Chemical Constituents from the Seeds of Amorpha fruticosa and Their Chemotaxonomic Significance

Xin Wu¹, Hongbo Liao^2*, Kefeng Wu¹, Liao Cui¹

¹Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical University, Zhanjiang, China.
²Department of Pharmacology, Guangdong Medical College, Zhanjiang, China.

DOI: 10.4236/oalib.1102740                

Abstract

Seventeen compounds, including six rotenoids (1-6), seven isoflavones (7-13), one stilbene (14) and three benzoic acid derivatives (15-17) were isolated from the seeds of Amorpha fruticosa. Their structures were elucidated by spectroscopic methods and by comparison of their reported spectral data. Among them, compound 4 was firstly purified as a natural product, compounds 5, 8 - 12 and 15 - 17 were isolated from the genus Amorpha for the first time, and compound 17 was obtained from the Fabaceae family initially. The presence of these compounds suggests that genus Amorpha and Dalbergia may have very close chemotaxonomic relationship, and shows the relationship between this plant and other species from the Fabaceae family.

Keywords

Amorpha fruticosa, Rotenoid, Isoflavone, Chemotaxonomy

Subject Areas: Plant Science

1. Introduction

The genus Amorpha (Fabaceae) including 16 species is native to North America with a center of diversity in the southeastern United States [1] . Among them, A. fruticosa (Figure 1) which has been used as a Chinese folk medicine for the treatment of burn, ambustion, carbuncle and eczema [2] is the only one species introduced into China (Zhao, 1982) [3] . Up to now, chemical investigations of genus Amorpha mainly focused on A. fruticosa, which resulted in the isolation of more than forty compounds, including stilbenes [4] , rotenoids [5] and flavanones

Figure 1. The photos of A. fruticosa (left) and its seeds (right).

[6] , as well as isoflavones [7] . The three former groups are also found in the other species of this genus [8] - [10] . Our previous study on the EtOAc partition of 95% EtOH extract of the seeds of A. fruticosa has resulted in the isolation of eight rotenoid glycosides [11] . In this paper, the isolation and characterization of seventeen compounds from its n-hexane partition were reported. Among them, one compound was firstly purified as a natural product, nine were isolated from the genus Amorpha for the first time, and one was obtained from the Fabaceae family initially. The presence of these compounds suggests that genus Amorpha and Dalbergia may have very close chemotaxonomic relationship, and shows the relationship between this plant and other species from the Fabaceae family.

2. Materials and Methods

2.1. Plant Material

The seeds of A. fruticosa were collected in Shangqiu City, Henan Province of China. After authenticated by Professor Zhangpin Gou (Department of Pharmacology, Guangdong Medical College), a voucher specimen (No. 20130910) was deposited in the Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical University, Zhanjiang, China.

2.2. General

1D and 2D NMR spectra were recorded on Bruker AV-500 spectrometer. CD spectra were measured on a Bio-Logic MOS-450 circular dichroism spectrometer. Column chromatographies (CC) were carried out using silica gel (Qingdaohaiyang, China) and Sephadex LH-20 (Pharmacia Biotech AB, Sweden). Analytical high- performance liquid chromatography (HPLC) was carried out on a Agilent 1200 series and a C₁₈ reversed-phase column (Cosmosil, 4.6 mm × 250 mm , 5.0 μm). Preparative HPLC were carried out on a Gilson 305 pump, a Varian Prostar 345 UV detector and a C₁₈ reversed-phase column (Cosmosil, 20 mm × 250 mm , 5.0 μm).

2.3. Extraction and Isolation

Fresh and powdered seeds of A. fruticosa (4.0 kg) were extracted three times with 95% EtOH at room temperature (3 × 40 L). The solution was removed under vacuum at 50˚C to yield the residue (495 g). The residue was suspended in distilled water, and then successively partitioned with n-hexane, EtOAc and n-BuOH respectively. After removing the solvent, then-hexane partition (120 g) was chromatographied by silica gel column using gradient mixtures of cyclohexane-acetone (92:8 → 50:50) as eluants to yield eleven fractions (Fr. H1-Fr. H11). Fr. H3 (3.3 g) was separated by Sephadex LH-20 column using CH₃OH as eluent and further purified by preparative HPLC (CH₃OH-H₂O, 15:85) to yield compounds 15 (1210.0 mg) and 16 (1040.0 mg), 17 (110.0 mg). Fr. H4 (4.2 g) was separated by Sephadex LH-20 column using CH₃OH as eluent to give four subfractions (Fr. H4a-Fr. H4d). Then, Fr. H4a (132.0 mg) was further purified by Sephadex LH-20 column as CH₃OH to yield compounds 13 (5.0 mg) and 6 (18.0 mg). Fr. H4b (1550.0 mg) was purified by preparative HPLC (CH₃OH-H₂O, 45:55) to yield compounds 14 (190.1 mg), 2 (324.0 mg), 1 (740.0 mg) and 3 (66.0 mg). Fr. H4c (250.0 mg) was purified by preparative HPLC (CH₃OH-H₂O, 60:40) to yield compound 5 (100.0 mg). Fr. H5 (500.0 mg) was separated as the same method as Fr. H4to yield three subfractions (Fr. H5a-Fr. H5c), which were purified by preparative HPLC (CH₃OH-H₂O, 45:55) to give compounds 4 (10.1 mg), 10 (5.3 mg) and 11 (6.2 mg), respectively. The same procedure was applied to Fr. H6 (430.0 mg) to get four subfractions (Fr. H6a-Fr. H6d). Fr. H6b, Fr. H6c and Fr. H6d were purified by preparative HPLC (CH₃OH-H₂O, 40:60) to yield compounds 7 (10.1 mg), 8 (5.5 mg) and 9 (10.4 mg), respectively. Fr. H9 (536.0 mg) was isolated by preparative HPLC (CH₃OH-H₂O, 25:75) to yield compound 12 (200.5 mg).

3. Results and Discussion

The isolated compounds were identified as 6aR, 12aR-dalbinol (1) [12] , amorphigenin (2) [12] , 12a-hydrox- ydalpanol (3) [12] , 6aS, 12aS-dalbinol (4) [12] , 12a-hydroxymunduserone (5) [13] , amorphispironone (6) [14] , ononin (7) [15] , isoformonentin (8) [16] , daidzein (9) [17] , prunetin (10) [18] , pratensein (11) (Dixit et al., 2012) [19] , 7-O-β-D-glucopyranosyl-7-hydroxy-2’,4’,5’-trimethoxyisoflavone (12) [20] , 7-hydroxy-2’,4’,5’-trimetho- xyisoflavone (13) [21] , amorfrutin B (14) [22] , gallic acid (15) [23] , 4-methoxygallic acid (16) [24] and 3,4-dimethoxygallic acid (17) [25] , respectively, on the basis of their ¹H NMR and ¹³C NMR spectra analysis and comparison with those reported data in the related literatures (Figure 2). The absolute configurations of 6a and 12a of 1-3 and 5 were assigned as R,R based on the CD data which revealed a negative Cotton effect at 326 nm, while 4 was S,S with mirror image CD spectrum of 1 (Figure 3) [11] .

The present study reported the isolation and structure elucidation of seventeen secondary metabolites from the seeds of A. fruticosa, including six rotenoids (1-6), seven isoflavones (7-13), one stilbene (14) and three benzoic acid derivatives (15-17). Among them, compound 4 was firstly purified as a natural product, compounds 5, 8-12 and 15-17 were isolated from the genus Amorpha for the first time, and compound 17 was obtained from the

Figure 2. The structures of compounds 1-17.

Figure 3. The CD spectra of compounds 1-5.

Fabaceae family initially. Although compound 12 has been once reported, it has no CAS number yet. Compounds 1-3, along with the previous reported compounds amorphin and 8’-O-β-D-glucopyranosyl-amorphigenin, have been isolated or detected in ten species of the genus Amorpha (A. fruticosa, A. angustifolia, A. canescens, A. frangrans, A. californica, A. glabra, A. mierophylla, A. nana, A. croceotanata, and A. caroliniana) [9] at the same time, which provides a chemotaxonomic evidence to support the morphological classification of this genus.

Rotenoids 1-2 and isoflavones 12-13, as well as our previous reported rotenoid glucosides 8’-O-β-D-gluco- glucopyranosyl-amorphigenin and dalbin [11] , were also isolated from Dalbergia monnetaria (Fabaceae) simultaneously [20] . Such a coincidence should be explained by the fact that the isoflavone 13 was the key intermediate during the biosynthesis of its corresponding rotenoids [26] . In addition, some compounds obtained in this studyalso exist in other species of genus Dalbergia, as follows: compound 1 in D. nitidula [27] ; compound 7 in D. paniculata [28] and D. odorifera [15] ; compound 9 in D. odorifera [17] , D. parviflora [29] , D. frutescens [30] , D. ecastophyllum [31] , D. stevensonii [32] and D. ecastophylla [33] ; compound 10 in D. odorifera [34] , D. sympathetic [18] , D. sissoo [35] and D. spinosa [36] ; compound 11 in D. sissoo [19] , D. parviflora [37] and D. odorifera [38] ; compound 13 in D. vacciniifolia [21] . All these suggested that genus Amorpha and Dalbergia may have very close chemotaxonomic relationship.

Furthermore, compounds 1 and 2 were also synchronally isolated from Berchemia discolor [39] , compound 3 was once obtained from Aeschynomene indica [40] , and compound 5 was obtained from Pachyrrhizus erosus [13] , Tephrosia fulvinervis [41] and Tephrosia pentaphylla [41] , which exhibit the relationships between A. fruticosa and these species, all belonging to family Fabaceae except Berchemia discolor (Rhamnaceae). Compound 4 was isolated naturally for the first time, while compounds 6 and 14 both only existed in A. fruticosa. Compounds 15 and 9 widely distributed in the family Fabaceae, and compound 16 has only been isolated from one species of it (Canavalia gladiate) [42] , while it is the first time to obtain compound 17 from this family. Therefore, compounds 4, 6, 14 and 17 should be served as chemotaxonomic markers for A. fruticosa.

4. Conclusion

According to the above results, our present study extends the knowledge about the compounds of genus Amorpha and confirms Dalbergia as its chemotaxonomic related genus.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81503226) and the Doctoral Fund of Guangdong Medical College (XB1301).

NOTES

^*Corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Straub, S.C.K. and Doyle, J.J. (2014) Molecular Phylogenetics of Amorpha (Fabaceae): An Evaluation of Monophyly, Species Relationships, and Polyploid Origins. Molecular Phylogenetics Evolution, 76, 49-66. http://dx.doi.org/10.1016/j.ympev.2014.02.025
[2]	Chinese P.U. (1997) A Dictionary of Chinese Materal Medica. 3th Edition, Chinese Science and Technology Press of Medicine, Beijing.
[3]	Zhao, H.K. (1982) A Dictionary of the Families and Genera of Chinese Seed Plants. 2th Edition, Science Publishing House, Beijing.
[4]	Mitscher, L.A., Park, Y.H., Alshamma, A., Hudson, P.B. and Haas, T. (1981) Amorfrutin-A and Amorfrutin-B, Bibenzyl Antimicrobial Agents from Amorpha fruticosa. Phytochemistry, 20, 781-785. http://dx.doi.org/10.1016/0031-9422(81)85174-6
[5]	Somleva, T. and Ognyanov, I. (1985) New Rotenoidsin Amorpha fruticosa Fruits. Planta Medica, 51, 219-221. http://dx.doi.org/10.1055/s-2007-969462
[6]	Rozsa, Z., Hohmann, J., Szendrei, K., Reisch, J. and Mester, I. (1982) Amoritin, Amorisin, and Amorilin-3 New Prenylated Flavanones from Amorpha fruticosa L. Heterocycles, 19, 1793-1796. http://dx.doi.org/10.3987/R-1982-10-1793
[7]	Lee, H.J., Lee, O.K., Kwon, Y.H., Choi, D.H., Kang, H.Y., Lee, H.Y., Paik, K.H. and Lee, H.J. (2006) Isoflavone Glycosides from the Bark of Amorpha fruticosa. Chemistry of Natural Compounds, 42, 415-418. http://dx.doi.org/10.1007/s10600-006-0169-4
[8]	Kemal, M., Khalil, S.K.W., Rao, N.G.S. and Woolsey, N.F. (1979) Isolation and Identification of a Cannabinoid-Like Compound from Amorpha Species. Journal of Natural Products, 42, 463-468. http://dx.doi.org/10.1021/np50005a004
[9]	Kasymov, A.U., Kondrate.Es, Rashkes, Y.V. and Abubakir, N.K. (1970) Amorphigenol-Beta-D-Glycopyransid from Amorpha. Khimiya Prirodnykh Soedinenii, 6, 192-195.
[10]	Mitscher, L.A., Gollapudi, S.R., Drake, S. and Oburn, D.S. (1985) Amorphastilbol, an Antimicrobial Agent from Amorpha nana. Phytochemistry, 24, 1481-1483. http://dx.doi.org/10.1016/S0031-9422(00)81048-1
[11]	Wu, X., Liao, H.B., Li, G.Q., Liu, Y., Cui, L., Wu, K.F., Zhu, X.H. and Zeng, X.B. (2015) Cytotoxic Rotenoid Glycosides from the Seeds of Amorpha fruticosa. Fitoterapia, 100, 75-80. http://dx.doi.org/10.1016/j.fitote.2014.11.015
[12]	Konoshima, T., Terada, H., Kokumai, M., Kozuka, M., Tokuda, H., Estes, J.R., Li, L.P., Wang, H.K. and Lee, K.H. (1993) Studies on Inhibitors of Skin Tumor Promotion. 12. Rotenoids from Amorpha fruticosa. Journal of Natural Products, 56, 843-848. http://dx.doi.org/10.1021/np50096a006
[13]	Kalra, A.J., Krishnamurti, M. and Nath, M. (1977) Chemical Investigation of Indian Yam Beans (Pachyrrhizus-Erosus)-Isolation and Structures of 2 New Rotenoids and a New Isoflavanone, Erosenone. Indian Journal of Chemistry Section B Organic Chemistry Including Medicinal Chemistry, 15, 1084-1086.
[14]	Shibata, H. and Shimizu, S. (1978) Amorphaquinone, a New Isoflavanquinone from Amorpha fruticosa L. Heterocycles, 10, 85-86. http://dx.doi.org/10.3987/S-1978-01-0085
[15]	Choi, C.W., Choi, Y.H., Cha, M.-R., Yoo, D.S., Kim, Y.S., Yon, G.H., Hong, K.S., Kim, Y.H. and Ryu, S.Y. (2010) Yeast Alpha-Glucosidase Inhibition by Isoflavones from Plants of Leguminosae as an in Vitro Alternative to Acarbose. Journal of Agricultural Food Chemistry, 58, 9988-9993. http://dx.doi.org/10.1021/jf101926j
[16]	Biegasiewicz, K.F., St Denis, J.D., Carroll, V.M. and Priefer, R. (2010) An Efficient Synthesis of Daidzein, Dimethyldaidzein, and Isoformononetin. Tetrahedron Letters, 51, 4408-4410. http://dx.doi.org/10.1016/j.tetlet.2010.06.078
[17]	Lee, C., Lee, J.W., Jin, Q., Jang, D.S., Lee, S.J., Lee, D., Hong, J.T., Kim, Y., Lee, M.K. and Hwang, B.Y. (2013) Inhibitory Constituents of the Heartwood of Dalbergia odorifera on Nitric Oxide Production in RAW 264.7 Macrophages. Bioorganic Medicinal Chemistry Letters, 23, 4263-4266. http://dx.doi.org/10.1016/j.bmcl.2013.04.032
[18]	Nagarajan, N.S., Sethuraman, M.G., Manoj, C.N. and Rao, R.P. (2006) Dalsympathetin—A New Isoflavone Gentiobioside from Dalbergia sympathetica (Dennst.). Natural Product Research, 20, 195-200. http://dx.doi.org/10.1080/14786410500046513
[19]	Dixit, P., Chillara, R., Khedgikar, V., Gautam, J., Kushwaha, P., Kumar, A., Singh, D., Trivedi, R. and Maurya, R. (2012) Constituents of Dalbergia sissoo Roxb. Leaves with Osteogenic Activity. Bioorganic Medicinal Chemistry Letters, 22, 890-897. http://dx.doi.org/10.1016/j.bmcl.2011.12.036
[20]	Abe, F., Donnelly, D.M.X., Moretti, C. and Polonsky, J. (1985) Isoflavanoid Constituents from Dalbergia monetaria. Phytochemistry, 24, 1071-1076. http://dx.doi.org/10.1016/S0031-9422(00)83185-4
[21]	Innocent, E. (2012) A New Isoflavone glycoside from Dalbergia vacciniifolia (Fabaceae). Scientia Pharmaceutica, 80, 469-474. http://dx.doi.org/10.3797/scipharm.1112-23
[22]	De Groot, J.C., Weidner, C., Krausze, J., Kawamoto, K., Schroeder, F.C., Sauer, S. and Buessow, K. (2013) Structural Characterization of Amorfrutins Bound to the Peroxisome Proliferator-Activated Receptor Gamma. Journal of Medicine Chemistry, 56, 1535-1543. http://dx.doi.org/10.1021/jm3013272
[23]	Ma, J., Yang, H., Basile, M.J. and Kennelly, E.J. (2004) Analysis of Polyphenolic Antioxidants from the Fruits of Three Pouteria Species by Selected Ion Monitoring Liquid Chromatography-Mass Spectrometry. Journal of Agricultural Food Chemistry, 52, 5873-5878. http://dx.doi.org/10.1021/jf049950k
[24]	Chaubal, R., Mujumdar, A.M., Misar, A. and Deshpande, N.R. (2005) Isolation of Phenolic Compounds from Acacia Nilotica with Topical Antiinflammatory Activity. Asian Journal of Chemistry, 17, 1595-1599.
[25]	Liu, S.P., Tian, W., Xue, Y.H., Zeng, W., Kong, Y.S. and Qiu, M. (2013) Method for Extracting Antioxidant from Myricaria Laxiflora, and Applications Thereof. Faming Zhuanli Shenqing, Patent No. CN 103301102 A 20130918.
[26]	Crombie, L., Dewick, P.M. and Whiting, D.A. (1973) Biosynthesis of Rotenoids-Chalcone, Isoflavone, and Rotenoid Stages in Formation of Amorphigenin by Amorpha fruticosa Seedlings. Journal of the Chemical Society Perkin Transactions, 1, 1285-1294. http://dx.doi.org/10.1039/p19730001285
[27]	Van Heerden, F.R., Brandt, E.V. and Roux, D.G. (1980) Synthesis of the Pyranoisoflavonoid, Heminitidulan-Isoflavanoidand Rotenoid Glycosides from the Bark of Dalbergia nitidula Welw Ex Bak. Journal of the Chemical Society Perkin Transactions, 1, 2463-2469. http://dx.doi.org/10.1039/p19800002463
[28]	Parthasa, M.R., Seshadri, T.R. and Varma, R.S. (1974) Minor Isoflavonoid Glycosides of Stem Bark of Dalbergia paniculata Isolation of a New C-Glycoside. Current Science (India), 43, 74-75.
[29]	De Eknamkul, W., Umehara, K., Monthakantirat, O., Toth, R., Frecer, V., Knapic, L., Braiuca, P., Noguchi, H. andMiertus, S. (2011) QSAR Study of Natural Estrogen-Like Isoflavonoids and Diphenolics from Thai Medicinal Plants. Journal of Molecular Graphics Modelling, 29, 784-794. http://dx.doi.org/10.1016/j.jmgm.2011.01.001
[30]	Khan, I.A., Avery, M.A., Burandt, C.L., Goins, D.K., Mikell, J.R., Nash, T.E., Azadegan, A. and Walker, L.A. (2000) Antigiardial Activity of Isoflavones from Dalbergia frutescens Bark. Journal of Natural Products, 63, 1414-1416. http://dx.doi.org/10.1021/np000010d
[31]	Deabreumatos, F.J., Gottlieb, O.R. and Souzaandrade, C.H. (1975) Flavonoids from Dalbergia ecastophyllum. Phytochemistry, 14, 825-826. http://dx.doi.org/10.1016/0031-9422(75)83053-6
[32]	Donnelly, D.M., Thompson, J.C., Whalley, W.B. and Ahmad, S. (1973) Dalbergia Species Part 9. Phytochemical Examination of Dalbergia stevensonii Stand. Journal of the Chemical Society Perkin Transactions, 1, 1737-1745. http://dx.doi.org/10.1039/p19730001737
[33]	De Abreu, M., Gottlieb, O.R., Ollis, W.D. and Souza Andrade, C.H. (1970) The Chemistry of Brazilian Leguminosae-D Part 24 the Flavanoids of Dalbergia ecastophylla-D. Boletim Do INPA Pesquisas Florestais, 1-5.
[34]	Liu, R.X., Ye, M., Guo, H.Z., Bi, K.S. and Guo, D.A. (2005) Liquid Chromatography/Electrospray Ionization Mass Spectrometry for the Characterization of Twenty-Three Flavonoids in the Extract of Dalbergia odorifera. Rapid Communicationsin Mass Spectrometry, 19, 1557-1565. http://dx.doi.org/10.1002/rcm.1936
[35]	Sarg, T., Ateya, A.M., Abdel-Ghani, A., Badr, W. and Shams, G. (1999) Phytochemical and Pharmacological Studies of Dalbergia sissoo Growing in Egypt. Pharmaceutical Biology, 37, 54-62. http://dx.doi.org/10.1076/phbi.37.1.54.6310
[36]	Narayanan, V. and Nagarajan, N.S. (1988) Isoflavone Galactosides from Dalbergia spinosa. Phytochemistry, 27, 2364-2365. http://dx.doi.org/10.1016/0031-9422(88)80167-5
[37]	Umehara, K., Nemoto, K., Matsushita, A., Terada, E., Monthakantirat, O., De-Eknamkul, W., Miyase, T., Warashina, T., Degawa, M. and Noguchi, H. (2009) Flavonoids from the Heartwood of the Thai Medicinal Plant Dalbergia parviflora and Their Effects on Estrogenic-Responsive Human Breast Cancer Cells. Journal of Natural Products, 72, 2163-2168. http://dx.doi.org/10.1021/np900676y
[38]	Ha, H.G., Kim, J.S., Kim, J.S., Lee, J.H. and Song, G.Y. (2002) Repub Korean Kongkae Taeho Kongbo. KR 2002091404 A 20021206.
[39]	Chin, Y.W., Mdee, L.K., Mbwambo, Z.H., Mi, Q., Chai, H.B., Cragg, G.M., Swanson, S.M. and Kinghorn, A.D. (2006) Prenylated Flavonoids from the Root Bark of Berchemia Discolor, a Tanzanian Medicinal Plant. Journal of Natural Products, 69, 1649-1652. http://dx.doi.org/10.1021/np060418w
[40]	Latorre, A.O., Borghi, G.A., Lopes, P.L., Higa, K.C., Lopes, L.M.X., Maiorka, P.C., Gorniak, S.L. and Haraguchi, M. (2011) First Report on Rotenoids as Neurotoxic Principles of Seeds from Aeschynomene indica (Leguminosae). Journal Animal Veterinary Advances, 10, 26-29. http://dx.doi.org/10.3923/javaa.2011.291.294
[41]	Dagne, E., Yenesew, A. and Waterman, P.G. (1989) Flavonoids and Isoflavonoids from Tephrosia fulvinervis and Tephrosia pentaphylla. Phytochemistry, 28, 3207-3210. http://dx.doi.org/10.1016/0031-9422(89)80308-5
[42]	Jeon, K.S., Na, H.J., Kim, Y.M. and Kwon, H.J. (2005) Antiangiogenic Activity of 4-0-Methylgallic Acid from Canavalia Gladiata, a Dietary Legume. Biochemical Biophysical Research Communication, 330, 1268-1274. http://dx.doi.org/10.1016/j.bbrc.2005.03.109