Therapeutic effect of yinchenhao decoction on cholelithiasis via mucin in the gallbladder and intestine.

Cholelithiasis is a common and frequently occurring disease worldwide that belongs to the category of jaundice in traditional Chinese medicine. Yinchenhao decoction (YD) consists of Artemisia capillaris Thunb., Gardenia jasminoides J.Ellis, and Rheum palmatum L., and is traditionally used to treat jaundice, which has a significant therapeutic effect on cholelithiasis. Our study aimed to investigate the pathological mechanism of cholelithiasis and the therapeutic mechanism of YD via mucin in the gallbladder and intestine. YD was prepared and analyzed using HPLC. The supersaturation stability experiment was designed by the solvent-shift method. The cell transport experiment was conducted by coculture monolayers. The animal experiment was performed using a cholelithiasis model with a high-cholesterol diet. The related indicators were detected by automatic biochemical analyzer, PCR, western blot, or ELISA. Statistics were analyzed using χ2-tests and t-tests. As the results, in cholelithiasis, MUC5AC highly expressed in the gallbladder shortened cholesterol supersaturation and promoted cholesterol crystallization via the inflammatory cytokine signaling pathway; MUC2 highly expressed in the small intestine prolonged cholesterol supersaturation and promoted cholesterol absorption via the inflammatory cytokine signaling pathway. YD inhibited mucin expression in the gallbladder and intestine in a concentration-dependent manner for cholelithiasis treatment by inhibiting the inflammatory cytokine signaling pathway, which was attributed to the active components, including chlorogenic acid, geniposide, and rhein.

A fatal case of self-poisoning through the ingestion of oleander leaves.

BACKGROUND: Benefits and even dangers of plants are known since time began. The ancients used plants and herbs because of their effects on the human body. Poisoning is a logical consequence of their use: history is full of episodes of plants and herbs poisoning, whether intentional or accidental. AIM: Oleander poisoning is generally accidental; an intentional assumption of its leaves to commit suicide is uncommon because the population is not aware of the harmfulness of its cardiotoxic glycosides, therefore we report a fatal case of self-poisoning through the voluntary ingestion of oleander leaves. METHODS: A diagnosis of oleander self-poisoning was highly suspected on the basis of the circumstantial evidence and the autopsy findings. Toxicological investigations were performed on the samples collected during the autopsy and aimed at confirm the presence of oleandrin at a toxic level. RESULTS: The autopsy revealed a piece of oleander leaf on the posterior third of the tongue's body and several plant residues, similar to the one recovered on the tongue, into the gastric content; petechiae on the deep surface of the scalp, multi-organ congestion, and pulmonary edema were also observed. The histological study corroborated the pulmonary edema macroscopically observed but did not provide any other information. The detection of oleandrin in biological cadaveric samples revealed high, fatal, concentrations. CONCLUSIONS: Cases of voluntary ingestion of oleander with a suicidal intent prove to be uncommon: in the case reported the victim was aware about the possibility to commit suicide through the ingestion of oleander leaves.

Determination of neuroprotective oxysterols in Calculus bovis, human gallstones, and traditional Chinese medicine preparations by liquid chromatography with mass spectrometry.

So far, the components responsible for the neuroprotective effects of Calculus bovis are unclear. Cholesterol, one of the major components in Calculus bovis, is easily oxidized into oxysterols, which possess direct or indirect neuroprotective effects proved by our and others' previous studies. Therefore, a liquid chromatography with mass spectrometry method coupled with ultrasonic extraction and solid-phase extraction was developed for the determination of neuroprotective oxysterols in Calculus bovis, human gallstones, and traditional Chinese medicine preparations. Chromatographic separation was achieved on a C18 column with isocratic elution at a flow rate of 1 mL/min. The established method showed good linearity (R(2) > 0.998), sensitivity with low limits of detection (0.06-0.39 µg/g), acceptable precisions (relative standard deviations ≤ 7.4%), stability (relative standard deviations ≤ 5.9%), and satisfactory accuracy (92.4-102.9%) for all analytes identified by different retention times, which could be applied for the determination of oxysterols. Five kinds of oxysterols proved to function as neuroprotectants were detected at different concentrations. Among them, 7ß-hydroxycholesterol and cholestane-3ß,5α,6ß-triol were rather abundant in the samples. It could be concluded that the potential neuroprotective components in Calculus bovis may be these oxysterols.

[Technological parameter optimization method for washing of coarse bear gall powder extracts based on NIR spectroscopy].

During the washing process of coarse bear gall powder extracts, it is necessary to adjust the amount of ethyl acetate according to the properties of raw materials, which aims to improving the yield and purity of the final product. In the research, using NIR spectra to reflect the comprehensive properties of coarse bear gall powder extracts, the process is optimized in a flexible way. Forty batches experiments are designed according to the weight ratio of ethyl acetate and coarse extracts of bear gall powder. The NIR spectra of the coarse extracts of bear gall powder are collected and processed using principal component analysis (PCA) method. The first 8 principal components combined with the amount of the ethyl acetate are used as the input variables, and calibration models are established to predict the yield and purity of the final product 30 batches are used as calibration set, which is used to establish the models, and other 10 batches are used as validation set, which is used for the performance appraisal of the established models. The correlation coefficients of the calibration, inner cross-validation and external validation for the purity model are 0.902, 0.896 and 0.883, respectively, and the RMSEC, RMSECV and RMSEP are 1.22%, 1.48% and 1.59%, respectively. The correlation coefficients of the calibration, inner cross-validation and external validation for the yield model are 0.921, 0.859 and 0.916, respectively, and the RMSEC, RMSECV and RMSEP are 1.39%, 1.65% and 1.53% respectively. This work demonstrated that NIR spectra combined with technology parameter could be used to predict the yield and purity of the final product. Using the established models, the most appropriate amount of the ethyl acetate can be determined according to the properties of the coarse bear gall powder extracts, and the yield and purity of the final product can be improved.

[Chemical constituents of bear bile].

OBJECTIVE: To study the chemical constituents of bear bile. METHOD: The compounds were isolated by repeated column HP20 macroporous adsorption resin, Sephadex LH-20, ODS and silica gel as packing materials. The structures were identified on the basis of extensive spectroscopic data analysis and by comparison of their spectral data reported. RESULT: Nine compounds were identified as 4',7-dihydroxyisoflavone (1), 4',7-dihydroxy-6-methoxyisoflavone (2), 4',6,7-trihydroxyisoflavone (3), 4'-methoxy-7-hydroxyisoflavone (4), tauroursodeoxycholic acid (5), taurochenodeoxycholic acid (6), ursodeoxycholic acid (7), chenodeoxycholic acid (8), cholesterol (9). CONCLUSION: Compounds 1-4 were separated from bear bile for the first time.

[The analysis of the content of bile acids in ox-bile powder and goat gall powder of Mongolian medicine].

The present study was to detect and compare the content of bile acids in ox bile powder and goat gall powder of Mongolia medicine by UV. Cholic acid with sulphuric acid were heated and dehydrated, and they produced conjugated double bond. The conjugated bond showed the same absorption peak in the ultraviolet range. The method of ultraviolet spectrophotometry can be used to detect and compare the content of bile acids in ox bile powder and goat gall powder. The result showed that the linear range was 0.003 3-0.016 7 mg x mL(-1) (r = 0.999 7). The average recovery (n = 5) of standard addition method of ox bile powder and goat gall powder was 98.48% (RSD = 1.79%) and 96.46% (RSD = 2.50%) respectively. The result of determination of five different samples showed that the content of bile acids in ox bile powder and goat gall powder was 40.85%-43.03% and 30.88%-32.64% respectively. The RSD of the analysis of ox bile powder and goat ball powder was 2.40% and 2.92% respectively, the RSD of stationary test of ox bile powder and goat ball powder in eight hours was 0.55% and 0.59% respectively, and the RSD of reproducibility of the analysis of ox bile powder and goat ball powder was 2.11% and 2.68% respectively. The method was simple, accurate, fast and easy to generalize and apply in many fields. It can be used to control the quality of ox bile powder and goat gall powder.

A discovery of low hydraulic resistance channel along meridians.

A hydro-mechanic model was put forward to study the fundamental nature of acupuncture meridians. The basic state of low hydraulic resistance was tested on humans and mini pigs using three methods. The first, a modified Guyton's method, proved that there was lower hydraulic resistance on meridians compared with nonmeridians. The second scanning method involved a single pressure transducer that can find the lowest resistance point in tissue, and the third method used two transducers and provided a more stable measurement. Using the latter method, low hydraulic resistance points were found very close to low impedance points along meridians. The transmission of artificial interstitial fluid pressure waves was measured to examine their connection to the low resistance points, with the result that a good connection between the points was confirmed. This means the points form channels along the meridians that we refer to as low hydraulic resistance channels. The channel was imaged through isotopic tracing and a migration of isotope (99m)Te could be found along the channel. The layer of the channel was detected by injecting Alcian blue and the track was found beneath the skin. All of the above experiments suggest the existence of a new type of channel in living tissues that has not yet been described in modern science, but coincides quite well with the Qi channel theory of traditional Chinese medicine.

Micronutrients, antioxidants, and carcinoma of the gallbladder.

OBJECTIVES: Nutrient deficiency in developing countries can be considered a significant contributory factor modifying the multistage process of carcinogenesis. Studies from different parts of the world have shown the deficiency of various micronutrients to be significantly associated with cancer. This study was undertaken to test the above hypothesis in patients with carcinoma of the gallbladder. METHODS: Selenium (Se), zinc (Zn), copper (Cu), manganese (Mn), ascorbic acid (vitamin C), and alpha-tocopherol (vitamin E) were estimated in the serum, bile, and gallbladder tissue of 30 patients each of carcinoma of the gallbladder (group-I), cholelithiasis (group-II), and only in the serum of 30 age- and sex-matched healthy controls (group-III). The minerals were analyzed by atomic absorption spectrophotometer and vitamins by spectrophotometry. RESULTS: The mean serum levels of Se, Zn, Mn, vitamin E, and vitamin C were significantly lower (P < 0.001) in group-I when compared with groups II and III. The mean biliary levels of Se and Zn (0.29, 3.45 mg/L) were reduced significantly (P < 0.001) in group-I when compared with group II (0.51, 5.2 mg/L). Mean tissue levels of Se and Zn were also significantly lower (P < 0.001) in group I (2.75, 43.09 microg/g) compared to group II (3.90, 61.37 microg/g). However, no significant difference was observed in tissue concentration of Mn, vitamin C, and vitamin E. Cu levels and Cu/Zn ratio showed a highly significant (P < 0.001) increase in serum, bile, and gallbladder tissue in carcinoma of the gallbladder compared to the other two groups. CONCLUSIONS: The data supports an association between lower levels of Se, Zn, vitamin E, and risk of carcinoma of the gallbladder and suggest that Cu/Zn ratio could be a useful parameter in evaluating the patients of carcinoma of the gallbladder.

[Effect of cholesterol in bile on cholecystokinin receptor in the gallbladder].

OBJECTIVE: To assess the effect of cholesterol in bile on cholecystokinin receptor (CCK-R) in the gallbladder. METHODS: One hundred Guinea pigs were randomly divided into four groups, 25 animals for each. The control group was fed a standard diet, and the cholesterol group fed a diet containing 2% cholesterol. After taking the 2% cholesterol diet for two weeks, the natural group persisted on the standard diet, and the treated group was perfused by traditional Chinese medicine. Serum cholecystokinin (CCK) level in the portal vein and maximal binding capacity (B(max)) and Kd of CCK-R in the gallbladder were measured in the four groups by RIA and RBA, and the concentrations of cholesterol in bile were also observed. RESULTS: Compared with the control group, after high-cholesterol feeding for two weeks, the gallbladder emptying rate [(65.83 +/- 7.32)% approximately (47.22 +/- 5.24)%] and B(max) of CCK-R [(60 +/- 27) approximately (32 +/- 13) fmol/mg protein] and in decreased fasting gallbladder volume (FV) [(0.89 +/- 0.26) approximately (1.34 +/- 0.61) cm(3)] and concentration of cholesterol [(0.44 +/- 0.11) approximately (0.60 +/- 0.13) mmol/L] in bile increased, but no change was in the serum CCK level and Kd of CCK-R in the cholesterol group. Compared with the natural group, after two-week in take of herb decoction of qingre lidan and liqi huoxue, FV [(1.27 +/- 0.60) approximately (0.90 +/- 0.27) cm(3)], RV [(0.85 +/- 0.45) approximately (0.32 +/- 0.12) cm(3)], FB [(0.92 +/- 0.35) approximately (0.73 +/- 0.21) cm(3)], RB [(0.76 +/- 0.34) approximately (0.29 +/- 0.08) cm(3)] in the treated group decreased significantly; but gallbladder emptying rate [(43.06 +/- 4.27)% approximately (67.01 +/- 6.82)%] increased significantly. The concentration of cholesterol in bile was lower in the treated group than in the natural group [(0.59 +/- 0.14) approximately (0.43 +/- 0.10) mmol/L], but no change was found in the serum CCK level. Bmax of CCK-R in the treated group increased significantly [(39 +/- 19) approximately (59 +/- 11) fmol/mg protein], Kd of CCK-R showed no significant changes between the treated group and natural group. CONCLUSION: High cholesterol in gallbladder bile causes defective muscle contraction by down-regulating CCK-R in the gallbladder, so the reduction of cholesterol concentration of bile may contribute to gallbladder contraction.

[Identification of Chinese crude drug snake gallbladder by DNA molecular marker].

AIM: It is difficult to identify the Chinese crude drug snake gallbladder accurately by morphological and microscopical characteristics or chemical components only. In order to solve the problem, the technique based on DNA molecular marker was introduced into the authentication of snake gallbladder. METHODS: DNA templates were extracted from the membrane or the bile of snake gallbladder, and also from the muscle of the original animal Elaphe schrenckii. About 400 bp DNA fragments of 12S rRNA gene were amplified from the templates and sequenced subsequently. RESULTS: Enough amounts of DNA templates could be extracted from a bit of membrane or bile of snake gallbladder. The sequence of amplicons from the membrane, bile and muscle of the same individual were identical completely. CONCLUSION: The technique of DNA molecular marker could be used for the authentication of snake gallbladder and bile. The results indicate that the technique could be used for the identification of crude drugs from other animal secretion. DNA sequence analysis also demonstrated that the origins of commercial snake gallbladder were complicated and more efficient quality control was necessary for supervising the crude drug in the market.

Apical Na+-Cl- symport in rabbit gallbladder epithelium: a thiazide-sensitive cotransporter (TSC).

Cl- apically enters the epithelium of rabbit gallbladder by a Na+-Cl- symport, sensitive to hydrochlorothiazide (HCTZ). Since HCTZ also activates an apical SITS-sensitive Cl- conductance (G(Cl)), the symport inhibition might be merely due to a short circuit of the symport by G(Cl) rather than to a direct action of HCTZ on the symporter. To examine whether the symport is directly inhibited by HCTZ and whether the symporter belongs to the family of thiazide-sensitive cotransporters (TSC), radiochemical measurements of the apical Cl- uptake, electrophysiological determinations of intracellular Cl- and Na+ activities (a(i,Cl) and a(i,Na)) with selective theta microelectrodes and molecular biology methods were used. The 13Cl- uptake proved to be a measurement of the apical unidirectional Cl- influx (Jmc) and of the symport only (without backflux components), with measuring times of 45 sec under all experiment conditions; its inhibition by HCTZ was unaffected by G(Cl) activation or abolition. After HCTZ treatment the decrease in a(i,Cl) (measured as the initial rate or in 3 min) was larger than the decrease in a(i,Na). The difference was reduced to one third in a group of epithelia in which the elicited G(Cl) was reduced to one third; moreover it was abolished in any case when G(Cl) was abolished with 10(-4) M SITS. The SITS-insensitive rate of a(i,Cl) decrease was equal to that of the a(i,Na) decrease in any case. Thus the a(i,Cl) decrease displays a component dependent on G(Cl) activation and a second component dependent on symport inhibition. Using the RT-PCR technique a cDNA fragment was obtained that was 99% identical to the corresponding region of the rabbit renal TSC isoform. The results indicate that in rabbit gallbladder epithelium HCTZ displays a dual action, namely G(Cl) activation and Na+-Cl- symport inhibition. This Na+-Cl- symporter is the first TSC found to be functionally expressed in a nonrenal or nonrenal-like epithelium.

[Studies on the stability of sodium taurocholate in different media and in the course of sterilization].

OBJECTIVE: To investigate the stability of the main active constituent of snake gall sodium taurocholate, TCANa in 25% alcoholic solution and artificial gastric juice, and in the course of hot pressurized sterilization. METHOD: The stability of the solution was predicted by an accelerated isothermal test, and the content of TCANa was determined by TLC-colorimetry. RESULT: The half-time(t1/2) of TCANa in 25% alcoholic solution is 60.57 d at 25 degrees C, and in artificial gastric juice 11.37 d at 37 degrees C, the loss rate under the condition(115 degrees C, 30 min) of sterilization about 3%. CONCLUSION: TCANa in 25% alcoholic solution is unstable. Therefore it is suggested that in order to raise the stability of TCANa the alcoholic concentration be increased and the solution be stored at temperatures below 0 degree C.

Identification and differentiation of bear bile used in medicinal products in Taiwan.

One hundred eighty-three suspect bear bile used in medicinal products, collected in Taiwan as gall bladders or dried powder forms, were analyzed using FTIR, HPTLC, and HPLC techniques to identify whether they are indeed bear bile. Those confirmed were further examined to determine whether the observed analytical parameters can be reliably used for source inference, i.e., differentiating products among North American black bear, farmed Asiatic black bear, polar bear, etc. Our data suggested that North American and polar bears contain a higher concentration of TC (relative to TUDC and TCDC), whereas the relative concentration of TC in Asiatic bears (wild or farmed) is much lower. Thus, the relative concentration of TC can potentially be used for differentiating Asiatic bear bile from North American and polar bear products, but it cannot be used for the differentiation of wild and farmed bear bile as suggested in an earlier report by Espinoza et al. The origin of the 183 samples analyzed were found to be as follows: 118 (64%), bile salts, or gall bladders were of domestic pig; 56 (31%), bile products of Asiatic bear; 4 (2.2%), Asiatic bear mixed with pig bile salts; 3 (1.6%) goat gall bladders; 1 (0.55%) water buffalo bile salts; and 1 (0.55%), pig bile salts mixed with water buffalo bile salts.

Post-natal changes in biliary lipids in suckling goat kids.

The post-natal composition of biliary lipids in the bile of suckling goat kids has been studied during the first month of life. Samples of hepatic and vesicular bile were obtained from animals fed with milk. Bile flow increased with age and the concentration of bile acids also rose up to 14 days of age. Cholesterol reached maximum values immediately after birth and then decreased. Bile acids conjugated with taurine predominated and this pattern of preferential tauroconjugation persisted at all ages, as has also been found in carnivores and sheep. Comparison of vesicular and hepatic bile composition revealed a poor concentration capacity of the gallbladder in pre-ruminant kids.

Study on the bile salt, sodium scymnol sulfate, from Rhizoprionodon acutus. IV. Structures of chimaerol and sodium chimaerol sulfate in the bile of Lamna ditropis and Rhizoprionodon acutus.

Sodium chimaerol sulfate (1) was isolated from the bile of Lamna ditropis and Rhizoprionodon acutus by chromatography on silica gel and Sephadex LH-20, together with sodium scymnol sulfates, (24R,25S)- and (24R,25R)-(+)-3 alpha,7 alpha,12 alpha,24,26-pentahydroxy-5 beta-cholestan-27-yl sodium sulfates (3 and 4) and 3, respectively. On acid hydrolysis, compound 1 afforded chimaerol (2), which was identified as (24R,25R)-(+)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol by direct comparison with an authentic sample, prepared by reduction of (24R,25S)-(+)-24,26-epoxy-5 beta-cholestane-3 alpha,7 alpha,12 alpha,27-tetrol (5) with LiAlH4. The structure of 1 was concluded to be (24R,25R)-(+)-3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-yl sodium sulfate, based on the chemical transformation and spectral data.

[Studies on chemical constituents of the gall of Python molurus bivittatus Schlegel].

Two constituents were isolated from the gall of Python molurus bivittus Schlegel, one is sodium taurodeoxycholate (I). The other is a new compound--sodium tauropythocholate (II). Its structure was elucidated as 3 alpha, 12 alpha, 16 alpha-trihydroxy-5-cholan-24-oic acid N-[2-sulfoethyl] amide by IR, 1HNMR, 13CNMR, MS 13C-1H COSY, and chemical reaction.

Selenium contents in human gallbladder bile.

The selenium contents in human gallbladder bile were analyzed. Thirty-seven subjects were studied; 22 patients with cholelithiasis in Niigata Prefecture and 15 patients (13 with cholelithiasis and 2 with gallbladder polypus) in Kochi Prefecture. Five ml of bile was withdrawn with a syringe from the gallbladder during the operation and stored at -20 degrees C until analysis. For the analysis by gas chromatograph with an electron-capture detector, 0.2 ml of sample was used. The mean selenium contents in bile were 269 +/- 39.0 (mean +/- S.D.) ng/ml for the subjects in Niigata and 285 +/- 84.4 ng/ml in Kochi; without significant difference. Of 37 samples analyzed, the mean content was 276 +/- 61.0 ng/ml.