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Proximate Chemical Composition and Antimicrobial Activities of Fixed oils from <em>Diospyros lotus</em> L. | OMICS International
ISSN: 2161-0444
Medicinal Chemistry

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Proximate Chemical Composition and Antimicrobial Activities of Fixed oils from Diospyros lotus L.

Ghias Uddin1, Abdur Rauf1*, Bina Shaheen Siddiqui2, Mohammad Arfan1, Inayat-Ur-Rahman3 and Inamullah Khan4

1Institute of Chemical Sciences, University of Peshawar, Peshawar, KPK, Pakistan

2H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan

3Medicinal Botanic Centre PCSIR Laboratories Complex Peshawar, Peshawar, KPK, Pakistan

4Department of Pharmacy, University of Peshawar, Peshawar, KPK, Pakistan

*Corresponding Author:
Abdur Rauf
Institute of Chemical Sciences
University of Peshawar, Peshawar, KPK, Pakistan
Tel: +923469488944
E-mail: [email protected]; [email protected]

Received date: August 16, 2013; Accepted date: November 06, 2013; Published date: November 08, 2013

Citation: Uddin G, Rauf A, Siddiqui BS, Arfan M, Rahman IU, et al. (2013) Proximate Chemical Composition and Antimicrobial Activities of Fixed oils from Diospyros lotus L. Med chem 3:282-285. doi:10.4172/2161-0444.1000152

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Abstract

Diospyros lotus roots are traditionally used in various diseases including its use in microbialinfections. We designed a study to identify chemical constituents of oil from D. lotus and its antimicrobial activities. Gas chromatography coupled to mass spectrometer(GC-MS) was used for chemical analyses. Results showed that oil contained saturated as well as unsaturated compounds. Oil was investigated for its antimicrobial properties. Oil showed moderate antibacterial activity against two gram positive and two gram negative bacterial strains however oil showed low activity against four fungal strains. Interestingly no cytotoxicity was observed in Brine shrimp model; these encouraging results indicate further yet extensive studies to explore therapeuticpotential in microbial infections.

Keywords

Diospyros lotus; Antibacterial; Antifungal

Introduction

Diospyros lotus belongs to family Ebenaceae, which consists of about 500 species. Genus Diospyros is widely distributed in tropical and subtropical region throughout world. Characteristic feature of Diospyros species is that all members of the genus are trees [1]. D. lotus tree grows up to 9 m in height and around 6 m in width. This plant is rarely available in Britain and it is native to Himalayan region. In folk medicine, Diospyros species are known for their multiple medicinal uses. In many traditional and folk medicinal systems all over the world, Diospyros plants are well known for their medicinal and therapeutic value. All parts of these plants have been used for medicinal purposes e.g. the leaves are used for lumbago; the fruits are carminative, astringent and cure biliousness the seeds are sedative and the bark is bitter, astringent and febrifuge [2]. Leaf extract of Japanese persimmon D. kaki in combination with jasmine is used in Japan for making antitobacco smokingcandies. Triterpenoids belonging to lupane, oleanane and ursane series have been isolated and showed anti-inflammatory activity [3]. Diospyros species are used in folk medicinal systems for various medicinal uses such as their use used as antifungal for internal hemorrhage, for bedwetting in children, Woman’s drug for insomnia hiccough, as an antihypertensive, dysponea, vermicide and vermifuge, sedative, antifebrile, to promote secretions, astringent, bactericidal [4,5]. There are only a few reports on the studies of the roots of this plant [6,7] hence in the current study we have made an effort to analyze the chemical constituents of this part and evaluate its antimicrobial and cytotoxicity activity.

Material and Methods

Plant material

D. lotus roots were collected from Razagram, Toormang distic Dir Khyber Pakhtunkhwa (KPK) province of Pakistan in the month of august, 2009. The plant was identified by Prof. Dr. Abdur Rahsid, Department of Botany, University of Peshawar, Peshawar, KPK, Pakistan and a Voucher specimen No Rauf (6645) [8] was deposited at the herbarium of the said department.

Extraction of oil

Shade dried and crushed roots of D. lotus (14 kg) were subjected to cold extraction with MeOH (3×10L) at room temperature. Extract was then concentrated under reduced pressure at temperature below 55°C. Final residue obtained was 400 g. This methanolicextract was suspended in water and successively partitioned with hexane, chloroform, EtOAc. Hexane fraction (8 g) was subjected to Column chromatography on silica gel (Merck, 5×60 cm). Column was eluted with hexane/EtOAc (100:0→0:10) as solvent system. A total of 30 fractions, RF-1 to RF- 30 were obtained based on TLC profiles. Fraction RF-10 to RF-13 contained yellow colored oil. Extracted oil was subjected GC-MS analysis and injected 1 μl to GCMS using auto injector system.

Chemicals and reagents for GC-MS

Boron triflouride solution in methanol (10%) was purchased from Fluka Chemie (Buchs, Switzerland). Sodium hydroxide solution (methanolic; 0.5N) and sodium chloride (analytical grade) were obtained from Merck (Darmstadt, Germany) while methanol (HPLC grade), n-hexane (HPLC grade) were from Fischer Scientific (Leicestershire, UK). Helium gas (99.9999%) from Pak gas (United Arab Emirates) was procured. Tridecanoic acid methyl ester and Fatty acid methyl esters (FAMEs) having 37 components (Table 1) standard mixture were obtained from AccuStandard (Newhaven, Connecticut USA).

Preparation of standard for GC-MS

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 this solution further working standard solutions were prepared.

Chromatographic separation of FAMEs

A gas chromatographfrom Shimadzu hyphenated to a mass spectrometer QP 2010 plus (Tokyo, Japan) equipped with an autosampler (AOC-20S) and auto-injector (AOC-20i) was used. 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): 250oC, interface temperature: 240°C, pressure: 100 KPa, solvent cut time; 1.6 min. 1 μL 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. 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 comparing the mass spectra obtained with those of standard mass spectra from the NIST library (NIST 05).

Antimicrobial activities

In this study six fungaland five bacterial strains were used as reported earlier [9,10]. Bacterial strains used were Escherrchia coli, Bacilus subtilis, Staphylococcus aureus, Klebsiella pneumonia and Straptococcus epidermis. Fungal strains chosen for this study were Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microspoum canis, Fusarium salani and Candida glaberata. All these strains were maintained on agar slant at 4°C. Slant was allowed to activate at 37°C for 24 hours on Müller-Hinton agar (for bacteria) and Sabouraud glucose agar (fungi) before any screening. Cultures were taken in triplicates at incubation temperature of 37°C for 24 to 72 hours. Broth culture (0.6 mL) of the test organism was placed in a sterile Petri-dish to which 20 ml of the sterile molten MHA 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°C for 24 h and the diameters of the zone of inhibition of microbial growth were measured in the plate in millimeters. Extent of antimicrobial activity was obtained by measuring the diameter of zone of inhibition around the hole. Bioassay was repeated three times and then the mean diameter was determined. In this study streptomycin, miconazole and amphotericin B were used as standard antibiotics to compare extract and fraction with it.

Cytotoxicity activity

A shallow rectangular plastic dish (22×32 cm), filled with artificial sea water was taken. The sea water was prepared with commercial salt mixture mixed with double distilled water. Brine shrimp (Artemia salina leach) eggs were hatched the dish. Dish was made unequally partitioned by using an artificial perforated device. About 50 mg of the eggs were sprinkled in to large compartment which becomes darken. Minor compartment was exposed to the ordinary light. After two days, nauplii were collected and removed by a pipette from lighted side. A sample of the compounds to be tested was prepared by dissolving 20 mg of each compound in DMF (2 ml). Three different stock solution i.e., 550, 50, and 5 mg/mL were transferred to 9 vials (three for every dilution were used for each test sample and LD50 is the average of the three values) with one vial containing DMF was reserved as a control. Solvent was allowed to evaporate keeping overnight. Two days, later when the shrimp larvae were ready, 1 mL of sea water and 10 shrimp were added to each vial (30 shrimps/ dilution) with a volume adjusted with sea water to 5 mL per vial. After 24 h, the numbers of survivors were counted using standard procedure [11-13]. The data was analyzed by the use of finny computer program to determine LD50 values (Figure 1).

medicinal-chemistry-Antibacterial-activity

Figure 1: Antibacterial activity of oil isolated from the roots of D. lotus.

Results and Discussion

GC-MS analysis

Table 2 presents the results obtained from GC-MS analysis indicating relative concentration of individual FAMEs based on the external standard method. Analysis was repeated three times and values of area and concentration are given in Table 2. Quantification of FAMEs was performed using three points calibration curve with R2 value less than 0.99 (R2>0.99) in each case. Table 2 shows the Quantification results obtained from fatty acid standard mixture of 37 components while Table 1 is the GC-MS chromatogram of D. lotus roots oil with properly labeled signals of analytes detected (Figure 2).

ID# Name R. Time Area Conc. (%) Std. Dev.
1 C6:0; Hexanoic acid, methyl ester 3.028 2637 40.00 0.001
2 C8:0; Caprylic acid, methyl ester 4.911 43548 40.00 0.002
3 C10:0; Capric acid, methyl ester 6.743 59804 40.00 0.002
4 C11:0; Undecanoic acid, methyl ester 7.608 32300 20.00 0.003
5 C12:0; Lauric acid, methyl ester 8.493 67697 40.00 0.002
6 C13:0; Tridecanoic  acid, methyl ester 9.554 35734 20.00 0.003
7 C14:0; Myristic acid, methyl ester 10.897 72212 40.00 0.003
8 C14:1c; Myristoleic acid, methyl ester 11.466 8237 20.00 0.003
9 C15:0; Pentadecanoic acid, methyl ester 12.548 38432 20.00 0.003
10 C15:1; Pentdecanoic acid, methyl ester 13.234 7517 20.00 0.002
11 C16:0; Palmitic acid, methyl ester 14.533 118819 60.00 0.002
12 C16:1c; Palmitoleic acid, methyl ester 15.066 7253 20.00 0.002
13 C17:0; Margaric acid, methyl ester 16.829 34317 20.00 0.003
14 C17:1; Heptadecenoic acid, methyl ester 17.417 7643 20.00 0.003
15 C18:0; Stearic acid, methyl ester 19.500 67063 40.00 0.001
16 C18:1c; Oleic acid, methyl ester 20.038 17841 40.00 0.003
17 C18:1n9T; Elaidic acid, methyl ester 20.115 7232 20.00 0.003
18 C18:2T; Linoleic acid, methyl ester 21.608 8777 20.00 0.004
19 C18:2C; Octadecadionoic  acid, methyl ester 21.856 9261 20.00 0.001
20 C18:3n6; G-linoleic acid , methyl ester 22.762 5708 20.00 0.002
21 C18:3n3; Linolenic acid , methyl ester 24.160 6455 20.00 0.003
22 C20:0; Arachidic acid, methyl ester 27.058 66297 40.00 0.004
23 C20:1; Eicosenoic acid , methyl ester 27.659 8757 20.00 0.003
24 C20:2; Eicosadienoic acid , methyl ester 29.395 6480 20.00 0.002
25 C20:3n6; 8,11,14-Eicosatrienoic acid , methyl ester 30.312 6128 20.00 0.004
26 C21:0; Heneicosanoic acid , methyl ester 30.745 30613 20.00 0.004
27 C20:4n6; Arachidonic acid , methyl ester 31.073 5846 20.00 0.003
28 C20:3n3; Eicosatrienoic acid , methyl ester 31.717 9586 20.00 0.001
29 C20:5N3; (EPA) Eicosapentaenoic acid , methyl ester 33.359 6262 20.00 0.001
30 C22:0; Behenic acid methyl ester 34.188 62867 40.00 0.005
31 C22:1; Eruccic acid methyl ester 34.753 6847 20.00 0.001
32 C22:2; Locosadienoic acid , methyl ester 36.323 9365 20.00 0.002
33 C23:0; Tricosanoic acid methyl ester 37.440 29002 20.00 0.002
34 C24:0; Tetracosanoic acid methyl ester 40.521 60828 40.00 0.003
35 C22:6n3; (DHA) Docosahexaenoic acid , methyl ester 40.859 5333 20.00 0.002
36 C24:1; Tetracosenoic acid methyl ester 41.098 9144 20.00 0.002

Table 1: Quantification results of 37 components standard.

Peak # Name Relative area Conc. R. Time m/z Std. Dev
1 C12:0; Lauric acid, methyl ester 2878 2.21 8.265 87.00 0.0001
2 C14:0;Myristic acid, methyl ester 1916 1,47 10.989 87.00 0.0001
3 C16:0;Palmitic acid, methyl ester 75624 58.07 14.657 87.00 0.0000
4 C18:0; Stearic acid, methyl ester 12414 9.53 19.670 87.00 0.0002
5 C18:1c; Oleic acid, methyl ester 13481 10.35 20.216 97.00 0.0002
6 C18:1n 9T; Elaidic acid, methyl ester 845 0.65 20.457 97.00 0.0002
7 C18:2c; Linoleic acid, methyl ester 23066 17.71 21.822 97.00 0.0003

Table 2: Quantification results of fixed oil isolated from D. lotus roots.

medicinal-chemistry-oil-isolated

Figure 2: Antifungal activity of oil isolated from the roots of D. lotus.

Both the saturated and unsaturated FAMEs were detected in sample under investigations. Palmitic acid, methyl ester was found in highest concentration (58.07%) among the identified analyzes of interest which shows anti-inflammatory and antibacterial activities. Second FAME with higher concentration was Linoleic acid methyl ester (17.71%). Among the other FAMES with concentrations more than 1% were: Oleic acid, methyl ester (10.35%), Stearic acid, methyl ester (9.53%), Lauric acid, methyl ester (2.21%) and Myristic acid, methyl ester (1.47%) were found. Concentrations of Llaidic acid methyl ester were less than 1% (Table1). Fixed oil revealed moderate antibacterial activity against all test bacterial strain except Staphylococcus aureus and E. coli was found to be the most susceptible bacterial strain. However streptomycin(standard) exhibited comparatively better activity than oil. In case of antifungal activity, only low activities were observed against. Candida albicans, Aspergillus flavus, Microspoum canis, Fusarium salani and Candida glaberata. Interestingly oil showed no considerable toxicity in brine-shrimp lethality assay which indicates safety of oils for pharmacological use. This study highlighted potential of D. lotus to be investigated further for its antibacterial effects at cellular and molecular levels.

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