Phytochemicals Screening, Phenolic Estimation and Evaluation for Anti-Oxidant, Anti-Inflammatory and Anti-Microbial Activities of Sequentially Soxhlet Extracted Coconut Testa — Oak Academic Publishing
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Phytochemicals Screening, Phenolic Estimation and Evaluation for Anti-Oxidant, Anti-Inflammatory and Anti-Microbial Activities of Sequentially Soxhlet Extracted Coconut Testa
Department of Biotechnology, North Orissa University, Baripada, Odisha, India
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Department of Biotechnology, North Orissa University, Baripada, Odisha, India
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Department of Biotechnology, North Orissa University, Baripada, Odisha, India
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Department of Biotechnology, North Orissa University, Baripada, Odisha, India
1 Department of Biotechnology, North Orissa University, Baripada, Odisha, India
2 Department of Biotechnology, North Orissa University, Baripada, Odisha, India
3 Department of Biotechnology, North Orissa University, Baripada, Odisha, India
4 Department of Biotechnology, North Orissa University, Baripada, Odisha, India
Background: In many coconut industries, the outer layer of thin brown skin of coconut kernel known as testa is peeled out as a byproduct. Despite the testa is rich in fat and plenty of polyphenolic compounds, it has been underutilized either as animal feed, serving as raw materials for bio-diesel production or discarded directly. Anticipating coconut testa (CT) as a natural source of multiple phyto-chemicals, its exploitation for the pharmacological activity or utilization as value added product is required which may reduce the disposal costs as well. Methods: Secondary metabolites from CT were extracted sequentially with different organic solvents based on polarity in the soxhlet apparatus followed by extraction with sterilized water. The crude dried extracts thus prepared were evaluated for qualitative screening of phytochemicals and quantitative estimation of total phenols, flavonoids and tannin content. Moreover, the antioxidant, anti-inflammatory and anti-microbial activities were also investigated. Results: Phytochemicals screening revealed the presence of polyphenolic compounds in methanolic fraction including phenols (822.60 ± 16.36 mg/g), flavonoids (103.30 ± 9.78 mg/g) and tannin (663.50 ± 19.26 mg/g), whereas non-phenolic compounds were present in other fractions. While methanolic fraction showed invariably the highest anti-oxidant activity in multiple assay methods, non-phenolic compounds in aqueous and chloroform fractions exhibited high anti-inflammatory activity. Antimicrobial activity was observed by both phenolic and non-phenolic compounds. Conclusion: The findings of the study reveal that CT is a rich source of various polyphenolic and non-phenolic natural antioxidants, anti-inflammatory and antimicrobial compounds. These findings are promising and form the basis to identify the number of active components and their characterization.
Manivannan, A., Bhardwaj, R., Padmanabhan, S., Suneja, P., Hebbar, K.B. and Kanade, S.R. (2018) Biochemical and Nutritional Characterization of Coconut (Cocos nucifera L.) Haustorium. Food Chemistry, 238, 153-159. https://doi.org/10.1016/j.foodchem.2016.10.127
DebMandal, M. and Mandal, S. (2011) Coconut (Cocos nucifera L.: Arecaceae): In Health Promotion and Disease Prevention. Asian Pacific Journal of Tropical Medicine, 4, 241-247. https://doi.org/10.1016/S1995-7645(11)60078-3
Yong, J.W.H., Ge, L.-Y., Ng, Y.F. and Tan, S.N. (2009) The Chemical Composition and Biological Properties of Coconut (Cocos nucifera L.) Molecules, 14, 5144-5164. https://doi.org/10.3390/molecules14125144
Zhang, L., Ravipati, A.S., Koyyalamudi, S.R., Jeong, S.C., Reddy, N., Smith, P.T., Bartlett, J., Shanmugam, K., Munch, G. and Wu, M.-J. (2011) Antioxidant and Anti-Inflammatory Activities of Selected Medicinal Plants Containing Phenolic and Flavonoid Compounds. Journal of Agricultural and Food Chemistry, 59, 12361-12367. https://doi.org/10.1021/jf203146e
Lathika, M. and Kumar, C.E.A. (2009) Indian Stakes in the Global Coconut Scenario by the Turn of the Century: An Empirical Investigation. South Asia Economic Journal, 10, 209-221. https://doi.org/10.1177/139156140901000109
Marikkar, J.M.N. and Madhrapperuma, W.S. (2012) Chapter 9. Coconut. In: Siddiq, M., Ed., Tropical and Subtropical Fruits: Postharvest Physiology, Processing and Packaging, John-Wiley Publishing Co., Ames, Iowa, USA, 159-177. https://doi.org/10.1002/9781118324097.ch9
Appaiah, P., Sunil, L., Prasanth Kumar, P.K. and Gopala Krishna, A.G. (2014) Composition of Coconut Testa, Coconut Kernel and Its Oil. Journal of the American Oil Chemists’ Society, 91, 917-924. https://doi.org/10.1007/s11746-014-2447-9
Adekola, K.A., Salleh, A.B., Zaidan, U.H., Azlan, A., Chiavaro, E., Paciulli, M. and Marikkar, J.M.N. (2017) Total Phenolic Content, Antioxidative and Antidiabetic Properties of Coconut (Cocos nucifera L.) Testa and Selected Bean Seed Coats. Italian Journal of Food Science, 29, 741-753.
Arivalagan, M., Roy, T.K., Yasmeen, A.M., Pavithra, K.C., Jwala, P.N., Shivasankara, K.S., Manikantan, M.R., Hebbar, K.B. and Kanade, S.R. (2018) Extraction of Phenolic Compounds with Antioxidant Potential from Coconut (Cocos nucifera L.) Testa and Identification of Phenolic Acids and Flavonoids Using UPLC Coupled with TQD-MS/MS. LWT-Food Science and Technology, 92, 116-126. https://doi.org/10.1016/j.lwt.2018.02.024
Pandey, K.B. and Rizvi, S.I. (2009) Plant Polyphenols as Dietary Antioxidants in Human Health and Disease. Oxidative Medicine and Cellular Longevity, 2, 270-278. https://doi.org/10.4161/oxim.2.5.9498
Manach, C., Scalbert, A., Morand, C., Rémésy, C. and Jiménez, L. (2004) Polyphenols: Food Sources and Bioavailability. The American Journal of Clinical Nutrition, 79, 727-747. https://doi.org/10.1093/ajcn/79.5.727
Everitt, A.V., Hilmer, S.N., Brand-Miller, J.C., Jamieson, H.A., Truswell, A.S., Sharma, A.P., Mason, R.S., Morris, B.J. and Le Couteur, D.G. (2006) Dietary Approaches That Delay Age-Related Diseases. Clinical Interventions in Aging, 1, 11-31.
Dzialo, M., Mierziak, J., Korzun, U., Preisner, M., Szopa, J. and Kulma, A. (2016) The Potential of Plant Phenolics in Prevention and Therapy of Skin Disorders. International Journal of Molecular Sciences, 17, 160. https://doi.org/10.3390/ijms17020160
Razak, D.L.A., Jamaluddin, A., YuhaslizaAbd. N., Rashid, Sharifudin, S.A. and Long, K. (2016) Comparative Study of Antioxidant Activities, Cosmeceutical Properties and Phenolic Acids Composition of Fermented Rice Bran and Coconut Testa. Jurnal Teknologi (Sciences & Engineering), 78, 11-12. https://doi.org/10.11113/jt.v78.9940
Swaroop, C., Jose, T.K. and Ramesh, A. (2016) Property Testing of Biodiesel Derived from Coconut Testa Oil and Its Property Comparison with Standard Values. International journal of Advances in Production and Mechanical Engineering, 2, 37-41.
Khoddami, A., Meredith, A.W. and Thomas, H.R. (2013) Techniques for Analysis of Plant Phenolic Compounds. Molecules, 18, 2328-2375. https://doi.org/10.3390/molecules18022328
Sun, T. and Ho, C.-T. (2005) Antioxidant Activities of Buckwheat Extracts. Food Chemistry, 90, 743-749. https://doi.org/10.1016/j.foodchem.2004.04.035
Turkmen, N., Sari, F. and Velioglu, Y.S. (2006) Effects of Extraction Solvents on Concentration and Antioxidant Activity of Black and black Mate Tea Polyphenols Determined by Ferrous Tartrate and Folin-Ciocalteu Methods. Food Chemistry, 99, 835-841. https://doi.org/10.1016/j.foodchem.2005.08.034
Hayouni, A., Abedrabba, M., Bouix, M. and Hamdi, M. (2007) The Effects of Solvents and Extraction Method on the Phenolic Contents and Biological Activities in Vitro of Tunisian Quercus coccifera L. and Juniperus phoenicea L. Fruit Extracts. Food Chemistry, 105, 1126-1134. https://doi.org/10.1016/j.foodchem.2007.02.010
Appaiah, P., Sunil, L., Gopala Krishna, A.G. and Suresh Kumar, G. (2016) Phytochemicals and Antioxidant Activity of Testa Extracts of Commercial Wet and Dry Coconuts and Cakes. International Research Journal of Pharmacy, 7, 9-13. https://doi.org/10.7897/2230-8407.079106
Zhao, X.-L., Chen, W.-J., Zhao, S.-L. and Tang, M.-M. (2012) Antioxidative Activity of the Extractives from Coconut Testa Oil. Chinese Journal of Tropical Crops, 33, 162-165.
Sashya, M.M.M., Meedeniya, D. and Coorey, R.V. (2012) Quality Assessment of Commercially Available Coconut Oils in Sri Lanka Using Refractometry. Proceedings of the Technical Sessions, 28, 37-44.
Seneviratne, K.N., HapuarachchI, C.D. and Ekanayake, S. (2009) Comparison of the Phenolic-Dependent Antioxidative Properties of Coconut Oil Extracted under Cold and Hot Conditions. Food Chemistry, 114, 1444-1449. https://doi.org/10.1016/j.foodchem.2008.11.038
Sheel, R., Nisha, K. and Kumar, J. (2014) Preliminary Phytochemical Screening of Methanolic Extract of Clerodendron infortunatum. IOSR-Journal of Applied Chemistry, 7, 10-13. https://doi.org/10.9790/5736-07121013
Nanna, R.S., Marka, R., Talari, S., Penchala, S. and Rudroju, S. (2013) Preliminary Phytochemical Analysis of Leaf, Stem, Root and Seed Extracts of Arachis hypogaea L. International Journal of Pharmaceutical Sciences Review and Research, 20, 134-139.
Saklani, S., Mishra, A.P., Sati, B. and Sati, H. (2012) Pharmacognostic, Phytochemical and Antimicrobial Screening of Aphanamixis polystachya, An Endangered Medicinal Tree. International Journal of Pharmacy and Pharmaceutical Sciences, 4, 235-240.
Sharma, V., Agarwal, A., Chaudhary, U. and Singh, M. (2013) Phytochemical Investigation of Various Extracts of Leaves and Stems of Achyranthes aspera L. International Journal of Pharmacy and Pharmaceutical Sciences, 5, 317-320.
Ebrahimzadeh, M.A., Pourmorad, F. and Bekhradnia, A.R. (2008) Iron Chelating Activity, Phenol and Flavonoid Content of Some Medicinal Plants From Iran. African Journal of Biotechnology, 7, 3188-3192.
Islam T., Hasan, M.R., Roy, A., Islam, M.S., Uddin, M.A., Islam, M.A., Neon, M.N. and Rana, M.S. (2015) Screening of In-Vitro Antioxidant, Brine Shrimp Lethality Bioassay and Antimicrobial Activities of Extracts of Bridelia retusa (L.) Spreng. Fruit. International Journal of Pharmacy, 5, 1058-1067.
Makkar, H.P.S., Dawra, R.K. and Singh, B. (1988) Determination of Both Tannin and Protein in a Tannin-Protein Complex. Journal of Agricultural and Food Chemistry, 36, 523-525. https://doi.org/10.1021/jf00081a600
Chang, S.-T., Wu, J.-H., Wang, S.-Y., Kang, P.-L., Yang, N.-S. and Shyur, L.-F. (2001) Antioxidant Activity of Extracts from Acacia confusa Bark and Heartwood. Journal of Agricultural and Food Chemistry, 49, 3420-3424. https://doi.org/10.1021/jf0100907
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. and Rice-Evans, C. (1999) Antioxidant Activity Applying an Improved ABTS Radical Cation Decolorization Assay. Free Radical Biology and Medicine, 26, 1231-1237. https://doi.org/10.1016/S0891-5849(98)00315-3
Prieto, P., Pineda, M. and Aguilar, M. (1999) Spectrophotometric Quantitation of Antioxidant Capacity through the Formation of a Phosphomolybdenum Complex: Specific Application to the Determination of Vitamin E. Analytical Biochemistry, 269, 337-341. https://doi.org/10.1006/abio.1999.4019
Dinis, T.C.P., Madeira, V.M.C. and Almeida, L.M. (1994) Action of Phenolic Derivatives (Acetoaaminophen, Salycilate and 5-Aminosalycilate) as Inhibitors of Membrane Lipid Peroxidation and as Peroxyl Radical Scavengers. Archieves of Biochemistry and Biophysics, 315, 161-169. https://doi.org/10.1006/abbi.1994.1485
Oyaizu, M. (1986) [Studies on Products of the Browning Reaction Prepared From Glucose Amine]. The Japanese Journal of Nutrition and Dietetics, 44, 307-315. https://doi.org/10.5264/eiyogakuzashi.44.307
Karaman, S., Tutem, E., Baskan, K.S. and Apak, R. (2010) Comparison of Total Antioxidant Capacity and Phenolic Composition of Some Apple Juices with Combined HPLC-CUPRAC Assay. Food Chemistry, 120, 1201-1209. https://doi.org/10.1016/j.foodchem.2009.11.065
Perez, C., Paul, M. and Bazerque, P. (1990) An Antibiotic Assay by the Agar Well Diffusion Method. Acta Biologiae et Medicinae Experimentalis, 15, 113-115.
Shinde, U.A., Kulkarni, K.R., Phadke, A.S., Nair, A.M., Dikshit, V.J.M. and Saraf, M.N. (1999) Mast Cell Stabilizing and Lipoxygenase Inhibitory Activity of Cedrus deodara (Roxb.) Loud. Wood Oil. Indian Journal of Experimental Biology, 37, 258-261.
Mizushima, Y. and Kobayashi, M. (1968) Interaction of Anti-Inflammatory Drugs with Serum Proteins, Especially with Some Biologically Active Proteins. Journal of Pharmacy and Pharmacology, 20, 169-173. https://doi.org/10.1111/j.2042-7158.1968.tb09718.x
Geetha, V., Bhavana, K.P., Chetana, R., Gopala Krishna, A.G. and Suresh Kumar, G. (2016) Studies on the Composition and In-Vitro Antioxidant Activities of Concentrates from Coconut Testa and Tender Coconut Water. Journal of Food Processing & Technology, 7, 588-592.
Lamdande, A.G., Garud, S.R. and Raghavarao K.S.M.S. (2017) Processing Methods for Increasing the Yield, Nutritional Value and Stability of Coconut Milk. International Journal of Nutrition and Food Engineering, 11, 12.
Zhao, H.-F., Dong, J., Lu, J.-J., Chen, J., Li, Y., Shan, L.-J., Lin, Y., Fan, W. and Gu, G.-X. (2006) Effect of Extraction Solvent Mixtures on Antioxidant Activity Evaluation and Their Extraction Capacity and Selectivity for Free Phenolic Compounds in Barley (Hordeum vulgare L.). Journal of Agricultural and Food Chemistry, 54, 7277-7286. https://doi.org/10.1021/jf061087w
Sulaiman, S.F., Sajak, A.A.B., Supriatno, K.L.O. and Seow, E.M. (2011) Effect of Solvents in Extracting Polyphenols and Antioxidants of Selected Raw Vegetables. Journal of Food Composition and Analysis, 24, 506-515. https://doi.org/10.1016/j.jfca.2011.01.020
Wijekoon, M.M.J.O., Bhat, R. and Karim, A.A. (2011) Effect of Extraction Solvents on the Phenolic Compounds and Antioxidant Activities of Bunga kantan (Etlingera elatior Jack.) Inflorescence. Journal of Food Composition and Analysis, 24, 615-619. https://doi.org/10.1016/j.jfca.2010.09.018
Chavan, U.D. and Amarowicz, R. (2013) Effect of Various Solvent Systems on Extraction of Phenolics, Tannins and Sugars from Beach Pea [Lathyrus maritimus L.]. International Food Research Journal, 20, 1139-1144.
Vyas, S., Kachhwaha, S. and Kothari, S.L. (2015) Comparative Analysis of Phenolic Contents and Total Antioxidant Capacity of Moringa oleifera Lam. Pharmacognosy Journal, 7, 44-51. https://doi.org/10.5530/pj.2015.1.5
Srinivasan, R., Chandrasekar, M.J.N., Nanjan, M.J. and Suresh, B. (2007) Antioxidant Activity of Caesalpinia digyna Root. Journal of Ethnopharmacology, 113, 284-291. https://doi.org/10.1016/j.jep.2007.06.006
Robbins, R.C. (1980) Medical and Nutritional Aspects of Citrus Bioflavonoids. Chapter 3. In: Citrus Nutrition and Quality, 43-59. https://doi.org/10.1021/bk-1980-0143.ch003
Pinelo, M., Franco, D., Sineiro, J., Rubilar, M., Sánchez, M., Jerez, M., Costoya, N. and Nunez, M.J. (2008) Polyphenols from Plant Materials: Extraction and Antioxidant Power. Electronic Journal of Environmental, Agriculture and Food Chemistry, 7, 3210-3216.
Antolovich, M., Prenzler, P., Robards, K. and Ryan, D. (2000) Sample Preparation in the Determination of Phenolic Compounds in Fruits. Analyst, 125, 989-1009. https://doi.org/10.1039/b000080i
Soler-Rivas, C., Espín, J.C. and Wichers, H.J. (2000) An Easy and FAST Test to Compare Total Free Radical Scavenger Capacity of Foodstuffs. Phytochemical Analysis, 11, 330-338. https://doi.org/10.1002/1099-1565(200009/10)11:5 3.0.CO;2-G
Toh, J.J., Khoo, H.E. and Azrina, A. (2013) Comparison of Antioxidant Properties of Pomelo [Citrus grandis (L) Osbeck] Varieties. International Food Research Journal, 20, 1661-1668.
Ramful, D., Tarnus, E., Aruona, O.I., Bourdon, E. and Bohorun, T. (2011) Polyphenol Composition, Vitamin C Content and Antioxidant Capacity of Mauritian Citrus Fruit Pulps. Food Research International, 44, 2088-2099. https://doi.org/10.1016/j.foodres.2011.03.056
Cano, A., Hernandez-Ruiz, J., Garcia-Canovas, F., Acosta, M. and Arnao, M.B. (1998) An End-Point Method for Estimation of the Total Antioxidant Activity in Plant Material. Phytochemical Analysis, 9, 196-202. https://doi.org/10.1002/(SICI)1099-1565(199807/08)9:4 3.0.CO;2-W
Apak, R., Guclu, K., Ozyurek, M. and Karademir, S.E. (2004) Novel Total Antioxidant Capacity Index for Dietary Polyphenols and Vitamins C and E, Using Their Cupric Ion Reducing Capability in the Presence of Neocuproine: CUPRAC Method. Journal of Agricultural and Food Chemistry, 52, 7970-7981. https://doi.org/10.1021/jf048741x
Mekni, M., Azez, R., Tekaya, M., Mechri, B. and Hammami, M. (2013) Phenolic, Non-Phenolic Compounds and Antioxidant Activity of Pomegranate Flower, Leaf and Bark Extracts of Four Tunisian Cultivars. Journal of Medicinal Plants Research, 7, 1100-1107.
Mortada, M.E., Abdel-Hameed, E.S., Ahmed, W.S. and El-Wakil, E.A. (2008) Non-Phenolic Antioxidant Compounds from Buddleja asiatica. Zeitschrift für Naturforschung C, 63, 483-491. https://doi.org/10.1515/znc-2008-7-803
Umapathy, E., Ndebia, E.J., Meeme, A., Adam, B., Menziura, P., Nkeh-Chungag, B.N. and Iputo, J.E. (2010) An Experimental Evaluation of Albuca setosa Aqueous Extract on Membrane Stabilization, Protein Denaturation and White Blood Cell Migration during Acute Inflammation. Journal of Medicinal Plant Research, 4, 789-795.
Mounnissamy, V.M., Kavimani, S., Balu, V. and Drlin, Q.S. (2008) Evaluation of Anti-Inflammatory and Membrane Stabilizing Properties of Ethanol Extract of Canjera rehedi. Iranian Journal of Pharmacology and Therapeutics, 6, 235-237.
Chaitanya, R., Sandhya, S., David, B., Vinod, K.R. and Murali, S. (2011) HRBC Membrane Stabilizing Property of Roor, Stem and Leaf of Glochidion velutinum. International Journal of Research in Pharmaceutical and Biomedical Sciences, 2, 256-259.
Mellors. A., Tappel, A.L., SawantI, P.L. and Desa, D. (1967) Mitochondrial Swelling and Uncoupling of Oxidative Phosphorylation by Lysosomes. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 143, 299-309. https://doi.org/10.1016/0005-2728(67)90084-9
Alhakmani, F., Kumar, S. and Khan, S.A. (2013) Estimation of Total Phenolic Content, In-vitro Antioxidant and Anti-Inflammatory Activity of Flowers of Moringa Oleifera. Asian Pacific Journal of Tropical Biomedicine, 3, 623-627. https://doi.org/10.1016/S2221-1691(13)60126-4
Kassim, M., Achoui, M., Mustafa, M.R., Mohd, M.A. and Yusoff, K.M. (2010) Ellagic Acid, Phenolic Acids, and Flavonoids in Malaysian Honey Extracts Demonstrate in Vitro Anti-Inflammatory Activity. Nutrition Research, 30, 650-659. https://doi.org/10.1016/j.nutres.2010.08.008
Masuda, T., Jitoe, A., Isobe, J., Nakatani, N. and Yonemori, S. (1993) Anti-Oxidative and Anti-Inflammatory Curcumin-Related Phenolics from Rhizomes of Curcuma domestica. Phytochemistry, 32, 1557-1560. https://doi.org/10.1016/0031-9422(93)85179-U
Claeson, P., Panthong, A., Tuchinda, P., Reutrakul, V., Kanjanapothi, D., Taylor, W.C. and Santisuk, T. (1993) Three Non-Phenolic Diarylheptanoids with Anti-Inflammatory Activity from Curcuma xanthorrhiza. Planta Medica, 59, 451-454. https://doi.org/10.1055/s-2006-959730
Claeson, P., Pongprayoon, U., Sematong, T., Tuchinada, P., Reutrakul, V., Soontornsaratune, P. and Taylor, W.C. (1996) Non-Phenolic Linear Diarylheptanoids from Curcuma xanthorrhiza: A Novel Type of Topical Anti-Inflammatory Agents: Structure-Activity Relationship. Planta Medica, 62, 236-240. https://doi.org/10.1055/s-2006-957867
Oyedapo, O.O., Akinpelu, B.A., Akinwunmi, K.F., Adeyinka, M.O. and Sipeolu, F.O. (2010) Red Blood Cell Membrane Stabilizing Potentials of Extracts of Lantana camara and Its Fractions. International Journal of Plant Physiology and Biochemistry, 2, 46-51.
Dodson, R.A., Hinds, T.R. and Vincenzi, F.F. (1987) Effects of Calcium and A23187 on Deformability and Volume of Human Red Blood Cells. Blood Cells, 12, 555-564.
Friederichs, E. and Meiselman, H.J. (1994) Effects of Calcium Permeabilization on RBC Rheologic Behavior. Biorheology, 31, 207-215.
Ahmad, I., Mehmood, Z. and Mohammad, F. (1998) Screening of Some Indian Medicinal Plants for Their Antimicrobial Properties. Journal of Ethnopharmacology, 62, 183-193. https://doi.org/10.1016/S0378-8741(98)00055-5
Eloff, J.N. (1998) Which Extractan Should Be Used for Screening and Isolation of Antimicrobial Components from Plants? Journal of Ethnopharmacology, 60, 1-8. https://doi.org/10.1016/S0378-8741(97)00123-2
Bhakuni, D.S., Dhar, M.L., Dhawan, B.N. and Mehrotra, B.N. (1969) Screening of Indian Plants for Biological Activity, Part II. Indian Journal of Experimental Biology, 7, 250-262. https://doi.org/10.1016/S0168-1605(02)00169-1
Alzoreky, N.S. and Nakahara, K. (2003) Antibacterial Activity of Extracts from Some Edible Plants Commonly Consumed in Asia. International Journal of Food Microbiology, 80, 223-230.
Olano, I., Alonso Paz, E., Cerdeiras, M.P., Fernandez, J., Ferreira, F., Moyna, P., Soubes, M., Vázquez, A., Vero, S. and Bassagoda, M.J. (1996) Screening of the Uruguayan Medicinal Plants for Anti-Microbial Activity. Part II. Journal of Ethnopharmacology, 53, 111-115.