Traditional Chinese medicine extract without glycyrrhizic acid, preparation method, medicinal preparation and application
阅读说明：本技术 不含甘草酸的中药提取物、制备方法、药物制剂及用途 (Traditional Chinese medicine extract without glycyrrhizic acid, preparation method, medicinal preparation and application ) 是由 李宁 郭筱璐 王丽 江亚勇 赖秋梅 孙彦平 韩丽娟 于 2021-09-06 设计创作，主要内容包括：本发明公开了一种不含甘草酸的中药提取物、制备方法、药物制剂及用途。该中药提取物为重量比8:3:3:2的葛根、黄芩、黄连和甘草的水提取物。本发明提供的中药提取物既保留了葛根芩连组合物的有效成分,又不含甘草酸,且药效优于含甘草酸的葛根芩连初提物。(The invention discloses a traditional Chinese medicine extract without glycyrrhizic acid, a preparation method, a medicinal preparation and application thereof. The traditional Chinese medicine extract is a water extract of radix puerariae, scutellaria baicalensis, coptis chinensis and liquorice in a weight ratio of 8:3:3: 2. The traditional Chinese medicine extract provided by the invention not only retains the effective components of the Gegenqinlian composition, but also does not contain glycyrrhizic acid, and the efficacy is better than that of the Gegenqinlian primary extract containing glycyrrhizic acid.)
1. A traditional Chinese medicine extract is characterized in that the traditional Chinese medicine extract is a water extract of radix puerariae, scutellaria baicalensis, coptis chinensis and liquorice in a weight ratio of 8:3:3:2, and the traditional Chinese medicine extract does not contain glycyrrhizic acid.
2. The method for preparing a herbal extract as claimed in claim 1, wherein the method comprises the steps of:
1) extracting radix puerariae, scutellaria baicalensis, coptis chinensis and liquorice in a weight ratio of 8:3:3:2 with water to obtain a primary extract of radix puerariae, scutellaria baicalensis and coptis chinensis;
2) removing glycyrrhizic acid from the initial extract of radix Puerariae and Scutellariae radix to obtain traditional Chinese medicine extract.
3. The preparation method according to claim 2, wherein in the step 1), the extraction method is decoction extraction, heating reflux extraction, soaking extraction or ultrasonic extraction.
4. The preparation method according to claim 3, wherein in the step 1), the mixture is heated and refluxed with water for 1 to 3 times, the amount of water added for each time is 5 to 20 times of the total weight of the radix puerariae, the radix scutellariae, the rhizoma coptidis and the liquorice, the extraction time is 0.5 to 3 hours, and the extracting solution is separated from the dregs of a decoction to obtain the extracting solution; concentrating the extractive solution at below 85 deg.C under reduced pressure, and recovering solvent to obtain radix Puerariae, Scutellariae radix and Coptidis rhizoma primary extract.
5. The method according to claim 2, wherein the step 2) comprises performing preparative liquid chromatographyRemoving glycyrrhizic acid from the initial extract of radix Puerariae, Scutellariae radix and Coptidis rhizoma, and preparing liquid chromatogram with C18Silica gel is used as a filling agent; detecting by using an ultraviolet detector, wherein the detection wavelength is 254 nm; elution was carried out in the following manner:
taking 0.1% formic acid-water as a mobile phase A and ethanol as a mobile phase B, and carrying out gradient elution according to the following gradient:
removing the glycyrrhizic acid-containing eluate, collecting all glycyrrhizic acid-free eluates, and recovering solvent to obtain the Chinese medicinal extract.
6. A pharmaceutical preparation, comprising the Chinese medicinal extract of claim 1 or the Chinese medicinal extract prepared by the method of any one of claims 2 to 5, and a pharmaceutically acceptable carrier.
7. The pharmaceutical preparation according to claim 6, wherein the traditional Chinese medicine extract accounts for 0.1-99.9% of the total weight of the pharmaceutical preparation, and the balance is a pharmaceutically acceptable carrier.
8. The pharmaceutical preparation of claim 7, wherein the pharmaceutical preparation is in the form of at least one of granules, tablets, capsules, pills, sprays, films, ointments, suppositories, gels, lyophilizates or injections.
9. Use of the traditional Chinese medicine extract according to claim 1 or the traditional Chinese medicine extract prepared by the method according to any one of claims 2 to 5 for preparing a medicament for preventing and/or treating metabolic syndrome.
10. Use of the herbal extract according to claim 1 or the herbal extract prepared by the method according to any one of claims 2 to 5 for the preparation of a medicament for the prevention and/or treatment of metabolic syndrome without adverse effects caused by glycyrrhizic acid.
Technical Field
The invention relates to a traditional Chinese medicine extract without glycyrrhizic acid, a preparation method, a medicinal preparation and application thereof.
Background
The Gegenqinlian decoction is clinically used for preventing and treating diabetes and metabolic syndrome. The metabolic syndrome is a group of clinical syndromes which are mainly characterized by obesity (or overweight), hyperglycemia, hypertension, dyslipidemia and the like. Insulin resistance is considered by the medical community to be the core of its pathogenesis, but is the result of the combined action of multiple types of histiocytes and signaling pathways, and is difficult to highly generalize by one mechanism. Conventional western medicine treatment (including novel insulin, hypoglycemic drugs, metabolic surgery, etc.) adopts a single disease treatment mode to intervene in different ways on each symptom of the metabolic syndrome, lacks an overall intervention scheme, and has limitations. The pueraria root, scutellaria and coptis decoction serving as a traditional Chinese medicine compound has advantages and application prospects in the aspects of preventing and treating metabolic syndrome.
Glycyrrhizic acid is derived from licorice in the formula, is a characteristic chemical component and an important bioactive component of licorice and related preparations, and is often used as an index component for quality control. The existing research considers that glycyrrhizic acid is one of the main effective components in the Gegenqinlian decoction, the combination of glycyrrhizic acid and berberine compound has similar drug effect with the Gegenqinlian decoction, and glycyrrhizic acid can promote the absorption of berberine.
The Gegenqinlian decoction is used for preventing and treating metabolic syndrome and needs to be taken for a long time. However, according to the report of the literature, the preparation containing glycyrrhizic acid has certain potential safety hazard after being taken for a long time. Glycyrrhizic acid is hydrolyzed in vivo into glycyrrhetinic acid, which has a chemical structure similar to that of steroid hormones. Glycyrrhizic acid and glycyrrhetinic acid are inhibitors of steroid hormone metabolic inactivating enzymes, and can improve the activity of endogenous and exogenous corticoids. Meanwhile, glycyrrhizic acid and glycyrrhetinic acid can be used as ligands and can be combined with a cortical hormone receptor to show glucocorticoid and mineralocorticoid effects. Glycyrrhiza uralensis has been found to have mineralocorticoid-like, i.e., aldosterone-like, effects as early as the 60's in the 20 th century. A large number of studies show that glycyrrhizic acid and glycyrrhetinic acid can reduce urine volume and sodium excretion and increase potassium excretion of human and experimental animals, and continuous use can cause hypertension, edema, hypokalemia, myasthenia, and the like.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a herbal extract without glycyrrhizic acid. The inventor surprisingly finds that when glycyrrhizic acid is not contained, the traditional Chinese medicine extract still has good effect on preventing and/or treating metabolic syndrome, and certain aspects are even better than the original formula of the radix puerariae and the radix scutellariae, and adverse reaction caused by long-term administration of glycyrrhizic acid is avoided. The invention also aims to provide a preparation method of the traditional Chinese medicine extract without glycyrrhizic acid. It is a further object of the present invention to provide a pharmaceutical formulation. The invention also aims to provide application of the traditional Chinese medicine extract in preparing a medicine for preventing and/or treating metabolic syndrome. The invention adopts the following technical scheme to achieve the purpose.
The invention provides a traditional Chinese medicine extract which is a water extract of radix puerariae, scutellaria baicalensis, coptis chinensis and liquorice in a weight ratio of 8:3:3:2, and the traditional Chinese medicine extract does not contain glycyrrhizic acid.
In the present invention, Glycyrrhizic acid (Glycyrrhizic acid) has a molecular formula of C42H62O16Molecular weight of 822.93, and structural formula as shown in formula (I):
the invention also provides a preparation method of the traditional Chinese medicine extract, which comprises the following steps:
1) extracting radix puerariae, scutellaria baicalensis, coptis chinensis and liquorice in a weight ratio of 8:3:3:2 with water to obtain a primary extract of radix puerariae, scutellaria baicalensis and coptis chinensis;
2) removing glycyrrhizic acid from the initial extract of radix Puerariae and Scutellariae radix to obtain traditional Chinese medicine extract.
The kudzu root, the scutellaria baicalensis, the coptis chinensis and the liquorice in the weight ratio of 8:3:3:2 are medicinal materials of a traditional Chinese medicine prescription, namely kudzu root, scutellaria baicalensis and coptis chinensis decoction. The radix puerariae and radix scutellariae composition is a medicinal composition which is prepared by extracting radix puerariae, radix scutellariae, rhizoma coptidis and liquorice according to the weight ratio of 8:3:3:2 with water and removing glycyrrhizic acid. The invention obtains the traditional Chinese medicine extract without glycyrrhizic acid by extracting, preparing liquid chromatographic separation and selectively removing glycyrrhizic acid on the basis of not changing the original composition of the radix puerariae qinlian decoction.
In the present invention, the pueraria root scutellaria baicalensis georgi extract can be prepared by adopting the conventional water extraction methods in the field, such as decoction extraction, heating reflux extraction, ultrasonic extraction, soaking extraction, etc., as long as the water-soluble chemical components in the medicinal materials can be fully extracted, and the method is not particularly limited. When the water is added for extraction, the kudzu root, the scutellaria baicalensis, the coptis chinensis and the liquorice can be respectively extracted, or the four medicinal materials can be mixed and then extracted, and preferably the four medicinal materials are mixed and then extracted. The kudzu root, the scutellaria baicalensis, the coptis chinensis and the liquorice can be fed in the form of decoction pieces, and the decoction pieces can also be fed after being crushed, and are not particularly limited. In one embodiment, the herbs are fed in pieces, mixed and extracted under heating and reflux. The Glycyrrhrizae radix may be processed Glycyrrhrizae radix Preparata, or unprocessed Glycyrrhrizae radix decoction pieces, preferably Glycyrrhrizae radix Preparata. The radix Glycyrrhizae Preparata can be obtained commercially, or processed by conventional processing method in the art such as adding Mel.
According to the preparation method of the invention, preferably, in the step 1), the kudzu root, the scutellaria baicalensis, the coptis chinensis and the liquorice are taken according to the weight ratio of 8:3:3:2, water is added for extraction, and the extracting solution is separated from the dregs to obtain the extracting solution; concentrating the extractive solution under reduced pressure, and recovering solvent to obtain radix Puerariae, Scutellariae radix and Coptidis rhizoma primary extract.
According to the preparation method of the present invention, preferably, in step 1), the extraction is decoction extraction, heating reflux extraction, soaking extraction or ultrasonic extraction. The decoction extraction method is suitable for preparing the extract in small batches. The heating reflux extraction can achieve the effect of decoction extraction by adjusting parameters such as water addition amount, extraction time and the like, and is suitable for large-scale industrial production. The soaking extraction energy consumption is low, the operation is simple, but the extraction rate is low, and the time consumption is long. Therefore, the invention preferably adopts heating reflux extraction or ultrasonic extraction to realize the high-efficiency and maximized retention of water-soluble chemical components in the Gegen Qinlian composition.
According to a preferred embodiment of the invention, in the step 1), radix puerariae, scutellaria baicalensis, coptis chinensis and liquorice are taken according to the weight ratio of 8:3:3:2, the heating reflux extraction is carried out for 1-3 times by using water, the water addition amount is 5-20 times of the total weight of the raw materials each time, the extraction is carried out for 0.5-3 hours each time, and the extracting solution is separated from the dregs to obtain the extracting solution; concentrating the extractive solution at below 85 deg.C under reduced pressure, and recovering solvent to obtain radix Puerariae, Scutellariae radix and Coptidis rhizoma crude extract. The water amount added in each time can be 5-20 times, preferably 5-15 times and more preferably 5-12 times of the total weight of the raw materials. The extraction time per time can be 0.5-3 hours, preferably 1-3 hours, and more preferably 1-2 hours.
According to the preparation method of the present invention, preferably, in the step 2), glycyrrhizic acid in the primary extract of pueraria radix scutellariae and coptis chinensis is removed by preparative liquid chromatography. Preparative liquid chromatography with C18Silica gel is used as a filling agent; preparing a liquid chromatogram with a gradient elution mode, and detecting by adopting an ultraviolet detector; the detection wavelength is 254 nm; elution was carried out in the following manner:
taking 0.1% formic acid-water as a mobile phase A and ethanol as a mobile phase B, and carrying out gradient elution according to the following gradient:
the flow rate was 10ml/min and the column temperature was room temperature. Injecting sample with glycyrrhizic acid reference substance solution to obtain glycyrrhizic acid retention time; then using the radix puerariae and radix scutellariae primary extract obtained by the separation of the preparative liquid chromatography to selectively switch the eluent containing the glycyrrhizic acid into waste liquid according to the retention time of the glycyrrhizic acid, and collecting all the eluent without the glycyrrhizic acid; recovering solvent to obtain Chinese medicinal extract.
More preferably, the preparative liquid chromatography is performed on octadecyl bonded silica gel (C)18Silica gel) as a filler.
According to one embodiment of the present invention, the preparation method comprises the steps of:
1) taking radix puerariae, scutellaria baicalensis, coptis chinensis and radix glycyrrhizae preparata according to a weight ratio of 8:3:3:2, heating and refluxing the radix puerariae, the scutellaria baicalensis, the coptis chinensis and the radix glycyrrhizae preparata with water for 2 times, adding water 5-10 times of the total weight of the raw materials each time, extracting for 1-2 hours each time, and separating an extracting solution from dregs to obtain an extracting solution; concentrating the extractive solution at below 85 deg.C under reduced pressure, and recovering solvent to obtain radix Puerariae, Scutellariae radix and Coptidis rhizoma primary extract;
2) preparative liquid chromatography with C18Silica gel is used as a filling agent, 0.1 percent formic acid-water is used as a mobile phase A, ethanol is used as a mobile phase B, and gradient elution is carried out according to the following gradient:
wherein the flow rate is 10ml/min, and the column temperature is room temperature; detecting by using an ultraviolet detector, wherein the detection wavelength is 254 nm; injecting sample with glycyrrhizic acid reference substance solution to obtain glycyrrhizic acid retention time; then using the radix puerariae and radix scutellariae primary extract obtained by the separation of the preparative liquid chromatography to selectively switch the eluent containing the glycyrrhizic acid into waste liquid according to the retention time of the glycyrrhizic acid, and collecting all the eluent without the glycyrrhizic acid; recovering solvent to obtain Chinese medicinal extract.
In the invention, C is used18The preparation liquid chromatography is used for separating the primary extract of the Gegen Qinlian, and glycyrrhizic acid in the primary extract of the Gegen Qinlian can be selectively and effectively removed through optimized process conditions. Meanwhile, the method has small dead adsorption to other chemical components in the initial extract of the Gegenqinlian, and basically does not influence the content and proportion of other components. At present, no report on a method for selectively removing glycyrrhizic acid in the primary extract of radix puerariae qinlian is available. The activity test shows that the traditional Chinese medicine extract keeps the effective components of the Gegen Qinlian composition, the effect of preventing and treating metabolic syndrome is not lower than that of the Gegen Qinlian primary extract, the effect of certain aspects is better than that of the Gegen Qinlian primary extract, the potential safety hazard caused by long-term administration of glycyrrhizic acid can be avoided, and the safety is better.
The invention also provides a pharmaceutical preparation which comprises the traditional Chinese medicine extract and a pharmaceutically acceptable carrier. The effective components in the traditional Chinese medicine extract are medicinal active components, the traditional Chinese medicine extract shows good efficacy on metabolic syndrome related disease models, has stronger effect than the initial extract of the radix puerariae and the radix scutellariae without glycyrrhizic acid removal, can eliminate the potential adverse reaction risk caused by long-term glycyrrhizic acid intake, and has better safety. Therefore, the traditional Chinese medicine extract without glycyrrhizic acid provided by the invention is more suitable for being developed into a medicinal preparation for preventing and treating metabolic syndrome.
The pharmaceutical preparation according to the invention preferably comprises the traditional Chinese medicine extract and a pharmaceutically acceptable carrier, wherein the traditional Chinese medicine extract accounts for 0.1-99.9% of the total weight of the pharmaceutical preparation, and the balance is the pharmaceutically acceptable carrier.
The pharmaceutical formulation of the present invention may be in any pharmaceutically acceptable dosage form. The pharmaceutical preparation comprises granules, tablets, capsules, pills, sprays, films, ointments, suppositories, gels, freeze-dried preparations or injections, the tablets comprise orally disintegrating tablets, dispersible tablets and the like, the capsules comprise soft capsules, hard capsules and the like, the pills comprise dropping pills, micro-pills and the like, and the ointments comprise ointments, plasters and the like.
The invention also provides application of the traditional Chinese medicine extract in preparation of a medicine for preventing and/or treating metabolic syndrome. The traditional Chinese medicine extract has good effects of preventing and treating metabolic syndrome, and the effect is not lower than that of the original formula and is better than that of the original formula in certain aspects. The traditional Chinese medicine extract disclosed by the invention has a particularly remarkable effect on metabolic syndrome induced by high-fat diet, can effectively reduce weight increase caused by high-fat diet, and has remarkable improvement and treatment effects on fatty liver caused by high-fat diet. Because the glycyrrhizic acid is not contained, the traditional Chinese medicine extract avoids potential adverse reaction risks caused by long-term administration of the glycyrrhizic acid and the glycyrrhetinic acid which is a metabolite in vivo thereof, has better safety, is easier to be accepted by patients, and is suitable for being developed into traditional Chinese medicine innovative medicines for preventing and treating metabolic syndrome. Therefore, the compound can be used for preparing medicines for preventing and/or treating metabolic syndrome.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the scope of the present invention is not limited thereto.
The following describes the drug substances used in the examples and comparative examples:
the kudzu root, the coptis root, the scutellaria baicalensis and the liquorice are medicinal materials which accord with the standard of national formulary.
Radix Puerariae is dried root of Pueraria lobata (Willd.) Ohwi of Leguminosae.
The Coptidis rhizoma is dried rhizome of Coptis chinensis Franch, Coptis deltoidea C.Y.Cheng et Hsiao or Coptis Teeta wall of Ranunculaceae.
The Scutellariae radix is dried root of Scutellaria baicalensis Georgi of Labiatae.
The Glycyrrhrizae radix is dried root and rhizome of Glycyrrhiza uralensis Fisch, Glycyrrhiza inflata Bat, or Glycyrrhiza glabra L.
Example 1
Taking radix puerariae, scutellaria baicalensis, coptis chinensis and radix glycyrrhizae preparata according to the weight ratio of 8:3:3:2, heating and refluxing the radix puerariae, the scutellaria baicalensis, the coptis chinensis and the radix glycyrrhizae preparata with water for 2 times, wherein the water amount added for each time is 8 times and 6 times of the total weight of the raw materials, the extraction time for each time is 1.5 hours and 1 hour respectively, filtering, and combining the two extracting solutions to obtain the total extracting solution. Concentrating the total extractive solution at below 85 deg.C under reduced pressure, and recovering solvent to obtain radix Puerariae, Scutellariae radix and Coptidis rhizoma primary extract.
Removing glycyrrhizic acid from the primary extract of radix Puerariae, Scutellariae radix and Coptidis rhizoma by liquid chromatography. Preparative liquid chromatography with C18Silica gel as a filler (Agilent Prep-C18, 21.2 mm. times.250 mm, 5 μm), 0.1% formic acid-water as mobile phase A, ethanol as mobile phase B, and gradient elution according to the following Table 1:
TABLE 1 preparation of gradient elution table for liquid chromatography
Time (min)
A％
B％
0
90
10
20
75
25
40
50
50
50
5
95
60
5
95
The flow rate was 10ml/min and the column temperature was room temperature.
And detecting by using an ultraviolet detector, wherein the detection wavelength is 254 nm. Injecting sample with glycyrrhizic acid reference solution to obtain glycyrrhizic acid retention time (retention time is 38.5 min). And selectively switching the glycyrrhizic acid-containing eluate to waste liquid according to the glycyrrhizic acid retention time of the primary extract of radix Puerariae and Scutellariae radix, collecting all glycyrrhizic acid-free eluates, and recovering solvent to obtain Chinese medicinal extract.
Comparative example 1
The primary extract of Gegen Qinlian in example 1 is filtered and added with C18Silica gel preparative liquid chromatography (Agilent Prep-C18, 21.2mm multiplied by 250mm, 5 μm) by taking methanol as a mobile phase A and water as a mobile phase B, performing gradient elution (0-60 min, 20-100% A) at the flow rate of 10ml/min and the column temperature of room temperature, and detecting by adopting an ultraviolet detector at the detection wavelength of 254 nm. And (4) measuring the separation degree of the glycyrrhizic acid and adjacent chromatographic peaks.
Comparative example 2
The primary extract of Gegen Qinlian in example 1 is filtered and added with C18Silica gel preparative liquid chromatography (Agilent Prep-C18, 21.2mm multiplied by 250mm, 5 μm) by taking ethanol as a mobile phase A and water as a mobile phase B, performing gradient elution (0-60 min, 20-100% A), wherein the flow rate is 10ml/min, the column temperature is room temperature, and the detection wavelength is 254nm by adopting an ultraviolet detector for detection. And (4) measuring the separation degree of the glycyrrhizic acid and adjacent chromatographic peaks.
Comparative example 3
The primary extract of Gegen Qinlian in example 1 is filtered and added with C18Silica gel preparation liquid chromatography (Agilent Prep-C18, 21.2mm multiplied by 250mm, 5 μm), acetonitrile is used as a mobile phase A, water is used as a mobile phase B, gradient elution (0-60 min, 20% -100% A) is carried out, the flow rate is 10ml/min, the column temperature is room temperature, an ultraviolet detector is adopted for detection, and the detection wavelength is 254 nm. And (4) measuring the separation degree of the glycyrrhizic acid and adjacent chromatographic peaks.
Comparative example 4
The primary extract of Gegen Qinlian in example 1 is filtered and added with C18Silica gel preparative liquid chromatography (Agilent Prep-C18, 21.2mm × 250mm, 5 μm) with 80% methanol water as mobile phase, isocratic elution at flow rate of 10ml/min and column temperature of room temperature, and ultraviolet detection at wavelength of 254 nm. And (5) observing the separation effect of the glycyrrhizic acid and the adjacent chromatographic peaks.
Comparative example 5
The primary extract of Gegen Qinlian in example 1 is filtered and added with C18Silica gel preparative liquid chromatography (Agilent Prep-C18, 21.2mm × 250mm, 5 μm) with 70% ethanol water as mobile phase, isocratic elution at flow rate of 10ml/min and column temperature of room temperature, and ultraviolet detection at wavelength of 254 nm. And (5) observing the separation effect of the glycyrrhizic acid and the adjacent chromatographic peaks.
Comparative example 6
The primary extract of Gegen Qinlian in example 1 is filtered and added with C18Silica gel preparative liquid chromatography (Agilent Prep-C18, 21.2 mm. times.250 mm, 5 μm) on 70% acetonitrile in waterThe eluent is a mobile phase, is eluted at an isocratic rate, has a flow rate of 10ml/min and a column temperature of room temperature, and is detected by an ultraviolet detector with a detection wavelength of 254 nm. And (5) observing the separation effect of the glycyrrhizic acid and the adjacent chromatographic peaks.
Example 2
Taking 10 parts by weight of the traditional Chinese medicine extract prepared in the example 1, adding 6 parts by weight of starch and 2 parts by weight of talcum powder, mixing uniformly, granulating by using 80% (volume percentage concentration) ethanol, drying at the temperature of below 60 ℃, adding 0.2 part by weight of magnesium stearate, mixing uniformly, and pressing into tablets to obtain the tablets containing the traditional Chinese medicine extract.
Example 3
Taking 50 parts by weight of the traditional Chinese medicine extract prepared in the example 1, adding 63 parts by weight of sucrose powder and 31.5 parts by weight of dextrin, uniformly mixing, granulating by 80% (volume percentage concentration) ethanol, drying at the temperature of below 60 ℃, grading, and subpackaging to obtain the granules containing the traditional Chinese medicine extract.
Example 4
Taking 30 parts by weight of the traditional Chinese medicine extract prepared in the example 1, adding 12 parts by weight of starch and 8 parts by weight of talcum powder, mixing uniformly, granulating by using 80% (volume percentage concentration) ethanol, drying at the temperature of below 60 ℃, and encapsulating to obtain capsules containing the traditional Chinese medicine extract.
Example 5
Taking 1 part by weight of the traditional Chinese medicine extract prepared in the embodiment 1, adding 21 parts by weight of polyethylene glycol-6000, uniformly mixing, heating and melting, transferring the melted material into a dripping pill, carrying out drip irrigation, dripping the medicine into liquid paraffin with the temperature of 6-8 ℃, removing oil, and preparing the dripping pill to obtain the dripping pill containing the traditional Chinese medicine extract.
Example 6
Taking 10 parts by weight of the traditional Chinese medicine extract prepared in the example 1, 90 parts by weight of glucose, 18 parts by weight of sodium thiosulfate and 20 parts by weight of distilled water, uniformly mixing, freeze-drying and subpackaging to obtain the freeze-drying agent containing the traditional Chinese medicine extract.
Experimental example 1
Example 1 and comparative examples 1-3 employ a gradient elution process with different mobile phases. Using the elution method of example 1, the degree of separation of glycyrrhizic acid from adjacent chromatographic peaks was 1.5. By adopting the elution method of comparative examples 1 to 3, the degrees of separation of glycyrrhizic acid from adjacent chromatographic peaks were 1.1, 1.2, and 1.1 in this order.
Comparative examples 4 to 6 adopt isocratic elution, baseline separation of glycyrrhizic acid from adjacent chromatographic peaks could not be achieved, and the strongly retained components could not be eluted from the chromatographic column, and the purpose of selectively removing glycyrrhizic acid without affecting other components could not be achieved.
The gradient elution method of embodiment 1 has better separation effect, the separation degree of glycyrrhizic acid and adjacent chromatographic peaks is good, and all components can be eluted from the chromatographic column, so that the aim of selectively removing glycyrrhizic acid without influencing other components can be fulfilled. The addition of formic acid effectively adjusts the peak shape of the chromatographic peak and improves the separation effect.
In addition, the inventors have also tried the following removal methods of glycyrrhizic acid:
firstly, 5g of the radix puerariae qinlian primary extract in example 1 is taken, silica gel column (packed with 100g of silica gel, diameter-height ratio of 1:10) is carried out, chloroform methanol 10:1, chloroform methanol 5:1, chloroform methanol 2:1, chloroform methanol 1:1 and methanol are sequentially used for gradient elution, 4 column volumes are carried out on each gradient elution, eluent is collected, and solvent is recovered. This experimental method does not achieve effective separation of glycyrrhizic acid.
And (II) taking 5g of the initial extract of the Gegen Qinlian prepared in the step 1) in the example 1, loading the initial extract on a Sephadex LH-20 chromatographic column (300 g of Sephadex LH-20 filler with the diameter-height ratio of 1:50), eluting by methanol for 15 column volumes, collecting eluent, and recovering the solvent. This experimental method does not achieve effective separation of glycyrrhizic acid.
Experimental example 2
Weighing 0.1g of the traditional Chinese medicine extract prepared in the example 1, weighing, adding 20mL of 70 vol% methanol, ultrasonically extracting for 2 times, 30min each time, combining the supernatants, 12000r/min, centrifuging for 10min, taking 300 mu L of the supernatant, and drying by nitrogen. Redissolving with 300. mu.L of 70 vol% methanol, 13000r/min, centrifuging for 10min, and sucking 2. mu.L of supernatant to inject into LC-MS.
The chemical composition of the herbal extract prepared in example 1 was analyzed using UHPLC-Q-TOF-MS/MS. UHPLC-Q-TOF-MS/MS chromatographic conditions were as follows:
a chromatographic column: waters Acquity BEH C18 column(2.1×100mm，1.7μm)；
Column temperature: 35 ℃;
mobile phase a was 0.1% formic acid-water;
the mobile phase B is acetonitrile;
flow rate: 0.3 ml/min;
sample introduction volume: 2 mu l of the solution;
the elution gradient is shown in table 2 below:
TABLE 2
Time (min)
A％
B％
0
95
5
3
95
5
10
70
30
15
60
40
25
46
54
28
46
54
34
5
95
38
5
95
39
95
5
UHPLC-Q-TOF-MS/MS mass spectrum conditions were as follows:
ESI, positive and negative ion scan mode, scan range m/z 100-1500, see Table 3.
TABLE 3
Ion mode
ESI(+)
ESI(-)
GS1
55
55
GS2
55
55
CUR
35
35
TEM
550
550
ISVF
5500
-4500
DP(MS)
60
-60
CE(MS)
5
5
DP(MS/MS)
50
50
CE(MS/MS)
30
-30
CES
5
5
Combining the results of MS accurate molecular weight, MS/MS secondary cleavage rule and open databases of Pubchem and the like, 16 compounds are identified. The results of the identification and the assignment of chromatographic peaks are shown in tables 4 and 5 below.
TABLE 4 identification of anion mode and chromatographic peak assignment
TABLE 5 Positive ion mode identification and chromatographic peak assignment
As can be seen from the table, glycyrrhizic acid was not detected in the herbal extract prepared in example 1.
Experimental example 3
The in vivo efficacy function of the radix puerariae radix scutellariae and rhizoma coptidis primary extract and the traditional Chinese medicine extract without glycyrrhizic acid is verified through a mouse model of high-fat diet induced metabolic syndrome, and the efficacy is evaluated.
C57BL/6J Male mice were 4 weeks old.
Metformin: Sigma-Aldrich;
physiological saline: henan Kolun pharmaceutical Co., Ltd;
common feed: beijing, Australian cooperative fodder Co., Ltd;
high-fat feed: one mouse and two biotech ltd, yozhou;
PCR kit: beijing Quanjin Biotechnology Ltd;
c57BL/6J male mouse, 4 weeks old (weight 16 ~ 18 g). Mice had free water diet, 12 hours light/dark cycle, temperature controlled at 21 + -2 deg.C, humidity 50-70%. One week after adaptive feeding, the animals were randomly divided into 5 groups of 6 animals each, and divided into a blank control group, a model group, a metformin group (positive drug control group), a complete group (initial extract of Gegen Qinlian of example 1), and a glycyrrhizic acid-removed group (extract of Chinese medicinal herbs of example 1). The placebo group was on a normal diet and the other 4 groups were on a high fat diet (HFD, 60% energy from fat).
The blank control group is fed with normal diet, each time is infused with 0.2ml of physiological saline, 5 days per week, and 14 weeks;
the model group was given a high fat diet, each with 0.2 ml/time saline, 5 days per week for 14 weeks;
the metformin group is given high-fat diet, metformin is given, the drug dose is 200mg/kg, and the intragastric dose is 0.2 ml/time, 5 days per week, and 14 weeks;
the whole formula group is given high fat diet, the Gegen Qinlian primary extract is used for intragastric administration, the intragastric administration is carried out according to 0.2ml per mouse, the single dose is 2.01g/kg, the administration is carried out once in the morning and at night, and the total dose is 4.027g/kg/d for 14 weeks;
removing glycyrrhizic acid, administering high fat diet, intragastrically administering the Chinese medicinal extract without glycyrrhizic acid, and intragastrically administering to each mouse by 0.2ml, with single dose of 2.01g/kg, once in the morning and once in the evening, and total dose of 4.027g/kg/d for 14 weeks;
animal body weights were measured weekly. At the end of the experiment, mice were sacrificed and liver tissue samples were collected for pathological analysis in example 4.
The increase in body weight of each group of animals over the course of the experiment is shown in Table 6. The results show that both the whole group and the glycyrrhizic acid-removed group can inhibit the abnormal weight increase caused by high fat diet, have obvious function of controlling weight gain, and have better effect than the whole group.
TABLE 6 results of body weight gain of animals in each group in pharmacological experiments
Packet/time point
Blank control group
Glycyrrhizic acid removing group
Metformin hydrochloride
Complete set
Model set
D0
0
0
0
0
0
D7
1.57±0.48
1.77±0.24
1.88±0.73
1.70±0.38
1.98±0.65
D14
2.65±0.92
2.60±0.27
3.00±0.81
3.27±0.79
3.72±1.24
D21
3.73±0.85
3.58±0.12
3.72±0.89
4.13±1.11
4.93±1.65
D28
3.60±1.04
4.13±0.65
4.78±0.95
4.75±1.33
6.15±1.89
D35
4.28±1.24
5.97±0.67
6.60±1.34
6.77±1.84
6.62±1.87
D42
4.68±1.46
6.87±1.09
7.95±1.59
7.72±1.46
9.27±2.31
D49
6.01±1.37
8.87±1.31
9.00±1.91
8.70±1.72
11.20±2.59
D56
5.73±1.43
9.68±1.86
9.05±2.38
10.18±2.24
12.83±2.93
D63
6.58±1.39
10.00±2.06
10.20±2.44
11.07±2.30
13.87±3.12
D70
7.05±1.47
12.05±2.43
12.27±3.31
11.85±4.51
16.98±3.58
D77
7.20±1.49
13.40±2.96
13.72±3.59
14.83±2.61
19.03±3.82
D84
7.22±1.19
14.68±3.24
14.73±4.64
15.83±3.32
20.75±3.77
D91
7.90±1.24
14.90±3.65
15.17±5.28
17.15±3.53
21.95±3.93
D98
7.77±1.34
14.67±3.59
14.48±5.63
16.25±3.49
21.23±3.88
Note: d represents days and weight gain in grams.
Experimental example 4
And observing the influence of the radix puerariae qinlian primary extract and the traditional Chinese medicine extract without glycyrrhizic acid on the liver of a mouse with the metabolic syndrome induced by high-fat diet.
The liver tissues of the mice collected in the pharmacological test of the above Experimental example 3 were used.
Paraformaldehyde: shanghai Michelin Biochemical technology, Inc. (MACKLIN);
anhydrous ethanol: guangdong Guanghua science and technology Co., Ltd. (JHD);
dewaxing agent: pearl oyster Soy Biotechnology Ltd;
embedding paraffin: shanghai Hualing rehabilitation apparatus factories;
hematoxylin: shenzhen, Peng, medical device, Inc.;
eosin: shenzhen, Peng, medical device, Inc.;
neutral gum: shanghai, China model plant;
tissue dehydrator: tyvit science and technology, TC-120C;
an embedding machine: tai Wei science and technology, TB-718D;
slicing machine: leica, HistoCore BIOCUT;
and (3) constant-temperature spreading and baking machine: tai Wei science and technology, TK-218 II;
an optical microscope: ZEISS, Primo Star;
the tissue is fixed by 4% paraformaldehyde for more than 24 hours, dehydrated by a conventional method and embedded by paraffin. After embedding, the sections were sliced with a microtome to a thickness of 3 μm. Sections were pretreated according to the sequence of table 7 below, then HE stained according to the sequence of table 8 below, air dried and mounted with neutral gum, and observed under a microscope after the gum had set.
TABLE 7
TABLE 8
Reagent
Time
Hematoxylin
8min
Washing with running water
3 times of
0.5% hydrochloric acid alcohol (differentiation)
5s
Washing with running water
3 times of
Tap water (blue)
10min
95% ethanol
1min
Eosin
10min
100% ethanol I
25s
100% ethanol II
25s
100% ethanol III
25s
Dewaxing agent III
2min
As can be seen from the liver tissue section, the mice in the blank control group with normal diet have normal liver shape, the liver cells in the normal control group have large volume, compact arrangement and acidophilic property, liver blood sinuses are arranged among liver cell cables, and endothelial cells and liver macrophages can be seen on the blood sinus wall. The liver of the mouse in the high-fat feeding model group is swollen with liver cells, lipid droplets with different sizes, roundness and tension appear in the cells, are mainly positioned in the peripheral area of liver lobules, and have inflammatory cell infiltration. The number of the lipid droplets in the model group is the largest, the size of the lipid droplets is 0.4-1.0 mu m, and the volume of the lipid droplets is larger.
The size of the metformin lipid drops is 0.4-0.7 mu m, and the quantity of the metformin lipid drops is large.
The fat droplets in the whole group are reduced, but the fat droplets still exist obviously, the size of the fat droplets is 0.3-0.6 mu m, and the volume of the fat droplets is larger.
The glycyrrhizic acid-removed group (the traditional Chinese medicine extract group without glycyrrhizic acid) has obviously reduced lipid droplets with the size of 0.3-0.5 μm, obviously reduced quantity and slightly reduced volume.
The result shows that the radix puerariae and radix scutellariae extract has obvious improvement and treatment effects on the fatty liver caused by high-fat diet, and the traditional Chinese medicine extract without glycyrrhizic acid has no influence on the treatment effect of the fatty liver, and is even better.
The literature reports that some sensitive patients (such as primary hypertension patients or mineralocorticoid sensitive individuals) can generate pseudo-aldosteronism by applying a small dose of glycyrrhizic acid drugs to cause hypertension (Zhangfang, Shenyaqin, research on the mineralocorticoid action of glycyrrhizic acid and glycyrrhetinic acid aglycone is advanced, modern drugs and clinics, 2011, 26 (6): 448-452.). Obviously, the traditional Chinese medicine extract without glycyrrhizic acid can basically eliminate potential safety hazards caused by long-term administration of glycyrrhizic acid on the basis of not reducing the treatment effect of metabolic syndrome, and has better safety and pharmacy.
The present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.