Abstract: The objectives of this study were to analyze the lipid content of 10 oil seeds of different plant families and the antimicrobial activities of these plant seeds that can add a new dimension in the alternative medicinal field of Indian origin. Chemical analysis reveals that the major components of all the seeds were myristic acid, ricinoleic acid, linoleic acid, palmitic acid, lauric acid and oleic acid. The other 4 components i.e., linolenic acid, palmitoleic acid, steric acid and arachidic acid present less than 30% of plant seeds. The primary screening tests for antibacterial and antifungal activities were shown positive for all the compounds. Escherichia coli and Candida albicans has shown a zone of clearance ranging 11-15 mm whereas Pseudomonas aeroginosa, Salmonella typhi, Staphylococcus aureus, Aspergillus niger and Penicillium notatum showed the range between 16-20 mm. It is assumed that oxidative effect could plausibly play an important role in the antimicrobial function of fatty acids. The higher oil content were found to be on Aegle marmelos (49%), Prunus amygdalus (48%), Cardiospermum halicacabum (47%), Brassica alba (38%) and can suggest as an agent of conservation in the cosmetic and/or food industries, as an active compound in medical preparations and as a disinfectants.
INTRODUCTION
The antiseptic qualities of aromatic and medicinal plants and their extracts have been recognized since antiquity, while attempts to characterize these properties in the laboratory date back to the early 1900s (Dorman and Deans, 2000).
Infectious diseases are the leading cause of death world-wide. Antibiotic resistance has become a global concern. The clinical efficacy of many existing antibiotics is being threatened by the emergence of multidrug-resistant pathogens. Many infectious diseases have been known to be treated with herbal remedies throughout the history of mankind. Natural products, either as pure compounds or as standardized plant extracts, provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity. There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action for new and re-emerging infectious diseases (Parekh and Chanda, 2007). Therefore, researchers are increasingly turning their attention to folk medicine, looking for new leads to develop better drugs against microbial infections (Benkeblia, 2004). The increasing failure of chemotherapeutics and antibiotic resistance exhibited by pathogenic microbial infectious agents has led to the screening of several medicinal plants for their potential antimicrobial activity (Colombo and Bosisio, 1996).
India is a varietals emporium of medicinal plants and is one of the richest countries in the world in regard to genetic resources of medicinal plants. It exhibits a wide range in topography and climate, which has a bearing on its vegetation and floristic composition (Parekh and Chanda, 2007).
Hydroxy fatty acids shows wide range of antifungal (Sjogren et al., 2003) and antibacterial (Mundt et al., 2003) activities. Considering the extensive applications of hydroxy fatty acids in medicinal importance, an attempt has been made to evaluate the antibacterial and antifungal activity of crude hydroxy fatty acids containing oil seeds of different plant seeds. Previous investigations showed the antimicrobial activities of different plant part of Mimusops hexandra, Prunus amygdalus (Yosef et al., 2007) Brassica alba (Ceylan and Fung, 2004), Cardiospermum halicacabum (Parekh and Chanda, 2007), Aegle marmelos, Anacardium occidentale (Ojewole, 2004), Jatropha gossypifolia (Ogundare, 2007), Hevea brasiliensis, Psoralea corylifolia (Prasad et al., 2004) and Bauhinia retusa.
In this study we describe in details the lipid contents of 10 oil seeds of different plant families and the antimicrobial activities of these plant seeds on common human pathogens including food borne, intestinal, nosocomial pathogens and dermatophytes.
MATERIALS AND METHODS
Plant Materials
The seed samples belonging to different plant families were collected
from Bidhan Chandra Krishi Viswavidalaya, Mohanpur, West Bengal and Pusa
Agricultural Research Institute, Bihar, from the full grown vegetative
plants during rainy season and tabulated in (Table 1)
with the sample Number.
Extraction of Oils (Soxhlet Method)
Lipid extraction was carried out by Soxhlet extraction method using
petroleum ether (40-60°C). A known weight of seeds were kept in the
Soxhlet`s apparatus and 250 mL of petroleum ether (40-60°C) was added
and extracted for 10 h the solvent was recovered by distillation and the
fat recovered in a minimum volume of ether was transferred into a pre-weighed
clean beaker. The solvent was evaporated at 55°C in incubator. Finally
oil was weighed. The direct thin layer chromatography of seed oils revealed
the presence of hydroxy fatty acids (Prost and Wrebiakowski, 1972).
Fatty acid Analysis by Gas Chromatography
Preparation of Sample for Gas Chromatography
One milliliter of 0.5 M KOH was added to 0.5 mL of sample and kept
it at 86°C in water bath for 10 min. Cooled and added 2 mL of hexane,
vortex for 15 min and centrifuged to take out the hexane phase. To aqueous
layer 1 mL of 0.7 NH4Cl and 2 mL of hexane centrifuge at 5000
rpm for 15 min. The hexane layer was collected. After that the hexane
layers were evaporated. 0.2 mL of benzene and 0.5 mL of BF3
was added and kept at 86°C in water bath for 10 min. After that cool
and add 1 mL of
| Table 1: | Medicinal plant of different plant families |
water. The aqueous phase was extracted 3 times with 2 mL of hexane. The hexane layer was pooled and 5 mL of water was added. The hexane layer was separated after centrifugation. The hexane was evaporated and benzene was added to ready the sample for gas chromatography (Goud et al., 2007).
Gas Chromatography
After the gas chromatography the fatty acids were identified by using
authentic fatty acid standards obtained from Sigma, USA. The relative
percentages of the fatty acids were determined using Shimadzu C-R 3A integrator
connected to gas chromatography.
Preparation of Drugs Solution
Each test compound (10 mg) was dissolved in dimethyl formamide (10
mL) to give solution of 1000 μg mL-1 and 0.1 mL of this
solution was used.
Microorganisms
The organisms Escherichia coli, Pseudomonas aeroginosa,
Salmonella typhi, Staphylococcus aureus were obtained from
Vaidehi Medical College, Bangalore. Three fungi culture Aspergillus
niger, Candida albicans and Penicillium notatum were
collected from different colleges in Bangalore.
Antimicrobial Assay
The antimicrobial properties were evaluated by agar well diffusion
method (Perez et al., 1990) using Mueller Hinton agar (Hi-media)
for bacteria and Sabouraud`s dextrose agar for fungi. The microorganisms
were activated by inoculating a loopful of the strain in the nutrient
broth (25 mL) and Sabouraud`s dextrose broth (25 mL). The culture flasks
were incubated at 37°C for 24 h (bacteria) and 25°C for 7 days
(fungi), respectively.
One milli liter of inoculums was inoculated into the 45-50°C cooled agar and plated. Using the cork borer wells was made and different extracts having 100 μg mL-1 concentration were transferred using a micropipette. Then the plates were kept in the refrigerator for 5 min for diffusion and incubated at 37°C for 24 h and 25°C for 7 days, respectively. The control experiment was carried out with tetracycline. Zone of inhibition was measured in millimeter. The experiments were carried out for ten successive trials and the average values are presented.
RESULTS AND DISCUSSION
The oil contents of different oil seeds containing hydroxy fatty acids are tabulated in (Table 2) after extraction of oil by Soxhlet Method.
The highest oil contains of the oil was found in Aegle marmelos
(49%) and the lowest in Hevea brasiliensis and Psoralea corylifolia
(14%). Direct thin layer chromatography revealed the presence of hydroxy
fatty acids in all the plant species.
| Table 2: | The oil contents of different oil seeds containing hydroxy fatty acids |
| Table 3: | Fatty acid composition of different oil seeds represented in (%) |
| - : Absent | |
| Table 4: | Screening results for antimicrobial activity of the oil seeds by the agar well diffusion method |
| N.B. diameter of the well: 10 mm | |
After the gas chromatography the composition and percentage of fatty acid was tabulated in the Table 3.
In all tested oils 10 components were identified. The major components of all the seeds were myristic acid and ricinoleic acid, linoleic acid, palmitic acid, lauric acid and oleic acid. The other 4 components i.e., linolenic acid, palmitoleic acid, steric acid and arachidic acid present less than 30% of plant seeds. Antibacterial activity of the different plant oil seeds containing are presented in (Table 4).
All the bacterial strains demonstrated some degree of sensitivity to the oil seeds tested. Escherichia coli and Candida albicans showed a zone of clearance ranging 11-15 mm whereas Pseudomonas aeroginosa, Salmonella typhi, Staphylococcus aureus, Aspergillus niger and Penicillium notatum showed the range between 16-20 mm. The control showed the zone diameter ranging 20-26 mm.
Natural hydroxy fatty acids isolated from different plant seeds showed a moderate antibacterial and antifungal activity. The antifungal property of fatty acids is due to its detergent-like activity which affects the structure of cell membranes of the target organisms. Indeed, cis-9-heptadecenoic acid, a compound similar to the 3-hydroxy fatty acids identified that readily partitions into the lipid bilayers of fungal membranes (Avis and Belanger, 2001). This increases membrane permeability and the release of intracellular electrolytes and proteins and, eventually, leads to cytoplasmic disintegration of fungal cells. It is assumed that this oxidative effect could plausibly play an important role in the antimicrobial function of fatty acids (KimYoung et al., 2006).
Gram-positive bacteria are more resistant then Gram negative bacteria to the antibacterial properties of plant volatile oils (Fredj et al., 2007). However, Brassica alba, Cardiospermum halicacabum, Jatropha gossypifolia and Bauhinia retusa oils appeared preferentially more active with respect to Gram reaction, exerting greater inhibitory activity against Gram-positive organisms Staph. aureus. In our study E. coli showed the more resistant. It may be due to the presence of plasmid conferring resistance. Among the fungi Candida albicans was more resistant compared to the other two.
Chemotherapeutic agents, used orally or systemically for the treatment of microbial infections of humans and animals, possess varying degrees of selective toxicity. Although the principle of selective toxicity is used in agriculture, pharmacology and diagnostic microbiology, its most dramatic application is the systemic chemotherapy of infectious disease (Dorman and Deans, 2000). The tested plant products appear to be effective against a wide spectrum of microorganisms, both pathogenic and nonpathogenic bacteria and fungi. Administered orally, these compounds may be able to control a wide range of microbes but there is also the possibility that they may cause an imbalance in the gut microflora, allowing opportunistic pathogenic coliforms to become established in the gastrointestinal tract with resultant deleterious effects. Further studies on therapeutic applications of volatile oils should be undertaken to investigate these issues, especially when considering the substantial number of analytical studies carried out on these natural products.
As a conclusion, among the all species the higher oil content were found to be on Aegle marmelos (49%), Prunus amygdalus (48%), Cardiospermum halicacabum (47%), Brassica alba (38%) and can be suggested as an agent of conservation in the cosmetic and/or food industries, as an active compound in medical preparations and as a disinfectants.
ACKNOWLEDGMENTS
We are very much thankful to Dr. Anjan Bhattacharjee, Officer in Charge, MAP Project, Dept. of Agriculture Chemicals, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur for providing the plant materials. Dr. Seemanthini Desai, Apollo Hospitals, Bangalore, India and Miss Hithyashi C B, Lecturer, Dayanand Sagar Institution, Bangalore for their valuable guidance and support.