New Alkaloids from Green Vegetable Soybeans and Their Inhibitory

Letter pubs.acs.org/JAFC

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A‑Activated Lymphocytes ABSTRACT: A comprehensive phytochemical study of the chemical constituents of green vegetable soybeans resulted in the isolation of two new alkaloids, soyalkaloid A, 1, and isoginsenine, 2, together with four known ones, ginsenine, 3, (1S,3S)-1methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, 4, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, 5, and indole-3-carboxylic acid, 6. The structures of compounds 1−6 were elucidated on the basis of spectroscopic and chemical analyses. All of the alkaloids were isolated from soybeans for the first time, and compound 1 was a new indole-type alkaloid with a novel carbocyclic skeleton. Their inhibitory activities on the proliferation of concanalin A-activated lymphocytes were assessed by CCK8 assay. KEYWORDS: soybean, Glycine max, alkaloids, structure identification, immunosuppression

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INTRODUCTION Soybean plant [Glycine max (L.) Merr., Fabaceae] is an important economic crop as a major source of healthy food in many Asian countries. Green vegetable soybeans, called “maodou” in China, are vegetable-type fruits of soybean plants that are harvested at the green stage for use as a vegetable before they mature and dry.1,2 Green vegetable soybeans are rich in protein, ω-3 fatty acids, carbohydrates, fiber, and micronutrients such as folic acid, manganese, vitamin K, potassium, and zinc.3 Micronutrients are nutrients that humans and livestock require in small quantities to assemble a whole range of physiological functions.4 Isoflavones, saponins, and phenolic compounds have been reported from soybeans. The extract of green vegetable soybeans exhibited various degrees of antioxidative and anti-inflammatory activities according to the published literature. However, to date, the ingredients from green vegetable soybeans have not been

Figure 1. Structures of compounds 1−6.

reported to exhibit immunosuppression activity. A comprehensive phytochemical study on green soy fruits led us to find the presence of alkaloids. We herein report the isolation and identification of six indole alkaloids, including two new alkaloids and four known ones. To the best of our knowledge, all of the alkaloids were isolated from soybeans for the first time. Meanwhile, their inhibitory activities on the proliferation of concanavlin A (Con A)-activated lymphocytes were first assessed in our group, using the cell counting kit-8 (CCK8) assay.

Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data of Compounds 1 and 2 (DMSO-d6, δ) 1 δC 1 1a 2 3 3a 4 5 5a 6a 6 7 8 9 9a 10 10a 11 a

2 δH

δC 50.0

155.2 37.1 22.5 55.7 174.4 104.8 126.2 117.8 118.7 121.2 111.1 136.3

4.852 m, 4.33 m 3.19 m, 2.75 m 4.34 t (5.5)a

7.46 d (8.0) 6.99 t (8.0) 7.07 t (8.0) 7.33 d (8.0)

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172.3 53.6 22.5 105.3 126.2 119.2 117.9 122.1 110.9 137.2

11.06 s 129.8

δH 4.87 q (7.5)

4.11 dd (12.0, 4.5) 3.36 m, 3.16 m

7.47 d (8.0) 7.03 t (8.0) 7.12 t (8.0) 7.31 d (8.0) 11.10 s

130.3 17.3

MATERIALS AND METHODS

General Experimental Procedures. Melting points were determined by the XT5 micromelting-point apparatus (XT5, Beijing families instrument light instrument plant, China) and are uncorrected. UV spectra were determined on a UV2401 spectrometer (Shimadzu Corp., Kyoto, Japan). The specific optical rotation values were determined with a PerkinElmer model 241 polarimeter (PerkinElmer Inc., Waltham, MA, USA). IR spectra were determined on a PerkinElmer 983 G spectrometer. 1H, 13C, and 2D NMR spectra were recorded on a Varian Inova 500 spectrometer (Varian Inc., Palo Alto, CA, USA) in DMSO-d6 using tetramethylsilane (TMS) as the internal Received: December 25, 2015 Revised: January 31, 2016 Accepted: January 31, 2016

1.70 d (7.5)

Data in parentheses are J values (in hertz). © XXXX American Chemical Society

A

DOI: 10.1021/acs.jafc.5b06107 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. HR-ESI-MS spectrum of compounds 1 and 2.

Figure 3. 1H NMR spectra of compounds 1 and 2. (250 × 9.4 mm i.d., 5 μm; Agilent Corp., Palo Alto, CA, USA) on a Shimadzu HPLC system composed of an LC-20AT pump with an SPD-20A detector (Shimadzu Corp.), and the wavelength for detection was 203 nm. Medium-pressure liquid chromatography (MPLC)

standard. HR-ESI-MS spectra were determined on a Micromass Q-TOF2 spectrometer (Micromass Corp., London, UK). Chromatography. HPLC analysis and purification were performed with an Agilent Zorbax SB-C18 semipreparative HPLC column B


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Figure 4. 13C NMR spectra of compounds 1 and 2.

Figure 5. HSQC spectra of compounds 1 and 2. purification was performed on a Büchi Flash Chromatography system composed of a C-650 pump and a flash column (460 × 26 mm i.d.; Büchi Corp., Flawil, Switzerland). Silica gel (200−300 mesh) used for column chromatography and precoated silica gel for TLC plates were purchased

from Qingdao Marine Chemical Factory (Qingdao, China). A 10% sulfuric acid alcohol solution was used for TLC colorization. Sephadex LH-20 for column chromatography was purchased from GE Corp. (Piscataway, NJ, USA). D101 macroporous resin for column C


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Figure 6. H−H COSY spectra of compounds 1 and 2. Experimental Animals approved by the State Council of People’s Republic of China. Materials. Fresh soy fruits (whole pods of green vegetable soybeans) were collected in Suzhou city (Jiangsu province, China) in September 2012 and authenticated by Prof. Xiao-ran Li (Soochow University). In this study, 5.0 kg of fresh fruits were cut into flakelets, spread on bamboo mats, and sun-dried. A voucher specimen (no. 12-09-16-01) was deposited in the herbarium of the College of Pharmaceutical Science at Soochow University. Extraction and Isolation. The dried plant material (1.2 kg) was extracted twice with MeOH at 80 °C under reflux. The solvent was subsequently removed under reduced pressure to yield a residue (225 g), which then was dissolved in distilled water and fractionated in petroleum ether, CHCl3, and EtOAc successively. The EtOAc fraction (32.5 g) was further vacuum chromatographed on a silica gel (60−100 mesh) column (30 × 18 cm i.d.), eluted with a gradient of CHCl3/ MeOH (90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 0:100, each 6.0 L). The CHCl3/MeOH (90:10) eluate (380 mg) was separated by MPLC over a silicon gel column and eluted with CHCl3/MeOH (95:5, 90:10, 85:15, 80:20, 100:0, each 500 mL) to afford five fractions. Fraction 3 (7.5 mg) was subjected to Sephadex LH-20 gel column chromatography (100 cm × 3 cm i.d.) and eluted with pure MeOH, resulting in compound 1 (3.0 mg). The CHCl3/MeOH (70:30) eluate (3.2 g) from the vacuum chromatography provided five fractions after separation by MPLC. Fraction 5 (30 mg) was further subjected to semipreparative RP-HPLC and eluted with MeOH/H2O (80:20), which yielded compounds 2 (3.2 mg, tR 28.75 min) and 3 (6.1 mg, tR 31.08 min). In a similar way, the CHCl3/MeOH (60:40) eluate (5.2 g) from the vacuum chromatography was first separated into six fractions by MPLC

Figure 7. Key H−H COSY, HMBC, and NOESY correlations of compounds 1 and 2. chromatography was purchased from Xi’an Sunresin New Materials Co. Ltd. (Xi’an, China). Animals. ICR mice of similar age and weight (20−25g) were used for this study. They were purchased from the Experimental Animal Center of Soochow University and were housed under specific pathogen-free conditions. The animal room was controlled for temperature (22 ± 2 °C), light (12 h light/dark cycle), and humidity (50 ± 10%). All laboratory feed pellets and bedding were autoclaved. The mice were anesthetized using diethyl ether, and blood samples (0.5 mL) were withdrawn from the tail vein of the mice. The animal study proposal was approved by the Institutional Animal Care and Use Committee of Soochow University with the Permit IACUC2007-13. All mouse experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning D


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Figure 8. HMBC spectra of compounds 1 and 2. The viability of the cells was assessed by CCK8 assay,5 which is based on the reduction of CCK8 by the mitochondrial dehydrogenase of intact cells to an orange formazan product. Briefly, 1 × 104 cells/well lymphocytes was dispensed within 96-well culture plates (Costar, USA) in 100 μL. Then different concentrations of compounds (0, 0.25, 0.5, 1.0, and 2.0 μg/mL) were put in different wells. Each of the concentrations above was regarded as one treated group, whereas there was no compound in the negative control group. The active control group was treated with 10 μg/mL Con A (Sigma-Aldrich, St. Louis, MO, USA), and 1 μM cyclosporine A (CsA) (Sigma-Aldrich, St. Louis, MO) as the positive control. Each of the treated or nontreated groups contained six parallel wells. Culture plates were then incubated for 24 h prior to the addition of 10 μL of CCK8 (Dojindo Molecular Technologies, Kumamoto, Japan) to each well, and cells were incubated for 3 h at 37 °C. The optical density (OD) was measured at a wavelength of 450 nm. Statistical Analysis. Data were expressed as the mean ± SD and evaluated using one-way ANOVA. The post hoc test was done with Student’s Dunnett test. (*) p < 0.05 was considered statistically significant. Calculations were performed using the SPSS 16.0 statistical package.

over the ODS column, and then these fractions were further separated by using semipreparative HPLC to yield compounds 4 (8.2 mg), 5 (7.5 mg), and 6 (31.8 mg). Soyalkaloid A (1): yellowish powder; [α]25 D 0.0 (c 0.12, MeOH); UV λmax (MeOH) 306, 351 nm; IR(KBr) νmax 3369, 2946, 1718, 1610, 1405, 1047, 882 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) spectroscopic data, see Table 1; HR-ESI-MS (positive ionization mode) m/z 227.0813 ([M + H]+, calcd for C13H11O2N2 227.0821). Isoginsenine (2): white amorphous powder; [α]25 D −92.1 (c 0.11, MeOH); UV λmax (MeOH) 220, 272, 290 nm; IR(KBr) νmax 3356, 3288, 1645, 1449, 1055, 814 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) spectroscopic data, see Table 1; HR-ESIMS (negative ionization mode) m/z 229.0964 ([M − H]−, calcd for C13H13O2N2 229.0977). Inhibitory Activities on the Proliferation of Con A-Activated Lymphocytes. Mice were sacrificed by cervical dislocation, and their spleens were removed aseptically. Spleens were placed in cold Hanks solution and teased apart with a pair of forceps and a needle. A single-cell suspension from the teased tissue was obtained by passing it through a 200-mesh sieve and hemolyzed by the buffer solution containing 1 mmol/L Tris-HCl and 1% NH4Cl (pH 7.2). Cells were washed twice with RPMI 1640 medium and subsequently suspended in complete RPMI 1640 culture medium. Cell viability was determined by Trypan blue dye exclusion.

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RESULTS AND DISCUSSION The MeOH extract of the dried soy fruits was fractionated by repeated silicon gel CC and ODS CC to yield two new alkaloids, 1 and 2, as well as four known ones, which were identified as E


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Figure 9. NOESY spectra of compounds 1 and 2.

Table 2. Inhibitory Activities of Compounds 1−6 on the Proliferation of Con A-Activated Lymphocytes compound concn (μM) 0 0.25 0.5 1.0 2.0

group A B A B A B A B A B

a

1

2

3

4

5

6

0.35 ± 0.04 0.43 ± 0.03 0.36 ± 0.02 0.40 ± 0.05 0.33 ± 0.05 0.36 ± 0.03*c 0.38 ± 0.04 0.32 ± 0.02** 0.40 ± 0.06# 0.29 ± 0.03**

0.35 ± 0.04 0.43 ± 0.03 0.34 ± 0.03 0.41 ± 0.02 0.31 ± 0.04 0.40 ± 0.05 0.33 ± 0.03 0.41 ± 0.03 0.37 ± 0.04 0.44 ± 0.06

0.35 ± 0.04 0.43 ± 0.03 0.35 ± 0.02 0.42 ± 0.04 0.32 ± 0.03 0.43 ± 0.02 0.36 ± 0.05 0.45 ± 0.06 0.33 ± 0.03 0.47 ± 0.05

0.35 ± 0.04 0.43 ± 0.03 0.34 ± 0.03 0.43 ± 0.05 0.34 ± 0.02 0.41 ± 0.06 0.33 ± 0.02 0.47 ± 0.08 0.35 ± 0.02 0.46 ± 0.05

0.35 ± 0.04 0.43 ± 0.03 0.36 ± 0.03 0.45 ± 0.04 0.32 ± 0.05 0.42 ± 0.05 0.34 ± 0.04 0.43 ± 0.02 0.37 ± 0.06 0.48 ± 0.08

0.35 ± 0.04 0.43 ± 0.03 0.36 ± 0.04 0.43 ± 0.03 0.31 ± 0.05 0.44 ± 0.05 0.37 ± 0.05 0.46 ± 0.05 0.39 ± 0.05# 0.45 ± 0.04

cyclosporine Ab 0.35 ± 0.04 0.43 ± 0.03

0.35 ± 0.03 0.28 ± 0.02**

a Group A, nontreated with Con A; group B, treated with Con A. bPositive control. c#, p < 0.05 group A vs 0 μM group A; *, p < 0.05; **, p < 0.01 group B vs 0 μM group B.

ginsenine, 3,6 (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline3-carboxylic acid, 4,7 (1R,3S)-1-methyl-1,2,3,4-tetrahydro-βcarboline-3-carboxylic acid, 5,7 and indole-3-carboxylic acid, 68 (Figure 1). Compound 1 was obtained as yellowish powder. The molecular formula of C13H10O2N2 was determined on the basis

of its HR-ESI-MS [M + H]+ ion peak at m/z 227.0813 (calcd 227.0821) (Figure 2), indicating 10 degrees of unsaturation. The IR spectrum showed absorption bands at 3369, 1718, and 1610 cm−1, assignable to secondary amine, α,β-unsaturated lactone, and α,β-unsaturated imide functions,7 respectively. The 1 H NMR spectrum of 1 (Table 1; Figure 3) indicated the F


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(with or without 10 μg/mL Con A activation) survival and proliferation were examined using the CCK8 assay. As shown in Table 2, no significant differences of the proliferation of lymphocytes were observed for the groups treated with different concentrations of alkaloids in comparison with the nontreated group (negative control). Con A promoted lymphocyte survival and proliferation at 24 h (Table 2); however, there were significant decreases in the groups treated with compound 1 at concentrations from 0.5 to 2.0 μmol/mL in Con A-activated lymphocytes (Table 2). Specifically, the inhibitory effect of compound 1 at the dosage of 2.0 μmol/mL could even be 50%, on par with that of cyclosporine A, a known immunosuppressant, used as the positive control in this assay, which could significantly reduce the proliferation of Con A-activated lymphocytes (Table 2). The other alkaloids did not exhibit significant immunosuppressive activity. These results demonstrated that compound 1 could not affect the proliferation of lymphocytes; however, it could dramatically reduce the proliferation of activated lymphocytes induced by Con A. Therefore, the related mechanism of compound 1 should be explored in further research as a promising natural immunosuppressant.

presence of one NH group as a singlet at δ 11.06 (1H, s, H-10), and four aromatic protons at δ 7.46 (1H, d, J = 8.0 Hz, H-6), 7.33 (1H, t, J = 8.0 Hz, H-9), 7.07 (1H, t, J = 8.0 Hz, H-8), and 6.99 (1H, t, J = 8.0 Hz, H-7), indicating the presence of an indole skeleton of an unsubstituted moiety in the aromatic ring. The 1H NMR spectrum also showed one triplet methine signal at δ 4.34 (1H, t, J = 5.5 Hz, H-3a), as well as signals of two methylene groups at δ 4.85, 4.33 (1H each, m, H-2) and δ 3.19, 2.75 (1H each, m, H-3). The 13C NMR spectrum (Table 1; Figure 4) exhibited resonances for 13 C atoms, substitution patterns of which were deduced from DEPT and HSQC (Figure 5) experiments as two CH2, five CH groups, and six quaternary C atoms. The correlated spectroscopy (H−H COSY) (Figures 6 and 7) indicated the four aromatic protons located at C-6, C-7, C-8, and C-9, respectively, and the two methylene groups were attributed to C-2 and C-3, whereas the methine group was established at C-3a. In the HMBC spectrum (Figures 7 and 8), correlations between δH 4.85, 4.33 (H-2) and δC 155.2 (C-1a) and between δH 3.19, 2.75 (H-3) and δC 155.2 (C-1a) indicated that the C−N double bond was established at C-1a and C-1, whereas the α,β-unsaturated lactone group was established at C-3a and C-5 on the basis of the HMBC correlation between δH 4.34 (H-3a) and δC 174.4 (C-5). Thus, compound 1 was identified as 3,3a-dihydro-2H-pyrrolo[2′,3′:5,6]pyrano[4,3-b]indol-5(10H)-one, named soyalkaloid A. Compound 2 was obtained as a white amorphous powder. The molecular formula was determined as C13H14O2N2 on the basis of its HR-ESI-MS [M − H]− ion peak at m/z 229.0964 (calcd 229.0977) (Figure 2), indicating 8 degrees of unsaturation. The IR spectrum showed absorption bands at 3356, 3288, and 1645 cm−1, assignable to hydroxy, secondary amine, and secondary amine functions,7 respectively. The 1H NMR spectrum (Table 1 and Figure 3) showed four aromatic proton signals at δ 7.47 (1H, d, J = 8.0 Hz, H-6), 7.31 (1H, t, J = 8.0 Hz, H-9), 7.12 (1H, t, J = 8.0 Hz, H-8), and 7.03 (1H, t, J = 8.0 Hz, H-7), one doublet methyl signal at δ 1.70 (1H, d, J = 7.5 Hz, H-11), two heteroatomic substituted methine signals at δ 4.87 (1H, q, J = 7.5 Hz, H-1) and 4.11 (1H, dd, J = 4.5, 12.0 Hz, H-4), and one methylene signal at δ 3.16 and 3.36 (1H each, m, H-5). The NMR spectral data of compound 2 were similar to those of ginsenine,7 except for the chemical shift changes for C-3 (+3.0 ppm) at δ 172.3, C-4 (−4.0 ppm) at δ 53.6 and for C-10a (−2.0 ppm) at δ 130.3, suggesting that compound 2 has α-OH instead of the β-OH in ginsenine. The observation of the NOE correlation between H-4 at δ 4.11 and H-11 at δ 1.70 in the NOESY spectrum (Figure 9) confirmed the assignment (Figure 2). Thus, the structure of compound 2 was determined to be 4α-hydroxy-1β-methyl-1,2,4,5-tetrahydroazepino[3′,4′-b]indol-5(10H)-one, named isoginsenine. Inhibitory Activities on the Proliferation of Con A-Activated Lymphocytes. During the course of removal of pathogenic microorganisms, the activation of T lymphocytes will play an important role in the immune response.9 However, the overproliferation of T lymphocytes is also harmful to healthy tissue,10 which may contribute to systemic lupus erythematosus,11 rheumatoid arthritis,12 Kawasaki disease,13 transplant reaction,14 and certain types of neurodegenerative diseases.15 The investigation of T lymphocyte proliferation inhibitor from natural products will be helpful to the development of safe therapeutic approaches against inflammatory and immune disorders.16−18 To determine whether alkaloids isolated from green soybeans have immune suppression activity, we tested the direct effects of them on mouse lymphocytes. The effects of different concentrations of compounds 1−6 on lymphocytes

Taoyun Wang† Jianping Zhao§ Xiaoran Li† Qiongming Xu*,† Yanli Liu*,‡ Ikhlas A. Khan†,§ Shilin Yang†

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† Department of Pharmacognosy and ‡Department of Pharmacology, College of Pharmaceutical Science, Soochow University, Suzhou 215123, China § National Center for Natural Products Research, and Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677, United States

AUTHOR INFORMATION

Corresponding Authors

*(Q.X.) Phone: 86-512-69561421. Fax: 86-512-65882089. E-mail: [email protected]. *(Y.L.) Phone: 86-512-65882080. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Jon Parcher at the University of Mississippi for helpful discussions. REFERENCES

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New Alkaloids from Green Vegetable Soybeans and Their Inhibitory

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