CN113082082A - Mechanochemical extraction method of mulberry leaf extract with alpha-glucosidase inhibitory activity - Google Patents
Mechanochemical extraction method of mulberry leaf extract with alpha-glucosidase inhibitory activity Download PDFInfo
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
- A61K36/605—Morus (mulberry)
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- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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- A61K2236/50—Methods involving additional extraction steps
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Abstract
The invention discloses a mechanochemical extraction method of mulberry leaf extract with alpha-glucosidase inhibitory activity, which comprises the following steps: the mulberry leaves are collected, dried, crushed and sieved, and the obtained mulberry leaf powder and a ball milling auxiliary agent are placed in a planetary ball mill for ball milling. Adding an extraction solvent into the mulberry leaf powder after ball milling, performing ultrasonic extraction to obtain a mulberry leaf extract, and performing rotary evaporation to remove the solvent to obtain a yellow-green extract. The content of 1-Deoxynojirimycin (DNJ) in the mulberry leaf extract is used as an index, and the content is detected by using an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry method, and the yield is calculated. The activity of the mulberry leaf extract is further verified by adopting an alpha-glucosidase activity inhibition test. The method has the advantages of simple preparation method of the extract, low cost, high content of active ingredient DNJ and high alpha-glucosidase inhibitory activity, and can provide technical reference data for development and comprehensive utilization of mulberry leaf products.
Description
Technical Field
The invention relates to the technical field of extraction of active ingredients of botanical drugs, in particular to a mechanochemical method for preparing a mulberry leaf extract from mulberry leaves, and the extract has alpha-glucosidase inhibitory activity.
Background
Mulberry LeavesIs dried leaf of Morus alba L (Morus alba L.) belonging to Moraceae, and has wide oval shape, heart-shaped base, slightly pointed top, dense white and soft leaf vein with sawtooth at edge. Old leaves are thick and dark green. Thin tender leaves, yellow green. The mulberry, also known as "Huangsang, Jia sang, Jing sang, Tie Fan", etc., is mainly grown in the northern temperate zone, native to China, and is now planted in Europe, Korea, Japan, etc. (wangting, chemical components and biological activity research progress of mulberry leaves [ J ].Food products and process for their preparation Medicine and food additive, 2018(20): 390-393.）。
The root, cortex mori radicis, leaves and fruits of mulberry can be used as medicines, and are common traditional Chinese medicines in China. Wherein, the mulberry leaves are not only the main food of the silkworm, but also the first Chinese medicine recorded in the Shennong herbal Jing, and can remove cold and heat to generate sweat, expel qi and nourish yin, nourish yin and enrich the blood, relieve cough and moisten dryness, clear liver and improve vision (the content determination and the multivariate statistical analysis of amino acid, nucleoside and alkaloid components of the mulberry leaves in different producing areas of the land city Yu. [ J ].Chinese modern applied medicine2020(37) 1052-1057, which is a commonly used herb for clearing away heat and toxic materials in traditional Chinese medicine. The main active ingredients of mulberry leaves include flavone, flavonoid glycoside, polyphenol, alkaloid, polysaccharide and the like (the chemical ingredients and development and utilization progress of mulberry leaves are Naja, Zulihua.Agricultural science of Hubei16-19. in 2020, (59), wherein, flavonoids and phenolic compounds and alkaloid compounds are the main effective components of mulberry leaves. Modern pharmacological studies show that mulberry leaves have the effects of reducing blood sugar, resisting oxidation, resisting inflammation, resisting bacteria, resisting viruses and the like, wherein alkaloid 1-Deoxynojirimycin (DNJ) in the mulberry leaves has a strong alpha-glucosidase inhibition effect, DNJ can reversibly compete with disaccharide in vivo by combining with alpha-glucosidase to inhibit the transformation of the disaccharide, and the blood sugar reducing effect is achieved (Wuxiahong, research progress of a mulberry leaf alkaloid determination method [ J ]. Guangzhou chemical industry, 2020(48): 24-26). In different growth periods, the content of effective components in the mulberry leaves is obviously changed, and the pharmacological activity shows different strengths. (Zhalii, dynamic analysis and evaluation of accumulation of alkaloids and flavonoid chemical components in mulberry leaves of different varieties in different growth periods [ J ]. Chinese traditional medicine J.2014(39）:4822-4828）。
The molecular structural formula of DNJ is:
the extraction method of DNJ in mulberry leaves mainly comprises heating reflux extraction, ultrasonic-assisted extraction, enzyme fermentation extraction and the like. According to the prior art (CN 102675188A), the DNJ extraction efficiency is low in both common solvent and acid extraction methods. Chinese patent (CN 101209284A) discloses a method for extracting alkaloid from mulberry leaves by heating and refluxing, the DNJ extraction rate is 0.12-0.13%, but the solvent consumption is large and the extraction time is long. Chinese patent (CN 110141602A) discloses a method for ultrasonically extracting mulberry leaf alkaloid, which is simple and efficient, and the content of total alkaloids in mulberry leaves is measured to be between 87% and 99.9%. Chinese patent (CN 102675188A) discloses a method for extracting DNJ from mulberry leaves by cellulase fermentation, the extraction efficiency of the method is about 0.299%, the method is green and environment-friendly, but the process needs to ferment, acidify and further purify the mulberry leaves, and further tests are needed in large-scale production.
In recent years, mechanochemical-assisted extraction techniques have been successfully applied to the extraction of active ingredients from plants (CN 110066305A, CN 110615854A). The method uses high-speed rotation collision of ball milling beads in a small tank to make mechanical force act on the surface of the plant powder to generate a wall breaking effect, so that effective ingredients in the plant ingredients can be efficiently released. And the auxiliary agents with different properties in the ball milling process can also change the extraction rate of the target compound so as to achieve high-efficiency and quick extraction. At present, a method for improving the extraction efficiency of DNJ in mulberry leaves by using a mechanochemical auxiliary extraction technology is not found. Therefore, the invention adopts mechanochemical assistance to extract DNJ from mulberry leaves so as to obtain the mulberry leaf extract with high DNJ content on the premise of simplicity and environmental protection.
Disclosure of Invention
The invention aims to provide a mechanochemical extraction method of mulberry leaf extract with alpha-glucosidase inhibitory activity, which improves the yield of DNJ (deoxyribose nucleic acid) which is a main component of the extract by a simple and rapid method.
The mechanochemical extraction method of the mulberry leaf extract with alpha-glucosidase inhibitory activity is characterized by comprising the following steps of:
1) collecting folium Mori, drying, pulverizing, and sieving to obtain folium Mori powder;
2) carrying out mechanochemical treatment on the mulberry leaf powder obtained in the step 1), wherein the treatment process comprises the following steps: ball-milling the mulberry leaf powder in a planetary ball mill together with a ball-milling auxiliary agent at the ball-milling rotation speed of 50-400 rpm for 5-20 min to obtain ball-milled mulberry leaf powder;
3) mixing the ball-milled mulberry leaf powder obtained in the step 2) with an extraction solvent according to a material-liquid ratio of 1: 30-50, wherein the material-liquid ratio unit is g/mL, then placing the mixture in a water bath at 30-40 ℃ for ultrasonic extraction for 5-20 min, cooling to room temperature after the ultrasonic extraction is finished, filtering, and removing the solvent from the filtrate through rotary evaporation to obtain a yellow-green mulberry leaf extract;
4) re-dissolving the mulberry leaf extract obtained in the step 3) with methanol, centrifuging, taking supernatant for dilution, filtering by a 0.22 mu m PTFE organic filter membrane, carrying out quantitative analysis on DNJ in the extract by adopting an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer, and calculating the yield of the crude extract according to the content of DNJ.
The mechanochemical extraction method of the mulberry leaf extract with the alpha-glucosidase inhibitory activity is characterized in that in the step 2), the ball milling filling rate in a planetary ball mill is 5-21%, and preferably 10.5%.
The mechanochemical extraction method of the mulberry leaf extract with the alpha-glucosidase inhibitory activity is characterized in that in the step 2), the ball milling rotating speed is 100-300 rpm, and the ball milling time is 5-15 min.
The mechanochemical extraction method of the mulberry leaf extract with the alpha-glucosidase inhibitory activity is characterized in that in the step 2), the ball milling auxiliary agent is citric acid; the mass of the ball-milling auxiliary agent is 3-12% of the mass of the mulberry leaf powder, and the preferred mass is 10%.
The mechanochemical extraction method of the mulberry leaf extract with the alpha-glucosidase inhibitory activity is characterized in that in the step 3), an extraction solvent is any one of ethyl acetate, methanol, ethanol, petroleum ether and water, or a mixed solvent of ethanol and water, and preferably ethanol water solution with the mass fraction of 70-80%; the material-liquid ratio is 1: 40, the unit of the material-liquid ratio is g/mL, the ultrasonic extraction temperature is 35 ℃, and the ultrasonic extraction time is 10 min.
The mechanochemical extraction method of the mulberry leaf extract with alpha-glucosidase inhibitory activity is characterized in that in the step 3), the obtained yellow-green mulberry leaf extract is analyzed and detected, and the specific process is as follows: and (3) redissolving the mulberry leaf extract by using methanol, centrifuging, taking supernatant for dilution, filtering by using a 0.22 mu m PTFE organic filter membrane, detecting filtrate by using an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer, carrying out quantitative analysis on DNJ in the extract, and calculating the yield of the crude extract by using the content of DNJ.
The mechanochemical extraction method of the mulberry leaf extract with the alpha-glucosidase inhibitory activity is characterized in that in the step 4), in the process of analyzing by using an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer, the liquid phase conditions are as follows: the chromatographic column is Waters Acquity UPLC BEH HILIC (100X 2.1 mm, 1.7 μm); the flow rate is 0.2 mL/min; the sample injection amount is 1.0 mu L; the column temperature is 30 ℃; the mobile phase A is 0.1wt% formic acid water solution, and the mobile phase B is acetonitrile; gradient elution conditions: 80-61% B for 0-1 min; 1-4.5 min, 61% B; 4.5-4.6 min, 61% -80% B; 4.6-13 min, 80% B. The mass spectrum conditions are as follows: electrospray ion source (positive ion mode ESI +); spraying gas: nitrogen gas; ion source temperature: 150 ℃; the detection mode is as follows: multiple reaction monitoring mode (MRM); taper hole voltage: 56V; capillary voltage: 2.97 kV; desolventizing gas temperature: 350 ℃; desolventizing agent gas flow: 650L/h; and (3) qualitative ion pair: 164.24 > 145.99; and (3) quantitative ion pair: 164.24 > 68.80.
The invention has the following advantages:
1) the mulberry leaves are rich in resources in China, and are one of plants containing a large amount of DNJ in nature. The mulberry leaf extract with high DNJ content is extracted from easily cultivated mulberry leaves, which is beneficial to the development of the medicinal value of the mulberry leaves in the aspect of reducing blood sugar and improves the comprehensive utilization of mulberry leaf resources.
2) According to the method, the DNJ component in the mulberry leaves is extracted by adopting mechanochemical-assisted ultrasonic for the first time, the mechanochemical parameters are optimized, and the yield is obviously improved compared with the DNJ extracted from the mulberry leaves by the traditional ultrasonic method.
3) And comparing and evaluating the alpha-glucosidase inhibitory activity of the mulberry leaf extract to obtain the mulberry leaf extract with higher inhibitory activity.
4) The invention has simple process, environmental protection and low cost.
Drawings
FIG. 1 is a graph showing the results of the alpha-glucosidase inhibitory activity assay of mulberry leaf extract.
Detailed Description
The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.
1. The specifications of the instruments and the medicines related to the invention are as follows:
XS205 dual range analytical balance (Mettler Toledo, switzerland); KH5200DE ultrasonic cleaner (kunshan ultrasonic instruments ltd); waters Acquity UPLC ultra high performance liquid chromatograph (Waters, usa); waters Xevo TQ-XS triple quadrupole mass spectrometer (Waters, USA); barnstead TII ultrapure water system (Thermo Scientific, usa); r-215 rotary evaporator (Buchi, Switzerland); microplate reader (Tecan, switzerland).
Chromatographic grade acetonitrile and ethanol were purchased from merck-stocky, germany, chromatographic grade formic acid and 1-Deoxynojirimycin (DNJ) were purchased from shanghai alatin reagent, ltd, acarbose and p-nitrophenyl-alpha-D-glucopyranoside (PNPG) were purchased from shanghai leaf biotechnology, ltd; the remaining reagents were analytical grade.
The mulberry leaves used in the experiment are collected from Deqing in Zhejiang.
2. The determination of the DNJ content in mulberry leaves described in the following examples was performed by using an ultra performance liquid chromatography tandem triple quadrupole mass spectrometer.
In the process of analyzing by using an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer, the liquid phase conditions are as follows: the chromatographic column is Waters Acquity UPLC BEH HILIC (100X 2.1 mm, 1.7 μm); the flow rate is 0.2 mL/min; the sample injection amount is 1.0 mu L; the column temperature is 30 ℃; the mobile phase A is 0.1wt% formic acid water solution, and the mobile phase B is acetonitrile; gradient elution conditions: 80-61% B for 0-1 min; 1-4.5 min, 61% B; 4.5-4.6 min, 61% -80% B; 4.6-13 min, 80% B. The mass spectrum conditions are as follows: electrospray ion source (positive ion mode ESI +); spraying gas: nitrogen gas; ion source temperature: 150 ℃; the detection mode is as follows: multiple reaction monitoring mode (MRM); taper hole voltage: 56V; capillary voltage: 2.97 kV; desolventizing gas temperature: 350 ℃; desolventizing agent gas flow: 650L/h; and (3) qualitative ion pair: 164.24 > 145.99; and (3) quantitative ion pair: 164.24 > 68.80.
The DNJ content in mulberry leaves is calculated by using an external standard method by taking 1-deoxynojirimycin (more than or equal to 98%) as a reference substance. And (3) drawing a standard curve by taking the peak area y as a vertical coordinate and taking the mass concentration x (ppb) of the reference substance as a horizontal coordinate, wherein a regression equation is as follows:
y = 393.19x + 3009.1，R2=0.9990
the calculation formula of the DNJ yield in mulberry leaves is as follows:
the yield was% (mg/g) = [ mass of DNJ (mg)/mass of mulberry leaf raw material (g) ]. 100%.
Example 1:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder is added into a stainless steel ball milling tank, 5 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 5.2%), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
Weighing 20 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operations, wherein the steps comprise adding 4 mL of 75% ethanol aqueous solution, mixing, then placing in a 35 ℃ water bath for ultrasonic extraction for 10min, filtering, and removing the solvent from the filtrate through rotary evaporation to obtain a yellow-green mulberry leaf extract; redissolving with 4 mL of methanol, centrifuging, collecting the supernatant 100 μ L, mixing with 1 mL of methanol, diluting, and filtering with 0.22 μm PTFE organic filter membrane. And (3) carrying out quantitative analysis on DNJ in the filtered extract by using UPLC-MS/MS (ultra high performance liquid chromatography-tandem triple quadrupole mass spectrometer).
The calculated yield of 1-deoxynojirimycin is 86% (mg/g).
Example 2:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder is added into a stainless steel ball milling tank, 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5%), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
Weighing 20 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operations, and repeating the operation steps in example 1. The final calculated yield of 1-deoxynojirimycin was 95% (mg/g).
Example 3:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder is added into a stainless steel ball milling tank, 15 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 15.7 percent), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
Weighing 20 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operations, and repeating the operation steps in example 1. The yield of 1-deoxynojirimycin was calculated to be 82% (mg/g).
Example 4:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder is added into a stainless steel ball milling tank, 20 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 20.9%), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
Weighing 20 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operations, and repeating the operation steps in example 1. The yield of 1-Deoxynojirimycin (DNJ) was calculated to be 68% (mg/g).
As can be seen from comparison of examples 1-4, with the increase of the ball milling filling rate, the mechanical acting force is increased, the wall breaking effect is enhanced, and the extraction yield is increased; however, when the number of the ball-milling beads is more than 10, the yield is not increased any more and is decreased as the number of the ball-milling beads is increased, mainly because the movement of the ball-milling beads is limited due to an excessively high ball-milling packing rate, and the mechanical action is reduced, thereby decreasing the yield.
Example 5:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder is added into a stainless steel ball milling tank, 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5%), the ball milling time is 5 min, and the ball milling rotating speed is 200 rpm.
Weighing 20 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operations, and repeating the operation steps in example 1. The yield of 1-deoxynojirimycin was calculated to be 81% (mg/g).
Example 6:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder is added into a stainless steel ball milling tank, 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5%), the ball milling time is 15 min, and the ball milling rotating speed is 200 rpm.
Weighing 20 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operations, and repeating the operation steps in example 1. The yield of 1-deoxynojirimycin was calculated to be 66% (mg/g).
It is understood from comparative examples 2, 5 and 6 that the DNJ yield can be improved by accumulating the energy in the ball mill tank by appropriately increasing the ball milling time. However, when the ball milling time is increased to 15 min, the yield is not increased any more, and the yield is in a descending trend, mainly because longer ball milling time generates higher energy, so that the heat in the ball milling tank is concentrated, the target components are damaged, and the yield is reduced.
Example 7:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder is added into a stainless steel ball milling tank, 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5%), the ball milling time is 10min, and the ball milling rotating speed is 100 rpm.
Weighing 20 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operations, and repeating the operation steps in example 1. The yield of 1-deoxynojirimycin was calculated to be 73% (mg/g).
Example 8:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder is added into a stainless steel ball milling tank, 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5%), the ball milling time is 10min, and the ball milling rotating speed is 300 rpm.
Weighing 20 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operations, and repeating the operation steps in example 1. The yield of 1-deoxynojirimycin was calculated to be 74% (mg/g).
As can be seen from comparison of examples 2, 7 and 8, when the rotation speed is increased from 100 rpm to 200 rpm, the mechanical action force is enhanced, the wall-breaking effect is improved, and the yield is improved. However, when the rotation speed reaches 300 rpm, the yield tends to decrease, mainly because the excessively high rotation speed generates high energy, which destroys effective components, resulting in a decrease in yield.
Example 9:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder.
Weighing folium Mori powder 20 mg, adding 4 mL 75% ethanol water solution, mixing, placing in 35 deg.C water bath, ultrasonic extracting for 10min, cooling to room temperature, filtering, rotary evaporating filtrate to remove solvent to obtain yellowish green folium Mori extract, re-dissolving the extract with methanol, centrifuging, diluting supernatant, filtering with 0.22 μm PTFE organic filter membrane, and quantitatively analyzing DNJ in the extract with UPLC-MS/MS. The yield of 1-deoxynojirimycin was calculated to be 66% (mg/g).
As is clear from comparison of example 2 and example 9, the mulberry powder after the ball milling operation has a higher DNJ yield than the simply pulverized mulberry powder under the same extraction conditions. The main reason is that the release of effective components is increased and the yield is improved under the action of proper ball milling mechanical force.
Example 10:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder and 10 mg of citric acid are added into a stainless steel ball milling tank, and the dosage of the mulberry leaf powder is 3.3 percent of the mass of the mulberry leaf powder. 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5 percent), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
Weighing 20.6 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operation, and repeating the operation steps in example 1. The yield of 1-deoxynojirimycin was calculated to be 98% (mg/g).
Example 11:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder and 10 mg of tartaric acid are added into a stainless steel ball milling tank, and the dosage of the mulberry leaf powder is 3.3 percent of the mass of the mulberry leaf powder. 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5 percent), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
Weighing 20.6 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operation, and repeating the operation steps in example 1. The yield of 1-deoxynojirimycin was calculated to be 53% (mg/g).
Example 12:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder and 10 mg of neutral alumina are added into a stainless steel ball milling tank, and the dosage of the mulberry leaf powder is 3.3 percent of the mass of the mulberry leaf powder. 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5 percent), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
Weighing 20.6 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operation, and repeating the operation steps in example 1. The yield of 1-deoxynojirimycin was calculated to be 97% (mg/g).
Example 13:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder and 10 mg of cyclodextrin are added into a stainless steel ball milling tank, and the dosage of the cyclodextrin is 3.3 percent of the mass of the mulberry leaf powder. 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5 percent), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
Weighing 20.6 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operation, and repeating the operation steps in example 1. The yield of 1-deoxynojirimycin was calculated to be 97% (mg/g).
Example 14:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder and 30 mg of citric acid are added into a stainless steel ball milling tank, and the dosage of the mulberry leaf powder is 10% of the mass of the mulberry leaf powder. 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5 percent), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
22 mg of ball-milled mulberry leaf powder is weighed, then solvent extraction and quantitative analysis operations are sequentially carried out, and the operation steps are repeated in example 1. The yield of 1-deoxynojirimycin was calculated to be 150% (mg/g).
Example 15:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder and 30 mg of neutral alumina are added into a stainless steel ball milling tank, and the dosage of the mulberry leaf powder is 10 percent of the mass of the mulberry leaf powder. 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5 percent), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
22 mg of ball-milled mulberry leaf powder is weighed, then solvent extraction and quantitative analysis operations are sequentially carried out, and the operation steps are repeated in example 1. The yield of 1-deoxynojirimycin was calculated to be 87% (mg/g).
Example 16:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder and 30 mg of cyclodextrin are added into a stainless steel ball milling tank, and the dosage of the cyclodextrin is 10 percent of the mass of the mulberry leaf powder. 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5 percent), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
22 mg of ball-milled mulberry leaf powder is weighed, then solvent extraction and quantitative analysis operations are sequentially carried out, and the operation steps are repeated in example 1. The yield of 1-deoxynojirimycin was calculated to be 60% (mg/g).
Example 17:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder and 5 mg of cyclodextrin are added into a stainless steel ball milling tank, and the dosage of the cyclodextrin is 1.7 percent of the mass of the mulberry leaf powder. 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5 percent), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
Weighing 20 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operations, and repeating the operation steps in example 1. The yield of 1-deoxynojirimycin was calculated to be 70% (mg/g).
Example 18:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder and 5 mg of neutral alumina are added into a stainless steel ball milling tank, and the dosage of the mulberry leaf powder is 1.7 percent of the mass of the mulberry leaf powder. 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5 percent), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
Weighing 20 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operations, and repeating the operation steps in example 1. The yield of 1-deoxynojirimycin was calculated to be 57% (mg/g).
Example 19:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. 0.30 g of mulberry leaf powder and 50 mg of citric acid are added into a stainless steel ball milling tank, and the dosage of the mulberry leaf powder is 17% of the mass of the mulberry leaf powder. 10 stainless steel balls with the diameter of 10 mm are added (the ball milling filling rate is 10.5 percent), the ball milling time is 10min, and the ball milling rotating speed is 200 rpm.
Weighing 20 mg of ball-milled mulberry leaf powder, then sequentially carrying out solvent extraction and quantitative analysis operations, and repeating the operation steps in example 1. The yield of 1-deoxynojirimycin was calculated to be 68% (mg/g).
Example 20:
freezing folium Mori collected in 3 months of 2020 at-80 deg.C, vacuum freeze drying, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain folium Mori powder. Weighing 20 mg of mulberry leaf powder, and adding 2mg of citric acid solid, wherein the dosage of the citric acid solid is 10 percent of the mass of the mulberry leaf powder. And (4) uniformly mixing. Then, the solvent extraction and quantitative analysis were performed in this order, and example 1 was repeated. The yield of 1-deoxynojirimycin was calculated to be 86% (mg/g).
The mechanochemical technology applies mechanical forces such as grinding, extrusion, shearing, friction and the like to act on a sample to induce the change of the internal physicochemical properties of the sample in a short time. As can be seen from comparison of examples 1-9, different ball milling times, ball milling speeds and filling rates all have an influence on the DNJ yield. The DNJ yield is increased and then decreased along with the increase of the ball milling time, the ball milling rotating speed and the filling rate. The probable reason is that under the action of a certain mechanical force, the cell wall is favorably broken, and effective substances are released; however, when the ball milling time, the rotational speed and the filling rate are too high, the effective components are destroyed due to too large mechanical force, so that the DNJ extraction rate tends to be reduced. As can be seen from comparative examples 10 to 15, different types and contents of auxiliaries also have an influence on the DNJ yield. After citric acid, neutral alumina and cyclodextrin are added, the influence of different additive types and additive contents on the DNJ yield is relatively large. Wherein the citric acid additive increases the DNJ yield to the maximum when the content is 10 percent. Presumably, DNJ is alkaline and reacts with acidic auxiliary agents under the action of high-energy mechanical force to form water-soluble substances, thereby improving the extraction rate. If the citric acid content is continuously increased, the DNJ structure may be changed due to the over-high acidity, and the yield is reduced. Tartaric acid is also an organic acid, but the amount of 3.3% tartaric acid did not increase the DNJ yield, but decreased it, probably because tartaric acid is more acidic than citric acid, which decreased the DNJ yield.
Example 21 mulberry leaf extract α -glucosidase inhibitory activity assay:
a series of sample solutions with the extract concentrations of 0.07 mg/mL, 0.15 mg/mL, 0.31 mg/mL, 0.62mg/mL, 0.82 mg/mL and 1.10 mg/mL were prepared from the mulberry leaf extract obtained in example 14 using 75% ethanol aqueous solution as a solvent.
When the mulberry leaf extract is subjected to alpha-glucosidase inhibition activity analysis, the following four groups of experiments are performed in parallel:
sample group: 20 μ L of the sample solution prepared above was pipetted into a 96-well plate using a pipette gun, 20 μ L of an α -glucosidase solution (prepared by adding α -glucosidase to PBS buffer) having an activity of 0.219U/mL was added, 40 μ L of PBS solution was added, incubation was performed at 37 ℃ for 10min, 40 μ L of a PNPG substrate solution (prepared by adding PNPG substrate to PBS buffer) having a concentration of 2.5mM was added to the well, reaction was continued at 37 ℃ for 30 min, and 80 μ L of sodium carbonate solution was finally added to terminate the reaction. The absorbance was measured at 405 nm.
Positive control group: a series of positive control solutions with different acarbose concentrations are prepared by taking acarbose as a control medicament and 75% ethanol water solution as a solvent. 20 μ L of the positive control solution prepared above was pipetted into a 96-well plate using a pipette gun, 20 μ L of an α -glucosidase solution (prepared by adding α -glucosidase to PBS buffer) having an activity of 0.219U/mL was added, 40 μ L of PBS solution was added, incubation was performed at 37 ℃ for 10min, 40 μ L of a PNPG substrate solution (prepared by adding PNPG substrate to PBS buffer) having a concentration of 2.5mM was added to the well, reaction was continued at 37 ℃ for 30 min, and 80 μ L of sodium carbonate solution was finally added to terminate the reaction. The absorbance was measured at 405 nm.
Blank group: the reaction was stopped by pipetting 20. mu.L of PBS into a 96-well plate using a pipette gun, adding 20. mu.L of an α -glucosidase solution (prepared by adding α -glucosidase to PBS) having an activity of 0.219U/mL, adding 40. mu.L of PBS solution, incubating at 37 ℃ for 10min, adding 40. mu.L of a PNPG substrate solution (prepared by adding PNPG substrate to PBS) having a concentration of 2.5mM to the well, continuing the reaction at 37 ℃ for 30 min, and finally adding 80. mu.L of sodium carbonate solution. The absorbance was measured at 405 nm.
Background group: 20 μ L of the sample solution prepared above was pipetted into a 96-well plate using a pipette gun, 20 μ L of PBS solution was added, 40 μ L of PBS solution was added, incubation was carried out at 37 ℃ for 10min, 40 μ L of a 2.5mM PNPG substrate solution (prepared by adding PNPG substrate to PBS buffer) was added to the well, the reaction was continued at 37 ℃ for 30 min, and 80 μ L of sodium carbonate solution was added to terminate the reaction. The absorbance was measured at 405 nm.
In the preparation process of the reaction solution in the sample group, the positive control group, the blank group and the background group, the concentration of the PBS buffer solution is 0.1M, and the concentration of the sodium carbonate solution is 1M.
The alpha-glucosidase inhibitory activity test in mulberry leaves is carried out in four groups of experiments in parallel, wherein A is1The absorbance result measured by the sample group or the positive control group; a. the0Absorbance results measured for the blank group; a. the2The absorbance results were obtained for the background group. The inhibitory activity was calculated as follows:
α -glucosidase inhibitory activity (%) = [1- (a)1-A2）/A0]*100%。
The results of the mulberry leaf extract and acarbose control drug on the alpha-glucosidase inhibitory activity according to the above alpha-glucosidase inhibitory activity test procedure are shown in FIG. 1.
The statements in this specification merely set forth a list of implementations of the inventive concept and the scope of the present invention should not be construed as limited to the particular forms set forth in the examples.
Claims (7)
1. A mechanochemical extraction method of mulberry leaf extract with alpha-glucosidase inhibitory activity is characterized by comprising the following steps:
1) collecting folium Mori, drying, pulverizing, and sieving to obtain folium Mori powder;
2) carrying out mechanochemical treatment on the mulberry leaf powder obtained in the step 1), wherein the treatment process comprises the following steps: ball-milling the mulberry leaf powder in a planetary ball mill together with a ball-milling auxiliary agent at the ball-milling rotation speed of 50-400 rpm for 5-20 min to obtain ball-milled mulberry leaf powder;
3) mixing the ball-milled mulberry leaf powder obtained in the step 2) with an extraction solvent according to a material-liquid ratio of 1: 30-50, wherein the material-liquid ratio unit is g/mL, then placing the mixture in a water bath at 30-40 ℃ for ultrasonic extraction for 5-20 min, cooling to room temperature after the ultrasonic extraction is finished, filtering, and removing the solvent from the filtrate through rotary evaporation to obtain a yellow-green mulberry leaf extract.
2. The mechanochemical extraction method of mulberry leaf extract with α -glucosidase inhibitory activity as claimed in claim 1, wherein in step 2), the ball milling filling rate in a planetary ball mill is 5% to 21%, preferably 10.5%.
3. The mechanochemical extraction method of mulberry leaf extract with α -glucosidase inhibitory activity as claimed in claim 1, wherein in step 2), the rotation speed of the ball mill is 100 to 300 rpm, and the ball milling time is 5 to 15 min.
4. The mechanochemical extraction method of mulberry leaf extract with α -glucosidase inhibitory activity as claimed in claim 1, wherein in step 2), the ball milling aid used is citric acid; the mass of the ball-milling auxiliary agent is 3-12% of the mass of the mulberry leaf powder, and the preferred mass is 10%.
5. The mechanochemical extraction method of mulberry leaf extract with α -glucosidase inhibitory activity as claimed in claim 1, wherein in step 3), the extraction solvent is any one of ethyl acetate, methanol, ethanol, petroleum ether and water, or a mixed solvent of ethanol and water, preferably an ethanol aqueous solution with a mass fraction of 70-80%; the material-liquid ratio is 1: 40, the unit of the material-liquid ratio is g/mL, the ultrasonic extraction temperature is 35 ℃, and the ultrasonic extraction time is 10 min.
6. The mechanochemical extraction method of mulberry leaf extract with α -glucosidase inhibitory activity as claimed in claim 1, wherein in step 3), the obtained yellow-green mulberry leaf extract is analyzed and detected by the following specific processes: and (3) redissolving the mulberry leaf extract by using methanol, centrifuging, taking supernatant for dilution, filtering by using a 0.22 mu m PTFE organic filter membrane, detecting filtrate by using an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer, carrying out quantitative analysis on DNJ in the extract, and calculating the yield of the crude extract by using the content of DNJ.
7. The mechanochemical extraction method of mulberry leaf extract with α -glucosidase inhibitory activity as claimed in claim 6, wherein during the detection by the ultra high performance liquid chromatography-tandem triple quadrupole mass spectrometer, the liquid phase conditions are as follows: the chromatographic column is Waters Acquity UPLC BEH HILIC (100X 2.1 mm, 1.7 μm); the flow rate is 0.2 mL/min; the sample injection amount is 1.0 mu L; the column temperature is 30 ℃; the mobile phase A is 0.1wt% formic acid water solution, and the mobile phase B is acetonitrile; gradient elution conditions: 80-61% B for 0-1 min; 1-4.5 min, 61% B; 4.5-4.6 min, 61% -80% B; 4.6-13 min, 80% B; the mass spectrum conditions are as follows: electrospray ion source, positive ion mode ESI +; the spraying gas is nitrogen; the ion source temperature is 140-160 ℃; the detection mode is a multi-reaction monitoring mode; the voltage of the taper hole is 50-60V; the capillary voltage is 2.9-3.0 kV; the temperature of the desolventizing gas is 330-360 ℃; the flow rate of the desolvation gas is 600-700L/h.
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