Discovery of a Potent Anti-Yeast Triterpenoid Saponin, Clematoside-S from Urena lobata L.

Urena lobata has been used as a traditional medicinal plant in India and China. In this study, we investigated the antimicrobial activity and isolated the active compound from the leaves of U. lobata. The 80% ethanol extract from U. lobata leaves showed an effective anti-yeast activity against Saccharomyces cerevisiae (S. cerevisiae) strains. Using a combination of chromatographic methods, (−)-trachelogenin (1) and clematoside-S (2) were isolated from this plant for the first time, and their chemical structure was identified by mass spectrometry (MS) and extensive nuclear magnetic resonance (NMR) data analysis. In addition, 1 was found to be inactive against all of the test microorganisms in the antimicrobial assay, whereas 2 exhibits a specific anti-yeast activity against S. cerevisiae strains with diameter of inhibition zones in the range from 11 to 20 mm. Furthermore, the MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) values of 2 against S. cerevisiae strains were detected to be in the ranges of 0.61 to 9.8 μg/mL and 2.42 to 9.8 μg/mL, respectively. This is the first report of 2 with a specific anti-yeast activity. The above result suggests the potential application of U. lobata to be used as a natural anti-yeast agent in food preservation.


Introduction
Food spoilage by the food-related yeast causes the deterioration of a wide range of foodstuffs such as wines, milk, fruit and vegetable juices, soft drink or meat. Spoilage yeasts not only significantly influence the cost and availability of foods and beverages, but also lead to economic losses in food industry [1][2][3]. Notably, Saccharomyces cerevisiae is listed on one of the most significant spoilage yeasts for fruit juices and soft drinks [4]. Although chemical preservatives can exclude yeast spoilage, there is a strong consumer demand to avoid or diminish the use of artificial preservatives. Therefore, much effort has been expended in the search for effective natural compounds from herbs and spices in order to control food spoilage caused by yeast and replace existing synthetic antibiotics in foodstuffs [5,6].
Urena lobata L., indigenous in China, is a number of the Malvaceae family. The plant, commonly known as Ye-Mian-Hua in China, is a popular folk medicine as diuretic, febrifuge, and also as a remedy for dysentery, cough, dropsy and rheumatism to exhibit a variety of biological activities, including antioxidant, anti-inflammatory, anti-proliferative, and antibacterial activities [7][8][9]. Recently, it has been reported that the methanolic extract from U. lobata leaves showed antibacterial activity against Micrococcus roseus and Mycobacterium smegmatis [10]. Some phytochemical compounds such as flavonoids, triglycerides and lignans have been isolated from this plant [11][12][13]. In the course of our ongoing program on identifying antimicrobial principles from natural materials, we found that the aqueous ethanolic extract from the leaves of U. lobata showed significant anti-yeast activity using S. cerevisiae as an indicator. In the present study, we have attempted to isolate the anti-yeast substance(s) from the leaves of U. lobata. On bioassay guided fractionation of aqueous ethanolic extract, further work led to the isolation of (−)-trachelogenin (1) and clematoside-S (2) (Figure 1). Herein, we describe the isolation and structural elucidation of compounds 1-2, together with evaluating their antibacterial and antifungal activities.

Isolation of Compounds 1-2 from the Leaves of Urena lobata
Scheme 1 shows the extract and isolation scheme of compounds 1-2 from the leaves of U. lobata. Powdered leaves of U. lobata were extracted with 80% ethanol and further fractionation was performed with a guidance of inhibitory zone diameter against S. cerevisiae (ATCC 204508). At a concentration of 10 mg/mL, the crude extract exhibited 15 mm of inhibitory zone diameter against S. cerevisiae. The extract was subjected to MCI gel column chromatography to give four fractions with a step gradient elution of water-methanol. The active fraction 4 (16 mm of inhibitory zone diameter against S. cerevisiae at a concentration of 10 mg/mL) eluted with 100% methanol, was further purified by preparative HPLC to afford compounds 1 and 2. This is the first report of compounds 1-2 isolated from U. lobata. (-)-Trachelogenin (1) Figure 1. Chemical structure of (−)-trachelogenin (1) and clematoside-S (2).

Identification of Isolated Compounds 1-2
The chemical structures of the isolated compounds 1 and 2 were identified by spectroscopic analyses consisting of MS, 1 H-NMR, 13 C-NMR and 2D-NMR data analyses. By comparison with literature data [14][15][16], compound 1 was identified as (−)-trachelogenin: pale-yellow gum; electron spray ionization-mass spectrometry (ESI-MS) (negative), m/z 387. 25  The 1 H-NMR spectrum apparently showed six tertiary and one secondary methyl signals. The 13 C-NMR spectrum in CD3OD showed 47 signals except for those overlapping with CD3OD signals, two of which were assigned to those of acetic acid (δC 29.09 ppm, δC 175.76 ppm). The polarization transfer (DEPT)-135 and DEPT-90 NMR spectra enclosed one methine carbon at δC 49.17 ppm among the signals overlapping with those of CD3OD, and showed 7 methyl, 13 methylene, 18 methine, 7 quaternary, and one carbonyl carbon signals to be assigned to the active principle. Since the active principle contains a pentose-deoxyhexose-pentose chain, 30 carbon signals comes from an aglycone, which is expected to be a triterpene. The heteronuclear single quantum coherence (HSQC) spectrum showed the signals of six tertiary and one secondary methyl carbons, the latter of which was assigned to a deoxyhexose by heteronuclear multiple-bond correlation (HMBC) spectrum analysis. The assignments of the protons and carbons are discussed in the following sessions and summarized in Table 1.
The HMQC spectrum showed that a carbinol carbon at δC 82.28 ppm, which was assigned to C-3 of an aglycone, carries a proton at δH 3.61 (H-3), and these showed long range couplings to the anomeric proton and carbon of a sugar, suggesting the linkage between the aglycone and a sugar chain. The proton at δH 3.61 ppm (H-3) showed long range couplings to methyl carbon (C-23) at δC 13.79 ppm and a quaternary carbon (C-4) at δC 43.95 ppm. The methyl proton on C-23 long-range coupled to C-4 and a primary carbinyl carbon (C-24) at δC 64.58 ppm in addition to the carbinol carbon, C-3, and a methine carbon (C-5) at δC 48.15 ppm. The methine proton at δH 1.26 ppm on C-5 showed coupling to C-6 at δC 18.80 ppm, C-10 at δC 37.61 ppm, and C-25 at δC 16.40 ppm in addition to C-4, C-23, and C-24.
The methyl proton on C-25 showed a coupling to a methylene carbon at δC 39.67 ppm, which was finally assigned to C-1, in addition to two quaternary carbons at δC 37.61 ppm (C-10) and δC 49.0 (C-9). The carbinyl proton at δH 3.61 ppm (H-3) on C-3 showed vicinal couplings to methylene protons at δH 1.74 ppm (H-2a, J = 4.5 Hz) and δH 1.85 ppm (H-2b, J = 12.2 Hz). These two protons are on the carbon at δC 26.58 ppm (C-2), showed a geminal coupling (J = 17.4 Hz), and vicinal couplings to a proton at δH 1.60 ppm (H-1a) on the carbon at δC 39.67 ppm (C-1). H-1 shows a geminal coupling to H-1b at δH 0.98 ppm, which shows vicinal couplings to H-2a and H-2b. Taking all the information mentioned above, a partial structure (ring A) of the aglycone was determined as shown in Figure 1.
Of the two olefinic carbon signals at δC 123.60 ppm and δC 145.21 ppm which are later assigned to C-12 and C-13, respectively, C-12 carries an olefinic proton at δH 5.23 ppm (H-12) which showed long-range couplings to carbons at δC 24.52 ppm (secondary), δC 49.0 ppm (C-9), and δC 42.72 ppm (tertiary, C-18). The HMQC spectrum showed that the carbon at δC 24.52 ppm carries protons at δH 1.87 ppm and δH 1.89 ppm, both of which showed couplings to the olefinic proton (H-12) at δH 5.23 ppm in the HMBC spectrum. In addition, one or both of the protons appearing at δH 1.87 ppm and δH 1.89 ppm showed couplings to both olefinic carbons. These H-H and C-H couplings suggest that the carbon at δC 24.52 ppm is vicinal to the carbon at δC 123.60 ppm (C-12) to be assigned to C-11. A long rage coupling from the proton at δH 1.62 ppm (H-9) on the methine carbon at δC 49.00 ppm (C-9) to C-11 was observed. To C-9, were observed long range couplings from H-12 at δH 5.23 ppm and methyl protons at δH 0.96 ppm (H3-25) and δH 0.80 ppm (H3-26). H-9 couples to C-11 in addition to the carbons at δC 37.61 ppm (C-10), δC 48.15 ppm (C-5), and δC 40.50 ppm (C-8). These couplings enclosed the sequence of C-10/C-9/C-11/C-12/C-13, and the attachment of C-25 on C-10. The methyl protons (H3-26) shows a long range coupling to a quaternary carbon at δC 42.96 ppm, which was assigned to C-14, in addition to C-9, C-10, and the methylene carbon at δC 33.39 ppm assigned to C-8 through HMQC and H 1 -H 1 COSY spectrum analyses. Based on the above information, the partial structure constructing rings A, B, and C appeared, and hederagenin came out as the candidate for the aglycone. Comparing our NMR data with those in literatures [17][18][19], the aglycone was unambiguously identified to be hederagenin.
The linked scan MS suggests the sequence of pentosyl-deoxyhexosyl-pentosyl chain. The D configurations of the arabinose and ribose and the L configuration of rhamnose were established after hydrolysis of 2 followed by GC analysis [20][21][22]. GC-MS analysis of the trimethylsilylates of N-methoximes of the sugars obtained by acid hydrolysis of 2 showed the major peaks at tRs of 15.29, 15.81, and 17.18 min, respectively. The tRs of these peaks were identical to those of standard silylated samples, showing that the sugar components of the active principle were D-arabinose, D-ribose, and L-rhamnose. The HMBC showed that the carbinyl proton (H-3) at δH 3.61 ppm long-range coupled to an anomeric carbon (C-1') at δC 104.63 ppm, and the anomeric proton (1'-H) at δH 4.51 ppm to C-3 of hederagenin at δC 82.28 ppm. The J value between H-1' and H-2' (δH 3.69 ppm) on the carbon (C-2') at δC 76.33 ppm was 6.0 Hz, suggesting the axial-equatorial relation of the two protons, and all signals of the protons and the carbons of the pentose directly attached to hedragenin were assigned as shown in Figure 1 and Table 1. The C-2' and C-3' were distinguished by the long-range coupling from H-5a' and H-5b' to the carbon at δC 74.21 ppm (C-3'). Based on the chemical shifts of all signals and the J value (6.0 Hz) between H-1' and H-2', the pentose was determined to be α-D-ribose (Figure 1). Similarly, all the signals of α-L-rhamnose were assigned as shown in Table 1 and the ether linkage from C-2' of ribose to C-1' of rhamnose was determined by the long-range coupling from H-2' of ribose to C-1'' of rhamnose and that from H-1'' of rhamnose to C-2' of ribose. The third sugar, arabinose, was determined to be β-anomer based on the J value (4.3 Hz) between H-1''' and H-2''', and all signals of β-arabinose were assigned as shown Table 1. The long-range coupling from H-3'' to C-1''' and that H-1''' to C-3'' showed the ether linkage between C-3'' of rhamnose and C-1''' of arabinose. Finally, the chemical structure of 2 was determined to be β-D-arabinosyl-(1-3)-α-L-rhamnosyl-(1-2)-α-D-ribosyl-(1-3β)-hederagenin, namely clematoside-S.

Antimicrobial Activity of Isolated Active Compound
The antimicrobial activity of the 80% ethanol extract from U. lobata leaves was evaluated using Oxford plate method against five strains of food-borne bacteria (Eschericha coli, Salmonella typhimurium, Staphylpcocuus aureus, Bacillus subtilis, and Bacillus laterosporus), four strains of fungi (Aspergillus flavus, Aspergillus niger, Rhizopus oryzae, and Pencicillum citrinum), and six yeast strains (Candida albicans, Saccharomyces cerevisiae ATCC 204505, Saccharomyces cerevisiae\ AY529515.1, Saccharomyces cerevisiae\AJ746340.1, Saccharomyces cerevisiae\JX103178.1, and Saccharomyces boulardii\KG254081.1) as shown in Table 2. The solvent (95% methanol) used as the negative control did not show any activity. At a concentration of 10 mg/mL, the crude extract showed no antibacterial activity. Meanwhile, the extract was inactive against three species of fungi (A. flavus, R. oryzae, and P. citrinum) even at 10 mg/mL. Among the test fungi, the most sensitive strain was A. niger with the diameter of inhibition zone of 9 mm. The extract showed no activity (10 mg/mL) against C. albicans. It was worth noting that the extract exhibited remarkable anti-yeast activities against S. cerevisiae ATCC 204505, S. cerevisiae\AY529515.1, S. cerevisiae\AJ746340.1, S. cerevisiae\JX103178.1, and S. boulardii\KG254081.1 with the diameter of the inhibition zone in the range from 14 to 17 mm. The result suggest that U. lobata leaves inhibits selectively the growth of some yeast strains. So, we selected out S. cerevisiae ATCC 204505 as the indicator for detecting the main anti-yeast substance(s) in U. lobata leaves. Using a combination of chromatographic methods, (−)-trachelogenin (1) and clematoside-S (2) were isolated from the 80% ethanol extract of U. lobata leaves. It was found that 1 showed no activity against all of the selected microorganisms in the antimicrobial assay (Table 2). However, 2 showed promising activity against S. cerevisiae ATCC 204505 as compared to standard anti-yeast reagent, streptomycin. In addition, 2 exhibited a potent inhibitory effect against the five test yeast strains, except for C. albicans, with diameter of inhibition zones in the range from 11 to 20 mm.
Further study was carried out to investigate the anti-yeast effect of 2 against S. cerevisiae ATCC 204505, S. cerevisiae\AY529515.1, S. cerevisiae\AJ746340.1, S. cerevisiae\JX103178.1, and S. boulardii\KG254081.1 by measuring the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The MICs and MBCs of 2 to the five test yeast strains were shown in Table 3. These results demonstrated that 2 had certain antibacterial and bactericidal property. In general, the MICs of 2 against the test yeasts were in range from 0.61 to 9.80 μg/mL, and MBCs from 2.42 to 9.80 μg/mL, respectively. Associated with the results of disc diameter of inhibition zone in Table 2, it was clearly indicated that 2 showed the strongest activity against S. cerevisiae ATCC 204505, whereas it showed the moderate activity against the other yeast strains (Table 3). clematoside-S (2) isolated from clematotic species has been reported to exhibit cytotoxic activity against several cancer cells [23]. However, compound 2 has never been studied for antimicrobial activity. Our finding is the first report on the isolation of 2 with anti-yeast activity from U. lobata. Therefore, it may be proposed that U. lobata can be used as a natural anti-yeast agent to control food spoilage caused by yeast. However, further study is warranted to provide clear evidence for toxicity profile.

Plant Material and Regents
The leaves of U. lobata used in this study were purchased from Chengdu Medicinal Materials (Chengdu, China) and properly identified at the Department of Pharmacology, Hua Xi Medicinal Center of Sichuan University, China. A voucher specimen is deposited in the Rice Research Institute, Sichuan Agricultural University (No. 20090603). Penicillin and streptomycin were obtained from Sichuan Changwei Pharmaceutical Co., Ltd. (Chengdu, China). Agar, beef extract, sucrose and peptone were purchased from Chengdu Best Reagents Co., Ltd. (Chengdu, China). All other reagents used were of analytical grade.

Antimicrobial Activity
The tested microorganisms contained Gram-negative bacteria ( 1, and Saccharomyces boulardii\KG254081.1) were isolated from spoiled grapes and identified by morphology, biochemical tests and ITS sequence analysis. All the microorganisms were maintained on nutrient agar at 4 °C and were sub-cultured every month in our laboratory. In the present test, nutrient agar culture medium was for bacteria and the medium of potatoes was for fungi and yeasts strains.
Antimicrobial activity of the test sample was determined by Oxford plate method [24]. In short, bacterial cultures were diluted to obtain a bacterial suspension of 10 6 CFU/mL with sterile water. Petri plates containing 20 mL of nutrient agar were inoculated with 0.2 mL of bacterial culture and were allowed to dry in sterile chamber. The Oxford plates (6 mm in diameter) were impregnated with 0.1 mL of test sample in 95% methanol and placed on the inoculated agar. Penicillin and streptomycin were used as the positive control for bacteria and fungus, respectively. The inoculated plates of bacteria were incubated at 37 °C for 24 h, and fungi were incubated at 35-37 °C for 48 h. The antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms.
The minimum inhibitory concentration (MIC) of the test sample was evaluated for the yeast strains which were determined by the method of broth dilution [25]. An aliquot of 2 mL of the medium of potatoes was placed into each tube, and all tubes were autoclaved at 121 °C. Test sample (filtered, 0.22 μm) was added to the tubes to keep the final concentrations ranging from 0.06 to 40 μg/mL. The test yeast suspension was added into to the inoculum size of 10 6 CFU/mL. Then, the inoculated tubes were incubated at 37 °C for 18-24 h. The MIC was evaluated by measuring the turbidity of inoculated culture media. Another liquid medium without adding any yeast was prepared as the negative control. The minimum inhibitory concentration at which no microorganism grew in the culture media was defined as the value of MIC. The minimum bactericidal concentration (MBC) of the test sample was determined according to the MIC values. The sample showing no increases in turbidity were streaked on potatoes medium and incubated at 37 °C for 18-24 h. The lowest concentration of the test sample where was no viable yeasts was identified as the value of MBC.

Conclusions
In the present study, (−)-trachelogenin (1) and clematoside-S (2) were isolated from the leaves of U. lobata for the first time by a combination of chromatographic methods and their chemical structure was identified by MS and extensive NMR data analysis. Furthermore, to the best of our knowledge, this is the first study demonstrating the antimicrobial activity of clematoside-S. In addition, clematoside-S shows a specific anti-yeast activity against S. cerevisiae strains, which are one of the most significant spoilage yeasts for juice and soft drinks.