International Journal of Drug Development and Research

Key words

Eclipta alba, aquous extract, antimicrobial

Introduction

The traditional use of plant derived medicinal compounds received much attention against the multifactorial antibiotic resistance as they are well tested for their efficacy and being used to treat various microbial diseases[13].Medicinal plants have been conventionally used as antimicrobial and antiinflammatory agents. Eclipta alba commonly known as False daisy, yerba de tago, and bhringraj, is a plant belonging to the family Asteraceae. The leaf extract is considered as a powerful liver tonic, rejuvenative and especially good for the hair. It is used as anti-venom memory disorders treatment, general tonic, edema, fevers and rheumatic joint pain treatment [7, 13].

The shoot extract of E. alba showed antimicrobial [3,15], antifungal activity [6] and weak cytotoxicity against the M-109 cell lines by alkaloids Verazine [1], antiviral activity against Ranikhet disease virus [9], effective against internal and external parasites. The herb has been used in the treatment of infective hepatitis in India [14] and snake venom poisoning in Brazil [10]. It has been reported that the leaves of this herb are used in the case of gastritis and respiratory disorders like cough and asthma [8], digestion, hepatitis, enlarged Plant is bitter, hot, sharp, dry in taste and is used in ayurveda & "siddha" for the treatment of Kapha and Vata imbalances. The herb Eclipta alba contains mainly coumestans i.e. wedelolactone (I) and demethylwedelolactone (II) polypeptides, polyacetylenes, thiophene-derivatives, steroids, triterpenes and flavonoids. Wedelolactone possesses a wide range of biological activities and is used for the treatment of hepatitis and cirrhosis[13,14], as an antibacterial, anti-hemorrhagic And for direct inhibition of IKK complex resulting in suppression of LPS-induced caspase-11 expression [8] .The antimicrobial assays were performed by MIC determination and diffusion disc method [2]. The antimicrobial activity of essential oils against Escherichia coli, Salmonella enteritidis, and Salmonella typhimurium was conducted by Methanol, ethyl acetate, and hexane extracts of Bridelia ferruginea leaves exhibit good antimicrobial activity against various pathogenic bacteria [12].

Material and Methods

Sample Collection

The plant samples were collected from North India region mainly from Duhai village locality, surrounding the campus of Institute of Applied Medicines and Research, Duhai, Ghaziabad, U.P. Fresh plant leaves were washed separately under running tap water as well as distilled water and shade drying of leaves was carried out at room temperature and then, homogenized to very fine powder and stored at 40C till the extraction was carried out [12].

Preparation of Aqueous Extract

20 grams of shade dried plant leaves powdered, was added to 100 ml autoclaved distilled water in 250 ml conical flasks and kept at room temp on a rotators shaker at 180 rpm for three days. The extracts were filtered through muslin cloth and then centrifuged at 10,000 rpm for 10 minutes .The supernatants obtained were filter sterilized The filtrates were made conc. And dried by vacuum drier and responded in sterile distilled water to make a conc. Of 400mg /ml, and stored at 4 0 C for further use.

Microorganism Used

The antimicrobial activity was carried out by using the organis E. coli, E. cloacae, E. faecali, P. vulgaris, S. aureus, K. pneumoniae, S. saprophyticus Were obtained from The Department of microbiology Institute of Applied Medicines and Research, Ghaziabad U.P. India. The microorganisms were maintained by sub-culturing on nutrient agar medium and used at regular intervals to carry out the antimicrobial activity. A 24 h old culture of each indicator strain with the O.D590 =0.650 was used for the preparation of bacterial suspension.

Assay Methods

The antimicrobial activity was evaluated by disc diffusion method. The media used for maintaining, culturing of microorganism and to carry out the antimicrobial susceptibility testing was prepared as - Dissolved 5 gm of peptone, 3 gm of beef extract, 5 gm of NaCl and 20 gm of agar in 0.5 L of distilled water. pH was adjusted to about 6.8-7.2 and final volume was adjusted to 1 L and autoclaved at 121°C and 15 Lbs for 15 minutes. The antimicrobial activity of leaves extract was evaluated by growing the broth of each tested microorganism till the late log phase developed by taking the absorbance (O.D.at 590nm=0.650 Water was taken as negative control and Ampicillin, Cephotoxime, Streptomycin and Tetracycline were taken as positive control. Using a sterile glass spreader, the nutrient broth cultures were spreaded on the surface of sterile nutrient agar plates.

Disc Diffusion Method

Briefly, nutrient broth / or agar was used to culture bacteria. Fresh overnight cultures of inoculum (0.1 ml) of each culture, was spread on agar plate. The plates were kept for 10 min and then the prepared sterilized discs (5 mm diameter) soaked with the concentrated extract were impregnated over the surface of the plate inoculated with the microorganisms and allowed to incubate at 37 0C for the 24 h. The zone of inhibition in mm was determined after incubation period. The microbes were plated in triplicates and average zone diameter was noted [5].

Determination of Minimum Inhibitory Concentration (MIC)

Some idea of the effectiveness of a chemotherapeutic agent against a pathogen can be obtained from the minimal inhibitory concentration. The MIC is the lowest concentration of a drug that prevents growth of a particular pathogen. For determination of MIC, Dilution susceptibility test was applied which was carried out by using the extract as 100%, (400mg/ml) 75% (300mg per ml) and 50% (200mg per ml) and 20micro L of each extract were applied on dics. The lowest concentration of plant extract resulting in no inhibition after required incubation is determined as the MIC.

Result and Discussion

The antimicrobial susceptibility was carried out by disc diffusion method. At the end of incubation period, the diameter of Zone of inhibition was measured with a glass ruler. The aqueous extract of leaf showed reliable Zone of Inhibition against E. cloacae and K. pneumonia while E. coli, E. faecalis, P. vulgaris, S. aureus and S. saprophyticus resistant to extract. The antimicrobial activity of aqueous leaf extract was also performed at different concentration (100%, 75% and 50%) indicated that activity of extract was decreased as conc was decreased and found effective up to the 50% against both the sensitive strain.

The aqueous extract of stem showed reliable Zone of Inhibition against E.cloacae E.faecalis , K. pneumoniae and S. saprophyticus while resistant against all other microbes. The aqueous extract of flower showed ZOI against P.vulgaris ,S.aureus and S.saprophyticus while resistant against all other microbes.

The antibacterial activity of antibiotics of cephotoxime the maximum ZOI was observed against E. cloacae minimum ZOI was observed against S .aureus .In case of chloramphenicol the maximum ZOI was observed against P.vulgaris and minimum against E.faecalis. In case of Tetracycline the maximum ZOI was observed against S. aureus and minimum against P.vulgaris. while the Streptomycin the maximum ZOI was observed against E.coli and minimum ZOI against E. faecalis..

Conclusion

From the results of present work. It had been concluded that leaves and stem aqueous extract of plant did not show significant antimicrobial activity against isolated tested microbes except E.clocae and K. Pneumoniae the antimicrobial susceptibility testing carried out by using standard drugs which were shown that cephatoxime has maximum antimicrobial effect against all tested organism except S. saprophyticus which was found resistant. Chloramphenicol and Tetracycline were shown moderate inhibitory effect while streptomycin shown only moderate zone of inhibition against E.coli and E. faecalis and other strains were found resistant.

Conflict of Interest

NIL

Source of Support

NONE

ACKNOWLEDGMENT

Authors are thankful to the management of Lovely Professional University for providing financial support and Institute of Applied Medicines and Research , Ghaziabad and CCRUM Patna for procurement of herbs.

Tables at a glance

Table 1 Table 2 Table 3 Table 4

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4

References

1. Abdal, K.M.S., Malone, B.D.B., Werkhoven, S., Van, M.C., David, T.F., Wisse, J.H., Bursuker, I., Neddermann, K.M., Mamber, S.W., and Kingston, D.G. DNA damaging steroidal alkaloids from Eclipta alba from the Surinam rainforest. J Natl Prod. 61(10), (1998), 1202-1208.
2. Ali-Shtayeh, M.S.; Yaghmour, M-R.; Faidi, Y.R.; Salem, K.; Al-Nurys, M.A Antimicrobial activity of 20 plants used in folkloric medicine in the Palestinian area. J. Ethnopharmacol., v. 60, p. 265- 270, (1998).
3. Anonymous (1952). The Wealth of India - Raw Materials, Council of Scientific and Industrial Research, New Delhi, vol. III.pp.127.
4. Cremer A., Microbiological methods, Butterworth and Co., London, 6th Ed, (1991),235.
5. Elizabeth, K.M. Antimicrobial activity of Allium sativum on some pathogenic bacteria. Indian J. Microbiol. 41, (2001), 321-323.
6. Hoffman BR, Delas Atlas H, Blanco K, Wiederhold N, Lewis RE, Williams L (2004). Screening of antibacterial and antifungal activities of ten medicinal plants from Ghana. J. Pharm. Biol. 1(42), (2004), 13-17.
7. Karthikumar, S., Vigneswari, K. and Jegatheesan, K. Screening of antibacterial and antioxidant activities of leaves of Eclipta prostrate Scientific Research and Essay Vol. 2 (4), pp. 101-104, April (2007)
8. Kobari M, Yang Z, Gong D, Heissmeyer V, Zhu H, Jung YK, Angelica M,Gakidis M, Rao A, Sekine T, Ikegami F, Yuan C, Yuan J. Wedelolactone suppress LPS- induced caspase-11 expression by directly inhibiting the IKK complex. J. Cell Death and Differentiation 11(1), (2004), 123-130.
9. Lans C, Harper T, Georges K, Bridgewater E. Medicinal and ethnoveterinary remedies of hunters in Trinidad. BMC Compl (2001).
10. Melo PA, Do Nascimento MC, Mors WB, Suarez- Kurtz G. Inhibition of the myotoxic and hemorrhagic activities of crotalid venoms by Ecliptaprostrata (Asteraceae) extracts and constituents. Toxicon. 32, (1994), 595-603.
11. Mi-HyangLee,HyunAe Kwon, Dong-Yeul Kwon , Hyun Park, Dong-Hwan Sohn , Youn-Chul Kim , Seong-KugEo , Ho-Young Kang , Sam-Woong Kim , John Hwa Lee Antibacterial activity of medicinal herb extracts against Salmonella. International Journal of Food Microbiology 111, (2006), 270 – 275.
12. Parekh J. and Chanda S. In vitro screening of antimicrobial activity of aqueous and alcoholic extracts of various Indian plant species against selected pathogens from Enterobacteriaceae., African Journal of Microbial Resarch, 1,(6),(2007), 92-99.
13. Thorat R.M,Jadhav,V.M.,GaikwadD.D , Jadhav S. L. Phytochemiacal and pharmacological potentials of Eclipta alba. Internaltional journal of pharmacy (1), (2010), 77-80.
14. Wagner, H., Geyer, B., Kiso, Y. and Rao, G.S. Coumestans as the Main Active Principles of the Liver Drugs Eclipta alba and Wedeliacalendulacea. PlantaMedica, 52, (1986), 370-373.
15. Wiart C, Mogana S, Khalifah S, Mahan M, Ismail S, Buckle M, Narayana AK, Sulaiman M. Antimicrobial screening of plants used for traditional medicine in the state of Perak, Peninsular Malaysia. Fitoterapia 75, (2004), 68-73.