Studies On The Chemical Constituents Of Isodon Nervosus And Their Antitumor Activity In Vitro

The genus Isodon is a perennial herbage, semi-shrub or shrub of Labiatae. There are about 150 species in the genus mainly distributing in Asia and Africa,90 species together with 21 varieties of them widely distributing in China, especially in the southwest of China. About 30 species of the genus Isodon have been used as traditional Chinese medicine for treatment of fever and inflammation along with detoxication, treatment of splenitis and mobilization of blood circulation, anti-bacteria and diminish inflammation. And some of them have been used as folk medicine for anti-tumor. In recent decades, a number of researches were carried out in many different kinds of Isodon plants, and people paid more attention on these kinds of plants possessed some biological activity such as anti-tumor, anti-bacterial, anti- inflammatory and antioxidant. A variety of Isodon plants were systematic studied by scholars, and many chemical constituents were isolated from them, including some diterpenoids with novel structure, which enriched the chemical composition database of this genus. Also many biological activity researches indicated that some compounds from these plants possessed widely biological activities, such as some diterpenoids exhibited significant anti-tumor activity. These studies have promoted the development and use of Isodon plant resources.Isodon nervosus (Hemsl.) C.Y.Wu et H.W.Li, also known as Dayeshezongguan or Lanhuachaihu or Maiyexiangchacai, a perennial herb belongs to Isodon genus of Labiatae, is widely distributed in Henan, Jiangxi, Guangdong, Guangxi, Guizhou, Sichuan and some other provinces of China, and their stems and leaves have been used satisfactorily for the treatment of fever and inflammation along with detoxication, acute infectious hepatitis, snakebite, skin itching. Few researches have been intensively performed on this plant so far, so it is of great practical significance to do systematic study to illustrate the material basis of Isodon nervosus in its anti-tumor activity and to further promote the development and utilities of this abundant resource in our country.In this Ph. D. thesis, we examined the chemical constituents of the stems and leaves of Isodon noversus collected from Yifeng area of Jiangxi province, summarized the structure and spectral characteristics of ent-kaurane diterpenoids isolated from this plant; evaluated the anti-tumor activity in vitro of 31 diterpeniods by using MTT method and analyzed the relationship between the structure and anti-tumor activity. The air-dried and milled plant (10 kg) was extracted with 95% ethanol (3x20 L, each 2 hours) in DTQ-100L multi-function extractor and filtered. The filtrate was dissolved in methanol and filtrated, and then decolorated by adding 4‰(w/v) of activated carbon for 3 times and filtrated, evaporated to give a residue (1.2 kg), which was suspended in diatomite (1.2 kg) and then extracted with petroleum ether (b.p.60-90℃,6 L), EtOAc (10 L), acetone (8 L) and methanol (6 L) by Soxhlet’s extractor, successively. Four partitions were evaporated to give the petroleum ether extraction, the EtOAc extraction, the acetone extraction and the methanol extraction. The petroleum ether and EtOAc extraction were separated and purified by gel silica column, RP-C18, Sephadex LH-20, preparative thin layer chromatography, recrystallization and some others techniques, which led to the isolation of 55 compounds. On the basis of spectroscopic data (IR, UV, HR-ESI-MS,’H-NMR,13C-NMR, DEPT-NMR,1H-1H COSY, HSQC, HMBC, NOESY and ROESY) interpretation,51 of them were identified, and their structures were elucidated as rabdonervosin D (WXM-1), rabdonervosin E (WXM-2), rabdonervosin F (WXM-3), rabdonervosin G (WXM-4), rabdonervosin H (WXM-5), rabdonervosin I (WXM-6), rabdonervosin J (WXM-7), rabdonervosin K (WXM-8), enmelol (WXM-9), rabdoternin A (WXM-10), rabdoternin B (WXM-11), odonicin (WXM-12), effusanin A (WXM-13), isodoternfolin B (WXM-14), effusanin E (WXM-15), neorabdosin (WXM-16), maoecrystal D (WXM-17), rubescensin Q (WXM-18), parvifoline G (WXM-19), isodocarpin (WXM-20),11β-hydroxy-6,7-seco-6,19:6,20-diepoxy-1α,7-olide-ent-kaur-15-one (WXM-21), longikaurin E (WXM-22), sculponeatin A (WXM-23), sculponeatin B (WXM-24), lasiokaurin (WXM-25), lasiokaurinol (WXM-26), oridonin (WXM-27), 6β,7β,14β,15β-tetra-hydroxy-7a,20-epoxy-ent-kaur-16-ene (WXM-28), nodosin (WXM-29), nervosin (WXM-30), rabdosichuanin D (WXM-31), lihsienin A (WXM-32), nervosanin B (WXM-33), epinodosinol (WXM-34), epinodosin (WXM-35), 8β,13β-oxidoeperu-14-en-18-oic-acid (WXM-36), friedelin (WXM-37),β-sitosterol (WXM-38), oleanolic acid (WXM-39), vanillic acid (WXM-40),3β-hydroxy-oleanolic-11,13-dien-28-acid (WXM-41), stigmasterol (WXM-42), ursolic acid (WXM-43), stearic acid (WXM-44),4-hydroxy-benzoic acid (45), palmitic acid (WXM-46), fumalic acid (WXM-47),2a,3β-dihydroxy-oleanolic-12-en-28-acid (WXM-48), caffeic acid (WXM-49),5,6,7,4’-tetrahydrexy-3’-methoxy-flavone (WXM-50),β-daucosterol (WXM-51).Compounds 1-3,9-36 were screened for their cytotoxicity against HepG2 (Human HCC cell line), CNE2 (nasopharyngeal carcinoma), PC-9/ZD, PC-9/CDDP (lung cancer), HCT116 (colon cancer) and HeLa (cervical cancer) cell lines by MTT method. Compounds 13,15,20,22-23,25-27,29-30,35 exhibited great cytotoxicity against one or more cell lines. Compared with other test cell lines, all compounds is of great selectivity in HepG2. Especially, compound 22 displayed significant anti-tumor activity with IC50 less than 10μmol/L against HeLa, PC-9/ZD, HepG2, CNE2, PC-9/CDDP cell lines.The relationship between structure and anti-tumor activity suggested that the carbonyl conjugated with an exomethylene group was essential for the inhibition of tumor cell proliferation. If the conjugated system unit was broken, the activity would be significantly weakened or even disappeared. Besides that, the side chain which attached to-OAc/-OH affected the activity. The -OAc/-OH located at C-11/C-14 enhanced the activity, while the -OH/-OAc substituted at C-1 reduced the activity, and if a lactone placed on C-1, activity was further diminished.In addition, recent advances in the research of chemical constituents and pharmacological roles of Isodon plants were reviewed.