Antibacterial studies of Catharanthus roseus of Jazan province against the selected bacterial strains

 

Mohammed M. Safhi, Maksood Ali, S.M. Sivakumar*, Aamena Jabeen, Manohara Y.N.

College of Pharmacy, Jazan University, Jazan, KSA

*Corresponding Author E-mail: drsmsivakumar@gmail.com

 

The objective of this preliminary work is to identify the phyto constituents and antibacterial activity of Catharanthus roseus of Jazan province, KSA. In Saudi Arabia the plant is used for the ornamental purpose and has been found well grown in Jazan province. Active constituents from the leaves of Catharanthus roseus were isolated by cold maceration process and confirmed by respective chemical test. The anti bacterial activity of methanolic extract on various human pathogens viz Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Klebsiella planticola and Proteus vulgaris was performed.  The extracts predominately show the presence of alkaloids and flavonoids. However, there is no significant anti bacterial spectrum of activity against the tested human pathogens.

 

 

INTRODUCTION:

Microorganisms develop resistance to various antibiotics treatment against infectious diseases. Drug resistance to emerging infectious disease is a great challenge and the development of new antibiotics is potentially very important towards the betterment of human health care¹. Globally a new trend has been blossomed that “Green medicine” is safer and thus public attitude is focusing towards the older system of medicine. Based on this aspect for past two decades the trend of global research has been focused towards herbs for the development of newer drugs and biomolecules that can offer potential advantage such as more specific and lesser side effects^2&3. Catharanthus roseus (L.) G. Don., originates from Madagascar well known as Madagascar periwinkle and Vinca rosea, is a perennial tropical plant belonging to the family Apocynaceae. Traditionally, Catharanthus roseus has been used as folk medicine especially for diabetes, blood pressure, diarrhoea, and etc^4-6. However, the medicinal uses of the plant vary with country to country⁷.  In Saudi Arabia the plant is used for the ornamental purpose and has been found well grown even at high temperature. More than 100 biomolecules have been isolated from various parts of plant among which vincristine and vinblastin are important alkaloids used in cancer treatment. Jazan is the smallest province of Saudi Arabia situated on the coast of Red Sea and has tropical climate with an average annual temperature of 30.1°C.

 

Catharanthus roseus has been found well grown in Jazan province throughout the year. The present study was undertaken to evaluate the various phytoconstituents present in Vinca rosea of Jazan province. The crude extract was screened to investigate the presence of phytoconstitutents and anti bacterial activity against certain human pathogens.

 

Table 1      various constituents of crude extract

S.no	Constituents	Presence or absence
1	Alkaloids	+++
2	Flavonoids	+++
3	Carbohydrates	+
4	Phenolic compounds	+
5	Proteins and amino acids	+
6	Glycosides	-
7	Saponins	-
8	Resins	-
9	Lipid Fats	-

*  (+ + +) Strongly positive, (+) Positive, (-) Negative.

 

MATERIALS AND METHODS:

Collection and extraction

The plant was collected during October 2012 in Jazan province, Saudi Arabia. Collected plant material was washed thoroughly in running tap water, rinsed in distilled water and shade dried for one week in open air. After drying, the leaves were powdered in a mixer grinder and stored in air tight bottle. Cold maceration process was used to extract the constitutents by macerating 10 gm of powder in 200 ml of petroleum ether for 24 hours. During maceration process shaking was frequently done for every six hours. The extract was filtered and the marc was used to extract the remaining constituents in methanol by the same cold maceration process.

Table 2 The anti bacterial activity of Catharanthus roseus against selected bacterial strains

Organisms	Zone of inhibition (mm)
	50 µg / 100 µl	100 µg / 100 µl	200 µg / 100 µl	300 µg / 100 µl	Ciprofloxacin disc (5 µg /disc)
Staphylococcus aureus	-	-	-	+	26 ± 1.2
Streptococcus pyogenes	-	-	-	+	28 ± 2.6
Bacillus subtilis	-	-	-	-	24 ± 1.4
Escherichia coli	-	-	-	-	21 ± 2.6
Proteus vulgaris	-	-	-	-	20 ± 0.2

 

 

 

After three days it was filtered and filtrate was evaporated to dryness in a to dryness in a flat bottom dish and dried at 105⁰C and then weighed. The extraction was subjected to phytochemical analysis and anti bacterial screening.

 

Strains used

24h culture of Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Bacillus subtilis, Klebsiella planticola and Proteus vulgaris.

 

Antibacterial screening

The cultures were sub cultured for 24 h and the concentration of working stock culture was assessed as      10^{- 6} CFU/ml. Specified quantity of Muller Hinton agar was prepared and plated in aseptic condition. The agar well diffusion technique was used for testing anti bacterial susceptibility of crude extract and methanol (solvent control) whereas agar disc diffusion method was followed for testing antibacterial susceptibility for standard ciprofloxacin disc (5 µg/ disc). The extracts were dissolved in methanol to get the known concentrations of 50 µg / 100 µl, 100µg/ 100 µl, 200µg/ 100 µl and 300µg/ 100 µl. After 24h of incubation at 37⁰ C the zone of inhibition was measured and tabulated.

 

Statistical analysis

All the experiment were performed six times (n=6) and the data were subjected to one way analysis of variance (ANOVA), the level of significance is P<0.0001 using Graph pad Instat software, USA. The test values were compared with standard drug values by using Dunnet’s test (post test).

 

RESULTS AND DISCUSSION:

The present antibiotic therapy for infectious diseases may lead to multiple drug resistance¹. The phytochemical analysis of petroleum ether extract of leaves of Catharanthus roseus by cold maceration process showed the presence of the various phyto constituents. Mohammed Ibrahim et al. (2011)⁸ reported that whole plant extraction of Catharanthus roseus by using mixture solvent of dichloromethane and methanol in 1: 1 proportion in soxhlet apparatus exhibit phytoconstituents such as carbohydrate and alkaloids. In our study, the crude extract of leaves showed positive test for alkaloids, flavonoids, carbohydrates, proteins, amino acids and phenolic compounds (Table 1). Therefore, it is clearly demonstrating that cold maceration process is better to obtain phyto constitutents rather than using hot continuous extraction. The results of anti bacterial activity showed no significant activity against the tested pathogens. However, in 2012, Govindasamy and Srinivasan⁹ reported that the ethanolic extracts of leaves of Catharanthus roseus collected from Thondi coastal region, India showed potential anti microbial activity. Ramya et al. (2008)¹⁰ reported that the anti bacterial potential of ethanol extract of different parts Catharanthus roseus exhibit significant activity from all the parts of plant. Among all, leaf extract was showing maximum activity against various human pathogens. During past two decades many attempts have been made to isolate a better anti microbial compounds from various herbs but most of them are under study level only. However, due to the successful isolation of vincristine, vinblastin and some of the earlier reports showed promising antimicrobial effect, we focused towards isolating the bio active substance from Catharanthus roseus of Jazan, Province, Saudi Arabia. In our study, the spectrum of activity of the crude extract showed the effect only against S.aureus and S.pyogenes when compared with the rest (Table 2). However, the activity was exhibited merely at the concentration of 300 µg / 100 µl and the zone of inhibition was found to be very less when compared to standard ciprofloxacin disc. Goyal et al., (2008)¹¹ stated that the antibacterial activity is dependent upon the polarity of the compounds. Therefore, our methodology for isolating vital bioactive substances is yet to be standardized.

 

CONCLUSION:

This preliminary research work demonstrates that the cold maceration process is a better technique to isolate various phytoconstitutents but the crude extract did not exhibit antibacterial activity. This might be due to climatic condition. However, further studies are under process in order to overcome the lacunae that we experienced in this work.

 

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Received on 20.02.2013       Modified on 28.02.2013

Accepted on 10.03.2013      © RJPT All right reserved