RU2818185C2 - Method of extracting from plants - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions relates to pharmaceutical industry, namely to a method for producing an extract, and to agents based on such an extract for reducing an increase in blood glucose. Method of extraction from plants includes the following stages: stage 1): obtaining a crude extraction solution from plants by using vegetable raw materials with the addition of water, alcoholic water or acidic water and extraction by boiling in a vessel with a reflux condenser, method of boiling or using ultrasonic extraction, and concentration of crude vegetable extraction solution; step 2): separation of the concentrated crude extraction solution using a cation-exchange resin and an anion-exchange resin to obtain a collected solution, wherein: cation-exchange resin is one or a combination of several selected from strongly acidic cation-exchange resin, weakly acidic cation-exchange resin and strongly alkaline cation-exchange resin of quaternary ammonium type; amounts of cation-exchange resin and plant material used have a weight ratio in range of 1:1 to 1:30; eluent for cation-exchange resin is a salt solution containing cations, or an alkaline solution containing cations, wherein the cations in the eluent have a concentration in range of 0.04 to 5 mol/l, wherein the collection from the cation-exchange resin is started, when the pH of effluent exceeds 7, or the initial collection point of the effluent is determined in accordance with the properties of the separated components using a precipitation reaction, and the collection from the cation-exchange resin is stopped when the volume of the collected solution reaches 0.1–10 times the weight of the plant material; weight of eluent used in separation with the help of cation-exchange resin is 0.1–30 times the weight of plant material, anion-exchange resin is one or a combination of several, selected from strongly alkaline anion-exchange resin and weakly alkaline anion-exchange resin; and the amount of the anion-exchange resin and the vegetal raw material used have a weight ratio in range of 1:1 to 1:80; collection from anion-exchange resin is stopped, when the volume of the collected solution reaches 0.05–10 times the weight of the plant material; step 3): concentrating the collected solution obtained in step 2); step 4): precipitating the concentrated solution obtained in step 3) with an alcohol using ethanol as the alcohol, wherein the ethanol used for the alcohol precipitation, and vegetal raw materials have a weight ratio in range of 1:4 to 1:600; and optionally, step 5): concentration and drying, wherein the plant is a Morus plant, wherein the plant extract obtained by the extraction method from plants contains each component in the following weight ratios with respect to the total weight of the plant extract: from 30% to 99% alkaloids; from 0.2% to 35% of polysaccharides; from 0.05% to 2% of flavones; from 0% to 25% of amino acids and from 0% to 20% of other components; wherein the content of heavy metals in the plant extract is not more than 10 ppm. Herbal extract, which reduces blood glucose level, obtained using the method described above. Pharmaceutical composition for reducing an increase in the blood glucose level containing the above plant extract. Use of the herbal extract or pharmaceutical composition in the preparation of a hypoglycemic drug. Medical product for reducing an increase in the blood glucose level containing a herbal extract. Use of the herbal extract when preparing a medical product for reducing an increase in the blood glucose level.

EFFECT: above-described method of producing the extract enables to effectively reduce the content of heavy metals in plant extracts, while significantly reducing the amount of ethanol used in the extraction method, thereby improving the quality of the product while reducing production costs and increasing the efficiency and safety of industrial production to a certain extent.

11 cl, 1 dwg, 12 tbl, 14 ex

Description

Field of technology to which the invention relates

The present invention relates to the field of extraction from plants, in particular to a method of extraction from plants.

State of the art

Natural plant extracts refer to products obtained using plants as raw materials and, in particular, to the preparation or concentration of one or more active components from plants without changing their active structure through physical and chemical extraction and separation according to the purpose of the final extracted product. Plant extracts contain rich and complex organic components, and most of the organic components have biological activities such as antibacterial activity, bacteriostatic activity, antioxidant activity and body immunity regulating activity. Due to the fact that they are environmentally friendly, healthy, safe, effective, waste-free, etc., plant extracts are widely used throughout the world in fields such as drugs, health products, cosmetics, food supplements, pesticides, feed and basic necessities. China is the cradle of silk weaving. Moraceae plants have been considered precious materials used as medicine and food since ancient times due to their high nutritional and medicinal value, which has always been reported in the classic works of traditional Chinese medicine, for example, even in the Compendium of Materia Medica there are the following words : “with a decoction of mulberry leaves you can quench your thirst instead of tea” and “with a drink after preparation and infusion you can quench your thirst instead of tea.” Modern science has also provided evidence for the presence of nutritional and pharmacologically active components in Moraceae plants, and has also studied the mechanisms of their action. Research has shown that the chemical components in Moraceae plants mainly include flavone compounds, polysaccharide compounds, alkaloids and amino acids, in addition to some volatile oils, tannin, succinic acid, adenine, vitamins, etc., and they have antifungal effects. anti-inflammatory effect, hypoglycemic effect, antioxidant effect, etc.

Extraction methods from plants can be divided into classical extraction methods and modern extraction methods. Classic extraction methods do not require special tools, are simple and easy to implement, do not require large extraction costs, and mainly include the solvent extraction method, steam distillation method, etc. Modern extraction methods are extraction methods based on modern advanced tools or recently developed extraction methods, and these mainly include ultrasonic extraction method, microwave extraction method, enzymatic extraction method, solid phase extraction method, etc. The components of plant extracts can be divided into lipophilic components and hydrophilic components, which can be obtained by selecting various solvents and/ or extraction methods for various plant extracts. For example, for lipophilic plant components, organic solvents can be used to perform percolation, cold leaching, ultrasonic extraction, microwave extraction, reflux extraction, etc.; As for water-soluble components, the commonly used extraction method is the water extraction and alcohol precipitation method, which is of great importance in the context of processing and purification, increasing the content of active components, reducing the administration dose and convenience in the production and molding of extracts in traditional Chinese medicine. Specific steps include aqueous extraction, concentration, alcohol precipitation, drying, etc., wherein alcohol precipitation refers to the addition of ethanol to the crude extraction solution until the concentration of ethanol reaches the appropriate concentration to ensure the precipitation of ethanol-insoluble impurities, and then performing solid-liquid separation to achieve the processing goal. For example, Xingjie Chang and colleagues extracted the active components of Forsythia according to the steps of decoction in water, concentration, ethanol precipitation and ethanol removal (Xingjie Chang, Qian Ding. Forsythiaside A, Forsythin and changes of the bacteriostatic activities thereof in the preparation process of water extraction and alcohol precipitation of Forsythia [J]. Guiding Journal of Traditional Chinese Medicine and Pharmacy, 2018, 24(9):39-41.); Aiying Shen and colleagues obtained mulberry leaf polysaccharides using a water extraction and ethanol precipitation method (Aiying Shen, Ziyu Zhu, Wenliang Zhang. Study on the extraction process of water-soluble polysaccharides from mulberry leaves [J]. Acta Sericologica Sinica, 2004, 30(3):277-279.). For further concentration and enrichment of the active components after precipitation with alcohol, resin separation, membrane separation, ultrafiltration, dialysis, etc. can also be carried out to achieve the purification goal.

The growing consumer demand for herbal extract products has prompted the in-depth development of plant extraction method, and the quality of products has been improved. However, compared with developed countries, there are still some serious shortcomings in Chinese herbal extracts. Among others, food safety issues such as heavy metal and pesticide residues have received much attention. During research, it was discovered that some plants are capable of enriching the environment with heavy metals (such as copper, lead, cadmium, zinc, etc.). For example, according to the results of a study conducted by Xing Zhang and colleagues, it was found that in case of growing mulberry tree in soil containing the following heavy metals, i.e. Cu (593.56 mg/kg), Pb (825.41 mg/kg), Cd (8.11 mg/kg), Zn (705.41 mg/kg), heavy metal content measured in roots is up to the following values: Cu: 33.13 mg/kg, Pb: 33.13 mg/kg, Cd: 4.53 mg/kg, Zn: 317.72 mg/kg, the content of heavy metals in leaves up to the following values: Cu: 13.18 mg/kg, Pb: 10.32 mg/kg, Cd: 1.90 mg/kg, Zn: 186.53 mg/kg, which is much higher than the relevant standards (according to the Standards for Green Trade in Medicinal Plants and preparations for import and export”, in raw materials, decoctions, extracts and preparations from plants, the total content of heavy metals should be 20.0 mg/kg or less, the Pb content should be 5 mg/kg or less, the Cd content should be 0, 3 mg/kg or less, Hg content should be 0.2 mg/kg or less, Cu content should be 20.0 mg/kg or less, and As content should be 2.0 mg/kg or less). An important aspect to consider when researching plant extraction methods is how to reduce residues of harmful substances such as heavy metals as much as possible.

Brief description of the invention

In general research and industrial production methods, water-soluble plant extracts are usually obtained by water extraction and alcohol precipitation, after alcohol precipitation, resin separation, membrane separation, ultrafiltration, dialysis, etc. are carried out for further enrichment and purification when considering increasing the content of a particular component. However, As for plant extracts, especially plants such as Moraceae, which are prone to the accumulation of heavy metals from the environment, the use of this extraction method to obtain extracts is insufficient to remove heavy metal residues in the extracts. The authors of the present invention, based on the results of repeated research, found that in the course of implementing the method of extraction from a plant, sequentially subjecting the crude extraction solution to resin separation, concentration and precipitation with alcohol can not only improve the separation efficiency of the active components, but can also significantly reduce heavy metal residues in plant extracts. Based on these results, the authors of the present invention have proposed a new method of extraction from plants, which can effectively reduce the content of heavy metals in plant extracts, while significantly reducing the amount of ethanol used in the extraction method, thereby improving the quality of the product while reducing production costs and up to a certain degree of increase in the efficiency and safety of industrial production.

In view of this, the first aspect of the present invention provides a plant extraction method comprising the following steps:

stage 1): obtaining a crude extraction solution from plants;

step 2): separating the crude extraction solution using a catine exchange resin and collecting the eluate, optionally separating the eluate using an anion exchange resin and collecting the effluent;

stage 3): concentration of the collected solution obtained in stage 2);

stages 4): precipitation of the concentrated solution obtained in stage 3) with alcohol; And

optional, step 5): concentration and drying.

1) Obtaining crude extraction solution from plants

In the present invention, the plant is preferably a plant from the family Moraceae, Liliaceae, Campanulaceae or Commelinaceae, further preferably a plant from the genus Moms, Hyacinthus, Adenophora or Commelina, and more preferably the plant is any plant or combination thereof 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 Hem si., 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, a mulberry variety bred from the above mulberry species, a mulberry hybrid obtained as a result of selective intraspecific and interspecific selection of the above mulberry species, Hyacinthus orientalis, Adenophora. triphylla var. japonica and Commelina communi; preferably the plant is Morus atropurpurea Roxb., Morus multicaulis Perrott., Morus alba L., Morus serrata Roxb., Morus bombycis Koidz. or a mulberry hybrid, the mulberry hybrid is preferably Yuesang No. 11, Guisangyou No. 62 or Sangteyou No. 2. Various parts such as leaf, root, branch, bark, bud, stem and fruit of the plant can be used.

The plant can be subjected to rough extraction with a solvent such as alcohol-water, water, an alkaline aqueous solution or an acidic aqueous solution, preferably the solvent used for the rough extraction is water.

During extraction, it is preferable to grind the plant and then add the resulting mass to water for thermal extraction, the extraction duration is preferably from 0.5 to 3 hours for each extraction, and the extraction itself is carried out from 1 to 3 times.

In a preferred embodiment, the crushed plant may be placed in an extraction tank to perform the extraction.

During extraction, the greater the amount of solvent added, the higher the rate of extraction from the plant. However, adding too much solvent may complicate subsequent separation and purification. The amount of solvent added is preferably 3-20 times and more preferably 4-15 times the weight of the loaded plant material, which allows for the greatest possible production of plant extract without excessively increasing the volume of the solution and increasing the complexity of subsequent processing.

The extraction can be carried out by a boiling method, an ultrasonic extraction method or a reflux extraction method, preferably it is carried out by a boiling method or a reflux extraction method, and more preferably it is carried out by a boiling method for which there is a more mature industrial equipment.

Optionally, the extraction can be repeated and the extraction solutions combined.

Preferably, the extraction solution is filtered to remove insoluble substances to obtain a crude plant extraction solution.

2) Separation using cation exchange resin and optional anion exchange resin

In the present invention, the components in the crude plant extraction solution are separated using an ion exchange resin.

In step 2), the crude plant extraction solution is loaded onto a cation exchange resin and separated through the cation exchange resin. Preferably, after packing into the column, the cation exchange resin is activated by successively washing with an acidic solution, an alkaline solution, and an acidic solution. The resin activation method also allows for adjustment of the pH value of the resin medium, thereby optimizing the selectivity of adsorption by the cation exchange resin and enhancing the separation effect.

Preferably, the resin is washed with an alkaline solution until the pH of the eluate is 8.0-9.5, preferably 8.5-9.5.

Preferably, the alkaline solution is an ammonia solution, a sodium hydroxide solution, a potassium hydroxide solution or a sodium carbonate solution, preferably an ammonia solution or a sodium hydroxide solution. Preferably, the concentration of the alkaline solution is 0.5-4 mol/L, preferably 1-2 mol/L.

Preferably, the resin is washed with an acidic solution until the pH of the eluate is 3.0-7.0, preferably 4.5-6.5.

Preferably, the acidic solution is selected from a hydrochloric acid solution, a phosphoric acid solution and a sodium phosphate-citric acid buffer, and more preferably a sodium phosphate-citric acid buffer. Preferably, the concentration of the acidic solution is 0.5-4 mol/L, preferably 1-2 mol/L.

When using sodium phosphate-citric acid buffer as an acidic solution, the pH of the sodium phosphate-citric acid buffer is preferably 4.0 to 6.5, and more preferably 4.5 to 5.0.

Optionally, the cation exchange resin can also be washed with 3 to 5 times the column volume of deionized water after the final acid wash.

Preferably, the cation exchange resin is one or a combination of a strong acid cation exchange resin, a weak acid cation exchange resin and a strong alkaline quaternary ammonium type cation exchange resin.

Preferably, the cation exchange resin is one or a combination of several selected from 732 type strong acid styrene cation exchange resin, 734 type strong acid styrene cation exchange resin, 002SC type strong acid styrene cation exchange resin, macroporous and strong acid cation exchange resin based on styrene type D001, macroporous and weakly acidic phenylpropene based cation exchange resin type D113 and macroporous and strongly alkaline cation exchange resin type D254 quaternary ammonium type.

Preferably, the cation exchange resin is a 732 type strong acid styrene cation exchange resin, a 734 type strong acid styrene cation exchange resin, and a D001 type macroporous and strong acid styrene cation exchange resin.

As a result of the study, it was found that, without being limited to any theory, the loading method of the cation exchange resin, especially the concentration of the loading solution and the amount of resin applied, has a significant impact on the adsorption and separation effects of plant components.

Preferably, the amount of the cation exchange resin used and the plant material charged has a mass ratio in the range of 1:1 to 1:30, preferably 1:1 to 1:25, and more preferably 1:2 to 1:20.

After loading the crude plant extraction solution onto the cation exchange resin, the loaded cation exchange resin is eluted with an eluent. Preferably, the eluent is a salt solution or an alkaline solution containing cations, preferably one or more selected from sodium chloride, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonia water, potassium chloride and sodium hydroxide.

Preferably, the concentration of cations in the eluent varies in the range of 0.04-5 mol/L, preferably 0.2-3 mol/L and more preferably 0.5-2.5 mol/L.

Preferably, the eluent flow rate is 1-15 BV/h (flow rate of the ion exchange filter in relative volumes per hour), preferably 5-10 BV/h.

Preferably, the mass of the eluent used for separation using the cation exchange resin is 0.1 to 30 times the mass of the loaded plant material. Preferably, elution is carried out with an eluent whose weight is 0.5-10 times greater than the weight of the loaded plant material.

Collection begins when the eluate begins to flow out of the cation exchange resin. The starting point of collection can be determined according to the pH of the effluent stream obtained from the cation exchange resin, for example, if an alkaline solution such as ammonia water is used to perform elution, collection begins when the pH of the effluent stream obtained from the cation exchange resin exceeds 7. Starting point collection can also be determined according to the properties of the components being separated, for example, the starting point for collection of the effluent can be determined using a chromogenic reaction or a precipitation reaction. Also, the starting point of collection can be determined using detection methods such as high performance liquid chromatography. Preferably, the collection is stopped when the volume of the collected solution reaches 0.1-10 times the weight of the loaded plant material, and more preferably, the collection is stopped when the volume of the collected solution reaches 0.2-5 times the weight of the loaded plant material.

During separation using the cation exchange resin, a fixed-bed ion exchange method can be used, and a continuous ion exchange method can also be used. Preferably, a continuous ion exchange method with a higher degree of automation is used.

To improve the separation effect of the cation exchange resin, it is also possible to perform multiple separations with the cation exchange resin, such as 2-5 times separation.

Optionally, the collected eluate is separated using an anion exchange resin. In separation using an anion exchange resin, the anion exchange resin, after packing into the column, is activated by successively washing with an alkaline solution, an acidic solution and an alkaline solution.

Preferably, the anoine exchange resin is washed with an acidic solution until the pH of the eluate is 3.0-7.0, preferably 4.5-6.5.

Preferably, the acidic solution is selected from a hydrochloric acid solution, a phosphoric acid solution and a sodium phosphate-citric acid buffer, and more preferably a sodium phosphate-citric acid buffer. Preferably, the concentration of the acidic solution is 0.5-4 mol/L, preferably 1-2 mol/L. When using sodium phosphate-citric acid buffer as an acidic solution, the pH of the sodium phosphate-citric acid buffer is preferably 4.0 to 6.5, and more preferably 4.5 to 5.0.

Preferably, the anoine exchange resin is washed with an alkaline solution until the pH of the eluate is 8.0-9.5, preferably 8.5-9.5.

Preferably, the alkaline solution is an ammonia solution, a sodium hydroxide solution, a potassium hydroxide solution or a sodium carbonate solution, preferably a sodium hydroxide solution. Preferably, the concentration of the sodium hydroxide solution is 0.5-4 mol/L, preferably 1-2 mol/L.

Preferably, the anion exchange resin is one or a combination of a strong alkali anion exchange resin, a weakly alkaline anion exchange resin and a weakly acidic anion exchange resin.

Preferably, the anion exchange resin is one or a combination of several selected from a 717 type high alkaline styrene anion exchange resin, a 711 type high alkaline styrene anion exchange resin, a macroporous resin, and a D201 type high alkaline styrene anion exchange resin, macroporous and D218 type acrylic based strong alkali anion exchange resin, D301-G type macroporous and weakly acidic styrene based anion exchange resin and D301 type D301 type macroporous and weakly alkaline styrene based anion exchange resin.

Preferably, the anion exchange resin is a 717 type high alkaline styrene anion exchange resin, a D201 type macroporous and high alkaline styrene anion exchange resin, and a D218 type macroporous and high alkaline acrylic anion exchange resin.

Preferably, the amount of the anion exchange resin used and the plant material loaded has a mass ratio in the range of 1:1 to 1:80, preferably 1:1 to 1:64, and more preferably 1:1 to 1:32.

Collection begins when liquid begins to flow out of the anion exchange resin. Preferably, the collection is stopped when the volume of the collected solution reaches 0.05-10 times the weight of the loaded plant material, and more preferably, the collection is stopped when the volume of the collected solution reaches 0.1-5 times the weight of the loaded plant material.

Optionally, to improve the separation effect of the anion exchange resin, multiple separations can also be performed with the anion exchange resin, for example, 2-4 times separation.

Preferably, the extraction method further includes the step of concentrating the crude plant extraction solution before separating in step 2).

Methods for concentrating the crude extraction solution include heat concentration, nanofiltration membrane concentration, reverse osmosis membrane concentration, and a combination thereof.

Concentration is preferably carried out by heat concentration, reverse osmosis membrane concentration, or a combination thereof so as to increase the concentration of the crude plant extraction solution.

Preferably, when using a reverse osmosis membrane and a nanofiltration membrane for concentration, in order to improve the concentration efficiency, the impurities can be removed by performing centrifugation, ultrafiltration membrane filtration or microfiltration membrane filtration before concentration with the reverse osmosis membrane and nanofiltration membrane .

Preferably, the crude plant extraction solution is concentrated until the mass concentration of solids in the solution is from 1% to 15%, preferably from 2% to 10%. Solids content refers to the solid matter remaining after removing water from solution.

Optionally, the concentrated crude extraction solution can also be subjected to alcohol precipitation prior to resin separation in step 2). During alcohol precipitation, ethanol is added to the crude extraction solution, the mixture is shaken and stirred evenly, stirring is stopped and the resulting mixture is kept for a certain period of time to precipitate insoluble substances in it. Preferably, ethanol is added to the crude plant extraction solution, the volume of ethanol being 0.2 to 20 times the volume of the crude plant extraction solution, preferably 0.4 to 10 times the volume of the crude plant extraction solution. More preferably, alcohol precipitation is carried out using an alcohol precipitation tank. Preferably, the stirring speed for alcohol precipitation is from 10 to 600 rpm, preferably from 40 to 500 rpm, and more preferably from 80 to 400 rpm.

3) Concentration of the collected solution obtained in step 2)

The concentration treatment methods in step 3) include heat concentration, nanofiltration membrane concentration, reverse osmosis membrane concentration, and a combination thereof. Preferably, the impurities can be removed by performing centrifugation, ultrafiltration membrane filtration or microfiltration membrane filtration before concentration through a reverse osmosis membrane and a nanofiltration membrane.

Preferably, the specific gravity of the concentrated liquid obtained in step 3) is from 1.0 to 1.3. Specific gravity refers to the mass ratio of a concentrated liquid to water under conditions in which the concentrated liquid and water have the same volume.

4) Precipitation with alcohol

During alcohol precipitation in step 4), the concentrated solution from step 3) is treated with ethanol. In particular, ethanol is added to the concentrated solution from step 3), the mixture is evenly shaken and stirred, the stirring is stopped and the resulting mixture is kept for a certain period of time to precipitate insoluble substances in it.

Preferably, in step 4, the ethanol used for alcohol precipitation and the loaded plant material have a mass ratio in the range of 1:4 to 1:600, preferably 1:20 to 1:300.

The alcohol precipitation is preferably carried out in an alcohol precipitation tank.

Preferably, in the alcohol precipitation in step 4, the stirring speed is from 10 to 600 rpm, preferably from 40 to 500 rpm, and more preferably from 80 to 400 rpm.

5) Concentration and drying

Optionally, the extraction method further comprises a concentration and drying step 5).

The solution, which has been precipitated with alcohol, is filtered to remove insoluble substances and concentrated under reduced pressure to obtain a plant extract as extractum or dried to obtain a dry product.

In a second aspect, the present invention provides a plant extract obtained in accordance with the extraction method described above.

Preferably, the plant extract obtained according to the above extraction method of the present invention contains alkaloids with a mass content of 3% or more (preferably contains alkaloids with a mass content of 3% to 99%, more preferably contains alkaloids with a mass content of 15% or more up to 99% and even more preferably contains alkaloids with a mass content of from 45% to 99%, such as from 35% to 70% or from 60% to 75%), and/or contains polysaccharides with a mass content of not more than 70% (preferably contains polysaccharides with a mass content of from 0.2% to 50%), and more preferably contains polysaccharides with a mass content of from 0.2% to 35%), and/or contains flavones with a mass content of not more than 10% (preferably contains flavones with mass content from 0.05% to 5% and more preferably contains flavones with a mass content from 0.05% to 2%), and/or contains amino acids with a mass content of not more than 50%) (preferably contains amino acids with a mass content from 0 %) to 40%) and more preferably contains amino acids with a mass content of from 0% to 25%), and/or other components (with a mass content of preferably from 0% to 25% and more preferably from 0% to 20%). The total content of all components is 100%, where “all components” refers generally to all components of the plant extract, including alkaloids, polysaccharides, flavones and amino acids. That is, in addition to alkaloids, polysaccharides, flavones and amino acids, the plant extract also contains other components.

Preferably, the plant extract obtained by the above-described extraction method of the present invention contains all components in the following mass ratios:

alkaloids from 3% to 99%; polysaccharides from 0.2% to 70%; flavones from 0% to 10%; amino acids from 0% to 50%; other components from 0% to 25%.

Preferably, the plant extract obtained by the above-described extraction method of the present invention contains all components in the following mass ratios:

alkaloids from 5% to 99%; polysaccharides from 0.2% to 50%; flavones from 0.05% to 5%; amino acids from 0% to 40%; other components from 0% to 20%.

Even more preferably, the plant extract obtained by the above-described extraction method of the present invention contains all components in the following mass ratios:

alkaloids from 30% to 99%; polysaccharides from 0.2% to 35%; flavones from 0.05% to 2%; amino acids from 0% to 25%; other components from 0% to 20%.

Preferably, the alkaloids contain 1-deoxynoirimicin (1-DNJ) in a weight content of 30% to 99%, preferably 50% to 95%, more preferably 55% to 90%, and more preferably 60% to 90%.

In a third aspect, the present invention provides a pharmaceutical composition comprising the above herbal extract and an optional pharmaceutically acceptable excipient.

An excipient is an inactive component that corresponds to the route of administration or method of administration and does not have a toxic effect on the human body.

The excipient may be a solid excipient or a liquid excipient. Solid excipients include, for example, sodium lactate, poloxamer, sodium dodecyl sulfate, sodium carboxymethylcellulose, gelatin, xanthan gum, povidone, starch, magnesium stearate, sodium carboxymethyl starch and talc. Liquid excipients include, for example, water, ethanol, syrup and glycerin.

Preferably, the dosage form of the pharmaceutical composition includes a preparation for oral administration.

Preferably, the dosage form of the pharmaceutical composition includes tablet, capsule, oral solution, oral emulsion, pill and granule.

There may be individual differences in the specific dose of administration depending on the patient's age, body weight, health status, diet, route of administration, drugs used in combination, treatment period, etc.

In a fourth aspect, the present invention provides the use of the above plant extract or the above pharmaceutical composition in the preparation of a hypoglycemic drug.

In another aspect, the present invention provides the use of the above plant extract or the above pharmaceutical composition in the preparation of a medicament for the treatment of abnormal glucose tolerance.

In yet another aspect, the present invention provides the use of the above herbal extract or the above pharmaceutical composition in the preparation of a medicament for preventing the development and/or treating a disease associated with abnormal blood glucose levels. Diseases include, but are not limited to, diabetes, diabetic nephropathy, diabetic foot, eye complications caused by diabetes, hyperglycemia, hyperuricemia, hyperlipidemia, changes in intestinal flora, and cardiovascular and cerebrovascular diseases such as cerebral infarction, cerebral hemorrhage, coronary heart disease and hypertension.

The present invention further provides the use of the above plant extract or the above pharmaceutical composition in the preparation of a lipid-lowering drug.

The present invention further provides the use of the above plant extract or the above pharmaceutical composition in the preparation of a medicament for regulating intestinal flora.

In yet another aspect, the present invention provides a food, medical product or beverage that contains the above herbal extract and an optional excipient suitable for the food, medical product or beverage.

The present invention further provides the use of the above plant extract in the preparation of a food product, medical product or beverage. Preferably, the food, health product or drink is a food, health product or drink with a hypoglycemic effect.

The plant material of the present invention refers to the plant material used for extraction, including, but not limited to, fresh or processed plant or fresh or processed plant parts.

In the present invention, the crude plant extraction solution is subjected to the processing steps of resin separation, concentration and alcohol precipitation, which produces the following beneficial effects compared to traditional extraction methods.

1. The content of heavy metals is significantly reduced.

2. The weight of ethanol used in the traditional water extraction and alcohol precipitation method is ¼ to 5 times the weight of the loaded plant material, while the amount of ethanol used in the extraction method of the present invention can be only 1/600 of the weight of the loaded plant material raw materials, which significantly reduces the amount of ethanol used, reduces production costs to a certain extent, facilitates industrial production and increases the safety of the production process.

Brief description of drawings

Figure 1 shows the results of a test of plant extracts obtained in examples 1, 2, 3, 5 and 6 in rats for sucrose tolerance.

Detailed description

The present invention will now be described in detail in connection with the accompanying drawings and examples. The features and advantages of the present invention will become better understood and defined from the given illustrative descriptions.

As used herein, the specific term “illustrative” means “used by way of example, or example, or explanation.” Any "illustrative" example presented herein should not necessarily be construed as superior or better than other examples.

Moreover, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

The present invention covers the following detection methods:

1. Determination of alkaloid content

An appropriate amount of extract was taken and water was added to dissolve it using ultrasound to obtain the test solution. Along with this, an appropriate amount of 1-deoxynoirimicin as a reference sample was accurately weighed out, and water was added to dissolve it to obtain a reference solution. Accurately measure the appropriate volumes of the reference solution and test solution respectively, add sodium bicarbonate solutions and mix thoroughly by shaking. Thereafter, a solution of 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) in acetone was added and heated at 30°C for 30 minutes. Acetic acid was added to stop the reaction. The reaction mixture was thoroughly mixed by shaking and filtered. The resulting filtrate was accurately collected and injected into a liquid chromatograph. Based on the peak area, the content of 1-deoxynoirimicin and the content of the total alkaloids in the test sample were calculated by calculation with an external standard (calculating chromatographic peaks with relative retention times in the range from 0.4 to 1.7 in terms of 1-deoxynoirimicin) (reference literature: Xuejun XIA, Renyun WANG, Yuling LIU. "Determination of mulberry twig alkaloids by RP-HPLC with pre-column derivatization" [J]. Chinese Journal of New Drugs, 2008, 17(23): 2044-2047).

2. Determination of amino acid content

An appropriate amount of extract was taken and water was added to dissolve it using ultrasound to obtain the test solution. Along with this, an appropriate amount of the amino acid mixture as a reference sample was accurately weighed out, and water was added to dissolve it to obtain a reference solution. The remaining operations were the same as for determining the alkaloid content.

3. Determination of polysaccharide content

The appropriate amount of extract was accurately weighed out, water was added, extracted with ultrasound and centrifuged at 4000 rpm for 10 min. The supernatant liquid was taken as the test solution. 2 ml of the above test solution was measured and placed in a stoppered test tube to which 6 ml of 0.1% anthrone-sulfuric acid reagent was added. The test tube was heated in a boiling water bath for 15 minutes and left in an ice bath for 15 minutes. The corresponding reagent was taken as a blank experiment. The absorbance value at 625 nm was immediately measured. The concentration of polysaccharides in the test sample relative to glucose was calculated using the linear regression equation of glucose, and their content was calculated using the following equation: Content = C*D*f/W, where W is the mass of the sample, C is the concentration of the polysaccharide relative to glucose, f - conversion factor (3.38), and D - dilution factor (reference literature: Zuofa ZHANG, Jie JIN, Liangen SHI. "Method for content determination of polysaccharide in Ramulus MorF", China Journal of Chinese Materia Medica [J]. 2018, 33(4): 462-464).

4. Determination of flavone content

An appropriate amount of rutin reference sample was weighed and dissolved in 60% ethanol to obtain a rutin reference stock solution. Accurately measured 0.5 ml, 1.0 ml, 3.0 ml, 5.0 ml and 7.0 ml of standard stock solutions of rutin, respectively, into 25 ml volumetric flasks, into which 10 ml of 5% sodium nitrite solution was added, respectively. 10%) solution of aluminum nitrate and 1 N. NaOH solution and then diluted with water to the desired scale and mixed thoroughly by shaking as a reference solution. A blank standard solution was used as a reference. The absorbance value was measured at 500 nm and a linear regression equation was constructed.

The appropriate amount of extract was accurately weighed, dissolved in a 60% ethanol solution using ultrasound, mixed thoroughly by shaking and centrifuged at 4000 rpm for 10 minutes. Thereafter, the supernatant was collected as a flavone extraction solution. 2.0 ml of flavone extraction solutions were accurately measured, to which were added, respectively, 10 ml of 5% sodium nitrite solution, 10% aluminum nitrate solution and 1 N. NaOH solution, then diluted with water to the required scale, mixed thoroughly by shaking, left for 15 minutes and centrifuged at 5000 rpm for 5 minutes. The supernatant was then measured for determination. An additional 2.0 mL of flavone extraction solution was accurately measured and diluted with water only to 25 mL as a blank reference solution in case of no color reaction. The absorbance values of the reaction solutions were measured at 500 nm. Flavone concentrations were calculated using a linear regression equation. Then the content of flavones in the sample was calculated in terms of rutin based on the weight of the sample and the dilution factor.

5. Determination of heavy metal content

The total content of heavy metals was determined by the second method from the General Rules section 0821, Volume IV, Chinese Pharmacopoeia, 2015 edition. The specific method was as follows:

(1) Obtaining a standard lead solution. 0.1599 g of lead nitrate was weighed out and placed in a 1000 ml volumetric flask. After dissolution with 5 ml of nitric acid and 50 ml of water, the solution was diluted with water to the desired scale and mixed thoroughly by shaking as a mother liquor. Accurately measure 0.5 ml, 1 ml, 2 ml, 5 ml and 8 ml of stock solutions into 5 ml volumetric flasks, respectively, dilute with water to the required scale and mix thoroughly by shaking, thus obtaining standard solutions of lead with a concentration of 5 ppm, 10 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm and 80 ppm.

(2) Sample analysis. 2 g of the sample was taken, slowly calcined until completely charred, cooled, moistened with exactly 0.5-1 ml of sulfuric acid, heated at low temperature until the sulfuric acid was completely removed, 0.5 ml of nitric acid was added, then evaporated to dryness, cooled after complete removal. removal of nitrogen oxide vapor, calcined at a temperature from 500°C to 600°C until complete ashing, cooled, added 2 ml of hydrochloric acid and evaporated to dryness in a water bath, followed by the addition of 15 ml of water. Ammonia solution was added dropwise until the phenolphthalein indicator solution turned slightly pink, and then 2 ml of acetate buffer (pH 3.5) was added. After dissolving the materials under low heat, the solution was transferred into Nessler tubes and the contents were diluted with water in 25 ml quantities as test tubes. Another reagent was taken to prepare the test solution and evaporated to dryness in a porcelain bowl, then 2 ml of acetate buffer (pH 3.5) and 15 ml of water were added, dissolved with gentle heating, then transferred to Nessler tubes, into which a certain amount of standard solution was added lead, described respectively in (1), and then diluted with water to 25 ml as reference tubes. Then, 2 ml of the thioacetamide test solution was added to the test tubes and reference tubes, respectively, mixed thoroughly by shaking, left for 2 minutes and simultaneously placed on white paper. Test tubes and reference tubes were viewed from a top-down perspective. The color in the test tubes was compared with the color in the reference tubes to determine the heavy metal content of the samples.

Content of heavy metals, i.e. lead, cadmium, mercury and arsenic can also be determined using induction plasma mass spectrometry (ICP-MS method), which is described in General Rule 0412 of Volume IV of the Chinese Pharmacopoeia, 2015 edition.

The heavy metal results were compared with data documented in the publication Green Trade Standards for Medicinal Plants and Preparations for Import and Export. The “Standards” state that plant materials, decoctions, extracts and preparations must have a total content of heavy metals ≤20.0 mg/kg, Pb≤5 mg/kg, Cd≤0.3 mg/kg, Hg≤2 mg /kg, Cu≤20.0 mg/kg and As≤2.0 mg/kg.

The present inventors compared two methods for detecting heavy metals. From the results, it was clear that the results of both methods were consistent, that is, the content of heavy metals in the plant extracts obtained by the method of the present invention complied with the provisions of the “Green Trade Standards for Medicinal Plants and Preparations for Import and Export”. Preferably, the content of heavy metals in the plant extracts obtained by the method of the present invention is not more than 20 ppm, more preferably not more than 10 ppm and even more preferably not more than 5 ppm.

Example 1

100 g of fresh mulberries (Morus alba L.) were taken and crushed, then 300 ml of alcoholic water was added in 2 times and extracted by boiling in a container with a reflux condenser each time for 1 hour. The extraction solutions were combined and filtered to remove insoluble substances, in resulting in a crude extraction solution. The crude extraction solution contained 5 to 10 ppm of heavy metals, including 5.44 ppm lead, 0.38 ppm cadmium, 0.06 ppm mercury, and 0.47 ppm arsenic. The crude extraction solution was concentrated by heating to 2% solids, kept at 25° C., and used as a loading solution for the cation exchange resin column.

The column was loaded with 5 g of a strongly acidic cation exchange resin based on type 732 styrene and washed with a 2.5 mol/L hydrochloric acid solution until the eluate pH was 3.5; washed with 1.5 mol/l sodium hydroxide solution until the eluate pH was 8.0; washed with 2.5 mol/l hydrochloric acid solution until the eluate pH was 3.5; and then washed with 3 times the column volume of deionized water to complete the activation. The concentrated extraction solution was loaded and then eluted with 3 L of 0.1 mol/L ammonia water at an elution rate of 10 BV/h. The eluate was collected when the effluent from the cation exchange resin column was found to have a pH >7. When the collected solution reached 1 L, collection was stopped. The collected solution was purified directly on a column with an anion exchange resin.

The column was loaded with 1.25 g of macroporous and highly alkaline acrylic-based anion exchange resin type D218, washed with 1.5 mol/L sodium hydroxide solution until the eluate pH was 9.0; washed with 1.5 mol/l hydrochloric acid solution until the eluate pH was 3.5; and washed with 1.5 mol/l sodium hydroxide solution until the eluate pH was 9.0; and completed the activation. The eluate collected from the cation exchange resin was loaded onto the anion exchange resin. The effluent was collected and collection was stopped when the volume of the effluent reached 1 L.

The collected solution obtained from separation on an anion exchange resin column was centrifuged to remove impurities and then concentrated through a reverse osmosis membrane. The specific gravity of the concentrated liquid was 1.0. It was transferred to an alcohol precipitation tank and 25 g of anhydrous ethanol was added at a stirring speed of 100 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and dried under vacuum to obtain an extract.

In addition, fresh mulberry branches, mulberry bark and mulberry leaves (Morus alba L.) were prepared and extracted. The extraction method and parameters were the same as described above. The content of heavy metals in the crude extraction solutions obtained from mulberry branches, mulberry bark and mulberry leaves ranged from 5 to 10 ppm, with the lead content being 5.51, 5.87 and 6.12 ppm, respectively, cadmium content were 0.37, 0.35 and 0.41 ppm, respectively, mercury content was 0.07, 0.08 and 0.06 ppm, respectively, arsenic content was 0.57, 0.55 and 0.61 ppm, respectively.

The content of components and the content of heavy metals in the obtained extracts of mulberry, mulberry branches, mulberry bark and mulberry leaves are shown in Table 1.

Example 2

100 g of fresh mulberry leaves (Morus atropurpurea Roxb) were taken and crushed, then 2000 ml of acidic water was added in 2 times and extracted by boiling each time for 1 hour. The extraction solutions were combined and filtered to remove insoluble substances, resulting in a crude extraction solution. The crude extraction solution contained 10 to 20 ppm of heavy metals, including 13.6 ppm lead, 0.84 ppm cadmium, 0.16 ppm mercury and 0.56 ppm arsenic. The crude extraction solution was centrifuged to remove impurities and then concentrated by reverse osmosis membrane filtration until the solids content reached 14.5%. The concentrated crude extraction solution was transferred to an alcohol precipitation tank and 80 g (approximately 100 ml) of anhydrous ethanol was added at a stirring speed of 300 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected as a loading solution for the cation exchange resin column.

The column was loaded with 10 g of a strongly acidic cation exchange resin based on type 734 styrene and washed with a 2 mol/L hydrochloric acid solution until the eluate pH was 4.5; washed with 1 mol/l sodium hydroxide solution until the eluate pH was 8.5; washed with 2 mol/l hydrochloric acid solution until the eluate pH was 4.5; and then washed with 5 times the column volume of deionized water to complete the activation. The extraction solution, after concentration and precipitation with alcohol, was loaded and then eluted with 2 L of 0.5 mol/L ammonia water at an elution rate of 8 BV/h. The eluate was collected when the effluent from the cation exchange resin column was found to have a pH >7. When the collected solution reached 800 ml, collection was stopped. The collected solution was purified directly on a column with an anion exchange resin.

8 g of a strongly alkaline anion exchange resin based on styrene 717 type was loaded into the column, washed with a 1.5 mol/L sodium hydroxide solution until the eluate pH was 9.0; washed with 1.5 mol/l hydrochloric acid solution until the eluate pH was 3.5; and washed with 1.5 mol/L sodium hydroxide solution until the eluate pH was 9.0, completing activation. The eluate collected from the cation exchange resin was loaded onto the anion exchange resin. The effluent was collected and collection was stopped when the effluent volume reached 750 mL.

The collected solution obtained by separation on an anion exchange resin column was concentrated by heating. The specific gravity of the concentrated liquid was 1.05. It was transferred to an alcohol precipitation tank, and 12.5 g of anhydrous ethanol was added at a stirring speed of 200 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and dried under vacuum to obtain an extract.

In addition, fresh mulberry branches and mulberry bark (Morus atropurpurea Roxb) were prepared and extracted. The extraction method and parameters were the same as described above. The content of heavy metals in the crude extraction solutions obtained from mulberry branches and mulberry bark ranged from 10 to 20 ppm, with the lead content being 14.5 and 15.8 ppm, respectively, and the cadmium content being 0.78 and 0.77 ppm respectively, the mercury content was 0.17 and 0.18 ppm, respectively, the arsenic content was 0.57 and 0.55 ppm, respectively.

The content of components and the content of heavy metals in the obtained extracts of mulberry branches, mulberry bark and mulberry leaves are given in Table 2.

Example 3

1000 kg of fresh mulberry branches (Yuesang No. 11) were taken and crushed, then 4000 L of water were added and extracted by refluxing for 2 hours. The extraction solutions were combined and filtered to remove insoluble materials, resulting in a crude extraction solution. The crude extraction solution contained 40 to 80 ppm of heavy metals, including 52 ppm lead, 1.94 ppm cadmium, 0.88 ppm mercury and 1.11 ppm arsenic. The crude extraction solution was concentrated by heating to 4% solids, kept at 50° C., and used as a loading solution for the cation exchange resin column.

The column was loaded with 150 kg of macroporous and weakly acidic cation exchange resin based on phenylpropene type D113, washed with a 2 mol/l hydrochloric acid solution until the eluate pH was 4.5; washed with 1 mol/l sodium hydroxide solution until the eluate pH was 8.5; washed with 2 mol/l hydrochloric acid solution until the eluate pH was 4.5; and then washed with 5 times the column volume of deionized water to complete the activation. The concentrated extraction solution was charged and then eluted with 1000 L of 2.5 mol/L ammonia water at an elution rate of 6 BV/h. The eluate was collected when the effluent from the cation exchange resin column was found to have a pH >7. When the collected solution reached 900 L, collection was stopped. The collected solution was purified directly on a column with an anion exchange resin.

The column was loaded with 62.5 kg of macroporous and highly alkaline acrylic-based anion exchange resin type D218, washed with 1.5 mol/L sodium hydroxide solution until the eluate pH was 9.0; washed with 1.5 mol/l hydrochloric acid solution until the eluate pH was 3.5; and washed with 1.5 mol/L sodium hydroxide solution until the eluate pH was 9.0, completing activation. The eluate collected from the cation exchange resin was loaded onto the anion exchange resin. The effluent was collected and collection was stopped when the volume of the effluent reached 870 L.

The collected solution obtained from separation on an anion exchange resin column was filtered through a microfiltration membrane to remove impurities and then concentrated through a reverse osmosis membrane. The specific gravity of the concentrated liquid was 1.1. It was transferred to an alcohol precipitation tank and 15 kg of anhydrous ethanol was added at a stirring speed of 400 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract.

In addition, fresh mulberry bark and mulberry leaves (Yuesang No. 11) were prepared and extracted. The extraction method and parameters were the same as described above. The content of heavy metals in the crude extraction solutions obtained from mulberry bark and mulberry leaves ranged from 40 to 80 ppm, with the lead content being 48 and 53 ppm, respectively, the cadmium content being 1.78 and 1.77 ppm, respectively, the content mercury was 0.77 and 0.78 ppm, respectively, arsenic was 0.87 and 0.95 ppm, respectively.

The content of components and the content of heavy metals in the obtained extracts of mulberry branches, mulberry bark and mulberry leaves are given in Table 3.

Example 4

1000 kg of air-dried mulberry bark (Guisangyou No. 62) was taken and crushed, then 10,000 liters of water were added in 2 times and extracted at reflux for 2.5 hours each time. The extraction solutions were combined and filtered to remove insoluble materials, resulting in a crude extraction solution. The crude extraction solution contained less than 5 ppm of heavy metals, including 1.57 ppm lead, 0.23 ppm cadmium, 0.09 ppm mercury and 0.58 ppm arsenic. The crude extraction solution was filtered through a microfiltration membrane to remove impurities and then concentrated through a reverse osmosis membrane until the solids content reached 6% and served as loading solution to the cation exchange resin column.

The column was loaded with 100 kg of macroporous and strongly acidic styrene-based cation exchange resin type D001. The cation exchange resin was activated according to the method described in example 3. The concentrated extraction solution was loaded and then eluted with 500 L of 0.2 mol/L ammonium chloride at an elution rate of 5 BV/h. The effluent was detected using 20% silicotungstic acid and began to be collected when a white precipitate formed. Collection was stopped when the volume of collected solution reached 200 L. The collected solution was purified directly on a column with an anion exchange resin.

The column was loaded with 32 kg of macroporous and highly alkaline styrene-based anion exchange resin type D201. The anion exchange resin was activated according to the method described in example 3. The eluate collected from the cation exchange resin was loaded onto the anion exchange resin. The effluent was collected and collection was stopped when the volume of the effluent reached 100 L.

The collected solution obtained by separation on an anion exchange resin column was concentrated by heating. The specific gravity of the concentrated liquid was 1.2. It was transferred to an alcohol precipitation tank and 3 kg of anhydrous ethanol was added at a stirring speed of 350 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract.

In addition, air-dried mulberry branches were prepared and extracted (Guisangyou No. 62). The extraction method and parameters were the same as described above. The crude extraction solution obtained from mulberry branches contained 5 to 10 ppm of heavy metals, with lead content being 1.66 ppm respectively, cadmium content being 0.25 ppm respectively, mercury content being 0.07 ppm respectively and arsenic was 0.60 ppm, respectively.

The content of components and the content of heavy metals in the obtained extracts of mulberry branches and mulberry bark are given in Table 4.

Example 5

10 kg of fresh mulberry branches (Sangteyou No. 2) were taken and crushed, then 150 liters of acidic water were added in 2 times and extracted by boiling each time for 3 hours. The extraction solutions were combined and filtered to remove insoluble substances. The extraction solution was concentrated by heating until the solids content reached 8%. It was transferred to a tank for precipitation with alcohol. 2367.9 g of anhydrous ethanol (3 L) was added while stirring with a stirrer at 300 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected as a loading solution for the cation exchange resin column.

The column was loaded with 5 kg of strong acid styrene-based cation exchange resin type 002SC. The cation exchange resin was activated according to the method described in example 3. The extraction solution, after concentration and precipitation with alcohol, was loaded and then eluted with 100 l of 5 mol/l potassium chloride at an elution rate of 5 BV/h. The effluent was detected using 20% silicotungstic acid and began to be collected when a white precipitate formed. Collection was stopped when the volume of collected solution reached 25 L. The collected solution was purified directly on a column with an anion exchange resin.

The column was loaded with 10 kg of a strong alkaline anion exchange resin based on type 711 styrene. The anion exchange resin was activated according to the method described in example 3. The eluate collected from the cation exchange resin was loaded onto the anion exchange resin. The effluent was collected and collection was stopped when the volume of the effluent reached 15 L. The collected solution was reloaded onto the cation exchange resin and separated sequentially through the cation exchange resin and the anion exchange resin twice in accordance with the methods described above.

The collected solution obtained after three column separations was centrifuged to remove impurities and then concentrated through a reverse osmosis membrane. The specific gravity of the concentrated liquid was 1.25. It was transferred to an alcohol precipitation tank and 125 g of anhydrous ethanol was added at a stirring speed of 1000 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract. In addition, fresh mulberry bark and mulberry leaves were prepared and extracted (Sangteyou No. 2). The extraction method and parameters were the same as described above. The content of components and content of heavy metals in the obtained extracts of mulberry branches, mulberry bark and mulberry leaves are given in Table 5.

Example 6

1 kg of fresh mulberry roots (Yuesang No. 11) was taken and ground, then 6 L of alcohol water was added 3 times and extracted using ultrasonic extraction for 1 hour each time. The extraction solutions were combined and filtered to remove insoluble substances to obtain the crude extraction solution. solution. The crude extraction solution served as the loading solution for the cation exchange resin column.

The column was loaded with 1 kg of macroporous and highly alkaline D254 quaternary ammonium cation exchange resin. The cation exchange resin was activated according to the method described in example 3. The crude extraction solution was loaded and then eluted with 15 L of 3 mol/L sodium chloride at an elution rate of 5 BV/h. The effluent was detected using 20% silicotungstic acid and began to be collected when a white precipitate formed. Collection was stopped when the volume of collected solution reached 5 L. The collected solution was purified directly on a column with an anion exchange resin.

The column was loaded with 1 kg of macroporous and weakly alkaline styrene-based anion exchange resin type D301. The anion exchange resin was activated according to the method described in example 3. The eluate collected from the cation exchange resin was loaded onto the anion exchange resin. The effluent was collected and collection was stopped when the volume of the effluent reached 5 L. The collected solution was reloaded onto the cation exchange resin and again separated sequentially through the cation exchange resin and anion exchange resin in accordance with the methods described above.

The collected solution obtained after two column separations was centrifuged to remove impurities and then concentrated through a reverse osmosis membrane. The specific gravity of the concentrated liquid was 1.2. It was transferred to an alcohol precipitation tank, and 6.3 g of anhydrous ethanol was added at a stirring speed of 600 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract. In addition, fresh mulberry branches and mulberry leaves were prepared and extracted (Yuesang No. 11). The extraction method and parameters were the same as described above. The content of components and content of heavy metals in the obtained extracts of mulberry branches, mulberry leaves and mulberry roots are given in Table 6.

Example 7

1000 kg of fresh mulberry branches (Morus atropurpurea Roxb) were taken and crushed, then 11500 liters of water were added and extracted by refluxing for 2 hours. The extraction solutions were combined and filtered to remove insoluble substances to obtain a crude extraction solution. The crude extraction solution was centrifuged to remove impurities and then concentrated through a reverse osmosis membrane until the solids content reached 1% and served as loading solution to the cation exchange resin column.

The column was loaded with 150 kg of macroporous and strongly acidic styrene-based cation exchange resin type D001. The cation exchange resin was activated according to the method described in example 3. The concentrated crude extraction solution was charged and eluted with 5000 L of 0.04 mol/L ammonium nitrate at an elution rate of 5 BV/h. The effluent was detected using 20% silicotungstic acid and began to be collected when a white precipitate formed. Collection was stopped when the volume of collected solution reached 1000 L.

The collected solution obtained by separation on a cation exchange resin column was concentrated through a nanofiltration membrane. The specific gravity of the concentrated liquid was 1.3. It was transferred to an alcohol precipitation tank and 1.7 kg of anhydrous ethanol was added at a stirring speed of 600 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract.

In addition, fresh mulberry bark and mulberry leaves (Morus atropurpurea Roxb) were prepared and extracted. The extraction method and parameters were the same as described above. The content of components and content of heavy metals in the obtained extracts of mulberry branches, mulberry bark and mulberry leaves are given in Table 7.

Example 8

1000 kg of fresh mulberry branches (Yuesang No. 11) were taken and crushed, then 8000 L of acidic water was added in 2 times and extracted by boiling method each time for 2 hours. The extraction solutions were combined and filtered to remove insoluble substances to obtain a crude extraction solution. The crude extraction solution was filtered through a microfiltration membrane to remove impurities, then concentrated through a reverse osmosis membrane until the solids content reached 1% and served as loading solution into the cation exchange resin column.

The column was loaded with 41.7 kg of a strong acid 732 type styrene cation exchange resin. The cation exchange resin was activated according to the method described in example 3. The crude extraction solution was charged and eluted with 1000 L of 0.1 mol/L sodium chloride at an elution rate of 5 BV/h. The effluent was detected using 20% silicotungstic acid and began to be collected when a white precipitate formed. Collection was stopped when the volume of collected solution reached 500 L.

The collected solution obtained by separation on a cation exchange resin column was concentrated using a nanofiltration membrane. The specific gravity of the concentrated liquid was 1.25. It was transferred to an alcohol precipitation tank and 15 kg of anhydrous ethanol was added at a stirring speed of 600 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract.

In addition, fresh mulberry bark and mulberry leaves (Yuesang No. 11) were prepared and extracted. The extraction method and parameters were the same as described above. The content of components and content of heavy metals in the obtained extracts of mulberry branches, mulberry bark and mulberry leaves are given in Table 8.

Example 9

100 g of fresh mulberry branches (Moms atropurpurea Roxb) were taken and crushed, then 600 ml of water was added, extracted by refluxing for 1 hour and filtered to remove insoluble matter to obtain a crude extraction solution. The crude extraction solution was first concentrated by heating to a solids content of 5% and used as a loading solution for the cation exchange resin column.

The column was loaded with 3.85 g of a strong acid 732 type styrene cation exchange resin. The cation exchange resin was activated according to the method described in example 3. The crude extraction solution was loaded and eluted with 700 ml of 0.15 mol/l ammonium chloride at an elution rate of 5 BV/h. The effluent was detected using 20% silicotungstic acid and began to be collected when a white precipitate formed. Collection was stopped when the volume of collected solution reached 100 ml.

The collected solution obtained by separation on a cation exchange resin column was concentrated by heating. The specific gravity of the concentrated liquid was 1.3. It was transferred to an alcohol precipitation tank and 25 g of anhydrous ethanol was added at a stirring speed of 600 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract.

In addition, fresh mulberry bark and mulberry leaves (Morus atropurpurea Roxb) were prepared and extracted. The extraction method and parameters were the same as described above. The content of components and the content of heavy metals in the obtained extracts of mulberry branches, mulberry bark and mulberry leaves are given in Table 9.

Example 10

1000 kg of fresh mulberry branches (Sangteyou No. 2) were taken and crushed, then 5000 L of water was added, extracted by refluxing for 1 hour, and filtered to remove insoluble matter to obtain a crude extraction solution. The crude extraction solution was first concentrated by heating to a solids content of 10% and used as a loading solution for the cation exchange resin column.

The column was loaded with 3.5 g of macroporous and highly alkaline D254 quaternary ammonium type cation exchange resin. The cation exchange resin was activated according to the method described in example 3. The crude extraction solution was loaded and then eluted with 200 L of 0.15 mol/L potassium chloride at an elution rate of 5 BV/h. The effluent was detected using 20% silicotungstic acid and began to be collected when a white precipitate formed. Collection was stopped when the volume of collected solution reached 100 L.

The collected solution obtained by separation on a cation exchange resin column was concentrated by heating. The specific gravity of the concentrated liquid was 1.05. It was transferred to an alcohol precipitation tank and 15 kg of anhydrous ethanol was added at a stirring speed of 600 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract.

In addition, fresh mulberry bark (Sangteyou No. 2) was prepared and extracted. The extraction method and parameters were the same as described above. The content of components and the content of heavy metals in the obtained extracts of mulberry branches and mulberry bark are given in Table 10.

Example 11

1000 g of fresh mulberry branches (Morus bombycis Koidz.) were taken and crushed, then Yul of acidic water was added in 3 times, extracted by ultrasonication for 2 hours each time and filtered to remove insoluble substances to obtain a crude extraction solution. The crude extraction solution was filtered through a microfiltration membrane to remove impurities, then concentrated through a reverse osmosis membrane until the solids content reached 4% and used as a loading solution into a cation exchange resin column.

The column was loaded with 66.67 g of macroporous and strongly acidic styrene-based cation exchange resin type D001. The cation exchange resin was activated according to the method described in example 3. The crude extraction solution was loaded and then eluted with 12 L of 1.5 mol/L ammonia water at an elution rate of 5 BV/h. The eluate was collected when the effluent from the cation exchange resin column was determined to contain alkaloids using high performance liquid chromatography. Collection was stopped when the volume of collected solution reached 100 ml. The collected solution was purified directly on a column with an anion exchange resin.

The column was loaded with 13.3 g of macroporous and highly alkaline styrene-based anion exchange resin type D218. The anion exchange resin was activated according to the method described in Example 3. The eluate collected from the cation exchange resin was loaded onto an anion exchange resin column. The effluent was collected and collection was stopped when the effluent volume reached 50 mL.

The collected solution obtained from separation on an anion exchange resin column was filtered through a microfiltration membrane to remove impurities and then concentrated through a reverse osmosis membrane. The specific gravity of the concentrated liquid was 1.15. It was transferred to an alcohol precipitation tank and 25 g of anhydrous ethanol was added at a stirring speed of 600 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract.

In addition, fresh mulberry bark and mulberry leaves (Morus bombycis Koidz.) were prepared and extracted. The extraction method and parameters were the same as described above. The content of components and the content of heavy metals in the obtained extracts of mulberry branches, mulberry bark and mulberry leaves are given in Table 11.

Example 12

100 g of fresh mulberry bark (Guisangyou No. 62) was taken and ground, then 1.2 L of alcohol water was added, extracted by boiling for 1 hour, and filtered to remove insoluble substances to obtain a crude extraction solution. The crude extraction solution was first concentrated by heating to a solids content of 8% and used as a loading solution for the cation exchange resin column.

The column was loaded with 33.34 g of a strong acid 734 type styrene cation exchange resin. The cation exchange resin was activated according to the method described in example 3. The crude extraction solution was loaded and then eluted with 50 ml of 2.5 mol/l ammonia water at an elution rate of 5 BV/h. The eluate was collected when the effluent from the cation exchange resin column was found to have a pH >7. Collection was stopped when the volume of collected solution reached 10 ml.

The collected solution obtained after separation on a cation exchange resin column was centrifuged to remove impurities and then concentrated through a reverse osmosis membrane. The specific gravity of the concentrated liquid was 1.2. It was transferred to an alcohol precipitation tank and 15 g of anhydrous ethanol was added at a stirring speed of 600 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract.

In the mulberry bark extract, the alkaloid content was 15%, the polysaccharide content was 38%, the flavone content was 2%, and the amino acid content was 40%. In alkaloids, the content of 1-DNJ was 52%.

Total heavy metal content was less than 5 ppm, including 0.29 ppm lead, 0 ppm cadmium, 0.02 ppm mercury and 0.10 ppm arsenic.

Example 13

100 g of fresh mulberry (Morus alba L.) branches were taken and crushed, then 300 ml of alkaline water was added, extracted by refluxing for 0.5 hour and filtered to remove insoluble substances to obtain a crude extraction solution. The crude extraction solution was centrifuged to remove impurities and then concentrated through a reverse osmosis membrane until the solids content reached 6% and used as a loading solution into a cation exchange resin column.

The column was loaded with 3.34 g of a strong acid 732-type styrene cation exchange resin. The cation exchange resin was activated according to the method described in example 3. The crude extraction solution was loaded and then eluted with 1.0 mol/L ammonia water at an elution rate of 5 BV/h. The effluent was detected using 20% silicotungstic acid and began to be collected when a white precipitate formed. Collection was stopped when the volume of collected solution reached 400 ml.

The collected solution obtained by separation on a cation exchange resin column was concentrated by heating. The specific gravity of the concentrated liquid was 1.25. It was transferred to an alcohol precipitation tank and 5 g of anhydrous ethanol was added at a stirring speed of 600 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract.

In the resulting extract of mulberry branches, the alkaloid content was 3%, the polysaccharide content was 60%, the flavone content was 5%, and the amino acid content was 30%. In alkaloids, the content of 1-DNJ was 47%.

Total heavy metal content was less than 5 ppm, including 0.31 ppm lead, 0 ppm cadmium, 0.01 ppm mercury and 0.14 ppm arsenic.

Example 14

100 g of fresh mulberry leaves (Morus multicaulis Perrott.) were taken and crushed, then 500 ml of alcoholic water was added, extracted by boiling for 0.5 hour and filtered to remove insoluble substances to obtain a crude extraction solution. The crude extraction solution was concentrated by a nanofiltration membrane to 12% solids and used as a loading solution for the cation exchange resin column.

The column was loaded with 25 g of a strong acid cation exchange resin based on type 732 styrene. The cation exchange resin was activated according to the method described in example 3. The crude extraction solution was loaded and then eluted with 2 L of 2.0 mol/L ammonia water at an elution rate of 5 BV/h. The eluate was collected when the effluent from the cation exchange resin column was determined to contain alkaloids using high performance liquid chromatography. Collection was stopped when the volume of collected solution reached 800 ml.

The collected solution obtained by separation on a cation exchange resin column was concentrated by heating. The specific gravity of the concentrated liquid was 1.14. It was transferred to an alcohol precipitation tank and 5 g of anhydrous ethanol was added at a stirring speed of 600 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract.

In the resulting mulberry leaf extract, the alkaloid content was 10%, the polysaccharide content was 43%, the flavone content was 1%, and the amino acid content was 37%. In alkaloids, the content of 1-DNJ was 50%.

Total heavy metal content was less than 5 ppm, including 0.33 ppm lead, 0.01 ppm cadmium, 0.02 ppm mercury and 0.15 ppm arsenic.

Example 15

100 g of fresh oriental hyacinth (Hyacinthus orientalis) bulbs were taken and crushed, then 700 ml of water was added in 2 times and extracted under ultrasound for 0.5 hours. The extraction solutions were combined and filtered to remove insoluble substances to obtain a crude extraction solution. The crude extraction solution was concentrated by a nanofiltration membrane to 10% solids and used as a loading solution for the cation exchange resin column.

The column was loaded with 3.5 g of a strong acid 732 type styrene cation exchange resin. The cation exchange resin was activated according to the method described in example 3. The concentrated crude extraction solution was charged and eluted with 10 ml of 1.75 mol/l sodium hydroxide solution at an elution rate of 10 BV/h. The eluate was collected when the effluent from the cation exchange resin column was found to have a pH >7. Collection was stopped when the volume of collected solution reached 10 ml. The collected solution was purified directly on a column with an anion exchange resin.

The column was loaded with 4 g of macroporous and highly alkaline styrene-based anion exchange resin type D218. The anion exchange resin was activated according to the method described in example 3. The eluate collected from the cation exchange resin was loaded onto the anion exchange resin. The effluent was collected and collection was stopped when the effluent volume reached 5 mL.

The collected solution obtained by separation on an anion exchange resin column was concentrated by heating. The specific gravity of the concentrated liquid was 1.1. It was transferred to an alcohol precipitation tank, and 0.4 g of anhydrous ethanol was added at a stirring speed of 100 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and dried under vacuum to obtain oriental hyacinth bulb extract.

In the resulting extract of oriental hyacinth bulbs, the alkaloid content was 3%, the polysaccharide content was 68%, the flavone content was 2%, and the amino acid content was 25%.

In alkaloids, the content of 1-DNJ was 30%.

The total content of heavy metals did not exceed 5 ppm, including 0.08 ppm lead, 0.01 ppm mercury and 0.15 ppm arsenic; no cadmium was detected.

Example 16

100 g of fresh leaves of Commelina communi were taken and crushed, then 500 ml of alcoholic water was added in 2 times and extracted by boiling in a container with a reflux refrigerator each time for 1 hour. The extraction solutions were combined and filtered to remove insoluble substances. The extraction solution was concentrated by heating to 10% solids, kept at 30° C., and used as a loading solution for the cation exchange resin column.

The column was loaded with 3.5 g of a strong acid 732 type styrene cation exchange resin. The cation exchange resin was activated according to the method described in example 3. The concentrated extraction solution was loaded and then eluted with 800 ml of 2.3 mol/l ammonia water at an elution rate of 10 BV/h. The eluate was collected when the effluent from the cation exchange resin column was found to have a pH >7. Collection was stopped when the volume of collected solution reached 300 ml.

The collected solution obtained from separation on a cation exchange resin column was filtered through an ultrafiltration membrane to remove impurities and then concentrated through a reverse osmosis membrane. The specific gravity of the concentrated liquid was 1.2. It was transferred to an alcohol precipitation tank and 5 g of anhydrous ethanol was added with a stirring speed of 500 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and dried under vacuum to obtain Commelina vulgaris leaf extract.

In the resulting extract of Commelina vulgaris leaves, the alkaloid content was 10%, the polysaccharide content was 27%, the flavone content was 10%), the amino acid content was 50%.

In alkaloids, the content of 1-DNJ was 50%.

The total content of heavy metals did not exceed 5 ppm, including 0.05 ppm lead and 0.17 ppm arsenic; cadmium and mercury were not detected.

Comparative example 1

1000 kg of fresh mulberry branches (Morus serrata Roxb.) from the same batch as in Example 3 were taken and subjected to crude extraction in accordance with the method described in Example 3. The crude extraction solution was concentrated by heating. 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 a stirring speed of 400 rpm.

After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain mulberry twig extract. The alkaloid content was 15%, the polysaccharide content was 40%, the flavone content was 5.2%, and the amino acid content was 30%.

In alkaloids, the content of 1-DNJ was 55%.

The total content of heavy metals ranged from 30 to 40 ppm, including 29.11 ppm lead, 1.50 ppm cadmium, 0.76 ppm mercury, 1.01 ppm arsenic, while the content of lead, cadmium and mercury exceeded the content standards.

Without the cation exchange resin column and anion exchange resin column separation steps, reduced alkaloid content, significantly increased heavy metal content, and a 3-fold increase in ethanol content were observed in this comparative example compared to Example 3.

Comparative example 2

1000 kg of fresh mulberry branches (Moms serrata Roxb.) from the same batch as in Example 3 were weighed and subjected to crude extraction, heating concentration and separation by cation exchange resin and anion exchange resin in accordance with the method described in Example 3. 870 L of the collected solution obtained by separation on an anion exchange resin column was transferred to an alcohol precipitation tank, and 135 kg of anhydrous ethanol was added at a stirring speed of 400 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain mulberry twig extract. The alkaloid content was 62%, the polysaccharide content was 18%, the flavone content was 1.1%, and the amino acid content was 12%.

In alkaloids, the content of 1-DNJ was 68%.

The total content of heavy metals ranged from 10 to 20 ppm, including 10.01 ppm lead, 0.70 ppm cadmium, 0.44 ppm mercury, 0.83 ppm arsenic, while the content of lead, cadmium and mercury exceeded the content standards.

Without the concentration step between resin separation and alcohol precipitation, this comparative example showed increased heavy metal content and an 8-fold increase in ethanol compared to example 3.

Comparative example 3

1000 kg of fresh mulberry branches (Morus serrata Roxb.) from the same batch as in Example 3 were weighed and subjected to crude extraction, heating concentration and separation by cation exchange resin and anion exchange resin in accordance with the method described in Example 3. 870 L of the collected solution obtained by separation on an anion exchange resin column was concentrated under reduced pressure to obtain mulberry twig extract. The alkaloid content was 52%, the polysaccharide content was 22%, the flavone content was 0.8%, and the amino acid content was 20%.

In alkaloids, the content of 1-DNJ was 60%.

The total content of heavy metals ranged from 20 to 40 ppm, including 22.15 ppm lead, 1.45 ppm cadmium, 0.65 ppm mercury, 0.89 ppm arsenic, while the content of lead, cadmium and mercury exceeded the content standards.

Without the alcohol precipitation step after resin separation, in this comparative example, a reduced alkaloid content and a significantly increased heavy metal content were observed compared to Example 3.

Comparative example 4

1000 kg of fresh mulberry branches (Morus serrata Roxb.) from the same batch as in example 3 were taken and crushed, then 4 times the volume of alcoholic water (4000 l) was added and extracted by heating in a container under reflux for 2 hours. The extraction solutions were combined and filtered to remove insoluble substances. The crude extraction solution was concentrated by heating. 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 a stirring speed of 400 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and loaded onto a cation exchange resin. According to the method described in Example 3, 150 kg of cation exchange resin was loaded onto the column and subjected to cation exchange resin separation and anion exchange resin separation. The collected solution obtained by separation on an anion exchange resin column was concentrated under reduced pressure to obtain mulberry twig extract. The alkaloid content was 51%, the polysaccharide content was 25%, the flavone content was 0.5%, and the amino acid content was 20%.

In alkaloids, the content of 1-DNJ was 55%.

The total content of heavy metals ranged from 10 to 20 ppm, including 11.11 ppm lead, 0.82 ppm cadmium, 0.50 ppm mercury, 0.53 ppm arsenic, while the content of lead, cadmium and mercury exceeded the content standards.

In this comparative example, alcohol precipitation was carried out before the separation step on a cation exchange resin and a reduced alkaloid content, an increased heavy metal content and a 3-fold increase in the amount of ethanol were observed compared to Example 3.

Comparative Example 5

1000 kg of fresh mulberry branches (Morus serrata Roxb.) from the same batch as in example 3 were taken and subjected to crude extraction and heating concentration in accordance with the method described in example 3. The concentrated crude extraction solution was loaded directly onto the anion exchanger. resin and subjected to separation on an anion exchange resin in accordance with the method described in example 3. 3000 L of the effluent was collected. The collected solution obtained after separation on an anion exchange resin column was centrifuged to remove impurities and then concentrated through a nanofiltration membrane. The specific gravity of the concentrated liquid was 1.1. It was transferred to an alcohol precipitation tank and 46 kg of anhydrous ethanol was added at a stirring speed of 400 rpm. After adding ethanol, stirring of the solution was stopped and it was subjected to precipitation with alcohol for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain mulberry twig extract.

In the mulberry branch extract, the alkaloid content was 40%, the polysaccharide content was 35%, the flavone content was 1.5%, and the amino acid content was 22%.

In alkaloids, the content of 1-DNJ was 50%.

The total content of heavy metals ranged from 30 to 40 ppm, including 30.01 ppm lead, 1.24 ppm cadmium, 0.21 ppm mercury, 0.85 ppm arsenic, while the content of lead, cadmium and mercury exceeded the content standards.

Without the separation step on the cation exchange resin, in this comparative example, reduced alkaloid content, significantly increased heavy metal content, and a 2-fold increase in ethanol content were observed compared to Example 3.

Test Case 1: Stability Study

The mulberry extract obtained in Example 1, mulberry leaf extracts obtained in Examples 2, 8 and 9, mulberry twig extracts obtained in Examples 3 and 5-7, and mulberry bark extracts obtained in Examples 4 and 10 were packaged into bags made of composite film and sealed, then kept for 24 months at a temperature of 25 ° C ± 2 ° C and a relative humidity of 60% ± 10%, after which the content of alkaloids was checked. The results are shown in Table 1 below.

From Table 1 it can be seen that the plant extracts obtained by the extraction method of the present invention have good stability.

Test Case 2: Residual Organic Solvents

Gas chromatography was used to detect residual tars, including n-hexane, methylcyclohexane, divinylbenzene, toluene, benzene, xylene and styrene. The plant extracts obtained in Examples 1-9 were tested and none of them contained residual gum.

Test case 3. Efficiency test

Normal male ICR mice were randomly divided into 6 groups (n=10) based on body weight and fasted overnight before testing. One of the groups was orally administered sucrose solutions (4.0 g/kg) as a control group (norm), while the other 5 groups were orally administered sucrose, as well as a sample of mulberry bark extract obtained in Example 1, a sample of leaf extract mulberry obtained in Example 2, and samples of mulberry twig extract obtained in Examples 3, 5 and 6 (10 mg/kg each based on total alkaloids) as administration groups. Blood glucose levels were measured before administration (0 min) and 30 min, 60 min and 120 min after administration. A curve of blood glucose level versus time was plotted and the area under the curve (AUC) of blood glucose level was calculated. The results were as shown in Figure 1.

From the results, it can be seen that the plant extracts obtained by the plant extraction method of the present invention lead to a significant reduction in the increase in blood glucose levels in normal mice after sucrose loading.

The present invention has been explained above on the basis of preferred embodiments, which, however, are only illustrative and demonstrative. Based on this, various substitutions and improvements can be made to the present invention, all of which fall within the scope of protection of the present invention.

Claims (31)

1. A method of extraction from plants, involving the following stages: stage 1): obtaining a crude extraction solution from plants by using plant raw materials with the addition of water, alcoholic water or acidic water and extracting by boiling in a container with reflux, by boiling or using ultrasonic extraction, and concentrating the crude plant extraction solution; step 2): separating the concentrated crude extraction solution using a cation exchange resin and an anion exchange resin to obtain a collected solution, wherein: the cation exchange resin is one or a combination of several selected from a strong acid cation exchange resin, a weak acid cation exchange resin and a strong alkaline quaternary ammonium type cation exchange resin; the amounts of cation exchange resin and plant raw materials used have a mass ratio in the range from 1:1 to 1:30; the eluent for the cation exchange resin is a saline solution containing cations or an alkaline solution containing cations, the cations in the eluent having a concentration in the range of 0.04 to 5 mol/L; wherein collection from the cation exchange resin begins when the pH of the effluent stream exceeds 7, or the starting point of collection of the effluent stream is determined in accordance with the properties of the separated components using a precipitation reaction, and collection from the cation exchange resin is stopped when the volume of the collected solution reaches 0.1-10 multiple weight of plant raw materials; the mass of the eluent used for separation using a cation exchange resin is 0.1-30 times the mass of the plant material, the anion exchange resin is one or a combination of several selected from a strong alkali anion exchange resin and a weak alkali anion exchange resin; And the amounts of anion exchange resin and plant raw materials used have a mass ratio in the range from 1:1 to 1:80; collection from the anion exchange resin is stopped when the volume of the collected solution reaches 0.05-10 times the mass of the plant material; stage 3): concentration of the collected solution obtained in stage 2); step 4): precipitation of the concentrated solution obtained in step 3) with alcohol using ethanol as the alcohol, wherein the ethanol used for alcohol precipitation and the plant material have a mass ratio ranging from 1:4 to 1:600; And optional step 5): concentration and drying, wherein the plant is a Morus plant, wherein the plant extract obtained by extraction from plants contains each component in the following mass ratios relative to the total weight of the plant extract: from 30% to 99% alkaloids; from 0.2% to 35% polysaccharides; from 0.05% to 2% flavones; from 0% to 25% amino acids; and from 0% to 20% other components; and the content of heavy metals in the plant extract is no more than 10 ppm. 2. The extraction method according to claim 1, wherein the plant is any plant or combination thereof 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 noiabilis 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, a mulberry variety bred from the above mulberry species, a mulberry hybrid obtained as a result of selective intraspecific and interspecific selection of the above mulberry species; preferably the plant is Morus atropurpurea Roxb., Morus multicaulis Perrott., Morus alba L., Morus serrata Roxb., Morus bombycis Koidz. or a mulberry hybrid, the mulberry hybrid is preferably Yuesang No. 11, Guisangyou No. 62 or Sangteyou No. 2. 3. The extraction method according to claim 1 or 2, wherein the cation exchange resin is one or a combination of several selected from a 732 type strong acid styrene cation exchange resin, a 734 type strong acid styrene cation exchange resin, a strong acid 734 type styrene cation exchange resin styrene-based type 002SC, macroporous and strongly acidic styrene-based cation exchange resin type D001, macroporous and weakly acidic phenylpropene-based cation exchange resin type D113 and macroporous and strongly alkaline cation exchange resin type D254 quaternary ammonium type; preferably, the cation exchange resin is a 732 type strong acid styrene cation exchange resin, a 734 type strong acid styrene cation exchange resin, and a D001 type macroporous and strong acid styrene cation exchange resin; preferably, when separated by a cation exchange resin in step 2), the cation exchange resin and the plant material have a mass ratio in the range of 1:1 to 1:25, and more preferably 1:2 to 1:20; And preferably, the mass of the eluent used in the separation using the cation exchange resin in step 2) is 0.5 to 10 times the mass of the plant material. 4. Extraction method according to any one of paragraphs. 1-3, wherein the anion exchange resin is one or a combination of a 717 type high alkaline styrene anion exchange resin, a 711 type high alkaline styrene anion exchange resin, a macroporous resin, and a D201 high alkaline styrene anion exchange resin , macroporous and strongly alkaline acrylic-based anion exchange resin type D218h; macroporous and weakly alkaline styrene-based anion exchange resin type D301; And preferably, the anion exchange resin is a 717 type high alkaline styrene anion exchange resin, a D201 type macroporous and high alkaline styrene anion exchange resin, and a D218 type macroporous and high alkaline acrylic anion exchange resin; preferably, when separated by the anion exchange resin in step 2), the anion exchange resin and the plant material have a mass ratio in the range of 1:1 to 1:64, and more preferably from 1:1 to 1:32. 5. Extraction method according to any one of paragraphs. 1-4, and the ethanol used for alcohol precipitation in step 4 and the plant material have a mass ratio in the range from 1:20 to 1:300. 6. Plant extract that reduces blood glucose levels, obtained by the extraction method according to any one of paragraphs. 1-5. 7. A pharmaceutical composition for reducing the increase in blood glucose levels, containing the herbal extract according to claim 6 and an optional pharmaceutically acceptable excipient. 8. The pharmaceutical composition according to claim 7, wherein the dosage form of the pharmaceutical composition includes an oral solid preparation and a liquid preparation, preferably the dosage form of the pharmaceutical composition includes a tablet, capsule, oral solution, oral emulsion, pill and granule. 9. Use of a plant extract according to claim 6 or a pharmaceutical composition according to claim 7 or 8 in the preparation of a hypoglycemic drug. 10. A medical product for reducing the increase in blood glucose levels, containing the plant extract according to claim 6. 11. The use of the plant extract according to claim 6 when obtaining a medical product to reduce the increase in blood glucose levels.