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SCREENING OF ANTIMICROBIAL ACTIVITY OF BUTEA MONOSPERMA (LAM) SEED, Manjiri Kulkarni, POST GRADUATE DEPARTMENT OF DRAVYAGUNA, K. L. E.’s Shri. B. M. Kankanawadi Ayurved Mahavidyalaya, Post - Graduate …

SCREENING OF ANTIMICROBIAL ACTIVITY OF BUTEA MONOSPERMA (LAM) SEED, Manjiri Kulkarni, POST GRADUATE DEPARTMENT OF DRAVYAGUNA, K. L. E.’s Shri. B. M. Kankanawadi Ayurved Mahavidyalaya, Post - Graduate Studies Cum Research Center,
Shahapur, BELGAUM.

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  • 1. Introduction================================================================== “ SCREENING OF ANTIMICROBIAL ACTIVITY OF BUTEA MONOSPERMA (LAM) SEED ” By Dr. Manjiri Kulkarni B.A.M.S. Dissertation Submitted to The Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore. In Partial Fulfillment of the requirements for the Degree of AYURVEDA VACHASPATHI in DRAVYAGUNA under the guidance of Dr. S. K. Hiremath M. D. (Jamnagar) POST GRADUATE DEPARTMENT OF DRAVYAGUNA K. L. E.’s Shri. B. M. Kankanawadi Ayurved Mahavidyalaya, Post - Graduate Studies Cum Research Center, Shahapur, BELGAUM. ____________________________________________________________________ 2007-08. 0=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 2. Introduction================================================================== The Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore. DECLARATION BY THE CANDIDATE I here by declare that this dissertation / thesis entitled ‘SCREENING OFANTIMICROBIAL ACTIVITY OF BUTEA MONOSPERMA (LAM.)’. Is a bonafide andgenuine research work carried out by me under the guidance of Dr . S. K. HiremathM. D. Professor.Date : Signature of the CandidatePlace : Belgaum. Dr. Manjiri Kulkarni P. G. Scholar 1=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 3. Introduction================================================================== CERTIFICATE BY THE GUIDE This is to certify that this dissertation entitled ‘SCREENING OFANTIMICROBIAL ACTIVITY OF BUTEA MONOSPERMA (LAM.)’ is a bonafideresearch work done by Dr. Manjiri Kulkarni in partial fulfillment of the requirement for thedegree of AYURVEDA VACHASPATHI.Date : SignaturePlace : Belgaum. Dr. S. K. Hiremath, M. D. Professor P. G. DEPARTMENT OF DRAVYAGUNA K. L. E.s Shri. B. M. Kankanawadi Ayurved Mahavidyalaya, Post - Graduate Studies Cum Research Centre, Shahapur, BELGAUM. 2=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 4. Introduction================================================================== ENDORSEMENT BY THE HOD, PRINCIPAL OF THE INSTITUTION This is to certify that this dissertation entitled ‘ SCREENING OFANTIMICROBIAL ACTIVITY OF BUTEA MONOSPERMA (LAM.) ’ is a bonafideresearch work done by Dr. Manjiri Kulkarni under the guidance of Dr. S. K. HiremathM. D. Professor.Dr. S. K. Hiremath, (M. D. Jamnagar) Dr. B. S. Prasad M. D. Ph. D.Professor Principal,P. G. DEPARTMENT OF DRAVYAGUNA K. L. E.s Society.K. L. E.s Shri. B. M. Kankanawadi Belgaum.Ayurved Mahavidyalaya,Post - Graduate Studies Cum Research Cemtre,Shahapur, BELGAUM. 3=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 5. Introduction================================================================== COPYRIGHT DECLARATION BY THE CANDIDATE I hereby declare that the Rajiv Gandhi University of Health Sciences, Karnatakashall have the right to preserve, use and disseminate this dissertation / thesis in print orelectronic format for academic / research purpose.Date : Signature of the CandidatePlace : Belgaum. Dr. Manjiri Kulkarni P. G. Scholar 4=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 6. Introduction================================================================== CONTENTS TABLE OF CONTENTSSl. No. Contents Page No. 1 Introduction 1-2 2 Objectives 3 3 Review of Literature a) Drug review 4-27 b) Microbiology review 28-41 c) Extraction Procedure review 41-45 4 Methodology 4.1) Collection 46 4.2) Authenification 46 4.3) Collection of Test Drugs and storage 46 4.4) Pharmacognostic Study 47 4.5) Physico – Chemical Study 48-53 4.6) Schematic Chart of extraction 54 4.7) Ethanol extraction 55-57 4.8) Aqueous extraction 57-58 4.9) Preliminary phytochemical screening 59-62 4.10) Qualitative confirmation (T.L.C., H.P.T.L.C.) 63-72 4.11) Evaluation of Antimicrobial activity 72-78 5 Results 79-86 6 Discussion 87-90 7 Conclusion 91 8 Summary 92 9 Biblography references 93-98 10 Annexure I-V 5=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 7. Introduction================================================================== LIST OF TABLESl. No. Tables Page No. 1 Showing synonyms according to different authors 13 2 Rasa, Guna, Veerya, Vipak, Doshghanata 16 3 Prayojyange according to different authors 16 4 Karma of Palash 17 5 Prayoga (uses) of Palash 18 6 Doshaghnata according to different authors 18 7 Prayojyanya according to different authors 26 8 Organoleptic characters 46 9 Preliminary phyto-chemical screening 81-82 10 Anti microgial results 84 11 E-coli 85 12 Staphylococcus Aureus 85 13 Bacillus subtillis 85 14 Candida albicans 86 6=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 8. Introduction================================================================== LIST OF PHOTOS AND FIGURESSl. No. Plates Contents Page No.1 Plates No. 1 Palash plant with flowers, pods and seeds 12 Plates No. 2 Distillation apparatus 13 Plates No. 3 Soxhlet apparatus 14 Plates No. 4 Muffle furnace 15 Plates No. 5 Incubator 16 Plates No. 6 Ash of Palash seed 27 Plates No. 7 Water soluble extractive 28 Plates No. 8 Alcohol soluble extractive 29 Plates No. 9 Cold water extract of Palash Beeja 210 Plates No. 10 Culture medium 311 Plates No. 11 Micro organisum 312 Plates No. 12 Micro Pippete 313 Plates No. 13 Micro scopic view of Palash seed 314 Plates No. 14 Bacillus subtillus 415 Plates No. 15 Candida albicans 416 Plates No. 16 E-coli 417 Plates No. 17 Staphylococcus 418 Plates No. 18 Water sol. extractive 519 Plates No. 19 Alcohol soluble extractive 520 Plates No. 20, 21 Water bath evaporation of Alc.and water sol. ext. 521 Plates No. 22 Evaporate extractive 522 Plates No. 23 Water and alcohol extract of Palash seed 5 7=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 9. Introduction================================================================== ACKNOWLEDGEMENT A dissertation is an aim for every student. It is very tough and a field which is a veryailien for any student. Therefore during the compilation of this entire project. I have beenblessed with a lot of senior faculty members who have gone to extreme limits in helping mewhole heartedly to complete my research work project. I owe a lot of gratitude to them. Ihave strived to do my best of my knowledge. I hope I have achieved my success because ofeverybodys keen participation. It is my privilege for having worked under the guidance of Dr.ShivamurtiK.Hiremath M.D. (Jamanagar) (Professor) Head of Department, postgraduate department,DRAVYAGUNA for his valuable support and guidance. I express my deep sense ofgratitude for suggesting this study and for his constant encouragement with allmost patiencethroughout the course of my research work project. I express my sincere thanks to our Principal Dr B. S. Prasad M.D PhD, forprovided me valuable, timely given suggestions and proper guidance andencouragement for my research work . I thank Dr. Yogini. R. Kulkarni M.D. PhD,Dr.S.R.Kulkarni M.D. and Dr Arun Chougale M.D. for their valuable guidance throughout mystudies. I am very much thankful to Dr. R.V. Savadi and Dr Kalpana Patil M Phram PhD, ofK.L.Es Pharmacy College, Belgaum for their valuable support during Analytical studies. I am also thankful to Dr. S. D. Kolkute, Mr. Shripad Bhat and Dr Harsha Hegde,Research officer of RCMR Belgaum. I also thank Mr. Haneef of RCMR for carrying outAntimicrobial studies. I also thank Dr Anand. S. Ammanagi M.D.DNB, MNAMS for their valuable guidance incarrying out antimicrobial studies. I thank Mr. Prakash Kokate NAFARI Pune. For carrying out TLC and HPTLCanalysis. 8=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 10. Introduction================================================================== I express my sincere sense of gratitude to the management committee and PrincipalDr Gangadharan. (Ayurved Medical College, NIPANI) who gave me full moral support,encouragement and kind co-operation. I also would like to thank always cherish memories of my senior and juniorcolleagues Dr. Ramesh Konakeri, Dr. Deepak Mummigatti, Dr. Mahadev Gundakalle, Dr.Manisha, Dr. Poornima B, Dr. Poornima Undi, Dr Ajeet Herwade, Dr. Nayana.Patil whohelped whenever needed at the studies. I express my sincere heartfelt thanks to all the teaching staff members of otherdepartments for their valuable suggestion and support during my postgraduate studies. I Thank our college librarian Mrs. G.C. Gulla who provided me necessary books andjournals for my studies. I am very much thankful to my family friends Shailaja Katti and Mr. AnandDeshpande for their moral support and kind co-operation. I indebeted to my beloved parents and family members, family friends Shailaja Kattiand Mr. Anand Deshpande for their moral support and kind co-opration. Lastly Vighnaharta Graphics D.T.P. Center, Nipani. For bringing out the dissertation in the present form.Date : / / Signature of CandidatePlace : Belgaum (Dr. Manjiri Kulkarni) 9=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 11. Introduction================================================================== LIST OF ABBREVIATIONS1. A. H. - Ashtanga Hrudaya2. A. H. Ni - Ashtanga Hrudaya Nidana Sthana3. A. H. Su - Ashtanga Hrudaya Stura sthan4. A. K. - Amarakosha5. A. R. - Abhidana Ratnamala6. A. S. - Ashtanga Sangraha7. B. A. - Bruhat Dravyaguna Adarsha8. B. P. N / B. P. N - Bhavaprakash Nighantu9. B. R. - Bhaishajya Ratnavali10. Cha - Charaka11. Cha. Chi - Charaka Chikitsa12. Cha. Su - Charaka Chikitsa Sutra Sthana13. Cha. S. N. - Charaka Chikitsa Samhita Nidhana Sthana14. Cha. S. Vi - Charaka Chikitsa Vimana Sthana15. C. da/c. d. - Chakradata16. D. N. - Dhanvantri Nighantu17. D. G. H. - Dravya Guna Hastamalaka18. D. G. (V. M. G.) - Dravya Guna Hastamalaka Vijanana by V. M. Gogte19. H. S. - Haritha Samhita20. K. N. - Kaiyadeva Nighantu21. L. S. - Longitudinal Section22. M. N. - Madhanapala Nighantu23. Mau. N. - Mahaushadha Nighantu24. MIC. - Minimal inhibitory concentration25. N. A. - Nighantu Adarsha26. Sha - s - Sharangadhara Samhita27. S/k - Shloka28. Su - Sushruta29. Su. S. N. - Sushruta Samhita Nidan Sthana30. T. S. - Transverse Section31. T. L. C. - Thin layer chromatography32. H. P. T. L. C. - High performance Thin layer chromatography33. Y. R. - Yoga Ratnakara 10=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 12. Introduction================================================================== ABSTRACTA. Background and Objectives : In modern medical acrence various diseases are treated with antimicrobials, whichplays vital role in chemotherapy. It is necessary to develop such drugs from natural sources."Palash" is described as Krimighna therefore this work is under taken to evaluateantimicrobial effect of Palash seed.Objectives :1. Pharmacognostical study, preliminary phytochemical screening of Palash seed.2. Antimicrobial activity of ‘Palash’ seed extracts by different solvents.B. Methods :I. Organoleptic character such as colour, taste etc. are studied. Total ash value acid insoluble ash value, water and alcohol extractive values were determined.II. Extraction of Palash seed was done by wring ethinol and water.III. Preliminary phytochemical screening and TLC and HPTCL of extracts were also carried out. Antibacterial and antifungal studies were carried out by cup diffusion method at threeconcentrations. 11=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 13. Introduction==================================================================C. Results :1. Total Ash value of ‘Palash’ seed - 7.685 %2. Acid insoluble ash value - 0.03 gms.3. Water soluble extractive value - 38.72 %4. Alcohol soluble extractive value - 20.72 %5. Phytoconstituentes present in extract are - Glycosides, Carbohydrates, Terpenoids, Tannins present in both extract, Alkoloid present in aq. extract, steroils present in alcohol extract.6. T.L.C. and H.P.T.L.C. report of the samples showed different peaks for different Rf. Values and ethnol extract values are double than water extract.7. The antimicrobial tests were done by Mueller – Hinton agar cup diffusion method, which shows negetive result with water extract for all microbes and ethnol extract shows also registrant for Candida albicans, but sensitive for remaining microbes. 12=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 14. Introduction================================================================== 13=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 15. Introduction================================================================== 1. INTRODUCTION Potent substances are present in version plants and herbs. Modern medicines are notfree of adverse effects, so the use of herbal formulation is ever increasing in western world.People in large scale wanted potent drugs. But at the same time they want drugs to be safe.Because of this reason traditional system of medicine is regaining its past glory once again. According to Ayurvedic literature PALASH - ("Butea monosperma") is used to treatvarious diseases. It is widely and abundantly available. Different parts of Palash are used in various diseases. According to the classicalreference having a prime property of "Krimighna"(1). It can be used to treat various skindiseases. An antimicrobial agent is one that inhibits or kills microbes but causes minimumharm to normal tissues of human being. As many micro organisms have become resistant tothe newest antimicrobial agent. So now we have taken furthers step towards inventing new antimicrobial agents fromthe field of Ayurveda without isolating active constituents. Thus we have conducted preliminary study of “Palash” seed by phytochemicalanalysis and antimicrobial activity.(A) Botanical authentification of the test drug from the respective taxonomist export.(B) Pharmacogonostic study i.e. macroscopic study, microscopic study, ash value, water soluble extractive values should be determined.(C) Preliminary phytochemical screening extracts of “Palash beeja” (seeds).(D) Antimicrobial activity of the alcohol and water extract of Palash seed by Agar plate method. As we are not isolating active principle from plant drug. So dose tends to be littlehigher, as compared to the synthetic preparation. Advantages are more than disadvantages.We are administering drug in natural form which is widely and easily accepted by thepatient. We will be are giving these drugs without harming the patient and would be moreeffective without compromising with our basic principles. 1=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 16. Introduction==================================================================A) Review of Liturature :- Drug review, Microbiology review, extraction procedure review,B) Methodology :- Collection of the drug, Authentification, Storage, Pharmacognostic study, Phytochemical screening, TCL, Antimicrobial activity,C) Result :- Aqueous extract showed resistant activity to bacteria and fungi and Ethenol extract showed resitant activity to fungi and sensitive to bacteries. 2=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 17. Objective================================================================== 2=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 18. Objective================================================================== 2. OBJECTIVES(2.1) Pharmacognostic and physicochemical study of Palash seed. To study regarding phyto constituents of the drugs so that it maybe helpful to interpret regarding its pharmaco kinetic in further studies.(2.2) Preliminary phyto chemical screening of Palash seed. To know presence of phytochemicals which also may help in studying the pharmaco kinetic of the drug.(2.3) Anti microbial activity of Palash seed. To know the antimicrobial effect, so that we can interpret on the Krimighna effect which is stated in Ayurveda texts and then a step wise and complete antimicrobial study can be done for better therapeutic effects. 3=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 19. Review of Literature================================================================== 3=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 20. Review of Literature==================================================================(A) Drug Review(1) Vedic Review (2), (3) : We have got the references of use of ‘Palash’ since vedic period. In this kala single drug thearpy was present and since then single drug therapy is being used The use of Palash was common in vedic period not only to treat the ailments but also in routine life and in holy rituals. In vedic era leaves, stem and flowers were more used but there are references regarding use of the seed. In vedic kala Palash tree was known as Shant Vruskha and Bramha Varchass. Samidha of this plant were used at the time of different Homa and Yadgnyas. According to Koushika sutra, Palash is Medhajanan and lepa of Palash these leaves was applied in Jalodar (Ascitis). Keshav told it was Sarvaroga Bheshaja and also it was used in Krimi Roga ( Ke.P. 9. 4/25/20 ). In Rigveda Kinshuk was the synonym given for Palash. Kinshuk means who shines brightly. This synonym is given because of its bright attractive colour of the flower. In Rigveda kala, Palash leaves were used with Ashwath and we get the referenes of uses of Palash leaves with Nyagrodha in Atharvaveda. In Upanayan samskar Dand (Stick) which is used by Brahmachari was also made from Palash wood. Palash was used frequently because it had a power to destroy the Rakshasas so also its stem was being used in Yadgnyas and patras were used to prepare Abhishek paatra. In Atharaveda “Parnamani” of Palash patra was used to gain Bala, Aayu, Samruddhi and fame. According to Shrout sutra patra valkala of Palash was used in preparation of curds. The tree is considered sacred both by Hindus and Buddhist. Hindus consider it, as holy because of the trifoliate formation of leaves which represents the Holy trinity of Brahma (the creator) on the left, Vishnu (the preserver) in the middle and Shiva (the destroyer) on the right. The flower of this plant are offered especially to Goddess 4=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 21. Review of Literature================================================================== Kali. In Krishnasthami Vratam, wood of Palash is also being used. Because of curse of Goddess Parvati, Bramha was converted into the tree of Palash. In Navagraha Stotra written by Vyasa the character of Ketu has been compared with Palash flower.1) Samhita kala :a) Charak Samhita : In charak samhita Palash is not included in any Gana but it is said to be used in treating many diseases and in different ritual functions. We couldnot get any synonym for Palash in this samhita. Leaf, flower, skin, seed and kshar are the parts used in different yogas. Palash is used in kasa, Grahani, Arsh, Udararoga, kushtha and in skin diseases. But seed is mainly used in skin diseases Kushtha, Arsh and Udarroga (ChS.Chi 110/13) (ChS.Chi 92/14)b) Sushrut Samhita : As per Sushrutcharya Palash is included in Rodhradi, Mustakadi, Ambavashtadi and Nyagrodhadi Gana. Kinshuk is used as synonym for Palash in various explanation in Sushrut Samhita. Utility of Palash in kushtha, Gulma, Udar Roga, Arsh, Bhagna, Netraroga and Raktaprasadan (Su. Chi 7/2, 9/7, 18/42, 19/49).KASHYAP SAMHITA : In this Samhita kwath of Palash is used to give Mukti from Sheetputana Grah forkids.2) Sangraha Kala :a) Ashtang Sangraha and Ashtang Hridaya : Vagbhata also includes Palash in Rodharadi, Mustadi etc., (A.H.S 15/32, 15/38) Rogaghnata of Palash is given in 5=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 22. Review of Literature================================================================== Krimi gulma, Kushtha, Vaata vyadhi, Rasayan, Shwitra kushtha, Arsh (A.H. Chi 20/26, A.S. Chi 5/9).b) Chakra Datta : He has told the utility of Palash same as Charak but he used Kinshuk as synonym.(Ch.Chi.3/258) (4)c) Sharangdhar Samhita : According to Sharangdhar, Palash seed is used in Loha Rasayan Yoni Sankochanarth lepa. Kshar of Palash is used for kesh Nirharnana lepa and for Netra prasadan vidhi. (5)d) Bhavaprakash : In Bhavaprakash, Palash includes in Vatadi Varg. Many Synonyms given for Palash are found here. And it is mainly indicated in Krimi, Arsh, Prameha, Vataja and Kaphaja Vikar, Kushtha, Gulma and Udara roga.e) Bhaishajya Ratnavali : It is used in Krimi, Arsh and Vatrog many diseases. Seeds are used in preparation of many yogas.f) Yoga Ratnakar (6) : Palash beeja recommended in Krimi Roga, Arsh, Gulma etc.,III) Nighantu kala : Many drugs have been described in detail by giving different synonyms and their properties and uses. The drug Palash has been described in every Nighantu because it is well known drug since Vedic Period.1) Bhavaprakash Nighantu (7) : In this Nighantu Palash is mentioned in Vatadi Varg and given nine synonyms. Though it is used in many diseases mainly indicated in Krumi Rog. (8)2) Shaligram Nighantu : This nighantu includes Palash in Phala Varga and gives 23 synonyms. Author stating that it is Krimihar and mainly seeds are used in Skin diseases. 6=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 23. Review of Literature================================================================== (9)3) Dhanwantari Nighantu : In this Nighantu it is included in Amradi varg and 14 synonyms are given for it. Krimihar property of seeds is mentioned. (10)4) Kaiyadeva Nighantu : In this Nighantu it is included in Aushadhi varga and twelve synonyms are given for it. (11)5) Madanpal Nighantu : In this Nighantu it is included into the Vatadi Varga. Different Synonyms are given in this book. Types of flowers and its uses in different Rogas are explained. (12)6) Raj Nighantu : In this Nighantu it is included given in Karviryadi Varga 11 synonyms are given for it. Nighantukar also mention that it is Krimihar and its seeds are used to treat various skin disorders. He has mentioned 4 types of Palash 1) Peeta 2) Shweta 3) Rakta and 4) Neel pushpa.Though all 4 are having same quality, in those Shweta is the best for treatment.7) Shodhal Nighantu (13) : In this Nighantu seed is said to have Krimihar property.8) Nighantu Adarsha (14) : Palash is classified in “Palashadi varga” so many references of different authors regarding Palash are given in this Nighantu.9) Priya Nighantu (15): He mentioned it in “Haritakyadi Varga”. Its seeds are Krimihar in action and seven synonyms are given.ADHUNIK KALA : (16)1) Indian Mateia Medica : The author of this book has mentioned vernacular names and chemical constituents of this drug.. Seeds are indicated in many skin diseases. Internally for worms, externally for ringworm, boils, pimples, bubbles, tumours and also in various other diseases. 7=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 24. Review of Literature================================================================== (17)2) Data base on medicinal plants used in Ayurveda Vol 1 : Detail explanation regarding plant Palash has been given such as - Family, Classical names (Synonyms) vernacular names, morphology, useful parts, uses and doses.Along with that pharmacognosy, chemical constituents, pharmacological activities, toxicology and therapeutic evaluation is explained. (18)3) Indian Herbal Pharmacopoeia : Detail Information regarding Palash seed is given i.e. macroscopic, microscopic examinations etc., (19)4) Medicinal Plants Quality Standards of Indian : Description regarding plant is given and also all types of phytochemical tests are explained in detail. (20)5) Vanoushadi Nidarshika : Containts description of seed and Im ¶e«dœ¦d «ddÎdd is given. (21)6) Indian Medicinal Plants : Detail description like names, vernacular names distribution etc., are given. Also properties and uses of seeds are given.7) Wild medicinal plants of India (22) : pharmacognasy and phytochemical description and use of Palash seed in folk medicine8) Indian medicinal plant by Kirtikar and Basu. (23) : Properties of seed in detail have described that seeds are not dry digestible, antihelmentic, aperient used in urinary discharge, piles, cure, skin diseases, tumours, abdominal trouble and also used in scorpion stinge. (24)9) The Useful Plants of India : Herpes in dhobies itch seed are pounded with lemon juice and used in skin diseases. Seed yield a fatty oil (18%).ii) Synonyms and their Meanings and Interpretations : Morphology of the plant, prominent characters, utility all the things have been mentioned by way of synonyms. There is a class of Nighantus which describe the drugs by its synonyms. Acharya 8=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 25. Review of Literature================================================================== Narahari Pandit the Author of Raja Nighantu has tried to arrange the synonyms in a proper way and elaborated them into 7 categories.The various synonyms of “Palash” as given in Nighantus as follows :DIFFERENT SYNONYMS :1) nbm (^m àeñ m nbm Ý ` & e: .) V{Z em `ñ Leaves are more succulent and Fleshy.2) q$ H (^m "q$ Ho `_² ?> {V ^«Ý O H ew w>Ñ nw dm & H $ .) H $ @ B mV Z $ H Ê g e înË V ew : ew m p : $S V ² The flowers resembles beak of parrot3) H Z (gm H ²hÝ , nbm rO ` H m à`mV & $ ¥ : o ¥ {_¿ .) $ _rZ pV e~ ñ ¥ oo om $ J J ² {_a The seeds are used in krimirogahar4) j m o: (^m j m j o l ð: & að l > .) a f w o d¥ > Kshara prepared from this plant is superior.5) I a ©({Z néf § ©` & nU .) : nU ñ _ Leaves are rough.6) {Ì nÌ : (gm Ì r{U nÌ Hì`ñ nU & .) o $ ` } m Leaflets are present 3 in No.7) nU (^m àeñ m nU© ` & : .) © V{Z mÝ `ñ Plants having more leaves.8) nw þ(Y ny: n{dÌ m w & V : .) V Ð o _: Ð This plant is used in Rituals.9) ~ ñ o: (gm ~ § Z _w § & rO Zh .) o rO ñ o Š ñ h V` 9=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 26. Review of Literature================================================================== Seeds are oily and snehayukta.10) ~ d¥: (^m ~ Uo j : d{X $ ñ $ o à` m dm & « j ÷ .) ÷ m « d¥ ¡ H Ha w o`Ë V g§ m f Á ² Wood of the plant utilized in yajnya kunda as fuel.11) `m $ (^m `ko Á Z & {kH: .) à`w : `_m It is used in yagnyas.12) a V înH (^m a V{Z nw Ê ` & Š nw $ .) Š m înm : `ñ Flowers in red in colour.13) dH înH (gm dH nw_ñ & $ : .) § « $ o « în ` nw $ Flowers are curved.14) dm : (^m dm ` ha em $ dm m B Aï> {Z Ê>"dm o: B gm o R Vha .) V ñ : _H V o: {V m K Q V W {V ob nm :& aY § J m nm ¡ T> > : It is used in vata vikara.15) dm W (n.) dZ àñ m m o ñ & Z : àñ o W dgm ` o @ We get these plants more in forest.16) g{_Û: (^m `ko à`w m§ m o > & a .) f w Š Z g{_Y§ îR V m l : Wood is used in yadgnyakunda.17) dm o: dV nm`{V B & VW m o nm § W {V18) q$ H :- Its flowers resembele to nose of H $ ew parrot in colour and shape.19) j m o :- að l > Its alkali is best among alkaline materials.20) nbm :- e Its leaves are fleshy and beautiful or it develops the muscle. 10=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 27. Review of Literature==================================================================21) a V wH:- Š nn $ î Its flowers are red in colour.22) da g{_Ü o Z B (ñ § `V @ `m {V d.) It mesmerize every one with its qualities and it improves appetite. (25)DESCRIPTION OF PLANT ACCORDING TO ITS SYNONYMS : Palash is sacred tree (Putadru) used in religious rituals and sacrifices (Yajnika,brahmavruksha, Samidvara), It grows widely (Vanaprastha) has characteristic leaves (Palash,parna) with three rough leaflets and curved (Kharaparna, triparna). Flowers are red (raktapuspaka) and curved (Vakrapuspaka) typical of papilionate resembling parrots beak(Kimshuka). Seeds are oily (bijasneha) and make a potent antihelmintic drug (Krimighna). Italso pacify vata (vatahara). The plant is one of the best among the sources of alkalies(Ksarasrestha).SPECIFIC CHARACTERS :1) Plant grows wild.2) Leaves rough, trifoliate.3) Flowers red, curved.4) Seeds Oily.5) Pacifies vata and one of the best sources of alkali, seeds antihemintic.Synonmys according to different authors : 11=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 28. Review of Literature==================================================================a) {e. {Z :- Á Š nw {eå r, {V V rO $ H d¥ . . dbÐ V în, ~ Š~ H $ Ý , ¥ V îUb) A{^. _§:- d $$ _, a n n g{_Û, n w r, d « . Hw § î , « gy J w H a ¥e§ H. W¥J $ sc) H {Z § :- q$ H H ©`m $ JU H dQU, Û ñ o, $ Q ¡. K w H $ $ , kH o $ w u rO Z > ew _m , , é , > n h {Ì d¥, a V în, j m o, dm o, ~ d¥, g{_X a Îm Š nw a ð V W ÷ Îm l > nm « ² . dd) _X nm {Z §:- nbm H H $ ,` m $ « nm m ð,a V în dÎm ² Z b K w e,q$ $ {_© {kH ÷ X a o Š nw,{Ì ¥,g{_V Q> ew,{H ,~ n,j l > .e) Am {Z Êw nbm q$ H ~ nm , j m o, dm o, ~ d¥, g{_X a X K Q:- e© > § e, H $ ÷ X ew « n a ð V W ÷ Îm , l > nm « d ² .TABLE NO. 1 12=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 29. Review of Literature==================================================================SHOWING SYNONYMS ACC. TO DIFFERENT AUTHORSSynonym Sha Cha Su AS DN RN KN BP.N AS.H Sho.N + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +VERNACULAR NAMES: 13=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 30. Review of Literature==================================================================Sanskrit - PalashHindi - Dhaka, Tesu, Palas, Chichra, Dhara, Faras Kankeri, Chini agondBeng - Palash, Ganch, KamarkasMar - Palasa.Tree Guj - Khasathi Khakra.Flower- Guj - Kemuda.Seeds Guj - Palash Paprha.Tam - Paras, Patsan, Camala, Paladula Modug Mooduga.Tel - Modhung, Midug chettu.Kann - Muttuga, Muttala, Muttagamara.Mal - Palashin Samatha, Camata, Pilacham, MuraklamarC²V - nm aewd - I mQ H> $rJm - n g y bm $ bA§ - Downy Branch buter. Flame of the forest, parrot tree, Judas tree (Myths.and traditions)½{b`adm - Beespaknm§~Or - Tesh.Am [a ogm - Kinjuko Porasu.Cw X© - Palash Papra.Bheda (Varieties) :According to Madanpal - Rakta, Peeta, Shweta and Neel ( according to colour of the flowers)In Abhidhanmanjiri - 1) Palash and 2) Valli PalashShaligram - 1) Kinshulak 2) Hastikarnak.According to Raj Nighantu - Rakta, Peeta, Shweta and Neel.Guna and Doshagnata : 14=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 31. Review of Literature==================================================================PN - Kashay, Shaligram - Katu, Tikta, Kashay, Katu Vipak Ushnavirya. Vatakaphahar.Taxanomical Classification :Kingdom - PlantaeDivision - Magnoliophyta,Class - MagnoliopsidaOrder - FabaleaFamily - FabaceaeGenus - ButeaSpecies - MonospermaLatin Name - Butea monospermaMeaning of Butea Monosperma (LAM) :Butea = Named after John Ear of Butea, patron of BotanyMono = One, sperma= seed.Before Butea monosperma, Butea frondosa was the name, frondosa means leafy.Palash is famous for its leaves.Flesh, like flesh or bloodOr - Demon.TABLE NO. 2Rasa, Guna, Verrya, Vipak, Doshaghnata 15=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 32. Review of Literature================================================================== Rasa B.P.N MN RN DN NA KNTikta + + +Katu + + + +Kashay + + + +GunaLaghu + +Rukha + +Snigdha + +VeeryaUshna + + + + + +VipakKatu + + + + +Doshagnataa) Kaphaghna + + + + + +b) Vataghna + + + + +c) Pittakar + + +TABLE NO. 3Prayojyanga according to different authors :Prayojyanga PN PV.S RN DG(VMG) DGH MN NA DN Sha.NBark + + + + +Flowers + + + + + + + + +Leaves + + + + + + +Seeds + + + + + + + + +Gum + + + + + + + + +TABLE NO. 4 : Karma of “Palash” 16=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 33. Review of Literature==================================================================Karma Bpn DN RW MN KN PNDeepan + + + +Vrushya +Krimihar + + + + + +Sangrahi + +Sarak + + +Vranahar + + + +Gulmahar + + +Grahani + +Arshya +Kusthahar +Rasayan +Veeryavardhak +USE OF "PALASH" SEED ACCORDING TO DIFFERENT AUTHORS : n eíV H `m : H Xo {d m : & bm w$ o ¥ m ZeZ fmîU $ f _r V ² rO n_mÊ> X wXo ZeH ²& (em m {Z Êw X § mH SV Xd m m ¥ & {bJ« K Q ~ $y Ë f { $ V _ > ) H `§ a J« n n gw`§ ¥ Z M~ § § nQ & ([à`{Z Êw $ n § m w§ a H § rO _V n ©^_² & fm {h î å ${_¿ > m KQ> ) ~ § w$w pZY îU H bm V &(Yd {Z Ê> rO V H >§ ñ ½ § ¥ g{O ² ݧ K Q Q$ H _w $ {_~ . w ) $ bKU _oe© ¥ m $ m _²& b§ w h : H V $ h î m $ H n {_d {d m H w éj § w Jw o àU ²& (^m nH $>§ H > ë X V & .à.) $ H o Q$ $ _m w ð a V ² rO H ܧ HÊ>a `Z {h : &(gm .) X § ¥ dg $ So gm o V ~ $ {_{d { m m o .{Z $ rO MpZYo § $> ¥ $ m O V & ({Z .) b~ § ñ ½îU H w $ $ ² `o² & .a m QH {_H Z V ² rO H ܧ {h : HÊ>a `Z &em . X § ¥ dg V $ So gm o ob ~ $ {_{d { mm T>TABLE NO. 5 17=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 34. Review of Literature==================================================================XII) Prayoga (Uses) : Prayoga BPN DN RN KN MN SNPrameha + + + +Arsha + + +Krimi + + + + + +Kushtha + + + +Gulma + + + +Udar Roga + +Twakvikar + + +Kandu + +Pleeha +Shool +Vatarakta + + +Raktapitta +TABLE NO. 6XIII) Doshagnatha according to different authers : Doshaghnata ShaN KN PN BPN DN RNKaphavata Shamak + + + + +Pittavardhak + +(VII) INTRODUCTION OF PLANT (GENUS AND SPECIES) (27) : This plant was identified as Butea monosperma (Lam) belonging to family Fabaceae.It was found that this genus Butea consist of around 35 species. Out of which literatureregarding the phytochemical and pharmacological profiles was available on only fewspecies. Most of the reports available were on the Butea monosperma and very few reportsare available on Butea superba and Butea parviflora. But as per literature seeds are welldocumented for various activities, 18=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 35. Review of Literature==================================================================BUTEA SPECIES : There are 35 species available in the Butea genus. Generally they are trees orscandent shrubs. The general characters of Butea species are as below :Leaves - Pinnately, 3-foliolate, stipellate.Flowers - Orange, purple, rose or white, densely fasciculate; Fascicles recemose or fasciculate paniculate, or ample paniculate.Calyx - Campanulate, the upper two teeth or lobes connate.Petals - Subequal or unequal; keen incurved and acute, or Straight and obtuse.Stamens - Ten, Vexillar stamen free, the other connate; anthers uniform.Ovary - Sessible or stipitate, 2-4 (-7) ovulate; style incurved not bearded; stigma capitate or truncate.Pod - Pod is one seeded.Botanical description of the important species of Butea are as follows :1) Butea Superba : Butea superba is a gigantic climbing shrub and its characters are mentioned in the following paragraphs.Trunk - Trunk is climbing from left to right, attaining 0.6 0.9 m girth.Leaflets - The leaflets attain 30-45 cm and sometimes 50 cm in young plants.Flowers - The flowers are larger than other species, 4.5-6.3 cm long, orange or orange scarlet, borne in great profusion along the leaflets branches on racemes which are 30 cm long. 19=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 36. Review of Literature==================================================================Pod - Pod is about 12.5 15 cm long.Distribution of Butea superba : Butea superba is distributed in the forest of Oudh and Bundelkhand, Chota Nagpur,Burma, Konkan, N. Kanara and Central. It is also found in South India.Distribution : Throughout India, in deciduous forests in areas up to 1,200 m elevation, also in openareas.The Plant : A medium-sized deciduous tree, with a somewhat crooked trunk,10-15 in height and5-6 in girth. The bark is bluish-grey or light-brown and yields a gum. Its bright orange-redflowers (1.5-2” long) bloom in great profusion at the beginning of the hot season before theappearance of new leaves. The pod contains a single seed (1”x3/4”) at its apex. Butea monosperma is common throughout India, Burma and Ceylon (up to 4,000),except in very arid parts. Generally, it grows gregariously on open grasslands and scatteredin mixed forests along with Shal (Shorea robusta). It is frost-hardy and drought-resistant andis a valuable species for reclaiming saline soils.Parts used : Bark, leaves, flowers, seeds, gums. Butea monosperma bark is white or yellowish-brown, but very liable to sap -stainand often turns greysh-brown or grey. It is a light wood and can be seasoned without muchdifficulty, but it contracts considerable during seasoning. It is not strong or durable inexposed situations, but is said to last much longer under water. It is easy to work either by 20=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 37. Review of Literature==================================================================hand or machine, and is used mainly for well-curbs, water-scoops and for fuel. It can also beemployed as a cheap board-wood . The leaves are much used throughout the country for making plates, cups, etc. Theyare eaten by buffaloes and elephants. The flowers yield a brilliant but very fugitive yellow colouring matter. This iscontained in the sap and may be obtained in the form of a decoction or infusion from driedflowers. The addition of alum, lime or an alkali deepens the colour to orange and also makesit less fugitive. The sap contains the chalcone, butein C15H12O5 (0.3%), orange yellowneedles, m.p., 2130c 2150c, and small quantities of butin, the colourless isomeric flavanone,and its glucoside, butrin.USES : Bark furnishes a very important exudation which hardens into a red brittle resinknown as butea-gum or Bengal kino or magugo, largely used “as a substitute for the Kino inIndia and to a limited extent in Europe also”. A red juice exudes from natural cracks and artificial incisions in the bark, and ithardens into a vitreous, ruby-red gum, known as Butea gum or Bengal kino. It isdistinguished from Malabar kino from Pterocarpus marsupium by its greater solubility inwater, and by the presence of corky particles. Butea gum contains a large proportion oftannin and mucilaginous material. On dry distillation, it is reported to yield pyrocatechin. Itis a powerful astringent and is given in many forms of chronic diarrhoea. (Dymock, Wardenand Hooper, I, 454) The seeds have long been valued as anthelmintic. Birdwood prescribes them forround worms and tapeworms. According to Chopra freshly powdered new seeds give fairlygood results against Ascaris, but old worm-eaten ones, like those frequently found in themarket, shows little activity. The oil, powdered seeds, and an alcoholic extract of seeds,provide quite ineffective against hookworms, etc. When pounded with lemon juice and 21=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 38. Review of Literature==================================================================applied, the seeds act as a powerful rubifacient and they have been successfully used incuring a form of herpes, known as dhobies itch. (Dymock et al., loc. Cit.) The seed is alsouseful in skin diseases Dadru, Pama, Kandu, ringworm infections etc. The seeds contain 18% of a yellow, tasteless oil (sp. Gr. 25o/25oC, 0.8983; sap. Val.,178; iod. Val., 67.2, Katti and Manjunath, J. Indian Chem. Soc., 1929, 6, 639). Fresh seedsare reported to contain proteolytic and lypolytic enzymes. The former is a mixture of plantproteinase and polypeptidase, and behaves like yeast trypsin (Chatterjee, Ghosh andChopra). The leaves are astringent, anti-inflammatory, anodyne and aphrodisiac, and are usefulin pimples, boils, flatulence, colic, worm infestations, inflammations, arthralgia andhaemorrhoids. The flowers are astringent, sweet, cooling, constipating, aphrodisiac, haemostatic,diuretic, febrifuge, depurative and tonic. They are useful in vitiated conditions of pitta andkapha, diarrhoea, haemorrhoids, menorrhagia, strangury, fever, leprosy, skin diseases,swellings, hyperdipsia, haemoptysis, arthritis, burning sensation, bone fractures, and are veryefficacious in birth control. Gum combined with other astringents and rock-salt is recommended byCHAKRADATTA, as an external application for “Pterygium” and opacities of the cornea. The seeds are purgative, ophthalmic, anthelmintic, rubefacient, depurative and tonic.They are useful in herpes, skin diseases, ringworm, ophthalmopathy, epilepsy, round worm,arthritis, flatulence, constipation and diabetes. As anthelmintic and aperient, BHAVAPRAKASH recommends new seeds to begiven in powder, 10 to 20 grains or as paste with honey added (“because the seeds are veryunpleasant to take and often produce retching pain in the abdomen and occasionallyvomiting and giddiness”) thrice daily for three successive days (especially for ascaris roundworms) and followed on the fourth day by a dose of castor oil. For this, the seeds are soaked 22=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 39. Review of Literature==================================================================in water, shells removed and kernels powdered after being dried. “Some medical menconsider that the seeds can be advantageously substituted for “santonin” against roundworms”. Externally the powder is a remedy for ringworm; it may be applied better in theform of a paste being pounded with lemon juice; also for herpes (Dhobis itch). “Moodooga oil is said to be practically inert and does not possess any anthelminticactivity. Active principle of the nature of alkaloid, neutral principle or glucoside could not beisolated from the seeds.USE OF PALASH IN DIFFERENT YOGAS :- According to Charak Samhita. Yogas Indication References Palash beeja. Udar rog. Ch.chi (110/13.) Palash beeja. Arsha Ch.chi (92/14.) Choorna. Kasa. Ch.chi (77/18.) P. Beeja + Ghruta. Atisaara, Ch.chi (27/19.) P.Beeja. Visha Ch.chi (51/23.) Palash Kshara Dwivruna Ch.chi (54/25.)ADHYAYA : According to Sushrut Samhita. YOGAS Indication References Palash Valkal Vruna bandhan. Su.chi. (3/6) Palash Choorna. Vaata Vyadhi Su.chi (4/32) PalashKshara. Ashmari Su.chi (7/22) Beeja lepa. Kushta. Su.chi (9/10) Beeja choorna. Prameha. Su.chi (11/8) Beeja choorna. Udar rog. Su.chi (14/13) Beeja choorna. Maha vyadhi. Su.chi (31/5)ACCORDING SHARANGDHAR SAMHITA : 23=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 40. Review of Literature================================================================== Palash beeja choorna Krumihar, 5/26. Palash beeja choorna Yoni sankochanartha lepa. 11/110 (Uttarakhanda) Palash pushapa swaras. Netrapasadan 13/78. (Uttarakhanda)ACCORDING TO ASHTANG SANGRAHA. (A.S) : P. Vrunta Swaras. Kasa. A.S.Chi (3/65) P. Churna. Raj Yakshma. A.S.Chi (5/9) P. Churna Arsha A.S.Chi (8/63) P. Choorna Vaatshonita. A.S.Chi (22/45)ACCORDING TO ASHTANG HRUDAYA. (A.H) P. VruntaGhruta. Raktapitta. AH.Chi (2/44) P. Ghruta Arsha AH.Chi (3/101) P. Beeja. Krumi AH.Chi (20/26) P.Kshara. Rasayan AH Kalpa Sthana (39/37)COLLECTION AND STORAGE OF DRUG : In the present study the matured seeds of Butea monosperma were collected in themonth of April and May near Belgaum forest area. The seeds were authenticated byTaxonomist. Soon after authentification all the seeds were dried at room temp until theywere free from the moisture and subjected to physical evaluation with different parameterslike nature, colour, taste, odour, size, shape width, length etc. Nearly 1.5kg seeds werecollected and kept in air tight jars to avoid fungal infections. Veerya period is 1 year.ADULTRATION AND SUBSTITUTION (28) : 24=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 41. Review of Literature================================================================== Butea monasperma seeds are adulterated with the seeds of B. Superba (Roxb). Thesecan be identified on the basis of the size of seeds i.e the seed of B. monosperma arecomparatively larger in size. But in this case the seeds were collected by own. So there is nochance of adulteration.(XI) CHEMICAL CONSTITUENTS REVIEW (29) : A Nitrogenous acidic compound (I) along with Palasonin isolated from seeds. α Amyrin, β Sitosterol, its glycoside and sucrase isolated from seeds Glycerides of Palmitic stearic, Lignoceric, Oleic, and, linoleic acids from seed oil. Seed contain 18% of fixed oil called moodooga oil small quantity of resin and largequantity of water soluble albuminoids. The compound (I) showed Antimicrobial activity against fungi which arepathogenius and gram +ve bacteria strepto coccus pyogences, staphylo coccus aureus,Bacillus substilis gram ve bacteria ,E. Coli, Pseudomonas, maximum inhibitary effect wasshown by gram +ve bacteria,ACTION AND USES IN SIDDHA : • Uses and Action are mentioned in Siddha. • Action and uses in Unani medicine. The fruits and seeds are bitter and oily and mare administered against piles, Eyediseases and Enlargement of spleen (Spleenomegaly).TABLE NO. 7 25=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 42. Review of Literature==================================================================Prayojyanga according to different authors: Parts used Names of the authors Or S.H.N D.N K.N N.A P.N BP.N R.N Prayojyanga Patra (Leaf) + + + + + + + Stem + + + + + Flower + + + + + + + Seed + + + + + + + Kshara + + + + + + +NOTE : Studies carried out on Palash seed.Pharmacological activities and clinical trails as:- - Antihelminthic activity. - Antifungal and Antimicrobial activity. - Antidiabetic activity. - Antioestrogenic activity. - Ophthalmic disorder. - Anti implantation activity in female rats. - Anti hepato toxic activity. - Uterotropic and uterine perioxidised activities in female rats. - Antioxidant activity.Internet information –(1) Kumari,N, Ehaum ,V, Mikosch, M, 2005 Seasonal photosynthetic performance and nutrient realation Butea monosperma (Fabacea) in comparison to two other woody species of a seasonal deciducos foresty in Nw India and planted tree in this area , Indian journal of forestry 2005 28:116-126.Yadava R. N. Tiwali L. 26=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 43. Review of Literature==================================================================(2) A potential antiwiral flavone glycoside from the seeds of Butea monosperma O. Kuhne J-Acian-Nal-Pro-Res-2005 Apr. : 7(2) : 185-8.Sexcena, Ajit Kumar, Gupta(3) A pharmacentical composition (extract of Butea monosperma flower) useful for the treatment of the hepatocellular caricinoma world intellectual property organization 2006?A. Khan, T. H. Prasad.(4) Butea monosperma and chemomodulation protective role against thioacetamide medicated hepatic alternation in wistar rates. Physiotherapy & Phytopharmacology 02/01/06.USES AND ACTIONS ARE NOT GIVEN IN SIDDHA : 27=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 44. Review of Literature================================================================== (30)Actions and Uses in Unani : The fruits and seeds are bitter and oily and are administered against piles, eyediseases and enlargement of spleen. (Spleenomegaly).B) MICROBIOLOGY REVIEW :I. Microbes (Krimi) In Vedas (Ayurveda) (31) : From the vedic period itself acharyas have been aware of minute organisms of thenature / environment. They have also described krimis, their habitats types, synonyms andtreatment. The acharyas had knowledge about jantus etc. that had harmful and harmless tobeings. under the heading of krimis they have described about both Ñï> (drustha) AÑï>H¥${_ (Adrushta krimis).HABITAT : `o « n © wZ w ² Y wewAß Ý & {H $_`: d o d o A¡ r~ n f w gd m V f f f : `o _m Vd {d ew ©X {_ {H m & (A. 2/31/5) Añ H Ý_m{d gd V ² $ § Y « _² & $ _rU In atharvveda it is told that krimis are present in all factors of the nature. They arepresent on parvath, vana aushadhi, pashu, etc.Minuteness of Krimis :(1) gy_Ë À ¡ o^dÝ í` m&(C.S.VI 7/11) ú dm H Ë : & M$ `Ñ(2) gm`m H X e©: &(vaghbhata) ¡å V $ X Z & ú o {M m 28=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 45. Review of Literature================================================================== Here Acharyas says that Krimis are not visible to our naked eyes.(1) gm`m H X e©: & ¡å V $ X Z & ú o {M m Here Drushta and Adrushta types are specified.Interpretation by Acharyas :1) H o Ü {V B {H & $ `m {V « & « _o ì` $ _r: One which survives on raw meat is krimi.2) H Vm $ o© md One which has desires.3) H om `m ga H : & $ « d© V U $ _V ñ ² _© One which crawls and moves.CAUSES OF INFECTION BY KRIMS :1) C H ~ d àmmnW _²M$ f w & X o h : U: ¥ ì`m $ { bo M gy_ `m ^ym V © `{Z ^m & ú or{Z V{Z H Jå Z $ a & V n _Uo {n {Z m `o§ W ²ñ $Y n ` : & ú m Am nV f m m H `© o Z ñ V Ý `o Z {d Ü pV nÌo {n Vo ZZ & AÝo {d `m m w ~ m m f Ý f O ² (` .d. 16/ 62) o2) àg§V Jm ñ em {Z mV gh oZV & Jm Ìg§ © :ídgm ^m m ² n V² ² O ² gh `m ƒm d Ì_m Z o m & eægZ {n ñ ë w ZV & `m n ² b H > d íMemMZ m X E M& $Á w§ a ð o o{^î`§ d fû Ì Am H aoü g§_pV Z m & ¡ g{J© m H Ý aZ & n $ Jm « $ _² (gw . 5/ 33- 34) .g.{Z3) ñ e}Hha eæX go m àm o m& n $ m `m d V ``m : m r Z ² JX gV g§[a Uo o ËH {d $ m eo : & } M Z m Ì d ² Ha {d f V Z $ m (A.h.{Z 14/ 441) . 29=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 46. Review of Literature================================================================== By dushita, vayu, jala annasevana, bhumi and by maithuna infected rogi gatrasparsha of infected pt. infection by nishwasa etc. use of contiminated clothes, bed etc. ofinfected patient / sahabhojana etc. krimis are said to be entering the body.ORIGIN OF KRIMIS : g§bom {H Š XV « ñ ^dpV C m : &(M . 17/ 38) ² ${_í`m` Ý nhVË _Z .g.gw Charaka says kleda in the body is one of main factors for the production of Krimi’s.ROUTE OF ENTRY : Am gw V e~ o n V `m {n M AeZ X å & _o n o i {d Š o o emo o X` Š _m m V m m ` {n M {d `m V_JXo V & (E5/ 29) XË àO em _Z m §`Ým m w V X`_ñ & Krimis enter the body through annapanadi and vruna mukhas. `m ¡n gn m o m n gd© & ¡Aí`m [a ©o Zgo [a m V `m {V d m o `§ NV V {H O {g & (A. 5/ 23) § `m JÀ > § « ^`^m & V _Ü { $_rThere they also enter through eyes, nostrils mouth to produce diseases. In sharangdhar samhita, he mentioned main two types of krimis are given (1)external (2) Internal * Internal krimis are further divided into 20 types eg. Kaphaja, Raktaja,Pureeshaga etc. a V {h {` ao Z a V m Ý dm d: &(A.h . 14/ 42) Š dm m mŠ O OV o ñ`m AU .gw Krimis presenting in blood are the minutest.VYAKT KRIMI LAKSHANAS : 30=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 47. Review of Literature================================================================== em V m§ w bww>g_m g_w m ñ m a V mÝ o {U m o OZ V I H ¡ Z ñ Z W § Š d{h `m $: îR § W§ Z Y `: g§ m AU m V nnX gw ¡Ëm ¡o _Ý ñ Z do ¥ñ m í` ú dÀ H W d m _m M$ ^dÝ AÑ : dU m§ h H S Vo pV í`m «JVZ Mf©$ w m m Ê> X g§ n m A{V ¥YZ§ ËH {gañ m _mV mW`m {_V & ñ ©{Z dX m M d w m `w § éUp VU ² m $ Z g ñ gU r & (M . 7/ 11) .g.d gmm C o U§d w § eo V b{gH Ü Z n `_mm H _m {U d {n ú Ë$ g $ m Ho H Xg§ o J: {H o {^_w>V $ $ ñX m bo d O « X À © W $ ² _`m NÝ p V ^ú Ý ËJm ²&{H V ËJX V : & o `pV d X rZ $ w ríMw «_`ñ d a {ea: ñ m Mñ MV m X V & (M . 5/ 10) m Z`w mW éUÝ X o& .g.{Z í p `m Raktaja krimis that are minute and because of the size mynuteners they aresometimes visible or invisible when vranagata they cause kandu, toda etc. when excessivelygrown they eat away twak, sira, snayu, mamsa tarunasthi. Thus krimis minutest in the twak, mamsa lasika, sharira in kleda and sweda. In vedas explanation regarding sun as a krimighna is stated (Ri/191/18), (A 2/32/1)(R1/28/1). Acharya Sushruta also clearly mentioned that 13 krimis are described as pureeshajaand shleshmaja are drushta krimis, while remaining 7 described as raktaja are invisible i.e.they are adrushta krimis, But acharya Charaka terms some krimis to be sukshma.INFERENCE : This brief explanation suggests that there was tremendous knowledge regardingmicrobes right from the vedic kala. Only the way of approach has been changed. 31=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 48. Review of Literature==================================================================II) INTRODUCTION :Antimicrobials (32) :- The agents which have a capacity to kill the microbes but which causes minimumharm to the human body tissue. It is not sufficient to report the name of microbe to the physician after isolating andidentifying clinically in the laboratory. Although physicians may have knowledge of antimicrobial agents and a generalpattern of susceptibility of certain antimicrobial agents. Susceptibility of many bacteria, fungi, viruses to antimicrobial agents cann’t bepredicted that many micro organisms including bacteria and fungi, become resistant to thenewest antimicrobial agent.SELECTION OF DRUG :-(1) Has the most activity against the pathogen.(2) Has the least toxicity towards the patient.(3) Has the least impact on the normal flora of the patient. Not only antimicrobial agents are made by natural products but also micro organismsthem selves. Antimicrobial activity can improve by antibiotics which are synthetics, semisyntheticand modification of natural compounds. 32=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 49. Review of Literature================================================================== Antibiotic which kills the organism is said to be “cidal” and which inhibit the growthof organism know as “static”. Antimicrobial agents have specific activity against bateria, fungi, and viruses. The antibiotic which act on both gran + ve and -ve bacteria in known as broadspectrum antibiotic. High dose of antibiotics leads to disturb the natural defense mechanism in human.The normal flora can become disrupted in the intestine cauring diarrhoea. Vaginal flora canbe disrupted cauring fungal infections of genital tract. To over come infection antimicrobial agent must attain effective level in infectedsite. Some antibiotic enters easily in to C.S.F. Eye or prostate. Some organisms cann’t be given through the mouth because of distruction by thestomach. Excretion of must of the druges are through kidney and some are broken down by theliver. Fixation of dose based on two criteria which kills micro organisms but does’nt harmto the patient. It is very difficult and tough to treat fungal diseases. Synergic action can be seen by giving drugs in combination than they actindependently.(3) Has the least impact on the normal flora of the patient. - Not completely destroying the intestinal flora. Not only antimicrobial agents aremade by natural products but also micro organisms them selves. 33=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 50. Review of Literature================================================================== One which kills the organism is said to be “cidal” and which inhibit the growth oforganism know as “static”. Antimicrobial agents have specific activity against bacteria, fungi and viruses. Theantibiotic which act on both gram +ve and -ve bacteria is known as broad spectrumantibiotic. High dose of antibiotics leads to disturb the natural defense mechanism inhuman. The normal flora can become disrupted in the intestine curing diarrhoea. Vaginalflora can be disrupted cauring fungal infections of genital tract. To over come infection antimicrobial agent must attain effective level in infectedsite, some antibiotic enters easily in to CSF. Eye or Pro state. Some organisms cannot begiven through the mouth because of distractions by the stomach. Exception of must of thedrugs are through kidney and some are broken down by the livers. Fixation of dose based ontwo criteria which kills micro organisms but doesn’t harm to the patient. It is very difficultand tough to treat fungal diseases. Suberic action can be seen by giving drugs in combinationthen they act independently.MINIMAL INHIBITORY CONCENTRATION (MIC) (33) :- A test that determines the lowest concentration of the antimicrobial agent that inhibitthe growth of an organism.Sensitivity :- Sensitivity test, in which an organism is placed with antibiotics to determine whichantibiotic will effectively kill the organism with smallest dose, also refers to the ability ? Anantibiotic to inhibit growth of an organism.Resistance :- When an antibiotic dose not inhibit the growth of organism. 34=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 51. Review of Literature==================================================================Static :- Inhibiting growth of an organism.Synergy :- Two or more drugs in combinations having a great effect that the sum of the twoacting independently.III) BACTERIOLOGY (34) :-1) Scientific Classification :(i) Staphylococcus (Gram+ve) Domain - Bacteria Kingdom - Bacteria Phylum - Fermicutes Claso - Cocci Urder - Bacillales Family - Staphylococcaceae Species - S. Aures. S. Aureus is a gram + ve cocci which appears as grape like cluster when seen throughmicroscope and has lagered, round golden yellow colonies, often withy B-hemolysis whengrown on blood agar plates. 35=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 52. Review of Literature================================================================== The bacteria’s name aureus means “golden” in Lactic. The most common cause ofstaph infections is a special bacterium, frequently living on the skin or in the nose of healthyperson, that can cause minor skin infection as pimples boils, c, Celluloids and abscesses. S. Aureus Has about 2600 genues. The species has been subdivided in to two subspecies S. Aureus and S. Aureus anaerobes. S. Aureus may occur as a commensal on humanskin scalp, armpit, groin. It also seen in the nose, throat, and less commonly seen in urine. S. Aureus can infect other tissues when normal barriers have been breached Eg. Skinor mucosa lining this leads to biols and carbuncle I infected it can cause severe diseasestaphylococcal scalded skin syndrome. Infection of this can be spread through contact with pus from an infected wound, skinto skin contact with an infected person and things. Which are used by this person.(ii) Bacillus subtilis Kingdom - Bacteria Phytum - Firmicutes Class - Bacilli Order - Bacillaceae Family - Bacillaceae Species - Subtilis It is a gram +ve, commonly found in soil. A member of genus bacillus. It is notconsidered as a human pathogen. It has approximately 4100 genus, only 192 were shown tobe indispensable. 36=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 53. Review of Literature==================================================================(iii) Escherichia Coli (Gram -ve) : This genus is named after Escherich who was the first to be deseribe the colonbacillus under the name bacteritim coloc commune (1885) based on minor differences in biochemical characteristics, colon bacilli were described under various names, but in view ofthe mutability of the bio chemical properties in this group, they have all been included in theColi which is further subdivided in to biotypes and serotypes. A few other species are E.forgusonii, E hermonii, E-vineries which are of less medical importance.MORPHOLOGY :- E-Coli is gram - ve, straight rod measuring 1-3x 0.7 mm arranged singly or in pairs.It is motile by peritricatate Hagelly, though some strains may be non motile capsules andfimbriae are found some strains, spores are not formed.VIRULENCE FACTORS : Theere are two types of virulence factors have been recognized in E-coli namely(1) Surface antigen (2) Toxins E-coli produces 2 types of toxins (a) Haemolysines and (b) Entero toxins. Heomolysines dont appear to be relevant in pathogenesis through they are producedmore commonly by virulent strains. Enterotoxins are very important in pathogenesis of diarrhoea. These distinct types ofE-coli, enterotoxins has been identified (i) Heat liable toxin (ii) Heat stable toxin (iii) Verotoxin. E-coli causes wound infection, abscesses and also septicemia. 37=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 54. Review of Literature================================================================== Because of it UTI. diarrhoea pathogenic infection like meningitis, peritonitis mayoccur.(IV) CANDIDA ALBICANS :- Kingdom - Fungi Phylum - Ascomycota Subphylum - Saccharo mycotina Class - Saccharaomycetoes Order - Saccharo my cetales Family - Saccaromycetales Genus - Candida Species - C-albicans. Synonim - Candida stellatoidea It is diploid a sexual fungus (a form of yeast. It causes (skin), oral and vaginalinfection in humans. These are many species of candida. The clinical spectrum of candida albicans rangefrom superficial infection of the skin to the life thereatening infection. They can be isolatedfrom healthy mucosal surfaces of oral cavity, vagina, G.T. tract and malaria.CLINICAL SYNDROMES :- The infection caused by organisms in the genus candida includes localised disease ofthe skin and nails, diseases that affect the mucosal surfaces of oral cavity, vagina,oesophagus and brochealial trees. 38=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 55. Review of Literature================================================================== Because of this fungal infection causes most areas or skin causing itchy lesions onthe perinium, including deeper rash in. Enterotoxins are very important in pathogenesis of diarrhoea. The distinct types of E-coil entero toxins has been identified . (a) Heatliable toxin (b) Heat stable toxin (c) Vero toxin E-coli causes wound infection , abscesses and also septicemia . Because of UTI,diarrhoea, pathogenic infection like Meningitis peritonitis may occur .(IV) CANDIDA ALBICANS :- Kingdom - Fungi Phylum - Ascomycota Subphylum - Saccharo mycotina Class - Sacch aromycetes Order - Saccharo mycetales Family - Sacchro mycetaceae Genus - Candida Species - C - albicans. Scaling and ulceration in candida is also notorious for invading subcutaeneous tissuesand causing serious infections. It resides in most health care facilities. It will grow in most laboratory media, including blood agar and sabouroud agar. Itappears after 24 hours has tiny dots and by 48 hours the dots are soft, creamy colonies. Itsmells like bakers yeast. 39=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 56. Review of Literature==================================================================(V) SKIN DISEASES (35) :- Severel infectional diseases are seen because of the various types of microorganisms. Palash seed is used in many skin diseases like Kustha, Pama, dadru and kandu.and also infection can be seen in. So in skin diseases ring worm infection is also present. This infections are commanlyfound in male, female and in children also. According to our references palash seed isusesful in skin manifestation. Fungal infections are difficult to cure fast. There is indirectrole of Bacillus subtilis in skin infetion.(VI) Microorganisms Frequently Encountered In Body Sites :1) Blood : Staphylococci, Coliform bacilli, Pseudomonas sp., streptococci, Haemophilus influenzae.2) Genital Tract : Coliform bacilli, Enterococci, Bacteriodes sp., Lactobacillus, Gardnerell vaginallis, Trichomonas vaginalis, Candida albicans, B-Hemolytic streptococci A,B and D. Neisseria gonorrohoeae, Treponema pallidum, Chlamydia tractomatis, Herpes simplex.3) Wounds : Staphylococcus aureus, Streptococcus pyogenes, Coliform bacilli, Bacteriodes sp., Pseudomonas sp., Clostridium sp., Anaerobic cocci.4) Respiratory Tract : Group A and B streptococci, Corynebacterium diptherise, streptococcus pneumonicae, Haemophilus influenzae, Mycobacterium tuberculosis, Klebsiella pneumoniae, Pseudomonas aeruginosa.5) Urinary Tract : Staphylococci, Diphtheroid Bacilli, Coliform bacilli, Enterococci, Proteus sp., Lactobacilli and B-hemolytic streptococci, Bacillus sp., E.Coli. 40=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 57. Review of Literature==================================================================6) Eye, Ear : Staphylococcus aureus, Hemophilus sp., Streptococcus-pneumoniae, Neisseria gonorrhoeae, and B-hemolytic streptococcus, Psedomonas species, Corynebaterium diphtheriae.7) Gastrointestinal Tract : Escherichia Coli, Clostridium sp., Salmonella sp., Shigella sp., Yersinia sp., Campylobacter sp., Vibrio sp.8) Cerebrospinal Fluid : Hemophilus influenzae, Neisseria meningitis, streptococcus pneumonia.INFERENCE : Here we can see that E.coli and staph.aureus are commonly encountered organisms invarious sites of the body. (36,37)C) EXTRACTION PROCEDURE REVIEW :-There are there types of extraction procedures are present.(a) Solid-Solid in this type solid components extracted from solid substance. This extraction always carried out before any further separation / processing.(b) Solid liquid extraction :- Where the solid drug is extracted with a liquid medium.(c) Liquid - Liquid extraction - In this process any of the two liquids that arent miscible includes that substance to be extracted (solvent extraction). To get specific drug extract herbal drugs are extracting of certain particular size withsuitable extraction medium. For extraction fresh or dried parts of plants are used. The partswhich are used for extraction must be dried before the procedure taking place. To avoidchemical changes drying operation should be performed under controlled condition. Dry 41=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 58. Review of Literature==================================================================these parts as quick as possible. Drying procedure must be going on in room temperature andin airy place High temperature must be avoid for drying. This part must be kept free frominfection. The extraction depends on the texture and water content of the plant material and onthe type of substance that being isolated. Alcohol is good solvent for preliminary extraction. After exhaustive extraction subsequently the material can be macerated in a blenderand filtered. After extraction of green tissues with alcohol chlorophyl is removed in to thesolvent at the extent. When the tissue on repeated extraction is completely free of greencolour, it can be assumed that all the low molecular wt. compounds have been extractedwhen we extract continuously dried powdered plant material such as dried seeds, root, leaf,heartwood in a soxhlet apparatus with a range of solvents we obtain organic constituents. There are short cut methods in extracting procedures that one can learns withpractice.SELECTIVITY OF SOLVENTS : Selection of the solvent is very important aspect, not only for the yield of one moreprinciple substances but also for the qualitative and quantitative composition of theacompanying substance.(i) Hot continuous extraction - soxhlation :- By the help of available soxhlet extractor one can prepare crude plant extract. Theprocedure is done by the help of pure solvents. The actual solvent proportions in the extracting chamber differ from that originallyused in the collector. 42=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 59. Review of Literature================================================================== Advantages of this apparatus is that it is fully automatic continuous method thatdoesnt require further manipulation other than concentration of the extractive and saves.INSTRUMENTATION AND PROCEDURE : In this procedure the material to be extracted is placed in a thimble. This thimble ismade up off cellulose or cloth in a central compartment with a siphoning device and sidearm both connected to a lower compartment. Reflux condenser is attached above the centralsample compartment. Solvent container, sample compartment and reflux condenser are separate item ofglass which is assembled together with the appropriate contents, to make the completeapparatus. The solvent in the lower container which is usually round bottemed flask is heated toboiling, and the vapour passes through the side arm up into the reflux condenser. The vapourliquities and drips into the thimble containing the material to be extracted. The warm solventpercolates through the material and the wall of the thimble and the extract gradually collectsthe central compartment when the height of the extract reaches the top of the siphon, theentire liq. in the central compartment flows through this and back into the lower solventcontainer. The process gets repeated then. The extract is collected in the lower vessels, gradually be coming more and moreconcentrated. It is assumed that no volatile substance is present. The vapour rising from theheated extract is pure solvent vapour and so the liquid dripping into the material from thecondenser is essentially pure solvent, which get from the extract. Small volume of solvent isneeded, the effective volume of solvent used for the extraction is proportional to the time forwhich the process is allowed to continue. 43=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 60. Review of Literature================================================================== The soxhlet processes is useful for exhaustive extraction of the plant material withparticular component is desired.LIMITATIONS :-(i) In this procedure solvent is recycled on and often the extract that collects in the lower container is continuously being heated and it may suffer thermal degradation reaction.(ii) Total extract will exceed their solubility in that particular solvent so it may precipitate in the lower container and require much greater volume of solvent for latter desolation.(iii) It is not possible when we are adopting the them procedure on a large scale it is not suitable before use of solvents with relatively high boiling point such as methanol or water, the whole apparatus below the condenser needs to be at this temperature for effective movement of the solvent vapour.(iv) This procedure is carried out with single solvent and we are unable to extract with any mixture solvent. (38,39)(B) AQUEOUS EXTRACTION :- (Maceration Principle) Maceration is the process of extraction of drugs with a solvent with several dailyshakings at room temperature. 44=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 61. Review of Literature==================================================================INSTRUMENTATION AND PROCEDURE :- A conical flask of required measurement is taken. Then required drug plant materialis choosen and poured stated quantity of water at room temperature to it and kept it for 7-8days. (Minimum 5 days). The vessel must be sealed highly. After 7 days residual liquid isdecanting and staining is expressed from the solid and extract kept to evaporate the watercontent. at certain temperature (300c-400c) on water bath. 45=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 62. Methodology================================================================== 45=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 63. Methodology================================================================== 4. METHODOLOGY(4.1) COLLECTION :- Plant seeds were collected from around Belgaum area.(4.2) AUTHENTIFICATION FROM :- I C M R - Belgaum.(4.3) COLLECTION TEST DRUG AND STORAGE. (i) 1.5kgs of Palash seeds were collected and dried in shade and stored in moisture free container (ii) Then as required it is used.(4.4) PHARMACOGNOSTIC STUDY(i) Organoleptic Characters :Table No. 8Sl. No. Parameters I.P. / B.P. / As per Literature Observation I Physical Tests Nature Flat dull red nearly black Flat Dark Brown Colour Dull red nearly black Dark Brown Odour Characteristic Characteristic Taste Bitter Bitter II Seed Length 2.0 - 5.0 cm. 2.5 - 4.0 cm. III Seed Width 1.0 - 3.0 cm. 1.5 - 2.5 cm. 46=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 64. Methodology==================================================================(ii) Macroscopic and Microscopic Characters.Macroscopic characters of Palash seed : The seed is reddish brown thin, flat, reniform, longer axis, 3-4cms and shorter 2-2.5cms. seed coat reddish brown waxy, faint odour, taste slightly acrid and bitter. Wt. of 100seeds nearly 80-115gms. The hylum of the seed is conspicuous and situated near the middleof the concave age of the seed.Microscopic Characters of Palash seed :- The seed taken for the study were very hard. So they were soaked in brine (saltwater) till they set softened and enable for easy sectioning. The transverse section (T.S.) or*Longi – Section as required are taken and viewed under microscope.OBSERVATION :- The seed has an outer festa and inner endosperm region. The outer festa contains asingle layer of pigment cells. The endosperm region has parenchymatous cells containaleurone grains and starch grains. The cells of the endosperm are filled with oil globules.The starch grains are rod shaped or ovoid. Near starch cell spiral form of proteins arepresent.POWER - MICROSCOPIC STRUCTURE OF POWDER : Powder contain yellowish brown colour, acrid and bitter with oil flavor andtypical smell, small fragments of festa. Cotyledonary parmanchyma containing a few starchgrains, hour glass cells, abundant spiral protein bodies, mucilage and oil globules.Note :- It is tough to take seed section. 47=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 65. Methodology==================================================================(4.5) PHYSIO-CHEMICAL STUDY (40) :-(a) Ash value :(i) Use to determine quality and purity of drug(ii) Ash contains in organic radical like phosphates, carbonates and silicates of Sodium, Pottasium Magnesium and Calcium etc.(iii) Some times inorganic variables like calcium oxalate, silica, carbonate content of the crude drug effects, total Ash value. Such variables are then by treated with acid and acid insoluble Ash value is determined.DETERMINATION OF TOTAL ASH VALUE :-(i) Weigh the silica crucible(ii) Powdered drug is weighed and put in to the crucible.(iii) Then it is placed in the Muffle furnace at 4500c for about 1/2 - 1 hour. (i.e. : untill all carbon particles get burnt off.)(iv) Cool it in dessicator(v) Then weigh the ash and calcutale the percentage of total ash with reference to the air dried sample of the crude drug. ( Standard ash value of "Palash" seed is not more than 8%).ASH VALUE :- (OBSERVATIONS)Crude powder :- (a) Weight of empty crucible = 18.227 gms. - (A) (b) Weight of drug taken (2 gms) = 2.000 gms. - (B) (c) Weight of crucible + ash = 18.642 gms. - (C) (d) Weight of ash = C - (A+B) = 1.485 gms ∴ For 100 gms of drug ash value = C - (A+B) * 100/2 = 7.425% % 48=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 66. Methodology==================================================================FINE POWDER :- (a) Wt. of empty crucible = 16.272 gms. - (A) (b) Wt. of drug taken (2 gms) = 2.000 gms. - (B) (c) Wt. of empty crucible + drug taken = A + B = (C) = 18.272 gms. (d) Wt. of crucible + ash = 16.735 gms. = (D) (e) Wt. of ash = C - D = 1.537 gms. 98 ∴ 100 gms of ash value is 1.537 x 50 = 7.685 %(B) ACID INSOLUBLE ASH VALUE :- (i) Using 25 ml. dil HCL (0.5 N) wash the ash from the dish used for total ash in to a beaker.ACID IN SOLUBLE ASH VALUE :-1) Using 25 ml of dil HCL (0.5N) wash the ash from the dish used for total ash into a beaker.2) Place wire gauze over a bunshen flame and boil for 5 minutes.3) Filter through a Whatman filter paper - wash the residue twice with hot water.4) Place it in a crucible Ignite the crucible (placing it in a muffle furnace)5) Heat it until all carbon is removed (at 2500c for half an hour to 45 minutes)6) Cool it and weigh the residue7) Calculate acid insoluble ash of the crude drug with reference to the air dried sample of crude drug. 49=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 67. Methodology==================================================================ACID INSOLUBLE ASH VALUE :- Wt. of crucible = 40.775 gms Wt. of drug = 02.007 gms. Wt. of ash = 01.73 gm Acid insoluble ash = 0.030 gm. (Standard acid insoluble ash value - > not more than 0.5 %)C) DETERMINATION OF WATER SOLUBLE EXTRACTIVE :- Ingredients :- a) Powdered drug - 5 gm. b) Water + Chloroform - 100 ml. c) 500 ml water + 1.25 ml chloroform Equipments :- Iodine flask ( conical flask C stopper ) (250 ml.) :- Beakers - 50 ml. :- Funnel - 1 in no. graduated cylinders (100 and 25 ml) :- Filter papers :- Water bath :- Incubators (oven) :- Weighing MachinePROCEDURE :-1) Weigh about 5 gm of the powered drug in a beaker and transfer it to a dry 250 ml. of iodine flask.2) Fill a 100 ml graduated cylinder to the required mark with the solvent (water + chloroform) wash out the weighing bottle and pour the washing together C the reminder of the solvent into the conical flask. 50=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 68. Methodology==================================================================3) Cork (stopper) the flask and set aside for 24 hrs. shaking frequently (maceration).4) Filter it into a 50 ml cylinder - When sufficient filtrate has been collected transfer 25 ml. of the filtrate to a weighed 25 ml. beaker as used for the ash values determinations.5) Evaporated to dryness on water bath and complete the drying in an oven at 1000c for about 10-15 min.6) Cool in desiccator and weigh.7) Calculated the percentage w/w of extractive with reference to the air dried drug.OBSERVATION :- Wt. of drug and beakar before drying = 52.659 gm Wt. of drug and beakar after drying = 52.175 gm 0.484 gm L 25 ml gives = 0.484 gm 100 ml gives = 0.484 x 4 1.936 gm L 5 : 100 : 1.936 gm 100 x 1.936 = 38.720 % 5 (standard value = not less than 25 % ) 51=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 69. Methodology==================================================================Determination of alcohol soluble extractive :Ingredients :- a) powdered drug - 5 gm b) alcohol (90 % ) - 100 ml.Equipments :- Iodine flask - 250 ml - 1 in no. :- Beakers - 50 ml., 25 ml. :- Measuring cylinder - 100 ml and 25 ml (graduated) :- Funnel - 1 in no. :- Filter papers :- Water bath :- Incubator (hot air oven) :- Weighing machinePROCEDURE :-1) Weigh about 5 gm of the powdered drug in a beaker and transfer it to a dry 250 ml. Iodine flask.2) Fill of 100 ml graduate cylinder to the required. Mark the solvent (90% alcohol) washout bottle and pour the washing, together with the remainder of the solvent into the conical flask.3) Cork (stopper) the flask and set aside for 25 hrs. Shaking frequently (maceration)4) Filter into a 50 ml cylinder when sufficient filtrate has been collected, transfer 25 ml. of the filtrate to the weighed 25 ml .5) Evaporated to dryness on water bath and complete the drying in an oven at 1000c for about 10 -15 mins. 52=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 70. Methodology==================================================================6) Cool in desiccator and weight.7) Calculate the percentage w/w of extractive with reference to the air dried drug.Calculation :- 1) Wt. of empty beaker = 49.241 gm 2) Wt. of beaker + 25 ml of substance evaporated = 49.500 gm. Diff. = 0.259 gm. L 25 ml gives = 0.259 gm. 100 ml gives = 0.259 x 4 = 1.036 gm. L 5 : 10 : 1.036 100 x 1.036 = 20.72 % 5 (Standard value not less than 19.70)E) Determination of Extractive values : * Useful for the evaluation of crude drug. * Give idea about the nature of chemical constituent present in a crude drug. * Useful for the estimation of specific constituent soluble in that particular solvent used for extraction. 53=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 71. Methodology==================================================================(4.6) Methodology (41) Extraction (Schematic Presentation) Extraction Aqueous Extract Alcohol Extract Hot Water Extract Cold Water extract Hot continuous extractAbbreviations for extract sample :- Cold water - A Hot Water - B Ethanol Extract - CExtractions :-INTRODUCTION :- As it is preliminary study, extractions selected were cold water extracted by maceration and hot continuous extraction by soxhlation (Ethanol) To start with we have taken alcohol i.e. ethanol from distilling rectified spirit by distillation procedure. Then the procured ethanol was used for the extraction of the following drug.(i) Seed powder of Palash : 54=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 72. Methodology==================================================================4.7) ETHANOL EXTRACTION (42) :-(i) Seeds Alcoholic Extraction : Instrumentation : (i) Soxhlet Apparatus. (a) Thimble - 400 ml. (b) Reflux condenser (c) 2 rubber pipes (inlet and outlet) (d) 1 lit round bottom flask.(ii) Heating Mantle :- 1 lit capacity. Other materials : (a) Filter paper (b) 500 ml beaker (c) Glass funnel. Ingredients : 1) Palash seed powder - 500 gms. 2) Alcohol (Ethenol) - 1000 ml.PRINCIPLE : Extraction involves the separation of bioactive portion of the plant tissues fromthe inactive components by using selective solvent in standard extraction. The process at atemperature approximately that of the boiling point of the solvent apparatus permits theuniform percolation of the drug and the continuous flow of vapour of the solvent around thepercolator is best for this type of extraction. The process is generally applied to the removal of natural products from driedtissues originating from plant fungi etc. The non stream volatile components may beremoved by solvent extraction using a batch or continuous process of extraction of a solid bya hot solvent it is better to use a soxhlet apparatus. 55=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 73. Methodology==================================================================PROCEDURE : The solid substance (the seed powder of palash) is placed in a porous thimble (i.e.made of tough filter paper or cotton) and the latter is placed in the inner tube (thimble) of thesoxhlet app. The apparatus is then fitted to a round bottom flask of appropriate sizecontaining the boiling chips and then to a reflux condenser (preferably the double surfacetype) Before joining condenser or even after joining, pour the solvent in the thimble slowlyand fill it. Then allow the thimble to soak along with the solvent over night. Then next day pour solvent (alcohol/ethanol) and fill the round bottom flask withappropriate quantity of solvent. The solvent is boiled gentely, the vapour passes up through the tube and is condensedby condenser and then condensed solvent falls in the thimble and slowly fills the body ofsoxhlet. When the solvent reaches the top of the tube, it siphons over into the flask and thisportion of substance, which it has extracted, gets collected in the round bottom flask. The process is repeated automatically, untill complete extraction is attained. Fifteento eighteen hours is needed for complete extraction of constituents.2nd Part :1) After extraction the solvent collected in round bottom flask is collected in a beaker and then evaporated.2) The extract was then concentrated on hot water bath and finally reduced to dryness (or till the alcohol smell in lost completely).3) After drying the respective extract was weghed weight and yield recorded.4) The physical characters were noted. yield of extract : 20.72 gms. 56=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 74. Methodology==================================================================OBSERVATIONS : Seed powder taken - 500 gm. solvent ethanol - 1000 ml. yield of extract - 20.72 gm. Time taken - 22 hrs. Cycles - 25 cycles. Colour - Changes from - dark yellow to whitish yellow. Inference - At first cycle extract is thick then thickness is decreases andalso colour changes.2) Hot Water Extraction : Palash seed powder - 250 gm Procedure - soxhlation Solvent - Water Distilled 750 ml. Yield of extract - 20.09 gm. Time taken - 20 hr.OBSERVATION : Colour changes as ethanol extract but yellowish tinge is more than that. (43)AQUEOUS EXTRACTION (WATER EXTRACTS) :Equipments :- Conical Flask - 2 lit capacity. Measuring cylinder - 25 ml capacity Silver foil and filter paper Emulsion cloth. 57=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 75. Methodology==================================================================Solvent :- Water, chloroform ( Preservatory )Ingredients :- Seed powder - 200 gm Water C. 1000 ml. Chloroform - 20 ml.PROCEDURE :-1) Required amount of the drug powder is taken in a conical flask.2) Pour some water (required amount of water) in a certain ratio.3) Add some chloroform as preservatory.4) Close the mouth of the conical flask with a filter paper and silver foil.5) Shake it vigorously with frequently intervals.6) Then place the conical flask in a warm place for 7 days with frequent vigorous shakings.7) After 7 days filter the content and the filtrate is dried and the liquid part is kept on a water bath and evaporated.8) It is evaporated till the required concentrate is achieved.9) And stored in a dry containers. Seed powder - 200 gm. Water - 1000 ml. Chloroform - 20 ml. 7 days it is keep as it is kept. After 7 days they were filtered respectively and later. Were kept on a water bath and evaporated to their required concentration. Observation - before evaporation - ml. After evaporation - 25.32 mg. 58=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 76. Methodology==================================================================(4.9) PRELIMINARY PHYTOCHEMICAL SCREENING (44) :PROCEDURE :-I. TESTS FOR PHYTOSTEROLS :(a) Salkowaski Test : To the test extract solution added few drops of conc. H2SO4 shaken and allowed to stand, lower layer turn red indicating the presence of sterols.(b) Liebermann - Burchard test : The test solution treated with few drops of acetic anhydride and mixed, when conc. H2SO4 is added from the sides of the test tube, it show a brown ring at the junction of the two layers and the upper layer turns green.(c) Sulphur test : Sulphur when added in to the test solution, it sinks in it.II. TEST FOR TRITERPENOIDS :(a) Salkowski test : Few drops of concentrated sulphuric acid was added to the test solution of the extract, shaken and on standing lower layer turns golden yellow.(b) Liebermann Burchard Test : To the test solution of the extract, few drops of acetic anhydride was added and mixed well. Then 1 ml of concentrated sulphuric acid was added from the sides of the test tube, a red colour is produced in the lower layer indicate Triterpenes.(c) Tschugagiu test : Excess of acetyl chloride and pinch of zinc clroride added to the test solution of extract, kept aside for reaction to subside and warmed on water bath, eosin red colour produced.(d) Briekorn and Brinar test : To the test solution of extract, few drops of chlorosuphonic acid in glacial acetic acid (7.3) red colour is produced. 59=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 77. Methodology==================================================================III. TESTS FOR GLYCOSIDES :(a) Baljets test : The test solution treated with sodium picrate gives yellow to orange colour.(b) Bromine water test : Test solution dissolved in brominoine water gives yellow precipitate.(c) Raymonds test : Test solution treated with dinitrobenzene in hot methanolic alkali gives violet colour.(d) Legals test : Test solution when treated with Pyridine (made alkaline by adding sodium nitroprusside solution) gives pink to red colour.IV. TEST FOR ANTHRAQUINONES :(a) Borntragers test : Boiled test extract solution with 5 ml of 10% sulphuric acid for 5 mins. Filtered while hot, cooled the filterate shaked gently with equal volume of benzene. Separated the benzene layer and when treated with half of its volume solution of ammonia (10%). Allowed to separate ammoniacal layer aquires rose pink colour due to the presence of anthraquinones.V. TESTS FOR SAPONINS :(a) Foam test : Test solution and shaken shows the formation of foam, which is stable an least for 15 mins. 60=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 78. Methodology==================================================================VI) TESTS FOR CARBOHYDRATES :(a) Molischs test : Test solution with few drops of Molischs reagent and 2ml of concentration H2SO4 added showly from the sides of the test tube shows a purple ring at the junction of 2 liquids.(b) Barfoeds test : Test solution treated with Barfoeds reagent, on boiling on a water bath shows brick red colour precipitate.(c) Benedicts test : The test solution treated with Benedicts reagent and boiling on water bath shows reddish brown precipitate.(d) Fehlings test : The test solution when heated with equal volume Fehlings A and B solutions, gives orange red precipitate presence of reducing sugars.(e) Selivinoffs test : A crystal of resorcinol is added to the test solution and warmed on a water bath equal volume of conc. HCL a rose colour is produced that showed ketone is present.(f) Tests for pentoses : Heat the test solution with a equal volume of HCL containing little phloroglucinol. Formation of red colour lidicates the present pentoses.VIII. TESTS FOR ALKALOIDS :(a) Mayers test : Test solution with Mayers reagent (Potassium Mercuric iodide) gives cream coloured precipitate.(b) Hagers test : The acidic solution with Hagers reagent (Saturated picric acid solution) gives yellow precipitate. 61=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 79. Methodology==================================================================(c) Dragendorffs test : The acidic solution with Drogendroffs reagent (Potassium bismuth iodide) shows reddish brown precipitate.IX. TESTS FOR FLAVONOIDS :(a) Shinoda test : Test solution with few fragments of magnesium ribbon and conc. HCL shows pink to magenta red colour.(b) Zn/HCL reducing test : Test solution with Zinc dust and few drops HCL shows magenta red colour.(b) Alkaline reagent test : Test solution when treated with sodium hydroxide solution shows increase in the intensity of yellow colour, which becomes colourless on addition of few drops of dilute acid.IX) TEST OF PHENOLICS / TAINS :(a) FeCl3 test : Test solution treated with few drops of FeCl3 solution gives dark colour.(b) Gelatin test : Test solution treated with gelatin gives white precipitate.X) TESTS FOR PROTEINS :(a) Millons test : Test solution when treated with Millons reagent and heated on a water bath, Protein is stained red on warming.(b) Xanthoproteic test : Test solution treated with conc. HNO3 and boiled gives yellow precipitate. 62=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 80. Methodology==================================================================(c) Biuret test : Test solution treated with 40% NaOH and dilute CuSO4 solution gives blue colour.XI) TEST FOR AMINO ACIDS :(a) Ninhydrin test : Test solution treated with Ninhydirn reagent gives blue colour.(A) THIN LAYER CHROMATOGRAPHY (TLC.) (45,46) :Introduction :- With the help of Thin Layer Chromatography (TLC.) One can determine particularvalue? Small amount of impurities. We get principle of TLC. and application of thetechnique in pharmaceutical analysis in volume I of the internal pharamocopoeia (5) This isthe earliest method to perform. And equipments which are required is introversive, so thetechnique is used frequently to evaluate medical plant material and their preparations. Parameters should be determined on the basic of published pharmacopeiamonographs established experimentally for the analysis of each individual plant material.* Type of absorbate and method of activation of no information on the letter can be obtained, heat at 1100c for 30 minutes.* Method of preparation and concentration of the test and reference solution.* Volume of the solution to be applied on the plate.* Mobile phase and the distance of migration.* Drying condition (including temperature) and modern of detection. For the spots obtained.* Number of approximate position, or the Rf values if necessary.* Fluorescence and colour. 63=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 81. Methodology==================================================================Types of thin layer chromatography methods are described as follow : (i) Classical method and (ii) Micro method. Which uses different size of plates and hence different quantities of solvent.Classical Method :Recommended Procedures : The method outlined below assumes that chromatographic plates prepared in thelaboratory are used but precoated plates, activated if necessary, may be used provided thatthey have proved suitable for application concerned. A powdered specimen of pharmacopoeial quality may be used as the referencematerial, if a test for the presence of known active principle of a medicinal plant material isto be carried out, a chemical reference substance identical to that principle should be used.The test and reference solution should be prepared simultaneously in exactly the same way.The reference solution should be of known concentration. If the relative concentrations ofthe chemical substances in the reference solution are selected in accordance with thecomposition of typical material, comparison of the spot size offers valuable additionalinformation. The solvent system should be specified in the test procedure of the individualmaterial being examined. A three colour mixture (e.g. 0.01% solution in toluene ofindophenol blue, sudan red G and dimethyl yellow) run together, permits a rapid check onthe prevailing chromatographical conditions. If it is suspected that the materials being examined are unstable, the chamber inwhich chromatography takes place should be protected from light. In any case, thechromatographic chamber should always be kept out of direct sunlight. Otherwise, the raysof the may be refracted to different degrees owing to imperfections on the glass walls of the 64=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 82. Methodology==================================================================chamber, giving rise to area of elevated temperature on the chromatographic plate and erraticflow of the mobile phase.Preparation of samples : Prior to testing, prepare an extract of the plant material to be examined, using a rapidextract process, as follows, To 0.0-1.0 g of the powdered plant matetial add 1-10 ml ofsolvent and extract either by stirring, shaking the mixture of 3-30 minutes, or heating toboiling and allowing to cool. Remove the insoluble matter by centrifugation, or filter througha small funnel with filter paper or a cotton plug. If necessary, evaporate the filtrate on awater-bath for just as long as is required to remove the solvent, then re-dissolve the residuein a small volume of solvent (e.g. 0.1 to 0.2 ml). If necessary, purify the test solution byrepeating the extraction with solvent at a different pH, or by sublimation, distillation, orother appropriate method.APPARTUS :The equipment consists of : Glass plates of uniform thickness throughout entire area, 15-20 cm long, and wideenough to accommodate the required number of test and reference solutions. A device for spreading uniform layer of coating material of desired thickness ontothe glass plates; A rack to hold the prepared plates (normally 10 plates with set spacing) during thedrying period or for transportation and storage; the rack should be small enough to fit in adrying oven and desiccators; A chromatographic chamber of transparent material, usually glass, with a tightlyfitting lid, of suitable size to accommodate the test plates; 65=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 83. Methodology================================================================== A suitable spraying implement with a fine spray nozzle, made of material resistant tothe reagents to be used; An ultraviolet light source emitting short (254mm) and ling(365mm) wavelengths. Before use, clean the plates scrupulously by immersing in a suitable cleaning liquidand rinsing thoroughly until the water runs off the plates without leaving any visiblewatermarks or oily spots, and then dry. The plates must be completely free of lint or dustwhen the coating material is applied.METHOD :Preparation of the adsorbent : Unless otherwise specified in the procedure for the plant material concerned preparea slurry of the coating material and water on an aqueous solution (see section 21,"specifications for adsordents") and, using the spreading device, coat the cleaned plates witha layer about 0.25 mm thick. Dry the coated plates in air, heat to activate 1100C for minutes,and then allow to cool. Inspect the uniformity of the coating in transmitted light and thetexture in reflected light. If the plates are not to be used immediately, store them in adesiccator containing silica gel, desiccant, R. To form and edge remove a narrow strip (2-5mm) of the coating material from the sides of the plate. If acid, alkaline or buffered layers are required, use diluted acid, base or salt mixturesinstead of water for the preparation of the slurry, As specified in the test procedure. Anaqueous solution of 5-7 g of sodium carboxymethylcellouse R could replace the water, if theabsorbent dose not already contain a binder. 66=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 84. Methodology==================================================================Saturation of the chromatographic chamber : Unless otherwise specified in the test procedure, the chromatography is carried out ina saturated chamber. To achieve saturation, line at least half of the total area of the insidewalls of the chamber with filter-paper, into the chamber a sufficient quantity of the mobilephase to saturate the filter-paper and form a layer about 5 mm dep. Close the chamber andallow to stand for at least 1 hour at room temperature. All operations during which the plate is exposed to the air should preferably becarried out at a relative humidity of 50 - 60%, and the plates should be handled with care.Application of the test and reference solution : Using a micropipette or a syringe graduated in ml, place spots of the test andreference solution onto the starting line, which should be parallel to and about 15 mm abovethe lower edge. The spots should be at least 15 mm from the sides of the plate, and at least15 mm. apart. They should be as possible, preferably not more than 4 mm in diameter, ifnecessary, apply the solution in portions, drying between applications. Mark the distance themobile phase in intended to ascend as specified in the rest procedure, usually 10-15 cm fromthe starting line. The results of speration can often by improved by applying the solutions to form ahorizontal band 10-15 mm long and not more than 5 mm wide.Development of chromatograms : Allow the spots to dry, place the plate - as nearly vertical as possible - into thechamber, ensuring that the points of application are above the surface of the mobile phase.Close the chamber. Develop the chromatogram at room temperature, unless otherwisespecified in the test procedure, allowing the solvent to ascend the specified distance. Remove 67=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 85. Methodology==================================================================the plate, mark the position of the solvent front and allow the solvent to evaporate at roomtemperature or as specified.Observation and interpretation of the chromatograms : Observe the spots produced in daylight, then under short-wave and long-waveultraviolet. Mark the centre of each spot with a needle. Measure and record the distance fromthe centre of each spot to the point of application, and indicate for each spot the wavalengthunder which it was observed. If indicated in the test procedure, spray the spots with thespecified reagent, and observe and compare the spots with those of a reference material. If required calculate the ratio of the distance traveled on the adsorbent by a givencompound to that traveled by the leading edge of the solvent (the Rf value) or the ratio of thedistance moved by a compound and a stated reference substance (the Rf value) as follows : a a Rf = Rf = b cwhere a = the distance between the point of application and the centre of the spot of the material being examined ; b = the distance between the point of application and the solvent front; c = the distance between the point of application and the centre of the spot of reference material. Rf value may vary with each experiment depending on the saturation conditions inthe chromatographic chamber, the activity of the adsorbent layer, and the composition of themobile phase.Note : The above mentioned procedures are as per W.H.O. criteria. 68=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 86. Methodology==================================================================1. Thin layer Chromatographic - Analysis (TLC) of Drugs : Of the many chromatographic methods presently available, thin layerchromatography has become widely adopted for the rapid and positive analysis of drugs anddrug preparations.There are several reasons for the popularity of this method :- The time required for the demonstration of most of the characterstic constituent of a drug by TLC is very short.- In addition to qualitative detection, TLC also provides semi-quantitative information on the chief active constituents of a drug or drug preparation, thus enabling as assessment of drug quality.- TLC provides a chromatographic drug fingerprint. It is therefore suitable for monitoring the identity and purity of drugs, and for detection adulteration and substitutions.- With the aid of appropriate separation procedures, TLC can be used to analyses drug combinations and phyto-chemical preparations.- Thin layer chromatograms can be documented.II. Documentation of Thin Later chromatograms :Various methods of documentation are possible :- Description of the Rf value and colour of the characteristic main zones, with reference to a standard substance or test mixture. This method has been adopted in 69=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 87. Methodology================================================================== the 8th edition of the German pharmacopoeia, the European pharmacopoeia, USP XX and other.- Construction of a scale diagram of the thin layer separation, showing migration distance and intensities of the characteristic zones. The zones observed in visible light (vis.) in UV-254 nm and UV-365 nm are described.- Colour photography in daylight or UV-light, under conditions that give the most authentic reproduction of the colours and intensities of the separated zones.- Densitometry or fluromety of the chromatogram at certain wavelenghts. Under favourable conditions, this procedure also yields a drug fingerprint, and enables the quantification of certain chief constituents. If suffers from the disadvantage that a calibration graph constructed at one wavelength is applicable to only some of the constituents. (47)B) High Power Thin layer chromatography (H.P.T.L.C.) : The high performance thin layer chromatography commonly known as H.P.T.L.C. issophisticated from of thin layer chromatography. It involves the same theoretical principlesof T.L.C. where in substances are separated on the basis of their differential migration insystem of 2 phases of special types of plates.The important steps involved are :1. Sample preparation 2. Selection of chromatographic layers.3. Plates. 4. Pre washing5. Conditioning 6. Sample application7. Pre conditioning 8. Mobile phase9. Chromatographic development. 10. Detection of spots.11. Scanning and documentation. 70=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 88. Methodology==================================================================1. Sample Preparation : For normal phase chromatography using silica gel pre-coated plates, solvents should be non polar of volatile type. For reversed phase chromatography usually polar solvents are used for dissolving the sample.2. Selection of chromatographic layers : The solution depends on the nature of material to be separated commonly used material are silica gel 60F 253, alumina cellulose etc.3. Plates : Plates of 20 x 20 cms or 5 x 7.5 cms having 100-200 mm adsordent thickness is used for qualitative analysis. Silica el 60F254 having a pore size 6 mm with flurescent indicator is a coat material. The solvent layer is fixed with the help of glue. The basic difference between T.L.C. and H.P.T.L.C. plates is partial size of coated material, which is 5-20 mm of T.L.C. and 4-8 mm for H.P.T.L.C.4. Pre-washing : Plates need to be washed to remove water vapours and volatile impunities. The plates are cloned by methanol.5. Conditioning : The pre-washed plates are placed in oven at 1200C for 15 to 20 minutes. This process is known as conditioning.6. Sample application : Application of 1.5-5 ml is most satisfactory for H.P.T.L.C. application of the sample.7. Pre conditioning (Chambers saturation) :- For low polarity mobile phase there is no need of saturation, however saturation is needed for highly polar mobile phases.8. Mobile Phase :- The solution of appropriate mobile phase is by trial and error in which chemical of analytic absorbent layer etc. are considered.9. Detection of spots : Immediately after the development is completed. The plates are removed from the chamber and dried to remove trees of mobile phase, Generally detection can be known by iodine vapour in Jodive chamber. 71=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 89. Methodology==================================================================10. Scanning and Documentation : The H.P.T.L.C. equipments are supplied with computer, data recording and storing divice. The developement of H.P.T.L.C. plates are scanned at selected U.V. regions wavelength by the instrument and the detected spots are seen on computer in the form of peaks. The scanner converts bond into peaks and peak heights or area is related to the concentration of the substance on the spot. The peak heights and area under the spot (curves) are measured by the instrument and are recorded as the printer.INFERENCE : These are the general procedures explained according to W.H.O. standards. In thisstudy we are concentrating only on the qualitative estimations of the sample extracts. i.e. Bytheir finger printing we are observing for their Rf values, which indicate the presence ofcertain phyto-constituents in each sample extract. These can be further clarified by phyto-chemical studies of their fractions which can be procured by fractionation.4.11 EVALUATION OF ANTIMICROBIAL ACTIVITY (48,49) :Principle : The agar dilution technique is used to measure qualitatively the in vitro activity of anAntimicrobial agent against the test bacterial. In this method, the petridishes were filled with inoculated liquefied agar medium touniform thickness. Then graded amount of test samples (Ex. antibiotics) are incorporated inagar plates and inoculated in spots with the organisms under study. If the organism understudy is susceptible to the incorporated test samples. 72=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 90. Methodology================================================================== No bacterial growth is expected in agar plates with higher amount of the drugs.Bacterial growth is observed as the test sample concentration in the agar plate diminishes.Inhibition of growth at the minimum or lowest concentration of test sample.Antimicrobial activity : The antimicrobial activity of a drug is generally expressed as its inhibiting effecttowards the growth of bacterium in nutrient broth or on nutrient agar.For this study, following conditions are observed for :1) The substance or extract must be in contact with the test organism.2) Conditions must be favourable for the growth of microorganisms in the absence of Antimicrobial substances.3) There must be means of estimating the amount of growth and thereby percentage of growth of inhibition.4) The activity of extract should be observed and determined by the growth response of microorganisms. Different methods employed in the study are agar streak. serial agar diffusion, paperdisc, cup-plate, strip diffusion, cylinder plate and turbidimetric methods.Methods :- Tests was done by cup diffusion method. In present study "Palash" seed powder were extracted with water and ethanol assolvents and all extracts were subjected for antimicrobial activity. 73=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 91. Methodology==================================================================Experimental Procedure :- (50) In the present study, the antimicrobial screening was done by cup plate method orcup diffusion method. This is one of the authentic procedure in I.P. where the antimicrobialextract is differed from the cup through an agar layer in petriplate (dish). Petridish to anextent such that the growth of added microorganism are restricted entirely in circular area orzone arround cavity containings the solutions of the test sample. The antimicrobial activityexpressed as zone diameter in mm which is measured with a divider. All the extract of the seed were screened for antimicrobial acitivity against broadspectrum of microorganisms and the activity compared with appropriate standards.Standards used in the study :- For bacterias (Gream +ve, Gram -ve) (i) Amikacin (ii) Olfloxacin (iii) PenicillinFor fungi (iv) Griseofulvin.Test organism used for the study :-(1) Bacterias :- (a) Staphylococcus aureus (gram +ve) (b) Bacillus subtillis (Gram +ve) (c) Escherichia Coli (Gram -ve)(2) Fungi :- (a) Candida albicans 74=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 92. Methodology==================================================================Preparation of Antimicrobial agent stock solution :1) Remove the Antimicrobial agent from the freezer and warm to room temperature before opening to avoid condensation of water.2) Weigh appropriate amount of the powdered semi liquid Antimicrobial agent.3) Dissolve the Antimicrobial agent powdered / semi liquid in solvent to make 2 mg / ml and mg / ml concentrations.Preparation of standard solutions :1) Required amount of standard Ofloxacin - 2 micg / disc and norfloxacin - 10 micg / disc solutions are prepared.2) The concentration of standard equivalent to 100 micg/disc of griseofulvin was prepared as standard for antifungal activity.Preparation of Inoculum : Stock cultures were maintained at 40c on slopes of nutrient agar. Active cultures forexperiences were prepared by transferring a loopful of cell from the stock cultures to testtubes of nutrient broth for bacteria and that was incubated without agitation for 24 hours x at370c an 250c respectively.Culture Medium : Different types of media have been used according to the types of organism In thepresent investigations, Antibiotic medium No. 5 (Nutrient Agar-Himedia) 75=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 93. Methodology==================================================================Nutrient agar composition : Agar : 15. gm Beef extract : 1.5 g Yeast extract : 3.0 g Peptone : 6.0 g Distilled water to make : 1000 ml. About 27 g of above readymade medium was dissolved in freshly prepared distilledwater (1000 ml) by gentle heating.Sterlization : Sterlization of the medium, tubes for slants, borer etc. was done by autoclaving at 15lbs/spqure inch. for 20 min. The glasswares like syringes, petridishes, pipettes, empty testtubes, were sterlized by dry heat in an over at a temprature of 1600C for 1 hr.Preparation of agar plates : The sterlised medium was cooled at 400C and 0.5 ml of inocula per 100 ml. ofmedium were added to the conical flask. This was shaken gently to avoid the formation ofair bubbles and then transferred into petridishes so as to obtain 6 mm thickness of medium.The medium in the plate was allowed to solidify at room temperature. 76=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 94. Methodology==================================================================Experimental Procedure : The sterile borer was used to prepare nine cups of 8 mm diameter in the medium ofeach petri-dish. An accurately. measured 0.1 ml solution of each concentration of solutionsof extracts and standard samples were added to the cups with the help of micropipette. Allthe plates were kept at room temperature for effecting diffusion of drug extract and standard.Later, they were incubated at 37 + 10C for 24 hrs. The presence of definite zones around thecup of any size indicated antibacterial activity. The control were run simultaneously toassess the activity of petroleum ether, chloroform, acetone - water and water which wereused as a vehicle for extract. The diameter of the zone of inhibition was measured andrecorded. The zone of inhibition for the antibacterial and anti-fungal activities of extracts werecalculated by measuring the inhibitory effect towards the growth of bacteria and fungusaround nutrient agar cup. The results for antibacterial activity were shown in table no. andanti-fungal was shown no.Conclusion : From the results, it is concluded, all extracts are resistant to bacterials as well asfungi, when compared to the standards.M.I.C. Method : First the stock solution of the test sample i.e. ethanol and water extracts of "Palash"seed were prepared separatively i.e. 10 mg. ml. concentrated stock 501n is prepared as muchrequired. 77=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 95. Methodology==================================================================Produce :(1) Sterile tubes were selected and serially labeled from 1 to 7.(2) 0.5 mg. of Muller Hinton broth solution is added from tube no. 2 to no. 7.(3) Then in tube 1 and 2, 0.5 ml. of stock 501n (10 mg/ml.) added.(4) From tube 2, transfer 0.5 ml to tube 3 mix 8 transfer to tube 4 and transfer similarly till tube 7 Discard 0.5 ml. from tube 7.(5) Adjust the bacterial count 105 organisms/ml by comparing with Mac Farlands Standards.(6) Add 0.5 ml of culture broth to all 7 tubes(7) Incubate the tubes at 370c cover night.(8) Than observe for turbidity and record the values observation note - Turbidity in first tube is the M.I.C. value for that microbes.Result : M.I.C. value of the extract has been mentioned in Table No. 11 to 14.Conclusion : Sample share better results on e-coli and S. aureus and basilius subtillis but totallyresistant again candida albicans That means ethanol extract shows best activity againstbacteriaes but not effectively acting on fungis. 78=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 96. Results================================================================== 78=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 97. Results================================================================== RESULTS(5.1) MACROSCOPIC AND MICROSCOPIC STUDY : As per the figures and photos in Annexure :(5.2) PHYSIO CHEMICAL STUDY :(i) Total Ash value : Sl. No. Parameters Observation 1. Seed powder of crude drug 7.425 % 2. Fine seed powder 7.685 %(ii) Acid insoluble ash Sl. No. Parameters Observation 1. Seed powder 0.03 gms(iii) Water soluble extractive values 38.72 %(iv) Alcohol soluble extraxctive value of "Palash seed 20.72 % 79=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 98. Results==================================================================(5.3)(a) Yeild of Extract : Sl. No. Extract Wt. Yield W/W. (1) Ethanol 20.72 gms. 20 % (2) Hot Water 20.09 gms. 20 % (3) Cold Water 25.32 gms. 25 %(b) Organoleptic Characters of Extracts :Organoleptic Alcoholic Hot water Cold Water Extract Extract ExtractColour Yellowish Brownish Brownish Dark BrownConsistancy Oily Oily OilyOdour Characteristics Characteristics CharacteristicsTaste Bitter Bitter Bitter 80=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 99. Results================================================================== TABLE NO. 9 5.4 PRELIMINARY PHYTO-CHEMICAL SCREENING : Extraxts Chemical Tests Alcohol Aqueous 1) Test pf Sterols a) Salkowaski test + − b) Liebermann - Burchard test + − 2) Test for Steroidal glycosides + + 3) Test for Triterpenoids a) Salkowaski test + − b) Liebermann - Burchard test + − c) Tschugajew test + − d) Briekorn and Brinar test + − 4) Test for Glycosides i) Cardiac glycosides − − a) Baljets test − − b) Raymonds test − − c) Legals test − − ii) Anthraquinone Glycosides − − a) Borntragers test − − b) Modified Borntragers test − − 5. Test for Saponins a) Foam test − 6. Test for Flavonoids / Flavonoid Glycosides a) Shinoda test + + b) Zinc Hydrochloric acid reduction test + + 81=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 100. Results================================================================== c) Alkaline reagent test + + 7. Test for carbohydrates a) Molischs test + + b) Fehling test + + c) Barfoeds test + + d) Benedicts test + + e) Selvinoffs test + + f) Test for pentoses + + 8. Test for Alkoloids + a) Mayers test − + b) Wagners test − + c) Hagers test − + d) Dragendorffs test − + 9. Test for Phenolics and Tannins + a) Lead acetate test + + b) Ferric Chloride test + + c) Gelatin test + − 10. Test for Lipid + 11. Test for Proteins − + a) Millons test − + b) Xanthoproteic test − + c) Biuret test − + 12. Test for free amino acid a) Ninhydrin test − + Note : " + " Present, " - " Absent. Glycosides + in both extract. Carbohydrat + in both extract. Sterols + in alcohol Terpenoids + in both extract Alkaloids + present in aq. Tannins + present in both 82=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 101. Results==================================================================HPTLC ANALYSIS :-Qualitative analysis of aquesous extract and ethenolic extract :Sl. Sampled Detail Amount of Rf valuesNo. extract on plate @ 500 m.1. Ethanol extract of "palash seed" 200 mcg. 0.16, 0.30, 0.472. Aqueous extract of "Palash seed" 200 mcg. 0.08, 0.14, 0.24Result :- Rf values of ethanol extract are approximately double that of aqeous extract.Remark :- There were no bands observed @ 254 mm. and @ 366 mm, but, ethanol extractshowed bands @ 500 mm but we cannt observed any bands of aqueous extract @ 500 mm. 83=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 102. Results==================================================================(5.5) ANTIMICROBIAL RESULTS :Cup and Diffusion Method (Mueller and Hinton Agar).TABLE NO. 10 Zone of Zone of Zone of Drug / Zone of Inhibition of Inhibition of Inhibition of Compound Inhibition of Staphylococcus of Candida of Bacilus Tested E-coli Aureus. Albicans. Subtillus.Ofloxacin (2micg/disc) 20 mm 15 mm - -Amikacin (10micg/disc) 14 mm 14 mm - -Penicillin (10micg/disc) 12 mm 24 mm - -Greseofulvin (100micg/disc) - - 10 mm -Cold water extract 0 0 0 0for 4 mg / ml - A1Hot water extract 0 0 0 0for 4 mg/ml - B1Ethanol extract 0 11.2 mm 0 10 mmfor 4 mg/ml - C1Cold water extract 2 mm 0 0 0for 6 mg/ml - A2Hot water extract 0 0 0 0for 6 mg/ml - B2Ethanol extract 0 12.3 mm 0 10.5 mmfor 6 mg/ml. - C2Cold water extract 6 mm 2 mm 0 0for 8 mg/ml. A3Hot water extract 3 mm 0 0 4 mmfor 8 mg/ml B3Ethanol extract 12 mm 13 mm 2 mm 12 mmfor 8 mg/ml - C3Note : ‘Aqueous extract’ showed resistant activity to bacteria and fungi. ‘Ethanol extract’showed resitant activity to fungi and sensitive to bacteries.Indicates :* A-Cold water extract * B-Hot water extract * C-Ethanol extract 84=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 103. Results==================================================================ANTIBACTERIAL RESULTS : TABLE NO. 11a) Esherichia Coli : Sl. No. 10 mg/ml 5 mg/ml. 2.5 mg/ml. 1.25 mg/ml. 0.612 0.3 0.15 MIC Value A R R R R R R R > 10 B R R R R R R R > 10 C S S S R R R R 2.5 TABLE NO. 12b) Staphylococcus Aureus : Sl. No. 10 mg/ml 5 mg/ml. 2.5 mg/ml. 1.25 mg/ml. 0.612 0.3 0.15 MIC Value A R R R R R R R > 10 B R R R R R R R > 10 C S S S R R R R 2.5 TABLE NO. 13c) Bacillus Subtillis : Sl. No. 10 mg/ml 5 mg/ml. 2.5 mg/ml. 1.25 mg/ml. 0.612 0.3 0.15 MIC Value A R R R R R R R > 10 B R R R R R R R > 10 C S S S R R R R 2.5 85=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 104. Results==================================================================ANTIFUNGAL RESULTS : TABLE NO. 14D) CANDIDA ALBICANS : Sl. No. 10 mg/ml 5 mg/ml. 2.5 mg/ml. 1.25 mg/ml. 0.612 0.3 0.15 MIC Value A R R R R R R R > 10 B R R R R R R R > 10 C R R R R R R R > 10NOTE : "S" - Sensitive "R" – Resistant 86=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 105. Discussion================================================================== 86=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 106. Discussion================================================================== 6. DISCUSSION "Palash" plant is wellknown in vedic period (are having lot of convining references."Kinshuk" is more popular synonym is refered to "Palash". In vedic kala "Palash" is used fornot only to treat the disease but day today life also. In "Bruhatrayes" is also known as "Palash" and "Kinhuk". Controversy regarding thisplant is not seen. Different parts like stem, seed, leaves, flowers kshara are used to treatvarious diseases. In nighantus nomenclature of the plant is given according to their physical charactersand combating common ailement. "Palash was used widely in ritual functions like “yagaya’’etc. That is why it is also given a name as yajnika and it is supposed to have more holyproperties that is why it is termed as "Putadru" and "Sumidwara", "Krimighna" is another.Synonym which is used to treat in krimi use of "Palash" patra is more common andaccording to its physical character, it is said to be Kharaparna and Tripatra. "Palash beeja issneha yukta that is why it is termed as Beeja sneha. According to chassical texts seeds are used to treat various skin diseases, so in thisstudy more attention and concentration is given regarding this property of "Palash" seed. Charakacharya not classified in four Ganas . But according to Sushruta Charyaand Vagbhatacharya "Palash" is included in same ganas like "Ambhasthadi, Nyagrodhadigana etc. The use of "Palash" seed is given seperately and different yogas in different samhitas.In nighantus "Palash" was explained under "Phala varga" Haritakyadi Varga, Vatadi Varga,Aamradi Varga, Etc. In "Raj Nighantu" references regarding "Palash" classification is done on the basis ofdifferent colours of the flowers as Rakta, Peeta, Shweta and Neela. 87=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 107. Discussion================================================================== "Palash" seed posses katu - tikta and kinchit kashaya rasa. Veerya - Ushna, Vipak -Katu, Guna "Kapha – Vata Shamaka pittakar". Its main karmas are Krimighna,Vaatashamak Udar rog, Arsha and It is also used in various skin disorders like Dadru,Paama, Kandu and Kushta etc. Botanically first it is known as "Butea frondasa" but now it was also termed to be as"Butea monosperma Limm? Controversy regarding this plant is not present. Because "Buteamonosperma is a tree and Butea superba is another (type of) variety of this plant but that isclimber one ! Adulteration may taking place with Butea monosperma seed with Buteasuperba seed, but seed of Butea monosperma is larger than that of Butea superba. "Palash" seed has been considered as "Krimighna" by different acharyaas. The workis intended to study on inference that correlates microbes explained in modern terms. Inantimicrobial study is needed for the time. As the bacteria, fungi etc. Microrganisms havebecome resistant to the newest antimicrobial agents, antimicrobial agents have traditionallybeen made of natural products in cluding microganisms themselves. Before starting actualstudy the drug was authenticated by expert Botanist, and then pharmacognostic study of thedrug i.e. macro and microscopic study was carried out to study its morphology, internalstructures and their contents in detail. After that physico - chemical analysis was done i.e. ash value, acid insoluble ash,alcohol and water soluble extractives values were determined. The ash value of sample waswithin normal range as given in Ayurvedic herbal pharmacoepoeia, this represents inorganicsalts present in drug like carbonates - bicarbonates etc. In this procedure the carbon isremoved by heating at low temperature. Acid insoluble ash value was also near by standard by this process we ruled outpresence of excess foreign particles, that is sand or silica etc. which may not be absorbed byacid media in body and may give rise to further complications. 88=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 108. Discussion================================================================== Alcohol soluble and water soluble extractive values are done at pilot study. This ishelpful regarding the yield of extract, which can be acquired by doing extraction on extract,which can be acquired by doing extraction on larger scales. Then the extraction procedure were carried out by ethanol and water as solvent. Theprocedure adopted was soxhlet extraction and cold maceration procedure. Ethanol wasselected because it is standard during the new drug extraction. The ethanol which was usedprepared in laboratory by distilling rectified spirit. Then the procedure ethanols percentage95% was clarified by feasible laboratory procedure. The preliminary phytochemical screeing was done that reveals the presence ofalkaloids, tannines, reducing sugars, proteins, flavanoids, glucosides in all alcohol and waterextracts. Qualitative analysis of plant extracts were done by T.L.C. and H.P.T.L.C. In this testpresence of various compounds that were detected by their Rf. values. These Rf. values canbe directly and indirectly help for checking adultration in raw or compound drug. In T.L.C. 0.2 gm. of extract was diluted to 10 ml. of ethanol and used for T.L.C. nobands were observed with aqueous extract. But with ethanol extract bands were not observesat 254 and 366. But band were seen at @ 500 nm. Rf values of ethanol extract are (0.16,0.30, 0.47) It shows that ethanol extract of seed is more effective than aqueous extract.Antimicrobial Activity :- This study was carried out on Gram +ve bacterias (S. aureus and B. Septilis) Gram -ve bacteria E-coli and fungi (Candia albicans). It was carried out by cup diffusion method agar plates. 89=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 109. Discussion================================================================== The zone of inhibition were recorded. In our study aqueous extract was resistantwhen compared to the modern standard drugs but ethanol extract is sensitive to bacterias butit is also resistant to fungi. MIC values against E-coli and S. aureus were 2.5 mg/ml. but against candidaalbicans it was 10 mg or more than that. 90=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 110. Conclusion================================================================== 90=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 111. Conclusion================================================================== 7. CONCLUSION1) The physico chemical results i.e. ash value, acid insoluble ash, alcohol and water soluble extractive values are within parameters of Ayurvedic Herbal pharmacopoeia.2) Ethanol extract yield of Palash seed is less than the aqueous extract.3) The preliminary phytochemicals screening of both ethanolic and water extract show presence of tannins, carbohydrates, Terpenoids, Glycosides and Alkoloid present in aq. extract and Streroil present in alcohol extract.4) Rf. values of ethanol extract are double than that of aqueous extract also height of peak of ethanol extract in more than that of aqueous extract.5) In antimicrobial activity aqueous extract showed resistance to all bacterias and fungi but ethanol extract is sensitive to bacterias and resistant to fungi. Thus ethanolic extract of palash seed can be used in bacterial infections The aim of this study is to evaluate the antimicrobial activity of "Palash" seed andnot to check the incidence or prevalence of disease. The statistical analysis of date is notconsidered. 91=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 112. Summary================================================================== 91=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 113. Summary================================================================== 7. SUMMARY Seed of "Palash" is used as "Krimighna" but it is not included in krimighna ganaor any gana by Charkacharya. It is used in to treat skin diseases and its antihelmenticproperty is proved. In the present study preliminary study was done in context to study itsantimicrobial activity.The plant drug (seeds) were subjected to :(i) Pharmacognostic review.(ii) Water ethenol soxlet extraction.(iii) Preliminary phytochemical screening(iv) T.L.C. and H.P.T.L.C. (Finger printing)(v) Antimicrobial activities (Bacterias and Fungi) The phy to chemicals observed were Tannins glycosides, etc. Antimicrobial study by cup-diffusion method showed positive result of ethenolextract of "Palash" seed on bacterias but it is resistant to fungi and aqueous extract showedresistant to both bacteria and fungi.Scope of further study :-(a) To study more for their single drug efficacy clinically.(b) Comparative study with patra, flowers can be done.(c) Extraction by different solvents to find extract base for their antimicrobial acitivity.(d) In vitro study for their antimplantation, aphro disiac and antioxidant property.(e) It will be better to repeat the same procedure for 3 to 4 times and then arrive to conclusion.NOTE : In a way to standardization we can check for the microbes in the crude / raw drugand then proceed with antimicrobial study. Then we can compare the presence of amount ofmicrobes present in the drug with that to its antimicrobial activity. 92=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 114. Biblography================================================================== 92=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 115. Biblography================================================================== BIBLOGRAPHY1. Triphathi Indradev; Raja Nighantu; Varanasi , Krishnadas Acadami 3rd edition 2003; Page No.3042. Sharma P.V., Namarupa Vijnananam, Varanasi, Satyaprakashan, 1st edition, 2000, Page No.1233. Gupta Shakti M , Plant Myths & Traditions In India , Chaukhamba Publications , edition 2001 , Page No. 114. Shrivastav Shailaja , Sharandhar Samhita , Varanasi , Chawkhamba Orientilia , edition 2003 , Page no. 172.5. Chunekar Krishnachandr , Bhavaprakash Nighantu , Varanasi , Choukhambha Bharati Academy , edition 2004 , Page No. 335.6. Shastri Shrilaxmipati , Yogaratnakar , Varanasi . Choukhamba Sanskrant Santhan , 8th edition 2004 , Page No. 334,335.7. Chunekar Krishnachandr , Bhavaprakash Nighantu , Varanasi , Choukhambha Bharati Academy , edition 2004 , Page No. 335.8. Vaishya Lala Shaligramaji , Shaligram Nighantu , Bombay, Khemaraj Shrikrishnadas Prakashan , edition 2002 , page 515.9. Sharma P.V., Dhanavantari Nighantu , Varanasi , Chaukhamba Orientilia , 2nd edition 1998 , Page No. 177.10. Sharma P.V., Kayadeva Nighantu , Varanasi , Chaukhamba Orientilia , 1st edition 1979 , Page No. 177. 93=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 116. Biblography==================================================================11. Khemaraj Shrikrishnadas , Madanpal Nighantu, Bombay , Shri Venkateshwar., year 1966.12. Triphathi Indradeva , Raja Nighantu , Varanasi , Krishnadas Academy, 3rd edition 2003 , Page No.304.13. Sharma P.V. Sodhal Nighantu , Baroda , Oriental Institute 1st edition 1978 , Page No. 304.14. Vaidya Bapalal , Nighantu, Baroda ,Varansi, Chaukhamba Bharati Academy, third edition 2002, page355.15. Sharma P.V. , Priya Nighantu , Varanasi , Chaukhamba Surabharati Prakashan , edition 2004 , Page No. 35.16. Nadakarni A. K., Indian materia medica vol. 1 , Popular prakashan, page 222.17. Sharma P. C.,Data base on Medicinal plants used in Ayurveda vol 1, Central council for Research in Ayurveda and Siddha, page336.18. Indian Herbal Pharmacopia, Indian drug Manufactures association , Mumbai , Edition 2002, Page 98.19. Gupta A.K.; Indian Council of medical Research, Mew delhi, Quality standards of Indian Medicinal plant, 2003.20. Singh Ramshushil , Vanoushadi Nidarshika Ayurvediyapharmacopia Lahor, Uttar Pradash Hindi Santhan 2nd edition 1983, Page –21. Vaidyagrantham P.S. Arya Vaidya sala, Indian Medicinal Plants, Chenni, Orient Longman private limited, Page – 314. 94=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 117. Biblography==================================================================22. Dhiman Anil Kumar, Wild medicinal plant of India Deharadun, Bisher, sighnaendra Palsigh.23. Kirtikar K.R & Basu B.D., Indian medicinal Plant vol- I 2nd edition, Dehradun, International Book Distribution, 1999 Page- 785.24. Ambasta S. P, The useful plants of India, New Delhi publications & information directorate council of Scientific & Industrial Research, Page – 91.25. Sharma P.V., Namarupa Vijnananam, Varanasi, Satyaprakashan, 1st edition, 2000, Page No.12326. Pandey Gyanedra, Dravyaguna Vijnana(Vol III), Varanasi. Krishna Academy, 1st edition 2001 Page-27. Reviews on Indian Medicinal Plant. Vol –IV Page – 490.28. Gupta A.K. , Quality Standards of Indian Medicinal Plants. Sharma P.V., Kayadeva Nighantu , Varanasi ,Chaukhamba Orientilia , 1st edition 1979 , Page No. 177.29. Sharma P.C., Yelne M.B, Dennis T.J. , Data Base On Medicinal Plants Used In Ayurveda Vol- I, New Delhi, Central Council for Research in Ayurveda and siddha, Reprinted 2002, Page 338.30. Shastri J.L.N. Ayurvedokta Aushadhi niruktamala, Varanasi Choukhamba oriental, 1st edition 2001, Page – 66.31. Baghel M.S. Research in ayurveda past & present, article Ayurveda and Microorganism, Page- No. 216, 226. 95=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 118. Biblography==================================================================32. Marshall Jacquelyn, Microbilogy, The clinical laboratory, manual series, Tomson Delmar Learning, 1st Reprint 2003, Sigapore.33. Marshall Jacquelyn, Microbilogy, The clinical laboratory, manual series, Tomson Delmar Learning, 1st Reprint 2003, Sigapore.34. Gupta Satish, The Short Text book of Medical Microbiology, 8th edition, New Delhi, Jaypee brothers-2002, Reprint 2004.35. Gupta Satish, The Short Text book of Medical Microbiology, 8th edition, New Delhi, Jaypee brothers-2002, Reprint 2004.36. Makharjee Pulok K. Quality control of Herbal drugs New Delhi, And Business Horizons- Pharmaceuticals publishers 1st edition 2002, Page – 381, 42537. Choudhari R.D., Herbal drug Industry, 1st edtion 1996, New Delhi. Eastern publishers India Tt58, 34,147,160,173,192,24,350,370,464,477,598.38. Makharjee Pulok K. Quality control of Herbal drugs New Delhi, And Business Horizons- Pharmaceuticals publishers 1st edition 2002, Page – 381, 42539. Choudhari R.D., Herbal drug Industry, 1st edtion 1996, New Delhi. Eastern publishers India Tt58, 34,147,160,173,192,24,350,370,464,477,598.40. Controller of publication , Indian pharmacopeia, 1996.41. Mukharjee Pulok K.Quality control of Herbal Press, new Delhi, Business Horizons – Pharmaceutical Publishers.42. Mukharjee Pulok K.Quality control of Herbal Press, new Delhi, Business Horizons – Pharmaceutical Publishers. 96=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 119. Biblography==================================================================43. Mukharjee Pulok K.Quality control of Herbal Press, new Delhi, Business Horizons – Pharmaceutical Publishers.44. R. Khandelwal K. Practical Pharmacogonosy, Techniques & Expriments, Pune , Nirali Prakashan, 13th edition, April 2005, Page- 10-18, 149-156.45. Qulity Control Methods of medical Plant Materials, wt 0, Geneva, ATIBS publishers & Distributors (Regd.) E-Mail- aitbs@ndf.vsnl.net.in.46. Wagener H. Bladt. S.E.M. Zganiski , PLANT DRUG Analysis , A Thin layer Chromotography Atlas.47. Kasture A.V. Mehadik R. et.al. Pharmaceutical Analysis, VOL 5 , Pune Nirali Prakashan 8th edition May 2002, Page No. 18-13.48. Gupta Satish, The Short Text Book of Medical Microbiology, 8th Edition, New Delhi, jaypee Brothers – 2002, Reprint- 2004.49. Harry w.et.al. , Aniseptic & disinfectant action , Microbes in Action. & lab Manual of Microbiology 1982 Page No.75 – 76.50. R. Khandelwal K., Practical Pharmacogonsy, Techniques and experiments, Pune, Nirali Prakashan, 13th Edtion, April 1 -2005, Page No. 10-18, 149-156.Other Books and References :1) Pandey Gyanendra, Dravya Guna Vignana, Vol-I, 2nd Edition, Varanasi Krishnadas Academy, 2002,2) Jayengar M. A. Study of Crude Drugs, Manipal, 12th Edition – 2004. 97=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
  • 120. Biblography==================================================================3) Rajpal V., Standardization of Botanicals, Vol – I, 2002, Reprint – 2004, New Delhi, Estern Publishers.4) Pammel L. H., A Manual of Poisonous Plants, Torch Press Cedar rapids, lowa, 1911, Reprint 1991.5) Shastry J. L. N. Ayurvedokta Aushadhi Nirukta Mala, Edition – 2001, Varanasi, Chowkhamba Orientalia, India.6) Singh Ram Shusheel Vanaushadhi Nidarshika Nirukta Mala, Edition – 2001, Varanasi, Chowkhamba Orientalia, India.7) Uniyal Mayaram, Medicinal Plants and Minerals of Uttarakhanda, Himalaya, 1st Edition, May 1997, Bihar, Baidyanath Ayu. Shoodh Sanstan.8) Mukhypadhyaya, Girindranath, History of India Medicines, Vol-I and II, Munshiram Manoharlal Publishers, Reprint 2003.9) Sharma P. V., Dalhana and his comments on drugs, Ist Edition, 1982, Munshiram Manoharlal Publication Pvt. Ltd.10) Raja Radha Kanta Deva, Shabdha Kalpa Druma Vol-II, Varanasi, Chowkhamba Sanskrit Series, 3rd Edition, 1967,11) Sharma P. V. Dravyaguna Vijnana Vol-II Varanasi, Chokhamba Bharati Academy, Reprint – 2005.12) Chopra R. N., Chopra I. C. et.al., Indigenous Drugs of India; (Tt) Culcutta, Academic Publishers, 1st Edition, 1958, 2nd Reprint – 1994.13) Singh Thakur Balwant, Chunenar K. C. et.al; Glossary of Vegetable drugs in Brahatrayees, 2nd Edition, 1999, Varanasi, Chokambha Amarabharati Prakashan 98=============================================================================== “SREENING OF ANTIMICROBIAL ACTIVITY OF ‘PALASH SEED’ (BUTEA MONOSPERMA LIM.)”
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