| Melilotus officinalis?Linn.?Pall is a herbaceous plant of the family Melilotus officinalis,which grows one or two years.It is bitter and cool,has the effects of clearing away heat,detoxifying,reducing inflammation and swelling.In this paper,the chemical constituents of 70%ethanol extract from Melilotus officinalis?Linn.?Pall and its anti-oxidant,anti-inflammatory and anti-tumor activities were studied,and the fingerprint of the total saponin with good anti-tumor activity was established by high performance liquid chromatography?HPLC?.Firstly,this paper uses silica gel column chromatography,Sephadex LH-20 column chromatography and semi-preparative HPLC and other separation and purification methods and IR,MS,NMR and other structural identification methods to study the chemical composition of 70%ethanol extract from Melilotus officinalis?Linn.?Pall and 29 compounds were identified.Among them,eleven compounds of flavonoids:kaempferol-3-O-?-L-rhamnoside?12?,kaempferol-3-O-rutinoside?13?,and meditation rosewood-3-O-?-glucopyranoside?14?,rutin?15?,apigenin-7-O-?-D-rutin glycoside?16?,robinin?17?,clovin?18?,isoquercitrin?19?,luteolin?20?,apigenin?21?,5,7,4'-trihydroxy-6,3'-dimethoxyflavone?22?.Seven compounds of triterpenoid saponins:?-aromatic alcohol?6?,Melilotus-saponin O2?10?,22?,24-dihydroxy-?-amyrin-3-?-D-glucuropyranoside?11?,3?,22?,24,-trihydroxy-olean-12en-3-o-?-D-glucopyranosy1-?1?2?-o-?-D-glucurpyranoside?26?,3?,22?,24,-trihydroxy-olean-12en-3-o-?-D-glucopyranosy1-?1?2?-o-?-D-glucopyranosyl-?1?2?-?-L-rhamnopyranoside?27?,Soyasapogenols B?28?,soyasapogenols E?29?.Five compounds of phenolic acids:salicylic acid?1?,ferulic acid?5?,caffeic acid?9?,Benzoic acid 4-o-?-D-mannopyanosyl?1?3?-?-L-rhamnopyranoside?24?,Benzoic acid 4-o-?-L-rhamnopyranosyl?1?6?-?-D-mannopyanosyl?1?3?-?-L-rhamnopyranoside?25?.Three compounds of coumarin:coumarin?2?,sorghum lactone?3?,dimethyl esculin?4?.One compounds of alkaloid:betaine?23?.Two sterol compounds:?-sitosterol?7?,?-carotene?8?,in which compounds 11,24,25,and 26are new compounds.Secondly,the activities on anti-oxidant,anti-inflammatory and anti-tumor of the above 29 compounds were studied with the DPPH·and ATBS+clearance method,lipopolysaccharide?LPS?-induced RAW264.7 cell inflammation in vitro model and CCK-8 respectively.The results of antioxidant activity experiments showed that phenolic acids?1,9,24,25?and flavonoids?compounds 15,17,18,19,20,21,22?have good antioxidant activity.Further study on anti-inflammatory activity of phenolic acids and flavonoids,the result showed that phenolic acids and flavonoids have a significant inhibitory effects on the release of inflammatory factors?NO,TNF-?,IL-6?in RAW264.7 cells by LPS in the concentration range of 50?g·mL-1,Among them,compound 1 and compound 9 have significant inhibitory effects on inflammatory factors.The anti-tumor activity test results showed that the total saponins and total phenolic acid active parts of Melilotus officinalis?Linn.?Pall had good anti-tumor activity,and the anti-tumor activity of these compounds were studide further,the result showed two phenolic compounds?compounds 24,25?and six oleanolic saponin compounds?compounds 6,11,26,27,28,29?have a good inhibitory effect on human breast cancer and prostate cancer.Finally,aiming at the total saponin of Melilotus officinalis?Linn.?Pall,which has significant antitumor activity,Agilent-DAD HPLC was used to determine the chromatographic conditions of the best extract by HPLC fingerprinting technique,and HPLC fingerprint was established.Applying the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System?2004 A?issued by the National Committee to process the HPLC profiles of ten batches of samples,establishing 17 common peak evaluation models,and using the reference retention time comparison method to identify the common peaks,and three peaks were identified,which were peak 14?compound 11?,peak 15?compound 26?,and peak 17?compound 27?. |
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