| Diabetes is a chronic disease characterized by hyperglycemia due to insufficient insulin secretion or insulin resistance in the body.At present,it is mainly divided into type Ⅰ(T1DM),type Ⅱ(T2DM),pregnancy type and other types,of which type I and type Ⅱ are the most common,accounting for more than 95%of the total number of cases.Diabetes can’t be cured,the incidence is high,and it often affects other tissues and organs.The number of deaths caused by diabetes and its complications has been rising year after year.It is the third biggest threat to human health and has become a prominent public health problem in the world..Mulberry leaves are the dry leaves of Morus alba L.,which is widely distributed in the north and south of China.It has been used as a Chinese herbal medicine in China for more than 3,000 years.It possesses the functions of evacuating wind and heat,clearing liver and eyesight,clearing the lungs and moistening dryness.Modern pharmacological studies have shown that mulberry leaves have rich pharmacological effects such as lowering blood sugar,lowering blood fat,lowering blood pressure,anti-cancer,anti-oxidation,anti-inflammation,and regulating the body’s immunity.Among them,the hypoglycemic effect is more prominent,and the "Compendium of Materia Medica"recorded that "the juice decocted is used as tea and can cure diabetes".Mulberry leaves can be used as medicine or edible.It has been recorded into the drug and food homologous list by the National Health Commission of the People’s Republic of China,and is a promising source of medicine and functional food.The chemical components of mulberry leaves mainly include polysaccharides,alkaloids,flavonoids,proteins and the fraction of small polar components.In this study,the fractions of polysaccharides,flavonoids and proteins were differentially extracted from mulberry leaves using ultrasonic assisted extraction,and then the fractions of alkaloids and the small polar components were extracted by heat reflux method.The extraction and purification processes of several chemical fractions of them were optimized.Then,the chemical parts of mulberry leaves were extracted and purified according to this process,and the high purity extracts of chemical fractions from mulberry leaves were obtained.Diabetic model mice were established by alloxan injection.A blank control group,a model group and a positive drug control group(metformin)were set up.Compared with the model group,the hypoglycemic activity of the fractions of polysaccharides,alkaloids,flavonoids,proteins and the small polar components from mulberry leaves were evaluated by determining fasting blood glucose levels.The five chemical fractions of mulberry leaves were orally administered in three doses as follows:a high dose(20 g/kg),a medium dose(10 g/kg)and a low dose(5 g/kg),respectively,according to the raw materials.The mice were incesantly administered by gastric perfusion for 3 weeks.The effects of these chemical fractions on diabetic mice were determined by observing mice body weights,measuring fasting blood glucose levels and detecting serum biochemical parameters.The results displayed that the FBG levels and glycated serum protein(GSP)levels in DM mice were decreased after treatment with different mulberry leaf extracts except for the proteins group(P<0.05).And with the prolongation of treatment time,their effects became stronger and stronger.The polysaccharides,alkaloids and flavonoids of mulberry leaves could significantly reduce the levels of FBG and GSP in DM mice(P<0.05 or P<0.01).The high-dose group of the small polar components could significantly reduce FBG levels(P<0.05),but had no effect on GSP levels.Mulberry leaf protein had no effect on the levels of FBG and GSP.Mulberry leaf polysaccharides,alkaloids and flavonoids significantly increased HK secretion and hepatic glycogen synthesis in DM mice(P<0.05 or P<0.01),and the former two significantly increased PK secretion(P<0.05).But the proteins and the small polar components from mulberry leaves had no significant effect on the secretion of HK and PK and the synthesis of hepatic glycogen.In addition,mulberry leaf polysaccharides,flavonoids and alkaloids can participate in lipid metabolism at different degrees,which could reduce the levels of total cholesterol(T-CHO)and triglyceride(TG)in DM mice(P<0.05 or P<0.01).Mulberry leaf alkaloids,flavonoids and the small polar components could significantly reduce the levels of low density lipoprotein cholesterol(LDL-C)(P<0.05),while the latter two and mulberry leaf polysaccharides could also improve the levels of high density lipoprotein cholesterol(HDL-C)(P<0.05 or P<0.01).Mulberry leaf proteins had no significant effect on the lipid metabolism in DM mice.Therefore,mulberry leaf polysaccharides,alkaloids and flavonoids have hypoglycemic effects at different degrees,which may play a role via regulating the secretion of PK,HK and the synthesis of hepatic glycogen in DM mice,and the polysaccharides,alkaloids,flavonoids and the small polar components have improved effects on lipid metabolism in DM mice.The fractions of alkaloids and flavonoids with hypoglycemic effects were selected,and were investigated on the safety evaluation of them.The acute toxicity test was carried out by the national standard method,5 g/kg was administered by one-time gastric perfusion,and the mice were observed for two weeks to record the mortality.In the subacute toxicity test,the normal mice were divided into 7 groups(a control group and defferent dose groups:1.00 g/kg,0.50 g/kg,and 0.25 g/kgs for alkaloids or flavonoids,respectively),each group containing 20 mice,male and female in haif,and consecutively administered by gastric perfusion for two weeks.The toxicity of alkaloids and flavonoids were evaluated by observing the general vital signs,examing the blood routine,detecting serum biochemical parameters and dissecting the mice for tissue or organ section.The results showed that the mortality of mice in alkaloids and flavonoids groups were zero in the acute toxicity experiments,indicating that LD50 of both flavonoids and alkaloids were greater than 5 g/kg.The results of the subacute toxicity test showed that alkaloids can reduce the body weight of male and female mice with a tendency of dose-dependence,and no other side effects were displayed.Flavonoids could only significantly reduce the weight of female mice with a tendency of dose dependence.The flavonoids and alkaloids of mulberry leaves had no other adverse effects on blood routine and the levels of serum biochemical indicators such as alanine aminotransferase(ALT),aspartate aminotransferase(AST),urea/nitrogen(BUN),creatinine(CR),creatine kinase(CK),lactate dehydrogenase(LDH-L),glucose(GLU),triglyceride(TG)and total cholesterol(T-CHO)in normal mice.The above results indicated that mulberry leaf flavonoids and alkaloids have high safety.In summary,this study optimized the extraction and purification process of several chemical fractions of mulberry leaves,The results of experimriment showed that polysaccharides,alkaloids and flavonoids of mulberry leaf have significant hypoglycemic effects on diabetes mice,and the the results of safety evaluation of the alkaloids and flavonoids from mulberry leaves displayed that both of these fractions have high safety.All these results provided a theoretical basis for the comprehensive development of mulberry leaves. |
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