Study of bioactivities of lipid content of fresh Lagenaria siceraria seeds pulp and identification of its chemical constituents

The aim of the present work was to analyse antimicrobial activities of the lipid content of seeds pulp of the fresh fruit of Lagenaria siceraria and identification of its chemical constituents by gas chromatography-mass spectrometry (GC-MS). Methanolic extract of the seeds pulp exhibited moderate to good efficacy against Achromobacter xylosoxidans, Pseudomonas aeruginosa, Bacillus megaterium and Escherichia coli having zone of inhibition (ZOI) 21.5, 20.0, 17.5 and 15.0 mm, respectively, at concentration 100 mg/ml. It showed better potency than antibiotic amoxylin against Enterobacter aerogenes, P. aeruginosa and Bacillus megaterium. The hexane fraction, that contained most of the lipid content, exhibited highest lethality against all the test microorganisms with ZOI 16.0 to 30.0 mm. It displayed highly significant efficacy against B. megaterium and Salmonella typhi with ZOI 30.0 and 22.5 mm, respectively. Minimum inhibitory concentration (MIC) of hexane fraction against B. megaterium and A. xylosoxidans was 0.40 mg/ml, while for others it ranged from 0.80 to 2.80 mg/ml. Palmitic acid exhibited significant potency against Staphylococcus aureus (ZOI, 20.0 mm, at concentration of 40 mg/ml), and moderately good efficacy against Stenotrophomonas maltophilia, Salmonella typhi and Enterobacter cloacae. Most compounds identified in the seeds pulp through GC-MS analysis were fatty acids or their esters. The compounds present in considerable concentration included azelaic acid (31.0%), (Z,Z)-9,12-octadecadienoic acid methyl ester (27.75%), and palmitic acid methyl ester (13.0%). Major component of ethyl acetate fraction was 4-hydroxybenzenemethanol (19.28%), while chloroform fraction contained 4-methoxymethylphenol (6.42%) and pentanoic acid 2-hydroxy-4-methyl-methyl ester (6.75%) as major constituents. The lipid content of seeds pulp of Lagenaria siceraria fruit contains components which are capable to inhibit bacterial growth, and the ones with strong anti-oxidative potential.


INTRODUCTION
As pathogens soon develop resistance against a drug, continuous efforts are required on the part of scientists to discover new and more effective medicines (Iwalokun et al., 2004).The challenge has provided a fresh impetus to investigate plants for alternative therapies.As a result, the past few decades have witnessed numerous publications on the subject from all parts of the world (Dvorkin and Whelan, 2008;Dilhuydy, 2003;Gülçin and Nurten, 2003).
Plants not only constitute an endless reservoir of a wide variety of natural products, they also provide easily affordable remedies to common people.Lagenaria siceraria is an important plant of family Cucurbitaceae, which has 120 genera and 825 species (Perveen and Qaiser, 2008).L. siceraria (or, bottle gourd in English) is a vigorous, annual, running or climbing vine with large green leaves and white flowers.The fruit is green in appearance with size varying from 2 to 12 inches in diameter and from 4 to 40 inches in length (Stephens, 2014).Seeds are numerous which are imbedded in the fleshy pulp.The fruit of the plant is a choice vegetable in Pakistan and India.The fruit of L. siceraria is used, in traditional medicine, as remedy for different diseases, which include fever, jaundice, cough, pectoral, asthma and other bronchial disorders (Shah et al., 2010;Aslam and Najam, 2013;Prajapati et al., 2010).The leaf, fruit peel and seed extracts of the plant have been shown to possess antimicrobial activities against a number of microorganisms (Goji et al., 2006;Badmanaban and Patel, 2010;Rodge and Biradar, 2012;Nagaraja et al., 2011).In phytochemical studies, a variety of chemical constituents have been isolated, which include flavonoids, triterpenoids and glycosides (Kumar et al., 2012;Gangwal et al., 2010).Recently, a number of phenolic glycosides have also been reported (Jaiswal and Kuhnert, 2014).
The present work reports, for the first time, study of antimicrobial activities of lipid content from the seeds pulp (fleshy pulp with imbedded seeds) of the fresh fruit against different bacterial strains, and identification of phytochemicals present in it through gas-chromatographmass spectrometer (GC-MS).Isolation and characterization of palmitic acid and quercetin has also been done.

Collection and preparation of plant material
The young (edible age) and fresh fruit of L. siceraria was collected in Pattoki, Pakistan, in April, 2013.The seeds pulp (the interior fleshy pulp with numerous imbedded seeds) was carefully separated.It was ground to obtain a paste like material, which was soaked in methanol for 15 days to extract phytochemicals.The dried methanolic extract was fractionated into solvents of different polarity.As a result, hexane, chloroform, ethyl acetate, n-butanolic and residual aqueous fractions were obtained.The methanolic extract and its fractions were concentrated on rotary evaporator under reduced pressure, and were stored in refrigerator in airtight flasks.

Microorganisms
In this study, both Gram-positive and Gram-negative bacterial Chaudhery et al. 1015 strains were used.The Gram-positive bacteria included Staphylococcus aureus and Bacillus megaterium.The Gramnegative bacteria were Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Achromobacter xylosoxidans, Stenotrophomonas maltophilia, Enterobacter cloacae, and Enterobacter aerogenes.These bacterial strains were obtained from biotechnology lab, Forman Christian College, Lahore, and University of Veterinary Sciences, Lahore.The microorganisms maintained on nutrient agar were stored at 4°C.

Determination of zones of inhibition
The antimicrobial activities of the methanolic extract and its fractions were carried out using agar well diffusion assay according to the method recommended by Clinical and Laboratory Standards Institute (CLSI, 2005).Briefly, 4 to 5 freshly grown, identical bacterial colonies were transferred into a test tube containing 5 ml of 5% sterilized normal saline (NS) solution and its turbidity was matched with 0.5% McFarland solution as a standard.The bacterial suspensions were swabbed by using sterilized cotton swabs onto the well-mixed and autoclaved nutrient agar (NA) medium (20 ml each) in a Petri plate (90 mm).After this, four wells were made at equal distances with a sterilized cork borer (6 mm).Then, each of these wells was filled with 140 μl of the fruit extract/fraction in dimethyl sulphoxide (DMSO).Antibiotic drugs amoxicillin, cefixime and levofloxacin were used as positive controls while DMSO was employed as negative control.The zones of inhibition (ZOI) of the fruit samples were measured in millimeters after incubation for about 12 h at 37°C.

Determination of MIC by agar dilution assay
The minimum inhibitory concentration (MIC) of an antibiotic drug against a microorganism is its lowest quantity that is sufficient to inhibit the growth of that microorganism.The MICs of L. siceraria seeds pulp (fleshy pulp with imbedded seeds) in the present study were determined by using agar dilution assay according to the standard protocol (Anonymous, 2000).Solutions of fruit extract/fractions of different dilutions were prepared in agar (MHA) in 90 mm Petri plate.For inoculum, 4 to 5 colonies of a culture were introduced into a test tube containing 5 ml of 5% sterilized normal saline solution and the mixture was compared with 0.5% McFarland solution to adjust the density of bacterial suspension.The inoculum of each bacterium was poured into the wells dug in the sample containing MHA.The growth of the bacteria was observed after incubation for overnight at 37°C and the MICs were noted.

GC-MS analysis
To identify the nature of its volatile chemical constituents L. siceraria seeds were subjected to GC-MS analysis on Agilent Technologies GCMS (7890 A) equipped with an HP-5 (length 30 m, i.d.250 μm, film thickness 25 μm).MS fused sample (1/100, v/v in methanol) of 1.0 μl were injected manually in the split mode (1:100).The relative percentage amount of each component was calculated by comparing its average peak area to the total areas.Mass spectrometer was Agilent Technologies (5975C) system working at 70 eV; scan time 1.5 s and oven temperature was 35 to 320°C with injector temperature of 180°C; mass range 50 to 600 amu.Software for mass spectra and chromatograms was NIST 05 spectral library.

Isolation of palmitic acid and quercetin
Palmitic acid and quercetin were isolated from hexane fraction of  Elution started with pure hexane followed by a series of hexaneethyl acetate mixtures and ultimately pure ethyl acetate.The eluted fractions with similar thin layered chromatography (TLC) behavior were combined.As a result, 14 fractions (F1-F14) were collected.
The first sub-fraction (F1) obtained on elution with 100% hexane yielded palmitic acid as major component when chromatographed on TLC (silica gel; hexane: ethyl acetate, 7:3), with R F 0.58.Palmitic acid was converted into its methyl ester by treating with a mixture of methanol and boron trifluoride.The methyl ester was then characterized through gas chromatography-mass spectrophotometer (GC-MS).F6, obtained at mobile phase hexane-ethyl acetate (50:50), had considerable quantity (1.2 g).It was subjected to saponification with alcoholic KOH, and non-saponifiable component was extracted into hexane leaving behind aqueous saponified material.Upon evaporation of hexane, greenish viscous liquid (600 mg) was obtained (F6B).It was loaded on a small column with silica gel as adsorbent and hexane -ethyl acetate mixtures as mobile phase, to afford seven sub-fractions (F6B1-F6B7).F6B6, obtained at mobile phase hexane-ethyl acetate (1:4; v/v), produced a single spot on 2-D TLC, which subsequently proved to be quercetin upon further purification by TLC and characterization.

Statistical analysis
All assays to determine bioactivities were conducted in triplicate.
The data was subjected to one-way analysis of variance (ANOVA) and results were expressed (where appropriate) as mean ± standard deviation.

RESULTS AND DISCUSSION
Antibacterial activity of methanolic extract of the seeds pulp of the fresh fruit of L. siceraria and its fractions in different solvents were determined against a number of bacteria using standard assays and the results are displayed in Tables 1 to 3  xylosoxidans was 0.40 mg/ml, while for other test microbes it ranged from 0.80 to 2.80 mg/ml.The remaining fractions showed moderate efficacy.The significant antimicrobial activity of hexane fraction may be attributed, at least in part, to different fatty acids and their esters, which were shown to be abundantly present in the fraction by GC-MS analysis.These phytochemicals are known to have antimicrobial activity (Som and Radhakrishnan, 2011;Desbois and Smith, 2010).

Identification of palmitic acid
Identification of palmitic acid was established through GC-MS analysis of its methyl ester.Melting point of the acid was 63°C, and UV-Visible spectrum showed lambda max at 210 nm, which was quite close to the standard value.The IR spectrum of isolated palmitic acid showed strong peak at 1710 cm -1 which indicated the presence of carbonyl (C=O).C-H stretching appeared between 2800 to 2900 cm -1 , -OH at 3000 to 3100 cm -1 , and >CO at 1240 cm -1 .

Antibacterial activity of palmitic acid
Antibacterial activity of palmitic acid was studied against E.

Antioxidant activity of palmitic acid
The DPPH radical method was used to determine antioxidant or free radical scavenging activity of palmitic acid (Figure 1).The activity was 12.49%, which was quite low as compared to the standard ascorbic acid 90.37% value.Palmitic acid is, thus, not a good antioxidant.

Identification of quercetin
Quercetin was obtained as yellowish green powder.Its Rf value on preparative TLC [silica gel; ethyl acetate (100%)], high performance liquid chromatography (HPLC) profile, melting point (315°C) and spectral data were similar to that of the standard.The lambda max of the isolated compound was 359 nm which is quite close to the literature value.The IR spectrum of isolated compound showed the strong absorption at 3200 cm -1 (O-H), 1663 cm -1 (C=O), and 1240 cm -1 (C-O).

Identification of phytochemical through GC-MS
Gas chromatography-mass spectrometry (GC-MS) is a technique commonly used for identification of chemical compounds with low boiling points.Different fractions of seeds pulp (fleshy pulp with imbedded seeds) of L. siceraria were subjected to GC-MS analysis to identify volatile phytochemicals present in them, and the results are included in (Tables 5 and 6).As the table illustrate, most compounds identified in the seeds pulp through GC-MS analysis were different fatty acids or their esters.The compounds present in considerable concentration included azelaic acid (31.0%), (Z,Z)-9,12-octadecadienoic acid methyl ester (27.75%), and palmitic acid methyl ester (13.0%).Major component of ethyl acetate fraction detected by GC-MS was 4-hydroxybenzenemethanol (19.28%), while the chloroform fraction contained 4methoxymethylphenol (6.42%) and 2-hydroxy-4-methylpentanoic acid methyl ester (6.75%) as major constituents.As the present work displayed, the lipid content of seeds pulp of L. siceraria fruit contains components which are capable to inhibit bacterial growth.It also contained constituents with strong anti-oxidative potential.

Conclusions
The lipid content of seeds pulp of L. siceraria fruit contained a wide variety of fatty acids and their esters.The lipid content had components which were capable to inhibit bacterial growth.It also contained constituents with strong anti-oxidative potential.The study provides a rational ground for the ethnomedicinal use of the fruit for different therapeutic purposes.

Table 1 .
Comparison of antibacterial activity of methanolic extract of seeds pulp of L. siceraria with standard antibiotics against different bacterial pathogens in terms of zones of inhibition (mm) at concentration 100 mg/ml.

Table 2 .
Comparison of antibacterial activity of the different fractions of seeds pulp of L. siceraria against different bacterial strains in terms of zones of inhibition (mm) at the concentration of 100 mg/ml.

Table 3 .
Minimum inhibitory concentrations (MICs, mg/mL) of methanolic extract of L. siceraria seeds pulp and its fraction in different solvents against various bacterial strains.

Table 4 .
The antibacterial activities of palmitic acid isolated from hexane fraction of seeds pulp of L. siceraria against some bacteria in terms of zones of inhibition (mm).Concentration of the sample was 40 mg/ml.

Table 5 .
Identification of compounds in lipid content of hexane fraction of seeds pulp of L. siceraria through GC-MS analysis.Table5.Identification of compounds in lipid content of hexane fraction of seeds pulp of L. siceraria through GC-MS analysis.

Table 6 .
Identification of compounds in chloroform and ethyl acetate fractions of seeds pulp of L. siceraria through GC-MS analysis.