Biopotency Assays:an Integrated Application to Quality Control of Chinese Materia Medica

The current quality control（QC） pattern for Chinese materia medica（CMM） lacks suitable methods and indicators to evaluate their safety and efficacy effectively, which impedes the smooth development of CMM. In this review, main problems of the current QC pattern for CMM, principally focused on the content determination of constituents,were summarized and the inspiration from the QC of biological products was introduced. With the aim at introducing a suitable tool to the QC of CMM, biopotency assay and its feasibility in the QC pattern for CMM were analyzed and confirmed by relevant researches with years of practice. From the applications of biopotency assays in the QC of CMM in the last 10 years, we propose that biopotency assays should be an integral part of the QC pattern for CMM, for these assays can make the QC indicators related to the clinical safety and efficacy, supplementing the existed QC system of CMM.

Application of microcalorimetry of Escherichia coli growth and discrimin antanalysis to the quality assessment of a Chinese herbal injection(Yinzhihuang)

This paper describes a novel approach to the quality control of a Chinese herbal injection based on microca lorimetric determination of its effect on the biothermal active fingerprint(BTAF)of Escherichia coli(E.coli)growth.Yinzhihuang Injection(YZHI)was selected for this proof of concept study.Reference samples of Y ZHI were collected and compared with test(stressed)samples prepared under different stress conditions.The BTAF of E.coli growth was found to be affected by YZHI and the changes were analyzed on the basis of eleven biothermokinetic parameters.Similarity and multivariate statistical analysis were used to investi-gate the differences between reference and test samples and discriminant analysis was used to delineate the altered samples.Reference samples were found to have coincident BTAFs with similarity index>0.99.Stressed samples showed differences in the BTAF which increased in line with decreased quality.Discriminant formulae were developed based on a sensitivity parameter which could identify all altered samples.In conclusion,BTAF can be used to assess the quality of YZHI both qualitatively and quantitatively and has the potential to provide a sensitive method for quality control of Chinese herbal injections.

Dynamic monitoring of the cytotoxic effects of protoberberine alkaloids from Rhizoma Coptidis on HepG2 cells using the xCELLigence system

AIM: To investigate the cytotoxic effects of the six protoberberine alkaloids(PAs) from Rhizoma Coptidis on HepG2 cells. METHOD: A systematic screening was conducted to investigate the dynamic response of HepG2 cells to the PAs using the impedance-based xCELLigence system. Cisplatin was selected as the positive control. The real time, concentration-response curves and the 50% inhibitory concentrations(IC50) were acquired to evaluate the anticancer activity of the PAs. RESULTS: All of the six PAs inhibited cell growth and induce death in HepG2 cells in a time- and concentration-dependent manner. The IC50 values of cisplatin, berberine, columbamine, coptisine, epiberberine, jatrorrhizine, and palmatine were 5.13, 42.33, 226.54, 36.90, 302.72, 383.54, and 456.96 μg·mL-1, respectively. The results obtained using the xCELLigence system corresponded well with those of the conventional methods. CONCLUSION: The xCELLigence system is a reliable and efficient tool for real-time screening of the cytotoxic effect of compounds in cell-based in vitro assays. Coptisine and berberine, with methylenedioxy group at C2 and C3 on the phenyl ring showed stronger effect.than the other four PAs. However, compared with cisplatin, the six PAs didn't show obvious cytotoxic effect on HepG2 cells.These results provided some useful data for the evaluation of the anticancer compounds, and the clinical application of traditional Chinese medicine.

A quality evaluation strategy for Rhizoma coptidis from a variety of different sources using chromatographic fingerprinting combined with biological fingerprinting

Most of herbal medicines of different sources are used for the same treatment goal in clinic despite their tremendous differences of the quality.Because the clinical efficacy is closely related to the quality of medicines,the implementation of an effective quality control(QC)process is vital.Chemical methods based on content determination and chromatographic fingerprints(CFs)have been recommended as powerful tools.In spite of advances in QC,it is still difficult to determine that which of the chemical compositions can best reflect the correlation with the desired biological activity.Herein,we have focused on the biological activities of multi-source herbal medicines to establish an analytical method.By looking for correlations between the biological activities and their chemical compositions,the method can identify reasonable chemical indicators or chemical methods with biological activity information.In this study,high-performance liquid chromatography(HPLC)and microcalorimetry are combined as a useful method for evaluating the quality of Rhizoma coptidis from different sources.The results of biological fingerprints(BFs)showed that the antibacterial activity sequence of R.coptidis of different sources was Coptis teeta Wall.(CT)>Coptis chinensis Franch.(CC)>Coptis deltoidea C.Y.Cheng et Hsiao(CD).The"spectrum-activity"relationships between CFs and BFs were investigated using the canonical correlation analysis(CCA).Compared with the CCA result of CFs and BFs,the canonical correlation coefficient r1and r2were 0.9974 and 0.9752,respectively.It revealed that there are a close correlation between the spectrum-activity relationships and several of the major anti-bacterial components in the CFs,including jatrorrhizine,coptisine,palmatine,and berberine.Of course,R.coptidis was only a representative of multisource medical plants.The result of the current study will provide an effective reference for evaluating the quality of multisource herbal medicines.

The toxic effect of solubilizing excipients on Tetrahymena thermophila BF_5 growth investigated by microcalorimetry

The toxic effect of different solubilizing excipients on the growth and metabolism of Tetrahymena thermophila BF5(T.t.BF5) at various concentrations was investigated by microcalorimetry.The thermogenic curves of T.t.BF5 growth were determined at 28°C,and were evaluated by dynamic parameters.The results indicated that the values of growth rate constant(k),maximum power(P1,P2),peak time(T1,T2) and total quantity of heat(Q) varied for different excipients.There was a good linear relationship between k and concentrations(r>0.95,P<0.01).5% inhibition concentration(IC5) of poloxamer 188,Tween 80,PEG 600,PEG 400 and Tween 20 was 2.18,1.07,1.35,0.58,and 0.045 mg/mL,respectively.After the principal component analysis(PCA),Q,k and P1 could characterize the effect of these excipients on T.t.BF5 growth.Comprehensive evaluation indicated that compared with the control group,poloxamer 188 had the weakest toxicity and Tween 20 had the strongest toxicity.

Rational Daily Administration Times of Yinchenhao Decoctionin Rats with Jaundice Based on PD/PK

Objective To study the rational daily administration times of Yinchenhao Decoction(YCHD)when it was used to treat experimental jaundice in rats based on pharmacodynamics/pharmacokinetics model.Methods Rats were modeled by 4%1-naphthylisothiocyanate(75 mg/kg)for 48 h,then YCHD was drenched with doses of 0.324 g/kg (extract,calculated with the clinical dosage)once,0.162 g/kg twice,and 0.108 g/kg thrice a day,respectively.The total bile and the flow rate of bile were observed after the first administration;Blood samples collected from the orbital sinus at different intervals were used to investigate the levels of liver enzymes(ALT and AST)and bilirubins (TBIL and DBIL),and determine the concentration of 6,7-dimethoxycoumarin(DME)in the plasma using UPLC at the same time,then we obtained the time-effect and time-dose curves.The rational daily administration times of YCHD when treating experimental jaundice were determined based on the comprehensive analysis of time-effect and time-concentration relationships.Results Within 10 h the total bile of rats which were administered once daily(G1) was 1.65 and 1.33 times higher than that of twice and thrice(G2 and G3)a day,respectively,and the four biochemical indexes(TBIL,ALT,DBIL,and AST)of G1 decreased faster than those of G2 and G3(P<0.05).On the other hand, the blood drug level of DME when administrated once daily could maintain at a higher level for a longer time,and its Cmax and AUC0→t were higher than those of G2 and G3,which might be the main reason why its effect was the most significant.Conclusion It is more appropriate to administrate once daily when YCHD is used to treat jaundice.

Effects of Two Curcuminoids on Candida albicans

Objective To investigate and compare the action of curcuminoids on the causal pathogens of Candida albicans growth. Methods The effects of curcumin (CUR) and demethoxycurcumin (DMC) on C. albicans growth were first investigated and compared by microcalorimetry coupled with multiple analytical methods. The quantitative thermo-kinetic parameters obtained from these curves were analyzed to show difference of the actions. Results By analyzing the main parameters screened from principal component analysis together with 50% inhibiting concentration values, it was demonstrated that both CUR and DMC showed good antifungal activities and CUR was stronger. It was further concluded from structure-activity relationship that the existence of methoxy group might enhance lipophilicity of the mother nucleus, which made it easier for the molecular to enter into the cell membrane of fungi to inhibit its growth. Conclusion This study provides a new method for screening new antifungal agents with high efficacy and low toxicity. Meanwhile, it contributes to the application of curcuminoids as food additive, colorant, and drug. Microcalorimetry is real-time, online, and dynamic, and it could be used to characterize the subtle difference among the effects of synthetic and natural products on the vital process of fungi.

Simultaneous Determination of Six Quaternary Ammonium Alkaloids in Coptidis Rhizoma by UPLC

Objective To establish a new,rapid,and reliable reversed-phase ultra performance liquid chromatography (RP-UPLC)method for the simultaneous determination of six quaternary ammonium alkaloids(QAAs)in Coptidis Rhizoma.Methods The effect of different experimental parameters on the analysis of QAAs by RP-UPLC was evaluated.Results Optimal resolution was achieved with an Acquity UPLC BEH C18 column using a gradient elution profile and a mobile phase consisting of water spiked with 10 mmol/L ammonium bicarbonate(A,pH adjusted to 10.0 by ammonia water)and acetonitrile(B),at a flow rate of 0.30 mL/min and wavelength of 345 nm. The column temperature was set at 30℃.The proposed method was found to be reproducible,precise,and rapid according to the method validation.Conclusion The proposed method,which is compatible with MS analysis and the preparation of QAA,provides some helpful insights into the quality control of Coptidis Rhizoma.

Short-Term Efficacy of Treating Hepatitis B Virus-Related Acute-on-Chronic Liver Failure Based on Cold Pattern Differentiation with Hot Herbs: A Randomized Controlled Trial

Objective： To evaluate the clinical efficacy and safety of Yinchen Zhufu Decoction（茵陈术附汤, YCZFD） in the treatment of acute-on-chronic liver failure caused by hepatitis B virus（HBV-ACLF） with cold pattern in Chinese medicine（CM）. Methods： This is a multi-center randomized controlled trial of integrative treatment of CM and Western medicine（WM） for the management of HBV-ACLF patients. A total of 200 HBV-ACLF patients with cold pattern were equally randomly assigned to receive YCZFD and WM（integrative treatment） or WM conventional therapy alone respectively for 4 weeks. The primary end point was the mortality for HBV-ACLF patients. Secondary outcome measures included Model for End-Stage Liver disease（MELD） score, liver biochemical function, coagulation function and complications. Adverse events during treatment were reported. Results： The mortality was decreased 14.28% in the integrative treatment group compared with WM group（χ^2=6.156, P=0.013）. The integrative treatment was found to significantly improve the MELD score（t=2.353, P=0.020）. There were statistically significant differences in aspartate transaminase, total bilirubin, indirect bilirubin, direct bilirubin and prothrombin time between the two groups（P〈0.05 or P〈0.01）. The complications of ascites（χ^2=9.033, P=0.003） and spontaneous bacteria peritonitis（χ^2=4.194, P=0.041） were improved significantly in the integrative treatment group. No serious adverse event was reported. Conclusions： The integrative treatment of CM and WM was effective and safe for HBV-ACLF patients with cold pattern in CM. The Chinese therapeutic principle ＂treating cold pattern with hot herbs＂ remains valuable to the clinical therapy.（Trial registration No. Chi CTR-TRC-10000766）

Study on the Complex Prescription Compatibility Law of the Cold and Hot Nature of Mahuang Decoction(麻黄汤) and Its Categorized Formulae Based on the Cold-Hot Pad Differentiating Assay

Objective：To explore the complex prescription compatibility law of the cold and hot nature of Mahuang Decoction（麻黄汤,MHD） and Maxing Shigan Decoction（麻杏石甘汤,MXSGD）,both categorized formulas but with different hot/cold natures.Methods：Oxygen consumption of mice was determined among three groups：MHD,MXSGD and the control;a cold-hot pad differentiating assay was used to observe the variability of temperature tropism among the groups of mice which was treated with MHD,MXSGD,and their compositions. Meanwhile,the total anti-oxidant capability（T-AOC） activity were detected.Results：After administration of MHD, the mice showed increased oxygen consumption（P0.01）.Compared with MHD group,the remaining rate of MXSGD mice on the hot pad was found to be significantly increased with the cold-hot pad differentiating assay （P0.05）.There was no significant difference（P0.05） among the remaining rates of MXSGD,MXSGD with high dose Gypsum Fibrosum（MXHGF） group,and MXSGD with low dose Gypsum Fibrosum（MXLGF） group mice.Compared with the MHD group,T-AOC activity of the mice in the Consensus Compositons group was significantly decreased（P=0.0494）.Compared with the MXSGD group,T-AOC activity of Gypsum Fibrosum （GF） group was increased significantly（P=0.0013）.Conclusions：The differences in cold and hot nature could be represented objectively between MHD with a hot nature and MXSGD with a cold nature.The reason may be the Gypsum Fibrosum which decreased the efficacy of the consensus compositions.However,increasing or decreasing the dose of Gypsum Fibrosum will not change the cold and hot nature of MXSGD.

"Dose-effect-response" Relationships of Paeoniae Radix Rubra on α-Naphthylisothiocyanate-induced Acute Cholestatic Hepatitis in Rats

Objective To investigate the hepatoprotective effects of Paeoniae Radix Rubra(PRR)at different doses against α-naphthylisothiocyanate(α-NIT)-induced acute cholestatic hepatitis in rats.Methods Rats were ig admini-strated with vehicle or PRR[(1,9,18,36,54,72,and 81 g/(kg·d)]3 d before and 2 d afterα-NIT(60 mg/kg)ig administration.The general status of rats,histopathology of liver,serum alanine aminotransaminase,aspartate aminotransaminase,total bilirubin,direct bilirubin,and alkaline phosphatase levels,were observed at respective time points(24 and 48 h)afterα-NIT administration.Using cluster analysis and correspondence analysis,the 'dose-effect-response'relationships of PRR were evaluated.Results The results showed that compared with model group,the serum biochemistry index significantly decreased with the increasing of PRR dosage(P<0.01), and the change and necrosis of hepatic cellula,and inflammatory cell infiltration were gradually alleviated. However,the improvement was not obviously found in the low-dose group[1 g/(kg·d)].The cluster analysis and correspondence analysis results showed that different doses of PRR could significantly ameliorateα-NIT-induced acute cholestatic hepatitis of rats in a dose-dependent manner.Conclusion The experiments show that administration doses of PRR in clinical use should be added properly in order to gain the expectant therapeutic effect,especially in the treatment of heavy acute cholestasis hepatitis.

Microcalorimetric Investigation of Toxic Effects of Three Injectable Solubilizing Excipients on Tetrahymena thermophila BF5 Growth

Using microcalorimetry, thermal metabolic curves of Tetrahymena thermophila BF5 （T. thermophila BFs） growth at 28℃ affected by three injectable solubilizing excipients （ISE） including tween 80, hydroxypro- pyl-β-cyclodextrin （HP-β-CD） and poloxamer 188 were measured. Meanwhile, the toxicities of three ISE were evaluated by dynamic parameters of thermal metabolic curves. In addition, the irritative effects of the ISE on myoblast L6cells were investigated to show their cytotoxicities by biochemical method. The results indicated that the effects of the ISE on T. thermophila BF5 varied for different ISE. 5% inhibition concentration values （IC5） of the ISE were 1.33, 1.83 and 1.64 mg/mL for tween 80, HP-β-CD and poloxamer 188, respectively. By the principal component analysis （PCA）, the total quantity of heat （Q）, growth rate constant （k） and second maximum power （P2） were selected as the main characteristic parameters to present their toxicities, there were good linear relationships between Q, k, P2 and concentrations c, suggesting that the toxicities of the ISE on T. thermophila BF5 were closely linked to their concentrations. The results of creatine kinase （CK） bioassay of myoblast L6 cells indicated that the sequence of irritative effects of the ISE was HP-β-CD〈poloxamer 188〈tween 80, which added to the results ob- tained from microcalorimetry.

Hot Property of Aconiti Lateralis Radix Praeparata and its Compatibility with Zingiberis Rhizoma Based on Animal Temperature Tropism

Abstract: Objective To establish an objective method for evaluating the intrinsic characteristics between hot and cold properties of herbal drugs and to study the hot property of Aconiti Lateralis Radix Praeparata (ALRP) and its compatibility with Zingiberis Rhizoma (ZR) on animal temperature tropism. Methods The equipment with cold/hot pads was used to investigate the variety of temperature tropism among mice treated by ALRP and its compatibility with ZR. Meanwhile, the activities of adenosine triphosphatase (ATPase), total anti-oxidant capability (T-AOC), and total superoxide dismutase (T-SOD) were measured. Results Compared with the homologous ALRP group, the compatibility between ALRP and ZR (AZ) had a stronger action against the decreasing of remaining rate on hot pad (P 【 0.05), suggesting an enhancement of cold tropism. Meanwhile, the internal indexes of Na+-K+-ATPase activity, Ca2+-Mg2+-ATPase activity, and oxygen consuming volume increased significantly (P 【 0.05), suggesting an enhancement of energy metabolism. The changes of T-AOC and T-SOD activity suggested that AZ reinforced the anti-oxidative capability of mice. In addition, it could be inferred from cluster analysis that the activity of AZ was concretely stronger than that of homologous ALRP. Conclusion ALRP and ZR are both herbs with hot property, but AZ has an enhanced trend of hot property. This external behavior of cold tropism and the internal activity of energy metabolism and anti-oxidant might reflect the internal hot property in an intuitive and objective way. The changes of ATPase activity of liver tissue might be the mechanism of drug action.

Study on Hot Property of Aconiti Lateralis Radix Praeparata and Its Compatibility with Zingiberis Rhizoma Based on Animal Temperature Tropism

Objective To establish an objective method for evaluating the intrinsic characteristics between hot and cold properties of herbal drugs and to study the hot property of Aconiti Lateralis Radix Praeparata(ALRP) and its compatibility with Zingiberis Rhizoma(ZR) on animal temperature tropism.Methods The equipment with cold/hot pads was used to investigate the variety of temperature tropism among mice treated by ALRP and its compatibility with ZR.Meanwhile,the activities of adenosine triphosphatase(ATPase),total anti-oxidant capability(T-AOC),and total superoxide dismutase(T-SOD) were measured.Results Compared with the homologous ALRP group,the compatibility between ALRP and ZR(AZ) had a stronger action against the decreasing of remaining rate on hot pad(P < 0.05),suggesting an enhancement of cold tropism.Meanwhile,the internal indexes of Na+-K+-ATPase activity,Ca2+-Mg2+-ATPase activity,and oxygen consuming volume increased significantly(P < 0.05),suggesting an enhancement of energy metabolism.The changes of T-AOC and T-SOD activity suggested that AZ reinforced the anti-oxidative capability of mice.In addition,it could be inferred from cluster analysis that the activity of AZ was concretely stronger than that of homologous ALRP.Conclusion ALRP and ZR are both herbs with hot property,but AZ has an enhanced trend of hot property.This external behavior of cold tropism and the internal activity of energy metabolism and anti-oxidant might reflect the internal hot property in an intuitive and objective way.The changes of ATPase activity of liver tissue might be the mechanism of drug action.

Study on Drug Property Differences of Shexiang (Moschus) and Bingpian (Borneolum Synthcticum) Based on Analysis of Biothermodynamics

Objective: To study the drug property differences of Shexiang (Moschus) and Bingpian (Borneolum Synthcticum) with biothermodynamics, and to verify the objectivity and authenticity of the drug property. Methods: The growth-thermogram curves of Escherichia coli (E. coli) affected by Shexiang (Moschus) and Bingpian (Borneolum Synthcticum) at different concentrations were determined with microcalorimetry, and the power-time curves (thermogram curves) of E. coli metabolism and characteristic parameters, such as growth rate constant (k), maximum output power (Pm), peak time (tp), total heat-output (Qt), etc. were analyzed with the principal component analysis (PCA) to find the close correlative parameters, so as to objectively reflect the drug property differences of Shexiang (Moschus) and Bingpian (Borneolum Synthcticum). Results: The values of P2 in the second exponential growth phase increased with the increase of the concentrations of Shexiang (Moschus) and Bingpian (Borneolum Synthcticum), and the P2 of Shexiang (Moschus) was larger than that of Bingpian (Borneolum Synthcticum); Q2 increased with the increase of the concentrations of Shexiang (Moschus), but for Bingpian (Borneolum Synthcticum) it was opposite. It is indicated that they have different effects on P2 and Q2 of E. coli in the second exponential growth phase, and have differences in warm and cold natures. Conclusion: The microcalorimetry can accurately and objectively appraise differences of the drug property of Shexiang (Moschus) and Bingpian (Borneolum Synthcticum) and verify the objectivity and authenticity of the drug property, so as to provide a new and useful method for studies of the drug property of Chinese drugs.

Research on Fuzi based on animal thermotropism behavior to discover if it has fewer "hot" characteristics without Ganjiang

OBJECTIVE:To investigate whether Fuzi(Radix Aconiti Praeparata) has fewer "hot" characteristics when administered without Ganjiang(Rhizoma Zingiberis).METHODS:Differences in the thermotropism behaviors of mice treated either with Fuzi(Radix Aconiti Praeparata),Ganjiang(Rhizoma Zingiberis) or the combination of the two given intragastrically were investigated using the Animal Thermotropism Behavior Surveillance System.The water intake volume,oxygen consumption volume,adenosine triphosphatase(ATPase) activity,total antioxidant capacity(T-AOC) and total superoxide dismutase(T-SOD) activity were determined during the investigation.RESULTS:When Fuzi and Ganjiang were administered together,the rate at which mice remained on a warm plate("remaining rate") and the times and distances of their movement were all significantly reduced(P<0.05).Compared with the Normal group,the reduction was 55.1%,48.3% and 44.8%,while compared with the Fuzi group,the reduction was 57.6%,34.3% and 36.0%,indicating that "cold" tropism was significantly increased.Compared with the Normal and Fuzi groups,the ATPase activity and the respiratory oxygen consumption volume of the Fuzi + Ganjiang group were significantly increased(P<0.05),suggesting an improvement in energy metabolism and showing a "hot" characteristic when Fuzi and Ganjiang are present together.Additionally,the T-AOC and T-SOD activity were significantly enhanced(P<0.05).CONCLUSION:The behavior of mice tending toward "cold" tropism can be regarded as a quantitative reflection of Fuzi having fewer characteristics consistent w ith a "hot" nature when not used with Ganjiang,the functional mechanism of which may be a change in the ATPase activity in liver tissue.