Hong ZHANG,Qingfang WANG,Bo SUN,Chao XIONG,Zhongxu SONG

1.Institute of Economic Crops,Hubei Academy of Agricultural Sciences,Wuhan 430064,China;2.Institute of Animal Husbandry and Veterinary Science,Hubei Academy of Agricultural Sciences,Wuhan 430064,China

In recent years,the proportion of feed grains accounting for food consumption has maintained a rapid growth in China.Feed grain supply has become one of the most important issues and key factors of food security.It is necessary to develop"nonfood"feed and focus on the development and utilization of unconventional feed resources.

Mulberry (Morus albaL.)is a perennial deep-rooted species with strong adaptability and high resistance to barren and drought,which can be cultivated in barren hills and slopes,with the total planting area of approximately one million hm2in China.Currently,mulberry has become one of the most high-yielding woody plants,but the peasants have only used mulberry leaves as silkworm feed.With the development of breeding industry,using mulberry as a broad-spectrum feed has attracted increasingly attention.Moreover,mulberry contains more abundant nutrients than other pasture species,which can be prepared into an ideal unconventional feed source.In addition,mulberry has rare diseases and insect pests,which can be planted with pesticide-free techniques to provide green feed sources with no pesticide residues.

Generally,mulberry branches and leaves are harvested by artificial or mechanical stumping,cut into 1-2 cm segments using a mechanical guillotine,dried,mechanically crushed into powder,and added into normal feedstuffs.

However,the content of mulberry powder is usually below 6%in chicken diets and below 10%in pig diets[2].Excessively high concentrations of mulberry powder in feedstuffs will affect the growth of livestock,mainly causing diarrhea.So far,studies of mulberry feedstuffs are mainly focused on the recommended dosage and their improving effect on animal quality,but little information is available on the reason why the dosage can not be raised.Previous studies show that alkaloids are the main factors causing diarrhea in animals.A certain amount of alkaloids exhibit a positive effect on the growth and development of pigs,but overdose will lead to diarrhea.Morusplants are characterized by juice secretion,which ooze juice after being damaged.Mulberry juice contains three kinds of alkaloids:1,4-dideoxy-1,4-imino-d-arabinitol(D-AB1),1-deoxynojirimycin(DNJ)and 1,4-dideoxy-1,4-imino-D-ribitol[3],which all belong to α-glucosidase inhibitors that can inhibit sugar degradation in animal intestines and affect normal glucose metabolism.Alkaloids are soluble in water,and the percentage of alkaloids in mulberry juice accounts for about 2.4%of the wet weight,which is 100 times of that in other issues[4].In this study,the effects of of mulberry leaf extract with and without removal of mulberry juice on starch metabolism were analyzed and compared,aiming at providing the basis for the promotion and utilization of mulberry feedstuffs.

Materials and Methods

Materials

Mulberry leavesMulberry leaves were collected from Husang 32 plants cultivated in the mulberry germplasm nursery ofInstitute ofEconomic Crops,Hubei Academy of Agricultural Sciences.

Experimental animalsLocal 3-month-old broilers with consistent weight (500 g);Dabai piglets with consistent weight(approximately 25 kg).

Methods

Feeding trials of broilers with mulberry juiceAccurately 0.1 g of mulberry juice was added into 10 g of feedstuff(per day)and fed to each broiler for five consecutive days;the defecation of broilers was observed to calculate watery excrement rate(on average).

Watery excrement rate=Watery excrements/Total excrements×100%.

Feeding trials of piglets with mulberry leaf powderDabai piglets were divided into two groups.Group A:normal feedstuff was added with 10%mulberry leaf powder with removal of mulberry juice;group B:normal feedstuff was added with 10%mulberry leaf powder without removal of mulberry juice.Experimental piglets were reared in pens for a week and then fed with feedstuffs containing 10%mulberry leaf powder.The dietary preference and adaptability to mulberry leaf powder and diarrhea situation in piglets was observed.After feeding for 12 d,each piglet was weighed(on average).

Extraction of mulberry leaf extractAccurately 200 mg of mulberry leaf powder with and without removal of mulberry juice were weighed,added with 1 ml of 0.05 mol/L HCl solution,mixed with a vortex for 1 min,processed by ultrasonic treatment for 40 min,evaporated at 60℃for 30 min to remove HCl,and centrifuged at 12 000 r/min for 10 min.The supernatant was collected separately to obtain two different mulberry leaf extracts.

Inhibitory effects of mulberry leaf extract on starch metabolismThe efficiency of starch degradation into glucose was determined with p-nitrophenol method that involves amylase and glucosidase.In accordance with the introductions of amylase assay kit(Wuhan Life Origin Biotech Co.,Ltd.),100 U of enzyme was added into 250 μlenzymaticreaction system and added with 30 μl of mulberry leaf extract,with 30 μl of distilled water as a control(original enzyme activity assay).Each treatment was repeated three times and the determined values were averaged to calculate the inhibition rate of mulberry leaf extract on starch metabolism.

Inhibition rate=(Original enzyme activity-Enzyme activity in samples)/Original enzyme activity×100%.

Determination of 1-deoxynojirimycin(alkaloid)content in mulberryleavesBy using ultraviolet reversed-phase high performance liquid chromatography[5],the content of 1-deoxynojirimycin in mulberry leaves with and without removal of mulberry juice was determined.Each treatment was repeated twice and the determined values were averaged.

Results and Analysis

Feeding trials of broilers with mulberry juice

Broilers were fed with 10 g of normal feedstuff containing 0.1 g of mulberry juice.Results showed that the loose stool rate of broilers increased significantly from 10.2%to 21.2%during the experimental period,which indicated thatmulberry juice could cause diarrhea in broilers(Table 1).

Feeding trials of piglets with mulberry leaf powder

Piglets weighing 25 kg were selected for feeding trials because they are more sensitive to diets than adult pigs.Experimental piglets were fed with feedstuff containing 10%mulberry leaf powder for 12 d.According to the observation results, experimental piglets exhibited higher dietary preference to mulberry leaf powder with removal of mulberry juice (treatment A)than to mulberry leaf powder without removal of mulberry juice(treatment B);after feeding,the diarrhea rate in group A was significantly lower than that in group B;weight gain in group A was significantly higher than that in group B;to be specific,the weight of piglets in group A increased averagelyby 7.4 kg and that in group B increased averagely by 5.9 kg(Table 2).

Table 1 Effects of different feedstuffs on defecation in broilers

Table 2 Effects of mulberry leaf powder with and without removal of mulberry juice on weight gain in piglets

Determination of 1-deoxynojirimycin (alkaloid)content in mulberry leaves

Mulberry juice contains various alkaloids,including 1-deoxynojirimycin.The variation of1-deoxynojirimycin content in mulberry samples with and without removal of mulberry juice reflects the effects of different treatment measures on alkaloid content in samples.According to the determination results,1-deoxynojirimycin content in mulberry samples without removal of mulberry juice was 0.19%;1-deoxynojirimycin content in mulberry leaves with removal of mulberry juice was 0.15%that wasreducedby21%compared with untreated mulberry leaves.

Inhibitory effects of different mulberry leaf extracts on starch hydrolysis

α-glucosidase is required for starch hydrolysis,and alkaloids in mulberry leaves are α-glucosidase inhibitors.In this study,the effects of mulberry leaf extract with and without removal of mulberry juice on starch hydrolysisin vitrowere analyzed.According to the results,the original enzyme activity was 0.053;after the addition of extract A (mulberry leaf extract with removal of mulberry juice),the enzyme activity (represented by the changes in absorbance value)reached 0.046 and the inhibition rate on starch metabolism was 13.2%;after the addition of extract B(mulberry leaf extract without removal of mulberry juice),the enzyme activity reached 0.035 and the inhibition rate on starch metabolism was 34.0%,which indicated that mulberry leaf extract without removal of mulberry juice exhibited significantly higher inhibitory effects on starch metabolism than mulberry leaf extract with removal of mulberry juice.

Conclusion and Discussion

Alkaloids in mulberry juice are αglucosidase inhibitors.α-glucosidase can act on glycosides or oligosaccharides to hydrolyze glucosidic bonds into glucose.Excess α-glucosidase inhibitors in the feedstuff will inhibit the hydrolysis of carbohydrates in animals.Mulberry leaf extracts exhibit significantly strong inhibitory effects on the hydrolysis process of starch into glucose.

Clinical studies have shown that blood sugar levels in patients with type II diabetes can be reduced effectively by 20 mg/d DNJ[6].In this study,results show that high concentrations of alkaloids affect carbohydrate metabolism and cause diarrhea in animals.Due to lack of hydrolase or other reasons,some carbohydrates can not be absorbed by the intestinal mucosa,forming hypertonic intestinal contents to absorb large amounts of water,thus causing hypertonic diarrhea.In the presentstudy,10% mulberry leaf powder was added into piglet diets,which significantly declined the growth rate of piglets;however,after removal of partial DNJ in mulberry leaves,diarrhea situation in piglets was reduced significantly and weight gain increased remarkably.

High concentrations of α-glucosidase inhibitors restrict the feed function of mulberry.However,China is rich in mulberry resources.Mulberry varieties with relatively low content of α-glucosidase inhibitors and high protein content can be selected to accommodate feedstuff needs of mulberry industry.

[1]AL-KIRSHI R,et al.Utilization of mulberry leaf meal(Morus alba)as protein supplement in diets for laying hens[J].I-talian Journal of Animal Science,2010,9(3):265-267.

[2]LI YG(李有貴),ZHANG L(張雷),ZHONG S(鐘石),et al.Effects of dietary mulberry leaf on growth performance,fat metabolism and meat quality of finishing pigs(飼糧中添加桑葉對育肥豬生長性能、脂肪代謝和肉品質(zhì)的影響)[J].Acta Zoonutrimenta Sinica(動物營養(yǎng)學(xué)報(bào)),2012,24(9):1805-1811

[3]KONNO K,et al.Mulberry latex rich in antidiabetic sugar-mimic alkaloids forces dieting on caterpillars[J].PNAS,2006,103(5):1337-1341.

[4]ASANO N,et al.Polyhydroxylated alkaloids isolated from mulberry trees(Morus albaL.)and silkworms(Bombyx moriL.)[J].J.Agric.Food Chem.,2001,49:4208-4213.

[5]SHI XQ(施新琴),CUI WZ(崔為正),QIU LQ(裘立群),et al.Determination of 1-deoxynojirimycin content by ultraviolet reversed-phase high performance liquid chromatography(用反相高效液相色譜-紫外檢測法測定1-脫氧野尻霉素的含量)[J].Science of Sericulture(蠶業(yè)科學(xué)),2006,32(1):146-149.

[6]OK HM,et al.The effect of aqueous mulberry leaf extract on glucose metabolism in subjects with impaired fasting glucose[J].The FASEB Journal,2012,26:440-448.