- Email: [email protected]
Dezhou donkey (Equus asinus) milk a potential treatment strategy for type 2 diabetes
Journal of Ethnopharmacology 246 (2020) 112221
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Dezhou donkey (Equus asinus) milk a potential treatment strategy for type 2 diabetes
T
Yan Lia, Yumei Fanb,c, Abdul Sami Shaikhd, Zhendong Wanga, Dongliang Wanga,b,c,∗∗, Haining Tana,∗ a
National Glycoengineering Research Center, Shandong University, Jinan, 250012, China National Engineering Research Center for Gelatin-based Traditional Chinese Medicine, Done-E Country, Liaocheng, 252000, China c Dong-E E-Jiao Co. Ltd., Done-E Country, Liaocheng, 252000, China d Institute of Clinical Pharmacology, Qilu Hospital of Shandong University, Jinan, 250012, China b
ARTICLE INFO
ABSTRACT
Keywords: Donkey milk Digestion Treatment Diabetes Insulin
Ethnopharmacological relevance: Donkey (Equus asinus) milk has become a medical and nutrient product since ancient times. In addition, donkey milk was regarded as a medicinal food and substitute product for infant formula in some ancient western countries. Chinese ancient medical books documented the medicinal value of donkey milk, using donkey milk to treat diabetes, cough and jaundice. Aim of the study: To investigate the donkey milk’s components and anti-diabetic effect of donkey milk in vitro and in vivo and to study the molecular mechanism of donkey milk was an anti-diabetic medication. Materials and methods: In this study, the gastrointestinal digested donkey milk was simulated in vitro and its products of protein digestion were analyzed by SDS-PAGE. We then performed cell viability assay, insulin secretion assay, animal experiments and ELISA assays to study the anti-diabetic effect of donkey milk in vitro and in vivo. Donkey milk’s anti-diabetic molecular mechanism and specific targets were detected by using quantitative real time PCR. Results: Lysozyme (LZ) and α-lactalbumin (α-La) exhibited significantly lower digestibility and higher retention than the other components of donkey milk. In vitro, 500 μg/mL of donkey milk could improve damaged β-cells viability significantly (P < 0.0001). In vivo, the blood glucose and HOMA-IR of diabetic rats treated with donkey milk were 14.23 ± 5.18 mM and 74.94 ± 23.62, respectively, whereas the diabetic group were 22.18 ± 2.23 mM and 112.16 ± 18.44, respectively (P < 0.01). The SOD value of donkey milk group was 265.87 ± 21.29 U/L, while the SOD value of diabetic group was 193.20 ± 52.07 U/L (P < 0.05). These results indicated that the blood glucose was reduced, the ability of the body to eliminate free radicals was enhanced, antioxidant levels in the body was increased, insulin resistance was improved in type 2 diabetic rats after donkey milk powder fed for 4 weeks. Furthermore, donkey milk could treat diabetes through down-regulating phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6PC). Conclusions: Donkey milk has played an important role in the treatment of type 2 diabetes, and contributed to the development of the donkey milk products.
1. Introduction Donkey (Equus asinus) milk contains a complete range of amino acids and is rich in vitamins and minerals. The amount of selenium in donkey milk is 5.16 times that of the selenium content of cow milk, and the quantities of lactalbumin and lactoglobulin account for more than 60% of the total protein content. These data throw light on the composition of donkey milk, and may be of substantial value in the rational
∗
design of infant formula in the future (Chiavari et al., 2005; Herrouin et al., 2000; Horrobin, 2000; Oftedal and Jenness, 1988; Uniacke-Lowe et al., 2010). According to the Compendium of Materia Medica, volume 50, the category of animals and Thousand golden prescriptions, donkey milk is recorded as to cure diabetes, asthma and bronchitis (Lu et al., 2006). It has also been recently reported that the whey protein present in cow milk stimulates insulin secretion and lowers blood sugar levels (Hai-
Corresponding author. National Glycoengineering Research Center, Shandong University, Jinan, 250012, China. Corresponding author. E-mail addresses: [email protected] (D. Wang), [email protected] (H. Tan).
∗∗
https://doi.org/10.1016/j.jep.2019.112221 Received 17 February 2019; Received in revised form 31 August 2019; Accepted 4 September 2019 Available online 05 September 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
Journal of Ethnopharmacology 246 (2020) 112221
Y. Li, et al.
Abbreviations LZ α-La Pck1 G6PC STZ MTT GHbA1c IR HOMA
LF SA HcIG CNs β-Lg SOD GSH MDA T-AOC qPCR Met
lysozyme α-lactalbumin phosphoenolpyruvate carboxykinase 1 glucose-6-phosphatase streptozotocin methylthiazolyldiphenyl-tetrazolium bromide glycosylated hemoglobin insulin resistance homeostasis model assessment
Ming et al., 2015; Jakubowicz and Froy, 2013; Petersen et al., 2009; Sebely and Vanessa, 2010). In comparison to ordinary milk, donkey milk has a higher content of whey protein, which might aid the prevention and treatment of diabetes (Herrouin et al., 2000). The research studies also demonstrate that donkey milk administration improves glucose disposal and insulin resistance, and acts on glucose and lipid metabolism (Giovanna et al., 2018; Trinchese et al., 2015). Donkey milk is known to have high concentration of triacylglycerol fatty acid particularly palmitic acid. Recently, the anti-inflammatory action and beneficial role of donkey milk in glucose metabolism has already been reported (Giovanna et al., 2018). The causes of type 2 diabetes mainly include β-cells dysfunction and insulin resistance produced by peripheral tissues and cells (Weng et al., 2016). The aim to study the donkey milk anti-diabetic function, we have designed cell models in vitro to investigate donkey milk’s anti-diabetic effects in this study. The cell models are damaged mouse insulinoma beta-pancreatic (MIN6) cells. From the experiments in vitro, we can develop a solution towards the treatment of type 2 diabetes with donkey milk. In addition, the effects of donkey milk in vivo on insulin sensitivity, blood glucose, the levels of glycosylated hemoglobin and antioxidants in the plasma were investigated. It is reported that diabetes induced by streptozotocin (STZ) affect hepatic gluconeogenesis key enzymes Pck1 and G6PC of expression directly or indirectly (Bae et al., 2010; Corpe et al., 2013; Sung et al., 2014; Thorens, 2015). In this study, we investigated Pck1 and G6PC relative mRNA levels. Therefore, there is a need for making a good explanation for the molecular aspects of donkey milk in the treatment for diabetes.
lactoferrin serum albumin high-chain immunoglobulin casein β-lactoglobulin superoxide dismutase glutathione malondialdehyde total anti-oxidation capacity quantitative real time PCR metformin
Liaocheng city, China. The gastric juice and intestinal fluid were prepared and the reaction time for chemical digestion was estimated using the methods previously described (Oomen et al., 2003; Tidona et al., 2011). The composition and concentration of the chemical simulator are provided in Table 1. Donkey milk (Dong-E E-Jiao Co. Ltd, China) powder (7.5 g) was dissolved in 75 mL of double-distilled water, mixed with 0.6 mL of gastric digester, and shaken at 37 °C for 0.5 h. Duodenal fluid (2.25 mL) and bile fluid (0.675 mL) were subsequently added to this solution and shaken at 37 °C for 0.5 h. Digestion was ceased at 4 °C and the solution was freeze-dried. And then, samples were analyzed by SDS-PAGE. 2.2. Cell viability assay MIN6 cells were seeded into 96-well plates (6 × 103 cells/well). After incubation overnight, MIN6 cells were treated with 5 mM STZ (Sigma, USA) for 24 h. Subsequently, donkey milk and metformin (Met) of different concentrations (500 μg/mL,250 μg/mL,100 μg/mL,50 μg/ mL and 25 μg/mL) were added to each well for 48 h, respectively. And then, 10 μL methylthiazolyldiphenyl-tetrazolium bromide (MTT, Solarbio, China) was added to each well and further cultured for 4 h under dark conditions. After removing the medium, 150 μL DMSO was added to each well to dissolve the formazan crystal. The absorbance was measured at 490 nm (Tomasz et al., 2011). 2.3. Insulin secretion assay MIN6 cells were seeded into 96-well plates (1 × 104 cells/well). After incubation overnight, MIN6 cells were treated with 5 mM STZ for 24 h. After 48 h incubation with donkey milk 500 μg/mL and metformin 500 μg/mL, the cells were preincubated at 37 °C for 30 min in KrebRinger bicarbonate Hepes buffer (KRB; 129 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 5 mM NaHCO3, 10 mM Hepes, and 0.2% BSA). Cells were then treated in KRB buffer containing
2. Materials and methods 2.1. Digestion of donkey milk Donkey milk selected from a donkey from Dezhou, aged five years and with 2 parities in its late lactation, located in a local farm in Table 1 Composition and concentration of the chemical simulator.
Inorganic salt solution
Organic salt solution
pH
Gastric juice
Duodenal fluid
Bile
1.57 mLNaCl 175.3 g/L 0.3 mL NaH2PO4 88.8 g/L 0.92mLKCl 89.6g/L 1.8 mL CaCl2·2H2O 22.2 g/L 1 mL NH4Cl 30.6 g/L 530 μL HCl 37% g/g
4 mL NaCl 175.3 g/L 4 mL NaHCO3 84.7 g/L 1 mL KH2PO4 8g/L 0.63 mL KCl 89.6 g/L 1 mL MgCl2 5 g/L 18 μL HCl 37% g/g 0.9 mL CaCl2·2H2O 22.2 g/L 0.4 mL urea 25 g/L 0.1 g BSA 0.3 g Parenzyme 0.05 g Lipase
3 mL NaCl 175.3 g/L 6.83 mL NaHCO3 84.7 g/L
7.8 ± 0.2
8.0 ± 0.2
0.34 mL urea 25 g/L 1 mL glucose 65 g/L 1 mL Glucuronic acid 2 g/L 1 mL D-Glucosamine Hydrochloride 33 g/L 0.1 g BSA 0.1 g Pepsin 0.3 g Mucin 1.07 ± 0.07
2
0.42 mL KCl 89.6 g/L 20 μL HCl 37% g/g 1 mL CaCl2·2H2O 22.2 g/L 0.6 g pig Bile salts 1 mL urea 25 g/L 0.18 g BSA
Journal of Ethnopharmacology 246 (2020) 112221
Y. Li, et al.
30 mM glucose for 60 min (Green et al., 2016; Gu et al., 2013). Cell supernatants were collected. Insulin was measured by ELISA kit (JingMei Biotechnology, China).
2.7. Statistical analysis The data are expressed as the mean ± standard deviation. Statistical analyses were performed using the SPSS 16.0 software. Oneway analysis of variance (ANOVA) followed by Tukey post-hoc test was used to assess the results except the quantitative real-time PCR results. The quantitative real-time PCR results were estimated by t-test. A pvalue < 0.05 was considered to be statistically significant.
2.4. Animal experiments All animal experiments in this study were approved by the Laboratory Animal Ethical and Welfare Committee of Shandong University Cheeloo College of Medicine. Twenty four male Wistar rats, aged 5–6 weeks, were divided into 4 groups (n = 6/group) as follows: normal group, diabetic group, donkey milk group and metformin group. The rats in the diabetic group, donkey milk group and metformin group were fed a high-fat diet, the normal rats were fed normal diets for 3 weeks. The rats were starved for 24 h, but were allowed free access to drinking water prior to modeling. The rats were intraperitoneally injected with STZ at a dose of 50 mg/kg. The rats with the fasting blood glucose above 11.1 mmol/L were considered diabetic (Srinivasan et al., 2005). In previous studies, the treatment groups were administered donkey milk powder at a dose of 3 g/kg•d, while the other groups were administered double distilled water (Belobrajdic et al., 2004; Ebaid, 2014; Frid et al., 2005; Frestedt et al., 2008; Tong et al., 2011; Vincenzetti et al., 2005). The metformin group was administered metformin (Shanghai Yuanye Biotechnology, China) at a dose of 100 mg/ kg•d (EI-Hadidy et al., 2015; Khan et al., 2018). The rats were allowed to continue to feed on their respective diets until the end of the study.
3. Results 3.1. Digestion with simulated gastric fluid and duodenal fluid in vitro As shown in Fig. 1, the gray levels (volume) of lactoferrin (LF), serum albumin (SA), high-chain immunoglobulin (HcIG), casein (CNs), and β-lactoglobulin (β-Lg) in digested donkey milk were reduced in comparison to those in undigested milk. As shown in Table 3, LZ and α-La exhibited lower digestibility and higher retention than the other components of donkey milk. The digestibility of the remaining five proteins (LF, SA, HcIG, CNs, and βlactoglobulin) exceeded 82%, while the retention rates for these proteins were less than 18%. The results demonstrated that LF, SA, HcIG, CNs, and LF were digested by the artificial gastrointestinal fluid. β-Lg and CNs were almost completely digested. LZ (5.13%) and α-La (6.02%) displayed lower digestibility in comparison to the other components. These results provided material evidence and an experimental basis for the antidiabetic effects of the oral ingestion of donkey milk powder.
2.5. Insulin and blood glucose, antioxidant capacity, and glycosylated hemoglobin (GHbA1c) assays
3.2. Effect of donkey milk on damaged MIN6 cells
All the rats were fasted for 12 h and the fasting blood glucose was measured by tail-cutting method. Blood samples were collected from the jugular sinus following the measurement of blood glucose. The plasma samples were separated for analysis by centrifugation of the blood samples at 5000 rpm for 5 min at 4 °C. ELISA kits (Bio-Swamp, China) were used measure the levels of related indicators. The index of insulin resistance (IR) as evaluated by the homeostasis model assessment (HOMA) was calculated, according to the following equation: HOMA-IR= Blood glucose (mmol/L) × Insulin/22.5.
The effect of donkey milk on the viability of damaged MIN6 cells was assessed by MTT assay. From Fig. 2, when the concentrations of donkey milk were 25, 100, 200, 250 and 500 μg/mL, the cell viability were 86.60 ± 1.14 %, 82.92 ± 1.13 %, 78.56 ± 1.41 %, 66.83 ± 2.38 % and 56.35 ± 1.56 % , respectively. At the same concentrations of metformin, the cell viability were 75.09 ± 0.45 % , 72.78 ± 4.66 % , 63.90 ± 3.08 % , 59.73 ± 3.38 % and 56.20 ± 2.58 %. Compared to the model group, the cell viability of damaged MIN6 cells treated with 100, 200, 250 and 500 μg/mL donkey milk and metformin increased significantly. At the concentions of 100, 200, 250 and 500 μg/mL, the donkey milk showed better effect than the metformin. MIN6 cells viability increased in a dose-dependent manner as the concentration increased in all groups.
2.6. Quantitative real time PCR Quantification was performed with a two-step reaction process: reverse transcription (RT) and PCR. Each RT reaction has two steps. The first step is 0.5 μg RNA, 2 μL of 4×g DNA wiper Mix, add Nuclease-free H2O to 8 μL. The reactions were performed in a GeneAmp® PCR System 9700 (Applied Biosystems, USA) for 2 min at 42 °C. The second step is to add 2 μL of 5 × HiScript II Q RT SuperMix IIa. The reactions were performed in a GeneAmp® PCR System 9700 (Applied Biosystems, USA) for 10 min at 25 °C; 30 min at 50 °C; 5 min at 85 °C. The 10 μL RT reaction mix was then diluted × 10 in nuclease-free water and held at −20 °C. Real-time PCR was performed using LightCycler® 480 Ⅱ Realtime PCR Instrument (Roche, Swiss) with 10 μL PCR reaction mixture that included 1 μL of cDNA, 5 μL of 2 × QuantiFast® SYBR® Green PCR Master Mix (Qiagen, Germany), 0.2 μL of forward primer, 0.2 μL of reverse primer and 3.6 μL of nuclease-free water. The reactions were incubated in a 384-well optical plate (Roche, Swiss) at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s. Each sample was run in triplicate for analysis. At the end of the PCR cycles, melting curve analysis was performed to validate the specific generation of the expected PCR product. The primer sequences were designed in the laboratory and synthesized by Generay Biotech (Generay, PRC) based on the mRNA sequences obtained from the NCBI database as follows Table 2.
3.3. Effect of donkey milk on insulin secretion The effect of donkey milk on the insulin secretion of damaged MIN6 cells was measured by ELISA kit. The insulin of normal group, model group, donkey milk group and metformin group were 1.11 ± 0.08 mIU/L , 0.58 ± 0.04 mIU/L, 0.51 ± 0.13 mIU/L and 0.69 ± 0.15 mIU/L, respectively. The insulin levels of STZ-induced damaged cells displayed significantly decreased in comparison to the normal cells. As shown in Fig. 3, donkey milk and metformin had no significant effect on insulin secretion. Table 2 Primers for quantitative real-time PCR.
3
Gene
NCBI accession number
Forward/reverse primers
Pck1
NM_198780
G6PC
NM_013098
ACTB
NM_031144
5’-GTCCCTAAGGAAGACGCC-3’ 5’-ACCTGGTCCTCCAGATAC-3’ 5’-AAGAGCTGCAAAGGAGAAC-3’ 5’-AGATCGACTCAATCTGGGAC-3’ 5’-CCACCATGTACCCAGGCATT-3’ 5’-CGGACTCATCGTACTCCTGC-3’
Journal of Ethnopharmacology 246 (2020) 112221
Y. Li, et al.
Fig. 3. Effect of donkey milk with 500 μg/mL on insulin secretion in vitro (n = 3). ∗∗∗P < 0 .001, significantly different compared with the normal group. Table 4 Effect of the short-term administration of donkey milk on the plasma insulin concentration in diabetic rats. Fig. 1. SDS-PAGE of simulated digestive components in donkey milk. In lanes 1, 2, 3, 4, and 5, the volume of undigested donkey milk loaded was 2, 3, 4, 5, and 6 μL, respectively. In lanes 6, 7, 8, 9, and 10, the volume of digested donkey milk loaded was 2, 3, 4, 5, and 6 μL, respectively. Lane 11 represents the molecular-mass ladder (10–170 kDa).
Lactoferrin Serum albumin High chain immunoglobulins Caseins β-lactoglobulins Lysozyme α-lactalbumin
Volume after digestion
Digestibility
Retention
299440 463505 190190
51875 47476 25066
82.67% 89.76% 86.82%
17.33% 10.24% 13.14%
1297035 761710 2727285 1896200
13197 105161 2587223 1782093
98.98% 86.19% 5.13% 6.02%
1.02% 13.81% 94.87% 93.98%
Insulin (mU/L)
Normal group Diabetic group Donkey milk group Metformin group
22.99 16.29 17.19 16.01
± 1.18 ± 1.45**** ± 0.76 ± 0.62
Data represent the means ± SD (n = 6). ∗∗∗∗P < 0.0001, significantly different compared with the normal group.
Table 3 Changes in the volume and digestibility of donkey milk during in vitro simulated digestion. Volume before digestion
Groups
diabetic group decreased significantly (Table 4). The results suggested that STZ reduced insulin secretion, which is consistent with insulin secretion assay in vitro. Compared to the diabetic group, the HOMA-IR sore was significantly decreased in donkey milk group and the metformin group (Table 5). A decrease in the HOMA-IR score indicated that the target tissues were more insulin-sensitive following the administration of donkey milk. The α-lactalbumin presented in donkey milk increased the insulin sensitivity of the target organs after 4 weeks (Frid et al., 2005). Meanwhile, the blood glucose of donkey milk group and metformin group were significantly decreased in contrast to the diabetic group (Table 6). These results demonstrated that a short-term administration of donkey milk could effectively decrease the blood glucose, increase the insulin sensitivity of the target organs and decrease the IR index. 3.5. Effect of donkey milk on the level of glycosylated hemoglobin in diabetic rats The level of glycosylated hemoglobin in the group of diabetic rats that were administered with metformin was 338.94 nmol/L, which was close to that of the normal group, while the group of diabetic was 391.72 nmol/L (Table 7). The level of glycosylated hemoglobin was relatively high at a high concentration of blood glucose. The result Table 5 Effects of the short-term administration of donkey milk (4 weeks) on the IR index of the diabetic rats.
Fig. 2. Effect of donkey milk and metformin on STZ-induced damaged MIN6 cells in vitro. Data represent the means ± SD (n = 3). ∗P < 0.05; ∗∗∗ P < 0.001, significantly different compared with the metformin. # P < 0.05; ##P < 0.01; ###P < 0.001; ####P < 0.0001, significantly different compared with the model.
3.4. Effect of donkey milk on the IR index and blood glucose in diabetic rats
Groups
HOMA-IR
Normal group Diabetic group Donkey milk group Metformin group
32.25 ± 2.11 112.16 ± 18.44**** 74.94 ± 23.62## 51.06 ± 19.32####
Data represent the means ± SD (n = 6). ∗∗∗∗P < 0.0001, significantly different compared with the normal group. ##P < 0.01; #### P < 0.0001, significantly different compared with the diabetic group.
Compared to the normal group, the insulin concentrations of 4
Journal of Ethnopharmacology 246 (2020) 112221
Y. Li, et al.
3.7. Effect of donkey milk on the antioxidant ability of the plasma of diabetic rats
Table 6 Effect of the short-term administration of donkey milk (4 weeks) on the blood glucose in diabetic rats. Groups
Blood glucose (mmol/L)
Normal group Diabetic group Donkey milk group Metformin group
4.53 ± 0.23 22.18 ± 2.23**** 14.23 ± 5.18## 10.22 ± 3.58####
Following a short-term administration of donkey milk (4 weeks), the whey protein present in donkey milk was found to significantly increase the SOD activity of the plasma of diabetic rats. The GSH and T-AOC levels of donkey milk group increased slightly in comparison to the diabetic group (Table 9). The results reported herein confirmed that donkey milk could enhance the body’s ability to scavenge free radicals and enhance the levels of antioxidants in the body.
Data represent the means ± SD (n = 6). ∗∗∗∗ P < 0.0001, significantly different compared with the normal group. ## P < 0.01; ####P < 0.0001, significantly different compared with the diabetic group.
3.8. The effects of donkey milk on the expression of Pck1 and G6PC As shown in Fig. 4, Pck1 and G6PC relative mRNA decreased significantly, compared with the diabetic group. This result demonstrated that gene expression of the Pck1 and G6PC is inhibited. Donkey milk could act positively in the treatment of diabetes through down-regulating Pck1 and G6PC.
Table 7 Effect of the short-term administration of donkey milk on the levels of glycosylated hemoglobin in diabetic rats. Groups
GHbA1C(nmol/L)
Normal group Diabetic group Donkey milk group Metformin group
313.94 391.72 382.56 338.94
4. Discussion
± 19.13 ± 37.17*** ± 30.44 ± 16.98#
In our previous study, we found that donkey milk have a total of 216 whey proteins. And we conducted proteomic analysis of whey proteins between different milk yields of Dezhou donkey by using label-free mass spectrometry (Zhang et al., 2019). In this study, donkey milk was digested with simulated gastrointestinal fluid in vitro, and investigated by SDS-PAGE. It was found that the retention rates of lysozyme and αlactalbumin in the digested donkey milk were higher than 93%. Lysozyme in donkey milk could induce bacterial death and might provide a material basis for donkey milk to treat lung cough (Chiavari et al., 2005; Horrobin, 2000). The abundant α-lactalbumin in donkey milk provides a modern theoretical basis for the prevention and treatment of diabetes. On one hand, damaged MIN6 cells viability were promoted in vitro. On the other hand, the administration of donkey milk powder for 4 weeks significantly increased the insulin sensitivity of the target organs in the diabetic rats, reduced blood glucose levels, improved the insulin resistance, enhanced the body’s ability to scavenge free radicals, and effectively improved the antioxidant levels in the body. A long-term administration (more than 20 weeks) is expected to completely reverse IR in diabetic rats. Furthermore, we also studied the molecular mechanism and specific targets of donkey milk for treatment of diabetes. Diabetes induced by STZ affect hepatic gluconeogenesis key enzymes G6PC and Pck1 of expression directly or indirectly. Phosphoenolpyr-
Data represent the means ± SD (n = 6). ∗∗∗P < 0.001, significantly different compared with the normal group. #P < 0.05, significantly different compared with the diabetic group.
indicated that blood glucose had been controlled to some extent following the administration of metformin. The level of glycosylated hemoglobin in the group of donkey milk decreased slightly relative to the diabetic group. 3.6. Effect of donkey milk on the body weight of diabetic rats In the normal group, donkey milk group and metformin group, the body weight of rats were increased steadily during the experiment. At the week 4, the final body weight of diabetic group decreased significantly relative to the normal group and increased significantly in the donkey milk group relative to diabetic group (Table 8).
Table 8 Effect of the short-term administration of donkey milk (4 weeks) on the body weight of diabetic rats.
Week Week Week Week
1 2 3 4
Body weights of the normal group (g)
Body weights of the diabetic group (g)
Body weights of the donkey milk group (g)
Body weights of the metformin group (g)
254.75 262.90 274.88 286.17
235.15 239.75 245.75 241.83
237.47 262.08 268.75 271.72
232.37 243.12 253.72 258.33
± ± ± ±
9.65 13.23 10.88 13.09
Data represent the means ± SD (n = 6). ∗P < 0.05; compared with the diabetic group.
± 7.54* ± 12.60 ± 19.92* ± 10.99*** ∗∗∗
± ± ± ±
14.61 22.31 19.56 20.02#
± ± ± ±
8.01 8.17 13.24 18.09
P < 0.001, significantly different compared with the normal group. #P < 0.05, significantly different
Table 9 Effect of the short-term administration of donkey milk (4 weeks) on the antioxidant indices in diabetic rats. Groups
GSH (μmol/L)
MDA (μmol/L)
T-AOC (mmoL/L)
SOD (U/L)
Normal group Diabetic group Donkey milk group Metformin group
12.57 11.19 15.07 11.75
13.77 12.47 11.90 12.27
0.22 0.14 0.16 0.21
253.87 193.20 265.87 272.53
± ± ± ±
4.20 2.24 4.41 3.35
± ± ± ±
2.17 1.25 1.23 0.95
± 0.05 ± 0.01*** ± 0.02 ± 0.02##
± 28.04 ± 52.07* ± 21.29# ± 54.73##
Data represent the means ± SD (n = 6). ∗P < 0.05:**P < 0.01; ∗∗∗P < 0.001, significantly different compared with the normal group. #P < 0.05; ##P < 0.01, significantly different compared with the diabetic group. 5
Journal of Ethnopharmacology 246 (2020) 112221
Y. Li, et al.
Fig. 4. Pck1 and G6PC expression levels between diabetic group and donkey milk group. Data represent the means ± SD (n = 6). ∗P < 0.05; significantly different compared with the diabetic group.
∗∗∗
P < 0.001,
Development Program (2015GSF118099), and The Open Project of Shandong Collaborative Innovation Center for Antibody Drugs, No. CIC-AD1810. We are grateful to the Dr. Yunfeng Deng and Ms. Hui Jing of the Katharine Hsu International Research Center of Human Infectious Diseases (Shandong Provincial Chest Hospital, China) for their help during the experiments performed in this study.
uvate carboxykinase is a rate-limiting step that catalyzes the gluconeogenesis, converting the oxaloacetate to the phosphoenolpyruvate, and the key gluconeogenesis enzyme glucose-6-phosphatase catalyzes the final step in gluconeogenesis, converting glucose 6 phosphate to glucose. After donkey milk was administered, Pck1 and G6PC relative mRNA levels were decreased glycogen output and insulin resistance were also reduced in both. In this study, the metformin was used to the positive control drug. Metformin could decrease the blood glucose, improve the function of pancreatic β cells and reduce insulin resistance. It was the first first-line anti-diabetic agent for type 2 diabetes mellitus treatment (Yang et al., 2016). In our results, donkey milk had a better effect on damaged cells viability than metformin. The anti-diabetic effect of donkey milk was similar to metformin in some biochemical parameters. Therefore, donkey milk could be developed into a supplementary product to treat diabetes. The studies are being carried out to evaluate the anti-diabetic effects when donkey milk would be used in combination with other anti-diabetic drugs.
References Bae, J.S., Kim, T.H., Kim, M.Y., Park, J.M., Ahn, Y.H., 2010. Transcriptional regulation of glucose sensors in pancreatic β-cells and liver: an update. Sensors 10, 5031–5053. https://doi.org/10.3390/s100505031. Belobrajdic, D.P., Mcintosh, G.H., Owens, J.A., 2004. A high-whey-protein diet reduces body weight gain and alters insulin sensitivity relative to red meat in wistar rats. J. Nutr. 134, 1454–1458. https://doi.org/10.1093/jn/134.6.1454. Chiavari, C., Coloretti, F., Nanni, M., Sorrentino, E., Grazia, L., 2005. Use of donkey's milk for a fermented beverage with Lactobacilli. Lait 85, 481–490. https://doi.org/10. 1051/lait:2005031. Corpe, C.P., Eck, P., Wang, J., Al-Hasani, H., Levine, M., 2013. Intestinal dehydroascorbic acid (DHA) transport mediated by the facilitative sugar transporters, GLUT2 and GLUT8. J. Biol. Chem. 288, 9092–9101. https://doi.org/10.1074/jbc.M112.436790. Ebaid, H., 2014. Promotion of immune and glycaemic functions in streptozotocin-induced diabetic rats treated with un-denatured camel milk whey proteins. Nutr. Metab. 11, 1–13. https://doi.org/10.1186/1743-7075-11-31. EI-Hadidy, W.F., Mohamed, A.R., Mannaa, H.F., 2015. Possible protective effect of procainamide as an epigenetic modifying agent in experimentally induced type 2 diabetes mellitus in rats. Alex. J. Med. 51 (1), 65–71. https://doi.org/10.1016/j.ajme. 2014.02.004. Frid, A.H., Nilsson, M., Holst, J.J., Bjorck, I.M., 2005. Effect of whey on blood glucose and insulin responses to composite breakfast and lunch meals in type 2 diabetic subjects. Am. J. Clin. Nutr. 82, 69–75. https://doi.org/10.1093/ajcn/82.1.69. Frestedt, J.L., Zenk, J.L., Kuskowski, M.A., Ward, L.S., Bastian, E.D., 2008. A whey-protein supplement increases fat loss and spares lean muscle in obese subjects: a randomized human clinical study. Nutr. Metab. 5, 8. https://doi.org/10.1186/17437075-5-8. Giovanna, T., Gina, C., Chiara, D.F., Serena, A., Marina, P., Tai, C.J., 2018. Human milk and donkey milk, compared to cow milk, reduce inflammatory mediators and modulate glucose and lipid metabolism, acting on mitochondrial function and oleylethanolamide levels in rat skeletal muscle. Front. Physiol. 9. https://doi.org/10. 3389/fphys.2018.00032. Green, A.D., Vasu, S., Moffett, R.C., Flatt, P.R., 2016. Co-culture of clonal beta cells with glp-1 and glucagon-secreting cell line impacts on beta cell insulin secretion, proliferation and susceptibility to cytotoxins. Biochimie 125, 119–125. https://doi.org/ 10.1016/j.biochi.2016.03.007. Gu, J., Li, W., Xiao, D., Wei, S., Cui, W., Chen, W., 2013. Compound K, a final intestinal metabolite of ginsenosides, enhances insulin secretion in min6 pancreatic β-cells by upregulation of GLUT2. Fitoterapia 87, 84–88. https://doi.org/10.1016/j.fitote. 2013.03.020. Hai-Ming, Z., Feng-Xia, L., Rui, C., 2015. Ancient records and modern research on the mechanisms of Chinese herbal medicines in the treatment of diabetes mellitus. Evid. Based Complement. Alternat. Med. 2015 1–14. https://doi.org/10.1155/2015/ 747982. Herrouin, M., Molle, D., Fauquant, J., Ballestra, F., Maubois, J.L., Leonil, J., 2000. New genetic variants identified in donkey's milk whey proteins. J. Protein Chem. 19 (2), 105–115. https://doi.org/10.1023/A:1007078415595. Horrobin, D.F., 2000. Essential fatty acid metabolism and its modification in atopic eczema. Am. J. Clin. Nutr. 71, 367s–372s. https://doi.org/10.1093/ajcn/71.1.367s.
5. Conclusion Donkey milk will play an important role in treatment of type 2 diabetes. Donkey milk could improve damaged β-cells viability, but does not stimulate insulin secretion from damaged pancreatic β cells. Donkey milk acted on the gluconeogenesis pathway, Pck1 and G6PC relative mRNA levels were decreased. Glycogen output was reduced. Therefore, the blood glucose was decreased. Meanwhile, donkey milk increased the insulin sensitivity of insulin-targeted organs and antioxidant levels in the body. Conflicts of interest The authors declare no potential conflicts of interest. Author contributions Yan Li, Dongliang Wang and Haining Tan designed the experiments and drafted the manuscript. Zhendong Wang and Yumei Fan contributed to the experimental data collection. Abdul Sami Shaikh, Dongliang Wang and Haining Tan revised the manuscript critically for important intellectual content. All authors read and approved the manuscript. Acknowledgments This work was supported by National Natural Science Foundation of China (81473129), Shandong Provincial Key Research and 6
Journal of Ethnopharmacology 246 (2020) 112221
Y. Li, et al. Jakubowicz, D., Froy, O., 2013. Biochemical and metabolic mechanisms by which dietary whey protein may combat obesity and Type 2 diabetes. J. Nutr. Biochem. 24 (1), 1–5. https://doi.org/10.1016/j.jnutbio.2012.07.008. Khan, M.F., Rawat, A.K., Khatoon, S., Hussain, M.K., Mishra, A., Negi, D.S., 2018. In vitro, and in vivo antidiabetic effect of extracts of melia azedarach, zanthoxylum alatum and tanacetum nubigenum. Integr. Med. Res. 7 (2), 176–183. https://doi.org/10. 1016/j.imr.2018.03.004. Lu, D.L., Zhang, D.F., Liu, P.L., Dong, M.L., 2006. The nutrition value and exploitation of donkey milk. J. Dairy Sci. Technol. 29 (6), 11–18. Oftedal, O.T., Jenness, R., 1988. Interspecies variation in milk composition among horses, zebras and asses (Perissodactyla: equidae). J. Dairy Res. 55, 57–66. https://doi.org/ 10.1017/S0022029900025851. Oomen, A.G., Rompelberg, C.J.M., Bruil, M.A., Dobbe, C.J.G., Pereboom, D.P.K.H., Sips, A.J.A.M., 2003. Development of an in vitro digestion model for estimating the bioaccessibility of soil contaminants. Arch. Environ. Contam. Toxicol. 44 (3), 0281–0287. https://doi.org/10.1007/s00244-002-1278-0. Petersen, B.L., Ward, L.S., Bastian, E.D., Jenkins, A.L., Campbell, J., Vuksan, V., 2009. A whey protein supplement decreases post-prandial glycemia. Nutr. J. 8, 47. https:// doi.org/10.1186/1475-2891-8-47. Sebely, P., Vanessa, E., 2010. The acute effects of four protein meals on insulin, glucose, appetite and energy intake in lean men. Br. J. Nutr. 104 (8), 1241–1248. https://doi. org/10.1017/S0007114510001911. Sung, T.C., Huang, J.W., Guo, H.R., 2014. Association between arsenic exposure and diabetes: a meta-analysis. Biomed. Res. Int. 2014 368087. https://doi.org/10.1155/ 2015/368087. Srinivasan, K., Viswanad, B.L., Kaul, C.L., Ramarao, P., 2005. Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. Pharmacol. Res. 52, 313–320. https://doi.org/10.1016/j. phrs.2005.05.004. Thorens, B., 2015. GLUT2, glucose sensing and glucose homeostasis. Diabetologia 58, 221–232. https://doi.org/10.1007/s00125-014-3451-1.
Tidona, F., Sekse, C., Criscione, A., Jacobsen, M., Bordonaro, S., Marletta, D., Vegarud, G.E., 2011. Antimicrobial effect of donkeys' milk digested in vitro with human gastrointestinal enzymes. Int. Dairy J. 21, 158–165. https://doi.org/10.1016/j.idairyj. 2010.10.008. Tomasz, P., Ewa, S., Ewa, P., Wojciech, M., Cezary, W., 2011. Effects of 1-methylnicotinamide and its metabolite N-methyl-2-pyridone-5-carboxamide on streptozotocininduced toxicity in murine insulinoma MIN6 cell line. Acta Biochim. Pol. 58, 75–77. https://doi.org/10.1093/abbs/gmr019. Tong, X., Dong, J.Y., Wu, Z.W., Li, W., Qin, L.Q., 2011. Dairy consumption and risk of type 2 diabetes mellitus: a meta-analysis of cohort studies. Eur. J. Clin. Nutr. 65, 1027–1031. https://doi.org/10.1038/ejcn.2011.62. Trinchese, G., Cavaliere, G., Canani, R.B., Matamoros, S., Bergamo, P., De Filippo, C., 2015. Human, donkey and cow milk differently affects energy efficiency and inflammatory state by modulating mitochondrial function and gut microbiota. J. Nutr. Biochem. 26 (11), 1136–1146. https://doi.org/10.1016/j.jnutbio.2015.05.003. Uniacke-Lowe, T., Huppertz, T., Fox, P.F., 2010. Equine milk proteins: chemistry, structure and nutritional significance. Int. Dairy J. 20 (9), 609–629. https://doi.org/10. 1016/j.idairyj.2010.02.007. Vincenzetti, S., Polidori, P., Fantuz, F., Mariani, P.L., Cammertoni, N., Vita, A., Polidori, F., 2005. Donkey milk caseins characterization. Ital. J. Anim. Sci. 4 (Suppl 2), 427–429. Weng, J., Ji, L., Jia, W., Lu, J., Zhou, Z., Zou, D., Zhu, D., Chen, L., Chen, L., Guo, L., 2016. Standards of care for type 2 diabetes in China. Diabetes-Metab. Res. 32, 442–458. https://doi.org/10.1002/dmrr.2827. Yang, X., Xu, Z., Zhang, C., Cai, Z., Zhang, J., 2016. Metformin, beyond an insulin sensitizer, targeting heart and pancreatic β cells. BBA-Mol. Basis. Dis. 1863 (8), 1984–1990. https://doi.org/10.1016/j.bbadis.2016.09.019. Zhang, X.H., Li, H.J., Yu, J., Zhou, X.S., Ji, C.L., Wu, S.S., Chen, Y.Q., Liu, J.H., Zhao, F.W., 2019. Label-free based comparative proteomic analysis of whey proteins between different milk yields of Dezhou donkey. Biochem. Biophys. Res. Commun. 508, 1. https://doi.org/10.1016/j.bbrc.2018.11.130.
7
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Dezhou donkey (Equus asinus) milk a potential treatment strategy for type 2 diabetes
T
Yan Lia, Yumei Fanb,c, Abdul Sami Shaikhd, Zhendong Wanga, Dongliang Wanga,b,c,∗∗, Haining Tana,∗ a
National Glycoengineering Research Center, Shandong University, Jinan, 250012, China National Engineering Research Center for Gelatin-based Traditional Chinese Medicine, Done-E Country, Liaocheng, 252000, China c Dong-E E-Jiao Co. Ltd., Done-E Country, Liaocheng, 252000, China d Institute of Clinical Pharmacology, Qilu Hospital of Shandong University, Jinan, 250012, China b
ARTICLE INFO
ABSTRACT
Keywords: Donkey milk Digestion Treatment Diabetes Insulin
Ethnopharmacological relevance: Donkey (Equus asinus) milk has become a medical and nutrient product since ancient times. In addition, donkey milk was regarded as a medicinal food and substitute product for infant formula in some ancient western countries. Chinese ancient medical books documented the medicinal value of donkey milk, using donkey milk to treat diabetes, cough and jaundice. Aim of the study: To investigate the donkey milk’s components and anti-diabetic effect of donkey milk in vitro and in vivo and to study the molecular mechanism of donkey milk was an anti-diabetic medication. Materials and methods: In this study, the gastrointestinal digested donkey milk was simulated in vitro and its products of protein digestion were analyzed by SDS-PAGE. We then performed cell viability assay, insulin secretion assay, animal experiments and ELISA assays to study the anti-diabetic effect of donkey milk in vitro and in vivo. Donkey milk’s anti-diabetic molecular mechanism and specific targets were detected by using quantitative real time PCR. Results: Lysozyme (LZ) and α-lactalbumin (α-La) exhibited significantly lower digestibility and higher retention than the other components of donkey milk. In vitro, 500 μg/mL of donkey milk could improve damaged β-cells viability significantly (P < 0.0001). In vivo, the blood glucose and HOMA-IR of diabetic rats treated with donkey milk were 14.23 ± 5.18 mM and 74.94 ± 23.62, respectively, whereas the diabetic group were 22.18 ± 2.23 mM and 112.16 ± 18.44, respectively (P < 0.01). The SOD value of donkey milk group was 265.87 ± 21.29 U/L, while the SOD value of diabetic group was 193.20 ± 52.07 U/L (P < 0.05). These results indicated that the blood glucose was reduced, the ability of the body to eliminate free radicals was enhanced, antioxidant levels in the body was increased, insulin resistance was improved in type 2 diabetic rats after donkey milk powder fed for 4 weeks. Furthermore, donkey milk could treat diabetes through down-regulating phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6PC). Conclusions: Donkey milk has played an important role in the treatment of type 2 diabetes, and contributed to the development of the donkey milk products.
1. Introduction Donkey (Equus asinus) milk contains a complete range of amino acids and is rich in vitamins and minerals. The amount of selenium in donkey milk is 5.16 times that of the selenium content of cow milk, and the quantities of lactalbumin and lactoglobulin account for more than 60% of the total protein content. These data throw light on the composition of donkey milk, and may be of substantial value in the rational
∗
design of infant formula in the future (Chiavari et al., 2005; Herrouin et al., 2000; Horrobin, 2000; Oftedal and Jenness, 1988; Uniacke-Lowe et al., 2010). According to the Compendium of Materia Medica, volume 50, the category of animals and Thousand golden prescriptions, donkey milk is recorded as to cure diabetes, asthma and bronchitis (Lu et al., 2006). It has also been recently reported that the whey protein present in cow milk stimulates insulin secretion and lowers blood sugar levels (Hai-
Corresponding author. National Glycoengineering Research Center, Shandong University, Jinan, 250012, China. Corresponding author. E-mail addresses: [email protected] (D. Wang), [email protected] (H. Tan).
∗∗
https://doi.org/10.1016/j.jep.2019.112221 Received 17 February 2019; Received in revised form 31 August 2019; Accepted 4 September 2019 Available online 05 September 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
Journal of Ethnopharmacology 246 (2020) 112221
Y. Li, et al.
Abbreviations LZ α-La Pck1 G6PC STZ MTT GHbA1c IR HOMA
LF SA HcIG CNs β-Lg SOD GSH MDA T-AOC qPCR Met
lysozyme α-lactalbumin phosphoenolpyruvate carboxykinase 1 glucose-6-phosphatase streptozotocin methylthiazolyldiphenyl-tetrazolium bromide glycosylated hemoglobin insulin resistance homeostasis model assessment
Ming et al., 2015; Jakubowicz and Froy, 2013; Petersen et al., 2009; Sebely and Vanessa, 2010). In comparison to ordinary milk, donkey milk has a higher content of whey protein, which might aid the prevention and treatment of diabetes (Herrouin et al., 2000). The research studies also demonstrate that donkey milk administration improves glucose disposal and insulin resistance, and acts on glucose and lipid metabolism (Giovanna et al., 2018; Trinchese et al., 2015). Donkey milk is known to have high concentration of triacylglycerol fatty acid particularly palmitic acid. Recently, the anti-inflammatory action and beneficial role of donkey milk in glucose metabolism has already been reported (Giovanna et al., 2018). The causes of type 2 diabetes mainly include β-cells dysfunction and insulin resistance produced by peripheral tissues and cells (Weng et al., 2016). The aim to study the donkey milk anti-diabetic function, we have designed cell models in vitro to investigate donkey milk’s anti-diabetic effects in this study. The cell models are damaged mouse insulinoma beta-pancreatic (MIN6) cells. From the experiments in vitro, we can develop a solution towards the treatment of type 2 diabetes with donkey milk. In addition, the effects of donkey milk in vivo on insulin sensitivity, blood glucose, the levels of glycosylated hemoglobin and antioxidants in the plasma were investigated. It is reported that diabetes induced by streptozotocin (STZ) affect hepatic gluconeogenesis key enzymes Pck1 and G6PC of expression directly or indirectly (Bae et al., 2010; Corpe et al., 2013; Sung et al., 2014; Thorens, 2015). In this study, we investigated Pck1 and G6PC relative mRNA levels. Therefore, there is a need for making a good explanation for the molecular aspects of donkey milk in the treatment for diabetes.
lactoferrin serum albumin high-chain immunoglobulin casein β-lactoglobulin superoxide dismutase glutathione malondialdehyde total anti-oxidation capacity quantitative real time PCR metformin
Liaocheng city, China. The gastric juice and intestinal fluid were prepared and the reaction time for chemical digestion was estimated using the methods previously described (Oomen et al., 2003; Tidona et al., 2011). The composition and concentration of the chemical simulator are provided in Table 1. Donkey milk (Dong-E E-Jiao Co. Ltd, China) powder (7.5 g) was dissolved in 75 mL of double-distilled water, mixed with 0.6 mL of gastric digester, and shaken at 37 °C for 0.5 h. Duodenal fluid (2.25 mL) and bile fluid (0.675 mL) were subsequently added to this solution and shaken at 37 °C for 0.5 h. Digestion was ceased at 4 °C and the solution was freeze-dried. And then, samples were analyzed by SDS-PAGE. 2.2. Cell viability assay MIN6 cells were seeded into 96-well plates (6 × 103 cells/well). After incubation overnight, MIN6 cells were treated with 5 mM STZ (Sigma, USA) for 24 h. Subsequently, donkey milk and metformin (Met) of different concentrations (500 μg/mL,250 μg/mL,100 μg/mL,50 μg/ mL and 25 μg/mL) were added to each well for 48 h, respectively. And then, 10 μL methylthiazolyldiphenyl-tetrazolium bromide (MTT, Solarbio, China) was added to each well and further cultured for 4 h under dark conditions. After removing the medium, 150 μL DMSO was added to each well to dissolve the formazan crystal. The absorbance was measured at 490 nm (Tomasz et al., 2011). 2.3. Insulin secretion assay MIN6 cells were seeded into 96-well plates (1 × 104 cells/well). After incubation overnight, MIN6 cells were treated with 5 mM STZ for 24 h. After 48 h incubation with donkey milk 500 μg/mL and metformin 500 μg/mL, the cells were preincubated at 37 °C for 30 min in KrebRinger bicarbonate Hepes buffer (KRB; 129 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 5 mM NaHCO3, 10 mM Hepes, and 0.2% BSA). Cells were then treated in KRB buffer containing
2. Materials and methods 2.1. Digestion of donkey milk Donkey milk selected from a donkey from Dezhou, aged five years and with 2 parities in its late lactation, located in a local farm in Table 1 Composition and concentration of the chemical simulator.
Inorganic salt solution
Organic salt solution
pH
Gastric juice
Duodenal fluid
Bile
1.57 mLNaCl 175.3 g/L 0.3 mL NaH2PO4 88.8 g/L 0.92mLKCl 89.6g/L 1.8 mL CaCl2·2H2O 22.2 g/L 1 mL NH4Cl 30.6 g/L 530 μL HCl 37% g/g
4 mL NaCl 175.3 g/L 4 mL NaHCO3 84.7 g/L 1 mL KH2PO4 8g/L 0.63 mL KCl 89.6 g/L 1 mL MgCl2 5 g/L 18 μL HCl 37% g/g 0.9 mL CaCl2·2H2O 22.2 g/L 0.4 mL urea 25 g/L 0.1 g BSA 0.3 g Parenzyme 0.05 g Lipase
3 mL NaCl 175.3 g/L 6.83 mL NaHCO3 84.7 g/L
7.8 ± 0.2
8.0 ± 0.2
0.34 mL urea 25 g/L 1 mL glucose 65 g/L 1 mL Glucuronic acid 2 g/L 1 mL D-Glucosamine Hydrochloride 33 g/L 0.1 g BSA 0.1 g Pepsin 0.3 g Mucin 1.07 ± 0.07
2
0.42 mL KCl 89.6 g/L 20 μL HCl 37% g/g 1 mL CaCl2·2H2O 22.2 g/L 0.6 g pig Bile salts 1 mL urea 25 g/L 0.18 g BSA
Journal of Ethnopharmacology 246 (2020) 112221
Y. Li, et al.
30 mM glucose for 60 min (Green et al., 2016; Gu et al., 2013). Cell supernatants were collected. Insulin was measured by ELISA kit (JingMei Biotechnology, China).
2.7. Statistical analysis The data are expressed as the mean ± standard deviation. Statistical analyses were performed using the SPSS 16.0 software. Oneway analysis of variance (ANOVA) followed by Tukey post-hoc test was used to assess the results except the quantitative real-time PCR results. The quantitative real-time PCR results were estimated by t-test. A pvalue < 0.05 was considered to be statistically significant.
2.4. Animal experiments All animal experiments in this study were approved by the Laboratory Animal Ethical and Welfare Committee of Shandong University Cheeloo College of Medicine. Twenty four male Wistar rats, aged 5–6 weeks, were divided into 4 groups (n = 6/group) as follows: normal group, diabetic group, donkey milk group and metformin group. The rats in the diabetic group, donkey milk group and metformin group were fed a high-fat diet, the normal rats were fed normal diets for 3 weeks. The rats were starved for 24 h, but were allowed free access to drinking water prior to modeling. The rats were intraperitoneally injected with STZ at a dose of 50 mg/kg. The rats with the fasting blood glucose above 11.1 mmol/L were considered diabetic (Srinivasan et al., 2005). In previous studies, the treatment groups were administered donkey milk powder at a dose of 3 g/kg•d, while the other groups were administered double distilled water (Belobrajdic et al., 2004; Ebaid, 2014; Frid et al., 2005; Frestedt et al., 2008; Tong et al., 2011; Vincenzetti et al., 2005). The metformin group was administered metformin (Shanghai Yuanye Biotechnology, China) at a dose of 100 mg/ kg•d (EI-Hadidy et al., 2015; Khan et al., 2018). The rats were allowed to continue to feed on their respective diets until the end of the study.
3. Results 3.1. Digestion with simulated gastric fluid and duodenal fluid in vitro As shown in Fig. 1, the gray levels (volume) of lactoferrin (LF), serum albumin (SA), high-chain immunoglobulin (HcIG), casein (CNs), and β-lactoglobulin (β-Lg) in digested donkey milk were reduced in comparison to those in undigested milk. As shown in Table 3, LZ and α-La exhibited lower digestibility and higher retention than the other components of donkey milk. The digestibility of the remaining five proteins (LF, SA, HcIG, CNs, and βlactoglobulin) exceeded 82%, while the retention rates for these proteins were less than 18%. The results demonstrated that LF, SA, HcIG, CNs, and LF were digested by the artificial gastrointestinal fluid. β-Lg and CNs were almost completely digested. LZ (5.13%) and α-La (6.02%) displayed lower digestibility in comparison to the other components. These results provided material evidence and an experimental basis for the antidiabetic effects of the oral ingestion of donkey milk powder.
2.5. Insulin and blood glucose, antioxidant capacity, and glycosylated hemoglobin (GHbA1c) assays
3.2. Effect of donkey milk on damaged MIN6 cells
All the rats were fasted for 12 h and the fasting blood glucose was measured by tail-cutting method. Blood samples were collected from the jugular sinus following the measurement of blood glucose. The plasma samples were separated for analysis by centrifugation of the blood samples at 5000 rpm for 5 min at 4 °C. ELISA kits (Bio-Swamp, China) were used measure the levels of related indicators. The index of insulin resistance (IR) as evaluated by the homeostasis model assessment (HOMA) was calculated, according to the following equation: HOMA-IR= Blood glucose (mmol/L) × Insulin/22.5.
The effect of donkey milk on the viability of damaged MIN6 cells was assessed by MTT assay. From Fig. 2, when the concentrations of donkey milk were 25, 100, 200, 250 and 500 μg/mL, the cell viability were 86.60 ± 1.14 %, 82.92 ± 1.13 %, 78.56 ± 1.41 %, 66.83 ± 2.38 % and 56.35 ± 1.56 % , respectively. At the same concentrations of metformin, the cell viability were 75.09 ± 0.45 % , 72.78 ± 4.66 % , 63.90 ± 3.08 % , 59.73 ± 3.38 % and 56.20 ± 2.58 %. Compared to the model group, the cell viability of damaged MIN6 cells treated with 100, 200, 250 and 500 μg/mL donkey milk and metformin increased significantly. At the concentions of 100, 200, 250 and 500 μg/mL, the donkey milk showed better effect than the metformin. MIN6 cells viability increased in a dose-dependent manner as the concentration increased in all groups.
2.6. Quantitative real time PCR Quantification was performed with a two-step reaction process: reverse transcription (RT) and PCR. Each RT reaction has two steps. The first step is 0.5 μg RNA, 2 μL of 4×g DNA wiper Mix, add Nuclease-free H2O to 8 μL. The reactions were performed in a GeneAmp® PCR System 9700 (Applied Biosystems, USA) for 2 min at 42 °C. The second step is to add 2 μL of 5 × HiScript II Q RT SuperMix IIa. The reactions were performed in a GeneAmp® PCR System 9700 (Applied Biosystems, USA) for 10 min at 25 °C; 30 min at 50 °C; 5 min at 85 °C. The 10 μL RT reaction mix was then diluted × 10 in nuclease-free water and held at −20 °C. Real-time PCR was performed using LightCycler® 480 Ⅱ Realtime PCR Instrument (Roche, Swiss) with 10 μL PCR reaction mixture that included 1 μL of cDNA, 5 μL of 2 × QuantiFast® SYBR® Green PCR Master Mix (Qiagen, Germany), 0.2 μL of forward primer, 0.2 μL of reverse primer and 3.6 μL of nuclease-free water. The reactions were incubated in a 384-well optical plate (Roche, Swiss) at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s. Each sample was run in triplicate for analysis. At the end of the PCR cycles, melting curve analysis was performed to validate the specific generation of the expected PCR product. The primer sequences were designed in the laboratory and synthesized by Generay Biotech (Generay, PRC) based on the mRNA sequences obtained from the NCBI database as follows Table 2.
3.3. Effect of donkey milk on insulin secretion The effect of donkey milk on the insulin secretion of damaged MIN6 cells was measured by ELISA kit. The insulin of normal group, model group, donkey milk group and metformin group were 1.11 ± 0.08 mIU/L , 0.58 ± 0.04 mIU/L, 0.51 ± 0.13 mIU/L and 0.69 ± 0.15 mIU/L, respectively. The insulin levels of STZ-induced damaged cells displayed significantly decreased in comparison to the normal cells. As shown in Fig. 3, donkey milk and metformin had no significant effect on insulin secretion. Table 2 Primers for quantitative real-time PCR.
3
Gene
NCBI accession number
Forward/reverse primers
Pck1
NM_198780
G6PC
NM_013098
ACTB
NM_031144
5’-GTCCCTAAGGAAGACGCC-3’ 5’-ACCTGGTCCTCCAGATAC-3’ 5’-AAGAGCTGCAAAGGAGAAC-3’ 5’-AGATCGACTCAATCTGGGAC-3’ 5’-CCACCATGTACCCAGGCATT-3’ 5’-CGGACTCATCGTACTCCTGC-3’
Journal of Ethnopharmacology 246 (2020) 112221
Y. Li, et al.
Fig. 3. Effect of donkey milk with 500 μg/mL on insulin secretion in vitro (n = 3). ∗∗∗P < 0 .001, significantly different compared with the normal group. Table 4 Effect of the short-term administration of donkey milk on the plasma insulin concentration in diabetic rats. Fig. 1. SDS-PAGE of simulated digestive components in donkey milk. In lanes 1, 2, 3, 4, and 5, the volume of undigested donkey milk loaded was 2, 3, 4, 5, and 6 μL, respectively. In lanes 6, 7, 8, 9, and 10, the volume of digested donkey milk loaded was 2, 3, 4, 5, and 6 μL, respectively. Lane 11 represents the molecular-mass ladder (10–170 kDa).
Lactoferrin Serum albumin High chain immunoglobulins Caseins β-lactoglobulins Lysozyme α-lactalbumin
Volume after digestion
Digestibility
Retention
299440 463505 190190
51875 47476 25066
82.67% 89.76% 86.82%
17.33% 10.24% 13.14%
1297035 761710 2727285 1896200
13197 105161 2587223 1782093
98.98% 86.19% 5.13% 6.02%
1.02% 13.81% 94.87% 93.98%
Insulin (mU/L)
Normal group Diabetic group Donkey milk group Metformin group
22.99 16.29 17.19 16.01
± 1.18 ± 1.45**** ± 0.76 ± 0.62
Data represent the means ± SD (n = 6). ∗∗∗∗P < 0.0001, significantly different compared with the normal group.
Table 3 Changes in the volume and digestibility of donkey milk during in vitro simulated digestion. Volume before digestion
Groups
diabetic group decreased significantly (Table 4). The results suggested that STZ reduced insulin secretion, which is consistent with insulin secretion assay in vitro. Compared to the diabetic group, the HOMA-IR sore was significantly decreased in donkey milk group and the metformin group (Table 5). A decrease in the HOMA-IR score indicated that the target tissues were more insulin-sensitive following the administration of donkey milk. The α-lactalbumin presented in donkey milk increased the insulin sensitivity of the target organs after 4 weeks (Frid et al., 2005). Meanwhile, the blood glucose of donkey milk group and metformin group were significantly decreased in contrast to the diabetic group (Table 6). These results demonstrated that a short-term administration of donkey milk could effectively decrease the blood glucose, increase the insulin sensitivity of the target organs and decrease the IR index. 3.5. Effect of donkey milk on the level of glycosylated hemoglobin in diabetic rats The level of glycosylated hemoglobin in the group of diabetic rats that were administered with metformin was 338.94 nmol/L, which was close to that of the normal group, while the group of diabetic was 391.72 nmol/L (Table 7). The level of glycosylated hemoglobin was relatively high at a high concentration of blood glucose. The result Table 5 Effects of the short-term administration of donkey milk (4 weeks) on the IR index of the diabetic rats.
Fig. 2. Effect of donkey milk and metformin on STZ-induced damaged MIN6 cells in vitro. Data represent the means ± SD (n = 3). ∗P < 0.05; ∗∗∗ P < 0.001, significantly different compared with the metformin. # P < 0.05; ##P < 0.01; ###P < 0.001; ####P < 0.0001, significantly different compared with the model.
3.4. Effect of donkey milk on the IR index and blood glucose in diabetic rats
Groups
HOMA-IR
Normal group Diabetic group Donkey milk group Metformin group
32.25 ± 2.11 112.16 ± 18.44**** 74.94 ± 23.62## 51.06 ± 19.32####
Data represent the means ± SD (n = 6). ∗∗∗∗P < 0.0001, significantly different compared with the normal group. ##P < 0.01; #### P < 0.0001, significantly different compared with the diabetic group.
Compared to the normal group, the insulin concentrations of 4
Journal of Ethnopharmacology 246 (2020) 112221
Y. Li, et al.
3.7. Effect of donkey milk on the antioxidant ability of the plasma of diabetic rats
Table 6 Effect of the short-term administration of donkey milk (4 weeks) on the blood glucose in diabetic rats. Groups
Blood glucose (mmol/L)
Normal group Diabetic group Donkey milk group Metformin group
4.53 ± 0.23 22.18 ± 2.23**** 14.23 ± 5.18## 10.22 ± 3.58####
Following a short-term administration of donkey milk (4 weeks), the whey protein present in donkey milk was found to significantly increase the SOD activity of the plasma of diabetic rats. The GSH and T-AOC levels of donkey milk group increased slightly in comparison to the diabetic group (Table 9). The results reported herein confirmed that donkey milk could enhance the body’s ability to scavenge free radicals and enhance the levels of antioxidants in the body.
Data represent the means ± SD (n = 6). ∗∗∗∗ P < 0.0001, significantly different compared with the normal group. ## P < 0.01; ####P < 0.0001, significantly different compared with the diabetic group.
3.8. The effects of donkey milk on the expression of Pck1 and G6PC As shown in Fig. 4, Pck1 and G6PC relative mRNA decreased significantly, compared with the diabetic group. This result demonstrated that gene expression of the Pck1 and G6PC is inhibited. Donkey milk could act positively in the treatment of diabetes through down-regulating Pck1 and G6PC.
Table 7 Effect of the short-term administration of donkey milk on the levels of glycosylated hemoglobin in diabetic rats. Groups
GHbA1C(nmol/L)
Normal group Diabetic group Donkey milk group Metformin group
313.94 391.72 382.56 338.94
4. Discussion
± 19.13 ± 37.17*** ± 30.44 ± 16.98#
In our previous study, we found that donkey milk have a total of 216 whey proteins. And we conducted proteomic analysis of whey proteins between different milk yields of Dezhou donkey by using label-free mass spectrometry (Zhang et al., 2019). In this study, donkey milk was digested with simulated gastrointestinal fluid in vitro, and investigated by SDS-PAGE. It was found that the retention rates of lysozyme and αlactalbumin in the digested donkey milk were higher than 93%. Lysozyme in donkey milk could induce bacterial death and might provide a material basis for donkey milk to treat lung cough (Chiavari et al., 2005; Horrobin, 2000). The abundant α-lactalbumin in donkey milk provides a modern theoretical basis for the prevention and treatment of diabetes. On one hand, damaged MIN6 cells viability were promoted in vitro. On the other hand, the administration of donkey milk powder for 4 weeks significantly increased the insulin sensitivity of the target organs in the diabetic rats, reduced blood glucose levels, improved the insulin resistance, enhanced the body’s ability to scavenge free radicals, and effectively improved the antioxidant levels in the body. A long-term administration (more than 20 weeks) is expected to completely reverse IR in diabetic rats. Furthermore, we also studied the molecular mechanism and specific targets of donkey milk for treatment of diabetes. Diabetes induced by STZ affect hepatic gluconeogenesis key enzymes G6PC and Pck1 of expression directly or indirectly. Phosphoenolpyr-
Data represent the means ± SD (n = 6). ∗∗∗P < 0.001, significantly different compared with the normal group. #P < 0.05, significantly different compared with the diabetic group.
indicated that blood glucose had been controlled to some extent following the administration of metformin. The level of glycosylated hemoglobin in the group of donkey milk decreased slightly relative to the diabetic group. 3.6. Effect of donkey milk on the body weight of diabetic rats In the normal group, donkey milk group and metformin group, the body weight of rats were increased steadily during the experiment. At the week 4, the final body weight of diabetic group decreased significantly relative to the normal group and increased significantly in the donkey milk group relative to diabetic group (Table 8).
Table 8 Effect of the short-term administration of donkey milk (4 weeks) on the body weight of diabetic rats.
Week Week Week Week
1 2 3 4
Body weights of the normal group (g)
Body weights of the diabetic group (g)
Body weights of the donkey milk group (g)
Body weights of the metformin group (g)
254.75 262.90 274.88 286.17
235.15 239.75 245.75 241.83
237.47 262.08 268.75 271.72
232.37 243.12 253.72 258.33
± ± ± ±
9.65 13.23 10.88 13.09
Data represent the means ± SD (n = 6). ∗P < 0.05; compared with the diabetic group.
± 7.54* ± 12.60 ± 19.92* ± 10.99*** ∗∗∗
± ± ± ±
14.61 22.31 19.56 20.02#
± ± ± ±
8.01 8.17 13.24 18.09
P < 0.001, significantly different compared with the normal group. #P < 0.05, significantly different
Table 9 Effect of the short-term administration of donkey milk (4 weeks) on the antioxidant indices in diabetic rats. Groups
GSH (μmol/L)
MDA (μmol/L)
T-AOC (mmoL/L)
SOD (U/L)
Normal group Diabetic group Donkey milk group Metformin group
12.57 11.19 15.07 11.75
13.77 12.47 11.90 12.27
0.22 0.14 0.16 0.21
253.87 193.20 265.87 272.53
± ± ± ±
4.20 2.24 4.41 3.35
± ± ± ±
2.17 1.25 1.23 0.95
± 0.05 ± 0.01*** ± 0.02 ± 0.02##
± 28.04 ± 52.07* ± 21.29# ± 54.73##
Data represent the means ± SD (n = 6). ∗P < 0.05:**P < 0.01; ∗∗∗P < 0.001, significantly different compared with the normal group. #P < 0.05; ##P < 0.01, significantly different compared with the diabetic group. 5
Journal of Ethnopharmacology 246 (2020) 112221
Y. Li, et al.
Fig. 4. Pck1 and G6PC expression levels between diabetic group and donkey milk group. Data represent the means ± SD (n = 6). ∗P < 0.05; significantly different compared with the diabetic group.
∗∗∗
P < 0.001,
Development Program (2015GSF118099), and The Open Project of Shandong Collaborative Innovation Center for Antibody Drugs, No. CIC-AD1810. We are grateful to the Dr. Yunfeng Deng and Ms. Hui Jing of the Katharine Hsu International Research Center of Human Infectious Diseases (Shandong Provincial Chest Hospital, China) for their help during the experiments performed in this study.
uvate carboxykinase is a rate-limiting step that catalyzes the gluconeogenesis, converting the oxaloacetate to the phosphoenolpyruvate, and the key gluconeogenesis enzyme glucose-6-phosphatase catalyzes the final step in gluconeogenesis, converting glucose 6 phosphate to glucose. After donkey milk was administered, Pck1 and G6PC relative mRNA levels were decreased glycogen output and insulin resistance were also reduced in both. In this study, the metformin was used to the positive control drug. Metformin could decrease the blood glucose, improve the function of pancreatic β cells and reduce insulin resistance. It was the first first-line anti-diabetic agent for type 2 diabetes mellitus treatment (Yang et al., 2016). In our results, donkey milk had a better effect on damaged cells viability than metformin. The anti-diabetic effect of donkey milk was similar to metformin in some biochemical parameters. Therefore, donkey milk could be developed into a supplementary product to treat diabetes. The studies are being carried out to evaluate the anti-diabetic effects when donkey milk would be used in combination with other anti-diabetic drugs.
References Bae, J.S., Kim, T.H., Kim, M.Y., Park, J.M., Ahn, Y.H., 2010. Transcriptional regulation of glucose sensors in pancreatic β-cells and liver: an update. Sensors 10, 5031–5053. https://doi.org/10.3390/s100505031. Belobrajdic, D.P., Mcintosh, G.H., Owens, J.A., 2004. A high-whey-protein diet reduces body weight gain and alters insulin sensitivity relative to red meat in wistar rats. J. Nutr. 134, 1454–1458. https://doi.org/10.1093/jn/134.6.1454. Chiavari, C., Coloretti, F., Nanni, M., Sorrentino, E., Grazia, L., 2005. Use of donkey's milk for a fermented beverage with Lactobacilli. Lait 85, 481–490. https://doi.org/10. 1051/lait:2005031. Corpe, C.P., Eck, P., Wang, J., Al-Hasani, H., Levine, M., 2013. Intestinal dehydroascorbic acid (DHA) transport mediated by the facilitative sugar transporters, GLUT2 and GLUT8. J. Biol. Chem. 288, 9092–9101. https://doi.org/10.1074/jbc.M112.436790. Ebaid, H., 2014. Promotion of immune and glycaemic functions in streptozotocin-induced diabetic rats treated with un-denatured camel milk whey proteins. Nutr. Metab. 11, 1–13. https://doi.org/10.1186/1743-7075-11-31. EI-Hadidy, W.F., Mohamed, A.R., Mannaa, H.F., 2015. Possible protective effect of procainamide as an epigenetic modifying agent in experimentally induced type 2 diabetes mellitus in rats. Alex. J. Med. 51 (1), 65–71. https://doi.org/10.1016/j.ajme. 2014.02.004. Frid, A.H., Nilsson, M., Holst, J.J., Bjorck, I.M., 2005. Effect of whey on blood glucose and insulin responses to composite breakfast and lunch meals in type 2 diabetic subjects. Am. J. Clin. Nutr. 82, 69–75. https://doi.org/10.1093/ajcn/82.1.69. Frestedt, J.L., Zenk, J.L., Kuskowski, M.A., Ward, L.S., Bastian, E.D., 2008. A whey-protein supplement increases fat loss and spares lean muscle in obese subjects: a randomized human clinical study. Nutr. Metab. 5, 8. https://doi.org/10.1186/17437075-5-8. Giovanna, T., Gina, C., Chiara, D.F., Serena, A., Marina, P., Tai, C.J., 2018. Human milk and donkey milk, compared to cow milk, reduce inflammatory mediators and modulate glucose and lipid metabolism, acting on mitochondrial function and oleylethanolamide levels in rat skeletal muscle. Front. Physiol. 9. https://doi.org/10. 3389/fphys.2018.00032. Green, A.D., Vasu, S., Moffett, R.C., Flatt, P.R., 2016. Co-culture of clonal beta cells with glp-1 and glucagon-secreting cell line impacts on beta cell insulin secretion, proliferation and susceptibility to cytotoxins. Biochimie 125, 119–125. https://doi.org/ 10.1016/j.biochi.2016.03.007. Gu, J., Li, W., Xiao, D., Wei, S., Cui, W., Chen, W., 2013. Compound K, a final intestinal metabolite of ginsenosides, enhances insulin secretion in min6 pancreatic β-cells by upregulation of GLUT2. Fitoterapia 87, 84–88. https://doi.org/10.1016/j.fitote. 2013.03.020. Hai-Ming, Z., Feng-Xia, L., Rui, C., 2015. Ancient records and modern research on the mechanisms of Chinese herbal medicines in the treatment of diabetes mellitus. Evid. Based Complement. Alternat. Med. 2015 1–14. https://doi.org/10.1155/2015/ 747982. Herrouin, M., Molle, D., Fauquant, J., Ballestra, F., Maubois, J.L., Leonil, J., 2000. New genetic variants identified in donkey's milk whey proteins. J. Protein Chem. 19 (2), 105–115. https://doi.org/10.1023/A:1007078415595. Horrobin, D.F., 2000. Essential fatty acid metabolism and its modification in atopic eczema. Am. J. Clin. Nutr. 71, 367s–372s. https://doi.org/10.1093/ajcn/71.1.367s.
5. Conclusion Donkey milk will play an important role in treatment of type 2 diabetes. Donkey milk could improve damaged β-cells viability, but does not stimulate insulin secretion from damaged pancreatic β cells. Donkey milk acted on the gluconeogenesis pathway, Pck1 and G6PC relative mRNA levels were decreased. Glycogen output was reduced. Therefore, the blood glucose was decreased. Meanwhile, donkey milk increased the insulin sensitivity of insulin-targeted organs and antioxidant levels in the body. Conflicts of interest The authors declare no potential conflicts of interest. Author contributions Yan Li, Dongliang Wang and Haining Tan designed the experiments and drafted the manuscript. Zhendong Wang and Yumei Fan contributed to the experimental data collection. Abdul Sami Shaikh, Dongliang Wang and Haining Tan revised the manuscript critically for important intellectual content. All authors read and approved the manuscript. Acknowledgments This work was supported by National Natural Science Foundation of China (81473129), Shandong Provincial Key Research and 6
Journal of Ethnopharmacology 246 (2020) 112221
Y. Li, et al. Jakubowicz, D., Froy, O., 2013. Biochemical and metabolic mechanisms by which dietary whey protein may combat obesity and Type 2 diabetes. J. Nutr. Biochem. 24 (1), 1–5. https://doi.org/10.1016/j.jnutbio.2012.07.008. Khan, M.F., Rawat, A.K., Khatoon, S., Hussain, M.K., Mishra, A., Negi, D.S., 2018. In vitro, and in vivo antidiabetic effect of extracts of melia azedarach, zanthoxylum alatum and tanacetum nubigenum. Integr. Med. Res. 7 (2), 176–183. https://doi.org/10. 1016/j.imr.2018.03.004. Lu, D.L., Zhang, D.F., Liu, P.L., Dong, M.L., 2006. The nutrition value and exploitation of donkey milk. J. Dairy Sci. Technol. 29 (6), 11–18. Oftedal, O.T., Jenness, R., 1988. Interspecies variation in milk composition among horses, zebras and asses (Perissodactyla: equidae). J. Dairy Res. 55, 57–66. https://doi.org/ 10.1017/S0022029900025851. Oomen, A.G., Rompelberg, C.J.M., Bruil, M.A., Dobbe, C.J.G., Pereboom, D.P.K.H., Sips, A.J.A.M., 2003. Development of an in vitro digestion model for estimating the bioaccessibility of soil contaminants. Arch. Environ. Contam. Toxicol. 44 (3), 0281–0287. https://doi.org/10.1007/s00244-002-1278-0. Petersen, B.L., Ward, L.S., Bastian, E.D., Jenkins, A.L., Campbell, J., Vuksan, V., 2009. A whey protein supplement decreases post-prandial glycemia. Nutr. J. 8, 47. https:// doi.org/10.1186/1475-2891-8-47. Sebely, P., Vanessa, E., 2010. The acute effects of four protein meals on insulin, glucose, appetite and energy intake in lean men. Br. J. Nutr. 104 (8), 1241–1248. https://doi. org/10.1017/S0007114510001911. Sung, T.C., Huang, J.W., Guo, H.R., 2014. Association between arsenic exposure and diabetes: a meta-analysis. Biomed. Res. Int. 2014 368087. https://doi.org/10.1155/ 2015/368087. Srinivasan, K., Viswanad, B.L., Kaul, C.L., Ramarao, P., 2005. Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. Pharmacol. Res. 52, 313–320. https://doi.org/10.1016/j. phrs.2005.05.004. Thorens, B., 2015. GLUT2, glucose sensing and glucose homeostasis. Diabetologia 58, 221–232. https://doi.org/10.1007/s00125-014-3451-1.
Tidona, F., Sekse, C., Criscione, A., Jacobsen, M., Bordonaro, S., Marletta, D., Vegarud, G.E., 2011. Antimicrobial effect of donkeys' milk digested in vitro with human gastrointestinal enzymes. Int. Dairy J. 21, 158–165. https://doi.org/10.1016/j.idairyj. 2010.10.008. Tomasz, P., Ewa, S., Ewa, P., Wojciech, M., Cezary, W., 2011. Effects of 1-methylnicotinamide and its metabolite N-methyl-2-pyridone-5-carboxamide on streptozotocininduced toxicity in murine insulinoma MIN6 cell line. Acta Biochim. Pol. 58, 75–77. https://doi.org/10.1093/abbs/gmr019. Tong, X., Dong, J.Y., Wu, Z.W., Li, W., Qin, L.Q., 2011. Dairy consumption and risk of type 2 diabetes mellitus: a meta-analysis of cohort studies. Eur. J. Clin. Nutr. 65, 1027–1031. https://doi.org/10.1038/ejcn.2011.62. Trinchese, G., Cavaliere, G., Canani, R.B., Matamoros, S., Bergamo, P., De Filippo, C., 2015. Human, donkey and cow milk differently affects energy efficiency and inflammatory state by modulating mitochondrial function and gut microbiota. J. Nutr. Biochem. 26 (11), 1136–1146. https://doi.org/10.1016/j.jnutbio.2015.05.003. Uniacke-Lowe, T., Huppertz, T., Fox, P.F., 2010. Equine milk proteins: chemistry, structure and nutritional significance. Int. Dairy J. 20 (9), 609–629. https://doi.org/10. 1016/j.idairyj.2010.02.007. Vincenzetti, S., Polidori, P., Fantuz, F., Mariani, P.L., Cammertoni, N., Vita, A., Polidori, F., 2005. Donkey milk caseins characterization. Ital. J. Anim. Sci. 4 (Suppl 2), 427–429. Weng, J., Ji, L., Jia, W., Lu, J., Zhou, Z., Zou, D., Zhu, D., Chen, L., Chen, L., Guo, L., 2016. Standards of care for type 2 diabetes in China. Diabetes-Metab. Res. 32, 442–458. https://doi.org/10.1002/dmrr.2827. Yang, X., Xu, Z., Zhang, C., Cai, Z., Zhang, J., 2016. Metformin, beyond an insulin sensitizer, targeting heart and pancreatic β cells. BBA-Mol. Basis. Dis. 1863 (8), 1984–1990. https://doi.org/10.1016/j.bbadis.2016.09.019. Zhang, X.H., Li, H.J., Yu, J., Zhou, X.S., Ji, C.L., Wu, S.S., Chen, Y.Q., Liu, J.H., Zhao, F.W., 2019. Label-free based comparative proteomic analysis of whey proteins between different milk yields of Dezhou donkey. Biochem. Biophys. Res. Commun. 508, 1. https://doi.org/10.1016/j.bbrc.2018.11.130.
7