Chemical Constituents and Antifungal Activity of Ficus hirta Vahl. Fruits

Phytochemical investigation of Ficus hirta Vahl. (Moraceae) fruits led to isolate two carboline alkaloids (1 and 2), five sesquiterpenoids/norsesquiterpenoids (3–7), three flavonoids (8–10), and one phenylpropane-1,2-diol (11). Their structures were elucidated by the analysis of their 1D and 2D NMR, and HR-ESI-MS data. All of the isolates were isolated from this species for the first time, while compounds 2, 4–6, and 8–11 were firstly reported from the genus Ficus. Antifungal assay revealed that compound 8 (namely pinocembrin-7-O-β-d-glucoside), a major flavonoid compound present in the ethanol extract of F. hirta fruits, showed good antifungal activity against Penicillium italicum, the phytopathogen of citrus blue mold caused the majority rotten of citrus fruits.


Introduction
The genus Ficus (Moraceae) contains more than 1000 species, most of them are distributed in tropical, sub-tropical, and Mediterranean regions [1]. There are around 98 species distributed in the South of China. Ficus hirta Vahl. is mainly distributed in Yunnan, Guizhou, Guangxi, Guangdong and Hainan province, China [1]. The fruits of F. hirta were used as medicine and food resource by the local people of Guangdong province, China. Previous chemical investigations on the F. hirta focused on its roots, which led to isolate and identify the predominant chemical constituents, flavonoids, and coumarins. To date, the total of 31 flavonoids [2][3][4][5][6][7] and 7 coumarins [2,3,5,8] have been reported from this species. Except the flavonoids and coumarins, there are some other compounds reported from this species, such as steroids [2,7] and benzoic acid derivatives [7].
Several studies on the pharmacological activities of F. hirta showed its antioxidation [9], cytotoxicity, and apoptosis of HeLa cells [10]; anti-inflammation and analgesia [11], antitussive and antiasthmatic [12], hepatoprotective [13], and radioresistance effects [14]. The fruits of F. hirta consumed as a plant-derived food that showed potential tonic effects [15]. Besides mentioned above, F. hirta also showed antibacterial activity against Escherichia coli, Staphylococcus aureus [16], and Penicillium italicum, a phytopathogenic cause of citrus blue mold resulted in the destructive fruit rotten of citrus. The fruits of F. hirta and several other medicinal plants were also used to control the phytopathogen in order to decrease the loss of citrus rotten [17,18]. The fruits of F. hirta showed promising antifungal activity against P. italicum and prolonged the Nanfeng mandarin preservation period [19], while the major antifungal constituents were not clear until now.
In order to continue our studies on isolation and identification of the antifungal compounds from plants. We have elucidated the antifungal constituents of F. hirta fruits. Fortunately, our previous studies have identified nine monosubstituted benzene derivatives from the extracts of F. hirta fruits and some of them showed good antifungal activities [17,18], while they were not the major antifungal constituents for their low content in the plant. In continuation, the current study was aimed to discover the major antifungal compounds with diverse structures from this species.

Results
As described previously, the ethanol extracts (FH) of the fruits of F. hirta and the fractions (FH1-FH4) fractionated by D101 macro resin column were evaluated for their antifungal activities against P. italicum. Fractions FH2-FH4 showed stronger antifungal activities in a concentration-dependent manner than that of FH crude extract [18]. The further isolation was focused on the fractions with potent antifungal activities to find more active compounds present in the fruits of F. hirta.
As a result, 11 compounds (1-11) ( Figure 1) were isolated from those fractions, and their structures were elucidated based on the analysis of spectroscopic data (including HR-ESI-MS, 1 H-NMR, 13 C-NMR, and 2D NMR) and comparison of these data to previous published paper.

Results
As described previously, the ethanol extracts (FH) of the fruits of F. hirta and the fractions (FH1-FH4) fractionated by D101 macro resin column were evaluated for their antifungal activities against P. italicum. Fractions FH2-FH4 showed stronger antifungal activities in a concentration-dependent manner than that of FH crude extract [18]. The further isolation was focused on the fractions with potent antifungal activities to find more active compounds present in the fruits of F. hirta.
structure units (including a sugar moiety). Analysis of the HMBC spectrum then enabled the connectivity of these spin coupling fragments and the other functional groups. The HMBC correlations ( Figure 2) from H3-12 (H3-13) to C-1, C-2, C-6, and C-13 (C-12); from H-11 to C-4, C-5, and C-6; from H-7 and H-8 to C-6; and from H3-10 to C-8 and C-9, allowed the construction of the planar structure of aglycone. The glucopyranose was linked to C-3 by the HMBC correlation from H-1′ to C-3. Searching the structure with SCIFINDER revealed it has the same planar structure as the NMR data of 5 with those of icariside B2 indicated they had the same stereochemistry. Therefore, compound 5 was determined as icariside B2.   Figure 3). The HMBC correlations ( Figure 3) from H2-12 to C-1, C-2, C-5, C-6, and C-13; from H3-14 to C-4, C-5, and C-6; from H-7 and H-8 to C-6; from H3-15 to C-8, C-9 and C-10; and from H-10 to C-11, constructed the planar structure of 6. Compared the NMR data of 6 with those of dihydrophaseic acid revealed they had the same structure. Therefore, compound 6 was elucidated as dihydrophaseic acid.    Figure 3). The HMBC correlations ( Figure 3) from H 2 -12 to C-1, C-2, C-5, C-6, and C-13; from H 3 -14 to C-4, C-5, and C-6; from H-7 and H-8 to C-6; from H 3 -15 to C-8, C-9 and C-10; and from H-10 to C-11, constructed the planar structure of 6. Compared the NMR data of 6 with those of dihydrophaseic acid revealed they had the same structure. Therefore, compound 6 was elucidated as dihydrophaseic acid. structure units (including a sugar moiety). Analysis of the HMBC spectrum then enabled the connectivity of these spin coupling fragments and the other functional groups. The HMBC correlations ( Figure 2) from H3-12 (H3-13) to C-1, C-2, C-6, and C-13 (C-12); from H-11 to C-4, C-5, and C-6; from H-7 and H-8 to C-6; and from H3-10 to C-8 and C-9, allowed the construction of the planar structure of aglycone. The glucopyranose was linked to C-3 by the HMBC correlation from H-1′ to C-3. Searching the structure with SCIFINDER revealed it has the same planar structure as the NMR data of 5 with those of icariside B2 indicated they had the same stereochemistry. Therefore, compound 5 was determined as icariside B2.  Figure 3). The HMBC correlations ( Figure 3) from H2-12 to C-1, C-2, C-5, C-6, and C-13; from H3-14 to C-4, C-5, and C-6; from H-7 and H-8 to C-6; from H3-15 to C-8, C-9 and C-10; and from H-10 to C-11, constructed the planar structure of 6. Compared the NMR data of 6 with those of dihydrophaseic acid revealed they had the same structure. Therefore, compound 6 was elucidated as dihydrophaseic acid.  The antifungal activities of all of the isolates were tested at two concentrations (2.0 and 4.0 mg/mL). The results showed that none of them except compound 8 showed antifungal activity with the DIZs of 19.0 ± 0.5 mm and 24.0 ± 0.5 mm at 2.0 and 4.0 mg/mL, respectively, which are more powerful than that of FH (11.0 ± 0.6 mm at 2.0 mg/mL).
Moreover, compound 8 was also evaluated, its antifungal activity using mycelia growth method, the results are shown in Table 1. The inhibition rate was shown as concentration-dependent. Compound 8 exhibited more than 90% inhibitory effect against P. italicum at 400 µg/mL, while 100% inhibition rate was achieved at 800 µg/mL.
The discovery of compounds 1 and 3 showed the relevance between this species and other Ficus species such as F. platypoda and F. pumila. Compound 6 is a derivative of plant hormone abscisic acid that can be classified as carotenoid sesquiterpenoid, which is different from other sesquiterpenoids isolated from the genus Ficus [32,33]. Compound 7 is a norsesquiterpenoids with 11C skeleton with different linkage with other 11C skeleton norsesquiterpenoids previously isolated from Ficus microcarpa [34,35]. Previously, two isomers of "Eriodictyol hexoside" and other flavonoids were tentative identified in the fruits of F. carica using HPLC-MS approaches [36]. The identification of eriodictyol-7-O-β-D-glucoside (10) in F. hirta confirmed these results and showed the relevance between this species and F. carica.
Pinocembrin-7-O-β-D-glucoside (8) showed good antifungal activity while the compounds 9 and 10 showed none activity, which indicated the antifungal activity of flavonoids maybe effected by the number of hydroxyl in the C loop. This is the first time the antifungal activity of compound 8 against P. italicum has been reported. However, some references have already revealed the antifungal activity of its aglycone, namely pinocembrin [37,38].
Overall, these results indicated that carboline alkaloid, megastigmanes, and flavonoids could be regarded as a chemotaxonomic marker of F. hirta. Also, while only one carotenoid sesquiterpenoid and a norsesquiterpenoid with 11C skeleton were identified herein, whether they may be regarded as a chemotaxonomic marker of F. hirta species remains to be established. Pinocembrin-7-O-β-D-glucoside (8) was the major antifungal constituent against P. italicum existed in the fruits of Ficus hirta.

Plant Material
The fruits of F. hirta were bought from Zhangshu medicinal market, Jiangxi Province, China, and authenticated by Prof. Shouran Zhou (College of Basic Medicine, Jiangxi University of Traditional Chinese Medicine). A voucher specimen (no. FH-201406) was deposited in the herbarium of Jiangxi Key Laboratory for Postharvest Technology and Nondestructive Testing of Fruits & Vegetables, Jiangxi Agricultural University (Nanchang, Jiangxi, China).

Extraction and Chromatography
The air dried fruits of F. hirta (4.9 kg) were ground and extracted using ultrasonic-assisted method with 95% ethanol (3 × 25 L) at 25 • C for 90 min. The extract were evaporated to remove ethanol solvent and yielded the dried ethanol extract (345.1 g), which was subjected to D101 macro rein column chromatography eluted with water, 30% ethanol (v/v), 50% ethanol, and 95% ethanol, respectively, to yield four fractions (FH1-FH4). Antifungal activity test indicated that three fractions (FH2-FH4) were the active fractions. Activity-guided isolation were performed accordingly, as follows.

Antifungal Activity Test
The antifungal activity of FH extracts and isolates against P. italicum was evaluated by the Oxford Cup method as described previously [17,18].
The antifungal activity of the pure compound 8 (pinocembrin-7-O-β-D-glucoside) against P. italicum were further examined by the mycelia growth method as described previously [17]. Briefly, the pure compound 8 were dissolved in 95% ethanol, and then added to the sterile PDA (potato dextrose agar) culture medium at the specified concentrations. The mixed media were then poured into plastic Petri dishes (90 mm). The agar-mycelial plugs (6 mm) infected with pathogens were incubated at the center of the Petri dishes sealed with parafilm and incubated in the dark. Mycelium colony growth diameters were measured when the fungal mycelium of the control group had completely covered the Petri dishes. All treatments were tested in six replicates. The inhibition of mycelial growth (IMG, %) was calculated as the following formula: IMG (%) = 100 × (dc − dt)/(dc − 6), where dc and dt were the mycelium diameters (mm) of the control and the treatment, respectively.

NMR and MS Data of Compounds 1-11
The 1 H-and 13 C-NMR data of these compounds (1-11) were listed as follows.