Dataset Information

Species identification of Asini Corii Collas (donkey glue) by PCR amplification of cytochrome b gene.

ABSTRACT: Asini Corii Collas (ACC; donkey glue) is a crude drug used to promote hematopoiesis and arrest bleeding. Because adulteration of the drug with substances from other animals such as horses, cattle, and pigs has been found, we examined PCR methods based on the sequence of the cytochrome b gene for source species identification. Two strategies for extracting DNA from ACC were compared, and the ion-exchange resin procedure was revealed to be more suitable than the silica-based one. Using DNA extracted from ACC by the ion-exchange resin procedure, PCR methods for species-specific detection of donkey, horse, cattle, and pig substances were established. When these species-specific PCR methods were applied to ACC, amplicons were obtained only by the donkey-specific PCR. Cattle-specific PCR detected as little as 0.1% admixture of cattle glue in the ACC. These results suggest that the species-specific PCR methods established in this study would be useful for simple and easy detection of adulteration of ACC.

SUBMITTER: Kumeta Y

PROVIDER: S-EPMC4353880 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Species identification of Asini Corii Collas (donkey glue) by PCR amplification of cytochrome b gene.

Kumeta Yukie Y Maruyama Takuro T Asama Hiroshi H Yamamoto Yutaka Y Hakamatsuka Takashi T Goda Yukihiro Y

Journal of natural medicines 20130627 1

Asini Corii Collas (ACC; donkey glue) is a crude drug used to promote hematopoiesis and arrest bleeding. Because adulteration of the drug with substances from other animals such as horses, cattle, and pigs has been found, we examined PCR methods based on the sequence of the cytochrome b gene for source species identification. Two strategies for extracting DNA from ACC were compared, and the ion-exchange resin procedure was revealed to be more suitable than the silica-based one. Using DNA extract ...[more]

PMID: 23807625

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Project description:Background:Asini Corii Colla (ACC) (namely donkey hide gelatin, E'jiao in Chinese) was one of the most valuable tonic traditional Chinese medicines which is an infallible remedy to promote hematopoiesis. It should be produced by fresh or dried donkey hide according to Chinese Pharmacopeia (2015 edition) with a long-time decoction, while as donkey and horse (or mule) all belong to equids so their hides or their hide gelatins are share much in common, that cause the difficult in distinguishing raw materials donkey hide from horse/mule hide for manufacturer, and the challenge in the quality evaluation of ACC for regulatory authority to identify the adulterated with horse hide. Objective:To establish an effective quality evaluation methods for ACC focused on the qualitative-based identification of the raw material's authenticity, mainly to identify the species origin of the gelatins. Materials and Methods:DNA extracted from (1) Raw materials (hides of donkey, horse, mule, bovine and pig); (2) Five hide-glues (bovine, pig, donkey, horse and mule hide-glue); (3) 11 batches of ACC commercial products made by different manufactures from local drug stores. Polymerase chain reaction (PCR) method with newly designed horse-specific primers I and primer pair II. Results:Use the primer pair I, a 234 bp target product could be amplified sensitively from the DNA sample of horse/mule adulterated commercial ACC products, though the DNA in commercial products is severely degraded. A 219 bp product could be amplified specifically from the DNA sample of horse/mule hide, while the results were all negative for the DNA templates of donkey hide, its gelatin and ACC products without adulteration. Conclusion:The developed PCR method based on primer I and II provide an effective approach to identify the species origin of highly processed product ACC (primer pair I) as well as to distinguish the raw material donkey hide (primer pair II), which might enlighten a new strategy to the Quality Evaluation of ACC. SUMMARY:Though the quality of commercial Asini Corii Colla (ACC) products varies greatly and produce with nondonkey hide was one the most common adulteration, the effective method to constrain such adulteration remains to be establishedThe gelatins made by donkey, horse, bovine, pig, mule shares much in common with each other, not only in contents of amino acids but also the profiles of protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focusing, gel filtration chromatography and two-dimensional electrophoresisThe adulteration in ACC by using horse/mule hide, which is most difficult to detect, could be identified by Polymerase chain reaction methods with newly designed horse/mule-specific primer. Abbreviations Used: ACC: Asini Corii Colla; TCMs: Traditional Chinese Medicines; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; IEF: Isoelectric focusing; GFC: Gel filtration chromatography; 2-DE: Two-dimensional electrophoresis; PCR: Polymerase chain reaction.

Project description:As an aging-associated degenerative disease, Alzheimer's disease is characterized by the deposition of amyloid beta (Aβ), oxidative stress, inflammation, dysfunction and loss of cholinergic neurons. Colla Corii Asini (CCA) is a traditional Chinese medicine which has been used for feebleness-related diseases and anti-aging. CCA might delay aging-induced degenerative changes in neurons. In the present study, we evaluated antioxidant activity, cytoprotective effects, and Aβ removability of enzyme-digested Colla Corii Asini (CCAD). Oxygen radical absorbance capacity (ORAC) activity assay showed that, as compared to gelatins from the skin of porcine, bovine and cold water fish, CCA exhibited the highest ORAC activity. The ORAC activity of CCA and CCAD was increased gradually by the length of time in storage. Ultrastructure analysis by scanning electron microscopy showed that among CCA manufactured in 2008, 2013, 2017 and gelatin from cold water fish skin, CCA manufactured in 2008 presented the smoothest surface structure. We further tested the protective effects of CCAD (manufactured in 2008) and enzyme-digested gelatin from cold water fish skin (FGD) on hydrogen peroxide (H2O2)-induced cell death in nerve growth factor-differentiated neuronal-like PC12 cells. Presto blue assay showed that both FGD and CCAD at 0.5 mg/mL increased cell viability in H2O2-treated neuronal-like PC12 cells. The protection of CCAD was significantly superior to that of FGD. Acetylcholinesterase (AchE) assay showed that both FGD and CCAD inhibited AchE activity in nerve growth factor-differentiated neuronal-like PC12 cells to 89.1% and 74.5% of that in non-treated cells, respectively. The data suggest that CCAD might be able to increase the neurotransmitter acetylcholine. Although CCAD inhibited AchE activity in neuronal-like PC12 cells, CCAD prevented H2O2-induced abnormal deterioration of AchE. ELISA and neprilysin activity assay results indicated that CCAD reduced amyloid beta accumulation and increased neprilysin activity in Aβ1-42-treated neuronal-like PC12 cells, suggesting that CCAD can enhance Aβ clearance. Our results suggest that CCA might be useful for preventing and treating Alzheimer's disease.

Dataset Information

Species identification of Asini Corii Collas (donkey glue) by PCR amplification of cytochrome b gene.

Publications

Species identification of Asini Corii Collas (donkey glue) by PCR amplification of cytochrome b gene.

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