1 Research Paper
In vitro antibacterial screening of Cuscuta reflexa ethanolic extract Shweta Tiwari1, Dr. R.C.Agrawal2, Dr. Pawan Jain3, Dr. Shama
J.P.Khanam4
1. Ojaswini Institute Par Excellence, Damoh (M.P.) India 2. Priyamvada Birla Cancer Hospital & research Center, Satna(M.P.) India
3. Ojaswini Institute Par Excellence, Damoh (M.P.) India 4. Ojaswini Institute Par Excellence, Damoh (M.P.) India
Abstract
The present study was carried out to evaluate the antibacterial activity of 50%
ethanol extract of stem of Cuscuta reflexa on selected three Gram positive and three Gram negative bacterial strains. The stem extract of Cuscuta reflexa showed potential antibacterial activities against all of the selected strains of microorganisms and the greatest activity was observed against Shigela flexeneri. For antibacterial test, Disc diffusion technique was used and the zone of inhibition of microorganisms was measured in mm.
Key words: Cuscuta reflexa, antibacterial screening.
Introduction:
Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources, many of which based on their use in traditional medicine. It has been noted that the original source of many important pharmaceuticals currently in use have been plants used by indigenous people (Balick et al., 1996). Herbal medicine or phytomedicine refers to the use of any plant’s seeds, berries, roots, leaves, stem, or flowers for medicinal purposes (Barrett et al., 1999). The potential of medicinal plants can be assessed by finding new chemical entities of wide structural diversity. These new chemical substances can also serve as templates for producing more effective drugs through semi-synthetic and total synthetic procedure. According to World Health Organization (WHO), about 74% of 119 plant-derived pharmaceutical medicines or biotechnology medicines are used in modern medicine in ways that correlate directly with their traditional uses (Newman et al., 2003; Barrett et al., 1999). In order to promote the use of medicinal plants as potential sources of antibacterial compounds, it is pertinent to thoroughly investigate their composition and activity and thus validate their use (Nair et al., 2006). Some phytochemicals produced by plants have antibacterial activity allowing these plants to be studied and used for the development of new antibacterial drugs (Nascimento et al., 2000). Secondary plant metabolites are largely unexplored in
‘conventional’ animal production systems. In the past, plant metabolites were
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generally considered as sources of nutritional factors. Recent bans and restrictions on the use of animal antibiotic growth promoters stimulated interest in bioactive secondary metabolites of plant source as alternative performance enhancers (Greathead, 2003).
Cuscuta (Dodder) (family - Cuscutaceae), is a genus of about 100-170 species of yellow, orange or red parasitic plants. The genus is found throughout the temperate to tropical regions of the world. There are very few reports regarding to its antibacterial and cytotoxic effects using various parts of this plant. Some scientists used leaves and stem extracts to screen its antibacterial activity. In 1981 L.P. Awasthi isolated anti viral substances from aquous exteact of C.reflexa plant. The aim of the present work was to evaluate the antibacterial assays to support the pharmacological effects of this plant. Although numerous studies have shown the medicinal values of this plant, there still remains ample scope for further in depth research. So far, for the first time an attempt was taken to investigate the antibacterial effect of Cusscuta reflexia by using 50% ethanol extract of the stem. Accordingly, we disclose herein the antibacterial effects of the stem of Cusscuta reflexa to further establish the scientific basis of the traditional uses of this plant.
Materials and methods:- Microorganisms used:
The test organisms (Pseudomonas aureogenosa, Staphylococcus aureus, Staphylococcus epidermis, Shigella flexineri, Bacillus substilis & E.Coli.) were obtained from the Department of Research, JN Cancer Hospital and Research Center, Bhopal (M.P.).Antibacterial Sensitivity test is done by Disk Diffusion Susceptibility Testing (Kirby-Bauer Method)
Objective:
Antibacterial screening was done to find out the antibacterial properties of different concentration of 50% ethanolic extract of Cuscuta reflexa.
Principle:
Kirby-Bauer Method (Disc diffusion method) was followed to test the antibacterial activity of different concentration of plants extract. The paper disc having the same diameter absorbed the concentration of extract. After impregnated the disc on agar plate, it diffuses their drug and in case of drug sensitive bacteria make a clear area that is known as Inhibitory area or Zone of inhibition.
Culture media:
The medium used for the activation of the microorganisms was nutrient broth.
The nutrient agar media was used for the antibacterial test. All the culture media were prepared and treated according to the manufacturer guidelines (Hi Media Laboratories Ltd., Mumbai, India).
Procedure:
Made the disc of Whattmann’s filter paper no.1 of same diameter (0.5mm) by the help of punching machine.
Autoclaved the disc in a Petri dish for sterilization.
Prepared each concentration of discs by pouring the disc in different concentration.
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Dried the discs and preserved in different concentration of Petri dishes at 2-8°C.
All of the required apparatus and materials were sterilized in autoclave and placed in a laminar airflow cabinet under pathogen free conditions.
Test organisms were collected from department’s microbial standard stock.
By streaking with loop, microorganisms were inoculated in nutrient broth and incubated at 35 ºC for 12 hr.
Nutrient agar media was prepared and poured in Petri plates and kept for drying.
Swab (cotton) was dipped in broth having microbial growth and gently squeezed against the inside of the tube to remove excess fluid.
Inoculated the dried surface of agar plate by streaking the swab over the entire sterile agar surface. Repeated this procedure two more times, and rotated the plate 60° each time to ensure an even distribution of inoculums.
Replaced the plate top and allow 3 to 5 minutes, but no longer than 15 minutes, for any excess surface moisture to be absorbed before applying the test and antibiotics disks.
Disks were dipped in drug ethanolic extract of Cuscuta reflexa at different concentration ( 25%, 50%, 75% & 100%) and air dried in laminar air flow before this step.
Placed the appropriate disks (Drugs and antibiotics) evenly (no closer than 24 mm from center to center) on the surface of the agar plate by using a sterile forcep.
Inverted the plate and placed them in an incubator at 35°C within 15 minutes after disks were applied.
After 6-8 hrs. Of incubation, examined each plate at an interval of 2 hours and measured the diameters of the zones of complete inhibition, including the diameter of the disk.
Data was analyzed by ANOVA.
Result:
The ethanol extract of the Cusscuta reflexa were screened against six human pathogenic bacteria to check antibacterial activities by disc diffusion method which showed valuable zone of inhibition. The specific zone of inhibition against various types of pathogenic bacteria was shown in table.
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Antibacterial activity of ethanolic extract of C.reflexa
Photographs showeing Zone of inhibition of Terminalia arjuna extract in different strains of bacteria
The present study indicates that the extract of C.reflexa (50% ethanol) has got profound antibacterial effect and may have potential use in medicine. From the previous studies and our current investigation it may be concluded that the cuscuta reflexa have antibacterial activity. This novel finding will aid us to conduct bioactivity guided isolation and characterization of leading compounds in due course.
Discussion:
It is one of the most versatile plants having a wide spectrum of medicinal activities. This versatile medicinal plant is the unique source of various types of compounds having diverse chemical structure. Very little work has been done on the plausible medicinal applications of these compounds and hence extensive investigation is needed to exploit their therapeutic utility to combat diseases. A drug development programme should be undertaken to develop modern drugs with the compounds isolated from Cuscuta reflexa. Although crude extracts from Cuscuta have medicinal applications from time immemorial, modern drugs can be developed after extensive investigation of its
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bioactivity, mechanism of action, pharmaco therapeutics, toxicity and after proper standardization and clinical trials.
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