cinobufagin and bufogenin

Chemical defences are widespread in nature, yet we know little about whether and how climatic and geographic factors affect their evolution. In this study, we investigated the natural variation in the concentration and composition of the main bufogenin toxin in adult Asian toads (Bufo gargarizans Cantor) captured in twenty-two regions. Moreover, we explored the relative importance of eight climatic factors (average temperature, maximum temperature, minimum temperature, average relative humidity, 20-20 time precipitation, maximum continuous precipitation, maximum ground temperature, and minimum ground temperature) in regulating toxin production. We found that compared to toads captured from central and southwestern China, toads from eastern China secreted higher concentrations of cinobufagin (CBG) and resibufogenin (RBG) but lower concentrations of telocinobufagin (TBG) and cinobufotalin (CFL). All 8 climatic variables had significant effects on bufogenin production (r

Topics: Animals; Bufanolides; Bufo bufo; Geography; Temperature; Toxins, Biological

Cinobufagin and resibufogenin are two major effective bufadienolides of Chan su (toad venom), which is a Chinese medicine obtained from the skin venom gland of toads and is used as a cardiotonic and central nervous system (CNS) respiratory agent, an analgesic and anesthetic, and as a remedy for ulcers. Many clinical cases showed that Chan su has severe side-effects on the CNS, causing shortness of breath, breathlessness, seizure, coma and cardiac arrhythmia. We used whole-cell recordings from brain slices to determine the effects of bufadienolides on excitability of a principal neuron in main olfactory bulb (MOB), mitral cells (MCs), and the cellular mechanism underlying the excitation. At higher concentrations, cinobufagin and resibufogenin induced irreversible over-excitation of MCs indicating a toxic effect. At lower concentrations, they concentration-dependently increased spontaneous firing rate, depolarized the membrane potential of MCs, and elicited inward currents. The excitatory effects were due to a direct action on MCs rather than an indirect phasic action. Bufadienolides and ouabain had similar effects on firing of MCs which suggested that bufadienolides activated neuron through a ouabain-like effect, most likely by inhibiting Na+/K+-ATPase. The direct action of bufadienolide on brain Na+ channels was tested by recordings from stably Nav1.2-transfected cells. Bufadienolides failed to make significant changes of the main properties of Nav1.2 channels in current amplitude, current-voltage (I-V) relationships, activation and inactivation. Our results suggest that inhibition of Na+/K+-ATPase may be involved in both the pharmacological and toxic effects of bufadienolide-evoked CNS excitation.

Topics: Amphibian Venoms; Animals; Bufanolides; Cardiotonic Agents; Central Nervous System; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Ion Channel Gating; Membrane Potentials; Mice, Inbred C57BL; NAV1.2 Voltage-Gated Sodium Channel; Neurons; Olfactory Bulb; Ouabain; Patch-Clamp Techniques; Sodium-Potassium-Exchanging ATPase

To develop a sensitive and accurate assay for detecting cinobufagin and resibufogenin in liver tissue using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).. The homogenization of liver tissue with internal standard dexamethasone was extracted with dichloromethane. The extracts with methanol were purified through ProElut C18 solid phase extraction and tested in positive electrospray ionization with multiple reaction monitoring of HPLC-MS/MS.. The good linear relationship of cinobufagin and resibufogenin in liver tissue were 1-204 ng/g and 1-206 ng/g, respectively. The minimal detection threshold (S/N > or = 3) of this method was 0.3 ng/g for both cinobufagin and resibufogenin. The matrix effect was 96.5%-126.7%. The extraction recovery coefficient was 70.0%-82.3%. The precision of intra-day and inter-day was less than 10%.. This method is sensitive and reliable, and can be used in forensic toxicological analysis.

Topics: Bufanolides; Chromatography, High Pressure Liquid; Forensic Toxicology; Humans; Liver; Sensitivity and Specificity; Solvents; Tandem Mass Spectrometry; Tissue Distribution

Cinobufacini has been traditionally used in China for the treatment of tumor since hundreds years ago. For recent years, its modern preparation,cinobifucini injection has also obtained satisfactory therapeutic functions for cancer.. High performance liquid chromatography (HPLC) analysis was applied to determine the content of cinobufagin, resibufogenin and bufothionine in cinobufacin extract liquid and injection; MTT assay and flow cytometric analysis were also respectively used to study the effect of cinobufacini extract liquid, injection and three chemical structures on cells and cell cycles.. HPLC results demonstrated that in cinobufacini extract liquid three ingredients (cinobufagin, resibufogenin and bufothionine) were all monitored while in cinofacini injection only bufothinone was detected; MTT assays showed bufothionine could obviously inhibit the proliferation of human hepatocellular carcinoma cell lines such as SMMC-7721 and BEL-7402 in a dose- and time-dependent manner as well as cinobufagin and resibufogenin; further flow cytometric analysis indicated obvious increases in G2/M phase and decrease in G0/G1 phase when SMMC-7721 cell line exposure to bufothionine (480 μg/ml).. These results suggested bufothionine could be involved in treatment of human cancer for cinobufacini injection and the mechanism might be relative to induce G2/M phase cell cycle arrest.

Topics: Amphibian Venoms; Antineoplastic Agents; Bufanolides; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Flow Cytometry; G2 Phase Cell Cycle Checkpoints; Humans; Indole Alkaloids; Injections; Liver Neoplasms; Medicine, Chinese Traditional; Quinolinium Compounds; Time Factors

A simple, accurate, sensitive, and robust reversed-phase high-performance liquid chromatography (HPLC) method employing cyclodextrins as mobile phase additives has been developed in order to separate and determine resibufogenin and cinobufagin. Various factors affecting the separation for them, such as the nature of cyclodextrins, organic solvent, the concentration of γ-cyclodextrin, and temperature, were systematically studied. γ-cyclodextrin, as an effective mobile phase additive, can markedly improve the separation for resibufogenin and cinobufagin. The role of γ-cyclodextrin in the developed HPLC method is attributed to the formation of the inclusion complex between resibufogenin (or cinobufagin) and γ-cyclodextrin. So, the apparent formation constant (K(f) ) of the resibufogenin (or cinobufagin)/γ-cyclodextrin inclusion complex and the thermodynamic parameters of the inclusion process also were investigated. Resibufogenin (or cinobufagin) forms the 1:1 inclusion complexes with γ-cyclodextrin, and the resibufogenin/γ-cyclodextrin complex is more stable than the cinobufagin/γ-cyclodextrin complex. The K(f) values of resibufogenin and cinobufagin decrease with the increase of the temperature. The thermodynamic parameters of the inclusion reveal that the inclusion process between resibufogenin (or cinobufagin) and γ-cyclodextrin is spontaneous, exothermic, and enthalpically driven. Finally, the optimized method was successfully applied to separate and determine of resibufogenin and cinobufagin in the different Chansu (Bufonis venenum) samples.

Topics: Animals; Bufanolides; Bufonidae; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Cyclodextrins

The aim of this paper is to report the development of a method for the determination of the binding rates of cinobufagin (CBG) and resibufogenin (RBG) with different plasma proteins. Equilibrium dialysis method was used to imitate the binding process between cinobufagin, resibufogenin and plasma proteins. HPLC was employed to determine the concentration of cinobufagin and resibufogenin inside and outside of the dialysis membrane, and then on the basis of which protein binding rates were calculated. The calibration curves of both cinobufagin and resibufogenin with human, rats' and Beagle dogs' plasma were linear in the range of 0.50-40.00 microg x mL(-1), while the buffer solution was 0.25-4.00 microg x mL(-1). The extract recovery of cinobufagin was in the range of (68.73 +/- 2.16)%--(79.27 +/- 1.62)%, while that of resibufogenin was (71.59 +/- 4.31)%--(83.47 +/- 2.63)%. The average plasma protein binding rates with cinobufagin were 85.63%, 80.21% and 70.10% in human, rats' and Beagle dogs' plasma, whereas those binding rates with resibufogenin were 84.51%, 75.11% and 70.60%, respectively. The results suggested that the protein binding rates of cinobufagin and resibufogenin were of middle strength, which in rats', Beagle dogs' and human plasma were decreased in the following order: human plasma > rats' plasma > Beagle' dogs plasma.

Topics: Animals; Blood Proteins; Bufanolides; Chromatography, High Pressure Liquid; Dialysis; Dogs; Humans; Molecular Structure; Protein Binding; Rats; Species Specificity

Outward delayed rectifier potassium channel and outward transient potassium channel have multiple important roles in maintaining the excitability of hippocampal neurons. The present study investigated the effects of two bufadienolides, Resibufogenin (RBG) and Cinobufagin (CBG), on the outward delayed rectifier potassium current (IK) and outward transient potassium current (IA) in rat hippocampal neurons. RBG and CBG have similar structures and both were isolated from the venom gland of toad skin. RBG inhibited both IK and IA, whereas CBG inhibited IK without noticeable effect on IA. Moreover, at 1 μM concentration both RBG and CBG could alter some channel kinetics and gating properties of IK, such as steady-state activation and inactivation curves, open probability and time constants. These findings suggested that IK is probably a target of bufadienolides, which may explain the mechanisms of bufadienolides' pathological effects on central nervous system.

Topics: Amphibian Venoms; Animals; Bufanolides; CA1 Region, Hippocampal; Cells, Cultured; Membrane Potentials; Patch-Clamp Techniques; Potassium Channels; Pyramidal Cells; Rats; Rats, Sprague-Dawley

To establish the quality standards of Linmaoxiang Jiedu pills.. Cinobufagin and bufogenin were determined by HPLC simultaneously.. The average recoverys of Cinobufagin and bufogenin was 100.5% and 100.3%, their linear range were 48.74-731.10 ng and 49.90-748.50 ng, respectively.. The method for quantification is reproducible and realizable. The method can be used to control quality of Linmaoxiang Jiedu pills as the quality standards.

Topics: Bufanolides; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Reproducibility of Results

A plasma pharmacochemistry analysis of the bioactive constituents in rat plasma after oral administration of ChanSu was performed. High performance liquid chromatography coupled with the electrospray ionization mass spectrometry techniques of HPLC/ESI-IT-MS/MS and RRLC/ESI-Q-TOF-MS were used for the chemical profiling of samples of dosed plasma, control plasma and ChanSu extract. Comparative analysis of the resulting chemical profiles revealed 20 prototype compounds and 4 metabolites derived from ChanSu as potential bioactive components. By MS/MS analysis and accurate molecular weight assessments, the majority of these bioactive components (19 prototype compounds and 2 metabolites) were structurally identified. Moreover, seven were confirmed by comparing their retention behaviors using MS and MS/MS analysis. This study proposes a series of potential bioactive components of ChanSu, which we hope will lead to new drug discovery based on ChanSu and other traditional Chinese medicines.

Topics: Animals; Bufanolides; Chromatography, High Pressure Liquid; Male; Medicine, Chinese Traditional; Rats; Rats, Sprague-Dawley; Spectrometry, Mass, Electrospray Ionization

Toad venom, called Chansu in China, has been widely used for the treatment of heart failure, sores, pains, and various cancers for a long time in clinic.. The aim of the study is to investigate the chemical differences among a variety of toad venoms from different geographic locations and related Bufo species.. Ten batches of commercial toad venom collected from different regions in China, one batch of fresh toad venom obtained from Bufo bufo gargarizans, and six batches of related Bufo species were analyzed by HPLC and LC-DAD-MS/MS. Individual components were identified by comparison of retention times, UV spectra, and mass spectra with authentic compounds, standard addition, as well as summarized MS fragmentation rules. Based on the profile of identified constituents and the content of cinobufagin and resibufogenin, the chemical differences observed among different samples are discussed.. Overall, 43 compounds were identified in the methanolic extracts of the different samples of toad venom. Besides of suberoyl arginine, several free bufadienolides, bufadienolide sulfates, and suberoyl esters of bufadienolides were found. The total amounts of cinobufagin and resibufogenin, which are the only two control markers according to the current Chinese Pharmacopoeia, varied widely from 0.7% to 10.9% in the commercial Chansu samples collected in the different locations in China. Low levels of resibufogenin, but no cinobufagin was observed in the samples from Bufo melanosticus and Bufo marinus, and even neither of both compounds was found in the sample from Bufo viridis.. The chemical profiles of the different commercial and collected toad venoms from related Bufo species differed significantly, not only in the absolute and relative contents, but also in the number and type of the constituents. The main reason for this variation are species-specific differences, but additional factors, such as the harvest and post-harvest processing, and adaption to environmental factors in different geographic locations, also seem to contribute.

Topics: Amphibian Venoms; Animals; Arginine; Bufanolides; Bufonidae; Chromatography, High Pressure Liquid; Mass Spectrometry

A preparative, high-speed, counter-current chromatographic (HSCCC) method for the isolation and purification of bufadienolides from Chansu was successfully developed by using stepwise elution with a two-phase solvent system composed of n-hexane: chloroform: methanol: water (4:1:2.5:5 and 4:1:4:5, v/v). A total of 7.5 mg of cinobufotalin (1), 8.0 mg of bufalin (2), 14.0 mg of cinobufagin (3) and 9.5 mg of resibufogenin (4) were obtained in a one-step separation from 80 mg of the crude extract with purities of 93.2%, 98.7%, 99.2%, and 99.4%, respectively. The chemical structures were determined from 1H NMR and 13C NMR spectroscopic data.

Topics: Bufanolides; Chromatography, High Pressure Liquid; Countercurrent Distribution; Drugs, Chinese Herbal; Molecular Structure

A 24-year-old male died suddenly following the intravenous injection of what was believed to be the ring-derivate amphetamine 'ecstasy' (MDMA). Toxicological analyses of the victim's blood and the injected material, however, failed to reveal MDMA, but showed instead low levels of bufotenine, a tryptamine derivative alkaloid found in the secretions of various toads. In addition, resibufogenin, cinobufagin and bufalin, bufadienolides that are also found in toad venom, were identified in the injected material. While these substances also occur in certain South American plants, the finding of paracetamol, promethazine and diclofenac would be in keeping with ingredients found in the traditional Chinese herbal product Chan Su that derives from the skin glands and secretions of toads and that is often adulterated with standard pharmaceutical drugs. This case demonstrates the problems that users and sellers may encounter from the unknown composition of street drugs and herbal medicines, and the danger that may be incurred from the injection of such materials. It also shows the difficulties that may be associated with attempting to identify low levels of organic toxins in postmortem specimens necessitating a targeted screening approach guided by information obtained at the death scene.

Topics: Amphibian Venoms; Animals; Anura; Bufanolides; Bufotenin; Death, Sudden; Forensic Toxicology; Hallucinogens; Humans; Illicit Drugs; Injections, Intravenous; Male; N-Methyl-3,4-methylenedioxyamphetamine; Young Adult

To investigate apparent aqueous solubility and apparent oil-water partition coefficients of three bufadienolides composition.. A high performance liquid chromatography method was established to determine apparent aqueous solubility of three bufadienolides composition; apparent oil-water partition coefficients of three bufadienolides composition was determined by shaking flask method.. The solubility of three bufadienolides composition reached the top value, 76.29 microg x mL(-1) (RBG), 51.85 microg x mL(-1) (CBG), 32.76 microg x mL(-1) (BL) respectively and a total of 160.9 microg x mL(-1) in the condition of 37 degrees C and pH 7.0, therefore three bufadienolides composition can be defined as insoluble products. The solubility of the three decreased in weak acid or base environment to some extent. RBG, CBG, BL are liposoluble components and the sequence of log P is RBG > BL > CBG.. RBG, CBG, BL is water-insoluble and hydrophobic. Surfactants can be applied to increase the solubility of three bufadienolides composition.

Topics: Bufanolides; Calibration; Hydrogen-Ion Concentration; Logistic Models; Oils; Solubility; Temperature; Water

Resibufogenin, cinobufagin, and bufalin are cytotoxic steroids isolated from the Chinese drug Chan'su. Biotransformation of these three bufadienolides by Nocardia sp. NRRL 5646 was investigated. Notably, resibufogenin was converted to 3-acetyl 15beta-hydroxyl bufotalin, via an unprecedented 14beta,15beta-epoxy ring cleavage and a regio-selective acetoxylation. This product showed significantly increased cytotoxic activity. The regio-selective acetylation of the 3-OH was also involved in the other reactions. The structures of metabolites were established by ESI-LC/MS and 2D NMR techniques. The in vitro cytotoxic activities against human cancer cell lines of the substrates and the transformed products were determined by the MTT method and their structure-activity relationship (SAR) was discussed. This investigation provided a useful approach to prepare new bufadienolides and the SAR research.

Topics: Acetylation; Antineoplastic Agents; Biotransformation; Bufanolides; Cell Line, Tumor; Drug Screening Assays, Antitumor; Humans; Molecular Structure; Nocardia; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Structure-Activity Relationship

A rapid cyclodextrin modified micellar electrokinetic chromatography (CD-MEKC) method was proposed for the determination of resibufogenin and cinobufagin in the Chinese herbal extracts from toad venom and its medicinal preparation (Liushen tablet). The two components have the close structural similarity and similar hydrophobicity, which result in poor resolution in normal MEKC. The addition of neutral beta-CD to the MEKC system was found to improve the separation of the studied compounds. The effects of several CD-MEKC parameters on the resolutions were evaluated systematically. Based on the investigation, a background electrolyte solution consisting of 10 mM borate buffer adjusted to pH 8.5, 40 mM sodium dodecyl sulfate (SDS), 12 mM beta-CD and 10% (v/v) of methanol was found to be optimal conditions for the fast separation. The contents of resibufogenin and cinobufagin were successfully determined within 5 min, with satisfactory repeatability and recovery.

Topics: Amphibian Venoms; Animals; beta-Cyclodextrins; Bufanolides; Chemistry, Pharmaceutical; Chromatography; Electrochemistry; Hydrogen-Ion Concentration; Medicine, Chinese Traditional; Methanol; Models, Chemical; Ranidae; Tablets; Technology, Pharmaceutical

A microemulsion electrokinetic chromatographic (MEEKC) method has been developed and validated for determination of resibufogenin and cinobufagin in toad venom and in traditional Chinese medicine prepared from the venom. The MEEKC method involved use of sodium dodecyl sulfate (SDS) as surfactant, heptane as organic solvent, and butan-1-ol as co-solvent. To improve the separation, the effect of temperature and running buffer pH were evaluated. The optimized conditions (heptane 0.81% (w/w), SDS 3.31% (w/w), butan-1-ol 6.61% (w/w), and 10 mmol L(-1) sodium tetraborate buffer, pH 9.2, and 298 nm as the detection wavelength) enabled useful and repeatable separation of the analytes.

Topics: Amphibian Venoms; Animals; Anura; Bufanolides; Capillary Electrochromatography; Emulsions; Hydrogen-Ion Concentration; Medicine, Chinese Traditional; Molecular Conformation; Pharmaceutical Preparations; Reproducibility of Results; Sensitivity and Specificity; Sodium Dodecyl Sulfate; Stereoisomerism; Temperature; Time Factors

Hydroxylation is an important route to synthesize more hydrophilic compounds of pharmaceutical significance. Microbial hydroxylation offers advantages over chemical means for its high specificity. In this study, a fungal strain Alternaria alternata AS 3.4578 was found to be able to catalyze the specific 12beta-hydroxylation of a variety of cytotoxic bufadienolides. Cinobufagin and resibufogenin could be completely metabolized by A. alternata to generate their 12beta-hydroxylated products in high yields (>90%) within 8 h of incubation. A. alternata could also convert 3-epi-desacetylcinobufagin into 3-epi-12beta-hydroxyl desacetylcinobufagin as the major product (70% yield). C-3 dehydrogenated products were detected in these reactions in fair yields, while their accumulation was relatively slow. The 12beta-hydroxylation of bufadienolides could be significantly inhibited by the substitution of 1beta-, 5-, or 16alpha-hydroxyl groups, and the 14beta,15beta-epoxy ring appeared to be a necessary structural requirement for the specificity. For the biotransformation of bufalin, a 14beta-OH bufadienolide, this reaction was not specific, and accompanied by 7beta-hydroxylation as a parallel and competing metabolic route. The biotransformation products were identified by comparison with authentic samples or tentatively characterized by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionization-mass spectrometry analyses.

Topics: Alternaria; Biotransformation; Bufanolides; Cholenes; Chromatography, High Pressure Liquid; Hydroxylation; Kinetics; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of resibufogenin and cinobufagin in traditional Chinese medicine (heart-protecting musk pill, shexiang baoxin wan in Chinese) was developed. A reversed-phase system with a Hypersil (ODS2) C(18) column and tetrahydrofuran: methanol: water (8:31:61) mobile phase was employed for the separation of resibufogenin and cinobufagin. The detection was set at 299 nm and ethinyl estradiol was chosen as the internal standard. The limit of detection was 1.5 ng for resibufogenin and 2.0 ng for cinobufagin at a signal-to-noise ratio of 4:1. It is a rapid, simple and accurate method for quantitative analysis of resibufogenin and cinobufagin in heart-protecting musk pill.

Topics: Bufanolides; Calibration; Cardiotonic Agents; Chromatography, High Pressure Liquid; Medicine, Chinese Traditional; Sensitivity and Specificity; Spectrophotometry, Ultraviolet

To establish a HPLC method to determine the contents of active constituents in Venenum Bufonis.. In determining the contents of resibufogenin and cinobufagin in the traditional Chinese medicine Venenum Bufonis, the mobile phase was acetonitrile-0.5% KH2PO4 solution(50:50)(pH adjusted with value phosphoric acid to 3.25 +/- 0.02).. The constituents thus determined have good linearity and separation. The average recovery of resibufogenin was 100.35%, RSD 1.86%; the average recovery of cinobufagin was 100.38%, RSD 2.09%.. The method was convenient, rapid, accurate and practicable.

Topics: Amphibian Venoms; Animals; Bufanolides; Bufo bufo; Bufonidae; Chromatography, High Pressure Liquid; Materia Medica

To study the chemical constituents of Bufo Siccus.. Based on silica column chromatography six compounds were obtained from the alcoholic extract of Bufo Siccus and identified by physico-chemical and spectroscopic analyses.. The compounds were identified as cholesterol, beta-sitosterol, resibufogenin, cinobufagin, bufalin and gamabufotalin.. Studies on the chemical constituents of Bufo Siccus were reported for the first time.

Topics: Animals; Bufanolides; Bufo bufo; Materia Medica; Molecular Structure

The method of enzyme immunoassay (EIA) was developed for measurement of anti-resibufogenin IgG reactive substance (RRS) in plasma and urine of healthy volunteers receiving a Senso containing drug, Kyushin (KY). Unchanged bufalin (BF), cinobufagin (CB), and resibufogenin (RB) and their metabolites in plasma and urine were also measured by EIA after the separation of these compounds by high performance liquid chromatography. In a single dosing study, the concentration of BF, CB and RB in plasma reached maximum at 1-2 hr after drug administration, and the half-lives (T1/2 of these were 15.8, 8.94 and 11.5 hr, respectively. In a multiple dosing study, the concentration of BF, CB and RB measured fitted on the estimated curve calculated from data of the single dosing study. These results suggest that BF, CB and RB, important components of Senso, do not have a cumulative nature.

Topics: Bufanolides; Cardiotonic Agents; Humans; Male; Medicine, Chinese Traditional

The bufadienolide compounds (bufalin, cinobufagin and resibufogenin), major constituents of Chansu in Liu-Shen-Wan (LSW), were determined by reverse phase high performance liquid chromatography. The procedure involves a preliminary extraction of the bufadienolides from LSW with chloroform using ultrasonication and subsequent evaporation to dryness of the chloroform extract. The residue of the chloroform extract was dissolved in methanol and separated on a Merck LiChrosorb RP-18 column. Methanol: water (74:26) was used as mobile phase. The compounds were satisfactorily separated with good chromatographic peaks. Good coefficients of correlation (r > 0.999) were obtained from the calibration of peak areas with concentrations for the 3 bufadienolides. Results of analysis showed that there were differences between the contents of bufadienolides in 11 LSW samples of different origin available to the public in Hong Kong where at present there is no legal control over the sale of traditional Chinese medicines. The variability of quantities of bufadienolides in Chansu may be a hazard to the public.

Topics: Bufanolides; Cardiotonic Agents; Chromatography, High Pressure Liquid; Medicine, Chinese Traditional