A method for preparing bone augmentation and reduction

Abstract

The present invention provides a method for preparing bone augmentation and reduction, and belongs to the technical field of traditional Chinese medicine preparations. The raw materials of the bone augmentation and reduction include: Radix Rehmanniae Preparata, Rhizoma Epimedii, Caulis Spatholobi, Cibotium Barbarum, Rhizoma Drynariae, Cynomorium Songaricum, Dipsacus Asper, Fructus Lycii and Semen Raphani; the method includes: choline salt, 1-octyl-3-methylimidazolium bromide and acid hydrogen bond donor or alcohol hydrogen bond donor are heated and mixed to prepare multi-component deep eutectic solvent A or multi-component deep eutectic solvent B; Radix Rehmanniae Preparata powder, multi-component deep eutectic solvent A and water are evenly mixed, and microwave heating is performed to obtain Radix Rehmanniae Preparata extract and Radix Rehmanniae solid residue; the Radix Rehmanniae solid residue after drying, the remaining raw material powder and the multi-component deep eutectic solvent B are mixed with the alcohol solution, and an extraction supernatant is obtained by short-term multiple ultrasonic-assisted extraction; the Radix Rehmanniae Preparata extract and the extraction supernatant are combined and concentrated to obtain a bone augmentation and reduction stock solution; and a bone augmentation and reduction preparation is prepared. The present invention can quickly and efficiently extract the effective active ingredients of each raw material of bone augmentation and reduction, and the conditions are mild and the time consumption is short.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a preparation method of bone augmentation and reduction. Background Art

[0002] Gu Zengxiao is a traditional Chinese medicine preparation used to treat bone hyperplasia such as low back and knee joint pain caused by liver and kidney deficiency. Its main ingredients are: Rehmannia root, roasted epimedium, Millettia reticulata, Cibotium barbarum, Drynaria fortunei, Cynomorium songaricum, Dipsacus asper, Lycium barbarum, and Radish seed.

[0003] Currently, traditional bone augmentation and reduction preparation processes include the following methods: first, all raw materials are crushed and decocted multiple times, the filtrates are combined and concentrated, and then made into corresponding dosage forms, such as tablets or capsules; second, dog spine and roasted epimedium are crushed into fine powder; the remaining raw materials are decocted multiple times, the filtrates are combined and concentrated, and then mixed with the fine powder to make the corresponding dosage form; third, each raw material is extracted separately using different extraction methods to obtain the extracted concentrate, which is then combined to make the corresponding dosage form.

[0004] Among the above methods, the first and second ones are traditional processes with simple processes, but both have defects such as low utilization rate of effective active ingredients in raw materials and large amount of medicinal materials used, especially Rehmannia root, which, as the main raw material, contains multiple effective active ingredients such as polysaccharides, saponin compounds, and flavonoid compounds; the third method uses enzymatic hydrolysis, carbon dioxide supercritical extraction and other technical means to extract the active ingredients in medicinal materials respectively, which has the problems of harsh extraction conditions, long time consumption, high cost, and large amount of waste liquid. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a method for preparing bone augmentation and reduction, aiming to solve at least one technical problem in the background technology.

[0006] The present invention is achieved in that:

[0007] A method for preparing bone augmentation and reduction medicine, wherein the raw materials of the bone augmentation and reduction medicine include prepared rehmannia root, roasted epimedium, millettia reticulata, cibotrya japonica root, drynaria root, cynomorium songaricum, dipsaccharum officinale, wolfberry fruit and radish seed;

[0008] The preparation method comprises the following steps:

[0009] Mixing choline salt, 1-octyl-3-methylimidazolium bromide and an acidic hydrogen bond donor by heating according to a preset molar ratio to prepare a multi-component deep eutectic solvent A;

[0010] Mixing choline salt, 1-octyl-3-methylimidazolium bromide and an alcohol hydrogen bond donor by heating according to a preset molar ratio to prepare a multi-component deep eutectic solvent B;

[0011] A solution of a multi-component deep eutectic solvent A is used as an extractant A, and Rehmannia root powder is treated with microwaves in a closed environment to obtain a Rehmannia root extract and Rehmannia root solid residue;

[0012] The dried Rehmannia root solid residue and the remaining raw material powder are mixed to form a mixture, and a solution of a multi-component deep eutectic solvent B is used as an extractant B, and ultrasound-assisted extraction is performed to obtain an extraction supernatant;

[0013] The prepared Rehmannia root extract and the supernatant are combined and concentrated to obtain the bone-increasing and eliminating stock solution;

[0014] The bone-increasing and quenching stock solution and adjuvants are prepared into the bone-increasing and quenching preparation.

[0015] Preferably, the specific steps of obtaining the Rehmannia root extract and Rehmannia root solid residue are:

[0016] The Rehmannia root powder, multi-element low eutectic solvent A and water are mixed evenly in a preset ratio, and the Rehmannia root powder is heated and extracted in a closed environment with microwave. After the extraction, it is cooled to room temperature and then centrifuged. After liquid phase distillation and impurities are removed, the Rehmannia root extract is obtained, and the solid phase is the Rehmannia root solid residue.

[0017] Preferably, the specific steps of obtaining the extraction supernatant are:

[0018] The dried Rehmannia glutinosa solid residue is mixed with the remaining raw material powder, and a mixed solution of a multi-component low eutectic solvent B and an alcohol solution is used as the extractant B. After sufficient stirring, ultrasonic-assisted extraction is performed, and then solid-liquid separation is performed. The obtained solid is added with the extractant B and ultrasonic-assisted extraction and solid-liquid separation are repeated at least once. The filtrates after each solid-liquid separation are combined to obtain an extraction supernatant.

[0019] Preferably, in the multi-component deep eutectic solvent A, the molar ratio of choline salt: 1-octyl-3-methylimidazolium bromide: acid hydrogen bond donor is 1:1:2-4; the choline salt is choline chloride or choline bromide; the acid hydrogen bond donor is citric acid or acetic acid; and the heating temperature during the preparation of the multi-component deep eutectic solvent A is 40°C-60°C.

[0020] Preferably, in the multi-component deep eutectic solvent B, the molar ratio of choline salt: 1-octyl-3-methylimidazolium bromide: alcohol hydrogen bond donor is 1:1:2-4; the choline salt is choline chloride or choline bromide; the alcohol hydrogen bond donor is 1,4-butanediol, 1,2-propylene glycol or triethylene glycol; and the heating temperature during the preparation of the multi-component deep eutectic solvent B is 40°C to 60°C.

[0021] Preferably, the Rehmannia root powder, multi-element deep eutectic solvent A, and water = 1 g: (10-15) g: (15-25) mL.

[0022] Preferably, the heating extraction time of Rehmannia glutinosa powder is 10 min to 20 min; the heating extraction temperature is 60° C. to 120° C.; and the microwave power is 150W to 250W.

[0023] Preferably, the steps of preparing the extraction supernatant specifically include:

[0024] Mix the dried Rehmannia root solid residue with the remaining raw material powder, and stir evenly to form a mixed powder;

[0025] The mixed powder and the extractant B are mixed in a preset ratio, stirred thoroughly, and then ultrasonic-assisted extraction is performed, followed by solid-liquid separation to obtain a first filtrate and a first solid residue;

[0026] The first solid residue is dried and mixed with the extractant B in a preset ratio, fully stirred, and then ultrasonic-assisted extraction is performed, followed by solid-liquid separation to obtain a second filtrate and a second solid residue; this step is repeated for the second solid residue to obtain a third filtrate and a third solid residue;

[0027] The first filtrate, the second filtrate and the third filtrate are combined and centrifuged, and impurities are removed by distillation to obtain an extraction supernatant;

[0028] The power of the ultrasonic-assisted extraction is 200W~300W; the temperature is 40°C~60°C; and the time of a single ultrasonic-assisted extraction is 5min~10min.

[0029] Preferably, in a single ultrasonic-assisted extraction process, the mass ratio of solid material: multi-component deep eutectic solvent B: alcohol solution is 1:1-2:30-60, and the solid material is a mixed powder, the first solid residue or the second solid residue.

[0030] Preferably, the alcohol solution is a methanol aqueous solution or an ethanol aqueous solution, and the mass concentration of alcohol in the alcohol solution is 40% to 80%.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention adopts a ternary deep eutectic solvent made of two different hydrogen bond donors, which can quickly and efficiently extract the effective active ingredients of various raw materials for bone augmentation and absorptivity under mild conditions and in a short time.

[0033] 2. The present invention uses choline salt + 1-octyl-3-methylimidazolium bromide + acid hydrogen bond donor to treat Rehmannia glutinosa, and cooperates with microwave treatment to fully extract multiple effective biologically active ingredients such as polysaccharides and saponin compounds in Rehmannia glutinosa in a short time; the present invention uses a composite acidic ternary deep eutectic solvent as an extractant. During the mixing process of the composite acidic ternary deep eutectic solvent with Rehmannia glutinosa powder, saponin compounds such as Rehmannia glutinosa polysaccharides and rehmannia glutinosa glycoside D can be quickly dissolved, form hydrogen bonds with the multi-component deep eutectic solvent A, and then dissolve in the solvent; at the same time, based on the thermal effect generated by microwaves and the non-thermal effect of the electric field, electromagnetic waves are used to penetrate Rehmannia glutinosa into the interior and act on the molecules, promoting molecular motion to generate a large amount of heat energy, accelerating the molecular motion speed of the biologically active ingredients, thereby accelerating their transfer to the solvent, and achieving efficient and rapid precipitation of the biologically active ingredients.

[0034] 3. The present invention uses choline salt + 1-octyl-3-methylimidazolium bromide + alcohol hydrogen bond donor to treat Rehmannia glutinosa solid residue and other raw materials, and uses a composite alcohol ternary deep eutectic solvent as an extractant. The hydroxyl groups in the alcohol can form intermolecular hydrogen bonds with the bioactive components in the raw materials, thereby increasing the solubility of components such as flavonoids, phenolic compounds, sinapines, and betaine. In addition, ultrasonic treatment is used in combination, and based on the thermal and mechanical effects of ultrasound, the penetration of the multi-component deep eutectic solvent B is enhanced, the transfer of the target components is accelerated, and the extraction efficiency of the target components is improved.

[0035] 4. The present invention is green and environmentally friendly, produces little waste liquid, and has a simple process. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific implementation cases described herein are only used to explain the present invention and are not intended to limit the present invention.

[0037] A method for preparing bone augmentation and reduction, comprising the following ingredients in parts by weight: 160-210 parts of Rehmannia root; 100-150 parts of roasted Epimedium; 100-150 parts of Millettia spatholobi; 100-150 parts of Cibotium barometz; 100-150 parts of Drynaria rhizome; 100-150 parts of Cynomorium songaricum; 50-80 parts of Dipsacus root; 50-80 parts of Lycium barbarum; and 50-80 parts of Radish seed. In practice, the amount of each ingredient can be adjusted based on actual needs and is not specifically limited herein.

[0038] The preparation method includes steps S1 to S5.

[0039] S1. Raw material pretreatment

[0040] The raw materials of bone augmentation and reduction are crushed separately and passed through 80-100 mesh sieves to prepare powder;

[0041] Weigh the materials according to the molar ratio of choline salt: 1-octyl-3-methylimidazolium bromide: acid hydrogen bond donor = 1:1:2-4, heat the three to 40°C-60°C and mix to prepare a multi-component deep eutectic solvent A;

[0042] Weigh the materials according to the molar ratio of choline salt: 1-octyl-3-methylimidazolium bromide: alcohol hydrogen bond donor = 1:1:2-4, heat the three to 40°C-60°C and mix to prepare a multi-component deep eutectic solvent B;

[0043] Among them, in the polyvalent deep eutectic solvent A and the polyvalent deep eutectic solvent B, the choline salt is choline chloride or choline bromide, the acid hydrogen bond donor is citric acid or acetic acid, and the alcohol hydrogen bond donor can be a polyol such as 1,4-butanediol, 1,2-propylene glycol or triethylene glycol.

[0044] S2. Rehmannia root powder, multi-element deep eutectic solvent A, and water are uniformly mixed in a preset ratio, and the Rehmannia root powder is heated and extracted in a closed environment in conjunction with microwave treatment; after the extraction, the powder is cooled to room temperature and then centrifuged, and the liquid phase is distilled to remove impurities to obtain Rehmannia root extract, and the solid phase is Rehmannia root solid residue;

[0045] Because Rehmannia root contains multiple active ingredients such as polysaccharides and saponin compounds, single water extraction and alcohol extraction require several hours of processing and cannot effectively extract the biologically active ingredients. The present invention uses a composite acidic ternary deep eutectic solvent as the extractant A. During the mixing process of the composite acidic ternary deep eutectic solvent with Rehmannia root powder, the bioactive ingredients such as Rehmannia root polysaccharides and saponin compounds (mainly rehmannoside D) can be dissolved from Rehmannia root. These bioactive ingredients form hydrogen bonds with the multi-component deep eutectic solvent A and are then dissolved in the multi-component deep eutectic solvent A.

[0046] In the above process, microwave treatment is used in conjunction with the thermal effect generated by microwaves and the non-thermal effect of the electric field. Electromagnetic waves penetrate the prepared Rehmannia root and act on its molecules, promoting molecular movement to generate a large amount of heat energy, thereby accelerating the molecular movement speed of the bioactive ingredients, thereby accelerating their transfer to the multi-component deep eutectic solvent A, and achieving efficient and rapid precipitation of the bioactive ingredients.

[0047] During the extraction process, the addition of water as a solvent increases the wettability of Rehmannia glutinosa powder and reduces the viscosity of the reaction system, thereby facilitating the molecular transfer of bioactive components. However, if the amount of water is too high, it will reduce the effectiveness of the internal hydrogen bond structure of the multi-component deep eutectic solvent A in dissolving the bioactive components.

[0048] By optimizing the ratio of raw materials to solvents, the extraction rate of bioactive components in Rehmannia glutinosa can be effectively improved. In a specific implementation, the ratio of Rehmannia glutinosa powder, multi-element deep eutectic solvent A, and water = 1g:(10-15)g:(15-25)mL; for example, it can be 1g:10g:15mL, 1g:12g:15mL, 1g:15g:15mL, 1g:15g:18mL, 1g:15g:20mL, 1g:15g:25mL; but the values are not limited to the listed values, and other values within the numerical range not listed are also applicable.

[0049] Microwaves can promote the precipitation of active medicinal components in Rehmannia glutinosa, but other components of Rehmannia glutinosa can also be precipitated under high-power microwave conditions, resulting in an increase in impurities. Therefore, the present invention adopts low-power microwaves; the power of the microwave treatment is 150W to 250W, for example, it can be 150W, 170W, 190W, 210W, 230W, 250W; but it is not limited to the listed values, and other values within the numerical range not listed are also applicable;

[0050] The heating extraction time is 10 min to 20 min; for example, it can be 10 min, 12 min, 15 min, 18 min, 20 min; but it is not limited to the listed values, and other values not listed within the numerical range are also applicable;

[0051] The temperature during the extraction process is 60°C to 120°C, for example, 60°C, 80°C, 100°C, or 120°C; but is not limited to the listed values, and other values not listed within the numerical range are also applicable.

[0052] S3, mixing the dried Rehmannia glutinosa solid residue with the remaining raw material powder, using a multi-element deep eutectic solvent B and an alcohol solution as an extractant B, stirring thoroughly, performing ultrasonic-assisted extraction, and then performing solid-liquid separation. After drying the obtained solid, adding the extractant B, repeating the ultrasonic-assisted extraction and solid-liquid separation at least once, combining the filtrates after each solid-liquid separation to obtain an extraction supernatant;

[0053] During the extraction of the bioactive components of Rehmannia root in step S2, the extraction of the flavonoid compounds contained therein is limited. In order to further extract the effective components in Rehmannia root, step S3 is to mix the solid residue of Rehmannia root separated in step S2 with the remaining raw material powders such as roasted Epimedium, Millettia reticulata, Cibotium barometz, Drynaria fortunei, Cynomorium songaricum, Dipsacus root, Lycium barbarum fruit, and Radish seed for extraction;

[0054] The multi-component deep eutectic solvent B is composed of a choline salt, 1-octyl-3-methylimidazolium bromide, and an alcohol hydrogen bond donor. The alcohol hydrogen bond donor is a polyol such as 1,4-butanediol, 1,2-propylene glycol, or triethylene glycol. The hydroxyl groups in the polyol can form intermolecular hydrogen bonds with the bioactive ingredients in the raw materials, thereby increasing the solubility of ingredients such as flavonoids, phenolic compounds, sinapines, and betaine.

[0055] This step uses ultrasound as an aid. Based on the thermal and mechanical effects of ultrasound, on the one hand, sound waves are converted into mechanical energy, generating cavitation, enhancing the permeability of the multi-component deep eutectic solvent B, and accelerating the extraction of the target components. On the other hand, when ultrasound propagates through the media such as powder and liquid in the reaction system, a portion of the ultrasonic energy is converted into heat energy due to the mutual movement between the media particles, the mutual friction at the interface, and the absorption of the media, thereby accelerating the transfer of bioactive ingredients and increasing their precipitation efficiency.

[0056] Research has found that as the extraction time increases, the amount of each component in the raw material increases. However, as time goes by, the structure of some compounds dissolved in the multi-component deep eutectic solvent B will be destroyed. Therefore, the ultrasonic-assisted extraction in this step uses short-term multiple extractions to extract the effective components in the solid. The specific operation is as follows:

[0057] (1) Mixing the dried Rehmannia root solid residue with the remaining raw material powder and stirring evenly to form a mixed powder;

[0058] (2) The mixed powder and extractant B are mixed in a preset ratio, stirred thoroughly, and then ultrasonic-assisted extraction is performed, followed by solid-liquid separation to obtain a first filtrate and a first solid residue;

[0059] (3) The first solid residue is dried and mixed with the extractant B in a preset ratio, fully stirred and then ultrasonically extracted, and then solid-liquid separation is performed to obtain a second filtrate and a second solid residue; this step is repeated for the second solid residue to obtain a third filtrate and a third solid residue;

[0060] (4) The first filtrate, the second filtrate, and the third filtrate are combined and centrifuged, and the extracted supernatant is obtained after distillation and impurities are removed.

[0061] During a single ultrasonic-assisted extraction process, the mass ratio of the mixed powder, the first solid residue or the second solid residue and other solid materials: the multi-element deep eutectic solvent B: the alcohol solution is 1:1~2:30~60, for example, it can be 1:1:30, 1:1.2:40, 1:1.5:45, 1:1.8:50, 1:2:60; but it is not limited to the listed values, and other values not listed within the numerical range are also applicable; when the number of ultrasonic-assisted extractions exceeds three times, the effective components extracted subsequently are limited and the effect is not obvious. Based on cost considerations, the number of ultrasonic-assisted extractions is controlled to three times;

[0062] The alcohol solution is a methanol aqueous solution or an ethanol aqueous solution, and the mass concentration of the alcohol in the alcohol solution is 40% to 80%; for example, it can be 40%, 50%, 60%, 70%, or 80%; but it is not limited to the listed values, and other values not listed within the numerical range are also applicable;

[0063] During the extraction process, the addition of an alcohol solution to the multi-component deep eutectic solvent B serves the following purposes: on the one hand, it improves the wettability of the raw materials to be extracted and reduces the viscosity of the extraction reaction system, thereby facilitating the molecular transfer of the bioactive components; on the other hand, the bioactive components precipitated in this step are more easily soluble in methanol, ethanol, etc. However, if the amount of alcohol solution used is too high, the effectiveness of the hydrogen bonding structure will be reduced.

[0064] The power of the ultrasonic-assisted extraction is 200W~300W; for example, it can be 200W, 220W, 250W, 280W, 300W; but it is not limited to the listed values, and other values not listed within the numerical range are also applicable;

[0065] The temperature of the ultrasonic-assisted extraction is 40°C to 60°C; for example, it can be 40°C, 45°C, 50°C, 55°C, or 60°C; but it is not limited to the listed values, and other values not listed within the numerical range are also applicable;

[0066] The time for a single ultrasound-assisted extraction is 5 minutes to 10 minutes; for example, it can be 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes; but it is not limited to the listed values, and other values not listed within the numerical range are also applicable.

[0067] S4, combining the Radix Rehmanniae Preparata extract and the supernatant to form a mixed solution, and concentrating the solution to obtain a bone-increasing and eliminating solution;

[0068] S5. preparing the bone-increasing and expelling preparation with the bone-increasing and expelling stock solution and the auxiliary agent;

[0069] Bone-enhancing and -reducing preparations can be formulated in dosage forms acceptable in the art, such as tablets, capsules, and pills. Excipients acceptable in the art, such as fillers, surfactants, lubricants, disintegrants, wetting agents, and adhesives, are not specifically limited herein. The following examples illustrate tablets, but the invention is not limited to these dosage forms; other dosage forms not listed in the art are also applicable.

[0070] Example 1

[0071] Example 1 of the present invention provides a method for preparing bone augmentation and reduction, the method comprising steps 1 to 5:

[0072] 1. Pretreatment of bone augmentation and digestion raw materials

[0073] Weigh the following ingredients for bone augmentation and reduction according to weight: 200 parts of Rehmannia root, 120 parts of roasted Epimedium, 120 parts of Millettia reticulata, 120 parts of Drynaria rhizome, 120 parts of Cynomorium songaricum, 60 parts of Dipsacus root, 60 parts of Lycium barbarum, and 60 parts of Radish seed; grind the ingredients separately and pass through an 80-100 mesh sieve to prepare powder;

[0074] 2. Extract the active ingredients from Rehmannia root

[0075] Choline bromide: 1-octyl-3-methylimidazolium bromide: citric acid are mixed in a molar ratio of 1:1:2, and the three are heated to 40°C to 60°C to prepare a multi-component deep eutectic solvent A;

[0076] Rehmannia root powder, multi-component deep eutectic solvent A, and water were uniformly mixed in a ratio of 1 g:10 g:15 mL. The Rehmannia root powder was heated to 60°C-70°C in a sealed environment with a 150 W microwave and extracted for 20 minutes. After the extraction, the mixture was cooled to room temperature and then centrifuged at 5000 rpm. The centrifugation not only separated the solid and liquid but also dissociated the multi-component deep eutectic solvent A from the extract. The liquid phase (i.e., the supernatant) was subjected to reduced pressure distillation to remove impurities such as acidic hydrogen bond donors, thereby obtaining a Rehmannia root extract. The solid phase was a Rehmannia root solid residue.

[0077] 3. Preparation of other extracts

[0078] According to the molar ratio of choline bromide (choline salt): 1-octyl-3-methylimidazolium bromide: alcohol hydrogen bond donor 1,4-butanediol = 1:1:2, the three are heated to 40°C to 60°C and mixed to prepare a multi-component deep eutectic solvent B;

[0079] Mix the dried Rehmannia root solid residue with the remaining raw material powder, and stir evenly to form a mixed powder;

[0080] After being fully stirred in a mass ratio of 1:1:30, the mixed powder: multi-component deep eutectic solvent B: 70% ethanol solution were mixed and then subjected to a first ultrasonic-assisted extraction at 40°C for 10 minutes with an ultrasonic power of 200W, followed by solid-liquid separation to obtain a first filtrate and a first solid residue;

[0081] The dried first solid residue: multi-component deep eutectic solvent B: 70% ethanol solution were mixed and stirred in a mass ratio of 1:1:30, and then a second ultrasonic-assisted extraction was performed at 40°C for 10 minutes with an ultrasonic power of 200W. Then, solid-liquid separation was performed to obtain a second filtrate and a second solid residue.

[0082] The dried second solid residue: multi-component deep eutectic solvent B: 70% ethanol solution were mixed and stirred in a mass ratio of 1:1:30, and then a third ultrasonic-assisted extraction was performed at 40°C for 10 minutes with an ultrasonic power of 200W, followed by solid-liquid separation to obtain a third filtrate and a third solid residue;

[0083] The first filtrate, the second filtrate and the third filtrate are combined and centrifuged, and impurities are removed by distillation to obtain an extraction supernatant;

[0084] 4. The Radix Rehmanniae Preparata extract and the supernatant are combined into a mixed liquid, and the mixed liquid is concentrated to obtain the bone-increasing and eliminating stock solution;

[0085] 5. Add compressible starch and 70% ethanol to the bone-dense and sludge solution, dry, and compress into bone-dense and sludge tablets.

[0086] Example 2

[0087] Based on Example 1, only the component ratio of the multi-component deep eutectic solvent A in step 2 was changed, and other steps and reaction parameters remained unchanged. The changes in the extraction amounts of rehmannia glutinosa glycoside D and rehmannia polysaccharides in the prepared rehmannia glutinosa extract prepared in step 2 are shown in Table 1 below.

[0088] The content of rehmannia glutinosa D in the raw material of Rehmannia glutinosa was measured by high performance liquid chromatography, and the content of Rehmannia glutinosa polysaccharides was measured by the phenol-sulfuric acid method. The extraction rate is the ratio of the content of the extracted component to the content of the corresponding component in the raw material of Rehmannia glutinosa.

[0089] Table 1

[0090]

[0091] In Table 1, A1 refers to choline bromide, A1' refers to choline chloride; A2 refers to 1-octyl-3-methylimidazolium bromide; A3 refers to citric acid, A3' refers to acetic acid;

[0092] As can be seen from the data in Table 1, in the present invention, the use of multi-element deep eutectic solvent A as extractant A can simultaneously, efficiently and quickly extract effective glycosides and rehmannia polysaccharides from Rehmannia glutinosa. As the amount of acid hydrogen bond donor increases, the extraction amount of rehmannia glutinosa glycoside D and rehmannia polysaccharides will increase. However, when it increases to a certain value, the effect of increasing the extraction amount of rehmannia glutinosa glycoside D and rehmannia polysaccharides is not obvious. Based on cost considerations, in the multi-element deep eutectic solvent A, the molar ratio of choline salt: 1-octyl-3-methylimidazolium bromide: alcohol hydrogen bond donor is 1:1:2~4.

[0093] Example 3

[0094] On the basis of Example 1, only the ratio of Rehmannia glutinosa powder, multi-component deep eutectic solvent A, and water in step 2 was changed, and the other steps and reaction parameters remained unchanged. The changes in the extraction amounts of rehmannia glutinosa glycoside D and rehmannia polysaccharides in the Rehmannia glutinosa extract prepared in step 2 are shown in Table 2 below.

[0095] Table 2

[0096]

[0097] As shown in Table 3, the extraction yields of rehmannia glutinosa and rehmannia polysaccharides increased with increasing amounts of multi-component deep eutectic solvent A. However, after the amount of multi-component deep eutectic solvent A reached a certain value, the extraction yields of rehmannia glutinosa and rehmannia polysaccharides actually decreased. This is because as the amount of multi-component deep eutectic solvent A increased, the amount of water remained unchanged, resulting in an increase in the viscosity of the reaction system, which in turn reduced molecular motion and transport, leading to a decrease in the extraction yield. Further increasing the amount of water increased the extraction yields of rehmannia glutinosa and rehmannia polysaccharides. Therefore, the ratio of Rehmannia glutinosa powder, multi-component deep eutectic solvent A, and water was 1 g: (10-15) g: (15-25) mL.

[0098] Example 4

[0099] On the basis of Example 1, only the microwave parameters in step 2 were changed, and the change in microwave power would affect the reaction temperature of the system. Under the premise that other steps and reaction parameters remained unchanged, the changes in the extraction amounts of rehmannia glutinosa glycoside D and rehmannia polysaccharide in the prepared rehmannia glutinosa extract prepared in step 2 were shown in Table 3 below.

[0100] Table 3

[0101]

[0102] As shown in Table 3, the extraction yields of rehmannia glutinosa and rehmannia polysaccharides increased significantly with increasing microwave power and reaction temperature. This is because increasing microwave parameters promotes cell destruction in Rehmannia glutinosa, accelerating the extraction of components, and microwaves accelerate molecular motion and transport. However, after the microwave power and reaction temperature reach a certain value, the extraction yields of rehmannia glutinosa and rehmannia polysaccharides decrease. This is because increasing microwave power and reaction temperature causes the water to gradually convert from liquid to gaseous state, increasing the viscosity of the reaction system and reducing molecular motion and transport, resulting in a decrease in the extraction yields of rehmannia glutinosa and rehmannia polysaccharides. For cost considerations, the heating temperature for Rehmannia glutinosa powder extraction was set between 60°C and 120°C, and the microwave power was set between 150W and 250W.

[0103] Example 5

[0104] Based on Example 1, only the component ratio of the multi-component deep eutectic solvent B in step 3 was changed, and the other steps and reaction parameters remained unchanged. The changes in the extraction amount of each active ingredient (taking flavonoids as an example) in the extraction supernatant obtained in step 3 are shown in Table 4 below.

[0105] The flavonoid content in the extraction supernatant was determined by HPLC. Since the raw materials of the bone-increasing and scavenging preparation, such as roasted epimedium, Millettia reticulata, and Drynaria fortunei, contain the highest flavonoid content, other raw materials also contain active ingredients such as phenols, sinapines, betaine, and protocatechuic acid, but the content is not high, flavonoids were used as a reference amount in step 3 to reflect the effect of different parameters on the extraction amount of active ingredients in the raw materials. The flavonoid content mentioned below refers to the flavonoid content in the extraction supernatant.

[0106] Table 4

[0107]

[0108] In Table 4, B1 refers to choline bromide, B1' refers to choline chloride; B2 refers to 1-octyl-3-methylimidazolium bromide; B3 refers to 1,4-butanediol, B3' refers to 1,2-propylene glycol, and B3'' refers to triethylene glycol;

[0109] As shown in Table 4, the use of polyvalent deep eutectic solvent B as extractant B in the present invention allows for the simultaneous, efficient, and rapid extraction of the active ingredients from each raw material. Increasing the amount of alcohol hydrogen bond donor increases the extraction yield of flavonoids, but after reaching a certain level, the effect on the extraction yield of flavonoids is not significant. Furthermore, a comparison of the extraction efficiency of different alcohol hydrogen bond donors and choline salts follows the order: triethylene glycol > 1,2-propylene glycol > 1,4-butanediol; and choline bromide > choline chloride. Therefore, in polyvalent deep eutectic solvent B, the molar ratio of choline salt: 1-octyl-3-methylimidazolium bromide: acid hydrogen bond donor is 1:1:2-4.

[0110] Example 6

[0111] Based on Example 1, only the ratio of the solid material, the multi-component deep eutectic solvent B, and water in step 3 was changed, and the other steps and reaction parameters remained unchanged. The changes in the extraction amount of each active ingredient (taking flavonoids as an example) in the extraction supernatant obtained in step 3 are shown in Table 5 below.

[0112] Table 5

[0113]

[0114] The data in Table 5 show that the extraction yield of flavonoids increases with increasing the amount of multi-component deep eutectic solvent B. However, after reaching a certain value, the increase in the extraction yield of flavonoids is not significant. This is because as the amount of multi-component deep eutectic solvent B increases, the alcohol solution remains unchanged, resulting in an increase in the viscosity of the reaction system, which in turn reduces molecular movement and transport, leading to a decrease in the extraction yield. Further increasing the amount of alcohol solution gradually increases the extraction yield of flavonoids. Therefore, during single-pass ultrasonic-assisted extraction, the mass ratio of solid material: multi-component deep eutectic solvent B: alcohol solution is set at 1:1-2:30-60.

[0115] Example 7

[0116] Based on Example 1, only the ultrasonic parameters in step 3 and the temperature of the reaction system during extraction were changed, and the other steps and reaction parameters remained unchanged. The changes in the extraction amount of each effective component (taking flavonoids as an example) in the extraction supernatant obtained in step 3 are shown in Table 6 below.

[0117] Table 6

[0118]

[0119] As shown in Table 6, the extraction yield of flavonoids increases significantly with increasing ultrasonic power and system reaction temperature. This is because increasing ultrasonic parameters enhances cavitation, strengthens the penetration of the multi-component deep eutectic solvent B, and accelerates the extraction of target components. Microwaves also accelerate molecular motion and transfer. However, after the ultrasonic power reaches a certain value, the increase in the extraction yield of the components is not significant. This is because the increased ultrasonic power leads to excessive cavitation, which destroys the structure of the bioactive components. In addition, the increased temperature causes the alcohol solution to evaporate, increasing the viscosity of the reaction system, which in turn reduces the motion and transfer of the bioactive components and limits the increase in the extraction yield. Therefore, based on cost considerations, the power for ultrasonic-assisted extraction is 200W-300W and the temperature is 40℃-60℃.

[0120] Comparative Example 1

[0121] In this comparative example, based on Example 1, only the component of the multi-component deep eutectic solvent A in step 2 was changed, and other steps and reaction parameters remained unchanged. The changes in the extraction amounts of rehmannia glutinosa glycoside D and rehmannia polysaccharides in the prepared Rehmannia glutinosa extract prepared in step 2 are shown in Table 7 below.

[0122] In this comparative example, the multi-component deep eutectic solvent A does not contain choline bromide. Specifically, 1-octyl-3-methylimidazolium bromide and citric acid are mixed in a molar ratio of 1:1, and the two are heated to 40° C. to mix, thereby preparing the multi-component deep eutectic solvent A.

[0123] Comparative Example 2

[0124] In this comparative example, based on Example 1, only the component of the multi-component deep eutectic solvent A in step 2 was changed, and other steps and reaction parameters remained unchanged. The changes in the extraction amounts of rehmannia glutinosa glycoside D and rehmannia polysaccharides in the prepared Rehmannia glutinosa extract prepared in step 2 are shown in Table 7 below.

[0125] In this comparative example, the multi-component deep eutectic solvent A does not contain 1-octyl-3-methylimidazolium bromide. Specifically, choline bromide and citric acid are mixed in a molar ratio of 1:1, and the two are heated to 40° C. to 60° C. to prepare the multi-component deep eutectic solvent A.

[0126] Comparative Example 3

[0127] In this comparative example, based on Example 1, the multi-component deep eutectic solvent A in step 2 was replaced with a 75% ethanol solution. Under the premise that other steps and reaction parameters remained unchanged, the changes in the extraction amounts of rehmannia glutinosa glycoside D and rehmannia polysaccharides in the prepared rehmannia glutinosa extract prepared in step 2 are shown in Table 7 below.

[0128] Comparative Example 4

[0129] In this comparative example, based on Example 1, microwave treatment was not used in step 2. Under the premise that other steps and reaction parameters remained unchanged, the changes in the extraction amounts of rehmannia glutinosa glycoside D and rehmannia polysaccharide in the prepared Rehmannia glutinosa extract in step 2 are shown in Table 7 below.

[0130] In this comparative example, step 2 is specifically as follows:

[0131] Choline bromide: 1-octyl-3-methylimidazolium bromide: citric acid are mixed in a molar ratio of 1:1:2 and heated to 40°C-60°C to prepare a multi-component deep eutectic solvent A. Rehmannia root powder, multi-component deep eutectic solvent A, and water are uniformly mixed in a ratio of 1 g:10 g:15 mL. The Rehmannia root is heated to 60°C-70°C in a sealed environment and extracted for 20 minutes. After the extraction, the extract is cooled to room temperature and then centrifuged at 5000 rpm. The centrifugation not only separates the solid and liquid, but also dissociates the multi-component deep eutectic solvent A from the extract. The liquid phase (i.e., the supernatant) is subjected to reduced pressure distillation to remove impurities such as acidic hydrogen bond donors, thereby obtaining a Rehmannia root extract. The solid phase is a Rehmannia root solid residue.

[0132] Comparative Example 5

[0133] In this comparative example, based on Example 1, the multi-component deep eutectic solvent A in step 2 was replaced with the multi-component deep eutectic solvent B. Under the premise that other steps and reaction parameters remained unchanged, the changes in the extraction amounts of rehmannia glutinosa glycoside D and rehmannia polysaccharides in the prepared Rehmannia glutinosa extract prepared in step 2 are shown in Table 7 below.

[0134] Table 7

[0135]

[0136] As can be seen from the data in Table 7, the use of a deep eutectic solvent of 1-octyl-3-methylimidazolium bromide + a hydrogen bond donor or a choline salt + a hydrogen bond donor, an ethanol solution, or a multi-component deep eutectic solvent B as an extractant is less effective in extracting the active ingredients from Rehmannia glutinosa than the multi-component deep eutectic solvent A of the present invention; during the extraction process, the effect of microwave synergistic operation is better than that of operation without microwave.

[0137] Comparative Example 6

[0138] In this comparative example, based on Example 1, only the composition of the multi-component deep eutectic solvent B in step 3 was changed, while the other steps and reaction parameters remained unchanged. The changes in the extraction amounts of the active ingredients (taking flavonoids as an example) in the extraction supernatant obtained in step 3 are shown in Table 8 below.

[0139] In this comparative example, the multi-component deep eutectic solvent B does not contain choline bromide. Specifically, 1-octyl-3-methylimidazolium bromide and 1,4-butanediol are mixed in a molar ratio of 1:1, and the two are heated to 40° C. to 60° C. to prepare the multi-component deep eutectic solvent B.

[0140] Comparative Example 7

[0141] In this comparative example, based on Example 1, only the composition of the multi-component deep eutectic solvent B in step 3 was changed, while the other steps and reaction parameters remained unchanged. The changes in the extraction amounts of the active ingredients (taking flavonoids as an example) in the extraction supernatant obtained in step 3 are shown in Table 8 below.

[0142] In this comparative example, the multi-component deep eutectic solvent B does not contain 1-octyl-3-methylimidazolium bromide. Specifically, choline bromide and 1,4-butanediol are mixed in a molar ratio of 1:1, and the two are heated to 40° C. to 60° C. to prepare the multi-component deep eutectic solvent B.

[0143] Comparative Example 8

[0144] In this comparative example, based on Example 1, ultrasonic co-treatment was not used in step 3. Under the premise that other steps and reaction parameters remained unchanged, the changes in the extraction amount of each effective component (taking flavonoids as an example) in the extraction supernatant obtained in step 3 are shown in Table 8 below.

[0145] Comparative Example 9

[0146] In this comparative example, based on Example 1, a one-time ultrasonic co-treatment was used in step 3. Under the premise that other steps and reaction parameters remained unchanged, the changes in the extraction amount of each effective component (taking flavonoids as an example) in the extraction supernatant obtained in step 3 are shown in Table 8 below.

[0147] The specific steps of step 3 of this comparative example are:

[0148] According to the molar ratio of choline bromide (choline salt): 1-octyl-3-methylimidazolium bromide: alcohol hydrogen bond donor 1,4-butanediol = 1:1:2, the three are heated to 40°C to 60°C and mixed to prepare a multi-component deep eutectic solvent B;

[0149] Mix the dried Rehmannia root solid residue with the remaining raw material powder, and stir evenly to form a mixed powder;

[0150] After being fully stirred in a mass ratio of mixed powder: multi-component low eutectic solvent B: 70% ethanol solution = 1:3:90, ultrasonic-assisted extraction was performed at 40°C for 30 minutes with an ultrasonic power of 200w, and then solid-liquid separation was performed to obtain a filtrate and solid residue; the filtrate was centrifuged and distilled to remove impurities to obtain an extraction supernatant.

[0151] Comparative Example 10

[0152] In this comparative example, based on Example 1, the mixed powder in step 3 only contained the remaining raw material powder excluding Rehmannia glutinosa, and under the premise that other steps and reaction parameters remained unchanged, the changes in the extraction amount of each effective component (taking flavonoids as an example) in the extraction supernatant obtained in step 3 are shown in Table 8 below.

[0153] Comparative Example 11

[0154] In this comparative example, based on Example 1, only the multi-component deep eutectic solvent B in step 3 was replaced with the multi-component deep eutectic solvent A. With the other steps and reaction parameters remaining unchanged, the changes in the extraction amounts of the active ingredients (taking flavonoids as an example) in the extraction supernatant obtained in step 3 are shown in Table 8 below.

[0155] Table 8

[0156]

[0157] As can be seen from the data in Table 8, the use of a deep eutectic solvent or a multi-component deep eutectic solvent A comprising 1-octyl-3-methylimidazolium bromide + a hydrogen bond donor or a choline salt + a hydrogen bond donor as an extractant is less effective in extracting the active ingredients from the drug than the multi-component deep eutectic solvent B of the present invention. During the extraction process, the comparison of the effects of different ultrasonic treatments is as follows: fractional ultrasonic treatment > single ultrasonic treatment > no ultrasonic treatment. After removing the Rehmannia glutinosa solid residue in step 2, the amount of flavonoids extracted is slightly reduced.

[0158] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for preparing bone augmentation and reduction, characterized in that: The raw materials for bone augmentation and reduction include prepared rehmannia root, roasted epimedium, millettia reticulata, cibotrya japonica root, drynaria root, cynomorium songaricum, dipsaccharum officinale, wolfberry fruit and radish seed; The preparation method comprises the following steps: The choline salt, 1-octyl-3-methylimidazolium bromide and an acidic hydrogen bond donor are heated and mixed according to a preset molar ratio to prepare a multi-element deep eutectic solvent A; the acidic hydrogen bond donor is citric acid or acetic acid; The choline salt, 1-octyl-3-methylimidazolium bromide and an alcohol hydrogen bond donor are heated and mixed according to a preset molar ratio to prepare a multi-element deep eutectic solvent B; the alcohol hydrogen bond donor is 1,4-butanediol; The Rehmannia root powder, the multi-element deep eutectic solvent A, and water were uniformly mixed in a preset ratio, and the Rehmannia root powder was treated with microwaves in a closed environment at a temperature of 60° C. to obtain a Rehmannia root extract and Rehmannia root solid residue; The dried Rehmannia root solid residue and the remaining raw material powder are mixed to form a material, and a mixed solution of a multi-component deep eutectic solvent B and an alcohol solution is used as an extractant B, and ultrasound-assisted extraction is performed to obtain an extraction supernatant; The prepared Rehmannia root extract and the supernatant are combined and concentrated to obtain the bone-increasing and eliminating stock solution; The bone-increasing and quenching stock solution and adjuvants are used to prepare the bone-increasing and quenching preparation; According to the molar ratio, in the multi-component deep eutectic solvent A, choline salt: 1-octyl-3-methylimidazolium bromide: acid hydrogen bond donor = 1:1:2-4; the choline salt is choline chloride or choline bromide; According to the molar ratio, in the multi-component deep eutectic solvent B, the choline salt: 1-octyl-3-methylimidazolium bromide: alcohol hydrogen bond donor = 1:1:2-4; the choline salt is choline bromide.

2. The method for preparing bone augmentation and reduction according to claim 1, characterized in that: The specific steps of obtaining the Radix Rehmanniae Preparata extract and the Radix Rehmanniae Preparata solid residue are: The Rehmannia root powder, multi-element low eutectic solvent A and water are mixed evenly in a preset ratio, and the Rehmannia root powder is heated and extracted in a closed environment with microwave. After the extraction, it is cooled to room temperature and then centrifuged. After liquid phase distillation and impurities are removed, the Rehmannia root extract is obtained, and the solid phase is the Rehmannia root solid residue.

3. The method for preparing bone augmentation and reduction according to claim 1, characterized in that: The specific steps of obtaining the extraction supernatant are: The dried Rehmannia glutinosa solid residue is mixed with the remaining raw material powder, and a mixed solution of a multi-component low eutectic solvent B and an alcohol solution is used as the extractant B. After sufficient stirring, ultrasonic-assisted extraction is performed, and then solid-liquid separation is performed. The obtained solid is added with the extractant B and ultrasonic-assisted extraction and solid-liquid separation are repeated at least once. The filtrates after each solid-liquid separation are combined to obtain an extraction supernatant.

4. The method for preparing bone augmentation and reduction according to claim 1, characterized in that: The heating temperature for preparing the multi-component deep eutectic solvent A is 40°C to 60°C.

5. The method for preparing bone augmentation and reduction according to claim 1, characterized in that: The heating temperature for preparing the multi-component deep eutectic solvent B is 40°C to 60°C.

6. The method for preparing bone augmentation and reduction according to claim 2, characterized in that: The prepared Rehmannia root powder, multi-element deep eutectic solvent A, and water = 1 g: (10-15) g: (15-25) mL.

7. The method for preparing bone augmentation and reduction according to claim 2, characterized in that: The time for heating and extracting the Radix Rehmanniae Preparata powder is 10-20 minutes; the temperature for heating and extracting is 60-120° C.; and the power of the microwave is 150-250W.

8. The method for preparing bone augmentation and reduction according to claim 3, characterized in that: The steps of preparing the extraction supernatant specifically include: Mix the dried Rehmannia root solid residue with the remaining raw material powder, and stir evenly to form a mixed powder; The mixed powder and the extractant B are mixed in a preset ratio, stirred thoroughly, and then ultrasonic-assisted extraction is performed, followed by solid-liquid separation to obtain a first filtrate and a first solid residue; The first solid residue is dried and mixed with the extractant B in a preset ratio, fully stirred, and then ultrasonic-assisted extraction is performed, followed by solid-liquid separation to obtain a second filtrate and a second solid residue; this step is repeated for the second solid residue to obtain a third filtrate and a third solid residue; The first filtrate, the second filtrate and the third filtrate are combined and centrifuged, and impurities are removed by distillation to obtain an extraction supernatant; The power of the ultrasonic-assisted extraction is 200W~300W; the temperature is 40°C~60°C; and the time of a single ultrasonic-assisted extraction is 5min~10min.

9. The method for preparing bone augmentation and reduction according to claim 8, characterized in that: During the single ultrasonic-assisted extraction process, the mass ratio of solid material: multi-component deep eutectic solvent B: alcohol solution is 1:1~2:30~60, and the solid material is a mixed powder, the first solid residue, or the second solid residue.

10. The method for preparing bone augmentation and reduction according to claim 3, characterized in that: The alcohol solution is a methanol aqueous solution or an ethanol aqueous solution, and the mass concentration of alcohol in the alcohol solution is 40% to 80%.

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