GC-MS Analysis of Anti-Enterobacterial Dichloromethane Fraction of Mandukaparni (Hydrocotyle javanica Thunb.) – A plant from Ayurveda

Hydrocotyle javanica Thunb. (H. javanica) and Centella asiatica (L.) Urban. (C. asiatica) which are popularly known as Java pennywort and Indian pennywort respectively are commonly known as ‘Mandukaparni’ in Ayurveda for resemblance in their leaf shape and used for the ailments of indigestion, dysentery, nervousness and blood impurity as mutual substitute in herbal preparations.1 C. asiatica (known as Gotu kola in Hindi) belonging to the Apiaceae family is an important medicinal plant of the Ayurveda for improving the brain function and delaying ageing process since thousands of years back and listed in the historic ‘Sushruta Samhita’, an ancient Indian medical text.2 Fresh Gotu kola juice is taken along with cow’s milk in cough and asthma and for rejuvenation, decoction is taken in fever and dysentery and leaf is used as a vegetable also.3 The Hydrocotyle genus has almost 100 species that grow in tropical and temperate regions worldwide and belongs to the family Araliaceae, previouslyApiaceae. H. javanica grows abundantly in Himalaya and southern part of India known as ‘Golpatta’ in Nepali, and ‘Mahagotukola in Hindi.4 The leaves of the plant are being used to treat several diseases like gastritis, throat shore, eye and ear infection by various ethnic communities like Bhutia, Damai, Kami, Lepcha, Limbu, Rai, Sherpa and Tamang of Darjeeling Hills (27°3'15.88'' N and 88°15'28.10'' E, elevation 2034 m) of West Bengal, India.5 It is also used to treat jaundice. Different biomedical aspects of the species like antibacterial activity and physicochemical parameters of methanol extract had been reported.6 In Kaushikasutra, Mandukaparni is described as an Tikta (Bitter in taste) Aushadhi (Medicinal herb). Other ancient Ayurvedic texts described the medicinal properties of the herb as follows: It has a Kasaya (Astringent) after taste and a cooling effect. The Guna (Main quality) of the herb is Laghu (Light to digest); and its Virya (Potency) is Sheeta (Cold). Its Prabhava (Action) is Nootropic and neuroprotective. The Dhatu (Tissue) effect is through Rasa (Fluid), Rakta (Blood) and Mamsa (Muscle).7 Gastro-intestinal ailments like diarrhea and typhoid are one of the major public health problems in developing 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 remained unknown. Objective: The main objective of the study was to characterize antienterobacterial dichloromethane fraction of the volatile oils of it by GC-MS. Materials and Methods: In the present study, dichloromethane (DCM) fraction (MP-DCMf) of Mandukaparni 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 well diffusion assay, viability assay and LDH assay and SEM studies. Characterization of the active MP-DCMf fraction was performed by TLC and GC-MS analysis. Results: The MP-DCMf possessed bio-active compounds that have antibacterial potentiality against both the Grampositive and Gram-negative bacteria. The MIC and MBC values were in the range from 1.56 mg/ml to 0.78 mg/ml and 6.25 to 1.56 mg/ml, respectively. The time kill assay showed that at a dose of 3.12 mg/ml of MP-DCMf was lethal to the E. coli MTCC 723 at the 18th hr of treatment. LDH release had moderate positive correlation with the activity index and time of treatment whereas strong negative correlation with CFU count. It caused highest cell disruption in S. mutans. The principal compounds were D-carvon (30.949%);1H-Isoindole-1,3(2H)-dione; 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-dimethylacetate (6.684%); p-Cresol (0.551%), and Thymol (0.118%). Pub-chem database search also supports that these compounds have very strong bactericidal activity by membrane damage as evidenced by LDH and SEM studies. Conclusions: MP-DCMf contains many potential antibacterial compounds that can be used to combat the gastrointestinal and food poising bacterial pathogens.


Hydrocotyle javanica Thunb. (H. javanica) and
Centella asiatica (L.) Urban. (C. asiatica) which are popularly known as Java pennywort and Indian pennywort respectively are commonly known as 'Mandukaparni' in Ayurveda for resemblance in their leaf shape and used for the ailments of indigestion, dysentery, nervousness and blood impurity as mutual substitute in herbal preparations. 1 C. asiatica (known as Gotu kola in Hindi) belonging to the Apiaceae family is an important medicinal plant of the Ayurveda for improving the brain function and delaying ageing process since thousands of years back and listed in the historic 'Sushruta Samhita', an ancient Indian medical text. 2 Fresh Gotu kola juice is taken along with cow's milk in cough and asthma and for rejuvenation, decoction is taken in fever and dysentery and leaf is used as a vegetable also. 3 The Hydrocotyle genus has almost 100 species that grow in tropical and temperate regions worldwide and belongs to the family Araliaceae, previouslyApiaceae. H. javanica grows abundantly in Himalaya and southern part of India known as 'Golpatta' in Nepali, and 'Mahagotukola in Hindi. 4 The leaves of the plant are being used to treat several diseases like gastritis, throat shore, eye and ear infection by various ethnic communities like Bhutia, Damai, Kami, Lepcha, Limbu, Rai, Sherpa and Tamang of Darjeeling Hills (27°3'15.88'' N and 88°15'28.10'' E, elevation 2034 m) of West Bengal, India. 5 It is also used to treat jaundice. Different biomedical aspects of the species like antibacterial activity and physicochemical parameters of methanol extract had been reported. 6 In Kaushikasutra, Mandukaparni is described as an Tikta (Bitter in taste) Aushadhi (Medicinal herb). Other ancient Ayurvedic texts described the medicinal properties of the herb as follows: It has a Kasaya (Astringent) after taste and a cooling effect. The Guna (Main quality) of the herb is Laghu (Light to digest); and its Virya (Potency) is Sheeta (Cold). Its Prabhava (Action) is Nootropic and neuroprotective. The Dhatu (Tissue) effect is through Rasa (Fluid), Rakta (Blood) and Mamsa (Muscle). 7 Gastro-intestinal ailments like diarrhea and typhoid are one of the major public health problems in developing and developed countries. WHO estimates the diarrhea and typhoid fever diseases among infants and young children caused by various enteric pathogens, responsible for approximately 2.2 million and 0.6 million deaths each year. Some important pathogenic bacteria viz. Escherichia coli, Salmonella spp., Shigella sp., Vibrio cholera and V. parahaemolyticus are responsible for diarrhea causing more than two million deaths every year. 8 In Indian hospitals, one-third of the total pediatric patients admitted are diarrheal and 17% mortality in indoor pediatric patients are diarrhea related. 9 To combat such fatal disease many potent antibiotics have been used for the last few decades like Rifaximin, Metronidazole, Ciprofloxacin but the recurrent and rampant use of certain drugs lead to development of resistance among these pathogens. Antibiotics also have been associated with adverse effects which include hypersensitivity, allergic reactions and immune suppression. 10 Many pathogens like S. typhi show multidrug resistance (MDR) against antibiotics. 11 In these circumstances, plants have the major advantages for the cheapest, less side effect and effective alternative sources of drugs. Therefore, the pharmaceutical sector is in a need to develop potent alternative anti-enterobacterial compounds to treat these drug resistant pathogens. Most of the cases, to develop such potential drug, the scaffold of the molecules of natural product is used. 12 The aim of the present study is to characterize the antibacterial constituents of the bioactive DCM fraction of Mandukaparni and to evaluate its mechanism of antibacterial action.

Plant material
The Mandukaparni plant (H. javanica) was collected from Darjeeling hill areas of West Bengal, India. The voucher specimen (vide reference no. DGC/SP-02) was identified and authenticated at Central National Herbarium, Indian Botanic Garden, Shibpur, Howrah, India.

Extraction and fractionation of bioactive components
The bioactive components of the Mandukaparni powder were extracted by Soxhlet extractor using 80% aqueous methanol solvent and concentrated by rotary vacuum evaporator following the methodology of Mandal et al. 13 The MP-DCMf fraction from the methanol extract was recovered with 1/4 th volume of DCM using orbital shaker for 48 hours. The recovered fraction was evaporated to dryness and used for further study.

Anti-bacterial activity assay
The gastrointestinal pathogenic bacterial strains used in this study were procured from MTCC, Chandigarh, India, and MCC, Pune, India and were cultured in respective culture conditions. Antibacterial activity of the MP-DCMf (25 mg/ml) was evaluated by the agar wells diffusion method. 14 Ciprofloxacin (50 μg/ml) and DCM (100%) were used as positive and negative control, respectively. The potentiality was assessed by activity index by calculating the ratio of diameter of zone inhibition of the extract and diameter of inhibition zone of antibiotic.

Determination of MIC and MBC and time kill assay
MIC was determined using 1.3% (w/v) Mueller Hinton agar (MHA) medium with the conc. range of MP-DCMf (25 mg/ml to 0.75 mg/ ml). 13 MBC and bactericidal kinetic assay were done by applying greater than the MIC dose on the actively grown bacterial strain (log phase growth) of B. cereus MTCC 1272, E. coli MTCC 723, E. faecalis MCC 2041T and S. mutans MTCC 497 and the viability was evaluated by plate count method after incubating at 37°C for 96 hrs. 14 Time kill measurement was done at ½, 1, 2, 4, 8, 16 and 20hrs. 15 The culture without treatment was considered as negative control.

Study on mode of antibacterial action: cellular integrity and cell morphology
The effect of MP-DCMf on cellular integrity was evaluated by lactate dehydrogenase (LDH) assay. Sonicated (100% amplitude, at 0.9 cycles for 5 min) [Probe Sonicator, PKS-250F, PCI Analytics, USA] test strain was taken as the positive control. Cell-free supernatant was taken at ½ h, 1 h, 2 h, 8h and 16 h of treatment time and the amount of LDH released in the cell free supernatant was measured using standard procedure. One unit of LDH was calculated as 1 μmol of nicotinamide adenine dinucleotide reduction (NADH) per minute. The effect of MP-DCMf on the cell morphology was confirmed by SEM after treatment at 2 × MIC dose on L. monocytogenes MTCC 657 following the protocol of Mandal et al. 13

Statistical analysis
Antimicrobial activities were calculated as mean ± SE. The correlation between LDH release and activity index, LDH release and time of treatment, and LDH release and Log CFU count were analyzed by Spearman's 2-tailed bi-variate method and linear regression model using MS -Excel 2007.  Table 1.

MIC, MBC and bactericidal kinetic of the MP-DCMf
The MICs were in the range from 3.12 mg/ml to 0.78 mg/ml. The MIC value for B. cereus, E. coli, S. typhimurium was 0.78 mg/ml while for B. subtilis, L. monocytogenes, S. mutans, was 1.56 mg/ml, for the E. faecalis, S. aureus and V. parahaemolyticus it was 3.12 mg/ml. The MBC values were in the range from 6.25 to 1.56 mg/ml. MBC value for B. cereus, E. coli, S. typhimurium was 1.56 mg/ml while for B. subtilis, L. monocytogenes, S. mutans it was 3.12 mg/ml and for E. faecalis, S. aureus and V. parahaemolyticus it was 6.25 mg/ml.The time kill assay showed that at a dose of 3.12 mg/ml, the MP-DCMf was lethal to the E. coli MTCC 723 at 18 th hrs of treatment (Figure 1) which indicated that the active fraction had a potential bactericidal effect on the treated bacterial strain.

LDH activity and its correlation
The LDH activity of MP-DCMf on the test strains was shown in Figure  2. A gradual increase in the LDH activity was recorded after 3 hrs and reaching the highest after 5 hrs of treatment. This indicated the MP -DCMf causes damage to the bacterial cell membrane. The statistical analysis revealed that LDH release had moderate positive correlation with the activity index and time of treatment whereas strong negative correlation with CFU count.The regression analysis also strongly supported the correlation results as represented in Figure 3.

Effect on the cell morphology
MP-DCMf caused cellular damage as confirmed by SEM photomicrographs (Figure 4A-D). It showed changes in the morphology and shape of the bacterial cells, like the formation of notch (4B), blebbing (4C) and decay of the cell walls (4D) which is comparable to the untreated bacteria (4A).

TLC analysis
The comparative TLC analysis of the MP-DCMf showed the presence of alkaloids, flavonoids, and phenols in the active fraction as shown in Figure 5.The presence of alkaloid in the fraction was confirmed as orange spots after spraying with the Dragendoff 's reagent which had R f value of 0.55. In flavonoids test (Fig. 5B) quercetin (spot 1), MP-DCMf (spot 2) and MP-DCMf + quercetin (spot 3) showed the same R f values i.e. 0.254 (Fig. 5B, spot, whereas in phenol (Fig. 5A)  Here, DCM solvent shows negative inhibition zone against all the enteric pathogens.

DISCUSSION
MP-DCMf has potential broad spectrum antibacterial activity at a dose of 25 mg/ml against Gram-negative and Gram-positive bacteria with the average diameter of zone inhibition of 17.00 mm and 15.00 mm, respectively. The minimum and maximum MBC values (6.25 mg/ml and 1.56 mg/ml, respectively) were significantly lower than the MBC value of other species of Hydrocotyle. 16 Bactericidal activity of the MP-DCMf was the highest against the enteric pathogen E. coli, S. enterica serovar. typhimurium and another highly entero-pathogenic strain, E. faecalis. B. cereus, B. subtilis, L. monocytogenes, and S. mutans were also very susceptible to the active fraction at a lethal dose of 3.12mg/ml. The most of the bacterial strains showed their growth inhibition within the minimum concentration of 1.56mg/ml. Hence, the MP-DCMf fraction contains some potential bactericidal compounds that can be used against these pathogenic bacteria. The MP-DCMf also exhibited significant bactericidal activity against the dental carries pathogen, S. mutans. The antibacterial efficacy, MIC and MBC of the MP-DCMf was greater than the methanol extract 13 as shown in Table 3. LDH activity results indicated that the MP-DCMf causes extracellular leakage due to bacterial cell membrane damage. Time kill assay has the implication in the bactericidal kinetics of the antibacterial agent against specific bacterial strain. It demonstrates the degree of lethality of the particular antibacterial agent. The comparative TLC analysis shows the presence of some potent antibacterial alkaloids, phenols and flavonoids in the MP-DCMf ( Figure 5).
GC-MS analysis of the MP-DCMf showed eight alkaloids, six phenolics, one flavonoid and one glycoside compounds present at a proportion of 30.126%, 3.579%, 0.055% and 0.075% of total moles, respectively as shown in the Table 2. A GC-MS study of Hydrocotyle bonariensis also reported the presence of monoterpenes (53.6% mol) and sesquiterpenes (10.5% mol) as the major phytochemical classes. 17 The monoterpenes α-pinene, β-pinene, and limonene represent the main components of the oil. These indicate the genus Hydrocotyle possesses the biosynthetic machinary of novel antimicrobials. For large scale isolation, purification and pharmaceutical application this plant may provide a valuable tool. Further in-vitro propagation and metabolic engineering could provide life supporting drugs for industrial uses.
In the MP-DCMf p-Cresol was detected as the major phenolic compound which possesses significant antibacterial potentiality and was also available in the methanol extract of the same plant. 18 It might also increase the plasma membrane permeability that results in higher leakage of fluid material from bacterial cells and inhibit microbial respiration. 19 Iso-indole alkaloids possess good activity against the Gram-negative bacteria (E. coli, K. pneumonia and P. aeruginosa,) and yeasts (C. albicans, S. cerevisiae) with MIC ranging from 50-200µg/ ml, while it was less susceptible against the Gram-positive bacteria (B. megaterium, B. subtilis and S. aureus). Iso-indole alkaloids act as nucleic acid synthesis inhibitor. 20 Terpenoid shows antibacterial activity against E. coli, E. faecalis, K. pneumonia, P. aeruginosa and S.     aureus. 21 Thymol was detected as the major terpenoid compound in the tested sample. Thymol, a phenolic monoterpenoid having hydroxyl groups at the different positions of its phenolic ring disrupts the cell membranes by interaction with the membrane proteins of the bacterial cell. 22 This loss of cellular integrity and decay of the cell walls due to active compounds present in the DCM fraction was visualized by the SEM study. Statistical analyses validated the antibacterial phenomena indicating the active compounds had a very strong bactericidal activity. The activity index and LDH correlation analysis proved that the bacterial zone inhibition by the active fraction is positively correlated with LDH release through the cell membrane of the bacterial cell. The Dosha Karma (Effect on humor) of Mandukaparni is that it overcomes Pitta dosha and Kapha dosha. 23 Our present study specified its effect on Pitta dosha by combating the gastrointestinal infections and related diseases of the hepato-biliary system.

CONCLUSION
The study suggested that the MP-DCMf contained some potent antibacterial compounds belonging to the class alkaloids, monoterpenoid and phenol. The antibacterial efficacy of these three types of secondary metabolites among which the alkaloids have specific for one or a limited number of molecular targets whereas phenols are multi-target agents modulating the activity of proteins, nucleic acids and bio-membranes in a less specific way. Thus, it can be concluded that the Mandukaparni (H. javanica) which is used in ayurvedic preparations have the potential to treat against bacterial pathogens of gastrointestinal infections, food borne and topical diseases (including dental caries pathogens).This is the first scientific report on the antibacterial potentiality of DCM fraction of the high altitude folklore medicinal herb, H. javanica. So, further isolation, purification and structure elucidation of the active compounds, their acute as well as cytotoxicity tests, QSAR guided chemical study for drug development, and vivid mode of action might explore the better application of this novel repository herb for antienteric drug development.