CN116440290A - Berberine oxide cyclodextrin inclusion compound, and preparation method and application thereof - Google Patents

Berberine oxide cyclodextrin inclusion compound, and preparation method and application thereof Download PDF

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CN116440290A
CN116440290A CN202310409421.9A CN202310409421A CN116440290A CN 116440290 A CN116440290 A CN 116440290A CN 202310409421 A CN202310409421 A CN 202310409421A CN 116440290 A CN116440290 A CN 116440290A
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cyclodextrin
oxide
beta
obb
protoberberine
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陈建南
刘煜洪
苏子仁
谢友良
黄晓其
谢建辉
黎玉翠
黄梓葳
黎敏华
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Research Institute Of Dongguan Guangzhou University Of Traditional Chinese Medicine
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Abstract

The invention discloses a protoberberine oxide cyclodextrin inclusion compound, a preparation method and application thereof. The preparation method of the protoberberine oxide cyclodextrin inclusion compound comprises the following steps: adding a solvent into cyclodextrin, heating to dissolve and preparing into a saturated cyclodextrin solution for later use; adding protoberberine oxide into the cyclodextrin solution, continuously heating, and stirring or ultrasonic treating; removing solvent, and grinding to obtain original berberine oxide cyclodextrin clathrate. The preparation method has simple conditions and low cost, and is suitable for industrialized mass production. The clathrate prepared by the method can greatly improve the water solubility and bioavailability of original berberine oxides and improve the drug effect.

Description

Berberine oxide cyclodextrin inclusion compound, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of pharmacy, and in particular relates to a protoberberine oxide cyclodextrin inclusion compound, a preparation method and application thereof.
Background
The protoberberine alkaloid is isoquinoline alkaloid derived from cortex Phellodendri and Coptidis rhizoma, and comprises berberine, palmatine, berberine, epiberberine, jateorhizine, etc. Various protoberberine alkaloids represented by berberine have been proved to have various pharmacological activities such as lipid lowering, blood sugar lowering and anti-inflammatory. The protoberberine oxide is a metabolite generated by the oxidation and metabolism of protoberberine alkaloid in vivo, and has more remarkable pharmacological activity than the proto-drug. The oxidized berberine (OBB) is the most representative protoberberine oxide, has a molecular formula of C20H17NO5, is yellow or beige crystalline powder, is easily dissolved in dichloromethane, is slightly dissolved in methanol and ethanol, is almost insoluble in water, and is one of main active metabolites of berberine in intestinal flora and erythrocytes. The existing research shows that the OBB has more remarkable pharmacological activity than berberine under the same administration dosage, and has stronger curative effects on the aspects of reducing blood sugar, reducing blood fat, reducing uric acid, resisting inflammation and the like. The method suggests that the protoberberine oxide has development value and potential in the aspect of treating metabolic diseases compared with the protoberberine alkaloid. However, the protoberberine alkaloid oxide has extremely low water solubility and poor bioavailability, which limits the development and application thereof in the field of medicine. Therefore, there is an urgent need to develop a new formulation method to improve the water solubility and bioavailability thereof.
Cyclodextrin (CD) is a class of cyclic oligosaccharide compounds, formed by linking 6-12 glucose molecules, and has a hydrophilic outer surface and a hydrophobic central cavity. This particular structure allows the CD to form inclusion complexes with hydrophobic drug molecules of suitable size and shape, enhancing the solubility of the hydrophobic drug in water. CD is less acid stable but is more acid resistant than starch and acyclic small molecule sugars. Meanwhile, CD is very stable to alkali, heat and mechanical action, and is a good natural or synthetic inclusion material. However, existing studies indicate that natural CD is less water soluble and has some toxicity. In order to further improve the pharmaceutical properties of CD, the application range of CD inclusion technology is expanded, and derivatives of CD are currently widely used in the market to prepare inclusion compounds, such as hydroxypropyl- β -CD (HP- β -CD), sulfobutyl- β -CD, and methyl- β -CD. The HP-beta-CD has the advantages of high water solubility, low toxicity, low cost and the like, and is widely applied to increasing the solubility and oral bioavailability of the medicine.
However, the conventional cyclodextrin inclusion process mainly comprises a saturated aqueous solution method, an ultrasonic method, a grinding method, a freeze-drying method and a spray-drying method, and the materials are fed according to the proportion of host and guest molecules forming inclusion compound in the preparation process. However, the water solubility of the protoxide berberine alkaloids is too low, so that the inclusion compound prepared by adopting the traditional thought has low encapsulation rate and poor solubility. How to obtain the original berberine oxide cyclodextrin inclusion compound with high encapsulation efficiency and good solubility is a technical problem to be solved.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art, solve the problems of extremely low water solubility, poor bioavailability and difficult patent medicine of the original berberine oxide, and provide a preparation method of the original berberine oxide cyclodextrin inclusion compound.
The invention also aims to provide the berberine oxide cyclodextrin inclusion compound obtained by the preparation method.
Still another object of the present invention is to provide an application of the above berberine oxide cyclodextrin inclusion compound, to improve the potency of protoberberine oxide for treating metabolic diseases, and to reduce the dosage.
The aim of the invention is achieved by the following technical scheme:
the preparation method of the protoberberine oxide cyclodextrin inclusion compound comprises the following steps:
(1) Adding a solvent into cyclodextrin, dissolving and preparing into a saturated cyclodextrin solution for later use;
(2) Adding protoberberine oxide into the cyclodextrin solution obtained in the step (1), continuously heating, and stirring or performing ultrasonic treatment;
(3) Removing solvent, and grinding to obtain original berberine oxide cyclodextrin clathrate.
The cyclodextrin in step (1) includes, but is not limited to, at least one of α -cyclodextrin, β -cyclodextrin, γ -cyclodextrin, hydroxyethyl- β -cyclodextrin, methyl- β -cyclodextrin, sulfobutyl- β -cyclodextrin, and hydroxypropyl- β -cyclodextrin; preferably, the cyclodextrin is hydroxypropyl-beta-cyclodextrin.
The solvent in the step (1) is a mixture of water and an alcohol solvent; ethanol with the concentration of 50-95% is preferable; more preferably 95% ethanol.
The temperature of the continuous heating in the step (2) is preferably 20-60 ℃; more preferably 30 to 60 DEG C
The conditions of the ultrasound described in step (2) are preferably: the frequency is 10-40 KHz, and the ultrasonic wave is carried out for 0.5-4 h; more preferably: the frequency is 40KHz, and the ultrasonic treatment is carried out for 1-4 hours.
The stirring conditions described in step (2) are preferably: the rotating speed is 400 r/min-800 r/min, and stirring is carried out for 1-4 h.
The protoberberine oxides in the step (2) include, but are not limited to, oxidized berberine, oxidized palmatine, oxidized epiberberine and oxidized berberine; preferably, the protoberberine oxide is berberine oxide.
The molar ratio of the cyclodextrin in the step (1) to the protoberberine oxide in the step (2) is preferably 2:1-10:1; more preferably 5:1 to 10:1; most preferably 10:1.
The method for removing the solvent described in step (3) includes, but is not limited to, evaporation under reduced pressure and freeze-drying.
The protoberberine oxide cyclodextrin inclusion compound is prepared by the preparation method.
The application of the protoberberine oxide cyclodextrin inclusion compound in preparing medicaments for treating metabolic diseases.
The medicine contains the protoberberine oxide cyclodextrin inclusion compound.
The dosage forms of the medicine include, but are not limited to, solid dosage forms, semisolid dosage forms, liquid dosage forms, syrups, drop pills, sustained release preparations, injection, emulsion and suspension.
The metabolic diseases comprise at least one or more of blood sugar, hyperlipidemia, hyperuricemia, uric acid nephropathy, diabetes, fatty liver and gout.
Compared with the prior art, the invention has the following advantages and effects:
(1) The clathrate compound prepared by the method can solve the problems of poor water solubility and low bioavailability of the original berberine oxides, and solves the problem that the prior art is difficult to be suitable for the original berberine oxides due to the overlarge difference between the physicochemical properties of the original berberine alkaloids and the original berberine oxides.
(2) The clathrate prepared by the method can greatly improve the potency of the original berberine oxides against metabolic diseases such as hyperuricemia, hyperglycemia, hyperlipidemia and the like, and expand the clinical application of the clathrate.
(3) The preparation method of the protoberberine oxide-CD inclusion compound has the advantages of simple operation, high efficiency, high yield and low cost, and can be applied to large-scale industrial production.
Drawings
FIG. 1 is a phase solubility diagram of oxidized berberine (OBB) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD).
FIG. 2 is a graph of appearance, solubility, and encapsulation efficiency results; wherein, figure A is an appearance diagram of the physical mixture and the inclusion compound of the OBB and the OBB-HP-beta-CD, figure B is a soluble result diagram of the physical mixture and the inclusion compound of the OBB and the OBB-HP-beta-CD, and figure C is a result diagram of the encapsulation efficiency of the OBB-HP-beta-CD obtained in examples 1 to 4.
FIG. 3 is an infrared spectrum of OBB, HP-beta-CD, physical mixtures of OBB-HP-beta-CD and clathrates.
FIG. 4 is a differential scanning calorimetric spectrum of an OBB, HP-beta-CD, OBB-HP-beta-CD physical mixture and clathrate.
FIG. 5 is a powder X-ray diffraction pattern of OBB, HP-beta-CD, physical mixtures of OBB-HP-beta-CD, and clathrates.
FIG. 6 is a graph showing the results of the dissolution of physical mixtures of OBB, HP-beta-CD, OBB-HP-beta-CD with inclusion compounds; wherein, the graph A is a dissolution rate result graph of the artificial gastric juice, and the graph B is a dissolution rate result graph of the artificial intestinal juice.
FIG. 7 is a graph of drug versus time following administration; wherein, figure A is the medicine-time curve graph after the oral administration of the OBB bulk drug and the OBB-HP-beta-CD inclusion compound, and figure B is the medicine-time curve graph after the intravenous injection of the OBB-HP-beta-CD inclusion compound.
FIG. 8 is a graph showing the effect of various drugs on serum Uric Acid (UA), creatinine (CRE), urea nitrogen (BUN), and Kidney index (Kidney index) of potassium hypoxanthine/oxazinate-induced hyperuricemia mice; compared with Con group, #P < 0.01; p < 0.05 compared to the HUA group; * P < 0.01.
FIG. 9 is a graph showing the results of hematoxylin-eosin staining (HE staining) of the kidney after each group of mice was treated with the drug; wherein the black arrows indicate atrophic glomeruli and abnormal tubular structures, and the black triangles indicate normal glomeruli and tubular structures.
FIG. 10 is a graph showing the effect of different drugs on glucose (Glu) levels in rats with fructose-induced metabolic syndrome; p < 0.05 compared to the FRU group; * P < 0.01; compared to the N groups, #P < 0.01.
FIG. 11 is a graph showing the effect of different drugs on fructose-induced metabolic syndrome rat Uric Acid (UA), creatinine (CRE), urea (Urea); p < 0.05 compared to the FRU group; * P < 0.01; compared to the N groups, #P < 0.01.
FIG. 12 is a graph showing the effect of various drugs on fructose-induced metabolic syndrome rat total Triglyceride (TG), total Cholesterol (TC), density lipoprotein (LDL-C) cholesterol, and high density lipoprotein cholesterol (HDL-C) levels; p < 0.05 compared to the FRU group; * P < 0.01; compared with N groups, #P < 0.05; # P < 0.01.
FIG. 13 is a graph of the effect of different drugs on total Triglyceride (TG), total Cholesterol (TC) levels of the Free Fatty Acid (FFAs) induced lipid overload model of HepG2 cells; compared with N groups, #P < 0.05; # P < 0.01; p < 0.05 compared to group M; * P < 0.01.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
In the embodiment of the application: protoberberine oxides are prepared according to the documents Li C, ai G, wang Y, et al Oxyberberine, a novel gut microbiota-mediated metabolite of berberine, possesses superior anti-colitis effect: impact on intestinal epithelial barrier, gut microbiota profile and TLR-MyD 88-NF-Kb path, pharmacol Res.2020Feb;152:104603. The purity of the obtained products is greater than 99%. HP-beta-CD was purchased from Siandeli BioCo., ltd; febuxostat was purchased from Shanghai leaf biotechnology limited; hypoxanthine, potassium oxazinate, were purchased from Sigma-Aldrich. All reagents or equipment not described in detail are commercially available conventional products.
The indexes such as glucose (Glu), triglyceride (TG), cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), uric Acid (UA), creatinine (CRE), urea nitrogen (BUN), urea (Urea), glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT) and the like are all measured by using a commercial kit; all the kits are purchased from the institute of biological engineering built in Nanjing, and the specific operation steps are carried out according to the kit instruction. The remaining process steps or preparation methods not mentioned in detail are all those known to the person skilled in the art.
Example 1
The preparation of the OBB-HP-beta-CD inclusion compound comprises the following steps:
2.17g of HP-beta-CD was taken, 10mL of 90% ethanol was added, and the mixture was stirred with a glass rod until the HP-beta-CD was completely dissolved, to obtain an HP-beta-CD ethanol solution. Precisely weighing OBB according to the molar ratio of the OBB to the HP-beta-CD of 1:10, adding the OBB into the HP-beta-CD ethanol solution, screwing the bottle cap, putting the bottle cap into an ultrasonic cleaner for ultrasonic treatment (40 KHz,40 ℃ for 2 h), removing the solvent by rotary evaporation (90 rpm,60 ℃) and drying and grinding the obtained solid to obtain the OBB-HP-beta-CD inclusion compound.
3 sets of experiments were repeated.
Example 2
The preparation procedure is as in example 1, wherein the amount of OBB added is 2:1, 4:1, 6:1, 8:1 and 10:1, respectively, in terms of molar ratio of OBB to HP-beta-CD.
Example 3
The preparation procedure was the same as in example 1, wherein the temperatures at the time of ultrasound in the ultrasonic cleaning apparatus were 20 ℃,30 ℃,40 ℃,50 ℃ and 60 ℃, respectively.
Example 4
The preparation procedure is as in example 1, wherein the times of ultrasound in the ultrasonic cleaner are 0.5h, 1h, 2h, 3h and 4h, respectively.
Example 5
The procedure is as in example 1, wherein the ultrasound (40 KHz,40 ℃ C., 2 h) conditions are changed to stirring (600 r/min,40 ℃ C., 2 h).
Performance detection
1. Phase solubility diagram
The inclusion ratio of OBB to HP-beta-CD was determined by the phase solubility method. The excess OBB was taken, a series of HP-beta-CD concentrations were added, heated and stirred at 37℃for 48h, and left at 37℃for 12h after stirring to reach dissolution equilibrium. Subsequently, the sample was centrifuged, the supernatant was taken, insoluble components were removed by passing through a 0.22 μm microporous filter membrane, the concentration of OBB in the sample was calculated by high performance liquid chromatography, and a phase solubility chart was drawn.
As a result, as shown in FIG. 1, the obtained phase solubility curve has a good linear relationship (R 2 = 0.9991), suggesting that OBB and HP- β -CD molecules can be bound in a molar ratio of 1:1.
2. Appearance and solubility
The OBB-HP-beta-CD clathrate prepared in example 1 was a pale yellow or white powder, as shown in FIG. 2A.
The OBB-HP-beta-CD clathrate prepared in example 1 has excellent solubility, and can form clear supersaturated OBB water solution fast in water, and the result is shown in figure 2B.
3. Yield is good
The yield in the preparation of the inclusion compound was calculated by weighing.
Yield= (clathrate mass/total charge) x 100%.
The results of the yields of example 1 are shown in Table 1, and the average yield is 95.41.+ -. 1.03%.
4. Encapsulation efficiency
(1) Calculation method
Since OBB is insoluble in water, the encapsulation efficiency of the inclusion compound is calculated from the content of water-soluble OBB and the total OBB content. Two clathrate samples were weighed and added with water and methanol, respectively, to allow the samples to be fully dissolved. After the sample is dissolved, insoluble components are removed through a 0.22 mu m microporous filter membrane, and the content of water-soluble OBB and the total OBB content in the clathrate compound sample are calculated through high performance liquid chromatography.
Encapsulation efficiency (%) =1- (water-soluble OBB content/total OBB content) ×100%.
(2) Experimental results
The results of the encapsulation efficiency of example 1 are shown in table 1, with an average encapsulation efficiency of 95.35±1.77%.
Examples 2 to 4 examined the effect of different reaction conditions on the encapsulation efficiency of OBB and HP-beta-CD. As shown in fig. 2C, 2D and 2E, unlike the conventional cyclodextrin inclusion process, the temperature, ultrasonic time and other factors in the process of preparing the OBB inclusion compound by the method have no significant influence on the product encapsulation efficiency, and only the feeding ratio is an important factor influencing the product encapsulation efficiency. From the perspective of the traditional cyclodextrin inclusion process, only the theoretical molecular inclusion proportion is adopted for feeding or the HP-beta-CD is properly added, the encapsulation rate of the obtained product is extremely low, the water solubility is poor, and a clear solution cannot be obtained (as shown in figure 2C). When the feeding ratio is greatly increased, the visible encapsulation efficiency and the solubility are greatly increased along with the increase of the feeding ratio until a completely clear supersaturated solution can be formed in water.
Table 1: determination of OBB-HP-beta-CD clathrate yield and encapsulation efficiency
5. Identification of OBB-HP-beta-CD inclusion compound
The inclusion compound obtained in example 1 was analyzed by infrared spectroscopy, differential scanning calorimetry and powder X-ray diffraction and compared with the physical mixture of the OBB drug substance, HP-beta-CD and OBB-HP-beta-CD to verify whether the OBB forms inclusion compound with HP-beta-CD.
As can be seen from the infrared spectrum of FIG. 3, the infrared characteristic peak of the OBB is 1643cm -1 、1493cm -1 And 1276cm -1 In this case, after preliminary mixing with HP-beta-CD to form a physical mixture, the characteristic peaks of OBB with lower intensity remain in the spectrum, but after forming an inclusion compound, the characteristic peaks disappear, which indicates that OBB forms an inclusion compound with HP-beta-CD.
As can be seen from the differential scanning calorimetric profile of FIG. 4, the OBB has a sharp endothermic peak at 203℃and the HP-beta-CD has a broader endothermic peak in the range of 50-150 ℃. Compared with the OBB bulk drug, the OBB-HP-beta-CD physical mixture has both an endothermic peak of OBB and an endothermic peak of HP-beta-CD, and in the map of the OBB-HP-beta-CD inclusion compound, the endothermic peak of OBB completely disappears, which indicates that the OBB and the HP-beta-CD form the inclusion compound.
As can be seen from the powder X-ray diffraction pattern of fig. 5, OBB has multiple specific crystalline diffraction peaks, whereas HP- β -CD has no distinct crystalline diffraction peaks, indicating that HP- β -CD is itself in an amorphous form. Whereas for physical mixtures of OBB-HP-beta-CD, the diffraction pattern has both an amorphous state of HP-beta-CD and a crystalline diffraction peak of the less intense OBB. This shows that in the physical mixture, the OBB is still dispersed in the HP-beta-CD in crystalline form. Whereas the OBB crystal characteristic diffraction peak of the OBB-HP-beta-CD inclusion compound almost completely disappeared, which indicates that the OBB is included by the cavities of HP-beta-CD, and exists in an amorphous form.
6. Dissolution test of OBB-HP-beta-CD clathrate
The OBB-HP-beta-CD clathrate used in the experiment was prepared according to the preparation method of example 1.
(1) Dissolution rate measurement method
And (3) taking an OBB 40mg, an OBB-HP-beta-CD inclusion compound and a physical mixture (the molar ratio is 1:10, which is equivalent to OBB 40 mg), filling the mixture into a gelatin capsule as a sample for detection, and testing the dissolution of the medicine according to the second method (oar method) of the first appendix 160 of the edition of the first part of the pharmacopoeia 2020 of the people's republic of China. The dissolution medium is 500mL of artificial gastric juice or artificial intestinal juice, the temperature is 37+/-0.3 ℃, the rotating speed is 100r/min, 1mL is quantitatively sampled at the time of 2 nd min, 5min, 10min, 15min, 20min, 30min, 45min, 60min, 90min and 120min respectively, and 1mL of equivalent isothermal medium is supplemented. The obtained sample is filtered by a microporous membrane with the size of 0.22 mu m, the OBB content in the sample is detected by high performance liquid chromatography, and the dissolution rate is calculated by taking the corresponding dissolution medium as a blank. This experiment was repeated 3 times and the average was taken.
(2) Experimental results
The dissolution profile of each sample is shown in fig. 6. The OBB raw material medicine is insoluble in artificial gastric juice and artificial intestinal juice. The dissolution rates of the OBB-HP-beta-CD physical mixture in the artificial gastric juice and the artificial intestinal juice at 120min are 23.72% and 15.58%, respectively. Compared with the physical mixture of the OBB bulk drug and the OBB-HP-beta-CD, the OBB-HP-beta-CD inclusion compound can reach the dissolution rate of nearly 100% in different dissolution media at 20min, which indicates that the OBB-HP-beta-CD inclusion compound can obviously improve the dissolution rate of OBB.
7. Bioavailability assay of OBB-HP-beta-CD inclusion compound
The OBB-HP-beta-CD clathrate used in the experiment was prepared according to the preparation method of example 1.
(1) Establishment of HPLC analysis method for blood sample
1) Pretreatment method for blood sample
Taking 250 mu L of whole blood of a medicated rat, adding four times of acetonitrile, swirling for 1min, and centrifuging at 10000rpm/min for 10min. After centrifugation, the supernatant is sucked, and after nitrogen is blown dry, 200 mu L of acetonitrile is added for redissolution, thus obtaining a test sample.
2) HPLC chromatographic conditions
Chromatographic column: phenomenex Luna C18 (250 mm. Times.4.6 mm) mobile phase: acetonitrile (a) with 0.1% trifluoroacetic acid (B); 0-20min:10-90% of A,20-30min:90-10% A. The flow rate is 1.0mL/min, the detection wavelength is 345nm, and the sample injection amount is as follows: 10 mu L.
(2) Experimental animals and methods:
18 rats weighing 180-220g (purchased from the university of Chinese medicine laboratory animal center) were randomly divided into three groups of 6, which were respectively an OBB bulk drug oral administration group, an OBB-HP-beta-CD inclusion compound oral administration group and an OBB-HP-beta-CD inclusion compound intravenous administration group. All rats for the test are fed into SPF class laboratory, and the indoor temperature is 25 ℃ and the humidity is 60-65% and the rats can eat and drink water freely after 12 hours of circulating artificial lighting. All rats were fasted for 12 hours before the start of the experiment without water. An appropriate amount of OBB was precisely weighed by an analytical balance, and an appropriate amount of physiological saline was added to prepare OBB having a concentration of 50mg/mL and 1mg/mL, which were used for oral administration and injection administration, respectively. Blood was taken through the orbital venous plexus at 5min, 10min, 15min, 30min, 1h, 2h, 2.5h, 4h, 5h, 6h, 7h, 8h, 12h and 24h after administration via different routes of administration. The obtained blood samples were stored in heparinized anticoagulation blood collection tubes, the blood plasma concentrations of the OBB at different time points were determined by HPLC after the blood samples were processed, and relevant pharmacokinetic parameters were calculated by DAS2.0 software. All experimental data are expressed as mean±sd.
(3) Experimental results
The experimental results are shown in Table 2 and FIG. 7, the OBB-HP-beta-CD clathrate compound can significantly improve the AUC and C of OBB in rats max . Calculated, the bioavailability of the OBB bulk drug is 2.01%, and the bioavailability of the OBB-HP-beta-CD inclusion compound is 32.54%. This suggests that the OBB-HP-beta-CD inclusion compound may significantly improve the bioavailability of OBB.
TABLE 2 pharmacokinetic parameters of OBB and OBB-HP-beta-CD clathrate in rats
8. Therapeutic effect of OBB-HP-beta-CD inclusion compound on potassium hypoxanthine-oxonate induced hyperuricemia
The OBB-HP-beta-CD clathrate used in the experiment was prepared according to the preparation method of example 1.
(1) Experimental animals and methods:
male KM mice (purchased from university of Chinese medicine laboratory animal center, guangzhou), SPF grade, weighing 20+ -2 g, were randomly divided into 7 groups (9 per group): blank (Con), model (HUA), FEB, OBB drug substance (OBB, 10 mg/kg) and OBB-HP-beta-CD clathrate (OBB-CD) were used in low, medium and high dose (1, 2, 4 mg/kg). All mice were acclimatized for one week prior to the start of the experiment. The administration of the model was started at 9 a.m. every day after the start of the experiment, and the mice of each group except the blank control group were induced to hyperuricemia model by intraperitoneal injection of potassium oxazinate (300 mg/kg) and lavage of hypoxanthine (300 mg/kg), while the blank control group was administered with an equal volume of physiological saline by intraperitoneal injection and lavage. After molding for 1h, the drug test group perfuses the corresponding concentration of the drug, and the blank control group and the model group perfuse the same volume of physiological saline. The experiment was performed for 10 days. Mice were sacrificed after the end of the experiment, and blood samples and kidney samples were collected to determine the corresponding index. All experimental data are expressed in mean±sd.
And (3) observing the indexes:
1) Uric Acid (UA) content;
2) Renal function related index: creatinine (CRE), urea nitrogen (BUN);
3) Kidney index (Kidney index);
4) Kidney HE staining;
(2) Experimental results
The experimental results are shown in FIGS. 8 to 9. The results show that: the OBB and the positive drug febuxostat can obviously reduce the blood UA level of mice with high uric acid, wherein the action effect generated by 2mg/kg of OBB-HP-beta-CD inclusion compound is equivalent to the action effect of 10mg/kg of OBB bulk drug, which indicates that the OBB-HP-beta-CD inclusion compound can obviously improve the potency of OBB for reducing uric acid. Meanwhile, the action intensity of the OBB-HP-beta-CD inclusion compound is in a dose-dependent relationship
The mice in the model group have obviously increased levels of blood uric acid, and also obviously increased levels of kidney index, blood CRE and blood BUN, and also show obvious kidney injury. The OBB-HP-beta-CD inclusion compound can obviously reduce the indexes of blood CRE, blood BUN and kidney of mice while reducing uric acid level in serum, so that the OBB has obvious kidney protection effect. Meanwhile, the renal HE staining result also shows that the OBB-HP-beta-CD inclusion compound can obviously improve renal injury caused by hyperuricemia. In addition, the experimental result shows that the action intensity of the OBB-HP-beta-CD inclusion compound with the concentration of 2mg/kg is equivalent to that of the OBB bulk drug with the concentration of 10mg/kg, which indicates that the OBB-HP-beta-CD inclusion compound can obviously improve the potency of the OBB for resisting kidney injury.
In conclusion, the OBB-HP-beta-CD inclusion compound has remarkable uric acid reducing and kidney protecting effects, and the potency of the OBB-HP-beta-CD inclusion compound is remarkably superior to that of an OBB bulk drug.
9. Therapeutic effect of OBB-HP-beta-CD inclusion compound on fructose-induced metabolic syndrome
The OBB-HP-beta-CD clathrate used in the experiment was prepared according to the preparation method of example 1.
(1) Experimental animals and methods:
SPF-grade male SD rats (purchased from the university of Guangzhou traditional Chinese medicine laboratory animal center) were selected, and after 1 week of normal feeding, the rats were divided into a normal group (N), a model group (Fru), a febuxostat group (Feb, 5 mg/kg), a tribromone group (BEN, 5 mg/kg), an atorvastatin calcium group (AFT, 5 mg/kg), a metformin group (MEM, 300 mg/kg), a berberine group (BBR, 50 mg/kg), an OBB-HP-beta-CD clathrate group (2.5, 5, 10 mg/kg) and an OBB bulk drug group (20 mg/kg), each group being 9 animals. All rats except the N groups were modeled as a HUA by free drinking 30% fructose solution. The experiment was performed for a total of 12 weeks, the first 6 weeks without dosing, and the remaining rats, except for the Con group, were dosed once daily with the corresponding drug. All rats were fasted for 12 hours on the last day of the experiment, and then the rats were sacrificed to obtain the materials and the relevant index was determined. All experimental data are expressed in mean±sd.
And (3) observing the indexes:
1) Blood glucose (Glu) levels
2) Uric Acid (UA) content;
3) Renal function related index: creatinine (CRE), urea (Urea);
4) Triglyceride (TG), cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C)
(2) Experimental results
The experimental results are shown in FIGS. 10 to 12. Compared with normal rats, the rats in the model group have obviously increased weight, blood sugar, blood fat, uric acid and other levels, and are accompanied by liver and kidney injury, which shows that high-concentration fructose can successfully induce the metabolic syndrome model of the rats. The OBB-HP-beta-CD inclusion compound with different dosages and indexes such as rat weight, blood glucose, triglyceride, cholesterol, blood uric acid, blood creatinine and the like can be obviously reduced, so that the OBB-HP-beta-CD inclusion compound has obvious improvement effect on fructose-induced rat metabolic syndrome. In addition, the OBB-HP-beta-CD inclusion compound can obviously relieve kidney injury caused by the metabolic syndrome of rats. Meanwhile, the OBB-HP-beta-CD inclusion compound has dose dependency on the drug effect of the fructose-induced metabolic syndrome rat, and the potency of the OBB-HP-beta-CD inclusion compound is about 4-8 times of that of the OBB bulk drug.
10. Lipid lowering effect of protoberberine alkaloid oxide-cyclodextrin clathrate on HepG2 cell
The OBB-HP-beta-CD inclusion compound, the Oxidation Palmatine (OPAL) -HP-beta-CD inclusion compound, the Oxidation Epiberberine (OEPI) -HP-beta-CD inclusion compound and the Oxidation Coptisine (OCOP) -HP-beta-CD inclusion compound used for the experiment were prepared according to the preparation method of example 1; the oxidized berberine (OBB) is replaced with corresponding Oxidized Palmatine (OPAL), oxidized Epiberberine (OEPI) and Oxidized Coptisine (OCOP).
(1) The experimental method comprises the following steps:
1) Preparation of experimental drugs and reagents
Preparation of high-fat culture medium: a basal cell culture medium was obtained by adding 45mL of RPMI-1640 medium (Gibco) to 5mL of Fetal Bovine Serum (FBS) and 0.5mL of penicillin-streptomycin solution. A proper amount of sodium oleate and sodium palmitate are precisely weighed, and pure water is used for preparing a sodium oleate mother solution with the concentration of 40mM and a sodium palmitate mother solution with the concentration of 20mM respectively. An appropriate amount of Bovine Serum Albumin (BSA) was added to the PBS buffer to prepare a 30% BSA solution. After the completion of the preparation, 40mM sodium oleate mother liquor, 20mM sodium palmitate mother liquor and Bovine Serum Albumin (BSA) were mixed in a ratio of 1:1:2, and diluted 15 times with the prepared basal medium to obtain 1mM Free Fatty Acid (FFAs) medium (0.66 mM oleic acid+0.33 mM palmitic acid), i.e., a high fat medium.
0.0672g of berberine (BBR) is precisely weighed by an analytical balance, dissolved by a proper amount of DMSO, and fully transferred into a 10mL measuring flask for constant volume, thus obtaining 20mM BBR mother liquor. The prepared mother solution is stored in a refrigerator at the temperature of 4 ℃, and diluted with FFAs culture medium according to the requirement during experiments.
1.2916g of metformin (Met) is precisely weighed by an analytical balance, dissolved by a proper amount of PBS buffer solution, and fully transferred into a 10mL volumetric flask for constant volume, thus obtaining 20mM Met mother solution. The prepared mother solution is stored in a refrigerator at the temperature of 4 ℃, and diluted with FFAs culture medium according to the requirement during experiments.
0.152g of OBB-HP-beta-CD inclusion compound is precisely weighed by an analytical balance, dissolved by 1mL of PBS buffer solution and fully transferred to obtain 10mM OBB mother liquor. The prepared mother solution is stored in a refrigerator at the temperature of 4 ℃, and diluted with FFAs culture medium according to the requirement during experiments.
0.158g of the palmatine Oxide (OPAL) -HP-beta-CD inclusion compound is precisely weighed by an analytical balance, dissolved by 1mL of PBS buffer solution and fully transferred to obtain 10mM palmatine oxide mother liquor. The prepared mother solution is stored in a refrigerator at the temperature of 4 ℃, and diluted with FFAs culture medium according to the requirement during experiments.
0.152g of the berberine Oxide (OEPI) -HP-beta-CD inclusion compound is precisely weighed by an analytical balance, dissolved by 1mL of PBS buffer solution and fully transferred to obtain 10mM berberine oxide mother liquor. The prepared mother solution is stored in a refrigerator at the temperature of 4 ℃, and diluted with FFAs culture medium according to the requirement during experiments.
0.153g of coptisine oxide (OCOP) -HP-beta-CD inclusion compound is precisely weighed by an analytical balance, dissolved by 1mL of PBS buffer solution and fully transferred to obtain 10mM coptisine oxide mother liquor. The prepared mother solution is stored in a refrigerator at the temperature of 4 ℃, and diluted with FFAs culture medium according to the requirement during experiments.
2) Culture of HepG2 cells
Cell resuscitation: the frozen tube containing HepG2 cells was removed from the liquid nitrogen flask and heated in a 37℃water bath for 1min before transferring the solution in the tube to a defined amount of basal medium. Cells were washed with basal medium and centrifuged, and the supernatant was discarded. Fresh medium was added to the pellet and carefully blown down with a pipette to form a uniformly dispersed cell suspension. According to the experimental requirement, transferring a certain volume of cell fluid into a cell culture bottle, and slightly shaking to uniformly disperse the cell fluid. After observing and determining that the cell morphology is normal through a microscope and no foreign matters exist in the culture medium, the culture flask can be placed in an incubator (37 ℃ C., 5% CO 2) for culture.
Cell culture: groups were made according to table 3 and incubated with different media.
Cell passaging and cryopreservation: the old culture medium in the cell culture flask is discarded, a proper amount of PBS buffer solution is used for washing off the residual culture medium, 1mL of pancreatin is added for digestion for 1-2min, and the cell culture flask can be properly beaten in the digestion process to promote cell suspension. When sufficient suspension of cells in the medium was observed, the digestion was stopped by rapid addition of serum-containing medium. The cell suspension is removed from the culture medium, centrifuged, the supernatant is discarded, a certain amount of fresh medium is added to resuspend the cells, and the cells are transferred to a cell culture flask for culture.
TABLE 3 cell administration incubation dose
3) Determination of Total Cholesterol and Total Triglycerides
HepG2 cells were inoculated into 6-well plates, starved cultured with serum-free medium for 6 hours, dosed with the drug-containing medium according to table 3 and incubated for 24 hours. After completion of the incubation, the drug-containing medium was discarded, 500. Mu.L of PBS was added, and the cells at the bottom of the 6-well plate were sufficiently suspended with a cell scraper. Taking out the cell suspension, repeatedly freezing and thawing for three times at-80 ℃ in a refrigerator, collecting the lysate and the cells into a test tube, centrifuging, and taking the supernatant. The Total Cholesterol (TC), total Triglycerides (TG) and protein content of each sample were determined using a kit.
(2) Experimental results
As shown in fig. 13, both TG and TC levels were significantly higher in the M groups than in the N groups, and the gap between the groups was statistically significant, indicating successful modeling of the lipid overload model. There was no significant difference in intracellular TG and TC levels in the CD group compared to the M group, indicating that HP- β -CD had no significant effect on cellular lipid accumulation. After the incubation of the drug-containing high-fat culture medium, the intracellular TG and TC levels are reduced to different degrees in comparison with the M group, the comparison with the M group has statistical differences, and the reduction of the intracellular TC and TG contents of the HepG2 cells of the OBB group is more obvious than that of other drug groups. The results show that the different protoberberine alkaloid oxide-cyclodextrin inclusion compounds have the capacity of inhibiting the accumulation of the lipid of HepG2 cells, and the OBB-HP-beta-CD inclusion compound has stronger lipid-lowering effect.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. The preparation method of the protoberberine oxide cyclodextrin inclusion compound is characterized by comprising the following steps:
(1) Adding a solvent into cyclodextrin, heating to dissolve and preparing into a saturated cyclodextrin solution for later use;
(2) Adding protoberberine oxide into the cyclodextrin solution obtained in the step (1), continuously heating, and stirring or performing ultrasonic treatment;
(3) Removing solvent, and grinding to obtain original berberine oxide cyclodextrin clathrate.
2. The method of manufacturing according to claim 1, characterized in that:
the molar ratio of the cyclodextrin in the step (1) to the protoberberine oxide in the step (2) is 2:1-10:1.
3. The preparation method according to claim 2, characterized in that:
the molar ratio of the cyclodextrin in the step (1) to the protoberberine oxide in the step (2) is 5:1-10:1.
4. The method of manufacturing according to claim 1, characterized in that:
the cyclodextrin in the step (1) is at least one of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, hydroxyethyl-beta-cyclodextrin, methyl-beta-cyclodextrin, sulfobutyl-beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin;
the solvent in the step (1) is a mixture of water and an alcohol solvent.
5. The method of manufacturing according to claim 4, wherein:
the cyclodextrin in the step (1) is hydroxypropyl-beta-cyclodextrin;
the solvent in the step (1) is 50% -95% ethanol.
6. The method of manufacturing according to claim 1, characterized in that:
the protoberberine oxide in the step (2) is berberine oxide, palmatine oxide, epiberberine oxide or berberine oxide;
the temperature of the continuous heating in the step (2) is preferably 30-60 ℃;
the conditions of the ultrasound described in step (2) are preferably: the frequency is 10-40 KHz, and the ultrasonic wave is carried out for 1-4 h;
the stirring conditions described in step (2) are preferably: the rotating speed is 400 r/min-800 r/min, and stirring is carried out for 1-4 h.
7. The method of manufacturing according to claim 1, characterized in that:
the method for removing the solvent in the step (3) is reduced pressure evaporation or freeze drying.
8. A protoberberine oxide cyclodextrin inclusion compound obtained by the preparation method of any one of claims 1 to 7.
9. The use of the protoberberine oxide cyclodextrin inclusion compound according to claim 8 for preparing a medicament for treating metabolic diseases.
10. Use according to claim 9
The dosage forms of the medicine comprise solid dosage forms, semisolid dosage forms, liquid dosage forms, syrups, drop pills, sustained and controlled release preparations, sustained release preparations, injections, emulsions and suspensions;
the metabolic diseases comprise at least one or more of blood sugar, hyperlipidemia, hyperuricemia, uric acid nephropathy, diabetes, fatty liver and gout.
CN202310409421.9A 2023-04-17 2023-04-17 Berberine oxide cyclodextrin inclusion compound, and preparation method and application thereof Pending CN116440290A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118340222A (en) * 2024-06-18 2024-07-16 浙江农林大学 Nanometer inclusion compound for enriching fragrance of fresh flower cell sap and preparation method thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118340222A (en) * 2024-06-18 2024-07-16 浙江农林大学 Nanometer inclusion compound for enriching fragrance of fresh flower cell sap and preparation method thereof

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