International Journal of Green Pharmacy  
Official publication of  TIFAC CORE in Green Pharmacy  
Home About us Instructions Submission Subscribe Advertise Contact e-Alerts Login    Print this page  Email this page Small font sizeDefault font sizeIncrease font size 
Users Online: 16 


 
ORIGINAL ARTICLE Table of Contents   
Year : 2008  |  Volume : 2  |  Issue : 3  |  Page : 176-181
In vitro evaluation of crude extracts of Catharanthus roseus for potential antibacterial activity


1 Department of Biotechnology, Microbiology Laboratory, Jaipuria Institute, Vasundhara, Ghaziabad, UP, India
2 Department of Science, Thomas Jefferson High School for Science and Technology, Alexandria, Virginia, USA
3 Department of Botany and Microbiology, Gurukul Kangri University, Hardwar, India

Click here for correspondence address and email

Date of Submission06-Mar-2008
Date of Acceptance10-Jun-2008
 

   Abstract 

Context: Catharanthus roseus (periwinkle) is an important medicinal plant, mentioned in Ayurveda, an ancient Indian Sanskrit literature. The plant is selected to evaluate the possibility for novel pharmaceuticals since most of the bacterial pathogens are developing resistance against currently available antibiotics.
Aims: To determine the antibacterial activity of crude extracts from different parts of Catharanthus roseus against several bacterial species of clinical significance.
Materials and Methods: Extraction of each plant part in appropriate solvent followed by evaluation of antibacterial activity by agar well diffusion assay against a total of six bacterial stains. Further, minimum inhibitory concentration(s) was evaluated for active crude extracts.
Results: Data indicated that the pattern of inhibition depends largely upon the extraction procedure, the plant part used for extraction, state of plant part (fresh or dry), solvent used for extraction and the microorganism tested. Dry powder extracts of all plant parts demonstrated more antibacterial activity than extracts prepared from fresh parts. Furthermore, extracts prepared from leaves were shown to have better efficacy than stem, root, and flower extracts. Organic extracts provided more potent antibacterial activity as compared to aqueous extracts. Among all the extracts, the ethanolic extract was found to be most active against almost all the bacterial species tested. Hot water and cold water extracts were completely inactive. Gram-positive bacteria were found more sensitive than Gram-negative bacteria.
Conclusions: The study promises an interesting future for designing potentially active antibacterial agents from Catharanthus roseus.

Keywords: Agar well diffusion assay, antibacterial activity, Ayurveda, Catharanthus roseus, minimum inhibitory concentration, periwinkle

How to cite this article:
Goyal P, Khanna A, Chauhan A, Chauhan G, Kaushik P. In vitro evaluation of crude extracts of Catharanthus roseus for potential antibacterial activity. Int J Green Pharm 2008;2:176-81

How to cite this URL:
Goyal P, Khanna A, Chauhan A, Chauhan G, Kaushik P. In vitro evaluation of crude extracts of Catharanthus roseus for potential antibacterial activity. Int J Green Pharm [serial online] 2008 [cited 2014 Nov 20];2:176-81. Available from: http://www.greenpharmacy.info/text.asp?2008/2/3/176/42739



   Introduction Top


Emerging and reemerging infections and spread of deadly, drug-resistant strains of organisms pose a challenge to the global public health for their treatment. Bacterial resistance to antibiotics is a major therapeutic problem and the pace at which new antibiotics are being produced is slowing. [1] Thus, the search for novel antimicrobial agents is of the utmost importance in the current world. [2] Global attention has been shifted towards finding new chemicals, specifically herbals, for the development of new drugs. These natural products can provide unique elements of molecular diversity and biological functionality, which is indispensable for novel drug discovery. [3]

Natural products or natural product-derived drugs comprise about 28% of all new chemical entities launched onto the market. [4] A large proportion of natural products in drug discovery has stemmed from the diverse molecular structures and the intricate carbon skeletons of natural products. [5]

Plants have proved to be significant natural resources for medicines; documentation of their use in medicine originates from ancient times. Ethnobotanical and ubiquitous plants provide a rich resource for natural drug research and development. [6] Medicinal plant-based drugs have the added advantage of being simple, effective and offering a broad spectrum of activity with greater emphasis on preventive action. [7] Indigenous systems of medicine and plant-based drugs could provide both concepts of therapy as well as therapeutic agents to complement modern medicine, especially in management of lifestyle and communicable diseases. Medicinal plant products could also prove useful in minimizing the adverse effects of various chemotherapeutic agents as well as in prolonging longevity and attaining positive general health. [8] The global interest in the medicinal potential of plants during the last few decades is therefore quite logical.

India is one of the richest countries in the world with regard to diversity of medicinal plants. [9] Catharanthus roseus L. (periwinkle) is an important member of the family Apocynaceae. This short-lived perennial with dark green and glossy leaves is native to Madagascar. This plant is frequently mentioned in Ayurveda (an ancient Indian literature) and has traditionally been used to treat diseases including cancer and diabetes. The plant has more than 70 types of alkaloids (mostly monoterpene indole alklaloids), and some are known to be effective in treating various types of cancers including breast and lung cancer, uterine cancer, melanomas, and Hodgkin's and non-Hodgkin's lymphoma. [10] The anticancer drugs vincristine and vinblastine are synthesized from alkaloids of Catharanthus roseus . The plant is also known for its antihypertensive and antispasmodic properties. Considering the medicinal value that this plant has already been shown to have, we evaluated the antibacterial potential in crude extracts of different parts ( viz., leaves, stem, root and flower) of this plant against clinically significant bacterial strains.


   Materials and Methods Top


Plant Material

Catharanthus roseus was collected in late July and early August of 2006 from semi-arid, unshaded land near the Jaipuria Institute in Vasundhara, Ghaziabad (NCR), India. The plant was taken to the laboratory and was authenticated by Prof. P. Kaushik. Plant materials (leaves, stem, root and flower) were washed separately first under running tap water, followed by sterilized distilled water.

Extract Preparation

Extraction was performed by two different modes: (1) Extraction of fresh plant material without drying and (2) Extraction after drying each plant part.

Hot Water Extraction

10 g of each plant part was boiled in 100 ml distilled water with constant stirring for 30 min. The solution was allowed to cool to room temperature and then filtered using muslin cloth. The filtrate was centrifuged at 5000 rpm for 15 min. The supernatant was again filtered using Whattman's Filter No. 1 under strict aseptic conditions. The filtrate was collected in fresh sterilized glass tubes and stored at 4C until use.

Cold Water Extraction

10 g of each plant part was macerated in pestle and mortar with 100 ml distilled water at room temperature and then filtered using muslin cloth. Filtrate obtained was again filtered using Whattman's Filter No. 1 under strict aseptic conditions and the filtrate was collected in fresh sterilized glass tubes and used within 24h for evaluation of antibacterial activity.

Organic Solvent Extraction

10 g of each plant part was thoroughly mixed with 100 ml organic solvent (ethanol and methanol). The mixture thus obtained was filtered through muslin cloth and then re-filtered by passing through Whattman's filter No. 1. The filtrate was then concentrated by complete evaporation of solvent at room temperature to yield the pure extract. Stock solutions of crude extracts were prepared by mixing well the appropriate amount of dried extracts with appropriate solvent to obtain a final concentration of 100 mg/ml. Each solution was stored at 4C after collecting in sterilized glass tubes until use.

Dry Powder Extractions

Crude dry powder extracts were prepared by first air-drying the plant material and then powdered using sterilized pestle and mortar under strict aseptic conditions. The powder was further subjected for aqueous and organic solvent extraction protocols as described above.

Bacterial Strains

A total of six bacterial strains including both Gram-negative and Gram-positive bacteria (  Escherichia More Details coli MTCC-739,  Salmonella More Details paratyphi MTCC-735, Klebsiella pneumoniae MTCC-39, Bacillus cereus MTCC-430, Bacillus subtilis MTCC-736, and Staphylococcus aureus MTCC-740) were selected to assess susceptibility patterns against the extracts prepared in the present study. All strains were collected from the Microbial Type Culture Collection (MTCC), India. The bacterial cultures were maintained in nutrient agar slants at 37C. Each of the microorganisms was reactivated prior to susceptibility testing by transferring them into a separate test tube containing nutrient broth and incubated overnight at 37C.

Antibacterial Susceptibility Assay

Extracts obtained by various processes were evaluated for their potential antibacterial activities by the standard agar well diffusion assay. [11] All extracts were sterilized by sterile membrane syringe filter (pore size 0.45 m, manufactured by Pall Life Sciences).  Petri dish More Detailses (100 mm) containing 18 ml of Mueller Hinton Agar (MHA) were seeded with approximately 100 l inoculum of bacterial strain (inoculum size was adjusted so as to deliver a final inoculum of approximately 10 8 CFU/ml). Media was allowed to solidify. Wells of 6 mm diameter were cut into solidified agar media using a sterilized cup-borer. 100 l of each extract was poured in the respective well and the plates were incubated at 37C overnight. The experiment was performed in triplicate under strict aseptic conditions to ensure consistency of all findings. The antibacterial activity of each extract was expressed in terms of the mean of diameter of zone of inhibition (in mm) produced by each extract at the end of incubation period.

Sterilized distilled water and other solvents used in preparation of extracts were used as negative control. Tetracycline was used as a standard antibiotic ( i . e . positive control) in the present study for a comparative analysis with the effectiveness of various plant extracts against selected microflora. The antibiotic (ALCYCLIN-500, manufactured by Alembic Limited) was procured from local chemist. On the basis of claim i . e . 500 mg/capsule; an appropriate amount of tetracycline powder was dissolved in sterilized distilled water to obtain a final concentration of 5 g/ml; this solution was used as a standard antibiotic throughout the study.

Assessment of Minimum Inhibitory Concentration

Active extracts obtained by agar well diffusion assay were further subjected to determine the minimum inhibitory concentration (MIC) required for the bacteriostatic effects by standard two-fold microdilution broth methodology. [12] A stock solution of each active extract was serially diluted in 96-wells microtiter plate with Mueller Hinton broth to obtain a concentration ranging from 8.0 g/ml to 4096 g/ml. A standardized inoculum for each bacterial strain was prepared so as to give an inoculum size of approximately 5 x 10 5 CFU/ml in each well. Microtiter plates were then kept at 37C for an overnight incubation. Following incubation, the MIC was calculated as the lowest concentration of the extract inhibiting the visible growth of bacterial strain using reflective viewer. All the chemical ingredients used in present study were of analytical grade and were purchased from Hi Media, India.


   Results Top


Results of the present investigation reveal the antibacterial nature of extracts of different parts ( viz., leaf, stem, root, and flower) of Catharanthus roseus . Each plant part was extracted in its dry powder form [Table 1] and fresh state [Table 2] using both organic and aqueous solvents. Data obtained demonstrates that the antibacterial activity of plant parts depends largely upon the extraction procedure, type of solvent used for extraction, and the bacterial strains tested for susceptibility assay. Data indicate that extracts prepared from dried parts exhibited better antibacterial activities than those extracts prepared from fresh plant part. Almost all parts of the plant showed antibacterial potential in dried form [Table 1] while only leaf and root extracts showed zone(s) of inhibition in their fresh states [Table 2]. The dry leaf extracts were found to have maximum inhibition, followed by root, stem and flower extracts. Leaf extracts showed zone(s) of inhibition ranging from 5.24 mm to 21.83 mm in diameter, while flower extracts were found to be virtually inactive in inhibition of microbial strains. Organic extracts were found to be more inhibitory than aqueous extracts. Ethanol was found to be a more suitable solvent for the maximum extraction of active metabolites than methanol. Gram-positive bacteria were found more susceptible as compared to Gram-negative species. Staphylococcus aureus was found most susceptible, as it was inhibited by almost all the organic extracts except that of fresh stem and fresh flower. Other Gram-positive bacteria ( Bacillus cereus and Bacillus subtilis ) were shown to have moderate-to-mild resistance, as they were not inhibited to such an extent. Ethanolic extracts were found to have variable inhibitory patterns against the Gram-negative bacteria with maximum inhibition of Klebsiella pneumoniae followed by Escherichia coli . Control experiments using sterile distilled water and solvents used for extraction (negative control) showed no inhibition of any bacteria.

Extracts found to have inhibitory effects were further tested for determination of minimum inhibitory concentration (MIC) by two-fold broth micro-dilution method against susceptible bacterial species [Table 3]. Data indicated that dry powder extracts showed more inhibition than fresh extracts of each plant part. Leaf extracts were found to have very low MIC values as compared with stem, root and flower extracts. Furthermore, ethanolic extracts showed consistently better antibacterial activity than methanolic extracts. The best MIC value (256 g/ml) for the dried leaf extract was seen against Staphylococcus aureus while it was 1024 g/ml for Klebsiella pneumoniae and 4096 g/ml for Salmonella paratyphi , though end-points were not reached in case of Escherichia coli and Bacillus subtilis . Fresh leaf extracts had MIC values more than 2048 g/ml against all species of bacteria. Staphylococcus aureus was inhibited at 512 g/ml by both dry stem and dry root extracts, but the MIC was much higher for this bacterium for fresh root or dry flower extracts. Gram-negative bacteria were found most resistant, as end points observed were much higher (ranging from 1024 g/ml to > 4096 g/ml) against almost all extracts.

All antibacterial activities were observed to be concentration-dependent. The efficacies of all extracts were less than that of the standard antibiotic, tetracycline.


   Discussion Top


Herbal medicines are a valuable and readily available resource for primary health care and complementary health care systems. Undoubtedly, the plant kingdom still holds many species of plants containing substances of medicinal value that have yet to be discovered; though large numbers of plants are constantly being screened for their antimicrobial effects. [13],[14],[15] These plants may prove to be a rich source of compounds with possible antimicrobial activities, but more pharmacological investigations are necessary.

The present study reveals the antibacterial potential of crude extracts of different parts of Catharanthus roseus. It is clearly demonstrated that extracts prepared from dried parts revealed better antibacterial activity than those extracts prepared from fresh plant part. Almost all parts of the plant showed antibacterial potential in dried form while only leaf and root extracts showed zone(s) of inhibition in their fresh states. Extracts from leaves of this plant demonstrated maximum antibacterial activity thus; it should be further studied to determine the active component(s). In a similar study, the leaf extracts of this plant was found to have significant antibacterial activity against Xanthomonas campestris. [16] Furthermore, Gram-positive bacteria were found to have more susceptibility as compared to Gram-negative bacterial species. This is probably due to the differences in chemical composition and structure of cell wall of both types of microorganisms. The present study also confirms the use of organic solvents in the preparation of plant extracts as compared to aqueous extracts. The polarity of antibacterial compounds make them more readily extracted by organic solvents, and using organic solvents does not negatively affect their bioactivity against bacterial species. Data also showed that some antimicrobial substances could only be extracted by organic solvents, suggesting that organic solvents are clearly better solvents of antimicrobial agents. [17]

The results also confirm the validity of the use of this plant as medicine in ancient medicinal traditions. The results of the present study supports the traditional usage of the studied plants and suggests that some of the plant extracts possess compounds with antimicrobial properties that can be used as antimicrobial agents in new drugs for the therapy of infectious diseases caused by pathogens. The most active extracts can be subjected to isolation of the therapeutic antimicrobials and carry out further pharmacological evaluation by several methods such as NMR, Mass Spectrometry, UC-MS, LC-MS etc.

 
   References Top

1.Russell AD. Antibiotic and biocide resistance in bacteria: Introduction. J Appl Microbiol Symp Suppl 2002;92:1S-3S.  Back to cited text no. 1    
2.Gootz TD. Discovery and development of new antimicrobial agents. Clin Microbiol Rev 1990;3:13-31.  Back to cited text no. 2    
3.Nisbet LJ, Moore M. Will natural products remain an important source of drug research for the future? Curr Opin Biotechnol 1997;8:708-12.  Back to cited text no. 3    
4.Newman DJ, Cragg GM, Snader KM. Natural products as sources of new drugs over the period 1981-2002. J Nat Prod 2003;6:1022-37.  Back to cited text no. 4    
5.Koehn FE, Carter GT. The evolving role of natural products in drug discovery. Nat Rev Drug Discov 2005;4:206-20.  Back to cited text no. 5    
6.Kong JM, Goh NK, Chia LS, Chia TF. Recent Advances in traditional plant drugs and orchids. Acta Pharmacol Sin 2003;24:7-21.  Back to cited text no. 6    
7.Chin Y, Balunas MJ, Chai HB, Kinghorn AD. Drug discovery from natural sources. AAPS J 2006;8:E239-53.  Back to cited text no. 7    
8.Kaushik P, Dhiman AK. Medicinal Plants and Raw Drugs of India. Bishen Singh, Mahendra Pal Singh. Dehradun: New Cannaught Place; 2000. p. XII+623 (with colour plates).  Back to cited text no. 8    
9.Kaushik P. Indigenous Medicinal Plants including Microbes and Fungi. New Delhi: Today and Tomorrow's Printers and Publishers; 1988. p. VIII+344.  Back to cited text no. 9    
10.El-Sayed A, Cordell GA. Catharanthamine: A new antitumor bisindole alkaloid from Catharanthus roseus . J Nat Prod 1981;44:289-93.  Back to cited text no. 10    
11.Perez C, Pauli M, Bazerque P. An antibiotic assay by the agar-well diffusion method. Acta Biologiae et Medecine Experimentalis 1990;15:113-5.  Back to cited text no. 11    
12.NCCLS-National Committee for Clinical Laboratory Standards: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standards M7-A4, Wayne, Pa: 1997.  Back to cited text no. 12    
13.Kaushik P, Singh Y. Antibacterial activity of extract of rhizome of Curcuma longa (turmeric). J Indian Bot Soc 2000;79:191-2.  Back to cited text no. 13    
14.Kaushik P, Upadhyay V, Singh P. Antibacterial potential of bryophytes. The Botanica 2000;50:132-6.  Back to cited text no. 14    
15.Kizil M, Kizil G, Yavuz M, Aytekin C. Antimicrobial activity of the tar obtained from the roots and stems of Pinus brutia . Pharma Biol 2002;40:135-8.  Back to cited text no. 15    
16.Satish S, Raveesha KA, Janardhana GR. Antibacterial activity of plant extracts on phytopathogenic Xanthomonas campestris pathovars. Lett Appl Microbiol 1999;28:145-7.  Back to cited text no. 16    
17.Thongson C, Davidson PM, Mahakarnchanakul W, Weiss J. Antimicrobial activity of ultrasound-assisted solvent-extracted spices. Lett Appl Microbiol 2004;39:401-6.  Back to cited text no. 17    

Top
Correspondence Address:
Pankaj Goyal
Department of Biotechnology, Jaipuria Institute, Vasundhara, Ghaziabad, UP
India
Login to access the Email id


DOI: 10.4103/0973-8258.42739

Get Permissions




 
 
    Tables

  [Table 1], [Table 2], [Table 3]

This article has been cited by
1 The Apoptotic Effect of Plant Based Nanosilver in Colon Cancer Cells is a p53 Dependent Process Involving ROS and JNK Cascade
Shakti Ranjan Satapathy,Purusottam Mohapatra,Dipon Das,Sumit Siddharth,Chanakya Nath Kundu
Pathology & Oncology Research. 2014;
[Pubmed]
2 Correspondence between flowers and leaves in terpenoid indole alkaloid metabolism of the phytoplasma-infected Catharanthus roseus plants
Suchi Srivastava,Richa Pandey,Sushil Kumar,Chandra Shekhar Nautiyal
Protoplasma. 2014;
[Pubmed]
3 Comparison of Fresh with Dry Extracts for Antibacterial Activity of Vigna radiate L. on Pathogenic Bacteria
Ali Abdul Hussein S. AL-Janabi
Pharmacognosy Journal. 2010; 2(16): 39
[Pubmed]
4 Antimicrobial activity and total content of polyphenols of Rheum L. species growing in Poland
Urszula Kosikowska, Helena D. Smolarz, Anna Malm
Central European Journal of Biology. 2010; 5(6): 814
[VIEW]



 

Top
 
  Search
 
  
  
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
    Introduction
    Materials and Me...
    Results
    Discussion
    References
    Article Tables

 Article Access Statistics
    Viewed7923    
    Printed204    
    Emailed4    
    PDF Downloaded981    
    Comments [Add]    
    Cited by others 4    

Recommend this journal