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Hydrocotyle Javanica Thunb.) – a Plant from Ayurveda

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Hydrocotyle Javanica Thunb.) – a Plant from Ayurveda Pharmacogn J. 2020; 12(6)Suppl: 1494-1503 A Multifaceted Journal in the field of Natural Products and Pharmacognosy Original Article www.phcogj.com GC-MS Analysis of Anti-Enterobacterial Dichloromethane Fraction of Mandukaparni (Hydrocotyle javanica Thunb.) – A plant from Ayurveda Manab Mandal1, Debabrata Misra1, Narendra Nath Ghosh1, Sukhendu Mandal2, Vivekananda Mandal1* ABSTRACT Background: Mandukaparni (Hydrocotyle javanica Thunb.) is a well known medicinal herb used as folklore medcine in many chronic and infectious gastric and other diseases by the people of Estern Himalayan regions. However, the therapeutic active principles of this plant Manab Mandal1, Debabrata remained unknown. Objective: The main objective of the study was to characterize anti- Misra1, Narendra Nath Ghosh1, enterobacterial dichloromethane fraction of the volatile oils of it by GC-MS. Materials and Sukhendu Mandal2, Vivekananda Methods: In the present study, dichloromethane (DCM) fraction (MP-DCMf) of Mandukaparni Mandal1* was collected by phase separation of the methanol extract and tested for anti-enterobacterial potentiality against human pathogenic gastrointestinal and food poisoning bacteria by agar 1 University of Gour Banga, Malda, INDIA. well diffusion assay, viability assay and LDH assay and SEM studies. Characterization of the 2University of Calcutta, Calcutta, INDIA. active MP-DCMf fraction was performed by TLC and GC-MS analysis. Results: The MP-DCMf Correspondence possessed bio-active compounds that have antibacterial potentiality against both the Gram- Vivekananda Mandal positive and Gram-negative bacteria. The MIC and MBC values were in the range from 1.56 University of Gour Banga, Malda, INDIA. mg/ml to 0.78 mg/ml and 6.25 to 1.56 mg/ml, respectively. The time kill assay showed that at a th E-mail: [email protected] dose of 3.12 mg/ml of MP-DCMf was lethal to the E. coli MTCC 723 at the 18 hr of treatment. History LDH release had moderate positive correlation with the activity index and time of treatment • Submission Date: 18-06-2020; whereas strong negative correlation with CFU count. It caused highest cell disruption in S. • Review completed: 01-08-2020; mutans. The principal compounds were D-carvon (30.949%);1H-Isoindole-1,3(2H)-dione; • Accepted Date: 08-08-2020. 2-(2-chlorophenyl) (28.483%);Cyclohexanone; 2-methyl-5-(1-methylethen) trans (10.04%); D-Limnone (9.256%);2,6-Octadien-1-ol, 3,7-dimethyl- acetate (6.684%); p-Cresol (0.551%), DOI : 10.5530/pj.2020.12.205 and Thymol (0.118%). Pub-chem database search also supports that these compounds have Article Available online very strong bactericidal activity by membrane damage as evidenced by LDH and SEM studies. http://www.phcogj.com/v12/i6s Conclusions: MP-DCMf contains many potential antibacterial compounds that can be used to Copyright combat the gastrointestinal and food poising bacterial pathogens. © 2020 Phcogj.Com. This is an open- Key words: Anti-enterobacterial, Chemical profiling, Mandukaparni, Time kill assay, Volatile access article distributed under the terms oils. of the Creative Commons Attribution 4.0 International license. INTRODUCTION in Himalaya and southern part of India known as ‘Golpatta’ in Nepali, and ‘Mahagotukola in Hindi.4 Hydrocotyle javanica Thunb. (H. javanica) and The leaves of the plant are being used to treat Centella asiatica (L.) Urban. (C. asiatica) which several diseases like gastritis, throat shore, eye and are popularly known as Java pennywort and ear infection by various ethnic communities like Indian pennywort respectively are commonly Bhutia, Damai, Kami, Lepcha, Limbu, Rai, Sherpa known as ‘Mandukaparni’ in Ayurveda for and Tamang of Darjeeling Hills (27°3'15.88'' N and resemblance in their leaf shape and used for the 88°15'28.10'' E, elevation 2034 m) of West Bengal, ailments of indigestion, dysentery, nervousness India.5 It is also used to treat jaundice. Different and blood impurity as mutual substitute in herbal biomedical aspects of the species like antibacterial 1 preparations. C. asiatica (known as Gotu kola activity and physicochemical parameters of methanol in Hindi) belonging to the Apiaceae family is an extract had been reported.6 In Kaushikasutra, important medicinal plant of the Ayurveda for Mandukaparni is described as an Tikta (Bitter in improving the brain function and delaying ageing taste) Aushadhi (Medicinal herb). Other ancient process since thousands of years back and listed Ayurvedic texts described the medicinal properties in the historic ‘Sushruta Samhita’, an ancient of the herb as follows: It has a Kasaya (Astringent) Indian medical text.2 Fresh Gotu kola juice is after taste and a cooling effect. The Guna (Main taken along with cow’s milk in cough and asthma quality) of the herb is Laghu (Light to digest); and and for rejuvenation, decoction is taken in fever its Virya (Potency) is Sheeta (Cold). Its Prabhava and dysentery and leaf is used as a vegetable also.3 (Action) is Nootropic and neuroprotective. The The Hydrocotyle genus has almost 100 species Dhatu (Tissue) effect is through Rasa (Fluid), Rakta that grow in tropical and temperate regions (Blood) and Mamsa (Muscle).7 Gastro-intestinal worldwide and belongs to the family Araliaceae, ailments like diarrhea and typhoid are one of previouslyApiaceae. H. javanica grows abundantly the major public health problems in developing Cite this article: Mandal M, Misra D, Ghosh NN, Mandal S, Mandal V. GC-MS Analysis of Anti- Enterobacterial Dichloromethane Fraction of Mandukaparni (Hydrocotyle javanica Thunb.) – A Phcogj.com plant from Ayurveda. Pharmacogn J. 2020;12(6)Suppl:1494-503. 1494 Pharmacognosy Journal, Vol 12, Issue 6(Suppl), Nov-Dec, 2020 Mandal, et al.: GC-MS Analysis of Anti-Enterobacterial Dichloromethane Fraction of Mandukaparni (Hydrocotyle javanica Thunb.) – A plant from Ayurveda and developed countries. WHO estimates the diarrhea and typhoid and cell morphology fever diseases among infants and young children caused by various enteric pathogens, responsible for approximately 2.2 million and The effect of MP-DCMf on cellular integrity was evaluated by lactate 0.6 million deaths each year. Some important pathogenic bacteria dehydrogenase (LDH) assay. Sonicated (100% amplitude, at 0.9 cycles viz. Escherichia coli, Salmonella spp., Shigella sp., Vibrio cholera and for 5 min) [Probe Sonicator, PKS-250F, PCI Analytics, USA] test strain V. parahaemolyticus are responsible for diarrhea causing more than was taken as the positive control. Cell-free supernatant was taken at two million deaths every year.8 In Indian hospitals, one-third of the ½ h, 1 h, 2 h, 8h and 16 h of treatment time and the amount of LDH total pediatric patients admitted are diarrheal and 17% mortality released in the cell free supernatant was measured using standard in indoor pediatric patients are diarrhea related.9 To combat such procedure. One unit of LDH was calculated as 1 μmol of nicotinamide fatal disease many potent antibiotics have been used for the last few adenine dinucleotide reduction (NADH) per minute. The effect of MP- decades like Rifaximin, Metronidazole, Ciprofloxacin but the recurrent DCMf on the cell morphology was confirmed by SEM after treatment and rampant use of certain drugs lead to development of resistance at 2 × MIC dose on L. monocytogenes MTCC 657 following the protocol 13 among these pathogens. Antibiotics also have been associated with of Mandal et al. adverse effects which include hypersensitivity, allergic reactions and GC-MS analysis immune suppression.10 Many pathogens like S. typhi show multidrug resistance (MDR) against antibiotics.11 In these circumstances, plants The active compounds present in the MP-DCMf was analysed by using have the major advantages for the cheapest, less side effect and effective the GC-MS instrument (Model 7890B GC-240 ION TRAP MS, Agilent alternative sources of drugs. Therefore, the pharmaceutical sector is in Technologies, USA) equipped with a capillary column, VF-5MS a need to develop potent alternative anti-enterobacterial compounds (Length-30m, ID-0.25 mm, Film-0.25 µm, Max temp-325 °C) with the to treat these drug resistant pathogens. Most of the cases, to develop mass detection range of m/z 10-1000.The instrument was operated in such potential drug, the scaffold of the molecules of natural product is electron impact mode at an emission current of 25 µAmps, maximum used.12 The aim of the present study is to characterize the antibacterial ion time of 65000 µSeconds, target TIC of 20000 counts, with injector constituents of the bioactive DCM fraction of Mandukaparni and to temperature at 250 °C, and detected at 300 °C.The identification evaluate its mechanism of antibacterial action. of compounds was done by matching the mass spectral records of NIST library and the Mole % of the compounds was determined by MATERIALS AND METHODS the formula: Mole % = Ai/Ac × 100, where, Ai = Peak area count of individual compound and A = Cumulative peak area count. Plant material c Thin layer chromatographic analysis The Mandukaparni plant (H. javanica) was collected from Darjeeling hill areas of West Bengal, India. The voucher specimen (vide reference TLC analysis was done in Silica Gel 60 F254 precoated aluminium sheet no. DGC/SP-02) was identified and authenticated at Central National (Merck, Germany), and developed in n-hexane-ethyl acetate (3:2, v/v) Herbarium, Indian Botanic Garden, Shibpur, Howrah, India. solvent system as mobile phase and detected under UV254 nm. The Extraction and fractionation of bioactive components chemical
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