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GC-MS Analysis of Fixed Oil from Nelumbo nucifera Gaertn Seeds: Evaluation of Antimicrobial, Antileishmanial and Urease Inhibitory Activities.

Byline: Shahnaz, Hidayatullah Khan, Farman Ali, Nasir Mehmood Khan, Afzal Shah and Shafiq Ur Rahman

Summary: In the present study, chemical composition of fixed oil (NnFO) obtained from Nelumbo nucifera seeds was determined by GC-MS analysis which revealed the presence of 39 compounds mainly comprised of 20.8 % keto fatty acids with high content of methyl ester of palmitic acid (13.59 %) and methyl ester of 9-oxo-nonanoic acid (11.89 %). The other major constituents identified were; fumaric acid-3-methylbut-3-enyl nonyl ester, 2-decenal and methyl ester of 9E- octadecenoic acid as 6.45 %, 5.09 %, 5.06 %, respectively. NnFO along with other fractions were tested for in vitro antimicrobial, antileishmanial and urease inhibitory assays. NnFO showed weak antibacterial activities against the tested bacteria while promising antifungal effect against Candida albicans (68 %), Candida glaberata (65 %) and Aspergillus flavus (64 %). NnFO showed strong antileishmanial activity with IC50 = 7.34 +-0.72 as compared to reference drug (5.1+- 0.29) probably due to the presence of keto-ene derivatives.

NnFO showed weak urease inhibitory activity while the ethyl acetate fraction (N3) strongly inhibited both J.B. urease (IC50= 21.45 %) and B.P. urease (IC50=28.65%) respectively. In conclusion, N. nucifera seeds fixed oil possess promising therapeutic potential as a new antifungal and antileishmanial agent.

Key words: Nelumbo nucifera, Fixed Oil, GC-MS analysis, Antimicrobial and antileishmanial activities, Jack Bean (J.B.) urease and Bacillus Pasteurii (B.P.) Urease inhibition.

Introduction

Nelumbo nucifera Gaertn (Nelumbonaceae) is commonly known as Indian lotus, Chinese water lily and sacred lotus. N. nucifera is widely dispersed in South East Asia [1]. Various parts of this plant have been used in Traditional Chinese Medicine and ayurvedic folk lore in treatment of various diseases e.g. epistaxis, haemoptysis, leucoderma, smallpox, dysentery, hepatopathy, spermatorrhoea, haematemesis, haematuria, metrorrhagia, hyperlipidaemia, skin diseases, leprosy and nervous exhaustion. It has also been used in the cure of tissue soreness, malignancy, skin diseases, and leprosy and as anticancer [2]. Different extracts of this plant exhibit anti-obesity, lipolytic, hypocholesterolaemic, antipyretic, hepatoprotective, hypoglycaemic, anti- inflammatory and diuretic activities [3].

It has been reported that seeds of this plant showed potent effect against enteritis, chronic diarrhea, insomnia, and find uses as antiemetic, diuretic and refrigerant [4]. Lotus seedpods and ethanolic extracts were found to reduce the cell explosion and cytokines in main individual tangential blood mononuclear cell and having haemostatic function [5]. Lotus seeds have also been used in Traditional Chinese Medicine in the remedy of neural diseases, restlessness fever and coronary heart diseases [6]. In our current investigation, we present the chemical composition of fixed oil (NnFO) obtained from seeds of Nelumbo nucifera Gaertn along with antimicrobial, antileishmanial and urease inhibitory activities compared to its various fractions (crude, N1-N4).

Experimental

Chemicals

Chloroform, n-butane, n-hexane and ethyl acetate, methanol, DMSO, Brine shrimps, sea salt, sabouraud's dextrose agar, glucantime, urea, thiourea, J. B. and B. P. urease etc were purchased from sigma. All the chemicals were of analytical grade.

Plant Collection and Identification

Mature and fully dried seeds of N. nucifera Gaertn were obtained from a local supplier (public market) in Bannu city. Further taxonomic identification was made by Prof. Abdur Rehman, Department of Botany, Govt. Post Graduate College, Bannu, KP, Pakistan. A voucher specimen (BG-201) has been submitted to the herbarium of University Science and Technology, Bannu, KP, Pakistan.

Extraction and Fractionation

The shade dried out material was chopped and ground to powder. The plant material (1 Kg) was soaked in methanol with occasional shaking, at room temperature for 15 days, followed by filtering off the methanolic extract. The filtrate concentration was carried out at low temperature (40 degC) and under vacuum in a rotary evaporator in order to obtain blackish gummy crude (250 g). The crude methanolic extract was suspended in distilled water (500 mL) and successively portioned with n-hexane (3 x 500 mL), chloroform (3 x 500 mL), ethyl acetate (3 x 500 mL), n-butanol (3 x 500 mL) to yield n- hexane (N1, 25 g), chloroform (N2, 45 g), ethyl acetate (N3, 55 g) and n-butanol (N4, 50 g) soluble fractions respectively.

Extraction of Fixed Oil

Dried and finely chopped plant material (10 g) of N. nucifera was extracted with n-hexane in a Soxhlet apparatus for 4 hrs. The extract was concentred under reduced pressure on a rotary evaporator and the residue (1 g) was dried in an oven at 105 degC to obtain blackish oil (NnFO, 100 mg) [7].

Sample Preparation

For GC-MS analysis, 1 mL of the sample was dissolved 10 mL n-hexane and 2 mL 2M methanolic KOH was added. The solution was mixed well and allowed to stand for 10 min. A blank sample was prepared and analyzed alongside the samples [8].

Gas Chromatography-Mass spectrometry (GC-MS)

GC-MS analysis was carried out on a 6890N Agilent gas chromatography coupled with a JMS 600 H JEOL mass spectrometer. The compound mixture was separated on a fused silica capillary SPBI column, 30 m x 0.32 mm, 0.25 um film thicknesses in a temperature program from 50 to 256 degC with a rate of 4 degC /min with 2 min hold. The injector was at 260degC and the flow rate of the carrier gas helium, was 1 mL/min. The EI mode JMS 600 H JEOL mass spectrometer have ionization volt of 70 eV, electron emission of 100 uA, ion source temperature of 250degC and analyzer temperature of 250 degC. Sample was injected manually in split mode. Total elution time was 45 min. MS scanning was performed from m/z 85 to m/z 390 [9].

Antibacterial Assay

Antibacterial assay was carried out by slight modification of agar disc diffusion process [10]. Bacterial strains used in this assay were Escherichia coli (ATCC 15224), Bacillus subtillis (ATCC 6663), Staphylococcus aureus (ATCC 29213), Shigella flexenari (ATCC 14028), Acinotobactor species (Clinically isolated, obtained from Microbiology Lab, Agha Khan University Hospital, Karachi), Klebsiella pneumonia (ATCC 138839), Solmenella typhi (ATCC 19430) and Micrococcus luteus (ATTC 10240). 1 mg/mL stock solution of experimental materials was prepared in sterilized DMSO and then diluted with 100 uL and 200 uL by addtion to particular wells. The control contained 100 uL and 200 uL DMSO. Clarithromycin was used as positive control. Petri plates were placed at room temperature to allow diffusion and then incubated at 37 degC for 24 hr. Diameter of the zones of inhibition was measured in mm. Minimum Inhibitory Concentration (MIC) of antibiotics was carried out by standard micro broth dilution method [11].

Antifungal Activity

The antifungal assay of the plant extracts was screened out by agar dilution method. Fungal strains used in this study were Aspergilus flavus (ATTC 32611), Aspergius niger, clinically isolated, obtained from Microbiology Lab, Agha Khan University Hospital, Karachi), Candida albicans (ATTC 2091), Trichophyton longifusus (ATTC 22397), Microsporum canis (ATTC 11622) and Candida glaberata (ATTC 90030). The samples (24 mg/mL) were dissolved in sterile DMSO [12]. Fungal growth was inhibited and was observed after 7 days at 37 degC. To control humidity, the samples were placed an open container of water in an incubator (40-50%). Fungal growth inhibition and linear growth was measured and calculated with respect to negative control.

Antileishmanial Assay

Solution for antileishmanial assay was prepared by dissolving 2 mg/mL of each N. nucifera extracts and derived fractions in 1 mL of DMSO (dimethyl sulfoxide). Stock solutions were further diluted serially (2000, 1000, 500, 250, 125, 62.5, 31.25 and 15.125 ug/mL) using DMSO. Samples were filtered by using a 0.2um syringe filter. The promastigotes form of the Leishmania tropica KMU 25 (isolated from a patient and identified by Khyber medical University, KP, Pakistan) were grown in M199 medium with 10% fetal bovine serum (FBS), HEPES buffer, streptomycin, and penicillin. Log phase promastigotes at 2x106/100 mL were used for the entire assay. Here, DMSO was used as a negative control while glucantime as positive control. Afterwards, microtitter plate was incubated at 24degC for 72 h. After incubation, about 20 ul of each dilution was pippeted on a neubauer counting chamber and viable cells were counted under a microscope [13].

Urease Inhibition Assay

Urease inhibitory assay was carried out by determination of ammonia production by indophenol's method. 100 uL of Jack bean urease (J.B. urease) and Bacillus Pasteurii urease (B.P urease) were mixed in 300 uL buffer and 100 mM urea and then incubated at 30 oC for 15 min. Briefly, 200 uL each of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 300 uL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The absorbance was measured after 50 min at 630 nm. Reactions were carried out in 1000 uL final volume in triplicate [14].

Results and Discussion

GC-MS Analysis of Fixed Oil

The TIC and GC chromatogram (Fig-1) shows the identification of thirty nine (39) compounds, nine of which including stigmasterol derivatives have <1 % abundance, were not included. The scan numbers, retention time, compound names and their percentage abundances/quantities as well as other related results have been provided in Table-1. Identification of the components of the fixed oil were based on the computer evaluation of mass spectra of sample through NIST based AMDIS (Automated mass spectral deconvolution and identification software), direct comparison of peaks and retention time with those for standard compounds as well as by following the characteristic fragmentation patterns of the mass spectra of particular class of compounds.

The assessments reveal the presences of various constituents of fatty acids including fatty acid ketones, aldehydes, ketodienes and methyl esters. The major constituent being was found methyl ester of palmitic acid (13.59 %) while methyl ester of 9- oxo-nonanoic acid, 3-methylbut-3-enyl nonyl ester of fumaric acid, 2-decenal , methyl ester of 9- octadecenoic acid, methyl ester of octanoic acid and 2-undecenal and were found to be 11.89 %, 6.45 %, 5.09 %, 5.06 %, 4.53 % 4.53 % and 3.964 respectively. The keto fatty acids comprised 20.8 % of the oil.

Table-1: GC-MS analysis of fix oil from seeds of Nelumbo nucifera (NnFO).

S. No###R.I###Compound name###M.F###%age abundance

1###6.029###2-Heptenal###C7H12O###1.12

2###9.326###4,5-dimethyl-2-Hepten -3-ol###C9H18O###1.69

3###9.527###Nonanal###C9H18O###2.26

4###9.92###Methyl ester of octanoic acid###C9H18O2###4.53

5###11.99###2-Sec-Butylcyclohexanone###C10H18O###1.58

6###12.259###2-Decenal###C10H18O###5.09

7###13.069###2,4-Decadienal###C10H16O###1.69

8###13.283###Methyl 8-oxooctanoate###C9H16O3###2.26

9###13.596###2-Ethyl-2-hexen-1-ol###C8H16O###1.58

10###13.743###2-Undecenal###C11H20O###3.96

11###13.954###3-methylbut-3-enyl nonyl ester of fumaric acid###C18H30O4###6.45

12###14.722###Methyl ester of 9-oxo-nonanoic acid###C10H18O3###11.89

13###16.631###Methyl 10-oxo-8-decenoate###C11H18O3###3.964

14###18.054###1-Ethyl-3,cis-(1,1-dimethylethyl)-4,cis-methoxycyclohexan-1-ol###C13H26O2###1.132

15###18.312###Methyl tetradecanoate###C15H30O2###1.35

16###18.438###1,6-Octadiene, 3-ethoxy-3,7-dimethyl-###C12H22O###1.69

17###22.644###Hexadecanoic acid, methyl ester###C17H34O2###13.59

18###23.698###Hexadecanoic acid###C16H32O2###1.359

19###29.265###9,12-Octadecadienoic acid (Z,Z)-, methyl ester###C19H34O2###2.81

20###29.745###9-Octadecenoic acid, methyl ester###C19H36O2###5.06

21###31.215###Octadecanoic acid, methyl ester###C19H38O2###1.79

22###36.141###9,12-Octadecadienoyl chloride###C18H31ClO###1.38

23###36.771###Oxiraneundecanoic acid, 3-pentyl-, methyl ester, cis###C19H36O3###1.77

24###37.23###Eicosanoic acid, methyl ester###C21H42O2###2.90

25###37.625###Z-8-Methyl-9-tetradecenoic acid###C15H28O2###2.36

26###38.054###cis-13-Eicosenoic acid###C20H38O2###1.12

27###39.837###Docosanoic acid, methyl ester###C23H46O2###1.46

28###40.02###1,2-Benzenedicarboxylic acid, diisooctyl ester###C24H38O4###3.37

29###40.81###21-methyldocosanoate###C24H48O2###1.57

30###41.7###Tetracosanoic acid, methyl ester###C25H50O2###1.34

###Others###----###6.15

Table-2: Antimicrobial activity of Nelumba nucifera crude, fractions (N1-N4) and fixed oil (NnFO).

###Bacteria###Z.I Standard###MIC mg/mL###Crude Extract###N1###N2###N3###N4###NnFO

###(Clarthromycin)###Z.I###Inh###Z.I###Inh###Z.I###Inh###Z.I###Inh###Z.I###Inh###Z.I###Inh

E. coli###34###0.175###17###50###5###15###----###----###11###32###22###65###5###16

B. subitilis###29###0.212###11###37###----###----###9###31###20###69###15###52###----###----

S. aurous###33###0.171###7###21###----###----###5###15###6###18###----###----###----###----

Shigella flexneri###22###0.236###5###23###----###----###----###----###4###18###8###36###04###10

Acenotobactor###32###0.322###9###28###----###----###3###9###----###----###5###16###---###---

K. pneumonia###30###0.184###----###----###3###10###5###17###----###----###2###7###4###10

S. typhi###35###0.111###5###17###9###26###----###----###7###20###----###----###---###----

M. leteus###28###0.439###----###----###----###----###----###----###9###32###----###----###----###----

Fungi###Standard Drug a (MIC g/mL)###L.G###Inh###L.G###Inh###L.G###Inh###L.G###Inh###L.G###Inh###L.G###Inh

A. Flavus###-----###38###62###80###20###65###35###32###68###45###55###36###64

A. niger###108###95###5###75###25 100###0###100###0###100###0###75###25

C. albicans###25###65###35###100###0###55###45###50###50###50###50###32###68

T. longifusus###78###40###60###90###10 100###0###60###40###90###10###100###0

M. Canis###98###80###20###100###0###98###2###90###10###100###0###100###0

C. glaberata###105###60###40###100###0###100###0###100###0###25###75###35###65

NnFO showed very weak zone of inhibition against the tested bacteria (Both Gram negative and positive) although the rest of fractions showed promising potential. The strongest antibacterial effect was displayed against by B. subtilis by ethyl acetate fractions (N3, 69%) and n-butanol fraction (N4, 65%) against E. coli, followed by moderate inhibition of both bacteria by the crude (37 % and 50%), respectively (Table-2). No work has been carried out on the chemical composition of the fixed oil from N. nucifera seeds up to our knowledge, but the oil from the flowers has shown promising antibacterial effects, especially against Gram positive bacteria. This effect was attributed towards the presence of alkaloids and flavonoids in the fixed oil of these flowers [15]. The results were in close comparison with anti-bacterial effect of other plant seed oils with a slight difference in chemical composition [16].

Most of the plant seeds contain resins and volatile oils such as borneol, camphene, citral, eucalyptol, linalool, phenllandrene, zingiberine and zingiberol phenols, responsible for its potent antimicrobial activities [17].

When tested against selected fungi, NnFO inhibited growth of both the Candida species by 68 % (C. albicans) and 65 % (C. glaberata) as well as Aspergillus flavus (64 %). N4 and N3 inhibited the growth of C. glaberata and A. flavus by 75% and 68% respectively while the crude extract inhibited both A. flavus (62 %) and T. longifusus 60 %. The other fraction showed mild to nil inhibition on the linear growth of various fungi. The results show that the fixed oil possess very promising antifungal activities against Candida species and may be effective in treatment of candidemia, particularly in immune-compromised patients suffered by HIV/HCV [10] and possible cure of dermatophytosis of nails [18].

NnFO showed strongest antileishmanial activity with IC50 = 7.34 +-0.72 (Table-3) as compared to reference drug Glucantime (5.1+- 0.29) against Leishmania tropica promastigotes while N3 exhibited the most potent activity (IC50= 68.88 +-1.27). Leishmaniasis is currently major parasitic problem in Pakistan hence our findings may have a significance importance of the use of N. nucifera seeds oil in treatment of it. The ethyl acetate fraction exhibited very strong activity (IC50 = 6.88) due to the high content of flavonoids and phenolics similar to the oils of Pluchea carolinensis [19] since polyphenols have been verified for their anti-parasitic activity [20]. The relatively higher antifungal and antileishmanial activities of NnFO may be attributed towards higher amount of these keto-enes derivatives in oil. Besides these, high amount of palmitic acid, oleic acid and behenic acid may contribute towards the potency of this oil [21].

Table-3: Antileishmanial activities of crude extract and fractions of N. nucifera (IC50 ug/mL).

###Extracts###IC50 (ug/mL)###Std. error

###Crude extract###252.3###+-38.22

###N1###400.1###+-66.39

###N2###426.3###+-53.32

###N3###6.88###+-1.27

###N4###11.92###+-3.72

###NnFO###7.34###+-0.72

###Glucantime *###5.1###+-0.29

It has been reported that various pathogenic diseases like hepatic coma urolithiasis, hepatic encephalopathy, pyelonephritis, ammonia and urinary catheter encrustation are due to urease inhibition. It plays a virulent role in certain human and animal pathogenesis such as in producing peptic ulcers, gastric carcinoma and development of kidney stones. It has been known that Helicobacter pylori causes gastrointestinal ulcers, under estimation, more than 50% people are infected with H. pylori worldwide [22]. The inhibition of urease activity can prevent H. pylori infection and is considered to be a hopeful remedy for ulcer. Urease inhibitors have been reported to decrease ecological problems [23]. NnFO was tested for urease inhibition activity (Table-4) along with crude and fractions (N1-N4) for further comparison.

No effect was observed for NnFO while N3 and N4 showed strong inhibition against both of the enzymes, Jack bean urease (J.B. urease) and Bacillus Pasteurii urease (B.P urease) with IC50 values of 21.45 +- 0.6, 35.76 +- 0.4 and 28.65 +- 0.3, 44.87 +- 0.2 respectively. Our previous investigation indicated the presence of steroids, terpenoids, alkaloids, and flavonoids in the seeds [24]. Hence these constituents might be responsible for the therapeutic potential of N. nucifera seeds. Use of medicinal plants has proved to be helpful in search for new antimicrobial agents and many useful antibiotics were already isolated from natural medicinal plant [25]. In conclusion, this study reports the antimicrobial, antileishmanial, urease inhibitory activities of the fixed oil from Nelumbo nucifera seeds; thus presenting a proof for diverse therapeutic applications of this medicinal plant as a cure in a broad range of ailments.

Table-4: Urease inhibition activities of crude extract and fractions of N. nucifera (IC50 ug/mL).

###Enzyme###Test Samples###Thiourea

###Crude###N1###N2###N3###N4###NnFO

J.B Urease 46.56+-0.4 --- 87.45+-0.6 21.45+-0.6 35.76+-0.4 ---- 14.13+-0.16

B.P Urease 58.77+-0.07 --- 94.35+-0.3 28.65+-0.3 44.87+-0.2 ---- 21.82+-0.31

Conclusion

This study reports the chemical composition of fixed oil from Nelumbo nucifera seeds and its antimicrobial, antileishmanial as well as urease inhibitory activities with comparison to other fractions. The results reveal that the fixed oil possess very promising antifungal activities against Candida species and may be effective in treatment of infections in bloodstream and major organs (candidemia), particularly in immune-compromised patients suffered by HIV/HCV. The oil also exhibit strong antileishmanial effect and may be used as external healing agent. Thus the report may be presented as a proof for diverse therapeutic applications of this medicinal plant as a cure in a broad range of ailments.

Acknowledgements

The authors are grateful to Higher Education Commission (HEC), Pakistan for providing partial financial support through HEC Indigenous 5000 Ph.D Fellowship Program.

Conflict of interest

The author declares no conflict of interest.

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