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Journal of Herbal Medicine and Toxicology 2 (2) 65-66 (2008) ISSN : 0973-4643 Original Article

ANTIMICROBIAL ACTIVITY OF THE LEAF EXTRACTS OF CALENDULA OFFICINALIS (LINN.)

CHAKRABORTHY, G. S.
SVKMS, NMIMS University, SPTM, Shirpur Campus, Babulde, Dist-Dhulia, Maharashtra- 425 405 (India) Corresponding author E-mail: phdgs77@indiatimes.com
Received : 12 May,2008; Revised : 17 June , 2008; Accepted : 22 June,2008

ABSTRACT : The present investigation was carried out to evaluate the antibacterial activities of Calendula officinalis Linn. Dried leaf powder of Calendula officinalis was successively extracted with petroleum ether, chloroform and ethanol using Soxhlet and macerated to form water extract. All extracts were screened for its antibacterial and antifungal activity using agar well diffusion method. The microorganisms used for antibacterial and antifungal were Bacillus subtulis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Candida albicans and Aspergillus niger. Gentamicin 5g/ml was used as standards. The extracts showed antimicrobial activity were subjected to minimum inhibitory concentration assay by two fold dilutions method. Petroleum ether, chloroform, ethanol and water extract exhibited in-vitro antibacterial activity. None of the extracts showed antifungal activity. Key words: Calendula officinalis (Linn.), Antibacterial activity.

INTRODUCTION
Alternative System of Medicine viz. Ayurveda, Siddha, and Traditional Chinese Medicine have become more popular in recent years. According to one estimate, more than 700 mono and poly-herbal preparations in the form of decoction, tincture, tablets and capsules from more than 100 plants are in clinical use. Calendula officinalis. Linn(Asteraceae), commonly known as Pot Marigold is an important Medicinal plant used in our Traditional System of Medicine to treat various diseases. Calendula is used in ayurveda for the treatment of fever and cancer [1]. It is used because of the broad area of biological activities like anti-inflammatory, anti-mutagenic, diuretic, antispasmodic activities. It is also used for in gastrointestinal, gynecological, eye diseases, skin injuries and in some cases of burns. The plant is rich in many pharmaceutical active ingredients like flavonoids, carotenoids, glycosides and sterols [2]. In present study antimicriboal activity of of different extracts of leaves of Calendula officinalis Linn was studied.

MATERIALS AND METHODS

Plant Material: The leaves of C.officinalis were collected from the local areas of Shirpur, Dist Dhulia, Maharashtra, India. It was authenticated from the proper source and a voucher specimen No: 01 was deposited in the department of Pharmacognosy, SPTM, NMIMS University, Shirpur, Maharashtra. Preparation of Extracts: Collected leaves were cleaned and shade-dried. The dried leaves were pulverized by a mechanical grinder and passed through a 20-mesh sieve. A powdered leaf (500 g) was successively extracted with petroleum ether, Chloroform and ethanol using a Soxhlet apparatus and water extracted by cold maceration. The extraction was carried out for 24 hrs at room temperature with mild shaking. The extracts were filtered and concentrated at 35 C, and the weight of each residue was recorded and percentage yield was calculated [3].

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Table 2: Minimum inhibitory Concentration of Calendula officinalis leaf extracts
Test Samples S.a. Ethanol Extract Chloroform Water Extract Petroleum Ether extract 13 14 12 16 Zone of Inhibition (mm)* Gram +ve/Gram ve B.s. 11 12 13 nt E.c. 12 13 12 nt K.p. 13 12 14 13

* Values are the means of three assays, nt- Not tested, S.a.- Staphylococcus aureus, B.s-. Bacillus subtulis, E.aEscherichia coli, K.p.- Klebsiella pneumonia.

Preliminary Phytochemical Screening: All the extracts were subjected to preliminary phytochemical qualitative screening for the presence or absence of various primary or secondary metabolites [4]. Screening for Antibacterial and Antifungal Activity: The antibacterial and antifungal activity was carried out by employing 24 h cultures of Bacillus subtulis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Candida albicans and Aspergillus niger. Activity of the above mentioned extracts was tested separately using Agar-Well diffusion method. The medium was sterilized by autoclaving at 120 C (15 lb/in2). About 30 ml of the medium (nutrient Agar Medium) with the respective strains of bacteria and fungi was transferred aseptically into each sterilized Petri plate. The Plates were left at room temperature for solidification. Each plate, a single well of 6 mm diameter was made using a sterile borer. The extracts were freshly reconstituted with suitable solvents (Dimethyl Sulphoxide) and tested at various concentrations. The samples and the control (0.1ml) were places in 6-mm diameter well. Antibacterial assay plates were incubated at 37 2C for 24 h, antifungal assay plates were incubated at 28 2C for 48 h. Standard disc (6 mm diameter) with Gentamicin (5g/ml) was used as a positive control for antibacterial activity, whereas Clotrimazole (5g/ml) was used as a positive control for antifungal activity. Each experiment was carried out in triplicates, and diameter of the zone of inhibition was measured. Observations and results are shown in Table 1. The extracts that showed antimicrobial activity were subjected to minimum inhibitory concentration (MIC) assay by serial two fold dilution method [5]. MIC was interpreted as the lowest concentration of the sample, which showed clear fluid without development of turbidity; observations and results are shown in Table 2.

RESULTS AND DISCUSSION

The antimicrobial assay showed that Chloroform, ethanol and water extracts of C.officinalis leaves exhibited in-vitro antibacterial activity against Grampositive and Gram-negative bacteria, whereas significant activity was not observed with petroleum ether extract. All the extracts did not show any activity with the antifungal organisms (Table 1). Minimum inhibitory concentration of the active extracts is shown in Table 2. The lowest MIC values were observed for ethanol extract, chloroform extract, water extract and petroleum ether extract against the bacteria. The results reveal that extracts of C.officinalis leaves were significantly effective against both Gram-positive and Gram-negative organism. Preliminary phytochemical screening of the extracts showed the presence of Alkaloids, carbohydrates, flavonoids, terpenoids, sterols and tannins. Thus further work can be carried on the isolation procedure for finding out the exact moiety responsible for the biological activity.

REFERENCES
[1]. Krag, K.: Plants used as contraceptives by the North American Indians: an ethnobotanical study, Botanical Museum, Cambridge, MA: Harvard University (1976) Pietta, P., Bruno, A.M.P., Rava, A.: J. Chromatogr, 593: 165-170 (1992) Kokate, C.K.: Practical Pharmacognosy, Vallabh Prakashan, New Delhi (2005) Harbone, J.B.: Phytochemical Methods-A Guide To Modern Techniques of Plant Analysis, Chapman and Hall London (1998) Florey, H.W., Chain, E. Florey, M.E.: The Antibiotic, Voli.I, Oxford University Press, New York (1989)

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