Constituents from Vigna vexillata and Their Anti-Inflammatory Activity

The seeds of Vigna genus are important food resources and there have already been many reports regarding their bioactivities. In our preliminary bioassay, the chloroform layer of methanol extracts of V. vexillata demonstrated significant anti-inflammatory bioactivity. Therefore, the present research is aimed to purify and identify the anti-inflammatory principles of V. vexillata. One new sterol (1) and two new isoflavones (2,3) were reported from the natural sources for the first time and their chemical structures were determined by the spectroscopic and mass spectrometric analyses. In addition, 37 known compounds were identified by comparison of their physical and spectroscopic data with those reported in the literature. Among the isolates, daidzein (23), abscisic acid (25), and quercetin (40) displayed the most significant inhibition of superoxide anion generation and elastase release.


Introduction
Phytochemicals from dietary and medicinal plants have been supposed as promising sources of potential anticancer agents with increasing anticancer evidences coupled with considerations of safety and efficacy [1,2]. Dietary and medicinal plants are importrant sources of phytochemicals for the treatment of cancers [3]. Several phytochemcials purified from natural plants, such as curcumin [4,5], epigallocatechin gallate [6], and soy isoflavones [7] had already been studied in various phases of clinical trials. The rapid population growth increase and inadequate supplies of foods had resulted in the nutrition deficiencies among the people living in the developing countries. The rigorous world food problem has presented an urgent condition for the nutritionists to investigate the choices of utilizing some less known crop seeds as additional sources of foods. With increasing interest in new food sources, the seeds of wild relatives of cultivated plants including the tribal pulses are now receiving more and more attention. The history of legumes is tied in closely with that of human civilization, appearing early in Asia, America, and Europe by thousand years ago, where they became essential for supplementing protein. Recently, there was a report regarding the chemical constituents in the wild soybean Glycine soja and their biological activity [8]. It initiated our interests to explore the chemical compositions of the wild relatives of edible legumes. For example, the seeds of Vigna vexillata are boiled and consumed by the tribal people living in the hilly region of Pune district, India [9,10]. The proximate composition, minerals, seed protein fractions, amino acids, fatty acids, and antinutritional factors of the seeds of V. vexillta were analyzed. However, information on the biochemical composition and chemical constituents of the wild relatives of grain legumes is very rare.
V. vexillata (L.) A. Rich (Fabaceae) is a perennial climbing or trailing herb belonging to the Vigna genus. This genus is widely distributed in tropical Africa, India, Indochina, Australia, Japan, Korea, China, and Taiwan. In Taiwan, the Vigna genus usually grows in grassland, margin of bush, at elevation between 1000 and 1800 m high, in the central mountain area [11]. The extracts of Vigna species have been reported to display the hypoglycemic [12,13], antihypertensive [14,15], cholesterol reduction [16], antioxidant [17,18], antibacterial [19,20], and anti-cancer bioactivities [21][22][23]. In the preliminary bioassay, at the tested concentration (10 µg/mL) the methanol extract, chloroform, and water fractions of V. vexillata displayed the inhibition of superoxide anion generation and elastase release with percentages of 22.06 ± 5.66%, 57.65 ± 3.69%, and 11.08 ± 5.19%; 19.35 ± 1.52%, 67.27 ± 3.53%, and 11.00 ± 3.37%, respectively (Table 1). Therefore, it is aimed to purify and identify the chemical constituents of the methanol extract V. vexillata in the present research. Furthermore, the anti-inflammatory bioactivities of these purified constituents were also examined to explore the new candidates of phytomedicinal lead compounds.

Anti-Inflammatory Activity
Overexpression of neutrophils had already been regarded to display significant correlations to various human diseases, such as rheumatoid arthritis, ischemia, reperfusion injury, chronic obstructive pulmonary disease, and asthma [54][55][56][57][58]. In response to diverse stimuli, activated neutrophils secreted a series of cytotoxins, such as superoxide anion and elastase [59]. Thus, in infected tissues and organs it was critical to maintain superoxide anion production and elastase release in physiological conditions. Nowadays only a few available agents could directly modulate neutrophil proinflammatory responses in clinical practice. Therefore, those purified compounds isolated in sufficient quantity were evaluated for inhibition of superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB (Table 2) and 1, 3, 19, 22, 23, 25, and 40 at 10 µM concentration exhibited the inhibition percentages higher than 50%. Among those examined constituents, daidzein (23), abscisic acid (25), and quercetin (40) displayed the most significant inhibition of superoxide anion generation and elastase release with IC 50 values ranged from 2.66 ± 0.85 to 5.51 ± 1.07 µM, compared with the reference compound LY294002 [60], which displayed IC 50 of 1.38 ± 0.22 and 1.95 ± 0.35 µM towards superoxide anion generation and elastase release, respectively. In addition, diphenyleniodonium (DPI), a NADPH oxidase inhibitor, was also used as a positive control for superoxide anion generation with IC 50 of 0.93 ± 0.52 µM. Therefore, the extracts and purified principles of V. vexillata have potential to be developed as new anti-inflammatory drugs or health foods.

General
All the chemicals were purchased from Merck KGaA (Darmstadt, Germany) unless specifically indicated. Melting points of purified compounds were determined by a Fisher Scientific melting point measuring apparatus without corrections. Optical rotations were measured with the Atago AP-300 automatic polarimeter. The UV spectra were obtained on a GBC Cintra 101 UV-Vis spectrophotometer. The IR spectra were obtained on a Bruker Tensor 27 FT-IR spectrometer. The mass and high-resolution mass spectra were obtained on a VG platform electrospray mass spectrometer and a Thermo Fisher Scientific LTQ orbitrap XL mass spectrometer (San Jose, CA) operated both in the negative-ion and positive-ion modes. 1 H-and 13 C-NMR, COSY, NOESY, HMQC, and HMBC spectra were recorded on the Bruker AV-500 and Avance III-400 NMR spectrometers with tetramethylsilane as the internal standard. Standard pulse sequences and parameters were used for the NMR experiments and all chemical shifts were reported in parts per million (ppm, δ). Column chromatography was performed on silica gels (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck KGaA). Thin layer chromatography (TLC) was conducted on precoated Kieselgel 60 F 254 plates (Merck) and the compounds were visualized by UV light or spraying with 10% (v/v) H 2 SO 4 followed by heating at 110 °C for 10 min. High performance liquid chromatography (HPLC) was performed on a Shimadzu LC-10ATVP series pumping system equipped with a Shimadzu SPD-M10AVP diode array detector.

Plant Materials
The whole plants of V. vexillata L. A. Rich (Fabaceae) were collected in the river shores of Chingshui River in Nantou, Taiwan, in March 2006. The plant materials were authenticated by C. S. Kuoh (Department of Bioscience, National Cheng Kung University, Tainan, Taiwan). A voucher specimen (PCKuo_2006002) was deposited in the herbarium of Department of Biotechnology, National Formosa University, Yunlin, Taiwan.

Extraction and Isolation
The whole plants of V. vexillata L. (4.9 kg) were powdered and exhaustively extracted with methanol under reflux (10 L × 5 × 8 h), and the combined extracts were concentrated under reduced pressure to give a dark brown syrup (900 g). The crude extract was partitioned between chloroform and water to afford chloroform (210 g) and water extracts (690 g), respectively.
The chloroform extract was subjected to a silica gel column eluted with n-hexane and a step gradient of acetone (100:1 to 1:1) to afford 13 fractions as monitored by TLC. There were no constituents identified from fractions 1-4. Fraction 5 was subjected to silica gel column chromatography with mixture of n-hexane and acetone (50:1) to yield a mixture of 4 and 5 (3.0 mg), and 6 (1.5 mg). Fraction 6 was further resolved on a silica gel column eluted with n-hexane and a step gradient of ethyl acetate (100:1 to 1:1) to give three subfractions (6.1-6.3). Subfraction 6.1 was recrystallized with chloroform and methanol to afford a mixture of 7 and 8 (900.0 mg). Subfraction 6.2 was purified with silica gel column chromatography eluted with the solvent mixture of n-hexane and ethyl acetate (8:1) to yield 9 (8.2 mg), 10 (10.0 mg), and 11 (4.0 mg). Subfraction 6.3 was separated through silica gel column chromatography eluted with chloroform and methanol (100:1) and further purified with preparative TLC on silica gel to afford 12 (1.0 mg). Column chromatography over silica gel of the seventh fraction by the mixture of benzene and ethyl acetate (8:1) and followed by purification with preparative TLC on silica gel to result in 13 (3.0 mg) and 14 (0.5 mg). Fractions 8 and 9 were combined and further separated by repeated column chromatography over silica gel eluted with chloroform and a step gradient with acetone (300:1 to 1:1) followed by purification with preparative TLC on silica gel to yield 1 (2. The water extract was applied to a reversed-phase Diaion HP-20 column eluted with water and methanol gradients to afford 14 fractions as monitored by C-18 TLC, however, no constituents were identified from fractions 1-3. Fractions 4 and 5 were combined and subjected into C-18 column chromatography eluted with water and methanol gradients and further recrystallization with chloroform-methanol to yield 28 (6.0 mg), 29 (2.5 mg), and 30 (5.0 mg), respectively. Fractions 6-8 were merged and purified by C-18 column chromatography eluted with water and methanol gradients followed by preparative TLC eluted with chloroform and methanol (20:1) on the resulted subfractions to afford 31 (20.0 mg), 32 (1.5 mg), 33 (2.0 mg), and 34 (15.0 mg), respectively. Fractions 7-9 were merged and purified by silica gel column chromatography eluted with chloroform and methanol (50:1) and further recrystallization of methanol of the subfractions to result in 35 (3.0 mg) and 36 (2.0 mg). Fractions 12 and 13 were combined and separated by silica gel column chromatography eluted with chloroform and methanol (50:1) to afford four subfractions (12.1-12.4). Subfraction 12.1 was recrystallized with chloroform and methanol to yield 35 (2.0 mg). Subfraction 12.2 was purified by reversed-phase HPLC with a Supelco Discovery ® HS C-18 (250 × 4.6 mm, 5µm) column eluted with 0.5 mL/min of MeOH-H 2 O (40:60) to give 37 (5.0 mg) and 38 (4.0 mg). Subfraction 12.3 was elastase release. The results were expressed as the percent of elastase release in the FMLP/ CB-activated, drug-free control system.

Statistical Analysis
Results were expressed as mean ± S.D. Computation of 50% inhibitory concentration (IC 50 ) was computer-assisted (PHARM/PCS v.4.2). Statistical comparisons were made between groups using Student's t test. Values of P less than 0.05 were considered to be statistically significant.

Conclusions
The present investigation on the methanol extracts of V. vexillata resulted in the isolation and characterization of totally three new principles (1-3) along with 37 known constituents . In the screening of their bioactivity, three compounds, daidzein (23), abscisic acid (25), and quercetin (40) from the active chloroform fraction demonstrated significant anti-inflammatory potentials compared with the PI3K inhibitor LY294002 and a NADPH oxidase inhibitor DPI. It is well known that the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) pathway plays an important role in neutrophil activation. The NADPH oxidase is also important in the inflammatory mechanism. Therefore, the purified principles of V. vexillata were potential to be developed as new anti-inflammatory drugs through the inhibition of PI3K or NADPH oxidase. This is the first report of complete chemical compositions of V. vexillata and it would provide the comprehensive knowledge related to the further discovery of phytochemical lead compounds from natural food sources.