CN116077679A - Ginsenoside RK1 cyclodextrin inclusion compound and preparation and sleep improvement application thereof - Google Patents
Ginsenoside RK1 cyclodextrin inclusion compound and preparation and sleep improvement application thereof Download PDFInfo
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Abstract
Ginsenoside RK1 cyclodextrin inclusion compound and application for preparing and improving sleep, and belongs to the technical field of natural compound preparation. In order to solve the technical problems of ginsenoside RK1 preparation in the prior art and further research on functions of ginsenoside RK1, the invention discloses a preparation method of a high-purity ginsenoside RK1 cyclodextrin inclusion compound, which comprises the steps of hydrolyzing total ginsenoside with acid, and preparing the ginsenoside RK1 cyclodextrin inclusion compound by utilizing the characteristic that cyclodextrin is selectively chelated with ginsenoside RK1 under specific acidic conditions for precipitation. The preparation method is simple to operate, environment-friendly, low in cost, suitable for industrial production and capable of promoting the development and utilization of ginseng plants. In addition, the ginsenoside RK1 cyclodextrin inclusion compound prepared by the method has the effect of improving cerebral energy metabolism disorder caused by sleep deprivation, and can be used for preparing medicaments for improving sleep.
Description
Technical Field
The invention belongs to the technical field of natural compound preparation, and particularly relates to a ginsenoside RK1 cyclodextrin inclusion compound and application thereof in preparation and sleep improvement.
Background
Ginsenoside RK1 is a special thin component of Ginseng radix RubriThere are ginsenosides (JO SK, KIM IS, YOON KS, et al preparation of ginsenosides Rg, RK1, and Rg5-selectively enriched ginsengs by a simple steaming process. Eur Food Res technology, 2015, 240 (1): 251-256). Ginsenoside RK1 has biological activities of resisting tumor, regulating blood sugar, protecting nervous system, etc. as novel rare saponin discovered in recent years. Ginsenoside RK1 can be prepared by hydrolyzing ginsenoside. The common hydrolysis modes at present are high-temperature thermal cracking, acid-base chemical method, enzyme method and the like. However, the current preparation method has some defects, such as insufficient hydrolysis or excessive hydrolysis when the strength of acid, the hydrolysis temperature and the hydrolysis time are not properly controlled, and thus has a problem of low yield. For example, chinese patent CN201610344506.3 discloses a method for producing ginsenoside RK1 by using protopanaxadiol saponins as raw material, which comprises adding protopanaxadiol saponins and water in a fermenter, and introducing N 2 Protecting, adding organic acid and catalytic amount of heteropoly acid HxYW with Keggin structure after on-line sterilization 12 O 40 ·nH 2 And (3) an O catalyst, wherein Y is selected from P, si, fe or Zn, x is 3 or 4, n is a positive integer of 0-30, reacting for 24-48 hours at 80-105 ℃, and finally collecting and purifying a reaction product to obtain the high-purity ginsenoside RK1. Chinese patent CN201710110137.6 discloses a preparation method and application of high purity compound ginsenoside RK1, the preparation method comprises dissolving panaxadiol saponins with glacial acetic acid aqueous solution, and then preparing ginsenoside RK1 by water bath reaction. CN202010007323.9 discloses a preparation method of ginsenoside RK1 and Rg5, wherein the content of ginsenoside Rb1 is 12.5-19.4%, the content of Rb2 is 10.3-13.4%, the content of Rb3 is 26.1-31.4%, and the content of Rd is 7.3-13.3%. The method comprises the steps of preparing ginsenoside by using a macroporous resin column, treating an aqueous solution of ginsenoside acid at ultrahigh pressure, treating at low temperature, centrifuging, heating supernatant after precipitation and filtration at high temperature, centrifuging again, filtering again to obtain precipitate, and drying the precipitate to obtain high-purity ginsenoside RK1 and ginsenoside Rg5. Chinese patent CN201710865039.3 discloses a method for extracting ginsenoside Rg5 and ginsenoside RK1, and its application, by reflux extracting steamed Ginseng plant material with solvent, and collecting extractive solutionSubjecting to macroporous adsorbent resin column chromatography to obtain total saponins containing ginsenoside Rg5 and RK1, subjecting total saponins, steamed ginsenoside, notoginseng radix total saponins or steamed ginsenoside Rb1 to macroporous adsorbent resin column chromatography to obtain group saponins with total ginsenoside Rg5 and RK1 content greater than 97%, and subjecting the group saponins to macroporous adsorbent resin column repeated column chromatography to obtain monomeric ginsenoside Rg5 and monomeric ginsenoside RK1 with purity greater than 90%.
As can be seen from the comparison, the ginsenoside RK1 is generally separated and purified at present, and the following problems are caused: (1) The method combines acid conversion and macroporous resin purification, has complex process, long separation time, needs desalting treatment, easily causes resource waste and is not friendly to the environment; (2) After the ginseng total saponins are separated into diol groups and triol groups, the diol group saponins are converted and prepared, and the diol groups are obtained with higher conversion efficiency, but more time and cost are often required, so that the complexity of the process is increased to a certain extent, and the resource waste is caused. (3) The ginsenoside is converted mostly to obtain the whole RK1, and impurities are contained, and the monomer RK1 is separated mostly by column chromatography such as silica gel, so that the separation cost is high and the environment is not friendly; is not suitable for industrialized mass production.
In addition, ginsenoside RK1 has poor stability and is easily degraded at normal temperature. It needs to be stored at low temperature and protected from light. So that the development and the utilization of the material are greatly limited. Cyclodextrin (CD) is a cyclic oligosaccharide of amylose produced by bacillus under the action of cyclodextrin glucosyltransferase, and is commonly three kinds of alpha-, beta-and gamma-cyclodextrin. Among them, beta-cyclodextrin is a research hotspot due to its cheapest and readily available and low toxicity properties. After the medicine molecule and cyclodextrin form inclusion compound, the water solubility of the medicine can be increased, the stability of the medicine can be improved, the irritation and adverse reaction of the medicine in gastrointestinal tract can be reduced, the release time of the medicine can be prolonged, and the bioavailability of the medicine can be improved. The greatest advantage of cyclodextrin inclusion is that it can change the properties of the drug from a molecular level and has little interference with the pharmacokinetic process of the drug. Currently, about 30 pharmaceutical formulations containing cyclodextrin are marketed in the world.
Sleep deprivation is a common sleep disorder that has become a ubiquitous problem in society. Investigation showed that more than 21% of the respondents had a daily sleep time of less than 6 h. Whereas recent studies indicate that sleep deprivation is causally related to energy metabolism disorders. Insufficient sleep increases the risk of developing type 2 diabetes and obesity. Further mechanical studies have found that sleep loss results in reduced insulin sensitivity and leptin levels, beta cell decompensation, reduced glucose tolerance, and the induction of diabetes or obesity. It was found that sleep insufficiency can cause insulin resistance by affecting clock gene expression, impairing mitochondrial function.
At present, methods such as neuroleptic drugs and vegetative nerves and the like are mostly adopted for treating insomnia, but the neuroleptic drugs cannot improve sleep structure and are easy to cause hangover after long-term administration. Therefore, the effective components for treating and improving insomnia are searched from the natural Chinese herbal medicines which are safe, reliable, low in cost and easy to obtain, and the effective components have important application value.
Disclosure of Invention
In order to solve the technical problems existing in the prior art for preparing ginsenoside RK1 and further research functions of the ginsenoside RK1, the invention provides a preparation method of a ginsenoside RK1 cyclodextrin inclusion compound, which comprises the following steps:
(1) Taking ginseng total saponins, adding 55% -65% citric acid aqueous solution according to a feed liquid ratio of 1 g to 5 mL, uniformly stirring, and cooling in a water bath at 90-120 ℃ at 4-6 h at room temperature to obtain hydrolysate;
(2) Adding cyclodextrin into the hydrolysate obtained in the step (1) according to the feed liquid ratio of 1.0 g to 1 mL to 1.6 g to 1 mL, stirring while adding, standing for 12-h, and centrifugally collecting precipitate to obtain a ginsenoside RK1 cyclodextrin inclusion compound crude product;
(3) Washing with 60% citric acid aqueous solution for 2 times, washing with 5% ethanol for 2 times, precipitating, and drying to obtain refined ginsenoside RK1 cyclodextrin clathrate.
The citric acid solution in the preparation method can be replaced by organic acid solution such as formic acid, acetic acid, acidic amino acid, malic acid, oxalic acid, lactic acid and the like with the same acidity or inorganic acid solution such as hydrochloric acid, sulfuric acid and the like with the same acidity.
Further defined, the ginseng total saponins in step (1) include ginseng stem and leaf total saponins, ginseng fruit total saponins, american ginseng stem and leaf total saponins, american ginseng fruit total saponins.
Further defined is that the aqueous citric acid solution of step (1) has a mass fraction of 60%.
Further defined, the water bath temperature of step (1) is 100 ℃ and the water bath time is 4 h.
Further defined, the feed to liquid ratio of step (2) is 1.2 g:1 mL.
Further defined, the cyclodextrin in step (2) is any one of alpha cyclodextrin, beta cyclodextrin and gamma cyclodextrin.
The invention also provides the ginsenoside RK1 cyclodextrin inclusion compound obtained by the preparation method.
The invention also provides application of the ginsenoside RK1 cyclodextrin inclusion compound obtained by the preparation method in preparing a medicament capable of improving sleep.
Further limited, the medicament is prepared by taking ginsenoside RK1 cyclodextrin inclusion compound as an active ingredient and matching auxiliary materials which can be used for medicaments.
Further defined, the medicament may act by oral or parenteral administration; the dosage forms of the medicine comprise tablets, capsules, powder, pills, granules, injections and patches.
The invention has the beneficial effects that:
the invention discloses a preparation method of a high-purity ginsenoside RK1 cyclodextrin inclusion compound (beta-CD-RK 1), which comprises the steps of hydrolyzing ginsenoside total saponins by acid, and preparing the ginsenoside RK1 cyclodextrin inclusion compound by utilizing the characteristic that cyclodextrin is selectively chelated with ginsenoside RK1 under a specific acidic condition, so as to realize the separation of ginsenoside RK1 and cyclodextrin inclusion in one step. The reaction substrates in the preparation method can be industrially obtained, can be purchased from the market, can be completely used for preparing the rare saponin RK1, is simple to operate, is environment-friendly and low in cost, is suitable for industrial production, and promotes the development and utilization of ginseng plants. The purity of the prepared ginsenoside RK1 cyclodextrin inclusion compound can reach more than 90%.
In addition, the invention also discovers that the ginsenoside RK1 cyclodextrin inclusion compound prepared has the effect of improving cerebral energy metabolism disorder caused by sleep deprivation, and specifically comprises (1) improving abnormal glycolipid metabolism and increasing ATP level; (2) Relieving mitochondrial structural damage, regulating mitochondrial biological functions, and improving AMPK/PGC-1/Nrf-1 pathway protein expression; (3) Improvement A 1 R and A 2A R expresses and regulates its expression level with mGluR5 and GABAA1 alpha, and plays a role in improving sleep in sleep deprived rats. The ginsenoside RK1 cyclodextrin inclusion compound obtained by the invention can be used for preparing medicaments capable of improving sleep. In addition, the oral absorption rate of the ginsenoside RK1 can be better improved by preparing the ginsenoside RK1 into cyclodextrin inclusion compound.
Drawings
FIG. 1 is a graph showing the effect of mass fraction of aqueous citric acid solution on purity of ginsenoside RK1 cyclodextrin inclusion compound in step (1);
FIG. 2 is a graph showing the effect of water bath temperature on purity of ginsenoside RK1 cyclodextrin inclusion compound;
FIG. 3 is a graph showing the effect of water bath time on purity of ginsenoside RK1 cyclodextrin inclusion compound;
FIG. 4 is a graph showing the effect of the ratio of beta cyclodextrin to the hydrolysate obtained in step (1) on the yield of ginsenoside RK1 cyclodextrin inclusion compound;
FIG. 5 is an HPLC chromatogram of RK1 after hydrolysis of total saponins from stems and leaves of American ginseng in example 2;
FIG. 6 is an HPLC chromatogram of β -CD-RK1 after isolation and purification in example 2;
FIG. 7 is an electron microscope image of β -CD-RK 1; wherein A in FIG. 7 is an electron microscope of beta-CD, B in FIG. 7 is a physical mixed electron microscope of beta-CD and ginsenoside RK1, C in FIG. 7 is an electron microscope of ginsenoside RK1, and D in FIG. 7 is an electron microscope of beta-CD-RK 1;
FIG. 8 is a graph showing the comparison of the relative expression amounts of the Nrf-1 proteins of rats in each group; wherein, compared with the blank group, * p<0.05， ** p<0.01; compared with the model group # p<0.05， ## p<0.01；
FIG. 9 is a graph showing the comparison of the relative expression level of the P-AMPK protein in each group of rats; wherein, compared with the blank group, * p<0.05， ** p<0.01; compared with the model group # p<0.05， ## p<0.01；
FIG. 10 is a graph showing the comparison of the relative expression amounts of PGC-1 alpha protein in each group of rats; wherein, compared with the blank group, * p<0.05， ** p<0.01; compared with the model group # p<0.05， ## p<0.01；
FIG. 11 is a graph showing the effect of high dose of beta-CD-RK 1 on rat hypothalamic pathology; wherein, A in FIG. 11 is blank group, B in FIG. 11 is model group, C in FIG. 11 is positive drug group, D in FIG. 11 is beta-CD-RK 1 high dose group;
FIG. 12A 1 R，A 2A WB panel of R, mGluR5, GABAA1 a and β -actin;
FIG. 13 is a graph showing comparison of protein expression levels of mGluR5 in each group of rats;
FIG. 14 shows rats A of each group 1 Comparing the protein expression level of R with a result graph;
FIG. 15 shows rats A of each group 2A Comparing the protein expression level of R with a result graph;
FIG. 16 is a graph showing the results of comparison of protein expression levels of GABAA 1. Alpha. In each group of rats;
FIG. 17 is a pharmacokinetic profile of RK1, beta-CD/RK 1 physical blend and beta-CD-RK 1 clathrate.
Detailed Description
The present invention is further illustrated by the following examples and drawings, which are not intended to be limiting, but any modifications, equivalents, improvements, etc. within the spirit and principles of the present invention are intended to be included within the scope of the present invention.
The method for preparing the ginsenoside RK1 cyclodextrin inclusion compound comprises the following steps:
(1) Weighing ginseng total saponins, adding citric acid aqueous solution according to a feed liquid ratio of 1 g to 5 mL, stirring uniformly, and then carrying out water bath and cooling at room temperature to obtain hydrolysate;
(2) Adding cyclodextrin into the hydrolysate obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain a ginsenoside RK1 cyclodextrin inclusion compound crude product;
(3) Washing twice by using a citric acid aqueous solution with the mass fraction of 60%, washing 2 times by using ethanol with the volume fraction of 5%, precipitating and drying to obtain the refined ginsenoside RK1 cyclodextrin inclusion compound.
In order to obtain the ginsenoside RK1 cyclodextrin inclusion compound with higher purity, the invention optimizes the corresponding parameters in the method, wherein the total ginsenoside is selected from the total ginsenoside of ginseng stems and leaves (the total content is about 85 percent) in the optimization process, and the beta cyclodextrin is used for the cyclodextrin selection, and the specific optimization process is as follows:
1. optimizing the mass fraction of the citric acid aqueous solution in the step (1):
different mass fractions of aqueous citric acid solutions (40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%) were prepared, the water bath temperature was set to 100 ℃ for the control variable, the water bath time was set to 1 h, and the feed-to-liquid ratio of beta-cyclodextrin to the hydrolysate obtained in step (1) was set to 1.0 g:1 mL. Detecting the purity of the ginsenoside RK1 cyclodextrin inclusion compound obtained in the step (3) by using an HPLC method.
The results are shown in FIG. 1, wherein the mass fraction of the aqueous citric acid solution in step (1) has a significant effect on the purity of the obtained beta-CD-RK 1. When the mass fraction of the aqueous solution of citric acid is 40% -50%, beta-CD-RK 1 can be obtained, but the purity of the beta-CD-RK 1 is lower than 70%; the purity of beta-CD-RK 1 is higher when the mass fraction of the aqueous citric acid solution is 55% -75%, wherein the purity of beta-CD-RK 1 is highest when the mass fraction of the aqueous citric acid solution is 60%, and is 92%. Thus, the mass fraction of the aqueous citric acid solution in step (1) is selected to be 55% -65%, preferably 60%.
2. The water bath temperature is optimized:
the water bath temperature was defined as 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃, 110 ℃, 120 ℃, the mass fraction of the aqueous citric acid solution in step (1) was set as 60% by mass, the water bath time was set as 1 h, and the feed-liquid ratio of the beta-cyclodextrin to the hydrolysate obtained in step (1) was set as 1.0 g:1 mL, respectively. Detecting the purity of the ginsenoside RK1 cyclodextrin inclusion compound obtained in the step (3) by using an HPLC method.
The results are shown in FIG. 2, where the water bath temperature has a significant effect on the purity of the resulting beta-CD-RK 1. The purity of beta-CD-RK 1 gradually increased when the water bath temperature increased from 60 ℃ to 90 ℃, and the purity of beta-CD-RK 1 was basically stable and the change was not obvious when the water bath temperature increased from 90 ℃ to 120 ℃, wherein the purity of beta-CD-RK 1 was the highest at the water bath temperature of 100 ℃, which was 93%. Thus, the water bath temperature is selected to be 90-120 ℃, preferably 100%.
3. The water bath time is optimized:
the water bath time was set to 0.5 h, 1 h, 2 h, 3 h, 4 h, respectively, the mass fraction of the aqueous citric acid solution in step (1) was set to 60% for the control variable, the water bath temperature was set to 100 ℃, and the feed liquid ratio of beta cyclodextrin to the hydrolysate obtained in step (1) was set to 1.0 g:1 mL. Detecting the purity of the ginsenoside RK1 cyclodextrin inclusion compound obtained in the step (3) by using an HPLC method.
The results are shown in FIG. 3, where the water bath time has a significant effect on the purity of the resulting beta-CD-RK 1. The purity of beta-CD-RK 1 gradually increased as the water bath time was extended from 0.5 h to 4 h, and the purity of beta-CD-RK 1 was substantially stable with insignificant change as the water bath time was extended from 4 h to 6 h, wherein the purity of beta-CD-RK 1 was highest at 93% for a water bath time of 4 h. Thus, a water bath time of 4 h-6 h, preferably 4 h, is selected.
4. Optimizing the feed liquid ratio of cyclodextrin to the hydrolysate obtained in the step (1):
the feed liquid ratio of the beta cyclodextrin to the hydrolysate obtained in the step (1) was set to 0.2 g:1 mL, 0.4 g:1 mL, 0.6 g:1 mL, 0.8 g:1 mL, 1.0 g:1 mL, 1.2 g:1 mL, 1.4 g:1 mL, respectively, the mass fraction of the aqueous citric acid solution in the step (1) was set to 60% as a control variable, the water bath temperature was set to 100℃and the water bath time was set to 3 h. Detecting the yield of the ginsenoside RK1 cyclodextrin inclusion compound obtained in the step (3) by using an HPLC method.
The results are shown in FIG. 4, and the ratio of the beta cyclodextrin to the hydrolysate obtained in the step (1) has a significant effect on the yield of the beta-CD-RK 1. When the feed liquid ratio of beta cyclodextrin to the hydrolysate obtained in the step (1) is increased from 0.2 g to 1 mL to 1.0 g to 1 mL, the yield of beta-CD-RK 1 is gradually increased, when the feed liquid ratio is 1.0 g to 1 mL, the yield of beta-CD-RK 1 reaches a higher value, when the feed liquid ratio is 1.2 g to 1 mL, the yield of beta-CD-RK 1 is highest and is 12%, and when the feed liquid ratio is increased from 1.2 g to 1 mL to 1.6 g to 1 mL, the yield of beta-CD-RK 1 is slightly reduced. Thus, the feed liquid ratio of beta cyclodextrin to the hydrolysate obtained in step (1) is selected to be 1.0 g:1 mL-1.6 g:1 mL, preferably 1.2 g:1 mL.
Through the optimization process, the invention obtains the preparation method of the ginsenoside RK1 cyclodextrin inclusion compound with higher purity, which comprises the following steps:
(1) Taking ginseng total saponins, adding 55% -65% citric acid aqueous solution according to a feed liquid ratio of 1 g to 5 mL, uniformly stirring, and cooling in a water bath at 90-120 ℃ at 4-6 h at room temperature to obtain hydrolysate;
(2) Adding cyclodextrin into the hydrolysate obtained in the step (1) according to the feed liquid ratio of 1.0 g to 1 mL to 1.6 g to 1 mL, stirring while adding, standing for 12-h, and centrifugally collecting precipitate to obtain a ginsenoside RK1 cyclodextrin inclusion compound crude product;
(3) Washing with 60% citric acid aqueous solution for 2 times, washing with 5% ethanol for 2 times, precipitating, and drying to obtain refined ginsenoside RK1 cyclodextrin clathrate.
The invention provides the following examples aiming at the optimized preparation method:
example 1:
(1) Taking ginseng fruit total saponins, adding a citric acid aqueous solution with the mass fraction of 55% according to the feed liquid ratio of 1 g to 5 mL, uniformly stirring, and cooling in a water bath of 4 h at 90 ℃ at room temperature to obtain hydrolysate;
(2) Adding alpha cyclodextrin into the hydrolysate obtained in the step (1) according to the feed liquid ratio of 1.6 g to 1 mL, stirring while adding, standing for 12 h, and centrifugally collecting precipitate to obtain a ginsenoside RK1 alpha cyclodextrin inclusion compound crude product;
(3) Washing with 60% citric acid aqueous solution for 2 times, washing with 5% ethanol for 2 times, precipitating, and drying to obtain refined ginsenoside RK1 alpha cyclodextrin clathrate.
The purity of the ginsenoside RK1 alpha cyclodextrin inclusion compound detected by an HPLC method is 90.1 percent.
Example 2:
(1) Taking total saponins of stems and leaves of American ginseng, adding a citric acid aqueous solution with the mass fraction of 65% according to the feed liquid ratio of 1 g to 5 mL, uniformly stirring, and cooling in a water bath of 6 h at 120 ℃ at room temperature to obtain hydrolysate;
(2) Adding beta cyclodextrin into the hydrolysate obtained in the step (1) according to the feed liquid ratio of 1.0 g to 1 mL, stirring while adding, standing for 12 h, and centrifugally collecting precipitate to obtain a ginsenoside RK1 beta cyclodextrin inclusion compound crude product;
(3) Washing twice by using a citric acid aqueous solution with the mass fraction of 60%, washing 2 times by using ethanol with the volume fraction of 5%, precipitating and drying to obtain the refined ginsenoside RK1 beta cyclodextrin inclusion compound.
The purity of the ginsenoside RK1 beta cyclodextrin inclusion compound detected by an HPLC method is 92.5 percent.
In this example, HPLC chromatogram of RK1 obtained after hydrolysis of total saponins of stems and leaves of American ginseng is shown in FIG. 5, HPLC chromatogram of beta-CD-RK 1 obtained after addition of beta-cyclodextrin to form clathrate compound and separation and purification is shown in FIG. 6, physical mixture of beta-CD and ginsenoside RK1 is shown in FIG. 7.
Example 3:
(1) Taking total saponins of American ginseng fruits, adding a citric acid aqueous solution with the mass fraction of 60% according to the feed liquid ratio of 1 g to 5 mL, uniformly stirring, and cooling in a water bath of 4 h at 100 ℃ at room temperature to obtain hydrolysate;
(2) Adding gamma cyclodextrin into the hydrolysate obtained in the step (1) according to the feed liquid ratio of 1.2 g to 1 mL, stirring while adding, standing for 12 h, and centrifugally collecting precipitate to obtain a ginsenoside RK1 cyclodextrin inclusion compound crude product;
(3) Washing with 60% citric acid aqueous solution for 2 times, washing with 5% ethanol for 2 times, precipitating, and drying to obtain refined ginsenoside RK1 gamma cyclodextrin clathrate.
The purity of the ginsenoside RK1 gamma cyclodextrin inclusion compound detected by an HPLC method is 90.2 percent.
Example 4: application of ginsenoside RK1 cyclodextrin inclusion compound
Experimental animals:
60 SPF-grade male Sprage Dawley rats, 6 weeks old, body mass (220-250) g were purchased from Liaoning Yingsi laboratory animal research center. The experiment maintains constant temperature (23+/-2 ℃) and constant humidity (50% -60%), and rats can freely move, drink water and eat.
Reagent:
kits for detecting Triglyceride (TG), total Cholesterol (TC), low density lipoprotein (LDL-C), high density lipoprotein (HDL-C) were all purchased from the institute of bioengineering, made in south-kyo; ATP content test box, na + -K + ATPase and Ca 2+ -Mg 2+ The ATPase kits were purchased from Shanghai enzyme-linked biotechnology Co., ltd; AMD, SOD, GSH-Px and ROS kit are all purchased from Nanjing's institute of biological engineering; hematoxylin eosin staining kit was purchased from south Beijing raw avionics limited; phosphorylated calcium/calmodulin-dependent protein kinase (p-CaMKK. Beta.), caMKK. Beta., phosphorylated AMP-dependent protein kinase (p-AMPK), AMPK, peroxisome proliferator-gamma co-activator-1α (PGC-1α) are all available from U.S. Cell Signaling Technology, A 1 R、A 2A R, mGluR5 and GABAA1 alpha antibodies were purchased from Abcam corporation, usa. The remaining reagents were all commercially available analytical.
Instrument:
sleep deprivation case, bayer medicine health Limited, multifunctional enzyme labeling apparatus (Spectramax M5, america Molecular Devices), inverted microscope (Olympus Japan), embedding machine, microtome, DVM6 transmission electron microscope (Leica Germany), electrophoresis tank, transfer tank, gelDocXR+gel imaging analysis system (Bio-Rad Co., USA).
Statistical methods:
SPSS 21.0 software package processed data, expressed as X+ -SEM. And graphic Prism 8.0 is used for graphic drawing. Comparing every two by using t test; two or more comparisons were performed using one-way anova and a post-hoc comparison was made between Bonferroni groups. The difference of P <0.05 is statistically significant.
(1) Sleep deprivation model preparation
And preparing a sleep deprivation model by adopting a multi-platform water environment method. The sleep deprivation box is a self-made 75 cm ×50 cm ×36 cm box body, and a conjoined metal round table (diameter 8 cm, height 8 cm, interval 15 cm) is placed inside the sleep deprivation box. The experimental process ensures that the water surface is below the round table 1 and cm, and the rats can drop into the water to cause REM stage sleep deprivation due to the decrease of muscular tension after entering a sleep state. The water environment of the blank group is similar, but the wire netting is placed on the platform and can move freely without falling into the water. The daily deprivation time was 12:00-the next day 8:00, the rest time is put back into the rearing cage for rest.
(2) Grouping and administration
The 60 rats were randomly divided into a blank group (without sleep deprivation), a model group, a β -CD-RK1 low dose group, a β -CD-RK1 high dose group and a positive drug group, each group having 12 rats. The dosage of the beta-CD-RK 1 low dosage group is 30mg/kg/d, the dosage of the beta-CD-RK 1 high dosage group is 60mg/kg/d, the positive drug group is administered by melatonin, the dosage of the positive drug group is 300mg/kg/d, and the model group and the blank group are administered with 0.9% physiological saline in equal volumes, and each group is continuously administered for 4 weeks.
The β -CD-RK1 used in this example is the β -CD-RK1 obtained by the preparation of example 3.
(3) Detection of beta-CD-RK 1 action
(1) Test of exhaustive swimming
Starting detection on the 28 th day of gastric lavage, and recording the time of the resis-tive swimming, the total swimming distance and the swimming speed of each group of rats respectively.
The test results are shown in table 1, and the model group has a significant decrease in swimming time, swimming speed and distance (P < 0.05) compared with the blank group; compared with the model group, the beta-CD-RK 1 group has obvious rise (P < 0.05) on the swimming time, swimming speed and distance at low dose and high dose, and the dose-effect relationship is presented. The beta-CD-RK 1 is suggested to be capable of significantly increasing the activity level of rats.
Table 1 results of the experimental tests on the exhaustive swimming of rats of each group
Note that: in contrast to the blank set of the cells, * p<0.05， ** p<0.01; compared with the model group # p<0.05， ## p<0.01。
(2) Sample collection
Blood was taken from the abdominal aorta and the whole brain was subsequently dissected off, hypothalamic tissue fixed in 2.5% glutaraldehyde solution, HE stained. The rest brain tissue such as hypothalamus is put in a refrigerator at-80 ℃ for standby.
(3) Blood glucose and blood lipid level detection
TC, TG, LDL-C, HDL-C detection is strictly carried out according to the instruction of the kit, and blood sugar is rapidly detected by blood sugar test paper.
The test results are shown in Table 2, and compared with the blank group, the model group FBS, TG, TC, LDL-C level is obviously increased, and HDL-C level is obviously reduced (P < 0.05); beta-CD-RK 1 significantly reduced the levels of rat FBS, TG, TC, LDL-C, increased HDL-C expression levels (P < 0.05) and exhibited dose-response relationships compared to the model group. The beta-CD-RK 1 is suggested to be capable of significantly improving abnormal glycolipid metabolism.
Table 2 blood glucose and blood lipid test results for rats of each group
Note that: in contrast to the blank set of the cells, * p<0.05， ** p<0.01; compared with the model group # p<0.05， ## p<0.01。
(4) ATP content and related enzyme content detection
ATP content level Na was performed according to the ATP kit instructions + -K + ATPase, ca 2+ -Mg 2+ ATPase Activity according to the instructions, the enzyme concentration was calculated by measuring absorbance after enzymatic reaction and phosphorus assay.
The test results are shown in Table 3, and the ATP content and Na content of the model group are compared with those of the blank group + -K + ATPase, ca 2+ -Mg 2+ -atpase decreases, suggesting increased sleep deprivation energy expenditure and accelerated metabolism; after intervention by beta-CD-RK 1, it is ATP, na + -K + -ATP and Ca 2+ -Mg 2+ -a significant increase in atpase levels, indicating that β -CD-RK1 improves energy expenditure due to sleep deprivation and maintains metabolic balance, suggesting that β -CD-RK1 can significantly increase ATP levels.
TABLE 3 detection results of ATP content and related enzyme content in rats of each group
Note that: in contrast to the blank set of the cells, * p<0.05， ** p<0.01; compared with the model group # p<0.05， ## p<0.01。
(5) Mitochondrial oxidative stress level detection
AMD, SOD, GSH-Px and ROS content levels were assayed according to the corresponding kit instructions.
The test results are shown in Table 4, and the oxidation stress level of the model group is obviously improved, which is shown by the reduction of SOD and GSH-Px levels and the increase of MDA; while beta-CD-RK 1 increases SOD and GSH-Px levels and decreases MDA levels. Sleep deprivation results in a significant increase in ROS, increased levels of oxidative stress in the body, which can exacerbate mitochondrial dysfunction, affect mitochondrial energy supply, and create metabolic imbalance. beta-CD-RK 1 relieves the excessive oxidative stress, improves active oxygen metabolism and provides a guarantee for maintaining the normal biological functions of mitochondria. The beta-CD-RK 1 is suggested to be capable of effectively relieving mitochondrial structural damage.
Table 4 results of mitochondrial oxidative stress level detection in rats of each group
Note that: in contrast to the blank set of the cells, * p<0.05， ** p<0.01; compared with the model group # p<0.05， ## p<0.01。
(6) Western blotting detection of AMPK pathway proteins and adenosine and neurotransmitter receptors
Extracting hypothalamic protein, selecting SDS-PAGE gel with corresponding concentration according to molecular weight, preparing separating gel and concentrating gel, loading, electrophoresis, transferring, labeling, sealing, and PBST cleaning for 3 times, adding p-AMPK, PGC-1α, A respectively 1 R、A 2A R, mGlu R5, GABAA1 alpha and beta-actin dilutions (dilution factor 1:1000), overnight at 4 ℃; secondary antibody (1:10000) was added dropwise and incubated at room temperature for 2 h, washed, ECL developed, photographed and analyzed.
As can be seen from the effect of beta-CD-RK 1 on the AMPK pathway proteins synthesized by rat mitochondria, compared with a blank group, the relative expression amount of the p-AMPK (see figure 9), PGC-1 alpha (see figure 10) and Nrf-1 (see figure 8) proteins in the model group is reduced, compared with the model group, the overall expression of the p-AMPK, PGC-1 alpha and Nrf-1 after the beta-CD-RK 1 is interfered is up-regulated, and the above study shows that the expression amount of the three proteins is reduced after the AMPK/PGC-1 alpha/Nrf-1 pathway key proteins and sleep deprivation is generally observed to show a trend of reduction. And the relative expression of p-AMPK, PGC-1α and Nrf-1 proteins increased following intervention by β -CD-RK1. The beta-CD-RK 1 is suggested to be capable of remarkably improving the expression of AMPK/PGC-1/Nrf-1 pathway protein and regulating mitochondrial biological functions.
The effect result of high dose beta-CD-RK 1 on rat hypothalamic pathology is shown in figure 11, the blank group has complete cell structure, a large number of cell-shaped circles, clear boundary with the surrounding, abundant cytoplasm, clear nucleus and large nuclear-plasma ratio; and the insomnia deprivation model group has reduced cell number, irregular morphology, concentrated cytoplasm, condensed cell nucleus and deep red partial cell staining. The morphological structure of cells is improved after the intervention of beta-CD-RK 1, and only a small amount of nuclear shrinkage and cell bad images appear, which suggests that the high dosage of beta-CD-RK 1 can improve the hypothalamic cell structure injury of the sleep deprived rat.
As can be seen from the effect of beta-CD-RK 1 on rat adenosine and neurotransmitter receptors, model group A compared with the blank group 1 Expression of R protein is significantly increased while A 2A Protein expression of R is significantly reduced; compared with the model group, A after beta-CD-RK 1 intervention 1 Protein expression of R is reduced while A 2A R protein expression was significantly enhanced (fig. 14 and 15). Glutamate (Glu) is the major excitatory transmitter of the central nervous system and is involved in the occurrence of sleep arousal. We examined the level of metabotropic glutamate receptor 5 (mGluR 5) expression. The results are shown in fig. 13, with the mGluR5 protein expression enhanced in the model group compared to the blank group; beta-CD-RK 1 reduced protein expression of mGluR5 compared to the model group. Gamma-aminobutyric acid (GABA) is also an important inhibitory neurotransmitter in the body, with a pronounced central inhibitory effect. As a result of detecting GABA receptor GABAA1 a, it was found that the protein expression of GABAA1 a in the model group was significantly reduced as compared with the blank group; compared to the model group, protein expression of GABAA1 a was significantly increased following β -CD-RK1 intervention (fig. 16). Suggesting that beta-CD-RK 1 can significantly improve A 1 R and A 2A R expression and modulation of its expression levels with mGluR5 and GABAA1 a.
The above study results show that: beta-CD-RK 1 can effectively improve the cerebral energy metabolism disturbance of the sleep deprived rat and improve the sleep. Mainly by (1) improving abnormal glycolipid metabolism, improving activity and ATP level; (2) Relieving mitochondrial structural damage, improving AMPK/PGC-1/Nrf-1 pathway protein expression, and regulating mitochondrial biological functions; (3) Improvement A 1 R and A 2A R expresses and regulates the expression level of mGluR5 and GABAA1 alpha, plays a role in improving sleep of sleep deprived rats, and beta-CD-RK 1 is a promising sleep improving drug.
In vivo bioavailability experiments of beta-CD-RK 1 in rats:
1. experimental apparatus and materials
The acquisition UPLC H-Class ultra-high performance liquid chromatograph, PDA detector, waters company, UK; high speed centrifuges (U.S. Thermo Fisher Scientific). Ginsenoside RK1 (98%), shanghai Yuan Ye Biotechnology Co., ltd.; beta-CD-RK 1 (obtained according to the preparation method described in example 3 of the invention); beta-CD/RK 1 physical mixture (98% ginsenoside RK1 from Shanghai Seiyaka Biotechnology Co., ltd. Was physically mixed with beta-cyclodextrin in the same molar ratio as beta-CD-RK 1); chromatographic methanol and chromatographic acetonitrile (Fisher, usa); male SD rats 18, body weight (220+ -20) g, supplied by Liaoning Changsheng Biotechnology Co., ltd., animal license number: SCXK (Liao) 2022-0001.
2. Experimental method
(1) Dosing regimen and blood sample collection
Male SD rats 18 were randomly divided into 3 groups, and were orally administered with no water withdrawal, no food withdrawal 12 h, oral gavage ginsenoside RK1 (20 mg/kg), beta-CD/RK 1 physical mix (containing ginsenoside RK 120 mg/kg) and beta-CD-RK 1 (containing ginsenoside RK 120 mg/kg), respectively; and 5, 10, 15, 30, 45, 60, 120, 240, 480, 720 min orbital blood was taken 0.2. 0.2 mL after administration, placed in heparinized EP tube, centrifuged (5000 rpm), and the supernatant was frozen at-20℃for measurement.
(2) Preparation of ginsenoside RK1 reference solution
Accurately weighing a proper amount of ginsenoside RK1 standard, dissolving with methanol, and preserving at 4 ℃ with constant volume concentration of 1 mu g/mL of stock solution. Before use, the standard working solutions with the concentrations of 10, 20, 50, 100, 200 and 500 ng/mL are diluted with methanol to a constant volume.
(3) Treatment of plasma samples
Taking a rat plasma sample of 100 mu L, placing the rat plasma sample in a 2 mL EP tube, adding 10 mu L of methanol, adding 200 mu L of acetonitrile, mixing for 3 min by vortex, centrifuging for 10 min (12000 rpm), and taking a supernatant for sample injection.
(4) Chromatographic conditions
The chromatographic column is an acquisition UPLC 18 column (50 mm multiplied by 2.1 mm,1.7 μm); mobile phase was water-acetonitrile, gradient elution procedure: 0-6.0 min, 13-22% acetonitrile; 6.0-18.0 min,22% -38% acetonitrile; 18.0-25.0 min,38% -40% acetonitrile; 25.0-30.0 min,40% -45% acetonitrile; 30.0-35.0 min,45% -58% acetonitrile; 35.0~40.00 min,58% -62% of acetonitrile. Column temperature is 35 ℃; volume flow rate is 0.4 mL/min; the sample injection amount is 3 mu L; the detection wavelength 203 nm.
(5) Data processing
The measured data were processed using DAS3.0 pharmacokinetic software, and the mean plasma concentration-time data were obtained by fitting the plasma concentration (C) to time (t) data using DAS 3.0.
3. Test results:
the pharmacokinetic profile is shown in FIG. 17 and the pharmacokinetic parameters are shown in Table 5. Compared with the ginsenoside RK1 group, the beta-CD-RK 1C Max and the AUC have significant differences (p is less than 0.01); no difference from the β -CD/RK1 physical mix group. As can be seen from Table 5, ginsenoside RK1 group AUC 0-24 h And AUC 0-∞ AUC of the β -CD-RK1 group were (559.3 + -21.6) ng.h/mL and (625.6 + -22.1) ng.h/mL, respectively 0-24 h And AUC 0-∞ The relative bioavailability of the beta-CD-RK 1 inclusion compound is improved by 213.3 percent compared with RK1, wherein the concentration is (1086.8 +/-35.3) ng.h/mL and (1334.6 +/-21.2) ng.h/mL respectively. Ginsenoside RK1 group CMax is (12.4+ -1.3) ng/mL, and beta-CD-RK 1 clathrate is (24.5+ -1.4) ng/mL, which is improved by more than 2.0 times. Compared with the ginsenoside RK1 group, the peak time is faster. T (T) 1/2 Is prolonged to 27.4 h from 18.2 h of ginsenoside RK1. Experimental results show that after the ginsenoside RK1 is included by beta-cyclodextrin, the absorption of the medicine in the body is obviously improved. The absorption peak concentration is obviously improved, and the half-life period of the prepared beta-cyclodextrin inclusion is obviously prolonged, so that the oral absorption rate of the ginsenoside RK1 can be better improved by preparing the ginsenoside RK1 into the cyclodextrin inclusion compound.
TABLE 5 pharmacokinetic parameters
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A preparation method of a ginsenoside RK1 cyclodextrin inclusion compound, which is characterized by comprising the following steps:
(1) Taking ginseng total saponins, adding 55% -65% citric acid aqueous solution according to a feed liquid ratio of 1 g to 5 mL, uniformly stirring, and cooling in a water bath at 90-120 ℃ at 4-6 h at room temperature to obtain hydrolysate;
(2) Adding cyclodextrin into the hydrolysate obtained in the step (1) according to the feed liquid ratio of 1.0 g to 1 mL to 1.6 g to 1 mL, stirring while adding, standing for 12-h, and centrifugally collecting precipitate to obtain a ginsenoside RK1 cyclodextrin inclusion compound crude product;
(3) Washing with 60% citric acid aqueous solution for 2 times, washing with 5% ethanol for 2 times, precipitating, and drying to obtain refined ginsenoside RK1 cyclodextrin clathrate.
2. The method according to claim 1, wherein the ginseng total saponins in step (1) include ginseng stem and leaf total saponins, ginseng fruit total saponins, american ginseng stem and leaf total saponins, american ginseng fruit total saponins.
3. The method according to claim 1, wherein the aqueous citric acid solution in step (1) has a mass fraction of 60%.
4. The method of claim 1, wherein the water bath temperature in step (1) is 100 ℃ and the water bath time is 4 h.
5. The method according to claim 1, wherein the feed liquid ratio in step (2) is 1.2 g/1 mL.
6. The method of claim 1, wherein the cyclodextrin in step (2) is any one of α -cyclodextrin, β -cyclodextrin and γ -cyclodextrin.
7. A ginsenoside RK1 cyclodextrin inclusion compound, wherein the RK1 cyclodextrin inclusion compound is obtained by the preparation method of any one of claims 1-6.
8. An application method of the ginsenoside RK1 cyclodextrin inclusion compound, which is characterized in that the ginsenoside RK1 cyclodextrin inclusion compound obtained by the preparation method of any one of claims 1-6 is used for preparing a medicament capable of improving sleep.
9. The application method according to claim 8, wherein the medicament is prepared by taking ginsenoside RK1 cyclodextrin inclusion compound as an active ingredient and matching auxiliary materials for medicaments.
10. The method of use according to claim 9, wherein the medicament is administered orally or parenterally; the dosage forms of the medicine comprise tablets, capsules, powder, pills, granules, injections and patches.
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