Methods for detection and quantification of gelatin from different sources.

Gelatin is a water-soluble protein obtained from the collagen of various animal origins (porcine, bovine, fish, donkey, horse, and deer hide) and has diverse applications in the food, pharmaceutical, and cosmetics industries. Porcine and bovine gelatins are extensively used in food and non-food products; however, their acceptance is limited due to religious prohibitions, whereas fish gelatin is accepted in all religions. In Southeast Asia, especially in China, gelatin obtained from donkey and deer skins is used in medicines. However, both sources suffer from adulteration (mixing different sources of gelatin) due to their limited availability and high cost. Unclear labeling and limited information about actual gelatin sources in gelatin-containing products cause serious concern among societies for halal and fraud authentication of gelatin sources. Therefore, authenticating gelatin sources in gelatin-based products is challenging due to close similarities between the composition differences and degradation of DNA and protein biomarkers in processed gelatin. Thus, different methods have been proposed to identify and quantify different gelatin sources in pharmaceutical and food products. To the best of our knowledge, this systematic and comprehensive review highlights different authentication techniques and their limitations in gelatin detection and quantification in various commercial products. This review also describes halal authentication and adulteration prevention strategies of various gelatin sources, mainly focussing on research gaps, challenges, and future directions in this research area.

Effects of Varying Ratios of Glycyrrhiza uralensis and Donkey Hide Gelatin Water Extracts on Dinitrochlorobenzene-Induced Atopic Dermatitis in NC/Nga Mice.

Atopic dermatitis is a chronic skin disease that affects millions of people all over the world. The objective of this study was to evaluate the inhibitory effects of the roots of Glycyrrhiza uralensis (GU) and Donkey Hide Gelatin (DHG) water extracts on DNCB-induced NC/Nga mice and TNF-α/IFN-γ treated keratinocytes or LPS-stimulated macrophages. The combined treatment using the water extracts of GU and DHG improved the skin symptom evaluation score and skin histology, with increased expression of the skin barrier proteins Claudin 1 and Sirt 1 in lesion areas. The IFN-γ activity was promoted in PBMCs, ALN, and dorsal skin tissue, while the absolute cell number was reduced for T cells so that the production and expression of serum IgE and cytokines were suppressed. In TNF-α/IFN-γ induced HaCaT cells, IL-6, IL-8, MDC, and RANTES were all inhibited by GU and DHG water extracts, while ICAM-1 and COX-2 levels were similarly downregulated. In addition, GU and DHG water extracts decreased LPS-mediated nitric oxide, IL-6, TNF-α, and PGE2 in RAW 264.7 cells, and the expression of iNOS and COX-2 also decreased. Notably, the DHG:GU ratio of 4:1 was shown to have the best effects of all ratios. In conclusion, GU and DHG have anti-skin inflammatory potentials that can be used as alternative ingredients in the formula of functional foods for people with atopic dermatitis.

Identification of Five Gelatins Based on Marker Peptides from Type I Collagen by Mass Spectrum in Multiple Reaction Monitoring Mode.

In this study, a novel pseudo-targeted peptidomics strategy, integrating the transition list generated by an in-house software (Pep-MRMer) and the retention time transfer by high-abundance ion-based retention time calibration (HAI-RT-cal), was developed to screen marker peptides of gelatins from five closely related animal species, including porcine, bovine, horse, mule, and donkey. Five marker peptides were screened from the molecular phenotypic differences of type I collagen. Furthermore, a simple and robust 10 min multiple reaction monitoring (MRM) method was established and performed well in distinguishing different gelatins, particularly in discerning horse-hide gelatin (HHG) and mule-hide gelatin (MHG) from donkey-hide gelatin (DHG). The market investigation revealed the serious adulteration of DHG. Meantime, the pseudo-targeted peptidomics could be used to screen marker peptides of other gelatin foods.

Inhibitory Effects of Donkey Hide Gelatin on DNCB-Induced Atopic Dermatitis in NC/Nga Mice.

The increase of atopic dermatitis has led to higher socio-economic cost and raised a need for alternative medicine as novel therapeutic agents. In this study, we aimed to evaluate the inhibitory effects of Donkey Hide Gelatin (DHG) water extract on DNCB-induced atopic dermatitis in NC/Nga mice and on tumor necrosis factor (TNF)-α/interferon (IFN)-γ-treated keratinocytes and to investigate its underlying molecular mechanisms. NC/Nga mice were induced by DNCB, administered Dexamethasone (3 mg/kg) or DHG water extracts (100-400 mg/kg) for 3 weeks. The skin symptom score, serum IgE and immune cells were measured, the ALN, spleen and dorsal skin tissue were extracted for FACS, quantitative real-time PCR and histology analysis. In vitro, HaCaT cells were induced by TNF-α/IFN-γ, the levels of pro-inflammatory cytokines and chemokines and its underlying mechanism were measured by ELISA and Western blot. As a result, DHG groups showed a significant decrease in the skin symptom score and the immune cell absolute number. It also showed a marked reduction of allergic and the levels of neutrophils and eosinophils in histology analysis. In TNF-α/IFN-γ induced HaCaT cells, DHG showed inhibition effects on IL-6, IL-8, TARC and RANTES, it also downregulated the expression of ICAM-1 and COX-2, up-regulated the expression of Filaggrin. Furthermore, DHG suppressed the activation of NF-κB and mitogen-activated protein kinases (MAPK) signaling pathway induced by TNF-α/IFN-γ. Taken together, DHG maybe a potential therapeutic agent or supplement for skin inflammatory disease such as atopic dermatitis.

An analysis of skin thickness in the Dezhou donkey population and identification of candidate genes by RNA-seq.

The aim of the present study was to analyze the main factors that have a significant impact on skin thickness, and to further identify the genes and signaling pathways regulating skin growth by RNA-seq in Dezhou donkeys. Skin samples from different body regions of 15 slaughtered donkeys were obtained to study variations in skin thickness over the bodies. Skin thickness data for another 514 donkeys was obtained by minimally invasive skin sampling from the back, and measurements of the donkeys' body size traits and pedigree data were also collected. These data were used to analyze changes in skin thickness and estimate genetic parameters. In addition, transcriptomic analysis was conducted on the skin tissues of individuals from two groups with significant differences in skin thickness. Our results showed that skin thickness over the bodies ranged from 1.08 to 4.36 mm. The skin from the back was the thickest and had the highest correlation with that of other regions of the body. The skin thickness decreased from the back to the side of the ventral abdomen, and the skin thickness on the limbs increased from the proximal end to the distal end. The results also showed that the skin from the same body regions of jacks was thicker than that of jennies in the same age group. The skin thickness of jennies increased from birth to the age of 2 and then clearly decreased after 2 years of age. The estimated heritability of skin thickness was 0.15, and the genetic correlations between skin thickness and body size traits were negligible. Transcriptome analysis showed that the thick-skin group had 65 up-regulated genes and 38 down-regulated genes compared with the thin-skin group. The differentially expressed genes were highly enriched in epidermal development and cell adhesion molecule signaling pathways. We identified the candidate genes responsible for variations in skin thickness in the Dezhou donkey, including KRT10, KRT1, CLDN9, MHCII and MMP28. These results contribute to a better understanding of the growth and development of donkey skin, reveal the molecular mechanism responsible for donkey skin thickness and suggest directions for genetic selection in the Dezhou donkey population.

Quantitative Detection of Donkey Hide Gelatin (Colla corii asini) Adulteration by Real-Time PCR on the Basis of Single-Copy Nuclear Genes.

ABSTRACT: Donkey hide gelatin (Colla corii asini) is well-known for its high nutritional value, especially for medicinal purposes. However, it is also a potential candidate for adulteration because of its low yield and high price. To quantitatively detect adulterated donkey hide gelatin with all possible mixed animal species, a real-time PCR approach on the basis of single-copy housekeeping nuclear reference primers was proposed in this study. For the system establishment, mixtures containing designated contents of pig hide with donkey hide were used to generate a calibration curve on the basis of the ratio of cycle threshold, CT (specificity/reference) with reasonable linearity (5 to 100%). Then, a set of experiments were performed on commercially available samples. The proposed PCR approach could specifically identify donkey hide from mixed animal products and quantify the content of donkey hide gelatin, thus facilitating control over this novel form of donkey hide gelatin adulteration.

How much is a donkey worth? / Qual o valor do jumento?

This study aimed to estimate the donkey unit cost of production and to contextualize it with the socio-economic condition of the Brazilian semiarid region. A study model was proposed in which the costs of maintenance and animal production were considered, assuming that the herd already existed on the property and the use of the available facilities. The production cycle of donkeys was estimated at 36 months, with an initial herd of 100 jennies and five jacks. For the estimation of forage production, native pastures to the semi-arid region were considered. The unit cost of production was estimated for donkey US$ 258.00/year. However, when the donkey is integrated into the production unit of the family farming system, this animal should be considered as a production asset (draught, pack, and ridden). In face of the growing demand for donkey hide in the international market, the absence of public policies for the protection and welfare of these animals, and even considering the possibility of integrating them into family farming systems, the following question must be asked: how long before Northeast donkeys are extinct? Estimating the unit cost of donkey production and considering them as an essential production asset in family farming could be a starting point to diminish their abandonment in several communities and the consequences of the exploitation of hides.(AU)

O estudo teve como objetivo estimar o custo de produção do jumento e contextualizar com a condição socioeconômica da região semiárida brasileira. Foi proposto um modelo de estudo no qual foram considerados os custos de manutenção e produção animal, admitindo que o rebanho já existisse na propriedade com utilização das instalações disponíveis. O ciclo de produção do jumento foi estimado em 36 meses, com rebanho inicial de 100 matrizes e cinco reprodutores. Para a estimativa de produção de forragem, foram consideradas pastagens nativas da região semiárida. Foi estimado o custo de produção do jumento em US$ 258,00/ano. Entretanto, quando o jumento está integrado à unidade produtiva do segmento da agricultura familiar, esse animal deve ser considerado como ativo de produção (tração, carga e transporte). Diante da crescente demanda da pele de jumentos no mercado internacional, da ausência de políticas públicas de proteção e bem-estar desses animais, e mesmo considerando a possibilidade de integrá-los ao sistema de agricultura familiar, permanece a pergunta: quanto tempo vai levar até que os jumentos nordestinos sejam extintos? Estimar o custo unitário da produção de jumentos e considerá-los como um ativo essencial de produção na agricultura familiar pode ser um ponto de partida para diminuir seu abandono às consequências da exploração de pele.(AU)

Specific, sensitive and rapid authentication of donkey-hide gelatine (Colla corii asini) in processed food using an isothermal nucleic acid amplification assay.

Donkey-hide gelatine (DHG) is a well-known, animal-derived traditional Chinese medicine material called Colla corii asini (known in Chinese as "E'jiao"). Because DHG is claimed to have properties that are beneficial to health, its consumption has increased, but its production has decreased. Thus, the incidence of DHG adulteration has become increasingly serious. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the authentication of DHG. Identification of donkey DNA from DHG was performed specifically and rapidly within one hour by LAMP primers. Moreover, the sensitivity of LAMP in authenticating DHG was 10-3 ng, which revealed a 105-fold higher sensitivity than that of conventional PCR. The relative detection limit was 0.1% DHG in the adulterants, including gelatines of horse, cow, pork, goat, sheep or chicken origins. When genomic DNAs extracted from heat-treated DHG samples, including boiling or autoclaving for 40 min, were used as templates, DHG detection by LAMP was unchanged and reproducible. In conclusion, the LAMP assay established herein could potentially be applied for the authentication of DHG and DHG-related products in herbal or food markets.

The recycling of the expired donkey-hide gelatin pulp for N/S co-doped hollow carbon nano-spheres anode in sodium ion battery.

How to reasonably manage and reutilize the waste expired liquid medicines has always been a puzzling public concern. For this reason, the waste expired medicine of donkey-hide gelatin pulp was recycled by hydrothermal carbonization and hard template for N/S co-doped hard carbon material, and its electrochemical Na-storage performances were also evaluated. The results showed that the resultant N/S co-doped hard carbon material manifested the morphology of hollow nano-spheres with the mean diameter of about 242.3 nm and the shell thickness of about 15 nm; N and S elements evenly distributed in carbon structure by in situ co-doping. Furthermore, N/S co-doped hard carbon also delivered the satisfactory electrochemical Na-storage capacities due to the synergistic effect of the unique hollow nano-spheres with thin shell and N/S co-doping. No doubt, the results would promote the circular economy mode of waste expired medicines.

Determination of Donkey-hide Gelatin in Lüjiao Buxue Granules by UPLC-QQQ/MS / 中国药房

OBJECTIVE:To establish a method for the determination of donkey-hide gelatin in Lüjiao buxue granules. METH-ODS:Trypsin was used for enzymatic hydrolysis of donkey-hide gelatin. UPLC-QQQ/MS was adopted for the determination of don-key-hide gelatin in the preparation:Hypersil GOLD C18 column was used with mobile phase consisted of 0.1% formic acid solu-tion-acetonitrile(gradient elution)at the flow rate of 0.3 mL/min. The column temperature was 30 ℃,and sample size was 5 μL. Mass chromatography condition was that ion source was electrospray ion source;ionization mode was ESI+;multiple response mon-itoring was adopted,and characteristic molecular ion peaks of donkey-hide gelatin with mass-to-charge ratio of m/z 539.8(double charge)→ 612.4 and m/z 539.8(double charge)→ 923.8 were used as detection ion pair. Spray voltage was 3100 V;sheath gas was 20 Arb;auxiliary gas was 8 Arb;ion transfer tube temperature was 350 ℃;evaporation temperature was 350 ℃;scan mode was SRM. Collision gas was high purity argon. RESULTS:The sample size of donkey-hide gelatin ranged 20.24-2024 μg/mL(r=0.9978). The detection limit was 1%. RSDs of precision,stability and reproducibility tests were all lower than 6.0%. The charac-teristic molecular ion peaks of donkey-hide gelatin could be detected in 27 batches of samples. CONCLUSIONS:The method is spe-cific and can be used for the determination of donkey-hide gelatin in Lüjiao buxue granules.

Qualitative Detection of Donkey-hide Gelatin in Fufang Ejiao Buxue Granule by UPLC-QQQ/MS / 中国药师

Objective:To establish an analytical method for the identification of donkey-hide gelatin in Fufang Ejiao Buxue gran-ule.Methods:The identification of donkey-hide gelatin in Fufang Ejiao Buxue granule was established by rapid resolution liquid chro -matography (UPLC) coupled with triple quadruple mass spectrometry (QQQ-MS).Results: The characteristic molecular peaks of donkey-hide gelatin were detected in ten batches of commercial samples .Conclusion:The present method is specific , precise and reli-able, and suitable for the identification of donkey-hide gelatin in Fufang Ejiao Buxue granule .The method provides scientific reference for the study of quality control method for gelatin ingredients in Chinese patent medicines .

Identification of donkey-hide and its counterfeits based on 1H-NMR metabolomics / 中草药

Objective: The aqueous extracts of donkey-hide and its counterfeits (horse-hide and mule-hide) were identified using 1H-NMR metabolomics to provide the basis for the identification and quality control of donkey-hide gelatin. Methods: The water-soluble components of the hairless donkey-hide and its counterfeits were extracted and the 1H-NMR technique was applied to analyzing the components. Moreover, the NMR data were analyzed and the component belongings were recognized using relevant software for the multivariate statistical analysis. Results: Forty-two water-soluble components were identified in donkey-hide by 1H-NMR. There were more acetate, leucine, valine, isoleucine and less lactate, serine, pyroglutamate, and creatine in the horse-hide than in the donkey-hide; While there were more acetate, creatine, alanine, choline and less glycerol and acetate in mule-hide than in donkey-hide. And they were all in a significant difference (P 1H-NMR metabolomics was established for the first time, which provides the experimental references for the donkey-hide identification and quality control study.

LC-MS/MS analysis of gelatine ingredients in chinese patent medicines / 中国药学杂志

OBJECTIVE: To establish an analysis method for identification of gelatine ingredients in Chinese patent medicines. METHODS: Trypsin was used for hydrolysis of gelatins. The characteristic getalins analyses were identified by rapid resolution liquid chromatography (RRLC) coupled to triple quadruple mass spectrometry (QQQ-MS). Trypsin was used for hydrolysis of gelatins. RESULTS: The established present method was specific, precise and reliable. Gelatine ingredients as prescribed wereas detected for several samples. However, instead of gelatin ingredients as prescribed, unprescribed gelatine ingredients were as detected for some samples. CONCLUSION: The methodology validation is performed and the results are satisfied. The method can be used for identification of donkey-hide gelatin, oxhide gelatin, deer-horn glue and tortoise-shell glue in Chinese patent medicines, and provides a scientific reference for the study of quality control method of gelatin ingredients in Chinese Patent Medicines.

Study on compound donkey-hide gelatin syrup in reducing adverse reactions of Qi-blood deficiency patients caused by clozapine / 国际中医中药杂志

Objective To investigate the compound donkey-hide gelatin syrup in reducing adverse reactions of qi-blood weakness patients caused by clozapine.Methods 132 patients from Psychiatric Hospital of Yunnan Province between January 2010 and June 2010 were randomly divided into a treatment group and a control group.Both groups were taken clozapine orally.On this basis,the treatment group was taken compound donkey-hide gelatin syrup and the control group was taken placebo syrup.After 8 weeks treatment for both groups,the PANSS,TESS,physical examination and experiment examination were observed to evaluate the clinical efficacy and safety.Results ① the total curative effect:the treatment group was 73.53％,the control group was 65.63％,showing statistical difference (x2=2.543,P＜0.05).② PANSS scores changes before and after the treatment:PANSS score at 2,4,6,8 weeks after the treatment of both groups were [(72.51 ±27.55),(60.54±24.03),(53.12± 15.27),(48.15± 11.88) in treatment group respectively,and (70.71 ±23.90),(58.89± 18.95),(53.06± 14.38),(48.98 ± 9.78) in the control group,respectively],both showing significant difference than the same group before the treatment [(103.99±39.12) in the treatment group,(99.78±34.35) in the control group] (P＜0.05).But there was no statistical significance between two groups (F=2.413,P＞0.05).③ adverse reactions:during the treatment liver function,blood cell analysis,dystonia,Parkinson's obstacle,akathisia,abnormal gastrointestinal reaction,heart rate,heart rate variability and blood pressure in the treatment group was significantly lower than the control group (x2=4.562,P＜0.05).Conclusion Compound donkey-hide gelatin syrup can effectively relieve adverse reactions in qi-blood weak psychosis patients after clozapine treatment and improve their drug tolerance.