Effects of drying and Eurotium Cristatum fermentation on the content and α-glucosidase inhibitory activity of flavonoid in mulberry leaves

: Developing new α-glucosidase inhibitors from mulberry leaves (ML) for the treatment of type II diabetes has significant advantages. Although content and activity of active substances is relatively higher in fresh ML (FML), it is not conducive to long-term storage and transportation. This article investigated the effects of drying and E. cristatum fermentation on the content and activity of flavonoids in mulberry leaves (MLF). The results indicated that hot-air drying (HD) was more beneficial for ML drying than natural air drying (AD). The flavonoid content in ML that dried at 60°C for 140 min (ML-HD60) was 36.98±2.8 mg/g dried ML just slightly lower than that in FML (39.29 ± 2.17 mg/g dried ML). Then different MLs were used as the substrate of E. cristatum YY-1, which indicated that the content and α-glucosidase inhibition rate of flavonoids extracted from the fermented ML-HD60 (MLF-HD60F) were increased obviously. The MLF-HD60F content was increased to 39.35±2.47 mg/g dried ML, which was comparable to that in unfermented FML. Meanwhile, fermentation by E. cristatum YY-1 would decrease the IC50 value of MLFs against α-glucosidase. And MLF-HD60F had the lowest IC50 value of 9.1 mg/L, which was 11.5 mg/L for FMLF-F. In conclusion, dried ML at 60°C for 140 min could maximize the content of flavonoids and their inhibition activity on α-glucosidase.


Introduction
Mulberry leaf (Mori Folium, ML) is the leaf of Morus alba L., a plant of the genus Morus L. in the family Moraceae.With the development of modern monitoring and analytic techniques, as well as the continuous progress of in vivo and ex vivo experimental methods, a variety of chemical components have been found in ML, such as flavonoids, alkaloids, polysaccharides, phenolic acids, coumarins, amino acids, vitamins, lipids, and minerals [1] .These chemical components not only give ML various efficiency, for example antioxidant, hypolipidemic, myocardial oxygen consumption reducing, hypoglycemic, anti-tumor, immunity improving, and other pharmacological effects, but also has the advantages of mild, long-lasting, and less toxic side effects [2] .In particular, ML are rich in 1deoxynojirimycin, a potent α-glucosidase inhibitor unique to the mulberry plants.Therefore, ML has attracted much attention in the treatment of diabetes mellitus [3,4] .
In recent years, diabetes mellitus has become the world's 3rd most common chronic disease that seriously threatens human health after cancer and cardiovascular diseases.According to the data provided by the International Diabetes Federation, the number of diabetic patients in the world were more than 460 million in 2019, which is expected to increase to 700 million by 2045 [5] .Therefore, the prevention and treatment of diabetes mellitus and its complications have become a major and difficult issue for the global medical community [6] .Among diabetes patients, the number of Type II diabetes patients accounts for more than 90%.Clinically, α-Glucosidase inhibitors are the first-choice hypoglycemic agents for type II diabetes mellitus, and adjuvant drugs for type I diabetes mellitus [7] .However, the major αglucosidase inhibitors currently used clinically, namely acarbose, voglibose, and miglitol, are often accompanied by various side effects [8] .Many researchers have begun to investigate novel α-glucosidase inhibitors with mild or no adverse effects.Flavonoids, polysaccharides, alkaloids, and other active ingredients in ML have all been confirmed with significant α-glucosidase inhibitory activity [9][10][11] .Therefore, the development of novel αglucosidase inhibitors from ML for the treatment of type II diabetes mellitus has significant advantages.
However, the extraction of natural α-glucosidase inhibitors from plants usually suffers from low content, low extraction efficiency and low inhibitory activity.Fermentation is one of the effective method to solve the above problems.At present, microbial fermentation technology has been widely used in ML.For example, Guo et al. [12] found that Monascus anka, Aspergillus niger and Aspergillus oryzae could increase the content of quercetin and kaempferol in fermented mulberry leaves.Total alkaloids and total flavonoids contents in the ML fermented by Lactobacillus plantarum CICC 21801 were increased by 26.53% and 29.56%, respectively, compared to unfermented ML [13] .Eurotium cristatum is a natural antibiotic with an extremely strong inhibitory effect.It has low nutritional requirements and can grow on different plant substrates [14] .At present, E.cristatum has shown outstanding advantages in the fermentation of some plant raw materials and has been applied in the fermentation of ML.Ma et al. [15] found that contents of flavonoids and polysaccharides in the mulberry leaves black tea that fermented by E. cristatum were higher than that of the original tea.Yang et al. [16] found that after been solid-state fermented by E. cristatum, the content of theabrownine in ML tea was increased and the α-glucosidase inhibition efficiency was significantly improved.Zheng et al. [17] reported that the contents of polysaccharide, flavonoid and polyphenol in the E. cristatum fermented mulberry leaves tea were decreased by 76.1%, 12% and 7.1%, respectively.However, the DNJ content in the fermented ML tea was increased by about three times of that in unfermented ML tea.
Currently, the fermentation of ML mainly focuses on its dried products.However, as for edible and medicinal homologous plants, fresh stuffs have superior efficacy, because the drying process usually causes qualitative and quantitative changes in the active ingredients [18] .For example, the total alkaloids content in fresh and dried amaranth was 0.948 mg/g and 0.657 mg/g, respectively, while the total flavonoids content in fresh amaranth was 15.55 mg/g, which decreased to 10.75 mg/g after drying [19]   .Wu et al. [20] found that drying at 40°C and 60°C for 240 min would decrease the concentration of flavonoids in ML by 1.372% and 1.740%, respectively.Liang et al. [21] reported that dried ML by hot air could improve the protein digestibility and solubility of ML, but it could also cause the degradation or denaturation of protein.However, dried ML by vacuum freeze-drying method would cause less demage of the protein structure and better preserve the protein properties.On the other hand, theoretically fresh stocks have more complex substances, especially volatile and antibiotic stuffs, which make the fermentaiton process greatly different from dried stocks.It is also known that different drying methods have remarkable differences in the content and activity of active substances in ML.For example, natural drying was favorable to increase the content of polyphenols and antioxidant activity, while drying after fermentation was favorable to increase the content of DNJ and flavonoids [22] .Therefore, in this paper, the ML dried by different drying method or parameters were used as the substrate of Eurotium cristatum.Then the effect of E. cristatum fermentation and drying on the content and α-glucosidase inhibitory activity of ML were investigated.

Materials
Fresh mulberry leaves (FML) were purchased from Shangluo City, Shaanxi Province.FML, free of pests and diseases, and without damage were selected, was cleaned and removed the surface water, and stored at 4℃ for use.Rutin standard, α-glucosidase, and 4-nitrophenyl-α-D-glucopyranoside (pNPG) were purchased from Yuanye Bio-Technology Co., Ltd.(Shanghai, China).4hydroxypiperidinol, saturated Reisner's salt solution, Dglucose, hydrochloric acid, acetone and other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China).Unless otherwise noted, all the reagents above are analytically pure.
The wet basis moisture content (M w , g/g) and dry basis moisture content (M d , g/g) of ML under different drying parameters were calculated according to formula (1) and (2), respectively.
Where, m t is the sample mass at moment t, g; m w is the initial wet mass of sample, g; m 0 is the oven dry weight of sample.

Preparation of inoculum E. Cristatum YY-1
E.cristatum YY-1 was kindly provided by the Institute of Process Engineering, Chinese Academy of Sciences (Beijng, China).Inoculated spores of E.cristatum YY-1 into potato-glucose-broth (PDB) medium, and cultured at 28℃ for 3 d to obtain the inoculum of E. cristatum YY-1.

The fermentation E. Cristatum YY-1 with different ML as substrates
Fresh ML (FML) and dried ML (DML) were prepared as the substrates of E. cristatum YY-1, respectively.The fermentation was conducted as described below.
Corn flour and bran were added to distilled water according to the solid-liquid ratio of 1:4, stirred well and sterilized at 121°C for 15 min.The fresh ML was crushed into slurry, the ratio of wet weigh of fresh ML (g) to the volume of distilled water (ml) was 1:3.The DML was pulverized to 40 mesh.20 g FML (wet weight, the moisture content of ML was 80%) were mixed with 5 g of sterilized mixture of corn flour and bran as described above, and stirred well to obtain FML substrate.Weighed 4 g dried ML, added with sterilized water at the solid-toliquid ratio of 1:3.Then added 5 g of sterilized corn flour and bran mixture, stirred evenly to obtain DML substrate.Inoculated 5 ml E. cristatum YY-1 inoculum into above ML substrate, stirred evenly and statically cultivated in an incubator at 28-30°C for 7d.Three parallel experiments were designed for each group.

Extraction and analysis of flavonoid in mulberry leaves
The mulberry leaves soaked in 70% ethanol solution and ultrasonically extracted at 70 kHz and 50°C for 30 min.The supernatant was collected and recorded as flavonoid extract of ML (MLF).The flavonoid content was determined by NaNO 2 -Al (NO 3 ) 3 -NaOH colorimetric method [23] .Rutin was used as the standard, and the absorbance was measured at 510 nm with a UV-Vis spectrophotometer (754PC, Jinghua Technology Instrument Co., Ltd., Shanghai, China).The standard curved line was plotted by taking the absorbance (A) as the vertical coordinate and the concentration of the control (C) as the horizontal coordinate, respectively.The contents of MLF in the samples were calculated according to the standard curved line.

Determination of α-glucosidase inhibitory activity
The determination of α-glucosidase inhibitory activity was conducted as describe by Kwon et al. [24] with slight modifications.The experiment was completed on a 96microplate reader (MB-96B, Suzhou Chenghuai Technology Co., Ltd.).Four groups were set up, blank group (B), control group (C), sample blank group (SB) and sample group (S).The blank group contained 80 μl PBS buffer.The control group consisted of 70 μl of PBS buffer and 10 μl of α-glucosidase solution.The sample blank group contained 60 μl PBS buffer and 20 μl inhibitor, and the sample group contained 50 μl PBS buffer, 20 μl inhibitor, and 10 μl α-glucosidase solution.All the groups were activated for 15 min at 37 °C.Then added 20 μl of pNPG and reacted for 30 min at 37 °C.The reaction was terminated with 1 mol/L sodium carbonate solution.The absorbance of each group was measured at 405 nm.And the α-glucosidase inhibition rate of different inhibitors was calculated according to formula (3).
Inhibitory rate of α-glucosidase (%) = (1 − ) Where, A B , A C , A S and A SB are the absorbance of the blank group, control group, sample group, and sample blank group, respectively.

Statistical analysis
In this paper, the experimental data was expressed as mean ± standard deviation and processed using Excel and Origin 8.0 software packages.Use one-way analysis of variance (ANOVA) with a probability level (P)≤0.05 to determine statistically significant differences between the three replicates.

Drying of mulberry leaves under different drying parameters
As shown in Fig. 1, the dehydration rate of ML at different drying temperatures was in the trend of decreasing rapidly at first and then slowly.When dried the ML at 40℃, the moisture content was rapidly decreased within 0-80 min, but tend to slow decreasing within 80-240 min.By dried for 240 min, the dry basis moisture content in ML was decreased to 0.74%.When drying at 50℃, the moisture content in ML initially decreased rapidly, but the decrease rate turned to slowly decrease after been dried for 80 min.The dry moisture content in ML was decreased to 0.44% after been dried for 180 min.For drying at 60°C, ML had a rapid drying phase from within the first 60 min, and the dry basis moisture content of ML would decrease to 0.27% at 140 min.For 70 °C drying, the moisture content in ML decreased rapidly to 12.24% within 30 min and to 0.43% within 60 min.In conclusion, drying at 40°C for 240 min, drying at 50°C for 180 min, drying at 60°C for 140 min, and drying at 70°C for 60 min were determined as the suitable parameters of drying ML to constant weight.

The variation of ML flavonoids content in drying and fermentation process
As seen in Fig. 2, drying had a significant effect on the total flavonoids (TF) content in ML.The TF content in FML was 39.29±2.17mg/g dried ML, which would be decreased in a different degree when dried at different parameters.Among the DML, the highest TF content of 36.98±2.8mg/g dried ML was obtained when dried the ML at 60°C for 140 min.The TF content in the DML that dried at lower temperatures (air drying and dried at 40°C and 50°C) had a pretty higher decrease than that dried at higher temperatures.This may be due to the fact that the enzymes present in ML still kept high catalytic activity at lower temperatures, which results in degradation or conversion of flavonoids, while, the enzymes would be denatured at high temperatures.However, when drying at 70°C for 1 h, the TF content in ML would decrease again, since flavonoids was a kind of heat-sensitive substance.
Therefore, 60°C for 140 min was the most suitable condition for the drying of ML.This result was different from that of Qin et al. [25] who reported that dried ML at 75°C for 60 min would result in the highest content of active substances, including TF, DNJ, and polysaccharide.FML and ML-AD are fresh mulberry leaves and air dried mulberry leaves, respectively.ML-HD40, ML-HD50, ML-HD60 and ML-HD70 are mulberry leaves dried by hot-air at 40℃, 50℃, 60℃, and 70℃, respectively.
E.Cristatum has been commonly used for the fermentation of mulberry leaves.In this paper, E.Cristatum YY-1 was used to investigate the effect of fermentation on the flavonoids content of mulberry leaves that dried by different conditions (Fig. 2).The results showed that the TF content in the extract of fermented FML and DML that dried below 60°C were reduced to different degrees.The decrease of TF content in FML was the most significant.At the end of fermentation, the TF content in the fermented FML (FMLF-F) was 30.33 ± 1.04 mg/g dried ML, which decreased by 22.8% compared to that in the unfermented FMLF.As for DML substrate, the TF content in the fermented ML-AD40 (MLF-AD40F) and fermented ML-AD50 (MLF-AD50F) were decreased to varying degrees, but increased in that of hightemperature dried The TF content of fermented ML-HD60 (MLF-AD60F) was 39.35 ± 2.47 mg/g dried ML, which was the highest among all fermented MLs and increased by 6.41% compared to that in the unfermented ML-HD60.This result was also related to the enzymes contained in ML.Considering the antibacterial properties of ML and E. Cristatum YY-1 themselves, the ML substrate was directly inoculated with E. Cristatum YY-1 and cultivated at 30°C.As a result, the microorganisms and enzymes inherent in ML might continue to play the biocatalytic effects during the fermentation process.In summary, dried at 60°C for 140 min and then fermented by E. Cristatum YY-1 was most beneficial for the retention of flavonoids in mulberry leaves.The TF content in fermented ML-HD60 was comparable to that in unfermented MLF, and was more conducive to preservation and transportation.

Effect of drying conditions on the αglucosidase inhibition activity of MLF
The α-glucosidase inhibition rate of flavonoids extracted from MLs (MLF) dried by different conditions was showed in Fig. 3.The results indicated that MLF-HD60 exhibited the highest inhibition rate of α-glucosidase.However, the maximum α-glucosidase inhibition rate of the flavonoids that extracted from other DMLs were all lower than that from FML, whereas flavonoids extracted from ML-AD (MLF-AD) showed the lowest αglucosidase inhibition rate.This might be due to the continuous catalytic reaction of enzymes and endophytic microbes in MLs at room temperature, consequently affect the α-glucosidase inhibitory activity of flavonoids.MLF-F and MLF-AD are flavonoids extracted from the fresh mulberry leaves and air dried mulberry leaves, respectively.MLF-HD40, MLF-HD50, MLF-HD60 and MLF-HD70 are flavonoids extracted from the mulberry leaves dried by hot-air at 40℃, 50℃, 60℃, and 70℃, respectively.
Further fitting on the α-glucosidase inhibition rate was performed by using different MLF concentrations, which indicated that all the fitting equations had high correlation coefficients (≥0.99) (Table 1).Calculated the IC50 value of different MLF based on the fitted equations.The results showed that the IC50 value of MLF-HD60 was 11.26 mg/L, which was the lowest value of all the tested MLF, followed by FMLF.While, ML-ADF had the highest IC50 value of 21.57mg/L.Interestingly, flavonoids extracted from lower temperature dried ML (MLF-HD40 and MLF-HD50) showed rather lower α-glucosidase inhibition rates than that extracted from ML dried at higher temperatures (MLF-HD70).It was suggested that high temperature would rapidly cause the activity loss of enzymes that can degrade flavonoids, thus better preserving the αglucosidase inhibition activity of MLF.

Effect of fermentation on the α-glucosidase inhibition activity of MLF
E. Cristatum YY-1 fermentation was carried out using ML as substrate, and the effect on α-glucosidase inhibition rate of MLF were investigated (Fig. 4).At lower MLF concentration (9~9.5 mg/L), the flavonoids extracted from fermented ML dried at 50-70°C showed higher αglucosidase inhibition rate.The α-glucosidase inhibition rates of 9.5 mg/L MLF-HD60F, 9 mg/L MLF-ADF and 9.3 mg/L FMLF-F were 52.2%, 27.0%, and 42.5%, respectively.The α-glucosidase inhibition rate of MLF-ADF that extracted from fermented ML-AD still was the lowest.Compared with low-temperature dried ML, flavonoids extracted from ML dried by high temperature and short time would result in higher α-glucosidase inhibitory rate.In particular, the MLF-HD60F showed the highest inhibition rate in all concentration ranges.As shown in Table 1, the IC50 value of MLF-HD60F against α-glucosidase was 9.1 mg/L, which was 19.84 mg/L for MLF-ADF.MLF-HD50F had a comparable IC50 value with FMLF-F.Compared with unfermented ML, the IC50 values of flavonoids extracted from the fermented DMLs was significantly decreased, especially when dried at 50-70°C.Therefore, drying ML with hot air at 60°C for 140 min would maximize the content of flavonoids and their inhibition activity on α-glucosidase.Moreover, it was most favorable for E.Cristatum YY-1 fermentation, which would not only result in high flavonoids content but also high α-glucosidase inhibition activity.FMLF-F and MLF-ADF are flavonoids extracted from the fermented fresh mulberry leaves and air dried mulberry leaves, respectively.MLF-HD40F, MLF-HD50F, MLF-HD60F, and MLF-HD70F are flavonoids extracted from the mulberry leaves that first hot-dried at 40℃, 50℃, 60℃, and 70℃, respectively and then fermented by E.Cristatum YY-1.

Conclusion
This article studied the effects of drying parameters and E.Cristatum fermentation on the content of flavonoids in hypoglycemic plant mulberry leaves, as well as its glucosidase inhibitory activity.The following conclusions were obtained: (1) High-temperature drying is more conducive to the preservation of flavonoids in mulberry leaves, but the temperature and time of drying should be controlled.The content of flavonoids in mulberry leave that hot air dried at 60 ℃ for 140 min was comparable to that in fresh mulberry leaves.
(2) After been fermented by E.Cristatum, the flavonoids content in fresh mulberry leaves and lowtemperature dried mulberry leaves were decreased obviously, especially fresh mulberry leaves, but in mulberry leaves dried at 60℃ and 70℃ were increased.
(3) The fermentation of E.Cristatum increased the inhibition efficiency of flavonoids extracts from mulberry leaves against α-glucosidase.The mulberry leaves dried at 60°C for 140 min was the most favorable substrate for E.Cristatum, which resulted in the highest flavoloids content and α-glucosidase inhibitory rate.
In conclusion, E.Cristatum fermentation combined with suitable drying conditions could bring the total flavonoids content and its α-glucosidase inhibition activity of dried mulberry leaves to a level comparable to that of fresh mulberry leaves, thus facilitating the preservation and transportation of mulberry leaves.

Fig. 1
Fig.1 Changes in moisture content of mulberry leaves at different drying temperatures (dried base moisture)

Fig. 2
Fig.2 Differences in total flavonoids content in the extracts of mulberry leaves dried under different parameters.

Fig. 3
Fig.3The effect of drying parameters on α-glucosidase inhibition rate of flavonoids extracts from mulberry leaves.

Fig. 4
Fig.4 The Effect of Eurotium Cristatum Fermentation on the α-Glucosidase Inhibition rate of total flavonoids that extracted from different Mulberry Leaves.

Table 1
Kinetics analysis of α-glucosidase inhibited by different inhibitors