Determination of Chemical Groups and Investigation of Anthelmintic, Cytotoxic, and Antibacterial Activities of Leaves of Cinnamomum Tamala (Family: Lauraceae)

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The present study was conducted for the characterization of possible chemical groups, …

The present study was conducted for the characterization of possible chemical groups,
evaluation of anthelmintic, cytotoxic and antibacterial activities of crude methanolic extract
of leaves of Cinnamomum tamala. The study revealed the presence of alkaloids, reducing
sugar, tannin, amino acids, glycosides and steroid in the crude extract. The extract showed
very potent anthelmintic activity while compared with the standard albendazole. To
investigate the cytotoxic activity, brine shrimp lethality bioassay was conducted, and the
extract showed significant activity while compared with the standard vincristine sulphate
(LC50 value 1.007 and 0.839μg/ml respectively). To evaluate the antibacterial activity, disc
diffusion method was followed, and the extract showed activity against Bacillus subtilis,
Staphylococcus aureus, Bacillus cereus, and Vibrio cholera, and resistant to Escherichia coli
and Salmonella typhi.

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  • 1. International Journal of Pharmamedix IndiaVolume-I, Issue-IIAvailable online on www.pharmamedix.in/Current-Issues.php Page 222Jamiuddin A. et al.; International Journal of Pharmamedix India, 2013, 1(2), 222-232.Note- This article is property of International Journal of Pharmamedix India [ISSN: 2320-1304].Published by: Pharmamedix IndiaTM[www.pharmamedix.in]This Open Access Article available on www.pharmamedix.in only for private and non-commercial use.“Determination of Chemical Groups and Investigation ofAnthelmintic, Cytotoxic, and Antibacterial Activities of Leaves ofCinnamomum Tamala (Family: Lauraceae)”.Jamiuddin Ahmed*, Nasrin Sultana, Syed M. R. Dewan, Mohammad N Amin, S. M. Naim Uddin.*Author for correspondenceJamiuddin AhmedLecturerDepartment of PharmacyNoakhali Science and Technology UniversitySonapur, Noakhali- 3814Bangladesh.E-mail: pharma.jamiahmed@gmail.comContact No.: +8801199113606
  • 2. International Journal of Pharmamedix IndiaVolume-I, Issue-IIAvailable online on www.pharmamedix.in/Current-Issues.php Page 223Introduction:Cinnamomum tamala Fr. Nees is an evergreentree up to 8.5m in height, belonging to familylauraceae. Lauraceae is a vast economicallyimportant family consisting mostly ofethnobotanical knowledge from ancient treesor tree-like shrubs. The genus Cinnamomumis represented by about 350 speciesworldwide. It is native to South-east Asia,some Pacific Islands and Australia growingmainly in tropical rain forests at varyingaltitudes [1].Due to its aroma, the leaves are kept inclothes and also chewed to disguise badmouth odor. The leaves of this tree have aclove like taste and a faintly pepper like odor.It is also used in Indian system of traditionalmedicines.Different extracts from leaves of C.tamala have shown anti-inflammatory [2],antioxidant [3], antiulcer [4], anticarcinogenic[5], antidiarrhoeal effects [6], antidiabeticwhich is mainly contributed byCinnamaldehyde (3-phenyl-2- propenal), apotential antidiabetic agent. It is also usedmedicinally as a carminative, an anti flatulent,a diuretic, treatment of cardiac disorders [7]analgesic in dental preparations, due topresence of eugenol (4-hydroxy-3-methoxyallylbenzene).Abstract:The present study was conducted for the characterization of possible chemical groups,evaluation of anthelmintic, cytotoxic and antibacterial activities of crude methanolic extractof leaves of Cinnamomum tamala. The study revealed the presence of alkaloids, reducingsugar, tannin, amino acids, glycosides and steroid in the crude extract. The extract showedvery potent anthelmintic activity while compared with the standard albendazole. Toinvestigate the cytotoxic activity, brine shrimp lethality bioassay was conducted, and theextract showed significant activity while compared with the standard vincristine sulphate(LC50 value 1.007 and 0.839μg/ml respectively). To evaluate the antibacterial activity, discdiffusion method was followed, and the extract showed activity against Bacillus subtilis,Staphylococcus aureus, Bacillus cereus, and Vibrio cholera, and resistant to Escherichia coliand Salmonella typhi.Keywords: Cinnamomum tamala, Lauraceae, cytotoxic, anthelmintic, antibacterial.
  • 3. International Journal of Pharmamedix IndiaVolume-I, Issue-IIAvailable online on www.pharmamedix.in/Current-Issues.php Page 224The main goal of our study was to evaluatethe presence of possible chemical groups,cytotoxic, anthelmintic, and antibacterialactivities of the crude methanolic extract ofthe leaves of Cinnamomum tamala to validateits folkloric use.Materials and Methods:Plant Material CollectionThe leaves of Cinnamomum tamala werecollected by the authors from the surroundingarea of Noakhali, a coastal region ofBangladesh in September, 2012. The plantwas identified and authenticated by expertbotanist of Bangladesh National Herbarium(DACB Accession No. 39290), Mirpur,Dhaka.Crude Extraction300 gm of the dried and powdered samplewas soaked in 1300 ml of 99.8% methanol(Merck KGaA, Germany). After 15 days thesolution was filtered using filter cloth andWhatman®filter paper No. 1. The resultingfiltrates were then evaporated in water bathmaintained at 40°c to dryness and thus ablackish green semisolid mass of the extractwas obtained.Chemical Group TestSmall quantity of freshly prepared methanolicextract of C. tamala leaves were subjected topreliminary quantitative phytochemicalinvestigation for the detection of chemicalconstituents using the following standardmethods [8].i. Detection of alkaloidsExtract was dissolved individually in diluteHydrochloric acid and the solutions werefiltered.a) Mayer’s Test: Filtrates were treatedwith Mayer’s reagent (PotassiumMercuric Iodide). Formation of ayellow colored precipitate marked thepresence of alkaloids.b) Hager’s Test: Filtered solutions weretaken in a test tube and Hager’sreagent (saturated picric acid solution)was added with it. Presence ofalkaloids was confirmed by theformation of yellow coloredprecipitate.ii. Detection of carbohydratesExtract was dissolved individually in 5 mldistilled water and filtered. The filtrates wereevaluated for the presence of carbohydrates.a) Benedict’s test: Filtrates were treatedwith Benedict’s reagent and heatedgently. Orange red precipitate pointedthe presence of reducing sugars.b) Fehling’s Test: Filtered solutionswere hydrolyzed with dil. HCl,
  • 4. International Journal of Pharmamedix IndiaVolume-I, Issue-IIAvailable online on www.pharmamedix.in/Current-Issues.php Page 225neutralized with alkali and heated withFehling’s A & B solutions. Formationof red precipitate specified thepresence of reducing sugars.iii. Detection of glycosidesExtract was hydrolyzed with dil. HCl, andthen subjected to test for glycosides.a) Legal’s Test: Extracts were mixedwith sodium nitropruside in pyridineand sodium hydroxide. Formation ofpink to blood red color indicated thepresence of glycosides.b) Modified Borntrager’s Test: Extractswere treated with Ferric Chloridesolution and immersed in boiling waterfor about 5 minutes. The mixture wascooled and extracted with equalvolumes of benzene. The benzene layerwas separated and treated withammonia solution. Formation of rose-pink color in the ammoniacal layershowed the presence of glycosides.iv. Detection of saponinsa) Froth Test: Extract was diluted withdistilled water to 20 ml and this wasshaken in a graduated cylinder for 15minutes. Formation of 1 cm layer offoam expressed the presence ofsaponins.b) Foam Test: 0.5 gm of extract wasshaken with 2 ml of water. Foam wasproduced which remained for 10minutes and pointed the presence ofsaponins.v. Detection of phytosterolsa) Salkowski’s Test: Extract was treatedwith chloroform and filtered. Thefiltrates were treated with few drops ofconc. sulphuric acid, shaken andallowed to stand. Appearance ofgolden yellow color showed thepresence of triterpenes.b) LibermannBurchard’s test: Extractwas mixed with chloroform andfiltered. The filtrates were treated withfew drops of acetic anhydride, boiledand cooled and then conc. sulphuricacid was added. Formation of brownring at the junction confirmed thepresence of phytosterols.vi. Detection of phenolsFerric Chloride Test: Extractsolution was taken in test tubes and 3-4 drops of ferric chloride solutionwere added to them. Formation ofbluish black color indicated thepresence of phenols.vii. Detection of tanninsGelatin Test: To the extract, 1%gelatin solution containing sodiumchloride was added. Formation of
  • 5. International Journal of Pharmamedix IndiaVolume-I, Issue-IIAvailable online on www.pharmamedix.in/Current-Issues.php Page 226white precipitate confirmed thepresence of tannins.viii. Detection of flavonoidsa) Alkaline Reagent Test: Extract wastreated with 4-5 drops of sodiumhydroxide solution. Formation ofintense yellow color, which becomescolorless on addition of dilute acid,indicated the presence of flavonoids.b) Lead acetate Test: 4-5 drops of leadacetate solution was added to theextract solution. Formation of yellowcolor precipitate marked the presenceof flavonoids.ix. Detection of proteins and aminoacidsa) Xanthoproteic Test: The extract wastreated with 4-5 drops of conc. Nitricacid. Formation of yellow colorindicated the presence of proteins.b) Ninhydrin Test: To the extract,0.25% w/v ninhydrin reagent wasadded and boiled for few minutes.Formation of blue color indicated thepresence of amino acid.x. Detection of fixed oils and fatsA few drops of 0.5N alcoholic potassiumhydroxide were added to a small quantity ofextract along with a drop of phenolphthalein.The mixture was heated on a water bath for 1-2 hours. Formation of soap or partialneutralization of alkali pointed the presenceof fixed oils and fats.xi. Detection of gums and mucilages1 ml of the extract was hydrolyzed using dil.HCl (3ml). Then Fehling’s solution wasadded drop by drop till the appearance of red.Test for mucilages were carried out bytreating 1 ml of extract with 2 ml ofruthenium red solution to get red colouredsolution.In-vitro Anthelmintic StudyThe anthelmintic study was carried out by themethod of Ajaiyeoba et al. [9]with minormodifications. Adult earthworms wereselected for the study of anthelmintic activitybecause of their anatomical and physiologicalresemblance with the intestinal roundwormparasites of human being [10]. Earthworms arewidely used as effective tools for anthelminticstudy due to their availability [11]. Adultearthworm (Pheretima posthuma) werecollected (3-5 cm in length and 0.1- 0.2 cm inwidth weighing about 0.8-3.04 g) from moistsoil of a road side field of Noakhali Scienceand Technology University, Sonapur,Noakhali. All the worms were properlywashed with normal saline in order to removeall fecal materials.Extract was weighed and dissolved in 10 mLof distilled water to obtain the solution of 20,
  • 6. International Journal of Pharmamedix IndiaVolume-I, Issue-IIAvailable online on www.pharmamedix.in/Current-Issues.php Page 22740, 60, and 80 mg/ml. Albendazole was usedas reference standard (20 mg/mL).Earthworms were divided into seven groups(each containing three worms) in petridish. Infive groups extract solution was applied, oneis for reference and one is for negativecontrol. Observations were made for thedetermination of paralysis time and deathtime of the worm. Paralysis was designated asthe occurrence where the worms do not moveeven in normal saline and death wasconfirmed when the worms lose their motilityfollowed with fading away of their bodycolor.In-vitro Cytotoxic StudyThe cytotoxic activity of the extract wasexamined using brine shrimp lethalitybioassay [12]. In this study vincristine sulphatewas used as the positive control. Measuredamount of the vincristine sulphate wassdissolved in DMSO to get an initialconcentration of 40µg/ml from which serialdilutions were made using DMSO to get20µg/ml, 10µg/ml, 5µg/ml, 2.5µg/ml,1.25µg/ml, 0.625µg/ml, 0.3125 µg/ml,0.15625µg/ml and 0.78125µg/ml solutionfrom the extract. Then the positive controlsolutions were added to the pre-marked vialscontaining ten living brine shrimp nauplii in 5ml simulated sea water to get the positivecontrol groups.100µl of DMSO was added toeach of three pre-marked glass vialscontaining 5 ml of simulated sea water and 10shrimp nauplii to use as control groups.Counting of NaupliiAfter 24 hours, by using a magnifying glass,the vials were inspected and the number ofsurvived nauplii in each vial was counted.From this data, the percent (%) of lethality ofthe brine shrimp nauplii was calculated foreach concentration.Antibacterial Activity TestTest OrganismsThree strains of Gram-positive (Bacilluscereus, Staphylococcus aureus, and Bacillussubtilis), and three strains of Gram negativebacteria (Escherichia coli, Salmonella typhi,Vibrio cholerae) were used to evaluate theantibacterial activity. The strains werecollected from the Department ofMicrobiology, Chittagong Veterinary andAnimal Sciences University. For theexperiment, the organisms were sub-culturedin nutrient broth and nutrient agar medium.Disc Diffusion Assay (DDA)Disc diffusion method is widely acceptablefor the evaluation of antimicrobial activity [13,14].In this method, an antibiotic was diffuse froma reliable source through the nutrient agar anda concentration gradient was created. Dried,sterilized filter paper discs (6 mm diameter,
  • 7. International Journal of Pharmamedix IndiaVolume-I, Issue-IIAvailable online on www.pharmamedix.in/Current-Issues.php Page 228HI-Media, China) containing the knownamounts of test samples (400 µg/disc) areplaced on nutrient agar medium consistentlyseeded with the test bacteria. As positive andnegative control, Standard antibiotic ofCiprofloxacin (5 µg/disc) and blank discswere used. For the maximum diffusion of thetest materials to the surrounding media theseplates were reserved at low temperature (4°C)for 24 hours. The plates were then incubatedat 37°C for 24 hours to allow optimumgrowth of the organisms. The test materialshaving antibacterial property inhibit microbialgrowth in plates and thereby yield a clear,distinct zone defined as zone of inhibition.The activity of the test sample was thendetermined by measuring the zone ofinhibition expressed in millimeter [15].Results and Discussion:Chemical Group testPhytochemical analysis of methanolic extractof leaves of C. tamala revealed the presenceof some important chemical constituents, e.g.,alkaloids, carbohydrates, glycosides, etc(Table 1).Table 1: Phytochemical screening of the methanolic extract of C. tamala leaves.Sl. No Group of phytoconstituents Methanolic extract1. Alkaloids +2. Carbohydrates +3. Glycosides +4. Saponins -5. Phytosterols +6. Phenols -7. Tannins +8. Flavonoids -9. Proteins and amino acids +10. Fats & fixed oils -11. Gum and mucilages -(+) = presence of constituents; (-) = absence of constituents
  • 8. International Journal of Pharmamedix IndiaVolume-I, Issue-IIAvailable online on www.pharmamedix.in/Current-Issues.php Page 229y = 34.02x + 52.58R² = 0.952020406080100120-2 -1 0 1 2Log C% Mortalityy = 21.75x + 50.07R² = 0.9060204060801001200 1 2 3mortalitylog c% MortalityLinear (%Mortality)Brine Shrimp Lethality BioassayLC50 (lethal concentration of half of the test organisms) and LC90 (lethal concentration of 90% ofthe test organisms) data (for establishing therapeutic index) of vincristine sulphate and all threeextracts have been given in table 2, and figure 1.Table 2: Cytotoxic effect of the test sample of C.tamalaSample LC50 (g/ml) LC90 (g/ml)Vincristine sulphate 0.839 12.59Crude extract 1.007 68.53(a) (b)Figure 1: Effect of (a) vincristine sulphate, (b) crude methanolic extract on brine shrimp nauplii.In-vitro Anthelmintic ActivityFrom the data (Table 3), we see that, the methanolic extract of C. tamala demonstrated paralysis aswell as death of worms in fewer times with the gradual increase of the sample concentration.
  • 9. International Journal of Pharmamedix IndiaVolume-I, Issue-IIAvailable online on www.pharmamedix.in/Current-Issues.php Page 230Table 3: Anthelmintic activity of crude methanolic extract of leaves of C. tamala against Pheretimaposthuma.Group Concentration(mg/ml)Paralysis time (min.) Death time (min.)Mean ±S.E.M. Mean ±S.E.M.Sample I 20 19.00±0.45 44.50±0.22Sample II 40 12.00±0.36 19.33±0.21Sample III 60 8.33±0.21 14.00±0.33Sample IV 80 5.33±0.21 8.50±0.22Standard 20 14.00±0.37 43.83±0.54n = 5, S.E.M. = Standard Error MeanAntibacterial Activity TestThe methanolic extract of the leaves showed moderate antibacterial activity against several testorganisms. The result of the antimicrobial activity in term of diameter of zone of inhibition in mm isshown in Figure 2. The zone of inhibition varied from 5 to 10 mm at this concentration. This extractdid not show any activity against E. coli and Salmonella typhi.Figure 2: Antibacterial activity of crude extract of Cinnamomum tamalaBacillussubtilisStaphylococcusaureusBacilluscereusEscherichia coliSalmonella typhiVibriocholeraecrude sample 5 5 6 0 0 10standard 20 18.5 23.5 16.9 16 19.30510152025Diameterofzoneofinhibitioninmm
  • 10. International Journal of Pharmamedix IndiaVolume-I, Issue-IIAvailable online on www.pharmamedix.in/Current-Issues.php Page 231Conclusion:From the above discussion it can be suggestedthat further in vivo investigation is needed toensure anthelmintic, antimicrobial, andanticancer activities of the leaves of C.tamala, and also it would be interesting tofind out responsible compound(s) and relativemechanisms for the mentioned activities.Acknowledgement:The authors are grateful to BNH to identifythe plant, and CVASU to supply themicrobes. Authors are also thankful toDepartment of Pharmacy, Noakhali Scienceand Technology University for providing thelaboratory facilities.References:1. Chakraborty U, Das H. Antidiabeticand antioxidant activities ofCinnamomum tamala leaf extracts inStz-Treated diabetic rats. GlobalJournal of Biotechnology &Biochemistry. 2010 (1): 12-18.2. Gambhire MN, Juvekar AR,Wankhede SS. Anti-inflammatoryactivity of aqueous extract ofCinnamomum tamala leaves by in vivoand in vitro methods. Journal ofPharmacy Research. 2009 (2) 9:1521-1524.3. Devi SL, Kannappan S, Anuradha CV.Evaluation of in vitro antioxidantactivity of Indian bay leaf,Cinnamomum tamala (Buch, -Ham,)T,Nees & Eberm using rat brainsynaptosomes as model system. IndianJournal of Experimental Biology.2007 (45) 9, 778-784.4. Eswaran MB, Surendran S,Vijayakumar M, Ojha SK, RawatAKS, Rao CV. Gastroprotectiveactivity of Cinnamomum tamalaleaves on experimental gastric ulcersin rats. Journal ofEthnopharmacology. 2010 (128):537–540.5. Rao AR, S Hashim. Chemopreventiveaction of oriental food-seasoningspices mixture Garam masala onDMBA-induced transplacental andtranslactational carcinogenesis inmice. Nutrition and cancer. 1995 (23)1: 91-101.6. Rao CV, Vijayakumar M, Sairam K,Kumar V. Antidiarrhoeal activity ofthe standardised extract ofCinnamomum tamala in experimentalrats. Journal of Natural Medicines.2008 (62) 4: 396-402.7. Chaurasia JK, Pandey N, Tripathi YB.Effect of hexane fraction of leaves ofCinnamomum tamala Linn onmacrophage functions.Inflammopharmacology. 2010 (18) 3:147-154.
  • 11. International Journal of Pharmamedix IndiaVolume-I, Issue-IIAvailable online on www.pharmamedix.in/Current-Issues.php Page 2328. Dewan SMR, Das A. Investigation ofin vitro thrombolytic potential andphytochemical nature of Crinumlatifolium L. leaves growing in coastalregion of Bangladesh. InternationalJournal of Biological andPharmaceutical Research. 2013 (4) 1:1-7.9. Ajaiyeoba EO, Onocha PA,Olarenwaju OT. In vitro anthelminticproperties of Buchholzia coriaceaeand Gynandropsis gynandries extract.Pharmaceutical Biology. 2001 (39):217-20.10. Chatterjee KD. Parasitology,Protozoology and Helminthology. 6thed. Calcutta: In Guha Ray SreeSaraswaty Press Ltd., Calcutta, India.1967.11. Sollman, T. Anthelmintic: Theirefficacy as tested on earthworms.Journal of Pharmacology andExperimental Therapeutics. 1918(112): 129-70.12. Meyer BN, Ferringni NR, Puam JE,Lacobsen LB, Nichols DE,McLaughlin JL. Brine shrimp: aconvenient general bioassay for activeconstituents. Planta Medica. 1982(45): 31-32.13. Bayer AW, Kirby WMM, Sherris JCand Turck M. Antibiotic susceptibilitytesting by a standardized single discmethod. American Journal of ClinicalPathology. 1966 (45): 493-496.14. Satheesh LS and Murugan K.Antimicrobial activity of proteaseinhibitor from leaves of Cocciniagrandis (L.) voight. Indian Journal ofExperimental Biology. 2011 (49): 366-374.15. Barry AL. Principle & Practice ofMicrobiology. 3rd Ed. Lea & Fabager,Philadelphia. 1976.