Authentication of Asini Corii Colla and Taurus Corii Colla based on UPLC-MS/MS and the discovery of antioxidant peptides associated with the PI3K-AKT pathway.

Asini Corii Colla (ACC) and Taurus Corii Colla (TCC) are well-known for their high nutritional value, especially for medicinal purposes. However, the aforementioned are also potential candidates for adulteration because of their low yield and high price. A UPLC-MS/MS approach based on the specific peptide was proposed to detect adulterated gelatin with possible mixed animal species. To explore the antioxidant activity, the peptides were separated to evaluate their effect on ·OH radical and DPPH· scavenging activity, together with PI3K-AKT pathway activation. The results showed that the peptides had excellent DPPH· and ·OH radical scavenging effects, and could alleviate H2O2-induced oxidative stress by promoting the phosphorylation of PI3K and AKT. According to the results of MALDI-TOF/MS, the shared mass-to-charge ratio (m/z) 1466, 1744 and 2382 may serve as a material basis for the antioxidant activity of both ACC and TCC, and contribute to their traditional tonic effects.

Herbal Textual Research on Asini Corii Colla in Famous Classical Formulas / 中国实验方剂学杂志

Through consulting the ancient and modern literature， this paper makes a textual research on the name， origin， producing area， harvesting and processing methods of Asini Corii Colla， so as to provide a basis for the development of the famous classical formulas containing the medicinal material. Before the Tang dynasty， cow leather was the main source of Asini Corii Colla， and donkey was rare as an introduced species. From the end of Tang dynasty to Song dynasty， due to the development of doctors' understanding of the properties and effects of medicines， with the increase of the number of donkeys and the limitation of the use of cow leather， the source of Asini Corii Colla changed from cow leather to donkey skin. During the Ming and Qing dynasties， the theory of medicine property was further developed， and all doctors basically agreed that black donkey skin and E-well water were two essential factors for making genuine Asini Corii Colla. Therefore， it is suggested that Asini Corii Colla should take Equus asinus as the authentic origin in the development of the famous classical formulas， attach importance to the quality of water source， take Liaocheng in Shandong province as the authentic producing area， and the processing should be carried out in accordance with the requirements of the 2020 edition of Chinese Pharmacopoeia.

[Determination of donkey skin ingredients in Asini Corii Colla by ultra-high performance liquid chromatography-tandem mass spectrometry].

In recent years, due to the shortage of donkey skin resources, the price of Asini Corii Colla has seen a rapid increase. Consequently, fake gelatin prepared from horse, mules, pig, and cow skin has appeared in the market, resulting in unreliable quality of Asini Corii Colla. Therefore, there is an urgent need to develop an efficient and accurate method for improving the quality of Asini Corii Colla. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to determine the donkey skin components in Asini Corii Colla. Accordingly, 0. l g of the evenly mixed sample was weighed and placed in a 50 mL volumetric flask; then, 1% ammonium bicarbonate solution was added to dissolve the sample, and the solution was diluted to the scale. Precisely 1.00 mL of the solution was extracted into a 5 mL volumetric flask, followed by the addition of 1.0 mL trypsin solution and 100 µL mixed internal standard working solution. This mixture was diluted to the scale using 1% ammonium bicarbonate solution, shaken, and placed in an incubator for 16 h to induce enzymolysis at a constant temperature of 37 ℃. The mixture was subsequently removed from the incubator, cooled to ambient temperature, filtered through a 0.22 µm membrane, and analyzed by LC-MS. Separation was performed on an UPLC system with a BEH C18 column (100 mm×2.1 mm, 2.5 µm) under gradient elution using acetonitrile containing 0.1% (v/v) formic acid (B) and water containing 0.1% (v/v) formic acid (A) as the mobile phases at a flow rate of 0.3 mL/min. The column temperature was 30 ℃, and the sample size was 2 µL. The gradient elution conditions are: 0-1 min, 10%B; 1-5 min, 10%B-30%B; 5-5.1 min, 30%B-70%B; 5.1-7 min, 70%B; 7-7.1 min, 70%B-10%B; 7.1-10 min, 10%B. The marker peptides were determined in positive electrospray ionization (ESI +) and multiple reaction monitoring (MRM) modes using the isotopic internal standard method. The optimized enzymolysis conditions were as follows: enzymolysis temperature, 37 ℃; enzymolysis time, 16 h; and amount of enzyme, 1 mL. The two marker peptides showed good linearities in the range of 50 to 1250 mg/L; the correlation coefficients (r) were greater than 0.996, and the limits of quantitation (S/N=10) were 20 mg/kg. At spiked levels of 300 mg/kg, 600 mg/kg, and 900 mg/kg, the average recovery ratios of the two marker peptides were 103.2% to 108.3%, while the relative standard deviations (RSDs) of 1.0%-3.0%. This method was favorable for testing actual samples. Asini Corii Colla from 29 production companies was detected by this method, and the sum contents of the two marker peptides was different because the production process and raw materials were different. The sum contents of the samples were 0.096% to 0.180% with an average of 0.151%. The developed method is simple, reliable, and reproducible, and it is suitable for detecting the donkey hide components Asini Corii Colla.

Efficacy and Safety of Ejiao (Asini Corii Colla) in Women With Blood Deficient Symptoms: A Randomized, Double-Blind, and Placebo-Controlled Clinical Trial.

Equus asinus L [Equidae; Asini Corii Colla] (donkey-hide gelatin, Ejiao), a well-known traditional Chinese medicine, has been widely used to nourish the blood, especially for women. The aim of this study was to assess the efficacy and safety of Ejiao in blood-deficient patients. A total of 210 participants were recruited and randomly allocated into the placebo control group and Ejiao-treated group (6 g/day). The primary outcomes on the efficacy of Ejiao included traditional Chinese medicine symptom scores, blood indicators, and SF-36. The secondary outcomes were changes in fireness and safety evaluation. Results showed that Ejiao treatment for 8 weeks had significantly improved dizziness symptoms. Among the tested 24 blood biochemical parameters, the hematocrit and red blood cell numbers decreased in the placebo control group, but decreased significantly less in the Ejiao treatment group. The white blood cell and neutrophil counts increased in the Ejiao group but were within the normal range. In addition, the quality of life improved as the scores in SF-36 domains were significantly higher in the Ejiao group. At the same time, there was no significant change in the fire-heat symptoms score or other safety parameters. Considering all these, our study showed that Ejiao has a promising effect in women suffering from blood deficiency without obvious adverse effects.

Collagen derived species-specific peptides for distinguishing donkey-hide gelatin (Asini Corii Colla).

Objective: As an important food therapy product with traditional Chinese medicine (TCM) applications, donkey-hide gelatin (Asini Corii Colla, ACC) has been used for thousands of years. However, till now few effective strategy had been proposed to distinguish ACC from other animal hide gelatins, especially closely related horse- and mule-hide gelatins, which was an embarrassment of ACC quality control. Methods: Combined mass spectrometry and bioinformatic methods have been applied to identify and verify two ACC-specific peptides (Pep-1 and Pep-2) capable of distinguishing ACC from other closely related animal gelatins with high selectivity. Results: It confirmed that these two peptides could be not only used for distinguishing ACC from highly homologous horse-hide and mule-hide gelatins as well as other animal hide gelatins. Conclusion: The present study provides a simple method for species-specific peptides discovery, which can be used for assessing the quality of animal gelatin products, and ensure they are authenticable and traceable.

[Analysis of peptides and proteins from Asini Corii Colla using nano LC-Q-Exactive-MS/MS].

This paper aims to systematically analyze the peptides and proteins from Asini Corii Colla(ACC) through shotgun proteomics. After high-pH reversed-phase fractionation, the proteins and peptides in the hydrolysate of ACC were further separated by nano LC-Q-Exactive-MS/MS under the following conditions: Thermo Scientific EASY column(100 µm×2 cm, 5 µm, C_(18)) as precolumn, Thermo Scientific EASY column(75 µm×100 mm, 3 µm, C_(18)) for solid phase extraction, gradient elution with 0.1% formic acid in water(mobile phase A) and 84% acetonitrile in water containing 0.1% formic acid(mobile phase B), and MS in positive ion mode. Based on Uniprot_Equus caballus, MS data, and literature, 2 291 peptides were identified from ACC by MaxQuant, with 255 Maillard reactions(AML, CML, CEL)-modified peptides identified for the first time. Through alignment, the peptides were found to belong to 678 equine proteins. In conclusion, the combination of nano LC-Q-Exactive-MS/MS and shotgun proteomics achieved rapid and accurate identification of the proteins and peptides in ACC, which provides the key information and new insights for further investigation of chemicals and effective substances in ACC.

Collagen derived species-specific peptides for distinguishing donkey-hide gelatin (Asini Corii Colla) / 中草药·英文版

Objective: As an important food therapy product with traditional Chinese medicine (TCM) applications, donkey-hide gelatin (Asini Corii Colla, ACC) has been used for thousands of years. However, till now few effective strategy had been proposed to distinguish ACC from other animal hide gelatins, especially closely related horse- and mule-hide gelatins, which was an embarrassment of ACC quality control. Methods: Combined mass spectrometry and bioinformatic methods have been applied to identify and verify two ACC-specific peptides (Pep-1 and Pep-2) capable of distinguishing ACC from other closely related animal gelatins with high selectivity. Results: It confirmed that these two peptides could be not only used for distinguishing ACC from highly homologous horse-hide and mule-hide gelatins as well as other animal hide gelatins. Conclusion: The present study provides a simple method for species-specific peptides discovery, which can be used for assessing the quality of animal gelatin products, and ensure they are authenticable and traceable.

Analysis of peptides and proteins from Asini Corii Colla using nano LC-Q-Exactive-MS/MS / 中国中药杂志

This paper aims to systematically analyze the peptides and proteins from Asini Corii Colla(ACC) through shotgun proteomics. After high-pH reversed-phase fractionation, the proteins and peptides in the hydrolysate of ACC were further separated by nano LC-Q-Exactive-MS/MS under the following conditions: Thermo Scientific EASY column(100 μm×2 cm, 5 μm, C_(18)) as precolumn, Thermo Scientific EASY column(75 μm×100 mm, 3 μm, C_(18)) for solid phase extraction, gradient elution with 0.1% formic acid in water(mobile phase A) and 84% acetonitrile in water containing 0.1% formic acid(mobile phase B), and MS in positive ion mode. Based on Uniprot_Equus caballus, MS data, and literature, 2 291 peptides were identified from ACC by MaxQuant, with 255 Maillard reactions(AML, CML, CEL)-modified peptides identified for the first time. Through alignment, the peptides were found to belong to 678 equine proteins. In conclusion, the combination of nano LC-Q-Exactive-MS/MS and shotgun proteomics achieved rapid and accurate identification of the proteins and peptides in ACC, which provides the key information and new insights for further investigation of chemicals and effective substances in ACC.

An optimized TaqMan real-time PCR method for authentication of ASINI CORII COLLA (donkey-hide gelatin).

In this study, probe/primers of high specificity and sensitivity were selected to analyze donkey-hide gelatin for donkey DNA and to look for horse, ox, and pig DNA as possible adulterants. The mitochondrial CO I genes in donkey, horse, and ox were selected as target sequences for design and synthesis of three pairs of specific probes and primers. In addition, eight pairs of probe/primers were obtained via literature search. Out of these eleven groups of probe/primers, those with the highest specificity and sensitivity were selected, which was fulfilled by the screening firstly with animal hide samples then the hide-glue samples. Other parameters that might affect detection specificity and efficiency-such as the amount of sampling and final concentration of primers-were also optimized. Replication tests were also conducted. The results showed that the selected probe/primers could accurately detect donkey DNA and horse, ox, and pig DNA in gelatin samples with good reproducibility. Analysis of four samples of on-market gelatin using this assay showed that two of the four samples indeed contained only donkey DNA, whereas the other two samples contained both donkey and horse DNA, indicating adulteration of these samples with horse hide. These results indicate that the TaqMan probe real-time PCR method can be used for identifying the purity of donkey DNA in gelatin samples, and can provide technical support for identifying adulterations in the gelatin market.

Study on anti-fatigue, anti-oxidative and hemostatic effects of small molecule Asini Corii Colla / 中国药理学通报

Aim: To study the anti-fatigue,anti-oxidative and hemostatic effects of small molecule Asini Corii Colla (SMACC). Methods: Rat model of complex blood deficiency was established to detect the exhausted time of swimming, hematology, superoxide dismutase (SOD), serum maleic dialdehyde (MDA), and lipid peroxide (LPO) levels, aiming to explore the anti-fatigue and anti-oxidative effects of SMACC. ICR mice were used to measure the blood clotting time (CT) and bleeding time (BT). Fevered and bleeding model and the heparinized bleeding model in rats were established to investigate the effect of SMACC in hemostasis and its possible mechanism. Results: Compared with model group, SMACC significantly prolonged the swimming time of model rats (P < 0. 05, P < 0. 01) and decreased the content of serum MDA, LPO (P < 0.05,P<0.01) at doses of 1. 500,0. 750,0. 375 g. kg-1,and increased the number of lymphocytes in the blood at doses of 1. 500,0. 375 g. kg-1 (P <0. 05). SMACC of 3. 00,1.50 g kg-1 significantly reduced the BT and CT (P < 0. 05). SMACC markedly reversed the prolonged prothrombin time (P < 0. 05) and the adverse changes of the hematological indicators. Conclusions: SMACC has the pharmacological effects of anti-fatigue, anti-oxidation and enhancing endurance, and also the effects of hemostasis and convergence.

Research progress on quality control of gelatinous Chinese materia medica / 中草药

Gelatinous Chinese materia medica (GCMM), as a valuable nourishing traditional medicine in China, has been used widely for a long time. The quality control for GCMM has undergone a long development, including physical and chemical tests, thin layer chromatography, electrophoresis, spectroscopy, HPLC, DNA identification techniques, and LC-MS with unique peptide as target. The article summarizes the progress of quality control system of GCMM in recent years, analyzes the remaining problems, puts forward the feasibility protocol to further improve the quality control system, and provides ideas for the comprehensive evaluation of GCMM and its related products.

Identification of the Adulterated Asini Corii Colla with Cytochrome c Oxidase Subunit I Gene-based Polymerase Chain Reaction.

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.

Medication Regularity Study on Prescriptions Containing Asini Corii Colla Based on Literature / 中国中医药信息杂志

Objective To discuss the medication regularity of prescriptions containing Asini Corii Colla;To provide data support for clinical compatibility application of Asini Corii Colla.Methods Totally 2635 articles about prescriptions containing Asini Corii Colla were retrieved from CNKI from Jan. 1995 to Dec. 2012. TCM Inheritance Support System V2.0 was employed to conduct frequency analysis and association rules analysis, with a purpose to determine the frequency, indications of disease, indications of syndrome and medicine core combination of common prescriptions containing Asini Corii Colla.Results The 2635 prescriptions containing Asini Corii Colla involved 1197 Chinese herbal medicines, 34 kinds of which were used in extremely high frequency (frequency>200). 30 kinds of indications of disease were treated in the high frequency (frequency>20). 32 kinds of indications of syndrome were treated in the high frequency (frequency>20). The combination of Glycyrrhizae Radix et Rhizoma, Angelicae Sinensis Radix and Asini Corii Colla showed the highest frequency in all three herbal combinations (486 times), and Atractylodix Macrocephalae Rhizoma, Asini Corii Colla, Codonopsis Radix and Astragali Radix showed the highest frequency in all four herbal combinations (272 times).Conclusion Most of compatibility them can enrich blood and tonify qi, and have major functions for gynecological diseases and major syndromes of qi-blood deficiency.

Study on Spray Drying Technology of Instant Asini Corii Colla / 世界科学技术-中医药现代化

This article was aimed to research the spray drying technology of instant Asini Corii Colla. HPLC was used. Contents of four kinds of amino acid and collection rate of powder were used as the indexes. The single factor experiment and orthogonal test were combined in the optimization of process conditions. The results showed that the best technology of spray drying technology was medicine liquid density of 1.10 (determined under the temperature of 60℃), inlet air temperature at 165℃, the pressed air speed was 45 L·h-1, the fluid speed was 15%. It was conclud-ed that the technology was reasonable and reliable, which can provide certain reference in the industrial production.

Determination of 13 amino acids in Asini Corii Colla by RP-HPLC with precolumn derivatization / 中草药

Objective: To establish a RP-HPLC method for the simultaneous determination of 13 amino acids in Asini Corii Colla. Methods: Asini Corii Colla was hydrolyzed for 3 h at 120°C using 6 mol/L hydrochloric acid, and the amino acids were determined by RP-HPLC after being derived with phenyl isothiocyanate. HPLC was performed on an Eclipse AAA column (75 mm × 4.6 mm, 3.5 μm) with acetonitrile-110 mmol/L ammonium acetate solution (1:99) as mobile phase A and acetic acid-acetonitrile-water (0.05:80:20) as mobile phase B in the gradient mode at a flow rate of 0.6 mL/min. The column temperature was 42°C, and the detection wavelength was set at 254 nm. Results: The 13 amino acids were separated within 36 min with a good linearity (r > 0.9977) in the range of the test concentration. The average recoveries (n = 3) were 95.2%-104.4% and the RSD values were less than 5.0%. Conclusion: The method is simple, accurate, and repeatable, which could be available for the quality control of Asini Corii Colla. It may also serve as a good reference for the determination of amino acids in other pharmaceuticals.