Mulberry leaf extract quality evaluation method and application thereof

Abstract

The invention discloses a mulberry leaf extract quality evaluation method and application thereof, and the method comprises the following steps: (1) preparation of a mulberry leaf reference extract, (2) establishment of a total flavone determination method based on the mulberry leaf reference extract, (3) establishment of a 1-DNJ content determination method based on the mulberry leaf reference extract, and (4) establishment of a mulberry leaf reference extract fingerprint spectrum and chromatographic peak identification. And (5) establishing a mulberry leaf extract quality evaluation method through combination of multi-component content determination and alpha-glucosidase inhibitory activity, and performing quality evaluation on mulberry leaf extracts sold in the market by different manufacturers. The method provided by the invention can realize comprehensive monitoring of the content and functional activity of the main active components 1-DNJ and flavonoid compounds in the mulberry leaf extract. The method not only can objectively and accurately evaluate the authenticity and quality of the mulberry leaf extract, but also can solve the problem that the efficacy activity of the mulberry leaf extract cannot be objectively and reasonably evaluated due to different preparation modes of mulberry leaf extracts from different manufacturers.

Description

Technical Field

[0001] The present invention relates to the technical field of quality detection of traditional Chinese medicine extracts, and specifically to a quality evaluation method and application of mulberry leaf extract. Background Art

[0002] Mulberry leaf is the dried leaf of the mulberry plant Marus alba L. in the Moraceae family. It tastes sweet, bitter, and cold, and belongs to the lung and liver meridians. Mulberry leaf has high medicinal value and has the effects of dispersing wind-heat, moistening the lung and relieving dryness, and clearing the liver and improving eyesight. Mulberry leaf contains a variety of bioactive components, such as alkaloids, flavonoids, polysaccharides, and polyphenols. Among them, alkaloid compounds (1-deoxynojirimycin, 1-DNJ) and their derivatives can play a role in reducing blood sugar by inhibiting the activity of α-glucosidase, and are being increasingly widely used in the development of various hypoglycemic products. Today, when related products of mulberry leaf extract have been recognized in the domestic and foreign traditional Chinese medicine and big health fields and have obtained a large market demand, the quality evaluation method of mulberry leaf extract is crucial. At present, in the quality standard of mulberry leaf in the Chinese Pharmacopoeia (2020 Edition), only the content of rutin is determined, and this method cannot fully reflect the accurate content of its main active ingredient 1-DNJ. At the same time, 1-DNJ not only exists in mulberry leaves, but also in the branches, leaves and roots of mulberry trees, as well as in the larvae and pupae of silkworms, etc., all contain this unique natural active ingredient.

[0003] However, at present, the quality of mulberry leaf extract products on the market is uneven, and most of them have a series of problems such as excessive dilution of excipients, adulteration with mulberry branches and mulberry bark, and process compliance. And the flavonoid compounds in mulberry leaf not only have various effects such as lowering blood pressure, reducing blood lipid, inhibiting fat accumulation and thrombus formation, but also are the most studied and have the most clearly defined chemical structure in mulberry leaf, and can be used to identify the authenticity of the raw materials of suppliers. Therefore, establishing a method that can simultaneously realize the semi-quantitative comparative analysis of the fingerprint of flavonoid components and the content determination of 1-DNJ can identify the authenticity of commercially available mulberry leaf extract and a series of problems such as whether there is dilution and weight gain of excipients in commercially available mulberry leaf extract or the use of non-compliant production processes such as alcohol extraction to increase the content of components.

[0004] In addition, for mulberry leaf extract, due to the differences in the extraction method and the final drying method of the extract, the natural active substances in mulberry leaf are affected by heat during the extraction and drying processes, resulting in losses or reductions. Conventional physical and chemical index detections, such as selecting a certain or certain types of chemical components representing natural active substances for determination, cannot intuitively make a quantitative evaluation of the changes in the activity of natural active substances themselves. Therefore, in order to better control the quality and efficacy of mulberry leaf extract, developing a combined method for simultaneously analyzing multiple active components and hypoglycemic efficacy evaluation in mulberry leaf extract has practical significance for the quality control of mulberry leaf extract. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies existing in the prior art, and provides a method for quality evaluation of mulberry leaf extract and its application, so as to achieve comprehensive monitoring of the contents and efficacy activities of the main active components 1-DNJ and flavonoid compounds in the mulberry leaf extract. This method is simple to operate and has good stability. It can objectively and accurately evaluate the authenticity, quality and superiority of the mulberry leaf extract, and can also solve the problem that it is impossible to objectively and reasonably evaluate the efficacy activity of the mulberry leaf extract due to different preparation methods of the mulberry leaf extract by different manufacturers, which is of great significance for controlling the quality of the mulberry leaf extract and ensuring its efficacy. The present invention is specifically realized through the following technical solutions: S1 Preparation of the mulberry leaf reference extract: Weigh the mulberry leaf control medicinal material and the mulberry leaf decoction pieces that meet the pharmacopoeia regulations, soak them in an appropriate amount of pure water, and then carry out two reflux extractions. Combine the extraction solutions, rotary evaporate and concentrate, freeze-dry, and pulverize and mix evenly to obtain the mulberry leaf reference extract.

[0006] S2 Establishment of the determination method for total flavonoids in the mulberry leaf reference extract: The determination of total flavonoids in the mulberry leaf extract adopts the ultraviolet spectrophotometry method. Using rutin as the reference substance, the content of total flavonoids in the mulberry leaf extract is determined at 510 nm by the sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method.

[0007] S3 Establishment of the determination method for the content of 1-DNJ in the mulberry leaf reference extract: Use FMOC-Cl to derivatize 1-DNJ in the mulberry leaf extract. Accurately inject the standard curve solution and the test sample solution into the liquid chromatograph, collect the liquid chromatogram at 254 nm, and calculate the peak area of the 1-DNJ derivative peak in all the chromatograms of the mulberry leaf reference extract according to the unified integration method. Take the concentration of the standard curve as the abscissa and the peak area of the 1-DNJ derivative as the ordinate, and linearly fit to obtain the peak area-concentration formula. According to the fitted peak area-concentration formula, calculate the concentration of 1-DNJ in the test sample solution, and calculate the content of 1-DNJ in the corresponding extract. Calculate the average content of 1-DNJ in the extract according to the determination results of parallel samples.

[0008] S4 Establishment of the fingerprint spectrum of the mulberry leaf reference extract and identification of chromatographic peaks: Using octadecylsilyl silica gel as the filler, pure methanol and 0.1% phosphoric acid solution as the mobile phase, adopt the gradient elution method, and the detection wavelength is 350 nm. Establish the HPLC fingerprint spectrum of the mulberry leaf reference extract and conduct a methodological investigation on this method. Subsequently, 6 components, namely neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, rutin and astragalin, are identified in the fingerprint spectrum of the mulberry leaf reference extract.

[0009] Establish a quality evaluation method for mulberry leaf extract by multi-component content determination of S5 combined with α-glucosidase inhibitory activity evaluation and evaluate the quality of commercially available mulberry leaf extracts from different manufacturers: Select fingerprint similarity, total flavonoids, 1-DNJ content and the contents of 6 index components as the main indicators for the quality evaluation of mulberry leaf extract. Among them, it meets the following criteria: fingerprint similarity > 0.9, the content ranges of total flavonoids and 1-DNJ are 8.30% ± 3.67% and 0 - 1.43% respectively, the proportion ranges of the 6 index components after conversion are ±30% of the benchmark after conversion, and the α-glucosidase inhibition rate is 71.98% ± 31.70% for first-class products; if it does not meet the above criteria, but fingerprint similarity > 0.9, the content ranges of total flavonoids and 1-DNJ are 8.30% ± 3.67% and 0 - 1.43% respectively, the proportion ranges of the 6 index components after conversion are within ±30% of the benchmark after conversion, and the α-glucosidase inhibition rate is 71.98% ± 31.70%, and any one or two of them are second-class products; if it does not meet the above criteria, but fingerprint similarity > 0.9, the content ranges of total flavonoids and 1-DNJ are 8.30% ± 3.67% and 0 - 1.43 respectively, the proportion ranges of the 6 index components after conversion are within ±30% of the benchmark after conversion, and the α-glucosidase inhibition rate is 71.98% ± 31.70%, and any three of them are third-class products; Prepare test solution of commercially available mulberry leaf extracts in multiple batches, and determine the contents of total flavonoids and 1-DNJ in the test solution of commercially available mulberry leaf extracts in multiple batches according to the detection methods of total flavonoids and 1-DNJ established based on the benchmark mulberry leaf extract. Then, accurately pipette the test solution and inject it into the liquid chromatograph for fingerprint determination; Import the fingerprints of 16 batches of commercially available mulberry leaf extracts (Y1 - Y16) into the similarity calculation software, perform peak matching with the average fingerprint of 3 batches of benchmark mulberry leaf extracts (S1 - S3), and calculate the similarity. Subsequently, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, rutin, and astragalin in commercially available mulberry leaf extracts from different manufacturers were identified. Use the average value of self-made mulberry leaf water extract as the benchmark for reference (black frame in the radar chart), and select 8 representative components for evaluation. Among them, total flavonoids and 1-DNJ are converted by proportion according to the content determination results, and the other 6 common peaks in the fingerprint are converted by relative peak area. The content differences of total flavonoids, 1-DNJ and the other 6 components in commercially available mulberry leaf extracts are visually displayed by radar charts for comparative study of mulberry leaf extracts from different manufacturers. Subsequently, use the in vitro activity screening model of α-glucosidase inhibitor to evaluate the α-glucosidase inhibitory activity of commercially available mulberry leaf extracts in multiple batches. Dissolve the mulberry leaf extract in phosphate buffer (pH 7.4), add α-glucosidase solution and substrate, and use an enzyme-labeling instrument to detect and calculate the activity of the extract against α-glycosidase inhibition.

[0010] Preferably, the mulberry leaf decoction pieces in step S1 should meet the pharmacopoeia standards. The specific method for preparing the reference extract of mulberry leaves is as follows: The reference medicinal materials of mulberry leaves and the mulberry leaf decoction pieces that meet the pharmacopoeia regulations are soaked in 10 times the amount of pure water and extracted by heating under reflux twice. Soak for 0.5 h before the first heating under reflux, and heat under reflux for 1 h each time. Combine the extraction solutions, filter through a 200-mesh filter cloth, concentrate, freeze-dry, pulverize, and mix evenly to obtain the product.

[0011] Preferably, the method for determining the total flavonoids in the reference extract of mulberry leaves in step S2 is as follows: The freeze-dried powder of the reference extract of mulberry leaves is fully mixed evenly. Weigh 1.0 g accurately and place it in a 125 mL conical flask. Add 50 mL of methanol accurately, stopper tightly, weigh, and ultrasonicate for 30 min until completely dissolved or dispersed (without lumps of insoluble matter). Let it cool, weigh again, make up the lost weight with methanol, and shake well. Transfer the solution to a 50 mL centrifuge tube, centrifuge (4000 r / min) for 5 min, accurately pipette 25 mL of the supernatant, place it in a 50 mL volumetric flask, dilute to the mark with pure water, and shake well. Accurately pipette an appropriate amount (adjusted according to the sample concentration, about 1 - 5 mL) and place it in a 25 mL volumetric flask, add 50% methanol to 6 mL, add 1 mL of 5% sodium nitrite solution, shake well, let stand for 6 min, then add 1 mL of 10% aluminum nitrate solution, shake well, let stand for 6 min, and finally add 10 mL of 4% sodium hydroxide test solution, make up the volume to the mark with 50% methanol, shake well, and let stand for 15 min. Take another 125 mL conical flask, and prepare it in the same way as the test solution preparation method except without adding the test sample.

[0012] Preferably, the method for determining the 1-DNJ content in the reference extract of mulberry leaves in step S3 is as follows: Pipette 200 μL of the test solution of the reference extract of mulberry leaves into a 5 mL centrifuge tube, successively add 350 μL of 0.4 mol / L potassium borate buffer solution (pH 8.5) and 500 μL of 5 mmol / L derivatization reagent FMOC-Cl acetonitrile solution, stopper tightly, vortex for 30 s to mix well, keep warm in a 25 °C constant temperature water bath for 25 min to fully react, then add 200 μL of 0.1 moL / L glycine solution to neutralize the excessive FMOC-Cl, react for 20 min, then add 150 μL of 1% acetic acid aqueous solution by volume and 600 μL of deionized water, mix well, let stand for 2 h, filter through a 0.45 μm filter membrane to obtain the derivatized sample solution.

[0013] Preferably, in step S3, the high performance liquid chromatography method is adopted in the determination method of 1-DNJ content in the mulberry leaf reference extract. An XSelect HSS C18 (4.6×250mm, 5μm) chromatographic column is used, with acetonitrile (mobile phase A) and 0.1% acetic acid / water (mobile phase B) as the mobile phases. The flow rate is 1.0 mL / min, and the gradient elution program is as follows: 0-18 min, mobile phase A: 32%-32%, mobile phase B: 68%-68%; 18-19 min, mobile phase A: 32%-90%, mobile phase B: 68%-10%; 19-25 min, mobile phase A: 90%-90%, mobile phase B: 10%-10%. The detection wavelength is 254 nm; the column temperature is 40 °C; the injection volume is 20 μL.

[0014] Preferably, in step S4, a Waters Tnature C18 chromatographic column (4.6 mm×250 mm, 5μm) is selected for establishing the HPLC characteristic fingerprint of the mulberry leaf reference extract; methanol is used as mobile phase A, and 0.1% phosphoric acid aqueous solution is used as mobile phase B; the flow rate is 1.0 mL / min; gradient elution is adopted, and the gradient elution program is as follows: 0-14 min, mobile phase A: 15%-70%, mobile phase B changes from 85%-30%; 14-35 min, mobile phase A: 30%-55%, mobile phase B: 70%-45%, 35-50 min, mobile phase A: 45%-40%, mobile phase B: 55%-60%. The detection wavelength is 350 nm; the column temperature is 30 °C; the injection volume is 10 μL.

[0015] Preferably, in step S5, the preparation method of the test solution for the quality evaluation of commercially available mulberry leaf extracts from different manufacturers in the determination of multi-component content is to accurately weigh 1.25 g of commercially available mulberry leaf extracts from different manufacturers, place them in a 25 mL volumetric flask, add 15 mL of 20% ethanol solution by volume ratio, shake for 2 min, perform ultrasonic treatment for 30 min, let it cool, add 20% ethanol solution by volume ratio to the scale, shake well, and filter through a microporous filter membrane to obtain the test sample.

[0016] Preferably, in step S5, the α-glucosidase inhibitory activity of commercially available mulberry leaf extracts from different manufacturers is evaluated on a 96-well plate. The sample determination group (including α-glucosidase solution + mulberry leaf extract solution + substrate solution), background group (including buffer solution + substrate solution, buffer solution + mulberry leaf extract solution), blank control group (including α-glucosidase solution + buffer solution + substrate solution), and positive control group (including α-glucosidase solution + acarbose solution + substrate solution) are set respectively.

[0017] Preferably, for the evaluation of the α-glucosidase inhibitory activity of commercially available mulberry leaf extracts from different manufacturers in step S5, each group was first placed in a constant temperature incubator at 37°C for pre-incubation for 5 min before the formal experiment. After that, except for the background group, 20 μL of the substrate solution was added to each of the remaining groups, and then each group was placed in a constant temperature incubator at 37°C for incubation for 15 min. After taking out, 60 μL of NaHCO3 solution was added to each group to terminate the reaction.

[0018] Preferably, for the evaluation of the α-glucosidase inhibitory activity of commercially available mulberry leaf extracts from different manufacturers in step S5, after the reaction was terminated, the OD value was measured at a wavelength of 400 nm using an enzyme-labeled instrument, and the inhibitory effect of the mulberry leaf extract on α-glucosidase was calculated. The calculation formula is: In the formula, ODblank represents the OD value of the blank control group; ODreaction represents the OD value of the sample measurement group; ODbackground represents the larger OD value of the first and second groups of the background group.

[0019] Advantages of the present invention: 1. Compared with the traditional method, the present invention realizes multi-index quality control, can accurately reflect the product quality of mulberry leaf extracts, and provides more basis for the construction of the internal control quality standard of mulberry leaf extracts and the screening of supplier raw materials; 2. The present invention combines the quantitative technology of index components with an in vitro efficacy evaluation model, and products that meet both the content requirements of index components and the activity requirements are confirmed as qualified products, which is of great significance for controlling the product quality consistency among products of different manufacturers and batches. Description of the drawings

[0020] Figure 1 HPLC fingerprint of 3 batches of mulberry leaf reference extracts in Example 1; Figure 2 Chromatographic peak identification of mulberry leaf reference extracts in Example 1; Figure 3 Fingerprint and chromatographic peak identification results of commercially available mulberry leaf extracts from 5 different manufacturers in Example 2; Figure 4 Radar chart of the analysis of 8 components of commercially available mulberry leaf extracts from 5 different manufacturers in Example 2; Figure 5 Fingerprint and chromatographic peak identification results of commercially available mulberry leaf extracts from 5 different manufacturers in Example 3; Figure 6 Radar chart of the analysis of 8 components of commercially available mulberry leaf extracts from 5 different manufacturers in Example 3; Figure 7 Fingerprint and chromatographic peak identification results of commercially available mulberry leaf extracts from 5 different manufacturers in Example 4; Figure 8Radar chart of the analysis of 8 components of commercially available mulberry leaf extracts from 5 different manufacturers in Example 4; Figure 9 Fingerprint spectrum and chromatographic peak identification results of the mulberry leaf extract from manufacturer Y16 in Example 5; Figure 10 Radar chart of the analysis of 8 components of the mulberry leaf extract from manufacturer Y16 in Example 5. Specific implementation manners

[0021] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are only for exemplarily illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention. Please refer to Figures 1-10 : Example 1 1. Establishment of the quality evaluation system for the reference mulberry leaf extract 1.1 Preparation of the reference mulberry leaf extract: Weigh 1 batch of reference medicinal materials (S1) and 2 batches of mulberry leaf decoctions (S2 - S3) of 100 g each into a round-bottom flask, add 1 L of pure water, soak for 30 min, reflux for 1 h, filter the extract through a 200-mesh filter cloth, add 1 L of pure water to the filter residue for secondary reflux extraction for 1 h, filter the extract through a 200-mesh filter cloth, combine it with the first extract, rotary evaporate and concentrate to about 200 mL, freeze-dry, pulverize and mix evenly to obtain 3 batches of reference mulberry leaf extracts (S1 - S3).

[0022] 1.2 Establishment of the determination method for the total flavonoids in the reference mulberry leaf extract 1.2.1 Preparation of the reference stock solution Precisely weigh rutin reference substance, dissolve and dilute it with 50% methanol to a concentration of about 0.2 mg / mL.

[0023] 1.2.2 Preparation of the standard curve solutions Precisely measure 0 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL of the reference stock solution respectively, place them in 25-mL volumetric flasks, make up to 6 mL with 50% methanol respectively, add 1 mL of 5% sodium nitrite solution to each, shake well, let stand for 6 min, then add 1 mL of 10% aluminum nitrate solution, shake well, let stand for 6 min, finally add 10 mL of 4% sodium hydroxide test solution, make up to the mark with 50% methanol, shake well, and let stand for 15 min.

[0024] 1.2.3 Determination of the standard curve According to the ultraviolet-visible spectrophotometry, measure the absorbance of each standard curve solution at a wavelength of 500 nm, take the absorbance as the ordinate and the rutin concentration as the abscissa to draw the standard curve.

[0025] 1.2.4 Preparation of the Test Solution for the Determination of Total Flavonoids in the Standard Extract of Mulberry Leaves Mix the freeze-dried powder of the standard extract of mulberry leaves evenly. Weigh accurately 1.0 g and place it in a 125 mL conical flask. Add precisely 50 mL of methanol, stopper tightly, weigh, sonicate for 30 min until completely dissolved or dispersed (no lumps of insoluble matter), let it cool, weigh again, make up the lost weight with methanol, and shake well. Transfer the solution to a 50 mL centrifuge tube, centrifuge (4000 r / min) for 5 min, accurately pipette 25 mL of the supernatant into a 50 mL volumetric flask, dilute to the mark with pure water, and shake well. Accurately pipette an appropriate amount (adjusted according to the sample concentration, about 1 - 5 mL) into a 25 mL volumetric flask, add 50% methanol to 6 mL, add 1 mL of 5% sodium nitrite solution, shake well, let stand for 6 min, then add 1 mL of 10% aluminum nitrate solution, shake well, let stand for 6 min, and finally add 10 mL of 4% sodium hydroxide test solution, make up to the mark with 50% methanol, shake well, and let stand for 15 min. Take another 125 mL conical flask, and prepare it in the same way as the test solution preparation method except without adding the test sample.

[0026] 1.2.5 Determination and Calculation of the Absorbance of the Test Solution According to the ultraviolet-visible spectrophotometry, measure the absorbance at a wavelength of 500 nm, substitute it into the standard curve to calculate the concentration of each test sample, and calculate the content. The results are shown in Table 1. 1.3 Establishment of the Method for Determining the Content of 1-DNJ in the Standard Extract of Mulberry Leaves 1.3.1 Preparation of the Test Solution for the Determination of the Content of 1-DNJ in the Standard Extract of Mulberry Leaves Mix the freeze-dried powder of the standard extract of mulberry leaves evenly. Weigh accurately about 0.2 g and transfer it to a 50 mL volumetric flask (2 parallel samples for each batch), dissolve with 0.01 mol / L hydrochloric acid solution and dilute to the mark, shake well, centrifuge and take the supernatant to obtain the test sample solution (if the actual content of 1-DNJ is relatively low, the concentration of the test sample should be adjusted to ensure that the liquid phase measurement value is within the linear range of the standard curve). Pipette 200 μL of the test sample solution into a 5 mL centrifuge tube, successively add 350 μL of 0.4 mol / L potassium borate buffer solution (pH 8.5) and 500 μL of 5 mmol / L derivatization reagent FMOC-Cl acetonitrile solution, stopper tightly, vortex for 30 s to mix well, incubate in a constant temperature water bath at 25 °C for 25 min to fully react, then add 200 μL of 0.1 mol / L glycine solution to neutralize the excessive FMOC-Cl, react for 20 min, then add 150 μL of 1% acetic acid aqueous solution by volume and 600 μL of deionized water, mix well, let stand for 2 h, and filter through a 0.45 μm filter membrane to obtain the derivatized sample solution.

[0027] 1.3.2 Preparation of 1-DNJ Standard Stock Solution (1.0 mg / mL) Accurately weigh 10 mg of 1-DNJ reference substance, place it in a 10 mL volumetric flask, dissolve it with 0.01 mol / L hydrochloric acid solution and dilute to the mark, shake well for standby.

[0028] 1.3.3 Preparation of 1-DNJ Standard Curve Solution Accurately pipette 0.8 mL, 0.6 mL, 0.4 mL, 0.2 mL, and 0.1 mL of 1-DNJ standard stock solution (1.0 mg / mL) into 10 mL volumetric flasks respectively, dilute to the mark with 0.01 mol / L hydrochloric acid aqueous solution, and shake well. Take 200 μL of each of the above solutions and place them in 5 mL centrifuge tubes. Then, successively add 350 μL of 0.4 mol / L potassium borate buffer solution (pH 8.5) and 500 μL of 5 mmol / L derivatization reagent FMOC-Cl acetonitrile solution. Seal tightly, vortex for 30 s to mix well, keep warm in a constant temperature water bath at 25 °C for 25 min to fully react. Then add 200 μL of glycine solution (0.1 mol / L) to neutralize the excess FMOC-Cl, react for 20 min, add 150 μL of 1% acetic acid aqueous solution by volume fraction and 600 μL of deionized water, mix well, let stand for 2 h, and filter through a 0.45 μm filter membrane to obtain a series of derivatized standard curve solutions.

[0029] 1.3.4 Determination of 1-DNJ Content in Mulberry Leaf Reference Extract by HPLC Use an XSelect HSS C18 (4.6×250 mm, 5 μm) chromatographic column, with acetonitrile (mobile phase A) and 0.1% acetic acid / water (mobile phase B) as the mobile phases, the flow rate is 1.0 mL / min, and the gradient elution program is as follows: 0 - 18 min, mobile phase A: 32% - 32%, mobile phase B: 68% - 68%; 18 - 19 min, mobile phase A: 32% - 90%, mobile phase B: 68% - 10%; 19 - 25 min, mobile phase A: 90% - 90%, mobile phase B: 10% - 10%. The detection wavelength is 254 nm; the column temperature is 40 °C; the injection volume is 20 μL.

[0030] Take the concentration of the standard curve as the abscissa and the peak area of the 1-DNJ derivative as the ordinate, and perform linear fitting for each to obtain the peak area-concentration formula (the fitting correlation coefficient should be ≥0.999).

[0031] Content calculation: According to the fitted peak area-concentration formula, calculate the 1-DNJ concentration of each test solution, and calculate the 1-DNJ content in the corresponding extract. Calculate the average content of 1-DNJ in the extract according to the determination results of parallel samples (the relative average deviation of parallel samples should be ≤2.0%). The results are shown in Table 2. 1.4 Establishment of Fingerprint of Mulberry Leaf Reference Extract and Identification of Chromatographic Peaks 1.4.1 Preparation of Test Solution for Determination of Fingerprint of Mulberry Leaf Reference Extract Precisely weigh 1.25 g of the freeze-dried powder of mulberry leaf reference extract, place it in a 25 mL volumetric flask, add about 20 mL of ethanol with a volume ratio of 20%, shake for 2 min, perform ultrasonic treatment (power 160 W, frequency 40 Hz) for 30 min, cool to room temperature, add 20% ethanol to the scale, shake well, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0032] 1.4.2 Preparation of Reference Substance Solution Precisely weigh an appropriate amount of rutin reference substance, dissolve it in methanol to prepare a solution containing 0.1 mg per 1 mL, shake well, and use it as the reference substance solution.

[0033] 1.4.3 Establishment of Flavonoid Fingerprint of Mulberry Leaf Reference Extract and Methodology Investigation The chromatographic conditions are as follows: Waters Tnature C18 chromatographic column (4.6 mm × 250 mm, 5 μm); methanol as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B; flow rate is 1.0 mL / min; gradient elution is adopted, and the gradient elution program is: 0 - 14 min, mobile phase A: 15% - 70%, mobile phase B from 85% - 30%; 14 - 35 min, mobile phase A: 30% - 55%, mobile phase B: 70% - 45%; 35 - 50 min, mobile phase A: 45% - 40%, mobile phase B: 55% - 60%. The detection wavelength is 350 nm; the column temperature is 30 °C.

[0034] Detect 3 batches (S1 - S3) of mulberry leaf reference extract under the established chromatographic conditions, calculate the theoretical plate number based on the rutin peak, and the results are all above 8000. Therefore, the theoretical plate number calculated based on the rutin peak should not be less than 8000.

[0035] Take 10 μL each of the reference substance solution and the test solution, inject them into the liquid chromatograph, record the chromatographic peaks, record the chromatogram, and use the peak at the corresponding position of the retention time of the rutin peak in the reference substance solution as the reference peak.

[0036] Methodology Investigation (1)Stability Experiment Precisely aspirate 10 μL of the same sample solution each time, repeat the determination every 4 hours for a total of 6 times. The RSDs of the relative retention times and peak area ratios of the common peaks are all less than 2% (Tables 3 and 4); use similarity calculation software (the "Operating Specifications of the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (Version 2004A)" recommended by the National Pharmacopoeia Commission, the same below) to calculate the similarity. The similarities of all fingerprint spectra are 1.000, meeting the technical requirements for chromatographic fingerprint spectrum research. The relative retention times of the common peaks in the stability test are shown in Table 3, and the relative peak areas of the common peaks in the stability test are shown in Table 4. (2)Precision test Precisely aspirate 10 μL of the same sample solution each time, inject the sample continuously for 6 times respectively. The RSDs of the relative retention times and peak area ratios of the common peaks are all less than 2% (Tables 5 and 6); use similarity calculation software to calculate the similarity. The similarities of all fingerprint spectra are 1.000, meeting the technical requirements for chromatographic fingerprint spectrum research. The relative retention times of the common peaks in the precision test are shown in Table 5, and the relative peak areas of the common peaks in the precision test are shown in Table 6. (3)Reproducibility test Prepare 6 test solution samples from the same sample, inject the samples for determination respectively. The RSDs of the relative retention times and peak area ratios of the common peaks are all less than 2% (see Tables 7 and 8); use similarity calculation software to calculate the similarity. The similarities of all fingerprint spectra are 1.000, meeting the technical requirements for chromatographic fingerprint spectrum research. 1.4.4 Establishment of the fingerprint spectrum of the reference extract of mulberry leaves and the results of chromatographic peak identification According to the determination results of 3 batches of reference extract samples of mulberry leaves, all the chromatographic peaks in the fingerprint spectra of each batch of samples elute within 45 minutes. For the fingerprint spectrum research of flavonoid components in the water extract of mulberry leaves, the peaks within 5 minutes of chromatographic retention time are generally solvent and impurity peaks. Therefore, the chromatographic peaks before 5 minutes of retention time are deducted when selecting common peaks. Combining the chromatograms of 3 batches of samples, after 5 minutes, there are 11 peaks that are common to 10 batches of samples. Among them, the 8th peak is the rutin peak. The proportion of the total area of the 11 peaks in each batch of samples to the total peak area is greater than 90%. Therefore, these 11 peaks are determined as common fingerprint peaks, and the analysis results are as Figure 1As shown below. Subsequently, the chromatographic peaks in the fingerprint were identified according to the reference substance. Among them, peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 4 is caffeic acid, peak 8 is rutin peak, and peak 10 is astragalin. The results of chromatographic peak identification are as Figure 2 shown below.

[0037] 1.5 Evaluation of the inhibitory activity of the reference mulberry leaf extract on α-glucosidase Four large groups were set up in a 96-well plate: the sample group, the background group (including 2 subgroups), the blank reference substance group, and the positive control group. Each subgroup had 3 parallel wells. In the sample determination group, 20 μL of α-glucosidase solution and 20 μL of mulberry leaf extract solution were added; in subgroup 1 of the background group, 40 μL of PBS was added, and in subgroup 2, 20 μL of PBS and 20 μL of mulberry leaf extract solution were added; in the blank control group, 20 μL of α-glucosidase solution and 20 μL of PBS were added; in the positive control group, 20 μL of α-glucosidase solution and 20 mL of acarbose solution were added. Except for subgroup 2 of the background group which added 20 mL of PBS, 20 μL of substrate solution was added to the rest of the groups, and then each group was placed in an incubator at 37 °C for 15 min. After taking out, 60 μL of NaHCO3 solution was added to each to terminate the reaction. The OD values of each solution were measured at a wavelength of 400 nm using an enzyme-labeling instrument, and the inhibitory effect of the mulberry leaf extract on α-glucosidase was calculated. The calculation formula is: Inhibitory rate of α-glucosidase activity (%) = (ODblank - (ODreaction - ODbackground)) / ODblank × 100% In the formula, ODblank represents the OD value of the blank control group; ODreaction represents the OD value of the sample determination group / positive control group; ODbackground represents the larger OD value of subgroup 2 of the background group; ODbackground1 represents the OD value of subgroup 1 of the background group. The results are shown in Table 9. 1.6 Quality evaluation system of mulberry leaf extract Select fingerprint similarity, total flavonoids, 1-DNJ percentage content (%), 6 index components and α-glucosidase inhibition rate as the quality evaluation indexes of mulberry leaf extract. Among them, it meets the following criteria: fingerprint similarity > 0.9, the content ranges of total flavonoids and 1-DNJ are 8.30% ± 3.67% and 0 - 1.43% respectively, the proportion range of 6 index components after conversion is ±30% of the benchmark after conversion, and the α-glucosidase inhibition rate is 71.98% ± 31.70% for first-class products; if it does not meet the above criteria but fingerprint similarity > 0.9, the content ranges of total flavonoids and 1-DNJ are 8.30% ± 3.67% and 0 - 1.43% respectively, the proportion range of 6 index components after conversion is within ±30% of the benchmark after conversion, and the α-glucosidase inhibition rate is 71.98% ± 31.70%, and one or two of them meet the requirements for second-class products; if it does not meet the above criteria but fingerprint similarity > 0.9, the content ranges of total flavonoids and 1-DNJ are 8.30% ± 3.67% and 0 - 1.43 respectively, the proportion range of 6 index components after conversion is within ±30% of the benchmark after conversion, and the α-glucosidase inhibition rate is 71.98% ± 31.70%, and any three of them meet the requirements for third-class products; as shown in Table 10 specifically. Example 2 1. Quality evaluation of commercially available mulberry leaf extracts from 5 different manufacturers 1.1 Collection of commercially available mulberry leaf extracts from different manufacturers Collect commercially available mulberry leaf extracts (Y1 - Y5) from 5 different manufacturers respectively 1.2 Determination of total flavonoids 1.2.1 Preparation of reference substance stock solution Same as 1.2.1 in Example 1 1.2.2 Preparation of standard curve solution Same as 1.2.2 in Example 1 1.2.3 Determination of standard curve Same as 1.2.3 in Example 1 1.2.4 Preparation of test solution of commercially available mulberry leaf extracts from 5 different manufacturers Mix the samples of commercially available mulberry leaf extracts from each manufacturer evenly, and prepare the test solutions of commercially available mulberry leaf extracts from different manufacturers with reference to 1.2.4 in Example 1 1.2.5 Determination and calculation of absorbance of test solution Same as 1.2.5 in Example 1, and the results of commercially available mulberry leaf extracts from 5 different manufacturers are shown in Table 11. 1.3 Determination of 1-DNJ content in commercially available mulberry leaf extracts from 5 different manufacturers 1.3.1 Preparation of test solution Mix the commercially available mulberry leaf extract samples from different manufacturers, and refer to 1.3.1 in Example 1 to prepare the test solution for the determination of 1-DNJ content in the commercially available mulberry leaf extracts from different manufacturers. 1.3.2 Preparation of 1-DNJ standard stock solution (1.0 mg / mL) Refer to 1.3.2 in Example 1.

[0038] 1.3.3 Preparation of 1-DNJ standard curve solution Refer to 1.3.3 in Example 1.

[0039] 1.3.4 Determination of 1-DNJ by HPLC Refer to 1.3.4 in Example 1. The determination results of 1-DNJ content in the commercially available mulberry leaf extracts from 5 different manufacturers are shown in Table 12. 1.4 Study on the fingerprint of flavonoid components in the commercially available mulberry leaf extracts from 5 different manufacturers It is intended to reflect the authenticity of the samples to a certain extent by comparing the similarity of the fingerprints and chromatographic peaks between the commercially available mulberry leaf extracts from different manufacturers and the reference mulberry leaf extract.

[0040] 1.4.1 Preparation of test solution for the commercially available mulberry leaf extracts from 5 different manufacturers Mix 5 batches of commercially available mulberry leaf extract samples from different manufacturers respectively. Weigh 1.25 g precisely and transfer it to a 25 mL volumetric flask. Add about 15 mL of 20% ethanol, shake, and ultrasonicate for 30 min until completely dissolved or dispersed. After cooling to room temperature, make up the volume with 20% ethanol, centrifuge, and take the supernatant to filter through a 0.22 μm filter membrane to obtain the solution.

[0041] 1.4.2 Preparation of reference substance solution Refer to 1.4.2 in Example 1.

[0042] 1.4.3 Determination of the fingerprint and similarity of flavonoids in the commercially available mulberry leaf extracts from 5 different manufacturers The chromatographic conditions refer to 1.4.3 in Example 1. After preparing the test solutions from 5 batches of commercially available mulberry leaf extracts (Y1 - Y5) and injecting them for high-performance liquid chromatography detection, import the fingerprints of the above 5 batches of commercially available mulberry leaf extracts into the similarity calculation software, perform peak matching with the average fingerprint of the fingerprints of 3 batches of mulberry leaf water extracts, and calculate the similarity. The similarity results are shown in Table 13. At the same time, identify the flavonoid compounds in the fingerprints of different commercially available mulberry leaf extracts. It is found that neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, rutin peaks and astragalin generally exist in different commercially available mulberry leaf extracts. The chromatographic peak identification results are as Figure 3 shown. 1.5 Radar chart analysis of mulberry leaf extracts sold by 5 different manufacturers In order to more intuitively reflect the quality differences of mulberry leaf extracts from different manufacturers, this study used radar charts to compare and evaluate the quality of mulberry leaf extracts. The average values ​​of three batches of homemade mulberry leaf benchmark water extracts were used as the benchmark for reference (black box in the radar chart), and 8 representative components were selected for evaluation. Among them, total flavonoids and 1-DNJ were proportionally converted using the content determination results, and the other 6 common peaks in the quantitative fingerprint were proportionally converted using relative peak areas. The content differences of total flavonoids, 1-DNJ and the other 6 components in commercially available mulberry leaf extracts were uniformly displayed using radar charts. The radar charts for the content comparison of the 8 components are shown in the figure. Figure 4 .

[0043] 1.6 Determination of the inhibitory activity of mulberry leaf extracts from five different manufacturers on α-glucosidase 1.6.1 Preparation of relevant solutions: (1) Accurately pipette 20 μL of 50 U / mL α-glucosidase solution and add 1980 μL of phosphate buffer (pH 7.4) to dilute it into 0.5 U / mL α-glucosidase solution; (2) Accurately weigh 3.01 mg of 4-nitrophenol-α-D-pyranoglucoside (PNPG), add 2 mL of phosphate buffer (pH 7.4) and dissolve it to 5 mmol / L to obtain the substrate solution; (3) Accurately weigh 3.23 mg of acarbose and add 1 mL of phosphate buffer (pH 7.4) to dissolve it to 1.5 mmol / L to obtain the acarbose solution; (4) Accurately weigh 1 mg of commercially available mulberry leaf extract and add 1 mL of phosphate buffer (pH 7.4) to dissolve it at 1 mg / mL to obtain a mulberry leaf extract solution; (5) Accurately weigh 0.42 g of NaHCO3 solid and add 5 mL of phosphate buffer (pH 7.4) to dissolve it to 1 mol / L to obtain NaHCO3 solution.

[0044] 1.6.2 Grouping of the inhibitory activity test of mulberry leaf extracts from 5 different manufacturers on α-glucosidase: Four large groups were set up on a 96-well plate: sample group, background group (including 2 groups), blank control group and positive control group, with 3 parallel wells in each group. The sample test group was added with 20 μL of α-glucosidase solution and 20 μL of mulberry leaf extract solution; background group 1 was added with 40 μL of PBS, and group 2 was added with 20 μL of PBS and 20 μL of mulberry leaf extract solution; the blank control group was added with 20 μL of α-glucosidase solution and 20 μL of PBS; the positive control group was added with 20 μL of α-glucosidase solution and 20 mL of acarbose solution.

[0045] 1.6.3 Inhibition Activity Test Experiment of Commercially Available Mulberry Leaf Extracts from 5 Different Manufacturers against α-Glucosidase: Except for the 2 groups in the background group that added 20 mL of PBS, each of the other groups added 20 μL of the substrate solution. Then, each group was placed in an incubator at 37 °C for 15 min. After taking them out, 60 μL of NaHCO3 solution was added to each group to terminate the reaction. The OD values of each solution were measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 400 nm, and the inhibitory effect of the mulberry leaf extract on α-glucosidase was calculated. The calculation formula is as follows: In the formula, ODblank represents the OD value of the blank control group; ODreaction represents the OD value of the sample determination group / positive control group; ODbackground represents the larger OD value of the 2 groups in the background group; ODbackground1 represents the OD value of the 1st group in the background group. The results are shown in Table 14. 1.7 Quality Evaluation Results of Commercially Available Mulberry Leaf Extracts from 5 Different Manufacturers It can be clearly seen from the radar chart that there is a large gap not only between the market extracts and the water extracts prepared from qualified decoction pieces in the laboratory, but also significant differences in the composition and content among different manufacturers. The fingerprint spectra of the samples from these 5 manufacturers showed an obvious chromatographic peak at a retention time of 13.2 min, which was different from the fingerprint spectrum of the mulberry leaf reference extract. After literature comparison, it was found that this peak was mulicandin A, proving that the mulberry leaf extracts from these manufacturers all contained mulberry bark and mulberry twig adulteration. In addition, according to Figure 4 (Radar chart) The results showed that the 1-DNJ content of manufacturers Y2, Y3, and Y4 was much higher than that of the mulberry leaf reference extract. At the same time, the results of the inhibition activity test of α-glucosidase showed that the hypoglycemic activity of these three manufacturers was quite strong, and the inhibition rate of α-glucosidase could reach more than 70%. 1-DNJ is the main active ingredient that inhibits α-glucosidase, and 1-DNJ not only exists in mulberry leaves, but also in the branches, leaves, and roots of mulberry trees, as well as in the larvae and pupae of silkworms. This indicates that the reason for the strong hypoglycemic activity of the mulberry leaf extracts from these manufacturers may be due to the adulteration of mulberry twigs and mulberry bark. Example 3 1. Quality Evaluation of Commercially Available Mulberry Leaf Extracts from 5 Different Manufacturers 1.1 Collection of Commercially Available Mulberry Leaf Extracts from Different Manufacturers Commercially available mulberry leaf extracts from 5 different manufacturers (Y6 - Y10) were collected respectively 1.2 Determination of Total Flavonoids 1.2.1 Preparation of Reference Stock Solution Same as 1.2.1 in Example 1 1.2.2 Preparation of Standard Curve Solution Same as 1.2.2 in Example 1 1.2.3 Determination of Standard Curve Same as 1.2.3 in Example 1 1.2.4 Preparation of Test Solutions of Mulberry Leaf Extracts Commercially Available from 5 Different Manufacturers Mix the mulberry leaf extract samples commercially available from each manufacturer evenly, and prepare the test solutions of mulberry leaf extracts commercially available from different manufacturers with reference to 1.2.4 in Example 1 1.2.5 Determination and Calculation of Absorbance of Test Solutions Same as 1.2.5 in Example 1. The results of mulberry leaf extracts commercially available from 5 different manufacturers are shown in Table 16 1.3 Determination of 1 - DNJ Content in Mulberry Leaf Extracts Commercially Available from 5 Different Manufacturers 1.3.1 Preparation of Test Solutions Mix the mulberry leaf extract samples commercially available from different manufacturers evenly, and prepare the test solutions for the determination of 1 - DNJ content in mulberry leaf extracts commercially available from different manufacturers with reference to 1.3.1 in Example 1 1.3.2 Preparation of 1 - DNJ Standard Stock Solution (1.0 mg / mL) With reference to 1.3.2 in Example 1

[0046] 1.3.3 Preparation of 1 - DNJ Standard Curve Solution With reference to 1.3.3 in Example 1

[0047] 1.3.4 Determination of 1 - DNJ by HPLC With reference to 1.3.4 in Example 1. The determination results of 1 - DNJ content in mulberry leaf extracts commercially available from 5 different manufacturers are shown in Table 17 1.4 Study on Fingerprint of Flavonoid Components in Mulberry Leaf Extracts Commercially Available from 5 Different Manufacturers 1.4.1 Preparation of Test Solutions of Mulberry Leaf Extracts Commercially Available from 5 Different Manufacturers With reference to 1.4.1 in Example 1 1.4.2 Preparation of Reference Solution With reference to 1.4.2 in Example 1

[0048] 1.4.3 Fingerprint and Similarity Determination of Flavonoids in Mulberry Leaf Extracts Commercially Available from 5 Different Manufacturers The chromatographic conditions refer to 1.4.3 in Example 1. Five batches of commercially available mulberry leaf extracts (Y6 - Y10) were prepared into test solutions and then injected for high - performance liquid chromatography detection. The fingerprint spectra of the above five batches of commercially available mulberry leaf extracts were imported into the similarity calculation software, and peak matching was carried out with the average spectrum of the fingerprint spectra of three batches of water - extracted mulberry leaf extracts, and the similarity was calculated. The similarity results are shown in Table 18. At the same time, flavonoids in the fingerprint spectra of different commercially available mulberry leaf extracts were identified. It was found that neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, rutin peaks and astragalin were commonly present in different commercially available mulberry leaf extracts. The chromatographic peak identification results are as Figure 5 shown. 1.5 Radar chart analysis of commercially available mulberry leaf extracts from 5 different manufacturers In order to more intuitively reflect the quality differences of mulberry leaf extracts from different manufacturers, this study used a radar chart to compare and evaluate the quality of mulberry leaf extracts. The average value of three batches of self - made reference water - extracted mulberry leaf extracts was used as a reference (the black frame in the radar chart), and 8 representative components were selected for evaluation. Among them, the total flavonoids and 1 - DNJ were proportionally converted using the content determination results, and the other 6 common peaks in the quantitative fingerprint spectrum were proportionally converted using the relative peak area. The content differences of total flavonoids, 1 - DNJ and the other 6 components in commercially available mulberry leaf extracts were uniformly and vividly displayed using a radar chart. The content comparison radar chart of 8 components is shown in Figure 6 .

[0049] 1.6 Determination of the inhibitory activity of commercially available mulberry leaf extracts from 5 different manufacturers on α - glucosidase Referring to 1.6.1 - 1.6.3 in Control Example 1 and substituting into the formula, the results of the determination of the inhibitory activity of commercially available mulberry leaf extracts from 5 different manufacturers on α - glucosidase are shown in Table 19. 1.7 Quality evaluation results of commercially available mulberry leaf extracts from 5 different manufacturers It can be clearly seen from the radar chart that the content of representative components in the mulberry leaf extracts of these 5 manufacturers is extremely low. It is speculated that in order to reduce the unit price of the products, these manufacturers used less mulberry leaves or a large amount of excipients for dilution. Among them, almost no representative components of mulberry leaves were detected in the products of manufacturer Y8, and there are major problems with the authenticity of its products. In addition, the radar chart shows that the content of 1 - DNJ in the mulberry leaf extracts of these 5 manufacturers is much less than that of the reference mulberry leaf extract. At the same time, the results of the inhibitory rate of commercially available mulberry leaf extracts from 5 manufacturers on α - glucosidase show that the inhibitory activity of the mulberry leaf extracts of these manufacturers on α - glucosidase is quite low (the inhibitory rate is less than 50%), proving that their hypoglycemic activity is poor, the content of active ingredients is low, and the product quality is poor. Example 4 1. Quality Evaluation of Commercially Available Mulberry Leaf Extracts from 5 Different Manufacturers 1.1 Collection of Commercially Available Mulberry Leaf Extracts from Different Manufacturers Commercially available mulberry leaf extracts from 5 different manufacturers (Y11 - Y15) were collected respectively. 1.2 Determination of Total Flavonoids 1.2.1 Preparation of Stock Solution of Reference Substance Same as 1.2.1 in Example 1 1.2.2 Preparation of Standard Curve Solution Same as 1.2.2 in Example 1 1.2.3 Determination of Standard Curve Same as 1.2.3 in Example 1 1.2.4 Preparation of Test Solution of Commercially Available Mulberry Leaf Extracts from 5 Different Manufacturers The samples of commercially available mulberry leaf extracts from each manufacturer were mixed evenly, and the test solutions of commercially available mulberry leaf extracts from different manufacturers were prepared with reference to 1.2.4 in Example 1. 1.2.5 Determination and Calculation of Absorbance of Test Solution Same as 1.2.5 in Example 1. The results of commercially available mulberry leaf extracts from 5 different manufacturers are shown in Table 21. 1.3 Determination of 1 - DNJ Content in Commercially Available Mulberry Leaf Extracts from 5 Different Manufacturers 1.3.1 Preparation of Test Solution The samples of commercially available mulberry leaf extracts from different manufacturers were mixed evenly, and the test solutions for the determination of 1 - DNJ content in commercially available mulberry leaf extracts from different manufacturers were prepared with reference to 1.3.1 in Example 1. 1.3.2 Preparation of 1 - DNJ Standard Stock Solution (1.0 mg / mL) Refer to 1.3.2 in Example 1.

[0050] 1.3.3 Preparation of 1 - DNJ Standard Curve Solution Refer to 1.3.3 in Example 1.

[0051] 1.3.4 Determination of 1 - DNJ by HPLC Refer to 1.3.4 in Example 1. The results of the determination of 1 - DNJ content in commercially available mulberry leaf extracts from 5 different manufacturers are shown in Table 22. 1.4 Study on Fingerprint of Flavonoid Components in Commercially Available Mulberry Leaf Extracts from 5 Different Manufacturers 1.4.1 Preparation of Test Solution of Commercially Available Mulberry Leaf Extracts from 5 Different Manufacturers Refer to 1.4.1 in Example 1.

[0052] 1.4.2 Preparation of reference substance solution Refer to 1.4.2 in Example 1.

[0053] 1.4.3 Fingerprint and similarity determination of flavonoids in commercially available mulberry leaf extracts from 5 different manufacturers The chromatographic conditions refer to 1.4.3 in Example 1. Five batches (Y11 - Y15) of commercially available mulberry leaf extracts were prepared into test solution and then injected for high - performance liquid chromatography detection. The fingerprint spectra of the above 5 batches of commercially available mulberry leaf extracts were imported into the similarity calculation software, and peak matching was carried out with the average spectrum of the fingerprint spectra of 3 batches of water - extracted mulberry leaf extracts, and the similarity was calculated. The similarity results are shown in Table 23. At the same time, the flavonoid compounds in the fingerprint spectra of different commercially available mulberry leaf extracts were identified. It was found that neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, rutin peak and astragalin generally existed in different commercially available mulberry leaf extracts. The chromatographic peak identification results are as Figure 7 shown. 1.5 Radar chart analysis of commercially available mulberry leaf extracts from 5 different manufacturers In order to more intuitively reflect the quality differences of mulberry leaf extracts from different manufacturers, this study used a radar chart to compare and evaluate the quality of mulberry leaf extracts. The average value of 3 batches of self - made reference water - extracted mulberry leaf extracts was used as a reference (black frame in the radar chart), and 8 representative components were selected for evaluation. Among them, total flavonoids and 1 - DNJ were proportionally converted using the content determination results, and the other 6 common peaks in the quantitative fingerprint spectrum were proportionally converted using the relative peak area. The content differences of total flavonoids, 1 - DNJ and the other 6 components in commercially available mulberry leaf extracts were visually displayed by a radar chart. The content comparison radar chart of 8 components is shown in Figure 8 .

[0054] 1.6 Inhibitory activity determination of commercially available mulberry leaf extracts from 5 different manufacturers against α - glucosidase Refer to 1.6.1 - 1.6.3 in Control Example 1, substitute into the formula. The results of the inhibitory activity determination of commercially available mulberry leaf extracts from 5 different manufacturers against α - glucosidase are shown in Table 24. 1.7 Quality evaluation results of commercially available mulberry leaf extracts from 5 different manufacturers It can be clearly seen from the radar chart that the content of 1-DNJ in the extract of manufacturer Y11 is greater than that of the mulberry leaf reference extract, corresponding to its strong inhibitory activity against α-glucosidase (inhibition rate 82.01%), but the contents of other flavonoid components are less than those of the mulberry leaf reference extract. For the extract of manufacturer Y12, the contents of individual components (caffeic acid, syringin, rutin) are much higher than those of the self-made water extract of mulberry leaves, and it is speculated that it may be a product prepared by alcohol extraction and purified and enriched by resin; the contents of flavonoid components and 1-DNJ in the mulberry leaf extracts of the other 3 manufacturers are less than the average value of the mulberry leaf reference extract, and it is speculated that these manufacturers use less mulberry leaf as raw material, corresponding to their weak inhibitory activity against α-glucosidase (inhibition rate < 50%). Example 5 1. Quality evaluation of commercially available mulberry leaf extracts from one manufacturer 1.1 Collection of commercially available mulberry leaf extracts Collect the commercially available mulberry leaf extract (Y16) from one manufacturer 1.2 Determination of total flavonoids 1.2.1 Preparation of stock solution of reference substance Same as 1.2.1 in Example 1 1.2.2 Preparation of standard curve solutions Same as 1.2.2 in Example 1 1.2.3 Determination of standard curve Same as 1.2.3 in Example 1 1.2.4 Preparation of test solution of commercially available mulberry leaf extract Mix the samples of the commercially available mulberry leaf extract of manufacturer Y16 evenly, and prepare the test solution of the commercially available mulberry leaf extract with reference to 1.2.4 in Example 1 1.2.5 Determination and calculation of absorbance of test solution Same as 1.2.5 in Example 1, and the results of the commercially available mulberry leaf extract of manufacturer Y16 are shown in Table 26 1.3 Determination of 1-DNJ content in the commercially available mulberry leaf extract of manufacturer Y16 1.3.1 Preparation of test solution Mix the samples of the commercially available mulberry leaf extract of manufacturer Y16 evenly, and prepare the test solution for the determination of 1-DNJ content in the commercially available mulberry leaf extract with reference to 1.3.1 in Example 1 1.3.2 Preparation of 1-DNJ standard stock solution (1.0 mg / mL) Refer to 1.3.2 in Example 1

[0055] 1.3.3 Preparation of 1-DNJ standard curve solutions Refer to 1.3.3 in Example 1.

[0056] 1.3.4 Determination of 1-DNJ by HPLC Refer to 1.3.4 in Example 1. The determination results of the 1-DNJ content in the commercially available mulberry leaf extract from manufacturer Y16 are shown in Table 27. 1.4 Study on the fingerprint of flavonoid components in the commercially available mulberry leaf extract from manufacturer Y16 1.4.1 Preparation of the test solution of the commercially available mulberry leaf extract from manufacturer Y16 Refer to 1.4.1 in Example 1. 1.4.2 Preparation of the reference solution Refer to 1.4.2 in Example 1.

[0057] 1.4.3 Fingerprint and similarity determination of flavonoids in the commercially available mulberry leaf extract from manufacturer Y16 The chromatographic conditions refer to 1.4.3 in Example 1. One batch of the commercially available mulberry leaf extract (Y16) was prepared into a test solution and then detected by high performance liquid chromatography. The fingerprint of the above-mentioned commercially available mulberry leaf extract was imported into the similarity calculation software, and peak matching was performed with the average fingerprint of 3 batches of water extracts of mulberry leaves, and the similarity was calculated. The similarity results are shown in Table 28. At the same time, the flavonoid compounds in the fingerprint of the mulberry leaf extract from manufacturer Y16 were identified. It was found that neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, rutin peak, astragalin were commonly present in different commercially available mulberry leaf extracts. The chromatographic peak identification results are as Figure 9 shown. 1.5 Radar chart analysis of the commercially available mulberry leaf extract from manufacturer Y16 In order to more intuitively reflect the quality of the mulberry leaf extract from manufacturer Y16, this study used a radar chart to compare and evaluate the quality of the mulberry leaf extract. The average value of 3 batches of self-made reference water extracts of mulberry leaves was used as the reference (the black frame in the radar chart), and 8 representative components were selected for evaluation. Among them, the total flavonoids and 1-DNJ were proportionally converted using the determination results, and the other 6 common peaks in the quantitative fingerprint were proportionally converted using the relative peak area. The content differences of the total flavonoids, 1-DNJ and the other 6 components in the commercially available mulberry leaf extract were visually displayed using a radar chart. The content comparison radar chart of the 8 components is shown in Figure 10 .

[0058] 1.6 Determination of the inhibitory activity of the commercially available mulberry leaf extract from manufacturer Y16 against α-glucosidase Refer to 1.6.1 - 1.6.3 in Control Example 1, substitute into the formula. The determination results of the inhibitory activity of the commercially available mulberry leaf extract from manufacturer Y16 against α-glucosidase are shown in Table 29. 1.7 Quality evaluation results of commercially available mulberry leaf extracts from 5 different manufacturers It can be clearly seen from the chromatogram that the mulberry leaf extract sample from manufacturer Y16 does not include the chromatographic peak of moracenin A and there is no adulteration. At the same time, the radar chart shows that the component contents of the mulberry leaf extract from manufacturer Y16 are relatively close to those of the self-made water extract, and the content of 1-DNJ is sufficient, with the α-glucosidase inhibition rate being greater than 50%. This indicates that both the raw materials and the process used are relatively compliant. The above results show that due to the lack of unified quality standard requirements, there may be huge differences in the raw materials and production processes used for mulberry leaf extracts by different manufacturers, resulting in great differences in their products. 。

Claims

1. A method for evaluating the quality of mulberry leaf extract, characterized in that, It includes the following steps: S1: Preparation of the reference mulberry leaf extract; S2: Establishment of the determination method for total flavonoids in the reference mulberry leaf extract; S3: Establishment of the determination method for 1-DNJ content in the reference mulberry leaf extract; S4: Establishment of the fingerprint of the reference mulberry leaf extract and identification of chromatographic peaks; S5: Establishment of a quality evaluation method for mulberry leaf extracts by determining the contents of multiple components and the α-glucosidase inhibitory activity, and quality evaluation of commercially available mulberry leaf extracts from different manufacturers.

2. The quality evaluation method of a mulberry leaf extract according to claim 1, wherein In the preparation process of the reference mulberry leaf extract in step S1, 1 batch of reference mulberry leaf medicinal materials is selected, and the other 2 batches are mulberry leaf decoction pieces that meet the requirements of the Chinese Pharmacopoeia. The mulberry leaf decoction pieces are soaked in pure water and then subjected to two reflux extractions. The extraction solutions are combined, rotary-evaporated and concentrated, freeze-dried, pulverized and mixed evenly to obtain the reference mulberry leaf extract.

3. The quality evaluation method of a mulberry leaf extract according to claim 2, wherein The ratio of the mulberry leaf decoction pieces to pure water is 1:

10.

4. The quality evaluation method of a mulberry leaf extract according to claim 1, wherein In step S2, the total flavonoids in the mulberry leaf extract are determined by ultraviolet spectrophotometry. Using rutin as the reference substance, the content of total flavonoids in the mulberry leaf extract is determined at 510 nm by the sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method.

5. A method for evaluating the quality of mulberry leaf extract according to claim 1, characterized in that, In step S3, FMOC-Cl is used to derivatize 1-DNJ in the mulberry leaf extract. The standard curve solution and the test sample solution are accurately injected into the liquid chromatograph, and the liquid chromatogram is collected at 254 nm. The peak areas of the 1-DNJ derivative peaks in all the chromatograms of the reference mulberry leaf extracts are calculated by a unified integration method; with the concentration of the standard curve as the abscissa and the peak area of the 1-DNJ derivative as the ordinate, a peak area-concentration formula is obtained by linear fitting; According to the fitted peak area-concentration formula, the concentration of 1-DNJ in the test sample solution is calculated, and the content of 1-DNJ in the corresponding extract is calculated. The average content of 1-DNJ in the extract is calculated according to the results of parallel sample determinations.

6. The quality evaluation method of a mulberry leaf extract according to claim 1, characterized in that In step S4, using octadecylsilyl silica gel as the filler and pure methanol and 0.1% phosphoric acid solution as the mobile phase, the HPLC fingerprint of the reference mulberry leaf extract is established and the methodology of this method is investigated; at the same time, 6 components including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, rutin and astragalin in the reference mulberry leaf extract are identified.

7. A method for evaluating the quality of mulberry leaf extract according to claim 1, characterized in that, In step S5, the quality evaluation of commercially available mulberry leaf extracts from different manufacturers is carried out. The average values of 8 representative components in the reference aqueous extract of mulberry leaves are used as the reference. Among them, the total flavonoids and 1-DNJ are proportionally converted by content determination, and the other 6 common peaks in the quantitative fingerprint are proportionally converted by relative peak area. The content differences of total flavonoids, 1-DNJ and the other 6 components in the commercially available mulberry leaf extracts are visually displayed by a radar chart. Subsequently, the α-glucosidase inhibitory activities of the commercially available extracts from different manufacturers are screened, and the mulberry leaf extracts from different manufacturers are compared and studied to comprehensively and systematically evaluate the quality of the commercially available extracts from different manufacturers.

8. Use of a method for evaluating the quality of mulberry leaf extract in evaluating the quality of mulberry leaf extract, characterized in that: Use a method for evaluating the quality of mulberry leaf extract as described in any one of claims 1 to 7 to judge the quality of commercially available mulberry leaf extracts from different manufacturers. The specific judgment criteria are as follows: Select fingerprint similarity, total flavonoids, 1-DNJ content, and the contents of 6 index components as the main indicators for evaluating the quality of mulberry leaf extract; if satisfied, fingerprint similarity > 0.9, the content ranges of total flavonoids and 1-DNJ are 8.30% ± 3.67% and 0 - 1.43% respectively, the range of the 6 index components after proportion conversion is plus or minus 30% of the benchmark conversion, and the α-glucosidase inhibition rate is 71.98% ± 31.70% for first-class products; if not satisfied, fingerprint similarity > 0.9, the content ranges of total flavonoids and 1-DNJ are 8.30% ± 3.67% and 0 - 1.43%, the range of the 6 index components after proportion conversion is within plus or minus 30% of the benchmark conversion, and the α-glucosidase inhibition rate is 71.98% ± 31.70%, and if one or two of them are not satisfied, it is a second-class product; if not satisfied, fingerprint similarity > 0.9, the content ranges of total flavonoids and 1-DNJ are 8.30% ± 3.67% and 0 - 1.43, the range of the 6 index components after proportion conversion is within plus or minus 30% of the benchmark conversion, and the α-glucosidase inhibition rate is 71.98% ± 31.70%, and if any three of them are not satisfied, it is a third-class product.

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