Lupeol acetate as a potent antifungal compound against opportunistic human and phytopathogenic mold Macrophominaphaseolina

Antifungal activity of Monotheca buxifolia methanolic extract and its various fractions were assessed against Macrophomina phaseolina, a soil-borne fungal pathogen of more than 500 vegetal species as well as rare and emerging opportunistic human pathogen. Different concentrations of methanolic extract (3.125 to 200 mg mL−1) inhibited fungal biomass by 39–45%. Isolated n-hexane, chloroform and ethyl acetate fractions suppressed fungal biomass by 32–52%, 29–50% and 29–35%, respectively. Triterpenes lupeol and lupeol acetate (1, 2) were isolated from n-hexane while betulin, β-sitosterol, β-amyrin, oleanolic acid (3–6) were isolated from chloroform fraction. Vanillic acid, protocatechuic acid, kaempferol and quercetin (7–10) were isolated from the ethyl acetate fraction and identified using various spectroscopic techniques namely mass spectroscopy and NMR. Antifungal activity of different concentrations (0.0312 to 2 mg mL−1) of the isolated compounds was evaluated and compared with the activity of a broad spectrum fungicide mancozeb. Different concentrations of mencozeb reduced fungal biomass by 83–85%. Among the isolated compounds lupeol acetate (2) was found the highest antifungal against M. phaseolina followed by betulin (3), vanillic acid (7), protocatechuic acid (8), β-amyrin (5) and oleanolic acid (6) resulting in 79–81%, 77–79%, 74–79%, 67–72%, 68–71% and 68–71%, respectively. Rest of the compounds also showed considerable antifungal activity and reduced M. phaseolina biomass by 41–64%.

www.nature.com/scientificreports/ Monotheca buxifolia (Falc.) A. DC. is monotypic genus of the family Sapotacea, grows mainly in Malakand Dir district, Pakistan. M. buxifolia is reported for wide-range of pharmacological activities including cough, wound healing, headache, analgesic, antipyretic, antiseptic and urinary tract infections 13 . The antibacterial activity of crude ethanolic extract and fractions of M. buxifolia using agar well diffusion technique confirmed their antibacterial potential 14 . Javed et al. 15 isolated and identified five triterpenes from the bioactive fractions of aerial parts of M. buxifolia and displayed potent cytotoxic activities in vitro. The other plants of family Sapotaceae are widely explored and reported for significant antimycotic activities in various experimental models, but there is not only a single report on antifungal activity and isolation of antifungal compounds from M. buxifolia 16 . Therefore, the present investigation was conducted to explore antifungal potential of M. buxifolia fractions and isolated compounds against M. phaseolina, a highly problematic phytopathogen for which there is not any registered fungicide up to now.

Discussion
Identification of isolated compounds. From M. buxifolia methanolic extract ten compounds were isolated from various sub-fractions. Various physical analysis and spectroscopic data co-ordinated from the already reported data and compound 1 was identified as lupeol, compound 2 as lupeol acetate, compound 3 as betulin, compound 4 as β-sitosterol, compound 5 as β-amyrin, compound 6 as oleanolic acid, compound 7 as vanillic acid, compound 8 as protocatechuic acid, compound 9 as kaempferol and compound 10 as quercetin [19][20][21][22][23][24][25][26][27][28] Structures of these compounds are presented in Fig. 2. Antifungal potential of fractions and isolated compounds. All the concentrations of methanolic extracts and its various fractions significantly reduced fungal biomass. Antifungal activity of both compounds lupeol acetate (2) 79-81% and betulin (3) 77-79% were found very close to that of fungicide mancozeb 83-84%. Some earlier studies also reported antifungal activity of lupeol acetate (2) against Aspergillus flavus. However, most of the previous studies showed antibacterial activity of this compound against Bacillus subtilis, Staphylococcus aureus and Escherichia coli 29,30 . In contrast to the present study, Manzano et al. 31  www.nature.com/scientificreports/ β-Amyrin (5) is known to inhibit growth of various clinical fungal species namely Candida stellatoidea, Candida krusei, Microsporum sp. and Trichophyton rubrum and Ascochyta rabiei, the cause of blight disease of chickpea 33,34 Oleanolic acid (6) is a pentacyclic triterpenoid that is widely spread in plant kingdom, with members of Oleaceae family as its main source. Apart from its various pharmaceutical properties, it also possesses antifungal activity against yeast and dermatophyte species 35,36 . Protocatechuic acid (8) is a type of phenolic acid that is found in many food plants and possesses a number of pharmaceutical properties and antifungal activity against Microsporum audouinii 37,38 .  www.nature.com/scientificreports/ Lupeol (1), a pentacyclic triterpenoid, has also been isolated from a number of plant species including green pepper, mangoes, strawberry, grapes, white cabbage and olive, and is known to have useful effect as a preventive and therapeutic agent against a number of ailments 39 . Although in the present study, it found comparatively less effective against M. phaseolina, however, it was highly effective against Penicillium notatum causing 90% growth inhibition when used in 200 µg mL −1 concentration 31 . It was also inhibitory to the growth of Fusarium solani, Aspergillus niger, Rhizoctoia phaseoli, Candida albicans, Penicillium chrysogenum, Cantharellus flavus and Microsporum canis, and its antifungal activity was comparable with miconazole 40 . β-Sitosterol (4) is known to inhibit growth of Aspergillus niger and Cladosporium cladosporioides at a concentration of 0.01 mg mL −1 . It also inhibited growth of germ tube of Fusarium verticillioides by 82% at 50 mg mL −141 . Quercetin (10) is a polyphenolic flavonoid and showed a low activity against different Candida species 42 . This compound is also known to increase efficacy of a fungicidal compound amphotericin B against a clinical fungal species Cryptococcus neoformans 43 . Kaempferol (9) is a phenolic compounds generally showing antibacterial activity 44 . Information regarding its antifungal activity are very rare. The present study concludes that aerial parts of M. buxifolia contain potentent antifungal constituents especially lupeol acetate (2) causing 79-81% reduction followed by betulin (3) 77-79% and vanillic acid (7) 74-79% for the management of M. phaseolina, a highly problematic phytopathogen for which there is not any registered fungicides so far.

Materials and methods
Chemicals and reagents. Chemicals were attained from Sigma-Aldrich Germany. For isolation work including chromatography various solvents were acquired from Fischer Scientific (Loughborough, UK). Silica and Flash column silica (70-230 mesh) and (230-400 mesh) were assimilated from E. Merck, Darmstadt, Germany.    (16 L × 4). Extracts were assembled and methanol was removed under reduced pressure at 50 °C temperature. Gummy mass (586 g) having dark brown colour was collected. This MeOH extract was added in water (410 mL) and further fractionation was done using n-hexane, chloroform and ethyl acetate (10 × 3 L) each to afford 151, 102 and 152 g of the fractions respectively. Aqueous fraction was collected 161 g and all the portions were refrigerated 17 . Antifungal bioassays with M. buxifolia extract/fractions. The methanolic extract and its four fractions were tested against the pathogenic fungus M. phaseolina in vitro. An amount of 1.2 g of methanolic extract and its various fractions was dissolved in 1 mL of dimethyl sulphoxide (DMSO) and added to 5 mL autoclaved malt extract broth to make stock solution of 200 mg mL −1 concentration. Six lower concentrations viz. 100, 50, 25, 12.5, 6.25 and 3.125 mg mL −1 were prepared from stock solution in series of double dilution. Similarly, control with respect to each concentration was prepared by dissolving 1 mL DMSO in 5 mL malt extract broth and double diluted in series. Experiment was performed in glass test tubes (10 mL), each containing 1 mL of growth medium. Tubes were inoculated aseptically with 20 µL of M. phaseolina suspension and each treatment was replicated three times. After 7 days of incubation at room temperature, fungal biomass was filtered, dried and weighed.

Isolation of
Isolation and purification of compounds. M. buxifolia lipophilic hexane fraction was subjected to vacuum liquid chromatography over silica gel and eluted with increasing order of solvent polarity as hexane-EtOAc (0 → 10). Hexane first sub-fraction, collected using n-hexane: EtOAc (7:3) was again chromatographed to isolate compound 1 (10 mg) using same polarity system. Hexane second sub-fraction collected using n-hexane-EtOAc (6:4) was again chromatographed to isolate compound 2 (8 mg) eluted with same solvent system. CHCl 3 fraction was subjected to silica gel column and eluted with n-hexane-chloroform (10:0 → 0:0) to chloroform-methanol (0:0 → 0:10). Chloroform fraction lead to isolation of three main sub-fractions. CHCl 3 sub-fraction 1 was further purified and compound 3 and 4 (13,10 mg) were obtained with n-hexane-CHCl 3 (7:3) n-hexane-dichloromethane (5:5) and (6:4) as eluent, respectively. Sub-fraction 2 eluted with n-hexane-chloroform (6:4) was purified with n-hexane:dichloromethane (5:5) to obtain compound 5 (15 mg). CHCl 3 sub-fraction 3 eluted with chloroform-methanol (9.5:0.5) was rechromatographed using dichloromethane-methanol (9:1) to afford compound 6 (30 mg). The EtOAc fraction was chromatographed using silica gel and eluted with solvent system of increasing polarity n-hexane, n-hexane: DCM and DCM:MeOH and three sub-fractions were collected. EtOAc sub-fraction 1, eluted by n-hexane: DCM (3:7) was refilled on silica gel and eluted by n-hexane:DCM (1:9) to attain compound 4 (4 mg) and 7 (6 mg). EtOAc sub-fraction 2 eluted by DCM 100%, was reloaded and eluted by same solvent system to separate compound 8 (8 mg). EtOAc sub-fraction 3 was isolated using DCM:MeOH (9:1) was rechromatographed and eluted with identical solvent system to collect compound 9 and 10 (6,8 mg). Quercetin (10). Melting point was recorded at 314-316 °C. Two characteristic absorption bands in UV spectrum at 250 and 360 nm were showed. HR-EI-MS spectra depicted molecular ion peak at m/z 302.0285 with relevant molecular formula C 15 H 10 O 6 . IR spectrum showed two characteristic bands at 3402 and 1610 cm −1 . Antifungal bioassays with pure compounds from M. buxifolia. Two compounds (1, 2) were isolated from n-hexane fraction, four compounds (3-6) from chloroform fraction and four compounds (7-10) from ethyl acetate fraction of the methanolic extract of M. buxifolia were tested for MIC values by microdilution assay. MIC values of mancozeb as reference synthetic fungicide (80%WP, KSS) and isolated compounds were tested by serial dilution in culture tubes. Six milligrams of each of the ten isolated compounds and mancozeb (active ingredient) were dissolved in 20 µL DMSO and added to autoclaved malt extract to raise the volume up to 3 mL, to prepare a growth medium of 2 mg mL −1 concentration. Further serial double dilutions viz. 1, 0.5, 0.25…0.0312 mg mL −1 were made using malt extract broth in culture tubes. Spore suspension was prepared by adding 10 day old fungal culture in double distilled water. DMSO (20 µL mL) was added in malt extract broth www.nature.com/scientificreports/ to prepare 3 mL of control that serially double diluted to make corresponding control treatments for each concentration. Each treatment was replicated thrice. Fugal suspension (20 µL) was added to each concentration (0.5 mL) of the growth medium and incubated at 27 °C. Fungal biomass was collected on filter papers after 3 days growth, dried and weighed.
Statistical analysis. All the data were analyzed by analysis of variance followed by Tukey's HSD Test using computer software Statistix 8.1.
Ethics approval. This article does not contain any studies with human participants or animal experiments.