aryl acetic derivatives e.g. Diclofenac.4. POOR ANTI-INFLAMMATORY AND GOOD ANALGESICS: i. Para amono phenyl derivatives e.g. Paracetamol or acetaminoidene. ii. Pyrozolon derivatives e. g. Metamezole.10. MANAGEMENT OF INFLAMMATION AND ACTION OF NON- STEROIDAL ANTI INFLAMMATORY DRUGS (NSAID) 48
  • 62. DISEASE REVIEW As stated earlier, the activation of cell membrane releases arachidonic acid, itis metabolized by cyclo-oxygenase pathway and produce prostaglandins (PGs).Theseinclude PGG2, PGH2, PGl2, PHE2 and PGE2 and cause vasodilatation and potentiateoedema. Some of these are supported to inhibit platelet aggregation. These further sensitize the chemical receptors of the afferent pain endings toother chemical mediators like bradykinin and histamine. Aspirin and asperin likeNSAID have been shown to inhibit release or synthesis of PG5 and thus producebeneficial anti-inflammatory condition where PGs are synthesized locally. It is unableto produce any analgesic effect where the sensory nerves are directly stimulated. Some of the NSAID produce anti-inflammatory effect indirectly,likeindomethacin. It inhibits phosphodiesterase and thus increases the intracellularconcentration of cyclic ATP, Cyclic ATP has been shown to stabilize membraneincluding lysosomal membrane in polymorphonuclear leukocytes. This prevent therelease of enzymes important in the inflammatory responses. Some drugs may inhibitthe activation of T-lymphocytes which release lymphokinase, which plays animportant role in mediating inflammation. 9. REVIEW ON SHOTA Nidana mean the causative factors which lead to the disease. nidanaparivarjana is the first line of treatment. According to different classics the nidanas ofshota can be classified as follows: 1. Ahara sambhandhi 2. Vihara sambhandhi 3. Anya.1. Ahara sambhandhi: Abhakta, Gunaheena Bhojana sevana by krisha and durbala person, kshara, Amla teekshana ushna, Guru padartha Adhika 49
  • 63. DISEASE REVIEW sevana,Dadhi, Apakwa Bhojana, Mrittikia, Shaka, Viruddha Bhojana, Dushta Bhojana, Gara Visha.2. Vihara sambhandhi: Nishkriyata, Ashodhita,3. Anya: Marmopaghata, Vishama Prasooti,POORVAROOPA Daha, Engorged Veins, Anga gourava.ROOPA Roopas are those which are well manifested by which diseases are diagnosed.The samanya laxana of shota are as fallows-Sagourava • Anavashtitatwa • UshnataUtsedha Wherever there is utseda it is considered as shota.NIDANA • Siratanutwa • Saloma harsha • VivarnataBHEDA All the shothas are of single type but according to hetu vishesha they are againdivided in to different types. Charaka in his chikista sthana has mentioned seven types of shotha. They areas fallows:1. Vataja2. Pittaja3. Kaphaja 50
  • 64. DISEASE REVIEW4. Dwandaja5. Sannipataja6. Abhigataja7. Vishaja. Where as Susruta has mentioned only five types of shotha, they are as follows:1. Vataja2. Pittaja3. Kaphaja4. Sannipataja5. Vishaja. Vagbhata in his Nidana sthana has mentioned Seven types of shotha1. Vataja2. Pittaja3. Kaphaja4. Dwandaja5. Sannipataja6. Abhigataja7. Vishaja. Where as in Madhava nidana we get again seven types of shotha1. Vataja2. Pittaja3. Kaphaja4. Dwandaja5. Sannipataja6. Abhigataja7. Vishaja. 51
  • 65. DISEASE REVIEWTable showing the different types of shota according to different classics. Table no:8Bhedha Ch Su Va MnVataja + + + +Pittaja + + + +Kaphaja + + + +Dwandaja + _ + +Sannipataja + + + +Abhigataja + _ + +Vishaja. + + + + Looking the types of Shothas according to the different classical texts, wecome across similarity in view of Charak, Vagbhata and Madhava nidana i.e. theSeven types of shothas are explained, where as in Sushruta we get only five types ofshotha.SAMPRAPTI Vata gets vitiated with its own vitiating factors which carries the rakta, pittaand kapha to utthana siras which gets obstructed there and settles in between the twakand mamsa. This sanchaya of rakta, pitta causes utseda which results in shotha.SADHYASADHYATA Before going to start the chikista of a particular disease, one should knowabout the sadhyasadhyata of a disease, i.e. whether it is easily curable, with efforts orincurable etc should be known. According to prabhava, the diseases are classified assadhya and asadhya. Sadhya is subdivided a sukha sadhya and krichra sadhya whereas asadhya is subdivided as yapya and pratyakhya. 52
  • 66. DISEASE REVIEW If the rogi is krisha or Durbala or if associated with upadravas, when it movesto the marma sthana, ruja yukta and associated with srava and sarvangashotha isconsidered to be asadhya. Where as in aheenamamsa, ekadoshaja, nava, balavanvyakti shotha is sukha saadhya.UPADRAVA Chardi, trishna aruchi,swasa, jwara, atisara,dourbalya, pipasa, hikka, kasa arethe updravas of shotha.CHIKITSA SOOTRA Depending on bala, dosha and kaala, one has to see the nidana, dosha, rutu andviparita chikitsa has to be done.CHIKITSASamanya chikitsa1. In ama dosha yukta firstly langhana and pachana2. In bahu dosha avastha shodana3. Shotha in adha pradesha-virechana4. Shotha in sira pradesha shiro-virechana.5. Shotha in urdhwa pradesha vamana6. If shotha has occurred due to excess sneha vastu sevan then ruksha kriya is to be done.7. In vataja shotha if vibhanda of mala is present niruhabasti to be given.Oushadhis used in Shotha1. Gandiradyarishta2. Astashataarista3. Punarnavadyarishta 53
  • 67. DISEASE REVIEW4. Phalatrikaadyarishta5. Kshragutika6. Kamsaharitaki7. Chitrakaghrita.PATHYA Kulatha yusha, Trikatu, Yavakshara choorna, Mudga, Vishakeira, Jangalapashu pakshi mamsa, Koorma, Shiki, Shallka mamsa rasa, Sauvarchala, Grinjana,Patola, Vayasi, Moolaka, Vetra, Nimba, one year old Yava and Shaali dhanya.APATHYA Gramya, Jaleeya, Anupa mamsa, Lavana, Shushka, Navanna, Gouda (GudaPadartha), Pishtanna, Dadhi, Tila, Vijjala, Madya, Amladravya vallura, Samashana,Guru,Asatmya, Vidahi anna, Divaswapna and Maithuna. 54
  • 68. MATERIALS AND METHODS MATERIALS AND METHODSA) Pharmacognostical study:Aim: The aim of present study was to see Morphological, Microscopical anddetermination of pH of Panchanga of Kakamachi.(Solanum nigrum linn.).1) MORPHOLOGICAL STUDY:Materials: The materials collected for the studies were.Drug: Solanum nigrum linn. (Kakamachi)Parts: LeavesCollection of Materials: The leaves of solanum nigrum linn were collected freshlyfrom local viscinity.Equipments: Sense organs.Chemicals: Formalin Aceto alcohol (F.A.A), Chloralhydrate, Conc HCL.Methods:i) Organoleptic Method ii) Extra features.i) Organoleptic Characteristics: In this method nature of the leaves, colour, taste, size, shape, odour, were studied with the help of sense organs.ii) Extra Features: The arrangement of leaves and its special characters were studied.2) MICROSCOPICAL STUDY:Materials: The materials collected for the study were.Drug: Fresh leaves and fine powder of leaves of Solanum nigrum linn.Equipments: Compound microscope, eye piece, camera lucida, Glass slides, coverslips, watch glass, camel brush, mountain brush, filter paper, blades, spirit lamp,pipettes.Chemicals: Fluroglycenol, Chloral hydrate, Conc HCL, Glycerin and Iodine. 55
  • 69. MATERIALS AND METHODSMethods: 1) Section Method 2) Staining process method.1) SECTION METHOD: • Put the sample in a test tube add sufficient chloral hydrate solution. • Hold the sample vertically in between the thumb and fore finger • With the help of a new blade, a thin transverse section of the sample is taken • 10-15, thin, sufficient sections were taken .Thick and oblique sections are rejected. • With the help of mountain brush the sections are transferred to a watch glass containing water.2) STAINING PROCESS: • Transverse section of the sample was taken and transferred it on to a slide with the help of mountain brush • Add a few drops of water • Add a few drops of chloralhydrate solution and allowed to heat for two to three minutes. • Add equal proportions of fluro-gylcenol and Concentrated Hydrochlroic Acid, warm gently on a flame and cool it , finally add a drop of glycerin and cover the section carefully with coverslip, focus the section under microscope and the arrangements of cells were studied.MATERIAL:Drug: Solanum nigrum linn.Parts: Leaves (Coarse powder) and thin sections of leaves.Equipments: Compound microscope, eye piece, camera lucida, Glass slides, coverslips, watch glass, camel brush, mountain brush, filter paper, blades, spirit lamp,micro meter. 56
  • 70. MATERIALS AND METHODSCHEMICALS: Fluro-glycenol, Chloral hydrate, Conc HCL, Glycerin.METHODS: Stomatal number, Stomatal index, Veinislet number, Vein terminationnumber, Palisade ratio.i) Vein Islet and Vein Termination:METHODOLOGY: • 3-4 pieces of fresh or dried leaf are cut from the middle portion of the lamina avoiding midrib and margin. • These are taken in a test tube and boiled with chloral hydrate solution in a water bath, until they are clean enough for observation. • Different cleaning methods are applied for individual leaves that mainly have very thick lamina • The pieces are taken on a watch glass and mounted on a glass slide in chloral hydrate solution with lower surface of the leaf facing upwards so that the veins which are more prominent in the lower surface are seen clearly under the microscope. • For this study 6x eye piece and low power objectives were used. The stage micrometer is focused (1 mm) and the camera lucida is fixed in such a way that the aperture of it is in the same line with that of the eye piece. • Black drawing sheet was placed on the same side of the microscope where camera lucida is fixed. • Using a white pencil the first and the last of the stage micrometer was removed and the slide was mounted with the leaf specimen and focused in the same way. 57
  • 71. MATERIALS AND METHODS • The square drawn in the paper was adjusted in such a way that it lies exactly in the middle of the field of vision and the image of the leaf piece mounted appears to be superimposed on the square of the drawing sheet. • Starting from one side all the vein islets inside the square as well as on the boundary were traced. The vein termination within the square only was taken in to account.ii) STOMATAL NUMBERS: The stomatal number is a very specific criteria for identification andcharacterization of leafy crude drugs.METHODOLOGY: • Stomatal number is the average number of stomata per mm2 of epidermis and the number on each surface of a leaf. Each stomata consist of two guard cells and the spore is counted as single unit. • It is indicated by the symbol S iii) STOMATAL INDEX: • Stomatal index is defined as the percentage of stomata from the total number of epidermis cells, which can be explained as S= The number of stomata in a given per unit area of leaf. E = Total number of epidermal cells including trichomes in the area of leaf.METHODOLOGY: • Fragments of leaf of 5x5 mm2 in size were taken in a test tube containing about 5 ml of chloral hydrate and heated on a water bath until the fragments are transparent, using the same procedure as described earlier in veinlet termination number determination. 58
  • 72. MATERIALS AND METHODS • A peeling was easily taken by partially cutting one of the veins with a blade on the lower epidermal region and pulling it. • This membranous layer which came out along with the vein was taken on a glass slide by cutting the thick vein section attached to it. • A few drops of chloral hydrate solution are added and the cover slip is placed on top. The peeling was sufficiently big enough to cover the entire field of view when mounted, A 10x eyepiece and high-powered objective were used. 1 mm2 of area is taken with the help of a stage micrometer as described in the previous section. On the tracing paper a circle smaller than that of the field of circle/square is clearly visible in the centre of the field of vision. Stomatal Index = S x 100 E+Siv) PALISADE RATIO:METHODOLOGY:• Small pieces of leaf from the apex taking the middle and basal portion of the lamina, were taken from the leaves, the lower epidermis was peeled off and then cut into pieces.• It was boiled gently in a test tube taking 4-5 leaf pieces in about 5 ml of strong solution of chloral hydrate till the green colour disappears. Then the pieces were kept separately on a glass slide with its upper epidermal layer kept upper most.• It was focused under high power objective with 15x eyepiece. Four clear continuous epidermal cells were observed and then the fine adjustment knob of the microscope is turned down slowly to observe the palisade cells.• After tracing four continuous epidermal cells, moving the fine adjustment of the microscope, palisade cells inside all the 4-epidermal cells were drawn. 59
  • 73. MATERIALS AND METHODS• The area of the sample was changed and the experiment should be repeated to those that are 50 % or more inside the outer boundary of 4 epidermal cells are taken in to account.• The number of total palisade cells were divided by 4 which give the average number of palisade cell under each epidermal cell.v) pH Value:EQUIPMENTS: A pH meter is used to analyze and measure the pH having a reading scaleextending from 1 to 14.Methodology: By electro metric method• In this method hydrogen electrode may be used to measure hydrogen ion concentration and also pH• The potential, which is created between electrode and the solution is reproducible and its magnitude depends upon the concentration of hydrogen ion.• A constant electrical potential against which the potential of the glass electrode can be measured is provided by the reference electrode. This consists of a metallic internal element immersed in a electrolyte saturated solution of KCL.PHYTOCHEMICAL STUDY:Aim: The main aim of the Phytochemical study was to know the chemicalconstituent in a trial drug, subjecting to different tests like extraction, preliminaryphytochemical tests and isolation of extracted fractions characterized by T. L. C, U .Vand I. R method. 60
  • 74. MATERIALS AND METHODSSOLUBILITY OF SOLANUM NIGRUM LINN. :Materials: Fine powder of panchanga of Solanum nigrum linn. (Kakamachi)Solvents:1) Petroleum ether, 2) Ethyl Acetate ,3) Chloroform, iv) Ethyl alcohol etcMETHODOLOGY: Taken in to the different filter paper in different funnels according to thedifferent solvents after seeing residue which was minimum that solubility of the drugin that solvent is maximum.1) EXTRACTION:MATERIALS:Drug: Coarse powder of panchanga of Solanum nigrum linn. (Kakamachi)Equipments: Soxhlet apparatus of 1,000 ml, round bottom flask, water condenser withdistillation apparatus, beakers 500 ml measuring cylinder, thermostat, electronicweighing machine, filter paper, magnetic stirrer, boiling chips etc.Chemicals: 90 % ethyl alcoholMethods: The air dried panchanga of Solanum nigrum linn. drug was subjected toexhaustive extraction, by soxhlet apparatus by around 18 hours with 90 % ethylalcohol, extraction was done in three batches of these , one batch of coarse powderwith ethyl alcohol. The extraction process is carried out for about 18 hours to eachbatch. After the extraction the solvents were distilled off to obtain semi solid extractand concentrated on magnetic stirrer, the weights of each batch of extract wererecorded. 61
  • 75. MATERIALS AND METHODS2) Preliminary phytochemical tests:MATERIALS;Drug: Extractive sample of Solanum nigrum linn.(Kakamachi)Equipments: Test tube with holder, stand, spirit lamp, pipette, glass rods, beaker 50ml to 250 ml, conical flask, waterbath, burner with stand.Chemicals: 10% conc H2SO4, chloroform solution, acetic unhydride, sulphur powder.soda lime, millions reagent, mercuric sulphate 10%, Sulphuric acid 1%, sodiumnitrate 5%, sodium hydroxide 1%, copper sulphate , 10% tannic acid, acetyl chloride,zinc chloride, Mayers reagent(Saturated picric acid) Solution, Dregendroffs reagent(Potassium bismoth iodide) Ammonium renicate, Molishs reagent, Barfords ,benedits reagent, saponin, ferric chloride, fragments pieces of magnesium ribbon andconcentrated hydrochloric acid, zinc dust, sodium hydroxide, 10% lead acetate,Bromine water etc.Methods:(i) Test for sterols:(a) Salkwoski’s test: A few drop of concentrated 10% sulphuric acid was added to the 5 ml sample solution, shaken and allowed to stand.(b) Liebermann- Burchardt test: The sample solution 5 ml of extract mixed with few drops of acetic unhydride. Where concentrated 10% sulphuric acid (H2SO4) sulphuric acid (H2SO4) was added from the sides of the test tube.(c) Sulphur test: Sulphur when added to the extract, it sinks in it showing the presence of sterols.Preparation of test solution: Dissolve 0.5 ml of test solution in 100 ml of water by heating and using thesolution for following test. 62
  • 76. MATERIALS AND METHODS(ii) Test for Proteins: (a) Test solution + Soda lime and heat (b) Test solution + Millon’s reagent mixed and allowed to stand (c) 1 ml test solution + 1 ml 10% mercuric sulphate in 10% sulphuric acid. Boiled gently for 30 seconds added 2 drops of 1% sodium nitrite solution. (d) Biuret test: 3 ml test solution + 1 ml 5% sodium hydroxide with + 2 drops of 1% CuSO4 solution mixed and allowed to stand. (e) Test solution + few drops of 10% tannic acid mixed and allowed to stand.(iii) Test of Triterpenoids: (a) Salkwoski’s test: A few drops of 10% concentrated sulphuric acid is added to the 5 ml of sample solution, shaken and allowed to stand. (b) Liebermann’s – Burchardt test: The sample solution mixed with few drops of acetic unhydride, in which concentrated sulphuric acid (H2SO4) is added from the sides of the test tube. (c) “Tschugajew test ”: Where excess of acetyl chloride and a pinch of zinc chloride are added to the sample containing, kept aside and warmed on water bath.(iv) Test for alkaloids: (a) Mayer’s test: The sample solution with mayer’s reagents ( potassium mercuric iodide) mixed and allowed to stand (b) Wagner’s test: sample solution with wagner’s reagents (iodine in potassium iodide) mixed and allowed to stand. (c) Hager’s test: sample solution with Hager’s reagents (Saturated picric acid) mixed and allowed to stand. 63
  • 77. MATERIALS AND METHODS (d) Dragendroff’s test: Sample solution with Dragendroff’s reagent ( Potassium Bismuth iodide) mixed and allowed to stand.v) Test for Carbohydrates: a) Molisch test: sample solution with few drops of molisch reagent and 2 ml of concentrated 10 % H2SO4 added slowly to the sides of the test tube. b) Barford’s test: sample solution with Barford’s reagent boiled on a water bath. c) Benedict’s test: sample solution with Benedict’s reagents and boiled on a water bath.vi) Test for Saponin’s: (a) Foam test: sample solution mixed with saponins and shaken, shows formation of froth. (b) Haemolysis test: 2 ml of 18% sodium chloride solution in two test tubes was taken to one test tube added distilled water and to the other test tube 2 ml of sample solution was added few drops of blood is added to both the test tubes.(vii) Test for flavonoids: (a) Ferric chloride test: sample solution with few drops of Ferric chloride solution mixed and allowed to stand. (b) Schinoda test: Sample solution with few fragments of magnesium ribbon and 10% concentrated hydrochloric acid mixed and allowed to stand. (c) Zinc-HCl reduction test: Sample solution with zinc dust and few drops of HCl mixed and allowed to stand. (d) Alkaline reagent test: Sample solution mixed with sodium hydroxide. 64
  • 78. MATERIALS AND METHODS (e) Lead acetate test: Sample solution mixed with few drops of 10% lead acetate. (f) Bromine water test: Sample solution mixed with bromine water and allowed to stand.(viii) Test for tannins: (a) 1 ml of sample solution when mixed with ferric chloride and allowed to stand. (b) Sample solution mixed with 10% lead acetate and allowed to stand. (c) Sample solution mixed with bromine water shows yellow colour.Identification by T.L.C.:Drug: Extraction of sample, which is treated with 1:10 ml solute: solvent like ethylalcohol with dilution method.Equipment: Silica gel, T.L.C. kit, hot air oven, standard glass, Wattaman glass plate,beakers, sprayer.Chemicals: Dragendroff’s reagent, silica gel, ethyl alcohol.METHODS:T.L.C. of the ethyl alcohol extract of the sample was carried out as follows: The silica gel powder mixed with water and made thin paste, then with thehelp of glass slide, the silica gel was spread on glass plates uniformly. Aftersometimes the air dried plate were kept in a hot oven at 1100C – 1200C heat was givencontinuously, then the prepared sample is kept on a side of the plate then immersed insolvents upto 30 minutes then Dragendroff’s solution is sprayed on the plates. A parameter called the Rf value is always used in TLC this is determined asfollows;Rf = Distance from the base line to the spot Distance from the base line to solvent 65
  • 79. MATERIALS AND METHODSMaterials for partial characterization by U.V. and I.R. spectroscopy:Drug: Extractive alkaloid sample of Panchanga of Solanum nigrum Linn.Equipments: Spectroscope potassium bromide disc.Chemical: Ethyl alcohol.METHODS:• Different chemicals when subjected for photometry in white light (including U.V) have specific affinity to absorber to transmit particular range of wavelength, which is related to that compound.• Spectrophotometer analysis involves the measurement of the ability of the dissolved solutes to absorb light of definite and narrows wavelength ranges.• These absorption are measured at a wavelength that are generally a characteristic of the chemical composition of a dissolved absorbing substance radiant allergy waves range from 200 nm to about 380 nm in the UV region and from 380 to around 780 nm in the visible region.• The UV or visible spectrum of a molecule is the result of change in energy of a molecule as shown or rather than of a particular bend. The UV and visible spectrum of a substance generally not have a high degree of specific but they are suitable for quantitative essays for many substances and useful as additional means of identification.• Hence, the UV spectral analysis was selected as one of the parameter. The UV visible spectra of the sample was recorded in a schematized double beam UV visible recording spectro-photometer (Model UV – 160A).UV Spectrophotometer analysis: The alkaloid fraction was subjected to spectral analysis. The details of spectralanalysis are as follows; 66
  • 80. MATERIALS AND METHODS The UV absorption spectrum of the alkaloid was recorded on schematizemodel 120 at medium. Scan speed the spectrum has shown a strong peak at 140 nmand a shoulder peak at 260 nm.UV spectrum:Ethyl alcohol (i) 240 nm (strong) (ii) 260 nm (shoulder) MaxIR Spectrum: The IR spectrum of the alkaloid was recorded on perkins Elumesk model 183at medium scan speed by applying potassium bromide disc. Peak at 3679.90 indicatesN = H stretch.And peak at 1518.68 indicate C = 0 stretchPeak at 627.27 indicate C – bond Max KBr disc(C) Experimental study:Aim: To evaluate the effect of extracted fraction of Panchanga of Solanum nigrumlinn by inducing to the albino rats to see its anti-inflammatory activity.(I) Selection of animals: Charles foster strain albino rats of either sex weighing between 110 – 180 gms for experiments with the following conditions.(1) The animals were obtained from the animal house attached to the medical college laboratory.(2) They were exposed to natural day and night cycles with ideal laboratory condition in terms of suitable temperature and humidity. They were feeded 67
  • 81. MATERIALS AND METHODS with Amrut brand rat pallet feed which are soaked in oil and which are supplied by Nava maharastra chakana oil mills and tap water.(3) They were kept in a good cage and supplied the tap water.(4) The rats being kept on standard diet to reduce the mortality of the rats and they can be maintained.(II) Sample size: 24 albino rats were taken for the experimental study distributed six in eachgroup.(III) Preparation of the test drug and administration: Table : 9Group I Standard Ibuprofen suspension was purchased and fed orally to the Group albino rats at the dosage of 162 mg/kg.Group II Trial The Solanum nigrum linn extracted was weighed and given Group to trial group in minimum dose to the albino rats at the dosage of 270 mg/kg.Group III Trial The Solanum nigrum linn extracted was weighed and given Group to trial group in maximum dose to the albino rats at the dosage of 270 mg/kg.Group IV Control 1% normal saline was fed orally to the albino rats at the group dosage of 1 ml each animal in the single dose.(IV) Grouping: The rats were grouped into four groups of six in each group.Group I: Standard group: Ibuprofen suspension in the dose of 162 mg/kg.Group II: Trial group (minimum dose): Extraction of Panchanga of Kakamachi withaqueous solution in the dose of 270 mg/kg. 68
  • 82. MATERIALS AND METHODSGroup III: Trial group (maximum dose): Extraction of Panchanga of Kakamachi withaqueous solution in the dose of 540 mg/kg.Group IV: Vehicle drug group or observation group.Preparation of Carragrinine: Here no further preparatory methods followed. The Carragrinine taken in thedosage 0.1 ml.(V) Induction method of Carragrinine: Carragriine is taken for the observational study purpose and this was given in the techniques hind rat paw oedema. At the doses of 0.1 ml to each animal at single dose for experimental study, to get the inflammation.(VI) Dose selection: The extracted fractions of Panchanga of Solanum nigrum linn was given withaqueous solution in the doses of 270 mg/kg Carrageen was induced in a dosage of 0.1ml in 150 mg/kg body weight to the albino rats. • Trial drug to the albino rats (Minimum dose) = 270 mg/kg. • Trial drug to the albino rats (Maximum dose) = 540 mg/kg. • Standard drug to the albino rats = 162 mg/kg • 1% normal saline water = 0.2 ml/200gms of rat.Conversion to 1kg body weight:Trial drug ( Minimum Dose) : = Human dose X 0.018 i.e., 3 gm x 0.018 = 0.054 gm or 54 mg. = 54 x 5 = 270 mg/kg. Rat: 270 mg/kg. 69
  • 83. MATERIALS AND METHODSTrial drug ( Maximum Dose ): = Human dose x 0.018 i.e., 6 gm x 0.018 = 0.108 gm. or 108 mg. = 108 x 5 = 540 mg/kg. Rat:540mg/kg.Standard Drug: = Human dose x 0.018 i.e., 1800 mg x 0.018 = 32.4 mg = 32.4 x 5 = 162 mg/kg Rat: 162 mg/kg(VII) Route of drug administration: The test drug was administered to the albino rats according to their bodyweights of the animals by oral route with the help of infant feeding tube.(VIII) Duration of treatment: The test drug was given in minimum and maximum doses for Group II and IIIrespectively, standard drug Ibuprofen was given to Group – I and for Group IV 1%normal saline was given to Carragrinine induced rats, later Anti-inflammatory effectwas evaluated.Statistical analysis: The data collected were statistically analyzed by using unpaired student ‘t’ testwith the consultation of bio-statistician. 70
  • 84. OBSERVATIONS AND RESULTS OBSERVATIONS 1) Observation of Pharmacognostical The pharmacognostical study includes, • Morphological study • Microscopical study • Physical evaluation of the drugA) Morphological observation:- In this study the Morphological characteristics were observed by Organoleptic method. Table No : 10Colour GreenTaste TiktaSize 2.5-9 by 2.5 cmsShape OvateOdour Characteristics acceptableNature of leaf Acuminate, glabrous, thin, entire sinuate toothed, tapering into the petiole,Touch SmoothBeside these extra features, the arrangement of the leaves and their specialcharacteristics were observed such as 1. Lanceolate 2. Membranous 3. Long petiole. 71
  • 85. OBSERVATIONS AND RESULTSMacro morphological evaluation of KAKAMACHI: While analyzing a herbal drug, an idea about the distribution of various celltypes within different plant organs is essential. After evaluation of the differentfragmented structures of a comminuted material, the charecterstics related to theinitial structures and thereby the identity of the original material can be revealed. • In the intact material the examination of the tissue distribution is based on the evaluation of different basic cell types and cell inclusion is described earlier is important for identifying KAKAMACHI. • Cytomorphological evaluation of KAKAMACHI is based on the examination of the basic cell types and inclusions that may not be botonically complete, in leaf where epidermis with stomata, cellulose, parenchyma, vascular eliments and chlorophyll were frequently present structures include epidermal trachomous glands and palisade cells, crystals of calcium oxalates, pericyclic fibers and collenchyma. For differentiating closely allied leaves, the determinations of differential characters like Vein Islet number, Stomatal Number, Palisade Ratio and Stomatal Index play a major role. 72
  • 86. OBSERVATIONS AND RESULTSShape of Leaf: Table No : 11Shape OvateLength and Width 2.5-9 by 2.5 cmMid rib Present at the centre of the leafGlabrous Surface is free from hairsLamina Structure The lamina is the flat part of the leaves which constitute the major portion in leaf drugs. It can show a very wide variation in its form.Shape of Lamina In case of a dried leaf where the original shape of the leaf was obscured it has to be soaked in warm water and spread. Here the shape of lamina was ovate.Composition of In leaves containing herbal drugs, they include true leaves ofLamina individual leaflets of compound leaves. This can be easily distinguished if the attachment of the leaf to the stem can be examined. Various compositions of the leaf have been imperipinnate, which depends on the presence of a terminal leaflet.Apex AcuminateBase The lower extremity of the lamina, is symmetrical.Venation Arrangement of the Veins on the laminaDorsiventral leaf These have a palisade layer below the upper epidermis and a spongy mesophyll above the lower epidermisMargin The margin of the leaf is entire, tapering sinuate toothed. 73
  • 87. OBSERVATIONS AND RESULTSB) Microscopical Observation:• Microscopical study includes the examination of the cell form and arrangement of the different cells in a drug. The KAKAMACHI were generally used in powdered or comminuted form where the macro- morphology is destroyed, so that the evaluation of the microscopical cell characters was essential indication.• The KAKAMACHI contain some basic cell types e.g. Parenchyma, Collenchyma, Sclerenchyma, Xylem, Phloem, Epidermis, Periderm etc.. along with some cell inclusion characteristics i.e. the presence of ergastic substances like starch, calcium oxalate, Calcium carbonate, alueronic, silica and different other cell contents. Analysis of the plant drugs based on the distribution of these various cell types within different organs is important to ensure the identification.• The basis of analysis by evaluating microscopical characters was that there were always sufficient differences in the same type or different type of plants as far as the cell characteristics were concerned. Standardization profiles of KAKAMACHI were not available for most drugs.As with any comparative procedure, the more information that usually unique, wereof greatest value, as they constitute the difference between adulterants and evaluationof the microscopical characters only can provide such information of a particulardrug can be stored and reviewed in various ways by section method and by stainingprocess method under compound microscope observed the following events such asParenchymal cells, Stomatal cells, Xylem, Phloem, Vascular bundles, Palisade cells,Starch grains, Fibres, Tracheids, Vessels, etc were seen. 74
  • 88. OBSERVATIONS AND RESULTSA) Observation of Parenchyma:• It occurs as general ground tissue in most plants. These are usually asymetric, thin walled and simplest type of cells. By course of maturity they may have intercellular spaces.• Secondary thickening may be present in reticulate or pitted form. Which can be lignified. The cytomorphology of different types of parenchyma has been shown.B) Observation of sclerenchyma: This is the hard supporting tissue with heavy secondary thickening. They are generally divided in to two categories in respect of their aspect ratio as follows• They are roundly isodiametric, although elongated and branched form may also occur.• They are found singly or as a complete layer or in groups with pitting and stratification often accompanying them.• They occur in the hard outer coat of seeds and fruits and pericycle regions of woody stem.C) Observation of Fibres: • They are thick walled with high length and width. Fibres are classified based on the area in which they occur In pericycle, Xylem or Phloem fibres. • Crystal sheaths are sometime formed , this feature together with different size frequency and distribution plays an important role as a diagnostic feature. The ground mass of secondary xylem of picroena excels in building up of compactly arranged thick walled wood fibres. 75
  • 89. OBSERVATIONS AND RESULTS • The Secondary Xylem contains wood fibres arranged in bundles. The Phloem fibres of liquorices resemble those of Xylem in being enclosed in a crystal sheath • The contribution, abundance, Size and shape of the phloem fibres constitute an important characters for the differentiation of medicinal bark.D) Observation of Collenchyma: • It is a supporting tissue directly derived from Parenchyma with greater mechanical strength. Secondary thickening is much more and composed of cellulose. The thickening may be stratified or unevenly distributed around the circumference of the cell. Collenchyma is present above and below the midrib bundle in many leaves.E) Observation of Xylem: • This is the principle water conducting tissue of a plant. They have lignified secondary thickened wall that can show a variety of forms. Secondary growth in thickness of stem and root is usually accompanied by the formation of secondary xylem.F) Observation of Tracheids: These are the basic cell type of xylem tissue with a lignified, thickened and pitted cell wall, it takes the form of a water conducting cell in a plant.G) Observation of Vessels: These constitute the fundamental conducting elements of the Xylem inangiosperm. The most primitive type of vessels consists of a vertical series of trachiedlike segments where as the advance type of vessels show complete dissolution of theend walls to give slit like opening. The essential difference between trachieds and 76
  • 90. OBSERVATIONS AND RESULTSvessels is that the former are imperforate, where as the latter have pores at the endwhich are connected to form a continuous file or tube.H) Observation of Xylem Parenchyma: These cells are axially elongated, sometimes thin walled but often withthickening and lignifications. It functions as a storage tissue and in some cases thecells are blocked with starch.I) Observation of Phloem: This compound tissue is responsible for the transport of food. It containscompanion cells, Sieve tubes, Phloem parenchyma and secretary cells. The sieve tubeis the conducting element in phloem. The sieve elements are the highly specializedcells in phloem and the main morphological characteristic is the occurrence of sievein their walls which may often be detected by recognition of callus pad which showsome staining characters (Chlor-zinc-Iodine solution stain callose) to a redish browncolouration. Anilineblue stains in to callose blue solution of ammoniacal coppernitrate (BP) does not dissolve cellulose. J) Observation of Epidermis: • This consists of a single layer of cells covering the whole plant i e. the outer most layer of the plant structure. The epidermis of the root constitutes the pilferers layer, shoots contain a compact layer of cells and that in contrast to the stomatal guard cells, are often devoid of chloroplast • The epidermal cell of the two surfaces of a leaf differs in form. Various diagnostic features including the shape of the anticlinal (Vertical) and Periclinal ( Horizantal) wall (Straight or wavy), the presence of thickening and occurrence of striations on the surface. Cuticle can play a major role in detecting the epidermis. 77
  • 91. OBSERVATIONS AND RESULTS • Epidermis has a specialized structure and most universal of these are stomata which control water loss from the plant. They occur most frequently on young leaves and stem. The structures of the epidermis and stomata are of first importance in the microscopical identification of leaves. • Different types of stomatas on the basis of their arrangement have been shown. Trichomes are the out growths of epidermal cells, which occur in all parts of a plant. • They are of the value in the analysis of KAKAMACHI with different distribution and frequencies. They are particularly useful in the examination of fresh material where the stomata and epidermal cells are not readily visible.K) Observation of Periderm: This is a protective tissue that replaces epidermis in stem and root. Typicalperiderm is usually present in root, of aquatic and subterranean stems and in the aerialstems of plants cork cambium on the inside has been shown.L) Observation of Starch:• This is the most common carbohydrate reserve and is found in varying amount in almost all plants.• It occurs in granules of varying sizes and found most abundantly in roots, rhizomes, fruits and seeds where they occur as larger grains.• The small granules are formed in chloroplasts by the condensation of sugar, which afterwards hydrolyzed; so that they pass in to the solutions of storage organs, where under the influence of leucoplasts. Large grains of reserve starch are formed.• Starch is considerable pharmaceutical importance. Various such starches include maize. 78
  • 92. OBSERVATIONS AND RESULTS• Starches of different categories, between crossed polaizer the granules appear bright on a black background and each usually shows a dark maltase cross due to the structure of the granule.• This type of appearance is completely specific to starch although it is shown by the granules of insulin. Starch occurs as irregular, angular masses or as a white powder. It is insoluble in cold water but forms a collidal solution on boiling with 15 parts(w/w) of water with it, on cooling this solution forms a translucent jelly, while heating with water the granules first swell and undergo gelatinization.• Starch granules also undergo gelatinization when treated with caustic potash. concentrated solution of calcium or zinc chloride or that of chloral hydrate.• For microscopical examination of starch in herbal starch solution in it. Starch is identified by its characteristic appearance in natural and polarized light and by the formation of bluish black coloured compounds (Starch Iodide) with N/50 iodine solution, where as insulin does not stain with this iodine.• Starch granules usually contains two carbohydrates.C) Observation of Physical evaluation: After collecting the materials and studied, the physical properties wereobserved as1) Observation of Vein Islet:• Vein Islet was the term used to indicate the minute of the photosynthetic tissues encircled by the ultimate divisions of the vascular stands.• A Vein Islet was the smallest unit of the tissue encircled by the ultimate division of the conducting strands of the leaves. The area of leaf considered was preferably taken from the lamina midway between the midrib and the margin. 79
  • 93. OBSERVATIONS AND RESULTS• The number Vein Islet per square mm was termed as Vein Islet number. This number per unit area of leaf was constant.2) Observation of Vein let termination:• An ultimate free end or termination of a Vein let was called Vein let termination and the number of the same per square mm of leaf surface was termed as Vein let termination number.• It was determined as per Vein let number and can be estimated at same time. It can be used as a distinguishing character for the leaf of the same species or different ones, more particularly when the Vein Islet numbers does not give distinguishing.• The leaf pieces were taken on to a watch glass and one each was mounted on a slide in chloral hydrate solution with lower surface of the leaf facing upwards so that the Veins which are more prominent in the lower surface are seen clearly under the microscope.• Usually for this study 6x eye piece and low power objectives were used. The stage micrometer was to be focused ( 1 mm) and the camera lucida was to be fixed in such a way that the aperture of it was in the same line with that of the eye piece.• Black drawing sheet was placed on the same side of the microscope where camera lucida was fixed.• Using a white pencil the first and last of the stage micrometer was removed and slide was mounted with the leaf specimen and focus in the same way.• The square drawn in the paper is adjusted in such a way that it lies exactly in the middle of the field of vision and the image of the leaf piece mounted appears to be superimposed on the square of the drawing sheet. 80
  • 94. OBSERVATIONS AND RESULTS• Starting from one side all the Vein Islet inside the square as well as on the boundary is to be traced. The Vein let termination within the square only was taken in to account. To get the exact values it was necessary to take reading from 4 such squares and trace the Vein Islet within it.• The Value obtained from the Vein Islet and Vein let termination was calculated as an average.3) Observation of Stomatal Index: • The stomatal number and the Stomatal Index was a very specific criteria for the identification and characterization of Kakamachi. • Stomatal number was the average number of stomata per mm2 of epidermis and the number on each surface of leaf. • Each stomata consists of two guard cells and the spore was counted as a single unit. Though this has significance in determining the quality of crude drugs, this number varies unfortunately, depending on the environmental condition and geographical sources where the plants were grown. • Stomatal Index was one of the more distinguishing characteristics for herbal leafy drugs. It was the percentage praportion of stomata on one side and epidermal cells plus stomata on the other side.• In other words, Stomatal Index is defined as the percentage of stomata from the total number of epidermal cells, which can be explained as; Stomatal index = S X 10 E+S S = the number of stomata in a given area of a leaf E = total number of epidermal cells including trichomes in the area of leaf. 81
  • 95. OBSERVATIONS AND RESULTS• A peeling can be easily taken by partially cutting one of the Veins with a blade on the lower epidermal region and pulling it. The membranous layer, which comes out along with the Vein was taken on a glass slide by cutting the thick Vein section attached to it.• A few drops of chloral hydrate solution were added and the cover slip was placed on the top. The peeling was to be sufficiently big enough to cover the entire field of view when mounted.• A 10x eyepiece and high powered objective used. 1mm2 of area was taken with the help of a stage micrometer as described in the previous section.• On the tracing paper a circle smaller than that of the field of view was drawn and the camera lucida is fixed in such a way that the circle /square was clearly visible in the centre of the field of vision.• For determining the stomatal number it was only to be marked and for each leaf sample, not lesser than ten determinations should be carried out and the average index was calculated by using this parameter, Identification and standerdization were done.4) Observation of Palisade Ratio: It can be defined as the average number of palisade cells present beneath eachupper epidermal cell. It can be determined on fine powder. This value remainsconstant within a range for a given plant species and was of diagnostic feature forcharacterization and identification of Kakamachi. Like this Stomatal Number, Stomatal Index, Vein Islet Number, VeinTermination Number, Palisade Ratio, Starch grain Determination.5) Observation of Extractive Values: During determination of extractivation following things were observed such as 82
  • 96. OBSERVATIONS AND RESULTS Total soluble constitute of the drug in any particular solvent or mixture ofsolvents was nothing but the extractive values that was observed and calculated. The Extraction of Kakamachi with a particular solvent yields a solventsolution containing different phyto-contituents. The composition of these phytoconstituents in that particular solvent depends upon the nature of the drug and solventused. The use of a single solvent can be the means of providing preliminaryinformation on the quality of a particular drug sample, for example in a drug wherethe extraction procedure for the constituents commences with water solvent, anysubsequent aqueous as the extraction on the re-dried residue will give a very low yieldof soluble matter.6) Observation of Water Soluble Extractive: During the determination of water soluble extractive following things wereobserved such as• Accurately weighed about 5 gm of powder sample was taken in a conical flask.• Correctly added 100 ml of water to it.• Shaken and kept over night• Next day observed it and filtered. 20 ml of filtrate was taken in a previously dried and weighed porcelain dish and evaporated on a hot water bath and carefully observed.• Later on weight of the residue was observed which was the water-soluble extraction.• After that, observe the percentage and calculated on the basis of air dried sample. 83
  • 97. OBSERVATIONS AND RESULTS7) Observation of pH Value: A potential is developed across the glass of electrode because of the differencein hydrogen activity in the solution on the two sides of the glass. The glass electrodesbehave like a concentration cell and so oxidizing and reducing agents do not disturb itas it is an oxidation reduction cell. The potential of the glass electrode is proportionalto the pH of the solution in which it is immersed. Connected a power pack of 230 V to the pH meter. Insert the combinedelectrode in the socket and adjust the reading of pH and temperature at 30 C, switchon the instrument. The instrument is filled with distilled water and warmed it. Dip theelectrode in a standard solution of pH and set temperature and the take the reading.Remove buffers wash and wipe the electrode and dip it in the unknown solution andtake the reading. 84
  • 98. OBSERVATIONS AND RESULTS Table No:12B) Phytochemical observationsTime Acidic Media pH Alkaline Media pH0 Hours 3.30 8.781 Hours 3.35 8.662 Hours 3.40 8.313 Hours 3.42 8.304 Hours 3.49 8.245 Hours 3.50 7.876 Hours 2.90 7.807 Hours 2.91 7.578 Hours 2.78 7.349 Hours 2.82 7.2010 Hours 2.83 7.2211Hours 2.80 7.0612 Hours 2.91 7.091) Observation on Choice of solvent for extraction: Leaves coarse powder of Solanum nigrum was taken in different solvents suchas petroleum ether, Butenol, Etyl acetate, Chloroform, Ethyl alcohol etc.. put on to thefilter paper directly in different funnels according to the different solvents after seeingthe residue, where residue was minimum that has solubility of the drug in that solventis maximum i.e. solubility of Solanum nigrum in ethyl alcohol. 85
  • 99. OBSERVATIONS AND RESULTS2) Observation of Extraction: During extraction the following were observed• By appropriate technique the coarse powder of Kakamachi is put in the round fold of filter paper in Soxhlet apparatus so that it can not obstruct any path ways of Soxhlet apparatus by thermostat mantle where uniform temperature is maintained.• During each batch, the cycles were continued till up to extractive factors of the leaves were get completely extracted in to the solvent then and then only every batch was stopped. Observation was done so that with this complete extractive factors were extracted in. After extraction solvents were distilled off, observation was done so that the solvent is completely distilled off from the total extraction.• Semi solid extraction taken off and put over the magnetic stirrer for concentration of extraction should not be so liquid or completely dried.3) Observations of Preliminary Phytochemical Test:1) Test for Sterols:a) Salkowiskis testExtract + Conc H2 SO4 + chloroform Solution Shaken Lower layer turns red. (Red P.P.T) Indicates the presence of Sterols.b) Libermann –Burschardt test:Extract + Conc H2 SO4 added Brown ring does notChloroform solution + at the sides of test tube shows the junctionAcetic anhydride + and upper layerIndicates the absence of Sterols dose not turns green 86
  • 100. OBSERVATIONS AND RESULTSc) Sulphur test::Extract + Sulphur Powder Shake downward Indicates the presence of Sterols2) Test for Proteins:a) Extract + Soda lime+ Ammonia Gas is evolved Test solution Indicates the presence of Proteins.b) Test solution + Millions reagent heated does not show white PPT does not become red. Indicate Absence of Proteinsc) Test Solution + 1 ml Mercuric Sulphate boil gently does not10 % H2 SO4 1 % sodium nitrate show red PPT Indicates the Absence of Proteins.d) Biuret test:Test Soln + 1 ml 5% NaOH heat does not show Violet or pink PPT+ 2 drops of 1% CuSO4 soln. Indicates Absence of Proteins.e) Test soln +Few drops 10% tannic acid heat does not show Violet or White P.P.T Indicates Absence of Proteins.3) Test for Triterpenoids:a) Salkwoskis test: Extraction+Conc H2 SO4+chloroform soln Shaken lower layer allow to stand does not turns red Indicates Absence of Triterpenoid. 87
  • 101. OBSERVATIONS AND RESULTSb) Libermann –Burschardt test: Chloroform soln + Extract Conc H2 SO4 lower layer doesn t + Acetic anhydride allow to stand turns red. Indicates Absence of Triterpenoidc) Tschegajew test: Extract test soln.+ Excess of water bath No eosin red colour forms. acetyl chloride and pinch of zinc chloride. Indicates Absence of Triterpenoid4) Test for Alkaloids:a) Mayers test: Test soln + Mayers reagent Potassium mercuric Observed Grey coloured iodide precipitate. Indicates the presence of Alkaloidsb) Wagners test: Acidic soln + Wagners reagent potassium iodide Gives brown of sample precipitate Indicates the presence of Alkaloids.c) Hagers test: Acid soln + Hagers reagent picric acid observed yellow of sample saturated precipitate Indicates the presence of Alkaloids 88
  • 102. OBSERVATIONS AND RESULTSd) Dragendroffs test: Acid soln + Drgendroffs potassium bismuth iodide Reddish brown of sample reagent precipitate Indicates the presence of Alkaloids.5) Test for Carbohydrates: a) Molisch test: Test soln +Molisch reagent conc H2 SO4 No purple ring at the junction Indicates the absence of Carbohydrate.b) Barfords test: Test soln + Barfords reagent boiling water bath No brick red PPT Indicates the absence of Carbohydrate.c) Benedicts test:Sample soln + Benedicts reagent boil water bath No reddish brown PPT Indicates the absence of Carbohydrate6) Test for Saponins:a) Foam test:Extract+Saponin Shake H2O Formation of stable froth for one minute. Indicate the presence of Saponin.b) Heamolysis test:18%Nacl+disH2O + drop of Blood No haemolysis 18% Nacl+ filterate+drop of Blood under microscope No haemolysis Indicates absence of Saponin. 89
  • 103. OBSERVATIONS AND RESULTS7) Test for Flavonoids:a) Ferric Chloride test Alcoholic soln +Few drops of Ferric ( Fecl2) Blackish red colour PPT chloride Indicates presence of Flavonoid.b) Schinoda test:Alcoholic soln + conc HCl show pink or magneta redfew fragments of magnesium colourribbon + conc HCL Indicates presence of Flavonoid.c) Zinc HCL reduction test: Alcoholic soln + Zinc few dropd of HCL No magneta red colour. Indicates Absence of Flavonoid.d) Alkaline reagent test: Alcoholic soln Treated with dilute Yellow colour dilute acid NaoH colourless Indicates presence of Flavonoid.e) Lead acetate test: Alcohol solution few drops of lead Yellow PPT. acetate 10% Indicates presence of Flavonoid.f) Bromine water test: Extraction + Bromine water dissolved Yellow precipitateIndicates presence of Flavonoid. 90
  • 104. OBSERVATIONS AND RESULTS8) Test for Tannins: a) 1 ml alcoholic soln treated with Fecl2 dark precipitate Indicates presence of Tannin. b) Alcoholic Soln treated with lead acetate white precipitate Indicates presence of Tannin. c) Alcoholic Solution treated with Bromine water Yellow colour. Indicates presence of tannins. OBSERVATIONS OF PRELIMNARY PHYTOCHEMICAL TESTS Table No 13Tests Observations1) Tests for Sterols:a) Salkowiskis test Turns Red Colourb) Libermann-Burchardt test No Brown ring at junctionc) Sulphur test Sinks down words2) Test for Proteins:a) Extract + Sodalime + Test soln. Ammonia gas was evolvedb) Test soln + Millions reagent No Red precipitatec) Test soln + Mercuric suphur No Red precipitated) Biuret test No violet precipitate seene) Test soln + 10% Tannic acid No white precipitate seen3) Test for Triterpenoids:a) Salkowiskis test Lower not turns redb) Libermann-Burchardt test Upper layer turns greenc) Tschegajew test. No eosin red colour formed.4) Test for Alkaloids: 91
  • 105. OBSERVATIONS AND RESULTSa) Mayers test Grey coloured precipitateb) Wagners test Gives Brown precipitatec) Hagers test Yellow precipitated) Dragendroff.s test Reddish Brown precipitate5) Test for Carbohydrate:a) Molischs test No purple ring at junction of two liquids.b) Barfords test No Brick red precipitatec) Benedicts test No Reddish Brown precipitate6) Test for Saponins:a) Foam test Formation of frothb) Haemolysis test No Haemolysis7) Test for Falavonoids:a) Ferric Chloride test Blackish red colour precipitateb) Schinoda test Pink or Magneta red colourc) Zinc-Hcl reduction test No Magneta red colourd) Alkaline reagent test Colourlesse) Lead acetate test Yellow precipitatef) bromine water test Yellow precipitate8) Test for Tannins:a) Solution + Fecl2 Dark precipitateb) Solution + Lead acetate White precipitatec) Solution + Bromine water Yellow precipitate 92
  • 106. OBSERVATIONS AND RESULTS4) Identification by TLC: About three to six centimeter solvent front migration is sufficient to effectproper separation Wattman TLC plates produced from 4-5 micro-meter, Silica gel,with an inert binder to form 200 mm layers about 7 cm development distance wasachieved sample preparation in TLC needs a concentrated solution as very lessamount to be applied and it had been developed using the technique of TLC.The details of TLC as follows Table No 14Plate Size : 20 x 8 cmTechnique : One way ascendingTemperature : 30 CExamination : Day light after sprayingPlate thickness : 3 mmActivation temperature : 110 CTime : 30 min- 1 hrDetecting Spraying Reagent : Dragendroffs reagentAbsorbent Layer : Silica gel G(Activated) percolated platesSolvent System : Chloroform • The Silica gel powder mixed with water and make thin paste ,then with the help of glass slide the silica was spread on glass plate uniformly. • After some times the air dried plates are kept in a hot oven at 110 C -120 C heat was given continuously. 93
  • 107. OBSERVATIONS AND RESULTS • For one hour then the prepared sample was kept on one of the plate then immersed in a solvent up to 10 minutes and then Dragendroffs solution is sprayed on the plates.During this procedure following observations were observed: • The Silica gel slurry taken on plates with the help of glass slide, the silica gel was spread on glass plates uniformly. • After sometimes the air dried plates were kept in hot oven at 110 C- 120 C. heat was given continuously. • After that, the prepared sample was kept inside the developing chamber with the plate, then immersed in a solvents up to 30 minutes , closing the plate with the lid. • After Dragendroffs reagent was sprayed on the plates. • Black spot was observed on TLC plate.5) Observation of U V and I R: During determination of partial characterization by UV and IR spectroscopy,the following observations were observed. • Absorption were measured at a wave length that were generally a characteristic of the chemical composition of a dissolved absorbing substance. • Radiant allergy waves range from 200 mm to about 380 mm in the U V region and from 380 to around 780 mm in the visible region. • The U V or Visible spectrum of a molecule was the result of change in energy of a molecule shown or rather than that of a particular bend. 94
  • 108. OBSERVATIONS AND RESULTS • The U V visible spectrum of a substance generally not have a high specific degree but were suitable for quantitative essays for many substances and useful in additional means of identification. • The U V spectral analysis was selected as one of the parameter. • The U V divisible spectra of the sample were recorded in a schimaduae double beam U V visible recording spectro-photoments ( Model UV-160 A) • Method extract of the samples after suitable dialatation, were scanned through 200-800 mm and the spectra was recorded.c) Observation of Experimental Study:Observation on Selection of Animals: • The handling of the selected laboratory animals involves two most imporatant responsibilities on the part of the experiment. • First, the animal was handled with utmost care so that it does not suffer pain and secondly, due regard was paid towards the health and well being of the animal colony. • Even when they were killed at the end of the experiment, INSA provides the following observations to followed by all research scholars engaged in the animal experimentations.1) Housing with breeding and maintenance of experimental animals to keep them inphysical comfort and good health and to permit them to grow and behave normally.2) Sources of experimental animals of known genetic health and nutritional status.3) Observation about technicians and other supportive staff with animals and their usein experiments. 95
  • 109. OBSERVATIONS AND RESULTS2) Sample size: Albino rat was one of the commonest animals suitable for experimental workbecause of its size and sensitivity to most of the drugs. It was also the moststandardized of all laboratory animals. It can be bred to obtain pure and uniformstrains and was found to be very useful in the studies which withstand long periods ofexperiments. Hence 24 Albino rats were selected for the study.I. Inclusive Criteria:i) Adult healthy Albino rats.ii) Male Albino rats weighing 110-180 gms.iii) Albino rats between 90-120 days old were included.II. Exclusive Criteria:i) Unhealthy Albino rats.ii) Weigh range between 110 gms above 180 gms.iii) Female Albino rats.iv) Albino rats of below 90 days and above 120 days were excluded.3) Observation on preparation of the test drug:Group I –Standard Group: Readily available Ibuprofen suspension was taken andgiven to the first group of rats from infant feeding tube and syringe in the dose of 162mg/kg, The Carragrinine was induced to rats and the inflammation was recorded.Group II- Trial Group ( Minimum dose) During the preparation of test to the Group II it was observed that the weighedextractive fraction was diluted with 1 ml of concentration of that of 1 ml of test drugis given to the second group of rats from infant feeding tube and syringe through oralroute, after six hours the carragrinine was induced to the rats and inflammation wasrecorded, before and after administering the prepared test drug. 96
  • 110. OBSERVATIONS AND RESULTSSample drug = 270 mg/kg.Group III- Trial Group (Maximum dose) During the preparation of test drug to the Group III it was observed that theweighed extractive fraction was diluted with 1 ml of concentration of that of 1 ml testdrug was given to the second group of rats from infant feeding tube and syringethrough oral route, after six hours the carraginine was induced to rats andinflammation was recorded before and after administering the prepared test drug.Sample drug= 540 mg/kg.Group IV (Normal Saline) 1% of normal is prepared with 100 ml of water and 1 ml was taken for theobservation purpose. This was fed orally to this group of rats in a single dose. AfterSix hours the already carraginine induced rats. The inflammation was recorded beforeand after administering normal saline.Normal Saline= 0.2 ml in 200 gms rat.4) Grouping: Table No : 15 Group I Standard Durg Ibuprofen 06 Rats Group II Trial drug sample Extraction of 06 Rats Kakamachi(Minimum dose-270 mg/kg) Group III Trial drug sample Extraction of Kakamachi 06 Rats (Minimum dose-540 mg/kg) Group IV Vehicle Normal Saline 06 Rats 97
  • 111. OBSERVATIONS AND RESULTS5) Induction Method: i) For all the 24 Rats, which were grouped into four groups of six each, thecarraginine taken with respective dose at 2 ml disposable syringe with needle wasadministered through hind paw of rat without disturbing the normal behaviour.ii) Initial inflammation was recorded.iii) Immediately just after the injection of carraginine, inflammation was recorded.6) Drug Administration:i) Simultaneously, the prepared test drug sample of Kakamachi was administered to the Albino rats orally.ii) At the same time the prepared standard trial drug and 1% normal saline was administered to Group I, Group II, Group III and Group IV orally respectively.iii) Three hours after administration of drugs again the inflammation was recorded in all the four groups by plathysmograph. The variation of inflammation between two groups was observed.7) Observation of selected dose:Trial drug minimum dose -270 mg/kgTrial drug maximum dose- 540 mg/kgNormal saline -0.2 ml/ 200 gm body weightCarraginine- 150 mg /kg body weight. i.e. 30 mg / ml yeast.Conversion of dose / kg body weight.Animal dose – Human dose x 0.0118 for 200 gm.Minimum dose- 3 gm x 0.018= 0.054 gm = 54 mg = 54 x 5 = 270 mg/kgTrial drug to Albino rats = 270 mg/kgMaximum dose = 6 gm x 0.018 = 0.108 gm =108 mg =108 x 5 =540 mg/kgTrial drug to Albino rats = 540 mg/kg. 98
  • 112. OBSERVATIONS AND RESULTS8. Duration of Treatment:i) The first three days observed for the natural behaviours with suitable housing.ii) 4th day the human –animal infection was observed.iii) 5th and 6th day kept for 48 hours starvation under observational study.iv) observed for the increased and decreased conditions of inflammation by recording.1) Observation of Anti-inflammatory activity:i) The Rats were held in a good position where the normal physiological functions should not affected.ii) The hind paw of the rat should be in the protruding position.iii) The hind–paw should be immersed in a Y shape tube of plathysmograph.iv) Carrageen induced hind-paw after immersing, the mercury level of Y shape tube suddenly raised .v) The raised mercury level was measured in mm.vi) That is the inflammation = raised mercury from Y shape tube in mm.vii) For group I treated with standard drug after induction of carraginine, reduction of inflammation was calculated.viii) For group II treated with trial drug ( Minimum dose ) after induction of carraginine reduction of inflammation was calculated.ix) For group III treated with trial drug ( Maximum dose ) after induction of carraginine, reduction of inflammation was calculated.x) For group II treated with 1% normal Saline after induction of carraginine the decreased inflammation was calculated.xi) At last which group was more potent and more significant that is also calculated. 99
  • 113. OBSERVATIONS AND RESULTSRESULTS OF PHARMACOGNOSTICAL STUDY1) Results of Morphological Study Table No :16 Colour Green Taste Bitter Size 2.5-9 cm Shape Ovate Odour Characteristics acceptable Touch Smooth2) Results of Microscopical Study: By the section method and by staining process, the following results are seenunder the microscope. Trachoma, Parenchyma cells, Xylem, Phloem, vascular Bundles, Stomatalcells, Palisade cells, Starch grains, Lower epidermis and Upper epidermis. Whileconsidering the cytomorphological aspects, in the case of whole drug the celldistribution can be determined by sectioning and in powders some degree of cellularaggregations and organization is retained. To evaluate all the cell parameters, thedistribution of the basic cell types as well as cell inclusion has to be studied. The basictypes include cellular parameters of plant cells as follows; 100
  • 114. OBSERVATIONS AND RESULTS Parameters of plant cells Table No: 17 Sl No Microscopical cells Values 1 Trachoma 15-20/mm2 2 Parenchyma cells 30-35/mm2 3 Xylem 20-25/mm2 4 Phloem 20-25/mm2 5 Vascular Bundles 10-15 in bundles 6 Stomatal cells 20-25/mm2 7 Palisade cells 15-20/mm2 8 Starch grains 20-25/mm2 9 Lower epidermal cells 20-25/mm2 10 Upper epidermal cells 20-25/mm2 11 Fibers 30-25/mm2 12 Collenchymal cells 20-25/mm2Physical evaluation: The sample of leaves of kakamachi are analyzed by employing variousparameters as mentioned in materials and methods and the data evolved had beenpresented here in the tabular form. 101
  • 115. OBSERVATIONS AND RESULTSIn physical evaluation the following results are seen as; Table No:18 Sl No Parameter Values 1 Stomatal number 16-25/mm2 2 Stomatal index 18.6 mm 3 Vein islet number 26/mm2 4 Vein termination number 15/mm2 5 Palisade 20-25/mm2 6 Starch grain 40-60/mm2 7 Epidermal 24/mm2 8 Starch grain determination 24/mm2 9 pH value 8(B) Results of phytochemical study:1) Choice of solvent: Table No :19 Sl. No. Solvent Soluble Sparingly soluble Insoluble 1 Distilled water + - - 2 Solvent ether - - + 3 Petroleum ether + + - 4 Acetone - - + 5 Benzene - + - 6 Toluene - - + 7 Chloroform - + - 8 Ethyl alcohol + + - 9 Xylene - - - 10 Carbon tetrachloride - - +Ethyl alcohol 90% 102
  • 116. OBSERVATIONS AND RESULTS(2) Extraction Table No: 20Kakamachi of each batch Solvent ExtractCoarse powder 120 gm Ethyl alcohol 650 ml 10 gms(3) Preliminary phytochemical tests Table No:21.Tests Results(1) Test for sterols (a) Salkowiski’s test +ve (b) Liberman’s-Burchard test -ve (c) Sulphur test +ve(2) Test for proteins (a) Extract + soda lime + test solution +ve (b) Test solution + Million’s reagent -ve (c) Test solution + mercuric sulphate -ve (d) Biuret test -ve (e) Test solution + 10% tannic acid -ve(3) Test for Triterpenoids (a) Salkowiski test -ve (b) Liberman’s-Burchard test +ve (c) Tschegajew test -ve(4) Test for alkaloids (a) Mayer’s test +ve (b) Wagner’s test +ve 103
  • 117. OBSERVATIONS AND RESULTS (c) Hager’s test +ve (d) Dragendroff’s test +ve(5) Test for carbohydrate (a) Molisch test -ve (b) Bar ford’s test -ve (c) Benedict’s test -ve(6) Test for Saponin’s (a) Foam test +ve (b) Haemolysis test -ve(7) Test for flavonoids (a) Ferric chloride test +ve (b) Schinoda test +ve (c) Zinc-HCl reduction test -ve (d) Alkaline reagent test +ve (e) Lead acetate test +ve (f) Bromine water test +ve(8) Test for tannin’s (a) Solution + FeCl2 +ve (b) Solution + lead acetate +ve (c) Solution + bromine water +ve(5) Identification by TLC* The T.L.C. study of the samples was carried out by using different conditions to evolve suitable T.L.C. pattern, and the data was observed. 104
  • 118. OBSERVATIONS AND RESULTS* When viewed under long U.V radiation showed only one spot at R 0.98 but no spot was seen in the sample.* T.L.C of the alcoholic extract on silica get ‘G’ plate using n-Butanol: Acetic acid-water (4:1:5) shows under UV (366 nm) three fluorescent zones of Rf. 0.54 (bright sky blue) 0.84 (light sky blue) and 0.93 (bright sky blue)* On exposure to Iodine vapor seven spots appear at Rf (0.15,0.27,0.54,0.67,0.78 and 0.93) all yellow.* On spraying with 5% Methanolix-sulphuric acid reagent and heating the plate at 1050 C for ten minutes, eight spots appear at Rf. 0.15,0.27,0.32,0.38 ( all grey), 0.54 (yellow) 0.67, 0.84 (light grey) and 0.93 (brown)* The chromatogram after spraying with Dragendroff’s reagent followed by heating at 1100 C for 5 minutes several spots at Rf. 0.12 (indistinct), 0.32 (violet), 0.58 ( faint violet) and 0.73 (pinkish violet), 0.80 (faint violet), 0.90 (faint violet), and 0.93 (violet).* Perusal of the evolved chromatograms clearly shows that the chromatographic patterns of the sample are quite different in all the conditions indicating wide difference in the chemical composition of Kakamachi.* The evolved chromatograms will be very useful for the analysis and identification of Kakamachi.5) U.V. Spectrophotometric Analysis:* The U.V Spectra of Ethyl alcohol extract, after suitable dilution of the samples were recorded and presented. The spectra of the Kakamachi shows absorption peak at 210 nm 207nm respectively.* The comparison of the spectra presented shows difference in indicating difference in chemical composition in Kakamachi. 105
  • 119. OBSERVATIONS AND RESULTSC) Results of Experimental study:I) Analytical values of inflammatory activity:Group- I treated with Standard Drug Table No: 22Shows the reading for the inflamed hind paw volume before and after thetreatment of Group-I Sample Initial Paw volume Paw volume after 3 hours Difference 1 1.44 1.24 +0.20 2 1.48 1.46 +0.02 3 1.40 1.30 +0.10 4 1.48 1.20 +0.28 5 1.40 1.30 +0.1 6 1.40 1.30 +0.1 Table no: 23Showing the statistical data of group – ISD SE t P0.091 0.037 13.548 <0.0001Graph showing the mean value of standard group 0.3 0.28 0.25 0.2 0.2 Difference 0.15 0.1 0.1 0.1 0.1 0.05 0.02 0 1 2 3 4 5 6 Samples 106
  • 120. OBSERVATIONS AND RESULTSGroup II treated with Trial drug Minimum Dose Table No: 24Shows the reading of the inflamed hind paw volume before and after treatmentof Group- IISample Initial paw volume Paw volume after 3 hrs Difference1 1.44 1.38 +0.062 1.46 1.39 +0.073 1.43 1.38 +0.054 1.40 1.34 +0.065 1.40 1.30 +0.16 1.40 1.36 +0.06 Table No: 25Showing the statistical data of group IISD SE t P0.42 0.017 3.438 <0.02Graph showing the mean value of Trial group (minimum dose) 0.12 0.1 0.1 0.08 0.07 Difference 0.06 0.06 0.06 0.06 0.05 0.04 0.02 0 1 2 3 4 5 6 Samples 107
  • 121. OBSERVATIONS AND RESULTSGroup III treated with Trial Drug (maximum dose) Table No: 26Shows the reading of the inflamed hind paw volume before and after treatmentof Group III Sample Initial paw volume Paw volume after 3 hrs Difference 1 1.44 1.26 +0.18 2 1.48 1.44 +0.04 3 1.40 1.38 +0.02 4 1.48 1.20 +0.28 5 1.40 1.30 +0.1 6 1.40 1.30 +0.1 Table No: 27Showing the statistical data of group IIISD SE t P0.091 0.037 11.548 <0.001Graph showing the mean value of standard group (maximum dose) 0.3 0.28 0.25 0.18 0.2 Difference 0.15 0.1 0.1 0.1 0.04 0.05 0.02 0 1 2 3 4 5 6 Samples 108
  • 122. OBSERVATIONS AND RESULTSGroup IV treated with control – Saline water Table No:28Shows the reading of the inflamed hind paw volume before and after treatmentof Group IV Sample Initial paw volume Paw volume after 3 hrs Difference 1 1.49 1.48 +0.01 2 1.33 1.31 -0.02 3 1.40 1.39 +0.01 4 1.38 1.37 +0.01 5 1.49 1.48 +0.01 6 1.32 1.31 +0.01 Table No: 29Showing the statistical data of group IVSD SE t P0.013 0.005 0.938 >0.10Graph showing the mean value of control group 0.015 0.01 0.01 0.01 0.01 0.01 0.01 0.005Difference 0 1 2 3 4 5 6 -0.005 -0.01 -0.015 -0.02 -0.02 -0.025 Samples 109
  • 123. OBSERVATIONS AND RESULTSSTATISTICAL ANALYSIS The data obtained through the experimental study as shown in the tables beinganalyzed statistically based on the student t-test values as follows. • In standard group the result was highly significant with p value <0.0001 • In the trial group (minimum dose) the result was moderately significant with P value <0.02 • In the trial group (maximum dose) the result was highly significant with P value 0.001 • In the control group the result was non significant with P value >0.10 Therefore, it is ascertained that Kakamachi can play a highly significant rolewith maximum dose and significant role with minimum dose in reducinginflammation being equally effective as the standard drug Ibuprofen. 110
  • 124. DISCUSSION DISCUSSION The title of the present study is “Pharmacognostical Studies and Anti-inflammatory effect of “Kakamachi” on Albino rats”. This protocol includes thesystematic study of the ideal drug Kakamachi, its specific characteristics,photochemical study for determination of different chemical components in the Trialdrug. Experimental study is done for Anti-inflammatory activity. Hence this titlerepresents the complete wholesome study of research work.In Ayurveda, there are so many drugs mentioned under the anti-inflammatoryproperty. In that, Kakamachi is one, which is a major one. The present study is ofPharmacognostical, phytochemical and experimental study of Kakamachi, with itsspecial reference to anti-inflammatory activity. Under this the literature review,disease review, aims and objects of the Kakamachi are studied, the materials andmethods, observations and results were discussed and concluded. Shotha, Shopha and Svayathu-the terms used may be different in differentcontexts, but all our acharyas has given much importance to this condition. Theyhave devoted separate chapters for the description of the Nidana Panchakas andChikisa. Sushruta, while describing the Nirukti of Sopha “Ekadeshothitha ShophaItyuchyate” meaning oedema arising in any one part of the body is Shopha. While,describing vishesa laxanas of Shopha, he uses the word Shvayathu, which has alsobeen used during description of sarvasara Shopha. So it may be understood that allthe three terms are synonymous. To understand the disease entity for experiments based on modern science, anattempt has been made by putting correlation between Shotha and inflammation. Thisis according to the respective explanations of the similarities in their symptoms.Inflammation is local response of living mammalian tissues elicited as a defense 111
  • 125. DISCUSSIONreaction in order to eliminate or limit the spread of injurious agents as well as toremove the consequent necrosed cells and tissues. A series of phenomenon includingincrease in the vascular permeability, accumulation of exudates and bringing intoaction of many mediators like the prostaglandin etc,. that plays an important role areset into action. Till the date a number of studies have been carried out to screen the variousherbs for anti-inflammatory action to put forth the alternative diseases. Herbalremedy, which could take the place of the synthetic drugs of the new era. Kakamachi is a drug popularly known for its efficacy in skin disorders andvruna shodana in various vruna. It has also been highlighted for its efficacy inHridaya roga, Rakta Shodaka, Shothahara and Hypertension and also other ailments.Many of the nighantukaras have mentioned shothahara property of Kakamachi in theirnighantus. Shweta and Rakta, the two varieties of Kakamachi are seen growing wildly inthe northern part of Karnataka. The drug is Tikta in Rasa, laghu, Tiksana and Virya is|Ushna and where as Vipaka is Katu. Panchanga of Kakamachi is considered to be itsofficial part. Solanum nigrum is taken up for the present study. The drug wasidentified by botanists and faculty members of PG department of Dravyaguna, beforebeginning the study. The present study includes the morphological, microscopical, physicalevaluations. Under the morphological discussions, the points were discussed such asmaterials, drugs useful parts, collection of materials, equipments, chemical, methodswere discussed . Under the discussion of microscopical study the section methods,staining process, chemical methods were discussed. 112
  • 126. DISCUSSIONDiscussions of Pharamacognostical Study: Present Pharmacognostical study includes morphological, physical evaluationof the drug. In the discussion of morphological study, following were observed i.etaste-Bitter, size, shape etc. In discussion of microscopical study, following were seen. The T.S. of leaf shows a non-glandulous trachoma especially on the mid ribregion. The trachoma are of two types i) Very slender, multi-cellular and uniseriatestraight or bend ii) Very smaller with thin cell wall, globose and conical apex, thevascular bundles are seen at the middle of the midrib region. the vascular bundles aresemi lunar in outline with an upper readily arranged xylem tissue and lower phloemtissue. It shows outline with margin. The epidermis possesses multi-cellularenumerous slender non-glandular trachomas. Below the bulge of the epidermis thereare small patches of collenchyma cells and matured tissues. There are parenchyma’s,cortex and small patches of sclerenchyma fibers are observed and also beneathsclerenchyma fibers there are distinct vascular bundles ranging from 8 to 10 innumbers. Below the cortical parenchyma there is a distinct endodermis consists oftangentially elongated parenchyama cells. The pericycle group of cells which aredistinct and comparatively wide, the phloem tissue is followed by a group of xylemtissue. The xylem is made up of large vessels and traechids most of parenchyma cellsof cortex and medullury rays contain small prismatic crystals of calcium oxalate andfew starch grains. By discussion of physical evaluation of the Kakamachi, the coarse powder ofthe same is mixed with water and shaken well, after keeping this solution over night isfiltered, the next day and on evaporation minimum solubility of the drug in water wasseen. Heaves coarse powder of the Solnum nigrum was taken in different solvent. 113
  • 127. DISCUSSIONThe solubility of solanum nigrum is maximum in ethyl alcohol was seen. Theextraction of coarse powder of Solanum nigrum was done by using soxhlet apparatusand uniformally maintaining the temperature by using a thermostat with ethyl alcoholof 90% as solvent. A magnetic stirrer is used. pH value of the drug was seen.Discussion of Phytochemical analysis: Here the phytochemical study performed to identify the active chemicalcomponents such as alkaloid, sterols, carbohydrates, tri-terpinoids and tannins etc. Before their chemical tests, the coarse powder of Solanum nigrum wassubjected to exhaustive extraction by soxhlet apparatus around 18 hours in differentbatches of 90 % ethyl alcohol. The extractive fractions of the component showsdifferent chemical constituents such as alkaloids with yellow precipitate,carbohydrates with brick red precipitate, Flavonoids with maganata red or pinkprecipitate colour. Sterols with red precipitate. Tritrepenoids with yellow precipitatesaponins with stable froth and tannins with white precipitate indicates presence ofactive components. In Ayruveda the medicinal values of the plants are mainly attributed to activechemical components such as sterols, carbohydrates triterpinoids and alkaloids,tannins etc. In the present study, all the phytochemical components of the Solanum nigrumwere tested qualitatively by improving specific chemical tests. Before these chemicaltests, the air dried seeds of Solanum nigrum were subjected to exhaustive extractionby Soxhlet apparatus around 18 hours with different batches with ethyl alcohol of90%. Which was the prime aim of the present protocol. The discussion of phytochemical analysis gives following findings such as 114
  • 128. DISCUSSIONDiscussion of UV and IR spectrum: Under the discussion of the physical evaluation, different things werediscussed, what are the different materials are used which drug was used, whatdifferent equipment is used which chemicals were used the methods which are to befollowed were discussed successfully. The methods for Vein Islet, Vein lettermination are discussed and also for the Stomatal Index, Palisade Ratio, the suitablemethods are discussed.Discussion on Identification by TLC method : By following the appropriate methodology T.L.C preparation Black spot wasobserved on TLC plate. This spot indicates the active chemical components presentinside the diluted solutions. It shows following findings as at 0.54 bright sky bluecolour. On exposure of Iodine vapour, it shows seven yellow colored spots. Onspraying with 5% methonolic-sulphuric acid reagent and heating plated at 1050 C forthirty minutes shows eight spots appears at Rf. Such 0.15, k 0.27, 0.32, 0.38 withshowing green colour. Like this different findings were discussed. The 20x8 cm plates are taken. Silica gel is the most commonly used absorbentand is evenly on these plate as slurry in water in a 250 mm thick layer by means of amechanical spreader or manually. The plates are then air dried and then activated at 0100 to 105 C for 30 to 45 minutes. Usually the solvent is allowed to ascend to adistance of 10 cm from the base line and therefore it is quite convenient to draw astraight line at this level by means of a mounted needle. The layer of stationary phasehas been rendered discontinuous, a Small amount of the sample in solution is appliedto the chromate plate using a template. Spot applications must be as small as possibleand it is best done by means of a capillary tube. 115
  • 129. DISCUSSION As the component of the mixture moves up the plate, it tends to diffuse andthus the size becomes increasingly larger. If therefore, the initial spot is larger, thanthe components which separate only slightly will merge together, and thus theresolution will not be discernable. The abbreviations indicating the particularsubstance applied are scratched with a mounted needle on the adsorbent layer wellabove the solvent front line at 10 cm level. The abbreviations must never be scratchedon the absorbent surface below the base line and below any solvent front line drawn.This will disturb the even flow of the solvent render the plate useless. The plate is then placed in the chroma tank. The solvent is now allowed toascent up the plate until H mark reaches the 10 cm line. Note the room temperaturecarefully because this is an important consideration. The plate is then removed fromthe tank dries with hair drier and examined first in day light and then finally sprayedwith suitable Dargendroff’s reagent. Any spots observed under these conditions are outlined with a mountedneedles. The line as we see is extremely important in calculating the chromatographicparameter. The Colour of each spot after spraying is noted. When the examination iscomplete, a copy of the TLC plate is made by means of a sheet of tracing papercarefully noting the spot abbreviations base line and solvent front. It is important to note the colours and the manner and conditions in which theyappear, as also other relevant particulars. The information should be recorded in thetracings.Discussion of UV and IR: Different chemicals when subjected for photometers in white light (includingUV) have specific affinity to absorb or to transmit a particular ranges of wavelengthwhich relates to that compound, speaks metric analysis which involves the 116
  • 130. DISCUSSIONmeasurement of the ability of the dissolved solutes to absorb light of definite andarrow wavelength ranges, these absorption are measured at a wavelength that aregenerally a characteristic of the chemical composition of a dissolved absorbingsubstance. Radiant energy waves range from 200 nm to about 380 nm in the UVregion and from 380 to round 780 nm in the visible region. The UV or visiblespectrum of a molecule is the result of change in energy of a molecule as a show orrather than of a particular bend, the UV and visible spectras of a substance generallydo not have a high degree of specific, but they are suitable for quantitative assays formany substances and useful as additional means of identification. Hence, the UVspectral analysis was selected as one of the parameter. The UV, visible spectra of thesample was recorded double beam UV visible recording spectrophotometer (ModelUV-160 A) The IR spectrum of the alkaloid was recorded on Perkins elumes model 183 atmedium scan speed by applying KBr disc. It involves the measurement of the abilityof the dissolved solutes to absorb light of definite and narrow wavelength ranges.Radiant energy waves range from200 nm to above 380 nm in the UV region from 380nm to around 780 nm in the visible region. The U.V and visible spectrum of asubstance generally do not have a high degree of specific radiation but they aresuitable for quantitative assay for many substances and useful in identification. The spectrum of powder of Solanum nigrum was seen. Hence the U.V spectralanalysis was selected as one of the parameter.Discussion on experimental study:The design of the study on inflammation was madeon animal experimentation. Albino rats weighing between 110-180 gms were selectedcarefully for the evaluation. Four groups of animals were selected for the studiesbased on the following pattern. 117
  • 131. DISCUSSIONGroup I : Standard Group : Ibuprofen suspension was purchased and fed orally to thealbino rats at the dosage of 162 mg/kg.Group II : Trial Group – The Solanum nigrum linn extracted was weighed and givento trial group in minimum dose to the albino rats at the dosage of 270 mg/kg.Group III : Trial Group – The Solanum nigrum linn extracted was weighed and givento trial group in maximum dose to the albino rats at the dosage of 540 mg/kg.Group IV : Control Group – 1% Normal saline was fed orally to the albino rats at thedosage of 1ml each animal in the single dose.Carraginine (0.1 ml of 1% w/v in saline) was used to induce inflammation in the lefthind paw of all the rats keeping the right paw as control. The paw volume of the ratsof four groups, before and after injecting Carraginine was measured usingPlethismograph. Paw volume was measured hourly for three hours after injecting theCarraginine and observations done to see the difference in paw oedema. Inflammation was observed between 15-30 minutes after induction ofCarraginine. On the analysis of observations it is found the albino rats in the controlgroup did not show any improvement by three hours. But the rats in the trial groupwith minimum dose have shown good response after three hours, where as trial groupwith maximum dose and standard drug showed improvement during second and thirdhour of injecting carraginine. Statistical analysis showed that the result in the trial group with maximumdose is almost nearer to that of standard group. Development of oedema induced by carraginine is commonly correlated withthe early exudative stage of inflammation, one of the important processes ofinflammatory pathology. In the beginning of carraginine injection, there is suddenelevation of paw volume in relation with histamine mediators. After one hour, the 118
  • 132. DISCUSSIONinflammation is increased gradually and was elevated during the later three hours.This second phase could be due to the liberation of prosto glandin and kinins, whichaccompanies leucocytes migration. Animal experimentation has its limitation in the evaluation of inflammation.All the symptoms cannot be evaluated as done in clinical study. Here only the utsedaor tumor can be evaluated through the aid of plethismography by which the reductionof inflammation can be calculated. All the statistical data and graphs are presented in the tables. Thus through animal experimentation, it is found that kakamachi has highlysignificant action as anti-inflammatory drug with maximum dose, where asmoderately significant action with minimum dose. The samprapti of shopha begins by vitiation of kapha, rakta and pitta, whichenter the bahya siras and in turn vitiates the vata located there. Thus sroto rodha iscaused which spreads to the areas in the viscinity and shopha results. The drug is tikta in rasa, laghu in guna ushna veerya and katu in vipaka. Ushnaveerya is known to act on vata and kapha and tikta rasa reduces pitta and cleans therakta. Laghu guna also act antagonistically on kapha. Katu in the drug contractsorganic tissues and lessens its secretions and in practice it is found that the katu effect,some times tend to overlap with anti inflammatory activity to some extent. Thus the Gunas of trial act all together upon pitta, rakta, kapha duringsamprapti vighatana and eventually pacifies these. Once the vitiated pitta, rakta, andkapha are brought to the normal state the vayu gets pacified thus clearing thesrotorodha relieving shotha. 119
  • 133. CONCLUSION CONCLUSION1) Review of classical and modern literatures shows that the trial drug Kakamachi is having significant Shothahara property.2) Shotha manifests as disease itself and also as associated symptom.3) Comparing the results from the control, trial groups especially makes out inhibitor activities of Kakamachi in inflammation.4) Statistically trial drug with maximum dose and standard drug are equipotent.5) Thus, through experimental studies, the trial drug has significant anti inflammatory action in Albino rats.6) No adverse effects were seen and well tolerated by Experimental animals. 120
  • 134. RECOMMONDATIONS FOR FUTURE STUDY RECOMMONDATIONS FOR FUTURE STUDY1. To get scientifically based description of Histopathology of cells or tissues, pharmacological study is necessary.2. To assess the claim made on Kakamachi in regarding inflammation, clinical study is necessary in all phases. 121
  • 135. SUMMARY SUMMARY The present dissertation entitled “Pharmacognostical studies and anti-Inflammatory effect of “Kakamachi” on albino rats”. It contains following parts suchas- I) Introduction. II) Reviews of Literature. III) Pharmacognostical study. IV) Phytochemical study. V) Experimental study. VI) Discussion. In the first part a brief introduction was given which deals with the importanceof plant, number of the plants in the Brihatrai and Nighantus in different Vedas,Samhita kala, Siddha, Unani and adhunik kala with its some reference for shothaharaactivity. Aim of selection of the plant kakamachi and the anti-inflammatory activity.The aim and objectives. Material and methods and plan of the study are given. In the second part comprises of Review of literature which is divided into twosection viz. Drug review and Disease references in different Nighantus, Samhitas,Veda and in different modern books along with its different synonyms, PropertiesDoshaghnata, Roghaghnata and also the Botanical description of plants such asvegetative characteristics, floral characteristics, floral formula, floral diagram etc andits family description with its medicinal value and chemical composition. And itsAmayika prayoga. 122
  • 136. SUMMARY Next section contains disease review. In this chapter, description ofinflammation with its introduction, definition and terminology, causes ofinflammation, classification of inflammation, General mechanism of inflammation,Complication of instantiation, Proliferative tissue changes in inflammation, Reviewon anti-inflammation, Classification of anti-inflammation, Management ofinflammation and action of Non-steroidal anti-inflammatory drugs. Third part deals with pharmacognostical study of the leaf which includesmacro morphological studies, microscopic studies and physical evaluation of theplant leaves. This part of the study deals with materials and methods which is the integralpart, which includes material and methods for pharmacognostical studyphytochemical study and experimental study. The aim of the materials assessment isto evaluate the morphological, microscopical and physical evaluation of the Solanumnigrum. Coarse powder of Leaves of solanum nigrum, sense organs, microscope,chemical test tubes, filter paper, pipette and weighing box, beakers, soxhlateapparatus, redistilled water condenser, conical flask, burner, ethyl alcohol, differenttest chemicals, T.L.C kit, hot oven, watt mann glass plate, sprayer, spectroscope,KBr.disc, Albino rats etc. Were some of the materials used for present study. To evaluate the effects of Solanum nigrum method, staining process methodfor identifying and to know the physical properties of chemical compound, physicalmethod, extraction methods, solvent selection procedure, different preliminaryphotochemical tests, identification by T.L.C method and characterization byspectroscopy method, animal selection, dose fixation, grouping method aresummarized in this part. 123
  • 137. SUMMARY The pharmacognostical study of coarse powder of kakamachi. It includes themacroscopic and microscopic study, which helps for evaluation of characteristics andidentification of Solanum nigrum with its nature, colour, taste, size, shape anddescription of physical constants of the present drug to evaluate their different values. The phytochemical study of this part includes choice of solvents for extractionwith different solvents like ethyl alcohol, petroleum either, ethanol, chloroform andwater to known the chemical constituents in the drug. Extraction of coarse powder ofSolanum nigrum, with soxhlate apparatus in different batches and the extractionprocess was carried out for about 18 hours. The extraction subjected to re-distillationfor recovery of solvents and to obtain a semi-solid extract and concentrated onmagnetic stirrer the weight of each batch was recorded in detailed examination. The sample drug was subjected to the T.L.C in which the separation takesplace in short time and better resolution and sensitivity can be obtained with smallersize which is very useful in qualitative and quantitative analysis of compounds aresummarized. The alkaloid fraction was subjected to spectral analysis, with differentchemicals. When subjected for photometry in UV light, which is having specificaffinity to absorb or to transmit a particular range of wave length which is related tothat compound are broadly explained in this part of study. Experimental study deals with animal experiments and anti-inflammatoryactivity. Here the efficacy of Solanum nigrum by its extraction was evaluated againstinflammation. Selection criteria of animals, preparation of test drug with divideddoses on 24 albino rats, grouping method of induction of carraginine. Route ofadministration of drug with respective doses of it etc. 124
  • 138. SUMMARY Determination of anti-inflammatory activity evaluated on albino ratsmaintaining all the procedures of selection, administration dose, route ofadministration. Carraginine induction method, duration of treatment etc. descriptiongiven in that part. Systematically procedure was done and description is given in thatpart of study. Thus by observing and analyzing the results obtained during this study aconclusion was drawn. 125
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