Characterization of mulberry leaf instant tea and evaluation of its hypolipidemia effect via regulation of intestinal microbiota

The purpose of this study was to characterize mulberry leaf instant tea(MLIT)powder prepared from the'Longsang No.1'(Morus abla L.cv.Longsang 1)mulberry leaves in Heilongjiang Province(China)and assess its obesity-preventing/relieving effects.A total of 174 compounds including quercetin,chlorogenic acid,1-deoxyecomycin(1-DNJ)related to antihyperlipidemia effects were identified from the MLIT powder.MLIT treatment reversed the Lee's index,fat coefficient,and serum biochemical parameters in both the obesity relieving and obesity preventing mice fed with high-fat diet.In the obesity relieving experiment,the relative abundance of Desulfovibrio in mouse feces decreased after both 0.5%and 1%MLIT treatments.In obesity preventing experiments,mouse with different amount of MLIT treatments showed increased relative abundance of Akkermansia,Bifidobacterium and Lactobacillus,while Deferribacteres,Desulfobacterota decreased.The beneficial bacteria in the intestinal tract of mice treated with MLIT increased.This study proved that MLIT had antihyperlipidemia potential via modulating intestinal microbiota in mice.

Structural elucidation of mulberry leaf oligosaccharide and its selective promotion of gut microbiota to alleviate type 2 diabetes mellitus

Two oligosaccharide fractions(MLO 2-1 and 2-2)were purified from enzymatic hydrolysate of mulberry leaf polysaccharide.The results of simulated digestion showed that MLO 2-2 was a digestible oligosaccharide,which could be degraded by human digestive juice;while MLO 2-1 possessed the non-digestible property in the upper gastrointestinal tract and performed the function by regulating the gut microbiota.Hence,MLO 2-1 was selected for the further analysis.The structure of MLO 2-1 was elucidated as follow:α-T-Glcp-(1→3)-β-Glcp-(1→5)-α-Araf-(1→5)-α-Araf-1→5)-α-Araf-(1→3)-α-(2-OAc)-Glcp-1.The in vitro fecal fermentation results showed that MLO 2-1 could modulate the composition of gut microbiota.Meanwhile,MLO 2-1 was effectively metabolized by fecal bacteria to produce lactate and short chain fatty acids,especially acetate and butyrate.The specific metabolic pathways of MLO 2-1 by gut microbiota were further illuminated.Gut microbiota analysis revealed that MLO 2-1 selectively promoted the growth of Ligilactobacillus murinus,a commensal bacterium presented a reduced level in T2DM mice.Animal experiments indicated that MLO 2-1 and L.murinus exhibited hypoglycemic activities.These results demonstrated that MLO 2-1 might alleviate T2DM by selectively accelerating the proliferation of L.murinus.

Synergistic effects of three traditional herbs green tea,mulberry leaf and corn silk on glucose uptake level of L6 myoblasts and the hypoglycemic mechanism

Background:Green tea,mulberry leaf and corn silk are traditional herbs used in the prevention and treatment of diabetes in China for a long time,but their synergistic hypoglycemic effects and mechanisms remain unclear.Methods:The effective components of green tea,mulberry leaf and corn silk were extracted and enriched.Mixture design of experiments was used to study the influences of different combinations on the cell viability and glucose uptake level of L6 myoblasts,so as to determine the optimal synergistic hypoglycemic combination.The possible hypoglycemic mechanism of the optimal synergistic combination was explored by cytotoxicity assay,glucose uptake assay,and western blot.Results:Three polyphenol enrichment fractions of the herbs,30%ethanol elution fraction of green tea(GT),50%ethanol elution fraction of mulberry leaf(ML)and 60%ethanol elution fraction of corn silk(CS)were obtained.The antioxidant activities of GT-30%,ML-50%and CS-60%were superior to those of crude extracts,and showed strong potential inα-amylase andα-glucosidase inhibition activities.The optimal synergistic combination of crude extracts G7(crude extract of green tea:crude extract of mulberry leaf:crude extract of corn silk=1:5:3),polyphenol enrichment fractions R3(GT-30%:ML-50%:CS-60%=1:7:1)and monomers X2(epigallocatechin gallate:morusin:formononetin=3:1:2)were selected,respectively.G7,R3,and X2 showed promoting effects on the cell viability and glucose uptake of L6 myoblasts within the detected concentration range.In addition,G7,R3,and X2 could increase the expression levels of p-PI3K/PI3K and p-Akt/Akt in L6 myoblasts,and promote the translocation of Glut4,but G7 and R3 showed more significant effects.Conclusion:The synergistic hypoglycemic effects of green tea,mulberry leaf and corn silk had the characteristics of multiple-components and multiple-targets with p-PI3K/PI3K,p-Akt/Akt and the translocation of Glut4 signal pathways involved.The three traditional herbs might have the potential to be combined used for the prevention and treatment of diabetes based on the synergistic hypoglycemic effects.

Effects of Nitrogen Amount and Nitrogen Form on 1-deoxynojimycin Content in Mulberry Leaf

[Objective] Effects of nitrogen amount and nitrogen form on 1-deoxynojimycin（DNJ）content in mulberry leaf was explored in order to provide a reference for improving DNJ content in mulberry leaf and its medicinal value.[Method] Effects of nitrogen amount（NO-3-N and NH＋4-N）and nitrogen form（NO-3-N/NH＋4-N）on DNJ content in mulberry leaf were studied by changing nitrogen amount and nitrogen form in water culture solution.[Result] DNJ contents in mulberry leaf both increased at first and then decreased with the increase of nitrogen amount,furthermore compared with NH＋4-N,NO-3-N was more beneficial to the accumulation of DNJ;DNJ content in mulberry leaf obviously increased at first and then decreased with the decrease of NO-3-N/NH＋4-N,among them when its rate was 25/75,DNJ content reached the highest.[Conclusion] Proper nitrogen amount and nitrogen form could effectively improve the DNJ content in mulberry leaf.

Research progress in mulberry leaf polysaccharide in treating diabetic nephropathy

Diabetic nephropathy(DN)is one of the most common complications of diabetes.It is an important cause of diabetes disability and death.DN is a systemic metabolic syndrome.In its pathogenesis,the interaction of various cell activities and a large number of cytokine biological activities,the activation of signal pathways and so on are involved in the development of DN.At present,the clinical treatment of DN is mainly Western medicine,but it has limitations such as strong toxicity,high side effects and poor compliance.Therefore,the discovery of natural anti-DN substances has also become an important means to treat DN.Mulberry leaves are the dry leaves of Morus alba L.It is not only a traditional Chinese medicine,but also a dual-purpose medicinal material for medicine and food.It has the effects of dispelling wind and clearing heat,cooling blood and brightening eyes,tonifying and so on.Mulberry leaf polysaccharide(MLP)is a kind of high molecular compound in mulberry leaves.It has many pharmacological effects,such as hypoglycemic,antioxidant,anti-stress,anti-virus and so on.Therefore,the pharmacological effects of mulberry leaf polysaccharides on diabetic nephropathy are reviewed in this paper,so as to provide references for further research and application.The pathogenesis of DN is complex,and the mechanism of renal injury has not been completely clarified.The current studies believe that DN is closely related to heredity,abnormal glucose metabolism,abnormal lipid metabolism,microcirculation disorder,cytokine action,oxidative stress and so on.Relevant studies show that the pharmacological effects of mulberry leaf polysaccharide in the prevention and treatment of DN mainly include:①Effect on transforming factor-β1(TGF-β1):TGF-β1 has become an important cytokine involved in the formation of renal fibrosis by regulating cell proliferation and differentiation and the production of extracellular matrix(ECM).MLP can significantly inhibit TGF-β1 protein,and then inhibit the synthesis of extracellular matrix by renal interstitial fibroblasts and inhibit the realization of fibrosis.②Effect on insulin receptor substrate(IRS-1):IRS-1 is an important signal molecule at the beginning of IR signal transduction.The decrease of IRS-1 gene expression or the decrease of expression can affect the effective transmission of IR signal and lead to the development and deterioration of diabetes. MPL can significantly increase the expression of IRS-1 mRNA in liver tissue of DN rats, so as to prevent and treat DN. ③ Effect on the expression of resistin protein in adipose tis sue. Resistin is a secretory polypeptide derived from adipose tissue and is specifically expressed in white adipose tissue and is closely related to type 2 diabetes mellitus (T2DM). Experimental studies show that MLP can effectively reduce the expression of resistin protein in white adipose tissue of T2DM rats, indicating that MLP may reduce the level of IR by inhibiting the expression of resistin in adipose tissue, thereby reducing the insulin resistance state of T2DM rats, so as to achieve the goal of treating diabetes. ④ Effect on adiponectin receptor 1 (AdipoR1): adiponectin can improve insulin resistance, reduce blood glucose and lipid. AdipoR1 is mainly expressed in skeletal muscle and kidney. Studies have shown that AdipoR1 is closely related to the occurrence and development of DN. The results showed that MLP could reduce the blood glucose and blood lipid level and up regulate the expression of AdipoR1 mRNA in DN rats, suggesting that MLP may delay the occurrence and development of DN. This article reviewed the pharmacological effects of mulberry leaf polysaccharides on diabetic nephropathy, and provided a useful basis for further development and utilization of mul berry leaf polysaccharides in the treatment of DN.

Mulberry leaf supplementation inhibits skatole deposition by regulating gut microbiota and upregulating liver cytochrome P4501A1 expression in finishing pigs

Skatole,a strong fecal odor substance,is generated through microbial degradation of tryptophan in the animal hindgut.It easily accumulates in adipose tissue and affects meat quality.In this study,the effect of mulberry leaf supplementation on skatole in finishing pigs was studied.In a 35-day trial,20 finishing pigs(barrows and gilts)were fed with a basal diet or basal diet with 6%mulberry leaves.Growth performance of the pigs(n=10)was automatically recorded by a performance-testing feeder system and 8 pigs in each treatment were slaughtered and sampled for the remaining tests.Skatole and short-chain fatty acids were detected using HPLC and gas chromatography,respectively.Fecal microbiota were analyzed using 16S r RNA gene sequencing.The metabolomics analysis of feces and serum was performed with UHPLC-MS/MS.The major cytochrome P450(CYP)enzymes that catalyze skatole degradation in the liver were tested by using RT-PCR and Western blot.Effects of major bioactive compounds in mulberry leaves on the CYP genes were verified in the hepatic cell line Hep G2 in an in vitro test(n=3).In finishing pigs,mulberry leaf supplementation had no significant effect on the average daily gain,average daily feed intake,and feed conversion ratio(P>0.05),but reduced skatole levels in feces,serum,and backfat(P<0.05),and increased acetic acid levels in feces(P=0.027).Mulberry leaf supplementation decreased the relative abundance of the skatole-producing bacteria Megasphaera and Olsenella(P<0.05).Indole-3acetic acid,the intermediate that is essential for skatole production,was significantly reduced in feces by mulberry leaf supplementation(P<0.05)and was positively correlated with skatole content in feces(P=0.004).In pigs treated with mulberry leaves,liver CYP1A1 expression was increased(P<0.05)and was negatively correlated with skatole content in backfat(P=0.045).The in vitro test demonstrated that mulberry leaf polyphenols and polysaccharides could directly stimulate CYP1A1 expression in hepatic cells.These findings suggest that mulberry leaf supplementation reduces skatole production and deposition in finishing pigs by regulating the gut microbiota and promoting skatole degradation in liver.

Mulberry leaf flavonoids activate BAT and induce browning of WAT to improve type 2 diabetes via regulating the AMPK/SIRT1/PGC-1α signaling pathway

Mulberry (Morus alba L.) leaf is a well-established traditional Chinese botanical and culinary resource. It has found widespread application in the management of diabetes. The bioactive constituents of mulberry leaf, specifically mulberry leaf flavonoids (MLFs), exhibit pronounced potential in the amelioration of type 2 diabetes (T2D). This potential is attributed to their ability to safeguard pancreatic β cells, enhance insulin resistance, and inhibit α-glucosidase activity. Our antecedent research findings underscore the substantial therapeutic efficacy of MLFs in treating T2D. However, the precise mechanistic underpinnings of MLF’s anti-T2D effects remain the subject of inquiry. Activation of brown/beige adipocytes is a novel and promising strategy for T2D treatment. In the present study, our primary objective was to elucidate the impact of MLFs on adipose tissue browning in db/db mice and 3T3-L1 cells and elucidate its underlying mechanism. The results manifested that MLFs reduced body weight and food intake, alleviated hepatic steatosis, improved insulin sensitivity, and increased lipolysis and thermogenesis in db/db mice. Moreover, MLFs activated brown adipose tissue (BAT) and induced the browning of inguinal white adipose tissue (IWAT) and 3T3-L1 adipocytes by increasing the expressions of brown adipocyte marker genes and proteins such as uncoupling protein 1 (UCP1) and beige adipocyte marker genes such as transmembrane protein 26 (Tmem26), thereby promoting mitochondrial biogenesis. Mechanistically, MLFs facilitated the activation of BAT and the induction of WAT browning to ameliorate T2D primarily through the activation of AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) signaling pathway. These findings highlight the unique capacity of MLF to counteract T2D by enhancing BAT activation and inducing browning of IWAT, thereby ameliorating glucose and lipid metabolism disorders. As such, MLFs emerge as a prospective and innovative browning agent for the treatment of T2D.

The regulatory effects of mulberry leaf and its extract on intestinal function

Since ancient times,mulberry leaf has a prominent intestinal regulatory effect,and the close relationship between intestinal diseases and other diseases has attracted more and more attention.This article reviewed the relevant articles on the mulberry leaf in recent years and summarized the research progress of mulberry leaf‘s biological activities and their effects on human intestinal regulation and the corresponding mechanisms.Multiple components in the mulberry leaf can individually or synergistically affect the human intestine,directly and indirectly promote the digestion,absorption,and transport function of the small intestine,adjust the balance of the flora,enhance the barrier function of the intestinal mucosa,improve the immunity of the part,and facilitate the intestine recovery from illness.

Effects of mulberry leaf flavonoid and resveratrol on methane emission and nutrient digestion in sheep

As a new type of methane control agent, natural plant extract has been widely studied in recent years,but in vivo studies are few. This study was to investigate the effects of the dietary supplementation of 2 different polyphenols on the methane（CH4） emission and digestion metabolism in sheep. Ten healthy crossbred sheep（Dorper（?）× small-tailed Han ♀; BW 60.0 ± 1.73 kg） were used in a change-over design.The sheep were fed the following 3 diets in the present study: the basal diet（CON） with no supplementation; the basal diet supplemented with 2 g mulberry leaf flavonoid（MLF） per day per sheep; the basal diet supplemented with 0.25 g resveratrol（RES） per day per sheep. Both MLF and RES reduced CH4 emission scaled to metabolic weight per kilogram of DMI and CO2 output scaled to metabolic weight, but the effect of RES was significant（P < 0.05）. Both MLF and RES significantly improved apparent digestibility of DM, OM, NDF, ADF, and nitrogen, but the effect of RES was significant（P < 0.05）. Both MLF and RES significantly improved ME（P < 0.05） and reduced energy losses in CH4 emission（P > 0.05）. In conclusion, MLF and RES can improve the digestibility of nutrients, the utilization of nutrients and energy, and reduce CH4 emission, but they are not conducive to nitrogen retention.

Scheme of Nitrogen Reasonable Fertilization in Mulberry Gardens of Sichuan Hilly Areas

Abstract In the research, five test groups were set, including urea at O, 120, 240, 360 and 480 kg/hm^2, respectively, and analysis was conducted on mulberry leaf yield and some characters of the quality in order to establish utility functions of ap-plied N in mulberry gardens with different production purposes and to provide schemes of rational N fertilization. The results indicated that yield of mulberry leaf and content and yield of active nutritional substances in the leaves would be reduced either by insufficient or excessive N quantity. In addition, 240 kg/hm^2 of applied N significantly improved active nutritional substances in leaf and 360 kg/hm^2 of applied N significantly improved yields of mulberry leaf and active nutritional sub-stances. Therefore, utility functions of applied N quantity with leaf yield, DNJ content and yield were established accordingly. The optimal N quantity in mulberry gardens for sericulture is recommended 360 kg/hm^2 and the optimal N quantities in mulberry gardens for increase of yield and content of DNJ are 299.56 and 294.72 kg/hm^2, respectively.

Free-radical scavenging properties of low molecular weight peptide(s) isolated from S1 cultivar of mulberry leaves and their impact on Bombyx mori(L.)(Bombycidae)

The mulberry leaves have been considered as a sole food source for silkworm, Bombyx mori （L.）. In present work an attempt was made to investigate the role of low molecular weight peptide（s） isolated from mulberry leaves on silkworm rearing. Also we have tried to find out the role of free-radical scavenging activities of isolated peptide（s） on silkworm growth. Larval growth rate was found effective under the influence of peptide（s）. Consumption rate of larvae after peptide（s） treatment on mulberry leaves was significantly enhanced over control. High antioxidant activity was found in Low molecular weight peptide（s） which have an effect on silkworm.

Herbaceous Cultivation Test of Mulberry in Sichuan Sericultural Area

With Guiteyou 2 as the test mulberry variety, mulberry herbaceous cultivation field was established at the planting densities of 60 000,90 000 and 120 000 plants/hm2, respectively. Hybrid mulberry herbaceous cultivation test was conducted, with Tongxiangqing mulberry field of mid-fist-form at conventional planting density of 4 995 plants/hm2 as the control. The results showed that the leaf yield of herbaceous cultivation mulberry was closely related to the planting density. With the increasing planting density, the leaf yield per plant was decreasing, but the leaf yield per unit area of mulberry field was increasing. The annual leaf yield per unit area in the mulberry field with the average planting density of 120 000 plants/hm2 was 37 560 kg/hm2, 2.14 times of that of the control field. The moisture content of mulberry leaf in herbaceous cultivation field was 4.74% higher than the control; the contents of crude protein, crude fat, crude fiber and crude ash were 1.264%, 0.014%, 0.744% and 0.002%lower than the control, respectively. Comparison of leaf rearing with herbaceous cultivation mulberry and control mulberry showed that there was no significant difference in growth status of silkworm. The cocoon weight, cocoon shell weight, cocoon yield per 10 000 larvae, cocoon filament length and non-broken filament length of Chuanshan × Shushui were 12.81%, 14.29%, 13.85%, 5.95% and 7.68% lower than the control, respectively; but the percentage of common cocoons was 0.16% higher than the control; the cleanness and neatness were 0.2 and 1.25 points higher than the control;the filament size was 0.196 dtex lower than the control. The cocoon weight, cocoon shell weight, cocoon yield per 10 000 larvae, cocoon filament length and non-broken filament length of Jingsong × Haoyue were 11.06%, 10.20%, 11.53%, 9.46% and 9.03% lower than the control, respectively;but the percentage of common cocoons was 1.77% higher than the control; the cleanness and neatness were 0.12 and 2.50 points higher the control;and the filament size was 0.196 dtex lower than the control.

Flavonoids from mulberry leaves inhibit fat production and improve fatty acid distribution in adipose tissue in finishing pigs

This study evaluated the effects of flavonoids from mulberry leaves(FML)on plasma biochemical indices,serum activities of lipid metabolism-related enzymes,fat morphology,fatty acid composition,and lipid metabolism in different adipose tissues of finishing pigs.We used 120 Chinese hybrid barrows of Berkshire and Bama mini-pigs with an average initial body weight of 45.11±4.23 kg.The pigs were randomly assigned to five treatment groups and fed a control diet based on corn,soybean meal,and wheat bran or a control diet supplemented with 0.02%,0.04%,0.08%,or 0.16%FML.Each experimental group had six replicates(pens),with four pigs per pen.After a 7-d adaptation period,the feeding trial was conducted for 58 d.Blood and adipose tissue samples were collected from 30 pigs(one pig per pen)at the end of the test.The results showed that FML supplementation significantly decreased the feed intake to body gain ratio,the plasma concentrations of total cholesterol and free fatty acids,and the serum activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase(linear or quadratic effects,P<0.05),and decreased the plasma triglyceride concentration(quadratic,P=0.07).Increasing FML supplementation increased the average daily gain and serum activities of lipoprotein lipase(linear and quadratic effects,P<0.05)and adipose triglyceride lipase(linear,P<0.05).Dietary FML supplementation decreased the adipocyte area in the dorsal subcutaneous adipose(DSA)tissue of finishing pigs(linear,P=0.05)and increased the adipocyte area in the visceral adipose tissue(quadratic,P<0.01).Increasing FML supplementation decreased the C20:1 content in DSA,abdominal subcutaneous adipose,and visceral adipose tissues of finishing pigs(P<0.05)and increased the C18:3n3 and n-3 PUFA contents(P<0.05).The lipid metabolism genes were regulated by the PPARγ-LXRa-ABCA1 signaling pathway,and their expressions differed in different adipose tissues.These findings suggest that FML improved growth performance,regulated lipid metabolism,inhibited fat production,and improved fatty acid distribution in the adipose tissue of finishing pigs,thereby improving pig fat's nutritional quality and health value.

Current anti-diabetes mechanisms and clinical trials using Morus alba L.

Backgrounds:Diabetes mellitus,especially type 2 diabetes,with its fast-rising prevalence,has become a global epidemic.Mulberry(Morus alba L.)leaf has been known to have hypoglycemic effects since ancient times.In Asia mulberry leaf is used as tea to complement the treatment of diabetes mellitus.The methods by which mulberry leaf affects the body and its mechanism when combined with chemical agents have been studied extensively.Conclusions:We summarize the possible mechanisms of the anti-diabetic effects of mulberry leaf based on extraction procedures,in vitro and in vivo experiments,and clinical trials.We also discuss the hypothesis that crosstalk and“critical nodes”may be useful for a deeper molecular understanding of the treatment and prevention of diabetes with mulberry leaf.