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

Abstract 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. Graphical Abstract


Introduction
The vast majority of traditional Chinese medicine comes from natural plants, followed by animals, minerals, and some artificial products (Pejin, Iodice, et al. 2012;Pejin, Kien-Thai, et al. 2012;Cyranoski 2018).Gelatinous Chinese medicines (GCMs) are made from animal skins, nails, horns, or bones following decoction with water and condensing into thick gelatin.GCMs represent a type of nourishing Chinese medicine, which show remarkable benefits, including replenishing blood, moistening dryness, and arresting bleeding (Hijikata et al. 2009;Lee et al. 2021).Asini Corii Colla (ACC, donkey-hide glue) and Taurus Corii Colla (TCC, cattle-hide glue) are two important and commonly used GCMs, which are widely used to treat dizziness, palpitation, insomnia and anemia (Zhang et al. 2021).Modern pharmacological studies have shown that both ACC and TCC have excellent anti-oxidation, anti-aging and anti-bacterial effects (Park et al. 2017;Xiao et al. 2020).
Although the consumption of GCMs has increased rapidly in recent years, the resources from medicinal animals are continuously decreasing.This has led to the emergence of adulterating ACC with cheaper swine skin and horse skin, mainly Scrofa Corii Colla (SCC, swine-hide glue) and Caballus Corii Colla (CCC, horse-hide glue), which not only endangers public health, but also causes unfair competition in the market (Chen et al. 2019).Traditional characteristic identification is highly dependent on experience, and it is difficult to accurately identify species.Moreover, immunochemical methods may be affected by the degree of proline hydroxylation, which is important in determining the antigenicity of collagen (Cheng et al. 2012).Although polymerase chain reaction (PCR) has high sensitivity and high taxonomic specificity, it has a limitation in that the DNA integrity is destroyed during gelatin processing (Merheb et al. 2016).Thus, the upgrading of identification methods is urgently required to ensure the safety and effectiveness of GCMs in a clinical setting.In contrast to DNA, the amino acid sequence of a protein is highly consistent in the processing of gelatin.The characteristic amino acid sequence of collagen is a repeating G-X-Y sequence, where G is glycine, and X and Y are primarily proline and hydroxyproline, respectively (Pawelec et al. 2016).This characteristic amino acid sequence has been used to identify the animal sources of gelatin by mass spectrometric detection of peptides after trypsin digestion (Han et al. 2022).
In this paper, UPLC-MS/MS was used to detect the specific peptides produced by enzymatic digestion of colloidal proteins, to reliably distinguish the common counterfeit and adulterated products of GCMs.The effect of the peptides on the PI3K-AKT signaling pathway was also detected given that the PI3K-AKT pathway is closely related to oxidative stress (Gao 2019).Active PI3K phosphorylates produce the second messenger phosphatidylinositol 3,4,5-triphosphate (PIP3); this causes the recruitment and phosphorylation of AKT, which is involved in the regulation of various life processes, including growth, apoptosis, and oxidative stress (Deng et al. 2019;Ediriweera et al. 2019).Several studies have confirmed that promoting the phosphorylation of PI3K and AKT can alleviate H 2 O 2 -induced oxidative stress of cells (Ma et al. 2021;Kang et al. 2022;Li et al. 2022).Meanwhile, the peptides were analyzed by MALDI-TOF/MS to trace the potential antioxidant active peptide, to clarify the material basis of the nourishing effect of ACC and TCC.We hope that this work provides new perspectives for the development and application of healthcare food containing ACC and TCC.

UPLC-MS/MS analysis of digested gelatins
As shown in Figures S1-S8 (Supplementary material), the four batches of self-made gelatin samples only presented the corresponding species-specific ion peaks, indicating that the selected ion pairs could be used to distinguish gelatin samples derived from different animals.The established UPLC-MS/MS method could be used for subsequent authentication of ACC and TCC.
Nine batches of commercial samples were tested by specific ion pairs, and the results are shown in Figures S9-S26 (Supplementary material).Samples D1-D6 all showed specific ion pairs of m/z 539.80 !m/z 612.40 and m/z 539.80 !m/z 923.80, indicating that donkey derived peptides were included in these samples.However, sample D1 showed specific ion pairs of m/z 641.30!m/z 726.20, m/z 641.30!m/z 783.30, m/z 774.00 !m/z 977.50 and m/z 774.00 !m/z 752.30, indicating the existence of both cattle-and swine-derived peptides.Specific ion pairs of m/z 386.25 !m/z 556.5 and m/z 386.25 !m/z 499.25 were detected in samples D5 and D6, indicating the presence of horse-derived peptides.Thus, samples D2-D4 were confirmed as genuine products, while samples D1, D5 and D6 were adulterated products.Samples C1-C3 showed specific ion pairs of m/z 641.30!m/z 726.20 and m/z 641.30!m/z 783.30, indicating the presence of cattle-derived peptides.As none of the other specific ion pairs were detected in these samples, C1-C3 were confirmed as genuine cattle-derived products.The authenticity of the above commercial products was judged, and the results were summarized in Table S1 (Supplementary material).

Antioxidant activities of peptides in ACC and TCC
Next, the antioxidant activities of ACC and TCC peptides were measured via DPPHÁ and ÁOH scavenging activity.The stronger the scavenging activity of free radicals, the stronger the antioxidant effect of peptides.
After being separated by Sephadex G-50, the antioxidant activity of the eluted fraction of ACC was tested, and the results are shown in Figure S27(a) (Supplementary material).Fractions 11-15 presented higher ÁOH scavenging activity than the other tubes; fractions 7-14 presented higher DPPHÁ scavenging activity.For TCC, as shown in Figure S27(b) (Supplementary material), fractions 13-15 presented higher ÁOH scavenging activity, and fractions 7-13 presented higher DPPHÁ scavenging activity.The fractions with higher activities were then analyzed by MALDI-TOF/MS.

Immunoblot analysis of the PI3K-AKT signaling pathway
The PI3K/AKT pathway regulates many cellular processes and has been suggested to be closely related to oxidative stress.Therefore, we explored whether ACC and TCC peptides could activate the PI3K/AKT pathway and mediate antioxidant activity.As shown in Figure S28 (Supplementary material), the p-PI3K and p-AKT levels in RAW264.7 cells decreased significantly after H 2 O 2 treatment, suggesting that oxidative stress caused by H 2 O 2 could inhibit PI3K and AKT activation.Supplementation with the total peptides of ACC and TCC significantly upregulated the p-PI3K and p-AKT levels in RAW264.7 cells.Higher doses of total peptides of ACC showed an improved ability to activate the PI3K/AKT pathway, while the total peptides of TCC presented similar results.
To further study the effect of different peptides separated by Sephadex G-50 on the PI3K-AKT pathway, the ACC and TCC peptides (fractions 7-15) with high ÁOH radical and DPPHÁ scavenging activity were collected to measure the effect on the PI3K/AKT pathway.As shown in Figure S29 (Supplementary material), compared to the H 2 O 2 model group, supplementing with ACC peptide fractions 7-15 significantly upregulated p-PI3K and p-AKT levels, with fractions 10 and 11 showing the most significant effects (p < 0.01), while for TCC, as shown in Figure S30 (Supplementary material), tubes 10-13 showed the most significant effects (p < 0.01).
The results of 3.2 and 3.3 showed that the ACC and TCC peptides had high antioxidant activity, which was closely related to the activation of the PI3K-AKT pathway.After separating by Sephadex G-50, ACC peptide fractions 10 and 11 had the strongest antioxidant activity and ability to mobilize the PI3K-AKT pathway, revealing the enrichment of highly antioxidant peptides in these fractions.Therefore, it is necessary to further analyze and identify the peptides from different fractions to identify antioxidant peptides, with the same holding true for TCC.

MALDI-TOF/MS analysis of antioxidant activity fractions
MALDI-TOF/MS was used to analyze the eluted fractions (peptides) in ACC and TCC of high antioxidant activity, results were shown in Table S2 and Figures S31-S48 (Supplementary material).For ACC, the ion m/z 1048 and 1466 were detected in fractions 8-12.The ion m/z 1195 and 853 were detected in fractions 9-11.The ion m/z 2382 was detected in fractions 8-9.The ion m/z 1744 and 701 were detected in fractions 10-11.The ion m/z 1069 and 704 were detected in fractions 14-15.The ion m/z 713 was detected in fractions 7-8.In conclusion, after separated by Sephadex G-50, the fraction with high antioxidant activity was composed of peptides with low molecular weight, and the potential high activity peptides shared the same MALDI-TOF/MS fragments, namely m/z 1048, 1466, 1195, 853, 2382, 1744, 701, 1069, 704 and 713.For TCC, the ion m/z 1466 was detected in fractions 8-11.The ion m/z 2382 was detected in fractions 8-10.The ion m/z 1744 was detected in fractions 9-11.The ion m/z 1105 was detected in fractions 8-9 and 11.The ion m/z 1077 was detected in fractions 12-13.The ion m/z 779 was detected in fractions 10-11.The ion m/z 770 was detected in fractions 8-9.The ion m/z 688 was detected in fractions 14-15.In conclusion, after separated by Sephadex G-50, the fraction with high antioxidant activity was composed of peptides with low molecular weight, and the potential high activity peptides shared the same MALDI-TOF/MS fragments, namely m/z 1466, 2382, 1744, 1105, 1077, 779, 770 and 668.It is notable that the same TOF/MS fragments of m/z 1466, 1744 and 2382 derived from the antioxidant peptides were both present in ACC and TCC samples, indicating that the m/z 1466, 1744 and 2382 may serve as a material basis for the antioxidant activity of both ACC and TCC, and contributed to their traditional tonic effects.

Experimental (see supplementary material)
All details are provided in the supplementary material.

Conclusions
Here, we focused on the authentication and antioxidant peptides of ACC and TCC.The results showed that UPLC-MS/MS technology could effectively identify specific peptides to establish the authenticity of ACC and TCC, which may provide a new research idea for the authentication of other drugs.It was also found that ACC and TCC had significant antioxidant activity, as well as the ability to induce the PI3K-AKT pathway, and the peptide with m/z 1466, 1744 and 2382 may be the main contributors to these effects.These findings provided a foundation for further study on antioxidant active substances of GCMs and a reference for improving the development and application of healthy food containing ACC.