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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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.
Diospyros lotus; Antibacterial; Antifungal
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 . 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 . 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 . 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.
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)  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).
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.
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).
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.
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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