Journal of Applied Botany and Food Quality 91, 114 - 119 (2018), DOI:10.5073/JABFQ.2018.091.016 Silk Biotechnology Laboratory, School of Biology and Basic Medical Sciences, Soochow University, Suzhou, PR China A novel estimation method of total flavonoids in edible medicinal mulberry leaves by ultrasound-assisted hydroalcohol-acid extraction and HPLC-DAD Jin-Ge Zhao, Yu-Qing Zhang* (Submitted: January 17, 2018; Accepted: March 29, 2018) * Corresponding author Summary Mulberry leaves have been widely used to produce various health products. In this paper, an efficient procedure of ultrasound-assisted hydroalcohol-acid extraction (UAHAE) was established to estimate the total flavonoids content in mulberry leaves by quantifying their resulting aglycones (quercetin and kaempferol) using HPLC-DAD. Effective hydrolysis of glycosides to aglycones was achieved in an ethanol-HCl-water (7/2/1, v/v/v) solution at 75 °C by ultrasound (40 kHz) for 60 min. The average contents of quercetin and kaemp- ferol were 7.12 mg/g and 2.13 mg/g, respectively, in the variety of cultivar MC308, and their recoveries were 105.48% and 105.81%, respectively. The total flavonoid content of most varieties was be- tween 4-10 mg/g, accounting for 80% in 86 species of mulberry leaves. With the increase in leaf maturity, the general trend of the change in flavonoid content was a decrease at first and then an in- crease. The highest total flavonoid content in commercial mulberry tea (CT 2) is 4.76 mg/g, and the DPPH radical scavenging activi- ties were similar to the distribution of total flavonoids measured by UAHAE in three types of mulberry tea. These finding provide a basis for industrial-scale manufacturing of edible medicinal mulberry leaf products. Key Words: mulberry leaves; total flavonoids; aglycones; quercetin; kaempferol; ultrasound; hydrolysis Introduction Mulberry (Morus alba L.) is an industrial crop with a high economic value (Zhang et al., 2016 ). Its fruit, leaves, branches and roots are good sources of bioactive compounds, and it has a long history of use in traditional Chinese medicine. Mulberry leaf is the main product of the mulberry tree, accounting for 64% of the ground part of the tree (Wang et al., 2005). It is nutritious and nontoxic and is usually used as feed for silkworms in most Asian countries. Some research- ers have found that mulberry leaves are rich in phenylpropanoids (hunyadi et al., 2012), flavonoids (Wanyo et al., 2011), polysaccha- ride (Thirugnanasambandham et al., 2015), alkaloids (sugiyama et al., 2016) and many other bioactive compounds. As a herbal medi- cine, extracts of mulberry leaves possess medicinal benefits against obesity (Lim et al., 2013), diabetes (ren et al., 2015), atherosclero- sis, hypertension (Lee et al., 2011) and tumours (yang et al., 2012). Because of their significant bioactivities, mulberry leaves have been widely used to produce various health products, such as beverages, health food and mulberry-leaf tea (Zhang et al., 2016 ). Flavonoids and flavonoids glycosides are the most important com- ponents in mulberry leaves. The total flavonoid content accounted for 1.0-3.0% of the mulberry leaf dry weight, which is higher than in other plants (Li et al., 2012). The major type of flavonoid in mul- berry leaves is glycoside, with mainly quercetin and kaempferol aglycones (Tab. 1). Kim et al. identified nine flavonoids from mul- berry leaves, kaempferol-3-O-β-D-glucopyranoside; kaempferol-3- O-(6”-O-acetyl)-β-D-glucopyranoside; quercetin-3-O-(6”-O- acetyl)-β-D-glucopyranoside; quercetin-3-O-β-D-glucopyranoside; kaempferol-3-O-α-L-rhamnopyranosyl-(1-6)-β-D-glucopyranoside; quercetin-3-O-α-L-rhamnopyranosyl-(1-6)-β-D-glucopyranoside; quercetin-3-O-β-D-glucopyranosyl-(1-6)-β-D-glucopyranoside; quercetin-3,7-di-O-β-D-glucopyranoside; and quercetin (Kim et al., 1999). Kayo et al. purified and identified two new flavonoids from a butanol extract of mulberry leaves, isopentenyl flavanone and iso- pentene flavanone glycoside (doi et al., 2001). Rutin, hesperetin, quercetin, kaempferol, naringenin, hydroxyflavin (Chang et al., 2016) and kaempferol (yang et al., 2012) have also been isolated from mulberry leaves. Many researchers have confirmed that flavo- noids extracted from mulberry leaves possess anti-hyperlipidemic (Kojima et al., 2000), anti-diabetic (Kobayashi et al., 2010), antican- cer (deepa et al., 2013), hepatoprotective (KaLanTari et al., 2009), and anti-Alzheimer’s disease (niidome et al., 2007) activities as well as have a synergistic effect on regulating the blood sugar level with alkaloids and polysaccharides in mulberry leaves. The level of total flavonoids in mulberry leaves is conventionally estimated by a colorimetric method using rutin as a standard. How- O OH O OH OR2 R1O OHa O OH O OH OR2 R1O b Tab. 1: Selected flavonoids with quercetin and kaempferol aglycones isolated from mulberry leaves R1 = H, R2 = H Quercetin Chang et al., 2016 R1 = H, R2 = glc Quercetin 3-O-β-D-glucopyranoside (Isoquercitrin) Kim et al., 1999 R1 = H, R2 = 6’’-acetyl-glc Quercetin 3-O-(6’’-O-acetyl)-β-D-glucopyranoside Kim et al., 1999 R1 = H, R2 = rha-glc Quercetin 3-O-β-D-rutinoside (Rutin) Chang et al., 2016 R1 = glc, R2 = H Quercetin 7-O-β-D-glucopyranoside Wang et al., 2013 R = glc, R2 = glc Quercetin 3,7-di-O-β-D-glucopyranoside Kim et al., 1999 R1 = H, R2 = H Kaempferol yang et al., 2012 R1 = H, R2 = glc Kaempferol 3-O-β-D-glucopyranoside (Astragalin) Kim et al., 1999 R1 = H, R2 = rha-glc Kaempferol 3-O-β-D-rutinoside Wang et al., 2013 Novel estimation method of total flavonoids in mulberry leaves 115 ever, due to the interference of other coexisting substances, such as chlorogenic acid and protocatechuic acid, in medicinal herbs and ex- tracts, the total flavonoids determined by the colorimetric method is higher than the actual value. Furthermore, most flavonoids standards are not commercially supplied, and there are difficulties in mea- suring the content of various flavonoids in mulberry leaves. After oral administration, flavonoid glycosides will be transferred to their aglycones by the action of hydrolytic enzymes and microorganisms. Therefore, the effective components of oral flavonoid glycosides are their aglycones (Liu et al., 2007). Ultrasound-assisted hydroalcohol-acid extraction (UAHAE) was de- veloped in this study to estimate the content of flavonoids in mul- berry leaves and mulberry tea. Quercetin and kaempferol, the major aglycones in mulberry leaves, were quantitatively detected by high performance liquid chromatography with a diode array detector (HPLC-DAD). Then, the total levels of the two aglycones were used to estimate or express the total flavonoids and their bioactivities in biosamples, especially mulberry leaves and tea. In this study, the fla- vonoid contents of 86 varieties of mulberry leaves and 3 species of mulberry leaves tea were examined by UAHAE. Materials and methods Chemicals and materials Different species of mulberry leaves were picked from the Mulberry Garden of Soochow University in May 2016. Then the mulberry leaves were freeze-dried on a lyophilizer (Modulyod, Thermo Elec- tron Corporation, USA). The resulting powder was stored at -4 °C and further used for the following experiments. Commercial tea 1 of mulberry leaves (CT1) and MC792 mulberry leaves were provi- ded from Shandong Sericulture Institute. Commercial teas 2 and 3 of mulberry leaves (CT2 and CT3) were purchased from Hangzhou Qingxin and Anhui Cunyutang Co. Ltd. (China). Rutin and chlo- rogenic acid were purchased from TCI Shanghai Co. Ltd (China). Quercetin, 5, 6-dihydroxycoumarin, isoquercitrin, astragalin and kaempferol standards were purchased from Sigma-Aldrich Co. Ltd. All other chemicals and solvents were of analytical grade, except those used for HPLC analysis, such as acetonitrile and methanol, which were of HPLC grade. To optimize hydrolysis conditions The extraction procedure was measured using Zhao’s method (Zhao et al., 2016) with some modifications. 250 mg of powder was suspended in 10 ml of an ethanol-HCl-water (V/V/V: 7/2/1) solution and kept at 75 °C and 40 kHz (Ultrasonic Bath Jiemeng Instruments, China) for different times for the hydrolysis. For comparison, 250 mg of powder was suspended in 10 ml of an ethanol-water (V/V: 7/3) solution and kept at 75 °C and 40 kHz for one hour. Then, the supernatant was used in the following analysis. Reproducibility analysis A 250-mg sample powder [leaf of mulberry cultivar 308# (shortly MC308), from middle position of the branch] was suspended in 10 ml of an ethanol-HCl-water (V/V/V: 7/2/1) solution and kept at 75 °C and 40 kHz for 1 h. Five repeated extractions of the samples were measured by HPLC-DAD to calculate the relative standard de- viation (RSD). Recovery assay Known levels of quercetin and kaempferol were added to the sample solution. Then, the mixture was extracted. The levels of quercetin and kaempferol were quantitatively detected by HPLC-DAD. The re- coveries of the two aglycones were calculated by the linear equation produced from the standard curves. HPLC-DAD analysis HPLC was performed with a Shimadzu HPLC system (Shimadzu, Japan), which consisted of a pump (LC-20AT), diode array detec- tor (DAD, SPD-M20A), C18 column (Agilent 250 × 4.6 mm) and LC-solution system manager program. The mobile phase was methyl alcohol-water-acetic acid at a ratio of 500/500/0.4 (v/v/v). The flow rate was 1 mL/min, and the eluate absorbance was monitored at 370 nm using a scanning range of 200 nm to 600 nm. The injection volume of the extract was 10 μl. HPLC analytical condition: mobile phase solvent A: acetonitrile, and solvent B: acetic acid / water (4:96, v/v). The flow rate was 1.0 mL/ min. The gradient for separation was 95% solvent B at 0 min, 25% solvent A and 75% solvent B at 40 min, 40% solvent A and 60% solvent B at 50 min, 47% solvent A and 53% solvent B at 60 min, and 60% solvent A and 40% solvent B at 65 min. The eluate absor- bance was monitored at 370 nm using a scanning range of 200 nm to 600 nm. The injection volume of the extract was 10 μL. Determination of various samples Mulberry leaves and tea were collected and immediately turned into powder through freeze-drying. Different leaf positions were collect- ed from Husang 32. The mulberry branches were divided into 1-10 from apical to basal. The extraction method was UAHAE. Assay of total flavonoids by colorimetric method For this section, 0.4 mL of the 70% ethanolic plant extract was mixed with 4.6 ml of ethanol. Then, 0.3 ml of 5% NaNO2 (for 5 min), 0.3 ml of Al(NO3)3 (for 6 min), 4 mL of 4% NaOH and 0.4 mL of 70% etha- nol were added to the mixture solution. The mixture was incubated for 10 min at room temperature. Then, the absorbance was measured at 510 nm (Hitachi UV-3000, Japan). The flavonoid content of the extracts was compared against the rutin standard calibration curve, which was plotted at 0, 0.01, 0.02, 0.04, 0.06 and 0.08 mg/mL. DPPH assay The 70% ethanolic extracts of cultivar Lu-792 and three commercial mulberry teas (CT1, CT2 and CT3) were concentrated in a vacuum and freeze-dried (Thermo, America). The DPPH radical scavenging activities of these extracts were measured using a previously reported method (Zhao et al., 2014). Statistical analysis The data are expressed as the mean ± SD. Comparisons were made using one-way ANOVA with Origin 9.1 software. P< 0.05 was con- sidered statistically significant. Results and discussion Comparison of hydroalcoholic and hydroalcohol-acid extraction High performance liquid chromatography (HPLC) was used for qualitative and quantitative research. The chromatogram showed several peaks; these peaks were identified by comparing the reten- tion times of various standards (Fig. 1). Chlorogenic acid, rutin, iso- quercitrin, and astragalin were identified from hydroalcoholic extrac- tion, but hardly any quercetin of kaempferol was detected. However, high levels of quercetin (52.88 min) and kaempferol (57.50 min) were identified from hydroalcohol-acid extraction, and the levels of ru- 116 J.-G. Zhao, Y.-Q. Zhang tin, isoquercitrin, and astragalin significantly decreased. This result means that flavonoid glycosides transformed to their resulting agly- cones (quercetin and kaempferol). On a C18 reverse-phase column, quercetin and kaempferol showed good retention behaviour. The concentrations and area under the chromatographic peak showed a good linear relationship. The linear regression equations for the two standard samples were as follows: quercetin, y= -149539.64 + 2100898.95x, R2 = 0.9954, and kaemp- ferol, y = -136852.43 + 2674291.47x, R2 = 0.9963. The levels of the two aglycones, quercetin and kaempferol, detected by HPLC-DAD were individually calculated by two linear regression equations in the following experiment. Optimisation of acid hydrolysis Different extraction times (10, 30, 50, 60, and 70 min) were inves- tigated to obtain all of the flavonoid aglycones. The levels of the quercetin and kaempferol aglycones were approximately 6.6 mg/ml and 1.9 mg/ml with the increase in extraction time from 10-70 min (40 kHz ultrasonic). The maximum extraction levels of flavonoid aglycones were 6.91 ± 0.16 mg/mL and 2.06 ± 0.05 mg/mL at 60 min, and the flavonoid concentration decreased with a longer extraction time. At 30 and 50 min, the levels of the quercetin and kaempferol aglycones were also high, but the higher error line indicated that hy- drolysis was not stable. Therefore, the best extraction condition was to suspend 250 mg of the sample powder in 10 mL of an ethanol- HCl-water (V/V/V: 7/2/1) solution and kept at 75 °C and 40 kHz for 60 min. Reproducibility of aglycone extraction by UAHAE-HPLC The mulberry leaf sample was extracted five times; then, these sam- ples were measured by HPLC-DAD (Tab. 2). According to the indi- vidual linear equations, the levels of quercetin and kaempferol were calculated to be an average of 7.12 mg/g and 2.13 mg/g with relative standard diversity (RSD) values of 6.44% and 6.86%, respectively. These data indicate that this method for measuring the contents of quercetin and kaempferol from mulberry leaves has good reprodu- cibility. Recovery of aglycone extraction by the UAHAE method To evaluate the accuracy of this analytical method, a recovery study of the extracts from mulberry leaves was performed by add- ing known levels of quercetin and kaempferol standards. The mean recovery was 105.48% for quercetin and 105.81% for kaempferol (Tab. 3). Recovery percentages higher than 100% could be explained by the interference of the sample matrix. The relative standard de- viation (RSD) determined the accuracy and stability of the methods. The RSD of the average recovery was 2.99% for quercetin and 2.62% for kaempferol. The recovery test showed that the UAHAE method for the hydrolysis and extraction of quercetin and kaempferol from mulberry leaves had very good precision. The content of total flavonoids at different leaf positions The mulberry branches were divided into several parts, from apical to basal (P1-P10). The total flavonoid levels of different mulberry leaves from the same branch also exhibited significant differences. At position 1 (P1), the highest level of total flavonoid content was 13.86 mg/ml (Fig. 2). Then, a significant decrease was observed from P2 to P5 (6.37 mg/g). From P5 to P7, the total flavonoid content in- creased to 9.63 mg/mL, and no significant differences were observed until P10. With the increase in leaf maturity, the general trend of the change in flavonoid content was a decrease at first and then an increase. The same results were found in bilberry leaves. A previous study reported that higher levels and increased expression of flavo- noid pathway genes were observed in bilberry apical leaves exposed to direct sunlight compared to basal leaves that received limited sun- light (jaaKoLa et al., 2004).The increase in the flavonoids from P5 to P7 may be due to synthesis and accumulation in basal leaves (marTZ et al., 2010). The difference between leaf positions may result from Fig. 1: HPLC chromatogram of hydroalcoholic (black line) and hydroalcohol-acid (blue line) extracts. Hydroalcoholic Extraction: extracted by ethanol-water (V/V: 7/3) solution. Hydroalcohol-acid Extraction: extracted by ethanol-HCL-water (V/V/V: 7/2/1) solution Tab. 2: Reproducibility for the determination of the two aglycones by the UAHAE method Aglycones Repeated measurements (mg/g) Means (mg/g) ± SD RSD (%) Quercetin 7.73 7.19 6.96 7.26 6.48 7.12 0.46 6.44 kaempferol 2.35 2.13 2.08 2.16 1.94 2.13 0.15 6.86 Experimental materials: The mulberry leaf variety is MC308. Novel estimation method of total flavonoids in mulberry leaves 117 different accumulation and allocation due to a variety factors that are responsive to the interaction between intrinsic, soil nutrient content and, environmental stimuli, including light, pathogens, temperature (Vagiri et al., 2015). The content of total flavonoids in different mulberry varieties The content of total flavonoids was estimated by the sum of querce- tin and kaempferol. As shown in Tab. 4, the content of total flavo- noids in different mulberry leaves ranged from 2.25-14.33 mg/g, which had a broad range for different varieties of mulberry leaves. This result also suggested that the total flavonoids had significant genotypic differences in different mulberry leaves. The minimum content of total flavonoids was found in LiuYe (2.25 mg/g) of all tested varieties. In the Lu-10 variety, the maximum flavonoid con- tent (14.33 mg/g), was six-fold higher than in LiuYe. The distribution of flavonoids in mulberry leaves also showed a normal distribution (Fig. 3). Most varieties were distributed between 4-10 mg/g, account- ing for 80% of the total determination. Additionally, 16% of mul- berry leaves were higher than 10 mg/g, and several of the highest varieties were LU-9, G60, LU10, indicating that they were potential resources for new product development. Tab. 3: Recoveries for the extraction of the two aglycones by the UAHAE method Aglycones Sample content Dosage Theoretical Measured Recovery RSD (mg/g) (mg) value (mg/g) value (mg/g) (%) (%) Quercetin 7.72 3.87 7.73 8.16 ± 0.24 105.48 ± 3.15 2.99 kaempferol 2.34 1.17 2.34 2.48 ± 0.07 105.81 ± 2.77 2.62 Experimental materials: The mulberry leaf variety is MC308. Fig. 2: The content of total flavonoids at different leaf positions 1-10: from apical to basal of the branch Tab. 4: Total flavonoids in 86 varieties of mulberry leaves Mulberry Total flavonoids Mulberry Total flavonoids Mulberry Total flavonoids Mulberry Total flavonoids cultivars (mg/g) cultivars (mg/g) cultivars (mg/g) cultivars (mg/g) MC206 5.76 T1 8.61 LU-10 14.33 G60 13.71 MC302 11.74 T10 10.68 LU-11 7.86 Boluo 6.18 MC303 8.56 T2 7.48 LU-12 5.19 CanZhuan4 7.53 MC304 7.2 T3 6.84 LU-14 8.3 DuoHu 9.38 MC307 5.39 T4 7.78 LU-2 5.47 FengWeiAnDu 5.45 MC308 9.61 T5 10.91 LU-3 6.61 FuYang 8.83 MC403 6.89 T6 11.78 LU-4 6.44 GaoGan 2.96 MC406 7.12 T7 10.03 LU-5 4.08 Gui 94 5.87 MC842 6.06 T8 9.56 LU-6 7.1 Gui D50 3.98 MC843 5.47 T9 11.31 LU-7 7.26 Gui Y50 11.57 MC3071 5.73 BeiDong2 8.78 LU-8 7.94 HeTian 8.53 D05 10.05 BeiDong 3 8.99 LU-9 12.87 HeiYou 5.99 G04 9.98 BeiDong 7 11.26 NO1 6.88 HongDing 7.04 HongQiao7 6.39 KangHan6 4.65 WoLong 8.73 HongPiMao 8.53 HongPi 7.61 JiuWenLong 7.68 HuSang4 8.77 RiZhou 9 6.86 Bei L6 10.12 GuaPiao 4.38 HuSang7 5.77 Sha2 6.92 LiuYe 2.25 HuSang 1 8.66 HuangBanSang 5.66 SuZhou 4.24 BingWU 8.94 HuSang32 6.38 NanD 7 9.32 Tang10 6.75 BaiPi 5.05 NO3 8.23 NiuJin 5.2 Yu71-1 5.48 WuHai 8.48 NanD1 9.07 Nong 10.51 YuanTouHeYe 4.25 WuLong 5.67 NanD2 5.79 QingPiGuaPiao 5.27 NanD 3 5.38 QingPiWaSang 5.16 NanD 4 4.49 118 J.-G. Zhao, Y.-Q. Zhang Total flavonoids in mulberry tea Mulberry leaf preparations are commonly used in commercial be- verages (especially mulberry tea) and health foods. Because mul- berry leaf tea does not contain tannic acid and caffeine, it cannot cause an increase in blood pressure or nervous excitation. It has be- come a popular functional beverage in some countries, such as China, Japan, and Korea. The quantitative analysis results revealed sub- stantial diversity in the total flavonoid contents in different species of mulberry tea. We measured three commercial teas of mulberry leaves (CT1, CT2, and CT3 (dark colour) ) by the traditional colori- metric method and UAHAE. The total flavonoid content measured by the traditional colorimetric method was 26.14 mg/ml in CT1; however, the total flavonoid content measured by the UAHAE me- thod was 3.15 mg/ml (Fig. 4). It was also found in mulberry tea that the contents measured by the traditional colorimetric method were much higher than those measured by UAHAE. Recently, Guo et al. had doubts about the traditional colorimetric method, which uses rutin as a standard to estimate the total flavonoid levels in bio- samples. Their results showed that non-flavonoids, such as caffeic acid, protocatechuic aldehyde, and chlorogenic acid, had a maximum absorption at 510 nm (guo et al., 2002). Chlorogenic acid (sánCheZ- saLCedo et al., 2015) and protocatechuic acid (Wu et al., 2013) have been identified from mulberry leaves, and these compounds react with NaNO2, Al(NO3)3, and NaOH with a maximum absorption at 507 nm. Anna also found that the procedure in the presence of NaNO2 in alkaline medium seemed to be specific for rutin, luteolin and catechins, but phenolic acids also exhibited considerable absor- bance at 510 nm. (Pękal et al., 2014). These reasons can lead to an increase in the total flavonoid content measured by the colorimetric method. Commercial mulberry leaf CT1 tea is made from Lu-792. The content of total flavonoids in Lu-792 is 8.74 mg/g; however, 3.15 mg/g is observed in CT1. For the Lu-792 ethanolic extract, the IC50 value of the DPPH radical scavenging abilities was 227.45 ug/ ml, which was significantly better than that of CT1 (470.24 μg/ml). Drying is one of the most critical processes in tea manufacturing, but this process may damage the antioxidant and nutritional qualities of the product (Wanyo et al., 2011). Kumar et al. found that a long drying time may cause degradation in food quality. In addition, this method results in undesirable qualities of the product, such as a dark colour and decreased antioxidant activity (praVeen Kumar et al., 2005). Flavonoids have been demonstrated to have good antioxidant activity (parhiZ et al., 2015). To evaluate the relationship between the flavo- noid content measured by the UAHAE method and antioxidant acti- vity, tea extracts were used to analyse the DPPH scavenging property. As shown in Fig. 4, the CT2 extract showed the maximum content (4.76 mg/g), while CT3 had the lowest content (2.13 mg/g). The IC50 values of the DPPH radical scavenging abilities of each mulberry tea extract were 470.24 μg/ml, 338.44 μg/ml and 651.39 μg/ml. The total flavonoid content of CT2 (59.84 mg/g) measured by the tradi- tional colorimetric method was higher than Lu-792 (54.45 mg/g), but the content measured by UAHAE was reversed in the two samples. The IC50 values of DPPH radical scavenging abilities of Lu-792 (227.45 μg/ml) were lower than CT2 (338.44 μg/ml), which means that UAHAE more accurately reflects the actual flavonoid content. The radical scavenging activities were similar to the distribution of total flavonoids measured by UAHAE in three types of mulberry tea. Conclusion Mulberry leaves have become a health product, especially mulberry tea, in some countries, such as China, Japan, and Korea. The es- tablished UAHAE method is efficient for flavonoid extraction and quantification in mulberry leaves and tea. The optimal hydrolysis of flavonoid glycosides and extraction of the resulting aglycones occur in an ethanol-HCl-water (7/2/1, V/V/V) solution at 75 °C (40 kHz ul- trasonic) over 1 h. The validated method is precise and stable. In this study, mulberry leaves of 86 varieties and mulberry leaf tea of 3 spe- cies were examined for their flavonoid content. These findings will provide a basis for better industrial-scale manufacture of mulberry tea and for a wide range of similar food products. Compliance with Ethical Standards Funding The authors gratefully acknowledge the earmarked fund (CARS-18-ZJ0502) for China Agriculture Research System (CARS) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, P. R. China. Conflict in Interests Jin-Ge Zhao declares that he has no conflict of interest. Yu-Qing Zhang declares that he has no conflict of interest. References Chang, y.C., yang, m.y., Chen, s.C., Wang, C.J., 2016: Mulberry leaf polyphenol extract improves obesity by inducing adipocyte apoptosis Fig. 3: Distribution diagram for the total flavonoid content in mulberry leaves Fig. 4: Determination of total flavonoids by the colorimetric method and UAHAE in mulberry tea extracts and their DPPH radical scaveng- ing activities. 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Zhao, j.g., yan, Q.Q., Xue, r.y., Zhang, j., Zhang, y.Q., 2014: Isolation and identification of colourless caffeoyl compounds in purple sweet potato by HPLC-DAD–ESI/MS and their antioxidant activities. Food Chem. 161, 22-26. Address of the author: Jin-Ge Zhao, Yu-Qing Zhang, Silk Biotechnology Laboratory, School of Bio- logy and Basic Medical Sciences, Soochow University; RM702-2303, No. 199, Renai Road, Dushuhu Higher Edu. Town, Suzhou 215123; PR China E-mail: sericult@suda.edu.cn © The Author(s) 2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative- commons.org/licenses/by-sa/4.0/).