CN111419912A - Red peony root polyphenol pharmaceutical preparation for treating acute hepatitis and preparation method thereof - Google Patents
Red peony root polyphenol pharmaceutical preparation for treating acute hepatitis and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a pharmaceutical preparation for treating acute hepatitis, which contains red paeony root total phenols, the red paeony root total phenols can reduce the expression of inflammatory factors in rat plasma, and red paeony root and total phenol components thereof can reduce the inflammatory reaction and the injury of the acute hepatitis by reducing the release of the inflammatory factors; the total phenols of red peony root of the present invention can be used for acute hepatitis.
Description
Technical Field
The invention relates to a medicament and a preparation method thereof, in particular to a red paeony root polyphenol medicinal preparation for treating acute hepatitis and a preparation method thereof.
Background
In modern clinical application, radix paeoniae rubrathe is often combined with radix paeoniae alba, radix rehmanniae, cortex moutan, radix et rhizoma rhei, semen aurantii immaturus, rhizoma cyperi, radix salviae miltiorrhizae, rhizoma ligustici wallichii and other medicines to form a medicine pair in a compound form, so as to play the effects of clearing heat and cooling blood, removing stasis and relieving pain and clearing liver fire, and is clinically used for treating cardiovascular and cerebrovascular diseases, liver and gallbladder diseases and inflammatory reaction diseases. Radix Paeoniae Rubra mainly contains monoterpene glycosides, phenols, polysaccharides, etc. Monoterpene compounds refer to terpenes and derivatives thereof containing two molecular isoprene units in the molecule, and studies on the basis of effective substances of radix Paeoniae Rubra drug properties by scholars at present mainly focus on radix Paeoniae Rubra monoterpene glycosides represented by higher content of paeoniflorin; the phenolic compounds refer to compounds in which hydrogen atoms on benzene rings in aromatic hydrocarbons are replaced by hydroxyl groups, and the research on other components such as red paeony phenols is less, so that the research on the basis of medicinal substances of the phenolic compounds is not comprehensive, and the action mechanism is not clear. Macroporous adsorption resin can adsorb monoterpene glycosides and phenols of radix Paeoniae Rubra, and polyamide resin only adsorbs phenols, and the research uses the difference of adsorption performance of two main chemical components of radix Paeoniae Rubra on the two adsorption resins, and adopts chromatography to perform component separation.
Disclosure of Invention
The invention aims to provide a red paeony root polyphenol pharmaceutical preparation for treating acute hepatitis and a preparation method thereof, and the invention aims to be realized by the following technical scheme:
a pharmaceutical preparation for treating acute hepatitis contains total phenols of radix Paeoniae Rubra.
The pharmaceutical preparation of the invention is prepared by the following method:
the preparation method of radix Paeoniae Rubra total phenols comprises decocting radix Paeoniae Rubra decoction pieces, extracting to obtain water extractive solution, passing through polyamide resin column with amount of 0.5-20 times of radix Paeoniae Rubra, washing with water with amount of 2-10 times of polyamide, collecting water washing solution (containing radix Paeoniae Rubra monoterpene glycosides and polysaccharides), eluting with 30-95% ethanol with amount of 1-20 times of polyamide, collecting ethanol eluate, and concentrating under reduced pressure until each m L is 2g crude drug to obtain radix Paeoniae Rubra total polyphenols extract;
the pharmaceutical preparation of the invention is preferably prepared by the following method:
the preparation method of total phenols of radix Paeoniae Rubra comprises decocting radix Paeoniae Rubra decoction pieces, extracting to obtain water extractive solution, passing through polyamide resin column at amount of 5, 10 or 15 times of radix Paeoniae Rubra by weight, washing with water at amount of 4 or 8 times of polyamide, collecting water washing solution, eluting with 40%, 60% or 95% ethanol at amount of 1, 10 or 15 times of polyamide, collecting ethanol eluate, and concentrating under reduced pressure until each m L is 2g crude drug to obtain total polyphenols extract of radix Paeoniae Rubra;
the medicine is prepared into pharmaceutically acceptable dosage forms by a conventional method, including oral dosage forms and injection dosage forms; the oral dosage form is capsule, oral liquid preparation or pill.
The invention also discloses application of the medicinal preparation in preparing a medicament for treating acute hepatitis.
Drawings
FIG. 1 shows chromatograms of HP L C sample containing radix Paeoniae Rubra water extract, radix Paeoniae Rubra total phenols, and radix Paeoniae Rubra total monoterpenes;
a: red peony root aqueous extract; b: red peony total phenols; c: red peony root total monoterpene; 1: gallic acid; 2: oxidizing paeoniflorin; 3: a catechin; 4: albiflorin; 5: 4, gallic acid ethyl ester; 6: paeoniflorin; 7: paeoniflorin benzoate.
Detailed Description
The following experimental examples and examples are intended to further illustrate but not limit the invention.
Experimental example 1:
1 Material
1.1 animals
Male SD rats 5, weighing 200 ± 20g, purchased from sbefu (beijing) laboratory animal science and technology limited, were housed in the SPF-grade animal house, beijing university of traditional Chinese medicine. During the experiment, rats freely drink and eat water, the room temperature of an animal house is 20-22 ℃, the relative humidity is 60% -70%, and the day and night alternation of 12h light illumination and darkness simulation is realized.
1.2 medicine and preparation
Bacterial lipopolysaccharide (L PS, available from Beijing Bainovei Biotech, Inc.) was prepared into 80 mg/L with physiological saline, and carrageenan (Ca, available from Beijing Bainovei Biotech, Inc.) was prepared into 10 g/L with physiological saline, all stored at 4 deg.C for use.
The radix Paeoniae Rubra water extract is prepared by soaking radix Paeoniae Rubra decoction pieces in water, decocting, filtering the extractive solution, mixing, concentrating under reduced pressure to obtain radix Paeoniae Rubra water extract containing about 2g crude drug per 1m L, and refrigerating at 4 deg.C.
The preparation method of radix Paeoniae Rubra total phenols comprises decocting radix Paeoniae Rubra decoction pieces with the above method to obtain water extractive solution, passing through polyamide resin column, washing polyamide column with water, collecting water lotion, eluting with ethanol, collecting ethanol eluate, concentrating under reduced pressure until the concentration of each 1m L is equal to 2g of crude drug, to obtain radix Paeoniae Rubra total polyphenols extract, and refrigerating at 4 deg.C.
The preparation method of radix Paeoniae Rubra total monoterpene comprises collecting water eluate of polyamide resin column, passing through D101 type macroporous resin column, washing macroporous resin with water, discarding water eluate, eluting macroporous resin with ethanol, collecting ethanol eluate, reducing pressure per 1m L to obtain radix Paeoniae Rubra total monoterpene extract, and refrigerating at 4 deg.C.
1.3 kits and instruments
The glutamic-pyruvic transaminase (A L T) kit (batch No. 20191110), the glutamic-oxalacetic transaminase (AST) kit (batch No. 20191110), the interleukin-1 β (I L-1 β) kit (batch No. 20191012), the interleukin-6 (I L-6) kit (batch No. 20191012), the tumor necrosis factor- α (TNF- α) kit (batch No. 20191012) and the interferon-gamma (IFN-gamma) kit (batch No. 20191012) are purchased from Beijing Huaying English biotechnological research institute.
Semi-automatic hemagglutination analyzer (model: PUN-208A), semi-automatic biochemical analyzer (manufacturer: Beijing Song technology Co., Ltd., model: A6), enzyme-labeled analyzer (Wuxi Waerde Lang Instrument Co., Ltd., model: Waerde Lang DR-200BS), polyamide resin and D101 macroporous resin (Shanghai Huazhen science and technology Co., Ltd., 30-60 mesh), Agilent 1100 type high performance liquid chromatograph (Agilent)
2 method
2.1 chromatographic conditions
The chromatographic conditions for distinguishing 7 main components of gallic acid, oxypaeoniflorin, catechin, albiflorin, ethyl gallate, paeoniflorin and benzoylpaeoniflorin in red paeony root are established by taking the chromatographic resolution of 7 main components of the gallic acid, the oxypaeoniflorin, the catechin, the paeoniflorin, the benzoylpaeoniflorin and the paeoniflorin in the red paeony root as indexes, wherein the chromatographic column is a DIKMA C18 column (4.6mm × 150 mm, 5 mu m), the mobile phase is acetonitrile (A) -0.05% glacial acetic acid aqueous solution (B), and the gradient elution conditions are 0 → 6min, 97% (B), 6 → 7min, 97% → 91% (B), 7 → 45min, 91% → 50min, 91% → 70 min, 50 → 69min, 70% (B), the flow rate is 1m L/min, the column temperature is 25 ℃, the detection wavelength is 235nm, and the sample introduction amount is 10 mu L.
2.2 grouping and administration
After the animals are fed for 3 days adaptively, the rats are randomly divided into 5 groups, namely a blank group, a model group, a red peony root water extract group (20g/kg), a red peony root total monoterpene group (20g/kg) and a red peony root total phenol group (20g/kg), 10 animals in each group are subjected to preventive administration every morning according to the weight of 10m L/kg, the administration is continuously carried out for 7 days, the gavage amount is properly adjusted according to the weight, and the normal group and the model group are subjected to gavage deionized water every day.
2.3 Molding
At 8 am 8d, except the blank group, L PS was intraperitoneally injected at 2.5mg/kg, and the same amount of physiological saline was injected into the blank group to prepare an acute hepatitis animal model.
2.4 obtaining materials and detecting indexes
6h after molding, rats are anesthetized with 10% chloral hydrate, blood is taken from the abdominal aorta, and the rats are placed in an anti-coagulation tube different from EDTA for storage.
The plasma A L T, AST, I L-1 β, I L-6, TNF- α and IFN-gamma are measured by an E L ISA method according to kit operation instructions.
2.5 statistical methods
Mean ± standard deviation for experimental data
Showing that SPSS Statistics 20.0 is adopted for statistical analysis, data is subjected to normal distribution and homogeneity of variance test, the data is in normal distribution, data among groups is compared and analyzed by single-factor variance analysis (One-way ANOVA), L SD is adopted for statistical analysis when two-by-two variances are equal, Dunnett's T3 is adopted for statistical analysis of variance, and each statistical result is P<A difference of 0.05 is statistically significant.
3 results
3.1 fingerprint of the drug
According to the chromatographic conditions under the item 2.1, taking red paeony root water extract, red paeony root total monoterpene and red paeony root total phenol samples, carrying out HP L C analysis, and establishing an HP L C chromatogram, wherein the results show that the component difference in the red paeony root total monoterpene and red paeony root total phenol samples is obvious, and the components of the prepared two extracts are not crossed with each other, as shown in figure 1.
3.2 comparison of the levels of A L T, AST in the plasma of rats in the groups
Compared with the blank group, the plasma A L T and AST contents of the rats in the model group are obviously increased (P <0.01, P <0.001), compared with the model group, the contents of A L T and AST in the Qingying decoction group, the red paeony root water extract group and the red paeony root total phenol group are all reduced, and the difference is obvious (P <0.05, P <0.01, P < 0.001).
TABLE 1 comparison of A L T, AST levels in plasma of rats in various groups
n＝10)
Note: comparison with blank group##P<0.01，###P<0.001; comparison with model group*P<0.05，**P<0.01，***P<0.01。
3.3 comparison of plasma PGE2 in rats in various groups
The plasma PGE2 content of the model group rats is increased compared with that of the blank group (P < 0.001); compared with the model group, the content of PGE2 in the red peony root water extract group, the red peony root total phenol group and the red peony root monoterpene group is reduced (P is less than 0.01, P is less than 0.001, and P is less than 0.001). See Table 2
Table 2 comparison of plasma PGE2 of rats in each group
n＝10)
Note: comparison with blank group###P<0.001; comparison with model group**P<0.01，***P<0.01。
3.4 comparison of inflammation indicators in plasma of rats in various groups
Compared with a blank group, the levels of the plasma TNF- α, IFN-gamma, I L-1 β and I L0-6 of rats in the model group are all increased (P <0.001), compared with the model group, the levels of the red paeony root water extract group, the red paeony root total phenolic group TNF-L1, IFN-gamma, I L-1 β and I L-6 are all reduced (P <0.05, P <0.01 and P <0.001), the levels of the red paeony root total monoterpene group IFN-gamma, I L-1 β and I L-6 are all reduced (P <0.01 and P <0.001), and the levels of the TNF- α are not obviously changed, which is shown in a table 3.
TABLE 3 comparison of inflammation indices in plasma of rats in various groups
n＝10)
Note: comparison with blank group###P<0.01; comparison with model group*P<0.05，**P<0.01，***P<0.001。
Experimental results show that the contents of PGE2, TNF- α, I L- β, I L-6 and IFN-gamma in the plasma of a rat after being injected with L PS in an abdominal cavity are all obviously increased, the expression of inflammatory factors in the plasma of the rat after being administered with red paeony root aqueous extract, red paeony root total monoterpene and red paeony root total phenol in a preventive manner is reduced, and the red paeony root and the total monoterpene and total phenol components thereof can reduce the inflammatory response and the injury of acute hepatitis by reducing the release of the inflammatory factors.
Experimental example 2:
1.1-1.2 same as Experimental example 1
1.3 kits and instruments
Prostaglandin E2(PGE2) kit (batch No.: 20181110), cyclic adenosine monophosphate (cAMP) reagent kit (batch No.: 20181110), interleukin-1 β (I L-1 β) kit (batch No.: 20181012), interleukin-6 (I L-6) kit (batch No.: 20181012), tumor necrosis factor- α (TNF- α) kit (batch No.: 20181012), interferon-gamma (IFN-gamma) kit (batch No.: 20181012), vascular endothelin (ET-1) kit (batch No.: 20181216), Nitric Oxide (NO) kit (batch No.: 20181216), thromboxane B2(TXB2) kit (batch No.: 20181216), 6-keto prostaglandin F1 α (6-keto-PGF1 α) kit (batch No.: 20181216), all of which are purchased from the institute of Biotechnology of Wako, North, Japan.
A semi-automatic hemagglutination analyzer (model: pulan-208A), a semi-automatic biochemical analyzer (manufacturer: beijing sonong technologies ltd., model: a6), an enzyme-labeled analyzer (tin-free wareward instruments ltd., model: wareward DR-200BS), a polyamide resin and D101 macroporous resin (shanghai warewandh technologies ltd., 30-60 mesh), an Agilent 1100 high performance liquid chromatograph (Agilent, usa).
2 method
2.1 chromatographic conditions
The chromatographic conditions for distinguishing 7 main components of gallic acid, oxypaeoniflorin, catechin, albiflorin, ethyl gallate, paeoniflorin and benzoylpaeoniflorin in red paeony root are established by taking the chromatographic resolution of 7 main components of the gallic acid, the oxypaeoniflorin, the catechin, the paeoniflorin, the benzoylpaeoniflorin and the paeoniflorin in the red paeony root as indexes, wherein the chromatographic column is a DIKMA C18 column (4.6mm × 150 mm, 5 mu m), the mobile phase is acetonitrile (A) -0.05% glacial acetic acid aqueous solution (B), and the gradient elution conditions are 0 → 6min, 97% (B), 6 → 7min, 97% → 91% (B), 7 → 45min, 91% → 50min, 91% → 70 min, 50 → 69min, 70% (B), the flow rate is 1m L/min, the column temperature is 25 ℃, the detection wavelength is 235nm, and the sample introduction amount is 10 mu L.
2.2 grouping and administration
After the animals are fed adaptively for 3 days, the rats are randomly divided into 6 groups, namely a blank group, a model group, a clear soup group (20g/kg), a red paeony root water extract group (20g/kg), a red paeony root total monoterpene group (20g/kg) and a red paeony root total phenol group (20g/kg), wherein 12 animals in each group are subjected to preventive administration every morning according to the weight of 10m L/kg, the gavage amount is adjusted properly according to the weight for 8 days continuously, and the normal group and the model group are subjected to the gavage deionized water every day.
2.3 Molding
At 8 am on 7d, rats were injected with carrageenan at a dose of 25mg/kg in addition to the blank group. After 24h, except for the blank group, the animal model of the toxic heat and blood stasis syndrome is prepared by injecting endotoxin into the tail vein of the rat according to the dose of 80 mug/kg. The blank group was injected with an equal amount of physiological saline and the time after completion of endotoxin injection was 0 h.
2.4 obtaining materials and detecting indexes
1h after molding, rats are anesthetized with 10% chloral hydrate, blood is taken from the abdominal aorta, and the rats are respectively stored in anticoagulation tubes containing sodium citrate and EDTA.
Serum PGE2, cAMP, I L-1 β, I L-6, TNF- α, IFN-. gamma.ET-1, NO, TXB2, 6-keto-PGF1 α were measured by the E L ISA method according to the kit protocol and Thrombin Time (TT), Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT) and Fibrinogen (FIB) values were measured by the chromogenic substrate method.
2.5 statistical methods
Mean ± standard deviation for experimental data
Showing that SPSS Statistics 20.0 is adopted for statistical analysis, data is subjected to normal distribution and homogeneity of variance test, the data is in normal distribution, data among groups is compared and analyzed by single-factor variance analysis (One-way ANOVA), L SD is adopted for statistical analysis when two-by-two variances are equal, Dunnett's T3 is adopted for statistical analysis of variance, and each statistical result is P<A difference of 0.05 is statistically significant.
3.2 PGE in plasma of various groups of rats2Comparison of cAMP levels
The levels of PGE2 and cAMP were significantly elevated in the model group rats compared to the blank group (P < 0.01); compared with the model group, the clear nutrient soup group, the red paeony root water extract group and the red paeony root total phenol group reduce the content of PGE2 and cAMP (P <0.01), the red paeony root total monoterpene group reduces the content of cAMP (P <0.01), and the red paeony root total phenol reduces PGE2 obviously (P <0.01) compared with the monoterpene group. See table 4.
TABLE 4 comparison of PGE2, cAMP levels in the plasma of rats in each group
n＝12)
Note: comparison with blank group##P<0.01; comparison with model group**P<0.01; compared with the radix Paeoniae Rubra Total monoterpene group△△P<0.01。
3.3 comparison of inflammation indicators in the plasma of rats in various groups
Compared with the blank group, the serum TNF- α, IFN-gamma, I L-1 β and I L0-6 of the rats in the model group are all increased (P <0.01), compared with the model group, the contents of the red paeony root water extract group, the red paeony root total phenol group TNF-L1, IFN-gamma, I L-1 β and I L-6 are all reduced (P <0.05 and P <0.01), the contents of the red paeony root total monoterpene group IFN-gamma, I L-1 β and I L-6 are all reduced (P <0.05 and P <0.01), and the content of the TNF- α is not obviously changed, which is shown in the table 5.
Table 5 comparison of the inflammation indicators in the plasma of rats in each group (ng/L;
n＝12)
note: comparison with blank group##P<0.01; comparison with model group*P<0.05，**P<0.01; compared with the radix Paeoniae Rubra Total monoterpene group△△P<0.01。
3.4 comparison of four clotting terms in groups of rats
Model rats were decreased in TT, PT, and APTT (P <0.01) and significantly increased in FIB content (P <0.05) compared to the blank group; compared with the model group, the red paeony root aqueous extract and the red paeony root total monoterpene group have prolonged TT, PT and APTT (P <0.01), obviously reduced FIB content of the red paeony root total monoterpene group (P <0.01), and no obvious change of TT, PT, FIB and APTT of the red paeony root total phenol group (P > 0.05). See table 6.
TABLE 6 comparison of four clotting terms in various groups of rats
n＝12)
Note: comparison with blank group#P<0.05##P<0.01; comparison with model group**P<0.01; compared with the radix Paeoniae Rubra Total monoterpene group△△P<0.01。
3.5 comparison of ET-1, NO/ET-1 in the plasma of rats in each group
Compared with a blank group, the NO content of the model group rats is reduced (P <0.01), the ET-1 content is increased (P <0.01), and the ratio of NO/ET-1 is obviously reduced (P < 0.001); compared with the model group, the NO content of the red peony root aqueous extract group, the red peony root total monoterpene group and the red peony root total phenol group is increased (P <0.05, P <0.01), the ET-1 content is reduced (P <0.01), and the ratio of NO/ET-1 is obviously increased (P <0.05, P < 0.01). See table 7.
TABLE 7 comparison of ET-1, NO, ET-1/NO in plasma of rats in each group
n＝12)
Note: comparison with blank group##P<0.01; comparison with model group*P<0.05**P<0.01; compared with the radix Paeoniae Rubra Total monoterpene group△P<0.05。
3.6 TXB in plasma of rats of each group2Comparison of 6-keto-PGF1 α, TXB2/6-keto-PGF1 α
Compared with the blank group, the rats in the model group have increased TXB2 content, the ratio of TXB2/6-keto-PGF1 α is increased (P <0.05, P <0.01), compared with the model group, the TXB2 content in the Qingying decoction group, the red paeony root water extract group, the red paeony root total monoterpene group and the red paeony root total phenol group is decreased (P <0.01), and the ratio of TXB2/6-keto-PGF1 α in the red paeony root water extract group, the red paeony root total monoterpene group and the red paeony root total phenol group is decreased (P <0.01) (see Table 8).
TABLE 8 comparison of TXB2, 6-keto-PGF1 α, TXB2/6-keto-PGF1 α in the plasma of rats in various groups
n＝12)
Note: comparison with blank group#P<0.05##P<0.01; comparison with model group**P<0.01; compared with the radix Paeoniae Rubra Total monoterpene group△P<0.05△△P<0.01。
The experimental result shows that the total paeonol and the total monoterpene of the red paeony root can maintain the dynamic balance of NO and ET-1 and prevent the formation of blood stasis by increasing the content of NO and reducing the content of ET-1, the total monoterpene of the red paeony root can also reduce the content of TXB2 and increase the content of NO and 6-keto-PGF1 α, and the balance of the total paeony root and the total monoterpene of the red paeony root is maintained to achieve the effect of removing blood stasis, and the effect of the total paeony root on ET-1, 6-keto-PGF1 α and TXB2/6-keto-PGF1 α is lower than that of the total monoterpene of the red paeony root by comparing the total paeony root and the total monoterpene of the red paeony root.
The invention improves the material basis of the action of the red paeony root by separating the total phenols of the red paeony root and the monoterpene of the red paeony root and researching the monoterpene of the red paeony root and the total phenols of the red paeony root on the aspect of treating acute hepatitis.
Lipopolysaccharide (L PS) is derived from an outer membrane of gram-negative bacteria, can stimulate macrophage cells in vivo to synthesize and release various inflammation mediators, and is the most common inflammation factor, so high-concentration bacterial endotoxin L PS is commonly used for inducing an acute hepatitis model, an organism is stimulated by an inflammation factor after being injected into an abdominal cavity with high concentration of L PS to activate neutrophil, eosinophil and mononuclear-phagocyte systems in vivo, so that release of various inflammation mediator prostaglandins (PGE2), tumor necrosis factor- α (TNF- α), interleukin-1L (I L1- β), interleukin-6 (I L-6), interferon-gamma (IFN-gamma) and the like is caused, permeability of a blood vessel is increased, inflammation cell chemotaxis is caused, so that generation of acute inflammation is induced [12, PGE2 is eicosyl unsaturated fatty acid, which is one of prostaglandin, which is used as an important inflammation mediator, blood vessel, blood flow and the like, when organism is stimulated by peripheral cells, the extracellular factors of peripheral cells, the Escherichia coli is stimulated by a macroreticular total paeonol, total paeonol synthesis and total paeonol synthesis of macrophage cell synthesis, total paeonol synthesis and total paeonol synthesis, the total paeonol release of the cholesterol, the total paeonol is not only a macroreticular cholesterol, but also can be used as a major inflammatory reaction of a macroreticular cholesterol, the total cholesterol of a macrogol, the total cholesterol of a macrogol in an endothelial cell, the total cholesterol of a macrogol of a total cholesterol of a macrogoldthread out, the total cholesterol of a total cholesterol of.
According to the traditional Chinese medicine, the syndrome of heat-toxin and blood stasis is that body fluid and blood are burned by warm diseases, blood is clotted and stagnated after being smoked, the primary factor is that warm heat-toxin enters nutrient-blood during the formation process, nutrient-yin depletion and collateral damage and body fluid damage are pathological, and the development trend is that heat-toxin is exuberant, heat-stasis is mixed and body resistance is weakened. Researches find that the toxic heat and blood stasis syndrome has close relationship with inflammation, fever and blood stasis of modern medicine in the aspects of pathology, pathogenesis and treatment, and a plurality of inflammatory diseases and complications thereof have the toxic heat and blood stasis syndrome manifestations clinically, such as rheumatic fever, viral hepatitis, allergic purpura, systemic lupus erythematosus and the like. The traditional Chinese medicine theory enables endotoxin to belong to heat evil and toxin evil, and the endotoxin is used for preparing the heat-toxin blood stasis syndrome animal model, so that the evolution process of diseases related to invasion of exogenous evil, heat entering nutrient blood, excessive heat being toxin and blood refining blood stasis in traditional Chinese medicine can be well simulated, the theory of formation of heat-toxin blood stasis syndrome in traditional Chinese medicine is met, and the change of objective biomedical indexes such as blood rheology, inflammatory factors and the like is provided for the heat-toxin blood stasis syndrome. Carrageenan is commonly used for causing animal inflammation models, and can be used in combination with endotoxin for preparing models with toxic heat and blood stasis syndrome.
After the body is injected with endotoxin and carrageenan, inflammatory cell factors such as I L-1 β, TNF- β, IFN-gamma, I L-6 and the like can be generated and released by activating a neutrophil, eosinophil and mononuclear phagocyte system in blood, on one hand, the cell factors can be used as endogenous pyrogens to transmit fever signals to a body temperature regulation center through various ways such as blood brain barrier specific transporter transport, endplate organelle and vagus nerve, on the other hand, the increase of the content of peripheral cell factors can induce the expression of prostaglandin synthase (mPGES-1) and cyclooxygenase (COX-2), further promote the synthesis of central fever positive regulation medium PGE2 and cAMP to increase the body temperature, and experimental results show that the PGE2, the cAMP, the I L-1 β, the TNF- α, the IFN-gamma and the I L-6 values are obviously reduced, the PGE L, the I L-6851, the I- α and the total phenols of the red peony root aqueous extract are reduced to further achieve the effect of reducing the release of the inflammatory cell factors and the total paeony phenol, but the total pyrogens the total phenols are better than the release of the fever signals and the inflammatory cell factors.
In addition, inflammatory cytokines such as I L-1 β, TNF- α and the like released by an organism can induce the expression of tissue factors and start an extrinsic coagulation path, vascular endothelial cells are damaged, and exposure of subendothelial collagen starts an intrinsic coagulation path, four coagulation items can reflect the anticoagulation and procoagulant capacity of the organism, and experimental results show that red paeony root water extract and red paeony root total monoterpene can obviously prolong TT, PT and APTT time and reduce FIB content, which indicates that the red paeony root water extract and the red paeony root total monoterpene can improve the blood stasis state of rats by activating corresponding coagulation factors, while red paeony root total phenols have NO obvious effect, NO, ET-1, TXB2 and 6-keto-PGF1 α are active substances closely related to vascular endothelial cell damage and platelet activation, ET-1 has a strong vasoconstriction effect, NO can dilate blood vessels and improve microcirculation, TXB2 can constrict blood vessels and promote platelet aggregation, and 6-keto-PGF1 α can dilate blood vessels and inhibit platelet aggregation, and the balanced disruption of the two can also cause the formation of thrombus.
The preparation of the total paeony glycoside mostly adopts a macroporous resin technology, and the total paeony glycoside prepared by the technology contains components with higher polarity of total phenols of the red paeony root according to the separation principle. The invention adopts a method of combining polyamide resin and macroporous resin, firstly, red paeony root total phenol is separated by a polyamide resin column, then, red paeony root total monoterpene is separated by a macroporous resin column, the red paeony root total phenol is separated from the red paeony root total monoterpene for the first time, and the prepared red paeony root total phenol and the prepared red paeony root total monoterpene are analyzed by fingerprint spectrums, and the chromatographic peaks of catechin and gallic acid in the red paeony root total phenol are found to be the main peaks; the paeoniflorin chromatographic peak is taken as the main component in the total monoterpene of red paeony root, which indicates that the chemical components of the total phenol of red paeony root and the total monoterpene of red paeony root are not crossed with each other. The red peony root total monoterpene and red peony root total phenol are used as important chemical components of red peony root, and animal experiments show that the two can play the roles of anti-inflammation and heat-clearing by reducing the generation and release of inflammatory factors and central fever positive regulation media, thereby achieving the effects of clearing heat and cooling blood; radix Paeoniae Rubra Total phenols and radix Paeoniae Rubra Total monoterpenesImproving the state of blood stasis by maintaining NO and ET-1 balance; however, radix Paeoniae Rubra total monoterpene can also maintain TXB by activating corresponding coagulation factors2Compared with the two, the effect of the total paeony phenol on inflammatory media such as central fever positive regulation media PGE2, I L-1 β, TNF- α and the like is superior to that of the total paeony monoterpene on the heat clearing and blood cooling effects, and the effect of the total paeony terpene on related indexes of blood vessel endothelial cell function and platelet activation such as four blood coagulation items TT, PT, APTT, FIB, ET-1, 6-keto-PGF1 α, TXB2/6-keto-PGF1 α and the like is superior to that of the total paeony phenol on the blood stasis dispelling effects.
Example 1:
the radix Paeoniae Rubra water extract is prepared by collecting radix Paeoniae Rubra decoction pieces 1.8kg, soaking in water for 1 hr, decocting and extracting for 2 times, each time for 1 hr, adding water 12 times and 10 times, filtering the extractive solution with filter cloth while it is hot, mixing, concentrating under reduced pressure to 900m L with crude drug content of 2g per 1m L to obtain radix Paeoniae Rubra water extract, and refrigerating at 4 deg.C for use.
The preparation method of total phenols of radix Paeoniae Rubra comprises decocting radix Paeoniae Rubra decoction pieces 1.8kg, extracting to obtain water extractive solution 36L, passing through polyamide resin column, washing with 15L water, collecting water extractive solution, eluting with 15L 95% ethanol, collecting ethanol eluate, concentrating under reduced pressure to 900m L (each 1m L is 2g crude drug), to obtain total polyphenols extract of radix Paeoniae Rubra, and refrigerating at 4 deg.C.
The preparation method of radix Paeoniae Rubra total monoterpene comprises collecting the above collected water solution of polyamide resin column, passing through D101 type macroporous resin column, washing macroporous resin with 2L water, collecting the effluent, eluting macroporous resin with 6L 95% ethanol, collecting ethanol eluate, concentrating under reduced pressure to 900m L, each 1m L is 2g crude drug, to obtain radix Paeoniae Rubra total monoterpene extract, refrigerating at 4 deg.C, and making into oral liquid by conventional method.
Example 2:
the radix Paeoniae Rubra water extract is prepared by collecting radix Paeoniae Rubra decoction pieces 1.8kg, soaking in water for 2 hr, decocting and extracting for 2 times, each time for 2 hr, respectively adding water 10 times and 8 times, filtering the extractive solution with filter cloth while it is hot, mixing, concentrating under reduced pressure to 900m L containing crude drug 2g per 1m L to obtain radix Paeoniae Rubra water extract, and refrigerating at 4 deg.C for use.
Preparing radix Paeoniae Rubra Total phenols by decocting radix Paeoniae Rubra decoction pieces with the above method to obtain water extractive solution 32L, passing through polyamide resin column, washing with 8L water, collecting water extractive solution, eluting with 10L 80% ethanol, collecting ethanol eluate, and concentrating under reduced pressure until each m L is 2g crude drug to obtain radix Paeoniae Rubra Total polyphenols extract;
the preparation method of radix Paeoniae Rubra total monoterpene comprises collecting polyamide resin column water eluate, passing through D101 type macroporous resin column, washing macroporous resin with 10L water, discarding water eluate, eluting macroporous resin with 580% ethanol, collecting ethanol eluate, concentrating under reduced pressure until each m L is equivalent to 2g crude drug content to obtain radix Paeoniae Rubra total monoterpene extract, and making into injection by conventional method.
Example 3:
the radix Paeoniae Rubra water extract is prepared by collecting radix Paeoniae Rubra decoction pieces 1.8kg, soaking in water for 2 hr, decocting and extracting for 2 times, each time for 0.5 hr, adding water 10 times and 10 times respectively, filtering the extractive solution with filter cloth while hot, mixing, concentrating under reduced pressure to 900m L containing crude drug 2g per 1m L to obtain radix Paeoniae Rubra water extract, and refrigerating at 4 deg.C for use.
The preparation method of total phenols of radix Paeoniae Rubra comprises collecting 1.8kg decoction pieces of radix Paeoniae Rubra, decocting and extracting to obtain water extractive solution 32L, passing through polyamide resin column, washing with 10L water, collecting water solution, eluting with 15L 95% ethanol, collecting ethanol eluate, concentrating under reduced pressure to 900m L, each 1m L is equivalent to 2g crude drug, to obtain total polyphenols extract of radix Paeoniae Rubra, and refrigerating at 4 deg.C for use.
Preparing radix Paeoniae Rubra total monoterpene by collecting the water solution from polyamide resin column, passing through D101 macroporous resin column, washing macroporous resin with 2L water, collecting the effluent, eluting macroporous resin with 6L 70% ethanol, collecting ethanol eluate, concentrating under reduced pressure to 900m L, wherein each 1m L is 2g of crude drug, and refrigerating at 4 deg.C to obtain radix Paeoniae Rubra total monoterpene extract, and making into capsule by conventional method.
Example 4:
the radix Paeoniae Rubra water extract is prepared by collecting radix Paeoniae Rubra decoction pieces 1.8kg, soaking in water for 0.5 hr, decocting and extracting for 3 times, 1 hr each time, with water amount of 8 times, 6 times and 6 times respectively, filtering the extractive solution with filter cloth while hot, mixing, concentrating under reduced pressure to 900m L, each 1m L contains about 2g crude drug to obtain radix Paeoniae Rubra water extract, and refrigerating at 4 deg.C for use.
The preparation method of total phenols of radix Paeoniae Rubra comprises collecting radix Paeoniae Rubra decoction pieces 1.8kg, decocting and extracting to obtain water extractive solution 32L, passing through polyamide resin column, washing with 8L water, discarding water solution, eluting with 8L 90% ethanol, collecting ethanol eluate, concentrating under reduced pressure to 900m L, each 1m L is equivalent to 2g crude drug, to obtain total polyphenols extract of radix Paeoniae Rubra, and refrigerating at 4 deg.C for use.
The preparation method of radix Paeoniae Rubra total monoterpene comprises collecting the above collected polyamide resin column effluent, passing through D101 type macroporous resin column, washing macroporous resin with 4L water, collecting effluent, eluting macroporous resin with 6L 70% ethanol, collecting ethanol eluate, concentrating under reduced pressure to 900m L, each 1m L is 2g crude drug, to obtain radix Paeoniae Rubra total monoterpene extract, refrigerating at 4 deg.C, and making into granule by conventional method.
Claims (8)
1. A pharmaceutical preparation for treating acute hepatitis contains total phenols of radix Paeoniae Rubra.
2. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation is prepared by a method comprising:
the preparation method of radix Paeoniae Rubra total phenols comprises decocting radix Paeoniae Rubra decoction pieces, extracting to obtain water extractive solution, passing through polyamide resin column at amount of 0.5-20 times of radix Paeoniae Rubra, washing with water at amount of 2-10 times of polyamide, discarding water eluate (containing radix Paeoniae Rubra total monoterpene and polysaccharide), eluting with 30-95% ethanol at amount of 1-20 times of polyamide, collecting ethanol eluate, and concentrating under reduced pressure until each m L is 2g crude drug to obtain radix Paeoniae Rubra total phenols extract.
3. The pharmaceutical formulation of claim 2, wherein the pharmaceutical formulation is prepared by a method comprising:
the preparation method of total phenols of radix Paeoniae Rubra comprises decocting radix Paeoniae Rubra decoction pieces, extracting to obtain water extractive solution, passing through polyamide resin column according to amount of 1, 10 or 15 times of radix Paeoniae Rubra, washing with water 4 or 8 times of polyamide, collecting water washing solution, eluting with 40%, 60% or 95% ethanol with amount of amide 5, 10 or 15 times of polyamide, collecting ethanol eluate, and concentrating under reduced pressure until each m L is 2g crude drug to obtain total polyphenols extract of radix Paeoniae Rubra.
4. The medicament of claim 1, 2 or 3, wherein the medicament is prepared into pharmaceutically acceptable dosage forms including oral dosage forms and injection dosage forms by conventional methods.
5. The medicament of claim 4, wherein the oral dosage form is a capsule, an oral liquid, or a pill.
6. Use of a pharmaceutical preparation according to any one of claims 1 to 5 for the preparation of a medicament for the treatment of acute hepatitis.
7. Use of a pharmaceutical formulation according to any one of claims 1 to 5 for the manufacture of a medicament for the treatment of the syndrome of toxic heat and blood stasis.
8. Use of a pharmaceutical formulation according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of inflammatory diseases and complications thereof.
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| CN1530112A (en) * | 2003-03-11 | 2004-09-22 | 北京金桥时代生物医药研究发展中心 | Medicine for preventing fibrous liver and preparing method thereof |
| CN1594343A (en) * | 2004-07-07 | 2005-03-16 | 姚仲青 | Method for removing tannin and benzoic acid from total glucoside of red peony |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1530112A (en) * | 2003-03-11 | 2004-09-22 | 北京金桥时代生物医药研究发展中心 | Medicine for preventing fibrous liver and preparing method thereof |
| CN1594343A (en) * | 2004-07-07 | 2005-03-16 | 姚仲青 | Method for removing tannin and benzoic acid from total glucoside of red peony |
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