SANTALUM ALBUM: CLINICAL ASPECTS FOR TREATMENT OF CANDIDA INFECTIONS

SANTALUM ALBUM: CLINICAL ASPECTS FOR TREATMENT OF CANDIDA INFECTIONS

Parveen Surain, Neeraj Kumar^*, Romika Dhiman and Vikas Meashi

Department of Microbiology, Kurukshetra University, Kurukshetra, Harayana, India

ABSTRACT: Background: In present study, we aim to assess the anticandidal activity of crude extracts and screening of bioactive compounds and their identification by NMR, IR and GC-MS. Results: A total of 192 isolates were isolated from patients having oral and vaginal candidiasis and a total of 134 isolates showed positive results with Gram staining. The selected isolates APKU-2, APKU-3 and APKU-4 were identified as C.albicans, C.lusitaniae and C.krusei by studying its morphological, biochemical, and molecular characteristics. Chemical characterization of the extracted compounds was done by H1NMR, Fourier transform infrared spectroscopy (FTIR), Gas chromatography–mass spectrometry (GC-MS) analysis to determine the structure, melting point, and molecular mass of the purified compounds. Conclusions: The crude extracts and purified compounds from the sandalwood holds potential as medications for various antifungal therapeutics. However, further research on their potential incorporation into different preparations, safety and cost- effectiveness remains to be done

Anticandidal activity,

S.album bark, Candida sp

INTRODUCTION: Candida is a diploid, dimorphic yeast producing three morphologic forms: yeast cells, pseudohyphae and true hyphae. It is an opportunistic pathogen causing various types of candidiasis which are on the increase around the globe. Candidiasis (or moniliasis) is an opportunistic fungal infection of humans caused by various species of Candida, especially Candida albicans. Superficial candidiasis of the oral cavity called thrush, seen as white patches, are common complications of AIDS, diabetes or prolonged antibiotic therapy ^{1, 2}. Candida can invade the lungs, kidneys, and heart or to be carrier in blood, where it causes a severe toxic reaction.

Of the 17 pathogenic species, C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei are often involved in 90% of invasive infections. C. kefyr, C. guilliermondii, C. lusitaniae, C. stellatoidea, and C. dubliniensis are the other major pathogens ^{3, 4}. Majority of the clinically used antifungals suffer from various drawbacks in terms of toxicity, drug-drug interactions, lack of fungicidal efficacy, high cost and emergence of resistant strains resulting from frequent usage ⁵.

The phenomenon of drug resistance has raised interest in substances of natural origin as a therapeutic alternative ^{6, 7}. Majority of the clinically used antifungals suffer from various drawbacks in terms of toxicity, drug-drug interactions, lack of fungicidal efficacy, high cost and emergence of resistant strains resulting from frequent usage. The phenomenon of drug resistance has raised interest in substances of natural origin as a therapeutic alternative ⁷. The known success of traditional medicine has guided the search for new chemotherapeutic alternatives to eliminate the infections caused by drug-resistant microbes and to reduce the harm caused by antibiotics. Medicinal plants and corresponding preparations have been used for a wide range of purposes and for many centuries people have been trying to treat diseases as well as alleviate symptoms by using different plant extracts and formulations ⁸. Medicinal plants are rich in a wide variety of secondary metabolites such as tannins, terpenoids, alkaloids, flavonoids and phenols, which have been used worldwide in traditional medicine to treat several diseases and infection. Among the plants investigated to date, one showing enormous potential is the pepper family, The Santalaceae, S.album, commonly known as sandalwood, Safed chandan, A small evergreen glabrous tree with slender drooping branches the sapwood white and odorless.

It generally occurs at altitudes of 2000-3000 feet. The tree attains the height of 60-65 feet and is actually an obligate hemi parasite plant on various hosts—“Cassia siamea, Pongamia glabra and Lantana acuminata” is a native of the highlands of southern India mainly Coorg, Chennai and Mysore? The chief constituents of the oil is santalol (90% or more) a mixture of two primary sesquiterpene alcohols, C15H24O viz, α-santalol (bp-166- 1670C) and β-santalol (b.p-177-1780C) in which α– form predominates. More than hundred constituents of sandalwood oil in categories of tannins, terpenes, resins and waxes have been reported ^{9, 10, 11, 12 13}.

METHODS:

Isolation of oral and vaginal candidiasis pathogens:

To diagnose fungal infection, debris/exudates samples were collected from the vaginal and oral sites, aseptically with the help of sterile cotton swabs and maintained at 4^ºC and brought to the Microbiology laboratory and processed to have immediately the exact nature of fungal flora involved. A detailed history was taken with particular reference to age, sex, presence of predisposing factors, onset and duration of complaints, initial appearance and progression of the lesions, treatment taken, marital status, exposure to sexually transmitted diseases and HIV status in relevant cases also noted. A detailed history regarding similar incidents in the family members were enquired into

The samples both from vaginal and oral sites were processed for direct isolation and examination of the Candida spp. on malt yeast extract agar (MEA) media. For direct microscopic examination, a small portion of the specimen was mounted in 10% KOH and Careful microscopic examination will reveal oval-shaped blastospores, pseudo hyphae and occasionally chlamydiospores. The size and shape of the yeast observed may also suggest the presence of non-albicans yeast. The budding cells of C. krusei for example are noticeably larger and more elongate than those of C. albicans. For selective isolation of a Candida spp.  the samples were inoculated on four different media: Malt yeast extract agar, Candidchrom agar, Biggy agar, Cornmeal with tween 80 agar. For isolation, sample swab was rolled and inoculated over the surface of agar plates of all the four media and incubated at 25⁰ C, 37⁰ C and 40⁰ C for 24-48 hrs. The plates were examined for the presence of growth and sporulating structures.

Microscopic and cultural characterization of isolates:

The purified isolates were identified on the basis of cultural characteristics (colour, exudates, texture of colony) by consulting various books, and available monographs. The identification of yeasts was done on the basis of morphological characteristics in lacto phenol cotton blue wet-mount microscopy (presence of budding). Direct examination of samples with KOH until enough dissolution of keratinized cells is achieved to microscopic visualization of the fungal cells can be performed. Gentle pressure applied to coverslip placed on sample, mixed with KOH solution, and should flatten the sample completely in order to allow good microscopic resolution. Different isolates were characterized with respect to growth on four different media i. e. MEA, Candidchrom agar, Biggy agar (containing fluorogenic or chromogenic substrates) and Corn meal agar for production of chlamydospores, Germ tube test, Carbohydrate assimilation and fermentation and Esterase production (lipolytic activity).

 Molecular characterization:

The molecular characterization of isolates has been done by Xcelris Labs Ltd.  Ahmedabad, India.

 Identification of a microbial culture using D1/D2 region of LSU based molecular technique:

DNA was isolated from the culture provided by the scientist. Its quality was evaluated on 1. 2% Agarose Gel, a single band of high-molecular weight DNA has been observed. Fragment of D1/D2 region of LSU (Large subunit 28S rDNA) gene was amplified by PCR from the above isolated plasmid DNA. A single discrete PCR amplicon band of 650 bp was observed when resolved on Agarose Gel (Gel Image-1). The PCR amplicon was purified to remove contaminants. Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with DF and DR primers using BDT v3. 1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. Consensus sequence of 596 bp of D2 region of 28S rDNA gene was generated from forward and reverse sequence data using aligner software. The D1/D2 region of LSU (Large subunit 28S rDNA) gene sequence was used to carry out BLAST with the database of NCBI gene bank database. Based on maximum identity score first ten sequences were selected the phylogenetic tree was constructed using MEGA 4. The purity of genomic DNA using agarose gel electrophoresis indicated the good quality and integrity of DNA.

Anticandidal activity of Santalum album plant extract:

The bark of S. album were collected from Ch. Devi lal park, Khizrabad. The taxonomic identity of this plant was confirmed by Dr. B. D. Vashishta, Kurukshetra University, Kurukshetra.

Extraction:

The bark samples were carefully washed under running tap water followed by sterile distilled water and air dried at 35-40°C for 4-5 days, homogenized to a fine powder using a sterilized mixer grinder and stored in air tight bottles. Six different solvents ethanol, methanol, acetone, petroleum ether, dichloromethane and sterile water were used for extraction. Ten grams of sample was separately soaked in conical flasks each containing 100ml of solvents ¹⁴. Each preparation was filtered through a sterilized Whatman No. 1 filter paper and finally concentrated to dryness under vacuum at 40°C using a Rota evaporator. The dried extracts, thus, obtained were sterilized by overnight UV-irradiation, checked for sterility on agar plates and stored at 4°C in labelled sterile bottles until further use ^15,16.

Purification and identification of active metabolites:

The purification and identification of the active compounds were done from the acetonic extracts of Santalum album (bark) showed best anticandidal activity against all the tested Candida pathogens. The purification of bioactive compounds was made by using thin layer chromatography (TLC- TLC Silica gel 60 F254 plates (Cat. No.1. 05554. 0007) of Merck, Germany were used for performing TLC. The TLC was performed at room temperature using properly equilibrated chromatography glass chamber). Column chromatography and Gas chromatography and Mass spectrometry (GC-MS). All the purified fractions collected were evaluated for their anticandidal efficacy against all the test organisms.

Structural elucidation of isolated metabolites:

Identification of compounds usually involves a combination of different techniques such as nuclear magnetic resonance (NMR) spectroscopy (The ¹H NMR spectra of pure, dried isolated compounds (5-10 mg) were done at the Department of Chemistry, Kurukshetra University, Kururkshetra using Bruker Advance DRX 300 FT-NMR in deuterated solvents, either dimethylsulphoxide (DMSO-d₆) or chloroform (CDCl₃) using tetramethylsilane (TMS) as internal standard). Infrared spectrometry (IR- IR analyses of three purified compounds was performed at Department of Chemistry, Kurukshetra University, Kururkshetra using ABB MB 3000 DTGS FT-IR Spectrophotometer using the KBr pellet technique. It has a resolution of 1 cm^-1 and can scan range of 450-4000 cm^-1).

Gas and mass spectrometry (GC-MS- GC-MS of the compounds was got done through Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University (JNU), New Delhi, India. For molecular analysis of purified polymer a coupled GCMS was performed using a GCMS-QP 2010 Plus model with capillary Column- Rtx-5 MS (30 mX 0. 25 mm i. d. X 0. 25 micrometer film thickness).  The samples were injected (1 ?L) in the split mode and the injection temperature was 270°C and column oven temperature was 100°C).

Test microorganisms:

The anticandidal activity was evaluated against different Candida spp. i.e. against three isolates (C. albicans strain-2 (KC139704) and C. krusei (KC616318) and one standard species (C. albicans strain-1 (MTCC No. 4748) as a control by agar well diffusion method. These were sub cultured on malt yeast agar (MEA) and incubated aerobically at 37°C.

Screening for anticandidal activity:

Anticandidal activity of six solvent extracts (acetone, methanol, ethanol, petroleum ether, dichloromethane and aqueous) of the leaves was determined by the agar well diffusion method. In this method, pure isolate of each yeast was sub cultured on the MEA plates at 37°C for 24h. Minimum of inoculum of the isolates were transferred into normal saline (0. 85%) under aseptic conditions. Density of each microbial suspension was adjusted equal to that of 106 cells/ml (standardized by 0. 5McFarland standard) and used as the inoculum for performing agar well diffusion assay. 100μl of inoculum of each test organism was spread onto the MEA plates.

The inoculated agar plates were allowed to dry, wells of 8mm were made with a sterile borer and the lower portion of each well was sealed with a little molten MEA medium. 100μl of it was propelled directly into each well (in triplicates) of the inoculated agar plates for each test organism.

The plates were allowed to stand for 10 minutes for diffusion of the extract to take place and incubated at 37°C for 24h. Sterile DMSO served as the negative control and ketoconazole as the positive control. The anticandidal activity, indicated by an inhibition zone surrounding the well containing the extract, was recorded if the zone of inhibition was greater than 8mm ¹⁵. The experiments were performed in triplicates and the mean values of the diameter of inhibition zones with ± standard deviation were calculated.

Determination of minimum inhibitory concentration (MIC):

MIC is defined as the lowest concentration of a compound/extract/drug that completely inhibits the growth of the microorganism in 24h. The MIC of all the extracts was determined following the modified agar well diffusion method of. A twofold serial dilution of each extract was prepared by first reconstituting the powder in DMSO followed by dilution in sterile distilled water to achieve a decreasing concentration range of 50mg/ml to 0. 39mg/ml.

A 100μl volume of each dilution was introduced into wells (triplicate) of the MEA plates already seeded with 100μl of standardized inoculum (106cells/ml) of the test microbial strain. All test plates were incubated aerobically at 37°C for 24 h and observed for the inhibition zones. The lowest concentration of the test extract showing a clear zone of inhibition (>8mm), considered as the MIC, was recorded for each test organism ¹⁶.

 RESULTS AND DISCUSSION:

Isolation of oral and vaginal candidiasis pathogens:

A total of 134 samples from vaginal (108) and oral (26) patients were found to be positive, of which 73. 4% from vaginal and 57. 7% from oral were found to be positive for the presence of yeasts during examination.

 Microscopic, cultural, biochemical and molecular characterization of selected isolates:

The selected isolate was classified up to genus level using the morphological and biochemical characteristics (Tables 1, 2 and 3). For further characterization, almost complete 28S rDNA gene sequences were determined. The obtained sequences were aligned and compared with the yeast sequences available in the Gene Bank database.  The phylogenetic analysis (Fig.1, 2 and 3) was done using MEGA 4 software by neighbor-joining tree and distance matrix-based nucleotide sequence homology which revealed that isolates APKU-2, 3 and 4 were C.albicans, C.lusitaniae and C.krusei.

TABLE 1: COLONIAL FEATURES OF CANDIDA SPP. ON FOUR DIFFERENT MEDIA.

TABLE 2: MORPHOLOGICAL FEATURES OF CANDIDA SPP.

 TABLE 3: BIOCHEMICAL FEATURES OF CANDIDA SPP.

Molecular characterization of Candida spp.: 

The purity of genomic DNA using agarose gel electrophoresis indicated the good quality and integrity of DNA. PCR amplification of 28S rDNA gene with DF and DR primers for all the Candida spp. viz.  APKU-2, APKU-3 and APKU-4 produced an amplification product of approximately 609 bp, 560 bp and 605 bp.

 FIG. 1: EVOLUTIONARY RELATIONSHIPS OF 11 TAXA

FIG.2: EVOLUTIONARY RELATIONSHIPS OF 11 TAXA

FIG. 3: EVOLUTIONARY RELATIONSHIPS OF 11 TAXA

TABLE 4: SIMILARITY PATTERN OF DIFFERENT ISOLATES WITH THE GENE BANK DATABASE SEQUENCES.

Anticandidal activity of Santalum album- bark

The organic extracts of S. album bark extracts showed inhibition of all the tested species. Acetonic extract with highest zone of inhibition against all the species. C. albicans strain-1 was most susceptible with maximum zone of inhibition 34. 6mm followed by C. albicans strain-2 (30. 6mm), C. krusei (22. 6mm). Aqueous extract showed activity against C. albicans strain-2.

TABLE 5: ANTICANDIDAL ACTIVITY OF PLANT EXTRACT OF SANTALUM ALBUM BARK.

Ca, C. albicans strain-1; Ca, C. albicans strain-2; Ck, C. krusei, - No activity; ^a Values, including diameter of the well (8mm), are means of three replicates, ± † Standard deviation. *Anticandidal activity was determined by agar well diffusion method on MEA medium.  

FIG. 4: ZONES OF INHIBITION PRODUCED BY THE ORGANIC AND AQUEOUS BARK EXTRACTS OF S. ALBUM AGAINST (a) C. ALBICANS STRAIN-1 (MTCC No. 4748), (b) C. ALBICANS STRAIN-2, Mt-METHANOL; Et-ETHANOL; Ac-ACETONE; PET- PETROLEUM ETHER; DCM- DICHLOROMETHANE; Aq-AQUEOUS.

FIG. 5: ZONES OF INHIBITION PRODUCED BY THE ORGANIC AND AQUEOUS BARK EXTRACTS OF S. ALBUM AGAINST (a) C. KRUSEI ; MT-METHANOL; Et-ETHANOL; Ac-ACETONE; Pet- PETROLEUM ETHER; DCM- DICHLOROMETHANe; Aq-AQUEOUMIC of Santalum album -bark

Excellent bioactivity has been found in all the organic extracts of S. album bark, the acetonic extract showing MIC values against all the species followed by dichloromethane, methanolic, ethanolic and petroleum ether extracts. The MIC of 0. 39mg/ml was found against C. albicans strain-1 followed by 1. 56mg/ml against C. glabrata and C. albicans strain-2.

TABLE 6: MIC OF SANTALUM ALBUM BARK EXTRACTS.

FIG. 6: PLATES SHOWING MINIMUM INHIBITORY CONCENTRATION (MIC) OF S. ALBUM BARK AGAINST C. ALBICANS STRAIN-1 (MTCC No. 4748) (1-50mg/ml; 2-25mg/ml; 3-6. 25mg/ml; 4-3. 12mg/ml; 5-1. 56mg/ml; 6-0. 39mg/ml).

Purification and identification of active compounds from Santalum album:

A total of 92 sub fractions, in 100ml each were collected from the acetonic extract of Santalum album by column chromatography. A gradient of benzene: acetone ranging between 100:0 and 0:100 were used to elute the compound. All the 92 sub fractions were analyzed by TLC. The fractions showing similar profile of Rf values were pooled and dried in Rota evaporator. Pooling of same fractions resulted in total of 6 sub fractions. The 6 sub fractions collected from column chromatography were evaluated for their anticandidal activity at 4.0 mg/ml by agar well diffusion method.

 GC-MS analysis of acetone extract of sandalwood bark:

GC-MS analysis helps in elucidating the structure of components. The key compounds of concern were identified based on their retention peak. A total of 107 compounds were identified from this plant with different retention time and area of percentage.

FIG. 7: GC-MS OF CRUDE ACETONIC BARK EXTRACT OF SANTALUM ALBUM.

Structural elucidation of pure compounds:

Identification of compound GP-3 and GP-2:

The two compounds, GP-3 from Santalum album were purified and were subjected to structural elucidation by NMR, IR and GC-MS spectroscopy. The compound GP-2, isolated from acetonic extract of S. album bark, was crystallized as green colored oil from the silica gel column after elution with benzene-acetone.

The compound GP-3, isolated from acetonic extract of S. album bark, was crystallized as colourless oil from the silica gel column after elution with benzene- acetone.

IR Spectroscopy:

IR (KBr) cm^-1: 3348 (O-H stretch), 1427-1597 (C-C). It was indicated from the spectrum that there was one functional groups present in the compound. The strong absorption bands at 3348 and 1427-1597 cm^-1 correspond to characteristic O-H and C-C stretch. The bands pointed towards the presence of aliphatic region. Thus, IR spectrum supported the results obtained from chemical analysis of GP-2 (Fig. 8-a).

IR (KBr) cm^-1: 1674 (C=C) stretch, 1481-1582 (C-C). It was indicated from the spectrum that there was one functional groups present in the compound. The strong absorption bands at 1674 and 1481-1582 cm^-1 correspond to characteristic C=C and C-C stretch. The bands pointed towards the presence of aromaticity. Thus, IR spectrum supported the results obtained from chemical analysis of GP-3 (Fig.9-a).

 NMR Spectroscopy:

¹H NMR (300MHz, CDCl₃): δ 1. 172-1. 219 (m, 15H), 3. 077-3. 202 (m, 3H), 5. 505 (s,O-H). The ¹H NMR spectrum showed a multiplet at δ 1. 172-1. 219 due to fifteen protons. A multiplet at δ 3. 077-3. 202 due to three protons. A singlet appearing at δ 5. 505 due to O-H was ascribed for hydroxyl group (Fig. 8-b).

 ¹H NMR (300MHz, CDCl₃): δ 8. 347-8. 329 (d,1H,J=Hz), 8. 224-8. 251 (d, 1H, J=8. 1Hz), 7. 633-7. 607 (d,1H,J=8. 1Hz),7. 827-7. 809 (d,1H,J=7. 2Hz), 5. 141 (s, 1H), 2. 564 (s, 3H). The ¹H NMR spectrum showed doublet at 8. 347, 8. 224, 7. 633 and 7. 827due to one proton each which was assigned for aromatic protons. A singlet appearing at 5. 141 and 2. 564 due to one and three protons (Fig. 9-b).

Based on IR, NMR and GC-MS spectrometry, the compound GP-2 from S. album bark was identified as Cis-myrtanol (Fig. 8-c).

Based on IR, NMR and GC-MS spectroscopy, the compound GP-3 from S. album bark was identified as Curcumen (Fig. 9-c).

Anticandidal activity of S. album bark purified compounds:

The results of anticandidal properties of S. album subfractions- cis-myrtanol, curcumen and the negative control (DMSO) are presented in Table. 7. A persual of the data reveals that of the two pure subfractions, cis-myrtanol showed good activity against all the tested species, the zone of inhibition ranging between 20.6mm and 23.6mm, with highest zone of inhibition 23.3mm against C. albicans strain-1 (MTCC No. 4748) followed by C. albicans strain-2 (22.6mm) and C. krusei (20.6mm). In case of curcumen, the zones of inhibition ranged between 15.6mm and 20.6mm with maximum against C. albicans strain-2 (20. 6mm) followed by C. albicans strain-1 (19. 3mm) and C. krusei (15. 6mm).

TABLE 7: ANTICANDIDAL ACTIVITY OF CIS-MYRTANOL AND CURCUMEN AGAINST CANDIDA SPP.

Ca, C. albicans strain-1 (MTCC No. 4748); Ca-,C. albicans strain-2; Ck, C. krusei, No activity; ^a Values, including diameter of the well (8mm), are means of three replicates. ± † Standard deviation.*Anticandidal activity was determined by agar well diffusion method on MEA medium.

Evaluation of synergistic/antagonistic anticandidal activity of pure compounds among themselves and with commercially available antifungal drugs:

The synergistic anticandidal potential of the two pure compounds isolated from S. album bark, determined by agar well diffusion method, are presented in Table 8. A persual of the data reveals that the combination of two pure compounds (cis-myrtanol + curcumen, 1:1) showed antagonistic effect against all the tested Candida spp. thus reducing the diameter of inhibition of microbial growth. The two compounds in combination produced zones in the range of 17. 3mm and 12. 3mm, maximum against C. krusei (17. 3mm) which were lesser than their individual zones of inhibition.

TABLE 8: SYNERGISTIC ACTIVITY OF PURE COMPOUNDS (INDIVIDUALLY AND IN COMBINATION) ISOLATED FROM S. ALBUM BARK.

The two compounds when tested in combination of 1:1 with the best active active (Neem ras) in order to determine their synergistic/antagonistic effects on Candida spp. through agar well diffusion method, revealed that antifungal neem ras showed antagonistic effect reducing the diameter of inhibition of Candida growth.

TABLE 9: EFFECT OF PURE COMPOUNDS ON THE ANTICANDIDAL ACTIVITY OF NEEM RAS ON CANDIDA SPP.

CONCLUSION: In this study, the crude extract of bark of Santalum album by GC-MS study results in the presence of 107 compounds. Santalum album was found to be the most potent plant possessing anticandidal activity against all the species of Candida in all the six solvents. C. albicans (4748) was most susceptible with maximum zone of inhibition 34. 6mm followed by C. glabrata (32. 6mm), C. tropicalis (14. 3mm). C. albicans (30. 6mm), C. krusei (22. 6mm) and C. lusitaniae (20. 3mm) were different isolates showing activity. On the basis of results obtained in the GC-MS analysis, the column chromatography of the plant part was done. The column sub fractions lead to the isolation of two and one active pure sub fractions each, GP-2 and GP-3 from S. album bark.The purified compounds isolated from the acetonic extract of S. album bark namely, GP-2 was crystallized as green coloured oil from the silica gel column after elution with benzene-acetone. Based on spectra obtained through IR, NMR and GC-MS spectrometry, the compound GP-2 was identified as cis-myrtanol, C10H18O. Another purified compound GP-3, was crystallized as colourless oil from the silica gel column after elution with benzene- acetone. Based on spectra obtained through IR, NMR and GC-MS spectrometry, the compound GP-3 was identified as curcumen, C15H22_.  Among the two pure subfractions, cis-myrtanol showed good activity against all the tested species, the zone of inhibition ranging between 15.0mm and 24.3mm, with highest zone of inhibition 24.3mm against C. tropicalis (Fig) followed by C. albicans (4748) (23.6mm), C. albicans (22.6mm), C. krusei (20.6mm), C. glabrata (20.3mm) and C. lusitaniae (15. 0mm). In case of curcumen, the zones of inhibition ranged between 15.6mm and 24.6mm with maximum against C. tropicalis (24.6mm) followed by C. albicans (20.6mm), C. albicans (4748) and C. lusitaniae (19.3mm) and C. krusei (15.6mm).

Furthermore, the integration of herbal medicine into modern medical practices must take into account the interrelated issues of quality, safety, and efficacy. Thus, after studying various plants for their anticandidal activity, the crude extracts and purified compounds from the plant can be incorporated into medications for various antifungal therapy. However, further studies for their incorporation into different preparations, safety and cost- effectiveness has to be conducted.

 COMPETING INTERESTS: The authors declare that they have no competing interests.

 ACKNOWLEDGEMENTS: We are grateful to Dr. B.D. Vashishta, Chairman, Department of Botany, Kurukshetra University, Kurukshetra, for helping with the identification of the plant samples. The authors are very thankful to the Department of Chemistry, Kurukshetra University, Kurukshetra, for providing the necessary facilities for NMR, FTIR analysis of the compounds and also thankful to AIRF (Advanced instrumentation research facility), JNU (Jawaharlal National University) for providing facilities for GC-MS.

CONFLICT OF INTEREST: With the submission of this manuscript I would like to undertake that all authors of this research paper have directly participated in the planning, execution, or analysis of this study. All authors of this paper have read and approved the final version submitted. The contents of this manuscript have not been copyrighted or published previously. The contents of this manuscript are not now under consideration for publication elsewhere. The contents of this manuscript will not be copyrighted, submitted, or published elsewhere, while acceptance by the Journal is under consideration. There are no directly related manuscripts or abstracts, published or unpublished, by any authors of this paper. My Institute’s representative is fully aware of this submission.

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How to cite this article:

Surain P, Kumar N, Dhiman R and Meashi V: Santalum Album: Clinical Aspects for Treatment of Candida Infections. Int J Pharm Sci Res 2016; 7(7): 2813-29.doi: 10.13040/IJPSR.0975-8232.7(7).2813-29.

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