CN115212241A - High-purity phenylpeptide rhizoma ligustici wallichii extract, preparation method thereof and application thereof in anti-hepatic fibrosis - Google Patents
High-purity phenylpeptide rhizoma ligustici wallichii extract, preparation method thereof and application thereof in anti-hepatic fibrosis Download PDFInfo
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to a high-purity phenylpeptide ligusticum wallichii extract, a preparation method thereof and application thereof in anti-hepatic fibrosis. The invention discovers a phthalide extract from ligusticum wallichii by research, compared with active extracts separated by other ligusticum wallichii traditional extraction processes, the phthalide extract can play a more remarkable role in resisting oxidation damage, relieving excessive collagen deposition, reducing the levels of serum glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, total cholic acid, alkaline phosphatase, total bilirubin, liver malondialdehyde and hydroxyproline, increasing superoxide dismutase and the like, thereby playing a more excellent anti-hepatic fibrosis role, further analyzing the active ingredients thereof, and laying a foundation for the practical application of ligusticum wallichii in the prevention and treatment of hepatic fibrosis, particularly cholestasis type hepatic fibrosis.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a high-purity benzene peptide type ligusticum wallichii extract, a preparation method thereof and application thereof in anti-hepatic fibrosis.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Liver fibrosis (Liver fibrosis) is a Liver injury caused by unbalanced stress degradation and diffuse excessive deposition of extracellular matrix, and is a common pathological change of various Liver diseases such as primary sclerosing cholecystitis, primary biliary cirrhosis, liver cancer and the like. The lack of occult and non-invasive diagnostic methods of early stage liver fibrosis increases the difficulty of liver fibrosis diagnosis and treatment. The activation of hepatic stellate cells is a central link for the formation of hepatic fibrosis, and in the early activation stage of the hepatic stellate cells, the permeability of endothelial cells of hepatic sinusoids is changed and interstitial fibers are proliferated, so that hepatic cells are ischemic, damage-related mode molecules are released, the induction of immune response is aggravated, and a large number of inflammatory mediators are released. Accumulation of inflammatory factors in the liver continues to activate hepatic stellate cells, further accelerating the progression of hepatic fibrosis.
At present, most of the hepatic fibrosis animal models are mice, which mainly include cholestasis type hepatic fibrosis, irritant-induced hepatic fibrosis, metabolic hepatic fibrosis, alcoholic hepatic fibrosis and immune hepatic fibrosis. Common bile duct ligation is a representative method for copying a biliary obstruction cholestasis hepatic fibrosis model, after bile duct ligation, liver tissues of a mouse can be seen in an obvious convergent area and liver cells around bile ducts are swollen and broken, and vacuole degeneration and fibrosis around portal veins and around sinuses are obvious; with the continuous and deep research of the gene-controlled mice, when the multidrug resistance gene 2 (MDR 2) is deleted, the insufficient transport of phosphatidylcholine to bile can be caused, non-suppurative cholangitis accompanied by portal phlebitis is induced, bile duct hyperplasia is caused, and the lumen is narrow and blocked; in addition, carbon tetrachloride or thioacetamide is a common tool medicine for inducing a fibrosis model, the model is mature, the success rate is high, and hepatic fibrosis characterization can be realized from 2 weeks. However, compared with the above molding methods such as chemical drug intervention, the liver injury induced by the bile duct ligation method is closer to the etiology and pathological manifestations of clinical hepatic fibrosis patients.
The traditional Chinese medicine is 'the ancient science of China, the key to open the Chinese civilization treasury'. A large number of clinical observations and experimental studies prove that the traditional Chinese medicine has exact curative effect and obvious advantages when treating various hepatic fibrosis. In recent years, research on the treatment of hepatic fibrosis by using the effective components of traditional Chinese medicines has made great progress. Therefore, screening more effective therapeutic drugs for hepatic fibrosis, especially for cholestatic hepatic fibrosis, based on traditional Chinese medicine raw materials becomes a current research hotspot.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a high-purity benzene peptide ligusticum wallichii extract, a preparation method thereof and application thereof in anti-hepatic fibrosis. The invention discovers a phthalide extract from ligusticum wallichii, compared with active extracts separated by other ligusticum wallichii traditional extraction processes, the phthalide extract can play a more remarkable role in resisting oxidation damage, relieving excessive collagen deposition, reducing the levels of serum alanine Aminotransferase (ALT), glutamic oxaloacetic transaminase (AST), total cholic acid (TBA), alkaline phosphatase (ALP), total bilirubin (T-BIL), liver Malondialdehyde (MDA) and Hydroxyproline (HYP), and increasing superoxide dismutase (SOD), so as to play a more excellent anti-hepatic fibrosis role, further analyze active ingredients of the extract, and lay a foundation for the actual application of ligusticum wallichii in particularly prevention and treatment of cholestasis type hepatic fibrosis. The present invention has been completed based on the above results.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides a high-purity phenylpeptide chuanxiong rhizome extract, which is obtained by leaching chuanxiong rhizome with ethanol; preferably, the ethanol leaching process can be performed by percolation.
The invention analyzes and identifies the active components of the medicine, and the main active component of the medicine is a benzene peptide compound (characteristic component: senkyunolide A).
The second aspect of the present invention provides a preparation method of the above phenylpeptide type chuanxiong rhizome extract, wherein the preparation method of the phenylpeptide type chuanxiong rhizome extract comprises the steps of taking chuanxiong rhizome as a raw material, taking ethanol as a solvent, and obtaining the extract by a percolation method.
Specifically, the preparation method comprises the following steps:
s1, smashing ligusticum wallichii to obtain micro powder, and soaking the micro powder in ethanol so as to ensure that the micro powder of the ligusticum wallichii is fully soaked;
s2, adding the ligusticum wallichii micro powder soaked in the step S1 into a percolation cylinder, and then adding ethanol from the upper part of the percolation cylinder to control the percolation liquid to flow out;
and S3, carrying out rotary evaporation on the percolate obtained in the step S2 to completely volatilize ethanol, and then carrying out freeze-drying treatment to obtain the product.
The third aspect of the invention provides the application of the above extract of the benzene peptide type ligusticum wallichii in the preparation of drugs for preventing and/or treating hepatic fibrosis.
Wherein the hepatic fibrosis is cholestatic hepatic fibrosis.
Specifically, the prevention and/or treatment of hepatic fibrosis is at least shown in any one or more of the following purposes:
(a) Resistance to oxidative damage;
(b) Relieving excessive collagen deposition;
(c) Reducing serum glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, total cholic acid, alkaline phosphatase, total bilirubin, liver malondialdehyde and hydroxyproline levels, and increasing superoxide dismutase;
(d) Preventing hepatic fibrosis.
In a fourth aspect of the present invention, a pharmaceutical composition for preventing and/or treating liver fibrosis is provided, wherein the pharmaceutical composition comprises the above extract of the phenylpeptide ligusticum wallichii and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.
Wherein the hepatic fibrosis is cholestatic hepatic fibrosis.
In a fifth aspect of the present invention, there is provided a method for preventing and/or treating hepatic fibrosis, the method comprising: administering the above-mentioned phenylpeptide rhizoma Ligustici Chuanxiong extract or pharmaceutical composition to a subject.
The beneficial effects of one or more of the above technical solutions are as follows:
the technical scheme provides the application of the phthalide traditional Chinese medicine extract in preparing the anti-hepatic fibrosis medicine. The phthalide Chinese medicinal extract is separated and purified by percolation method and identified by ultra-high performance liquid chromatography. Pharmacodynamic experiments show that the phthalide traditional Chinese medicine extract has the effects of obviously reducing serum glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase and liver malondialdehyde, recovering the activity of superoxide dismutase and improving cholestatic hepatic fibrosis. Compared with the component and extract of the hemlock parsley separated by the traditional extraction process, the phthalide traditional Chinese medicine extract shows obvious advantage effect of anti-fibrosis.
The phthalide traditional Chinese medicine extract prepared by the technical scheme can be used for further preparing anti-hepatic fibrosis medicines, and compared with the common anti-cholestasis hepatic fibrosis means in clinic at present, the extract has the advantages of wide sources of Umbelliferae plants, low price and obvious drug effect, and has extremely high clinical treatment and new drug conversion significance.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a UPLC diagram of a Ligusticum chuanxiong phthalide extract in an embodiment of the present invention; note: the UPLC of total phthalide of rhizoma Ligustici Chuanxiong containing standard cnidium lactone A has a No. 4 peak which is characteristic peak of phthalide of rhizoma Ligustici Chuanxiong: senkyunolide A.
FIG. 2 is a schematic diagram of bile duct ligation-type liver fibrosis injury modeling of a mouse in an embodiment of the invention; note: sham was Sham and BDL was bile duct ligation.
FIG. 3 is a diagram showing the pathological structural changes of the liver of a mouse according to an embodiment of the present invention; (A) H&E, staining pattern; (B) Masson staining pattern; note: sham is a Sham operation group, BDL is a bile duct ligation operation group, BDL + CX PHL BDL + CX as the treatment group of chuanxiong rhizome phthalides extract PA BDL + CX as the treatment group of the phenolic acid extract of chuanxiong rhizome AE Is a treatment group of rhizoma ligustici wallichii water extract.
FIG. 4 is a diagram illustrating the detection of an index of liver fibrosis damage in mouse serum according to an embodiment of the present invention; note: sham is a Sham operation group, BDL is a bile duct ligation operation group, BDL + CX PHL BDL + CX as the treatment group of chuanxiong rhizome phthalides extract PA BDL + CX as the treatment group of the phenolic acid extract of chuanxiong rhizome AE Is a treatment group of rhizoma ligustici wallichii water extract.
FIG. 5 is a diagram illustrating the detection of an index of liver fibrosis damage in a mouse liver according to an embodiment of the present invention; note: sham is a Sham operation group, BDL is a bile duct ligation operation group, BDL + CX PHL BDL + CX as the treatment group of chuanxiong rhizome phthalides extract PA BDL + CX as the treatment group of the phenolic acid extract of chuanxiong rhizome AE Is a treatment group of rhizoma ligustici wallichii water extract.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Unless otherwise specified, the experimental procedures for specific conditions in the following detailed description are generally in accordance with conventional procedures and conditions within the skill of the art, as such techniques and conditions are fully explained in the literature.
At present, 250ml of pentoxifylline injection is intravenous drip, colchicine and broad-spectrum antifibrosis drugs of pirfenidone and flufenidone which are accepted treatment methods aiming at cholestasis type hepatic fibrosis. However, the dosage of the pentoxifylline injection is not more than 0.3g, otherwise adverse reactions such as nausea, dizziness, headache, anorexia, abdominal distension, vomiting and the like are easily caused, and the pirfenidone and the flufenidone which are broad-spectrum antifibrosis drugs lack liver targeting property and have common treatment effect and side effects such as gastrointestinal reaction and the like. Therefore, the search for effective anti-cholestatic liver fibrosis drugs in natural products is urgent. The traditional Chinese medicine is a treasure in ancient science in China, and a large number of clinical observations and experimental studies prove that the traditional Chinese medicine has definite curative effect and obvious advantages in the treatment of hepatic fibrosis. In recent years, research on the treatment of hepatic fibrosis by using the effective components of the traditional Chinese medicine has made great progress and development. In view of this, the invention focuses on natural products, extracts and separates phthalide traditional Chinese medicine extracts, phenolic acid traditional Chinese medicine extracts and ligusticum wallichii traditional Chinese medicine water extracts, and compares the therapeutic action of the phthalide traditional Chinese medicine extracts, the phenolic acid traditional Chinese medicine extracts and the ligusticum wallichii traditional Chinese medicine water extracts on hepatic fibrosis through experimental means, thereby confirming the anti-fibrosis dominant effect of the phthalide traditional Chinese medicine extracts.
Specifically, in a typical embodiment of the present invention, a high-purity phenylpeptide chuanxiong rhizome extract is provided, wherein the extract is obtained by leaching chuanxiong rhizome with ethanol; preferably, the ethanol leaching process can be performed by percolation.
The ethanol is high concentration ethanol, specifically 80-95% ethanol, and in one embodiment of the invention, the ethanol is 95% ethanol; the high-concentration ethanol is selected, so that the benzene peptide compounds in the ligusticum wallichii can be fully leached.
The invention analyzes and identifies the active components of the medicine, and the main active component of the medicine is a benzene peptide compound (characteristic component: senkyunolide A).
The invention proves that the phenylpeptide ligusticum wallichii extract has obvious antioxidant damage, relieves excessive collagen deposition, reduces the levels of serum glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, total cholic acid, alkaline phosphatase, total bilirubin, liver malondialdehyde and hydroxyproline, and increases superoxide dismutase and the like, thereby playing more excellent effects of resisting hepatic fibrosis, particularly resisting cholestasis type hepatic fibrosis.
In another embodiment of the present invention, a preparation method of the above-mentioned phenylpeptide chuanxiong rhizome extract is provided, wherein the preparation method comprises using chuanxiong rhizome as a raw material, using ethanol as a solvent, and obtaining the extract by percolation.
Specifically, the preparation method comprises the following steps:
s1, smashing ligusticum wallichii to obtain micro powder, and soaking the micro powder in ethanol so as to ensure that the micro powder of the ligusticum wallichii is fully soaked;
s2, adding the ligusticum wallichii micro powder soaked in the step S1 into a percolation cylinder, and then adding ethanol from the upper part of the percolation cylinder to control the percolation liquid to flow out;
and S3, carrying out rotary evaporation on the percolate obtained in the step S2 to completely volatilize ethanol, and then carrying out freeze-drying treatment to obtain the product.
Wherein, in each step, the ethanol is high-concentration ethanol, specifically 80-95% ethanol, and in one embodiment of the invention, the ethanol is 95% ethanol; the high-concentration ethanol is selected, so that the full leaching of the phenyl peptide compounds in the ligusticum wallichii is facilitated.
In the step S1, all the micro powder of the ligusticum wallichii can pass through a No. 5 sieve (the aperture is 180 mu m); 95% pass through No. 6 sieve (pore size 150 μm);
in the step S2, the percolation cylinder is fully paved with absorbent cotton balls pre-wetted by 80-95% (preferably 95%) ethanol, and the thickness of the cotton ball filter layer is 1.5cm or more;
the mass ratio of the ligusticum wallichii micro powder to ethanol (comprising the premixed ethanol in the step S1 and the ethanol added in the step S2) is 1-20, preferably 1;
the percolation flow rate is controlled to be 0.5-2 drops/s, preferably 1 drop/s.
In the step S3, the rotary evaporation is performed by using a vacuum rotary evaporator, so that the solution ethanol is completely volatilized.
The lyophilization process specifically comprises: freezing the extract after evaporating ethanol at low temperature, and performing vacuum freeze-drying treatment;
wherein the low-temperature freezing is freezing for more than 12 hours at-80 ℃;
the vacuum freeze-drying treatment is carried out by adopting a vacuum freeze-dryer, the temperature of a cold trap of the vacuum freeze-dryer is-50 ℃, the temperature of a sample pool is-28 ℃, the pressure is 200pa, and whether the water-containing part is completely freeze-dried is observed after freeze-drying for 48 hours.
The invention further analyzes, identifies and researches the main active ingredients of the ligusticum wallichii extract, which mainly comprises phenyl peptide compounds (characteristic ingredient: senkyunolide A); and a method for identifying and analyzing the active ingredients is constructed, and comprises identifying and analyzing the active ingredients by using a UPLC method.
In another embodiment of the present invention, the identification analysis method comprises:
mixing the prepared rhizoma Ligustici Chuanxiong extract, adding methanol for dissolving and diluting, filtering to obtain a sample solution, and detecting and analyzing senkyunolide A in the sample solution based on UPLC.
Wherein the liquid chromatography conditions comprise: the chromatographic column is C18 column, preferably ACQUITY-UPLC-C18 column with specification of 150mm × 2.1mm i.d,5 μ M; gradient elution is adopted, and mobile phases are 0.1% formic acid water solution (phase A) and acetonitrile (phase B);
the specific procedure of gradient elution is: 0-0.2 min (5% A), 0.2-7 min (5% A-80% A), 7-8.0 min (80% A-100% A), 8.5-9.0 min (100% A-5% A), 9.0-10.0 min (5% A).
The flow rate is 0.1-1mL/min, preferably 0.4mL/min, the detection wavelength is 280nm, the sample amount is 1-5 μ L, preferably 2 μ L, and the column temperature is 30-40 ℃, preferably 35 ℃.
In another embodiment of the present invention, the application of the above-mentioned extract of cnidium officinale kimura et migo peptides in preparing drugs for preventing and/or treating hepatic fibrosis is provided.
According to the present invention, the concept of "prevention and/or treatment" means any measure suitable for the treatment of liver fibrosis related diseases, or the prophylactic treatment of such manifested diseases or manifested symptoms, or the avoidance of recurrence of such diseases, e.g. recurrence after the end of a treatment period or treatment of symptoms of already established diseases, or the pre-interventional prevention or inhibition or reduction of the occurrence of such diseases or symptoms.
Specifically, the prevention and/or treatment of hepatic fibrosis is at least shown in any one or more of the following purposes:
(a) Resistance to oxidative damage;
(b) Relieving excessive collagen deposition;
(c) Reducing serum glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, total cholic acid, alkaline phosphatase, total bilirubin, liver malondialdehyde and hydroxyproline levels, and increasing superoxide dismutase;
(d) Preventing hepatic fibrosis.
In another embodiment of the present invention, there is provided a pharmaceutical composition for preventing and/or treating liver fibrosis, the pharmaceutical composition comprises the above-mentioned extract of cnidium officinale having phenylpeptide type, and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.
In another embodiment of the present invention, the pharmaceutically inactive ingredient may be a pharmaceutically commonly used carrier, excipient, diluent, or the like. Further, the composition can be prepared into oral preparations such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and spray, external preparations, suppositories, and sterile injectable solutions according to a conventional method.
In yet another embodiment of the present invention, the pharmaceutically inactive ingredients such as carriers, excipients and diluents which may be included are well known in the art and can be determined by one of ordinary skill in the art to meet clinical standards.
In still another embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like.
In still another embodiment of the present invention, the medicament of the present invention may be administered into the body by a known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.
In still another embodiment of the present invention, the subject to which the medicament is administered may be a human or non-human mammal, such as a mouse, rat, guinea pig, rabbit, dog, monkey, orangutan, or the like.
In another embodiment of the present invention, there is provided a method for preventing and/or treating hepatic fibrosis, the method comprising: administering the above-mentioned phenylpeptide rhizoma Ligustici Chuanxiong extract or pharmaceutical composition to a subject.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Examples
1. Extracting different components of rhizoma Ligustici Chuanxiong
1.1 Chinese medicinal materials
The traditional Chinese medicine ligusticum wallichii adopted in the invention is provided by Beijing Hojingtang drugstore.
1.2 Experimental instruments
Milling machine (SS-1022, wailana electric appliances Co., ltd., wuyi), electronic balance (JY 5002, shunhui constant-level scientific instruments Co., ltd., shanghai), vacuum rotary evaporator (RE-52 AA, shanghai Yangrong Biochemical instruments factory), open type low-temperature cold water circulator (ZX-LSJ-10D, shanghai Zhixin), vacuum freeze-drying machine (FD-2, beijing Bo Yi kang laboratory instruments Co., ltd.), normal-temperature percolator, traditional Chinese medicine standard medicine sieve, etc.
1.3 Process for preparing phthalides extract of Chinese herbs
1) Weighing appropriate amount of rhizoma Ligustici Chuanxiong, and pulverizing with a pulverizer.
2) Sieving the medicinal powder with standard Chinese medicinal sieve to obtain rhizoma Ligustici Chuanxiong micropowder (all passing through No. 5 sieve, 95% passing through No. 6 sieve).
3) Weighing appropriate amount of rhizoma Ligustici Chuanxiong micropowder, and soaking in 95% ethanol for more than 1 hr to ensure that the powder is sufficiently soaked by solvent.
4) The bottom of the percolation cylinder is completely filled with absorbent cotton balls which are pre-wetted by 95% ethanol, and the thickness of the cotton ball filter layer is more than 1.5 cm.
5) Adding the rhizoma Ligustici Chuanxiong powder premixed with 95% ethanol into the percolating cylinder, and lightly knocking the percolating cylinder to keep the surface of the powder smooth.
6) Slowly adding 95% ethanol along the wall of the percolate cylinder, keeping the surface of the medicinal powder flat, premixing with the medicinal powder, and adding 95% ethanol 15 times of the medicinal powder.
7) Adjusting the sealing degree of the percolation cylinder, reducing the volatilization of the extraction solvent and simultaneously communicating with the atmosphere to ensure the normal operation of percolation.
8) Regulating the outlet flow of the percolation barrel, and keeping the percolation speed at about 1 drop/s.
9) If the overnight percolation is needed, the percolate which flows out needs to be stored at 4 ℃ in a sealing way, and meanwhile, the room temperature is ensured to be between 20 ℃ and 30 ℃ so as to ensure the stability of the extraction effect.
10 ) rotary evaporating the total extractive solution at room temperature with vacuum rotary evaporator until the ethanol solution is completely volatilized, and smelling without alcohol smell.
11 Freezing the above extractive solution at-80 deg.C for 12 hr or more, transferring to vacuum freeze dryer (cold trap temperature-50 deg.C, sample cell temperature-28 deg.C, pressure 200 pa), freeze drying for 48 hr, and observing whether the water-containing part is completely freeze dried.
12 ) completely freeze-drying to obtain a brown oily suspension which is a total phthalide freeze-dried solution, and calculating the yield.
13 ) if the suspension needs to be diluted, the suspension needs to be fully mixed, and then a proper amount of suspension needs to be sucked out by a weight reduction method and dissolved to the specified concentration by using methanol.
1.4 preparation process of phenolic acid extract of Chinese herbs
1) Weighing appropriate amount of rhizoma Ligustici Chuanxiong, and pulverizing with a pulverizer.
2) Sieving the medicinal powder with standard Chinese medicinal sieve to obtain rhizoma Ligustici Chuanxiong micropowder (all passing through No. 5 sieve, 95% passing through No. 6 sieve).
3) 13 volumes of 65% ethanol, sonicated 2 times for 55 minutes each.
4) After filtration, the filtrate was evaporated to dryness and the residue was collected.
5) Dissolving the residue with ammonia water, filtering, and adjusting pH to 2-3 with concentrated hydrochloric acid.
6) Extracting with ethyl acetate for 3 times, collecting ethyl acetate extractive solution, and concentrating.
7) And (3) performing rotary evaporation on the ethyl acetate extracting solution until the ethyl acetate is completely volatilized, adding water, performing ultrasonic redistribution, transferring and freeze-drying.
1.5 Process for preparing aqueous extract of Chinese medicinal materials
1) Weighing appropriate amount of rhizoma Ligustici Chuanxiong, and pulverizing with a pulverizer.
2) Sieving the medicinal powder with standard sieve to obtain rhizoma Ligustici Chuanxiong micropowder (all passing through No. 5 sieve, 95% passing through No. 6 sieve).
3) 10 times of water is added, and the reflux extraction is carried out for 2 times, and each time is 1.5 hours.
4) Mixing extractive solutions, and concentrating by rotary evaporation at 50-60 deg.C under reduced pressure.
5) And (6) freeze-drying.
2. Identification of main active components in ligusticum wallichii extract
2.1 Experimental instruments
Ultra high performance liquid chromatograph (LC-40A, shimadzu, japan), column: ACQUITY-UPLC-C18 with specification of 150mm x 2.1mm i.d,5 μ M, analytical electronic balance, ultrasonic cleaning instrument, etc.
2.2 drugs and standards
Senkyunolide A (B21461) was obtained from Shanghai leaf Biotech Co., ltd, acetonitrile (chromatographic grade, purity > 99.9%), and anhydrous formic acid (chromatographic grade, purity > 99.9%) were obtained from Beijing Zhengcheng Biotech Co., ltd, wahaha purified water, cellulose acetate filter (0.22. Mu.M), and Chinese medicinal materials were obtained from Beijing Hojingtang drugstore.
2.3 preparation scheme
1) Chromatographic separation conditions
The mobile phase is A phase: 0.1% aqueous formic acid, phase B: and (3) acetonitrile. The gradient elution procedure was: 0-0.2 min (5% A), 0.2-7 min (5% A-80% A), 7-8.0 min (80% A-100% A), 8.5-9.0 min (100% A-5% A), 9.0-10.0 min (5% A); the flow rate is 0.4 ml/min; the column temperature was 35 ℃. In order to better show the conditions of the compounds contained in all the standard substance and the total phthalide test solution, the detection wavelength is selected to be 280nm.
2) Test solution preparation and determination scheme
Weighing a proper amount of the traditional Chinese medicine ligusticum wallichii, and crushing to obtain the ligusticum wallichii micro powder which can completely pass through a No. 5 sieve. Soaking rhizoma Ligustici Chuanxiong powder with 95% ethanol for 1 hr, and percolating with 15 times of 95% ethanol to obtain rhizoma Ligustici Chuanxiong total phthalide extractive solution. The total phthalide extract of rhizoma Ligustici Chuanxiong is subjected to rotary evaporation at room temperature and low pressure until no alcohol smell is produced. Freeze-drying the alcohol-free extractive solution in vacuum freeze dryer (cold trap temperature-50 deg.C, sample pool temperature-28 deg.C, and pressure 200 pa) for 24 hr to obtain rhizoma Ligustici Chuanxiong total phthalide freeze-dried suspension.
And (3) fully and uniformly mixing the total phthalide suspension by using a vortex instrument, weighing a proper amount of suspension, dissolving the suspension by using methanol, and transferring the suspension to a 5ml constant volume bottle for constant volume. Then diluted to 10mg/ml (lyophilized suspension mass) using methanol gradient according to the post-volume concentration, and stored at 4 ℃ for use within one week. Diluting rhizoma Ligustici Chuanxiong total phthalide solution with methanol to 2mg/ml, and filtering with filter membrane to obtain liquid phase vial.
3) Control solution preparation and assay protocol
Accurately weighing senkyunolide A about 1mg by using an analytical balance, adding anhydrous methanol to fully dissolve, and fixing the volume. Taking the solution with constant volume, and diluting the solution to 0.2mg/ml by using methanol for later use. The control solution was filtered into a liquid phase vial using a 0.22 μ M cellulose acetate filter before loading.
4) Sample introduction and measurement
The samples were injected according to the above liquid chromatography conditions, each sample requiring 3 times of injection, each injection volume being 2. Mu.l. Measuring the retention time, peak area and peak height of senkyunolide A, and calculating the content of the sample peak in the sample solution according to the above information of the standard solution.
3. Bile duct ligation hepatic fibrosis operation model construction
1) The mice of 6-10 weeks are anesthetized, double ligation is carried out on the free common bile duct after the abdomen is opened, and the free common bile duct is separated between two ligatures to ensure that the common bile duct is completely blocked, and then the surgical thread is sutured to close the abdominal cavity.
2) After the common bile duct is ligated, the mouse has decreased food intake, scattered and lusterless fur, reduced action, reduced response to external stimuli (such as sound and light), deepened urine color, dark red with yellow color, and changed like thick tea. The liver begins to change obviously, the liver is enlarged obviously, and glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase rise to more than 10 times of the normal liver level.
3) After HE staining and sirius red staining, the hepatic tissues of common bile duct ligation mice were obviously changed: the liver cells in the area of the liver sink and around the bile duct swell, rupture, vacuolar degeneration, inflammatory cell infiltration is obvious, and fibrosis around the portal vein and around the sinuses is obvious.
The choledocholithiasis method is used for copying a mouse hepatic fibrosis model, and has the advantages of simple operation, high molding rate, obvious liver fibrosis, stable index and no self-reversal of fibrosis in a short time.
4. Determination of anti-fibrosis activity of rhizoma Ligustici Chuanxiong phthalides components
4.1 Ligusticum chuanxiong phtaloids component can significantly alleviate collagen deposition
The same position of the liver lobe of each group of mice is taken, the section processing is carried out, H & E staining and Masson staining are carried out, and the pathological change of the liver and the collagen deposition condition are recorded. H & E staining results show that liver tissues of the Sham group are complete, the hepatocytes are arranged in a cord shape around the central vein, and no obvious deformation, necrosis and the like are caused. Compared with the Sham group, the BDL group has poor liver tissue integrity, bile duct hyperplasia, inflammatory cell infiltration and cytoplasm vacuolation, and obvious collagen accumulation; in the phthalide treatment group, the structural integrity of the liver is better, and the liver sinus region has no obvious expansion and congestion phenomena; in the phenolic acid treatment group, the structural integrity of the liver is reduced, and obvious liver cell group damage, bile duct hyperplasia and collagen accumulation can be seen in liver tissues; in the treatment group of the ligusticum wallichii aqueous extract, the liver structure is relatively complete, a small amount of inflammatory cell infiltration phenomenon can be seen in interstitial substances, and partial collagen accumulation can still be seen outside cells. Compared with the treatment groups of the ligusticum wallichii phenolic acids and the water extract, the ligusticum wallichii phenyl peptides substance has better effects of resisting hepatic fibrosis and relieving liver injury.
4.2 Ligusticum chuanxiong phtaloids component can significantly reduce ALT, AST, ALP, TBA and T-BIL level in serum
Collecting mouse serum of sham operation group, bile duct ligation model group and rhizoma Ligustici Chuanxiong different extract intervention group, and detecting ALT, AST, ALP, TBA and T-BIL level in serum. The detection result shows that the levels of ALT, AST, ALP, TBA and T-BIL in the blood serum of the mice in the bile duct ligation group are obviously increased, and the intervention of the ligusticum wallichii phthalein type component can effectively reduce the levels of ALT, AST, ALP, TBA and T-BIL in the blood serum of the mice, and the result prompts that the ligusticum wallichii phthalein type component has a more obvious liver protection effect compared with the ligusticum wallichii phenolic acid type component and a ligusticum wallichii water extract.
4.3 rhizoma Ligustici Chuanxiong phthalides can relieve oxidative stress injury of liver
After the different components of the ligusticum wallichii are subjected to dry prognosis, collecting mouse liver tissues and detecting the levels of MDA, SOD, HYP and TBA in the liver tissues. The detection result shows that the MDA level in the liver tissue of the mouse with the bile duct ligation group is obviously increased, the SOD level is reduced, the unbalance of the redox system in the liver of the mouse with the model group is prompted, and compared with the phenolic acid component of the ligusticum wallichii and the water extract of the ligusticum wallichii, the benzene phthalein component of the ligusticum wallichii can obviously restore the redox balance system in the liver. Meanwhile, HYP is an oxidative stress product and an important component forming extracellular matrix in hepatic fibrosis and cirrhosis, can reflect oxidative damage of liver and hepatic fibrosis degree, and the results show that the ligusticum wallichii phthalide extract has more remarkable liver protection effect compared with ligusticum wallichii phenolic acid components and ligusticum wallichii water extract.
Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the present invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive efforts by those skilled in the art based on the technical solution of the present invention.
Claims (10)
1. A high-purity phenylpeptide rhizoma Ligustici Chuanxiong extract is prepared by leaching rhizoma Ligustici Chuanxiong with ethanol; preferably, the ethanol leaching process is performed by percolation.
2. The method for preparing the phenylpeptide chuanxiong rhizome extract as claimed in claim 1, wherein the specific preparation method of the phenylpeptide chuanxiong rhizome extract comprises using chuanxiong rhizome as a raw material, using ethanol as a solvent, and obtaining the extract by percolation.
3. The method of claim 2, comprising:
s1, smashing ligusticum wallichii to obtain micro powder, and soaking the micro powder in ethanol so as to ensure that the micro powder of the ligusticum wallichii is fully soaked;
s2, adding the ligusticum wallichii micro powder soaked in the step S1 into a percolation cylinder, and then adding ethanol from the upper part of the percolation cylinder to control the percolation liquid to flow out;
and S3, carrying out rotary evaporation on the percolate obtained in the step S2 to completely volatilize ethanol, and then carrying out freeze-drying treatment to obtain the product.
4. The method according to claim 3, wherein the ethanol in each step is high-concentration ethanol, specifically 80-95% ethanol, preferably 95% ethanol.
5. The method of claim 3, wherein in step S1, the entire fine powder of Chuan Xiong passes through No. 5 sieve, and 95% passes through No. 6 sieve.
6. The method according to claim 3, wherein in step S2, the percolation cylinder is completely filled with absorbent cotton balls pre-wetted with 80-95% (preferably 95%) ethanol, and the thickness of the cotton ball filter layer is 1.5cm or more;
the mass ratio of the ligusticum wallichii micro powder to ethanol (comprising the premixed ethanol in the step S1 and the ethanol added in the step S2) is 1-20, preferably 1;
the percolation flow rate is controlled to be 0.5-2 drops/s, preferably 1 drop/s.
7. The method according to claim 3, wherein in the step S3, the rotary evaporation is performed by a vacuum rotary evaporator;
the freeze-drying treatment specifically comprises: freezing the extract after evaporating ethanol at low temperature, and performing vacuum freeze-drying treatment;
preferably, the preparation method further comprises identifying and analyzing the prepared ligusticum wallichii extract, including identifying and analyzing characteristic component senkyunolide A in the ligusticum wallichii extract by using a UPLC method;
preferably, the identification analysis method comprises:
mixing the prepared rhizoma Ligustici Chuanxiong extract, adding methanol for dissolving and diluting, filtering to obtain a sample solution, and detecting and analyzing characteristic component senkyunolide A of benzene peptides in the sample solution based on UPLC;
wherein the liquid chromatography conditions comprise: the chromatographic column is C18 column, preferably ACQUITY-UPLC-C18 column, specification is 150mm × 2.1mm i.d,5 μ M; gradient elution is adopted, and mobile phases are 0.1% formic acid water solution (phase A) and acetonitrile (phase B);
the specific procedure of gradient elution is: 0-0.2 min (5% A), 0.2-7 min (5% A-80% A), 7-8.0 min (80% A-100% A), 8.5-9.0 min (100% A-5% A), 9.0-10.0 min (5% A);
the flow rate is 0.1-1mL/min, preferably 0.4mL/min, the detection wavelength is 280nm, the sample amount is 1-5 μ L, preferably 2 μ L, and the column temperature is 30-40 deg.C, preferably 35 deg.C.
8. Use of the extract of the Ligusticum wallichii with the phenylpeptide according to claim 1 or the extract of the Ligusticum wallichii with the phenylpeptide prepared by the preparation method according to any one of claims 2-7 in preparing a medicament for preventing and/or treating hepatic fibrosis.
9. Use according to claim 8, wherein the liver fibrosis is in particular cholestatic liver fibrosis;
preferably, the prevention and/or treatment of hepatic fibrosis is at least manifested by any one or more of the following:
(a) Resistance to oxidative damage;
(b) Relieving excessive collagen deposition;
(c) Reducing serum glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, total cholic acid, alkaline phosphatase, total bilirubin, liver malondialdehyde and hydroxyproline, and increasing superoxide dismutase;
(d) Preventing hepatic fibrosis.
10. A pharmaceutical composition for preventing and/or treating hepatic fibrosis, which comprises the extract of the cnidium officinale Makino of claim 1 and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient;
preferably, the liver fibrosis is cholestatic liver fibrosis.
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| CN116509840A (en) * | 2023-04-27 | 2023-08-01 | 四川省中医药科学院 | Application of senkyunolide I in preparation of products for preventing and treating organ fibrosis |
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| CN101978980A (en) * | 2010-11-04 | 2011-02-23 | 深圳市泰康制药有限公司 | Szechuan lovage rhizome total phthalide extract |
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| CN1580049A (en) * | 2003-07-31 | 2005-02-16 | 深圳市泰康制药有限公司 | Method for extracting total phenyl phthalein of ligusitcum wallichii |
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| CN116509840A (en) * | 2023-04-27 | 2023-08-01 | 四川省中医药科学院 | Application of senkyunolide I in preparation of products for preventing and treating organ fibrosis |
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