Chemistry and Bioactivity of the Seeds of Vaccaria segetalis - ACS

Chapter 21

Chemistry and Bioactivity of the Seeds of Vaccaria segetalis 1,4

1

1

2

Shengmin Sang , Aina Lao , Zhongliang Chen , Jun Uzawa , and Yasuo Fujimoto Downloaded by COLUMBIA UNIV on May 5, 2015 | http://pubs.acs.org Publication Date: August 5, 2003 | doi: 10.1021/bk-2003-0859.ch021

3

1

Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274, Japan Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, NJ 08901-8520 2

3

4

Vaccaria segetalis is an annual herb widely distributed in Asia, Europe and other parts of the world. The seeds of this plant, known as Wang-Bu-Liu-Xing in traditional Chinese medicine, have been used widely to promote diuresis and milk secretion, activate blood circulation and relieve carbuncle. Our research group recently isolated a wide range of chemical compounds, including triterpene saponins, alkaloids, cyclic peptide, phenolic acid,flavonoidsand steroids from the seeds of this herb. Their structures were identified by a combination of FABMS, ESIMS, ID and 2D-NMR (DQFCOSY, TOCSY, ROSEY, HMQC, HMBC and new techniques HMQC-TOCSY and HMBC-TOCSY). This paper summarizes the isolation, classification and biological activities of the components isolated from this species.

© 2003 American Chemical Society In Oriental Foods and Herbs; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

279

Downloaded by COLUMBIA UNIV on May 5, 2015 | http://pubs.acs.org Publication Date: August 5, 2003 | doi: 10.1021/bk-2003-0859.ch021

280 The plant Vaccaria segetalis (Neck.) Garcke (syn. V. pyramidata Medik) (Caryophyllaceae) is an annual herb widely distributed in Asia, Europe and other parts of the world. In Japan, it has been cultivated as a garden plant for several centuries. In China, it is distributed all over the country except the south. The seeds of this plant, known as Wang-Bu-Liu-Xing in traditional Chinese medicine have been widely used to promote diuresis and milk secretion, activate blood circulation and relieve carbuncle according to traditional Chinese medicine (/). Previous studies on the seeds of this species led to the isolation of several triterpenoid saponins, (2-7) seven cyclic peptides (8-11) and one flavonoid (Vacarin) (12). Recently, our research group re-investigated the components of V. segetalis (13-24). This paper mainly discusses the isolation, classification and biological activities of the components isolated from this species by our research group.

Materials and Methods General Experimental Procedures. Optical rotation: JASCO-DIP-181 polarimeter. IR:Perkin-Elmer 599 infrared spectrometer. H (600 MHz) and C (150 MHz) NMR: JEOL ct600 with NM-AFG type field gradient unit, TMS as internal standard. FAB-MS: MAT-95 Mass spectrometer. CC: silica gel 60H, TLC: HSGF254 (Qingdao Haiyang Chemical Group Co. of China). Plant Material. The seeds of Vaccaria segetalis were purchased at Shijia Zhuang, Hebei Province (China) in 1995. The botanical identification was made by Professor Xuesheng Bao (Shanghai Institute of Drug Control). A voucher specimen has been deposited at the Herbarium of the Department of Phytochemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Extraction and Isolation. The powdered seeds of V. segetalis (50 Kg) were extracted successively with petroleum ether χ 2 and 95% EtOH χ 3. After evaporation of ethanol in vacuo, the residue was suspended in water and then extracted successively with CH C1 , EtOAc and «-BuOH. Using silica gel column, Sephadex LH-20 column and prepared TLC plate, we obtained six pure compounds from the methylene chloride fraction and 10 compounds from the ethyl acetatefraction(Figure 1). The «-BuOH fraction was subjected to Diaion HP-20 using a EtOH-H 0 gradient system (0%-100%). Four fractions were obtained: waterfraction,30% ethanolfraction,70% ethanolfractionand ethanol fraction. By repeated column chromatographic purification over silica gel and RP C-18 gel, seven pure compounds were obtained from the 30% ethanol fraction and 20 compounds were obtained from the 70% ethanolfraction(Figure 1). !

2

2

2

In Oriental Foods and Herbs; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

13

281

Seeds of Vaccaria segetalis (50Kg) 1. Extracted with Petroleum ether 2. Extracted with Ethanol


Oil fraction

Ethanol fraction Extracted with Dimethyl chloride, Ethyl acetate and n-butanol, ^ f respectively

Dimethyl chloride fraction

Ethyl acetate fraction

Butanol fraction

Water fraction

Silica gel column Sephadex LH-20 column Prepared TLC plate 6 compounds

10 compounds Butanol fraction Diaion HP-20 column Water-Ethanol solvent systerm

I

Γ

τ 70%

Water fraction 30%

1

Ethanol fraction

Silica gel column RPC-18 column 7 compounds

20 compounds

Figure 1. Extraction and isolation procedure.


282

Phytochemistry The powdered seeds of V. segetalis (50 Kg) were extracted successively with petroleum ether χ 2 and 95% EtOH χ 3. After evaporation of ethanol in vacuo, the residue was suspended in water and then extracted successively with CH C1 , EtOAc and H - B U O H . 40 compounds were isolated and identified from these fractions. According to their structures, they can be classified as: triterpenoid saponins, alkaloids, cyclic peptides, phenolic acid, flavonoids, steroids and other compounds (Figures 2-10). 14 of them are new compounds. Among these new compounds, 12 are new triterpenoid saponins. Research has shown that saponins from a variety of sources, such as medicinal plants and foodstuffs, have considerable health benefits (25,26). Saponins occurring in V. segetalis seeds are glycosides of triterpenes. Triterpene aglycons can be substituted with several functional groups that result in a number of structurally different aglycons. In the triterpene saponins that we isolated from this seed, the aglycons received trivial names such as gypsogenin, quillaic acid, gypsogenic acid, 16hydroxygypsogenic acid and 3,4-secogypsogenic acid. The aglycons can be further substituted with 3-6 sugars, including glucopyranose, xylopyranose, arabinofiiranose, fucopyranose, galactopyranose, glucurono-pyranosic acid and rhamnopyranose. Using repeated column chromatography on Diaion HP-20, silica gel and RP-18 silica gel, nineteen triterpene saponins, including six gypsogenin type saponins (1-6), five quillaic acid type saponins (7-11), seven gypsogenic acid type saponins (12-18) and one 3,4-seco derivative of gypsogenic acid type saponins (19) were isolated from the butanolfraction.It is notable that we isolated four cyclic peptides (Figure 6) from the ethyl acetate fraction of this seed. Cyclic peptides are natural products exhibiting a wide variety of biological functions. Large numbers of cyclic peptides with unique structures and various pharmacological activities are reported from marine organisms and microorganisms (27), whereas only few examples are known from higher plants (28-32). These compounds were first reported by Itokawa et al (32). Among them compounds 24 and 25 showed estrogen-like activity assayed by the increment of uterus against ovariectomized rats (33). In addition, four alkaloids (Figure 5), three phenolic acids (Figure 7), four flavonoids (Figure 8), two steroids (Figure 9) and four other compounds (Figure 10) were also isolated and identified from this seed. The structures of the isolated compounds were elucidated by spectral methods which include FABMS, Ή-NMR, C-NMR, DQFCOSY, TOCSY, ROESY, HMQC, HMBC, HMQC-TOCSY, and HMBCTOCSY.


2

13


2


283

Figure 2. Structures of triterpene saponins (compounds 1-11).



284



22

23

Figure 5. Structures of alkaloids (compounds 20-23).



286

Figure 7. Structures ofphenolic acids (compounds 28-30).



287

32. R ι =Ara, R 2=H 33. Ri=Glc,R 2=H 34. R j =Ara, R 2=Glc

Figure 8. Structures offlavonoids (compounds 31-34).

35. R=H 36. R=Glc Figure 9. Structures ofsteroids (compounds 35 and 36).


288

CH OH


2

OH

40

Figure 10. Structures of compounds 37-40.

Bioactivities In order to find the bioactive compounds responsible for the traditional use of this seed, two bioassays were conducted to investigated the inhibitory effect on the growth of both the human HL-60 cell line and the Luteal cells of rats. One new flavonoid (31) exerted inhibitory effect on the growth of the HL-60 cell line with IC value of 10.9 μΜ. 18 compounds were tested for the inhibition of growth of Luteal cells of rats. Three new triterpenoids (compounds 4,10 and 11) exhibited strong inhibitory effect on the growth of these cells resulting in 100% inhibition at a concentration of 20 //g/mL. It is known that steroid saponins have an inhibitory effect on the growth of luteal cells (25). However, these compounds are unique examples of triterpenoid saponins that show inhibitory activity on the growth of luteal cells. 50


289


Conclusion In conclusion, 40 compounds were isolated and identified from the seeds of V. segetalis. 15 of them (2-11, 14, 17, 18, 28, 31) are new compounds, one of them (37) is a new natural product. Most of these new compounds have complex structures. New 2D techniques, HMQC-TOCSY and HMBC-TOCSY, were applied to determine their structures. 18 compounds were tested for the inhibition of Luteal cells in rats. Three new compounds showed very strong inhibition resulting in 100% at a concentration of 20 #g/mL. One new flavonoid showed inhibitory effect on the growth of the HL-60 cell line. Thus, the seeds of V. segetalis are a rich source of bioactive triterpene saponins, alkaloids, cyclic peptides, phenolic compounds and steroids.

Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 29632050).

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Jiangsu New Medical College, Zhong-yao-da-ci-dian, Shanghai Science and Technology Publisher, 1986; p 311. Litvinenko, V.I.; Amanmuradov, K.; Abubakirov, N.K. Khim. Prir. Soed.1961, 3, 159-164. Amanmuradov, K.; Abubakirov, N.K. Khim. Geol. Nauk. 1964, 6, 104-108. Morita, H.; Yun, Y.S.; Takeya, K.; Itokawa, H.; Yamada, K.; Shirota, O. Bioorg. Med. Chem. Letters 1997, 7, 1095-1096. Yun, Y.S.; Shimizu, K.; Morita, H.; Takeya, K.; Itokawa, H.; Shirota, O. Phytochemistry 1998, 47, 143-144. Koike, K.; Jia, Z.H.; Nikaido, T. Phytochemistry 1998, 47, 1343-1349. Jia, Z.H.; Koike, K.; Kudo, M.; Li, H.Y.; Nikaido, T. Phytochemistry 1998, 48, 529-536. Morita, H.; Yun, Y.S.; Takeya, K.; Itokawa, H. Tetrahedron 1995, 51, 5987-6002. Morita, H.; Yun, Y.S.; Takeya, K.; Itokawa, H. Tetrahedron 1995, 51, 6003-6014. Morita, H.; Yun, Y.S.; Takeya, K.; Itokawa, H. Phytochemistry 1996, 42, 439-441.



290 11. Yun, Y.S.; Morita, H.; Takeya, Κ.; Itokawa, Η. J. Nat. Prod. 1997, 60, 216218. 12. Baeva, R. T.; Karryev, M.O.; Litvinenko, V.I.; Abubakirov, N.K. Khim. Prir. Soedin. 1974, 10, 171-176. 13. Sang, S.M.; Lao, A.N.; Wang, H.C.; Chen, Z.L.; Uzawa, J.; Fujimoto, Y. Nat. Prod. Sci. 1998, 4, 268-273. 14. Sang, S.M.; Lao, A.N.; Wang, H.C.; Chen, Z.L.; Uzawa, J.; Fujimoto, Y. J. Asian. Nat. Prod. Res. 1999, 7, 199-201. 15. Sang, S.M.; Lao, A.N.; Wang, H.C.; Chen, Z.L.; Uzawa, J.; Fujimoto, Y. J. Asian. Nat. Prod. Res. 2000, 2, 187-193. 16. Sang, S.M.; Lao, A.N.; Wang, H.C.; Chen, Z.L.; Uzawa, J.; Fujimoto, Y. Chin. Chem. Lett. 2000, 11, 49-52. 17. Sang, S.M.; Lao, A.N.; Wang, H.C.; Chen, Z.L.; Uzawa, J.; Fujimoto, Y. Tetrahedron Lett. 2000, 41, 9205-9207. 18. Sang, S.M.; Lao, A.N.; Wang, H.C.; Chen, Z.L.; Uzawa, J.; Fujimoto, Y. J. Asian. Nat. Prod. Res. (in press). 19. Sang, S.M.; Lao, A.N.; Wang, H.C.; Chen, Z.L.; Uzawa, J.; Fujimoto, Y. J. Asian. Nat. Prod. Res. (Submitted) 20. Sang, S.M.; Lao, A.N.; Wang, H.C.; Chen, Z.L.; Uzawa, J.; Fujimoto, Y. Phytochemistry 1998, 47, 569-871. 21. Sang, S.M.; Lao, A.N.; Wang, H.C.; Chen, Z.L. Zhongcaoyao 2000, 31, 169-171. 22. Sang, S.M.; Lao, A.N.; Wang, H.C.; Chen, Z.L. Tianran Chanwu Yanjiu Yu Kaifa 2000,12,12-15. 23. Sang, S.M.; Lao, A.N.; Wang, H.C.; Chen, Z.L. Tianran Chanwu Yanjiu Yu Kaifa 1999, 10, 1-4. 24. Sang, S.M.; Xia, Z.H.; Lao, A.N.; Wang, H.C.; Chen, Z.L. Zhongguo Zhongyao Zazhi 2000, 25, 221-222. 25. Rao, A.V.; Gurfinkel, D.M. Drug Metabol. Drug Interact. 2000, 17, 211235. 26. Hostettmann, K.; Marston, A. Saponins. Cambridge University Press: Cambridge, England, 1995. 27. Matsubara, Y.; Yusa, T.; Sawabe, Α.; Lizuka, Y.; Takekuma, S.; Yoshida, Y. Agric. Biol. Chem. 1991, 55, 2923-2929. 28. Yahara, S.; Shigeyama, C.; Ura, T.; Wakamatsu, K.; Yasuhara, T.; Nohara, T. Chem. Pharm, Bull. 1993, 41, 703-709. 29. Witherup, K.M.; Bogusky, M.J.; Amderson, P.S.; Ramjit, H.; Ransom, R.W.; Wood, T.; Sardama, M . J. Nat. Prod. 1994, 57, 1619-1625. 30. Van den Berg, A.J.J.; Horsten, S.F.A.J.; Kettenes-van den Bosch, J.J.; Kroes, B.H.; Beukelman, C.J.; Leeflang, B.R.; Labadie, R.P. FEBS Lett. 1995, 358, 215-218.


291


31. Van den Berg, A.J.J.; Horsten, S.F.A.J.; Kettenes-van den Bosch, J.J.; Kroes, B.H.; Beukelman, C.J.; Leeflang, B.R.; Labadie, R.P. Phytochemistry 1996, 42, 129-133. 32. Itokawa, H.; Yun, Y. Morita, H.; Takeya K.;Yamada, K.. Planta Med. 1995, 61, 561-562. 33. Rae, M.T.; Menzies, G.S.; McNeilly, A.S.; Woad, K.; Webb, R. and Bramley, T.A. Biology of Reproduction, 1998, 59, 1016.


Chemistry and Bioactivity of the Seeds of Vaccaria segetalis - ACS

Recommend Documents