Plant extraction method

Abstract

Disclosed is a plant extraction method, comprising the following steps: 1) preparation of a crude plant extraction solution; 2) separation of the crude extraction solution through a cation resin and an optional anion resin to obtain a collection solution; 3) concentration of the collection solution obtained in step 2); 4) alcohol precipitation of the concentration solution obtained in step 3); and optionally, step 5) concentration and drying. The method can effectively reduce the content of heavy metals in an extract, and reduce the amount of ethanol in an extraction process, thereby improving the product quality and lowering the production cost, and industrial production efficiency and safety are improved. The extract obtained by means of the method can be used to prepare drugs for reducing fat, treating abnormal glucose tolerance, treating diseases related to abnormal blood glucose, and regulating intestinal flora, and can be processed into food products, health-care products or drinks.

Description

A plant extraction method Technical Field

[0001] This invention belongs to the field of plant extraction technology, and specifically relates to a plant extraction method. Background Technology

[0002] Natural plant extracts refer to products obtained by using plants as raw materials, through physicochemical extraction and separation according to the final intended use of the extracted product, to selectively obtain or concentrate one or more effective components from the plant without altering its effective structure. Plant extracts contain rich and complex organic components, most of which possess biological activities such as antibacterial, bacteriostatic, antioxidant, and immune-regulating effects. Due to their advantages of being green, healthy, safe, highly effective, and residue-free, plant extracts are widely used worldwide in medicine, health products, cosmetics, food additives, pesticides, feed, and daily necessities. my country is the birthplace of the sericulture industry. Moraceae plants, due to their high nutritional and medicinal value, have been recognized as precious materials for both medicinal and edible purposes since ancient times, as recorded in traditional Chinese medicine classics. For example, the *Compendium of Materia Medica* records that "mulberry leaf juice, when boiled and drunk as a tea, can quench thirst," and "roasted and boiled, it can quench thirst as a tea substitute." Modern science has also confirmed the nutritional components, pharmacologically active components, and mechanisms of action of Moraceae plants. Studies have shown that the chemical components in Moraceae plants mainly include flavonoids, polysaccharides, alkaloids, and amino acids. In addition, there are some volatile oils, tannins, succinic acid, adenine, vitamins, etc., which have antifungal, anti-inflammatory, hypoglycemic, and antioxidant effects.

[0003] Plant extraction methods can be divided into classical and modern methods. Classical extraction methods do not require special equipment, are simple to operate, and have low extraction costs. These mainly include solvent extraction and steam distillation. Modern extraction methods are based on advanced modern equipment or are newly developed methods, mainly including ultrasonic extraction, microwave extraction, enzyme extraction, and solid-phase extraction. Plant extracts can be classified as lipophilic or hydrophilic. Different solvents and / or extraction methods can be selected for different plant extracts. For example, for lipophilic plant components, organic solvents can be used for percolation, cold soaking, ultrasonic extraction, microwave extraction, and reflux extraction. For water-soluble components, a commonly used extraction method is water extraction followed by alcohol precipitation. This method is significant for the purification and impurity removal of traditional Chinese medicine extracts, increasing the content of active ingredients, reducing dosage, and facilitating production. Specific steps include water extraction, concentration, alcohol precipitation, and drying. Alcohol precipitation involves adding ethanol to an appropriate concentration to the crude extract to precipitate impurities that are poorly soluble in ethanol, followed by solid-liquid separation to achieve purification. For example, Chang Xingjie et al. extracted the effective components of Forsythia suspensa by following the steps of water decoction-concentration-ethanol precipitation-ethanol recovery (Chang Xingjie, Ding Qian. Changes in forsythoside A and forsythoside and antibacterial activity in the process chain of water extraction and alcohol precipitation of Forsythia suspensa [J]. Journal of Traditional Chinese Medicine, 2018, 24(9):39-41.); Shen Aiying et al. obtained mulberry leaf polysaccharides by water extraction and ethanol precipitation (Shen Aiying, Zhu Ziyu, Zhang Wenliang. Study on extraction process of water-soluble polysaccharides from mulberry leaves [J]. Sericultural Science, 2004, 30(3):277-279.). In order to further concentrate and enrich the effective components, resin separation, membrane separation, ultrafiltration, dialysis, etc. can be carried out after alcohol precipitation to achieve the purpose of purification.

[0004] The growing consumer demand for plant extract products has spurred significant advancements in plant extraction technology and improved product quality. However, compared to developed countries, my country's plant extracts still face some prominent issues, with product safety concerns such as heavy metals and pesticide residues drawing widespread attention. Studies have found that some plants have the ability to accumulate heavy metals (such as copper, lead, cadmium, and zinc) in the environment. For example, a study by Zhang Xing et al. showed that mulberry trees planted in soil with heavy metal content of copper (Cu) (593.56 mg / kg), lead (Pb) (825.41 mg / kg), cadmium (Cd) (8.11 mg / kg), and zinc (Zn) (705.41 mg / kg) had heavy metal contents as high as: Cu 33.13 mg / kg, Pb 33.13 mg / kg, Cd 4.53 mg / kg, Zn 317.72 mg / kg, and leaves as high as: Cu 13.18 mg / kg, Pb 10.32 mg / kg, Cd 1.90 mg / kg, Zn The concentration of 186.53 mg / kg far exceeds relevant regulations (according to the "Green Industry Standard for Import and Export of Medicinal Plants and Preparations," the total heavy metal content in plant raw materials, processed slices, extracts, and preparations should be ≤20.0 mg / kg, lead (Pb) ≤5 mg / kg, cadmium (Cd) ≤0.3 mg / kg, mercury (Hg) ≤0.2 mg / kg, copper (Cu) ≤20.0 mg / kg, and arsenic (As) ≤2.0 mg / kg). Minimizing the residue of heavy metals and other harmful substances is a crucial aspect to consider in research on plant extraction methods.

[0005] Summary of the Invention

[0006] In general research and industrial production processes, water-soluble plant extracts are typically obtained through water extraction and alcohol precipitation. When considering increasing the content of specific components, further enrichment and purification processes such as resin separation, membrane separation, ultrafiltration, and dialysis are often performed after alcohol precipitation. However, for plant extracts, especially those from plants like the Moraceae family that easily accumulate heavy metals from the environment, this extraction method is insufficient to remove heavy metal residues. Through repeated research, the inventors have discovered that sequentially performing resin separation, concentration, and alcohol precipitation on the crude extract during plant extraction not only improves the separation efficiency of effective components but also significantly reduces heavy metal residues in the plant extract. Based on this discovery, the inventors provide a novel plant extraction method that effectively reduces the heavy metal content in plant extracts while significantly reducing the amount of ethanol used in the extraction process. This improves product quality while reducing production costs and, to some extent, enhances industrial production efficiency and safety.

[0007] In view of this, in a first aspect, the present invention provides a plant extraction method, characterized by comprising the following steps:

[0008] Step 1): Prepare crude plant extract;

[0009] Step 2): The crude extract is separated by cation exchange resin, and the eluent is collected. Optionally, the eluent is separated by anion exchange resin, and the effluent is collected.

[0010] Step 3): Concentrate the collected liquid obtained in Step 2);

[0011] Step 4): Perform alcohol precipitation on the concentrated solution obtained in step 3);

[0012] Optional step 5) Concentration and drying process.

[0013] 1) Preparation of crude plant extract

[0014] In this invention, the plants are preferably from the Moraceae, Liliaceae, Campanulaceae, and Commelinaceae families, and more preferably from the genera *Morus*, *Hyacinthus*, *Adenophora*, and *Commelina*. More preferably, the plants are *Morus multicaulis* Perrott., *Morus alba* L., *Morus atropurpurea* Roxb., *Morus mizuho* Hotta, *Morus wittiorum* Hand Mazz., *Morus laevigata* Wall., *Morus nigra* Linn., *Morus cathayana* Hemsi., *Morus serrata* Roxb., *Morus mongolica* Schneid., *Morus bombycis* Koidz., and *Morus chuanxiong*. The species include *Notabilis Schneid.*, *Morus nigriformis* Koidz.*, *Morus yunnanensis* Koidz.*, *Morus australis* Poir.*, *Morus mongolica (Bur.) Schneid var. diabolica* Koidz.*, *Morus alba* var. *Pendula* Dippel, *Morus alba* var. *Pendula* Dippel, and *Morus alba* var. *Pendula*, as well as mulberry varieties bred from the above species, hybrid mulberry varieties selected from the above species within or between species, and any one or more combinations of *Hyacinthus orientalis*, *Adenophora triphylla* var. *japonica*, and *Commelina communis*; preferably, the plants are selected from *Morus dongnanensis*, *Morus rufosa*, *Morus alba* var. *truncatula ... var. *truncatula*, *Morus alba* var. *truncatula* var. *truncatula*, *Morus alba* var. *truncatula* var. *truncatula* var. *truncatula* var. *truncatula* var. *truncatula* var. *truncatula* var. *truncatula* var. *truncatula* var. *truncatula* var. *trun The leaves, roots, branches, bark, buds, stems, fruits, and other parts of the plant can be used.

[0015] The plant can be crudely extracted using solvents such as alcohol-water, water, alkaline aqueous solution, or acidic aqueous solution. Preferably, water is used as the solvent for crude extraction.

[0016] During extraction, it is preferable to crush the plant and add it to water for hot extraction. The extraction time is preferably 0.5-3 hours each time, and the number of extractions is 1-3 times.

[0017] In a preferred embodiment, the pulverized plant material can be added to an extraction tank for extraction.

[0018] The more solvent added during extraction, the higher the extraction rate of the plant. However, adding too much solvent will increase the difficulty of subsequent separation and purification. The preferred amount of solvent added is 3-20 times the weight of the plant raw material, and more preferably 4-15 times. This can maximize the extraction of plant extract without increasing the solution volume and thus increasing the difficulty of subsequent processing.

[0019] Extraction can be performed using decoction, ultrasonic extraction, or reflux extraction, with decoction or reflux extraction being preferred; decoction using more mature industrial equipment is even more preferred.

[0020] Optionally, extraction can be repeated and the extracts combined.

[0021] Preferably, the insoluble matter is removed by filtration of the extract to obtain a crude plant extract.

[0022] 2) Separation by cation exchange resin and optionally anion exchange resin

[0023] This invention uses ion exchange resin to separate components in crude plant extracts.

[0024] In step 2), the crude plant extract is loaded onto a cation exchange resin for separation. Preferably, after packing the cation exchange resin column, activation is performed in the order of washing with acidic solution, then alkaline solution, then acidic solution again. Resin activation also allows for adjustment of the pH value of the resin environment, thereby optimizing the adsorption selectivity of the cation exchange resin and improving the separation effect.

[0025] Preferably, the alkaline solution is used to wash until the pH of the washing solution is 8.0-9.5, more preferably 8.5-9.5;

[0026] Preferably, the alkaline solution is selected from ammonia solution, sodium hydroxide solution, potassium hydroxide solution or sodium carbonate solution, and more preferably ammonia solution or sodium hydroxide solution; preferably, the concentration of the alkaline solution is 0.5-4 mol / L, and more preferably 1-2 mol / L.

[0027] Preferably, the acidic solution is used to wash until the pH of the washing solution is 3.0-7.0, more preferably 4.5-6.5.

[0028] Preferably, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, and disodium hydrogen phosphate-citrate buffer solution, more preferably disodium hydrogen phosphate-citrate buffer solution. Preferably, the concentration of the acidic solution is 0.5-4 mol / L, more preferably 1-2 mol / L.

[0029] When using disodium hydrogen phosphate-citric acid buffer as an acidic solution, the pH value of the disodium hydrogen phosphate-citric acid buffer is preferably 4.0-6.5, more preferably 4.5-5.0.

[0030] Optionally, the cation exchange resin may be rinsed with 3-5 column volumes of deionized water after the final acidic solution wash.

[0031] Preferably, the cation exchange resin is selected from one or more combinations of strongly acidic cation exchange resins, weakly acidic cation exchange resins, and strongly basic quaternary ammonium cation exchange resins.

[0032] Preferably, the cation exchange resin is selected from one or more combinations of the following: type 732 strong acid styrene-based cation exchange resin, type 734 strong acid styrene-based cation exchange resin, type 002SC strong acid styrene-based cation exchange resin, type D001 macroporous strong acid styrene-based cation exchange resin, type D113 macroporous weak acid styrene-propylene-based cation exchange resin, and type D254 macroporous strong basic quaternary ammonium-based cation exchange resin.

[0033] Preferably, the cation exchange resin is a type 732 strong acid styrene-based cation exchange resin, a type 734 strong acid styrene-based cation exchange resin, or a type D001 macroporous strong acid styrene-based cation exchange resin.

[0034] Studies have found that, without being bound by any theory, the loading process of cation exchange resin has a significant impact on the adsorption and separation of plant components, especially the concentration of the loading solution and the amount of resin used.

[0035] Preferably, the ratio of the amount of cationic resin to the weight of the plant raw materials is 1:1-30, more preferably 1:1-25, and even more preferably 1:2-20.

[0036] After loading the crude plant extract onto the cation exchange resin, the cation exchange resin is eluted with an eluent. Preferably, the eluent is a salt solution or alkaline solution containing cations, preferably one or more of sodium chloride, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonia, potassium chloride, and sodium hydroxide.

[0037] Preferably, the concentration of cations in the eluent is 0.04-5 mol / L, more preferably 0.2-3 mol / L, and even more preferably 0.5-2.5 mol / L.

[0038] Preferably, the eluent flow rate is 1-15 BV / h, and more preferably 5-10 BV / h.

[0039] Preferably, the eluent used for cation exchange is 0.1-30 times the weight of the plant raw material, and more preferably, the eluent is 0.5-10 times the weight of the plant raw material.

[0040] Collection begins when the eluent flows out of the cation exchange resin. The starting point for collection can be determined based on the pH of the cation exchange resin eluent; for example, when using an alkaline solution such as ammonia for elution, collection begins when the pH of the cation exchange resin eluent is >7. Alternatively, the starting point can be determined based on the properties of the components to be separated; for example, colorimetric or precipitation reactions can be used to determine the starting point for collection. High-performance liquid chromatography (HPLC) and other detection methods can also be used to determine the starting point for collection. Preferably, collection is stopped when the volume of the collected liquid reaches 0.1-10 times the weight of the plant raw material input; more preferably, collection is stopped when the volume of the collected liquid reaches 0.2-5 times the weight of the plant raw material input.

[0041] Cation exchange resin separation can be performed using either a fixed-bed ion exchange process or a continuous ion exchange process. The more automated continuous ion exchange process is preferred.

[0042] To improve the separation effect of cation exchange resin, it can be used for multiple separations, such as 2-5 separations.

[0043] Optionally, the collected eluent is separated using an anion exchange resin. During separation with the anion exchange resin, after the anion exchange resin column is packed, activation is performed in the order of washing with an alkaline solution, then with an acidic solution, then with another alkaline solution.

[0044] Preferably, the acidic solution is used to wash until the pH of the washing solution is 3.0-7.0, more preferably 4.5-6.5;

[0045] Preferably, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, and disodium hydrogen phosphate-citrate buffer solution, more preferably disodium hydrogen phosphate-citrate buffer solution; preferably, the concentration of the acidic solution is 0.5-4 mol / L, more preferably 1-2 mol / L. When using disodium hydrogen phosphate-citrate buffer solution as the acidic solution, the pH value of the disodium hydrogen phosphate-citrate buffer solution is preferably 4.0-6.5, more preferably 4.5-5.0.

[0046] Preferably, the alkaline solution is used to wash until the pH of the washing solution is 8.0-9.5, more preferably 8.5-9.5;

[0047] Preferably, the alkaline solution is selected from ammonia solution, sodium hydroxide solution, potassium hydroxide solution or sodium carbonate solution, and is preferably sodium hydroxide solution; preferably, the concentration of the sodium hydroxide solution is 0.5-4 mol / L, and more preferably 1-2 mol / L.

[0048] Preferably, the anion exchange resin is selected from one or more combinations of strongly basic anion exchange resins, weakly basic anion exchange resins, or weakly acidic anion exchange resins.

[0049] Preferably, the anion exchange resin is selected from one or more combinations of the following: 717 type strong base styrene-based anion exchange resin, 711 type strong base styrene-based anion exchange resin, D201 type macroporous strong base styrene-based anion exchange resin, D218 type macroporous strong base acrylic anion exchange resin, D301-G type macroporous weak acid styrene-based anion exchange resin, and D301 type macroporous weak base styrene-based anion exchange resin.

[0050] Preferably, the anion exchange resin is a type 717 strong base styrene-based anion exchange resin, a type D201 macroporous strong base styrene-based anion exchange resin, or a type D218 macroporous strong base acrylic anion exchange resin.

[0051] Preferably, the ratio of the amount of anion exchange resin to the weight of the plant raw materials is 1:1-80, more preferably 1:1-64, and even more preferably 1:1-32.

[0052] Collection begins when the liquid flows out of the anion exchange resin. Preferably, collection is stopped when the volume of the collected liquid reaches 0.05-10 times the weight of the plant raw materials. More preferably, collection is stopped when the volume of the collected liquid reaches 0.1-5 times the weight of the plant raw materials.

[0053] Optionally, to improve the separation effect of the anion exchange resin, the anion exchange resin can be used for multiple separations, such as 2-4 separations.

[0054] Preferably, the extraction method further includes: a step of concentrating the crude plant extract before separation in step 2);

[0055] Methods for concentrating crude extracts include thermal concentration, nanofiltration membrane concentration, reverse ion permeation membrane concentration, and combinations thereof.

[0056] It is preferable to use thermal concentration, reverse ion permeation membrane, or a combination thereof for concentration to increase the concentration of the crude plant extract.

[0057] Preferably, when using reverse ion permeation membranes and nanofiltration membranes for concentration, in order to improve the concentration efficiency, centrifugation, ultrafiltration, or microfiltration can be performed to remove impurities before concentration using reverse ion permeation membranes and nanofiltration membranes.

[0058] Preferably, the crude plant extract is concentrated to a solids concentration of 1-15%, more preferably 2-10%. The solids refer to the solid substances remaining after water is removed from the solution.

[0059] Optionally, the concentrated crude extract can be subjected to alcohol precipitation before the resin separation treatment in step 2). During alcohol precipitation, ethanol is added to the crude extract, stirred until homogeneous, and then allowed to stand for a certain period to allow the insoluble substances to precipitate. Preferably, 0.2-20 times the volume of ethanol is added to the crude plant extract, more preferably 0.4-10 times the volume of ethanol. More preferably, an alcohol precipitation tank is used for alcohol precipitation. Preferably, the stirring speed during alcohol precipitation is 10-600 rpm, more preferably 40-500 rpm, and more preferably 80-400 rpm.

[0060] 3) Concentrate the collected liquid obtained in step 2).

[0061] The concentration process in step 3) includes thermal concentration, nanofiltration membrane concentration, reverse ion osmosis membrane concentration, and combinations thereof. Preferably, centrifugation, ultrafiltration membrane filtration, or microfiltration membrane filtration are performed to remove impurities before reverse ion osmosis membrane and nanofiltration membrane concentration.

[0062] Preferably, the specific gravity of the concentrated liquid obtained in step 3) is 1.0-1.3. This specific gravity refers to the mass ratio of the concentrated liquid to water under the same volume conditions.

[0063] 4) Alcohol precipitation treatment

[0064] In step 4), the alcohol precipitation process involves treating the concentrate from step 3) with ethanol. Specifically, ethanol is added to the concentrate from step 3), stirred until well mixed, and then the mixture is allowed to stand for a certain period of time after stirring is stopped, allowing the insoluble substances to precipitate.

[0065] Preferably, in step 4), the weight ratio of ethanol used for alcohol precipitation to plant raw materials is 1:4-600, more preferably 1:20-300.

[0066] Alcohol precipitation is preferably carried out in an alcohol precipitation tank.

[0067] Preferably, in step 4) alcohol precipitation, the stirring speed is 10-600 rpm, more preferably 40-500 rpm, and even more preferably 80-400 rpm.

[0068] 5) Concentration and drying process

[0069] Optionally, the extraction method further includes: step 5) concentration and drying.

[0070] The alcohol precipitation solution is filtered to remove insoluble matter, and concentrated under reduced pressure to obtain a plant extract extract or dried product.

[0071] Secondly, the present invention provides a plant extract obtained according to the above extraction method.

[0072] Preferably, the plant extract obtained by the extraction method described above according to the present invention contains alkaloids at a weight content of 3% or more (preferably alkaloids at a weight content of 3-99%, more preferably alkaloids at a weight content of 15-99%, and even more preferably alkaloids at a weight content of 45-99%, for example 35-70%, or 60-75%), and / or polysaccharides at a weight content of not more than 70% (preferably polysaccharides at a weight content of 0.2-50%, more preferably polysaccharides at a weight content of 0.2-35%), and / or flavonoids at a weight content of not more than 10% (preferably flavonoids at a weight content of 0.05-5%, more preferably flavonoids at a weight content of 0.05-2%), and / or amino acids at a weight content of not more than 50% (preferably amino acids at a weight content of 0-40%, more preferably amino acids at a weight content of 0-25%), and / or other components (preferably 0-25%, more preferably 0-20%). The sum of the contents of each component is 100%. Each component refers to all components in the plant extract, including alkaloids, polysaccharides, flavonoids and amino acids. That is, the plant extract contains other components besides alkaloids, polysaccharides, flavonoids and amino acids.

[0073] Preferably, the plant extract obtained by the extraction method of the present invention comprises the following components in the following weight ratios:

[0074]

[0075] Preferably, the plant extract obtained by the extraction method of the present invention comprises the following components in the following weight ratios:

[0076]

[0077] More preferably, the plant extract obtained by the extraction method of the present invention comprises the following components in the following weight ratios:

[0078]

[0079] Preferably, the alkaloid contains 30-99% by weight of 1-deoxynojirimycin (1-DNJ), more preferably 50-95%, more preferably 55-90%, and even more preferably 60-90%.

[0080] Thirdly, the present invention provides a pharmaceutical composition comprising the above-mentioned plant extract and optionally present pharmaceutically acceptable excipients.

[0081] The excipients are inactive ingredients that are non-toxic to the human body and conform to the route of administration or method of drug delivery.

[0082] The excipients may be solid or liquid. Solid excipients include, for example, sodium lactate, poloxamer, sodium lauryl sulfate, sodium carboxymethyl cellulose, gelatin, xanthan gum, povidone, starch, magnesium stearate, sodium carboxymethyl starch, and talc; liquid excipients include, for example, water, ethanol, syrup, and glycerin.

[0083] Preferably, the dosage form of the pharmaceutical composition includes an oral administration formulation;

[0084] Preferably, the dosage form of the pharmaceutical composition includes: tablets, capsules, oral solutions, oral emulsions, pills, and granules;

[0085] The specific dosage may vary depending on the patient's age, weight, health condition, diet, route of administration, combination of medications, and treatment duration.

[0086] Fourthly, the present invention provides the use of the above-mentioned plant extract or the above-mentioned pharmaceutical composition in the preparation of hypoglycemic drugs.

[0087] On the other hand, the present invention provides the use of the above-mentioned plant extract or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating glucose intolerance.

[0088] In another aspect, the present invention provides the use of the above-mentioned plant extracts or pharmaceutical compositions in the preparation of medicaments for the prevention and / or treatment of diseases related to abnormal blood sugar, including but not limited to diabetes, diabetic nephropathy, diabetic foot, diabetic retinopathy, hyperglycemia, hyperuricemia, hyperlipidemia, intestinal flora imbalance, and cardiovascular and cerebrovascular diseases such as cerebral infarction, cerebral hemorrhage, coronary heart disease, and hypertension.

[0089] The present invention also provides the use of the above-mentioned plant extract or the above-mentioned pharmaceutical composition in the preparation of lipid-lowering drugs.

[0090] The present invention also provides the use of the above-mentioned plant extract or the above-mentioned pharmaceutical composition in the preparation of a drug for regulating intestinal flora.

[0091] In another aspect, the present invention provides a food, health product or beverage, characterized in that the food, health product or beverage comprises the above-mentioned plant extracts and optional excipients acceptable to the food, health product or beverage.

[0092] This invention also provides the use of the above-mentioned plant extracts in the preparation of food, health products, or beverages. Preferably, the food, health product, or beverage is a functional food, health product, or beverage with hypoglycemic effects.

[0093] The plant raw materials mentioned in this invention refer to plant materials used for extraction, including but not limited to fresh or processed plants or plant parts.

[0094] This invention involves sequential resin separation, concentration, and alcohol precipitation of crude plant extracts, offering the following advantages compared to conventional extraction methods:

[0095] 1. Heavy metal content levels have significantly decreased;

[0096] 2. Conventional water extraction and alcohol precipitation methods use ethanol at a rate of 1 / 4 to 5 times the weight of the plant raw materials, while the extraction method of this invention can use as little as 1 / 600 of the weight of the plant raw materials, which greatly reduces the amount of ethanol used, reduces production costs to a certain extent, facilitates industrial production, and improves the safety of the production process. Attached Figure Description

[0097] Figure 1 shows the rat sucrose tolerance test of the plant extracts obtained in Examples 1, 2, 3, 5 and 6. Detailed Implementation

[0098] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Through these exemplary descriptions, the features and advantages of the present invention will become clearer and more apparent.

[0099] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments.

[0100] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0101] The detection method involved in this invention includes:

[0102] 1. Determination of alkaloid content:

[0103] Take an appropriate amount of extract, add water and sonicate to dissolve it to prepare the test solution; separately, accurately weigh an appropriate amount of 1-deoxynojirimycin reference standard, add water to dissolve and prepare the reference solution. Accurately measure appropriate amounts of the reference solution and the test solution, add sodium bicarbonate solution, shake well, then add 9-chloroformate methyl ester (FMOC-Cl) acetone solution, heat at 30℃ for 30 min, add acetic acid to terminate the reaction, shake well, filter, accurately pipette the filtrate and inject it into the liquid chromatograph, calculate the content of 1-deoxynojirimycin and total alkaloids in the test sample according to the peak area using the external standard method (calculated as 1-deoxynojirimycin, calculate the chromatographic peaks with relative retention times in the range of 0.4-1.7) (Reference: Xia Xuejun, Wang Renyun, Liu Yuling. Determination of total alkaloids in mulberry twigs by pre-column derivatization RP-HPLC method [J]. Chinese Journal of New Drugs, 2008, 17(23):2044-2047.).

[0104] 2. Amino acid content determination:

[0105] Take an appropriate amount of the extract, add water and sonicate to dissolve it to prepare the test solution; separately, accurately weigh an appropriate amount of the mixed amino acid reference standard, add water and dissolve to prepare the reference solution. The remaining procedures are the same as for the determination of alkaloid content.

[0106] 3. Polysaccharide content determination:

[0107] Accurately weigh an appropriate amount of extract, add water and sonicate, centrifuge at 4000 rpm for 10 min, and take the supernatant as the test solution. Accurately measure 2 ml of the above test solution, place it in a stoppered test tube, add 6 ml of 0.1% anthrone sulfuric acid reagent, heat in a boiling water bath for 15 min, place in an ice water bath for 15 min, use the corresponding reagent as a blank, and immediately measure the absorbance value at 625 nm. Calculate the concentration of polysaccharides in the test sample as glucose according to the glucose linear regression equation, and calculate the content according to the following formula: Content = C*D*f / W, where: W is the sample mass, C is the concentration of polysaccharides as glucose, f is the conversion factor (3.38), and D is the dilution factor (Reference: Zhang Zuofa, Jin Jie, Shi Liangen. Determination of content of mulberry twig polysaccharides, Chinese Journal of Traditional Chinese Medicine [J]. 2018, 33(4): 462-464).

[0108] 4. Flavonoid content determination:

[0109] Weigh an appropriate amount of rutin reference standard and dissolve it in 60% ethanol to obtain a rutin reference standard stock solution. Accurately measure 0.5, 1.0, 3.0, 5.0, and 7.0 ml into 25 ml volumetric flasks, respectively. Add 10 ml of 5% sodium nitrite solution, 10% aluminum nitrate solution, and 1N NaOH solution to each flask. Then dilute with water to the mark and shake well to obtain the reference solution. Using a blank standard solution as a reference, measure the absorbance at 500 nm and plot a linear regression equation.

[0110] Accurately weigh an appropriate amount of the extract, add 60% ethanol solution and sonicate to dissolve. Shake well, centrifuge at 4000 rpm for 10 min, and take the supernatant as the flavonoid extract. Accurately measure 2.0 ml of the flavonoid extract, add 10 ml each of 5% sodium nitrite solution, 10% aluminum nitrate solution, and 1N NaOH solution, then dilute with water to the mark, shake well, let stand for 15 min, centrifuge at 5000 rpm for 5 min, and take the supernatant for analysis. Separately, accurately measure 2.0 ml of the flavonoid extract (no color development), dilute with water to 25 ml as a blank standard solution. Measure the absorbance of each reaction solution at 500 nm, calculate the flavonoid concentration according to the linear regression equation, and then calculate the flavonoid content (calculated as rutin) in the sample based on the sample weight and dilution factor.

[0111] 5. Heavy metal content detection

[0112] The total heavy metal content was determined using Method II in General Chapter 0821 of Part IV of the 2015 edition of the Chinese Pharmacopoeia. The specific method is as follows:

[0113] (1) Preparation of standard lead solutions. Weigh 0.1599 g of lead nitrate and place it in a 1000 ml volumetric flask. Add 5 ml of nitric acid and 50 ml of water to dissolve it. Dilute with water to the mark and shake well to obtain the stock solution. Accurately measure 0.5, 1, 2, 5, and 8 ml of the stock solution and place them in 5 ml volumetric flasks. Dilute with water to the mark and shake well to obtain standard lead solutions of 5 ppm, 10 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm, and 80 ppm.

[0114] (2) Sample determination. Take 2g of sample, slowly ignite until completely carbonized, cool, add 0.5-1ml of sulfuric acid to just moisten, heat at low temperature until the sulfuric acid is removed, add 0.5ml of nitric acid, evaporate to dryness until the nitrogen oxide vapor is removed, cool, ignite at 500-600℃ to completely ashed, cool, add 2ml of hydrochloric acid, evaporate to dryness on a water bath, add 15ml of water, add ammonia test solution until a faint pink color appears to phenolphthalein indicator, then add 2ml of acetate buffer (pH 3.5), gently heat to dissolve, transfer to a Nessler tube, dilute with water to 25ml as a test tube; separately prepare the test solution. The reagents for the sample solution were evaporated to dryness in a porcelain dish, and then 2 ml of acetate buffer (pH 3.5) and 15 ml of water were added. After dissolving by gentle heating, the solution was transferred to Nessler tubes. A certain amount of standard lead solution from (1) was added to each tube, and then diluted with water to 25 ml as a control tube. Then, 2 ml of thioacetamide test solution was added to each of the test tubes and control tubes, shaken well, and left for 2 minutes. The tubes were then placed on white paper and viewed from top to bottom. The color of the test tubes was compared with that of the control tubes to determine the heavy metal content in the sample.

[0115] The content of heavy metals lead, cadmium, mercury and arsenic can also be detected by inductively coupled plasma mass spectrometry (ICP-MS) as described in General Chapter 0412 of Part IV of the 2015 edition of the Chinese Pharmacopoeia.

[0116] The heavy metal content test results were compared with the "Green Industry Standard for Import and Export of Medicinal Plants and Preparations". The standard stipulates that the total amount of heavy metals in plant raw materials, decoction pieces, extracts and preparations shall be ≤20.0mg / kg, lead (Pb) ≤5mg / kg, cadmium (Cd) ≤0.3mg / kg, mercury (Hg) ≤0.2mg / kg, copper (Cu) ≤20.0mg / kg and arsenic (As) ≤2.0mg / kg.

[0117] The inventors compared two heavy metal detection methods, and the results showed that the determination results of the two methods were consistent. The heavy metal content of the plant extract obtained by the method of the present invention complies with the provisions of the "Green Industry Standard for Import and Export of Medicinal Plants and Preparations". Preferably, the heavy metal content of the plant extract obtained by the method of the present invention does not exceed 20 ppm, more preferably, does not exceed 10 ppm, and even more preferably, does not exceed 5 ppm.

[0118] Example 1

[0119] Take 100g of fresh mulberries (white mulberry), crush them, add 300ml of alcohol-water mixture in two portions, and heat under reflux for 1 hour each time. Combine the extracts, filter to remove insoluble matter, and obtain a crude extract. The crude extract was found to contain 5-10 ppm of heavy metals, including 5.44 ppm of lead, 0.38 ppm of cadmium, 0.06 ppm of mercury, and 0.47 ppm of arsenic. The crude extract was then concentrated by heat to a solids content of 2% and kept at 25℃ as the loading solution for a cation exchange resin column.

[0120] 5g of a 732 type strong acid styrene-based cation exchange resin was packed into a column. The column was washed with 2.5mol / L hydrochloric acid until the eluent pH reached 3.5; then with 1.5mol / L sodium hydroxide solution until the eluent pH reached 8.0; and finally with 2.5mol / L hydrochloric acid solution until the eluent pH reached 3.5. Activation was then achieved by rinsing with 3 column volumes of deionized water. The concentrated extract was loaded onto the column, followed by elution with 3L of 0.1mol / L ammonia at a rate of 10 BV / h. The eluent was collected when the pH of the cation exchange column eluent was greater than 7. Collection was stopped when 1L of eluent was collected, and the collected eluent was directly purified using an anion exchange column.

[0121] Activation was completed by packing 1.25 g of D218 macroporous strong-basic acrylic anion exchange resin into a column, washing with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0; washing with 1.5 mol / L hydrochloric acid solution until the eluent pH reached 3.5; and washing with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0. The collected cation exchange resin eluent was then loaded onto the anion exchange resin, and the eluent was collected until it reached 1 L.

[0122] The collected solution obtained after anion exchange column separation was centrifuged to remove impurities and then concentrated through a reverse ion permeation membrane. The specific gravity of the concentrated liquid was 1.0. It was then transferred to an alcohol precipitation tank, and 25 g of anhydrous ethanol was added at 100 rpm with a stirring paddle. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and dried under vacuum to obtain the extract.

[0123] In addition, fresh mulberry branches, mulberry bark, and mulberry leaves (white mulberry) were extracted using the same methods and parameters as described above. The crude extracts obtained from mulberry branches, mulberry bark, and mulberry leaves contained 5-10 ppm of heavy metals, with lead contents of 5.51, 5.87, and 6.12 ppm, cadmium contents of 0.37, 0.35, and 0.41 ppm, mercury contents of 0.07, 0.08, and 0.06 ppm, and arsenic contents of 0.57, 0.55, and 0.61 ppm.

[0124] The components and heavy metal contents of the obtained mulberry, mulberry twig, mulberry bark and mulberry leaf extracts are shown in Table 1.

[0125] Table 1. Content of each component and heavy metal content in the plant extract obtained in Example 1

[0126]

[0127] Example 2

[0128] Take 100g of fresh mulberry leaves (Guangdong mulberry), crush them, add 2000ml of acidic water in two portions, and extract by decoction for 1 hour each time. Combine the extracts, filter to remove insoluble matter, and obtain crude extract. The crude extract was found to contain 10-20 ppm of heavy metals, with lead at 13.6 ppm, cadmium at 0.84 ppm, mercury at 0.16 ppm, and arsenic at 0.56 ppm. The crude extract was first centrifuged to remove impurities, then concentrated by reverse ion permeation membrane filtration until the solid content reached 14.5%. The concentrated crude extract was transferred to an alcohol precipitation tank, and 80g (about 100ml) of anhydrous ethanol was added at 300rpm. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was used as the loading solution for the cation exchange resin column.

[0129] 10g of 734 type strong acid styrene-based cation exchange resin was packed into a column. The column was washed with 2mol / L hydrochloric acid until the eluent pH reached 4.5; then with 1mol / L sodium hydroxide solution until the eluent pH reached 8.5; and finally with 2mol / L hydrochloric acid solution until the eluent pH reached 4.5. Activation was then achieved by rinsing with 5 column volumes of deionized water. The concentrated, alcohol-precipitated extract was loaded onto the column, followed by elution with 2L of 0.5mol / L ammonia at a rate of 8 BV / h. The eluent was collected when the pH of the cation exchange column eluent was greater than 7. Collection was stopped when 800ml of eluent was collected, and the collected eluent was directly purified using an anion exchange column.

[0130] Activation was completed by packing 8g of 717 type strong base styrene-based anion exchange resin into a column and washing with 1.5mol / L sodium hydroxide solution until the eluent pH reached 9.0; then washing with 1.5mol / L hydrochloric acid solution until the eluent pH reached 3.5; and finally washing with 1.5mol / L sodium hydroxide solution until the eluent pH reached 9.0. The collected cation exchange resin eluent was then loaded onto the anion exchange resin, and the eluent was collected until 750ml was reached.

[0131] The collected solution obtained after anion exchange column separation was thermally concentrated to a specific gravity of 1.05. This concentrated liquid was then transferred to an alcohol precipitation tank, and 12.5 g of anhydrous ethanol was added with a stirrer at 200 rpm. After the ethanol addition was complete, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and dried under vacuum to obtain the extract.

[0132] In addition, fresh mulberry branches and mulberry bark (Guangdong mulberry) were extracted using the same extraction methods and parameters as described above. The crude extracts obtained from mulberry branches and mulberry bark contained 10-20 ppm of heavy metals, with lead content of 14.5 and 15.8 ppm, cadmium content of 0.78 and 0.77 ppm, mercury content of 0.17 and 0.18 ppm, and arsenic content of 0.57 and 0.55 ppm, respectively.

[0133] The components and heavy metal contents of the obtained mulberry twig, mulberry bark and mulberry leaf extracts are shown in Table 2.

[0134] Table 2. Content of each component and heavy metal content in the plant extract obtained in Example 2

[0135]

[0136]

[0137] Example 3

[0138] 1000 kg of fresh mulberry branches (Yue Sang 11) were crushed, and 4000 L of water were added. The mixture was extracted using the reflux method for 2 hours. The extracts were combined, filtered to remove insoluble matter, and a crude extract was obtained. The crude extract was found to contain 40-80 ppm of heavy metals, including 52 ppm of lead, 1.94 ppm of cadmium, 0.88 ppm of mercury, and 1.11 ppm of arsenic. The crude extract was then thermally concentrated until the solids content reached 4%, and kept at 50°C as the loading solution for a cation exchange resin column.

[0139] A 150 kg column of D113 macroporous weakly acidic styrene-propylene cation exchange resin was packed. The column was washed with 2 mol / L hydrochloric acid until the eluent pH reached 4.5; then with 1 mol / L sodium hydroxide solution until the eluent pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluent pH reached 4.5. Activation was then achieved by rinsing with 5 column volumes of deionized water. The concentrated extract was loaded onto the column, followed by elution with 1000 L of 2.5 mol / L ammonia solution at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column eluent was greater than 7. Collection was stopped when 900 L of eluent was collected, and the collected eluent was directly purified using an anion exchange column.

[0140] Activation was completed by packing a column with 62.5 kg of D218 macroporous strong basic acrylic anion exchange resin. The column was washed with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0; then with 1.5 mol / L hydrochloric acid solution until the eluent pH reached 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0. The collected cation exchange resin eluent was then loaded onto the anion exchange resin, and the eluent was collected until a flow rate of 870 L was reached.

[0141] The collected solution obtained after anion exchange column separation was filtered through a microfiltration membrane to remove impurities and then concentrated using a reverse ion exchange membrane. The specific gravity of the concentrated liquid was 1.1. It was then transferred to an alcohol precipitation tank, and 15 kg of anhydrous ethanol was added at 400 rpm with a stirring paddle. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract.

[0142] In addition, fresh mulberry bark and leaves (Yue Sang 11) were extracted using the same extraction method and parameters as described above. The crude extracts obtained from the mulberry bark and leaves contained 40-80 ppm of heavy metals, with lead content of 48 and 53 ppm, cadmium content of 1.78 and 1.77 ppm, mercury content of 0.77 and 0.78 ppm, and arsenic content of 0.87 and 0.95 ppm, respectively.

[0143] The components and heavy metal contents of the obtained mulberry twig, mulberry bark and mulberry leaf extracts are shown in Table 3.

[0144] Table 3. Content of each component and heavy metal in the plant extract obtained in Example 3

[0145]

[0146]

[0147] Example 4

[0148] 1000 kg of dried mulberry bark (Gui Sang You 62) was crushed and mixed with 10000 L of water in two portions. Each extraction was performed using reflux for 2.5 hours. The extracts were combined, filtered to remove insoluble matter, and a crude extract was obtained. The crude extract was found to contain less than 5 ppm of heavy metals, with lead at 1.57 ppm, cadmium at 0.23 ppm, mercury at 0.09 ppm, and arsenic at 0.58 ppm. The crude extract was first filtered through a microfiltration membrane to remove impurities, and then concentrated using a reverse ion exchange membrane until the solids content reached 6%, which was then used as the loading solution for a cation exchange resin column.

[0149] A 100kg column of D001 type macroporous strong acid styrene-based cation exchange resin was packed, and the cation exchange resin was activated according to the method in Example 3. The concentrated extract was loaded onto the column, and then eluted with 500L of 0.2mol / L ammonium chloride at a rate of 5 BV / h. The eluent was monitored with 20% silicotungstic acid; collection began when a white precipitate formed and stopped when 200L of the collected solution was reached. The collected solution was then directly purified using an anion exchange column.

[0150] A 32kg column of D201 type macroporous strong base styrene-based anion exchange resin was packed, and the anion exchange resin was activated according to the method in Example 3. The collected cation resin eluent was loaded onto the anion exchange resin, and the eluent was collected until 100L was reached.

[0151] The collected solution obtained after anion exchange column separation was thermally concentrated to a specific gravity of 1.2. This concentrated liquid was then transferred to an alcohol precipitation tank, and 3 kg of anhydrous ethanol was added with a stirrer at 350 rpm. After the ethanol addition was complete, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract.

[0152] In addition, dried mulberry branches (Gui Sang You 62) were used for extraction. The extraction method and parameters were the same as those described above. The crude extract obtained from the mulberry branches contained 5-10 ppm of heavy metals, 1.66 ppm of lead, 0.25 ppm of cadmium, 0.07 ppm of mercury, and 0.60 ppm of arsenic.

[0153] The components and heavy metal contents of the obtained mulberry twig and mulberry bark extracts are shown in Table 4.

[0154] Table 4. Content of each component and heavy metal content in the plant extract obtained in Example 4

[0155]

[0156]

[0157] Example 5

[0158] Take 10 kg of fresh mulberry branches (Sangtuo No. 2), crush them, add 150 L of water in two portions, and extract by decoction for 3 hours each time. Combine the extracts and filter to remove insoluble matter. Concentrate the extract by heat until the solid content reaches 8%, transfer it to an alcohol precipitation tank, and add 2367.9 g of anhydrous ethanol (3 L) at 300 rpm. After the ethanol is added, stop stirring and precipitate for 24 hours. Use the supernatant as the loading solution for the cation exchange resin column.

[0159] A 5kg column of 002SC type strong acid styrene-based cation exchange resin was packed, and the cation exchange resin was activated according to the method in Example 3. The concentrated, alcohol-precipitated extract was loaded onto the column, followed by elution with 100L of 5mol / L potassium chloride at a rate of 5 BV / h. The eluent was monitored with 20% silicotungstic acid; collection began when a white precipitate formed and stopped when 25L of the collected solution was collected. The collected solution was then directly purified using an anion exchange column.

[0160] A 10 kg column of 711 type strong base styrene-based anion exchange resin was packed, and the anion exchange resin was activated according to the method in Example 3. The collected cation exchange resin eluent was loaded onto the anion exchange resin, and the eluent was collected until 15 L was reached. The collected eluent was then loaded back onto the cation exchange resin, and the separation was repeated twice more using cation exchange resin and anion exchange resin in sequence, following the method described above.

[0161] The collected liquid obtained after three column separations was centrifuged to remove impurities and then concentrated by reverse ion permeation membrane. The specific gravity of the concentrated liquid was 1.25. It was then transferred to an alcohol precipitation tank, and 125g of anhydrous ethanol was added at 1000rpm with a stirring paddle. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract. Fresh mulberry bark and mulberry leaves (Sangtuo No. 2) were also extracted using the same methods and parameters as described above. The components and heavy metal contents of the obtained mulberry twig, mulberry bark, and mulberry leaf extracts are shown in Table 5.

[0162] Table 5. Content of each component and heavy metal content in the plant extract obtained in Example 5

[0163]

[0164] Example 6

[0165] Take 1 kg of fresh mulberry root (Yue Sang 11), crush it, add 6 L of alcohol-water in three portions, and extract with ultrasound for 1 hour each time. Combine the extracts, filter to remove insoluble matter, and obtain crude extract. Use crude extract as the loading solution for cation exchange resin column.

[0166] One kg of D254 macroporous strong-basic quaternary ammonium cation exchange resin was packed into a column, and the resin was activated according to the method in Example 3. The crude extract was loaded onto the column, followed by elution with 15 L of 3 mol / L sodium chloride at a rate of 5 BV / h. The eluent was monitored with 20% silicotungstic acid; collection began when a white precipitate formed and stopped when 5 L of the collected solution was collected. The collected solution was then directly purified using an anion exchange column.

[0167] A column was packed with 1 kg of D301 type macroporous weakly basic styrene-based anion exchange resin, and the anion exchange resin was activated according to the method in Example 3. The collected cation exchange resin eluent was loaded onto the anion exchange resin, and the eluent was collected until 5 L of eluent was reached. The collected eluent was then loaded back onto the cation exchange resin, and the separation was repeated with cation exchange resin and anion exchange resin sequentially as described above.

[0168] The collected liquid obtained after two column separations was centrifuged to remove impurities and then concentrated through a reverse ion osmosis membrane. The specific gravity of the concentrated liquid was 1.2. It was then transferred to an alcohol precipitation tank, and 6.3 g of anhydrous ethanol was added under a stirring speed of 600 rpm. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract. Fresh mulberry branches and leaves (Yue Sang 11) were also used for extraction, and the extraction methods and parameters were the same as those described above. The components and heavy metal contents of the obtained mulberry branch, leaf, and root extracts are shown in Table 6.

[0169] Table 6. Content of each component and heavy metal content in the plant extract obtained in Example 6

[0170]

[0171] Example 7

[0172] Take 1000 kg of fresh mulberry branches (Guangdong mulberry), crush them, add 11500 L of water, heat and reflux for 2 h, combine the extracts, filter to remove insoluble matter, and obtain crude extract. The crude extract is first centrifuged to remove impurities, and then concentrated with a reverse ion permeation membrane until the solid content reaches 1%, which is used as the loading solution for the cation exchange resin column.

[0173] A column of 150 kg of D001 type macroporous strong acid styrene-based cation exchange resin was packed, and the cation exchange resin was activated according to the method in Example 3. The concentrated crude extract was loaded onto the column and eluted with 5000 L of 0.04 mol / L ammonium nitrate at a rate of 5 BV / h. The eluent was monitored with 20% silicotungstic acid; collection began when a white precipitate formed and stopped when 1000 L of eluent was collected.

[0174] The collected solution obtained after cation exchange column separation was concentrated by nanofiltration membrane. The specific gravity of the concentrated liquid was 1.3. It was then transferred to an alcohol precipitation tank, and 1.7 kg of anhydrous ethanol was added at 600 rpm with a stirring paddle. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract.

[0175] In addition, fresh mulberry bark and leaves (from Guangdong mulberry) were extracted using the same methods and parameters as described above. The components and heavy metal contents of the obtained mulberry twig, mulberry bark, and mulberry leaf extracts are shown in Table 7.

[0176] Table 7. Content of each component and heavy metal content in the plant extract obtained in Example 7

[0177]

[0178]

[0179] Example 8

[0180] Take 1000 kg of fresh mulberry branches (Yue Sang 11), crush them, add 8000 L of water in two portions, and extract by decoction for 2 hours each time. Combine the extracts, filter to remove insoluble matter, and obtain crude extract. The crude extract is first filtered through a microfiltration membrane to remove impurities, and then concentrated through a reverse ion permeation membrane until the solid content reaches 1%, which is then used as the loading solution for the cation exchange resin column.

[0181] A column was packed with 41.7 kg of 732 type strong acid styrene-based cation exchange resin, and the resin was activated according to the method in Example 3. The crude extract was loaded onto the column, followed by elution with 1000 L of 0.1 mol / L sodium chloride at a rate of 5 BV / h. The eluent was monitored with 20% silicotungstic acid; collection began when a white precipitate formed and stopped when 500 L of eluent was collected.

[0182] The collected solution obtained after cation exchange column separation was concentrated by nanofiltration membrane. The specific gravity of the concentrated liquid was 1.25. It was then transferred to an alcohol precipitation tank, and 15 kg of anhydrous ethanol was added at 600 rpm with a stirring paddle. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract.

[0183] In addition, fresh mulberry bark and leaves (Yue Sang 11) were extracted using the same methods and parameters as described above. The components and heavy metal contents of the obtained mulberry twig, mulberry bark, and mulberry leaf extracts are shown in Table 8.

[0184] Table 8. Content of each component and heavy metal content in the plant extract obtained in Example 8

[0185]

[0186]

[0187] Example 9

[0188] Take 100g of fresh mulberry branches (Guangdong mulberry), crush them, add 600ml of water, heat under reflux for 1 hour, filter to remove insoluble matter, and obtain crude extract. The crude extract is first concentrated by heat until the solid content reaches 5%, and then used as the loading solution for the cation exchange resin column.

[0189] 3.85 g of 732 type strong acid styrene-based cation exchange resin was packed into a column, and the cation exchange resin was activated according to the method in Example 3. The crude extract was loaded onto the column, and then eluted with 700 mL of 0.15 mol / L ammonium chloride at a rate of 5 BV / h. The eluent was monitored with 20% silicotungstic acid; collection began when a white precipitate formed and stopped when 100 mL of the collected solution was reached.

[0190] The collected solution obtained after cation exchange column separation was thermally concentrated to a specific gravity of 1.3. This concentrated liquid was then transferred to an alcohol precipitation tank, and 25 g of anhydrous ethanol was added with a stirrer at 600 rpm. After the ethanol addition was complete, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract.

[0191] In addition, fresh mulberry bark and leaves (from Guangdong mulberry) were extracted using the same methods and parameters as described above. The components and heavy metal contents of the obtained mulberry twig, mulberry bark, and mulberry leaf extracts are shown in Table 9.

[0192] Table 9. Content of each component and heavy metal content in the plant extract obtained in Example 9

[0193]

[0194]

[0195] Example 10

[0196] Take 1000 kg of fresh mulberry branches (Sangtuo No. 2), crush them, add 5000 L of water, heat and reflux for 1 hour, filter to remove insoluble matter, and obtain crude extract. The crude extract is first concentrated by heat until the solid content reaches 10%, and then used as the loading solution for the cation exchange resin column.

[0197] 3.5 g of D254 macroporous strong basic quaternary ammonium cation exchange resin was packed into a column, and the cation exchange resin was activated according to the method in Example 3. The crude extract was loaded onto the column, and then eluted with 200 L of 0.15 mol / L potassium chloride at a rate of 5 BV / h. The eluent was monitored with 20% silicotungstic acid; collection began when a white precipitate formed and stopped when 100 L of the collected solution was reached.

[0198] The collected solution obtained after cation exchange column separation was thermally concentrated to a specific gravity of 1.05. This concentrated liquid was then transferred to an alcohol precipitation tank, and 15 kg of anhydrous ethanol was added with a stirrer at 600 rpm. After the ethanol addition was complete, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract.

[0199] In addition, fresh mulberry bark (Sangtuo No. 2) was used for extraction, and the extraction method and parameters were the same as those described above. The components and heavy metal contents of the obtained mulberry twig and mulberry bark extracts are shown in Table 10.

[0200] Table 10. Content of each component and heavy metal in the plant extract obtained in Example 10

[0201]

[0202]

[0203] Example 11

[0204] Take 1000g of fresh mulberry branches (wild mulberry), crush them, add 10L of acidic water in three portions, and extract with ultrasound for 2 hours each time. Filter to remove insoluble matter to obtain a crude extract. The crude extract is first filtered through a microfiltration membrane to remove impurities, and then concentrated with a reverse ion permeation membrane until the solid content reaches 4%, which is then used as the loading solution for a cation exchange resin column.

[0205] 66.67 g of D001 type macroporous strong acid styrene-based cation exchange resin was packed into a column, and the resin was activated according to the method in Example 3. The crude extract was loaded onto the column, followed by elution with 12 L of 1.5 mol / L ammonia at a rate of 5 BV / h. The eluent was collected when alkaloids were detected in the cation exchange column eluent by high-performance liquid chromatography (HPLC). Collection was stopped when 100 mL of eluent was collected. The collected solution was then directly purified using an anion exchange column.

[0206] 13.3 g of D218 macroporous strong-base styrene-based anion exchange resin was packed into a column, and the anion exchange resin was activated according to the method in Example 3. The collected cation resin eluent was loaded onto the anion exchange column, and the eluent was collected until 50 ml was reached.

[0207] The collected solution obtained after anion exchange column separation was first filtered through a microfiltration membrane to remove impurities, and then concentrated using a reverse ion osmosis membrane. The specific gravity of the concentrated liquid was 1.15. It was then transferred to an alcohol precipitation tank, and 25 g of anhydrous ethanol was added at 600 rpm with a stirring paddle. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract.

[0208] In addition, fresh mulberry bark and leaves (wild mulberry) were extracted using the same methods and parameters as described above. The components and heavy metal contents of the obtained mulberry twig, mulberry bark, and mulberry leaf extracts are shown in Table 11.

[0209] Table 11. Content of each component and heavy metal in the plant extract obtained in Example 11

[0210]

[0211]

[0212] Example 12

[0213] Take 100g of fresh mulberry bark (Guisangyou 62), crush it, add 1.2L of alcohol-water mixture, and extract by decoction for 1 hour. Filter to remove insoluble matter to obtain crude extract. The crude extract is first concentrated by heat until the solid content reaches 8%, and then used as the loading solution for cation exchange resin column.

[0214] 33.34 g of 734 type strong acid styrene-based cation exchange resin was packed into a column, and the cation exchange resin was activated according to the method in Example 3. The crude extract was loaded onto the column, followed by elution with 50 mL of 2.5 mol / L ammonia solution at a rate of 5 BV / h. The eluent was collected when the pH of the cation exchange column effluent was greater than 7, and collection was stopped when 10 mL of eluent was collected.

[0215] The collected liquid obtained after cation exchange column separation was centrifuged to remove impurities, and then concentrated using a reverse ion osmosis membrane. The specific gravity of the concentrated liquid was 1.2. It was then transferred to an alcohol precipitation tank, and 15 g of anhydrous ethanol was added with a stirrer at 600 rpm. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract.

[0216] The obtained mulberry bark extract contained 15% alkaloids, 38% polysaccharides, 2% flavonoids, and 40% amino acids. Among the alkaloids, the content of 1-DNJ was 52%.

[0217] The total heavy metal content is less than 5 ppm, with lead content at 0.29 ppm, cadmium at 0 ppm, mercury at 0.02 ppm, and arsenic at 0.10 ppm.

[0218] Example 13

[0219] Take 100g of fresh mulberry branches (white mulberry), crush them, add 300ml of alkaline water, heat and reflux for 0.5h, filter to remove insoluble matter, and obtain crude extract. The crude extract is first centrifuged to remove impurities and then concentrated with a reverse ion permeation membrane until the solid content reaches 6%, which is then used as the loading solution for the cation exchange resin column.

[0220] 3.34 g of 732 type strong acid styrene-based cation exchange resin was packed into a column, and the cation exchange resin was activated according to the method in Example 3. The crude extract was loaded onto the column, followed by elution with 3 L of 1.0 mol / L ammonia at a rate of 5 BV / h. The eluent was monitored with 20% silicotungstic acid; collection began when a white precipitate formed and stopped when 400 mL of the collected solution was reached.

[0221] The collected solution obtained after cation exchange column separation was thermally concentrated to a specific gravity of 1.25. This concentrated liquid was then transferred to an alcohol precipitation tank, and 5 g of anhydrous ethanol was added with a stirrer at 600 rpm. After the ethanol addition was complete, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract.

[0222] The obtained mulberry twig extract contained 3% alkaloids, 60% polysaccharides, 5% flavonoids, and 30% amino acids. Among the alkaloids, the content of 1-DNJ was 47%.

[0223] The total heavy metal content is less than 5 ppm, with lead content at 0.31 ppm, cadmium at 0 ppm, mercury at 0.01 ppm, and arsenic at 0.14 ppm.

[0224] Example 14

[0225] Take 100g of fresh mulberry leaves (Lu mulberry), crush them, add 500ml of alcohol-water mixture, and extract by decoction for 0.5h. Filter to remove insoluble matter to obtain crude extract. Concentrate the crude extract through nanofiltration membrane until the solid content reaches 12%, and use it as the loading solution for cation exchange resin column.

[0226] 25g of 732 type strong acid styrene-based cation exchange resin was packed into a column, and the cation exchange resin was activated according to the method in Example 3. The crude extract was loaded onto the column, and then eluted with 2L of 2.0mol / L ammonia at a rate of 5 BV / h. The eluent was collected when alkaloids were detected in the cation exchange column eluent by high performance liquid chromatography (HPLC). Collection was stopped when 800mL of eluent was collected.

[0227] The collected solution obtained after cation exchange column separation was thermally concentrated to a specific gravity of 1.14. This concentrated liquid was then transferred to an alcohol precipitation tank, and 5 g of anhydrous ethanol was added with a stirrer at 600 rpm. After the ethanol addition was complete, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract.

[0228] The obtained mulberry leaf extract contained 10% alkaloids, 43% polysaccharides, 1% flavonoids, and 37% amino acids. Among the alkaloids, 1-DNJ accounted for 50%.

[0229] The total heavy metal content is less than 5 ppm, with lead content at 0.33 ppm, cadmium at 0.01 ppm, mercury at 0.02 ppm, and arsenic at 0.15 ppm.

[0230] Example 15

[0231] Take 100g of fresh hyacinth (Hyacinthus orientalis) bulbs, crush them, add 700ml of water in two portions, and extract with ultrasound for 0.5h each time. Combine the extracts, filter to remove insoluble matter, and obtain a crude extract. Concentrate the crude extract through a nanofiltration membrane until the solid content reaches 10%, and use it as the loading solution for a cation exchange resin column.

[0232] 3.5 g of a 732 type strong acid styrene-based cation exchange resin was packed into a column, and the cation exchange resin was activated according to the method in Example 3. The concentrated crude extract was loaded onto the column and eluted with 10 ml of 1.75 mol / L sodium hydroxide solution at a rate of 10 BV / h. The eluent was collected when the pH of the cation exchange column effluent was >7. Collection was stopped when 10 ml of eluent was collected, and the collected eluent was directly purified by an anion exchange column.

[0233] 4g of D218 macroporous strong-base styrene-based anion exchange resin was packed into a column, and the anion exchange resin was activated according to the method in Example 3. The collected cation resin eluent was loaded onto the anion exchange resin, and the eluent was collected until 5ml was reached.

[0234] The collected solution obtained after anion exchange column separation was thermally concentrated to a specific gravity of 1.1. This concentrated liquid was then transferred to an alcohol precipitation tank, and 0.4 g of anhydrous ethanol was added with a stirrer at 100 rpm. After the ethanol addition was complete, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and dried under vacuum to obtain the hyacinth bulb extract.

[0235] The hyacinth bulb extract contains 3% alkaloids, 68% polysaccharides, 2% flavonoids, and 25% amino acids.

[0236] The content of 1-DNJ in the alkaloids is 30%.

[0237] The total heavy metal content was less than 5 ppm, lead content was 0.08 ppm, cadmium was not detected, mercury was 0.01 ppm, and arsenic was 0.15 ppm.

[0238] Example 16

[0239] Take 100g of fresh Commelina communis leaves, crush them, add 500ml of alcohol-water in two portions, and heat under reflux for 1 hour each time. Combine the extracts and filter to remove insoluble matter. Concentrate the extract by heat until the solid content reaches 10%, and keep it at 30℃ as the loading solution for cation exchange resin column.

[0240] 3.5 g of 732 type strong acid styrene-based cation exchange resin was packed into a column, and the cation exchange resin was activated according to the method in Example 3. The concentrated extract was loaded onto the column, and then eluted with 800 ml of 2.3 mol / L ammonia water at a rate of 10 BV / h. The eluent was collected when the pH of the cation exchange column effluent was >7, and collection was stopped when 300 ml of eluent was collected.

[0241] The collected liquid obtained after cation exchange column separation was filtered through an ultrafiltration membrane to remove impurities, and then concentrated using a reverse ion permeation membrane. The specific gravity of the concentrated liquid was 1.2. It was then transferred to an alcohol precipitation tank, and 5 g of anhydrous ethanol was added with a stirrer at 500 rpm. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and dried under vacuum to obtain the extract of Commelina communis leaf.

[0242] The extract of Commelina communis leaf contains 10% alkaloids, 27% polysaccharides, 10% flavonoids, and 50% amino acids.

[0243] The content of 1-DNJ in the alkaloids is 50%.

[0244] The total heavy metal content was less than 5 ppm, the lead content was 0.05 ppm, cadmium was not detected, mercury was not detected, and arsenic was 0.17 ppm.

[0245] Comparative Example 1

[0246] 1000 kg of fresh mulberry branches (Mulberry twigs with fine teeth) from the same batch as in Example 3 were taken and crudely extracted according to the method in Example 3. The crude extract was then concentrated by heat, and the specific gravity of the concentrated liquid was 1.1. It was transferred to an alcohol precipitation tank, and 62 kg of anhydrous ethanol was added at 400 rpm with a stirring paddle. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain a mulberry branch extract. The extract contained 15% alkaloids, 40% polysaccharides, 5.2% flavonoids, and 30% amino acids.

[0247] The content of 1-DNJ in the alkaloids is 55%.

[0248] The total heavy metal content was 30-40 ppm, with lead at 29.11 ppm, cadmium at 1.50 ppm, mercury at 0.76 ppm, and arsenic at 1.01 ppm. The levels of lead, cadmium, and mercury exceeded the standards.

[0249] After removing the cation and anion column separation steps, the comparative example showed a decrease in alkaloid content, a significant increase in heavy metal content, and a 3-fold increase in ethanol usage compared to Example 3.

[0250] Comparative Example 2

[0251] 1000 kg of fresh mulberry branches (Mulberry twigs with fine teeth) from the same batch as in Example 3 were taken and subjected to crude extraction, thermal concentration, and separation by cation exchange and anion exchange resins according to the method in Example 3. The 870 L collected liquid obtained after anion exchange column separation was transferred to an alcohol precipitation tank, and 135 kg of anhydrous ethanol was added at 400 rpm. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 h. The supernatant was collected and concentrated, and then concentrated under reduced pressure to obtain mulberry branch extract. The content of alkaloids was 62%, the content of polysaccharides was 18%, the content of flavonoids was 1.1%, and the content of amino acids was 12%.

[0252] The content of 1-DNJ in the alkaloids is 68%.

[0253] The total heavy metal content was 10-20 ppm, with lead at 10.01 ppm, cadmium at 0.70 ppm, mercury at 0.44 ppm, and arsenic at 0.83 ppm. Lead, cadmium, and mercury exceeded the standards.

[0254] This comparative example did not have a concentration step after resin separation and before alcohol precipitation. Compared with Example 3, the heavy metal content was increased and the amount of ethanol used was increased by 8 times.

[0255] Comparative Example 3

[0256] Take 1000 kg of fresh mulberry branches (fine-toothed mulberry) from the same batch as in Example 3, and perform crude extraction, heating concentration and separation by cation exchange resin and anion exchange resin according to the method of Example 3. The 870 L collected liquid obtained after separation by anion exchange column is concentrated under reduced pressure to obtain mulberry branch extract, wherein the content of alkaloids is 52%, the content of polysaccharides is 22%, the content of flavonoids is 0.8%, and the content of amino acids is 20%.

[0257] The content of 1-DNJ in the alkaloids is 60%.

[0258] The total heavy metal content was 20-40 ppm, with lead at 22.15 ppm, cadmium at 1.45 ppm, mercury at 0.65 ppm, and arsenic at 0.89 ppm. The levels of lead, cadmium, and mercury exceeded the standards.

[0259] In this comparative example, the alcohol precipitation step was removed after resin separation. Compared with Example 3, the alkaloid content was reduced and the heavy metal content was significantly increased.

[0260] Comparative Example 4

[0261] 1000 kg of fresh mulberry branches (Mulberry arborescens) from the same batch as in Example 3 were taken, pulverized, and then 4 times the amount of alcohol and water (4000 L) were added. Extraction was performed by reflux for 2 hours. The extracts were combined, filtered to remove insoluble matter, and the crude extract was thermally concentrated to a specific gravity of 1.1. This concentrated liquid was transferred to an alcohol precipitation tank, and 62 kg of anhydrous ethanol was added with a stirrer at 400 rpm. After the ethanol addition was complete, stirring was stopped, and alcohol precipitation was performed for 24 hours. The supernatant was then loaded onto a cation exchange resin column. Separation was performed using 150 kg of cation exchange resin and anion exchange resin, following the method described in Example 3. The collected liquid after anion exchange column separation was concentrated under reduced pressure to obtain a mulberry branch extract. The extract contained 51% alkaloids, 25% polysaccharides, 0.5% flavonoids, and 20% amino acids.

[0262] The content of 1-DNJ in the alkaloids is 55%.

[0263] The total heavy metal content was 10-20 ppm, with lead at 11.11 ppm, cadmium at 0.82 ppm, mercury at 0.50 ppm, and arsenic at 0.53 ppm. Lead, cadmium, and mercury exceeded the standards.

[0264] In this comparative example, alcohol precipitation was performed before the cation exchange resin separation step. Compared with Example 3, the alkaloid content was reduced, the heavy metal content was increased, and the amount of ethanol used was increased by 3 times.

[0265] Comparative Example 5

[0266] Take 1000 kg of fresh mulberry branches (Fine-toothed mulberry) from the same batch as in Example 3, and perform crude extraction and thermal concentration according to the method in Example 3. The concentrated crude extract is directly loaded onto an anion exchange resin, and anion exchange resin separation is performed according to the method in Example 3. Collect 3000 L of the eluent. Centrifuge the collected liquid after anion exchange column separation to remove impurities, and then concentrate it using nanofiltration. The specific gravity of the concentrated liquid is 1.1. Transfer it to an alcohol precipitation tank, and add 46 kg of anhydrous ethanol at 400 rpm. After the ethanol is added, stop stirring and allow it to precipitate for 24 hours. Collect the supernatant and concentrate it under reduced pressure to obtain the mulberry branch extract.

[0267] The mulberry twig extract contains 40% alkaloids, 35% polysaccharides, 1.5% flavonoids, and 22% amino acids.

[0268] The content of 1-DNJ in the alkaloids is 50%.

[0269] The total heavy metal content was 30-40 ppm, with lead at 30.01 ppm, cadmium at 1.24 ppm, mercury at 0.21 ppm, and arsenic at 0.85 ppm. The levels of lead, cadmium, and mercury exceeded the standards.

[0270] This comparative example omits the cation exchange resin separation step. Compared with Example 3, the alkaloid content is reduced, the heavy metal content is significantly increased, and the ethanol usage is increased by 2 times.

[0271] Stability Study of Experiment Example 1

[0272] Mulberry extract obtained in Example 1, mulberry leaf extract obtained in Examples 2, 8, and 9, mulberry twig extract obtained in Examples 3, 5-7, and mulberry bark extract obtained in Examples 4 and 10 were sealed in composite film bags and stored for 24 months at a temperature of 25℃±2℃ and a relative humidity of RH 60%±10%. The alkaloid content was then measured, and the results are shown in Table 1 below.

[0273] Table 1:

[0274]

[0275] As shown in Table 1 above, the plant extract obtained by the extraction method of the present invention has good stability.

[0276] Test Example 2: Residual Organic Solvents

[0277] Resin residues were detected by gas chromatography, including n-hexane, methylcyclohexane, divinylbenzene, toluene, benzene, xylene, and styrene. The plant extracts prepared in Examples 1-9 were tested, and no resin residues were detected.

[0278] Trial Example 3: Therapeutic Effect Trial

[0279] Normal male ICR mice were randomly divided into 6 groups (n=10) according to their body weight. The mice were fasted overnight before the experiment. One group was given sucrose solution (4.0 g / kg) orally as the control group (Normal). The other 5 groups were given sucrose orally and mulberry bark extract prepared in Example 1, mulberry leaf extract prepared in Example 2, and mulberry twig extract samples from Examples 3, 5, and 6 (all at 10 mg / kg based on total alkaloids) orally as the treatment groups. Blood glucose levels were measured before administration (0 min) and at 30 min, 60 min, and 120 min after administration. Blood glucose curves over time were plotted and the area under the blood glucose curve (AUC) was calculated. The results are shown in Figure 1.

[0280] The results showed that the plant extract obtained by the plant extraction method of the present invention could significantly reduce the increase in blood glucose after sucrose loading in normal mice.

[0281] The present invention has been described above with reference to preferred embodiments; however, these embodiments are merely exemplary and illustrative. Various substitutions and modifications can be made to the present invention based on these embodiments, all of which fall within the scope of protection of the present invention.

Claims

1. A plant extraction method comprising the following steps of: step 1): preparing a crude plant extraction solution; concentrating the crude plant extraction solution;step 2): separating the concentrated crude extraction solution via a cation resin and an anion resin to obtain a collection solution; wherein the cation resin is one or a combination of more selected from a strongly acidic cation exchange resin, a weakly acidic cation exchange resin, and a strongly alkaline quaternary ammonium-type cation resin;the amount of the cation resin used and the charged plant raw material have a mass ratio ranging from 1:1 to 1:30;an eluent for the cation resin is a salt solution containing cations or an alkaline solution containing cations; the cations in the eluent have a concentration ranging from 0.04 to 5 mol / L; collection begins when the pH of the effluent from the cation resin is higher than 7 or the starting point of the collection of the effluent is determined according to a precipitation reaction; the collection is terminated when the volume of the collection solution from the cation resin reaches 0.1 to 10 times the mass of the charged plant raw material; a mass of the eluent used for the separation via the cation resin is 0.1 to 30 times the mass of the charged plant raw material; the anion resin is one or a combination of more selected from a strongly alkaline anion exchange resin, a weakly alkaline anion exchange resin, and a weakly acidic anion exchange resin;the amount of the anion resin used and the charged plant raw material have a mass ratio ranging from 1:1 to 1:80; and the collection is terminated when the volume of the collection solution from the anion resin reaches 0.05 to 10 times the mass of the charged plant raw material;step 3): concentrating the collection solution obtained in step 2); andstep 4): subjecting the concentrated solution obtained in step 3) to alcohol precipitation, wherein ethanol used for the alcohol precipitation and the charged plant raw material have a mass ratio ranging from 1:4 to 1:600;wherein the plant is one of the plants of Moraceae;a plant extract obtained by the plant extraction method contains each component in the following mass ratios: alkaloids 30% to 99%; polysaccharides 0.2% to 35%; flavones 0.05% to 2%; amino acids 0% to 25%; and other components 0% to 20%; the alkaloids contain 1-deoxynojirimycin with a mass content of 50% to 99%, and the heavy metal content of the plant extract is not more than 10 ppm.

2. The extraction method according to claim 1, wherein the plant is any one or a combination of more selected from Morus multicaulis Perrott., Morus alba L., Morus atropurpurea Roxb, Morusmizuho Hotta, Morus wittiorum Hand Mazz., Morus laevigata Wall, Morus nigra Linn., Morus cathayana Hemsi., Morus serrata Roxb., Morus mongolica Schneid., Morus bombycis Koidz., Morus notabilis Schneid., Morus nigriformis Koidz., Morusyunnanensis Koidz., Morus australis Poir., Morus mongolica (Bur.) Schneid var. diabolica Koidz., Morus alba L. var. macrophylla loud, Morus alba Var.Pendula Dippel, Morus alba L. var. venosa Delili, and a hybrid mulberry obtained from selective intra-species or inter-species breeding of the above mulberry species.

3. The extraction method according to claim 2, wherein the plant is Morus atropurpurea Roxb, Morus multicaulis Perrott., Morus alba L., Morus serrata Roxb., or Morus bombycis Koidz.

4. The extraction method according to claim 3, wherein the hybrid mulberry is Yuesang 11, Guisangyou 62 or Sangteyou 2.

5. The extraction method according to claim 1, wherein the cation resin is one or a combination of more selected from a strongly acidic styrene-based cation exchange resin, a macroporous and strongly acidic styrene-based cation exchange resin, a macroporous and weakly acidic phenylpropene-based cation exchange resin, and a macroporous and strongly alkaline quaternary ammonium-type cation exchange resin.

6. The extraction method according to claim 1, wherein the cation resin is a strongly acidic styrene-based cation exchange resin, and a macroporous and strongly acidic styrene-based cation exchange resin.

7. The extraction method according to claim 1, wherein upon separation via the cation resin in step 2), the cation resin and the charged plant raw material have a mass ratio ranging from 1:1 to 1:25.

8. The extraction method according to claim 7, wherein upon separation via the cation resin in step 2), the cation resin and the charged plant raw material have a mass ratio ranging from 1:2 to 1:20.

9. The extraction method according to claim 1, wherein the mass of the eluent used for the separation via the cation resin in step 2) is 0.5 to 10 times the mass of the charged plant raw material.

10. The extraction method according to claim 1, wherein the anion resin is one or a combination of more selected from a strongly alkaline styrene-based anion exchange resin, a macroporous and strongly alkaline acrylic-based anion exchange resin, a macroporous and weakly acidic styrene-based anion exchange resin, and a macroporous and weakly alkaline styrene-based anion exchange resin.

11. The extraction method according to claim 10, wherein the anion resin is a strongly alkaline styrene-based anion exchange resin, a macroporous and strongly alkaline styrene-based anion exchange resin and a macroporous and strongly alkaline acrylic-based anion exchange resin.

12. The extraction method according to claim 1, wherein upon separation via the anion resin in step 2), the anion resin and the charged plant raw material have a mass ratio ranging from 1:1 to 1:64.

13. The extraction method according to claim 1, wherein upon separation via the anion resin in step 2), the anion resin and the charged plant raw material have a mass ratio ranging from 1:1 to 1:32.

14. The extraction method according to claim 1, wherein ethanol used for the alcohol precipitation in step 4) and the charged plant raw material have a mass ratio ranging from 1:20 to 1:300.

15. The extraction method according to claim 1, wherein the method further comprises step 5): concentrating and drying.

16. A plant extract obtained by the extraction method of any one of claims 1 to 15.

17. A pharmaceutical composition comprising the plant extract of claim 16.

18. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

19. The pharmaceutical composition according to claim 17, wherein a dosage form of the pharmaceutical composition includes an oral solid preparation and a liquid preparation.

20. The pharmaceutical composition according to claim 17, wherein a dosage form of the pharmaceutical composition includes a tablet, a capsule, an oral solution, an oral emulsion, a pill and a granule.

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