| Gualouzi is the dried ripe seed of Trichosanthes kirilowii Maxim,or Trichosanthes rosthornii Harms (Fam.Cucurbitaceae). The seed is a common traditional Chinese medicine. It possesses the actions to resolve phlegm and to cause laxation, and is used to cure dry cough with sticky sputum and constipation. It is full of fatty oil. Some pharmacological tests have shown that the extracts of the seeds possess dilating coronary artery, protecting ischemic myocardium, anti-inflammatory, anti-tumor, and decanta activities. The seeds have the chemical composition such as protein and glucoprotein. Although there are some reports about the chemical composition, it still needs further to be studied. There is no report about making qualitative discrimination using any characteristic chemical constituent. There is still no report about quantitative study and establishing method of assaying.In order to consummate the studies of Gallous, in the paper, we carry out the research from three parts. (1) One new compound and four known compounds were isolated from the unsaponifiable matter of the seeds of Trichosanthes kirilowii. The structures were elucidated on the basis of MS,UV,IR and extensive NMR (1H,13C, 1H-1H COSY, DEPT, HMQC, HMBC and NOESY) studies. (2) We have established a method of qualitative discrimination on thin-layer chromatography (TLC) by using karounidiol as a standard substance. (3) We have established an HPLC method to determine the content of karounidiol in Gualouzi. The method is utilized to determine the contents of eighteen sources and the scope of content.The seeds of Trichosanthes kirilowii Maxim.were purchased from Jiangshu in 2003. The fatty oil was extracted from the ripe dried seeds. It was saponified with 5 % KOH in MeOH for 1h. The methanol extract was filtered and concentrated under reduced pressure. The methanol extract was suspended in water and the unsaponifiable fraction was extracted with diethyl ether for several times. The diethyl ether fraction was partitioned with water until pH to 7, then the diethyl ether solution was dried with anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The yield of the unsaponfiable matter is 1.425 % from the seeds. The unsaponifiable matter was subjected to chemical and chromatographic separations. Based on chemical methods and chromatographic evidences, four known compounds have been established as 7-oxodihydrokarounidiol (compound 2), isokarounidiol (compound 3), karounidiol (compounidiol 4), and stigmasta-7, 22-dien-3-ol (compound 5). One new compound, named as 6-hydroxydihydrokarounidiol (compound 1), was elucidated as (3α,6α,13α,14β,20α)-3,6,29-trihydroxy-13-methyl-26-norolean-8-ene, on the basis of extensive NMR (1H,13C,1H-1H COSY, DEPT, HMQC, HMBC and NOESY), IR and MS studies. Thirty-eight seeds were collected, which recorded as rich in fatty oil in The Pharmacopeia of the People's Republic of China. Their nonsaponifiable lipids were distinguished with thin-layer chromatography (TLC). The results shown that karounidiol is a characteristic composition of Gualouzi. A TLC method to determine karounidiol has been established. The method has supplied to analyze twelve samples that come from all over our country. It is proved that the method is feasible. The dried ripe seeds powder (1 g) was saponified with 5 % KOH in MeOH for 3 h directly. The unsaponifiable matter was extracted with diethyl-ether for 4 times (40 ml×1, 30 ml×3). The diethyl-ether phase was washed with distilled water until pH to 7, dried with anhydrous sodium sulfate, filtrated, and evaporated to dry. The residue was removed and dissolved in 1 ml of ethyl acetate. The sample solution was developed on TLC of silica gel and with petroleum ether-ethyl acetate (2:1) as mobile phase. Dry it in air. The TLC plate was sprayed with 5 % phosphomolybdic acid in EtOH, and heated at 105℃for 10 min. The fluorescent spot in the chromatogram obtained with the test solution corresponds in position and same color to the spot in the chromatogram obtained with the reference drug solution. This method was simple and reliable.We may establish an HPLC method with a UV detector to determine the continent of karounidiol, because of 239 nm absorption in ultraviolet of karounidiol. The method has been supplied to determine eighteen samples, which comes from over our country. The scope of content is 55.20 mg - 86.24 mg of karounidiol per 100 g of seeds. We suggest that the content of karounidiol must be not less than 0.05 %. Model C18 column (250 mm×4.6 mm,5μm) was used. Column temperature was kept at 40℃. Methanol-water (97:3, v/v) was used for the mobile phase. The detective wave length was 239 nm.10 g of the dried seed powder was saponified with 5 % KOH in MeOH for 1 h. The unsaponifiable matter was extracted with ethyl-acetate for 4 times (40 ml×1,30 ml×3). The ethyl-acetate phase was washed with distilled water until pH to 7, dried with anhydrous sodium sulfate, filtrated, and evaporated to dryness under reduced pressure. The residue was removed into 10 ml of volumetric flask with methanol. 0.1 ml HC1 (1 mol/L) was added into the flask, and then methanol was added to the scale. The solution was filtered through a 0.5μm syringe filter before use.Regression equation is y=1622458116x+50263 with 0.9999 of a correlation coefficient. Calibration graph were constructed in the range 1.07-5.33μg/ml.The average recovery was 100.16 % (n=6) and RSD was 1.91 %. Regression equation revealed a good linear relationship. This method is simple, accurate, reliable, and can be used to control the quality of Gualouzi. The chemical structure of new compound 6-hydroxydihydrokarounidiol... |
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