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Phytochemical Profile and Antifungal Activity of Leaves Methanol Extract from the Psydrax dicoccos (Gaertn) Teys. Binn. Rubiaceae Family

The present study was aimed to antifungal activity and phytoconstituents of leaves from the Psydrax dicoccos Gaertn. The antifungal activity of methanol extract from the P. dicoccos against Candida albicans, C. krusei, C. guilliermondii, C. parapsilosis, C. tropicalis, C. glabrata, four dermatophytes viz., Trichophyton rubrum, T. mentagrophytes, Microsporum gypseum and Epidermophyton flocossum. The methanol extract of the leaves were subjected to Fourier transform infrared spectroscopy (FT-IR) and Gas Chromatography-Mass Spectroscopic (GC-MS) analysis. The mean zones of inhibition produced by the tested extract in disc diffusion assays against fungal strains were ranged from 7.3 to 15.5 mm. The MIC values were between 125 and 500 μg/ml while, the MFC values were between 250 and 1000 μg/ml. The highest mean zones of inhibition (15.5± 0.6 mm) was observed with methanol extract of P. dicoccos against C. albicans. The GC-MS analysis of P. dicoccos leaves showed the presence of cinnamic acid, 2H-1-Benzopyran-2-one,5,7dimethoxy,(Z)6,(Z)9-Pentadecadien-1-ol, Benzofuran and n-Hexadecanoic acid as major compounds. Finally it can be concluded that the antifungal activity may be present in cinnamic acid, 2H-1-Benzopyran-2-one, 5, 7-, (Z) 6,(Z)9-Pentadecadien-1-ol, n-Hexadecanoic acid from the methanol extract from the P. dicoccos is highly valuable in medicinal usage and have fewer side effects.


Introduction
Fungi are ubiquitous in the environment and infection due to fungal pathogens has become more frequent [1,2]. With the rise of HIV, opportunistic fungal pathogens have become a common cause of morbidity and mortality [3]. Incidence of microbial infections has increased in recent decades, especially mycoses, which account for a high rate of death among patients with a weakened immune system. Opportunistic fungal infections are a serious threat to such patients and have been reported to occur at an alarming rate [4].
In the past two decades, the prevalence of candidiasis has been increased. Candida albicans is an opportunistic pathogen, causing mycoses in immunocompromised patients as well as long-term antibiotic users [5]. Also, other Candida species such as C. glabrata, C. parapsilosis, C. tropicalis and C. krusei are among the oral mucosal lesions suspected agents in AIDS patients [6]. Dermatophytic infections have increased considerably during the past several decades [7]. Traditionally, infections caused by dermatophytes have been named according to the anatomical locations involved by appending the Latin term designating the body site after the word tinea. The most common clinical manifestations are beard, glabrous skin, scalp, groin, hand, feet and nails [8].
Medicinal plants contain some organic compounds which provide definite physiological action on the human body and these bioactive substances include tannins, alkaloids, carbohydrates, terpenoids, steroids and flavonoids [9,10]. These compounds are synthesized by primary or rather secondary metabolism of living organisms. Secondary metabolites are chemically and taxonomically extremely diverse compounds with obscure function. They are widely used in the human therapy, veterinary, agriculture, scientific research and countless other areas [11]. A large number of phytochemicals belonging to several chemical classes have been shown to have inhibitory effects on all types of microorganisms in vitro [12]. Plant products have been part of phytomedicines since time immemorial. This can be derived from barks, leaves, flowers, roots, fruits and seeds [13]. Knowledge of the chemical constituents of plants is desirable because such information will be value for synthesis of complex chemical substances [14][15][16] .
Psydrax dicoccos Gaertn. (Syn. Canthium dicoccum (Gaertn.) Teys. & Binn a member of Rubiaceae. The plant is found in Deccan peninsula, Maharashtra southwards, and extending from Bihar eastwards to Assam and Meghalaya of Indian states. It is an unarmed shrub, grows up to 3m tall. In India the bark is used for fever and also applied as plasters, decoction of the root is used in diarrhea. Bark powder with sesame oil is used in rheumatic pains. Used in inflammation, during night boiled leaf extract is taken for 2 months [17].
Hence, the present research was conducted to investigate the phytochemical constituents of Psydrax dicoccos using FT-IR and GC-MS.

Collection of Plant Material
The fresh leaves of Psydrax dicoccos (Rubiaceae) were collected from Silambur (Lat, 11.35ºN; Long, 79.31ºE), Ariyalur District, Tamil Nadu, India. During the months from March to April 2014. The specimens were deposited in Department of Botany, Annamalai University (Herb-No-AUBOT# 263), Annamalai nagar. Collected leaves were initially washed with water, then surface sterilized with disinfectant solution of 10 % sodium hypochlorite solution and finally rinsed with sterile distilled water and shade dried under room temperature and grounded in to a coarse powder.

Preparation of Extraction
One hundred grams of powdered material of leaf, samples were extracted in a Soxhlet apparatus for 8 hours with methanol. The extracts were filtered, pooled and the solvent was evaporated with the help of rotary evaporator (Heidolph, Germany) under reduced pressure at 40 ºC and the crude extract was kept at 4 ºC in refrigerator for further analysis.

Preparation of Inocula
Twenty-four hour old cultures of selected Candida strains were mixed with physiological saline and turbidity was adjusted by adding sterile physiological saline until a McFarland turbidity standard of 0.5-2.5 × 10 3 cells/mL. The filamentous fungal strains were subcultured on SDA and incubated at 30°C for 4-7 days for dermatophytes. The growth was scraped aseptically, crushed and macerated thoroughly in sterile distilled water and inoculum of fungal strains were obtained according to reported procedures and adjusted to 0.4-5 × 10 4 cells/mL.

Disc-Diffusion Assay
The in vitro antifungal activities of methanol extract were screened using agar diffusion method [18]. For that assay, mass of Fungi for preparing the petriplates were prepared by pouring 20 ml of sabouraud dextrose agar and allowed to solidify for 20 minutes. The sstandardized inoculum suspension were swabbed on the top of the solidified media and allowed to dry for 54 Volume 7 10 minutes. Discs with different concentrations of extracts (1000, 500 and 250 µg/disc) were prepared and aseptically applied on the surface of the petriplates. Amphotercin-B (100 units/disc) for Yeast and Ketoconazole (5µg/disc) for dermatophytes were used as positive controls and 10 per cent DMSO was used as blind controls in all the assays. After that, the plates were incubated at 28 °C for 24 hours for yeast and 30 °C for 3 -5 days with dermatophytes. The zone of the inhibition was measured in millimeter. The experiments were carried out in triplicates.

Minimum Inhibitory Concentration (MIC) for Fungi
The MIC of the methanol crude extract of P. dicoccos was determined by using broth micro dilution technique as recommended by CLSI M27-A3 [19] and M38-A2 [20] for yeast and filamentous fungi respectively. The MIC values were determined in RPMI-1640 (Himedia, Mumbai) with L-glutamine without sodium bicarbonate, pH 7.0 with morpholine propane sulfonic acid (MOPS). Fifty milligram of crude extracts were dissolved in 1 mL of 10% DMSO and stock solution was obtained for the determination of MIC. For crude extracts, 20 µL of each plant extract, was dissolved with 980 µL of RPMI-1640 medium (2 mg/mL). From that, two fold serial dilutions in the range from 1000 to 15.7 µg/mL were prepared. 200 µL of solution was poured into first well of 96 well microtitre plates and then, 100 µL were transformed to the next well containing 100 µL of RPMI-1640. The same procedure was performed for all wells. 10 µL of fungal standardized inoculum suspensions containing 0.5-2.5×10 3 cfu/mL for yeast and 0.4-5×10 4 cfu/mL for dermatophytes were transferred to each well. The control well contained only sterile water and devoid of inoculum. The microtitre tray plates were incubated without agitation at 28 °C for 24 h for yeast and 30 °C for 4-7 days for dermatophytes. The MIC of the extract was recorded as the lowest concentration of extracts inhibited the growth of the Candida and dermatophytic strains when compared to that of control.

Minimum Fungicidal Concentration (MFC)
MFC of the extracts were determined by plating 100 µl of samples from each MIC assay well with growth inhibition. Wells were transferred to freshly prepared sabouraud dextrose agar plates and incubated in incubator at 28 °C for 24 hours for yeasts and 30 °C for 3 -5 hours for dermatophytes. The MFC was recorded as the lowest concentration of the extracts that did not permit any visible fungal growth after the period of incubation.

GC-MS Analysis
Gas chromatography (GC) analysis was carried out using Agilent 6890 N gas chromatography equipped with mass selective detector coupled to front injector type 1079. The chromatograph was fitted with DB 5 MS capillary column (30 m x 0.25 mm i.d., film thickness 0.25 µm). The injector temperature was set at 280 °C and the oven temperature was initially at 45 °C then programmed to 300 °C at the rate of 10 °C/min and finally held at 200 °C for 5 min. Helium was used as a carrier gas with the flow rate of 1.0 mL/min. One microlitre of the sample (diluted with acetone 1:10) was injected in the split mode in the ratio of 1:100. The percentage of sample was calculated by the GC peak area.
GC-mass spectrometry (GC-MS) analysis of sample was performed using Agilent gas chromatography equipped with JEOL GC MATE-II HR Mass Spectrometer. GC conditions were the same as reported for GC analysis and the same column was used. The mass spectrometer was operated in the electron impact mode at 70 eV. Ion source and transfer line temperature was kept at 250 °C. The mass spectra were obtained by centroid scan of the mass range from 40 to 1000 amu. The extract was identified based on the comparison of their retention indices (RI), Retention time (RT), mass spectra of WILEY, NIST library data of the GC-MS system and literature data [21].

Fourier Transform Infra-Red Spectra
IR spectrum was recorded in spectrophotometer (Thermo Scientific NICOLET-iS5). The active principle was mixed with KBr and pellet technique was adopted to record the spectra.

International Journal of Pharmacology, Phytochemistry and Ethnomedicine
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Statistical Analysis
The results are expressed as the mean  SD. All statistical analyses were performed using SPSS version 16.0 statistical software (SPSS Inc., Chicago, IL, USA). Student's t-test was performed to determine any significant difference between different extracts for in vitro antifungal assays. Comparison of means for in vivo antifungal assessment was carried out using one-way analysis of variance (ANOVA) and Duncan test. P value < 0.05 was considered statistically significant.

Results and Discussion
The methanol extract of Psydrax dicoccos leaves exhibited varied levels of antifungal activity against Candida albicans, C. krusei, C. guilliermondii, C. parapsilosis, C. tropicalis, C. glabrata, four dermatophytes viz., Trichophyton rubrum, T. mentagrophytes, Microsporum gypseum and Epidermophyton flocossum. The results revealed that the mean zones of inhibition ranged from 7.3 to 15.5 mm. The MIC values were between 125 and 500 µg/ml, while, the MFC values were between 250 and 1000 µg/ml and the results are presented in Table 1. Similar results were observed with present study Umaiyambigai et al. [22] reported that the antimicrobial activity of P. dicoccos leaves were extracted successively with different solvents viz., petroleum ether, chloroform, ethyl acetate and methanol and screened for their antimicrobial activity against Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Vibrio cholerae, Candida albicans, C. parapsilosis and C. tropicalis. The basis for varying degree of sensitivity of test microorganisms may be due to intrinsic tolerance of microorganism, their nature and combinations of phytochemicals present in the crude extracts [23]. Similarly, the differences in the antimicrobial activity of crude extracts may be due to the amount of antimicrobial agent present in the extract and their mode of action on different test microorganisms [24].

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In the present study, methanol extract used to test the antifungal activity of Psydrax dicoccos showed the highest antifungal activity (15.5 mm at 1000 µg/ml) against C. albicans. Earlier researcher reported that the same results in methanol extract of leaves of Lantana camara showed antifungal activity against Aspergillus fumigatus and A. flavus [25]. Ramirez et al. [26] recorded the similar results of methanol extract of leaves of Piper ecuadorense showed antifungal activity against Trichophyton mentagrophytes and T. rubrum.
The isolated compound, Pistagremic acid (-2-methyl-6-(4,4,10,13,14-pentamethyl-3-O-2, 3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]-phenanthren-17-yl)hept-2enoic acid) from Pistacia integerrima exhibited promising antibacterial activity against Klebsiella pneumoniae, Straptodirimu sp. and Bacillus stearothermophilus [34]. The fatty acids esters such as cyclopentane-tridecanoic acid, methyl ester, tartronic acid n-Hexadecanoic acid (p-ethoxyphenyl), diethyl ester, 7,10-octadecadenoic acid, methyl ester, heptadecanoic acid, 16-methyl methyl ester, and 9-Octadecenoic acid [Z]-, 2-hydroxyl-1-[hydroxymethyl], ethyl ester were isolated from petroleum ether extract of dried fruiting bodies of Pleurotus eous against Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumonia [35]. In the present study methanol extract showed the highest antifungal activity may be due to the existence of secondary metabolites with antifungal properties.  In the present study FT-IR analysis of methanol extract of leaves of P. dicoccos was carried out the compounds indicated shows that the band at 3408, 2924, 2853, 1761, 1662, 1627, 1384, 1308, 823, 801, 518, 500 and 483 cm -1 (Fig. 2). The broad band at 3408 cm-1 OH stretching in alcohol and phenol group, 2924 cm -1 to 2853 cm -1 attributed to C-H stretching vibration in alkanes group, the peaks around 1662 to 1627 cm -1 are due to the amide I and II region that are characteristic of protein and enzyme, Small bands at 1734 cm −1 are represented C=O stretching vibrations of carboxylic acid. 1384 cm-1 C-H stretching alkanes group, The weak band at 1038 cm -1 can be attributed to the glycoside/C-OH bonds in the polysaccharide / protein structure and 518 to 483 cm-1 C-H out of plane bending alkenes group. Similar results were compared with present study FT-IR analysis was used to identify the functional group of active components based on peak values in the region of infrared radiation [36]. Ragavendran et al. [37] screened the functional groups of carboxylic acids, amines, amides, sulphur derivatives, polysaccharides, organic hydrocarbons, halogens that are responsible for various medicinal properties of Aerva lanata. Hexadecanoic acid ethyl ester has been identified to have antibacterial and antifungal activity against range of species studied. Since natural substances have proved to have less side effects and less unwanted reaction with environment, using natural materials for biological prevention of microbes is more desirable [38].

Conclusion
Finally, it can be concluded that the study proved the antifungal activity, GC-MS and FT-IR analysis of methanol extract from the P. dicoccos and advocates the potentiality of the plant as a source of alternative medicine. So, use of natural products, especially methanol extracts of P. dicoccos leaves may be considered as a cinnamic acid, 2H-1-Benzopyran-2-one, 5,7-, (Z)6,(Z)9-Pentadecadien-1-ol, n-Hexadecanoic acid new source of natural antifungal agents. Natural substances have been proved to have fewer side effects and less unwanted reactions with the environment. However, a detailed pharmacological investigation of the plant is essential.