A kind of mulberry leaf extract and preparation method thereof

Abstract

The invention discloses a mulberry leaf extract and a preparation method thereof, and belongs to the technical field of plant extraction. The invention provides a preparation method for a mulberry leaf extract, which sequentially performs extraction in an acidic aqueous solution containing acidic protease, extraction in a neutral or slightly alkaline aqueous solution, and extraction in an alkaline aqueous solution, and cooperates with subsequent activated carbon decolorization, electrodialysis desalination, and reverse osmosis concentration, so as to obtain a mulberry leaf extract with a complete variety of effective components and a high extraction rate. At the same time, the preparation method provided by the invention does not use an organic solvent, i.e., is environmentally friendly, and the obtained product is conducive to human absorption and utilization. Moreover, the overall extraction method is simple to operate, has no requirements on equipment, and is conducive to actual production.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and in particular relates to a mulberry leaf extract and a preparation method thereof. Background Art

[0002] Mulberry leaf is the dried leaf of Morusalba L., a plant of the Moraceae family. It is produced in most parts of China, especially in the mulberry area of ​​the middle and lower reaches of the Yangtze River and the Sichuan Basin. It is a kind of medicinal and edible raw material approved by my country and widely used by the people. Since ancient times, Chinese medicine has used mulberry leaf as a traditional Chinese medicine for the treatment of thirst syndrome (equivalent to diabetes in modern medicine) in clinical practice. The Compendium of Materia Medica records that "mulberry leaf juice boiled instead of tea can stop thirst" and "moxibustion boiled and drunk instead of tea can stop thirst". Modern doctors also often use mulberry leaf in traditional Chinese medicine compound for clinical use, and most of them are effective. In recent years, researchers from various countries have conducted systematic research on the composition, function and mechanism of action of mulberry leaf. The results show that mulberry leaf contains rich functional substances such as polysaccharides, proteins, flavonoids, polyphenols and alkaloids (with 1-deoxynojirimycin DNJ as the main representative substance), which has multiple health care effects such as lowering blood sugar, lowering blood pressure, lowering blood lipids, and delaying aging. Domestic and foreign research data confirm that alkaloids and polysaccharides are the main blood sugar lowering active ingredients in mulberry leaves, and flavonoids also have the effects of lowering blood lipids, scavenging free radicals, and anti-inflammatory. Considering that many of the effects of mulberry leaves come from the combined effects of polysaccharides, flavonoids, alkaloids and other effective substances, it is necessary to study the extraction method that retains the effective ingredients in mulberry leaves as much as possible.

[0003] At present, mulberry leaf extraction mainly adopts water extraction or ethanol or other organic solvent extraction methods. For example, patent CN102524769B proposes a method for preparing mulberry leaf extract, wherein a certain amount of dried mulberry leaves are crushed, and 12 times the amount of raw medicine water is added to heat and reflux for extraction for 3 times, and the filtrate is combined, appropriately concentrated, and passed through an ultrafiltration membrane with a 20,000 Dalton ultrafiltration membrane, and the effluent is then passed through an ultrafiltration membrane with a cutoff value of 2,500 Dalton, and the effluent is collected, and concentrated through a nanofiltration membrane with a cutoff value of 300 Dalton, and the concentrate is spray dried and crushed to obtain a light yellow mulberry leaf extract, wherein the DNJ content is 1.2%. The product is light in color, tasteless, and has good water solubility, but the content of effective ingredients such as mulberry leaf flavonoids and mulberry leaf polyphenols is low. CN108635408B proposes a method for preparing mulberry leaf extract, which includes extracting a certain amount of mulberry leaf medicinal material with 20 times water twice, coarse filtering, and filtering through a disc centrifuge to obtain a clarified mulberry leaf extract, and then passing through a cation exchange resin to adsorb alkaloid components such as DNJ, and then eluting with ammonia water to obtain a mulberry leaf extract with a high DNJ content. This method has a complex process and uses ion exchange resin for treatment, which increases the difficulty and cost of treatment, and only uses DNJ as the target product, and cannot take other components into consideration. CN101278977A provides a method for preparing the main active ingredients of mulberry leaves, which extracts mulberry leaves with a mixed solvent of water and ethanol and uses resin for adsorption to obtain three main active ingredients in mulberry leaves, alkaloids, flavonoids and polysaccharides. This method uses organic solvents such as ethanol, and there are regulatory problems in its application in ordinary foods, and the cost is also high.

[0004] The shortcomings of the existing technology are: the effective ingredients in the mulberry leaf extracts prepared by most processes are single, and the alkaloid DNJ is the target product, while other effective ingredients such as mulberry leaf polysaccharides and mulberry leaf flavonoids cannot be effectively extracted, and the process is complicated and the cost is high; a few preparation methods with full ingredients as the main goal use organic solvents such as ethanol, which also have problems such as difficulty in application and high cost. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a mulberry leaf extract which is environmentally friendly, low-cost, has a complete variety of active ingredients in the extract and a high content, and a preparation method thereof.

[0006] To achieve the above object, in a first aspect of the present invention, a method for preparing a mulberry leaf extract comprises the following steps:

[0007] (1) adding the pretreated mulberry leaves to an acidic aqueous solution containing an acidic protease to perform a first heating extraction, followed by filtering to collect a first filter residue and a first filtrate;

[0008] (2) mixing the first filter residue with water and performing a second heating extraction, followed by filtering to collect a second filter residue and a second filtrate;

[0009] (3) mixing the second filter residue and the alkaline aqueous solution, performing a third heating extraction, and then filtering to collect a third filtrate;

[0010] (4) The first filtrate, the second filtrate and the third filtrate are mixed, decolorized, desalted, concentrated and dried to obtain a mulberry leaf extract.

[0011] In a preparation method of a mulberry leaf extract provided by the present invention, extraction in an acidic aqueous solution containing acidic protease, extraction in a neutral or slightly alkaline aqueous solution, and extraction in an alkaline aqueous solution are sequentially performed, so that a mulberry leaf extract with a full range of effective ingredients can be obtained; specifically, the total flavonoids content in the obtained mulberry leaf extract is above 14.3%, the mulberry leaf polysaccharide content is above 19.2%, and the mulberry leaf alkaloid content is above 0.69%; and the obtained extraction rate is relatively high, being above 15.2%.

[0012] As a preferred embodiment of the preparation method of the present invention, the pretreatment is: the dried mulberry leaf raw material is washed and dusted, then air-dried, crushed and sieved through a 30-40 mesh sieve to obtain the pretreated mulberry leaves.

[0013] As a preferred embodiment of the preparation method of the present invention, in the step (1), the pH value of the acidic aqueous solution is 1.8 to 2.5.

[0014] The inventors have found that when the pH value of the acidic aqueous solution is further selected to be 1.8-2.5, it can better cooperate with the added acidic protease to act, so that the content of effective ingredients in the obtained extract is higher and the extraction rate is better.

[0015] Preferably, the acidic aqueous solution is formed by adding an inorganic acid, and the inorganic acid may be at least one of hydrochloric acid, sulfuric acid, phosphoric acid, and glacial acetic acid.

[0016] As a preferred embodiment of the preparation method of the present invention, in the step (1), the amount of acid protease added is 0.01-0.2% based on the pretreated mulberry leaves.

[0017] Preferably, in the step (1), the amount of acid protease added is 0.04-0.1% based on the pretreated mulberry leaves.

[0018] The addition of acidic protease will not destroy polysaccharides, flavonoids and alkaloids, can improve the extraction rate, and can ensure the extraction amount of effective ingredients. The inventors have found that when the amount of acidic protease added is 0.01-0.2%, especially 0.04-0.1%, while ensuring the addition of as little protein as possible, a better extraction rate and effective ingredient content can be achieved.

[0019] As a preferred embodiment of the preparation method of the present invention, in the step (1), the acidic protease is selected from at least one of trypsin, pepsin, papain, ficin, bromelain, and acidic proteases of microbial origin.

[0020] Preferably, the acidic protease is an acidic protease of microbial origin.

[0021] For example, the acidic protease derived from a microorganism may be an acidic protease derived from Aspergillus niger, an acidic protease derived from Aspergillus usami, or an acidic protease derived from Aspergillus oryzae; and the acidic protease derived from Aspergillus niger may be FDG-2240.

[0022] The type of acidic protease will affect the content of effective ingredients in the extract, and further affect the extraction rate. The inventors have found that when the acidic protease is further selected as an acidic protease of microbial origin, especially an acidic protease of Aspergillus niger origin, the comprehensive effect is optimal.

[0023] As a preferred embodiment of the preparation method of the present invention, in the step (2), the pH value of water is 7.0-7.5.

[0024] As a preferred embodiment of the preparation method of the present invention, in the step (3), the pH value of the alkaline aqueous solution is 8.1 to 8.8.

[0025] The inventors have found that when the pH value range during extraction in step (2) and step (3) is further selected to be the above values, the overall effect is better.

[0026] Preferably, the alkaline aqueous solution is formed by adding an inorganic base, and the inorganic base may be at least one of sodium hydroxide, potassium hydroxide, and calcium hydroxide.

[0027] As a preferred embodiment of the preparation method of the present invention, the temperatures of the first heating extraction, the second heating extraction, and the third heating extraction are each independently 40 to 90° C., the solid-liquid ratio is each independently 6 to 20 times, and the extraction time is each independently 1 to 6 hours.

[0028] Preferably, the temperatures of the first heating extraction, the second heating extraction and the third heating extraction are each independently 60-65° C., the solid-liquid ratio is each independently 8-10 times, and the extraction time is each independently 2-3 hours.

[0029] The solid-liquid ratio of the first heating extraction refers to the mass ratio of the pretreated mulberry leaves to the corresponding extract, the solid-liquid ratio of the second heating extraction is the mass ratio of the first filter residue to the corresponding extract, and the solid-liquid ratio of the third heating extraction is the mass ratio of the second filter residue to the corresponding extract.

[0030] The inventors have found that when the extraction parameters of acidic aqueous solution extraction, water extraction, and alkaline aqueous solution extraction are within the range given above, especially within a further preferred range, a good balance can be established between cost and extraction effect; and the limitation of the extraction temperature can also ensure that the effective ingredients are fully extracted and not destroyed, thereby achieving excellent extraction rate and effective ingredient content.

[0031] As a preferred embodiment of the preparation method of the present invention, the decolorization is carried out using activated carbon at 30-70° C., and the amount of activated carbon added is 0.5-3% of the total mass of the first filtrate, the second filtrate and the third filtrate.

[0032] Preferably, the decolorization temperature is 40-50°C.

[0033] Preferably, the decolorization time is 20 to 120 min, more preferably, the decolorization time is 30 to 40 min.

[0034] Using activated carbon for decolorization can effectively remove a large number of small molecular organic pigment impurities, thereby improving the efficiency of subsequent purification and improving the color purity of the product.

[0035] As a preferred embodiment of the preparation method of the present invention, the desalination is carried out by electrodialysis. Specifically, the liquid after decolorization is centrifuged and filtered, the filtrate is collected and subjected to electrodialysis equipment, the desalination is operated at a certain voltage, the chloride ions in the effluent are detected, and the electrodialysis is stopped until there is no obvious white precipitate when the nitric acid-silver nitrate solution is added.

[0036] Preferably, the centrifugal rotation speed is 6000-12000 r / min, more preferably 8000-10000 r / min.

[0037] Preferably, the electrodialysis pressure is 15-30V, and the time is 30-90min.

[0038] More preferably, the electrodialysis pressure is 20-25 V and the time is 50-60 min.

[0039] Preferably, the electrodialysis membrane may be any one of a homogeneous membrane, a metal membrane, a selective permeable membrane, and a bipolar membrane.

[0040] More preferably, the electrodialysis membrane is an alloy membrane, specifically the LANRANEX4S membrane of Hangzhou Lanran Environmental Technology Co., Ltd.; when using this membrane for extraction, the extraction effect is higher.

[0041] The present invention uses electrodialysis to remove salt, without the need to use conventional anion and cation exchange resins, is easy to operate, low cost, and does not use a large amount of acid-base regeneration resins, thus avoiding secondary pollution; when dialysis is performed under the action of an electric field, charged solute particles (such as ions) in the solution can migrate through the membrane. Electrodialysis can effectively remove salt impurities without causing loss of organic matter.

[0042] As a preferred embodiment of the preparation method of the present invention, the concentration is to concentrate until the Brix of the system is 15-40%.

[0043] Preferably, the concentration is concentrated to a Brix of 25 to 30%.

[0044] The concentration performed in the present invention adopts a reverse osmosis membrane for concentration, and the electrodialysis desalted liquid directly enters the reverse osmosis membrane equipment for low-temperature concentration.

[0045] Preferably, the temperature of the low-temperature concentration is 40-60°C.

[0046] In the subsequent purification stage, a combination of electrodialysis desalination and reverse osmosis concentration is used to remove impurities and concentrate the mulberry leaf extract at a lower temperature, effectively avoiding the degradation of active ingredients.

[0047] As a preferred embodiment of the preparation method of the present invention, the drying is spray drying, the spray drying air inlet temperature is 170-180°C, and the air outlet temperature is 70-80°C.

[0048] In a second aspect of the present invention, the present invention provides a mulberry leaf extract, wherein the mulberry leaf extract is prepared by the preparation method provided by the present invention.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The preparation method of a mulberry leaf extract provided by the present invention sequentially performs extraction in an acidic aqueous solution containing acidic protease, extraction in a neutral or slightly alkaline aqueous solution, and extraction in an alkaline aqueous solution, and cooperates with subsequent activated carbon decolorization, electrodialysis desalination, and reverse osmosis concentration, so as to obtain a mulberry leaf extract with a complete range of effective components and a high extraction rate; specifically, the total extraction rate of the obtained mulberry leaf extract is above 15.2%, the total flavonoid content is above 14.3%, the mulberry leaf polysaccharide content is above 19.2%, and the mulberry leaf alkaloid content is above 0.69%; at the same time, the preparation method provided by the present invention does not use organic solvents, that is, it is environmentally friendly, and the extraction method provided by the present invention conforms to the characteristics of Chinese medicine compatibility and clinical medication and the characteristics of oral drugs in gastrointestinal transport and absorption, therefore, the obtained product is conducive to human absorption and utilization, and also avoids the problem of enrichment of toxic and harmful substances in the human body caused by organic solvent residues caused by organic solvent extraction. In addition, the extraction method provided by the present invention is simple to operate, has no requirements on equipment, and is conducive to actual production. DETAILED DESCRIPTION

[0051] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0052] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0053] The mulberry leaves used in the embodiment of the present invention and the comparative example are from the same batch of mulberry leaves, and the pretreatment method is also the same, specifically: the dried mulberry leaf raw materials are washed and dusted, then air-dried, crushed, and passed through a 40-mesh sieve to obtain the pretreated mulberry leaves.

[0054] Acid protease 1: FDG-2240, 2.5 million U / g, Xiasheng Industrial;

[0055] Acid protease 2: trypsin, 2500μ / mg, Geyuan Tianrun;

[0056] Acid protease 3: pepsin, 10,000 NF U / mg, Sichuan Feideli;

[0057] Acid protease 4: papain, 200,000 U / g, Pangbo Biotechnology;

[0058] Acid protease 5: Ficin, 200,000 U / g, Pangbo Biotechnology;

[0059] Acid protease 6: Bromelain, 200,000 U / g, Pangbo Biotechnology.

[0060] Example 1

[0061] The embodiment of the present invention provides a mulberry leaf extract, and the preparation method of the mulberry leaf extract comprises the following steps:

[0062] (1) First heating extraction: 100 g of pretreated mulberry leaves were weighed and added to 800 mL of deionized water. 30% hydrochloric acid was added while stirring to adjust the pH value of the system to 2.0. Then 0.05 g of acid protease 1 was added. The mixture was stirred and extracted at 55-60° C. for 1.5 h. After the extraction was completed, the mixture was filtered to collect the first residue and the first filtrate.

[0063] (2) Second heating extraction: add 800 mL of deionized water (pH value of water is 7.0) to the first filter residue (105 g), then stir and extract at 55-60° C. for 1.5 h. After the extraction is completed, filter and collect the second filter residue and the second filtrate;

[0064] (3) Third heating extraction: add 800 mL of deionized water to the second filter residue (104 g), add 30% sodium hydroxide aqueous solution while stirring to adjust the pH value of the system to 8.3, then stir and extract at 55-60° C. for 1.5 h. After the extraction is completed, filter and collect the third filtrate;

[0065] (4) Decolorization: The first filtrate, the second filtrate and the third filtrate are combined to obtain a mixed solution, and then activated carbon is added at 1% by weight of the mixed solution, the temperature is raised to 45° C., and the mixture is stirred for 45 min for decolorization. After the decolorization is completed, the mixture is filtered through a tubular centrifuge at a speed of 6000 r / min, and the centrifuge is collected;

[0066] (5) Desalination: The centrifuge liquid is fed into an electrodialysis device, and a constant voltage of 20 V is used. The electrodialysis time is 40 min. The chloride ions in the effluent are detected until no obvious white precipitate is added by dropping nitric acid-silver nitrate solution. The electrodialysis is stopped to obtain an electrodialysate;

[0067] (6) Concentration: Add the electrodialyzed liquid to the reverse osmosis membrane equipment and perform low-temperature concentration at 55±5°C until the Brix of the concentrated liquid reaches 28%, then stop the concentration to obtain the concentrated liquid;

[0068] (7) Drying: The concentrated solution is spray dried with the air inlet temperature controlled at 170-180°C and the air outlet temperature at 70-80°C to obtain a yellow-brown solid powder.

[0069] Example 2

[0070] The embodiment of the present invention provides a mulberry leaf extract, and the preparation method of the mulberry leaf extract comprises the following steps:

[0071] (1) First heating extraction: 500 g of pretreated mulberry leaves were weighed and added to 5000 mL of deionized water. 30% sulfuric acid was added to adjust the pH value of the system to 2.2 while stirring. Then 0.4 g of acid protease 1 was added. The mixture was stirred and extracted at 60-65° C. for 2 h. After the extraction was completed, the mixture was filtered to collect the first residue and the first filtrate.

[0072] (2) Second heating extraction: add 5000 mL of deionized water to the first filter residue (508 g), the pH value of the water is 7.0, and then stir and extract at 60-65° C. for 2 h. After the extraction is completed, filter and collect the second filter residue and the second filtrate;

[0073] (3) Third heating extraction: add 5000 mL of deionized water to the second filter residue (506 g), add 30% potassium hydroxide aqueous solution while stirring to adjust the pH value of the system to 8.4, then stir and extract at 60-65° C. for 2 h. After the extraction is completed, filter and collect the third filtrate;

[0074] (4) Decolorization: The first filtrate, the second filtrate and the third filtrate are combined to obtain a mixed solution, and then activated carbon is added at a weight percentage of 2% by weight of the mixed solution. The mixture is heated to 40° C. and stirred for 60 min for decolorization. After decolorization, the mixture is filtered through a tubular centrifuge at a speed of 8000 r / min, and the centrifuge is collected.

[0075] (5) Desalination: The centrifuge liquid is fed into an electrodialysis device, and a constant voltage of 25 V is used. The electrodialysis time is 35 min. The chloride ions in the effluent are detected until no obvious white precipitate is added after the nitric acid-silver nitrate solution is added. The electrodialysis is stopped to obtain an electrodialysis liquid.

[0076] (6) Concentration: Add the electrodialyzed liquid to the reverse osmosis membrane equipment and perform low-temperature concentration at 55±5°C until the Brix of the concentrated liquid reaches 22%, then stop the concentration to obtain the concentrated liquid;

[0077] (7) Drying: The concentrated solution is spray dried with the air inlet temperature controlled at 170-180°C and the air outlet temperature at 70-80°C to obtain a yellow-brown solid powder.

[0078] Example 3

[0079] The embodiment of the present invention provides a mulberry leaf extract, and the preparation method of the mulberry leaf extract comprises the following steps:

[0080] (1) First heating extraction: weigh 1000 g of the pretreated mulberry leaves and add them to 12 L of deionized water. Add 99% glacial acetic acid while stirring to adjust the pH value of the system to 2.5, then add 0.4 g of acid protease 1, and then stir and extract at 60-65° C. for 2.5 h. After the extraction is completed, filter and collect the first filter residue and the first filtrate;

[0081] (2) Second heating extraction: add 1.2 L of deionized water (pH value of water is 7.0) to the first filter residue (1010 g), then stir and extract at 60-65° C. for 2.5 h. After the extraction is completed, filter and collect the second filter residue and the second filtrate;

[0082] (3) Third heating extraction: 1.2 L of deionized water was added to the second filter residue (1005 g), and a 30% sodium hydroxide aqueous solution was added under stirring to adjust the pH value of the system to 8.8, followed by stirring and extraction at 60-65° C. for 2.5 h. After the extraction was completed, the third filtrate was filtered and collected;

[0083] (4) Decolorization: The first filtrate, the second filtrate and the third filtrate are combined to obtain a mixed solution, and then 3% of the weight of the mixed solution is added with activated carbon, the temperature is raised to 50° C., and the mixture is stirred for 40 min for decolorization. After the decolorization is completed, the mixture is filtered through a tubular centrifuge at a speed of 10,000 r / min, and the centrifuge is collected;

[0084] (5) Desalination: The centrifuge liquid is fed into an electrodialysis device, and a constant voltage of 28 V is used. The electrodialysis time is 60 min. The chloride ions in the effluent are detected until no obvious white precipitate is added by dropping nitric acid-silver nitrate solution. The electrodialysis is stopped to obtain an electrodialysate.

[0085] (6) Concentration: Add the electrodialyzed liquid to the reverse osmosis membrane equipment and perform low-temperature concentration at 55±5°C until the Brix of the concentrated liquid reaches 18%, then stop the concentration to obtain the concentrated liquid;

[0086] (7) Drying: The concentrated solution is spray dried with the air inlet temperature controlled at 170-180°C and the air outlet temperature at 70-80°C to obtain a yellow-brown solid powder.

[0087] Embodiments 4 to 8

[0088] Embodiments 4 to 8 of the present invention provide a mulberry leaf extract, wherein the preparation method of the mulberry leaf extract is different from that of Embodiment 1 in that different acidic proteases are used, and the specific differences are shown in Table 1;

[0089] Table 1

[0090] Example 4 Example 5 Example 6 Example 7 Example 8 Acid proteinase 2 Acid proteinase 3 Acid proteinase 4 Acid proteinase 5 Acid proteinase 6

[0091] Example 9

[0092] The embodiment of the present invention provides a mulberry leaf extract. The only difference between the preparation method of the mulberry leaf extract and that of Example 1 is that the extraction temperature in step (1), step (2) and step (3) is 65-70°C.

[0093] Example 10

[0094] The embodiment of the present invention provides a mulberry leaf extract, and the only difference between the preparation method of the mulberry leaf extract and that of Example 1 is that the pH value in step (1) is 1.

[0095] Embodiment 11

[0096] The embodiment of the present invention provides a mulberry leaf extract. The only difference between the preparation method of the mulberry leaf extract and that of Example 1 is that the pH value in step (3) is 9.5.

[0097] Comparative Example 1

[0098] The comparative example of the present invention provides a mulberry leaf extract, and the preparation method of the mulberry leaf extract is carried out by referring to the method of patent CN102524769B, specifically:

[0099] Weigh 100g of pretreated mulberry leaves, add 12 times the amount of raw medicine water, heat and reflux to extract 3 times, combine the filtrate, concentrate appropriately, pass through a 20,000 Dalton ultrafiltration membrane, and then pass the effluent through an ultrafiltration membrane with a cutoff value of 2,500 Daltons. Collect the effluent, concentrate through a nanofiltration membrane with a cutoff value of 300 Daltons, and spray dry the concentrate to obtain a light yellow mulberry leaf extract.

[0100] Comparative Example 2

[0101] The comparative example of the present invention provides a mulberry leaf extract, and the preparation method of the mulberry leaf extract is carried out by referring to the method of patent CN108635408B, specifically:

[0102] Take 300g of mulberry leaves, add water to extract twice, add 6000mL of water each time, extract at 90℃ for 2 hours, filter through a filter, filter through a disc centrifuge (speed is 10000r / min), and obtain a clarified mulberry leaf extract. The filtered mulberry leaf extract is loaded on LSI-010 type (H type) cation exchange resin at a flow rate of 2BV / h. After the loading is completed, let it stand for 2 hours to allow complete adsorption, then add 4 times the volume of pure water to elute the resin, and then add 3 times the volume of the resin 0.5mol / L ammonia water for elution. The elution rate is controlled at 2BV / h to obtain an ammonia eluate. After concentrating the eluate, the ammonia taste is more obvious. After adding an appropriate amount of 5% hydrochloric acid to neutralize it, continue to concentrate and dry to obtain a mulberry leaf extract.

[0103] Comparative Example 3

[0104] The comparative example of the present invention provides a mulberry leaf extract, and the only difference between the preparation method of the mulberry leaf extract and that of Example 1 is that acidic protease is not added in step (1).

[0105] Comparative Example 4

[0106] The comparative example of the present invention provides a mulberry leaf extract, and the only difference between the preparation method of the mulberry leaf extract and that of Example 1 is that there is no step (1).

[0107] Comparative Example 5

[0108] The comparative example of the present invention provides a mulberry leaf extract, and the only difference between the preparation method of the mulberry leaf extract and that of Example 1 is that there is no step (2).

[0109] Comparative Example 6

[0110] The comparative example of the present invention provides a mulberry leaf extract, and the only difference between the preparation method of the mulberry leaf extract and that of Example 1 is that there is no step (3).

[0111] Comparative Example 7

[0112] The comparative example of the present invention provides a mulberry leaf extract. The preparation method of the mulberry leaf extract is different from that of Example 1 in that no acidic protease is added in step (1), and a neutral protease is added in step (2), and specifically comprises the following steps:

[0113] (1) First heating extraction: 100 g of pretreated mulberry leaves were weighed and added to 800 mL of deionized water. 30% hydrochloric acid was added while stirring to adjust the pH value of the system to 2.0. The mixture was then stirred and extracted at 55-60° C. for 1.5 h. After the extraction was completed, the mixture was filtered to collect the first residue and the first filtrate.

[0114] (2) Second heating extraction: add 800 mL of deionized water to the first filter residue (105 g), the pH value of the water is 7.0, then add 0.05 g of neutral protease SUKAPro NE, then stir and extract at 55-60° C. for 1.5 h. After the extraction is completed, filter and collect the second filter residue and the second filtrate;

[0115] (3) Third heating extraction: add 800 mL of deionized water to the second filter residue (104 g), add 30% sodium hydroxide aqueous solution while stirring to adjust the pH value of the system to 8.3, then stir and extract at 55-60° C. for 1.5 h. After the extraction is completed, filter and collect the third filtrate;

[0116] (4) Decolorization: The first filtrate, the second filtrate and the third filtrate are combined to obtain a mixed solution, and then activated carbon is added at 1% by weight of the mixed solution, the temperature is raised to 45° C., and the mixture is stirred for 45 min for decolorization. After the decolorization is completed, the mixture is filtered through a tubular centrifuge at a speed of 6000 r / min, and the centrifuge is collected;

[0117] (5) Desalination: The centrifuge liquid is fed into an electrodialysis device, and a constant voltage of 20 V is used. The electrodialysis time is 40 min. The chloride ions in the effluent are detected until no obvious white precipitate is added by dropping nitric acid-silver nitrate solution. The electrodialysis is stopped to obtain an electrodialysate;

[0118] (6) Concentration: Add the electrodialyzed liquid to the reverse osmosis membrane equipment and perform low-temperature concentration at 55±5°C until the Brix of the concentrated liquid reaches 28%, then stop the concentration to obtain the concentrated liquid;

[0119] (7) Drying: The concentrated solution was spray dried with the air inlet temperature controlled at 170-180°C and the air outlet temperature at 70-80°C to obtain 24.6 g of yellow-brown solid powder.

[0120] Comparative Example 8

[0121] The comparative example of the present invention provides a mulberry leaf extract. The difference between the preparation method of the mulberry leaf extract and that of Example 1 is that the order of extraction is changed, and the method specifically comprises the following steps:

[0122] (1) First heating extraction: weigh 100 g of the pretreated mulberry leaves and add them to 800 mL of deionized water. Add 30% sodium hydroxide aqueous solution while stirring to adjust the pH value of the system to 8.3. Then, stir and extract at 55-60° C. for 1.5 h. After the extraction is completed, filter and collect the first filter residue and the first filtrate;

[0123] (2) Second heating extraction: add 800 mL of deionized water (pH value of water is 7.0) to the first filter residue (105 g), then stir and extract at 55-60° C. for 1.5 h. After the extraction is completed, filter and collect the second filter residue and the second filtrate;

[0124] (3) Third heating extraction: The second filter residue (104 g) was added to 800 mL of deionized water, and 30% hydrochloric acid was added to adjust the pH value of the system to 2.0 while stirring, and then 0.05 g of acid protease 1 was added, followed by stirring and extraction at 55-60° C. for 1.5 h. After the extraction was completed, the third filtrate was filtered and collected;

[0125] (4) Decolorization: The first filtrate, the second filtrate and the third filtrate are combined to obtain a mixed solution, and then activated carbon is added at 1% by weight of the mixed solution, the temperature is raised to 45° C., and the mixture is stirred for 45 min for decolorization. After the decolorization is completed, the mixture is filtered through a tubular centrifuge at a speed of 6000 r / min, and the centrifuge is collected;

[0126] (5) Desalination: The centrifuge liquid is fed into an electrodialysis device, and a constant voltage of 20 V is used. The electrodialysis time is 40 min. The chloride ions in the effluent are detected until no obvious white precipitate is added by dropping nitric acid-silver nitrate solution. The electrodialysis is stopped to obtain an electrodialysate;

[0127] (6) Concentration: Add the electrodialyzed liquid to the reverse osmosis membrane equipment and perform low-temperature concentration at 55±5°C until the Brix of the concentrated liquid reaches 28%, then stop the concentration to obtain the concentrated liquid;

[0128] (7) Drying: The concentrated solution is spray dried with the air inlet temperature controlled at 170-180°C and the air outlet temperature at 70-80°C to obtain a yellow-brown solid powder.

[0129] Effect example

[0130] The effect example of the present invention records the extraction rate of the mulberry leaf extracts prepared in Examples 1 to 11 and Comparative Examples 1 to 8 and the content of the effective ingredients mulberry leaf total flavonoids, mulberry leaf polysaccharides, and mulberry leaf alkaloids, wherein the content test method of mulberry leaf total flavonoids, mulberry leaf polysaccharides, and mulberry leaf alkaloids is as follows:

[0131] 1. Mulberry leaf total flavonoids: Detected by spectrophotometry, specifically:

[0132] 1) Drawing of standard curve

[0133] Accurately weigh 10.40 mg of rutin standard (bake to constant weight at 120°C), place in a 50 mL volumetric flask, dissolve and dilute with 50% ethanol to prepare a rutin standard solution. Accurately pipette 0, 1.0, 2.0, 3.0, 4.0, 5.0 mL of rutin standard solution respectively, place in a 10 mL volumetric flask, add 0.5 mL of 5% NaNO2 solution respectively, shake well, let stand for 5 minutes, add 0.5 mL of 10% Al(NO3)3 solution, and then add 4 mL of 4% NaOH solution after another 5 minutes, mix well, and dilute to the mark with 50% ethanol.

[0134] 2) Extraction of total flavonoids from samples

[0135] Accurately weigh 1.000 g of sample into a 100 mL conical flask, add 40 mL of 70% ethanol by volume, extract with ultrasonic power of 420 W at 60 ° C for 40 min, filter, transfer the filtrate to a 100 mL volumetric flask, wash the filter flask with 70% ethanol 3 to 5 times, add 70% ethanol to the scale, and shake to obtain the sample solution to be tested.

[0136] 3) Determination of total flavonoids content in mulberry leaves

[0137] Accurately draw 1mL of the sample solution to be tested, put it into a 10mL volumetric flask, add 0.5mL of 5% NaNO2 solution, shake well, let it stand for 5 minutes, add 0.5mL of 10% Al(NO3)3 solution, and then add 4mL of 4% NaOH solution after another 5 minutes, mix well, and dilute to the scale with 50% ethanol. Let it stand for 15 minutes, and measure the absorbance at 510nm. Calculate the total flavonoid content according to the standard curve.

[0138] 2. Mulberry leaf polysaccharide: Detected by spectrophotometry, specifically:

[0139] 1) Drawing of standard curve

[0140] Take 0.1 g of anhydrous glucose dried to constant weight, accurately weigh it, place it in a 100 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, accurately measure 10 mL, place it in a 100 mL volumetric flask, add water to the scale, shake well, and you have it.

[0141] 2) Extraction of mulberry leaf polysaccharides from samples

[0142] Accurately measure 3 mL of this product, place it in a 100 mL volumetric flask, add water to dilute to the scale, and shake well; accurately measure 5 mL, place it in a 50 mL volumetric flask, add water to the scale, and shake well.

[0143] 3) Determination of mulberry leaf polysaccharide content

[0144] Accurately measure 2 mL of reference solution and test solution, place them in 25 mL volumetric flasks, accurately add 1 mL of 5% phenol (freshly distilled) solution, and quickly and accurately add 5 mL of concentrated sulfuric acid, shake while adding, leave for 10 minutes, heat in a water bath for 15 minutes, immediately cool in an ice water bath for 5 minutes, take out, and let it cool to room temperature. At the same time, perform a blank test correction, measure the absorbance at a wavelength of 490 nm according to the UV-Vis spectrophotometry method, and calculate to obtain the result.

[0145] 3. Mulberry leaf alkaloids: Detection was performed using high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD), specifically:

[0146] 1) Preparation of reference solution

[0147] Accurately weigh 10 mg of DNJ (1-deoxynojirimycin) reference substance, add distilled water and ultrasonicate for 1 min to dissolve and dilute to 50 mL, shake well. Accurately measure 1 mL of the above solution into a 10 mL volumetric flask, add water to dilute to volume, and shake well.

[0148] 2) Preparation of sample solution

[0149] Accurately weigh 100 mg of the sample, add distilled water, ultrasonicate for 1 min to dissolve and dilute to 50 mL, shake well. Accurately measure 1 mL of the above solution into a 10 mL volumetric flask, add water to dilute to volume, shake well.

[0150] 3) Pre-column derivatization

[0151] Accurately measure 100 μL of the test solution and place it in a 1.5 mL centrifuge tube, accurately add 100 μL of potassium borate buffer solution (pH 8.5), then accurately add 200 μL of 5 mmol / L 9-fluorenylmethyl chloroformate (FMOC-Cl) acetonitrile solution, shake well, react in a 20°C water bath for 20 min, accurately add 100 μL of 0.1 mmol / L glycine, and finally add 500 μL of 0.1% glacial acetic acid solution, shake well, and filter through a 0.45 μm filter membrane to obtain the product.

[0152] 4) Chromatographic conditions

[0153] Chromatographic column: HiQSiL18 chromatographic column (4.6mm250mm, μm);

[0154] Mobile phase: acetonitrile-0.1% acetic acid (50:50);

[0155] Injection volume: 20 μL;

[0156] Flow rate: 0.8 mL / min;

[0157] ELSD drift tube temperature: 45°C;

[0158] Carrier gas flow rate: 2.5L / min;

[0159] 5) Determination of alkaloid content in mulberry leaves

[0160] After testing under the above chromatographic conditions, calculate the actual content.

[0161] The calculation method of the extraction rate is: extraction rate = the mass of the mulberry leaf extract obtained by extraction / the mass of the mulberry leaf after pretreatment * 100%. The test results are shown in Table 2;

[0162] Table 2

[0163]

[0164]

[0165] As can be seen from Table 2, when the extraction method of the present invention is used for extraction, the extraction rate of the obtained mulberry leaf extract is above 15.2%, which is higher than the extraction rate in the prior art (Comparative Example 1, Comparative Example 2), especially much higher than the extraction rate of 0.11% in Comparative Example 2, and the content of total flavonoids in the prepared mulberry leaf extract is above 14.3%, the content of mulberry leaf polysaccharides is above 19.2%, and the content of mulberry leaf alkaloids is above 0.69%. At the same time, the mulberry leaf extract obtained by the extraction method of the present invention also has a unique aroma of mulberry leaves; especially when FG-2240, which is a microbial source of acidic protease, is further selected and the pH value of the first heating extraction and the pH value of the third heating extraction are within the preferred range of the present invention, the extraction rate of the obtained mulberry leaf extract is above 22.1%, and the content of total flavonoids in the prepared mulberry leaf extract is above 20.8%, the content of mulberry leaf polysaccharides is above 19.2%, and the content of mulberry leaf alkaloids is above 1.07%;

[0166] It can be seen from Examples 1 and 4 to 8 that the type of acidic protease will also affect the extraction rate and the content of the effective ingredient. When the acidic protease is further preferably FG-2240, the best comprehensive effect is obtained.

[0167] It can be seen from Examples 1 and 9 that the extraction temperature will also affect the content of active ingredients in the mulberry leaf extract. When the extraction temperature is appropriately increased, the content of mulberry leaf polysaccharides shows a downward trend. It can be seen from Examples 1 and 10 that the pH value in the first heating extraction will also affect the content of active ingredients. When the pH value is too low, the extraction rate and the content of mulberry leaf polysaccharides will show a downward trend. It can be seen from Examples 1 and 11 that when the alkalinity is high during the third heating extraction, the mulberry leaf alkaloids will show a downward trend.

[0168] It can be seen from Example 1 and Comparative Example 3 that when the first heating extraction is performed without adding acidic protease, the obtained extraction rate and the total flavonoid content of mulberry leaves are significantly reduced. Compared with Example 1, the extraction rate in Comparative Example 3 is reduced by 38.21%, and the total flavonoid content is reduced by 60.89%;

[0169] It can be seen from Example 1 and Comparative Examples 4-6 that when any of the steps of acid extraction, water extraction or alkali extraction is missing, the extraction rate of mulberry leaves is significantly reduced, and the content of total flavonoids in mulberry leaves is also significantly reduced; compared with Example 1, the reduction in the extraction rate in Comparative Examples 4-6 is 33.33-68.29%, and the reduction in the content of total flavonoids in mulberry leaves is 38.22-62.50%; in particular, when the acid extraction is missing in Comparative Example 4, the alkaloids in mulberry leaves are also significantly reduced, and compared with Example 1, it has decreased by 54.78%;

[0170] It can be seen from Example 1 and Comparative Example 7 that when neutral protease is added in step (2) for extraction, the mulberry leaf extraction rate is significantly reduced, and the contents of mulberry leaf total flavonoids, mulberry leaf polysaccharides and mulberry leaf alkaloids are significantly reduced. Compared with Example 1, the extraction rate of Comparative Example 7 is reduced by 50%, and the contents of mulberry leaf total flavonoids, mulberry leaf polysaccharides and mulberry leaf alkaloids are reduced by 36.89%, 61.41% and 40%, respectively.

[0171] It can be seen from Example 1 and Comparative Example 8 that when the extraction order is changed, the obtained mulberry leaf extract decreases significantly, and the contents of mulberry leaf total flavonoids and mulberry leaf polysaccharides both decrease significantly. Compared with Example 1, the extraction rate of Comparative Example 8 decreases by 41.06%, and the contents of mulberry leaf total flavonoids and mulberry leaf polysaccharides decrease by 29.78% and 47.83%, respectively.

[0172] Finally, it should be noted that the above embodiments are intended to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a mulberry leaf extract, characterized in that: The preparation method comprises the following steps: (1) adding the pretreated mulberry leaves to an acidic aqueous solution containing an acidic protease to perform a first heating extraction, followed by filtering to collect a first filter residue and a first filtrate; (2) mixing the first filter residue with water and performing a second heating extraction, followed by filtering to collect a second filter residue and a second filtrate; (3) mixing the second filter residue and the alkaline aqueous solution, performing a third heating extraction, and then filtering to collect a third filtrate; (4) mixing the first filtrate, the second filtrate and the third filtrate, decolorizing, desalting, concentrating and drying to obtain a mulberry leaf extract; In the step (1), the pH value of the acidic aqueous solution is 1.8 to 2.5; In the step (1), the amount of acid protease added is 0.01-0.2% based on the pretreated mulberry leaves; In the step (1), the acidic protease is selected from at least one of FDG-2240, trypsin, and pepsin; In the step (3), the pH value of the alkaline aqueous solution is 8.1 to 8.8; The temperatures of the first heating extraction, the second heating extraction, and the third heating extraction are each independently 55 to 70° C., the solid-liquid ratio is each independently 8 to 10 times, and the extraction time is each independently 1.5 to 2.5 hours.

2. The preparation method according to claim 1, characterized in that: In the step (2), the pH value of water is 7.0 to 7.

5.

3. The preparation method according to claim 1, characterized in that: The decolorization is carried out by using activated carbon at 30-70° C., and the amount of activated carbon added is 0.5-3% of the total mass of the first filtrate, the second filtrate and the third filtrate.

4. The preparation method according to claim 1, characterized in that: The concentration is to concentrate until the Brix of the system is 15-40%.

5. A mulberry leaf extract, characterized in that The mulberry leaf extract is prepared by the preparation method according to any one of claims 1 to 4.

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