Proximate chemical composition and biological profile of fatty acids of Withania somnifera L dunal

The fixed oils extracted from the fruit (berry) of Withania somnifera were evaluated for their chemical composition. The gas chromatography-mass spectrometry (GC-MS) analysis shows the presence of various saturated and unsaturated fatty acids present in the roots of Withania somnifera. The total eight fatty acid compounds were identified by GC-MS with the library searches scale with varying percentage such as linoleic acid (11.247%), linoleic acid (4.000%), palmitic acid, (2.842%) and tetracosanoic acid (0.880%), palmitic acid (0.42%), lenoleic acid (0.23%), oleic acid (0.14%), elaidic acid (0.01%). The fixed oil was evaluated for different biological activities such as phytotoxicity, antibacterial, antifungal, cytotoxicity, antibacterial, and antiurease activities. The oil shows moderate percent inhibition against the entire activities. The oil shows dose dependent DPPH radical scavenging activity.


INDRODUCTION
Genus Withania belongs to the family Solanaceae, which comprises 84 genera and more than 3000 species dispersed throughout the world. Berry of Withania somnifera is ingredient of many Ayurveda's and produces successfully developed medicines for tumors, tubercular glands, carbuncles, and ulcers (Mirajalili et al., 2009). Phytochemical studies showed that Aswagandha possesses anti-inflammatory, antitumor, antistress, antioxidant, immunomodulatory, hemopoetic, rejuvenating properties (Lakshmi et al., 2000). It also appears to exert a positive influence on the endocrine, cardiopulmonary, and central nervous system (Lakshmi et al., 2000). The phytochemical screening gave positive results for carbohydrates, alkaloids, glycosides, and fixed oil and fats (Christina et al., 2004). The fruit (berry) of the plant have a milk coagulation property attributed to the pulp and husk of the berry, which has been used in the preparation of vegetable rennent ferment for cheese (Mirajalili et al., 2009). The fruit are reported to be sedative, emetic, and stomachic, a blood-purifier and febrifuge and alternative, diuretic and bitter tonic in dyspepsia as well as growth promoter in infants (Dhuley et al., 1997). Due to the extensively used of W. somnifera as a folk medicine for the treatment of various diseases, fixed oils have been extracted from the fruit of W. somnifera and checked their biological activities such as phytotoxicity, antibacterial, antifungal, cytotoxicity, antibacterial, and antiurease activities. Here, we are reported for the first time its urease inhibitory and antioxidant activity.

Plant
W. somnifera fruit (berry) was collected from Peshawar, Khyber Pakhtunkhwa, Pakistan in month of December, 2011. The plant was identified by taxonomist, from the Department of Botany, University of Peshawar, Pakistan.

Extraction and fractionation
Shade dried fruit of W. somnifera was filled in the flask and extracted successively with methanol solvent in soxhlet extractor for 48 h. The solvent extract was concentrated under reduce pressure at 40°C using rotavapor, and suspended in water and successively partitioned with n-hexane, chloroform, ethyl acetate and methanolic fraction according to standard protocol (Uddin et al., 2011a;.

Preparation of standard
Internal standard was prepared by dissolving 13.7 mg of tridecanoic acid methyl ester in 1 ml hexane. External standard was prepared by diluting 10 mg of 37 component FAMEs mix standard to 10 ml with dichloromethane. From the stock solution further working solutions were prepared.

Extraction of oil f r o m fruit and preparation of FAMEs
The shade dried and crushed fruit of W. somnifera (100 g) was subjected to cold extraction with MeOH (3 × 10 L) at room temperature. The extract was then concentrated under reduced pressure at temperature below 40°C. The final residue obtained was 25 g. This methanolic extract was suspended in water and successively partitioned with n-hexane, chloroform, EtOAc. The hexane fraction (4 g) was subjected to column chromatography on silica gel (Merck Silica gel 60 (0.063 -0.200 mm), 5 × 60 cm). The column was eluted with n-hexane: EtOAc (100:0 → 0:5) as solvent system. A total of 20 fractions, RO-1 to RO-20 were obtained based Uddin et al. 2035 on thin layer chromatography (TLC) profiles. Fraction RO-1 to RO-3 contained yellow coloured oil. Yield: (0.60 g).

Preparation of FAMEs
For gas chromatographic analysis, it is required that the sample being analyzed must be volatile. For the identifications of FAMES, along with other volatile organic compounds the oil were directly subjected to GC-MS. To identify fatty acid, the following procedures were adapted. In order to make fatty acids present in the oil volatile, derivitization of oil was performed prior to GC-MS analysis. Methylation general method was used for converting non-volatile fatty acids into volatile FAMEs (Vickery, 1981). Methylation of fatty acids was achieved with BF 3 -methanol as derivatizing reagent, which is the accepted procedure for converting fatty acids into FAMEs. Derivitization was done according to the AOAC standard reference method (Yue et al., 2010). To a known amount of sample (equivalent to 25 mg fat) was added 0.1 ml internal standard (1.37 mg) and 1.5 ml of sodium hydroxide solution in methanol (0.5 N), sealed and heated in boiling water bath for 5 min. The hydrolyzed sample was cooled and added 2.5 ml of boron triflouride solution in methanol (10%). The solution was then sealed and heated in boiling water bath for 30 min and then cooled. To the esterified solution was added 5 ml saturated sodium chloride solution and extracted twice with 1 ml hexane. The hexane extract was filtered through 0.45 m membrane filter and injected 1  l to GC-MS using auto injector system.

Chromatographic separation of FAMEs
Gas chromatograph (Shimadzu) hyphenated to a mass spectrometer QP 2010 plus (Tokyo, Japan) equipped with an autosampler (AOC-20S) and auto-injector (AOC-20i) was used for analysis of fatty acid. Helium was used as carrier gas. All chromatographic separations were performed on a capillary column (TRB-FFAP; Technokroma) having specifications: length, 30 m; ID, 0.35 mm; thickness, 0.250 µm; treated with polyethylene glycol. Other GC-MS conditions are ion source temperature (EI): 250°C, interface temperature: 240°C, pressure: 100 KPa, solvent cut time: 1.6 min. One microlitre of sample and standard were injected into the GC column. Injector was operated in a split mode with a split ratio 1:50. Injection temperature was 240°C. The column temperature program started at 50°C for 1 min and changed to 150°C at the rate of 15°C min -1 . The temperature was raised to 175°C at the rate of 2.5°C/min and held for 5 minutes. Then the temperature was increased to 220°C at the rate of 2.5°C/min and kept constant for 5 minutes. Total elution time was 45 minutes. MS scanning was performed from m/z 85 to m/z 380. GC-MS solutions software provided by the supplier was used to control the system and to acquire the data. Identification of the compounds was carried out by com-paring the mass spectra obtained with those of standard mass spectra from the NIST library (NIST 05) (Zhao et al., 2006).

Urease assay and inhibition
Reaction mixtures comprising 25 μl of enzyme (jack bean urease) solution and 55 μl of buffers containing 100 mM urea were incubated with 5 μl of test compounds (0.5 mM concentration) at 30°C for 15 min in 96-well plates. Urease activity was determined by measuring ammonia production using the indophenol method as described by weather burn. Briefly, 45 μl each phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprussside) and 70 μl of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The increasing absorbance at 630 nm was measured after 50 min, using a microplate reader (Molecular Device, USA). All reactions were performed in triplicate in a final volume of 200 μl. The results (change in absorbance per min) were processed by using softMax Pro software (molecular Device, USA). The entire assays were performed at pH 6.8. Percentage inhibitions were calculated from the formula 100-(ODtestwell/ODcontrol) × 100. Thiourea was used as the standard inhibitor of urease (Arfan et al., 2010).

Antibacterial assay
Three strains of Gram-positive bacteria (Staphylococcus aureus, Strap epidermis and Bacillus subtilis) and two of Gram-negative bacteria (Escherichia coli and Klebsiella pneumonia) is earlier discuss (Uddin et al., 2011b), obtained from PNRL laboratories, Institute of Chemical Sciences, University of Peshawar, Peshawar, Pakistan were used in the present study. These organisms were kept in Müller-Hinton agar in the refrigerator at 4°C, prior to subculture. Tests for susceptibility were done using modified agar well diffusion method to test the antibacterial activity of the oil. The Muller-Hinton agar was used as medium. The cultures were taken in triplicates at incubation temperature of 37°C for 24 to 72 h. The broth culture (0.6 ml) of the test organism was placed in a sterile Petri-dish to which 20 ml of the sterile molten Muller-Hinton agar was added. Holes were bored in to the medium using 0.2 ml of the oil. Streptomycin was the standard antimicrobial agent at a concentration of 2 mg/ml. Inoculation was done for 1 h to make possible the diffusion of the antimicrobial agent into the medium. Incubation was done at 37°Cfor 24 h and the diameters of the zone of inhibition of microbial growth were measured in the plate in millimeter.

Insecticidal activity
Oil isolated from fruits was investigated against various insects such as Tribolium castaneum, Rhyzopertha dominica and Callosobruchus analis according to standard protocol (Uddin et al., 2011c). The test sample was prepared by dissolving 1019.10 µg/cm 2 of oil in 3 ml acetone and loaded in a Petri dish covered with the filter papers. After duration of 24 h, 10 test insects were put in each plate and incubated at temperature 27°C for 24 h duration with 50% relative humidity in growth chamber. The results were evaluated as percentage mortality, calculated with reference to the positive and negative controls. In this bioassay study, permethrin was used as a standard drug, while permethrin, acetone and test insects were used as positive and negative controls. The percentage mortality was calculated by using the formula:

Cytotoxicity bioassay
Method used in current study is very simple for determination of cytotoxicity of oil of W. somnifera. In this bioassay, artificial "sea water" was prepared by dissolving 38 g sea salt per liter of double distilled water and filtered "Sea water" was put in a small tank, and brine-shrimp eggs (1 mg) (Artemia salina) was added and was kept covered by covering with aluminum foil to provide darken condition. It was allowed to stand for duration of 24 h at 25°C which provided a large number of larvae. Twenty milligrams of the concentrated sample was dissolved in 2 ml CHCl3 (20 mg/2 ml) and transferred to 500, 50 and 5 μl vials corresponding to 1000, 100 and 10 μg/ml, respectively. Then three replicates were prepared for each concentration making a total of nine vials. The material in these vials was concentrated, dissolved in DMSO (50 μl) and 5 ml "sea water" added to each. Then 10 shrimps were added to each vial, and were allowed to stand for 24 h duration, the result was calculated and the number of surviving shrimps was noted. Etoposide was used as positive control .

DPPH free radical scavenging assay
Twenty-five milligrams of oil was taken and dissolved in distilled methanol and diluted up to 50 ml as discussed earlier (Uddin et al., 2012). From this stock solution different micrograms solution of 10, 20, 40, 60, 80 and 100 µg/ml were prepared by dilution method. 5 ml of each solution was taken in a test tube and 1 ml of 0.001 M of DPPH solution was added to it. All these solutions were kept in dark for 30 min. Also, 5 ml methanol was taken and 1 ml of DPPH solution was added, for control solution. At the end of incubation period the mixtures were examined for the antioxidant activity using Optima UV-Visible spectrophotometer at wavelength of 517 nm (Uddin et al., 2011c). The experiments were performed with triplicate readings. Percent DPPH was determined using the formula as follows: RSA (%) = Control abs -Extract abs × 100/Control.

RESULTS AND DISCUSSION
The GC-MS analysis of the fruits of W. somnifera resulted in the identification of total 8 components as presented in the Tables. The major constituents among the identified constituents were linoleic acid, methyl ester (11.247%), linoleic acid, ethyl ester (4.000%), palmitic acid, methyl ester (2.842%) and tetracosanoic acid, methyl ester (0.880%). The remaining detail of the chemical composition is shown in Table 1. Total of eight components were identified using GC-MS analysis. The major constituent of the fruits fixed oils were palmitic acid (0.42%), lenoleic acid (0.23%), oleic acid (0.14%), stearic acid (0.10%), myristic acid (0.03%), hexanoic acid (0.01%), lauric acid (0.01%), and elaidic acid (0.01%). The IR spectroscopy of oils was recorded which it showed the presence of different functional groups. The IR gives broad signal at 33950, 3340 and 3299 which indicate the presence of OH stretching. The IR gives strong signals at 2997, and 2990 which indicated the presence CH stretching saturated. A weak signal observed at 2884 indicates the presence of carboxylic acid. The strong signal observed at the 1695 indicate the presence of C=O. After IR characterization the fixed oil was subjected to different biological activities such as antifungal, antibacterial, urease inhibition and DPPH radical scavenging activities (Table 2 to 7). The fixed oil shows dose dependent response in DPPH radical scavenging activity, which indicates that fixed oil, has good antioxidant activity ( Table 2). The percent inhibitions of fixed oil against antioxidant at different   concentrations are presented in Figure 1. The antifungal activities of the fixed oil are listed in Table 3, which showed weak inhibition against the two strains of fungi. The fixed oil showed percent inhibition of 25 and 20 against the Microsporum canis and Fusarium solani, respectively. The cytotoxicity and phytotoxicity profile of fixed oil shows that it is non-toxic as compared to standard toxic drug (Tables 4 and 5). The fixed oil was  Table 6. Antibacterial profile of oil extracted from Withania somnifera.  also evaluated for the antibacterial activity against the three strains of bacteria (Escherichia coli, Bacillus subtillis, Shigella flexenari). The fixed oil showed weak inhibition of 7 and 8 against E. coli and B. subtilis, respectively at 25 µg disc ( Table 6). The fixed oil showed good inhibition with 39.0% against urease enzyme inhibition studies. For the first time, the urease inhibitory activity of fixed oil from W. somnifera was reported. Further research on isolation of fixed oils may result in a leading research in the field of drug discovery.