Studies On Anti-Tumor Constituents Of Hedyotis Diffusa Willd

Hedyotis diffusa Willd.[Oldenlandia diffusa（Willd.） Roxb.],which belongs to the family of Rubiaceae,is well-known in Chinese folk medicine for the treatment of cancer, especially for the therapies against esophagus,stomach,liver and rectum malignant tumors. In this study,the chemical constituents,pharmacological activity in vitro,quality control methods and preliminary pharmacokinetics of Hedyotis diffusa were investigated by means described as follows.The chemical constituents of 90%ethanol extract of Hedyotis diffusa were isolated by using multiple column chromatographic techniques,including silica gel column chromatography,reversed-phased preparative high performance liquid chromatography （RP-PHPLC）,Polyamide column chromatography and Sephadex LH-20,and 17 compounds were obtained.The structures of 15 compounds were fully elucidated by chemical and spectroscopic methods（MS,UV,NMR）.Among them,one is a new compound, 2,7-dihydroxy-3-methyl anthraquinone;one compound,3,4-dihydroxy methyl benzoate,was isolated from Hedyotis diffusa for the first time.An HPLC-ESI/TOF-MS method in both positive and negative modes was developed and applied to study the chemical constituents of extract of Hedyotis diffusa.According to the retention time and fragmentation patterns of the four standard compounds,as well as the structure information obtained from the literatures,eight constituents were unambiguous/tentatively identified.The chemical names of the identified compounds are deacetylasperulosidic acid,deacetyl asperuloside,scandoside,10-acetyl scandoside, asperuloside,p-coumaric acid,E-6-O-p-coumaroyl scandoside methyl ester, Z-6-O-p-coumaroyl scandoside methyl ester.Both the crude extract and eight compounds isolated from Hedyotis diffusa were tested for their anticancer activity in vitro by an MTT assay.The growth inhibition study showed that the crude extract of Hedyotis diffusa exerted a significant toxic effect against MCF-7, BEL-7402 and SGC-7901 cell lines.Compared with the other two cancer cell lines, SGC-7901 cells were more sensitive to the cytotoxic action of Hedyotis diffusa extract,which exhibited an IC₍50 value of 4.81μg/mL.Among the compounds tested,only 2,7-dihydroxy-3-methyl anthraquinone exhibited dose dependent growth inhibition effect and showed a IC50 values of 53.72μM,54.22μM and 84.40μM against MCF-7,BEL-7402 and SGC-7901 cancer cell lines,respectively.Anthraquinones and iridoids are two major kinds of constituents in Hedyotis diffusa.An HPLC-UV method was developed for simultaneous determination of four anthraquinones in Hedyotis diffusa.An HPLC-DAD method using gradient elution was established for the simultaneous determination of p-coumaric acid and three iridoids in Hedyotis diffusa.These methods were applied to determine the amounts of these bioactive compounds in Hedyotis diffusa.The results indicated that the contents of those constituents in samples from different sources varied significantly.The methods established in this paper were specific,accurate, sensitive and were suitable for the quality control of Hedyotis diffusa.2-Hydroxy-3-methyl anthraquinone and deacetylasperulosidic acid were selected respectively as the representative compounds of anthraquinone and iridoid constituents in Hedyotis diffusa for the in vivo pharmacokinetic study.An HPLC method coupled with UV detection was developed and validated for the determination of 2-hydroxy-3-methyl anthraquinone in rat plasma.Ethinylestradiol was chosen as the internal standard. Chromatographic separation was conducted on a Diamonsil C₁₈ column（200 mm×4.6 mm,5μm）,using a mixture of methanol-water（78:22,v/v） as mobile phase with detection wavelength at 265 nm.The calibration curve of 2-hydroxy-3-methyl anthraquinone was linear over the range of 0.10-4.0μg/mL in rat plasma.The lower limit of quantification （LLOQ） was 0.10μg/mL.The intra- and inter-day precisions（RSDs） were less than 7.1%and 9.3%,respectively.The intra-and inter-day accuracies（REs） were within-5.1-5.4%.The developed method was applied to the pharmacokinetic study of 2-hydroxy-3-methyl anthraquinone in rats after intraperitoneal administration.The pharmacokinetic parameters obtained after intraperitoneal administration of 6mg/kg 2-hydroxy-3-methyl anthraquinone were as follows:C_max 2.89μg/mL,area under concentration-time curve(A∪C_0-∞) 159μg·min/mL,apparent elimination rate constant（K_e） 0.0164 min^-1,biological half life(T_1/2) 44.3min.An HPLC method coupled with UV detection was developed and validated for the determination of deacetylasperulosidic acid in rat plasma.Scandoside was chosen as the internal standard.Chromatographic separation was performed on a pinnacleⅡC₁₈ column （250mm×4.6mm,5μm）,using a mixture of methanol-5 mM ammonium acetate（3:97,v/v, pH adjusted to 3.7 with glacial acetic acid） as mobile phase with detection wavelength at 230 nm.The calibration curve of deacetylasperulosidic acid was linear over the range of 0.20-40μg/mL in rat plasma.The lower limit of quantification（LLOQ） was 0.20μg/mL.The intra and inter-day precisions（RSDs） were less than 6.6%and 10.1%,respectively.The intra- and inter-day accuracies（REs） were within-2.5-0.75%.The developed method was applied to the pharmacokinetic study of deacetylasperulosidic acid in rats after intravenous administration.The pharmacokinetic parameters obtained after intravenous administration of 4.2mg/kg deacetylasperulosidic acid were as follows:C_max 17.66μg/mL,A∪C_0-∞ 619μg·min/mL,K_e 0.0244min^-1,T_1/2 28.6min。In conclusion,by utilizing various professional knowledge and analytical techniques,the chemical constituents of Hedyotis diffusa were investigated,the crude extract and some compounds isolated from Hedyotis diffusa were tested for their growth inhibition effect against cancer cell lines in vitro,the quantitative determination of anthraquinones and iridoids in Hedyotis diffusa were developed,and pharmacokinetic processes of some representative constituents had also been studied.This study provided useful information for the interpretation of antitumor activity-related substance,quality control,exploration and utilization of Hedyotis diffusa.