| Objective:Rehmannia glutinosa Libosch belongs to the genus Rehmannia of Scrophulariaceae, of which root was important medicinal material in traditional Chinese medicine. The fresh root was called "xian di huang", and80%dry root was called "sheng di huang". Rehmannia, which consisted6species, distributes many provinces of China, as well as North Korea, and Japan. Modern pharmacological studies had shown that it had strong activities, such as anti-ulcer, anti-aging, anti-diabetic, anti-hypercholesterolemic, anti-tumor, hepato-protective activity. Rehmannia glutinosa Libosch also has estrogen-like effect as previously studies of our lab. Study of the chemical constituents of the leaves of Rehmannia glutinosa Libosch can further clarify the basis of its efficacy substances and find new active ingredient resources, which lay the foundation for its further development and utilization.Methods:1. The dry leaves of Rehmannia glutinosa Libosch in harvest time were extracted by50%aqueous acetone three times at room temperature and filtered. The combined extracts were concentrated under reduced pressure in a vacuum evaporator to the gross extracts. They were dissolved in water, yield the water soluble matter and the water insoluble matter, and then the water soluble matter were subjected to Diaion HP-20porous polymer resin and eluted with H2O,10%CH3OH,20%CH3OH,30%CH3OH,40%CH3OH,80%CH3OH, successively. The fractions eluted were subjected to Toyopearl HW-40column chromatography and eluted with H2O,10%CH3OH successively to afford subfractions. Then the subfractions were isolated and purified by Toyopearl HW-40, Sephadex LH-20, silica gel column chromatography, preparative liquid chromatography. The water insoluble matter were extracted with EtOAc to obtain the extract, and was chromatographed on silica gel by gradient elution with CH2C12-CH3OH(100:1,80:1,50:1), each stream seperated by Toyopearl HW-40, MCI Gel CHP-20, Sephadex LH-20column chromatography and recrystallized to give compound. The structures were identified on the basis of physiochemical characteristics and spectral methods such as IR, UV,1H-NMR,13C-NMR,1H-1H COSY, DEPT-135, HMBC, HSQC and so on.2. The MTT assay was used for the quantitative determination of cellular proliferation. The following human tumor cell lines were used: MCF-7(human breast cancer cells), MG63(human osteoblast-like cells), HepG2(human hepatocellular carcinoma cells). The cytotoxicity in vitro of the three new compounds Glutinosalactone A-C isolated from leaves of Rehmannia glutinosa Libosch were tested.Results:1.35compounds were isolated from leaves of Rehmannia glutinosa Libosch and the structures were elucidated by spectroscopic and chemical methods. They were p-hyoxybenzoic acid (DHY-1), gentisic acid (DHY-2), p-hydroxyphenylethyl alcohol (DHY-3), luteolin-7-O-β-D-glucuronide (DHY-2),6,7-dihydroxy-coumarin (DHY-5),3,4-dihydroxyphenylethyl alcohol (DHY-6),1,2,4-benzenetri (DHY-7), protocatechuic acid (DHY-8),6-O-vanillate ajugol (DHY-11-1), darendoside B (DHY-12-1),6-O-E-feruloyl ajugol (DHY-13-1), decaffeoylacteoside (DHY-14-1),8-epiloganic acid (DHY-16), catalpol (DHY-17), daucosterol (DHY-18),2β,3β,19α-trihydroxyolean-12-ene-13,28-dioic acid (DHY-19), ajugol (DHY-20), β-hydroxyacteoside (DHY-22), echinacoside (DHY-24),9,12,13-trihydroxy-10-octadecenoic acid (DHY-25),3β,19α,20β,24,30-pentahydroxyurs-12-en-28-oic acid δ-lactone (DHY-26-1),3β,19α,20β,30-tetrahydroxyurs-12-en-28-oic acid δ-lactone (DHY- 27), glutinolic acid (DHY-28), acteoside isomer (DHY-29), jionoside B1(DHY-30-1), acteoside (DHY-32), β-sitosterol (DHY-A), luteolin (DHY-E), diosmetin (DHY-L), apigenin (DHY-N), oleanolic acid (DHY-H), ursolic acid (DHY-F),12a,13α-epoxy-3β,19α,20β,30-tetrahydroxyurs-28-oic acid (5-lactone (DHY-O), oleanonic acid (DHY-Q),2α,3β-dihydroxyolean-12-ene-28-oic acid (DHY-R).2. The MTT assay was used for the quantitative determination of cellular proliferation, and DHY-0(Glutinosalactone C) exhibited moderate cytotoxicity against MCF-7(IC50=8.38±0.16μM), MG63(IC50=39.25±0.21μM), HepG2(IC50=17.29±2.16μM). DHY-26-1(Glutinosalactone A) shows weak cytotoxicity against MCF-7(IC50=74.65±0.60nM), MG63(IC50=89.27±093μM) and HepG2(IC50=45.36±1.62μM), DHY-B (Glutinosalactone B) showed no cytotoxicity of the selected three kinds of cancer cells.Conclusions: Three new compounds, DHY-26-1(Glutinosa-lactone A), DHY-27(Glutinosalactone B), DHY-0(Glutinosalactone C) were isolated from the leaves of Rehmannia glutinosa Libosch. The other seventeen known compounds DHY-1, DHY-2, DHY-3, DHY-4, DHY-5, DHY-6, DHY-7, DHY-8, DHY-19, DHY-22, DHY-25, DHY-L, DHY-N, DHY-H, DHY-F, DHY-Q, DHY-R were isolated from this plant for the first time.Triterpenoids isolated from this plant for the first time. This study provided sciebtific basis for the fundamental research of Rehmannia glutinosa Libosch. |
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