Mulberry leaf extract polycystic liposome, preparation method and hypoglycemic application thereof

Abstract

The present application relates to the field of food health care technology, and particularly relates to mulberry leaf extract polycapsule liposome, a preparation method, health food and a blood sugar reducing application thereof; the mulberry leaf extract polycapsule liposome comprises mulberry leaf extract and blank polycapsule liposome; the mulberry leaf extract comprises mulberry leaf flavone and mulberry leaf alkaloid; the blank polycapsule liposome is composed of lipid bilayer, and comprises a plurality of aqueous chambers inside and is separated by the lipid bilayer between the aqueous chambers; wherein the mulberry leaf flavone is located between the lipid bilayers, and the mulberry leaf alkaloid is located inside the aqueous chambers. The mulberry leaf extract polycapsule liposome provided by the present application can simultaneously achieve embedding of the mulberry leaf flavone and the mulberry leaf alkaloid, thereby better achieving a blood sugar reducing effect.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, specifically to a mulberry leaf extract multicyst liposome, its preparation method, and its hypoglycemic application. Background Technology

[0002] Mulberry leaves are from the plant *Morus alba* (Moraceae family). Mours albaL The dried leaves of mulberry (Mulberry leaf) have a long history of use as a common traditional Chinese medicine. The Compendium of Materia Medica records that "a decoction of mulberry leaves can be used as a tea substitute to treat diabetes." Modern pharmacological studies have also shown that mulberry leaves have various pharmacological effects, including lowering blood sugar, lowering blood lipids, lowering blood pressure, anti-oxidation, anti-inflammation, and regulating the body's immunity, with the hypoglycemic effect being the most prominent. Currently, mulberry leaves can be used both as medicine and food, and have been included in the list of medicinal and edible substances by the Chinese health authorities, possessing significant medicinal and edible value.

[0003] Existing reports have demonstrated that mulberry leaf flavonoids and mulberry leaf alkaloid DNJ are the main hypoglycemic active ingredients, which can inhibit the activity of disaccharidases (sucrase, isomaltase, maltase) in the small intestine, and are good α-glucosidase inhibitors. In addition, mulberry leaf flavonoids and DNJ can also stimulate the secretion of GLP-1 in the small intestine, thereby lowering blood sugar through multiple pathways, such as promoting insulin secretion, delaying gastric emptying, and strengthening central appetite suppression to reduce food intake.

[0004] Mulberry leaf flavonoids are mainly composed of rutin, quercetin, and kaempferol nuclei, and their poor water solubility and absorption result in extremely low bioavailability. Although mulberry leaf alkaloid DNJ is water-soluble, its short half-life of approximately 2 hours in the human body affects the sustained blood glucose control effect. The low bioavailability of mulberry leaf flavonoids and the short half-life of mulberry leaf alkaloid DNJ both contribute to the final blood glucose-lowering effect of mulberry leaves.

[0005] Chinese patent document CN109350746A discloses a method for preparing a sustained-release formulation of 1-deoxynojirimycin (i.e., mulberry leaf alkaloid DNJ) using mulberry leaf polysaccharide as a carrier, comprising the following steps: first synthesizing Pluronic-polyethyleneimine (PEI), then synthesizing Pluronic-polyethyleneimine (PEI)-phenylboronic acid (PBA), then synthesizing Pluronic-polyethyleneimine (PEI)-phenylboronic acid (PBA)-mulberry leaf polysaccharide nanocarrier, and finally obtaining a sustained-release formulation of 1-deoxynojirimycin; Chinese patent document CN115252677A discloses a mulberry leaf prebiotic based on intestinal probiotic regulation of blood glucose homeostasis and its preparation method, which encapsulates mulberry leaf alkaloids and mulberry leaf polysaccharides through a gel method to achieve the purpose of sustained release of mulberry leaf alkaloids. However, the above patents do not involve the encapsulation of dual-functional components of mulberry leaf flavonoids and mulberry leaf alkaloid DNJ; therefore, there is an urgent need for a method that can effectively encapsulate mulberry leaf flavonoids and mulberry leaf alkaloid DNJ. Summary of the Invention

[0006] The purpose of this invention is to provide a multi-capsule liposome of mulberry leaf extract, its preparation method, and its hypoglycemic application, so as to overcome the problems of existing technologies with dual-function components of mulberry leaf flavonoids and mulberry leaf alkaloid DNJ, such as insolubility or poor stability in aqueous solution systems, easy precipitation, organic solvent residue, long processing time, and high cost.

[0007] According to a first aspect of this invention, a mulberry leaf extract multicyst liposome is provided, the mulberry leaf extract multicyst liposome comprising mulberry leaf extract and blank multicyst liposome; the mulberry leaf extract comprising mulberry leaf flavonoids and mulberry leaf alkaloids; the blank multicyst liposome is composed of a lipid bilayer, the blank multicyst liposome containing a plurality of aqueous chambers separated by the lipid bilayer; wherein the mulberry leaf flavonoids are located between the lipid bilayers, and the mulberry leaf alkaloids are located within the aqueous chambers.

[0008] In one aspect of the invention, the blank multicyst liposomes have a particle size of 10-60 μm.

[0009] In one aspect of the invention, in the mulberry leaf extract polycystic liposomes, the mass ratio of the mulberry leaf extract to the blank polycystic liposomes is selected from 1:1 to 1:5.

[0010] In one aspect of the invention, the mulberry leaf flavonoids include compounds such as rutin (vitamin P, rutin), quercetin, isoquercitrin, and quercetin-3-triglucoside.

[0011] In one aspect of the present invention, the mulberry leaf alkaloids include mulberry leaf DNJ (1-deoxynojirimycin), N-methyl-1-DNJ (N-Me-DNJ), 2-oxo-α-D-galactopyranoside-1-DNJ, fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol, 1,4-dideoxy-1,4-imino-(2-oxo-β-D-glucopyranoside)-D-arabinitol, and 1α,2β,3α,4β-tetrahydroxy-norhydantoin (norhydantoin), etc.; specifically, the mulberry leaf alkaloids include mulberry leaf DNJ (1-deoxynojirimycin).

[0012] In one aspect of the invention, blank multivesicular liposomes, or multivesicular liposomes (MVLs), are composed of multiple non-concentric drug aqueous solution vesicles separated by a continuous non-concentric lipid bilayer. This results in a high encapsulation efficiency for both water-soluble and lipid-soluble drugs. Specifically, MVLs release the drug through ruptured vesicles, while intact vesicles remain intact. This non-concentric topological structure of MVLs allows them to form a "reservoir" at the release site. As the lipid bilayer degrades, the encapsulated drug is gradually released, producing a good sustained-release effect while avoiding burst release. Furthermore, by adjusting the formulation and process parameters, the drug release time can be controlled from several days to several weeks.

[0013] According to a second aspect of this invention, a method for producing mulberry leaf extract multicyst liposomes is provided, the method comprising the following steps:

[0014] Step 1: Prepare an aqueous extract containing mulberry leaf extract;

[0015] Step 2: Mix the aqueous extract containing mulberry leaf extract with the oil phase and prepare a water-in-oil double emulsion at a shear rate of 5000-9000 rpm.

[0016] Step 3: Slowly inject the water-in-oil double emulsion into the aqueous phase and prepare a water-in-oil double emulsion under stirring.

[0017] In one aspect of the invention, the aqueous extract containing mulberry leaf extract is prepared by the following steps:

[0018] Step 1-1: Provide mulberry leaves, wash and chop the mulberry leaves, add water and pulp them to obtain the first mulberry leaf pulp;

[0019] Step 1-2: Grind the first mulberry leaf pulp to obtain the second mulberry leaf pulp;

[0020] Steps 1-3: Add water to the second mulberry leaf slurry, then heat it, and reflux extract to obtain a crude extract containing mulberry leaf extract;

[0021] Steps 1-4: The crude extract containing mulberry leaf extract is subjected to centrifugation, flocculation and membrane filtration to obtain the extract containing mulberry leaf extract.

[0022] In one aspect of the invention, in steps 1-2, during the grinding process, a sand mill is used for grinding, the sand mill has a rotation speed of 5-10 m / s, a grinding time of 20-60 min, a grinding temperature of 40℃-50℃, and the grinding media is Φ0.65mm zirconia grinding beads.

[0023] In one aspect of the invention, in steps 1-2, the particle size of the mulberry leaves in the obtained second mulberry leaf slurry is 200-400 nm.

[0024] In one aspect of the invention, in steps 1-3, the ratio of the second mulberry leaf slurry to the added water is selected as 1:(1-2).

[0025] In one aspect of the invention, in steps 1-3, the reflux extraction temperature is 60℃-80℃, and the extraction time is 1-3h.

[0026] In one aspect of the invention, in steps 1-4, the centrifugation speed is 3000-5000 rpm / min.

[0027] In one aspect of the present invention, in steps 1-4, the crude extract containing mulberry leaf extract can be centrifuged to remove insoluble substances such as fiber and macromolecular proteins.

[0028] In one aspect of the invention, in steps 1-4, a flocculant is added to the flocculation process, the flocculant comprising at least one of chitosan, gum arabic, disodium hydrogen phosphate, trisodium phosphate, zinc sulfate, and ferrous sulfate.

[0029] In one aspect of the invention, in steps 1-4, the mass percentage of the flocculant used is selected from 0.5‰ to 3‰ based on the total mass of the crude extract containing mulberry leaf extract after centrifugation.

[0030] In one aspect of the present invention, in steps 1-4, after adding a flocculant to the crude extract containing mulberry leaf extract after centrifugation, the extract is stirred, allowed to stand, and centrifuged to obtain a clear mulberry leaf liquid containing mulberry leaf extract.

[0031] In one aspect of the invention, in steps 1-4, the membrane pore size of the membrane filter is 200-800D.

[0032] In one aspect of the invention, in step 2, the aqueous extract containing mulberry leaf extract is heated to fully dissolve its contents, then mixed with the oil phase and subjected to high-speed shearing to form a stable W / O (water-in-oil) emulsion.

[0033] In one aspect of the invention, in step 2, the shearing rate is selected from 5000-9000 rpm, and the shearing time is selected from 5-10 min.

[0034] In one aspect of the invention, in step 2, the oil phase comprises phospholipids, neutral lipids, and an organic solvent; wherein the neutral lipids are selected from at least one of triglycerides, diglycerides, sterols, sterols, and tocopherols.

[0035] In one aspect of the invention, the phospholipid is selected from at least one of lecithin, soybean phospholipid, cephalin, dioleoylphosphatidylcholine, hydrogenated soybean phospholipid, distearylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, and phosphatidylethanolamine.

[0036] In one aspect of the invention, the neutral lipid is selected from at least one of trioleic acid glyceride, tricaprylic acid glyceride, tridecanoic acid glyceride, vitamin E, and α-tocopherol.

[0037] In one aspect of the invention, the organic solvent is selected from diethyl ether, dichloromethane, chloroform, ethyl acetate, or cyclohexane.

[0038] In one aspect of the invention, in step 2, the oil phase contains 50%-70% soybean phospholipids, 20%-30% cholesterol, 5%-15% diglycerides, 1%-5% vitamin E, and 0.5%-2% ethyl acetate.

[0039] In one aspect of the invention, in step 3, under magnetic stirring conditions, a W / O (water-in-oil) emulsion is slowly injected into the aqueous phase to form a W / O / W (water-in-oil) emulsion.

[0040] In one aspect of the invention, the aqueous phase contains an osmotic pressure regulator selected from at least one of glucose, sucrose, mannitol, sodium chloride, trehalose, succinate, cyclodextrin, arginine, galactose, mannose, maltose, mannitol, glycine, lysine, citrate, sorbitol, and dextran.

[0041] In one aspect of the invention, in step 3, the aqueous phase contains glucose and lysine, wherein the glucose content is 3.5%-5.5% (W / W) and the lysine concentration is 20-30 nmol / L.

[0042] In one aspect of the present invention, after obtaining the water-in-oil-in-water double emulsion in step 3, the organic solvent is removed by a low-temperature negative pressure vacuum process and spray drying is performed to obtain mulberry leaf extract polycystic liposomes; the obtained mulberry leaf extract polycystic liposomes are in powder form.

[0043] The beneficial effects of this invention are as follows:

[0044] (1) The mulberry leaf extract multicyst liposomes provided by the present invention can simultaneously encapsulate mulberry leaf flavonoids and mulberry leaf DNJ, thereby achieving a better blood sugar lowering effect.

[0045] (2) The multi-capsule liposome encapsulation technology provided by this invention can improve the solubility and bioavailability of mulberry leaf flavonoids, while also enhancing the sustained-release effect of mulberry leaf DNJ. Multi-capsule liposomes can encapsulate water-soluble mulberry leaf DNJ in aqueous vesicles (i.e., aqueous chambers) and lipid-soluble mulberry leaf flavonoids in a phospholipid bilayer, achieving dual-component encapsulation of both water-soluble and lipid-soluble substances. This effectively solves the problems of low bioavailability of mulberry leaf flavonoids and short half-life of mulberry leaf DNJ, thus improving the overall hypoglycemic effect of mulberry leaves. The multi-capsule liposome is composed of multiple aqueous chambers arranged in a non-concentric circle, with each aqueous chamber separated by a lipid bilayer phospholipid membrane. This results in good drug loading, encapsulation efficiency, stability, and sustained-release effect. Compared to other encapsulation technologies, multi-capsule liposomes can exist more stably in aqueous systems.

[0046] (3) Compared with conventional pulverization and ultrafine pulverization processes in the prior art, the grinding method provided by the present invention can make the particle size of mulberry leaves during reflux extraction reach 200-400nm, which greatly increases the specific surface area of ​​mulberry leaves and extraction solvent, and can make the dissolution rate of mulberry leaf flavonoids and mulberry leaf DNJ reach more than 95% during water extraction; thus solving the problem of low extraction rate in the water extraction process in the prior art.

[0047] (4) The extraction aqueous solution is purified and impurities are removed by flocculation and membrane filtration. Compared with the conventional resin purification process (adsorption, elution, concentration) in the prior art, the purification process provided by the present invention is simplified and can avoid energy consumption and loss of active ingredients under high temperature conditions caused by resin purification and concentration. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the structure of the mulberry leaf extract multicyst liposomes obtained in Example 1 of the present invention.

[0049] Figure 2 This is a line graph showing the effect of different mulberry leaf treatments on blood glucose levels in rats in Experiment Example 1 of this invention.

[0050] Figure 3 This is a bar chart showing the effects of different mulberry leaf treatments on the gastrointestinal hormone GLP-1 in rats in Experiment Example 1 of this invention. Detailed Implementation

[0051] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the invention based on the disclosed content, which should also fall within the scope of protection claimed in this application.

[0052] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process was carried out at room temperature.

[0053] Example

[0054] Example 1:

[0055] Example 1 includes the following steps:

[0056] Wash and chop fresh mulberry leaves, add a certain proportion of water and blend until uniform to form the first mulberry leaf pulp.

[0057] Then, grinding media (Φ0.65mm zirconia grinding beads) are added to the grinding chamber of the sand mill. The first mulberry leaf slurry is poured into the grinding chamber through the feed pump. The rotation speed is set to 8m / s and the grinding time is 40min to perform nano-grinding and obtain the second mulberry leaf slurry with a particle size of 200-400nm.

[0058] Add 1.5 times the volume of water to the second mulberry leaf slurry, set the temperature to 60℃, and perform heating, stirring and reflux extraction for a total of 3 hours to obtain crude extract.

[0059] The crude extract was then centrifuged at 4000 rpm. The supernatant was collected and 0.8‰ chitosan and gum arabic were added for flocculation. The extract was then filtered to obtain a clear extract. The clear extract was filtered through a 600D filter membrane to obtain an extract containing mulberry leaf extract.

[0060] The oil phase was prepared by dissolving 60% soybean lecithin, 25% cholesterol, 12% diglyceride, and 2% vitamin E in 1% ethyl acetate.

[0061] The oil phase was then poured into the extraction aqueous solution and sheared at a high shear rate of 8000 rpm for 10 min to obtain a stable W / O type proemulsion. The proemulsion was then slowly injected into an aqueous phase containing 4% glucose and 25 nmol / L lysine to form a W / O / W type double emulsion. The ethyl acetate organic solvent in the double emulsion was removed by low-temperature negative pressure vacuum. The composite emulsion was then spray-dried to obtain the mulberry leaf extract multi-capsule liposome powder of Example 1; its structure is as follows. Figure 1 As shown, the multicystic liposome contains multiple aqueous chambers separated by a lipid bilayer; mulberry leaf flavonoids are located between the lipid bilayers, and mulberry leaf DNJ is located within the aqueous chambers.

[0062] Example 2:

[0063] Example 2 includes the following steps:

[0064] Wash and chop fresh mulberry leaves, add a certain proportion of water and blend until uniform to form the first mulberry leaf pulp.

[0065] Then, grinding media (Φ0.65mm zirconia grinding beads) are added to the grinding chamber of the sand mill. The first mulberry leaf slurry is poured into the grinding chamber through the feed pump. The rotation speed is set to 6m / s and the grinding time is 50min to perform nano-grinding and obtain the second mulberry leaf slurry with a particle size of 200-400nm.

[0066] Add the same volume of water to the second mulberry leaf slurry, set the temperature to 70℃, and perform heating, stirring and reflux extraction for a total of 2 hours to obtain crude extract aqueous solution.

[0067] The crude extract was then centrifuged at 5000 rpm. The supernatant was collected and 1.1‰ chitosan and disodium bicarbonate were added for flocculation. The extract was then filtered to obtain a clear extract. The clear extract was filtered through an 800D filter membrane to obtain an extract containing mulberry leaf extract.

[0068] The oil phase was prepared by dissolving 55% soybean lecithin, 30% cholesterol, 10% diglycerides, and 3.5% vitamin E in 1.5% ethyl acetate.

[0069] The oil phase was then poured into the extraction aqueous solution and sheared at a high shear rate of 7000 rpm for 10 min to obtain a stable W / O type promulgated emulsion. The promulgated emulsion was then slowly injected into an aqueous phase containing 3.5% glucose and 20 nmol / L lysine to form a W / O / W type double emulsion. The ethyl acetate organic solvent in the double emulsion was removed by low temperature negative pressure vacuum. The composite emulsion was then spray-dried to obtain the mulberry leaf extract multi-capsule liposome powder of Example 2.

[0070] Example 3:

[0071] Example 3 includes the following steps:

[0072] Wash and chop fresh mulberry leaves, add a certain proportion of water and blend until uniform to form the first mulberry leaf pulp.

[0073] Then, grinding media (Φ0.65mm zirconia grinding beads) are added to the grinding chamber of the sand mill. The first mulberry leaf slurry is poured into the grinding chamber through the feed pump. The rotation speed is set to 10m / s and the grinding time is 20min to perform nano-grinding and obtain the second mulberry leaf slurry with a particle size of 200-400nm.

[0074] Add twice the volume of water to the second mulberry leaf slurry, set the temperature to 80℃, and perform heating, stirring and reflux extraction for a total of 1 hour to obtain crude extract aqueous solution.

[0075] The crude extract was then centrifuged at 3500 rpm. The supernatant was collected and 2‰ sodium bicarbonate, octyl sulfate and ferrous sulfate were added for flocculation. The extract was filtered to obtain a clear extract. The clear extract was then filtered through a 400D filter membrane to obtain an extract containing mulberry leaf extract.

[0076] The oil phase was prepared by dissolving 70% soybean lecithin, 20% cholesterol, 6% diglycerides, and 2% vitamin E in 2% ethyl acetate.

[0077] The oil phase was then poured into the extraction aqueous solution and sheared at a high shear rate of 9000 rpm for 5 min to obtain a stable W / O type promulgated emulsion. The promulgated emulsion was then slowly injected into an aqueous phase containing 5.5% glucose and 30 nmol / L lysine to form a W / O / W type double emulsion. The ethyl acetate organic solvent in the double emulsion was removed by low temperature negative pressure vacuum. The composite emulsion was then spray-dried to obtain the mulberry leaf extract multi-capsule liposome powder of Example 3.

[0078] Comparative Example 1:

[0079] Comparative Example 1 includes the following steps:

[0080] Wash and chop fresh mulberry leaves, add a certain proportion of water and blend until uniform to form the first mulberry leaf pulp.

[0081] Then, grinding media (Φ0.65mm zirconia grinding beads) are added to the grinding chamber of the sand mill. The first mulberry leaf slurry is poured into the grinding chamber through the feed pump. The rotation speed is set to 8m / s and the grinding time is 40min to perform nano-grinding and obtain the second mulberry leaf slurry with a particle size of 200-400nm.

[0082] Add 1.5 times the volume of water to the second mulberry leaf slurry, set the temperature to 60℃, and perform heating, stirring and reflux extraction for a total of 3 hours to obtain crude extract.

[0083] The crude extract was then centrifuged at 4000 rpm. The supernatant was collected and 0.8‰ chitosan and gum arabic were added for flocculation. The extract was filtered to obtain a clear extract. The clear extract was then filtered through a 600D filter membrane to obtain an extract containing mulberry leaf extract. The extract containing mulberry leaf extract was then spray-dried to obtain mulberry leaf extract powder, as described in Comparative Example 1.

[0084] Experiment Example 1: Animal Experiment

[0085] 1.1 Animal husbandry:

[0086] Twenty-four SPF-grade male SD rats, 7-8 weeks old and weighing 180-200g, were selected. The rats were housed in a room with a temperature of 20℃-25℃, with regular daily water and feed changes. All rats had free access to tap water and food. After one week of acclimatization, healthy rats of similar weight were selected for subsequent experiments.

[0087] 1.2 Experimental Grouping:

[0088] Rats were randomly divided into a blank control group, a mulberry leaf powder group, and a mulberry leaf extract multicyst liposome group, with a total of 8 mice in each group.

[0089] 1.3 Experimental Samples

[0090] The mulberry leaf extract multicyst liposome powder obtained in Example 1 was selected as the mulberry leaf extract multicyst liposome group; the mulberry leaf extract powder obtained in Comparative Example 1 was selected as the mulberry leaf powder group.

[0091] 1.4 Experimental Procedure:

[0092] Mulberry leaf powder containing polycystic liposomes and conventional mulberry leaf powder were prepared with sterile water to form solutions of mulberry leaf flavonoids and DNJ at the same concentration. Sucrose was administered at a dose of 0.1 g / 100 g to rats in the control group, conventional mulberry leaf group, and polycystic liposome mulberry leaf group via gavage. Five minutes later, the conventional mulberry leaf group and the polycystic liposome mulberry leaf group were administered both conventional mulberry leaf powder solution and polycystic liposome mulberry leaf powder solution via gavage, at a volume of 1 mL / 100 g. Administering sucrose before the drugs prevents premature absorption and degradation of DNJ by the body. After gavage, blood was collected from the tail using a lancet, and blood glucose levels were measured at 0 h, 0.5 h, 1 h, and 2 h using a Roche glucometer. Once blood glucose levels returned to normal, a second gavage (using only sucrose solution) was administered. Blood glucose levels were measured hourly after 0.5 h until blood glucose levels returned to normal, and the data were recorded to observe differences in blood glucose changes. After the blood glucose measurement, rats were anesthetized by intraperitoneal injection of pentobarbital, and blood was collected from the abdominal aorta. The blood was allowed to stand at room temperature for 2 hours, then centrifuged at 3500 rpm for 10 minutes. The supernatant serum was collected and stored at -80℃ for later use. The GLP-1 content in the serum of each group of rats was measured using a glucagon-like peptide (GLP-1) ELISA kit according to the manufacturer's instructions.

[0093] Experimental results are as follows Figure 2 and 3 And as shown in Table 1;

[0094] Table 1. Effects of different groups on the gastrointestinal hormone GLP-1 in rats

[0095]

[0096] Note: Compared with the control group, *p<0.05, **p<0.01

[0097] according to Figure 2 As can be seen, after the first gavage administration of sucrose, the blood glucose level in the control group reached its maximum at 12.2 mmol / mL at 0.5 h, while the maximum blood glucose levels in Comparative Example 1 and Example 1 were significantly lower than those in the control group, at 11.2 mmol / mL and 10.3 mmol / mL, respectively. Within 0-3 h after the first gavage administration, the blood glucose level in Example 1 was < that in the conventional mulberry leaf group < that in the control group, indicating that the overall hypoglycemic effect of mulberry leaves was significantly improved after treatment with multicystic liposomes. After 3 h, a second gavage administration of sucrose was administered, and the blood glucose level in the control group rapidly increased to its maximum at 12.1 mmol / mL. The blood glucose level in Comparative Example 1 at 0.5 h was 11.6 mmol / mL, which was lower than that in the control group but higher than the blood glucose level of 11.2 mmol / mL at 0.5 h after the first gavage administration of sucrose. This indicates that the hypoglycemic effect of Comparative Example 1 was significantly reduced after the second sucrose intake, mainly due to the short half-life of DNJ and the partial metabolism of the DNJ. Compared to the blank group and Comparative Example 1, Example 1 showed the lowest blood glucose concentration at 3.5 hours, at 9.6 mmol / mL. This was lower than the blood glucose level 0.5 hours after the first gavage administration of sucrose, indicating that Example 1 had a relatively long-lasting effect in inhibiting blood glucose elevation, thus proving that the hypoglycemic components of mulberry leaf extract achieved a sustained-release effect.

[0098] GLP-1 lowers blood sugar through multiple pathways, including promoting insulin secretion, delaying gastric emptying, and strengthening central appetite suppression to reduce food intake. (Based on Table 1 and...) Figure 3 As can be seen, mulberry leaves can increase the concentration of the gastrointestinal hormone GLP-1 in rats; the GLP-1 concentration in Comparative Example 1 was 0.98 pmol / L, which was 10.2% higher than that in the control group; the GLP-1 concentration in Example 1 was 1.19 pmol / L, which was 35.2% higher than that in the control group and 21.4% higher than that in Comparative Example 1. This indicates that after treatment with multi-capsule liposomes, it can significantly increase the secretion of the gastrointestinal hormone GLP-1 in rats and enhance the efficacy of mulberry leaves.

Claims

1. A method for preparing a mulberry leaf extract polycystic liposome, characterized by, The method includes the following steps: Wash and chop fresh mulberry leaves, add a certain proportion of water and blend until uniform to form the first mulberry leaf pulp. Then, grinding media are added to the grinding chamber of the sand mill, and the first mulberry leaf slurry is poured into the grinding chamber through the feed pump. The rotation speed is set to 8m / s and the grinding time is 40min to perform nano-grinding and obtain the second mulberry leaf slurry with a particle size of 200-400nm. Add 1.5 times the volume of water to the second mulberry leaf slurry, set the temperature to 60℃, and perform heating, stirring, and reflux extraction for a total of 3 hours to obtain crude extract. The crude extract was then centrifuged at 4000 rpm. The supernatant was collected and 0.8‰ chitosan and gum arabic were added for flocculation. The extract was then filtered to obtain a clear extract. The clear extract was filtered through a 600D filter membrane to obtain an extract containing mulberry leaf extract. The oil phase was prepared by dissolving 60% soybean lecithin, 25% cholesterol, 12% diglyceride, and 2% vitamin E in 1% ethyl acetate. The oil phase was then poured into the extraction aqueous solution and sheared for 10 minutes at a high shear rate of 8000 rpm to obtain a stable W / O type primary emulsion. The primary emulsion was then slowly injected into an aqueous phase containing 4% glucose and 25 nmol / L lysine to form a W / O / W type double emulsion. The ethyl acetate organic solvent in the double emulsion was removed by low temperature negative pressure vacuum, and the composite emulsion was spray-dried and powdered.

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