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GCMS Analysis and In-Vitro Activities of Monotheca buxifolia (Falc.) A. DC. Fruit.

Byline: Irfan Ullah, Jamshaid Ali Khan, Achyut Adhikari, Peer Abdul Hannan, Faisal Shakeel, Muhammad Kifayatullah, Ajmal Khan and Shafiq Ur Rahman

Summary: The aim of the current study was to evaluate various fractions of Monotheca buxifolia fruit chemically using GC-MS and in vitro antibacterial, antifungal, and leishmanicidal potentials in order to provide scientific evidences for its folk uses. A variety of pharmacologically active compounds were identified including hexadecanoic acid, oleic acid, vaccinic acid, and pthanlic acid. All the tested samples were found fairly effective in antibacterial assay, P. aeruginosa was found more susceptible to ethyl acetate fraction with inhibition of 69.5 %, while B. subtilis was found completely resistant. Similarly the growth of M. canis and F. solani were moderately inhibited by M. buxifolia. The maximum (50%) antifungal effect was observed in case of ethyl acetate fraction against F. solani, While no leishmanicidal activity was found in the entire tested samples. Yet the use of this plant can be validated in the management of different bacterial and fungal infections.

Key words: Antibacterial; Antifungal; GC-MS; Leishmanicidal; Monotheca buxifolia.

Introduction

People in under developed countries are suffering more from infectious diseases as compared to developed countries. Many reports suggest that the disease burden is increasing with each passing day [1]. Attempts are made to introduce new drugs that are more potent, having comparatively low adverse effect(s) and economical [2]. In traditional medicine a number of plants have been used for the treatment of various infectious diseases including Berberis lyceum [3], and Azadirachta indica [4] etc. Drugs derived from plants are commonly used in developing countries by local practitioners called hakims as they are economical and easily available to maximum population, knowledge about these herbal drugs are transferred from generation to generation verbally without any valid scientific information [5], therefore, it is now essential to provide scientific rationale to the folk uses of these plants to validate them as best alternative(s) of the currently used drugs [6-7].

Monotheca buxifolia is a wild short tree of family Sapotaceae, which also bears an edible berry like fruit [8]. It is available in many areas of Pakistan (native) Bunair, Swat, Dir and Darra Adam Khail [9-11]. In local language the fruit of this plant is known as Gurgura [12]. In folk medicine, the fruit has been used as purgative, refrigerant, digestive, vermicidal, laxative, hematinic and antioxidant properties. While it has also been recommended to treat a number of infectious diseases including gastro-urinary disorders while antimony made of its gum in eye infections [8], [13-15]. Previously, the plant has been investigated for the presence of compounds of a number of pharmacologically active chemical classes, hepatoprotective [16], antinociceptive, antipyretic, anti-inflammatory [17], cytotoxic [18] and urease enzyme inhibitory potential(s) [19].

While considering various folk uses, the present study was intended to investigate M. buxifolia crude hydroethanolic extract (MBHE) and its various solvents soluble fractions for antibacterial, antifungal and leishmanicidal potentials.

Experimental

Plant material

Fruits of M. buxifolia were collected from different areas of District Dir, Khyber Pakhtunkhwa (Pakistan) in the month of August and were authenticated by Ghulam Jelani, a taxonomist of University of Peshawar, Peshawar, Pakistan. Specimen has been deposited in the herbarium of Department of Botany, University of Peshawar for future reference, under reference number: Bot. 20061 (PUP).

Extraction and fractionation

Fruits of Monotheca buxifolia were washed with water to remove dust. Seeds were removed and collected. The fleshy pulp was dried under shade in well ventilated place at room temperature. It was then pulverized in chopper (Moulinex, France). Dry powder was subjected for maceration, added sufficient hydroethanolic solvent (30:70), three times to get the maximum soluble portion and then filtered through filter paper (Whatman-1). Solvent was evaporated under reduced pressure using rotary evaporator (BUCHI) at 40AdegC, 1.8 kg semisolid extract was obtained. For fractionation 1.6 kg extract was soaked overnight in distilled water, solvent were added in ascending order of polarity i.e. n-hexane (5L x 3) was added shaken well for proper mixing and separated the n-hexane soluble layer in a separating funnel, other solvents used were choloroform, ethyl acetate, n-butanol while the remaining was considered as the aqueous soluble fraction.

GCMS Analysis

A valid and optimized GC method was applied before proceeding to MS and the same was repeated for each sample respectively. The GC used for analysis was installed with column (fused silica, ZB-5MS), which was connected with MS (JEOL JMS 600-H). The oven temperature for column was set initially as 70AdegC for 3 min. and raised to 150AdegC at a sequential speed of 5AdegC/min., while to 250AdegC at 10AdegC/minute for 10 min. and lastly to 275AdegC at 10AdegC/min. for 30 min. The front injector and detector temperature were set as 240AdegC and 260AdegC respectively. A mode of split ratio 80 eV was set for injection. The carrier gas used was Helium at a flow rate of 1.7 ml/min. The run time for chloroform fraction was 75.01 min, while for ethyl acetate and n-butanol were 75.00, 62.75 min. respectively.

Antibacterial Bioassay

For antibacterial activity, agar well diffusion method was used [20]. In brief, 28 g nutrient agar was dissolved in one liter distilled and de-ionized water and sterilized by autoclaving at 121AdegC for 15 minutes, cooled to 45AdegC and transferred to 14 cm diameter Petri-plates (8mm thick layer) and covered aseptically, allowed to solidify at room temperature.

24 hours old bacterial inoculum containing 104-106 CFU/ml was added and distributed uniformly. Wells were made in the agar with sterilized borer of 6 mm diameter, 100 ul of sample (3mg/ml of DMSO) was added to each agar well and incubated at 37AdegC for 24 hours. DMSO and Imipenim were used as control. All the procedures were performed in triplicate and mean of zones of inhibition was calculated.

Antifungal Bioassay

Antifungal potential of crude extract and fractions was performed against five fungal strains by agar tube dilution method [21]. 24 mg of each sample was dissolved in 1 ml sterile DMSO as a stock solution. Sabouraud dextrose agar (SDA) media was prepared and transferred to screw capped tubes and sterilized by autoclaving at 121AdegC for 15 minutes. Tubes were allowed to cool up to 50AdegC, to that non solidified media test samples 400 ug/ml (66.6 ul) were added from stock solution. Tubes were solidified at room temperature in slanting position. To each tube 4 mm diameter piece of a week old cultured fungus was added. Other media were supplemented with DMSO and standard antifungal drugs served as control. Tubes were incubated at 28 +- 1AdegC for 7 days. Upon completion of incubation period, the tubes were examined for the linear growth inhibition of fungi in millimeters. Percent inhibition was calculated with the reference to negative and positive controls.

Anti Leishmanial Activity

For anti leishmanial activity previously reported leishmanicidal protocol was followed [22], Leishmania major (DESTO) promastigotes were cultured at 22 to 25AdegC in RPMI-1640 (Sigma). The media was added with 10 percent temperature inactivated (56AdegC for 30 minutes) fetal bovine serum (FBS). Promastigote culture in the log phase of development was centrifuged at 2000 revolution per minute for 10 minutes, washed with sterile saline three-times in same condition of experiment. Parasites were diluted to 106 cells/ml by adding recently prepared medium. In a standard micro-titer plate of 96-wells, first row was supplemented with 180 ul of the recently prepared medium while other rows were added 100 ul. Crude extract and fractions (20 ul) was mixed in medium and diluted serially.

100 ul of parasite-culture was added to each well. DMSO and standard drugs were added to other rows served as control. They were incubated at 21-22AdegC for 3days (72 hrs). The living parasites were counted in Neubauer chamber under microscope. Experiment was done in triplicate and results were obtained. IC50 were calculated by using a Windows based EZ-Fit 5.03 Perrella Scientific computer Software.

Result and Discussion

Using GC-MS Chemical analysis, a total 8, 12, and 6 compounds were identified in chloroform, ethyl acetate, and n-butanol fractions respectively. Compounds found in chloroform fraction were n-hexadenoic acid in maximum with percent abundance of 30.33% followed by chondrillasterol, oleic acid, stigmast-7-3n-3-ol, and 13-tetradecenal with a percentage of 11.28, 13.02, 8.52 and 8.11% respectively, rest of the compounds were present in less than 5 percent relative abundance. Similarly the ethyl acetate fraction was dominated by cyclooctaneacetic acid, 2-oxo- with percentage of 11.76% followed by cis-vaccenic acid, cis-11-hexadecandeinal, 4H-pyan-4-one(5-hydroxy-2-(hydroxymethyl))-, Z,Z-10, 12-hexadecandien-1-ol acetate, and eicosanoic acid with percentage of 11.45, 9.35, 8.77, 6.71, and 5.78% respectively.

While in n-butanol fractionthe n-hexadecanoic acid was present in high concentration with 13.72% followed by ester of phtalic acid, cis-7, cis-11-hexadecandien-1-yl acetate, octadecanoic acid, and oxacycloheptadec-8-ene-2-one, (8Z) with relative abundance of 7.66 5.99, 5.88 and 5.11% respectively. Antibacterial potential of MBHE and subsequent solvents soluble fractions was evaluated against various bacterial strains. As shown in the results (tables) that all the samples were found (mild - moderate) active against tested bacterial strains except B. subtilits. Ethylacetate fraction was most active while aqueous fraction was least active comparatively. The percent inhibition was found by comparison of the zones of inhibition of tested samples with the zone of inhibition of imipenem (antibacterial drug, 10 ug/disc).

E. coli was most susceptible to ethylacetate fraction with inhibitory zones 15 mm percent affect 60%, followed by MBHE, n-butanol and aqueous one with inhibitory zones of 12, 11 and 8 mm respectively, while percent inhibition of these fractions were 48, 44, and 32% respectively. Chloroform fraction was least active against E. coli with a zone of inhibition of 6 mm and percent inhibition 24%. The maximum inhibitory potential against S. flexeneri was shown by n-butanol fraction followed ethyl acetate fractions with zones of inhibitions of 14, and 11 mm respectively and their percent inhibitory activity was 50, 46.43 and 22.92%. Mild inhibitory activity of against S. flexeneri was observed in case of MBHE and aqueous one with 7 mm inhibitory and 25% inhibitory potential. S. flexeneri was completely resistant to the antibacterial effect of chloroform fraction.

The maximum anti-bacterial potential in opposition to S. aureus was exhibited via chloroform fraction with zone of inhibition 15 mm and their percent inhibitory effect was 31.25%. S. aureus was mildly inhibited by n-butanol, MBHE and aqueous fraction with zones of inhibition of 11, 9 and 8 mm respectively and 22.91, 18.75 and 16.66% inhibitory effect. S. aureus was resistant to the antibacterial effect of ethylacetate fraction. The maximum antibacterial effect against P. aeruginosa was shown by ethylacetate fraction with inhibitory zone of 16 mm and percent inhibition 69.56%. Comparatively chloroform and n-butanol fractions were having better antibacterial effect against P. aeruginosa with zones of inhibition of 13 mm each and 56.52 percent inhibition respectively. In case of aqueous fraction and MBHE a very low inhibitory activity was observed against P. aeruginosa with zones of inhibition 6 and 5 mm and percent inhibitory effect 26.08 and 21.74% respectively.

MBHE and chloroform showed 10 mm zone of inhibition each with 35.71% effect respectively. Ethylacetate fraction showed a mild inhibitory effect against S. typhi with 5 mm zone of inhibition and percent effect 17.85%. S. typhi was completely resistant to the antibacterial effect of n-butanol and aqueous fraction. It is also clear from the results that B. subtilis was completely resistant to the antibacterial effect of MBHE and fractions. In the antifungal assay MBHE showed 20% inhibition against Fusarium solani, while other strains were resistant to its antifungal effect. n-Hexane fraction exhibited 25% inhibition against Microsporum canis, while no fungicidal activity was noticed against other tested strains. Chloroform extract showed 25% inhibition against F. solani; other strains were resistant to its antifungal property.

Ethylacetate fraction showed a maximum inhibitory activity against F.solani followed by M. canis with percent inhibition of 50 and 10% respectively, while no antifungal activity was observed against other strains. n-Butanol fraction showed maximum antifungal effect against M. canis followed by F. solani with inhibition of 30 and 20% respectively; growth of other strains were not affected by n-butanol fraction. Aqueous fraction was only active against M. canis with 30% inhibitory potential. The MBHE exhibited 20% antifungal potential against F. solani, while no antifungal potential was observed against other strains, while in the antileishmanial study the tested samples didn't show any significant leishmanicidal activity. Their IC50 were more than 100 and no appreciable inhibition was observed.

Table-1: Compounds identified in chloroform fraction.

###S.no###Compound Name###Molecular formula###Mol. Wt.###r.t###% abundance

###1###n-Hexadecanoic acid###C16H32O2###256###27.25###30.33

###2###Oleic acid###C18H34O2###282###29.55###11.28

###3###Octadecanoic acid###C18H36O2###284###29.75###1.77

###4###Pthalic acid, di(2-propylpently) ester###C24H38O4###390###32.98###1.78

###5###13-Tetradecenal###C14H26O###210###35.02###8.11

###6###Chondrillasterol###C29H48O###412###47.18###13.02

###7###Stigmast-7-en-3-ol, (3a, 5a, 24S)-###C29H50O###414###48.57###8.52

###8###A'-Neogammacer-22(29)-ene###C30H50###410###51.42###4.31

Table-2: Compounds identified ethyl acetate fraction.

S.no###Compound Name###Molecular formula###Mol. Wt.###r.t###% abundance

###1###Isoamyl Lactate###C8H16O3###160###6.38###5.35

###2###4H-Pyran-4-one, 5-hydroxy-2-(hydroxymethyl)-###C6H6O4###142###17.07###8.77

###3###Tetradecanoic acid###C14H28O2###228###27.55###3.28

###4###cis-Vaccenic acid###C18H34O2###282###29.63###11.45

###5###Octadecnoic acid###C18H36O2###284###29.82###2.61

###6###Ethyl 9,12-hexadecanoate###C18H32O2###280###29.98###3.24

###7###Z,Z-10, 12-Hexadecandien-1-ol acetate###C18H32O2###280###30.32###6.71

###8###Cyclooctaneacetic acid, 2-oxo-###C10H16O3###184###30.78###11.76

###9###Eicosanoic acid###C20H40O2###312###31.52###5.78

###10###cis,cis-7,10, -Hexadecadeinal###C16H28O###236###31.93###4.13

###11###cis-11-Hexadecenal###C16H30O###238###32.37###9.35

###12###Pthalic acid, di(2-propylpently) ester###C24H38O4###390###30.93###3.65

Table-3: Compounds identified in n-butanol fraction

###S.no###Compound Name###Molecular formula###Mol. Wt.###r.t###% abundance

###1###5-Hydroxymethyl furufural###C6H6O3###126###10.15###4.91

###2###n-Hexadecanoic acid###C16H32O2###256###27.52###13.72

###3###Oxacycloheptadec-8-ene-2-one, (8Z)###C16H28O2###252###29.48###5.11

###4###Octadecanoic acid###C18H36O2###284###29.7###5.88

###5###cis-7,cis-11-Hexadecadien-1-yl acetate###C18H32O2###280###30.25###5.99

###6###Phtalic acid, di92-propylpently) ester###C24H38O4###390###32.85###7.66

Table-4: Antibacterial activity of MBHE and subsequent fractions

###Standard###MBHE###Chloroform###Ethylacetate###n-Butanol###Aqueous

###Name of###Zone of inhibition###Zone of inhibition###inhibition###Zone of inhibition###inhibition###Zone of inhibition###inhibition Zone of inhibition###inhibition###Zone of inhibition###inhibition

###Bacteria###(mm)###(mm)###(%)###(mm)###(%)###(mm)###(%)###(mm)###(%)###(mm)###(%)

###E. coli###25###11###44###6###24###15###60###12###48###08###32

###S. flexeneri###28###7###25###--###--###11###22.92###14###50###7###25

###S. aureus###48###9###18.75###15###31.25###--###--###11###22.91###8###16.66

###P. aeruginosa###23###5###21.74###13###56.52###16###69.56###13###56.52###06###26.08

###S. typhi###28###10###35.71###10###35.71###05###17.85###--###--###--###--

###B. subtilis###50###--###--###--###--###--###--###--###--###--

Table-5: Antifungal activities of MBHE and subsequent fractions.

Name of fungi###Standard###MBHE###n-Hexane###Chloroform###Ethylacetate###n-Butanol###Aqueous

C. albicans###A###--###--###--###--###--###--

A. flavus###B###--###--###--###--###--###--

M. canis###C###--###25###--###10###30###30

F. solani###D###20###--###25###50###20###--

Candida glabrata###E###--###--###--###--###--###--

Table-6: Leishmanicidal activity of MBHE and subsequent fractions.

###Test organism###Sample###IC50 (ug/ml)

###MBHE###>100

###n-Hexane###>100

###Chloroform###>100

###Leishmania major###Ethyl acetate###>100

###(DESTO)###n-Butanol###>100

###Aqueous###>100

###Amphotericin B###0.29 +- 0.05

###Pentamidine###5.09 +- 0.09

The MBHE and subsequent fractions are the loaded sources of anti-bacterial agents. The chloroform fraction containing n-hexadonoic acid, oleic acid as a major components being previously studied for antibacterial effect [23]. The antibacterial effect in chloroform fraction against P. aeruginosa, S. aureus, and S. typhi may be due to presence of these compounds. Similarly antifungal activity against F. solani is also observed for chloroform fraction; which has been previously reported for oleic acid and hexadecanoic acid [24]. The ethylacetate fraction containing cylco-octadecanoic acid, cis-vaccenic acid, cis-11-hexadecanediol and eicosanoic acid being responsible for the antibacterial effect against P. aeruginosa, S. typhi, and S. flexineri [25]. Similarly the antifungal potential of against F.solani, and M. canis is also associated with the presence of fatty acid including cis-vaccinic acid etc [26].

While in n-butanol fraction the prime quantity observed was that of n-hexadecanoic acid, pthalic acid and octadecaoic acid previously found effective againt bacterial and fungal strains [25-26]. A variety of antibacterial are present in the market, these drugs are quite efficacious in diseases caused by pathogens except resistant species. Majority of them are synthetic or semi synthetic derivatives [27].

Studies on medicinal plants are highly appreciated to discover effective therapeutic antibacterial moieties that are safe, potent and efficacious against current resistant species. Many plants have been screened for their antibacterial activity [28]. The aforementioned protocol is significant in the identification of plants and their extracts for antibacterial potential [12],[29]. While the aforementioned fungal strains are significant in human point of view, as these strains are the root causes of various types of infections in human [30]. A variety of diseases caused by Microsporum canis such as Tinea corporis, Tinea faciei , Tinea capitis, dermatophytosis and various mycetomas in human and certain pets [31-33].

Fusarium solani causes invasive mycosis such as onychomycosis in both immunocompromised and immunosuppressed patients, cutaneous hyalo-hyphomycosis, systemic infections with high mortality rate, while in immunocompetent patients causes mycotic keratitis when the fungi get penetrates through wounds [34]. Studies of antifungal agents against Fusarium solani are also important in agriculture point of view as it is a major cause of fungal infection in various crops such as brown root rot, sudden death syndrome of soybean, disease of stored potatoes [35].

Conclusion

M. buxifolia fruit or extract/fractions can be used in the management of diseases caused by the above mentioned bacterial strains except B. subtilis. As the fruit is used forlklorically in the treatment of various infections including gastro-urinary disorders therefore, this anti-bacterial bioassay affords a valid scientific rationale to the traditional use(s) of fruit of this plant specie. Yet it is also evident from the results of the above mentioned antifungal assay that n-hexane, ethyl acetate, n-butanol and aqueous fractions of M. buxifolia fruit can be used against diseases caused by Microsporum canis, while MBHE, chloroform, ethylacetate and n-butanol fractions can used in the management of ailments caused by F. solani.

Acknowledgements

We (the authors) are thankful to University of Peshawar and H.E.J. Research Institute of Chemistry, University of Karachi for providing research facilities.

Conflict of Interest

We (the authors) declare that we don't have any conflict of interest.

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Title Annotation:gas chromatography, mass spectrometry
Author:Ullah, Irfan; Khan, Jamshaid Ali; Adhikari, Achyut; Hannan, Peer Abdul; Shakeel, Faisal; Kifayatulla
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Feb 28, 2019
Words:4670
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