Research progress in quality control of Bufonis Venenum in preparations / 中国中药杂志

Bufonis Venenum, an animal medicinal material, is widely used for treating cardiovascular diseases and pain induced by rheumatics or malignant tumors. In view of the high activity and high toxicity, it is of great significance to pay attention to the quality control of Bufonis Venenum to ensure the safety and effectiveness of its preparations. China's drug standards involve 102 preparations(474 batch numbers) containing Bufonis Venenum approved for sale, including 14 preparations in the Chinese Pharmacopoeia(2020 edition) and 68 preparations in the standards issued by the Ministry of Health Drug Standard of the People's Republic of China. Bufonis Venenum is mostly used in pill and powder preparations in the form of raw powder, with the main functions of clearing heat, removing toxin, relieving swelling and pain, replenishing qi, activating blood, opening orifice, and awakening brain. Except the high level of quality control for Bufonis Venenum in the preparations in the Chinese Pharmacopoeia(2020 edition), the quality control standards of Bufonis Venenum in other preparations are low or even absent. Therefore, it is urgent to conduct research on the improvement of quality standards for the preparations containing Bufonis Venenum. This study retrieved the reports focusing on the quality evaluation and quality control of the preparations containing Bufonis Venenum from CNKI, PubMed, and Web of Science. Qualitative and quantitative analysis methods for 64 preparations containing Bufonis Venenum have been reported, mainly including thin-layer chromatography, HPLC fingerprint, and multi-component content determination. The index components mainly involved bufadienolides, such as gamabufalin, arenobufagin, bufotalin, bufalin, cinobufagin, and resibufogenin. According to the literature information, this paper suggests that attention should be paid to the correlations between the analysis methods and detection indexes of medicinal materials, decoction pieces and preparations, the monitoring of indole alkaloids, and the content uniformity inspection for further improving the quality standards for the preparations containing Bufonis Venenum.

Systematic comparison of two kinds of Bufonis Venenum derived from different Bufo gargarizans subspecies based on metabolomics and antitumor activity / 中国中药杂志

This paper compared the differences between two kinds of Bufonis Venenum produced by Bufo gargarizans gargarizans and B. gararizans andrewsi, and verified the rationality of the market value orientation of Bufonis Venenum based on the zebrafish mo-del. Twenty batches of Bufonis Venenum from Jiangsu province, Hebei province, Liaoning province, Jilin province, and Liangshan, Sichuan province, including B. gargarizans gargarizans and B. gararizans andrewsi, were collected. The UHPLC-LTQ-Orbitrap-MS combined with principal component analysis was used to compare the differences between two kinds of Bufonis Venenum. According to the limiting conditions of VIP>1, FC<0.5 or FC>2.0, and peak total area ratio>1%, 9 differential markers were determined, which were cinobufagin, cinobufotalin, arenobufagin, resibufogenin, scillaredin A, resibufagin, 3-(N-suberoylargininyl)-arenobufagin, 3-(N-suberoylargininyl)-marinobufagin, and 3-(N-suberoylargininyl)-resibufogenin. The content of 20 batches of Bufonis Venenum was determined according to the Chinese Pharmacopoeia(2020 edition) by high-performance liquid chromatography, and the 2 batches of Bufonis Venenum, CS7(8.99% of total content) and CS9(5.03% of total content), with the largest difference in the total content of the three quality control indexes of the Chinese Pharmacopoeia(bufalin, cinobufagin, and resibufogenin) were selected to evaluate their anti-liver tumor activity based on the zebrafish model. The tumor inhibition rates of the 2 batches were 38.06% and 45.29%, respectively, proving that only using the quality control indexes of the Chinese Pharmacopoeia as the value orientation of Bufonis Venenum market circulation was unreasonable. This research provides data support for the effective utilization of Bufonis Venenum resources and the establishment of a rational quality evaluation system of Bufonis Venenum.

Comparative morphology and histology of the brain in Chinese toad ( Bufo gargarizans ) and chinese fire-billed newt ( Cynops orientalis ) / Morfología e histología comparada del cerebro en el sapo chino ( Bufo gargarizans ) y el tritón vientre de fuego chino ( Cynops orientalis )

The morphological and histological structure of the brains of Bufo gargarizans and Cynops orientalis were observed by anatomy and light microscopy. The results show that the brains of Bufo gargarizans and Cynops orientalis are divided into 5 parts which include the telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata. The telencephalon consists of the olfactory bulb and the cerebral hemisphere. The olfactory bulb is developed that has two pairs of olfactory nerve. Bufo gargarizan has a symmetrical oval hemisphere optic lobes; Cynops orientalis only has a spherical optic lobe. The cerebellum is situated behind the optic lobe and closely connected with the myelencephalon. In this paper, the morphological and histological differences between the two species are discussed. The proportion of cerebral hemisphere is gradually increasing, which correlated with a progressive increase in the number of neuronal cell classes, and reflected in behavior complexity.

La estructura morfológica e histológica de los cerebros de Bufo gargarizans y Cynops orientalis se observó mediante anatomía y microscopía óptica. Los resultados muestran que los cerebros de Bufo gargarizans y Cynops orientalis se dividen en 5 partes, que incluyen el telencéfalo, diencéfalo, mesencéfalo, cerebelo y mielencéfalo. El telencéfalo consiste en bulbo olfatorio y hemisferio cerebral. El bulbo olfatorio tiene dos pares de nervios olfatorios. Los lóbulos ópticos de Bufo gargarizans son ovalados y simétricos en ambos hemisferios cerebrales; Cynops orientalis tiene solo un lóbulo óptico esférico. El cerebelo está situado detrás del lóbulo óptico y está estrechamente conectado con el mielencéfalo. En este trabajo, se discuten las diferencias morfológicas e histológicas entre las dos especies. El tamaño del hemisferio cerebral aumenta gradualmente, lo que se correlaciona con un aumento progresivo de células neuronales en los núcleos, reflejándose en la complejidad del comportamiento.

Identification and bactericidal activity of a novel Cathelicidin family member from skin of Bufu bufo gargarizans / 中国中药杂志

The skin transcriptome of Bufu bufo gargarizans was determined by conventional methods. A novel full length cDNA coding for a Cathelicidin precursor was identified by transcriptomic data assembling, annotation and blast search of corresponding data banks. According to the known processing methods of Cathelicidin family members, present reported novel Cathelicidin precursor of B. bufo gargarizans might be cleaved at 2 possible sites of the same precursor and generate both BG-CATH25 and BG-CATH29 as mature molecules. The deduced BG-CATH25 and BG-CATH29 were synthesized with purity>95% to evaluate the properties and bactericidal activities. The secondary structural characteristics of both BG-CATH25 and BG-CATH29 in different solutions were determined by Circular Dichroism (CD) Analysis. CD results indicated that random coil conformation were the main structural elements for both BG-CATH25 and BG-CATH29 in different buffer systems. Antimicrobial activities against tested bacterial strains were carried out by plating method. Both BG-CATH25 and BG-CATH29 showed strong antibacterial activities against Aeromonas hydrophila, with MIC values of 1.25, 10 mg•L⁻¹, respectively. However, both of them showed weak bactericidal activities against human pathogenic bacteria, like Escherichia coli (ATCC25922）,Staphylococcus aureus (ATCC25923）and Pseudomonas aeruginosa (ATCC 27853).

Effects of different stocking density and diet on development of Bufo gargarizans tadpoles / 中国中药杂志

The effects of four different stocking densities and five different diets on the growth of Bufo gargarizans tadpoles were studied to determine the optimum stocking density and diet. For stocking density experiment, the tadpoles were fed respectively at different density of 200, 500, 1 000 and 2 000 tadpoles per square meter. For diet experiment, the tadpoles were divided into five groups fed respectively with five different diets. The body weight, snout-vent length and tail length were measured every seven days, and mortality was recorded. The results showed that: the survival rates of tadpole before metamorphosis and after metamorphosis were from 68.7% to 96.3% and from 5.7% to 36.0%, respectively; the optimum stocking density is 1 000 tadpoles per square meter for the stocking density had no effect on the survival rate of tadpole before metamorphosis, and the tadpoles had the relative large body weight and survival numbers in 1000 tadpoles per square meter; the diet Ⅱ(37.9% crude protein and 5.7% crude fat), Ⅳ (25.1% crude protein and 4.0% crude fat), and Ⅴ (egg yolk) were all the optimum diets for the diet had no effect on the survival rate of tadpole before metamorphosis and the tadpoles fed with three kinds of diet above had relatively large body weight, and one of these three diets based on their availability and cost should be adopted during breeding period.

Cloning, prokaryotic expression and function of the Bufo bufo gargarizans tryptophan hydroxylase I gene / 药学学报

Tryptophan hydroxylase I (TPH1) catalases the 5-hydroxylation reaction of L-Trp, which is the rate-limiting step in the synthesis of serotonin. Serotonin, a major component of Bufonis venenum, is involved in numerous physiological functions as an important neurotransmitter. In this study, BbgTPH1 cDNA was cloned from the parotid gland of Bufo bufo gargarizans. Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pMAL-BbgTPH1, and the induced conditions to express the recombinant BbgTPH1 in E. coli TB1 cells were optimized. The full length of BbgTPH1 is 1984 bp (GenBank accession No. JQ768313) with a 1443 bp open reading frame (ORF) encoding a 480 amino acid residues. The deduced protein molecular weight is 55.2 kDa and its theoretical isoelectric point is 5.58. The sequence includes conserved domain and special signal sequence of the aromatic amino acid hydroxylase (AAAH) superfamily. Homologous alignment showed that BbgTPH1 shared a high homology with other species. Phylogenetic tree showed the closest relative to BbgTPH1 was Xenopus tropical-TPH1. The best induction conditions of recombinant BbgTPH1 were 0.5 mmol·L-1 IPTG at 20℃ for 8 h. The function of BbgTPH1 was identified by in vitro enzymatic reaction and the recombinant BbgTPH1 was able to produce 5-hydroxytryptophan by catalyzing tryptophan. This study represents the first time of cloning and identification of the function of TPH1 in Bufo genus. The results of this study will be an important foundation for future studies of biosynthesis of bufotenines in the parotid gland of B. bufo gargarizans.

Identification of the skin of Bufo bufo gargarizans and its adulterants based on DNA barcoding / 中国药学杂志

OBJECTIVE: To provide new technique for quick and accurate identification of the skin of Bufo bufo gargarizans u-sing DNA barcoding method based on the COI sequence. METHODS: Fifty-four samples of the skin of Bufo bufo gargarizans and its four kinds of adulterants were collected from 16 different localities. The DNAs were extracted, the COI squences were amplified and sequenced bi-directionally. The obtained sequences were assembled using the CodonCode Aligner V 4.2. Then the genetic distances were accumulated using Kimura-2-Parameter(K2P) model, and the NJ tree was constructed by MEGA5.0. RESULTS: The intra-specific genetic distances were much smaller than inter-specific ones between the skin of Bufo bufo gargarizans and its adulterants. Furthermore, the NJ tree showed that the skin of Bufo bufo gargarizans can be distinguished from its adulterants clearly. CONCLUSION: The COI sequence can be used as the DNA barcode to identify the skin of Bufo bufo gargarizans and its adulterants with accuracy and efficiency.

Study on comparative histology of retinas in Ctenopharyngodon idella, Cynops orientalis, Bufo bufo gargarizans, Gekko japonicus, and Columba livia / Estudio histológico comparado de la retina en Ctenopharyngodon idella, Cynops orientalis, Bufo bufo gargarizans, Gekko japonicus, y Columba livia

The objective of this study was to explore the relationship between the retinal structure and its life adaptation to the environment of Ctenopharyngodon idella, Cynops orientalis, Bufo bufo gargarizans, Gekko japonicus and Columba livia . Measuring retinal thickness of each layer, the nuclei layer, and the diameter of each nuclear layer of the five animals, the statistical data analysis shows that: the nuclei layers of five animals are all 4, and their structures can be divided to 10 layers when observing with optical microscope. The retinal thickness of Ctenopharyngodon idella was 190.49 mm, Cynops orientalis was 173.07 µm, and the Bufo bufo gargarizans was 195.06 µm, Gekko japonicus was 224.32 µm and Columba livia was 174.10 µm. The number of retinal inner nuclear layers of Bufo bufo gargarizans and Gekko japonicus and Columba livia are more than their outer nuclear layers, on the contrary, retinal inner nuclear layers of Ctenopharyngodon idella and Cynops orientalis are less than their outer nuclear layers. The rod and cone layer of retina of Cynops orientalis were more advanced, but their nerve fiber layer (NFL) degraded highly, revealing a strong photosensitivity but a low visual sensitivity; to Columba livia , their NFL of retina are highly developed, so as their vision. The different structures and functions of the retina of Ctenopharyngodon idella, Cynops orientalis, Bufo bufo gargarizans, Gekko japonicus and Columba livia correspond with their behavioral characteristics and the living environment's change from aquatic to amphibious to land.

El objetivo de este estudio fue explorar la relación entre las estructuras de la retina y su adaptación al medioambiente en Ctenopharyngodon idella, Cynops orientalis, Bufo bufo gargarizans,Gekko japonicus y Columba livia . La medición del espesor de cada capa de la retina, la capa nuclear y su diámetro en los cinco animales, mostró a través del análisis estadístico que las capas nucleares en todos ellos fueron 4, y sus estructuras se pueden dividir en 10 capas cuando se observan con el microscopio óptico. El espesor de la retina de Ctenopharyngodon idella fue 190,49 µm, de Cynops orientalis fue 173,07 µm, de Bufo bufo gargarizans fue 195,06 µm, de Gekko japonicus fue 224,32 µm y de Columba livia fue 174,10 µm. El número de capas nucleares internas de la retina de Bufo gargarizans, Gekko japonicus y Columba livia fue mayor que sus capas nucleares externas, mientras que las capas nucleares internas de Ctenopharyngodon idella y Cynops orientalis fueron menos que las capas nucleares externas. La capa de conos y bastones de la retina de Cynops orientalis fue más desarrollada, pero su capa de fibras nerviosas presentó una elevada degeneración, lo que muestra una gran fotosensibilidad, pero una sensibilidad visual baja. En Columba livia, la capa de fibras nerviosas de la retina estuvo muy desarrollada, y de esta manera, su visión. El grado de desarrollo de las diferentes estructuras y funciones de la retina de Ctenopharyngodon idella, Cynops orientalis, Bufo bufo gargarizans, Gekko japonicus y Columba livia está relacionada con sus características de comportamiento y el cambio de las condiciones de las vidas acuática y anfibia en la tierra.

A research of peripheral blood cells annually in bufo bufo gargarizans / Investigación de las células sanguíneas periféricas en un periodo anual del bufo bufo gargarizans

The present paper deals with a histological study of the blood cells of Bufo Bufo gargarizans in different months: January, March, May, July and October. The methods used are by routine blood smear in Wright stain and observation in vivo. We found that in smears and in vivo two main types of cells of the red cells: mitotic as well as amitotic. While amitotic occurs all the year round, particularly in July, mitosis so far had been seen only in July. It is also found that there are plenty of neutrophils in the blood cells of Bufo Bufo gargarizans, furthermore, the nuclei of these cells are polymorphic, especially in January and March. Meanwhile, the concentration of red cells was lowest in May and highest in January; The concentration of white blood cells was highest in October and lowest in March; As to granulocytes, eosinophils in July and October had higher proportion, while neutrophils and basophils in July; in agranulocytes, mononuclear cells reached the highest value in March, lowest in January, lymphocytes and the maximum value appeared in May, the lowest value appeared in July. Morphological changes of thrombocytes were not obvious.

Se realizó el presente estudio histológico de las células sanguíneas de Bufo Bufo gargarizans en diferentes meses del año: enero, marzo, mayo, julio y octubre. Fueron utilizados métodos de rutina por frotis de sangre con tinción de Wright y observación in vivo. Encontramos dos tipos principales de células de glóbulos rojos al frotis como también en células in vivo: mitóticas y amitóticas. Por cuanto amitosis se produce durante todo el año, sobre todo en el mes de julio, la mitosis hasta el momento se había observado solamente en julio. Además, se encontró una gran cantidad de neutrófilos en los glóbulos de Bufo Bufo gargarizans. Los núcleos de estas células son polimórficos, especialmente en enero y marzo. La concentración de glóbulos rojos era más bajo en mayo y más alta en enero; la concentración de las células blancas de la sangre fue mayor en octubre y menor en marzo. En cuanto a los granulocitos, eosinófilos estos presentaron una mayor proporción en julio y octubre, mientras que los neutrófilos y basófilos registraran una mayor proporción en el mes de julio. Los agranulocitos y las células mononucleares alcanzaron el valor más alto en marzo, y el valor más bajo en enero. Los linfocitos y el valor máximo fue registrado en mayo, el valor más bajo fue registrado en julio. No fueron evidentes los cambios morfológicos de trombocitos, lo que podría tener relación con su estabilidad.

Rapid and quantitative determination of multiple quality indicators (indole alkaloids, etc.) in concentration process of Bufo bufo gargarizans Cantor extract using fourier transform near infrared spectroscopy and chemometrics / 中国药学杂志

OBJECTIVE: To investigate the application of near infrared spectroscopy in rapid and quantitative determination of multiple quality indicators in concentration process of Bufo bufo gargarizans Cantor extract. METHODS: NIR transmission spectra of concentrated solutions were collected in rectangular quartz cuvette with different optical pathlengths (2 and 5 mm). Calibration models of density, water content and indole alkaloids were developed by partial least squares regression (PLSR). The established models were applied to predict the unknown samples to test the performance of the models. RESULTS: The correlation coefficients of the density, water content and indole alka. The residual predictive deviations (RPD) were all above 3, and the relative standard errors of predictions(RSEP) were less than 10%, indicating the satisfactory model performance and predictive ability. Moreover, better calibration results were obtained with an optical path length of 2 mm for density and water content, and 5 mm for indole alkaloids. The results revealed that the optical path length has influence on the NIR analysis results, and the appropriate path length for the NIR analysis should be determined by comparison analysis. CONCLUSION: NIR spectroscopy technique provides a novel efficient and environmental friendly approach for the fast simultaneous determination of three key quality indicators (density, water content and indole alkaloids) in the concentration process of Bufo bufo gargarizans Cantor extract. Copyright 2012 by the Chinese Pharmaceutical Association.

Isolation and sequencing of doublesex/male abnormal 3 (DM) related transcription factor (Dmrt) genes from the Asian toad Bufo gargarizans (Cantor, 1842)

The doublesex/male abnormal 3 (dsx/mab-3 or DM) domain gene family involved in sexual development encodes putative transcription factors including a DNA-binding homology motif, the DM domain. We used highly degenerate primers to clone and sequence seven distinct DM related transcription factor (Dmrt) genes from the Asian toad (Bufo gargarizans Cantor, 1842). A database search for the cloned sequences revealed the following percentage identity with the homologous Dmrt genes of the human: BgDmrt1 = 97 percent, BgDmrt2 = 97 percent, three isoforms of BgDmrt3 (BgDmrt3a = 93 percent, BgDmrt3b = 95 percent, BgDmrt3c = 100 percent) and two isoforms of BgDmrt5 (BgDmrt5 = 97 percent, BgDmrt5 = 91 percent). Based on DM domain amino acid sequence similarities we constructed a phylogenetic tree which grouped vertebrate and invertebrate Dmrt genes into seven distinct subfamilies. The DM domains of both human and the newly-discovered Bufo gargarizans genes contained two conserved zinc-chelating sites (CCHC and HCCC), except BgDmrt3b, which contained the CCRC and HCCC sites.

Quality Standard of Bufo gargarizans / 中国药房

OBJECTIVE:To establish the quality standard of Bufo gargarizans.METHODS:The quality of Bufo gargarizans was identified by TLC and the contents of Cinobufagin and Bufogenin were determined by HPLC.RESULTS:The TLC spots were clear and well-isolated.The linear ranges of Cinobufagin and Bufogenin were 0.100 4～1.255 0 ?g(r=0.999 9)and 0.103 6～1.295 0 ?g(r=0.999 9),respectively,with average recovery rates at 98.35% and 98.26%,respectively and RSD at 1.48%(n=9) and 1.76%(n=9),respectively.CONCLUSION: The established standard is suitable for the quality control of Bufo gargarizans.

SIMULTANEOUSLY DEMONSTRATION OF TYROSINE-HYDROXYLASE(AND SOMATOSTATIN)GENE EXPRESSION AND PEPTIDE STORAGE IN SINGLE NEURON OF THE HEART / 解剖学报

Objective To demonstrate simultaneously on transcription and translate levels the tyrosine-hydroxylase(and somatostatin)gene expression and peptide storage in same intrinsic cardiac ganglionic neuron of bufo bufo gargarizans. Methods Using immunohistochemical method,in situ hybrodization method and in situ hybrodization/immunohistochemical double labeling method on freeze-sections of sinus venanum cavanum of bufo bufo gargarizans atrial posterior wall (30*!?m)to observe THmRNA and /or TH-IR and SSmRNA and /or SS-IR dyeing neurons of intrinsic cardiac ganglia. Results There are kinds of positive neurons:1.TH-IR positive neurons;2.THmRNA positive neurons;3.TH mRNA/TH-IR double positive neurons and 1.SS-IR positive neurons;2.SSmRNA positive neurons;3.SSmRNA/SS-IR double positive neurons.Conclusion 1.TH mRNA and TH appearing in same intrinsic cardiac ganglionic neuron demonstrate that a part of intrinsic cardiac ganglionic neurons are possibly adrenergic sympathetic neurons;2.SSmRNA positive neurons and SSmRNA/SS-IR double positive neurons indicat that SS could be a neurotransmitter of intrincic cardiac ganglinic neurons.It provides neuroanatomy evidence of regulating cardiac function(excitatory conduct、cardiac muscle secrete and contract)by intrinsic cardiac SS-nergic neuron.