Quantitative Determination of Two Flavonoids and Four Phenoquinones of Huoxue Cuyu Capsules by HPLC / 中国药房

OBJECTIVE：To establish a method for simultaneous determination of two flavonoids （rutin and kaempferol-3-O- rutinoside） and four phenoquinones （dihydrotanshinone Ⅰ，cryptotanshinone，tanshinone Ⅰ and tanshinoneⅡA） in Huoxue cuyu capsules. METHODS：HPLC method was adopted. The determination was performed on Welch Ultimate XB-C18 column with mobile phase consisted of acetonitrile-0.1％phosphoric acid solution（gradient elution）at the flow rate of 1.0 mL/min. The column temperature was set at 20 ℃，and detection wavelength was set at 270 nm. The sample size was 10 μL. RESULTS：The linear range of rutin，kaempferol-3-O-rutinoside，dihydrotanshinone Ⅰ，cryptotanshinone，tanshinone Ⅰ and tanshinoneⅡA were 172.13-860.66 μg/mL（r＝0.999 7），15.33-76.66 μg/mL（r＝0.999 8），12.81-64.06 μg/mL（r＝0.999 3），5.90-29.52 μg/mL（r＝0.999 3），5.12-25.60 μg/mL（r＝0.999 2），6.71-33.57 μg/mL（r＝0.999 7），respectively. The limits of detection were 0.08，0.01，0.01，0.01，0.01，0.01 μg/mL. The limits of quantitation were 0.27，0.02，0.03，0.03，0.03，0.03 μg/mL，respectively. RSDs of precision，stability test and repetition tests were all lower than 2.0％（n＝6）. The recoveries were 97.54％-100.25％（RSD＝1.07％，n＝6），96.90％-101.91％（RSD＝1.73％，n＝6），96.24％-102.89％（RSD＝2.32％，n＝6），97.04％-102.18％（RSD＝1.82％，n＝6），95.06％-97.73％（RSD＝1.18％，n＝6），95.59％-101.40％（RSD＝2.29％，n＝6），respectively. CONCLUSIONS：The method is sensitive，rapid，simple and has good reproducibility. It can be used for simultaneous determination of rutin， kaempferol-3-O-rutinoside， dihydrotanshinone Ⅰ， cryptotanshinone， tanshinone Ⅰ， tanshinoneⅡA in Huoxue cuyu capsules.

Metabolites of tanshinone Ⅰ and tanshinone ⅡA in rats / 中国中药杂志

An efficient method of ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap (UHPLC-LTQ-Orbitrap) mass spectrometer was established to elucidate the metabolites of tanshinone Ⅰ and tanshinone ⅡA in rats. Urine and plasma samples were collected after oral gavage. After processing biological sample by solid phase extraction, Waters ACQUITY HPLC BEH C₁₈ column (2.1 mm×100 mm, 1.7 μm) was used with 0.1% formic acid (A) - acetonitrile (B) solution as the mobile phase for gradient elution. The plasma, urine and the blank samples were then analyzed by ESI-LTQ-Orbitrap equipped with an ESI ion source under positive ion mode. On the basis of the accurate mass measurements, multiple mass spectra and comparison of data with published literature, a total of 26 metabolites were tentatively identified and characterized in the rat samples. Among them, 7 metabolites were derived from tanshinone Ⅰ through metabolic pathways of glucuronide conjugation, hydroxylation, reduction reaction, demethylation reaction, methylation, sulfate conjugation and their composite reactions. Nineteen metabolites were derived from tanshinone ⅡA through metabolic pathways of hydroxylation, reduction reaction, methylation, sulfate conjugation, glucuronidation, glucosylation and their complicated reactions. The results showed that the metabolism of tanshinone Ⅰ and tanshinone ⅡA in rats could be comprehensively clarified by using UHPLC-LTQ-Orbitrap mass spectrometer, providing material basis for the further research in terms of pharmacodynamics, toxicology, and secondary development of Chinese medicine.

Inhibitory effect of combination of tanshinoneⅠ,metformin and aspirin on malignant melanoma model mice / 中国药理学与毒理学杂志

OBJECTIVE To explore the antitumor effects of combined tanshinoneⅠ(TanⅠ),metformin (Met) and aspirin (Asp) on malignant melanoma in mice and the possible mechanisms. METHODS C57BL/6 mice were injected with 0.1 mL B16F10 cells(2.8×109L-1)to establish the subcutaneous trans-plantation tumor model at the right forelimbs axillary.Then,the mice were divided into 8 groups according to body mass,including model group, TanⅠgroup(20 mg·kg-1,ip),Asp group(210 mg·kg-1,orally in drinking water), Met group (70 mg·kg-1, orally in drinking water), Asp+Met group, TanⅠ+Asp group, TanⅠ+Met group and TanⅠ+Asp+Met group,10 mice in each group.Each mouse drank about 7 mL of water every day for a total of 18 d.The mouse body mass was measured every other day and the tumor diameter was calculated every day. The mice were sacrificed after treatment, the tumor mass was measured and the tumor inhibitory rates were counted. The histopathological changes of the liver and spleen were observed with HE staining. The percentage of lymphocytes in the tumor tissue such as CD8+T,CD4+T and Treg cells was detected by flow cytometry.Inflammatory factors such as interleukin-6 (IL-6),IL-1β and tumor necrosis factor-α (TNF-α) were detected by ELISA. RESULTS The body mass (including tumor mass)of mice in different groups increased during the experiment,but that of TanⅠ+Asp+Met group increased more slower than in model group(P<0.01).At the end of the experiment,no lesions were seen in any liver or spleen tissue by pathological observation,and the number of survivors was 8/10(model group),8/10(TanⅠgroup),7/10(Asp group),7/10(Met group),8/10(TanⅠ+Asp group), 8/10 (TanⅠ+Met group), 7/10 (Asp+Met group) and 5/10 (TanⅠ+Asp+ Met group), respectively. Compared with model group,there were no obvious changes in tumor volume or tumor mass in TanⅠ, Asp and Met groups and other two-two joint groups,but the tumor volume and tumor mass in TanⅠ+Asp+ Met group were significantly decreased (P<0.01, P<0.05), and the tumor inhibitory rate in this group was 46.2%.Compared with the model group,the percentage of CD8+T cells increased(P<0.05) in TanⅠ+Asp+Met group,but there were no significant changes in other groups.The contents of IL-6, IL-1β and TNF-α in tumor tissue of TanⅠ+Met group were much higher than in model group(P<0.01, P<0.05,P<0.05)and the content of IL-6 increased in TanⅠ+Asp+Met group(P<0.01).CONCLUSION Combination of TanⅠ,Asp and Met can effectively inhibit the growth of melanoma in mice,which may be related to the increasing percentage of CD8+T lymphocytes and IL-6 in tumor tissue.However there are possibly some side effects.

An inveatigation on adsorptive effect of attapulgite with Tanshinone Ⅰand Tanshinone Ⅱ_A in traditional Chinese medicine decoction / 中国生化药物杂志

Purpose To investigate the adsorptive effect of attapulgite on Tanshinone Ⅰ and TanshinoneⅡ_A in decoction of Radix et Rhizoma Salviae Mihiorrhizae and Composite Dansen Pill.Methods Using the amount of solid component and the content of Tanshinone Ⅰ and Tanshinone Ⅱ_A as index,the adsorptive effect of common attapulgite and acidified attapulgite on decoction of Radix et Rhizoma Salviae Miltiorrhizae and Composite Dansen Pill was investigated respectively and the content of Tanshinone Ⅰ and Tanshinone Ⅱ_A was determined by HPLC.Results Common attapulgite could not markedly decreased the amount of solid component and had a litfle effect on the content of Tanshinone Ⅰ and TanshinoneⅡ_A in decoction of Radix et Rhizoma Salviae Miltiorrhizae and Composite Dansen Pill.The acidified attapulgite could obviously decrease the amount of solid component and had a little effect on the content of Tanshinone Ⅰ and TanshinoneⅡ_A in decoction of Radix et Rhizoma Salviae Miltiorrhizae and Composite Dansen Pill.Conclusion Acidified attapulgite could be used as adsorptive clarifier in decoction of Radix et Rhizoma Salviae Miltiorrhizae and Composite Dansen Pill.