Microbial community structure and transformation of indoles in soaking and fermentation of Indigo Naturalis / 中国中药杂志

The soaking and fermentation of Baphicacanthus cusia( Nees),the important intermediate link of Indigo Naturalis processing,facilitates the synthesis of indigo and indirubin precursors and the dissolution of endogenous enzymes and other effective components,while the role of microorganisms in the fermentation is ignored. The present study investigated the changes of microbial community structure in Indigo Naturalis processing based on 16 S amplicon sequencing and bioinformatics. Meanwhile,the contents of indigo,indirubin,isatin,tryptanthrin,indole glycoside,etc. were determined to explore the correlation between the microorganisms and the alterations of the main components. As demonstrated by the results,the microbial diversity decreased gradually with the fermentation,which bottomed out after the addition of lime. Proteobacteria,Bacteroidetes,and Firmicutes were the main dominant communities in the fermentation. The relative abundance of Proteobacteria declined gradually with the prolongation of fermentation time,and to the lowest level after the addition of lime. The relative abundance of Firmicutes increased,and that of Bacteroidetes decreased first and then increased. The contents of effective substances in Indigo Naturalis also showed different variation tendencies. As fermentation went on,indole glycoside decreased gradually; indigo first increased and then decreased; indirubin and isatin first decreased and then increased; tryptanthrin gradually increased. Those changes were presumedly related to the roles of microorganisms in the synthesis of different components. This study preliminarily clarified the important role of microorganisms in the soaking and fermentation and provided a scientific basis for the control of Indigo Naturalis processing and the preparation of high-quality Indigo Naturalis.

HPLC-DAD Characteristic Chromatogram of Baphicacanthis Cusiae Rhizoma et Radix / 中国药学杂志

OBJECTIVE: To establish the characteristic chromatogram of Baphicacanthis Cusiae Rhizoma et Radix and its decoction pieces by HPLC for the identification of authentic and counterfeit products. METHODS: High performance liquid chromatography (HPLC) was used with Agilent Zorbax C18(4.6 mm×250 mm, 5 μm). The mobile phase was acetonitrile-water with gradient elution. The detector was a secondary tube array (DAD). The column temperature was maintained at 35 ℃, the flow rate was 1.0 mL•min-1, and the injection volume was 10 μL. RESULTS: Fifteen batches of genuine crude drug and twelve batches of genuine decoction pieces were determined. Five common peaks were found, among which three peaks were 2-benzoxazolinone, indigo and indirubin. CONCLUSION: The established characteristic chromatogram of Baphicacanthis Cusiae Rhizoma et Radix can effectively distinguish the authentic from the counterfeit. The methodological demonstration shows that the method is accurate, stable and reproducible.

Discovery of an Indirubin Derivative as a Novel c-Met Kinase Inhibitor with In Vitro Anti-Tumor Effects

The c-Met protein is a receptor tyrosine kinase involved in cell growth, proliferation, survival, and angiogenesis of several human tumors. Overexpression of c-Met has been found in gastric cancers and correlated with a poor prognosis. Indirubin is the active component of Danggui Longhui Wan, which is a traditional Chinese antileukemic recipe. In the present study, we tested the anti-cancer effects of an indirubin derivative, LDD-1937, on human gastric cancer cells SNU-638. When we performed the in vitro kinase assay against the c-Met activity, LDD-1937 inhibited the activity of c-Met. This result was confirmed by immunoblot and immunofluorescence of phosphorylated c-Met. Immunoblot analysis showed that LDD-1937 decreased the expression of the Erk1/2, STAT3, STAT5, and Akt, downstream proteins of c-Met. In addition, LDD-1937 reduced the cell viability and suppressed colony formation and migration of SNU-638 cells. Furthermore, LDD-1937 induced G2/M phase arrest in the SNU-638 cells by decreasing the expression levels of cyclin B1 and CDC2. Cleaved-PARP, an apoptosis-related protein, was up-regulated in cells treated with LDD-1937. Overall, this study suggests that LDD-1937 may be a novel small-molecule with therapeutic potential for selectively inhibiting c-Met and c-Met downstream pathways in human gastric cancers overexpressing c-Met.

Optimal extraction of indirubin from Isatidis Folium based on Plackett-Burman design combined with central composite design-response surface methodology / 中草药

Objective: To investigate the significance of each influencing factor and optimize the process of extracting indirubin from Isatidis Folium by Plackett-Burman design combined with central composite design-response surface methodology (CCD-RSM). Methods: Plackett-Burman experimental design was used to screen the main influencing factors, and CCD-RSM was used to optimize the extraction process of indirubin. With the concentration of ethanol, the ratio of material to liquid, and the extraction time as independent variables and the extraction amount of indirubin as dependent variable, the optimum extraction process of indirubin from Isatidis Folium was predicted and analyzed by multiple linear regression and binomial fitting models with independent and dependent variables and the three-dimensional surface graph. Results; The optimal extraction process of indirubin was as follows: ethanol concentration 62%, solvent/sample ratio of 26, and extraction time 9 min. Under these conditions, the maximal extraction rate of indirubin was 4.37 mg/g which was consistent with model predictions. Conclusion: The optimal process is simple and convenient for extracting indirubin from Isatidis Folium with high precision, reproducibility, and predictability.

The modification of natural products for medical use

Drug innovation is characterized by painstaking molecular-level syntheses and modifications as the basic components of research and development. Similarly, natural products are chemically tailored and modified based upon their structural and biological properties. To some extent, the modification of natural products is quite different fromstructure-based drug discovery. This review describes the general strategies and principles for the modification of natural products to drugs, as illustrated by several successful medicines that originated from natural products.

Characterization of the Indirubin Derivative LDD970 as a Small Molecule Aurora Kinase A Inhibitor in Human Colorectal Cancer Cells

Aurora kinase A plays an essential role in mitosis including chromosome separation and cytokinesis. Aberrant expression and activity of Aurora kinase A is associated with numerous malignancies including colorectal cancer followed by poor prognosis. The aim of this study is to determine the inhibitory effects of LDD970, an indirubin derivative, on Aurora kinase A in HT29 colorectal cancer cells. In vitro kinase assay revealed that, LDD970 inhibited levels of activated Aurora kinase A (IC₅₀=0.37 mM). The inhibitory effects of LDD970 on Aurora kinase A, autophosphorylation and phosphorylation of histone H3 (Ser10), were confirmed by immunoblot analysis. Moreover, LDD970 inhibited migration of HT29 cells and upregulated apoptosis-related protein cleaved PARP. In cell viability assay, LDD970 was observed to suppress HT29 cell growth (GI₅₀=4.22 µM). Although further studies are required, results of the present study suggest that LDD970 provide a valuable insight into small molecule indirubin derivative for therapeutic potential in human colorectal cancer.

Effects of Radix Isatidis and contained indigo and indirubin on organic cation transporters OCT1 and OCT2 in mouse kidney / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of Radix Isatidis and its constituents indigo and in?dirubin on two principal subtypes of organic cation transporters(OCT)OCT1,OCT2 in vivo in mice. METHODS Decoction of Radix Isatidis (DRI) 1.6 and 6.4 g · kg-1,granules of Radix Isatidis (GRI) 0.615 and 2.460 g·kg-1,indigo 0.008 and 0.640 mg·kg-1 and indirubin 0.0192 and 1.536 mg·kg-1 were ig given to NIH mice(60 mice per group),twice a day for 5 d. Four control groups were set up,including the vehicle of water,0.5% sodium carboxymethyl cellulose(CMC),additives of sucrose plus dextrin (1.5 g · kg-1)and positive control quinidine(0.025 g · kg-1). Sixty minutes after the last dosing,all the mice were iv given metformin(Met)5 mg·kg-1,and at 1.0,2.5,5.0,7.5,10.0 and 20.0 min after Met iv,10 mice in each group were sacrificed to collect whole blood and kidneys respectively. The right kidney was homogenized for Met accumulation test and the left one used to extract total RNA for analysis of OCT1 and OCT2 mRNA expressions by real-time PCR. The contents of Met in sera and kidneys were quantified by HPLC. Major pharmacokinetic parameters of Met in sera were analyzed by pharmacokinetic software(DAS 2.0). RESULTS There was no significant difference between water control group,0.5%CMC group and sucrose plus dextrin group in any examined item. Compared with vehicle control group (water and 0.5%CMC group),all the related pharmacokinetic parameters in DRI 6.4 g · kg- 1,GRI 2.46 g · kg-1,indigo 0.640 mg · kg-1 and indirubin 1.536 mg · kg-1 groups were changed significantly (P<0.05,P<0.01). The elimination half time (t1/2β) was prolonged 13%-97%,volume of distribution reduced by 13%-72%,clearance(Cl)reduced by 9%-65%,and the area under the concentration-time curve (AUC0-20 min) increased by 13%-135%. AUC0-20 min obtained from renal Met accumulations was significantly increased(P<0.01)while Met uptake by kidney slices was reduced(P<0.05,P<0.01). The expressions of OCT1 and OCT2 mRNA were obviously down-regulated(P<0.05,P<0.01). CONCLUSION The mouse renal OCT1 and OCT2 are significantly inhibited by DRI,GRI,indigo and indirubin. The inhibitory effect of Radix Isatidis on OCT1 and OCT2 probably arises from indigo and indirubin contained.

Simultaneous determination of six components in Baikening Granules by UPLC-MS/MS / 中草药

Objective: To develop an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous determination of six components (indirubin, peimine, peiminine, ginkalide A, ginkalide B, and ginkalide C) in Baikening Granules. Methods: Preparing the samples solution by Soxhlet's extraction using methanol-chloroform as solvent, and multiple reaction monitoring (MRM) with a polarity-switching electrospray ionization (ESI) source between positive and negative modes was used for the quantification of indirubin, two peimisines, and three bilobalides. The detection was performed on an Inertsutain C18 column (75 mm × 3.0 mm, 2 μm) using a mobile phase consisted of methanol-acetonitrile and 2 mmol/L ammonium acetate water solution with gradient elution lasting 6 min. Results: All of the analytes showed good linearity (r ≥ 0.995 8) in the tested ranges. The precision, repeatability, and stability of the method were good for the six components. The average recoveries were in the range of 96.5%-105.1% with relative standard deviations (RSD) ≤ 4.2%. Conclusion: The new established polarity-switching LC-MS-MS method has been proven to be highly sensitive and effective in evaluating the quality of Baikening Granules, and peimine, peiminine, ginkalide A, ginkalide B, and ginkalide C are quantified in this drug for the first time.

Correlation between chemical composition in processof Isatidis Radix preparation and its pharmacological activity / 中草药

Objective: To investigate the key factors in the process of IsatidisRadix (IR)preparation. The content changes of its main chemical ingredients (epigoitrin, uridine, guanosine, adenosine, and indirubin) and the correlation with its pharmacological activity had been studied, which would providetheoretical foundation for further optimization of process. Methods: The process of IR preparation (extracting-concentrating-alcohol sinking-drying-granulation) was simulated. HPLC was employed to determine the contents of the chemical ingredients in each step of IR preparation, and the clearance rate of OH•was used as the model to investigate the antioxidant activity. Cluster analysis, partial least sqμares (PLS), and multiple linear regression (MLR) were used to analyze the correlation between its chromatogram and pharmacological activity. Results: The content changes of chemical ingredients from IR preparation were generally decreased on the whole, and the highest loss rate of chemical ingredients occurred in the concentrating-drying process; Two metrological methods had been used toanalyze the correlationand the chromatogram ofindirubin, guanosine, and adenosine, which was positively related with the clearance rate of OH•.Conclusion: Each step in the process of IR preparation had the significant effect on the content of its chemical ingredients and pharmacological activity. The concentrating and drying process may be the key process which would finally influence the quality of preparation. Indirubin, guanosine, and adenosine may be the effective substances of IR with antioxidant activity.

Study of mechanism of indirubin derivative PHⅡ-7 in augmenting TRAIL-induced cytotoxicity in breast cancer cell line as well as its chemo-resistant counterpart / 中国药理学通报

Aim To investigate the effect of indirubin derivative PHⅡ-7 and TRAIL on proliferation in breast cancer cell MCF-7 and its MDR counterpart MCF-7/ADR and the mechanism.Methods Growth inhibition rate was examined respectively by MTT assay under treatment with TRAIL or PHⅡ-7 or in combination. Cell apoptosis and ROS production were examined by flow cytometry.The change of TRAIL receptors(DR4/DR5 )in mRNA was analysed by realtime PCR.Re-sults IC50 of PHⅡ-7 on MCF-7 and MCF-7/ADR was (4.49 ±1.55 ),(3.44 ±0.90 )μmol · L-1 respec-tively;MDA-MB-231 was TRAIL sensitive cell line, and apparently TRAIL induced apoptosis in MDA-MB-23 1 .Low concentration of PHⅡ-7 in combination with TRAIL could augment TRAIL-induced cytotoxic effect including apoptosis while TRAIL or PHⅡ-7 treatment alone had limited cytotoxity to those cells.Besides, PHⅡ-7 at this concentration had little toxicity to hu-man peripheral blood mononuclear cells even if in com-bination with TRAIL.PHⅡ-7 generated ROS produc-tion inside MCF-7 and MCF-7/ADR cells and up-regu-lated DR4/DR5 expression concentration dependently. Once upon ROS scavenger NAC involved,the effect of TRAIL receptors up-regualtion by expression was abro-gated.Conclusions PHⅡ-7 at low concentration could improve the sensitivities of breast cancer cell MCF-7 and MCF-7/ADR to TRAIL,the mechanism of which may be the ability of ROS production by PHⅡ-7 help up-regulated TRAIL receptor DR4,DR5 .Our re-search set a solid foundation for PHⅡ-7 in combination with TRAIL in future clinical application.

Determination of Indigo and Indirubin in Baphicacanthus cusia from Different Producing Areas and Medicinal Parts by RP-HPLC / 医药导报

Objective To establish a RP-HPLC method for determining indigo and indirubin in Baphicacanthus cusia from different producing areas and medicinal parts. Methods The separation was achieved by an Agilent TC-C18 Column (4.6 mm×250 mm, 5 μm) at 25 ℃ using methanol-water (75??25) as mobile phase at a flow rate of 1 mL??min-1.The detection wavelength was 290 nm. Results Indigo had a good linear relationship with peak area at range of 0. 051 3-0.820 8 μg (r=0.999 3).The recovery rate was 99.00% and RSD was 1.30% (n=6).Indirubin had a good linear relationship with peak area at range of 0.049 5-0.792 0 μg (r=0.999 9).The recovery rate was 98.88% and RSD was 1.51% (n=6). Conclusion The contents of the two components are obviously different in Baphicacanthus cusia because of different places or medicinal parts. The proposed method is simple, rapid and reliable. This method for determination of indigo and indirubin in Baphicacanthus cusia by RP-HPLC provides a basis for quality control of Baphicacanthus cusia.

Study on Quality Standard for Isatidis Folium Dispensing Granules / 中国药师

Objective:To establish the quality standard for Isatidis folium dispensing granules. Methods:Indigo and indirubin in the granules were identified by TLC. The extract content was determined by a hot dipping method for the determination of alcohol-solu-ble extract. The content of indirubin was determined by HPLC. Results:The spots in TLC were clear and well-separated. The extrac-tion limit no less than 12. 0% was preliminarily drawn up. Indirubin showed a good linear relationship within the range of 0. 50-8. 04μg·ml-1(r=0. 999 9) with the average recovery of 99. 41%(RSD=1. 21%, n=9). The content limit no less than 50. 0 μg per pack was preliminarily drawn up for indirubin. Conclusion: The method is easy-operated and accurate with good specificity for the quality control of Isatidis folium dispensing granules.

lnhibition of Radix lsatidis and its constituents indigo and indirubin on major organic anion transporters Oat1, Oat2 and Oat3 in mouse kidneys / 中国药理学与毒理学杂志

OBJECTlVE To investigate the inhibition of Radix lsatidis and its major constituents indigo and indirubin on three principal subtypes of organic anion transporters ( OATs) , Oat1, Oat2 and Oat3 in vivo in mice. METHODS Granules of Radix lsatidis ( GRl) 0.615 and 2.46 g·kg-1 , decoction of Radix lsatidis ( DRl) 1.6 and 6.4 g·kg-1 , indigo 0.008 and 0.64 mg·kg-1 and indirubin 0.0192 and 1.536 mg·kg-1 were ig given to the NlH mice (60 mice per group), twice a day, for 5 d while four control groups were set up, including vehicle of water, 0.5%sodium carboxymethyl cellulose ( CMC) , positive control probe-necid (0.05 g·kg-1) and additives of sucrose plus dextrin (1.5 g·kg-1 each) groups. After the last dosing of the test samples, para-aminohippuric acid ( PAH) clearance test was conducted. All the mice were iv given PAH 0.03 g·kg-1 and 1, 2.5, 5, 7.5, 10 and 20 min later before 10 mice per group were euthanized to collect whole blood and the kidneys were quickly removed. Each right kidney was homoge-nized to analyze the PAH accumulations and each left kidney to extract total mRNA for analysis of Oat1, Oat2 and Oat3 gene expressions using quantitative real-time PCR. The concentrations of PAH in sera and in kidney homogenates were determined by the method of Kiguchi. Major pharmacokinetic parame-ters of PAH in sera were calculated by pharmacokinetic software ( DAS2.0) . PAH uptake test for kidney slices was performed on another group of NlH mice according to the method of Nakakariya. RESULTS There was no significant difference between water control group and 0.5%CMC group in all the examined items. Compared with the vehicle control groups ( water and 0. 5%CMC group ) , elimination half time ( t1/2β) of PAH in GRl 2.46 g·kg-1 ,indigo 0.64 mg·kg-1 and indirubin 1.536 mg·kg-1 groups was signifi-cantly prolonged (P<0.05), the total clearance (Cl) and volume of distribution (Vd) were obviously reduced ( P<0.01) and the area under the curve ( AUC0-20 min ) of PAH in all the tested groups was signifi-cantly increased ( P<0.01) . AUC0-20 min obtained from renal PAH accumulations within the checked time was significantly higher ( P<0.05, P<0.01) than in the vehicle control group. But there was in no signifi-cant difference between all the study groups in kidney-to-plasma AUC ratios. PAH uptake results by kidney slices were significantly lower ( P<0. 05, P<0. 01 ) than in vehicle control group in every two dosages of all the four samples tested. Compared with vehicle control group, the mRNA expressions of Oat1, Oat2 and Oat3 were obviously ( P<0.05, P<0.01) and abnormally regulated in the groups of GRl 2.46 g·kg-1, DRl 6.4 g·kg-1, indigo 0.64 mg·kg-1 and indirubin 1.536 mg·kg-1. CONCLUSlON The renal Oat1, Oat2 and Oat3 of mice are significantly inhibited by GRl, DRl, indigo and indirubin. The inhibitory function of Radix lsatidis probably stems from indigo and indirubin contained in it.

Research progress on effects of indirubin and its structurally similar compounds on anticancer and neuroprotection / 中草药

Indirubin is the active ingredient in many Chinese materia medica and mainly used to treat leukemia. It has been founded as leading inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) by competing with ATP binding sites. Increasing new findings pointed out that the unique chemical structure of indirubin could contribute to the polypharmacological activities particularly against cancer and neurodegeneration therapy while these diseases shared common molecular link on abnormal phosphorylation of CDKs and GSK-3. In this review, the underlying mechanisms of dual actions of indirubin and its structurally similar compounds on therapy of cancer and neurodegenerative diseases are presented.

Effect of indirubin-3'-monoxime on proliferation and apoptosis of human HT-29 cells / 中国癌症杂志

Background and purpose: In recent years indirubin-3'-monoxime has been found to be capable of inhibiting some cell proliferation in vitro and in vivo studies, but human colon cancer HT-29 cells, therefore the purpose in this paper was to study the effect of indirubin-3'-monoxime on proliferation and apoptosis of HT-29 cells and its associated mechanism. Methods: HT-29 cells were treated with indirubin-3'-monoxime. The proliferative status of cells was measured by methabenzthiazuron (MTT) assay, flow cytometry (FCM) was used to measure the apoptosis rate. RT-PCR was used to measure the transcription of apoptosis suppressor gene bcl-2, survivin and apoptosis promoting gene Bar. Results: Indimbin-3'-monoxime inhibited growth of HT-29 cells in a dose-dependent and time-dependent manner (F=11.25, P<0.01). The apoptosis rate increased after the treatment by indirubin-3'-monoxime at 10 μmol/L. There were significant differences between different time groups (F=195.25, P<0.01). The transcription of survivin (F=78.75, P<0.01) and Bax (F=87.61, P<0.01) mRNA in HT-29 cells were increased; the transcription of bcl-2 was significantly decreased (F=95.82, P<0.01). Conclusion: Indirubin-3'-monoxime has obviously inhibited proliferation and induce apoptosis of colon cancer HT-29 cells, its mechanism may be related to decrease the bcl-2/Bax ratio.

Quality of Isatis indigotica of Different Growing Period / 中国药房

OBJECTIVE:To investigate the quality of Isatis indigotica of different growing periods.METHODS:The property and the microscopic characteristics of Isatis indigotica of 1 or 2 growing years were identified and the contents of adenosine,indigo and indirubin were determined by HPLC. RESULTS:Isatis indigotca of 1 growing year showed wide liber and sparsely arranged parenchyma cells while Isatis indigotca of 2 growing years showed narrow liber and tightly arranged parenchyma cells. Without timely open-air drying or proper storage after harvesting,the Isatis indigotca was likely to experience color change of darkening in cross-section,increase of the content of the indigo,and decrease of the contents of adenosine and indirubin. The Isatis indigotica of too short growing period or two growing years were low in contents of adenosine,indigo and indirubin. CONCLUSION:The planting of Isatis indigotica should be standardized and which should be given a quality control to avoid the great quality difference arose from differences of habitat,planting and harvesting,initial processing and storing method,etc.

Determination of Indirubin in Kangbingan Oral Liquid by Ultraviolet Spectrophotometry / 中国中医药信息杂志

Objective To determine the content of indirubin in Kangbingan Oral Liquid by UV spectrophotometer methods.Methods The content of indirubin was determined by Heltos Gamma ultravioletvisible spectrophotometer with chloroformic solution of 2～8 ?g/mL confected by standard sample of indigotin and indirubin.The sample of negative blank solution was confected by the prescription without Radix Isatidis.The standard sample and the blank sample were determined separately with the wavelength at 500～700 nm.Results Indirubin had the maximum absorption at wavelength of 540.0 nm and indigotin had the maximum absorption at wavelength of 634.5 nm.The linear correlation was available at the range of 0.20～1.40 ?g/mL,and the correct coefficient was 0.999 4.The average recovery rate was 99.05% and RSD was 0.87%(n=6).Conclusion This method is simple and accurate,and can be used to control the quality of Kangbingan Oral Liquid.

Study of Quality Standard for Ulter Zhuhuang Gel / 中国中医药信息杂志

Objective To establish the quality standard for Ulter Zhuhuang gel.Methods Indirubin,cholic acid and borneol were identified by TLC.The content of indirubin and cholic acid was determined by HPLC.Result The TLC sports developed was fairly clear.The HPLC method showed that the average recovery of indirubin and cholic acid was 100.3% with RSD=2.1% and 98.9% with RSD=1.8% respectively.Conclusion The method is convenient and accurate.It can be used for the quality control of Ulter Zhuhuang gel.

Preparation and Quality Control of Anti-inflammatory Dental Ulcer Film / 中国药房

OBJECTIVE: To prepare anti-inflammatory dental ulcer film and establish its quality standard. METHODS: Drug-containing mortar and blank mortar were prepared to prepare compound film agent. HPLC was used to the quantitative analysis of indirubin. RESULTS: The preparation had well appearance and its detection of index was up to the standard stated in Chinese Pharmacopeia. The linear range of indirubin was 0.8～6.4 ?g?mL-1 (r=0.999 8) with an average recovery rate of 100.65% (RSD=1.07%,n=6). CONCLUSION: The formula of preparation is reasonable, the preparation method is simple and applicable and determination method is accurate and reliable.

Simultaneous determination of four effective components in Guilin Watermelon Frost by HPLC-MS / 中成药

AIM: To establish an HPLC-MS method for simultaneously determining four effective components in Guilin Watermelon Frost(Mirabilitum Praeparatum,Rhizoma Coptidis,Radix Scutellariae,etc.). METHODS: The sample was extracted with methanol containing 20% chloroform under ultrasonication.The HPLC separation was performed on Zorbax SB C_(18)(3.0 mm?250 mm,5 ?m) column using water including 0.5% formic acid(A)-methanol(B) as mobile phase,with the gradient elution(0-2 min,60%B;2-5 min,60%→90%B;5 min to the end,90%B), at a flow rate of 0.40 mL/min.The compounds were analyzed by ESI-MS under ion monitoring mode(0-3 min,m/z 249;3-6 min,m/z 336;6-10 min,m/z 447;10-16 min,m/z 263). RESULTS: The linear ranges were 0.020-10.0 ?g/mL,0.010-40.0 ?g/mL,0.036-50.0 ?g/mL and 0.040-4.00 ?g/mL for matrine,berberine,baicalin and indirubin,with detection limits of 0.005,0.001,0.006 and 0.010 ?g/mL,respectively.The average recoveries ranged from 96% to 101% with all relative standard deviations less than 3%.CONCLUSION: (The method is rapid,accurate and suitable for the quality control of the four effective) components in Guilin Watermelon Frost.