Traditional Chinese medicine compound extract as well as extraction method and application thereof

Abstract

The invention discloses a traditional Chinese medicine compound extract as well as an extraction method and application thereof, and relates to the technical field of modern extraction and preparations of traditional Chinese medicines, the traditional Chinese medicine compound extract is prepared from a core medicine group, a synergistic component, a targeted strengthening component and preparation auxiliary materials in parts by weight: the core medicine group comprises 12-25 parts of ginseng under forest, 18-35 parts of radix ophiopogonis from Sichuan of China and 10-20 parts of fructus schisandrae; the synergistic components comprise 8-20 parts of dendrobium officinale, 5-15 parts of gynostemma pentaphylla and 3-10 parts of perilla leaves; a targeting strengthening component: 1-4 parts of a glutathione-selenium compound; according to the method, fat-soluble components are extracted through ultrasonic-microwave synergy, water-soluble polysaccharide is extracted through the enzymolysis coupling dynamic high-pressure microjet technology, and volatile active components are captured in combination with the vacuum low-temperature molecular distillation technology. The problems of component loss and degradation caused by a traditional single extraction method are solved, the extraction rate of core functional components such as ginsenoside, ophiopogon japonicus polysaccharide, dendrobium polysaccharide and schizandrin A is remarkably increased, and richness and integrity of chemical components of the extract are ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine modernization extraction and preparation technology, in particular to a traditional Chinese medicine compound extract, its extraction method, application and preparation method. BACKGROUND

[0002] With the in-depth study of the energy metabolism mechanism of modern medicine, mitochondria as the energy factory of cells, its dysfunction has been recognized as one of the core pathological links of many chronic diseases, such as type 2 diabetes, non-alcoholic fatty liver disease, Alzheimer's disease, etc. There are different degrees of mitochondrial damage. Traditional Chinese medicine compound has the characteristics of integrated regulation of multiple components, multiple pathways and multiple targets, and has significant potential in improving energy metabolism and cell function of the body. However, although traditional Chinese medicine compound has theoretical advantages and clinical basis in mitochondrial protection, its actual development and application are seriously limited by the backward extraction and preparation technology.

[0003] The current commonly used water extraction method, alcohol extraction method or simple mixed extraction method has obvious limitations, and it is difficult to simultaneously extract and retain multiple active ingredients with large polarity difference and different stability. For example, the fat-soluble components such as ginsenosides and the water-soluble components such as ophiopogon polysaccharide and dendrobium polysaccharide have completely different polarity requirements for solvents; and the heat-sensitive components such as schisantherin A and perilla volatile oil are easily degraded or lost by volatilization in the conventional high-temperature extraction process. This leads to incomplete types of effective components, low extraction rate, high impurity content, and unstable chemical properties in the final extract, which directly affects the reproducibility of drug efficacy and quality stability.

[0004] Further, the crude extract obtained by traditional process is easy to absorb moisture and caking, and a large amount of auxiliary materials often need to be added during the preparation process, which not only increases the process complexity, but also may affect the in vivo release and absorption behavior of the drug. In addition, the lack of targeted purification steps leads to incomplete removal of heavy metals, pesticide residues and high molecular impurities, which poses a safety hazard; and the lack of modern targeted delivery technology makes it difficult for active ingredients to be effectively enriched in mitochondria in the lesion site, resulting in low bioavailability and limiting the full play of its pharmacological effect. Therefore, we propose a traditional Chinese medicine compound extract, its extraction method, application and preparation method to solve the above problems.

[0005] The above information disclosed in this BACKGROUND section is only for the purpose of increasing the understanding of the background of the present application, and therefore, it can include information that is not prior art to those of ordinary skill in the art. SUMMARY

[0006] The present application aims to provide a traditional Chinese medicine compound extract, an extraction method and application thereof, to solve the problem that the traditional extraction process cannot simultaneously achieve efficient extraction and preservation of multiple polarity and multiple heat-sensitive active ingredients, resulting in low effective ingredient content, high impurity residue, poor stability and lack of targeting of the compound extract, thereby restricting its application in the field of precise treatment such as mitochondrial protection. The present application provides a traditional Chinese medicine compound extract with clear active ingredients, high purity and stability, and mitochondrial targeting delivery function, as well as an efficient and controllable preparation method thereof, and specific application thereof in preparing a drug for preventing or treating a mitochondrial dysfunction-related disease, by constructing a technical system covering formula design, phase separation targeted extraction, multi-stage purification and intelligent formulation.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] A traditional Chinese medicine compound extract is prepared from a core drug group, a synergistic component, a targeted reinforcing component and a preparation auxiliary material by weight:

[0009] The core drug group comprises 12-25 parts of understory ginseng, 18-35 parts of Sichuan ophiopogon, and 10-20 parts of Schisandra chinensis.

[0010] The synergistic component comprises 8-20 parts of Dendrobium officinale, 5-15 parts of Gynostemma pentaphyllum, and 3-10 parts of Perilla frutescens.

[0011] The targeted reinforcing component comprises 1-4 parts of glutathione-selenium complex.

[0012] The preparation auxiliary material comprises 2-6 parts of hydroxypropyl-beta-cyclodextrin and 3-8 parts of mesoporous silicon nanoparticles with a pore size of 20-50 nm.

[0013] The active ingredient content of the extract is as follows: the total content of ginsenoside Rg1 and Rb1 is greater than or equal to 4.2%, the total content of ophiopogon polysaccharide and dendrobium polysaccharide is greater than or equal to 18.0%, the content of schisandrin A is greater than or equal to 1.0%, and the content of glutathione-selenium complex is greater than or equal to 0.8%.

[0014] Among them, the content of ginsenoside Rg1 is greater than or equal to 2.0%, the content of ginsenoside Rb1 is greater than or equal to 2.2%, the content of ophiopogon polysaccharide is greater than or equal to 10.0%, and the content of dendrobium polysaccharide is greater than or equal to 8.0%.

[0015] An extraction method of a traditional Chinese medicine compound extract comprises three core steps of phase separation targeted extraction, multi-stage purification and intelligent formulation, and the specific steps are as follows:

[0016] Phase separation targeted extraction:

[0017] (1) Extraction of fat-soluble components: Take the undergrowth ginseng powder and gynostemma pentaphyllum powder, add 75% ethanol-ethyl acetate mixed solvent (volume ratio 4:1) according to the solid-liquid ratio of 1:10, and use ultrasonic-microwave synergistic extraction. The extraction parameters are: power 400W, microwave frequency 2450MHz, temperature 55℃, time 60min, ultrasonic interval 3s / 3s, extraction 2 times. The fat-soluble extract A is obtained by combining the extract;

[0018] (2) Extraction of water-soluble components: Take the radix ophiopogonis powder and dendrobium officinale powder, add deionized water according to the solid-liquid ratio of 1:12, add 0.2% cellulase (enzyme activity ≥5000U / g) and 0.1% pectinase (enzyme activity ≥3000U / g), adjust the pH to 5.0, and then perform enzymatic hydrolysis at 50℃ for 40min. Then, perform dynamic high-pressure microjet extraction 3 times at 120MPa, and filter to obtain the water-soluble extract B;

[0019] (3) Extraction of heat-sensitive components: Take the schisandra chinensis powder, perilla leaf powder, and alcohol extraction residues of step (1), add deionized water according to the solid-liquid ratio of 1:8, and use vacuum low-temperature molecular distillation extraction. The parameters are: distillation temperature 42℃, vacuum degree 5Pa, scraper membrane rotation speed 200r / min. Collect the volatile oil, and then perform inclusion with hydroxypropyl-β-cyclodextrin according to the mass ratio of 1:6 to obtain the volatile oil inclusion compound C;

[0020] Multi-stage purification:

[0021] (4) Resin enrichment: Concentrate the fat-soluble extract A under reduced pressure (-0.09MPa, 55℃) to remove the alcohol taste, mix with the water-soluble extract B, adjust the pH to 7.0, pump into an AB-8 macroporous resin column (diameter-height ratio 1:10), adsorb at a flow rate of 2BV / h, elute with 3BV of deionized water (3BV / h) to remove impurities, and elute with 65% ethanol (2BV / h). Collect the eluate to obtain the purified liquid D;

[0022] (5) Membrane separation and impurity removal: Filter the purified liquid D through a 0.2μm ceramic membrane and a 10kDa ultrafiltration membrane in sequence to obtain the clear purified liquid E;

[0023] (6) Removal of heavy metals: Add 0.5% sodium dithiocarbamate to the purified liquid E, stir at 40℃ for 30min, centrifuge at 10000r / min for 20min to remove the precipitate, and obtain the purified liquid F;

[0024] (7) Component compounding: Concentrate the purified liquid F to a relative density of 1.20-1.25 (60℃), add the volatile oil inclusion compound C and glutathione-selenium complex, stir to dissolve, and obtain the compound concentrated liquid G;

[0025] Intelligent formulation:

[0026] (8) Mesoporous silica loading: complex concentrate G and mesoporous silica nanoparticles are mixed at a mass ratio of 3:1, stirred at 60 DEG C for 90 min to obtain loading liquid H;

[0027] (9) drying and pill making: the loading liquid H is spray dried to obtain dry powder, mixed with microcrystalline cellulose at a ratio of 9:1, and prepared into 1-2mm pellets by an extrusion-spheronization machine to obtain finished products;

[0028] Wherein, the air inlet of the spraying machine is 170 DEG C, the air outlet is 75 DEG C, the atomization speed is 20000r / min, the extrusion of the extrusion-spheronization machine is 80r / min, and the rolling is 300r / min.

[0029] Compared with the prior art, the beneficial effects of the present application are:

[0030] The present application adopts ultrasonic-microwave synergistic extraction of fat-soluble ingredients, enzyme-coupled dynamic high-pressure microjet technology for extracting water-soluble polysaccharides, and combines vacuum low-temperature molecular distillation technology to capture volatile active ingredients. The problems of component loss and degradation caused by traditional single extraction method are overcome, the extraction rate of core active ingredients such as ginsenosides, ophiopogon polysaccharides, dendrobium polysaccharides and schisantherin A is significantly improved, and the richness and integrity of the chemical components of the extract are ensured.

[0031] Through the system integration of multi-stage purification technology, including selective enrichment of macroporous adsorption resin, fine impurity removal by membrane separation, and specific heavy metal removal process, a large amount of tannins, starch, proteins and heavy metals and other invalid or harmful impurities in the extract are effectively removed, so that the purity of the extract reaches more than 85%. At the same time, by introducing hydroxypropyl-beta-cyclodextrin inclusion technology for stabilizing volatile oil, and combining with mesoporous silica nanoparticle loading to construct a protective structure, the stability of heat-sensitive and easily-oxidized components is enhanced.

[0032] The present application scientifically adds glutathione-selenium complex in the prescription, and uses mesoporous silica nanoparticles with surface modified PEG as a delivery carrier, uses the high drug loading capacity of mesoporous silica and the long circulation characteristics of PEG, and the mitochondrial targeting of glutathione, to realize the synergistic effect of active ingredients and the directional enrichment to the mitochondria of diseased cells.

[0033] The above summary is only for the purpose of the description and is not intended to limit in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features will be readily apparent to those skilled in the art by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0034] Fig. 1 The flow chart of the phase separation targeting extraction in the present application;

[0035] Fig. 2Flow chart for multi-stage purification in the present application;

[0036] Fig. 3 Flow chart for intelligent formulation in the present application. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0038] Embodiment one, preparation of traditional Chinese medicine compound extract for protecting mitochondria

[0039] 1. Experimental raw materials

[0040] Undergrowth ginseng (produced in Jilin, Panax ginseng C. A. Mey.), Sichuan ophiopogon (the native medicinal material, produced in Mianyang, Sichuan, Ophiopogon japonicus (Linn. f.) Ker-Gawl.), Schisandra chinensis (pickled, produced in Liaoning, Schisandra chinensis (Turcz.) Baill.), Dendrobium officinale Kimura et Migo (fresh product, produced in Yunnan, Dendrobium officinale Kimura et Migo), Gynostemma pentaphyllum (Thunb.) Makino (tender leaves, produced in Shaanxi, Gynostemma pentaphyllum (Thunb.) Makino), Perilla frutescens (L.) Britt. (freeze-dried, produced in Jiangsu, Perilla frutescens (L.) Britt.); glutathione-selenium complex (reduced glutathione and selenomethionine are compounded at 5:1, purity ≥98%), hydroxypropyl-β-cyclodextrin (pharmaceutical grade, purity ≥99%), mesoporous silica nanoparticles (pore size 20-50 nm, surface modification 2000 Da PEG chain, particle size 50-100 nm); cellulase (enzyme activity 5000 U / g), pectinase (enzyme activity 3000 U / g), AB-8 macroporous resin (pore size Specific surface area 400-500 m 2 / g) are all pharmaceutical grade reagents.

[0041] 2. Preparation steps are shown in Figs. 1-3

[0042] (1) Raw material pretreatment: 20 parts of undergrowth ginseng, 25 parts of Sichuan ophiopogon, 15 parts of Schisandra chinensis, 12 parts of Dendrobium officinale Kimura et Migo, 8 parts of Gynostemma pentaphyllum, and 6 parts of Perilla frutescens (L.) Britt. are respectively pulverized by a high-speed pulverizer and passed through a 100-mesh sieve, and then placed in a dryer for standby. ​

[0043] (2) Phase separation targeted extraction

[0044] ① Extraction of fat-soluble components: Take the powder of undergrowth ginseng and gynostemma pentaphyllum, and add 75% ethanol-ethyl acetate mixed solvent (volume ratio 4:1) according to the solid-liquid ratio of 1:10. Put it into the ultrasonic-microwave synergistic extraction instrument, set the power to 400W, the microwave frequency to 2450MHz, the temperature to 55℃, the time to 60min, and the ultrasonic interval to 3s / 3s. Extract twice, centrifuge the combined extract at 8000r / min for 15min, and take the supernatant to obtain the fat-soluble extract A.

[0045] ② Extraction of water-soluble components: Take the powder of radix ophiopogonis and dendrobium officinale, and add deionized water according to the solid-liquid ratio of 1:12. Add 0.2% cellulase and 0.1% pectinase, and adjust the pH to 5.0 with 1mol / L citric acid. Enzyme hydrolysis at 50℃ constant temperature water bath for 40min. Then transfer to dynamic high pressure microjet extraction equipment, set the pressure to 120MPa, and extract for 3 cycles. The extract is filtered by plate and frame (pore size 0.45μm) to obtain the water-soluble extract B.

[0046] ③ Extraction of heat-sensitive components: Take the powder of schisandra chinensis and perilla frutescens, and the alcohol extraction residue of step ①, and add deionized water according to the solid-liquid ratio of 1:8. Use vacuum low-temperature molecular distillation device for extraction, set the distillation temperature to 42℃, the vacuum degree to 5Pa, and the scraper membrane rotation speed to 200r / min. Collect the volatile oil phase. Mix the volatile oil with hydroxypropyl-β-cyclodextrin according to the mass ratio of 1:6, stir at 50℃ for 60min to obtain the volatile oil inclusion compound C.

[0047] (3) Multistage purification

[0048] ① Resin enrichment: Put the fat-soluble extract A into a rotary evaporator and concentrate under reduced pressure at a vacuum degree of-0.09MPa and a temperature of 55℃ until there is no alcohol smell. Mix it evenly with the water-soluble extract B, adjust the pH to 7.0, and pump it into the pretreated AB-8 macroporous resin column (diameter-height ratio 1:10) at a flow rate of 2BV / h. First, elute with 3BV of deionized water (flow rate 3BV / h) to remove impurities, and then elute with 65% ethanol (flow rate 2BV / h). Collect the ethanol eluate to obtain the purified liquid D.

[0049] ② Membrane separation and impurity removal: Purified liquid D is sequentially filtered by 0.2μm ceramic membrane (to remove particulate impurities) and 10kDa ultrafiltration membrane (to remove macromolecular impurities) to obtain clear purified liquid E.

[0050] ③ Heavy metal removal: Add 0.5% sodium dithiocarbamate to the purified liquid E, stir at 40℃ for 30min, centrifuge at 10000r / min for 20min, discard the precipitate and take the supernatant to obtain the purified liquid F.

[0051] ④ Component compounding: Concentrate the purified solution F to a relative density of 1.20-1.25 (measured at 60℃), add the volatile oil inclusion complex C and 2 parts of glutathione-selenium complex, stir at 50℃ until completely dissolved, and obtain the compound concentrate G.

[0052] (4) Intelligent formulation

[0053] ① Mesoporous silica loading: The composite concentrate G was mixed with 5 parts of mesoporous silica nanoparticles at a mass ratio of 3:1. The mixture was stirred in a constant temperature water bath at 60℃ for 90 min. During the reaction, samples were taken every 15 min to observe the loading status to ensure that the active ingredients fully entered the mesoporous silica channels, thus obtaining the loading solution H.

[0054] ②Drying and pelleting: The loaded liquid H is passed into a centrifugal spray dryer, with the inlet air temperature set to 170℃, the outlet air temperature to 75℃, and the atomization speed to 20000r / min. The dried powder is collected. The dried powder is mixed with microcrystalline cellulose at a ratio of 9:1 and fed into an extruder and spheronizer. The extrusion speed is set to 80r / min and the spheronizer speed to 300r / min. Microparticles with a particle size of 1-2mm are prepared, which yields the finished product of the traditional Chinese medicine compound extract for mitochondrial protection.

[0055] 3. Quality Inspection Results

[0056] The ginsenoside content was determined by HPLC: Rg1 content was 2.3%, Rb1 content was 2.4%, and the total content was 4.7%. The polysaccharide content was determined by the phenol-sulfuric acid method: Ophiopogon japonicus polysaccharide was 11.2%, Dendrobium nobile polysaccharide was 8.5%, and the total content was 19.7%. The schisandrin A content was determined by UPLC-MS / MS: 1.3%. The glutathione-selenium complex content was determined by high performance liquid chromatography-atomic fluorescence spectrometry: 0.9%.

[0057] The moisture content of the dry powder was 3.2%, and the bulk density of the microspheres was 0.72 g / cm³. 3 The friability was 0.6%, and the levels of heavy metals (lead 0.2 mg / kg, arsenic 0.1 mg / kg, mercury not detected) and solvent residues (ethanol 280 mg / kg, ethyl acetate 150 mg / kg) all met the pharmaceutical grade standards.

[0058] Example 2: Preparation of mitochondrial-protective oral sustained-release capsules

[0059] 1. Experimental materials: Traditional Chinese medicine compound extract prepared in Example 1 (active ingredient content meets the standard), hydroxypropyl methylcellulose (K4M, pharmaceutical grade), microcrystalline cellulose (PH101, pharmaceutical grade), magnesium stearate (pharmaceutical grade), and capsule shells (No. 0 gelatin capsules, pharmaceutical grade).

[0060] 2. Preparation steps (1) Formulation design for a dosage of 1000 capsules

[0061] Traditional Chinese medicine compound extract 250 g, hydroxypropyl methyl cellulose 50 g, microcrystalline cellulose 30 g, magnesium stearate 5 g, total weight 335 g.

[0062] (2) Preparation process

[0063] ① Mixing: Traditional Chinese medicine compound extract, hydroxypropyl methyl cellulose, and microcrystalline cellulose were sequentially put into a three-dimensional mixer, the rotation speed was set to 15 r / min, and mixing was performed for 30 min to ensure uniformity of the materials and content uniformity RSD≤3%.

[0064] ② Granulation: The mixed materials were granulated through an 18-mesh sieve to remove caked particles, and uniform granules were obtained.

[0065] ③ Total mixing: Magnesium stearate was added to the granules after granulation, and the mixture was again put into a three-dimensional mixer, the rotation speed was set to 15 r / min, and mixing was performed for 5 min.

[0066] ④ Filling: The total mixed granules were filled into No. 0 capsule shells by a full-automatic capsule filling machine, the filling amount was adjusted so that each capsule contained 250 mg of extract, and the filling speed was 300 capsules / min.

[0067] ⑤ Polishing and screening: The filled capsules were polished by a capsule polishing machine to remove surface powder, and then screened by a capsule sorting machine to remove capsules with a weight difference exceeding ±5%, and the final product of the mitochondria-protecting oral sustained-release capsule was obtained.

[0068] 3. Preparation quality test results

[0069] (1) Sustained-release property test: The dissolution test method (paddle method) was used, 900 mL of pH 6.8 phosphate buffer was used as the dissolution medium, the rotation speed was 50 r / min, and the temperature was 37℃±0.5℃. The results showed that the dissolution amount was 25%±2% at 1 h, 58%±3% at 4 h, and 92%±2% at 8 h, which met the requirements of the dissolution curve of sustained-release preparations.

[0070] (2) Stability test: The accelerated stability test was set at 40℃ and RH 75% for 6 months: the capsules had no deformation or adhesion, and the contents were not hygroscopic; the content of active ingredients: the total content of ginsenoside Rg1+Rb1 was 4.6%, the total content of polysaccharides was 19.5%, and the degradation rate was less than 1%; the microbial limit met the pharmaceutical standards.

[0071] (3) Other indicators: weight difference ±3.2% (meeting the requirement of ±7.5% limit); friability: 0.3% (meeting the requirement of ≤1.0%); disintegration time: not detected (no disintegration time requirement for sustained-release capsules).

[0072] Example Three, Efficacy Verification of Traditional Chinese Medicine Compound Extract on Mitochondrial Dysfunction Model Mice

[0073] 1. Experimental materials

[0074] (1) Experimental animals: 60 SPF level db / db mice, male, 6-8 weeks old, weighing 25-30 g; 15 C57BL / 6J mice of the same strain were used as a normal control group, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The feeding environment: temperature 22℃±2℃, humidity 55%±5%, 12h light / dark cycle, free feeding and drinking.

[0075] (2) Experimental drugs: extract of the application (prepared in Example 1), sustained-release capsules of the application (prepared in Example 2, ground into powder for use), commercially available Shengmai capsules.

[0076] (3) Detection reagents: blood glucose detection kit (glucose oxidase method), mitochondrial membrane potential detection kit (JC-1 method), ATP detection kit (fluorescence method), TNF-α / IL-6 ELISA kit.

[0077] 2. Experimental method

[0078] (1) Model grouping and administration: 60 db / db mice were randomly divided into 4 groups, 15 in each group: model control group (gavaged with normal saline), extract low-dose group (gavaged with extract 200mg / kg), extract high-dose group (gavaged with extract 400mg / kg), commercially available drug control group (gavaged with Shengmai capsule contents 400mg / kg); the normal control group (C57BL / 6J mice) was gavaged with normal saline. Drug administration was performed once a day for 8 consecutive weeks, and changes in body weight and food intake of the mice were recorded.

[0079] (2) Index detection

[0080] ① Blood glucose detection: before administration and after 4 weeks and 8 weeks of administration, the mice were fasted for 12h without water, blood was taken from the tail vein, and fasting blood glucose (FBG) was measured using a blood glucose detector.

[0081] ② Mitochondrial function detection: after 8 weeks of administration, the liver tissue of the mice was taken out, and the liver mitochondria were extracted according to the instructions of the mitochondrial extraction kit; the mitochondrial membrane potential (Δψm) was detected by JC-1 method, and the red / green fluorescence intensity ratio was observed and calculated under a fluorescence microscope; the mitochondrial ATP production was measured using an ATP detection kit.

[0082] ③ Inflammatory factor detection: the mouse serum was taken, and the contents of TNF-α and IL-6 were detected according to the instructions of the ELISA kit.

[0083] 3. Experimental results

[0084] (1) Blood glucose regulation effect: the fasting blood glucose of each group of db / db mice before administration had no significant difference (P>0.05); after 8 weeks of administration, the fasting blood glucose of the high-dose extract group was reduced by 42.3% (P<0.01) compared with the model control group, the low-dose extract group was reduced by 28.5% (P<0.05), and the commercial drug control group was reduced by 25.1% (P<0.05); the blood glucose improvement effect of the high-dose extract group was significantly better than that of the commercial drug control group (P<0.05).

[0085] (2) Mitochondrial function improvement effect: compared with the normal control group, the mitochondrial membrane potential (red-green fluorescence ratio) of the model control group was reduced by 68.2%, and the ATP production was reduced by 72.5% (all P<0.01); after 8 weeks of administration, the mitochondrial membrane potential of the high-dose extract group was increased by 65.8% and the ATP production was increased by 68.3% (all P<0.01) compared with the model control group, the low-dose extract group was increased by 42.1% and 45.6% (all P<0.05), and the commercial drug control group was increased by 38.7% and 40.2% (all P<0.05); the mitochondrial function repair effect of the high-dose extract group was better than that of the commercial drug control group (P<0.05).

[0086] (3) Inflammatory factor inhibition effect: the contents of TNF-α and IL-6 in the serum of the model control group were 3.2 times and 2.8 times (all P<0.01) of the normal control group respectively; after 8 weeks of administration, the contents of TNF-α and IL-6 in the high-dose extract group were reduced by 58.6% and 52.4% (all P<0.01) compared with the model control group, which was significantly better than the commercial drug control group (reduced by 35.2% and 31.7%, P<0.05).

[0087] 4. Experimental conclusion: the traditional Chinese medicine compound extract can significantly reduce the blood glucose of type 2 diabetes model mice, improve the liver mitochondrial membrane potential and ATP production, and inhibit the expression of inflammatory factors, and the effect of the high-dose group is better than that of the commercial Shengmai capsule, which confirms that it has excellent mitochondrial protection effect and can be used for preparing therapeutic drugs for mitochondrial dysfunction related diseases.

[0088] Comparative Example One, preparation and efficacy comparison of traditional Shengmaisan extract

[0089] 1. Preparation scheme

[0090] (1) Group design: 20 parts of understory ginseng, 25 parts of Chuanmaidong and 15 parts of north schisandra, except for synergistic components, targeted strengthening components and preparation adjuvants, the rest conditions are the same as those of Example One.

[0091] (2) Preparation process: the phase separation targeted extraction-multistage purification-smart formulation process of Example One is followed, and the extraction steps of the corresponding components are deleted and reduced due to the adjustment of the group formula.

[0092] 2. Quality and efficacy test results

[0093] (1) Active ingredient content: total content of ginsenoside Rg1 + Rb1 3.1% (4.7% in the present application), content of ophiopogon polysaccharide 9.8% (11.2% in the present application), no dendrobium polysaccharide and glutathione-selenium complex, content of schizandrin A 0.7% (1.3% in the present application).

[0094] (2) Mitochondrial protection efficacy: after 8 weeks of administration, the fasting blood glucose was reduced by 22.4% compared with the model control group (42.3% in the high dose group of the present application); the liver mitochondrial membrane potential was increased by 35.6%, and the ATP production was increased by 32.8% (65.8% and 68.3% in the high dose group of the present application, respectively); the contents of serum TNF-α and IL-6 were reduced by 30.1% and 27.5% (58.6% and 52.4% in the high dose group of the present application, respectively).

[0095] Comparative Example 2, preparation and quality comparison of the extract of the prescription of the present application and the traditional water extraction process

[0096] 1. Preparation scheme

[0097] (1) The prescription design adopts the prescription of Example 1.

[0098] (2) Preparation process: adopt the traditional one-pot boiling water extraction process: grind all medicinal materials to 100 meshes, mix, add 10 times the amount of deionized water, decoct for 3 times (1.5 h each time), combine the decoction, reduce pressure to concentrate to a relative density of 1.20-1.25, and directly spray dry to obtain dry powder.

[0099] 2. Quality test results

[0100] (1) Active ingredient extraction rate and purity: total extraction rate of ginsenoside Rg1 + Rb1 58.2% (94.2% in the present application), total extraction rate of ophiopogon polysaccharide + dendrobium polysaccharide 62.5% (91.5% in the present application); purity of extract 42.3% (85.3% in the present application), content of impurities (tannin, starch) 48.6% (14.7% in the present application).

[0101] (2) Stability test: after accelerated test (40℃, RH75%, 6 months), the degradation rate of ginsenoside is 26.8% (1.2% in the present application), the degradation rate of schizandrin A is 32.5% (0.8% in the present application); the dry powder is seriously hygroscopic, and has poor flowability (bulk density 0.45g / cm 3 , 0.72g / cm 3 in the present application).

[0102] Comparative Example 3, preparation and efficacy comparison of the extract of the prescription of the present application and the process of the present application

[0103] 1. Preparation scheme

[0104] (1) Composition design: retain the core drug group and synergistic component, delete the targeted strengthening component and mesoporous silica nanoparticles, and the rest of the components and proportions are the same as Example One: 20 parts of understory ginseng, 25 parts of Sichuan ophiopogon, 15 parts of Schisandra chinensis, 12 parts of Dendrobium officinale, 8 parts of Gynostemma pentaphyllum, 6 parts of Perilla frutescens, 4 parts of hydroxypropyl-β-cyclodextrin.

[0105] (2) Preparation process: the extraction, purification and preparation process of Example One is used, and only the glutathione-selenium compound compounding and mesoporous silica loading steps are omitted.

[0106] 3.2 Pharmacodynamic test results: after 8 weeks of administration, the liver mitochondrial membrane potential increased by 41.2% compared with the model control group (the high dose group of the application increased by 65.8%), the ATP production increased by 40.5% (the high dose group of the application increased by 68.3%); the mitochondrial ROS level decreased by 42.3% (the high dose group of the application decreased by 70.1%), and the targeted mitochondrial protection effect was significantly weaker than that of the application.

[0107] In summary, the active ingredient content and mitochondrial protection efficacy of Comparative Example One are significantly lower than those of the application, confirming that components such as Dendrobium officinale and Gynostemma pentaphyllum can produce synergistic effects with the core drug group and enhance efficacy.

[0108] The extraction rate, purity and stability of the active ingredients of Comparative Example Two are far inferior to those of the application, indicating that the phase-targeted extraction-multistage purification process can selectively retain different polar components, reduce impurities and active ingredient degradation, and solve the core defects of traditional processes.

[0109] The mitochondrial function repair effect of Comparative Example Three is significantly decreased, verifying the mitochondrial targeting of glutathione-selenium compound and the delivery synergistic effect of mesoporous silica.

[0110] The embodiments of the application are given for the purpose of illustration and description, although embodiments of the application have been shown and described above, it will be understood that the above embodiments are exemplary and should not be construed as limiting the application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the application.

Claims

1. A compound extract of traditional Chinese medicine, characterized in that, By weight, it is composed of core drug components, synergistic components, targeted enhancement components, and formulation excipients: Core medicinal group: 12-25 parts of wild ginseng, 18-35 parts of Ophiopogon japonicus, and 10-20 parts of Schisandra chinensis; Synergistic effect components: Dendrobium officinale 8-20 parts, Gynostemma pentaphyllum 5-15 parts, Perilla frutescens leaf 3-10 parts; Targeted enhancement component: 1-4 parts of glutathione-selenium complex; Pharmaceutical excipients: 2-6 parts of hydroxypropyl-β-cyclodextrin, 3-8 parts of mesoporous silica nanoparticles with a pore size of 20-50 nm; The active ingredient content of the extract is as follows: total content of ginsenosides Rg1 and Rb1 ≥ 4.2%, total content of Ophiopogon japonicus polysaccharide and Dendrobium officinale polysaccharide ≥ 18.0%, schisandrin A content ≥ 1.0%, and glutathione-selenium complex content ≥ 0.8%. Among them, the content of ginsenoside Rg1 is ≥2.0%, the content of ginsenoside Rb1 is ≥2.2%, the content of Ophiopogon japonicus polysaccharide is ≥10.0%, and the content of Dendrobium nobile polysaccharide is ≥8.0%.

2. The traditional Chinese medicine compound extract according to claim 1, characterized in that: The glutathione-selenium complex is composed of reduced glutathione and organic selenium in a mass ratio of 5:1, wherein the organic selenium is selenomethionine. The surface of the mesoporous silica nanoparticles is modified with polyethylene glycol (PEG) chains, with a molecular weight of 2000-5000 Da.

3. A method for extracting a compound extract of traditional Chinese medicine, characterized in that: It includes three core steps: phase-separated targeted extraction, multi-stage purification, and intelligent formulation. The specific steps are as follows: Phase-specific targeted extraction: (1) Extraction of fat-soluble components: Take ginseng powder and gynostemma pentaphyllum powder from forests, add 75% ethanol-ethyl acetate mixed solvent (volume ratio 4:1) at a material-liquid ratio of 1:10, and use ultrasonic-microwave synergistic extraction. The extraction parameters are: power 400W, microwave frequency 2450MHz, temperature 55℃, time 60min, ultrasonic interval 3s / 3s, extraction twice, and combine the extracts to obtain fat-soluble extract A; (2) Extraction of water-soluble components: Take Ophiopogon japonicus powder and Dendrobium officinale powder, add deionized water at a material-to-liquid ratio of 1:12, add 0.2% cellulase (enzyme activity ≥5000U / g) and 0.1% pectinase (enzyme activity ≥3000U / g), adjust the pH to 5.0, enzymatically hydrolyze at 50℃ for 40 min, and then extract three times by dynamic high pressure microjet at 120MPa. Filter to obtain water-soluble extract B; (3) Extraction of heat-sensitive components: Take Schisandra chinensis powder, Perilla frutescens leaf powder and the alcohol extract residue from step (1), add deionized water at a material-to-liquid ratio of 1:8, and extract by vacuum low-temperature molecular distillation with the following parameters: distillation temperature 42℃, vacuum degree 5Pa, and scraping speed 200r / min. Collect the volatile oil and include it with hydroxypropyl-β-cyclodextrin at a mass ratio of 1:6 to obtain volatile oil inclusion complex C. Multi-stage purification: (4) Resin enrichment: The lipid-soluble extract A was concentrated under reduced pressure (-0.09 MPa, 55℃) until there was no alcohol odor. It was then mixed with the water-soluble extract B, and the pH was adjusted to 7.

0. The mixture was pumped into an AB-8 macroporous resin column (diameter-to-height ratio 1:10) and adsorbed at a flow rate of 2 BV / h. Impurities were removed by elution with 3 BV of deionized water (3 BV / h) and 65% ethanol (2 BV / h). The eluent was collected to obtain purified solution D. (5) Membrane separation and impurity removal: The purified solution D is filtered sequentially through a 0.2 μm ceramic membrane and a 10 kDa ultrafiltration membrane to obtain a clear purified solution E; (6) Removal of heavy metals: Add 0.5% sodium dithiocarbamate to the purification solution E, stir at 40°C for 30 min, centrifuge at 10000 r / min for 20 min to remove the precipitate, and obtain purification solution F; (7) Component compounding: Concentrate purified solution F to a relative density of 1.20-1.25 (60℃), add volatile oil inclusion complex C and glutathione-selenium complex, stir to dissolve and obtain compound concentrated solution G; Intelligent formulation: (8) Mesoporous silica loading: The composite concentrate G and mesoporous silica nanoparticles were mixed at a mass ratio of 3:1 and stirred at 60°C for 90 min to obtain the loading solution H. (9) Drying and pelleting: The loaded liquid H is spray-dried to obtain dry powder, which is mixed with microcrystalline cellulose at a ratio of 9:1, and then processed into 1-2 mm micro pellets by an extrusion spheronizer to obtain the finished product; The sprayer has an inlet temperature of 170°C, an outlet temperature of 75°C, and an atomization speed of 20,000 r / min. The extrusion and spheroidizing machine has an extrusion speed of 80 r / min and a spheroidizing speed of 300 r / min.

4. The extraction method of a traditional Chinese medicine compound extract according to claim 3, characterized in that: In step (1), the particle size of the ginseng powder and gynostemma pentaphyllum powder is 100 mesh; in step (2), the particle size of the ophiopogon japonicus powder and dendrobium officinale powder is 100 mesh; in step (3), the particle size of the schisandra chinensis powder and perilla leaf powder is 100 mesh.

5. The extraction method of a traditional Chinese medicine compound extract according to claim 3, characterized in that: In step (4), the pore size of the AB-8 macroporous resin is Specific surface area is 400-500 m² 2 / g, the pretreatment method is as follows: soak in 95% ethanol and deionized water for 24h in sequence, ultrasonically clean 3 times and then pack into column.

6. The extraction method of a traditional Chinese medicine compound extract according to claim 3, characterized in that: In step (9), the spray drying is performed using a centrifugal spray dryer, and the moisture content of the dry powder is ≤5%; the bulk density of the microparticles is 0.6-0.8 g / cm³. 3 , friability ≤ 1.0%.

7. The application of a traditional Chinese medicine compound extract according to any one of claims 1-2 in the preparation of mitochondrial protective drugs, characterized in that: The mitochondrial protective drug is used to improve mitochondrial dysfunction in patients with type 2 diabetes mellitus of qi and yin deficiency type, non-alcoholic fatty liver disease, and mild Alzheimer's disease.

8. The application according to claim 7, characterized in that: The drug is in the form of an oral sustained-release capsule, each containing 250mg of traditional Chinese medicine compound extract, and excipients including 50mg of hydroxypropyl methylcellulose, 30mg of microcrystalline cellulose, and 5mg of magnesium stearate.

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