MICROBICIDAL POTENTIALITY OF PURIFIED ANTHOCYANIN FROM IN VITRO CULTURE OF CLERODENDRON INFORTUNATUM L. AGAINST SELECTED PATHOGENS
Objective: Clerodendron infortunatum L. is a widely used medicinal herb over centuries for curing many skin-borne disorders. The present study was designed to validate the tribal knowledge by evaluating antimicrobial potential of purified anthocyanin extracted from in vitro cell suspension culture.
Methods: The explants were inoculated on murashige and skoog (MS) medium mixed with various combinations of 2, 4-D a+BAP for callus induction. Green compact callus was initiated within 30 d from the explants on MS medium fortified with benzylaminopurine (BAP) (2.0 mg/l)+2, 4-D (0.5 mg/l). Subsequently, anthocyanin was triggered from the compact callus by subculturing in the medium containing 2, 4-D and Kinetin. Cell suspension culture was also developed. Anthocyanin production was enhanced by elicitation using salicylic acid and others. Three chromatographic methods such as solid phase extraction by Sepharose C18 column, Oasis-MCX and Amberlite XAD 7+Sephadex LH 120 sorbents were used to purify the in vitro synthesized anthocyanin from the cell cultures. HPLC and molar absorptivity assay were carried to check the purity. Antimicrobial analysis was also carried using standard protocols to check minimum inhibitory concentration (MIC) and minimum killing concentration (MKC).
Results: The mean purity values obtained by high-performance thin layer chromatography (HPLC) were 90.9%Â±1.9, 80.60%Â±2.3 for Oasis MCX, Amberlite XAD-7+Sephadex LH-20 column respectively. However, the purity by molar absorptivity was found to be less. HPLC chromatogram revealed 12 fractions of anthocyanin. Inhibition zone diameter, MIC and MKC values obtained for the purified anthocyanin revealedÂ its antimicrobial potentiality but at different levels among the selected bacteria and fungi. C.Â albicans, S. aureus, P. aerugenosa showed significant values followed by MRSA, E. coli and A. flavus. The results are comparable with the synthetic antibiotics. However, E. faecalis was more resistance. Mode of action was confirmed from the results of intracellular potassium leakage and bacterial membrane integrity analysis.
Conclusion: Thus, the study confirms the efficacy of anthocyanin as natural antimicrobial and suggests the possibility of employing it as drugs for the treatment of infectious diseases caused by the pathogens.
2. Valcheva Kuzmanova SV, Belcheva A. Current knowledge of Aronia melanocarpa as a medicinal plant. Folia Med 2006;48:11â€“7.
3. Kokotkiewicz A, Jaremicz Z, Luczkiewicz M. Aronia plants: a review of traditional use, biological activities, and perspectives for modern medicine. J Med Food 2010;13:255â€“69.
4. Chattopadhyay RR, Bhattacharyya SK. Herbal spices as alternative antimicrobial food preservatives: an update. Pharmacogn Rev 2007;1:239â€“47.
5. Chitra W, Calderon P, Gagnon D. Evaluation of selected medicinal plants extracted in different ethanol concentrations for antibacterial activity against human pathogens. J Medical Active Plants 2012;1:60â€“8.
6. Bhattacharjee D, Das A, Kanti Das S, Chakraborthy GS. Clerodendrum Infortunatum Linn.: a review. J Adv Pharm Healthcare Res 2011;1:81-5.
7. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 1962;15:473-97.
8. Rodrigues APM, Vendrame WA. Micropropagation of tropical woody species. In: Jain SM. Ishii K. Micropropagation of woody trees and fruits. Dordrecht: Kluwer Academic Publishers; 2003. p. 153-79.
9. Giusti MM, Wrolstad RE. Acylated anthocyanins from edible sources and their applications in food systems. Biochem Eng J 2003;14:217-25.
10. Collins CH, Lyne PM. Microbiological Methods. Butterworth, London; 1970.
11. NCCLS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard-Fifth Edition. NCCLS document M7-A5. NCCLS: Wayne, PA, USA; 2000.
12. Demarsh PL, Gagnon RC, Hetzberg RP, Jaworski DD. Methods of screening for antimicrobial compounds. Smithkline Beccham Corporation. Publ World Intellectual Property Organization (WIPO); 2001.
13. Perez C, Pauli M, Bazerque P. An antibiotic assay by the agar-well diffusion method. Acta Biol Med Exp 1990;15:113-5.
14. Edris AE, Farrag ES. Antifungal activity of peppermint and sweet basil essential oils and their major aroma constituents on some plant pathogenic fungi from the vapor phase. Food 2003;47:117-21.
15. Simoes M, Pereira MO, Vieira MJ. Validation of respirometry as a short-term method to assess the efficacy of biocides. Biofouling 2005;21:9â€“17.
16. Sun J, Deng Z, Yan A. Bacterial multidrug efflux pumps: Mechanisms, physiology and pharmacological exploitations. Biochem Biophy Res Comm 2014;453:254â€“67.
17. Borges A, Simoes LC, Saavedra MJ, Simoes M. The action of selected isothiocyanates on bacterial biofilm prevention and control. Int Biodet Biodeg 2014;86:25â€“33.
18. Alberto MR, Canavosio MAR, Nadra MCM. Antimicrobial effect of polyphenols from apple skins on human bacterial pathogens. Electron J Biotechnol 2006;9:205â€“9.
19. Cisowska A, Wojnicz D, Hendrich AB. Anthocyanins as antimicrobial agents of natural plant origin. Nat Prod Commun 2011;6:149-56.
20. LiepiÅ†a I, Nikolajeva V, Jakobsone I. Antimicrobial activity of extracts from fruits of Aronia melanocarpa and Sorbus aucuparia. Environ Exp Biol 2013;11:195â€“9.
21. Burdulis D, Ã„arkinas A, Jasutien I, StackeviIen E, Nikolajevas L, Janulis V. Comparative study of anthocyanin composition, antimicrobial and antioxidant activity in bilberry (Vaccinium myrtillus L.) and blueberry (Vaccinium corymbosum L.) fruits. Acta Poloniae Pharm Drug Res 2009;66:399-408.
22. LiegiutÄ— S, MajienÄ— D, TrumbeckaitÄ— S, Liobikas J, Bendokas V, Stanys V, et al. Anthocyanin composition and antimicrobial activity of sour cherry (Prunus cerasus L.) fruit extracts. ZemdirbystÄ— (Agriculture) 2009;96:141-8.
23. Yoon BI, Bae WJ, Choi YS, Kim SJ, Syn Ha U, Hong S, et al. The anti-inflammatory and antimicrobial effects of anthocyanin extracted from black soybean on chronic bacterial prostatitis rat model. Chin J Integr Med 2013;23:1-6.
24. Srirangaraj S, Segar L, Kali A. Multidrug-resistant Acinetobacter baumannii from nosocomial urinary tract infection: a case report. Asian J Pharm Clin Res 2015;8:6-8.
25. Radhika D, Mohaideen A. Fourier transform infrared analysis of Ulva lactuca and Gracilaria corticata and their effect on antibacterial activity. Asian J Pharm Clin Res 2015;8:209-12.