Studies On The Fingerprinting Of Gegenqinlian Decoctioin And Serum Pharmacochemistry

Objective: To study the formulation rules and drug matching principles of traditional Chinese compound medicine, GQD, as a representative, was studied by using high efficiency liquid chromatography (HPLC) fingerprinting and serum pharmacochemistry. The current study set the basis for future research on the spectrum of Pharmacokinetics. In addition, the synergy-attenuation principle after drug matching was illustrated.Methods:To establish the analysis methods of liquid chromatography fingerprinting on GQD and precipitation, pueraria isoflavones, skullcapflavones, coptis alkaloids after drug matching; to establish isolation methods of pueraria isoflavones, skullcapflavones, coptis alkaloids from sera and to investigate their situations on blood-vessel penetration; and to study in vivo drug processes after drug matching.Sample preparation: Six replicates of each ingredient of GQD were obtained according to prescription (Radix Puerariae 15g,Radix Scutellariae 9g,Rhizoma Coptidis 9g,Radix Glycytthizae 6g). Each replicate was added 400 ml water followed by 15 min soak. Radix Puerariae was first decocted 20min, and the remaining ingredients were decocted 30min. After two times of decoction, decoction solution was filtered by 120 meshs. The filtrates were mixed together and metered to 1000mL at 90℃. Standing still at room temperature and at 4℃for 24 h, the filtrates were centrifuged at 3000r/min to obtain 800 ml supernatant. The supernatant was precipitated by 800ml dmso and filtered. In addition, each ingredient in GOD was respectively decoted following the same method as above. Mixture was metered to 1000ml for analysis. Decocting method was the same as above. Solution concentration was followed by metering to 200ml, and was kept at 30℃. Precipitates were ground and suspended in 200ml 0.5% CMC-Na as lavage sample. SD mice were bled 5 ml after 1h administration. The blood were stored in heparinized test tubes. Supernatant was obtained after centrifugation 30 min at 3000r/min, and 20%TFA(1 :8) was added to determine pueraria isoflavones and skullcapflavones serum components. Vibrated 5min and centrifuged 30 min at 3000r/min, supernatant was added MEOH(1:8) to determine coptis alkaloids serum components. Supernatant was metered to 1.0ml, filtered (0.22μm) and dissolved. Based on the literatures, the appropriate HPLC conditions were chosen for pueraria isoflavones, skullcapflavones, coptis alkaloids. Pueraria isoflavones HPLC: Agilent Zorbax SB C₁₈ column (4.6mm×250mm,5μm) and ternary gradient mobile phase were used (A: HPLC ultrapure water,B:MEOH,C:0.5%HAC aqu). The elution gradients included 0-10min(20%B,20%C),10-30min(20-30%B,20%-17.5%C),30-50min(30-80%B,17.5 -5%C),50-51min(80%B,5-0%C),51-60min(80-100%B),60-70min(100%B,). The other conditions included were speed:0.6ml/min, column temperature:25℃,λ,:250nm, Inj:10ul, and parsing time:70min. Skullcapflavones HPLC: The same chromatogram conditions were used as pueraria isoflavones except the wavelength wasλ,:280nm.Coptis alkaloids HPLC: The conditions were: Kromasil C₁₈ column(4.6mm×250mm,5μm), binary gradient mobile phase, C: buffer salt (0.02molKH₂PO₄+1.4mLH₃PO₄/L) aqu, affiliate triethyla-mine till PH=6.0,D:CH₃CN ,elution gradient:0-10min(90%C, 10%D), 10-25min(90-75%C, 10%-25%D),25-35min (75-65% C,25-35%D),35-45min(65-50%C,35-50%D),45-50min(50-10%C,90-100) %D, speed:0.8 ml/min, column temperature:25℃;λ,:268nm, Inj.10μ1, and parsing time:50min.(5) Peak origin analysisResult:Examination of the composition variations of pueraria isoflavones and confirmation of its origin:Comparison of Radix Puerariaea and GQD supernatant: Both contained similar chromatography fingerprinting peak number. However, the peak area of Radix Puerariaea is larger than GQD. Compared to the standard chromatography retention time and its ultraviolet spectrum, no significant peaks were observed for daidzein in the supernatant. In comparison with peak area, three pueraria isoflavones including daidzin area in the translucent part is lower than GQD.Compared toGQD, precipitation exhibited fewer chromatography fingerprinting peaks than GQD. Only five peaks representing pueraria isoflavones components including 3'-hydroxypuerarin,puerarin,puerarin-7-O-apioside,and daidzin were identified. There were aother three peaks whose ultraviolet absorption reaction were like pueraria isoflavones. However, the ultraviolet response value was too low resulting no significant the ultraviolet absorb reaction.Examinination of the composition variations of skullcapflavones and confirmation of its origin before and after compatibility:Comparison of Radix Scutellariae and GQD supernatant: The chromatography fingerprinting peaks were silimar, but Radix Scutellariae is larger than GQD for the peak area, Compared to the standard chromatography retention time and its comparative analysis, the baicalein and wogonin chromatography fingerprinting area after drug matching is smaller than the area by single drug, exhibiting a significant reduction.Compared to supernatant of GQD, GQD precipitation displayed less chromatography fingerprinting peaks with only four skullcapflavones components including ibaicalin, baicalein, wogonin. There are aother two locations whose ultraviolet absorption reactions were similar to skullcapflavone. However, the ultraviolet absorb reaction was insignificant due to the low ultraviolet response value.Examinination of the composition variations of coptis alkaloids and confirmation of its origin before and after compatibility:The chromatography fingerprinting peak was simaliar for Coptis Chinensis and GQD supernatant. Compared with the standard chromatography retention time and its comparative analysis, the jateorrhizine, palmatine, and berberine chromatography fingerprinting area in the GQD was half of coptis chinensis.In comparison of GQD with the precipitation, peaks numbers of precipitation was same as GQD , and chromatography fingerprinting area was almost identical to each other with a little difference in the ratio of each alkaloid. According to the berberine chromatography fingerprinting as a control, the chromatography fingerprinting area of jateorrhizine was 1.3-1.5 times larger after compatibility than the area before compatibility. Regardless of compatibility, the chromatography fingerprinting area of palmatine and berberine was similar. It seems like that the jateorrhizine was easier to react with pueraria isoflavones and skullcapflavones, and jateorrhizine reaction ability was stronger than palmatine.Serum pharmacochemistry: Based on the chromatography fingerprinting retention time and ultraviolet absorb reaction, two components in Radix Puerariaea penetrated blood vessel directly, and another two components were the metabolites in the analysis of pueraria isoflavones.. The analysis the skullcapflavones indicated that one components directly penetrated blood vessel, while another components could not be determined dut to the low ultraviolet response value. The analysis the coptis alkaloids indicated that four components were metabolites after the the penetration of GQD into blood. Other components could not be determined because fo the low ultraviolet response value.Conclusion:(1) Difference between GQD supernatant and drugs: the variation of pueraria isoflavones was not obvious with the coptis alkaloids varied most followed by skullcapflavones.(2) Reasons of precipitate formation :It was possible due to the fact that coptis alkaloids is alkaline, the pueraria isoflavones is acidic, and GQD is acidic. Therefore, precipitation reaction occurred because of the hydrogen power change during the decocting. Jateorrhizine, palmatine, berberine and coptisine were all of QAB. Nitrogen atoms in the QAB structure existed in ionic form, and such structure also contained OH". The7-Ph-OH provided by puerarin and 4'—Ph-OH provided,provided by daidzein may react with ionic nitrogen to form chelator. In addition, the coptis alkaloids in the Coptis Chinensis such as jateorrhizine, palmatine, berberine and coptisine can react with pueraria isoflavones and skullcap-flavones. The kinds of coptis alkaloids is multitude after compatibility.The contents in GQD and precipitates were similar. The deduction mechanisms of Coptis Chinensis "Bitter-cold action" after compatibility needed further research.The experimental results indicated that the Radix Puerariae reacted with other drugs in GQD after compatibility. The content of pueraria isoflavones was lower as a precipitate. The precipitation may result from the competitive mechanisms of pueraria isoflavones, skullcapflavones and glycyrrhizin. However, further studies are needed to illustrate the mechanisms. (3) Compared to supernatant of GQD and drugs, less components in the GQD than drugs penetrated into blood, We deduced that the attenuation principle after compatibility.Compared to GQD after blood-vessel penetration, the signals of precipitate chromatography were weaker after compatibility, and no significant ultraviolet absorption behavior was observed. The signals did not significantly change as administration time varied. We concluded that less components in the precipitates penetrated into blood, and the absorption speed was slow.