Feb 15, 2014 - pepper (Capsicum annuum L.), being highly effective for inhibition of .... Abelardo Meneses-García, Yolanda López-Vidal,. Aaron Blair.
Research Article
Vol: 2; Issue: 2
EVALUATION OF ANTIMICROBIAL ACTIVITY OF CAPSAICIN EXTRACT AGAINST B.SUBTILIS SPECIES Bjorn John Stephen1, *Dr. Vikram Kumar2 1
Biotech. Scholar, Amity Institute of Biotechnology, Amity University Rajasthan, Jaipur-303007 2
Sr. Lecturer, Amity Institute of Biotechnology, Amity University Rajasthan, Jaipur-303007
Date Received: 20
TH
Date of Accepted: th
Jan 2014
4 Feb 2014
Date Published: 15th Feb 2014
Abstract: Chillies contain the chemical group of alkaloid compounds called capsaicinoids, which are responsible for the pungency of the Capsicum species. These are produced as secondary metabolites by chilli peppers. In chillies, capsaicin and dihydrocapsaicin are the major components of capsaicinoids and generally represent 90% of the chillies pungency. The aim of the study was to evaluate capsaicin as a bioactive component and check its antimicrobial activity against various microorganisms. Capsaicin was extracted from red chillies using 95% ethanol as solvent. The measurement of growth inhibition of microorganisms by diffusion and dilution assays was used for screening capsaicin. The findings revealed that capsaicin exhibits antimicrobial properties against B.subtilis species.
Keywords: Capsaicin, extraction, B.subtilis, antimicrobial activity Introduction A compound is considered biologically active if it has direct effects on a living organism. Bioactive compounds are extra nutritional constituents that typically occur in small quantities in foods. Intense researches are being carried out to evaluate their effects on health. The demand for this scientific inquiry was the result of many epidemiologic studies that have shown protective effects of plant-based diets on cardiovascular disease (CVD) and cancer [1]. Many bioactive compounds have been discovered. These compounds vary widely in chemical structure and function and are grouped accordingly. Phenolic compounds, including their subcategory, flavonoids, are present in all plants and have been studied extensively in cereals, legumes, nuts, olive oil, vegetables, fruits, tea, and red wine. Many phenolic compounds have antioxidant properties, and some studies have demonstrated favourable effects on thrombosis and tumorogenesis and promotion.
element. They are nonvolatile alkaloids, which are acid amides of C9-C11 branched-chain fatty acids and vanillylamine, and are responsible for the pungency of the Capsicum species [2]. The major difference between the capsaicinoids is the length of the aliphatic side chain, the presence or absence of a double bond, the branching point and their relative pungency. Generally, capsaicin and dihydrocapsaicin are responsible for 90% of the pepper fruits pungency [3]. For this experiment dried red chillies were selected as the compound for capsaicin study. They were bought from a local store. In order to study about capsaicin, first the extraction of the compound was carried out. Extraction is the crucial first step in the analysis of medicinal plants, because it is necessary to extract the desired chemical components from the plant materials for further separation and characterization. The basic operation included steps, such as pre-washing, drying of plant materials or freeze drying, grinding to obtain a
Capsaicinoids a phenolic compound is a bioactive
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homogenous sample and often improving the kinetics of analytic extraction and also increasing the contact of sample surface with the solvent system. Proper actions must be taken to assure that potential active constituents are not lost, distorted or destroyed during the preparation of the extract from plant samples. The selection of solvent system largely depends on the specific nature of the bioactive compound being targeted. Different solvent systems are available to extract the bioactive compound from natural products. The extraction of hydrophilic compounds uses polar solvents such as methanol, ethanol or ethyl-acetate. For extraction of more lipophilic compounds, dichloromethane or a mixture of dichloromethane/methanol in ratio of 1:1 are used. In some instances, extraction with hexane is used to remove chlorophyll. For this experiment soxhlet extraction method was used. Soxhlet extraction is a traditional method which can reach the best combination of extraction efficiency and industry realization. Soxhlet extraction can realize continuously multiple extraction and can reach extraction and separation simultaneously. Soxhlet extraction is widely used [4,5,6] and its operation units are traditional and classic. Recently, new microwave-integrated Soxhlet extraction has been used to extract lipids from food product which augments the use of Soxhlet extraction [7]. The next part was to perform susceptibility test. The goal of a susceptibility test is to predict through an in vitro assessment of successfully finding sensitivity or resistance to a particular antimicrobial agent. This testing helps to better understand the kind of microbes the bioactive compound is active against and thus be useful for formulating medication for specific diseases [8]. Well diffusion method and dilution method was used in this experiment. Bacillus is a genus of gram positive, rod shaped bacteria and a member of the phylum Firmicutes. Bacillus species can be obligate aerobes of facultative anerobes and test positive for enzyme catalyses. Ubiquitous in nature, Bacillus includes both free-living and pathogenic species. Bacillus subtilis, known also as the hay bacillus or grass bacillus, is a Grampositive, catalase-positive bacterium [9] A member of the genus Bacillus, B. subtilis is rod-shaped, and has the ability to form a tough, protective endospore, allowing the organism to tolerate extreme environmental conditions. Most healthy individuals are resistant to this microorganism. However, persons with severely weakened immune systems are prone to suffer from food poisoning if they ingest food that contains these bacteria. The next experiment was to quantify the concentration of capsaicin in red chillies. Quantification of capsaicin is important as it is the major component in chillies responsible for pungency. Of all the methods used for
quantifying capsaicininoids, those based on colorimetric are most popularly and routinely used. Normally extracts are prepared first and this is followed by concentration of the chilly extract by solvent evaporations. Then the estimation would be carried out. In this experiment Folin-Ciocalteau reagent was used to perform the colorimetric estimation as it formed a stable blue colour. The experimental protocol was based on the findings in Bajaj & Kaur (1978). Due to the fact that plant extracts usually occur as a combination of various types of bioactive compounds or phytochemicals with different polarities, their separation still remains a big challenge for the process of identification and characterization of bioactive compounds. It is a common practice in isolation of these bioactive compounds that a number of different separation techniques such as TLC, column chromatography, flash chromatography, Sephadex chromatography and HPLC, should be used to obtain pure compounds. The pure compounds are then used for the determination of structure and biological activity. Besides that, non-chromatographic techniques such as immunoassay, which use monoclonal antibodies (MAbs), phytochemical screening assay, Fouriertransform infrared spectroscopy (FTIR), can also be used to obtain and facilitate the identification of the bioactive compounds. MATERIALS AND METHODS Preparation of sample Red chillies were bought from a local store of Amity University Rajasthan, Jaipur campus and. The chillies were cut and deseeded and grinded using a mixer. 20 gm were weighed and used for the experiment. 95% Ethanol 95ml of ethanol was taken in a beaker and to it 5 ml of distilled water was added and stored. Nutrient Agar 1gm of nutrient broth and 1gm of agar were added into a beaker. 100ml of distilled water was added and the solution was autoclaved and immediately used. Preparation of Bacterial sample 5ml of nutrient broth was prepared and B.subtilus which was got from a pure strain was inoculated into the nutrient broth and left overnight for growth. Saturated Sodium Hydroxide To 100 ml of water add 35 g of anhydrous sodium carbonate dissolve at 70-80 ºC and allow to cool overnight. The solution was filtered through glass wool.
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SOXHLET EXTRACTION Capsaicin extraction was carried out by using the soxhlet extraction apparatus.20 g of red chillies was bought from a local store. They were then deseeded and grinded. Only the flesh was used for the experiment. The apparatus was set up and the extraction was carried out for 20 cycles at 100ºC. The solvent used was 95% ethanol. ANTIMICROBIAL ACTIVITY
disc method. Ethanol was used as control. After 48 hours a 2cm zone of inhibition was observed around the capsaicin sample indicating a positive result for antimicrobial activity against B.subtilis species. Zone of inhibition is the zone where no microbial growth is observed and it’s directly related to the effect of the sample against the bacteria .It implies that capsaicin inhibits the growth of Bacillus species. The 2cm zone indicates a high level of growth inhibition.
The antimicrobial activity was checked by 1- Well Diffusion Assay 2- Dilution Test Assay Well Diffusion Assay [16] Diffusion assays are commonly used for screening antibacterial and antifungal activities of extracts from natural compounds. The well diffusion assay is an alternative to discs diffusion assay as they are suitable for aqueous extracts since they are difficult to dry on paper discs. B. subtilis was utilised in this experiment. • The strain B. subtilis was inoculated in nutrient broth. • 2% nutrient agar was prepared and poured into petriplates • 2ml of the inoculated broth was spread onto the plate. • Two wells were made by using a borer. • To one well 95%ethanol was added and to the other Extracted capsaicin sample was added. It was then incubated at 27ºC for overnight. • After Incubation the inhibition zone was measured. Dilution Test Assay [15] • • • • • •
3 test tubes were taken for the experiment. 5ml of nutrient broth was added to all the test tubes 1st test tube- No compound added (use as Blank) 2nd test tube-100µl of B.subtilis 3rd test tube-100µl of B.subtilis and 100ul of capsaicin The absorbance was read at 660nm after every 12 hours
RESULTS Well Diffusion Assay Well diffusion assay was preferred over disc diffusion as capsaicin is insoluble and will not diffuse when using the
Figure 1. B.subtilis zone of inhibition when treated with Capsaicin Dilution Test To further analyse the antimicrobial effect the dilution test was carried out. When B.subtilis strains were tested with capsaicin, varying degrees of antimicrobial activity was observed after 60 hours of incubation. Within 24 hours no considerable effect was observed on the growth of B.subtilus with or without capsaicin. After 36 hour’s slight decline in the growth of bacillus in the presence of capsaicin were observed. However after 48 hours approximately 40% decline in growth of Bacillus was observed in the presence of capsaicin as compared to in the absence of any bioactive compound, indicating an inhibitory effect against Bacillus species. Previous studies also reported capsaicin, extracted from sweet pepper (Capsicum annuum L.), being highly effective for inhibition of B.subtilis strains [15]. DISSCUSSION Capsaicinoids are the compounds responsible for the pungency of pepper fruits and their products. Peppers are the fruits of plants from the genus Capsicum and belong to the family Solanaceae. There are several domesticated species of chili peppers, among them Capsicum annuum, C. frutescens and C. chinense, which include many common varieties. These various peppers are widely used in many parts of the world for their valued and characteristic sensory properties: color, pungency and aroma.
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Pungency, a commercially important attribute of peppers, is due to the presence of chemicals from the characteristic capsaicinoids group. The two most abundant capsaicinoids in peppers are capsaicin (8methyl-N-vanillyl-trans-6-nonenamide) and dihydrocapsaicin, both constituting about 90%, with capsaicin accounting for ~71% of the total capsaicinoids in most of the pungent varieties. Capsaicin content of peppers is one of the major parameters that determine its commercial quality •
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Capsaicin is also considered as an active principle which accounts for the pharmaceutical properties of peppers. It has been used as an analgesic against arthritis pain and inflammation. It has also been reported to show anticancer effect. To be active against neurogenic inflammation (burning and stinging of hands, mouth and eyes). This property is the basis for the use of capsaicin in defensive pepper sprays. Capsaicin has also been reported to show protective effects against high cholesterol levels and obesity. Capsaicin and other members of the capsaicinoids group produce a large number of physiological and pharmacological effects on the gastrointestinal tract, the cardiovascular and respiratory system as well as the sensory and thermoregulation systems. These effects result principally from the specific action of capsaicinoids on primary afferent neurons of the C-fiber type. This specific influence provides the rationale for their use to treat some peripheral painful states, such as rheumatoid arthritis [34].
Capsaicin was extracted through soxhlet extraction. Though a classical method, soxhlet extraction is still efficient and widely used. However, may advances have been made to minimise interfering substance during the extraction process. 95% ethanol was considered the best solvent for extraction and hence used in the experiment. The antimicrobial effect of capsaicin was shown by the two test performed namely the well diffusion and dilution assay. The experiments proved that capsaicin has an inhibitory effect on Baccilus subtilus by preventing the growth of the micro organism. The dilution assay showed the inhibition takes place after 48 hrs incubation. FUTURE PROSPECTS In order to develop a better understanding of capsaicin, we would look into the antimicrobial sensitivity of capsaicin towards various kinds of gram positive and gram negative bacteria by performing well diffusion test. It has been reported that the growth of is enhanced by treating it against capsaicin. Thus the effect of capsaicin against fungal growth would also need to be looked into. HPLC would also be performed to for the separation and quantification of capsaicin. Now a day’s natural colour has great value. People are very conscious about health hazards caused by chemical colours, so natural colours would help overcome these. It produces great value in market for its usage in food, textile, and cosmetics. Hence further we would also be looking into the extraction of the colour component and see chemical extraction and estimation of colour.
CONCLUSION Numerous bioactive compounds appear to have beneficial health effects. Much scientific research needs to be conducted before we can begin to make sciencebased dietary recommendations. Despite this, there is sufficient evidence to recommend consuming food sources rich in bioactive compounds. From a practical perspective, this translates to recommending a diet rich in a variety of fruits, vegetables, whole grains, legumes, oils, and nuts. Capsaicin is said to be one such bioactive compound. Researchers claim it helps to prevent various types of cancers and hence it is recommended in our daily diet. Hence the need to study capsaicin is important.
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Table1. The antimicrobial activity of capsaicin of 15ppm against B. Subtilis Treatment/Absorbance
0 hrs
18hrs
24hrs
36 hrs
48 hrs
Nutrient Broth (NB)
0
0
0
0
0
NB+Bacteria(Bac)
0
0.2
0.3
0.8
1.2
NB+Bac+Capsaicin
0
0.3
0.3
0.7
0.8
1.4
Dilution assay antimicrobial activity
Absorbance (660 nm)
1.2 1 0.8 0.6 0.4 0.2 0 0 hrs 18hrs Nutrient Broth (NB)
24hrs 36 hrs 48 hrs NB+Bacteria(Bac) NB+Bac+Capsaicin
Time Figure2. The antimicrobial activity of capsaicin of 15ppm against B. Subtilis
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