Volume 17 Supplement 1
Quality and safety issues related to traditional animal medicine: role of taurine
© Takahashi et al; licensee BioMed Central Ltd. 2010
Published: 24 August 2010
Calculus Bovis (:C.Bovis) is one of the most precious and commonly-used medicinal materials in Japan and China. As the natural occurrence is very rare, a source of supply for C. Bovis is far behind the actual need and great efforts have been taken for some substitutes of natural C. Bovis. Unfortunately, very little information is available on the quality and/or clinical efficacy of medication based on C. Bovis. To ensure sustainable use of traditional therapeutic agents derived from C. Bovis, we felt that several issues needed to be addressed: 1) the source of the C. Bovis materials and quality control; 2) the role of taurine in the efficacy of C. Bovis.
Nine samples of natural C. Bovis and its substitutes were collected. ICP-MS was used for elemental analysis and the characterization was performed by principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) as multivariate approaches. The efficacy of C. Bovis was evaluated for morphology, viability and beating pattern on cultured cardiac myocytes and/or fibroblasts.
PCA and multi-elemental focus was effective in discriminating C. Bovis samples derived from different habitats. A satisfactory classification using SIMCA was obtained among Australia C. Bovis, other habitats and the substitutes. Australian samples had better batch uniformity than other habitats and were composed of fewer elements. We have used Australian C. Bovis for assessment on its bioactive compounds. Rat cardiac cells incubated with C. Bovis extract (0.01-0.1mg/ml) maintained normal morphology, viability and beating pattern. Cardiac myocytes and fibroblasts treated for 48 h with CA (0.5mM) or DCA (0.1mM) caused cell injury, as reflected by changes in appearance and a reduction of viability detected by the MTS assay. In cardiomyocytes, 0.5 h exposure of CA (0.5mM) markedly decreased the velocity ratio of beating, whereas the simultaneous addition of 1 mM taurine largely prevented the decrease.
The multi-elemental focus provided some references for the quality control and the efficacy of C. Bovis. Taurine partly attenuated the harmful actions of bile acids. It is plausible that the relationship between taurine and the bile acids contributes to therapeutic effect of C. Bovis.
Calculus Bovis (Goo in Japanese, Niuhuang in Chinese, the gallstone of Bos Taurus domesticus Gmmelin) is one of the most precious and commonly-used medicinal materials in Japan and China. Its use was first recorded in “Shennong Bencao Jing” (Divine Farmer’s Materia Medica Classic) more than two thousand years ago, and now it has been used in 650 out of the 4500 traditional Chinese medicines [1, 2]. In the 230 cardioactive types of Japanese OTC drugs, 228 drugs contain C. Bovis, which has the effects of sedation, anti-hyperspasmia, relieving fever, diminishing inflammation and normalizing function of the gallbladder [3, 4]. A source of supply for C. Bovis is far behind the actual need since the natural occurrence of C. Bovis is very rare. In China, great efforts have been taken for some substitutes of natural C. Bovis. The safety and efficacy of C. Bovis is closely correlated with quality of the source materials. Unfortunately, very little information is available on the quality and/or clinical efficacy of medication based on traditional animal sources.
We have previously reported on the anti-arrhythmic actions of C. Bovis as a traditional knowledge-product and suggested these effects are partly mediated by taurine . To ensure sustainable use of traditional therapeutic agents derived from C. Bovis, we felt that several issues needed to be addressed: 1) the source of the C. Bovis preparation and quality control; 2) the role of taurine in the cardio-active efficacy of C. Bovis.
Materials and methods
Samples and reagents
Summary of investigated samples
Natural, PW 1)
Takahashi K . 3)
Osaka univ. 4)
In vitro prepared, PW
Takahashi K .
In vitro prepared, PW
Artificial cultured C.bovis
In vitro cultured, CL
Artificial cultured C.bovis
In vitro cultured, CL
Preparation of Calculus. Bovis-extract and taurine measurement
Water or DMSO was used as the solvent to extract the desired components. Natural C. Bovis contains a high concentration of bile acid, which adversely affects cells by disrupting their cell membrane. Each concentration of major bile acids in C. Bovis (100% DMSO) was equivalent to 0.5 mM CA, 0.1 mM DCA and 0.5 mM taurocholic acid (:TCA), respectively . To minimize the extraction of these bile acids, water or DMSO/water (=2:1) was used as the solvent to extract the desired components. Ten mg of well-pulverized crude drug was extracted with 5 ml of water at room temperature. The extract was centrifuged at 12000 x g for 20 min at 4°C. A final concentration of C. Bovis extract to the culture medium was 0.01-0.1mg/ml according to the human OTC doses.
The taurine or bile acids content of C. Bovis derived samples was measured by the procedure using ultra performance liquid chromatography (LaChromUltra, HITACHI, Inc., Tokyo, Japan) equipped with a L-2485U fluorescence detector or L-2400U UV detector , respectively.
ICP-MS measurement and multivariate analysis
Major and trace elements in materials were measured by using inductively coupled plasma mass spectrometry (:ICP-MS) . The C. bovis powder (10 mg) was added to 1ml of HNO3 (Nacalai Tesque, Kyoto, Japan), vortexed and left standing overnight at room temperature. Then, 100μl of samples were diluted with 9.9 ml of water and filtered through 0.45 μm pore size hydrophilic PTFE membrane filter (Millipore, Billerica, MA). ICP-MS analysis was performed on the Agilent 7500 Series ICP-MS (Agilent Technologies, Inc., Santa Clara, CA). Among the attained data, the elements which were detected as more than 1μg/l in the solvent were selected. Each data in the chart was shown as relative concentration. Principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) were used as exploratory techniques and classification procedures, respectively . Variables such as Na, Mg, Al, K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ge, As, Mo, Hg and Pb have been used as discriminating factors.
Cell culture and assessment on bioactive compounds of Calculus Bovis
Primary cultures of cardiac cells from 1-day-old Wister rats were prepared according to the methods described previously . All experimental procedures were approved by the Animal Care Committee of Osaka University and conformed to international guidelines.
Cardiac cells were seeded in a 96-well plate and were determined by MTS assay using CellTiter 96® aqueous one solution reagent (Promega Corporation) . After 48 h incubation in serum-containing culture medium, medium was transferred to serum-free medium. After 24h incubation, cells were treated with C. Bovis, bile acids and taurine for 48h. Following removal of the C. Bovis, cells were washed with phosphate-buffered saline (PBS) twice. The cells were then incubated in serum free maintenance medium (100μl) with one solution reagent (20μl) for 3h. Cell viability was defined as the ratio (expressed as a percentage) of absorbance of treated cells to untreated cells at 490nm.
The beating status of cultured myocardial cells was monitored with an inverted phase-contrast microscope and was measured by using a photosenser equipped with a microcomputer (P-200, Adachi Co.,Osaka). The shape and location of each cell in the dish were recorded before initiating the experiments . The beating properties of the same cells were monitored following the chosen perturbation.
Statistical significance was determined by the Student's t-test. Each value was expressed as the mean ± S.E. Differences were considered statistically significant when the calculated P value was less than 0.05.
Quality characterization of Calculus Bovis using pattern recognition techniques and multiple element data
Charcteristics of Calculus Bovis in Chinese Pharmacopoeia 2005
Natural Calculus Bovis,
Bos taurus domesticus Gmelin
CA : ≥ 4.0%
Bilirubin ≥ 35.0%
Total ash ≤ 10.0%
Artificial Calculus Bovis
Ox bile-powder (CA: ≥ 42.0%)
Cholic acid:CA ( ≥ 80.0%)
Bilirubin ( ≥ 90%)
Cholesterol, Trace elements
CA : ≥ 13.0%
Bilirubin : ≥ 0.63%
Tiwai Peiyu Niuhuang
Fresh bile of Bos taurus
Cholic acid:CA ( ≥ 95.0%)
Ca Bilirubinacet complexes
( CA ≥ 43.0 %)
Deoxycholic acid:DCA ( ≥95.0 %)
CA : ≥ 6.0%
Bilirubin : ≥ 35.0%
On the other hand, reference values for C. Bovis in Japanese Pharmacopeia  is as follows: spherical or massive stone, 1-4 cm in diameter; externally yellow-brown to red-brown; light, fragile and easily broken. Fractured surface shows yellow-brown to red-brown annular rings, often containing white granular substances or thin layers in these annular rings. Odor, slight; taste, slightly bitter, followed by slight sweetness. Namely, the form, colour, smell and very importantly the taste are some of the sensory indicators, on the basis of traditional knowledge, to test for quality.
The role of taurine on the cardioactive efficacy of Calucus Bovis
In this study, we have used natural C. Bovis originated from Australia for ensuring its efficacy. The major constituents of this extract were bile acids (cholic acid: CA, deoxycholic acid: DCA, taurocholic acid:TCA) and taurine. The average bile acid concentration in Australian C. Bovis extract (0.1mg/ml ) was equivalent to 0.32 mM CA,0.32mM DCA and 0.24 mM TCA, respectively (not data shown).
Cardiac myocytes and fibroblasts treated for 48 h with CA or DCA caused cell injury, as reflected by changes in appearance and a reduction of viability detected by the MTS assay (Figure 4). The cell viability of both cells was reduced by approximately 70 % when the cells were treated with 0.1mM DCA, whereas TCA at a concentration of 0.5 mM had no effect over a 48-h period. Treatment with 0.5 mM CA caused a decrease in viability of fibroblasts by 66 % of control after 48 h. Cardiomyocyte was not significantly altered its viability by treatment with 0.5 mM CA for 48 h.
In China Pharmacopoeia, bile acids are the major active components and CA has been used as the chemical marker for quality control of C. Bovis and its substitutes . Taurine is also provided as an ingredient of artificial C. Bovis. In the present study, taurine content in several samples of natural C. Bovis and its substitutes varied widely between 0.035~104.7 mg/g. The mean taurine concentration for Australian natural C. Bovis in the study was 0.32 ±0.19 mg/g C.Bovis (2.56 μmol/g). The values for the highest and lowest taurine concentrations were 3.92 mg/g of Chinese and 0.035 mg/g of Mexican one, respectively.
Natural C. bovis is obtained as a valuable by-product from animals used for meat production. As the natural occurrence is very rare, great efforts have been taken for some substitutes of natural C. Bovis. In 2004, it was reported that 98 % of C. Bovis used in China was artificial . However, due to the different developmental conditions, chemical constituents of substitutes might be different from those natural C. Bovis, which thus may lead to the variation of therapeutic effects. In Japan, all materials of C. Bovis are imported from several countries such as Australia, Latin America, Africa, India and China . Empirically, Japanese importers evaluated Australian C. Bovis as a high quality product without scientific reasons. The disordered use and abuse result in the loss of original pharmaceutical actions and therapeutic values of this natural product. Therefore, to ensure the quality of C. Bovis and its substitutes, an efficient quality control approach is urgently needed. Correctly characterizing the traditional animal materials is the inevitable starting point for studying C. Bovis.
First, we have characterized Natural C. Bovis originating from Australia using pattern recognition techniques and major and trace elements data (Figure 1, 2). PCA and multi-elemental focus was effective in discriminating C. Bovis samples derived from different habitats. There is general agreement among the experts/importers on crude drugs that Australian C. Bovis is quite a good quality on the basis of traditional knowledge. Interestingly, natural grouping of Australian samples was observed (Figure 2). Namely, the Australian ones had better batch uniformity than other habitats and were composed of fewer elements. The result indicated the variation among C. Bovis samples under different developmental conditions such as natural or artificial.
Secondly, we have used Australian C. Bovis for assessment on its bioactive compounds. Chemical and pharmacological investigations on C. Bovis resulted in discovering several kinds of bioactive components, i.e. bile acids, bilirubin and some inorgamic salts [1, 12, 13]. CA has been used as the chemical marker for quality control of C. Bovis in the provisions of Chinese Pharmacopeia (Table 2) . Taurine and CA are also one of the ingredients for artificial substitutes (Table 2) . Since the content of CA,DCA and taurine in C. Bovis extract derived from different sources can vary enormously, its effect on cardiomyocytes needs to be analyzed after normalization based the value of taurine or CA/DCA. This is particularly important because CA/DCA and taurine have opposite effect on cardiomyocytes.
In the present study, cardiac cells treated with CA or DCA caused cell injury, as reflected by the morphological change and a reduction of cell viability (Figure 3, 4). Rat cardiac cells incubated with C. Bovis extract (0.01-0.1mg/ml) maintained a normal morphology, viability and beating pattern (Figure 3, 4, 5). However, cardiac cells treated with CA or DCA caused cell injury, as reflected by the morphological change and a reduction of cell viability. TCA, a major constituent in C. Bovis, also caused loss of synchronous beating, bradycardia and cessation of contraction in cultured rat cardiomyocytes [14, 15]. We demonstrated a reduced rate of contraction and proportion of beating cells when rat cardiomyocytes were exposed to CA. 0.5 h exposure of CA markedly decreased the velocity ratio of beating, whereas the simultaneous addition of 1 mM taurine significantly prevented the decrease (Figure 6). Our previous data reported that C. Bovis extract was effective in protecting against the abnormal beating induced high Ca2+, and its efficacy was interfered by an inhibitor of taurine transport, bata-alanine [3, 16]. It has been recognized that taurine may have some beneficial effects due to, for instance, its antioxidant or anti-apoptotic capacity [17–20]. It is plausible that the relationship between taurine and the bile acids contribute to the therapeutic effect of C. Bovis.
Finally, this work provided some references for the quality control of C. Bovis materials. For practical purposes and for conservation reasons , it is desirable to find acceptable substitutes to C. Bovis used in traditional medicines. And it is necessary to make use of modern scientific tools to establish verifiable synthetic substitutes as sustainable replacements for medicinal resources. Future work would be focused on the different pharmacological actions of these natural C. Bovis and the substitutes of some spurious breeds.
The present work provided some references for the quality control and the efficacy of C. Bovis. PCA and multi-elemental focus was effective in discriminating C. Bovis samples derive from different habitats. Taurine partly attenuated the harmful actions of bile acids. It is plausible that the relationship between taurine and the bile acids contribute to the therapeutic effect of C. Bovis.
- C.Bovis :
- ICP-MS :
inductively coupled plasma mass spectrometry
- PCA :
principal component analysis
- SIMCA :
soft independent modeling of class analogy
- CA :
- DCA :
- TCA :
- GCA :
- TDCA :
This article has been published as part of Journal of Biomedical Science Volume 17 Supplement 1, 2010: Proceedings of the 17th International Meeting of Taurine. The full contents of the supplement are available online at http://www.jbiomedsci.com/supplements/17/S1.
We thank Dr. Feng Qiu and Dr. Xiao-Long Hou for the support of the materials, and Dr. Takeshi Bamba for help with UPLC and multivariate analysis.
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