WO2024222408A1 - Composition of berberine vitamin c salt, and preparation method therefor and use thereof - Google Patents

Abstract

Provided is a composition of berberine vitamin C salt, which comprises the berberine vitamin C salt, and one or more of a gel base component, a pH regulator, a humectant, a chelating agent, an emulsifier, a permeation enhancer and a solvent. Further provided is the use of the composition. The composition has the effects of reducing blood sugar, blood fat and blood pressure, reducing body weight and reducing body fat. When used for external application, the composition can be used for effectively reducing body weight, and effectively enriches fat; moreover the composition can directly act on abdominal fat cells, so that the volume of fat cells is reduced and the browning of white adipose occurs. When used orally, the composition can also be used for effectively reducing body weight, body fat and blood fat.

Description

A berberine vitamin C salt composition, preparation method and application thereof Technical Field

The invention belongs to the technical field of drug research, and specifically relates to a berberine vitamin C salt composition, a preparation method and application thereof.

Background Art

Berberine, also known as berberine, is a quaternary ammonium alkaloid isolated from the Chinese herbal medicine Coptis chinensis and is the main active ingredient in the antibacterial effect of Coptis chinensis. It was first obtained by M.-E. Chavalier and G. Pertain from the bark of Xanthoxylonclava in 1826.

Pharmacological studies in recent years have found that berberine hydrochloride has multiple pharmacological activities, such as improving cardiovascular diseases and lowering blood sugar and blood lipids, in addition to its antibacterial and anti-inflammatory effects. However, berberine is a highly polar quaternary ammonium base, which makes it difficult to penetrate the lipophilic barrier of the intestine. In addition, its hydrochloride has very low water solubility, resulting in poor absorption in the human body after oral administration [Yu Chen, Zhang Hui, Pan Junfang, et al., Detection and preliminary study of metabolites in urine of healthy volunteers after oral administration of berberine hydrochloride, Chinese Journal of Clinical Pharmacology, 2000: (16) 36-39.].

Patent document CN 101323613 A discloses a berberine adduct, which acts as a prodrug to increase the blood concentration of berberine in the body, thereby improving the bioavailability of berberine.

Patent document US20220185807A1 discloses a berberine ascorbate (name: 9,10-dimethoxy-5,6-dihydro-[1,3]dioxane[4,5-g]isoquinolin[3,2-a]isoquinolin-7-ium.(R)-2-((S)-1,2-dihydroxyethyl)-4-hydroxy-5-oxo-2,5-dihydrofuran-3-ol ester, molecular weight: 367.14), the structure of which is as follows:

However, the document does not describe any further application of the salt.

Summary of the invention

The invention provides a berberine vitamin C salt composition, a preparation, a preparation method and an application.

A berberine vitamin C salt composition comprises berberine vitamin C salt and one or more of a gel base component, a pH regulator, a moisturizer, a chelating agent, an emulsifier, a penetration enhancer, a solvent and other additives.

A berberine vitamin C salt composition comprises berberine vitamin C salt, a moisturizer, an emulsifier and other additives.

As an embodiment, the berberine vitamin C salt composition comprises, by weight percentage:

The above composition can be prepared into a gel.

Another feasible solution is a berberine vitamin C salt composition, which comprises, by weight percentage:

The above composition can be used to prepare emulsion preparations and the like.

It is characterized in that, measured by weight percentage, it comprises:

Another feasible solution is a berberine vitamin C salt composition, according to weight percent Percentage:

The above composition can be used to prepare a patch and the like.

The berberine vitamin C salt is an effective ingredient of the composition. Preferably, the weight percentage of the berberine vitamin C salt is 5 to 20%. Further, the weight percentage of the berberine vitamin C salt is 10 to 20%.

The gel base component has multiple physiological functions such as biodegradability, biocompatibility, non-toxicity, antibacterial, anti-cancer, lipid-lowering, and immune enhancement. As an embodiment, the gel base component is selected from one or more of other high molecular natural active polysaccharides such as chitosan, chitin, chitosan derivatives, chitosan, pullulan, gum arabic, etc. As a further preference, the gel is chitosan. As a preference, the weight percentage content of the gel base component is 0.5-5%; further 1-5%. Further 2-5%.

The pH regulator is selected from one or more of other organic acid regulators such as lactic acid, citric acid, sodium citrate, succinic acid, disodium succinate, propionic acid, butyric acid, and azelaic acid. Preferably, the weight percentage content of the pH regulator is 0.1 to 8%; further, the weight percentage content of the pH regulator is 0.5 to 8%; further, the weight percentage content of the pH regulator is 1 to 8%; further, the weight percentage content of the pH regulator is 2 to 8%.

The moisturizer is selected from one or more of lactic acid, glycerin, hyaluronic acid, urea, emu oil, manuka oil, olive oil, glycine, isopropyl palmitate and other common moisturizers. Preferably, the weight percentage of the moisturizer is 0.1-5%; further, the weight percentage of the moisturizer is 0.5-5%; further, the weight percentage of the moisturizer is 1-5%; further, the weight percentage of the moisturizer is 2-5%. As a specific In the embodiment, when preparing a gel, the moisturizer is selected from lactic acid, and one or more of glycerol, hyaluronic acid, and urea. When preparing an emulsion, the moisturizer is selected from one or more of emu oil, manuka oil, olive oil, isopropyl palmitate, and glycine.

In some cases, the pH regulator and the moisturizing agent can be selected from the same substance, that is, this substance can not only adjust the pH of the composition, but also have a moisturizing effect. At this time, the total amount of the pH regulator and the moisturizing agent added is 0.1-8% (wt); further 1-8%.

The chelating agent plays a stabilizing role and has a synergistic antiseptic and antioxidative effect. Preferably, the chelating agent is selected from one or more of other common chelating agents such as ethylenediaminetetraacetic acid, sodium triphosphate, sodium hexametaphosphate, citric acid, sodium citrate, triethanolamine, diethanolamine, polyacrylic acid sodium salt, etc. Preferably, the weight percentage content of the chelating agent is 0.5-5%; more preferably, the weight percentage content of the chelating agent is 0.5-4%; further, the weight percentage content of the chelating agent is 0.5-3%.

Preferably, the emulsifier is selected from one or more of the common emulsifiers such as Tween 80, steareth-6, glyceryl stearate, behenyl alcohol, ceteareth-20, arachidyl alcohol, hydroxyethyl acrylate, glycerin, sorbic acid, polysorbate 20, etc. Preferably, the weight percentage content of the emulsifier is 0.1-5%; further 1-5%. As a specific embodiment, when preparing a gel, the preferred emulsifier is selected from one or more of Tween 80, steareth-6, glyceryl stearate, behenyl alcohol, ceteareth-20, arachidyl alcohol, hydroxyethyl acrylate, and glycerin; when preparing an emulsion, the preferred emulsifier is selected from one or more of sorbic acid and polysorbate 20 or a mixture of two.

Preferably, the penetration enhancer is selected from one or more of common penetration enhancers such as laurocapram, cyclohexane hexol, dimethyl isosorbide, tetrahydropiperine, N-n-alkylbenzisothiazolone, propylene glycol, urea, ethanol, benzalkonium chloride, polysorbate 80, etc. Preferably, the weight percentage of the penetration enhancer is 0.1-5%.

In some cases, some components of the emulsifier or penetration enhancer can play both an emulsifying role and a penetration enhancing role, and have multiple comprehensive effects.

Preferably, the solvent is one or more of water, ethanol, and glycerol. Similarly, some solvents, while serving as solvents, can also perform functions such as moisturizing, promoting penetration, emulsifying, and pH adjustment.

Preferably, the other additives include one or more of preservatives, antioxidants, solubilizers, thickeners, colorants, and adhesives. The preservatives include propylene glycol, benzoic acid, sorbic acid, parabens, dehydroacetic acid, and their corresponding salts. The antioxidants include one or more of phospholipid complex flavonoids (lecithin), flavonoid extracts (peony leaf extract or calendula extract) or vitamin C, methyl hydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and sodium benzoate. The solubilizer is selected from inulin lauryl carbamate, etc.; the thickener includes glycerol, etc. When preparing the emulsion, the other additives include preservatives, antioxidants, solubilizers, and thickeners. Further, the weight percentage of the preservative is 0.3-5%; further, 0.5-5%; and further, 0.8-4%. Further, the weight percentage of the antioxidant is 1-15%. The weight percentage of the solubilizer or thickener is 1-8%.

A preparation is prepared from the composition described in any one of the above technical solutions.

Preferably, the preparation is an external ointment.

Furthermore, it can be applied as gel, emulsion, or cream, etc. Furthermore, it can be applied as cream, gel, or emulsion to human skin, such as the abdomen, waist, back, buttocks, limbs, etc. where fat accumulation is obvious.

Preferably, the preparation is an oral aqueous solution, ointment, tablet, powder, capsule, etc.

A preparation method of the gel comprises: dissolving the gel base component in a solvent, adding a chelating agent, an emulsifier or a penetration enhancer, and then adding berberine vitamin C salt, a pH regulator or a moisturizer to the remaining solvent, stirring, and obtaining the preparation.

A use of the composition described in any of the above technical solutions in the preparation of drugs for treating or improving cardiovascular diseases and reducing blood sugar, blood lipids, body weight, and body fat.

A use of the composition described in any of the above technical solutions in the preparation of drugs for treating or improving cardiovascular diseases, blood sugar lowering drugs, blood lipid lowering drugs, weight reducing drugs, and body fat reducing drugs.

An application of the composition described in any one of the above technical solutions in treating or improving cardiovascular diseases, lowering blood sugar, reducing body weight, and reducing body fat.

The above composition can be used by external application, external patch or internal administration.

When used for animal treatment or improvement of the above symptoms (improvement of cardiovascular disease and reduction of blood sugar, blood lipids, body weight, and body fat), the dosage for external use (coating or patch, etc.) can be 100 mg/kg or more, and further, the dosage is 10 mg/kg to 1000 mg/kg. When taken orally, the dosage is The oral dosage is 50 mg/kg/day or more, and the further oral dosage is 50 mg/kg to 500 mg/kg/day.

When used for treating or improving the above symptoms (improving cardiovascular diseases and lowering blood sugar, blood lipids, body weight, and body fat), the dosage for external use (coating or patch, etc.) can be 3 mg/kg or more, and further, the dosage is 1 mg/kg to 30 mg/kg. When taken orally, the dosage is 3 mg/kg/day or more, and further, the oral dosage is 1 mg/kg to 30 mg/kg/day.

The composition of the present invention has the effects of lowering blood sugar, blood lipids and blood pressure, and when used for in vitro coating, it is effectively enriched in fat and reduces fat volume.

The composition of the present invention has the effects of lowering blood sugar, blood lipids, blood pressure, as well as reducing body weight and body fat. When used for in vitro coating, it can effectively reduce body weight, while effectively enriching in fat, and can directly act on abdominal fat cells, reducing the volume of fat cells and causing white fat to change to brown; when used orally, it can also effectively reduce body weight, body fat and blood lipids.

The invention discloses an application of berberine vitamin C salt, which is used for preparing drugs for improving cardiovascular diseases of humans or animals, lowering blood sugar, lowering blood lipids, lowering body weight and lowering body fat.

A berberine vitamin C salt is a single crystal with a space group of P21 (no.4) and a unit cell parameter of α＝90°, β＝94.3(3)°, γ＝90°, the minimum asymmetric unit contains 2 of each of the two target molecules, and the same molecules have different spatial conformations and have no symmetric relationship with each other. There are 4 of each of the two target molecules in each unit cell.

At the same time, the experimental results show that when high-fat model mice are orally administered with the new berberine salt, the new berberine salt can help the mice resist the weight gain induced by a high-fat diet, effectively reduce body weight, and reduce body fat at the same time. In addition, the new berberine salt directly participates in the energy metabolism of obesity at the fat cell level, causing the volume of white fat cells to decrease.

In addition, experiments have shown that under the condition of oral administration of berberine new salt, berberine new salt can reduce the weight of obese cats, and there is a certain dose dependence, and the effect of high dose is more obvious; at the same time, oral administration of berberine new salt can effectively lower the blood lipids of obese cats.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a graph showing the average plasma concentration-time curve after abdominal administration of 200 mg/kg berberine new salt to SD rats.

Fig. 2. Bar graph of average tissue drug concentrations after administration of berberine new salt.

Fig. 3 Schematic diagram of abdominal fat in mice after drug administration.

Figure 4 Schematic diagram of pathological staining of abdominal adipocytes in mice after drug administration (scale size is 50 μm).

Figure 5 is a graph showing changes in body weight of high-fat model mice after abdominal administration.

Figure 6 is a graph showing changes in body weight of high-fat model mice after oral administration.

Figure 7 Body fat data of high-fat model mice detected by nuclear magnetic resonance imaging after oral administration.

Figure 8 is a graph showing the body weight changes in the oral drug-discontinuation group and the drug-continuation group of high-fat model mice.

Figure 9 Electron microscopic images of iWAT tissue in high-fat model mice after oral administration (scale size is 500nm).

Figure 10 eWAT tissue staining images of high-fat model mice after oral administration (scale size is 50 μm).

Figure 11 iWAT tissue staining images of high-fat model mice after oral administration (scale size is 50 μm).

Figure 12 is a graph showing weight changes in cats in the control group.

Figure 13 is a graph showing changes in body weight of cats in the high-dose medication group.

Figure 14 is a graph showing changes in body weight of cats in the low-dose medication group.

Fig. 15 is a curve diagram of changes in TG triglycerides of cats.

Figure 16 is an ellipsoid diagram of the three-dimensional structure of the new salt molecule of berberine.

Figure 17 Molecular stacking diagram of the crystal cell of the new berberine salt (projected along the a-axis and b-axis).

DETAILED DESCRIPTION

Example 1 Preparation of external preparation (gel) of new berberine salt:

Berberine Vitamin C Salt Gel Preparation (10% w/w) Formula:

Among them, berberine vitamin C salt can be obtained according to the preparation method of US20220185807A1.

Preparation of berberine vitamin C salt gel preparation:

Weigh an appropriate amount of chitosan and dissolve it in water, add EDTA solution and Tween 80, and stir to form a mixed solution. Take an appropriate amount of berberine vitamin C salt and add it to the above mixed solution, then add an appropriate amount of lactic acid, add water to the final volume, stir to dissolve, and you have it.

Example 2 (emulsion)

Preparation of berberine vitamin C salt emulsion preparation:

Weigh appropriate amounts of vitamin C, inulin lauryl carbamate, glycine, lecithin and marigold extract and dissolve them in a mixed solvent of propylene glycol and water, then add berberine vitamin C salt and glycerol, and stir well to form a mixed solution. Separately take appropriate amounts of sorbic acid, olive oil and isopropyl palmitate and mix well, then slowly add the mixed solution into the above berberine vitamin C salt mixed solution, stirring while adding to make the system uniform. Then add polysorbate 20 and stir well to obtain the product.

Embodiment 3 (gel)

A gel was prepared in a similar manner to that of Example 1.

Example 4 (patch)

After obtaining the gel of Example 1, add an appropriate amount of sodium polyacrylate, stir evenly, dry, and then apply with the aid of patch to obtain a patch preparation of the salt.

Example 5 Preparation of the Oral Internal Preparation (Aqueous Solution) of the New Berberine Salt

Take an appropriate amount of berberine vitamin C salt solid, stir and dissolve it in water to obtain an aqueous solution of the salt.

The following performance tests were performed on the gel obtained in Example 1:

(I) This experiment investigated the pharmacokinetic behavior of berberine new salt in SD rats under the condition of abdominal epidermal administration of berberine new salt.

1. Sample configuration:

The test article was prepared to a final concentration of 109 mg/mL for abdominal administration.

The test sample is prepared in CMC-Na (sodium carboxymethyl cellulose) solvent and appears as a uniform yellow suspension after preparation.

2. Experimental system

(1) Experimental animals

Species & strain: SD rat

Source of experimental animals: Jinan Pengyue Experimental Animal Breeding Co., Ltd.

Planned age at dosing: 6-8 weeks at the start of dosing.

Planned body weight at dosing: 180-250 g at the start of dosing.

Number of animals and sex: 4, male, 1 of which was a spare rat.

(2) Feeding and management

The animals were housed in transparent resin plastic cages (530 mm x 400 mm x 200 mm).

Fluorescent lighting, 12 hours of lighting (08:00-20:00) and 12 hours of no lighting per day. The ambient temperature and relative humidity of the animal room should be controlled within the range of 20-26°C and 40-70%, respectively.

All animals had free access to drinking water during the experiment.

(3) Animal marking

Use a high-quality marker pen to write the number directly on the mouse's tail and use basic fuchsin to mark the back. Attach a label to the outside of the animal cage to mark the study number, animal number, group number, gender and other information.

3. Brief description of experimental methods

(1) Groups and doses

The animal body weight was measured before administration, and healthy animals with similar body weight were selected for inclusion in the experiment.

Dosing frequency: Single dose.

(2) Sample collection

After the experimental animals were anesthetized with ether, at least 0.2 mL of blood was collected from the jugular vein and an anticoagulant, sodium heparin, was added.

Collection time:

0min before administration, 15min, 30min, 45min, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 12h, 24h, and 36h after administration.

36 h after blood collection, liver and abdominal fat were collected and then stored in a -80°C refrigerator.

Sample processing

After the blood sample is collected, it is placed in a labeled centrifuge tube and quickly centrifuged to separate the plasma. The centrifugation conditions are:

3000 rpm, 15 min, 4°C, plasma was stored below -40°C until testing.

4. Standard curve and quality control samples

(1) Preparation of berberine standard curve and quality control samples: Prepare standard curve working solutions (concentrations of 4 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL) and quality control working solutions (concentrations of 8 ng/mL, 80 ng/mL, 800 ng/mL) using 50% methanol water as the diluent. Take 5 μL of each working solution and mix with 45 μL of blank plasma to prepare plasma standard curve and quality control samples. Add 300 μL of internal standard working solution (200 g/mL, methanol, methyldopa) to the prepared standard curve and quality control samples, vortex mix for 1 min, and centrifuge at 4°C and 15400g for 10 min. Take the supernatant for sample analysis. The standard curve and quality control results accompanying the sample analysis showed that the detection and analysis batch met the set standards.

(2) Sample pretreatment

Pretreatment of berberine new salt plasma samples: After the plasma samples were thawed at room temperature, 30 μL were taken, 180 μL of internal standard (200 ng/mL, methanol, methyldopa) was added, vortexed for about 1 min, centrifuged at 4 ° C, 15400g for 10 min, and the supernatant was taken for sampling and analysis.

(3) Pretreatment of liver, intestine, and abdominal fat samples: After the liver, intestine, and abdominal fat tissue samples were homogenized (tissue: 50% methanol water = 1 g: 5 mL), 60 μL of the tissue sample homogenate was taken, 360 μL of the internal standard working solution (200 ng/mL, methanol, methyldopa) was added, vortexed for 1 min, and centrifuged at 4°C, 15400g for 10 min. The supernatant was taken for sampling and analysis.

5. Data collection and statistical analysis

Analyst 1.6.3 software outputs original spectra, concentration, accuracy and other data.

Microsoft Excel 2007 software calculates mean, standard deviation, coefficient of variation, etc.

The WinNonlin software non-compartmental method (NCA) was used to calculate the main pharmacokinetic parameters such as AUC, C _max and t _1/2 .

GraphPad Prism 9 was used for C-T plotting.

Specific steps:

The experiment selected 4 SD rats of similar weight and divided them into 2 groups. Berberine new salt was smeared on the abdominal epidermis of rats 1-3, and the dosage was 200 mg/kg, a single dose. Blood was collected at 0min before administration, and 15min, 30min, 45min, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 12h, 24h, and 36h after administration. After 36h, liver tissue and abdominal fat were collected. The remaining rats were assigned to Group 2 as a spare group for backup and taking blank matrix. After blood collection, the samples were placed in labeled centrifuge tubes and centrifuged quickly (centrifugation conditions: 3000 rpm, 15 minutes, 4℃) to separate the plasma, and the plasma was stored at -40℃ for testing. The berberine content in plasma was detected by LC-MS/MS analysis, and the lower limit of quantification of the method was 4ng/mL. The plasma concentration data were statistically analyzed using the metabolic kinetic data analysis software WinNonlin 7.0, and the pharmacokinetic parameters were calculated using the non-compartmental model method (NCA).

The plasma drug concentrations of SD rats after application of berberine vitamin C salt gel preparation are shown in Table 1 ;

Table 1 Timetable of plasma drug concentrations in SD rats after administration of berberine (dose: 200 mg/kg)

After homogenizing liver, intestine, and abdominal adipose tissue samples (tissue: 50% methanol water = 1g: 5mL), take 60μL of tissue sample homogenate, add 360μL of internal standard working solution (200ng/mL, methanol, methyldopa), vortex mix for 1min, and centrifuge at 4℃, 15400g for 10min. Take the supernatant for sampling and analysis. The berberine content in the supernatant was also detected by LC-MS/MS analysis method, and the quantitative limit of the method was 4ng/mL. The plasma concentration data were statistically analyzed using the metabolic kinetic data analysis software WinNonlin7.0, and the pharmacokinetic parameters were calculated using the non-compartmental model method (NCA).

The drug concentration in animal tissues after administration of berberine vitamin C salt is shown in Table 2:

Table 2 Drug concentrations in animal tissues after administration

The main pharmacokinetic parameters of berberine vitamin C salt (berberine new salt) in SD rats are shown in Table 3:

Table 3 Main pharmacokinetic parameters of berberine new salt in SD rats

Conclusion: After SD rats were given 200 mg/kg of berberine new salt transdermal preparation in the abdomen, the in vivo pharmacokinetic data in Figure 1 showed that the berberine blood concentration increased significantly, and the berberine new salt can be absorbed into the body circulation through the skin; as shown in Figure 2, the drug concentration in the tissue showed that the berberine new salt transdermal preparation can reach the liver and fat, and can be enriched in fat. In addition, compared with oral administration, the peak concentration of berberine prototype in the blood is lower in the transdermal route, the absorption degree is lower, but the half-life is longer.

The following performance tests were performed on the patch obtained in Example 4:

(I) This experiment investigated the effect of berberine new salt gel on abdominal fat cells in mice under the condition of abdominal epidermal administration of berberine new salt.

1. Experimental subjects

Ordinary mouse.

Temperature and relative humidity were controlled (22±2°C, 55±5%) with 12-h light-dark cycle and free access to food and water.

2. Experimental plan

The control group was fed normally, and the experimental group was treated with the patch (dose of about 22 mg/mouse)

3. Detection indicators

Drug concentration, pathological analysis.

4. Experimental results

In the mouse experiment, after applying the patch containing the new salt of berberine (applied to the abdominal epidermis), we observed the enrichment of berberine (BBR) in the visceral fat of mice. After applying the patch containing the new salt of berberine for 1 day, the content of berberine in the fat was 0.036-0.14μg/g; after applying the patch for 5 consecutive days, the content of berberine in the fat could reach 1.44-10.92μg/g (see Table 4).

Table 4 Changes in berberine concentration of coptis chinensis extract in abdominal fat cells of mice after abdominal administration

Figure 3 is a schematic diagram of abdominal fat after patch administration in mice, where samples No. 1 and No. 2 are administered for 5 consecutive days, and samples No. 3 and No. 4 are administered for 1 day. From the color of the visceral fat, it can be seen that the fat of mice that have been applied with the new salt of berberine for 5 days is darker than the fat that has been applied for only 1 day, indicating that the fat "browns" as the berberine is applied. Therefore, the experimental results show that berberine can be absorbed by fat cells through the skin and directly acts on fat cells. The abdominal fat cells show brown changes of white fat cells, and the color deepens with the increase of administration time. Figure 4 is a schematic diagram of pathological staining of abdominal fat cells after administration in mice. After pathological staining, we observed the changes in fat cells before and after the use of berberine. It can be found that after the use of berberine, the abdominal fat cells of mice became significantly smaller, which is consistent with the expectation of browning of white fat cells.

5. Experimental conclusion

Our experimental results show that after berberine new salt gel was administered to the abdominal epidermis of mice, berberine was absorbed transdermally and could directly act on abdominal fat cells, reducing the volume of abdominal fat cells and causing white fat to turn brown, directly participating in the metabolism of obesity at the fat cell level.

(II) This experiment investigated the effect of berberine new salt gel on the body weight and abdominal fat cells of high-fat model mice under the condition of berberine new salt administration to the abdominal epidermis.

1. Experimental subjects

A total of 10 DIO model mice fed with HFD (high fat diet) were divided into a control group and a drug group, with 5 mice in each group.

Temperature and relative humidity were controlled (22±2°C, 55±5%) with 12-h light-dark cycle and free access to food and water.

2. Experimental plan

The mice in the control group were fed normally, and the mice in the medication group were treated with berberine new salt gel (dose of about 22 mg/mouse)

3. Detection indicators

weight.

4. Experimental results

At present, there are few reports on the mechanism of berberine, a Chinese herbal medicine, reducing weight by acting on fat cells, and there are no relevant literature reports on the study of berberine acting directly on abdominal fat cells through skin absorption. In a mouse model of obesity caused by a high-fat diet, we observed the weight changes of mice after applying the berberine new salt gel. The results showed that, as shown in Figure 5, the weight of mice with berberine new salt gel applied to the abdomen was significantly reduced after 4 days compared with the control group.

5. Experimental conclusion

Our experimental results show that in a mouse model of obesity induced by a high-fat diet, after berberine new salt gel is administered epidermally in the abdomen, berberine is transdermally absorbed and can effectively reduce the weight of mice.

The following performance tests were performed using the aqueous solution of berberine vitamin C salt prepared in Example 5 as an oral preparation:

(I) This study investigated the effects of berberine new salt oral administration on the body weight and fat cells of high-fat model mice under the condition of oral administration of berberine new salt solution.

1. Experimental subjects

There were 20 DIO model mice fed with HFD (high fat diet), 10 in each of the experimental group and the control group.

Temperature and relative humidity were controlled (22±2°C, 55±5%) with 12-h light-dark cycle and free access to food and water.

2. Experimental plan

The experimental group DIO mice were orally administered berberine new salt 100mg/kg/day (the drug was dissolved in the mouse drinking water), and after 50 days of continuous administration, the dose was adjusted to 500mg/kg/day. After 77 days of continuous administration, the experimental group was divided into two groups, the drug withdrawal group stopped drinking water administration, and the continued drug administration group continued normal administration.

3. Detection indicators

Weight, body fat, pathological analysis.

4. Experimental results

As shown in Figure 6, when the high-fat model mice were orally administered with the new salt of berberine, after 50 days, when the dosage was adjusted to 500 mg/kg/day, the weight of the experimental group (BBR) and the control group (NC) mice showed differentiation and a large weight difference, with a weight difference of 10% on day 77. The results showed that oral administration of the new salt of berberine in drinking water can help mice resist the weight gain induced by a high-fat diet and effectively reduce their body weight. At the same time, we used nuclear magnetic resonance to detect the body fat data of the two groups of mice, fat mass (fat weight) and lean mass (lean mass excluding adipose tissue). As shown in Figure 7, the body fat of the experimental group (BBR) that was orally administered with the new salt of berberine was significantly reduced compared with the control group (WT).

After 77 days, the experimental group was equally divided into two groups, the drug withdrawal group (WATER) and the continued drug administration group (BBR). As can be seen from the results in Figure 8, the body weight of the mice in the drug withdrawal group slightly recovered after drug withdrawal, while the body weight of the mice in the continued drug administration group continued to decrease, indicating that the weight loss was caused by the administration of the new salt of berberine, which once again verified the effect of oral administration.

After the experiment (77+5 days later), we collected epididymal white adipose tissue (eWAT) and abdominal adipose tissue (iWAT) samples from mice. We observed the iWAT tissue by electron microscopy. Figure 9 is an electron micrograph of iWAT tissue. As shown in the figure, the control group (WT) and the experimental group (BBR) each showed two electron micrographs of different regions, and it can be seen that the number and size of mitochondria in the abdominal white fat iWAT of high-fat model mice increased under the condition of oral administration of berberine new salt.

The iWAT and eWAT samples of mice were stained with HE: the samples were fixed with 4% paraformaldehyde, dehydrated with gradient sugar solution, embedded in OCT, frozen and sectioned, stained with conventional HE, and observed and photographed under an optical microscope after sealing. Figure 10 is the eWAT tissue staining diagram. The control group and the experimental group each show 2 pictures of different areas of vision. It can be seen that the inguinal white fat eWAT in the experimental group Figure 11 is a staining diagram of iWAT tissue. The control group and the experimental group each show 2 diagrams of different field of view. It can be seen that the cell size of abdominal white fat iWAT tissue in the experimental group is also significantly reduced. The results of Figures 10 and 11 both indicate that oral administration of the new berberine salt may affect the energy metabolism of fat cells and cause the volume of white fat cells to decrease.

5. Experimental conclusion

Our experimental results show that when high-fat model mice are orally administered with the new berberine salt, it can help the mice resist the weight gain induced by a high-fat diet, effectively reduce body weight, and reduce body fat. In addition, the new berberine salt directly participates in the energy metabolism of obesity at the fat cell level, causing the volume of white fat cells to decrease.

The following performance tests were performed using the berberine vitamin C salt powder as an oral preparation:

(I) This experiment investigated the effects of berberine new salt on cats' body weight and biochemical indices under the condition of oral administration of berberine new salt.

1. Experimental subjects

cat.

2. Experimental plan

The experimental cats were divided into a healthy control group, an obese control group, a high-dose medication group, and a low-dose medication group. The experimental groups are shown in Table 5:

Table 5 High and low dose grouping of experimental cats

The berberine new salt powder is directly packaged in capsules for oral administration.

The new salt of berberine was orally administered to the obese cats, with the high-dose group receiving 100 mg/day and the low-dose group receiving 50 mg/day. The cats in the healthy control group and the obese control group were raised normally.

3. Detection indicators

Body weight, blood biochemical indexes

4. Experimental results

After two months of medication, the weight of cats in each group was counted. As shown in Figure 12, the weight of cats in the obese control group and the healthy control group remained stable; as shown in Figure 13, the weight of cats in the high-dose medication group decreased over time; as shown in Figure 14, the weight of cats in the low-dose medication group decreased slowly over time. It can be seen that oral administration of the new berberine salt can reduce the weight of obese cats, and the effect of oral administration of high-dose new berberine salt (100 mg/day) is more obvious. During the experiment, we took blood samples from cats to test the TG triglyceride index in the blood. As shown in Figure 15, the triglyceride index of the obese control cat increased over time, and the triglyceride of the healthy control cat was stable at 0.33mmol/L. Under the condition of oral administration of the new berberine salt, the triglyceride index of the obese cat decreased. This result shows that oral administration of the new berberine salt can reduce the blood lipids of obese cats.

5. Experimental conclusion

Our experimental results show that under the condition of oral administration of berberine new salt, berberine new salt can reduce the weight of obese cats, and there is a certain dose dependence, and the effect of high dose is more obvious; at the same time, oral administration of berberine new salt can effectively reduce the blood lipids of obese cats.

Finally, the structure and crystal form of the microcrystals in the solid sample of berberine vitamin C salt were determined by microcrystal electron diffraction (MicroED) technology.

1. Test samples

Berberine Vitamin C Salt Powder

2. Testing equipment

Talos F200C cryo-EM, CetaD camera

3. Detection and analysis process

MicroED data were collected using a Talos F200C cryo-EM coupled with a CetaD detector. After checking the quality of the microcrystals, 19 crystals were selected and data were collected under liquid nitrogen freezing conditions. During data analysis, the 8 best sets of data were used to determine the unit cell parameters and merge the data.

During the structural analysis, the atomic scattering factor of electrons (waves) was used to calculate the theoretical structure factor, and the structural analysis was completed using the SHELXT software to obtain the initial structure. Subsequent refinements were completed using the SHELXL program based on the least squares refinement method. Combined with the molecular formula information of the compound, all non-hydrogen atoms were identified and anisotropically refined, and all hydrogen atoms were obtained by computational hydrogenation.

4. Experimental results and conclusions

We used microcrystal electron diffraction (MicroED) technology to determine the structure and crystal form of the microcrystals in the solid sample of berberine vitamin C salt. The results are shown in the figure. Figure 16 is the three-dimensional structure ellipsoid diagram of the berberine new salt molecule, and Figure 17 is the molecular stacking diagram of the berberine new salt unit cell (projected along the a-axis and b-axis). These structural refinement results show that the structure belongs to the monoclinic system, the space group is P21 (no.4), and the unit cell parameters are α＝90°, β＝94.3(3)°, γ＝90°, the minimum asymmetric unit contains 2 of each of the two target molecules, and the same molecules have different spatial conformations and have no symmetric relationship with each other. There are 4 of each of the two target molecules in each unit cell.

Claims (16)

A berberine vitamin C salt composition, characterized in that it comprises berberine vitamin C salt, and one or more of a gel base component, a pH adjuster, a moisturizer, a chelating agent, an emulsifier, a penetration enhancer, other additives and a solvent. The berberine vitamin C salt composition according to claim 1, characterized in that, in terms of weight percentage, it comprises:
The berberine vitamin C salt composition according to claim 1, characterized in that, in terms of weight percentage, it comprises:
The berberine vitamin C salt composition according to claim 1, characterized in that it comprises: berberine vitamin C salt, a moisturizer, an emulsifier and other additives. The berberine vitamin C salt composition according to claim 4, characterized in that, in terms of weight percentage, it comprises:

The berberine vitamin C salt composition according to any one of claims 1, 2, 4, and 5, characterized in that the weight percentage of the berberine vitamin C salt is 5 to 20%. The berberine vitamin C salt composition according to claim 6, characterized in that the weight percentage of the berberine vitamin C salt is 10-20%. The berberine vitamin C salt composition according to any one of claims 1 to 3 is characterized in that the gel base component is selected from one or more of chitosan, chitin, chitosan derivatives, chitosan, pullulan, gum arabic, carbomer 940 or other high-molecular-weight natural active polysaccharides; the pH regulator is selected from one or more of lactic acid, citric acid, sodium citrate, succinic acid, disodium succinate, propionic acid, butyric acid, azelaic acid or other organic acid regulators; the chelating agent is selected from one or more of ethylenediaminetetraacetic acid, sodium triphosphate, sodium hexametaphosphate, citric acid, sodium citrate, triethanolamine, diethanolamine, polyacrylic acid sodium salt, and other chelating agents; the penetration enhancer is selected from one or more of laurocapram, cyclohexanehexol, dimethyl isosorbide, tetrahydropiperine, N-alkylbenzisothiazolone, propylene glycol, urea, ethanol, benzalkonium chloride, and polysorbate 80; and the solvent is water. The berberine vitamin C salt composition according to any one of claims 1 to 5 is characterized in that the moisturizer is selected from one or more of lactic acid, glycerin, hyaluronic acid, urea, emu oil, manuka oil, olive oil, isopropyl palmitate, glycine or other common moisturizers; the emulsifier is selected from one or more of Tween 80, steareth-6, glyceryl stearate, behenyl alcohol, ceteareth-20, arachidyl alcohol, hydroxyethyl acrylate, glycerin, sorbic acid, and polysorbate 20. The berberine vitamin C salt composition according to any one of claims 1 to 5, characterized in that the other additives include one or more of preservatives, antibacterial agents, antioxidants, solubilizers, thickeners, colorants, and adhesives. A preparation, characterized in that it is prepared from the composition according to any one of claims 1 to 10. The preparation according to claim 11 is characterized in that it is an external gel, an external emulsion, an external ointment, an internal aqueous solution, an internal ointment, an internal tablet, an internal powder, or an internal capsule. A method for preparing the gel according to claim 12, characterized in that it comprises: dissolving the gel base component in a solvent, adding a chelating agent, an emulsifier or a penetration enhancer, and then adding berberine vitamin C salt, a pH adjuster or a moisturizer to the remaining solvent, stirring, and obtaining the preparation. A use of the composition according to any one of claims 1 to 10 in the preparation of drugs for improving cardiovascular diseases, lowering blood sugar, lowering blood lipids, lowering body weight, and lowering body fat in humans or animals. An application of berberine vitamin C salt, characterized in that it is used for preparing drugs for improving cardiovascular diseases in humans or animals, lowering blood sugar, lowering blood lipids, reducing body weight, and reducing body fat. A berberine vitamin C salt single crystal, characterized in that the space group is P21 (no.4), and the unit cell parameters are α=90°, β=94.3(3)°, γ=90°.