CN100493596C - A kind of ginger extract, its preparation method and application - Google Patents

Abstract

The invention discloses a ginger extract and a preparation method thereof. Combining supercritical carbon dioxide fluid extraction and column chromatography methods, the ginger extract with a gingerol content greater than 50% is obtained. The invention also discloses the application of the ginger extract in the preparation of medicines for preventing and treating liver fibrosis. Experiments show that the ginger extract obtained by the present invention has a very significant effect on liver protection and anti-hepatic fibrosis, and can be used to further develop new liver protection and anti-hepatic fibrosis drugs; and, due to the obtained gingerol content Ginger extract greater than 50%, thus can be used for the preparation of drop pill dosage form.

Description

A kind of ginger extract, its preparation method and application

technical field

The invention relates to a traditional Chinese medicine extract and its new pharmaceutical use, in particular to a ginger extract, its preparation method and its application in the preparation of medicines for preventing and treating liver fibrosis.

Background technique

Liver fibrosis refers to the excessive deposition of fibrous connective tissue in the liver, which is the result of an imbalance between fibroproliferation and fibrolysis. Fibrous hyperplasia is a repair response of the body to damage. Repeated or persistent chronic liver parenchymal inflammation and necrosis caused by various causes can lead to continuous fibrous hyperplasia and liver fibrosis. From many chronic liver diseases, especially chronic viral From the perspective of the clinical and pathological evolution of hepatitis, liver fibrosis and liver cirrhosis are continuous developmental processes, and it is difficult to separate them completely.

Anti-fibrosis therapy aims to reduce the degree of fibrosis, delay its development, and even reverse its pathological process. In the prior art, the following methods are mainly used: one is to use interferon and the like to inhibit collagen synthesis; the other is to use drugs such as colchicine to inhibit tubulin polymerization, thereby interfering with the collagen secretion of cells. Treatment by promoting collagen degradation and gene therapy are still under study. A variety of traditional Chinese medicines have been reported to have the effect of protecting the liver, but there is no report on the use of ginger in the treatment of liver fibrosis.

Ginger is a traditional Chinese medicine. It has the functions of dispelling cold and relieving the exterior, warming the middle and relieving vomiting, resolving phlegm, relieving cough and detoxification. It can be used for diseases such as wind-cold cold, cough, stomach cold and vomiting. Modern phytochemical and pharmacological studies have found that the main active ingredient in ginger is gingerol, including gingerol, zingerone and shogaol, among which gingerol is the most important. The content directly affects the quality and efficacy of ginger preparations.

When used to prepare medicine, it is necessary to extract the active ingredients of ginger. The common extraction method is ethanol circulation method, but its extraction rate is low, and the content of gingerol obtained is low. Therefore, in the past two years, there have been reports on the use of supercritical carbon dioxide to extract ginger oil. In the ginger oil obtained by adopting this method, the content of gingerol is usually between 10% and 20%.

As a drug dosage form, drop pills can dissolve the drug at a constant rate, and the effect of controlled release drop pills can reach several days, so it is suitable for the preparation of drugs that need to be used continuously for a long time. However, the drug loading of the drop pills is small, and the weight of most drop pills is less than 100 mg, thus requiring further enrichment of active ingredients or effective parts of the medicine. The content of gingerol in the above-mentioned ginger oil obviously can not be suitable for making the demand of dripping pill.

Contents of the invention

The purpose of the present invention is to provide a new application of ginger extract and a method for preparing ginger extract suitable for the application.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A preparation method of ginger extract, comprising the following steps,

(1) Grinding the dried ginger to a particle size of 30-50 mesh as a raw material for preparation;

(2) Extraction by supercritical carbon dioxide fluid extraction method, extraction pressure 24-30MPa, temperature 42-45°C, reaction for 3-6 hours, _CO2 flow rate of 9-10kg/h.kg raw material;

The extracted fluid undergoes two stages of analysis respectively, the pressure of the first stage of analysis is 12-14MPa, the temperature is 56-60°C, the pressure of the second stage of analysis is 6-7MPa, and the temperature is 38-42°C;

The light yellow ginger oil obtained by the above extraction is separated by column chromatography, so that the content of gingerol is greater than 50%, and the required ginger extract is obtained.

The present invention also claims to protect the ginger extract prepared by the above method.

And the application of the obtained ginger extract in the preparation of medicines for preventing and treating liver fibrosis.

A more preferred solution is that the pharmaceutical dosage form is a dropping pill.

In the above technical scheme, the supercritical CO ₂ fluid (SFE-CO ₂ ) extraction process is as follows:

_CO2 steel cylinder→refrigeration system→high pressure pump→extraction kettle→desorption kettle I→desorption kettle II→circulation.

Ginger oil obtained through supercritical carbon dioxide extraction is light yellow oil, which has a strong natural aroma and spicy taste of fresh ginger. 59 components have been qualitatively and quantitatively identified by GC-MS, and the content of gingerol is 13.09%, of which , gingerol (gingerol) 1.78%, shogaol (shogaol) 2.36%, zingerone (zingerone) 3.52%, gingerene (Zangiberene) 4.43%, other more than 50 kinds of monoterpenes and sesquiterpenoids, the relative content is 46.83%.

The above-mentioned ginger oil is further extracted by column chromatography to obtain refined ginger oil, and the content of gingerol in the extract is determined to be greater than 50%, which is the ginger extract required by the present invention.

In order to better solve the stability of gingerol, the main medicinal ingredient, part of the refined ginger oil was clathrated with β-CD. The formulation design of the sustained-release dropping pill contains both the extract and the clathrate, and the ratio of the two is adjusted to obtain different drug release effects.

The preparation method of dropping pills is as follows: weigh a certain amount of auxiliary materials, heat and melt, cover and seal and keep warm at 60 ° C, drop into methyl silicone oil to condense, after shrinking into pellets, collect pellets, and absorb the coolant adhered to the surface , put into the desiccator to dry.

It is preferable to use water-soluble carrier PEG6000 to make a solid dispersion, so that the drug molecules are not easy to form aggregates, or the particles are not easy to form aggregates, which ensures the high dispersibility of the drug, accelerates the dissolution and absorption of the drug, and constitutes the composition of the sustained-release drop pill. Quick release part. It is preferred to use water-insoluble stearyl alcohol and inclusion of β-CD to retard drug release, and constitute the sustained-release part of the sustained-release drop pill. Ginger oil sustained-release dripping pills are made by this method, so that the drug can be released slowly in the body for 12 hours. Therefore, the basis for prescription screening is to release about 40% of the drug in 2 hours, and more than 80% of the drug in 10 hours. The concentration quickly reaches the therapeutic range, and can continue to maintain the drug concentration for a period of time.

Studies on the efficacy of the above preparations have shown that:

1. In the whole animal acute liver injury experiment, refined ginger oil has obvious hepatoprotective effect, and it can inhibit the increase of animal serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) caused by carbon tetrachloride and acetaminophen (Table 1, Table 2); The elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels are significantly reduced (Table 3), and refined ginger oil can also reduce hepatocyte malonyltransferase caused by carbon tetrachloride damage. Aldehyde (MDA) content was elevated and superoxide dismutase (SOD) activity was elevated (Table 4). Pathological examination showed that refined ginger oil could significantly improve the liver microstructure of animals with acute liver injury. Refined ginger oil has a significant protective effect on carbon tetrachloride and H ₂ O ₂ induced BRL hepatocyte injury (Table 5), and can make alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the cell culture supernatant levels were significantly lower (Table 5).

2. In the experimental liver fibrosis model, treatment with refined ginger oil can significantly improve the degree of liver fibrosis, reduce alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), and The increase of laminin (LN) (Table 6, Table 7), and can increase the content of serum total protein (TP) and albumin (Alb) (Table 8), significantly reduce the deposition of collagen fibers and pseudolobules in liver tissue Formation.

3. The possible mechanism of the hepatoprotective effect of refined ginger oil is mainly related to its anti-oxidation, including reducing malondialdehyde (MDA), increasing the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) ( Table 9, Table 10). Its anti-hepatic fibrosis mechanism, in addition to anti-oxidation, also includes reducing the activation of hepatic stellate cells (HSCs) by transforming growth factor-β1 (TGF-β1) in vivo (Table 11), reducing collagen Synthesis and reduction of tissue inhibitor of metalloproteinase-1 (TIMP-1) inhibition of matrix metalloproteinases (MMPs) increased collagen breakdown (Table 12).

It has been confirmed by animal experiments that the LD ₅₀ of refined ginger oil administered to mice is 20 ml/kg, and the 95% credible limit is 18.7-21.2 ml/kg. In the autopsy of the poisoned mice, no obvious pathological changes were found in the heart, liver, spleen, lung, kidney, brain and other major substantive organs.

Due to the use of the above-mentioned technical solutions, the present invention has the following advantages compared with the prior art:

1. the present invention proposes the application of ginger extract in the medicine of preparing anti-hepatic fibrosis, and experiment shows, its hepatoprotective and the effect of anti-hepatic fibrosis are very remarkable, can be used for further developing new hepatoprotective and anti-hepatic fibrosis Drugs for liver fibrosis.

2. Since ginger extract is a pure natural substance, it will not produce other side effects in the process of fighting liver fibrosis.

3. The present invention obtains a ginger extract with a gingerol content greater than 50% by controlling the analytical parameters and further refining by column chromatography, which can be used for the preparation of dripping pill dosage forms.

Detailed ways

The present invention will be further described below in conjunction with embodiment:

Embodiment one: the preparation of ginger extract

Supercritical CO ₂ fluid (SFE-CO ₂ ) extraction process is as follows:

_CO2 steel cylinder→refrigeration system→high pressure pump→extraction kettle→desorption kettle I→desorption kettle II→circulation.

The extraction parameters are: the pressure of the extraction kettle is 24mpa, the temperature is 42°C, the pressure of the analysis kettle I is 12.4mpa, and the temperature is 60°C, the pressure of the analysis kettle II is 6.5mpa, the temperature is 40°C, and the flow rate _of CO is 10kg/ h.kg of raw materials, the time is 4-6 hours.

Ginger, a traditional Chinese medicinal material, is crushed and sieved, with a particle size of 30-50 mesh. Weigh 10 kg and put it into the extraction kettle. After 4-6 hours of extraction, the material is discharged from the analysis kettle I and the analysis kettle II, and it is 180 g of light yellow ginger oil. It has a strong natural aroma and spicy taste of fresh ginger, and after standing, there is a small amount of yellow precipitation at the bottom.

59 components were qualitatively and quantitatively identified by GC-MS, and the content of gingerol was 13.09%, wherein, gingerol (gingerol) 1.78%, shogaol (shogaol) 2.36%, zingerone (zingerone) 3.52%, zingiberene (Zangiberene) 4.43%, other more than 50 kinds of monoterpenes and sesquiterpenes, the relative content is 46.83%.

The above-mentioned ginger oil is further extracted by column chromatography to obtain refined ginger oil, and the content of gingerol in the extract is determined to be greater than 50%, which is the obtained ginger extract.

Embodiment two: the preparation of the drop pill that is used for preventing and treating liver fibrosis

The ginger extract obtained in Example 1 was used as the drug in the dropping pills. In order to better solve the stability of the main medicinal ingredient gingerol, part of the refined ginger oil was clathrated with β-CD. The formulation design of the sustained-release dropping pill contains both the extract and the clathrate, and the ratio of the two is adjusted to obtain different drug release effects.

Preliminary tests were carried out on the auxiliary materials and proportions of the dropping pills. Weigh a certain amount of excipients, heat and melt, cover and seal and keep warm at 60°C, drop into methyl silicone oil to condense, after shrinking into pellets, collect the pellets, absorb the coolant adhered to the surface, and put them in the dryer to dry. have to. The difficulty of making pills from each prescription is evaluated from the following aspects: roundness, hardness, viscosity of feed liquid, fluidity, and degree of ease of dripping.

Preliminary test results show that with the increase of the dosage of PEG4000 or PEG6000 in the selected prescription, the dosage of water-insoluble carrier should be appropriately increased, and the prescription of sustained-release dropping pills should be systematically investigated and screened. And the ratio of gingerol and β-CD inclusion compound in the refined ginger oil is the investigation factor, and each factor takes 4 levels respectively, and adopts the L ₁₆ (4 ⁵ ) orthogonal test table to test, with 0～12h drop pill The cumulative percent release (F) of internal gingerol was used as an index to investigate the slow-release effect of the dropping pills.

Release test:

With reference to "Chinese Pharmacopoeia) 2000 edition, the operation under the first part of the transfer method. Accurately weigh an appropriate amount of sample (equivalent to 60mg gingerol), measure 500mL of artificial gastric juice with pH 1.2 and pour it into the dissolution vessel, control the temperature at (37±0.5)°C, and the rotation speed at (100±1)r·min ^-1 , 1, Take 5mL samples after 2h, transfer to artificial intestinal juice with pH 6.8 after 2h to continue release, take 5mL samples regularly at 4, 6, 8, 10, and 12h respectively, filter with 0.8μm microporous membrane, and supplement the same volume and temperature for dissolution medium. The initial filtrate was discarded, and the content of the subsequent filtrate was determined according to the given chromatographic conditions, thereby obtaining the cumulative release amount of the drug at different times.

It is preferable to use water-soluble carrier PEG6000 to make a solid dispersion, so that the drug molecules are not easy to form aggregates, or the particles are not easy to form aggregates, which ensures the high dispersibility of the drug, accelerates the dissolution and absorption of the drug, and constitutes the composition of the sustained-release drop pill. Quick release part. It is preferred to use water-insoluble stearyl alcohol and inclusion of β-CD to retard drug release, and constitute the sustained-release part of the sustained-release drop pill. Ginger oil sustained-release dripping pills are made by this method, so that the drug can be released slowly in the body for 12 hours. Therefore, the basis for prescription screening is to release about 40% of the drug in 2 hours, and more than 80% of the drug in 10 hours. The concentration quickly reaches the therapeutic range, and can continue to maintain the drug concentration for a period of time.

Example 3: Application test of ginger extract dripping pills in the treatment of liver fibrosis

The ginger extract dripping pill that obtains with embodiment two carries out various comparative tests, and test result sees the following tables, shows:

1. In the whole animal acute liver injury experiment, refined ginger oil has obvious hepatoprotective effect, and it can inhibit the increase of animal serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) caused by carbon tetrachloride and acetaminophen (Table 1, Table 2); The elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels are significantly reduced (Table 3), and refined ginger oil can also reduce hepatocyte malonyltransferase caused by carbon tetrachloride damage. Aldehyde (MDA) content was elevated and superoxide dismutase (SOD) activity was elevated (Table 4). Pathological examination showed that refined ginger oil could significantly improve the liver microstructure of animals with acute liver injury. Refined ginger oil has a significant protective effect on carbon tetrachloride and H ₂ O ₂ induced BRL hepatocyte injury (Table 5), and can make alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the cell culture supernatant levels were significantly lower (Table 5).

2. In the experimental liver fibrosis model, treatment with refined ginger oil can significantly improve the degree of liver fibrosis, reduce alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), and The increase of laminin (LN) (Table 6, Table 7), and can increase the content of serum total protein (TP) and albumin (Alb) (Table 8), significantly reduce the deposition of collagen fibers and pseudolobules in liver tissue Formation.

3. The possible mechanism of the hepatoprotective effect of refined ginger oil is mainly related to its anti-oxidation, including reducing malondialdehyde (MDA), increasing the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) ( Table 9, Table 10). Its anti-hepatic fibrosis mechanism, in addition to anti-oxidation, also includes reducing the activation of hepatic stellate cells (HSCs) by transforming growth factor-β1 (TGF-β1) in vivo (Table 11), reducing collagen Synthesis and reduction of tissue inhibitor of metalloproteinase-1 (TIMP-1) inhibition of matrix metalloproteinases (MMPs) increased collagen breakdown (Table 12).

It has been confirmed by animal experiments that the LD ₅₀ of refined ginger oil to mice after oral administration is 20ml/kg, and the 95% credible limit is 18.7-21.2ml/kg. In the autopsy of the poisoned mice, no obvious pathological changes were found in the heart, liver, spleen, lung, kidney, brain and other major substantive organs.

Table 1 Protective effect of refined ginger oil on carbon tetrachloride-induced acute liver injury in mice (n=10, x±s)

Compared with the normal control group, ^△△ P<0.01; compared with the liver injury model group, *P<0.05, **P<0.01.

Table 2 Protective effect of refined ginger oil on acetaminophen-induced acute liver injury in mice (n=10, x±s)

Compared with the normal control group, ^△△ P<0.01; compared with the liver injury model group, *P<0.05, **P<0.01.

Table 3 Effects on ALT and AST after CCl ₄ hepatocyte injury in rats (n=4, x±s)

Compared with the normal control group, ^△△ P<0.01; compared with the liver injury model group, *P<0.05, **P<0.01.

Table 4 Effects on SOD and MDA after liver cell injury induced by CCl ₄ in rats (n=4, x±s)

Compared with the normal control group, ^△△ P<0.01; compared with the liver injury model group, *P<0.05, **P<0.01.

Table 5 Protective effect on rat _{H2O2 -} induced liver cell injury (n=4, x±s)

Compared with the normal control group, ^△△ P<0.01; compared with the liver injury model group, *P<0.05, **P<0.01

Table 6 Effects on serum ALT and AST of CCl ₄ liver fibrosis in rats (n=10, x±s)

Compared with the normal control group, ^△△ P<0.01; compared with the liver injury model group, *P<0.05, **P<0.01

Table 7 Effects on Serum HA and LN of CCl ₄ Hepatic Fibrosis in Rats (n=8, x±s)

Compared with the normal control group, ^△△ P<0.01; compared with the liver injury model group, *P<0.05, **P<0.01

Table 8 Effects on Serum TP and Alb of CCl ₄ Hepatic Fibrosis in Rats (n=10, x±s)

Compared with the normal control group, ^△ P<0.01; compared with the liver injury model group, *P<0.05, **P<0.01

Table 9 Effects of serum SODMDA on rat CCl ₄ liver fibrosis (n=10, x±s)

Compared with the normal control group, ^△△ P<0.01; compared with the liver injury model group, ^* <0.05, **P<0.01

Table 10 Effects on Serum GSH-Px of CCl ₄ Hepatic Fibrosis in Rats (n=10, x±s)

Compared with the normal control group, ^△△ P<0.01; compared with the liver injury model group, *P<0.05, **P<0.01

Table 11 Effects on Serum TGF-β1 of CCl ₄ Hepatic Fibrosis in Rats (n=8, x±s)

Compared with the normal control group, ^△△ P<0.01; compared with the liver injury model group, *P<0.05, **P<0.01

Table 12 Effects on serum TIMP-1mRNA of rat CCl ₄ liver fibrosis (n=10, x±s)

Compared with the normal control group, ^△△ P<0.01; compared with the liver injury model group, *P<0.05, **P<0.01

Claims (2)

1. Rhizoma Zingiberis Recens extract is as the application of unique crude drug in the medicine of preparation control hepatic fibrosis, and the preparation method of wherein said Rhizoma Zingiberis Recens extract comprises the following steps:

(1) ginger powder is broken to granularity 30-50 orders, as the preparation raw material;

(2) adopt the extraction of supercritical carbon dioxide extraction method, extracting pressure 24-30MPa, 42-45 ℃ of temperature were reacted CO 3-6 hours ₂Flow velocity is 9-10kg/h.kg raw material;

Wherein resolve through two-stage respectively through the fluid of extraction, the pressure that the described first order is resolved is 12-14MPa, and temperature is 56-60 ℃, and the pressure that resolve the second level is 6-7MPa, and temperature is 38-42 ℃;

The faint yellow oil of ginger that above-mentioned extraction is obtained separates with column chromatography, makes the content of gingerol greater than 50%, obtains required Rhizoma Zingiberis Recens extract.

2. Rhizoma Zingiberis Recens extract according to claim 1 is as the application of unique crude drug in the medicine of preparation control hepatic fibrosis, and it is characterized in that: described pharmaceutical dosage form is a drop pill.