CN108379495B - Application of galangal extract in preparation of preparation for preventing and/or treating non-alcoholic fatty liver disease - Google Patents

Application of galangal extract in preparation of preparation for preventing and/or treating non-alcoholic fatty liver disease Download PDF

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CN108379495B
CN108379495B CN201810501185.2A CN201810501185A CN108379495B CN 108379495 B CN108379495 B CN 108379495B CN 201810501185 A CN201810501185 A CN 201810501185A CN 108379495 B CN108379495 B CN 108379495B
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赵玲
杨博
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Wuhan Polytechnic University
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Abstract

The invention belongs to the technical field of biology, and relates to application of a galangal extract in preparation of a preparation for preventing and/or treating non-alcoholic fatty liver disease. The galangal extract contains more than 99wt% of total flavone, and contains 67-85 wt% of galangin, 7-21 wt% of galangin-3-O-methyl ether and 2-13 wt% of kaempferol-4' -O-methyl ether, and the total content of the galangin extract is not more than 100%. The galangal extract disclosed by the invention can reduce the levels of triglyceride, cholesterol, ALT and AST of non-alcoholic fatty liver mice, and has the effects of increasing high-density lipoprotein and reducing low-density lipoprotein.

Description

Application of galangal extract in preparation of preparation for preventing and/or treating non-alcoholic fatty liver disease
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a galangal extract in preparation of a preparation for preventing and/or treating non-alcoholic fatty liver disease.
Background
The diagnosis rate of Non-alcoholic fatty liver disease (NAFLD) is increasing worldwide, and is considered to be one of the most common liver diseases in western countries, accounting for about 20% -30% of the total population. Meanwhile, the medicine also gradually becomes an increasingly serious problem of chronic liver diseases in China, and the morbidity is about 15 percent. Non-alcoholic fatty liver disease refers to the clinical syndrome of diffuse hepatocellular fat becoming pathological, except for large drinking and other factors clearly damaging the liver, and includes simple fatty liver, non-alcoholic steatohepatitis and related cirrhosis and hepatocellular carcinoma thereof. In recent years, NAFLD has increased in incidence year by year due to improvements in living standards, changes in lifestyle and dietary structure, and relative delays in preventive measures, and has become a worldwide health problem. The development of NAFLD is a complicated process related to inflammation, oxidative stress, autophagy, endoplasmic reticulum stress and insulin resistance, at present, no medicine for effectively treating the non-alcoholic fatty liver is available, traditional Chinese medicines and natural medicines are treasure in China, the material basis of the traditional Chinese medicines and the natural medicines are all natural products, compared with western medicines, the medicine has the characteristics of rich structure, unique activity, low toxicity and side effects, diversified action targets and the like, is an important source of innovative medicines in China, and the medicine for preventing and treating the non-alcoholic fatty liver, which is safe, effective and low in toxic and side effects, is selected and developed in the traditional Chinese medicines, so that the medicine has extremely wide market prospect and can create good social and economic benefits.
Galangal (Alpinia officinarum Hance) is a plant of the genus Alpinia in the family Zingiberaceae (Zingibergeraceae), is also called as lesser galangal and lesser galangal, is a plant resource used as both medicine and food, is often used as a seasoning in Guangdong and southeast Asia countries, is a plant used as both medicine and food, has the effects of warming stomach, dispelling cold, promoting qi circulation and relieving pain when root and stem are used as medicines. Galangal has been cultivated and used for hundreds of years in China, and contains a large amount of flavone compounds and diphenyl heptane compounds. Modern pharmacological research shows that the galangal and the extract have the effects of resisting tumors, bacteria, viruses and obesity, gastric ulcer and gastric mucosa protection, but no relevant literature report is available at present on the prevention and treatment of the non-alcoholic fatty liver disease by using the galangal extract.
Disclosure of Invention
The invention aims to provide a new application of a galangal extract, and particularly relates to an application of the galangal extract in preparation of a preparation for preventing and/or treating non-alcoholic fatty liver disease.
In order to achieve the above object, the present invention provides a use of a galangal extract in the preparation of a preparation for preventing and/or treating non-alcoholic fatty liver disease, wherein the galangal extract has a total flavone content of 99wt% or more, and comprises 67wt% to 85wt% of galangin, 7wt% to 21wt% of galangin-3-O-methyl ether, and 2wt% to 13wt% of kaempferol-4' -O-methyl ether, the total of which is not more than 100%.
In the invention, the content of each component is preferably measured by a high performance liquid chromatography, and specifically, the content is measured by referring to the following documents: zhao Xiao 38932, Chengxiao, Gunn celluloid, etc. and HPLC method to determine the content of 3 kinds of flavone components in galangal simultaneously. Journal of drug analysis, 2009, 29 (12): 2036-2039.
The galangal extract of the present invention is preferably prepared by a method comprising the steps of: pulverizing rhizoma Alpiniae Officinarum, adding methyl tert-butyl ether for flash extraction, filtering to obtain residue, adding ethyl acetate into the residue for extraction, recovering ethyl acetate extract to obtain rhizoma Alpiniae Officinarum extract, subjecting the extract to column chromatography separation, petroleum ether-ethyl acetate gradient elution, thin layer chromatography monitoring, mixing the extracts containing galangin, galangin-3-O-methyl ether, and kaempferol-4' -O-methyl ether, removing petroleum ether-ethyl acetate solvent from the extract, and recrystallizing with ethanol to obtain the rhizoma Alpiniae Officinarum extract.
In particular, flash extraction may be performed twice. The extraction with ethyl acetate can be carried out at room temperature. The amount of the methyl tert-butyl ether and the ethyl acetate can be 4-6 times of the mass of the extract.
According to the invention, the petroleum ether-ethyl acetate gradient elution is preferably carried out using petroleum ether-ethyl acetate gradient eluents in the volume ratio of 10: 1, 8: 1, 5: 1, 3: 1, 1: 1.
According to the invention, the column chromatography is preferably a polyamide column chromatography.
According to a specific embodiment of the present invention, the galangal extract is prepared by the following steps: pulverizing rhizoma Alpiniae Officinarum, adding 4-6 times of methyl tert-butyl ether solvent, extracting for 2 times with flash extractor, and filtering to obtain residue. Extracting the residue with 4-6 times of ethyl acetate for 2 times to obtain ethyl acetate extractive solution, recovering ethyl acetate to obtain rhizoma Alpiniae Officinarum extract, subjecting the extract to polyamide column chromatography separation, gradient eluting with petroleum ether-ethyl acetate at volume ratio of 10: 1, 8: 1, 5: 1, 3: 1, 1: 1, monitoring by thin layer chromatography, mixing the fractions containing galangin, galangin-3-O-methyl ether, and kaempferol-4' -O-methyl ether, recovering petroleum ether-ethyl acetate solvent from the fractions, and recrystallizing with ethanol.
In the invention, the preparation can be a medicament, and can also be food, such as health-care food.
The medicament may include one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" refers to excipients and/or adjuvants that facilitate processing of the active ingredients of the present invention into pharmaceutically acceptable formulations. Suitable excipients for use in the present invention include fillers such as sugars (e.g., lactose, sucrose, mannitol, sorbitol); a cellulose derivative; magnesium sulfate; calcium phosphates (e.g., tricalcium phosphate, calcium hydrogen phosphate); binders such as starch paste (e.g., corn starch, wheat starch, rice starch, potato starch), gelatin, tragacanth, or polyvinylpyrrolidone. Suitable adjuvants that may be used in the present invention include flow restrictors and lubricants, such as talc, silica, stearic acid or its derivatives (e.g., magnesium stearate), and/or polyethylene glycol.
The formulation of the medicament may be administered by any desired route of administration, including oral, topical, intramuscular, intraperitoneal, subcutaneous, intratumoral or intravenous routes. In the invention, the dosage form of the medicine can be various conventional and appropriate dosage forms in the field, including but not limited to tablets, capsules, granules, dripping pills and oral solutions.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
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The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
Fig. 1 shows the liver structure of the normal control group observed under a light microscope.
Figure 2 shows the structure of the model group of livers observed under the light microscope.
Fig. 3-5 show the liver structures of the galangal extract low dose group, the galangal extract medium dose group, and the galangal extract high dose group observed under a light microscope.
Fig. 6 shows the liver structure of the positive drug group observed under a light microscope.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.
The intervention and treatment effects of the galangal extract on nonalcoholic fatty liver mice are further illustrated by specific efficacy experiments.
1. Medicine
And (3) testing a sample: the galangal extract disclosed by the invention is prepared by the following preparation method: pulverizing rhizoma Alpiniae Officinarum, adding 5 times of methyl tert-butyl ether solvent, extracting for 2 times with flash extractor, and filtering to obtain residue. Extracting the residue with 5 times of ethyl acetate for 2 times to obtain ethyl acetate extractive solution, recovering ethyl acetate to obtain rhizoma Alpiniae Officinarum extract, separating by polyamide column chromatography, gradient eluting with petroleum ether-ethyl acetate at volume ratio of 10: 1, 8: 1, 5: 1, 3: 1, 1: 1, monitoring by thin layer chromatography, mixing the fractions containing galangin, galangin-3-O-methyl ether, and kaempferol-4' -O-methyl ether, recovering petroleum ether-ethyl acetate solvent, and recrystallizing with ethanol.
Positive control drug: fenofibrate, produced by libofir pharmaceutical, france.
2. Animals: an SPF-level Kunming mouse model, which is 8 weeks old, 20-22g in weight and male; provided by the university of science and technology experimental center in Huazhong.
3. Experimental methods
60 male Kunming mice of SPF class were randomly divided into 6 groups, which were: a normal control group, a positive drug group (fenofibrate is 10mg/kg), a galangal extract high dose group (200mg/kg), a galangal extract medium dose group (100mg/kg), a galangal extract low dose group (50mg/kg) and a model group, wherein each group comprises 10 galangal extracts. After 1 week of adaptive feeding, the other 5 groups except the normal control group were fed with high fat diet (self-prepared: 73% basal diet, 25% lard, 2% cholesterol) continuously for 16 weeks, the food intake and body mass of mice were recorded every week from the first week of high fat diet feeding, the mice were fed in clean animal rooms at room temperature: 22 ± 2 ℃, humidity: 30-70%, and ventilating once in 20 minutes for 12 hours in light and shade. Dissolving rhizoma Alpiniae Officinarum extract and fenofibrate in 0.5% CMC-Na, respectively adding high, medium and low dosages of 50mg/kg, 100mg/kg and 200mg/kg, and intragastrically administering once a day for 16 weeks; the mice in the blank control group and the high-fat model group are filled with corresponding 0.5 percent CMC-Na solution. After the last administration, fasting is carried out for 12 hours without water supply, the weight is weighed, blood is taken from the orbit, and the liver is quickly dissected and taken. A liver tissue with the size of about 1.5cm multiplied by 1.5cm is taken from the same position of the left lobe of the liver and is completely soaked in 10 percent paraformaldehyde solution for fixation for subsequent section HE staining. HE stained sections were observed under 200 x light.
As shown in figure 1, the normal control group had intact liver structure, radial arrangement of hepatocytes, normal size and morphology of hepatocytes, and no abnormal lesions such as hepatic steatosis and edema. As shown in FIG. 2, most of the hepatocytes under the liver histoscope of the model group showed obvious steatosis, enlarged cells and larger lipid droplets. As shown in fig. 3-5, the fat change degree of the liver cells of the galangal extract is reduced from the low dose to the high and low dose groups, and the obvious dose correlation is shown. As can be seen in FIG. 5, the amount of hepatic cell fat was significantly reduced in the high galangal dose group, and the liver cells were almost uniform in size and normal in morphology. As shown in FIG. 6, the histopathology and histology of the liver of the mice in the fenofibrate group are similar to those of the normal control group, and no abnormal lesion is found. In conclusion, it can be seen that the galangal extract has a significant improvement effect on non-alcoholic fatty liver disease.
Blood biochemical indexes such as blood fat, transaminase and the like are detected according to the operation instructions of the biochemical detection kit.
The experimental data are preliminarily sorted by office excel, expressed as mean value +/-standard deviation/standard error (mean +/-SD/SEM), analyzed by SPSS 17.0 statistical software, and the experimental results are all analyzed by
Figure BDA0001670383490000051
Showing that the comparison among groups adopts one-way analysis of variance.
The results are shown in Table 1.
TABLE 1 Effect of Alpinia galanga extract on liver function in non-alcoholic fatty liver disease mice
Figure BDA0001670383490000061
As can be seen from table 1, the galangal extract of the present invention can reduce the levels of triglyceride, cholesterol, ALT, and AST in non-alcoholic fatty liver mice, and has the effects of increasing high density lipoprotein and reducing low density lipoprotein. And the galangal high dose group had an effect comparable to the positive drug.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims (8)

1. The application of the galangal extract in preparing the preparation for preventing and/or treating the non-alcoholic fatty liver disease is characterized in that the content of total flavonoids in the galangal extract is more than 99wt%, and the galangal extract contains 67-85 wt% of galangin, 7-21 wt% of galangin-3-O-methyl ether and 2-13 wt% of kaempferol-4' -O-methyl ether, wherein the total content is not more than 100%;
the galangal extract is prepared by a method comprising the following steps: pulverizing rhizoma Alpiniae Officinarum, adding methyl tert-butyl ether for flash extraction, filtering to obtain residue, adding ethyl acetate into the residue for extraction, recovering ethyl acetate extract to obtain rhizoma Alpiniae Officinarum extract, subjecting the extract to column chromatography separation, petroleum ether-ethyl acetate gradient elution, thin layer chromatography monitoring, mixing the extracts containing galangin, galangin-3-O-methyl ether, and kaempferol-4' -O-methyl ether, removing petroleum ether-ethyl acetate solvent from the extract, and recrystallizing with ethanol to obtain the rhizoma Alpiniae Officinarum extract.
2. The use of the galangal extract according to claim 1, for the preparation of a preparation for the prevention and/or treatment of non-alcoholic fatty liver disease, wherein the content of each component is measured by high performance liquid chromatography.
3. Use of galangal extract according to claim 1 for the preparation of a preparation for the prevention and/or treatment of non-alcoholic fatty liver disease, characterized in that flash extraction is performed twice.
4. The use of the galangal extract according to claim 1, for the preparation of a formulation for the prevention and/or treatment of non-alcoholic fatty liver disease, characterized in that petroleum ether-ethyl acetate gradient elution is performed with a petroleum ether-ethyl acetate gradient elution ratio of 10: 1, 8: 1, 5: 1, 3: 1, 1: 1 by volume.
5. The use of the galangal extract according to claim 1, for the preparation of a preparation for the prevention and/or treatment of non-alcoholic fatty liver disease, wherein the column chromatography is polyamide column chromatography.
6. Use of an extract of galangal as claimed in claim 1, for the preparation of a preparation for the prevention and/or treatment of non-alcoholic fatty liver disease, wherein the preparation comprises a medicament.
7. Use of an extract of galangal as claimed in claim 6, for the preparation of a preparation for the prevention and/or treatment of non-alcoholic fatty liver disease, wherein said medicament comprises one or more pharmaceutically acceptable carriers.
8. The use of the galangal extract according to claim 6, for the preparation of a preparation for the prevention and/or treatment of non-alcoholic fatty liver disease, wherein the dosage form of the medicament is selected from tablets, capsules, granules, dripping pills and oral solutions.
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CN1879613A (en) * 2005-06-16 2006-12-20 广州汉方现代中药研究开发有限公司 Medicine containing galangin and preparation method thereof
CN101637503A (en) * 2009-08-19 2010-02-03 大连中植环境生物科技有限公司 Total flavonoids of broussonetia papyrifera extract and preparation method and application thereof
CN104906483A (en) * 2015-05-26 2015-09-16 南京泽朗医药科技有限公司 Technology for extracting galangal total flavonoids

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CN1879613A (en) * 2005-06-16 2006-12-20 广州汉方现代中药研究开发有限公司 Medicine containing galangin and preparation method thereof
CN101637503A (en) * 2009-08-19 2010-02-03 大连中植环境生物科技有限公司 Total flavonoids of broussonetia papyrifera extract and preparation method and application thereof
CN104906483A (en) * 2015-05-26 2015-09-16 南京泽朗医药科技有限公司 Technology for extracting galangal total flavonoids

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