Structural characterization of a new amino acid derivatives, Maillard Reaction product of red ginseng and potential protective activity against cisplatin-evoked intestinal injury

Structural characterization of a new amino acid derivatives, Maillard Reaction product of red ginseng and potential protective activity against cisplatin-evoked intestinal injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Structural characterization of a new amino acid derivatives, Maillard Reaction product of red ginseng and potential protective activity against cisplatin-evoked intestinal injury Wei Liu, Yi-nan Zheng, Shuang Jiang, Shen Ren, Shan Tang, Jing Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2627146/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Based on the Maillard reaction principle of red ginseng ( Panax ginseng C.A. Meyer), this study innovatively synthesized a new amino acid derivative by combining arginine with lactose through simulated synthesis, and was separated and purified through repeated silica gel and polyacrylamide gel (Bio-gel P-II) column chromatography. The purity of the product was determined to be 99.86% and its molecular weight was determined to be 497.3612 (negative ion mode) by electrospray ionisation mass spectrometry (ESI-MS). The chemical structure was identified to be 1-(arginine-N α group)-1-deoxy-4-O-(α- D -galactopyranosyl)- D -fructose, named Argininyl-fructosyl-galactose (AFGA, C 18 H 34 N 4 O 12 ). Subsequently, by establishing cisplatin-induced intestinal injury in vivo and IEC-6 cell model, the results showed that pretreatment with AFGA significantly ameliorated cisplatin induced oxidative stress by reducing levels of reactive oxygen species (ROS) in IEC-6 cells ( p <0.05, p <0.01), and could effectively reduce the secretion of pro-inflammatory factors in serum and the expression level of NF-κB protein in intestinal tissues ( p <0.01). Meantime, AFGA also inhibited the expression of p-PI3K/p-Akt, caspase 3, 9, cytochrome C and Bax protein intestinal tissue in mice ( p <0.01), and promoted the expression of Bcl-2 protein ( p <0.01). Importantly, the molecular docking results of AFGA also suggested a better binding ability with the above-mentioned related target proteins, and further revealed AFGA as a potential multifunctional therapeutic agent with clear protective effect against cisplatin-induced intestinal injury. Argininyl-fructosyl-galactose structure identification safety evaluation cisplatin intestinal toxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1 Introduction Maillard reaction (MR), also called non-enzymatic browning reaction, the main reaction in food processing and storage (Li and Liu, 2022 ), an important ingredient in the food industry due to its changes in colour, aroma and nutritive value of Maillard reaction products (MRPs)(Nooshkam et al., 2019a). MR are a series of sequential and complex reactions starting with the condensation of carbonyl compounds (usually reducing sugars) and amino compounds such as proteins, peptides, and amino acids (Silvan et al., 2006). This spontaneous chemical reaction involves the covalent attachment (aldol condensation) of protein with carbonyl groups from reducing sugars, as well as the formation of Schiff bases and Amadori compounds (Anton et al., 2012). Amino acid derivatives are often studied in model systems consisting of reducing sugars and amino acids, are also an Amadori compound (Chen et al., 2019). In recent years, there has been growing interest in the development of amino acid derivatives because of the increasingly prominent application of MR in newly-type functional foods (Silvan, van de Lagemaat, 2006). This process is common in medicinal plants rich in amino acids and reducing sugars, such as P. ginseng , P. notoginseng and Cibotium barometz . Particularly, ginseng contains 17 kinds of amino acids and high contents of glucose and maltose, which provides the material basis for the MR (Du et al., 2012). The process conditions such as steaming and drying in the process of red ginseng also provide sufficient external conditions for the reaction. Simultaneously, milk is rich in lactose, and lactose also reacts primarily with the free amino groups of the milk proteins to proceed to the early, intermediate, and advanced stages of the MR, and forms enormous amounts of MRPs (Shimamura et al., 2011, van Boekel, 2006 ). The reactions of lactose have therefore frequently been investigated and many researchers have demonstrated the potential of lactose during the MR-related processing field. Generally, the steaming and heating treatment process of ginseng will be accompanied by MR, especially ginseng is rich in amino acids and reducing sugars, which will produce amino acid derivatives. However, since Amadori compound is a primary product of MR, it has the characteristics of small polarity, easy solubility and instability. The production process of amino acid derivatives in red ginseng can be well represented by the simulated synthesis method. Previous research has shown that at a certain temperature, arginine with glucose or maltose in glacial acetic acid are the reaction media, and when pH < 3.0, there will be obvious browning reaction (Du, Liu, 2012), and through Amadori rearrangement to produce Arginyl-fructose (AF) and Arginyl-fructose–glucose (AFG)(Kim et al., 2010). the production and content of these compounds are closely related to the ginseng processing (Matsuura et al., 1994). It’s worth noting that the MR also has inevitable connection with the types of reducing sugars, glucose and galactose are epimers, maltose is composed of two glucoses, and lactose is composed of galactose and glucose, this revealed the lactose in potential application of MR. There is increasing evidence that red ginseng or their active components have conspicuous protective effects on the side effects caused by cisplatin(Zhang et al., 2020), especially the low polar substances, such as AFG and maltol in red ginseng are the most representative(Li et al., 2019, Mi et al., 2019, Xing et al., 2022). Compared with ginsenosides(Li et al., 2021), non-saponin ingredients contains fewer sugar and lower polarity, which is more conducive to human intestinal absorption. In addition, previous studies have reported that AFG has a protective effect on cisplatin-induced kidney injury through anti-oxidant stress, anti-inflammatory and anti-apoptosis pathway(Li, Zhang, 2019). Simultaneously, AF and AFG in red ginseng also have a variety of nutritional value and pharmacological effects such as immune modulatory activities (Kim, Lee, 2010 , Yu et al., 2021), anti-hyperglycemic(Ha et al., 2011), anti-diabetes and its complications (Liu et al., 2020a) and antioxidant properties (Ide et al., 1999) in vitro and in vivo . With the increasing recognition of the various therapeutic effects of MRPs, amino acid derivatives as a major non-saponin health food or alternative medicine needs to be thoroughly evaluated. Based on the above facts, this study combined arginine in red ginseng with lactose for the first time to synthesize novel amino acid derivatives, and then carried out separation and purification, structural identification and safety evaluation, as well as investigate the protective activity of action and effect of AFGA against cisplatin-induced intestinal toxicity in vivo and in vitro. 2 Materials And Methods 2.1 Chemicals and Reagents L-arginine (CAS. 74-79-3), D-(+)-Maltose (CAS. 6363-53-7), D-Lactose (CAS. 64044-51-5) and acetic acid (CAS. 64-19-7) monohydrate were provided from aladdin biochemical reagent Co. Ltd. (Shanghai, China). RHAWN001 column-layer chromatographic silica gel, polyacrylamide gel (Bio-gel P-II) column chromatography and ethanol absolute were provided from Shanghai Yi-en Chemical Technology Co., Ltd (Shanghai, China). Cisplatin (Pt > 99%) was provided from Sigma-Aldrich (St. Louis, MO, USA). Hematoxylin and eosin (H&E), Glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). dimethyl methylene sulfone (DMSO) were provided from Sigma-Aldrich (St Louis, MO, USA). ROS staining kit were provided from Wan-lei Biotechnology, (Shenyang, China). The ELISA kits of mouse TNF-alpha (TNF-α), IL-1 beta (IL-1β) and Diamine Oxidase (DAO) were provided from R&D systems (Minneapolis, MN, USA). Hoechst 33258 dye kits were provided from Beyotime Co., Ltd. (Shanghai, China). Immunofluorescence staining and SABC Daylight488-labeled secondary antibodies were provided by BOSTER Bio-Engineer Co., Ltd. (Wuhan, China). Primary antibodies against caspase 3, cleaved-caspase 3, caspase 9, cleaved-caspase 9, PI3K (p85), Akt, p-PI3K (p-p85), p-Akt, and phospho-NF-κB (p-NF-κB, p-p65) were provided from Cell Signaling Technology (Danvers, MA, USA), and the antibodies for Bax, Bcl-2 and β-actin were provided by Abcam (Cambridge, U.K.). 2.2 Chemical synthesis and purity determination of novel amino acid derivatives The novel amino acid derivatives are prepared according to the MR mechanism in red ginseng and slightly modified according to the previous AFG synthesis method (Li, Zhang, 2019). In brief, the method amino acid derivatives was synthesized by heating the dissolving of L-arginine (2.0g) with Lactose (4.0g) in 20 mL of glacial acetic acid in a water bath at 85 ◦C for 120 min. After the reaction, the novel amino acid derivatives crude product was obtained by vacuum drying (50 ◦C) and alcohol precipitation with absolute ethanol. Moreover, the crude product of amino acid derivatives (mixed with 100–200 mesh silica gel) was appended to the RHAWN001 column-layer chromatographic silica gel (200 ~ 300 mesh) with 1/5 times of silica gel volume (Matsuura, Zheng, 1994) and eluted by the with 70% ethanol-water solution, using thin-layer chromatography (TLC) for tracking target ingredient. The developing solvent is n-butanol: water: acetic acid = 2:1:1, and the color reaction is carried out with 0.2% ninhydrin ethanol solution. The component was collected with value of the retention factor (Rf) of 0.174. A portion of ethanol was removed by a rotary evaporator, and then the lyophilized powder was diluted with sterile water to a 0.5 g/mL super polyacrylamide gel column (Biogel P-II). The amino acid derivatives single point was collected and freeze-dried. Chromatographic analyses were performed on Waters e2695 (Waters, USA), coupled with an ELSD instrument (Shimadzu Technologies ELSD-16, Japan). The drift tube temperature for the ELSD was set at 120 ◦C with a nitrogen flow rate of 1.8 L/min, Yue-Xu Ultimate® amino acid plus amino acid column (4.6 mm×300 mm, 5µm), The column temperature was set at 30°C. The flow rate was 1.0 mL/min, and the injection volume was 20 µL. The mobile phase consisted of 0.3% heptafluorobutyric acid solution (A) and acetonitrile (B) with gradient elution: 0 ~ 10 min, 100% A; 10 ~ 15 min, 100%~93% A; 15 ~ 40 min, 93%~50% A; 40 ~ 41 min, 50%~100% A, 41 ~ 60 min, 100% A. 2.3 Structural characterization of AFGA The nuclear magnetic resonance ( 1 H-NMR and 13 C-NMR), and heteronuclear multiple-bond connectivity (HMBC) and heteronuclear single quantum correlation (HSQC) spectra were measured using a Bruker AV600 NMR spectrometer (Bruker Co., Karlsruhe, Germany; 600 MHz for 1 H and 150 MHz for 13 C) with tetramethylsilane as an internal standard. Chemical shifts (δ) are expressed in ppm, with the coupling constants ( J ) reported in Hertz (Hz). The electrospray ionization mass spectrometry (ESI-MS) was recorded using an Agilent 1200 HPLC with a 6300 lon-trap liquid chromatography/mass spectrophotometry (LC/MS; Agilent Technologies; ionization mode, negative; nebulizing gas [N 2 ] pressure, 35 psi; drying gas [N 2 ] flow, 8 L/min; temp, 350ϘC) and Q-Exactive mass spectrometer (Thermo Scientific, Bremen, Germany). 2.4 Culture of rat intestinal crypt epithelial (IEC-6) cells The IEC-6 cells were maintained in DMEM medium supplemented with 10% FBS, 1% penicillin/streptomycin in humidified atmosphere of 5% CO 2 at 37°C. For passaging, the medium was changed every 48 h until the cells reached 80 ~ 90% confluent. Cell viability was detected using MTT assays. The cells were seeded in a 96-well culture plates and cultured at 37°C for 24 h, then the cells were pretreated with cisplatin (0.25, 0.5, 1, 2, 4, 8, 16, 32 µM) for 24 h, and then 20 µL MTT solution (5 mg/mL) was added to each well, after incubating for 3.5 h at 37°C, the culture supernatant was discarded, 150 µL DMSO was added to each well and shake for 10 min. Finally, the absorbance was measured at 490 nm using a microplate reader (Nano, Germany), and cisplatin of appropriate concentration were selected. Similarly, the cells were seeded into 96-well culture plates and the cells were pretreated with AFGA at 3.125, 6.25, 12.5, 25, 50, 100 and 200 µM for 24 h, and then exposure to cisplatin (1.5 µM) or not for 24 h, MTT was used to detect cell survival. 2.5 ROS staining The relative levels of intracellular ROS were determined by a fluorometric assay (DCF-DA assay) as previously described with some modifications (Hu et al., 2021a). IEC-6 cells were seeded in 6-well culture plates, and the supernatants were discarded after AFGA and cisplatin treatment, the plates were rinsed two times with PBS buffer, and incubate with 1.0 µM DCFH-DA at 37°C in the dark after 30 min, the culture medium was removed, washed twice with PBS, and the fluorescence intensity of ROS was observed using a Leica microscope (Leica TCS SP8, Solms, Germany). 2.6 Animals and experiments design 40 males ICR mice, weighting 22 ~ 25 g, were provided by Changchun YISI Experimental Animals Co., Ltd. (Changchun, Jilin province, China) with a Certificate of Quality (SCXK (JI)-2019-0015). The mice were given a standard laboratory diet and water ad libitum and maintained at 12 h light/dark cycle at constant temperature (22 ± 2°C). All experimental animals’ processing project were strictly performed according to the Guide for the Care and Use of Laboratory Animals (2016). All the animal experiments were carried out consistent with experimental practices and standards, which were authorized by Ethical Committee of Jilin Agricultural University (Permit Number: JLAU-21-003). After acclimation for one week, the mice were randomly divided into 4 groups (n = 10): Normal group, AFGA (100mg/kg), cisplatin group, cisplatin + AFGA (100 mg/kg) respectively. Since there is currently no therapeutic agent for cisplatin-induced intestinal injury in the clinic, a group of positive drug was not set up in the present work. Mice were administered with AFGA by intragastric administration once daily for 10 days. All mice were allowed to take water freely during the experiment. On the 7th day of treatment, intestinal injury was induced by intraperitoneal injection of cisplatin (20 mg/kg), except for normal group, 1 h after the final AFGA treatment. The experiment was terminated 72 h after injection of cisplatin. Body weights, blood and organ samples were collected immediately. 2.7 Biochemical parameters determination The intestine tissues used to estimate the antioxidant activities (Zhang, Wang, 2020). Lipid peroxides were measured by MDA kits, and CAT activity, and GSH and SOD content were determined by related assay kits according to the manufacturer’s instructions. Serum was separated by centrifugation at 1000 g for 10 min twice from blood collection. According to the instructions of the manufacturer, samples were added into a 96-well plate coated with antibodies specific for mouse DAO, TNF-α and IL-1β, according to the manufacturer in ELISA reader are specified in the protocol provided under 450 nm conditions. 2.8 Histopathological staining Duodenum was collected and then washed with phosphate-buffered saline. The tissues were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, cut into 5 µm thickness. The histopathological changes were mounted with neutral gum and representative images were captured used light microscope (Olympus BX-60, Tokyo, Japan). In order to evaluate the apoptosis in intestinal cells in vivo and vitro , apoptotic cells were measured by Hoechst 33258 staining, as described above (Ma et al., 2017). Liver tissues were fixed were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, cut into 5 µm thickness, and stained using the Hoechst 33258 solution with 10 µg/mL. Hoechst 33258 staining of nuclei under ultraviolet excitation was observed and photographed under a fluorescence microscope (Leica DM750, Germany). Quantification of apoptosis by Image-Pro plus 6.0. Immunofluorescence staining for evaluating the degree of inflammation in intestinal tissue, duodenum tissues were incubated with p-NF-κB antibody (1:300) in a humidified chamber at 4°C overnight, followed by SABC-Dylight448-labeled secondary antibody (BOSTER Biological Technology, Wuhan, China) incubation for 30 min at 37°C. Nucleus in testicular tissues was carried out using 4, 6 diamidino-2-phenylindole (DAPI) staining. The degree of inflammation in intestine tissues observed under a fluorescence microscope (Leica TCS SP8, Germany). 2.9 Molecular docking studies The molecular structures of AFGA were drawn by Chem Bio Draw. The 2D structure was processed and transformed into a three-dimensional (3D) structure by Chem Bio 3D Ultra 14.0.0.117, and the MM2 algorithm was used for energy minimization. PyMOL was used to remove the water molecules and organic matter of the target protein receptor. AutoDockTools-1.5.6 was used to add the nonpolar hydrogen and calculate Gasteiger charges for the structure and save it as PDBQT file. AutoDock Vina was run for virtual docking It is generally accepted that the lower the energy is, the more likely the binding is to occur. Finally, the conformation with the best affinity was selected as the final docking conformation and visualized in PyMOL. 2.10 Western blot analysis Firstly, the intestine tissues were cracked by using Radio Immunoprecipitation Assay (RIPA) buffer, and the total protein concentrations (Liu et al., 2020b) were measured using a BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). The proteins (50-µg) were separated with 12%SDS polyacrylamide gels and transferred to a polyvinylidene difluoride (PVDF) membrane. Then, the membrane was blocked using 5% non- loaded on the 12% SDS-PAGE and transferred onto a PVDF membranes. And then blocked with 5% (w/v) skim milk in Tris-buffered saline (TBS) with 0.1% Tween-20 for approximately 2 h, and incubated overnight at 4°C with primary antibodies Subsequently, membranes were incubated with the HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the protein band intensities were assessed with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). The immunoreactive bands were quantified via densitometry using Image J (Version 1.8.0, National Institutes of Health, USA) and standardized to β-actin and were expressed as fold changes relative to the normal value. 2.11 Statistical analysis All data were expressed as the mean ± standard deviation (Mean ± S.D.) and analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post-test. Statistical graphs were performed using GraphPad Prism 8.0 software (San Diego, CA, USA). p < 0.001, p < 0.01 or p < 0.05 were considered to be significant. 3 Results 3.1 Synthesis and structural characterization of AFGA According to the chemical mechanism of initial MR, AFG was obtained from L-arginine with maltose under acidic condition by chemical synthesis. Similarly, maltose and lactose are epimers (Fig. 1 A). In this study, a novel amino acid derivative was synthesized from L-arginine and lactose by simulating the synthesis method of AFG, and then purified and separated by column chromatography of silica gel and Bio-gel P-II column chromatography. After HPLC-ELSD analysis, a novel amino acid derivatives purity was 99.86% (Fig. 1 B-C). The novel amino acid derivative was obtained as a white amorphous powder, easily soluble in water, methanol, ethanol, etc., with a melting point (mp) of 158 ~ 160°C. By normal-phase silica gel TLC (n-butanol: water: acetic acid = 2:1:1), the color reaction of 0.2% ninhydrin ethanol solution was purple. The molecular weight was determined as 497.3612 by ESI-MS (Fig. 2 B). The combination of 13 C-NMR (Supplementary Fig. 1) and HMBC showed eighteen carbons, and the specific signal attribution was shown in Fig. 2 A and Table 1 . It was found that the sugar group has 12 carbons, δ 101.38 and δ 96.19 showed two sugar groups. The 1 H-NMR spectrum (Supplementary Fig. 2) showed [ δ H 3.510 (1H, dd), 3.590 (1H), 3.725 (1H, dd), 3.82 (1H, d), 4.03 (1H, dd), 2.75 (2H, d, J = 6.6Hz)]; [δppm 3.232 (1H), 3.518 (1H, d), 3.775 (1H, d), 2.95 (1H, dd), 4.24 (1H, dd), 3.483 (2H, d, J = 6.6 Hz]. It could be inferred that this compound contains twelve -OH single bonds in the sugar group, further utilized the HMBC spectrum, the following coupled relationship can be observed C-1′ δ 4.47 (1H, d, J = 7.8 Hz), C-6 δ H 3.84 (1H, d, J = 3.0 Hz), C-1′′ δ H 3.92 (1H, d, J = 12.0Hz), C-2′ ( δ C 96.19 ), H-1′′ ( δ H 5.04, d, J = 7.7 Hz ), C6′′-glc (δ H 3.64) and C-4′′ (δ C 70.52). In 1 H-NMR, J 1, 2 < 7 Hz, and α-glucose is enzymatically hydrolyzed to yield galactose and glucose. Therefore, it’s inferred that the connection sequence of the novel amino acid derivatives was: arginine-fructose-galactose, and molecular formula was C 18 H 34 N 4 O 12 . Table 1 13 C-NMR chemical shifts of novel amino acid derivatives in D 2 O C δppm C δppm C δppm 1 173.50(s) 1’ 53.13(t) 1” 101.51(d) 2 63.15(d) 2’ 96.17(s) 2’’ 70.48(d) 3 27.35(t) 3’ 69.74(d) 3’’ 73.55(d) 4 24.73(t) 4’ 78.35(d) 4’’ 70.52(d) 5 41.26(t) 5’ 70.07(d) 5’’ 73.22(d) 6 157.59(s) 6’ 64.73(t) 6’’ 61.36(d) Based on previous reports of AFG studies, The 13 C-NMR structure of fructose contains configurations such as α-furan, β-pyran and β-furan. The fructose in this compound was produced from glucose by Amadori rearrangement. The 13 C-NMR and 1 H-NMR spectrum showed C-1' of fructose is linked to the α-amino nitrogen atom of arginine to form a C-N bond. Its structure was determined as: 1-(arginine-Nα group)-1-deoxy-4-O-(α-D-galactopyranosyl)-D-fructose (Fig. 2 C). named as Argininyl-fructosyl-galactose (AFGA) (Fig. 2 D). The synthesis mechanism of AFGA is shown in Fig. 3 . 3.2 Activity screening of AFGA and protective activity of cisplatin-induced intestinal injury In this study, the activity of AFGA was screened through cell experiments to establish the injury of IEC-6 cells by cisplatin, and clarify the pharmacodynamic effect of AFGA. As shown in Fig. 4 A-D, AFGA application increased the viability of IEC-6 cells in a dose-dependent manner after 24 h exposure to cisplatin (1.0 µM), AFGA with concentration range from 3.125 to 200 µM exerted conspicuous protective effect on cell viability ( p < 0.05 or p < 0.01). According to the MTT assay results, 25, 50 and 100 µM was chosen for the subsequent experiments. As shown in Fig. 5 A-B, after single injection of cisplatin (20 mg/kg) caused a noticeable body weight loss, and the morphology of the intestinal tissue was observed to become white and thin. Meantime, histopathological examination staining showed the intestinal tissue injury was serious, and the villi of intestinal wall were uneven, cell degeneration, part of the villi atrophy, shedding and disappearance, glands and crypts disappear in cisplatin group. However, AFGA treatment prevented the changes in body weight and histomorphology were significantly improved at the doses of 100 mg/kg, and AFGA (100mg/kg) was not toxic in mice (Fig. 5 C-D). Moreover, compared with the cisplatin group, the abnormal increase of DAO activity in serum was significantly improved after AFGA pretreatment ( p < 0.05), indicating that AFGA could effectively maintain the integrity of intestinal barrier function (Fig. 5 E). 3.3 AFGA ameliorated oxidative stress and inflammation response in cisplatin-induced intestinal injury in vivo and in vitro Since cisplatin was prone to cause oxidative damage to the body, this study tested some oxidative indicators to verify the effect of AFGA supplementation on cisplatin-induced oxidative stress. As shown in Fig. 5 F-I, cisplatin exposure resulted in prominent increased MDA content, and significantly decreased GSH, SOD and CAT levels in intestinal homogenates compared with the normal group ( p < 0.01). Interestingly, AFGA pretreatment dramatically reverse these changes ( p < 0.05). To further explore the activity of AFGA ameliorated cisplatin-induced intestinal oxidative injury, we also studied the production of ROS in IEC-6 cells. As shown in Fig. 5 J, the expression of ROS in cells exposed to cisplatin was significantly increased. AFGA could significantly decreased the green fluorescence intensity and decreased the expression of ROS in cells in a dose-dependent manner ( p < 0.05, p < 0.01). The AFGA alone did not cause significant increase of ROS in IEC-6 cells, indicated that AFGA was non-toxic and further supporting the assay results in vivo . Together, these results clearly demonstrated that AFGA was effective in reducing cisplatin-induced ROS accumulation. As shown in Fig. 6 A-B, two pro-inflammatory factors, TNF-α and IL-1β were detected in serum. Compared with the normal group, cisplatin exposure could significantly increase the levels of TNF-α and IL-1β, these changes were reversed after AFGA treatment ( p < 0.05). There was no significant change in AFGA alone group. To directly demonstrated that cisplatin could induce intestinal inflammation in mice, this study used immunofluorescence analysis to evaluated the protein expression of p-NF-κB antibodies. As summarized in Fig. 6 C, there was no significant difference between AFGA alone and normal group, but cisplatin can significantly increase the nucleus and cytoplasm of cisplatin-induced intestinal tissues ( p < 0.01). However, AFGA could antagonize p-NF-κB overexpression ( p < 0.05). 3.4 AFGA ameliorated cisplatin-induced intestinal cell apoptosis To evaluated the effect of AFGA on the apoptosis of intestinal cells induced by cisplatin. Hoechst 33258 staining (Fig. 6 F) has shown nuclei intense fluorescence chromatin condensation and nuclear shrinkage were clearly observed in cisplatin group ( p < 0.01). Interestingly, administration of AFGA showed normal regular outline of intestinal epithelium and remarkably reduced apoptosis ( p < 0.05). In cultured IEC-6 cells, Hoechst 33258 staining (Fig. 6 E) also showed that after AFGA pretreatment, nuclear agglutination and abundant bright red or blue fluorescence were observed, which was consistent with the in vivo results. As shown in Fig. 7 A-B, western blot analysis result confirmed the significant decrease in p-PI3K and p-Akt expression in mice by cisplatin. Inclusion of AFGA diminished the inhibition of cisplatin on p-PI3K and p-Akt protein expressions ( p < 0.05, p < 0.01). Furthermore, the protein expression of the anti-apoptotic protein Bcl-2 in the cisplatin group decreased, and the expression of the pro-apoptotic proteins Bax and cleaved-caspase 3, 9 increased ( p < 0.05, p < 0.01). AFGA could significantly improve the apoptosis of intestinal induced by cisplatin ( p < 0.05, p < 0.01). These results demonstrated that AFGA could inhibit cisplatin-induced intestinal cells apoptosis by regulating the PI3K/Akt signaling pathway. 3.5 Molecular docking of AFGA with target proteins Molecular docking was used to verify if AFGA have a significant role in the regulation of proteins related to apoptosis pathway (PI3K, Akt and caspase 3). The results showed the potential binding capacity of the compounds to core target proteins (Fig. 8 A-C). With the screening criteria of binding energy ≤-6.0 kJ/mol, the results all ranged from − 8.8 to -6.3 kJ/mol which indicated a strong and table binding between the compound and the protein. Compared with AFG, AFGA has stronger binding ability to apoptosis-related target proteins (Fig. 8 D-G). These results showed that AFGA had a good medicinal reference value, and exerted higher pharmacological activity than AFG. 4 Discussion Amino acid derivatives one of the important products of MR, especially AF and AFG found in the process of red ginseng production are the most active and high content (Kim, Lee, 2010 ). Previous studies have reported that ARPs have high application value in food development and also have a variety of pharmacological effects in vitro and in vivo (Kim and Lee, 2010 , Nooshkam et al., 2019b). Meantime, strengthening the in-depth study of MR of red ginseng and clarifying the synthesis mechanism of amino acids and reducing sugars can well promote the enrichment and development of MRPs (Du, Liu, 2012). Based on the clear production mechanism of AFG, this study innovatively combined lactose with the abundant arginine in red ginseng to synthesize novel amino acid derivatives. The chemical structure was determined to be 1-(arginine-N α group)-1-deoxy-4-O-(α- D -galactopyranosyl)- D -fructose, named AFGA. Through activity screening, the medicinal value of AFGA was expounded from the perspective of in vivo and in vitro . In addition, the protective effect of AFGA on cisplatin-induced intestinal injury was revealed from the aspects of oxidative stress, inflammatory response and apoptosis, and it was demonstrated that AFGA can exert intestinal protective activity through PI3K/Akt and downstream caspase pathway. Extensive research suggested that cisplatin can cause the destruction of intestinal mucosal cells and changes in the structure of the intestinal epithelium(Khan et al., 2012, Shahid et al., 2018), which was related to the accumulation of ROS and the generation of oxidative stress(Akhter et al., 2021, Zhao et al., 2022). Meantime, previous studies have established that ROS plays important biological roles in cell homeostasis (Hu et al., 2021b). The main forms of ROS include superoxide, hydrogen peroxide and hydroxyl radicals(Liao et al., 2019, Liu et al., 2022). In current study, the mice single injected with cisplatin (20 mg/kg), resulted in abnormally elevated levels of MDA and decreased GSH, SOD and CAT content of the intestinal tissue in mice, and IEC-6 cell assays also showed that cisplatin treatment excessive accumulation of ROS, and further supporting the assay results in vivo . These were consistent with previously reported results (Hu, Yang, 2021b). Indeed, ROS has been shown to promote the up-regulation of pro-inflammatory cytokines (Wang et al., 2015). This study shown that TNF-α and IL-1β in the serum of mice significantly increase, and immunofluorescence analysis also indicated that AFGA could antagonize p-NF-κB overexpression and play an important role in cisplatin-induced intestinal toxicity. On the other hand, ROS accumulation also feeds back into the apoptosis pathway, reduce ROS production may inhibits intestinal cell apoptosis and promotes its differentiation (Maradagi et al., 2022). When a large amount of ROS accumulated it increased mitochondrial permeability, stimulated pro-apoptotic factor release and initiated apoptosis (Liu et al., 2021). Consistently, this study indicated that Hoechst 33258 staining and western blot analysis to further detect apoptosis both in mice and in IEC-6 cells. Compared with the cisplatin group, AFGA significantly reduced the pro-apoptotic cytochrome C, cleavage caspases 3, 9 and Bax proteins intestinal tissue in mice, and promote the expression of Bcl-2 protein. Previous research has shown that targeting PI3K/Akt by chemosensitizer may improve the anticancer efficacy of cisplatin and decrease the side-effects (Mi et al., 2018), and the abnormal activation of PI3K/Akt is generally associated with the development of drug resistance (Zhang et al., 2021). Consistent with these results, the current study demonstrated that AFGA treatment can significantly reverse these apoptosis-related proteins expression. Meantime, this study also performed molecular docking experiments on the key proteins in PI3K/Akt signaling pathway and found that AFGA (binding energy ≤-7.7 kJ/mol) has stronger binding ability to apoptosis-related proteins than AFG (binding energy ≤-6.5 kJ/mol). These study demonstrated the protective effect of AFGA on cisplatin-induced intestinal injury. In conclusion, based on the MR principle of red ginseng, this study innovatively synthesized a novel amino acid derivative by combining arginine in red ginseng with lactose by the method of simulated synthesis, the structure was determined to be 1-(arginine-N α group)-1-deoxy-4-O-(α- D -galactopyranosyl)- D -fructose, named AFGA. Through in vitro and in vivo experiments, it was shown that AFGA has good intestinal injury protection activity. Abbreviations AFGA , Argininyl-fructosyl-galactose; AFG, Arginyl-fructose-glucose; Akt (PKB) , Protein Kinase B; Bcl-2 , B-cell-lymphoma-2; CAT , Catalase; DAO , Diamine oxidase; ESI-MS , electrospray ionization tandem mass spectrometry; GSH , Glutathione; HMBC , Heteronuclear multiple-bond connectivity; HSQC , Heteronuclear single quantum correlation; HPLC-ELSD, High performance liquid chromatography-evaporative light scattering detection; IL-1β , Interleukin-1β; MDA , Malondialdehyde; MR, Maillard reaction; MRPs, Maillard reaction products; NF-κB , Nuclear transcription factor-κB; NMR, nuclear magnetic resonance; PI3K , phosphatidylinositol 3-kinase; SOD, Superoxide dismutase, TNF-α , Tumor necrosis factor-α Declarations Conflicts of Interest The authors declare that there are no conflicts of interest. Author Contributions: Wei Li conceived and designed the experiments; Wei Liu and Zi Wang performed the experiments; Shuang Jiang, Shen Ren and Shan Tang contributed analysis tools; Wei Liu wrote the paper. Yi-nan Zheng and Jing Zhang contributed to the improvement of the writing. All authors reviewed and approved the contents of the manuscript. Data Availability The datasets generated and analyzed during the current study are available in the corresponding author upon request. Ethical Approval All experimental animals’ processing project were strictly performed according to the Guide for the Care and Use of Laboratory Animals (2016). All the animal experiments were carried out consistent with experimental practices and standards, which were authorized by Ethical Committee of Jilin Agricultural University (Permit Number: JLAU-21-003). Funding This work was supported by the grants of National Natural Science Foundation of China (No. 82104465), Changchun Science & Technology Development Plan (No. 21ZGY11), Jilin Science & Technology Development Plan (No. 20200301037RQ), and the Scientific Research Planning Project of Jilin Provincial Department of Education (No. JJKH20220371KJ). References Akhter N, Wilson A, Thomas R, Al-Rashed F, Kochumon S, Al-Roub A, et al. ROS/TNF-alpha Crosstalk Triggers the Expression of IL-8 and MCP-1 in Human Monocytic THP-1 Cells via the NF-kappaB and ERK1/2 Mediated Signaling. Int J Mol Sci. 2021;22, 3-12. Anton PM, Craus A, Niquet-Leridon C, Tessier FJ. Highly heated food rich in Maillard reaction products limit an experimental colitis in mice. Food Funct. 2012;3:941-9. Chen K, Zhao J, Shi X, Abdul Q, Jiang Z. Characterization and Antioxidant Activity of Products Derived from Xylose-Bovine Casein Hydrolysate Maillard Reaction: Impact of Reaction Time. Foods. 2019;8, 115-127. 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The protective effects of maltol on cisplatin-induced nephrotoxicity through the AMPK-mediated PI3K/Akt and p53 signaling pathways. Sci Rep. 2018;8:15922. Nooshkam M, Varidi M, Bashash M. The Maillard reaction products as food-born antioxidant and antibrowning agents in model and real food systems. Food chemistry. 2019a;275:644-60. Shahid F, Farooqui Z, Khan F. Cisplatin-induced gastrointestinal toxicity: An update on possible mechanisms and on available gastroprotective strategies. Eur J Pharmacol. 2018;827:49-57. Shimamura T, Kurogi Y, Katsuno S, Kashiwagi T, Ukeda H. Demonstration of the presence of aminoreductone formed during the Maillard reaction in milk. Food Chem. 2011;129:1088-92. Silvan JM, van de Lagemaat J, Olano A, Del Castillo MD. Analysis and biological properties of amino acid derivates formed by Maillard reaction in foods. J Pharm Biomed Anal. 2006;41:1543-51. van Boekel MA. Formation of flavour compounds in the Maillard reaction. Biotechnol Adv. 2006;24:230-3. Wang W, Zhou PH, Xu CG, Zhou XJ, Hu W, Zhang J. Baicalein attenuates renal fibrosis by inhibiting inflammation via down-regulating NF-kappaB and MAPK signal pathways. J Mol Histol. 2015;46:283-90. Xing JJ, Mi XJ, Hou JG, Cai EB, Zheng SW, Wang SH, et al. Maltol mitigates cisplatin-evoked cardiotoxicity via inhibiting the PI3K/Akt signaling pathway in rodents in vivo and in vitro. Phytother Res. 2022;36:1724-35. Yu J, Lee J, Kim T, Kang H, Lee S, Mitiku H, et al. Immune Modulatory Activities of Arginyl-Fructose (AF) and AF-Enriched Natural Products in In-Vitro and In-Vivo Animal Models. Molecules (Basel, Switzerland). 2021;26-42. Zhang JJ, Wang JQ, Xu XY, Yang JY, Wang Z, Jiang S, et al. Red ginseng protects against cisplatin-induced intestinal toxicity by inhibiting apoptosis and autophagy via the PI3K/AKT and MAPK signaling pathways. Food Funct. 2020;11:4236-48. Zhang JJ, Zhou YD, Liu YB, Wang JQ, Li KK, Gong XJ, et al. Protective Effect of 20(R)-Ginsenoside Rg3 Against Cisplatin-Induced Renal Toxicity via PI3K/AKT and NF-κB Signaling Pathways Based on the Premise of Ensuring Anticancer Effect. Am J Chin Med. 2021;49:1739-56. Zhao Q, Shi Q, Zhu Q, Hu Y, Zhang X. A mini-review of advances in intestinal flora and necrotizing enterocolitis. Lett Appl Microbiol. 2022; 75:2-9. Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2627146","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":179439091,"identity":"d388059d-49b7-49e9-a94f-93688abe0048","order_by":0,"name":"Wei Liu","email":"","orcid":"","institution":"Beihua University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Liu","suffix":""},{"id":179439093,"identity":"d72c4326-c109-4afe-9ecc-13244df33921","order_by":1,"name":"Yi-nan Zheng","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yi-nan","middleName":"","lastName":"Zheng","suffix":""},{"id":179439095,"identity":"0c6ecf1f-54c3-4af4-988d-34bdb8fc6e45","order_by":2,"name":"Shuang Jiang","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Jiang","suffix":""},{"id":179439097,"identity":"515a8b44-0949-4871-8fca-b741e5b632be","order_by":3,"name":"Shen Ren","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shen","middleName":"","lastName":"Ren","suffix":""},{"id":179439100,"identity":"e82538f9-e201-4192-b438-d60865b65ca4","order_by":4,"name":"Shan Tang","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Tang","suffix":""},{"id":179439101,"identity":"72e73505-5947-489f-8f23-5f14eaebeddc","order_by":5,"name":"Jing Zhang","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhang","suffix":""},{"id":179439102,"identity":"8f39ec47-ef6d-46be-bb69-74b3037d866f","order_by":6,"name":"Zi Wang","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zi","middleName":"","lastName":"Wang","suffix":""},{"id":179439103,"identity":"28b96d1b-c673-42f0-8c15-c81bdc3df985","order_by":7,"name":"Wei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBACPmYgkfjvv5z98cbGBx+I0cIG0pLAxmzMcOZws+EMorRANSY23Ehvk+YgSgs7j+GDBzxsjI0zHzZIMzDYyek2EHQYj7FBggQPM7N0YoNxAUOysdkBglp4t0kkGEiwsQG1JM9gOJC4jQgt238kJBjw8EgebDjMQ6SWbQwJBxIkJCQYG5uJ1ML/WSKx4YCBAU9iM+MMAyL8ws9/LPHjz4YD9RvYjz//8aHCTo6gFjRgQJryUTAKRsEoGAU4AAAZ+Dw0nyVZoAAAAABJRU5ErkJggg==","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2023-02-25 07:44:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2627146/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2627146/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":33714917,"identity":"e02c9b9c-b52d-42c5-8292-e0eed1afeb32","added_by":"auto","created_at":"2023-03-02 23:49:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":309896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynthesis and HPLC-ELSD analysis of novel amino acid derivatives.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) the chemical structures of maltose and lactose and synthesis of novel amino acid derivatives; (B) HPLC-ELSD analysis of novel amino acid derivatives before and after separation.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2627146/v1/23f40f04985fe86f09020ef8.png"},{"id":33714163,"identity":"6330e097-8d2f-404f-af8f-9f0e9a804352","added_by":"auto","created_at":"2023-03-02 23:41:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":546731,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural identification of novel amino acid derivatives.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) heteronuclear multiple-bond connectivity (HMBC) and (B) ESI-MS analysis of novel amino acid derivatives (negative ion mode); (C) HMBC and \u003csup\u003e1\u003c/sup\u003eH-HMR coupling correlations for novel amino acid derivatives; (D) chemical and 3D structures of novel amino acid derivatives (AFGA).\u003c/p\u003e","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-2627146/v1/f9e7d4a43af3269fd45d6091.png"},{"id":33714916,"identity":"50073899-18ab-4881-8a71-ca1e47f7e1d7","added_by":"auto","created_at":"2023-03-02 23:49:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":119314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of the synthesis mechanism of AFGA.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-2627146/v1/f6163c2dd6d33c7ecadce13b.png"},{"id":33714169,"identity":"d945352c-6980-4bc4-9d7f-5d188d94c3de","added_by":"auto","created_at":"2023-03-02 23:41:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":320215,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivity screening of AFGA. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003ecell viability were detected by MTT assay, cisplatin toxicity dose screening and (B) AFGA toxicity assay; (C) cell viability were detected by MTT assay after treatment with AFGA and cisplatin (1.0 μM) for 24 h in advance and (D)\u003cstrong\u003e \u003c/strong\u003ePretreatment time dependence of AFGA activity. All data were expressed as mean ± S.D. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 or \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with normal group. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 or **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01 compared with cisplatin group.\u003c/p\u003e","description":"","filename":"Onlinefloatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-2627146/v1/89c51e6736112722598eff04.png"},{"id":33714171,"identity":"273f910d-c54c-47af-9aa2-c362bd70802d","added_by":"auto","created_at":"2023-03-02 23:41:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2476987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAFGA ameliorated cisplatin-induced intestinal histomorphological changes and oxidative stress injury.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Effects of AFGA on body weight changes in mice with cisplatin-induced intestinal injury; (B) Effect of AFGA on cisplatin-induced morphological changes in intestinal tissues of mice; (C-D) Representative images revealed intestinal tissue \u0026nbsp;cross-sectional areas in normal, AFGA (100mg/kg), cisplatin (20 mg/kg), cisplatin + AFGA (100 mg/kg) groups (H\u0026amp;E ×200); (E) Serum DAO activity; (F) Oxidative stress in intestinal tissue of related markers MDA; (G) GSH; (H) SOD; (I) CAT; (J) Effects of AFGA with different concentrations (25, 50 and 100 μM) on ROS generation in cisplatin-induced IEC-6 cells (200×) and relative fluorescence intensity. All data were expressed as mean ± S.D. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 or \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with normal group. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 or **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01 compared with cisplatin group.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2627146/v1/d8544799fbed39039244fd33.png"},{"id":33714168,"identity":"25f223b8-8268-4ea9-9048-95a7ffcca5b5","added_by":"auto","created_at":"2023-03-02 23:41:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":586217,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAFGA ameliorated cisplatin-induced intestinal injury by alleviating ROS-mediated apoptosis and inflammation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B)\u003cstrong\u003e \u003c/strong\u003eSerum TNF-α and IL-1β levels; \u003cstrong\u003e(C) \u003c/strong\u003eFluorescent immunostaining of p-NF-κB (red) in tissue section and quantification of fluorescence intensity. Representative immunofluorescence images were taken at 200 ×, 6-Diamidino-2-phenylindole (DAPI) (Blue) acted as a nuclear counterstain; (E) Hoechst 33258 staining of IEC-6 cells (200×) and relative fluorescence intensity; (F) Analysis of apoptosis in intestinal tissues were evaluated by Hoechst 33258 and the relative levels of fluorescence intensity. All data were expressed as mean ± S.D. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 or \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with normal group. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 or **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01 compared with cisplatin group.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2627146/v1/e49874d63e24bae03d34147e.png"},{"id":33714167,"identity":"c7bed493-e428-4b3f-aadf-8ebda25b6b2a","added_by":"auto","created_at":"2023-03-02 23:41:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":467147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAFGA ameliorated cisplatin-induced intestinal injury \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e PI3K/Akt and downstream Caspase apoptosis pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B)\u003cstrong\u003e \u003c/strong\u003eWestern blots analysis for protein levels of PI3K, p-PI3K, Akt, p-Akt, Bax, Bcl-2, Cytochrome C, caspase 3, cl-caspase 3, caspase 9 and cl-caspase 9 in intestinal tissues and quantification of relative protein expression. All data were expressed as mean ± S.D. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 or \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with normal group. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 or **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01 compared with cisplatin group.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-2627146/v1/45db9a43b552e6ee65a7545b.png"},{"id":33714172,"identity":"4fecfbd5-8986-4bee-9bfc-26795a27fbbf","added_by":"auto","created_at":"2023-03-02 23:41:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2892370,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding of AFGA interacting with target proteins related to apoptosis pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe binding mode of AFGA for the proteins of PI3K (A), Akt (B), Caspase 3 (C) and the binding of AFG for the proteins of PI3K (D), Akt (E), Caspase 3 (F). Different colors represents the associated apoptosis-related protein, and the yellow dotted line represents the hydrophobic interactions between the compound and the associated protein. The scoring of binding force between compound and protein (G).\u003c/p\u003e","description":"","filename":"Onlinefloatimage17.png","url":"https://assets-eu.researchsquare.com/files/rs-2627146/v1/86b39d7303d369912666e700.png"},{"id":33714918,"identity":"5fa6f7d2-9498-4dea-9c15-57816116a0b9","added_by":"auto","created_at":"2023-03-02 23:49:38","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2024471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe molecular mechanism of AFGA antagonizing cisplatin-induced intestinal toxicity.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-2627146/v1/cab1cd51e3acaff455bd49c3.png"},{"id":33991871,"identity":"d4cd07e5-15c3-44b7-97d2-d8f9cd56ecb7","added_by":"auto","created_at":"2023-03-09 03:44:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3943158,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2627146/v1/cba7aec3-238a-4d57-8303-c966c75c7b0e.pdf"},{"id":33714164,"identity":"24ab4ab7-fbeb-470f-ad00-5049193fab0f","added_by":"auto","created_at":"2023-03-02 23:41:38","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":982364,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2627146/v1/ef93a6e18b14a329e25cb387.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structural characterization of a new amino acid derivatives, Maillard Reaction product of red ginseng and potential protective activity against cisplatin-evoked intestinal injury","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eMaillard reaction (MR), also called non-enzymatic browning reaction, the main reaction in food processing and storage (Li and Liu, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), an important ingredient in the food industry due to its changes in colour, aroma and nutritive value of Maillard reaction products (MRPs)(Nooshkam et al., 2019a). MR are a series of sequential and complex reactions starting with the condensation of carbonyl compounds (usually reducing sugars) and amino compounds such as proteins, peptides, and amino acids (Silvan et al., 2006). This spontaneous chemical reaction involves the covalent attachment (aldol condensation) of protein with carbonyl groups from reducing sugars, as well as the formation of Schiff bases and Amadori compounds (Anton et al., 2012). Amino acid derivatives are often studied in model systems consisting of reducing sugars and amino acids, are also an Amadori compound (Chen et al., 2019). In recent years, there has been growing interest in the development of amino acid derivatives because of the increasingly prominent application of MR in newly-type functional foods (Silvan, van de Lagemaat, 2006). This process is common in medicinal plants rich in amino acids and reducing sugars, such as \u003cem\u003eP. ginseng\u003c/em\u003e, \u003cem\u003eP. notoginseng\u003c/em\u003e and \u003cem\u003eCibotium barometz\u003c/em\u003e. Particularly, ginseng contains 17 kinds of amino acids and high contents of glucose and maltose, which provides the material basis for the MR (Du et al., 2012). The process conditions such as steaming and drying in the process of red ginseng also provide sufficient external conditions for the reaction. Simultaneously, milk is rich in lactose, and lactose also reacts primarily with the free amino groups of the milk proteins to proceed to the early, intermediate, and advanced stages of the MR, and forms enormous amounts of MRPs (Shimamura et al., 2011, van Boekel, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The reactions of lactose have therefore frequently been investigated and many researchers have demonstrated the potential of lactose during the MR-related processing field.\u003c/p\u003e \u003cp\u003eGenerally, the steaming and heating treatment process of ginseng will be accompanied by MR, especially ginseng is rich in amino acids and reducing sugars, which will produce amino acid derivatives. However, since Amadori compound is a primary product of MR, it has the characteristics of small polarity, easy solubility and instability. The production process of amino acid derivatives in red ginseng can be well represented by the simulated synthesis method. Previous research has shown that at a certain temperature, arginine with glucose or maltose in glacial acetic acid are the reaction media, and when pH\u0026thinsp;\u0026lt;\u0026thinsp;3.0, there will be obvious browning reaction (Du, Liu, 2012), and through Amadori rearrangement to produce Arginyl-fructose (AF) and Arginyl-fructose\u0026ndash;glucose (AFG)(Kim et al., 2010). the production and content of these compounds are closely related to the ginseng processing (Matsuura et al., 1994). It\u0026rsquo;s worth noting that the MR also has inevitable connection with the types of reducing sugars, glucose and galactose are epimers, maltose is composed of two glucoses, and lactose is composed of galactose and glucose, this revealed the lactose in potential application of MR.\u003c/p\u003e \u003cp\u003eThere is increasing evidence that red ginseng or their active components have conspicuous protective effects on the side effects caused by cisplatin(Zhang et al., 2020), especially the low polar substances, such as AFG and maltol in red ginseng are the most representative(Li et al., 2019, Mi et al., 2019, Xing et al., 2022). Compared with ginsenosides(Li et al., 2021), non-saponin ingredients contains fewer sugar and lower polarity, which is more conducive to human intestinal absorption. In addition, previous studies have reported that AFG has a protective effect on cisplatin-induced kidney injury through anti-oxidant stress, anti-inflammatory and anti-apoptosis pathway(Li, Zhang, 2019). Simultaneously, AF and AFG in red ginseng also have a variety of nutritional value and pharmacological effects such as immune modulatory activities (Kim, Lee, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Yu et al., 2021), anti-hyperglycemic(Ha et al., 2011), anti-diabetes and its complications (Liu et al., 2020a) and antioxidant properties (Ide et al., 1999) in \u003cem\u003evitro\u003c/em\u003e and in \u003cem\u003evivo\u003c/em\u003e. With the increasing recognition of the various therapeutic effects of MRPs, amino acid derivatives as a major non-saponin health food or alternative medicine needs to be thoroughly evaluated.\u003c/p\u003e \u003cp\u003eBased on the above facts, this study combined arginine in red ginseng with lactose for the first time to synthesize novel amino acid derivatives, and then carried out separation and purification, structural identification and safety evaluation, as well as investigate the protective activity of action and effect of AFGA against cisplatin-induced intestinal toxicity in \u003cem\u003evivo\u003c/em\u003e and in \u003cem\u003evitro.\u003c/em\u003e\u003c/p\u003e"},{"header":"2 Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Chemicals and Reagents\u003c/h2\u003e\n \u003cp\u003eL-arginine (CAS. 74-79-3), D-(+)-Maltose (CAS. 6363-53-7), D-Lactose (CAS. 64044-51-5) and acetic acid (CAS. 64-19-7) monohydrate were provided from aladdin biochemical reagent Co. Ltd. (Shanghai, China). RHAWN001 column-layer chromatographic silica gel, polyacrylamide gel (Bio-gel P-II) column chromatography and ethanol absolute were provided from Shanghai Yi-en Chemical Technology Co., Ltd (Shanghai, China). Cisplatin (Pt\u0026thinsp;\u0026gt;\u0026thinsp;99%) was provided from Sigma-Aldrich (St. Louis, MO, USA). Hematoxylin and eosin (H\u0026amp;E), Glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). dimethyl methylene sulfone (DMSO) were provided from Sigma-Aldrich (St Louis, MO, USA). ROS staining kit were provided from Wan-lei Biotechnology, (Shenyang, China). The ELISA kits of mouse TNF-alpha (TNF-\u0026alpha;), IL-1 beta (IL-1\u0026beta;) and Diamine Oxidase (DAO) were provided from R\u0026amp;D systems (Minneapolis, MN, USA). Hoechst 33258 dye kits were provided from Beyotime Co., Ltd. (Shanghai, China). Immunofluorescence staining and SABC Daylight488-labeled secondary antibodies were provided by BOSTER Bio-Engineer Co., Ltd. (Wuhan, China). Primary antibodies against caspase 3, cleaved-caspase 3, caspase 9, cleaved-caspase 9, PI3K (p85), Akt, p-PI3K (p-p85), p-Akt, and phospho-NF-\u0026kappa;B (p-NF-\u0026kappa;B, p-p65) were provided from Cell Signaling Technology (Danvers, MA, USA), and the antibodies for Bax, Bcl-2 and \u0026beta;-actin were provided by Abcam (Cambridge, U.K.).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Chemical synthesis and purity determination of novel amino acid derivatives\u003c/h2\u003e\n \u003cp\u003eThe novel amino acid derivatives are prepared according to the MR mechanism in red ginseng and slightly modified according to the previous AFG synthesis method (Li, Zhang, 2019). In brief, the method amino acid derivatives was synthesized by heating the dissolving of L-arginine (2.0g) with Lactose (4.0g) in 20 mL of glacial acetic acid in a water bath at 85 ◦C for 120 min. After the reaction, the novel amino acid derivatives crude product was obtained by vacuum drying (50 ◦C) and alcohol precipitation with absolute ethanol. Moreover, the crude product of amino acid derivatives (mixed with 100\u0026ndash;200 mesh silica gel) was appended to the RHAWN001 column-layer chromatographic silica gel (200\u0026thinsp;~\u0026thinsp;300 mesh) with 1/5 times of silica gel volume (Matsuura, Zheng, 1994) and eluted by the with 70% ethanol-water solution, using thin-layer chromatography (TLC) for tracking target ingredient. The developing solvent is n-butanol: water: acetic acid\u0026thinsp;=\u0026thinsp;2:1:1, and the color reaction is carried out with 0.2% ninhydrin ethanol solution. The component was collected with value of the retention factor (Rf) of 0.174. A portion of ethanol was removed by a rotary evaporator, and then the lyophilized powder was diluted with sterile water to a 0.5 g/mL super polyacrylamide gel column (Biogel P-II). The amino acid derivatives single point was collected and freeze-dried.\u003c/p\u003e\n \u003cp\u003eChromatographic analyses were performed on Waters e2695 (Waters, USA), coupled with an ELSD instrument (Shimadzu Technologies ELSD-16, Japan). The drift tube temperature for the ELSD was set at 120 ◦C with a nitrogen flow rate of 1.8 L/min, Yue-Xu Ultimate\u0026reg; amino acid plus amino acid column (4.6 mm\u0026times;300 mm, 5\u0026micro;m), The column temperature was set at 30\u0026deg;C. The flow rate was 1.0 mL/min, and the injection volume was 20 \u0026micro;L. The mobile phase consisted of 0.3% heptafluorobutyric acid solution (A) and acetonitrile (B) with gradient elution: 0\u0026thinsp;~\u0026thinsp;10 min, 100% A; 10\u0026thinsp;~\u0026thinsp;15 min, 100%~93% A; 15\u0026thinsp;~\u0026thinsp;40 min, 93%~50% A; 40\u0026thinsp;~\u0026thinsp;41 min, 50%~100% A, 41\u0026thinsp;~\u0026thinsp;60 min, 100% A.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.3 Structural characterization of AFGA\u003c/h2\u003e\n \u003cp\u003eThe nuclear magnetic resonance (\u003csup\u003e1\u003c/sup\u003eH-NMR and \u003csup\u003e13\u003c/sup\u003eC-NMR), and heteronuclear multiple-bond connectivity (HMBC) and heteronuclear single quantum correlation (HSQC) spectra were measured using a Bruker AV600 NMR spectrometer (Bruker Co., Karlsruhe, Germany; 600 MHz for \u003csup\u003e1\u003c/sup\u003eH and 150 MHz for \u003csup\u003e13\u003c/sup\u003eC) with tetramethylsilane as an internal standard. Chemical shifts (\u0026delta;) are expressed in ppm, with the coupling constants (\u003cem\u003eJ\u003c/em\u003e) reported in Hertz (Hz). The electrospray ionization mass spectrometry (ESI-MS) was recorded using an Agilent 1200 HPLC with a 6300 lon-trap liquid chromatography/mass spectrophotometry (LC/MS; Agilent Technologies; ionization mode, negative; nebulizing gas [N\u003csub\u003e2\u003c/sub\u003e] pressure, 35 psi; drying gas [N\u003csub\u003e2\u003c/sub\u003e] flow, 8 L/min; temp, 350ϘC) and Q-Exactive mass spectrometer (Thermo Scientific, Bremen, Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.4 Culture of rat intestinal crypt epithelial (IEC-6) cells\u003c/h2\u003e\n \u003cp\u003eThe IEC-6 cells were maintained in DMEM medium supplemented with 10% FBS, 1% penicillin/streptomycin in humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. For passaging, the medium was changed every 48 h until the cells reached 80\u0026thinsp;~\u0026thinsp;90% confluent.\u003c/p\u003e\n \u003cp\u003eCell viability was detected using MTT assays. The cells were seeded in a 96-well culture plates and cultured at 37\u0026deg;C for 24 h, then the cells were pretreated with cisplatin (0.25, 0.5, 1, 2, 4, 8, 16, 32 \u0026micro;M) for 24 h, and then 20 \u0026micro;L MTT solution (5 mg/mL) was added to each well, after incubating for 3.5 h at 37\u0026deg;C, the culture supernatant was discarded, 150 \u0026micro;L DMSO was added to each well and shake for 10 min. Finally, the absorbance was measured at 490 nm using a microplate reader (Nano, Germany), and cisplatin of appropriate concentration were selected. Similarly, the cells were seeded into 96-well culture plates and the cells were pretreated with AFGA at 3.125, 6.25, 12.5, 25, 50, 100 and 200 \u0026micro;M for 24 h, and then exposure to cisplatin (1.5 \u0026micro;M) or not for 24 h, MTT was used to detect cell survival.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.5 ROS staining\u003c/h2\u003e\n \u003cp\u003eThe relative levels of intracellular ROS were determined by a fluorometric assay (DCF-DA assay) as previously described with some modifications (Hu et al., 2021a). IEC-6 cells were seeded in 6-well culture plates, and the supernatants were discarded after AFGA and cisplatin treatment, the plates were rinsed two times with PBS buffer, and incubate with 1.0 \u0026micro;M DCFH-DA at 37\u0026deg;C in the dark after 30 min, the culture medium was removed, washed twice with PBS, and the fluorescence intensity of ROS was observed using a Leica microscope (Leica TCS SP8, Solms, Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.6 Animals and experiments design\u003c/h2\u003e\n \u003cp\u003e40 males ICR mice, weighting 22\u0026thinsp;~\u0026thinsp;25 g, were provided by Changchun YISI Experimental Animals Co., Ltd. (Changchun, Jilin province, China) with a Certificate of Quality (SCXK (JI)-2019-0015). The mice were given a standard laboratory diet and water \u003cem\u003ead libitum\u003c/em\u003e and maintained at 12 h light/dark cycle at constant temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C). All experimental animals\u0026rsquo; processing project were strictly performed according to the Guide for the Care and Use of Laboratory Animals (2016). All the animal experiments were carried out consistent with experimental practices and standards, which were authorized by Ethical Committee of Jilin Agricultural University (Permit Number: JLAU-21-003).\u003c/p\u003e\n \u003cp\u003eAfter acclimation for one week, the mice were randomly divided into 4 groups (n\u0026thinsp;=\u0026thinsp;10): Normal group, AFGA (100mg/kg), cisplatin group, cisplatin\u0026thinsp;+\u0026thinsp;AFGA (100 mg/kg) respectively. Since there is currently no therapeutic agent for cisplatin-induced intestinal injury in the clinic, a group of positive drug was not set up in the present work. Mice were administered with AFGA by intragastric administration once daily for 10 days. All mice were allowed to take water freely during the experiment. On the 7th day of treatment, intestinal injury was induced by intraperitoneal injection of cisplatin (20 mg/kg), except for normal group, 1 h after the final AFGA treatment. The experiment was terminated 72 h after injection of cisplatin. Body weights, blood and organ samples were collected immediately.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003e2.7 Biochemical parameters determination\u003c/h2\u003e\n \u003cp\u003eThe intestine tissues used to estimate the antioxidant activities (Zhang, Wang, 2020). Lipid peroxides were measured by MDA kits, and CAT activity, and GSH and SOD content were determined by related assay kits according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n \u003cp\u003eSerum was separated by centrifugation at 1000 g for 10 min twice from blood collection. According to the instructions of the manufacturer, samples were added into a 96-well plate coated with antibodies specific for mouse DAO, TNF-\u0026alpha; and IL-1\u0026beta;, according to the manufacturer in ELISA reader are specified in the protocol provided under 450 nm conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003e2.8 Histopathological staining\u003c/h2\u003e\n \u003cp\u003eDuodenum was collected and then washed with phosphate-buffered saline. The tissues were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, cut into 5 \u0026micro;m thickness. The histopathological changes were mounted with neutral gum and representative images were captured used light microscope (Olympus BX-60, Tokyo, Japan).\u003c/p\u003e\n \u003cp\u003eIn order to evaluate the apoptosis in intestinal cells in \u003cem\u003evivo\u003c/em\u003e and \u003cem\u003evitro\u003c/em\u003e, apoptotic cells were measured by Hoechst 33258 staining, as described above (Ma et al., 2017). Liver tissues were fixed were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, cut into 5 \u0026micro;m thickness, and stained using the Hoechst 33258 solution with 10 \u0026micro;g/mL. Hoechst 33258 staining of nuclei under ultraviolet excitation was observed and photographed under a fluorescence microscope (Leica DM750, Germany). Quantification of apoptosis by Image-Pro plus 6.0.\u003c/p\u003e\n \u003cp\u003eImmunofluorescence staining for evaluating the degree of inflammation in intestinal tissue, duodenum tissues were incubated with p-NF-\u0026kappa;B antibody (1:300) in a humidified chamber at 4\u0026deg;C overnight, followed by SABC-Dylight448-labeled secondary antibody (BOSTER Biological Technology, Wuhan, China) incubation for 30 min at 37\u0026deg;C. Nucleus in testicular tissues was carried out using 4, 6 diamidino-2-phenylindole (DAPI) staining. The degree of inflammation in intestine tissues observed under a fluorescence microscope (Leica TCS SP8, Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003e2.9 Molecular docking studies\u003c/h2\u003e\n \u003cp\u003eThe molecular structures of AFGA were drawn by Chem Bio Draw. The 2D structure was processed and transformed into a three-dimensional (3D) structure by Chem Bio 3D Ultra 14.0.0.117, and the MM2 algorithm was used for energy minimization. PyMOL was used to remove the water molecules and organic matter of the target protein receptor. AutoDockTools-1.5.6 was used to add the nonpolar hydrogen and calculate Gasteiger charges for the structure and save it as PDBQT file. AutoDock Vina was run for virtual docking It is generally accepted that the lower the energy is, the more likely the binding is to occur. Finally, the conformation with the best affinity was selected as the final docking conformation and visualized in PyMOL.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003e2.10 Western blot analysis\u003c/h2\u003e\n \u003cp\u003eFirstly, the intestine tissues were cracked by using Radio Immunoprecipitation Assay (RIPA) buffer, and the total protein concentrations (Liu et al., 2020b) were measured using a BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). The proteins (50-\u0026micro;g) were separated with 12%SDS polyacrylamide gels and transferred to a polyvinylidene difluoride (PVDF) membrane. Then, the membrane was blocked using 5% non- loaded on the 12% SDS-PAGE and transferred onto a PVDF membranes. And then blocked with 5% (w/v) skim milk in Tris-buffered saline (TBS) with 0.1% Tween-20 for approximately 2 h, and incubated overnight at 4\u0026deg;C with primary antibodies Subsequently, membranes were incubated with the HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the protein band intensities were assessed with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). The immunoreactive bands were quantified \u003cem\u003evia\u003c/em\u003e densitometry using Image J (Version 1.8.0, National Institutes of Health, USA) and standardized to \u0026beta;-actin and were expressed as fold changes relative to the normal value.\u003c/p\u003e\n \u003ch2\u003e\u003cstrong\u003e2.11 Statistical analysis\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eAll data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D.) and analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post-test. Statistical graphs were performed using GraphPad Prism 8.0 software (San Diego, CA, USA). \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01 or \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 were considered to be significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003e3.1 Synthesis and structural characterization of AFGA\u003c/h2\u003e\n \u003cp\u003eAccording to the chemical mechanism of initial MR, AFG was obtained from L-arginine with maltose under acidic condition by chemical synthesis. Similarly, maltose and lactose are epimers (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). In this study, a novel amino acid derivative was synthesized from L-arginine and lactose by simulating the synthesis method of AFG, and then purified and separated by column chromatography of silica gel and Bio-gel P-II column chromatography. After HPLC-ELSD analysis, a novel amino acid derivatives purity was 99.86% (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB-C).\u003c/p\u003e\n \u003cp\u003eThe novel amino acid derivative was obtained as a white amorphous powder, easily soluble in water, methanol, ethanol, etc., with a melting point (mp) of 158\u0026thinsp;~\u0026thinsp;160\u0026deg;C. By normal-phase silica gel TLC (n-butanol: water: acetic acid\u0026thinsp;=\u0026thinsp;2:1:1), the color reaction of 0.2% ninhydrin ethanol solution was purple. The molecular weight was determined as 497.3612 by ESI-MS (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). The combination of \u003csup\u003e13\u003c/sup\u003eC-NMR (Supplementary Fig. 1) and HMBC showed eighteen carbons, and the specific signal attribution was shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. It was found that the sugar group has 12 carbons, \u0026delta; 101.38 and \u0026delta; 96.19 showed two sugar groups. The \u003csup\u003e1\u003c/sup\u003eH-NMR spectrum (Supplementary Fig.\u0026nbsp;2) showed [\u003cem\u003e\u0026delta;\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e 3.510 (1H, dd), 3.590 (1H), 3.725 (1H, dd), 3.82 (1H, d), 4.03 (1H, dd), 2.75 (2H, d, J\u0026thinsp;=\u0026thinsp;6.6Hz)]; [\u0026delta;ppm 3.232 (1H), 3.518 (1H, d), 3.775 (1H, d), 2.95 (1H, dd), 4.24 (1H, dd), 3.483 (2H, d, J\u0026thinsp;=\u0026thinsp;6.6 Hz]. It could be inferred that this compound contains twelve -OH single bonds in the sugar group, further utilized the HMBC spectrum, the following coupled relationship can be observed C-1\u0026prime; \u0026delta; 4.47 (1H, d, J\u0026thinsp;=\u0026thinsp;7.8 Hz), C-6 \u0026delta;\u003csub\u003eH\u003c/sub\u003e 3.84 (1H, d, J\u0026thinsp;=\u0026thinsp;3.0 Hz), C-1\u0026prime;\u0026prime; \u0026delta;\u003csub\u003eH\u003c/sub\u003e 3.92 (1H, d, J\u0026thinsp;=\u0026thinsp;12.0Hz), C-2\u0026prime; ( \u0026delta;\u003csub\u003eC\u003c/sub\u003e 96.19 ), H-1\u0026prime;\u0026prime; ( \u0026delta;\u003csub\u003eH\u003c/sub\u003e 5.04, d, J\u0026thinsp;=\u0026thinsp;7.7 Hz ), C6\u0026prime;\u0026prime;-glc (\u0026delta;\u003csub\u003eH\u003c/sub\u003e 3.64) and C-4\u0026prime;\u0026prime; (\u0026delta;\u003csub\u003eC\u003c/sub\u003e 70.52). In \u003csup\u003e1\u003c/sup\u003eH-NMR, J\u003csub\u003e1, 2\u003c/sub\u003e \u0026lt; 7 Hz, and \u0026alpha;-glucose is enzymatically hydrolyzed to yield galactose and glucose. Therefore, it\u0026rsquo;s inferred that the connection sequence of the novel amino acid derivatives was: arginine-fructose-galactose, and molecular formula was C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e.\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003csup\u003e13\u003c/sup\u003eC-NMR chemical shifts of novel amino acid derivatives in D\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026delta;ppm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026delta;ppm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026delta;ppm\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e173.50(s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.13(t)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026rdquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e101.51(d)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.15(d)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.17(s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u0026rsquo;\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70.48(d)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.35(t)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.74(d)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026rsquo;\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.55(d)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.73(t)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.35(d)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u0026rsquo;\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70.52(d)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.26(t)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70.07(d)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.22(d)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e157.59(s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.73(t)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u0026rsquo;\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61.36(d)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eBased on previous reports of AFG studies, The \u003csup\u003e13\u003c/sup\u003eC-NMR structure of fructose contains configurations such as \u0026alpha;-furan, \u0026beta;-pyran and \u0026beta;-furan. The fructose in this compound was produced from glucose by Amadori rearrangement. The \u003csup\u003e13\u003c/sup\u003eC-NMR and \u003csup\u003e1\u003c/sup\u003eH-NMR spectrum showed C-1\u0026apos; of fructose is linked to the \u0026alpha;-amino nitrogen atom of arginine to form a C-N bond. Its structure was determined as: 1-(arginine-N\u0026alpha; group)-1-deoxy-4-O-(\u0026alpha;-D-galactopyranosyl)-D-fructose (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). named as Argininyl-fructosyl-galactose (AFGA) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). The synthesis mechanism of AFGA is shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003e3.2 Activity screening of AFGA and protective activity of cisplatin-induced intestinal injury\u003c/h2\u003e\n \u003cp\u003eIn this study, the activity of AFGA was screened through cell experiments to establish the injury of IEC-6 cells by cisplatin, and clarify the pharmacodynamic effect of AFGA. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-D, AFGA application increased the viability of IEC-6 cells in a dose-dependent manner after 24 h exposure to cisplatin (1.0 \u0026micro;M), AFGA with concentration range from 3.125 to 200 \u0026micro;M exerted conspicuous protective effect on cell viability (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). According to the MTT assay results, 25, 50 and 100 \u0026micro;M was chosen for the subsequent experiments.\u003c/p\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-B, after single injection of cisplatin (20 mg/kg) caused a noticeable body weight loss, and the morphology of the intestinal tissue was observed to become white and thin. Meantime, histopathological examination staining showed the intestinal tissue injury was serious, and the villi of intestinal wall were uneven, cell degeneration, part of the villi atrophy, shedding and disappearance, glands and crypts disappear in cisplatin group. However, AFGA treatment prevented the changes in body weight and histomorphology were significantly improved at the doses of 100 mg/kg, and AFGA (100mg/kg) was not toxic in mice (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). Moreover, compared with the cisplatin group, the abnormal increase of DAO activity in serum was significantly improved after AFGA pretreatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that AFGA could effectively maintain the integrity of intestinal barrier function (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e\n \u003ch2\u003e\u003cstrong\u003e3.3 AFGA ameliorated oxidative stress and inflammation response in cisplatin-induced intestinal injury in\u003c/strong\u003e \u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003evivo\u003c/span\u003e \u003cstrong\u003eand in\u003c/strong\u003e \u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003evitro\u003c/span\u003e\u003c/h2\u003e\n \u003cp\u003eSince cisplatin was prone to cause oxidative damage to the body, this study tested some oxidative indicators to verify the effect of AFGA supplementation on cisplatin-induced oxidative stress. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF-I, cisplatin exposure resulted in prominent increased MDA content, and significantly decreased GSH, SOD and CAT levels in intestinal homogenates compared with the normal group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Interestingly, AFGA pretreatment dramatically reverse these changes (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eTo further explore the activity of AFGA ameliorated cisplatin-induced intestinal oxidative injury, we also studied the production of ROS in IEC-6 cells. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eJ, the expression of ROS in cells exposed to cisplatin was significantly increased. AFGA could significantly decreased the green fluorescence intensity and decreased the expression of ROS in cells in a dose-dependent manner (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The AFGA alone did not cause significant increase of ROS in IEC-6 cells, indicated that AFGA was non-toxic and further supporting the assay results in \u003cem\u003evivo\u003c/em\u003e. Together, these results clearly demonstrated that AFGA was effective in reducing cisplatin-induced ROS accumulation.\u003c/p\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA-B, two pro-inflammatory factors, TNF-\u0026alpha; and IL-1\u0026beta; were detected in serum. Compared with the normal group, cisplatin exposure could significantly increase the levels of TNF-\u0026alpha; and IL-1\u0026beta;, these changes were reversed after AFGA treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no significant change in AFGA alone group.\u003c/p\u003e\n \u003cp\u003eTo directly demonstrated that cisplatin could induce intestinal inflammation in mice, this study used immunofluorescence analysis to evaluated the protein expression of p-NF-\u0026kappa;B antibodies. As summarized in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, there was no significant difference between AFGA alone and normal group, but cisplatin can significantly increase the nucleus and cytoplasm of cisplatin-induced intestinal tissues (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, AFGA could antagonize p-NF-\u0026kappa;B overexpression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003e3.4 AFGA ameliorated cisplatin-induced intestinal cell apoptosis\u003c/h2\u003e\n \u003cp\u003eTo evaluated the effect of AFGA on the apoptosis of intestinal cells induced by cisplatin. Hoechst 33258 staining (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF) has shown nuclei intense fluorescence chromatin condensation and nuclear shrinkage were clearly observed in cisplatin group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Interestingly, administration of AFGA showed normal regular outline of intestinal epithelium and remarkably reduced apoptosis (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In cultured IEC-6 cells, Hoechst 33258 staining (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE) also showed that after AFGA pretreatment, nuclear agglutination and abundant bright red or blue fluorescence were observed, which was consistent with the in \u003cem\u003evivo\u003c/em\u003e results.\u003c/p\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA-B, western blot analysis result confirmed the significant decrease in p-PI3K and p-Akt expression in mice by cisplatin. Inclusion of AFGA diminished the inhibition of cisplatin on p-PI3K and p-Akt protein expressions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, the protein expression of the anti-apoptotic protein Bcl-2 in the cisplatin group decreased, and the expression of the pro-apoptotic proteins Bax and cleaved-caspase 3, 9 increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). AFGA could significantly improve the apoptosis of intestinal induced by cisplatin (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results demonstrated that AFGA could inhibit cisplatin-induced intestinal cells apoptosis by regulating the PI3K/Akt signaling pathway.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\n \u003ch2\u003e3.5 Molecular docking of AFGA with target proteins\u003c/h2\u003e\n \u003cp\u003eMolecular docking was used to verify if AFGA have a significant role in the regulation of proteins related to apoptosis pathway (PI3K, Akt and caspase 3). The results showed the potential binding capacity of the compounds to core target proteins (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA-C). With the screening criteria of binding energy \u0026le;-6.0 kJ/mol, the results all ranged from \u0026minus;\u0026thinsp;8.8 to -6.3 kJ/mol which indicated a strong and table binding between the compound and the protein. Compared with AFG, AFGA has stronger binding ability to apoptosis-related target proteins (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD-G). These results showed that AFGA had a good medicinal reference value, and exerted higher pharmacological activity than AFG.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eAmino acid derivatives one of the important products of MR, especially AF and AFG found in the process of red ginseng production are the most active and high content (Kim, Lee, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Previous studies have reported that ARPs have high application value in food development and also have a variety of pharmacological effects in \u003cem\u003evitro\u003c/em\u003e and in \u003cem\u003evivo\u003c/em\u003e(Kim and Lee, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Nooshkam et al., 2019b). Meantime, strengthening the in-depth study of MR of red ginseng and clarifying the synthesis mechanism of amino acids and reducing sugars can well promote the enrichment and development of MRPs (Du, Liu, 2012). Based on the clear production mechanism of AFG, this study innovatively combined lactose with the abundant arginine in red ginseng to synthesize novel amino acid derivatives. The chemical structure was determined to be 1-(arginine-N\u003csup\u003eα\u003c/sup\u003egroup)-1-deoxy-4-O-(α-\u003cem\u003eD\u003c/em\u003e-galactopyranosyl)-\u003cem\u003eD\u003c/em\u003e-fructose, named AFGA. Through activity screening, the medicinal value of AFGA was expounded from the perspective of in \u003cem\u003evivo\u003c/em\u003e and in \u003cem\u003evitro\u003c/em\u003e. In addition, the protective effect of AFGA on cisplatin-induced intestinal injury was revealed from the aspects of oxidative stress, inflammatory response and apoptosis, and it was demonstrated that AFGA can exert intestinal protective activity through PI3K/Akt and downstream caspase pathway.\u003c/p\u003e \u003cp\u003eExtensive research suggested that cisplatin can cause the destruction of intestinal mucosal cells and changes in the structure of the intestinal epithelium(Khan et al., 2012, Shahid et al., 2018), which was related to the accumulation of ROS and the generation of oxidative stress(Akhter et al., 2021, Zhao et al., 2022). Meantime, previous studies have established that ROS plays important biological roles in cell homeostasis (Hu et al., 2021b). The main forms of ROS include superoxide, hydrogen peroxide and hydroxyl radicals(Liao et al., 2019, Liu et al., 2022). In current study, the mice single injected with cisplatin (20 mg/kg), resulted in abnormally elevated levels of MDA and decreased GSH, SOD and CAT content of the intestinal tissue in mice, and IEC-6 cell assays also showed that cisplatin treatment excessive accumulation of ROS, and further supporting the assay results in \u003cem\u003evivo\u003c/em\u003e. These were consistent with previously reported results (Hu, Yang, 2021b). Indeed, ROS has been shown to promote the up-regulation of pro-inflammatory cytokines (Wang et al., 2015). This study shown that TNF-α and IL-1β in the serum of mice significantly increase, and immunofluorescence analysis also indicated that AFGA could antagonize p-NF-κB overexpression and play an important role in cisplatin-induced intestinal toxicity.\u003c/p\u003e \u003cp\u003eOn the other hand, ROS accumulation also feeds back into the apoptosis pathway, reduce ROS production may inhibits intestinal cell apoptosis and promotes its differentiation (Maradagi et al., 2022). When a large amount of ROS accumulated it increased mitochondrial permeability, stimulated pro-apoptotic factor release and initiated apoptosis (Liu et al., 2021). Consistently, this study indicated that Hoechst 33258 staining and western blot analysis to further detect apoptosis both in mice and in IEC-6 cells. Compared with the cisplatin group, AFGA significantly reduced the pro-apoptotic cytochrome C, cleavage caspases 3, 9 and Bax proteins intestinal tissue in mice, and promote the expression of Bcl-2 protein. Previous research has shown that targeting PI3K/Akt by chemosensitizer may improve the anticancer efficacy of cisplatin and decrease the side-effects (Mi et al., 2018), and the abnormal activation of PI3K/Akt is generally associated with the development of drug resistance (Zhang et al., 2021). Consistent with these results, the current study demonstrated that AFGA treatment can significantly reverse these apoptosis-related proteins expression. Meantime, this study also performed molecular docking experiments on the key proteins in PI3K/Akt signaling pathway and found that AFGA (binding energy \u0026le;-7.7 kJ/mol) has stronger binding ability to apoptosis-related proteins than AFG (binding energy \u0026le;-6.5 kJ/mol). These study demonstrated the protective effect of AFGA on cisplatin-induced intestinal injury.\u003c/p\u003e \u003cp\u003eIn conclusion, based on the MR principle of red ginseng, this study innovatively synthesized a novel amino acid derivative by combining arginine in red ginseng with lactose by the method of simulated synthesis, the structure was determined to be 1-(arginine-N\u003csup\u003eα\u003c/sup\u003egroup)-1-deoxy-4-O-(α-\u003cem\u003eD\u003c/em\u003e-galactopyranosyl)-\u003cem\u003eD\u003c/em\u003e-fructose, named AFGA. Through in \u003cem\u003evitro\u003c/em\u003e and in \u003cem\u003evivo\u003c/em\u003e experiments, it was shown that AFGA has good intestinal injury protection activity.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eAFGA\u003c/strong\u003e, Argininyl-fructosyl-galactose; \u003cstrong\u003eAFG,\u003c/strong\u003e Arginyl-fructose-glucose; \u003cstrong\u003eAkt (PKB)\u003c/strong\u003e, Protein Kinase B;\u0026nbsp;\u003cstrong\u003eBcl-2\u003c/strong\u003e, B-cell-lymphoma-2; \u003cstrong\u003eCAT\u003c/strong\u003e, Catalase; \u003cstrong\u003eDAO\u003c/strong\u003e, Diamine oxidase; \u003cstrong\u003eESI-MS\u003c/strong\u003e, electrospray ionization tandem mass spectrometry;\u003cstrong\u003e\u0026nbsp;GSH\u003c/strong\u003e, Glutathione; \u003cstrong\u003eHMBC\u003c/strong\u003e, Heteronuclear multiple-bond connectivity; \u003cstrong\u003eHSQC\u003c/strong\u003e, Heteronuclear single quantum correlation; \u003cstrong\u003eHPLC-ELSD,\u0026nbsp;\u003c/strong\u003eHigh performance liquid chromatography-evaporative light scattering detection;\u0026nbsp;\u003cstrong\u003eIL-1\u0026beta;\u003c/strong\u003e, Interleukin-1\u0026beta;; \u003cstrong\u003eMDA\u003c/strong\u003e, Malondialdehyde; \u003cstrong\u003eMR,\u003c/strong\u003e Maillard reaction; \u003cstrong\u003eMRPs,\u003c/strong\u003e Maillard reaction products; \u003cstrong\u003eNF-\u0026kappa;B\u003c/strong\u003e, Nuclear transcription factor-\u0026kappa;B; \u003cstrong\u003eNMR,\u003c/strong\u003e nuclear magnetic resonance; \u003cstrong\u003ePI3K\u003c/strong\u003e, phosphatidylinositol 3-kinase; \u003cstrong\u003eSOD,\u0026nbsp;\u003c/strong\u003eSuperoxide dismutase, \u003cstrong\u003eTNF-\u0026alpha;\u003c/strong\u003e, Tumor necrosis factor-\u0026alpha;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe authors declare\u0026nbsp;that there are no conflicts of interest.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eWei Li conceived and designed the experiments; Wei Liu and Zi Wang performed the experiments; Shuang Jiang, Shen Ren and Shan Tang contributed analysis tools; Wei Liu wrote the paper. Yi-nan Zheng and Jing Zhang contributed to the improvement of the writing. All authors reviewed and approved the contents of the manuscript.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe datasets generated and analyzed during the current study are available in the corresponding author upon request.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAll experimental animals\u0026rsquo; processing project were strictly performed according to the Guide for the Care and Use of Laboratory Animals (2016). All the animal experiments were carried out consistent with experimental practices and standards, which were authorized by Ethical Committee of Jilin Agricultural University (Permit Number: JLAU-21-003).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThis work was supported by the grants of National Natural Science Foundation of China (No. 82104465), Changchun Science \u0026amp; Technology Development Plan (No. 21ZGY11), Jilin Science \u0026amp; Technology Development Plan (No. 20200301037RQ), and the Scientific Research Planning Project of Jilin Provincial Department of Education (No. JJKH20220371KJ).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\u003cbr\u003e\u003c/div\u003e"},{"header":"References","content":"\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003col\u003e\n \u003cli\u003eAkhter N, Wilson A, Thomas R, Al-Rashed F, Kochumon S, Al-Roub A, et al. 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Food Funct. 2020;11:4236-48.\u003c/li\u003e\n \u003cli\u003eZhang JJ, Zhou YD, Liu YB, Wang JQ, Li KK, Gong XJ, et al. Protective Effect of 20(R)-Ginsenoside Rg3 Against Cisplatin-Induced Renal Toxicity via PI3K/AKT and NF-\u0026kappa;B Signaling Pathways Based on the Premise of Ensuring Anticancer Effect. Am J Chin Med. 2021;49:1739-56.\u003c/li\u003e\n \u003cli\u003eZhao Q, Shi Q, Zhu Q, Hu Y, Zhang X. A mini-review of advances in intestinal flora and necrotizing enterocolitis. Lett Appl Microbiol. 2022; 75:2-9.\u003c/li\u003e\n \u003c/ol\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\u003cbr\u003e\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Argininyl-fructosyl-galactose, structure identification, safety evaluation, cisplatin, intestinal toxicity","lastPublishedDoi":"10.21203/rs.3.rs-2627146/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2627146/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBased on the Maillard reaction principle of red ginseng (\u003cem\u003ePanax ginseng\u003c/em\u003e C.A. Meyer), this study innovatively synthesized a new amino acid derivative by combining arginine with lactose through simulated synthesis, and was separated and purified through repeated silica gel and polyacrylamide gel (Bio-gel P-II) column chromatography. The purity of the product was determined to be 99.86% and its molecular weight was determined to be 497.3612 (negative ion mode) by electrospray ionisation mass spectrometry (ESI-MS). The chemical structure was identified to be 1-(arginine-N\u003csup\u003eα\u003c/sup\u003egroup)-1-deoxy-4-O-(α-\u003cem\u003eD\u003c/em\u003e-galactopyranosyl)-\u003cem\u003eD\u003c/em\u003e-fructose, named Argininyl-fructosyl-galactose (AFGA, C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e). Subsequently, by establishing cisplatin-induced intestinal injury in \u003cem\u003evivo\u003c/em\u003e and IEC-6 cell model, the results showed that pretreatment with AFGA significantly ameliorated cisplatin induced oxidative stress by reducing levels of reactive oxygen species (ROS) in IEC-6 cells (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), and could effectively reduce the secretion of pro-inflammatory factors in serum and the expression level of NF-κB protein in intestinal tissues (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Meantime, AFGA also inhibited the expression of p-PI3K/p-Akt, caspase 3, 9, cytochrome C and Bax protein intestinal tissue in mice (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), and promoted the expression of Bcl-2 protein (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Importantly, the molecular docking results of AFGA also suggested a better binding ability with the above-mentioned related target proteins, and further revealed AFGA as a potential multifunctional therapeutic agent with clear protective effect against cisplatin-induced intestinal injury.\u003c/p\u003e","manuscriptTitle":"Structural characterization of a new amino acid derivatives, Maillard Reaction product of red ginseng and potential protective activity against cisplatin-evoked intestinal injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-02 23:41:33","doi":"10.21203/rs.3.rs-2627146/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"944e1e15-0b58-495c-a069-f3ffb949c581","owner":[],"postedDate":"March 2nd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-03-09T03:44:22+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-02 23:41:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2627146","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2627146","identity":"rs-2627146","version":["v1"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}