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6J mice
Journal of Functional Foods 63 (2019) 103505
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Mulberry leaf aqueous extract ameliorates blood glucose and enhances energy expenditure in obese C57BL/6J mice
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Xiaoyun Hea,b,1, Haoyu Lib,1, Ruxin Gaob, Chuanhai Zhanga,b, Fei Liangc, Yao Shenga, ⁎ Shujuan Zhenga, Jia Xua, Wentao Xua,b, Kunlun Huanga,b, a Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China b Key Laboratory of Safety Assessment of Genetically Modified Organism (Food Safety), Ministry of Agriculture, Beijing 100083, China c Department of Reproductive Physiology, Zhejiang Academy of Medical Sciences, Hangzhou 310013, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Obesity Mulberry leaf aqueous extract Blood glucose Insulin resistance Energy expenditure
Mulberry leaf garners much attention from researchers because of its hypoglycemic effect. This study’s objective was to determine the impact of mulberry leaf aqueous extract (MLAE) on decreasing blood glucose and promoting energy expenditure in obese C57BL/6J mice (induced by a high-fat diet). The mice were fed with high fat diet and treated with MLAE for 12 weeks. The results showed that MLAE significantly ameliorated the hyperglycemia and insulin resistance in mice that were induced by their high-fat diet. The improved blood glucose metabolism accompanied by elevated energy expenditure was found. Abundant flavonoids, polyphenols, and alkaloids in MLAE may have contributed to the above results. Also, MLAE restored gut microbiota imbalance in obese mice and was beneficial to their liver and kidney functioning. Together, these results suggest MLAE may be a potent ingredient in dietetics and can serve as a safe hypoglycemic ingredient in health product development.
1. Introduction Obesity is a global health concern, and according to a recent study from the Centers for Disease Control and Prevention (CDC) of the USA, 69% of adults in the USA are overweight, and more than 35% of all adults there are obese (Health, 2014). This high rate of obesity is probably caused by high-fat and high-sugar diets and the lack of necessary physical exercise common in modern society (Brundtland, 2002; Storlien, James, Burleigh, Chisholm, & Kraegen, 1986). Obesity predisposes the body to onset of cardiovascular disease, diabetes mellitus, hyperlipidemia, and arteriosclerosis (Mehta & Farmer, 2007). To treat and prevent obesity and obesity complications, an increasing number of people choose hypoglycemic or weight-loss drugs. However, their long-term use will damage the liver and kidney. Not surprisingly, finding safe and effective weight-loss and hypoglycemic ingredients is becoming increasingly urgent. Diabetes is now a major modern disease threatening humans. According to the latest data available from the International Diabetes Federation, ca. 415 million adults have diabetes worldwide, with one in every 11 adults globally suffering from diabetes (Cho, Whiting, &
Forouhi, 2016); this suggests it has become one of the main threats to human life. Diabetes is divided into type 1 and type 2; the latter is more common, however, for which obesity is one of the important causes (Lim, Lee, Kim, Yang, & Lim, 2013). A key feature of type 2 diabetes is insulin resistance, caused by inflammation that is induced by macrophage infiltration into adipose tissue (Shoelson, Lee, & Goldfine, 2006; Weisberg, 2003). High-fat intake is considered a major factor in developing insulin resistance and obesity (Jr, 1985). Therefore, ameliorating insulin resistance and reducing blood glucose are crucial means of treating type 2 diabetes. High-fat diet induction is a common way to establish an obesity model, with mounting evidence of insulin resistance and glucose metabolism disorder in animals fed a high-fat diet (Cani et al., 2007; Riant et al., 2013; Shoelson, Lee, & Goldfine, 2006). In this way, using such diets lets us build models for studying obesity and hyperglycemia. Many studies have shown that mulberry leaf extract and dry powder are rich in a variety of active compounds, including alkaloids, phenolic acids, and flavonoids, such as DNJ, chlorogenic acid and rutin respectively (Hunyadi, Martins, Hsieh, Seres, & Zupkó, 2012; Kim & Jang, 2011; Tsuduki, Kikuchi, Nakagawa, & Miyazawa, 2013). DNJ, a kind of
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Corresponding author at: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China. E-mail address: [email protected] (K. Huang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jff.2019.103505 Received 7 April 2019; Received in revised form 15 July 2019; Accepted 5 August 2019 1756-4646/ © 2019 Published by Elsevier Ltd.
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To prepare the MLAE, the mulberry leaf powder was mixed with distilled water in a ratio of dried mulberry leaf powder:water = 1:10 (mass ratio), and the mixture was placed in a 60 °C-water bath for 2 h. The mixture was then centrifuged for 10 min at 2000 rpm, and the supernatant was inspissated on a vacuum rotary evaporator (at 48 °C, 30 rpm). The concentrated solution was lyophilized for 37 h, and the lyophilized power was stored at −80 °C until its use. Following the methods used by other laboratories with some modifications (Aliakbarian, Fathi, Perego, & Dehghani, 2012; JeszkaSkowron, 2014; Kim & Jang, 2011; Xu, 2015), the total phenolic contents and flavonoid contents (respectively, TPC and TFC), as well as the rutin, chlorogenic acid, and DNJ contents, of MLAE and the dry mulberry leaves (prepared according to the method described previously) were determined by liquid chromatography on an Agilent 1100 system (Agilent Technologies Inc, Santa Clara, USA) and a spectrophotometer (UVIKON XL-172, Secomam AG, France). We mainly optimized the chromatographic conditions. The specific modifications are as follows: The optimal derivatization conditions were: 100 μL of leaf extract was mixed with 100 μL of 0.4 M potassium borated buffer (pH 8.0) in a 5 mL microtube. 700 μL of 1 g/L FMOC-Cl in acetonitrile was mixed and allowed to react at room temperature for 10 min. The optimized chromatographic conditions were: the samples were eluted with a mobile phase of ACN–10 mM ammonium acetate(pH 4.0) (40:60, v/v) at 0.8 mL/min for 15 min.
alkaloid, is a characteristic constituent of mulberry leaves; it is a potent R-glucosidase inhibitor with low toxicity (Ju, Kim, Sung, & Kim, 2016), and many experiments have demonstrated that treatment with DNJ can significantly reduce the blood sugar of hyperglycemia-induced mice (Asano, 2001; Li, 2013). Chlorogenic acid, one of the most abundant phenylpropanoids compounds in human diets, was reported to modulate glucose and lipid metabolism in vivo, in both healthy individuals and those with conditions related to genetic metabolic disorders (Meng, Cao, Feng, Peng, & Hu, 2013). Rutin, a compound present in mulberry leaf extract, is a kind of flavonoid and possesses anti-inflammatory and antioxidant activities and reduces insulin resistance in insulin-resistant mice (Hsu, 2014). Other work has shown that mulberry leaf extracts had hypoglycemic and antiatherogenic effects in certain animal models and in humans (Miyahara, Miyazawa, Satoh, Sakai, & Mizusaki, 2004; Murata, 2004). Finally, according to several studies, mulberry leaves and their ethanol extracts can improve insulin secretion, which is the mechanism by which mulberry leaves can treat type 2 diabetes (Ji, 2011; Ren, 2015). To our best knowledge, there is limited information available on preventing type 2 diabetes in obese mice using mulberry leaf aqueous extract. In our study, the C57BL/6J mouse model was established by high-fat diet induction. To explore its effects and how MLAE improves energy metabolism and reduces blood glucose, the body weight, energy metabolism, glucose metabolism, and insulin resistance were measured in these obese mice treated with mulberry leaf aqueous extract for 12 weeks. Some indicators of liver and kidney functioning were also monitored to ensure there is no liver or kidney damage of MLAE treatment.
2.3. Animals The experimental design was approved by the Animal Ethics Committee of China Agricultural University, Beijing (approval ID: KY20150017). MLAE, 3- to 4-week-old C57BL/6J mice were purchased from Vital River Laboratories (Beijing, China) and housed (4 animals/ cage) under controlled temperature (22 ± 2 °C), humidity (55% ± 10%), and light (12-h light-dark cycle) conditions in a specific pathogen-free (SPF) animal room at the Supervision, Inspection and Testing Center for Genetically Modified Organisms of the Ministry of Agriculture (Beijing, China; license number SYXK [Beijing] 20150045).
2. Materials and methods 2.1. Diet composition Fresh mulberry leaf samples were obtained from Danyang City, Jiangsu Province, China. The high-fat diet (60% calories from fat, D12492) and low-fat diet (10% calories from fat, D12450B) for the mice were both prepared by Beijing Hua Fukang Bioscience Co., LTD (Beijing, China). All reagents used in the current study were of analytical grade (Table 1).
2.4. Experimental protocols Animals were allowed to acclimate to their room environment for 7 days. Thirty-six C57BL/6J MLAE mice were randomized into two groups: one group had 12 mice that were fed a low-fat diet, while the other group had 24 mice that were fed a high-fat diet. After 8 weeks of feeding, 6 and 12 mice were appropriately selected from the low-fat diet group and high-fat diet group according to weight, respectively; the difference in the average body weight between these two groups exceeded 15%. Then, the high-fat diet group was randomly divided into the MLAE and HFD groups (n = 6 per group), and the low-fat diet group was renamed the LFD group (n = 6). The LFD and HFD groups served as the vehicle control and negative control, respectively. The mice in the MLAE group were treated with a mulberry leaf aqueous extract (1000 mg/kg body weight, BW) solution dissolved in distilled water, administered by stomach gavage daily at the same time and kept high-fat diet until the end of the experiment. Both LFD and HFD groups were treated with distilled water by stomach gavage, as done for the MLAE group. The stomach gavage treatment period lasted 12 weeks, and the diets provided to each group were the same as prior to gavage. Table 1 gives the composition of both experimental diets, and their respective weight ratio and energy ratio information are given in Table 2. The body weights and food intake were recorded weekly. Energy intake was calculated for the mice in all treatment groups using the same formula: food intake of the low-fat chow (g) [or high-fat chow (g)] × 3.58 [(or 5.24 for high-fat chow)]. Body fat percentage and FFM were calculated based on the ratio of white adipose tissue weight and
2.2. Chemicals Fresh mulberry leaves were first dehydrated at a constant temperature of 60 °C in a convection dryer (YF-6CHZ-2, Yongfengjixie, Fujian, China). Then, a grinder was used to powder the dried mulberry leaves, so they could pass through a 40-mesh sieve. Table 1 Composition and percentage content of the low-fat diet (LFD) and high-fat diet (HFD) of experimental mice.
Casein Cystine Corn starch Maltodextrin Sucrose Cellulose Bean oil Lard M1002 DCP Calcium carbonate Potassium citrate monohydrate V1001 Choline bitartrate Edible dye Total:
LFD
HFD
18.985 0.284 29.859 3.318 33.177 4.740 2.370 1.896 0.948 1.232 0.520 1.564 0.948 0.190 0.005 100.000
25.845 0.388 0.000 16.153 8.891 6.461 3.231 31.660 1.292 1.680 0.711 2.132 1.291 0.258 0.007 100.000
2
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functioning in the experimental mice, their serum samples were used for blood biochemical tests of high-density lipoprotein (HDL), lowdensity lipoprotein (LDL), triglycerides (TG), blood urea nitrogen (BUN), creatinine (CREA), aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and dehydrogenase (LDH). All were quantified on a HITACHI 7020 instrument (Hitachi Ltd, Japan)
Table 2 Weight ratio and energy ratio in low-fat diet (LFD) and high-fat diet (HFD) fed to the experimental mice. LFD
Protein Carbohydrate Fat Total: kcal/g
HFD
Weight ratio g%
Energy ratio kcal%
Weight ratio g%
Energy ratio kcal%
19.2 67.3 4.3
20 70 10 100
26 26 35
20 20 60 100
3.85
2.10. Intestinal microflora analysis Mouse feces were collected and stored at −80 °C. Microbial genomic DNA was extracted from each fecal sample using an extraction kit. The V3 + V4 region of the 16 S rRNA was amplified by PCR and sequenced on a Hi-Seq platform (Illumina, San Diego, USA) at The Novogene Bioinformatics Institute (Beijing, China). Sequence analyses were performed using the UPARSE v7.0.1001 software tool (UPARSE); those sequences with ≥97% similarity were assigned to the same operational taxonomic unit (OTU). Taxonomic annotation was carried out using an RDP classifier (v2.2). Nonmetric multidimensional scaling (NMDS) plots and alpha and beta diversity indices were used to analyze the variation between each group, utilizing the PAST v2.17 software program. Heat maps were drawn using heatmap illustrator software (v1.0.3.7).
5.24
body weight of the mouse. 2.5. Blood glucose and intraperitoneal glucose tolerance test Blood glucose was determined from mice tail blood samples, taken after 16 h of fasting every four weeks, by blood glucose meters (ACCUCHEK, Shanghai, China). In the 12th week of the MLAE treatment, intraperitoneal glucose tolerance tests (GTT) were performed on the mice of all groups. Each animal was fasted for 16 h and then given an intraperitoneal injection of glucose (1.5 g/kg BW). Blood glucose levels were measured at 0, 15, 30, 60, and 120 min post-glucose injection. The area under the curve of blood glucose concentration change from 0 to 120 min was calculated (Kong, 2008).
2.11. Statistics Single-factor analysis of variance (ANOVA) followed by two-tailed Student’s t tests was used to compare the groups’ means, using MS Office Excel 2013. Graphs were plotted using GraphPad Prism 5 (GraphPad Software, California, USA). Almost all data are presented as the mean ± SEM. Significant differences were considered found at P < 0.05.
2.6. Plasma insulin and HOMA-IR After completing the mice experiment, blood samples were taken from the mice and their plasma insulin assayed by a mouse INS ELISA kit (Huaxiabio, Beijing, China). The homeostasis model assessment-insulin resistance (HOMA-IR) was used to measure insulin sensitivity of individual mice of all groups as follows: [fasting plasma insulin (μg/ L) × fasting blood glucose (mg/dL)]/22.5 (Ding et al., 2010).
3. Results 3.1. Component analysis of MLAE and mulberry leaves
2.7. Rectal temperature and energy expenditure The contents of the major components in MLAE and dry mulberry leaf powder are shown in Table 3. The most abundant active ingredients in MLAE mainly included rutin (44.38 mg/g), chlorogenic acid (7.33 mg/g), and DNJ (3.29 mg/g).
An Oxylet system (Panlab, Spain) was prepared for each mouse at 11 week of the MLAE treatment, for which free access to food and water was provided in their cages. After 12 h of acclimation, oxygen consumption was measured for 24 h, from which volume of oxygen consumption (VO2) and the respiratory exchange ratio (RER) were obtained. Energy expenditure was calculated, using VO2 and RER, as follows: {VO2 × [3.815 + (1.23 × RER)] × 40.1868}, expressed as kilojoules per kilogram fat-free mass (FFM) per hour (Stewart, 2008; Xu, 2009). The mice were also placed in a cold room (4 °C) for 4 h, and their cold-induced thermogenesis was evaluated with a temperature sensor (AT210, Zhongyi Dapeng, Shenzhen, China). Finally, each mouse was photographed using a handheld infrared camera (FLIR T600, FLIR, Oregon, USA) against a blank whiteboard.
3.2. Effect of MLAE on mice body weight Mice with high-fat diet-induced obesity (DIO) were successfully obtained at 8 weeks post-treatment, at which time they weighed at least 15% more than the control mice. After 12 weeks, the mice fed the HFD had body weights 26% higher than the LFD control mice (31.53 compared with 39.74 g, Fig. 1A). In addition, the MLAE-treated animals underwent a 6% decrease in body weight compared with the HFD group (Fig. 1A). No significant differences were found in the body weight, fat mass, lean mass, and body fat percentage between the HFD and MLAE groups (Fig. 1B–D).
2.8. Preparation of adipose tissues and blood samples At the end of 12 weeks, blood was collected from the orbital venous plexus in the eyes of the mice. For the biochemical characterization of this blood, serum samples were isolated from it by centrifugation (5000 rpm, 10 min) and collected. Then, all mice were euthanized, and their abdominal adipose tissues and epididymal adipose tissues were dissected out and weighed. All the blood and tissues samples were stored at −80 °C until further analysis.
Table 3 Quantification of the major components in lyophilized MLAE and dry mulberry leaves (mg/g dry extract or mulberry leaves powder).
2.9. Biochemical characterization
Sample
TFC (mg/g)
TPC (mg/g)
Rutin (mg/g)
Chlorogenic acid (mg/g)
DNJ (mg/g)
MLAE Dry mulberry leaves
44.38 29.86
29.32 16.94
44.38 29.86
7.33 6.89
3.29 1.28
TPC: Total phenolic contents. TFC: Total flavonoid contents.
To detect lipid metabolism and determine liver and kidney 3
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Fig. 1. The MLAE treatment did not significantly affect mice body weight but did improve their mean daily food intake compared with the HFD group. (A) The mean body weights of the LFD, HFD, and MLAE groups in the 12-week experiment. (B) Fat mass, (C) lean mass, (D) fat mass ratio, and (E) food intake of the LFD, HFD, and MLAE groups were measured and calculated after 12 weeks of treatment. Bars represent the mean + SEM, n = 6. The significance level is represented by “a,” “b,” and “c” (P < 0.05). There are no significant differences between “ab” and “a” or “b.”
3.3. MLAE enhanced food intake
3.4. Hypoglycemic effect of MLAE on DIO mice
Food intake was recorded weekly, and daily food intake was calculated by averaging the accumulated food intake at 12 weeks. Daily food intake was significantly higher in the MLAE than HFD group (P < 0.01, Fig. 1E), but it was similar between the LFD and MLAE groups (Fig. 1E).
At the treatment’s start, blood glucose levels in the MLAE and HFD groups significantly exceeded those in the LFD group, a difference induced by the 8-week-feeding of a high-fat diet. In this 12-week experiment, the MLAE group’s blood glucose consistently decreased, while that of the HFD group continued to increase, whereas that of the LFD group remained essentially unchanged. From weeks 4 and 5 4
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Fig. 2. The blood glucose level and insulin resistance were both lower in the MLAE group than those in the HFD group. (A) Blood glucose was measured every 4 weeks. (B) A glucose tolerance test was performed by injecting 15%-glucose solution into C57BL/6J mice. (C) The area under the curve of the glucose tolerance test was calculated for the C57BL/6J mice. (D) The plasma insulin concentration was measured in the 12th week. (E) The HOMA-IR values were calculated by fasting plasma insulin (μg/L) × fasting blood glucose (mg/dL)]/22.5. Bars represent the mean + SEM, n = 6. P < 0.05 and P < 0.01 when comparing the MLAE group with the HFD control. The significance level is represented by “a,” “b,” and “c.” There are no significant differences between “ab” and “a” or “b.”
groups (P < 0.05, Fig. 2B). Variation in fasting blood glucose levels occurred over time, with those in MLAE-treated mice being significantly lower than those in the HFD group at 60, 90, and 120 min post-injection of the glucose solution (P < 0.05, Fig. 2B). The area under the curve (AUC) of the HFD group was significantly greater than that of either the LFD or MLAE group (54.0% and 17.5%, Fig. 2C). The effects of MLAE on plasma insulin concentration were also evaluated by GTT after 12 weeks of daily stomach gavage with the MLAE solution. There was a significant reduction in plasma insulin concentrations in the MLAE group relative to those of the HFD group (P < 0.05, Fig. 2D), and likewise for their corresponding HOME-IR values (P < 0.05, Fig. 2E). However, no significant differences were
through week 12, a significant difference emerged in blood glucose levels between the MLAE and HFD groups (P < 0.05; Fig. 2A), where blood glucose levels in the LFD and MLAE groups were similar at the 12th week. Hence, the hypoglycemic effect of MLAE was significant (Fig. 2A). 3.5. MLAE ameliorated carbohydrate metabolism and insulin resistance GTT was performed in all three groups after 12 weeks of treatment. Mice in the LFD group served as the normal glucose metabolism group (the control). Initial fasting blood glucose levels (time = 0 min) tended to be significantly higher in the HFD group than either MLAE or NCD 5
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Fig. 3. The MLAE treatment promoted thermogenesis and energy metabolism in C57BL/6J mice. The daily energy intake calculated by food intake of low-fat chow (g) [or high-fat chow (g)] × 3.58 [or 5.24 for high-fat chow]. (A) Infrared imaging, (B) infrared imaging values, (C) rectal temperatures, (D) energy expenditures calculated from VO2 and RER {VO2 × [3.815 + (1.23 × RER)] × 40.1868} in (E) the LFD, HFD, and MLAE groups were measured and determined at the 12th week. Bars represent the mean + SEM, n = 6. The significance level is represented by “a,” “b,” and “c.” There are no significant differences between “ab” and “a” or “b.”
3.7. MLAE improved energy metabolism of mice
evident between the HFD and MLAE groups in terms of their plasma insulin concentrations or HOMA-IR values (P < 0.05, Fig. 2E).
To explore whether mulberry leaves could ameliorate energy metabolism and enhance energy expenditure, the rectal temperature, conducted infrared thermal imaging, and calculated EE values (based on VO2 and RQ) of the experimental mice have been measured. Rectal temperatures of the MLAE group were significantly higher than those of the HFD and LFD groups (P < 0.05, Fig. 3B). Infrared thermal images were taken with a handheld infrared camera (Fig. 3C), and their resulting values were greater in the MLAE than the HFD group (P < 0.05, Fig. 3D). Furthermore, calculated EE values of the MLAE group were significantly higher than those of the LFD and HFD groups
3.6. MLAE improved energy intake in DIO mice To intuitively study the energy metabolism of mice, their individual energy intake values were derived from their monitored food intakes. Energy intake of the MLAE treatment group significantly exceeded those of the other two groups (P < 0.05, Fig. 3A). Additionally, energy intake in the HFD group was higher than that in the LFD group (P < 0.05, Fig. 3A). 6
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The MLAE treatment decreased the AST, ALP, ALT, and LDH levels relative to the HFD group, however no significant differences have been found (Fig. 4B). No significant differences in these levels occurred between the LFD and HFD groups. Concerning the BUN and CREA levels, treatment with MLAE significantly reduced the CREA content in DIO mice (P < 0.05; Fig. 4C) but negligibly affected the CREA or BUN contents of the control mice. 3.10. MLAE treatment restored gut microbiota imbalance in DIO mice The intestinal flora data of mice were categorized into an HFD group (high-fat-fed obese model mice); LFD group (low-fat-fed mice), MLAE group (added mulberry-fed obese model mice), and Initial State group (all mice, just after acclimation to the room environment when not yet grouped). Differences between the above groups in their mice gut flora were analyzed by non-metric multidimensional scaling analysis (NMDS) (Fig. 5A). Evidently, the HFD group was most different from the LFD group and Initial State group, indicating the intestinal flora of obese mice differed significantly from that of the pre-fed and LFD-fed mice at the end of the experiment. Compared with the LFD and the Initial Sate groups, the MLAE group has a large area intersection in the two-dimensional plane distribution of the NMDS map, and the difference is not significant. Comparing the α- and β-diversity between the four groups, as in Fig. 5B and C, both measures of microbial diversity in the HFD group differed significantly from those of the LFD group and the mulberry leaf treatment group, and these latter two groups had similar α-diversity. This indicated mulberry leaf feeding was capable of restructuring the intestinal flora community of DIO mice back to its normal composition. According to the heat map-cluster analysis (Fig. 5D), differences between the HFD and LFD groups were significant, in that the mulberry leaf treatment could restore most of the changed flora to some extent. Compared with the LFD group, Zymomonas, Helicobacter, Bilophila in Proteobacteria; Anaerrotruncus, Anaerovorax, Ruminococcus, Oscillospira, Dehalobacterium, Coprococcus, Anaerostipes, Lactobacillales, Roseburia in Firmicutes; Odoribacter in Bacteroidetes; Desulfovibrio in Nitrospirae and Mucispirillum in Deferribacteres of HFD group all significantly increased, whereas the mulberry leaf treatment group was able to reverse these changes to gut microflora. The microorganisms that were significantly reduced in DIO mice—such as Enterococcus in Firmicutes; Devosia in Proteobacteria; Paraprevotella, Prevotella in Bacteroides; Bifidobacterium in Actinobacteria—were also increased by feeding mulberry leaves to the mice. Therefore, the mulberry leaf treatment greatly improved the intestinal flora of DIO mice on the whole.
Fig. 4. The MLAE treatment ameliorated the blood lipid profile and have no damage to liver and kidney function, based on the blood biochemistry of mice. The lipid indexes HDL, LDL, and TG (A), markers of liver function ALT, AST, ALP, and LDH (B), and markers of kidney function BUN and CREA (C), in the C57BL/6J mice. Bars represent the mean + SEM, n = 6. The significance level is represented by “a,” “b,” and “c.” There are no significant differences between “ab” and “a” or “b.”
(P < 0.05, Fig. 3E). These results indicated MLAE indeed improved energy metabolism in the mice and promoted thermogenesis.
4. Discussion In China, mulberry leaves are used as a traditional medicinal herb (Miyahara, Miyazawa, Satoh, Sakai, & Mizusaki, 2004). The present research shows that mulberry leaves can decrease blood glucose and blood pressure in mice and improve their insulin sensitivity. These impacts on bodily health may have been caused by the high content of alkaloids, polyphenols, and flavonoids characterizing mulberry leaves (Ji et al., 2011; Ren et al., 2015; Yang, Jhon, & Tseng, 2012). Compared with the reported constituents of edible vegetables and fruits, the total polyphenol and flavonoid contents of the mulberry leaves used in our study are much higher, suggesting that mulberry leaves and their aqueous extract harbor hypoglycemic potential (Miean & Mohamed, 2001; Turkmen, Sari, & Velioglu, 2005). In our study, the body weights of the mice treated with MLAE by stomach gavage for 12 weeks were on par with those of the HFD group (Fig. 1A). Similarly, Lim, Lee, Kim, Yang, and Lim (2013) found that treatment with mulberry leaf extract had no significant effect on the body weight of mice fed a high-fat diet. In addition, some investigators have suggested that black tea, green tea, and mulberry extracts in
3.8. MLAE ameliorated the lipid profile of DIO mice To gauge the effect of MLAE on lipid metabolism, we took blood serum samples from the mice for their biochemical testing. A high-fat diet significantly increased the levels of several metabolic risk factors, namely TG and LDL (P < 0.05, Fig. 4A). However, the MLAE treatment ameliorated the high-fat diet-induced high LDL and increased the HDL in DIO mice compared with the corresponding levels of the HFD group; this indicated that MLAE improved the lipid profile of DIO mice and reduced their risk of cardiovascular disease (Nardelli, Ribeiro, & Balbo, 2011; Stefanick, 1998). Stomach gavage with the MLAE solution did not significantly influence the TG in mice. 3.9. MLAE have no damage to liver and kidney function To assess the safety of MLAE for liver and kidney functioning, clinical biochemical analyses of mice serum samples were performed. 7
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Fig. 5. The MLAE treatment restored the gut microbiota imbalance in DIO mice. NMDS plot of the Bray–Curtis similarity coefficients calculated from the 16 S rRNA sequencing data of the gut microbiota (A). Mulberry leaf influences intestinal flora of type 2 diabetes rats at phylum (B). Alpha diversity of each group represented by the Simpson index (C). Beta diversity of each group as quantified by the Bray–Curtis index (D). Heat map-A cluster analysis of the effects of MLAE on intestinal flora of DIO mice (E). Bars represent the mean + SEM, Initial state: n = 8, HFD: n = 10, LFD: n = 6, MLAE: n = 7. *P < 0.05 between compared groups, **P < 0.01 between compared groups.
reduced blood glucose compared with the HFD group, and because the MLAE and LFD groups had similar blood glucose levels, this demonstrated that MLAE has a measurable hypoglycemic effect (Fig. 2). These results are consistent with other studies that reported similar decreases in blood glucose levels in diabetic rats treated with the ethanol or acetone extracts of mulberry leaves (Jeszka-Skowron, 2014). The hypoglycemic effect in our study could be due to the high levels of DNJ (Jimin, 2009), flavonoids (Jadhav & Puchchakayala, 2012), and polyphenols (Ji, Chung, Jung, Wee, & Kwon, 2013) in mulberry leaves. Of these bioactive substances, DNJ in particular has received special attention as a competitive inhibitor of intestinal α-glucosidases, which can affect carbohydrate digestion and absorption and lead to suppressed postprandial hyperglycemia (Chung, Kim, JI, & Kwon, 2013).
combination do not influence weight loss (e.g., Fallon, Zhong, Furne, & Levitt, 2008). In our work here, the HFD group had higher energy intake than did the LFD group, and mice in the HFD group were 22% heavier than those in the LFD group at week 12, on average. However, daily energy intake and food intake of the MLAE group surpassed that of the HFD group, yet the former had a lower mice weight and body fat percentage than did the latter group. These results may indicate that although the difference was not significant, MLAE still contributed to weight loss. In subsequent experiments, MLAE can significantly improve the energy expenditure of mice, which neatly explains these results. We also focused on exploring the hypoglycemic effect of MLAE treatment by stomach gavage. In this respect, the MLAE treatment 8
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organs. Studies show that the content and proportion of Firmicutes and Bacteroidetes in the intestinal flora can change significantly in diabetic patients or obese patients. It is known that Bacteroidetes and Firmicutes have important effects on body metabolism in the intestinal tract, where there exists a mutualism in the form of a symbiotic relationship, which together promotes the body’s absorption or storage of energy. Changes in the gut proportions of Bacteroidetes and Firmicutes can promote obesity and type 2 diabetes (Larsen, 2010; Rios-Covian, 2013; Turnbaugh, 2006). In our study, mulberry leaves restored the reduced proportion of Bacteroides and sclerenchyma in intestinal flora of obese mice (Fig. 5B). Bifidobacterium are a kind of intestine-beneficial microorganisms, which act as biological barriers capable of not only nutritional, anti-tumor, and immune enhancement effects but also improved gastrointestinal function, anti-aging responses, and other important physiological functions (Kootte, 2012; Le, 2014). Studies have shown that Bifidobacterium can affect the body’s insulin sensitivity and glucose tolerance (Ding et al., 2010). In our experiment, the number of intestinal Bifidobacterium in DIO mice was reduced, but mulberry leaves restored their abundance. Intestinal inflammation is closely related to the development of obesity and diabetes in the body. Ding et al. found that the local inflammatory reaction of intestinal tissue is related to obesity and insulin resistance in mice and may be involved in the occurrence and development of type 2 diabetes. In DIO mice, their intestinal flora has an increased content of Desulfovibrio, which is a gram-negative genus associated with intestinal inflammation (Sawin, 2015), but mulberry leaves can lower Desulfovibrio abundance. Therefore, mulberry leaves may regulate glucose metabolism and energy consumption by increasing the ratio of intestinal Bacteroidetes and Firmicutes, thereby augmenting the content of beneficial bacteria and reducing intestinal inflammation in DIO mice. Recently, studies have shown that mulberry leaf extract can improve blood sugar levels in type 2 diabetes mellitus through clinical trials (Lown et al., 2017; Riche, Riche, East, Barrett, & May, 2017). This shows that mulberry leaf extract as a natural and safe food functional factor has more application prospects.
Work by Kimura (2007) reported that healthy volunteers who took 0.8 or 1.2 g of DNJ-enriched mulberry powder had markedly suppressed elevation of their postprandial blood glucose levels. In our glucose tolerance tests, at 0 min, the lower mean fasting blood glucose levels of the MLAE group suggested mulberry leaves contributed to significantly reducing blood glucose in mice after 12 weeks of the MLAE treatment (Fig. 2B). Blood sugar levels similar between LFD and MLAE groups at 0 min further indicated mulberry leaves can decrease blood sugar to approximately normal levels. At 60, 90, and 120 min, the significant differences found vis-à-vis the HFD group suggested that MLAE treatment can improve glucose tolerance, and these effects were confirmed by the AUC. The plasma insulin results indicated that administering the MLAE for 12 weeks considerably decreased the fasting insulin concentration and HOMA-IR in the DIO mice. This effect improved glucose tolerance and quantitatively reduced plasma insulin concentrations and HOMA-IR values, which confirmed the diminished insulin resistance of DIO mice was a direct response to MLAE. Similar findings were reported by Ji Yeon Kim, who found the same effect in obese rats when 5% mulberry leaf water extract was added to their diet for 6 weeks (Ji, 2011). DNJ reportedly has an antidiabetic effect in type 2 diabetic rats, for which notable improvements occurred in fasting blood glucose levels and glucose tolerance, but especially increased insulin sensitivity (Kong, 2008). However, some flavonoids and polyphenols, such as rutin and chlorogenic acid, can also improve glucose tolerance and decrease insulin resistance (Hsu et al., 2014; Johnston, Clifford, & Morgan, 2003). The synergistic effects of these active ingredients in mulberry leaves may even be stronger than those arising from pure DNJ (Kwon, Ji, Ji, & Kwon, 2011). In our study, although MLAE improved glucose metabolism, the AUC values of the MLAE and LFD groups were significantly different. Hence, the duration and dosage of the MLAE treatment were likely insufficient, in that a simple MLAE treatment cannot completely restore glucose metabolism to its normal levels. Importantly, fat feeding decreases energy expenditure and causes obesity (Storlien, James, Burleigh, Chisholm, & Kraegen, 1986). Indeed, the infrared thermal imaging values and rectal temperature were lowest in the HFD group, which indicated that HFD decreased energy expenditure in mice; however, since the EE values in obese mice were enhanced by the MLAE treatment (Fig. 3A-E), this suggested that MLAE not only reduced the extent EE’s decline as induced by a high-fat diet but also improved EE to above-normal levels. This effect may be due to the flavonols and quercetin in mulberry leaves, which are known to elevate oxygen consumption in human skeletal myocytes (Stewart, 2008). The mechanism of this effect is thought to involve upregulation of type 2 deiodinase expression, which can increase energy expenditure by increasing the formation of triiodothyronine (Da-Silva, 2007). Obesity can lead to liver damage, which mainly manifests as elevated ALT and AST (Fraulob, Ogg-Diamantino, Fernandes-Santos, Aguila, & Mandarim-de-Lacerda, 2010). Here, because ALT, ASL, ALP, and LDH levels—all biomarkers of liver injury—were decreased in the MLAE group (Fig. 4B) this suggests MLAE have no damage on mouse liver Tang, Huang, Lee, Tang, & Wang, 2013 have reported similar outcomes, showing that mulberry water extracts can significantly inhibit the levels of ALT, AST, and ALP. Furthermore, no differences among the BUN levels of the three groups and a significant decrease in CREA in the MLAE group relative to the HFD group together indicate that MLAE has a potential protective effects on kidney functioning (Fig. 4C) (Diwan, Brown, & Gobe, 2017; Hemmati, Jalali, Rashidi, & Hormozi, 2010). The MLAE’s protection of the liver and kidney likely depends on the flavonoids and polyphenols, such as rutin and chlorogenic acid, in the mulberry leaves, since these components can suppress oxidative stress and inflammation while also improving kidney regeneration (Domitrović, Cvijanović, Šušnić, & Katalinić, 2014; Korkmaz & Kolankaya, 2013; Lee, Shen, Lai, & Wu, 2013; Shi, 2013). In summary, our experiment proved that MLAE imparts a protective effect on liver and kidney functioning rather than being toxic to these vital
5. Conclusion This study investigated the beneficial effects of MLAE for decreasing insulin resistance and blood glucose levels, as well as improving energy metabolism, in obese mice. The safety of our MLAE treatment was also been confirmed, proving it caused no damage to liver and kidney function but instead simply protected them. Although the MLAE treatment did not significantly reduce the weight of the mice, combined with the energy intake, MLAE did impede the process of weight gain. MLAE’s high content of bioactive molecules, namely, DNJ, flavonoids, and polyphenols, may be primarily responsible for the multiple physiological functions of MLAE, yet specific molecular mechanisms still require elucidation. This study demonstrated the various physiological functions of MLAE and provides guidance for developing functional foods and medicines based on MLAE. Author disclosures The National Natural Science Foundation of China (81700684 to C. Zhang). Practical application Our research suggests MLAE can serve as a safe hypoglycemic ingredient for use in the development of health products, and perhaps even drugs. The hypoglycemic effect of mulberry leaves/MLAE was significant and sustained, indicating its great potential in food applications; currently, these applications include using mulberry leaves/ MLAE as a food additive to make health drinks or bread for people with 9
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diabetes or diabetic nephropathy. The specific composition and functions of MLAE thus merit further investigation and practical study.
Food Chemistry, 132(4), 1796–1801. Cho, N., Whiting, D., & Forouhi, N. (2016). IDF Diabetes Atlas, 2015. Fallon, E., Zhong, L., Furne, J. K., & Levitt, M. (2008). A mixture of extracts of black and green teas and mulberry leaf did not reduce weight gain in rats fed a high-fat diet. Alternative Medicine Review, 13(1), 43–49. Fraulob, J. C., Ogg-Diamantino, R., Fernandes-Santos, C., Aguila, M. B., & Mandarim-deLacerda, C. A. (2010). A mouse model of metabolic syndrome: Insulin resistance, fatty liver and non-alcoholic fatty pancreas disease (NAFPD) in C57BL/6 mice fed a high fat diet. Journal of Clinical Biochemistry and Nutrition, 46(3), 212–223. Health, U. D. O. (2014). Health, United States, 2013: With special feature on prescription drugs. Claitors Publishing. Hemmati, A. A., Jalali, M. T., Rashidi, I., & Hormozi, T. K. (2010). Impact of aqueous extract of black mulberry (Morus nigra) on liver and kidney funcion of diabetic mice. Jundishapur Journal of Natural Pharmaceutical Products, 5(1), S48–S49. Hsu, C. Y., Shih, H. Y., Chia, Y. C., Lee, C. H., Ashida, H., Lai, Y. K., & Weng, C. F. (2014). Rutin potentiates insulin receptor kinase to enhance insulin-dependent glucose transporter 4 translocation. Molecular Nutrition & Food Research, 58(6), 1168–1176. Hunyadi, A., Martins, A., Hsieh, T. J., Seres, A., & Zupkó, I. (2012). Chlorogenic acid and rutin play a major role in the in vivo anti-diabetic activity of Morus alba leaf extract on type II diabetic rats. PLoS ONE, 7(11), e50619. Jadhav, R., & Puchchakayala, G. (2012). Hypoglycemic and antidiabetic activity of flavonoids: Boswellic acid, Ellagic acid, quercetin, Rutin on StreptozotocinNicotinamide induced Type 2 Diabetic. International Journal of Pharmacy & Pharmaceutical Sciences, 4(2), 251–256. Jeszka-Skowron, M., Flaczyk, E., Jeszka, J., Krejpcio, Z., Król, E., & Buchowski, M. S. (2014). Mulberry leaf extract intake reduces hyperglycaemia in streptozotocin (STZ)induced diabetic rats fed high-fat diet. Journal of Functional Foods, 8(1), 9–17. Ji, Y. K., Choi, B. G., Jung, M. J., Wee, J. H., Chung, K. H., & Kwon, O. (2011). Mulberry leaf water extract ameliorates insulin sensitivity in high fat or high sucrose diet induced overweight rats. Applied Biological Chemistry, 54(4), 612–618. Ji, Y. K., Chung, H. I., Jung, K. O., Wee, J. H., & Kwon, O. (2013). Chemical profiles and hypoglycemic activities of mulberry leaf extracts vary with ethanol concentration. Food Science and Biotechnology, 22(5), 1–5. Jimin, P., Hayoon, B., Hyein, J., Yeonkyoung, K., Jiyeon, K., & Oran, K. (2009). Postprandial hypoglycemic effect of mulberry leaf in Goto-Kakizaki rats and counterpart control Wistar rats. Nutrition Research & Practice, 3(4), 272–278. Johnston, K. L., Clifford, & Morgan, L. M. (2003). Coffee acutely modifies gastrointestinal hormone secretion and glucose tolerance in humans: Glycemic effects of chlorogenic acid and caffeine. American Journal of Clinical Nutrition, 78(4), 728. Jr, D. E. (1985). Diet and obesity. American Journal of Clinical Nutrition, 41(5 Suppl), 1132. Ju, W. T., Kim, H. B., Sung, G. B., & Kim, Y. S. (2016). Comparison of optimal temperature and time conditions for highest a-glucosidase inhibitory activity from various of Korea mulberry teas, 33, 31–35. Kim, G. N., & Jang, H. D. (2011). Flavonol content in the water extract of the mulberry (Morus alba L.) leaf and their antioxidant capacities. Journal of Food Science, 76(6), 869–873. Kimura, T., Nakagawa, K., Kubota, H., Kojima, Y., Goto, Y., Yamagishi, K., ... Miyazawa, T. (2007). Food-grade mulberry powder enriched with 1-deoxynojirimycin suppresses the elevation of postprandial blood glucose in humans. Journal of Agricultural and Food Chemistry, 55(14), 5869–5874. Kong, W. H., Oh, S. H., Ahn, Y. R., Kim, K. W., Kim, J. H., & Seo, S. W. (2008). Antiobesity effects and improvement of insulin sensitivity by 1-deoxynojirimycin in animal models. Journal of Agricultural & Food Chemistry, 56(8), 2613–2619. Kootte, R. S., Vrieze, A., Holleman, F., Dallinga-Thie, G. M., Zoetendal, E. G., de Vos, W. M., ... Nieuwdorp, M. (2012). The therapeutic potential of manipulating gut microbiota in obesity and type 2 diabetes mellitus. Diabetes, Obesity & Metabolism, 14(2), 112–120. Korkmaz, A., & Kolankaya, D. (2013). Inhibiting inducible nitric oxide synthase with rutin reduces renal ischemia/reperfusion injury. Canadian Journal of Surgery Journal Canadien De Chirurgie, 56(1), 6–14. Kwon, O., Kwon, H. J., Ji, Y. C., & Ji, Y. K. (2011). Comparison of 1-deoxynojirimycin and aqueous mulberry leaf extract with emphasis on postprandial hypoglycemic effects. In vivo and in vitro studies. Journal of Agricultural & Food Chemistry, 59(7), 3014–3019. Larsen, N., Vogensen, F. K., van den Berg, F. W., Nielsen, D. S., Andreasen, A. S., Pedersen, B. K., ... Jakobsen, M. (2010). Gut microbiota in human adults with type 2 diabetes differs from non-diabetic adults. PLoS ONE, 5(2), e9085. Le, T. K., Hosaka, T., LE, T. T., Nguyen, T. G., Tran, Q. B., Le, T. H., & Pham, X. D. (2014). Oral administration of Bifidobacterium spp. improves insulin resistance, induces adiponectin, and prevents inflammatory adipokine expressions. Biomedical Research, 35(5), 303–310. Lee, C. C., Shen, S. R., Lai, Y. J., & Wu, S. C. (2013). Rutin and quercetin, bioactive compounds from tartary buckwheat, prevent liver inflammatory injury. Food & Function, 4(5), 794–802. Li, Y. G., Ji, D. F., Zhong, S., Lin, T. B., Lv, Z. Q., Hu, G. Y., & Wang, X. (2013). 1deoxynojirimycin inhibits glucose absorption and accelerates glucose metabolism in streptozotocin-induced diabetic mice. Scientific Reports, 3(7442), 1377. Lim, Y., Lim, H. H., Lee, S. O., Kim, S. Y., & Yang, S. J. (2013). Anti-inflammatory and antiobesity effects of mulberry leaf and fruit extract on high fat diet-induced obesity. Experimental Biology and Medicine, 238(10), 1160. Lown, M., Fuller, R., Lightowler, H., Fraser, A., Gallagher, A., Stuart, B., ... Lewith, G. (2017). Mulberry-extract improves glucose tolerance and decreases insulin concentrations in normoglycaemic adults: Results of a randomised double-blind placebocontrolled study. PLoS ONE, 12(2), e0172239. Mehta, S., & Farmer, J. A. (2007). Obesity and inflammation: A new look at an old
Ethics statement The experimental design was approved by Animal Ethics Committee of China Agricultural University, Beijing, and the approval ID of this study is KY20150017. Male, 3–4 weeks old, C57BL/6J mice were purchased from Vital River Laboratories (Beijing) and housed (4 animal/ cage) under controlled temperature (22 ± 2 °C) and humidity (55% ± 10%) and light (12 h light-dark cycle) in an Specific Pathogen Free (SPF) animal room at the Supervision, Inspection and Testing Center for Genetically Modified Organisms of the Ministry of Agriculture (Beijing, China; license number SYXK (Beijing) 2015-0045). Acknowledgments This research was supported in part by the National Natural Science Foundation of China (81700684 to C. Zhang) and the Key Research and Development Program of Shandong Province (No. 2018YYSP019). Declaration of Competing Interest The authors report that there are no conflicts of interest to declare. Author Contributions C.Z. wrote the main manuscript; H.L. and K.H. designed the study; Y.S., H.L., and S Z. performed the animal trials; W X. performed the component analysis; H.L. and C.Z. performed the data analyses. X.Y. revised the manuscript. All authors reviewed the manuscript. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.103505. References Aliakbarian, B., Fathi, A., Perego, P., & Dehghani, F. (2012). Extraction of antioxidants from winery wastes using subcritical water. The Journal of Supercritical Fluids, 65(65), 18–24. Asano, N., Yamashita, T., Yasuda, K., Ikeda, K., Kizu, H., Kameda, Y., ... Ryu, K. S. (2001). Polyhydroxylated alkaloids isolated from mulberry trees (Morus alba L.) and silkworms (Bombyx mori L.). Journal of Agricultural and Food Chemistry, 49(9), 4208–4213. Brundtland, G. H. (2002). From the World Health Organization. Reducing risks to health, promoting healthy life. Jama the Journal of the American Medical Association, 288(16), 1974. Cani, P. D., Amar, J., Iglesias, M. A., Poggi, M., Knauf, C., Bastelica, D., ... Chabo, C. (2007). Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes, 56(7), 1761–1772. Chung, H. I., Kim, J., JI, Y. K., & Kwon, O. (2013). Acute intake of mulberry leaf aqueous extract affects postprandial glucose response after maltose loading: Randomized double-blind placebo-controlled pilot study. Journal of Functional Foods, 5(3), 1502–1506. Da-Silva, W. S., Harney, J. W., Kim, B. W., Li, J., Bianco, S. D., Crescenzi, A., ... Bianco, A. C. (2007). The small polyphenolic molecule kaempferol increases cellular energy expenditure and thyroid hormone activation. Diabetes, 56(3), 767–776. Ding, S., Chi, M. M., Scull, B. P., Rigby, R., Schwerbrock, N. M. J., Magness, S., ... Lund, P. K. (2010). High-fat diet: Bacteria interactions promote intestinal inflammation which precedes and correlates with obesity and insulin resistance in mouse. PLoS ONE, 5(8), e12191. Diwan, V., Brown, L., & Gobe, G. C. (2017). The flavonoid rutin improves kidney and heart structure and function in an adenine-induced rat model of chronic kidney disease. Journal of Functional Foods, 33, 85–93. Domitrović, R., Cvijanović, O., Šušnić, V., & Katalinić, N. (2014). Renoprotective mechanisms of chlorogenic acid in cisplatin-induced kidney injury. Toxicology, 324(10), 98. Meng, S., Cao, J., Feng, Q., Peng, J., & Hu, Y. (2013). Roles of Chlorogenic Acid on Regulating Glucose and Lipids Metabolism: A Review. Evidence-Based Complementray and Alternative Medicine,2013,(2013-8-25), 2013, 801457. Yang, N. C., Jhon, K. Y., & Tseng, C. Y. (2012). Antihypertensive effect of mulberry leaf aqueous extract containing γ-aminobutyric acid in spontaneously hypertensive rats.
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Shoelson, S. E., Lee, J., & Goldfine, A. B. (2006). Inflammation and insulin resistance. Diabetes Care, 116(7), 1793–1801. Stefanick, M. L., Mackey, S., Sheehan, M., Ellsworth, N., Haskell, W. L., & Wood, P. D. (1998). Effects of diet and exercise in men and postmenopausal women with low levels of HDL cholesterol and high levels of LDL cholesterol. New England Journal of Medicine, 339(1), 12–20. Stewart, L. K., Soileau, J. L., Ribnicky, D., Wang, Z. Q., Raskin, I., Poulev, A., ... Gettys, T. W. (2008). Quercetin transiently increases energy expenditure but persistently decreases circulating markers of inflammation in C57BL/6J mice fed a high-fat diet. Metabolism-clinical & Experimental, 57(7 Suppl 1), S39. Storlien, L. H., James, D. E., Burleigh, K. M., Chisholm, D. J., & Kraegen, E. W. (1986). Fat feeding causes widespread in vivo insulin resistance, decreased energy expenditure, and obesity in rats. American Journal of Physiology, 251(5 Pt 1), E576–E583. Tang, Y. H., Tang, C. C., Huang, H. P., Lee, Y. J., & Wang, C. J. (2013). Hepatoprotective effect of mulberry water extracts on ethanol-induced liver injury via anti-inflammation and inhibition of lipogenesis in C57BL/6J mice. Food & Chemical Toxicology An International Journal Published for the British Industrial Biological Research Association, 62(2Pt1), 786–796. Tsuduki, T., Kikuchi, I., Nakagawa, K., & Miyazawa, T. (2013). Intake of mulberry 1deoxynojirimycin prevents diet-induced obesity through increases in adiponectin in mice. Food Chemistry, 139(1–4), 16–23. Turkmen, N., Sari, F., & Velioglu, Y. S. (2005). The effect of cooking methods on total phenolics and antioxidant activity of selected green vegetables. Food Chemistry, 93(4), 713–718. Turnbaugh, P. J., Ley, R. E., Mahowald, M. A., Magrini, V., Mardis, E. R., & Gordon, J. I. (2006). An obesity-associated gut microbiome with increased capacity for energy harvest. Nature (London), 444(7122), 1027–1131. Weisberg, S. P., Mccann, D., Desai, M., Rosenbaum, M., Leibel, R. L., & Ferrante, A. W. (2003). Obesity is associated with macrophage accumulation in adipose tissue. Journal of Clinical Investigation, 112(12), 1796–1808. Xu, J., Lloyd, D. C., Stanislaus, S., Chen, M., Sivits, G., Vonderfecht, S., ... Chen, J. (2009). Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice. Diabetes, 58(1), 250–259. Xu, C., Yang, B., Wei, Z., Li, X., Tian, J., & Lin, Z. (2015). Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities. Food Chemistry, 186, 83–89.
problem. Current Atherosclerosis Reports, 9(2), 134–138. Miean, K. H., & Mohamed, S. (2001). Flavonoid (myricetin, quercetin, kaempferol, luteolin, and apigenin) content of edible tropical plants. Journal of Agricultural and Food Chemistry, 49(6), 3106–3112. Miyahara, C., Miyazawa, M., Satoh, S., Sakai, A., & Mizusaki, S. (2004). Inhibitory effects of mulberry leaf extract on postprandial hyperglycemia in normal rats. Journal of Nutritional Science & Vitaminology, 50(3), 161–164. Murata, K., Yatsunami, K., Fukuda, E., Onodera, S., Mizukami, O., Hoshino, G., & Kamei, T. (2004). Antihyperglycemic effects of propolis mixed with mulberry leaf extract on patients with type 2 diabetes. Alternative Therapies in Health and Medicine, 10(3), 78–79. Nardelli, T. R., Ribeiro, R. A., & Balbo, S. L. (2011). Taurine prevents fat deposition and ameliorates plasma lipid profile in monosodium glutamate-obese rats. Amino Acids, 41(4), 901–908. Ren, C., Zhang, Y., Cui, W., Lu, G., Wang, Y., Gao, H., ... Mu, Z. (2015). A polysaccharide extract of mulberry leaf ameliorates hepatic glucose metabolism and insulin signaling in rats with type 2 diabetes induced by high fat-diet and streptozotocin. International Journal of Biological Macromolecules, 72, 951. Riant, E., Waget, A., Cogo, H., Arnal, J. F., Burcelin, R., & Gourdy, P. (2013). Estrogens protect against high-fat diet-induced insulin resistance and glucose intolerance in mice. Endocrinology, 150(5), 2109–2117. Riche, D. M., Riche, K. D., East, H. E., Barrett, E. K., & May, W. L. (2017). Impact of mulberry leaf extract on type 2 diabetes (Mul-DM): A randomized, placebo-controlled pilot study. Complementary Therapies in Medicine, 32, 105–108. Rios-Covian, D., Arboleya, S., Hernandez-Barranco, A. M., Alvarez-Buylla, J. R., RuasMadiedo, P., Gueimonde, M., & de los Reyes-Gavilan, C. G. (2013). Interactions between bifidobacterium and bacteroides species in; Cofermentations are affected by carbon sources, including; Exopolysaccharides produced by bifidobacteria. Applied & Environmental Microbiology, 79(23), 7518–7524. Sawin, E. A., De Wolfe, T. J., Aktas, B., Stroup, B. M., Murali, S. G., Steele, J. L., & Ney, D. M. (2015). Glycomacropeptide is a prebiotic that reduces Desulfovibrio bacteria, increases cecal short-chain fatty acids, and is anti-inflammatory in mice. American Journal of Physiology. Gastrointestinal and Liver Physiology, 309(7), G590. Shi, H., Dong, L., Jiang, J., Zhao, J., Zhao, G., Dang, X., ... Jia, M. (2013). Chlorogenic acid reduces liver inflammation and fibrosis through inhibition of toll-like receptor 4 signaling pathway. Toxicology, 303(1), 107–114.
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Mulberry leaf aqueous extract ameliorates blood glucose and enhances energy expenditure in obese C57BL/6J mice
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Xiaoyun Hea,b,1, Haoyu Lib,1, Ruxin Gaob, Chuanhai Zhanga,b, Fei Liangc, Yao Shenga, ⁎ Shujuan Zhenga, Jia Xua, Wentao Xua,b, Kunlun Huanga,b, a Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China b Key Laboratory of Safety Assessment of Genetically Modified Organism (Food Safety), Ministry of Agriculture, Beijing 100083, China c Department of Reproductive Physiology, Zhejiang Academy of Medical Sciences, Hangzhou 310013, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Obesity Mulberry leaf aqueous extract Blood glucose Insulin resistance Energy expenditure
Mulberry leaf garners much attention from researchers because of its hypoglycemic effect. This study’s objective was to determine the impact of mulberry leaf aqueous extract (MLAE) on decreasing blood glucose and promoting energy expenditure in obese C57BL/6J mice (induced by a high-fat diet). The mice were fed with high fat diet and treated with MLAE for 12 weeks. The results showed that MLAE significantly ameliorated the hyperglycemia and insulin resistance in mice that were induced by their high-fat diet. The improved blood glucose metabolism accompanied by elevated energy expenditure was found. Abundant flavonoids, polyphenols, and alkaloids in MLAE may have contributed to the above results. Also, MLAE restored gut microbiota imbalance in obese mice and was beneficial to their liver and kidney functioning. Together, these results suggest MLAE may be a potent ingredient in dietetics and can serve as a safe hypoglycemic ingredient in health product development.
1. Introduction Obesity is a global health concern, and according to a recent study from the Centers for Disease Control and Prevention (CDC) of the USA, 69% of adults in the USA are overweight, and more than 35% of all adults there are obese (Health, 2014). This high rate of obesity is probably caused by high-fat and high-sugar diets and the lack of necessary physical exercise common in modern society (Brundtland, 2002; Storlien, James, Burleigh, Chisholm, & Kraegen, 1986). Obesity predisposes the body to onset of cardiovascular disease, diabetes mellitus, hyperlipidemia, and arteriosclerosis (Mehta & Farmer, 2007). To treat and prevent obesity and obesity complications, an increasing number of people choose hypoglycemic or weight-loss drugs. However, their long-term use will damage the liver and kidney. Not surprisingly, finding safe and effective weight-loss and hypoglycemic ingredients is becoming increasingly urgent. Diabetes is now a major modern disease threatening humans. According to the latest data available from the International Diabetes Federation, ca. 415 million adults have diabetes worldwide, with one in every 11 adults globally suffering from diabetes (Cho, Whiting, &
Forouhi, 2016); this suggests it has become one of the main threats to human life. Diabetes is divided into type 1 and type 2; the latter is more common, however, for which obesity is one of the important causes (Lim, Lee, Kim, Yang, & Lim, 2013). A key feature of type 2 diabetes is insulin resistance, caused by inflammation that is induced by macrophage infiltration into adipose tissue (Shoelson, Lee, & Goldfine, 2006; Weisberg, 2003). High-fat intake is considered a major factor in developing insulin resistance and obesity (Jr, 1985). Therefore, ameliorating insulin resistance and reducing blood glucose are crucial means of treating type 2 diabetes. High-fat diet induction is a common way to establish an obesity model, with mounting evidence of insulin resistance and glucose metabolism disorder in animals fed a high-fat diet (Cani et al., 2007; Riant et al., 2013; Shoelson, Lee, & Goldfine, 2006). In this way, using such diets lets us build models for studying obesity and hyperglycemia. Many studies have shown that mulberry leaf extract and dry powder are rich in a variety of active compounds, including alkaloids, phenolic acids, and flavonoids, such as DNJ, chlorogenic acid and rutin respectively (Hunyadi, Martins, Hsieh, Seres, & Zupkó, 2012; Kim & Jang, 2011; Tsuduki, Kikuchi, Nakagawa, & Miyazawa, 2013). DNJ, a kind of
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Corresponding author at: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China. E-mail address: [email protected] (K. Huang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jff.2019.103505 Received 7 April 2019; Received in revised form 15 July 2019; Accepted 5 August 2019 1756-4646/ © 2019 Published by Elsevier Ltd.
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To prepare the MLAE, the mulberry leaf powder was mixed with distilled water in a ratio of dried mulberry leaf powder:water = 1:10 (mass ratio), and the mixture was placed in a 60 °C-water bath for 2 h. The mixture was then centrifuged for 10 min at 2000 rpm, and the supernatant was inspissated on a vacuum rotary evaporator (at 48 °C, 30 rpm). The concentrated solution was lyophilized for 37 h, and the lyophilized power was stored at −80 °C until its use. Following the methods used by other laboratories with some modifications (Aliakbarian, Fathi, Perego, & Dehghani, 2012; JeszkaSkowron, 2014; Kim & Jang, 2011; Xu, 2015), the total phenolic contents and flavonoid contents (respectively, TPC and TFC), as well as the rutin, chlorogenic acid, and DNJ contents, of MLAE and the dry mulberry leaves (prepared according to the method described previously) were determined by liquid chromatography on an Agilent 1100 system (Agilent Technologies Inc, Santa Clara, USA) and a spectrophotometer (UVIKON XL-172, Secomam AG, France). We mainly optimized the chromatographic conditions. The specific modifications are as follows: The optimal derivatization conditions were: 100 μL of leaf extract was mixed with 100 μL of 0.4 M potassium borated buffer (pH 8.0) in a 5 mL microtube. 700 μL of 1 g/L FMOC-Cl in acetonitrile was mixed and allowed to react at room temperature for 10 min. The optimized chromatographic conditions were: the samples were eluted with a mobile phase of ACN–10 mM ammonium acetate(pH 4.0) (40:60, v/v) at 0.8 mL/min for 15 min.
alkaloid, is a characteristic constituent of mulberry leaves; it is a potent R-glucosidase inhibitor with low toxicity (Ju, Kim, Sung, & Kim, 2016), and many experiments have demonstrated that treatment with DNJ can significantly reduce the blood sugar of hyperglycemia-induced mice (Asano, 2001; Li, 2013). Chlorogenic acid, one of the most abundant phenylpropanoids compounds in human diets, was reported to modulate glucose and lipid metabolism in vivo, in both healthy individuals and those with conditions related to genetic metabolic disorders (Meng, Cao, Feng, Peng, & Hu, 2013). Rutin, a compound present in mulberry leaf extract, is a kind of flavonoid and possesses anti-inflammatory and antioxidant activities and reduces insulin resistance in insulin-resistant mice (Hsu, 2014). Other work has shown that mulberry leaf extracts had hypoglycemic and antiatherogenic effects in certain animal models and in humans (Miyahara, Miyazawa, Satoh, Sakai, & Mizusaki, 2004; Murata, 2004). Finally, according to several studies, mulberry leaves and their ethanol extracts can improve insulin secretion, which is the mechanism by which mulberry leaves can treat type 2 diabetes (Ji, 2011; Ren, 2015). To our best knowledge, there is limited information available on preventing type 2 diabetes in obese mice using mulberry leaf aqueous extract. In our study, the C57BL/6J mouse model was established by high-fat diet induction. To explore its effects and how MLAE improves energy metabolism and reduces blood glucose, the body weight, energy metabolism, glucose metabolism, and insulin resistance were measured in these obese mice treated with mulberry leaf aqueous extract for 12 weeks. Some indicators of liver and kidney functioning were also monitored to ensure there is no liver or kidney damage of MLAE treatment.
2.3. Animals The experimental design was approved by the Animal Ethics Committee of China Agricultural University, Beijing (approval ID: KY20150017). MLAE, 3- to 4-week-old C57BL/6J mice were purchased from Vital River Laboratories (Beijing, China) and housed (4 animals/ cage) under controlled temperature (22 ± 2 °C), humidity (55% ± 10%), and light (12-h light-dark cycle) conditions in a specific pathogen-free (SPF) animal room at the Supervision, Inspection and Testing Center for Genetically Modified Organisms of the Ministry of Agriculture (Beijing, China; license number SYXK [Beijing] 20150045).
2. Materials and methods 2.1. Diet composition Fresh mulberry leaf samples were obtained from Danyang City, Jiangsu Province, China. The high-fat diet (60% calories from fat, D12492) and low-fat diet (10% calories from fat, D12450B) for the mice were both prepared by Beijing Hua Fukang Bioscience Co., LTD (Beijing, China). All reagents used in the current study were of analytical grade (Table 1).
2.4. Experimental protocols Animals were allowed to acclimate to their room environment for 7 days. Thirty-six C57BL/6J MLAE mice were randomized into two groups: one group had 12 mice that were fed a low-fat diet, while the other group had 24 mice that were fed a high-fat diet. After 8 weeks of feeding, 6 and 12 mice were appropriately selected from the low-fat diet group and high-fat diet group according to weight, respectively; the difference in the average body weight between these two groups exceeded 15%. Then, the high-fat diet group was randomly divided into the MLAE and HFD groups (n = 6 per group), and the low-fat diet group was renamed the LFD group (n = 6). The LFD and HFD groups served as the vehicle control and negative control, respectively. The mice in the MLAE group were treated with a mulberry leaf aqueous extract (1000 mg/kg body weight, BW) solution dissolved in distilled water, administered by stomach gavage daily at the same time and kept high-fat diet until the end of the experiment. Both LFD and HFD groups were treated with distilled water by stomach gavage, as done for the MLAE group. The stomach gavage treatment period lasted 12 weeks, and the diets provided to each group were the same as prior to gavage. Table 1 gives the composition of both experimental diets, and their respective weight ratio and energy ratio information are given in Table 2. The body weights and food intake were recorded weekly. Energy intake was calculated for the mice in all treatment groups using the same formula: food intake of the low-fat chow (g) [or high-fat chow (g)] × 3.58 [(or 5.24 for high-fat chow)]. Body fat percentage and FFM were calculated based on the ratio of white adipose tissue weight and
2.2. Chemicals Fresh mulberry leaves were first dehydrated at a constant temperature of 60 °C in a convection dryer (YF-6CHZ-2, Yongfengjixie, Fujian, China). Then, a grinder was used to powder the dried mulberry leaves, so they could pass through a 40-mesh sieve. Table 1 Composition and percentage content of the low-fat diet (LFD) and high-fat diet (HFD) of experimental mice.
Casein Cystine Corn starch Maltodextrin Sucrose Cellulose Bean oil Lard M1002 DCP Calcium carbonate Potassium citrate monohydrate V1001 Choline bitartrate Edible dye Total:
LFD
HFD
18.985 0.284 29.859 3.318 33.177 4.740 2.370 1.896 0.948 1.232 0.520 1.564 0.948 0.190 0.005 100.000
25.845 0.388 0.000 16.153 8.891 6.461 3.231 31.660 1.292 1.680 0.711 2.132 1.291 0.258 0.007 100.000
2
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functioning in the experimental mice, their serum samples were used for blood biochemical tests of high-density lipoprotein (HDL), lowdensity lipoprotein (LDL), triglycerides (TG), blood urea nitrogen (BUN), creatinine (CREA), aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and dehydrogenase (LDH). All were quantified on a HITACHI 7020 instrument (Hitachi Ltd, Japan)
Table 2 Weight ratio and energy ratio in low-fat diet (LFD) and high-fat diet (HFD) fed to the experimental mice. LFD
Protein Carbohydrate Fat Total: kcal/g
HFD
Weight ratio g%
Energy ratio kcal%
Weight ratio g%
Energy ratio kcal%
19.2 67.3 4.3
20 70 10 100
26 26 35
20 20 60 100
3.85
2.10. Intestinal microflora analysis Mouse feces were collected and stored at −80 °C. Microbial genomic DNA was extracted from each fecal sample using an extraction kit. The V3 + V4 region of the 16 S rRNA was amplified by PCR and sequenced on a Hi-Seq platform (Illumina, San Diego, USA) at The Novogene Bioinformatics Institute (Beijing, China). Sequence analyses were performed using the UPARSE v7.0.1001 software tool (UPARSE); those sequences with ≥97% similarity were assigned to the same operational taxonomic unit (OTU). Taxonomic annotation was carried out using an RDP classifier (v2.2). Nonmetric multidimensional scaling (NMDS) plots and alpha and beta diversity indices were used to analyze the variation between each group, utilizing the PAST v2.17 software program. Heat maps were drawn using heatmap illustrator software (v1.0.3.7).
5.24
body weight of the mouse. 2.5. Blood glucose and intraperitoneal glucose tolerance test Blood glucose was determined from mice tail blood samples, taken after 16 h of fasting every four weeks, by blood glucose meters (ACCUCHEK, Shanghai, China). In the 12th week of the MLAE treatment, intraperitoneal glucose tolerance tests (GTT) were performed on the mice of all groups. Each animal was fasted for 16 h and then given an intraperitoneal injection of glucose (1.5 g/kg BW). Blood glucose levels were measured at 0, 15, 30, 60, and 120 min post-glucose injection. The area under the curve of blood glucose concentration change from 0 to 120 min was calculated (Kong, 2008).
2.11. Statistics Single-factor analysis of variance (ANOVA) followed by two-tailed Student’s t tests was used to compare the groups’ means, using MS Office Excel 2013. Graphs were plotted using GraphPad Prism 5 (GraphPad Software, California, USA). Almost all data are presented as the mean ± SEM. Significant differences were considered found at P < 0.05.
2.6. Plasma insulin and HOMA-IR After completing the mice experiment, blood samples were taken from the mice and their plasma insulin assayed by a mouse INS ELISA kit (Huaxiabio, Beijing, China). The homeostasis model assessment-insulin resistance (HOMA-IR) was used to measure insulin sensitivity of individual mice of all groups as follows: [fasting plasma insulin (μg/ L) × fasting blood glucose (mg/dL)]/22.5 (Ding et al., 2010).
3. Results 3.1. Component analysis of MLAE and mulberry leaves
2.7. Rectal temperature and energy expenditure The contents of the major components in MLAE and dry mulberry leaf powder are shown in Table 3. The most abundant active ingredients in MLAE mainly included rutin (44.38 mg/g), chlorogenic acid (7.33 mg/g), and DNJ (3.29 mg/g).
An Oxylet system (Panlab, Spain) was prepared for each mouse at 11 week of the MLAE treatment, for which free access to food and water was provided in their cages. After 12 h of acclimation, oxygen consumption was measured for 24 h, from which volume of oxygen consumption (VO2) and the respiratory exchange ratio (RER) were obtained. Energy expenditure was calculated, using VO2 and RER, as follows: {VO2 × [3.815 + (1.23 × RER)] × 40.1868}, expressed as kilojoules per kilogram fat-free mass (FFM) per hour (Stewart, 2008; Xu, 2009). The mice were also placed in a cold room (4 °C) for 4 h, and their cold-induced thermogenesis was evaluated with a temperature sensor (AT210, Zhongyi Dapeng, Shenzhen, China). Finally, each mouse was photographed using a handheld infrared camera (FLIR T600, FLIR, Oregon, USA) against a blank whiteboard.
3.2. Effect of MLAE on mice body weight Mice with high-fat diet-induced obesity (DIO) were successfully obtained at 8 weeks post-treatment, at which time they weighed at least 15% more than the control mice. After 12 weeks, the mice fed the HFD had body weights 26% higher than the LFD control mice (31.53 compared with 39.74 g, Fig. 1A). In addition, the MLAE-treated animals underwent a 6% decrease in body weight compared with the HFD group (Fig. 1A). No significant differences were found in the body weight, fat mass, lean mass, and body fat percentage between the HFD and MLAE groups (Fig. 1B–D).
2.8. Preparation of adipose tissues and blood samples At the end of 12 weeks, blood was collected from the orbital venous plexus in the eyes of the mice. For the biochemical characterization of this blood, serum samples were isolated from it by centrifugation (5000 rpm, 10 min) and collected. Then, all mice were euthanized, and their abdominal adipose tissues and epididymal adipose tissues were dissected out and weighed. All the blood and tissues samples were stored at −80 °C until further analysis.
Table 3 Quantification of the major components in lyophilized MLAE and dry mulberry leaves (mg/g dry extract or mulberry leaves powder).
2.9. Biochemical characterization
Sample
TFC (mg/g)
TPC (mg/g)
Rutin (mg/g)
Chlorogenic acid (mg/g)
DNJ (mg/g)
MLAE Dry mulberry leaves
44.38 29.86
29.32 16.94
44.38 29.86
7.33 6.89
3.29 1.28
TPC: Total phenolic contents. TFC: Total flavonoid contents.
To detect lipid metabolism and determine liver and kidney 3
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Fig. 1. The MLAE treatment did not significantly affect mice body weight but did improve their mean daily food intake compared with the HFD group. (A) The mean body weights of the LFD, HFD, and MLAE groups in the 12-week experiment. (B) Fat mass, (C) lean mass, (D) fat mass ratio, and (E) food intake of the LFD, HFD, and MLAE groups were measured and calculated after 12 weeks of treatment. Bars represent the mean + SEM, n = 6. The significance level is represented by “a,” “b,” and “c” (P < 0.05). There are no significant differences between “ab” and “a” or “b.”
3.3. MLAE enhanced food intake
3.4. Hypoglycemic effect of MLAE on DIO mice
Food intake was recorded weekly, and daily food intake was calculated by averaging the accumulated food intake at 12 weeks. Daily food intake was significantly higher in the MLAE than HFD group (P < 0.01, Fig. 1E), but it was similar between the LFD and MLAE groups (Fig. 1E).
At the treatment’s start, blood glucose levels in the MLAE and HFD groups significantly exceeded those in the LFD group, a difference induced by the 8-week-feeding of a high-fat diet. In this 12-week experiment, the MLAE group’s blood glucose consistently decreased, while that of the HFD group continued to increase, whereas that of the LFD group remained essentially unchanged. From weeks 4 and 5 4
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Fig. 2. The blood glucose level and insulin resistance were both lower in the MLAE group than those in the HFD group. (A) Blood glucose was measured every 4 weeks. (B) A glucose tolerance test was performed by injecting 15%-glucose solution into C57BL/6J mice. (C) The area under the curve of the glucose tolerance test was calculated for the C57BL/6J mice. (D) The plasma insulin concentration was measured in the 12th week. (E) The HOMA-IR values were calculated by fasting plasma insulin (μg/L) × fasting blood glucose (mg/dL)]/22.5. Bars represent the mean + SEM, n = 6. P < 0.05 and P < 0.01 when comparing the MLAE group with the HFD control. The significance level is represented by “a,” “b,” and “c.” There are no significant differences between “ab” and “a” or “b.”
groups (P < 0.05, Fig. 2B). Variation in fasting blood glucose levels occurred over time, with those in MLAE-treated mice being significantly lower than those in the HFD group at 60, 90, and 120 min post-injection of the glucose solution (P < 0.05, Fig. 2B). The area under the curve (AUC) of the HFD group was significantly greater than that of either the LFD or MLAE group (54.0% and 17.5%, Fig. 2C). The effects of MLAE on plasma insulin concentration were also evaluated by GTT after 12 weeks of daily stomach gavage with the MLAE solution. There was a significant reduction in plasma insulin concentrations in the MLAE group relative to those of the HFD group (P < 0.05, Fig. 2D), and likewise for their corresponding HOME-IR values (P < 0.05, Fig. 2E). However, no significant differences were
through week 12, a significant difference emerged in blood glucose levels between the MLAE and HFD groups (P < 0.05; Fig. 2A), where blood glucose levels in the LFD and MLAE groups were similar at the 12th week. Hence, the hypoglycemic effect of MLAE was significant (Fig. 2A). 3.5. MLAE ameliorated carbohydrate metabolism and insulin resistance GTT was performed in all three groups after 12 weeks of treatment. Mice in the LFD group served as the normal glucose metabolism group (the control). Initial fasting blood glucose levels (time = 0 min) tended to be significantly higher in the HFD group than either MLAE or NCD 5
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Fig. 3. The MLAE treatment promoted thermogenesis and energy metabolism in C57BL/6J mice. The daily energy intake calculated by food intake of low-fat chow (g) [or high-fat chow (g)] × 3.58 [or 5.24 for high-fat chow]. (A) Infrared imaging, (B) infrared imaging values, (C) rectal temperatures, (D) energy expenditures calculated from VO2 and RER {VO2 × [3.815 + (1.23 × RER)] × 40.1868} in (E) the LFD, HFD, and MLAE groups were measured and determined at the 12th week. Bars represent the mean + SEM, n = 6. The significance level is represented by “a,” “b,” and “c.” There are no significant differences between “ab” and “a” or “b.”
3.7. MLAE improved energy metabolism of mice
evident between the HFD and MLAE groups in terms of their plasma insulin concentrations or HOMA-IR values (P < 0.05, Fig. 2E).
To explore whether mulberry leaves could ameliorate energy metabolism and enhance energy expenditure, the rectal temperature, conducted infrared thermal imaging, and calculated EE values (based on VO2 and RQ) of the experimental mice have been measured. Rectal temperatures of the MLAE group were significantly higher than those of the HFD and LFD groups (P < 0.05, Fig. 3B). Infrared thermal images were taken with a handheld infrared camera (Fig. 3C), and their resulting values were greater in the MLAE than the HFD group (P < 0.05, Fig. 3D). Furthermore, calculated EE values of the MLAE group were significantly higher than those of the LFD and HFD groups
3.6. MLAE improved energy intake in DIO mice To intuitively study the energy metabolism of mice, their individual energy intake values were derived from their monitored food intakes. Energy intake of the MLAE treatment group significantly exceeded those of the other two groups (P < 0.05, Fig. 3A). Additionally, energy intake in the HFD group was higher than that in the LFD group (P < 0.05, Fig. 3A). 6
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The MLAE treatment decreased the AST, ALP, ALT, and LDH levels relative to the HFD group, however no significant differences have been found (Fig. 4B). No significant differences in these levels occurred between the LFD and HFD groups. Concerning the BUN and CREA levels, treatment with MLAE significantly reduced the CREA content in DIO mice (P < 0.05; Fig. 4C) but negligibly affected the CREA or BUN contents of the control mice. 3.10. MLAE treatment restored gut microbiota imbalance in DIO mice The intestinal flora data of mice were categorized into an HFD group (high-fat-fed obese model mice); LFD group (low-fat-fed mice), MLAE group (added mulberry-fed obese model mice), and Initial State group (all mice, just after acclimation to the room environment when not yet grouped). Differences between the above groups in their mice gut flora were analyzed by non-metric multidimensional scaling analysis (NMDS) (Fig. 5A). Evidently, the HFD group was most different from the LFD group and Initial State group, indicating the intestinal flora of obese mice differed significantly from that of the pre-fed and LFD-fed mice at the end of the experiment. Compared with the LFD and the Initial Sate groups, the MLAE group has a large area intersection in the two-dimensional plane distribution of the NMDS map, and the difference is not significant. Comparing the α- and β-diversity between the four groups, as in Fig. 5B and C, both measures of microbial diversity in the HFD group differed significantly from those of the LFD group and the mulberry leaf treatment group, and these latter two groups had similar α-diversity. This indicated mulberry leaf feeding was capable of restructuring the intestinal flora community of DIO mice back to its normal composition. According to the heat map-cluster analysis (Fig. 5D), differences between the HFD and LFD groups were significant, in that the mulberry leaf treatment could restore most of the changed flora to some extent. Compared with the LFD group, Zymomonas, Helicobacter, Bilophila in Proteobacteria; Anaerrotruncus, Anaerovorax, Ruminococcus, Oscillospira, Dehalobacterium, Coprococcus, Anaerostipes, Lactobacillales, Roseburia in Firmicutes; Odoribacter in Bacteroidetes; Desulfovibrio in Nitrospirae and Mucispirillum in Deferribacteres of HFD group all significantly increased, whereas the mulberry leaf treatment group was able to reverse these changes to gut microflora. The microorganisms that were significantly reduced in DIO mice—such as Enterococcus in Firmicutes; Devosia in Proteobacteria; Paraprevotella, Prevotella in Bacteroides; Bifidobacterium in Actinobacteria—were also increased by feeding mulberry leaves to the mice. Therefore, the mulberry leaf treatment greatly improved the intestinal flora of DIO mice on the whole.
Fig. 4. The MLAE treatment ameliorated the blood lipid profile and have no damage to liver and kidney function, based on the blood biochemistry of mice. The lipid indexes HDL, LDL, and TG (A), markers of liver function ALT, AST, ALP, and LDH (B), and markers of kidney function BUN and CREA (C), in the C57BL/6J mice. Bars represent the mean + SEM, n = 6. The significance level is represented by “a,” “b,” and “c.” There are no significant differences between “ab” and “a” or “b.”
(P < 0.05, Fig. 3E). These results indicated MLAE indeed improved energy metabolism in the mice and promoted thermogenesis.
4. Discussion In China, mulberry leaves are used as a traditional medicinal herb (Miyahara, Miyazawa, Satoh, Sakai, & Mizusaki, 2004). The present research shows that mulberry leaves can decrease blood glucose and blood pressure in mice and improve their insulin sensitivity. These impacts on bodily health may have been caused by the high content of alkaloids, polyphenols, and flavonoids characterizing mulberry leaves (Ji et al., 2011; Ren et al., 2015; Yang, Jhon, & Tseng, 2012). Compared with the reported constituents of edible vegetables and fruits, the total polyphenol and flavonoid contents of the mulberry leaves used in our study are much higher, suggesting that mulberry leaves and their aqueous extract harbor hypoglycemic potential (Miean & Mohamed, 2001; Turkmen, Sari, & Velioglu, 2005). In our study, the body weights of the mice treated with MLAE by stomach gavage for 12 weeks were on par with those of the HFD group (Fig. 1A). Similarly, Lim, Lee, Kim, Yang, and Lim (2013) found that treatment with mulberry leaf extract had no significant effect on the body weight of mice fed a high-fat diet. In addition, some investigators have suggested that black tea, green tea, and mulberry extracts in
3.8. MLAE ameliorated the lipid profile of DIO mice To gauge the effect of MLAE on lipid metabolism, we took blood serum samples from the mice for their biochemical testing. A high-fat diet significantly increased the levels of several metabolic risk factors, namely TG and LDL (P < 0.05, Fig. 4A). However, the MLAE treatment ameliorated the high-fat diet-induced high LDL and increased the HDL in DIO mice compared with the corresponding levels of the HFD group; this indicated that MLAE improved the lipid profile of DIO mice and reduced their risk of cardiovascular disease (Nardelli, Ribeiro, & Balbo, 2011; Stefanick, 1998). Stomach gavage with the MLAE solution did not significantly influence the TG in mice. 3.9. MLAE have no damage to liver and kidney function To assess the safety of MLAE for liver and kidney functioning, clinical biochemical analyses of mice serum samples were performed. 7
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Fig. 5. The MLAE treatment restored the gut microbiota imbalance in DIO mice. NMDS plot of the Bray–Curtis similarity coefficients calculated from the 16 S rRNA sequencing data of the gut microbiota (A). Mulberry leaf influences intestinal flora of type 2 diabetes rats at phylum (B). Alpha diversity of each group represented by the Simpson index (C). Beta diversity of each group as quantified by the Bray–Curtis index (D). Heat map-A cluster analysis of the effects of MLAE on intestinal flora of DIO mice (E). Bars represent the mean + SEM, Initial state: n = 8, HFD: n = 10, LFD: n = 6, MLAE: n = 7. *P < 0.05 between compared groups, **P < 0.01 between compared groups.
reduced blood glucose compared with the HFD group, and because the MLAE and LFD groups had similar blood glucose levels, this demonstrated that MLAE has a measurable hypoglycemic effect (Fig. 2). These results are consistent with other studies that reported similar decreases in blood glucose levels in diabetic rats treated with the ethanol or acetone extracts of mulberry leaves (Jeszka-Skowron, 2014). The hypoglycemic effect in our study could be due to the high levels of DNJ (Jimin, 2009), flavonoids (Jadhav & Puchchakayala, 2012), and polyphenols (Ji, Chung, Jung, Wee, & Kwon, 2013) in mulberry leaves. Of these bioactive substances, DNJ in particular has received special attention as a competitive inhibitor of intestinal α-glucosidases, which can affect carbohydrate digestion and absorption and lead to suppressed postprandial hyperglycemia (Chung, Kim, JI, & Kwon, 2013).
combination do not influence weight loss (e.g., Fallon, Zhong, Furne, & Levitt, 2008). In our work here, the HFD group had higher energy intake than did the LFD group, and mice in the HFD group were 22% heavier than those in the LFD group at week 12, on average. However, daily energy intake and food intake of the MLAE group surpassed that of the HFD group, yet the former had a lower mice weight and body fat percentage than did the latter group. These results may indicate that although the difference was not significant, MLAE still contributed to weight loss. In subsequent experiments, MLAE can significantly improve the energy expenditure of mice, which neatly explains these results. We also focused on exploring the hypoglycemic effect of MLAE treatment by stomach gavage. In this respect, the MLAE treatment 8
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organs. Studies show that the content and proportion of Firmicutes and Bacteroidetes in the intestinal flora can change significantly in diabetic patients or obese patients. It is known that Bacteroidetes and Firmicutes have important effects on body metabolism in the intestinal tract, where there exists a mutualism in the form of a symbiotic relationship, which together promotes the body’s absorption or storage of energy. Changes in the gut proportions of Bacteroidetes and Firmicutes can promote obesity and type 2 diabetes (Larsen, 2010; Rios-Covian, 2013; Turnbaugh, 2006). In our study, mulberry leaves restored the reduced proportion of Bacteroides and sclerenchyma in intestinal flora of obese mice (Fig. 5B). Bifidobacterium are a kind of intestine-beneficial microorganisms, which act as biological barriers capable of not only nutritional, anti-tumor, and immune enhancement effects but also improved gastrointestinal function, anti-aging responses, and other important physiological functions (Kootte, 2012; Le, 2014). Studies have shown that Bifidobacterium can affect the body’s insulin sensitivity and glucose tolerance (Ding et al., 2010). In our experiment, the number of intestinal Bifidobacterium in DIO mice was reduced, but mulberry leaves restored their abundance. Intestinal inflammation is closely related to the development of obesity and diabetes in the body. Ding et al. found that the local inflammatory reaction of intestinal tissue is related to obesity and insulin resistance in mice and may be involved in the occurrence and development of type 2 diabetes. In DIO mice, their intestinal flora has an increased content of Desulfovibrio, which is a gram-negative genus associated with intestinal inflammation (Sawin, 2015), but mulberry leaves can lower Desulfovibrio abundance. Therefore, mulberry leaves may regulate glucose metabolism and energy consumption by increasing the ratio of intestinal Bacteroidetes and Firmicutes, thereby augmenting the content of beneficial bacteria and reducing intestinal inflammation in DIO mice. Recently, studies have shown that mulberry leaf extract can improve blood sugar levels in type 2 diabetes mellitus through clinical trials (Lown et al., 2017; Riche, Riche, East, Barrett, & May, 2017). This shows that mulberry leaf extract as a natural and safe food functional factor has more application prospects.
Work by Kimura (2007) reported that healthy volunteers who took 0.8 or 1.2 g of DNJ-enriched mulberry powder had markedly suppressed elevation of their postprandial blood glucose levels. In our glucose tolerance tests, at 0 min, the lower mean fasting blood glucose levels of the MLAE group suggested mulberry leaves contributed to significantly reducing blood glucose in mice after 12 weeks of the MLAE treatment (Fig. 2B). Blood sugar levels similar between LFD and MLAE groups at 0 min further indicated mulberry leaves can decrease blood sugar to approximately normal levels. At 60, 90, and 120 min, the significant differences found vis-à-vis the HFD group suggested that MLAE treatment can improve glucose tolerance, and these effects were confirmed by the AUC. The plasma insulin results indicated that administering the MLAE for 12 weeks considerably decreased the fasting insulin concentration and HOMA-IR in the DIO mice. This effect improved glucose tolerance and quantitatively reduced plasma insulin concentrations and HOMA-IR values, which confirmed the diminished insulin resistance of DIO mice was a direct response to MLAE. Similar findings were reported by Ji Yeon Kim, who found the same effect in obese rats when 5% mulberry leaf water extract was added to their diet for 6 weeks (Ji, 2011). DNJ reportedly has an antidiabetic effect in type 2 diabetic rats, for which notable improvements occurred in fasting blood glucose levels and glucose tolerance, but especially increased insulin sensitivity (Kong, 2008). However, some flavonoids and polyphenols, such as rutin and chlorogenic acid, can also improve glucose tolerance and decrease insulin resistance (Hsu et al., 2014; Johnston, Clifford, & Morgan, 2003). The synergistic effects of these active ingredients in mulberry leaves may even be stronger than those arising from pure DNJ (Kwon, Ji, Ji, & Kwon, 2011). In our study, although MLAE improved glucose metabolism, the AUC values of the MLAE and LFD groups were significantly different. Hence, the duration and dosage of the MLAE treatment were likely insufficient, in that a simple MLAE treatment cannot completely restore glucose metabolism to its normal levels. Importantly, fat feeding decreases energy expenditure and causes obesity (Storlien, James, Burleigh, Chisholm, & Kraegen, 1986). Indeed, the infrared thermal imaging values and rectal temperature were lowest in the HFD group, which indicated that HFD decreased energy expenditure in mice; however, since the EE values in obese mice were enhanced by the MLAE treatment (Fig. 3A-E), this suggested that MLAE not only reduced the extent EE’s decline as induced by a high-fat diet but also improved EE to above-normal levels. This effect may be due to the flavonols and quercetin in mulberry leaves, which are known to elevate oxygen consumption in human skeletal myocytes (Stewart, 2008). The mechanism of this effect is thought to involve upregulation of type 2 deiodinase expression, which can increase energy expenditure by increasing the formation of triiodothyronine (Da-Silva, 2007). Obesity can lead to liver damage, which mainly manifests as elevated ALT and AST (Fraulob, Ogg-Diamantino, Fernandes-Santos, Aguila, & Mandarim-de-Lacerda, 2010). Here, because ALT, ASL, ALP, and LDH levels—all biomarkers of liver injury—were decreased in the MLAE group (Fig. 4B) this suggests MLAE have no damage on mouse liver Tang, Huang, Lee, Tang, & Wang, 2013 have reported similar outcomes, showing that mulberry water extracts can significantly inhibit the levels of ALT, AST, and ALP. Furthermore, no differences among the BUN levels of the three groups and a significant decrease in CREA in the MLAE group relative to the HFD group together indicate that MLAE has a potential protective effects on kidney functioning (Fig. 4C) (Diwan, Brown, & Gobe, 2017; Hemmati, Jalali, Rashidi, & Hormozi, 2010). The MLAE’s protection of the liver and kidney likely depends on the flavonoids and polyphenols, such as rutin and chlorogenic acid, in the mulberry leaves, since these components can suppress oxidative stress and inflammation while also improving kidney regeneration (Domitrović, Cvijanović, Šušnić, & Katalinić, 2014; Korkmaz & Kolankaya, 2013; Lee, Shen, Lai, & Wu, 2013; Shi, 2013). In summary, our experiment proved that MLAE imparts a protective effect on liver and kidney functioning rather than being toxic to these vital
5. Conclusion This study investigated the beneficial effects of MLAE for decreasing insulin resistance and blood glucose levels, as well as improving energy metabolism, in obese mice. The safety of our MLAE treatment was also been confirmed, proving it caused no damage to liver and kidney function but instead simply protected them. Although the MLAE treatment did not significantly reduce the weight of the mice, combined with the energy intake, MLAE did impede the process of weight gain. MLAE’s high content of bioactive molecules, namely, DNJ, flavonoids, and polyphenols, may be primarily responsible for the multiple physiological functions of MLAE, yet specific molecular mechanisms still require elucidation. This study demonstrated the various physiological functions of MLAE and provides guidance for developing functional foods and medicines based on MLAE. Author disclosures The National Natural Science Foundation of China (81700684 to C. Zhang). Practical application Our research suggests MLAE can serve as a safe hypoglycemic ingredient for use in the development of health products, and perhaps even drugs. The hypoglycemic effect of mulberry leaves/MLAE was significant and sustained, indicating its great potential in food applications; currently, these applications include using mulberry leaves/ MLAE as a food additive to make health drinks or bread for people with 9
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diabetes or diabetic nephropathy. The specific composition and functions of MLAE thus merit further investigation and practical study.
Food Chemistry, 132(4), 1796–1801. Cho, N., Whiting, D., & Forouhi, N. (2016). IDF Diabetes Atlas, 2015. Fallon, E., Zhong, L., Furne, J. K., & Levitt, M. (2008). A mixture of extracts of black and green teas and mulberry leaf did not reduce weight gain in rats fed a high-fat diet. Alternative Medicine Review, 13(1), 43–49. Fraulob, J. C., Ogg-Diamantino, R., Fernandes-Santos, C., Aguila, M. B., & Mandarim-deLacerda, C. A. (2010). A mouse model of metabolic syndrome: Insulin resistance, fatty liver and non-alcoholic fatty pancreas disease (NAFPD) in C57BL/6 mice fed a high fat diet. Journal of Clinical Biochemistry and Nutrition, 46(3), 212–223. Health, U. D. O. (2014). Health, United States, 2013: With special feature on prescription drugs. Claitors Publishing. Hemmati, A. A., Jalali, M. T., Rashidi, I., & Hormozi, T. K. (2010). Impact of aqueous extract of black mulberry (Morus nigra) on liver and kidney funcion of diabetic mice. Jundishapur Journal of Natural Pharmaceutical Products, 5(1), S48–S49. Hsu, C. Y., Shih, H. Y., Chia, Y. C., Lee, C. H., Ashida, H., Lai, Y. K., & Weng, C. F. (2014). Rutin potentiates insulin receptor kinase to enhance insulin-dependent glucose transporter 4 translocation. Molecular Nutrition & Food Research, 58(6), 1168–1176. Hunyadi, A., Martins, A., Hsieh, T. J., Seres, A., & Zupkó, I. (2012). Chlorogenic acid and rutin play a major role in the in vivo anti-diabetic activity of Morus alba leaf extract on type II diabetic rats. PLoS ONE, 7(11), e50619. Jadhav, R., & Puchchakayala, G. (2012). Hypoglycemic and antidiabetic activity of flavonoids: Boswellic acid, Ellagic acid, quercetin, Rutin on StreptozotocinNicotinamide induced Type 2 Diabetic. International Journal of Pharmacy & Pharmaceutical Sciences, 4(2), 251–256. Jeszka-Skowron, M., Flaczyk, E., Jeszka, J., Krejpcio, Z., Król, E., & Buchowski, M. S. (2014). Mulberry leaf extract intake reduces hyperglycaemia in streptozotocin (STZ)induced diabetic rats fed high-fat diet. Journal of Functional Foods, 8(1), 9–17. Ji, Y. K., Choi, B. G., Jung, M. J., Wee, J. H., Chung, K. H., & Kwon, O. (2011). Mulberry leaf water extract ameliorates insulin sensitivity in high fat or high sucrose diet induced overweight rats. Applied Biological Chemistry, 54(4), 612–618. Ji, Y. K., Chung, H. I., Jung, K. O., Wee, J. H., & Kwon, O. (2013). Chemical profiles and hypoglycemic activities of mulberry leaf extracts vary with ethanol concentration. Food Science and Biotechnology, 22(5), 1–5. Jimin, P., Hayoon, B., Hyein, J., Yeonkyoung, K., Jiyeon, K., & Oran, K. (2009). Postprandial hypoglycemic effect of mulberry leaf in Goto-Kakizaki rats and counterpart control Wistar rats. Nutrition Research & Practice, 3(4), 272–278. Johnston, K. L., Clifford, & Morgan, L. M. (2003). Coffee acutely modifies gastrointestinal hormone secretion and glucose tolerance in humans: Glycemic effects of chlorogenic acid and caffeine. American Journal of Clinical Nutrition, 78(4), 728. Jr, D. E. (1985). Diet and obesity. American Journal of Clinical Nutrition, 41(5 Suppl), 1132. Ju, W. T., Kim, H. B., Sung, G. B., & Kim, Y. S. (2016). Comparison of optimal temperature and time conditions for highest a-glucosidase inhibitory activity from various of Korea mulberry teas, 33, 31–35. Kim, G. N., & Jang, H. D. (2011). Flavonol content in the water extract of the mulberry (Morus alba L.) leaf and their antioxidant capacities. Journal of Food Science, 76(6), 869–873. Kimura, T., Nakagawa, K., Kubota, H., Kojima, Y., Goto, Y., Yamagishi, K., ... Miyazawa, T. (2007). Food-grade mulberry powder enriched with 1-deoxynojirimycin suppresses the elevation of postprandial blood glucose in humans. Journal of Agricultural and Food Chemistry, 55(14), 5869–5874. Kong, W. H., Oh, S. H., Ahn, Y. R., Kim, K. W., Kim, J. H., & Seo, S. W. (2008). Antiobesity effects and improvement of insulin sensitivity by 1-deoxynojirimycin in animal models. Journal of Agricultural & Food Chemistry, 56(8), 2613–2619. Kootte, R. S., Vrieze, A., Holleman, F., Dallinga-Thie, G. M., Zoetendal, E. G., de Vos, W. M., ... Nieuwdorp, M. (2012). The therapeutic potential of manipulating gut microbiota in obesity and type 2 diabetes mellitus. Diabetes, Obesity & Metabolism, 14(2), 112–120. Korkmaz, A., & Kolankaya, D. (2013). Inhibiting inducible nitric oxide synthase with rutin reduces renal ischemia/reperfusion injury. Canadian Journal of Surgery Journal Canadien De Chirurgie, 56(1), 6–14. Kwon, O., Kwon, H. J., Ji, Y. C., & Ji, Y. K. (2011). Comparison of 1-deoxynojirimycin and aqueous mulberry leaf extract with emphasis on postprandial hypoglycemic effects. In vivo and in vitro studies. Journal of Agricultural & Food Chemistry, 59(7), 3014–3019. Larsen, N., Vogensen, F. K., van den Berg, F. W., Nielsen, D. S., Andreasen, A. S., Pedersen, B. K., ... Jakobsen, M. (2010). Gut microbiota in human adults with type 2 diabetes differs from non-diabetic adults. PLoS ONE, 5(2), e9085. Le, T. K., Hosaka, T., LE, T. T., Nguyen, T. G., Tran, Q. B., Le, T. H., & Pham, X. D. (2014). Oral administration of Bifidobacterium spp. improves insulin resistance, induces adiponectin, and prevents inflammatory adipokine expressions. Biomedical Research, 35(5), 303–310. Lee, C. C., Shen, S. R., Lai, Y. J., & Wu, S. C. (2013). Rutin and quercetin, bioactive compounds from tartary buckwheat, prevent liver inflammatory injury. Food & Function, 4(5), 794–802. Li, Y. G., Ji, D. F., Zhong, S., Lin, T. B., Lv, Z. Q., Hu, G. Y., & Wang, X. (2013). 1deoxynojirimycin inhibits glucose absorption and accelerates glucose metabolism in streptozotocin-induced diabetic mice. Scientific Reports, 3(7442), 1377. Lim, Y., Lim, H. H., Lee, S. O., Kim, S. Y., & Yang, S. J. (2013). Anti-inflammatory and antiobesity effects of mulberry leaf and fruit extract on high fat diet-induced obesity. Experimental Biology and Medicine, 238(10), 1160. Lown, M., Fuller, R., Lightowler, H., Fraser, A., Gallagher, A., Stuart, B., ... Lewith, G. (2017). Mulberry-extract improves glucose tolerance and decreases insulin concentrations in normoglycaemic adults: Results of a randomised double-blind placebocontrolled study. PLoS ONE, 12(2), e0172239. Mehta, S., & Farmer, J. A. (2007). Obesity and inflammation: A new look at an old
Ethics statement The experimental design was approved by Animal Ethics Committee of China Agricultural University, Beijing, and the approval ID of this study is KY20150017. Male, 3–4 weeks old, C57BL/6J mice were purchased from Vital River Laboratories (Beijing) and housed (4 animal/ cage) under controlled temperature (22 ± 2 °C) and humidity (55% ± 10%) and light (12 h light-dark cycle) in an Specific Pathogen Free (SPF) animal room at the Supervision, Inspection and Testing Center for Genetically Modified Organisms of the Ministry of Agriculture (Beijing, China; license number SYXK (Beijing) 2015-0045). Acknowledgments This research was supported in part by the National Natural Science Foundation of China (81700684 to C. Zhang) and the Key Research and Development Program of Shandong Province (No. 2018YYSP019). Declaration of Competing Interest The authors report that there are no conflicts of interest to declare. Author Contributions C.Z. wrote the main manuscript; H.L. and K.H. designed the study; Y.S., H.L., and S Z. performed the animal trials; W X. performed the component analysis; H.L. and C.Z. performed the data analyses. X.Y. revised the manuscript. All authors reviewed the manuscript. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.103505. References Aliakbarian, B., Fathi, A., Perego, P., & Dehghani, F. (2012). Extraction of antioxidants from winery wastes using subcritical water. The Journal of Supercritical Fluids, 65(65), 18–24. Asano, N., Yamashita, T., Yasuda, K., Ikeda, K., Kizu, H., Kameda, Y., ... Ryu, K. S. (2001). Polyhydroxylated alkaloids isolated from mulberry trees (Morus alba L.) and silkworms (Bombyx mori L.). Journal of Agricultural and Food Chemistry, 49(9), 4208–4213. Brundtland, G. H. (2002). From the World Health Organization. Reducing risks to health, promoting healthy life. Jama the Journal of the American Medical Association, 288(16), 1974. Cani, P. D., Amar, J., Iglesias, M. A., Poggi, M., Knauf, C., Bastelica, D., ... Chabo, C. (2007). Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes, 56(7), 1761–1772. Chung, H. I., Kim, J., JI, Y. K., & Kwon, O. (2013). Acute intake of mulberry leaf aqueous extract affects postprandial glucose response after maltose loading: Randomized double-blind placebo-controlled pilot study. Journal of Functional Foods, 5(3), 1502–1506. Da-Silva, W. S., Harney, J. W., Kim, B. W., Li, J., Bianco, S. D., Crescenzi, A., ... Bianco, A. C. (2007). The small polyphenolic molecule kaempferol increases cellular energy expenditure and thyroid hormone activation. Diabetes, 56(3), 767–776. Ding, S., Chi, M. M., Scull, B. P., Rigby, R., Schwerbrock, N. M. J., Magness, S., ... Lund, P. K. (2010). High-fat diet: Bacteria interactions promote intestinal inflammation which precedes and correlates with obesity and insulin resistance in mouse. PLoS ONE, 5(8), e12191. Diwan, V., Brown, L., & Gobe, G. C. (2017). The flavonoid rutin improves kidney and heart structure and function in an adenine-induced rat model of chronic kidney disease. Journal of Functional Foods, 33, 85–93. Domitrović, R., Cvijanović, O., Šušnić, V., & Katalinić, N. (2014). Renoprotective mechanisms of chlorogenic acid in cisplatin-induced kidney injury. Toxicology, 324(10), 98. Meng, S., Cao, J., Feng, Q., Peng, J., & Hu, Y. (2013). Roles of Chlorogenic Acid on Regulating Glucose and Lipids Metabolism: A Review. Evidence-Based Complementray and Alternative Medicine,2013,(2013-8-25), 2013, 801457. Yang, N. C., Jhon, K. Y., & Tseng, C. Y. (2012). Antihypertensive effect of mulberry leaf aqueous extract containing γ-aminobutyric acid in spontaneously hypertensive rats.
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Journal of Functional Foods 63 (2019) 103505
X. He, et al.
Shoelson, S. E., Lee, J., & Goldfine, A. B. (2006). Inflammation and insulin resistance. Diabetes Care, 116(7), 1793–1801. Stefanick, M. L., Mackey, S., Sheehan, M., Ellsworth, N., Haskell, W. L., & Wood, P. D. (1998). Effects of diet and exercise in men and postmenopausal women with low levels of HDL cholesterol and high levels of LDL cholesterol. New England Journal of Medicine, 339(1), 12–20. Stewart, L. K., Soileau, J. L., Ribnicky, D., Wang, Z. Q., Raskin, I., Poulev, A., ... Gettys, T. W. (2008). Quercetin transiently increases energy expenditure but persistently decreases circulating markers of inflammation in C57BL/6J mice fed a high-fat diet. Metabolism-clinical & Experimental, 57(7 Suppl 1), S39. Storlien, L. H., James, D. E., Burleigh, K. M., Chisholm, D. J., & Kraegen, E. W. (1986). Fat feeding causes widespread in vivo insulin resistance, decreased energy expenditure, and obesity in rats. American Journal of Physiology, 251(5 Pt 1), E576–E583. Tang, Y. H., Tang, C. C., Huang, H. P., Lee, Y. J., & Wang, C. J. (2013). Hepatoprotective effect of mulberry water extracts on ethanol-induced liver injury via anti-inflammation and inhibition of lipogenesis in C57BL/6J mice. Food & Chemical Toxicology An International Journal Published for the British Industrial Biological Research Association, 62(2Pt1), 786–796. Tsuduki, T., Kikuchi, I., Nakagawa, K., & Miyazawa, T. (2013). Intake of mulberry 1deoxynojirimycin prevents diet-induced obesity through increases in adiponectin in mice. Food Chemistry, 139(1–4), 16–23. Turkmen, N., Sari, F., & Velioglu, Y. S. (2005). The effect of cooking methods on total phenolics and antioxidant activity of selected green vegetables. Food Chemistry, 93(4), 713–718. Turnbaugh, P. J., Ley, R. E., Mahowald, M. A., Magrini, V., Mardis, E. R., & Gordon, J. I. (2006). An obesity-associated gut microbiome with increased capacity for energy harvest. Nature (London), 444(7122), 1027–1131. Weisberg, S. P., Mccann, D., Desai, M., Rosenbaum, M., Leibel, R. L., & Ferrante, A. W. (2003). Obesity is associated with macrophage accumulation in adipose tissue. Journal of Clinical Investigation, 112(12), 1796–1808. Xu, J., Lloyd, D. C., Stanislaus, S., Chen, M., Sivits, G., Vonderfecht, S., ... Chen, J. (2009). Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice. Diabetes, 58(1), 250–259. Xu, C., Yang, B., Wei, Z., Li, X., Tian, J., & Lin, Z. (2015). Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities. Food Chemistry, 186, 83–89.
problem. Current Atherosclerosis Reports, 9(2), 134–138. Miean, K. H., & Mohamed, S. (2001). Flavonoid (myricetin, quercetin, kaempferol, luteolin, and apigenin) content of edible tropical plants. Journal of Agricultural and Food Chemistry, 49(6), 3106–3112. Miyahara, C., Miyazawa, M., Satoh, S., Sakai, A., & Mizusaki, S. (2004). Inhibitory effects of mulberry leaf extract on postprandial hyperglycemia in normal rats. Journal of Nutritional Science & Vitaminology, 50(3), 161–164. Murata, K., Yatsunami, K., Fukuda, E., Onodera, S., Mizukami, O., Hoshino, G., & Kamei, T. (2004). Antihyperglycemic effects of propolis mixed with mulberry leaf extract on patients with type 2 diabetes. Alternative Therapies in Health and Medicine, 10(3), 78–79. Nardelli, T. R., Ribeiro, R. A., & Balbo, S. L. (2011). Taurine prevents fat deposition and ameliorates plasma lipid profile in monosodium glutamate-obese rats. Amino Acids, 41(4), 901–908. Ren, C., Zhang, Y., Cui, W., Lu, G., Wang, Y., Gao, H., ... Mu, Z. (2015). A polysaccharide extract of mulberry leaf ameliorates hepatic glucose metabolism and insulin signaling in rats with type 2 diabetes induced by high fat-diet and streptozotocin. International Journal of Biological Macromolecules, 72, 951. Riant, E., Waget, A., Cogo, H., Arnal, J. F., Burcelin, R., & Gourdy, P. (2013). Estrogens protect against high-fat diet-induced insulin resistance and glucose intolerance in mice. Endocrinology, 150(5), 2109–2117. Riche, D. M., Riche, K. D., East, H. E., Barrett, E. K., & May, W. L. (2017). Impact of mulberry leaf extract on type 2 diabetes (Mul-DM): A randomized, placebo-controlled pilot study. Complementary Therapies in Medicine, 32, 105–108. Rios-Covian, D., Arboleya, S., Hernandez-Barranco, A. M., Alvarez-Buylla, J. R., RuasMadiedo, P., Gueimonde, M., & de los Reyes-Gavilan, C. G. (2013). Interactions between bifidobacterium and bacteroides species in; Cofermentations are affected by carbon sources, including; Exopolysaccharides produced by bifidobacteria. Applied & Environmental Microbiology, 79(23), 7518–7524. Sawin, E. A., De Wolfe, T. J., Aktas, B., Stroup, B. M., Murali, S. G., Steele, J. L., & Ney, D. M. (2015). Glycomacropeptide is a prebiotic that reduces Desulfovibrio bacteria, increases cecal short-chain fatty acids, and is anti-inflammatory in mice. American Journal of Physiology. Gastrointestinal and Liver Physiology, 309(7), G590. Shi, H., Dong, L., Jiang, J., Zhao, J., Zhao, G., Dang, X., ... Jia, M. (2013). Chlorogenic acid reduces liver inflammation and fibrosis through inhibition of toll-like receptor 4 signaling pathway. Toxicology, 303(1), 107–114.
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