CN110882319A

CN110882319A - Application of thoroughfare bitter orange, thoroughfare bitter orange extract and products containing thoroughfare bitter orange extract in preventing and/or treating metabolic liver diseases

Info

Publication number: CN110882319A
Application number: CN201911132599.3A
Authority: CN
Inventors: 张峰; 王思为; 白永凤; 宋剑锋
Original assignee: Quzhou Peoples Hospital
Current assignee: Quzhou Peoples Hospital
Priority date: 2019-11-19
Filing date: 2019-11-19
Publication date: 2020-03-17

Abstract

The invention relates to the technical field of metabolic liver diseases, in particular to application of thoroughfare bitter orange, thoroughfare bitter orange extract or a composition containing thoroughfare bitter orange extract in preparation of products for preventing and/or treating metabolic liver diseases, particularly non-alcoholic fatty liver diseases. The thoroughfare type fructus aurantii and thoroughfare type fructus aurantii extract or the composition containing the thoroughfare type fructus aurantii extract and thoroughfare type fructus aurantii extract have the effects of reducing liver lipid accumulation or improving and/or relieving liver fatty lesion, and are strong in pharmacological effect, safe and non-toxic. The product has the advantages of rich raw material sources, low price, safety, no toxic or side effect, simple preparation process route, low production cost, capability of being prepared into conventional dosage forms such as oral dosage forms, tablets and the like, convenience in use and great prospect and development value, and is a product for preventing and/or treating metabolic liver diseases, particularly non-alcoholic fatty liver diseases.

Description

Application of thoroughfare bitter orange, thoroughfare bitter orange extract and products containing thoroughfare bitter orange extract in preventing and/or treating metabolic liver diseases

Technical Field

The invention relates to the technical field of metabolic liver diseases, in particular to the application of thoroughfare bitter orange, thoroughfare bitter orange extracts and products containing the thoroughfare bitter orange extracts in preventing and/or treating metabolic liver diseases, particularly non-alcoholic fatty liver diseases.

Background

Non-alcoholic fatty liver disease (NAFLD) refers to a chronic inflammatory liver disease characterized by steatosis and fat storage in liver parenchymal cells, causing impairment of liver function without history of excessive alcohol consumption. Studies have shown that the progression of NAFLD leads to cirrhosis and even liver cancer. In China, the prevalence rate of NAFLD is remarkably increased, and NAFLD becomes the second largest liver disease next to viral hepatitis, and seriously threatens the life health of people in China. However, there is currently no definitive effective treatment for NAFLD.

Therefore, there is an urgent and need to find safe, effective and economical products for preventing and/or treating metabolic liver diseases, especially non-alcoholic fatty liver diseases.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provides the application of thoroughfare bitter orange, thoroughfare bitter orange extract and products containing the thoroughfare bitter orange extract in preventing and/or treating metabolic liver diseases, in particular non-alcoholic fatty liver diseases. The invention takes thoroughfare bitter orange as a main component, is used for preventing and/or treating metabolic liver diseases, particularly non-alcoholic fatty liver diseases, and has remarkable curative effect.

In order to achieve the above objects, the first aspect of the present invention provides the use of thoroughfare bitter orange, thoroughfare bitter orange extract or a composition containing the same for the preparation of a product for the prevention and/or treatment of metabolic liver diseases, particularly non-alcoholic fatty liver diseases.

The second aspect of the invention provides the application of thoroughfare bitter orange, thoroughfare bitter orange extract or a composition containing the thoroughfare bitter orange extract in preparing products for reducing liver lipid accumulation or improving and/or relieving fatty lesions of the liver.

Preferably, the thoroughfare bitter orange extract is thoroughfare bitter orange alcohol extract prepared by heating reflux extraction.

Preferably, the thoroughfare bitter orange crushed product is soaked in an alcohol solvent for 0.5 to 2 hours before reflux extraction.

The invention develops new medical application of thoroughfare bitter orange creatively and develops a new application field. The thoroughfare type fructus aurantii and thoroughfare type fructus aurantii extract or the composition containing the thoroughfare type fructus aurantii extract and thoroughfare type fructus aurantii extract have the effects of reducing liver lipid accumulation or improving and/or relieving liver fatty lesion, and are strong in pharmacological effect, safe and non-toxic. The product has the advantages of rich raw material sources, low price, safety, no toxic or side effect, simple preparation process route, low production cost, capability of being prepared into conventional dosage forms such as oral dosage forms, tablets and the like, convenience in use and great prospect and development value, and is a product for preventing and/or treating metabolic liver diseases, particularly non-alcoholic fatty liver diseases.

Drawings

FIG. 1 shows the liver cell morphology (HE staining and oil red O staining) of mice of DBM group, DB-QFA group, DB-QFAE group and DB-QFAW group.

FIG. 2 shows liver cell morphology (HE staining and oil red O staining) of mice of CON group, HFD-QFA group, HFD-QFAE group and HFD-QFAW group.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

According to the first aspect of the present invention, there is provided the use of thoroughfare bitter orange, thoroughfare bitter orange extract or a composition containing the same in the manufacture of a product for the prevention and/or treatment of metabolic liver diseases, particularly non-alcoholic fatty liver diseases.

In a second aspect of the present invention, there is provided a use of thoroughfare bitter orange, thoroughfare bitter orange extract or a composition containing the same in the preparation of a product for reducing liver lipid accumulation or improving and/or alleviating liver fatty pathological changes.

Thoroughfare fructus aurantii, i.e. Hu shaddock slices, is dried immature fruit of Citrus changshan-huyou Y.B.Chang, which is a Rutaceae plant, is harvested when 7 months of pericarp is green, cut into two halves from the middle, and dried in the sun or at low temperature. Bitter, pungent and sour, slightly cold in nature, entering spleen and stomach meridians, with the effects of regulating qi, relieving epigastric distention, activating stagnancy and relieving flatulence, and can be used for treating qi stagnation in chest and hypochondrium, fullness and pain, indigestion, phlegm and fluid retention, gastroptosis, proctoptosis, and uterine prolapse. It was recorded in 2015 edition Zhejiang province Standard for processing Chinese herbs in 2016 (8 months).

According to the invention, when the thoroughfare type bitter orange is directly used for medicine, the thoroughfare type bitter orange can be directly ground into thoroughfare type bitter orange powder for medicine taking, the granularity of the thoroughfare type bitter orange powder is not particularly limited as long as the thoroughfare type bitter orange powder can be easily taken by a patient. In addition, the thoroughfare bitter orange powder can also be taken together with other food materials or medicinal materials.

According to the present invention, when the thoroughfare type fructus aurantii extract is used as a medicine, the thoroughfare type fructus aurantii extract can be prepared by various methods known in the art, for example, the thoroughfare type fructus aurantii extract can be prepared by brewing and extracting in a tea bag-replacing manner, can be prepared by brewing and extracting with alcohol (such as wine) as a solvent, can be prepared by decocting the medicine with water, and can also be prepared by extracting in a heating reflux manner, an ultrasonic extraction manner, a supercritical fluid extraction method, a macroporous resin adsorption method, and the like.

According to the invention, the thoroughfare bitter orange extract can be an alcohol extract of thoroughfare bitter orange and can also be water extract of thoroughfare bitter orange. Among them, the alcohol is preferably ethanol.

According to a preferred embodiment of the present invention, the thoroughfare bitter orange extract is a thoroughfare bitter orange alcohol extract prepared by heating reflux extraction, and the extraction method comprises:

(1) crushing thoroughfare bitter orange, and performing reflux extraction on the crushed product by using an alcohol solvent to obtain an extracting solution;

(2) carrying out solid-liquid separation on the extracting solution, and recovering an alcohol solvent to obtain a concentrated solution;

(3) drying the concentrated solution to obtain the thoroughfare bitter orange extract.

According to the present invention, in the step (1), the particle size of the thoroughfare bitter orange pulverized product can be selected within a wide range.

Wherein, the dosage of the alcoholic solvent can be selected in a wide range, preferably, the dosage of the alcoholic solvent is 10 to 20 times, more preferably 12 to 18 times, even more preferably 14 to 16 times, and most preferably 15 times relative to each kilogram of thoroughfare bitter orange.

Also, the concentration of the alcohol solvent is not particularly limited, and it is preferable to use a high concentration of the alcohol solvent, for example, 70 to 90% by weight, more preferably 75 to 85% by weight, and most preferably 80% by weight.

The heating reflux condition can be selected in a wide range, preferably, the heating reflux temperature is 50-90 ℃ and the time is 0.5-6 hours, more preferably, the reflux temperature is 60-80 ℃ and the time is 1-3 hours, most preferably, the reflux temperature is 70 ℃ and the time is 2 hours.

According to a preferred embodiment of the present invention, in order to further improve the extraction efficiency and the content of active substances in the extract, the method further comprises immersing the thoroughfare bitter orange crushed product in an alcohol solvent for 0.5 to 2 hours before the heating reflux extraction.

Further preferably, in order to further enhance the extraction efficiency, the heating reflux extraction may be performed in a plurality of times, for example, 2 times, 3 times, 4 times, 5 times, etc., and then the extract liquids extracted each time are combined to be the final extract liquid. The skilled person can select the extraction effect according to the actual extraction effect, as long as the total time of the reflux extraction is controlled within the above range, and each extraction can be performed in equal time.

According to the present invention, in the step (2), the method of subjecting the extract to solid-liquid separation may be various methods conventional in the art, for example, filtration, centrifugation and the like, as long as the solid residue in the extract can be sufficiently removed. The conditions for the solid-liquid separation can be appropriately selected by those skilled in the art according to the desired solid content of the filtrate.

Wherein the solid-liquid separation can be carried out at normal temperature.

According to the present invention, the method for recovering the alcohol solvent from the filtrate in the step (2) may be performed according to various methods known in the art, for example, a freezing condensation method, etc.

According to the present invention, in the step (3), the drying method of the concentrated solution may be performed according to various methods known in the art, for example, a freeze-drying method, a spray-drying method, and the like.

According to the invention, the product is a medicament, health product or food. The medicament contains thoroughfare bitter orange or thoroughfare bitter orange extract and pharmaceutically acceptable adjuvant, preservative or stabilizer; the health care product contains thoroughfare bitter orange or thoroughfare bitter orange extract and auxiliary materials acceptable in the health care product; the food contains thoroughfare bitter orange or thoroughfare bitter orange extract and acceptable auxiliary materials or adjuvants in food. The types and contents of adjuvants, preservatives or stabilizers are well known to those skilled in the art and are not described herein.

According to the present invention, the pharmaceutical or nutraceutical may be prepared in various dosage forms, for example, may be prepared in the form of, but not limited to, tablets, capsules (including soft and hard capsules), powders, oral liquids, granules and pills. The food product may be prepared in a form conventional in the art.

According to the present invention, when used for reducing liver lipid accumulation (e.g., reducing liver TG levels and reducing liver lipid droplet number), or improving and/or alleviating liver steatosis (e.g., reducing serum ALT, AST, NEFA, TG and improving liver cell morphology), the thoroughfare bitter orange or thoroughfare bitter orange extract can be administered in a dosage of 0.1-0.3g per kilogram body weight per day, and can be administered in multiple divided doses.

The present invention will be described in detail below by way of examples. In the following examples of the present invention,

1. materials and reagents

Thoroughfare bitter orange: provided and identified by traditional Chinese medicine Limited in south pore of Quzhou.

Mouse high fat diet (Research diet D12492): purchased from Research die, usa.

Normal feed: is provided by animal experiment center of Zhejiang Chinese medicine university.

Alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), free fatty acid (NEFA), Triglyceride (TG) detection kit: purchased from Nanjing institute of bioengineering.

Oil red O staining kit: purchased from Solarbio, china.

2 laboratory animals

Male spontaneous type 2 diabetes C57BLKS/J db/db mice (db/db mice), Stock No:000642, 7-8 weeks old, body weight 30-40 g; the same genetic background C57BLKS/J db wild type mouse (db/m), 7-8 weeks old, weight 18-22g, the above animals purchased from Nanjing university-Nanjing biomedical research institute, animal production license number: SYXK (threo) 2015-0001. The feed is raised in the animal experiment center of Zhejiang university of traditional Chinese medicine, and free to eat and drink water. The animal experiments were approved by the medical ethics committee of the people's hospital in Quzhou City.

Male C57BL/6 mice, 7-8 weeks old, 22-25g in body weight, SPF grade, provided by the animal testing center of traditional chinese medicine university, chekiang, production license number: SCXK (Shanghai) 2008-0016.

Preparation example

Preparation example 1

The preparation example is used for explaining the preparation of thoroughfare bitter orange dry powder

1kg of thoroughfare bitter orange medicinal material is weighed and cleaned, and is crushed into coarse powder, thus obtaining thoroughfare bitter orange dry powder (QFA).

Preparation example 2

The preparation example is used for explaining the preparation of thoroughfare bitter orange alcohol extract

Weighing 1kg of thoroughfare bitter orange medicinal material, cleaning, crushing, adding 15 times of 80% ethanol (15000mL), soaking for 1h, heating and refluxing for 3 times, extracting for 2h each time, combining the extracting solutions, cooling and filtering, recovering the solvent from the filtrate, and drying to obtain thoroughfare bitter orange alcohol extract (QFAE).

Preparation example 3

The preparation example is used for explaining the preparation of thoroughfare bitter orange water extract

Weighing 1kg of thoroughfare bitter orange medicinal material, cleaning, crushing, adding 15 times of water (15000mL), soaking for 1h, heating and refluxing for 3 times, extracting for 2h each time, combining the extracting solutions, cooling, filtering, concentrating and drying to obtain thoroughfare bitter orange water extract (QFAW).

Comparative preparation example 1

This comparative preparation example is illustrative of the preparation of alcohol extract of ripe grapefruit

The preparation of a mature grapefruit alcohol extract (HYE) was carried out in accordance with the method of preparation example 2, except that the thoroughfare bitter orange was replaced with an equal amount of mature grapefruit.

Comparative preparation example 2

This comparative preparation example is intended to illustrate the preparation of an alcohol extract of ripe citrus grandis peel

The preparation of an alcohol extract (HYPE) of mature citrus grandis peel was carried out according to the method of preparation example 2, except that the amount of thoroughfare bitter orange was replaced with the same amount of mature citrus grandis peel.

Examples

1. Animal grouping and administration

(1)54 male spontaneous type 2 diabetes mice (db/db mice), 7-8 weeks old, after being acclimatized for one week, were randomly divided into 6 groups of 9 mice each:

in DB group, DB/DB mice were gazed with distilled water;

performing intragastric administration on DB-QFA group, DB/DB mice with 300mg/kg/d thoroughfare bitter orange dry powder;

in DB-QFAAE group, DB/DB mice are drenched with 300mg/kg/d thoroughfare bitter orange alcohol extract;

performing intragastric administration on DB-QFAW group, DB/DB mouse with 300mg/kg/d thoroughfare fructus Aurantii water extract;

in DB-HYE group, DB/DB mice were gavaged with 300mg/kg/d of mature grapefruit alcohol extract;

in DB-HYPE group, DB/DB mice are filled with 300mg/kg/d mature alcohol extract of citrus grandis peel;

taking another 9 wild type db/m mice as DBM group, and perfusing the mice with distilled water to obtain a normal control group;

all mice were fed normal diet and water for 6 weeks.

(2) 84C 57BL/6 mice, 7-8 weeks old, were acclimatized for one week and then randomized into 7 groups of 12 mice each:

CON group, normal control group, was fed with normal feed and gavaged with distilled water;

HFD group, fed with high-fat diet and gavage with distilled water;

feeding HFD-QFA group with high-fat feed, and intragastrically administering 300mg/kg/d of thoroughfare fructus Aurantii dry powder;

feeding HFD-QFAE group with high-fat feed, and intragastrically infusing 300mg/kg/d of thoroughfare fructus Aurantii alcohol extract;

feeding HFD-QFAW group with high-fat feed, and intragastrically irrigating with 300mg/kg/d thoroughfare fructus Aurantii water extract;

HFD-HYE group, fed with high-fat diet, and intragastrically administered with 300mg/kg/d of ripe grapefruit alcohol extract;

HFD-HYPE group, which is fed with high-fat feed, and the extract of ripe citrus grandis peel alcohol of 300mg/kg/d is intragastric;

all mice had normal water and were dosed for 14 weeks.

2. Body weight and liver weight determination

After 24h from the last dose, the body weight of each group of mice was measured, then the mice were sacrificed, livers were removed and weighed, and the results are averaged for each group as shown in tables 1 and 2.

3. Serum alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), free fatty acid (NEFA), and Triglyceride (TG) assays

24h after the last administration, pentobarbital sodium anesthetized mice, blood was collected from the abdominal aorta, serum was separated, and serum ALT, AST, NEFA and TG levels were measured in each group using alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), free fatty acid (NEFA) and Triglyceride (TG) detection kits, respectively, and averaged for each group, and the results are shown in tables 3 and 4.

4. Liver Triglyceride (TG) assay

The liver tissues are taken, the liver tissue oil of each group of mice is extracted by adopting a conventional organic solvent extraction method, the Triglyceride (TG) level of each group of mice is determined by adopting a Triglyceride (TG) detection kit, the average value of each group is taken, and the result is shown in Table 5.

5. Histopathological observation and oil red O staining of liver

Taking liver tissues, fixing with 10% formalin, embedding the liver tissues into the conventional paraffin, HE staining, and observing under a 400-fold microscope, wherein the HE staining of mice in a DBM group, a DB-QFA group, a DB-QFAE group and a DB-QFAW group is shown in figure 1; HE staining of mice in CON, HFD-QFA, HFD-QFAE and HFD-QFAW groups is shown in FIG. 2.

Dyeing with oil red O: freezing and slicing liver tissues, washing with distilled water, washing away embedding agents, washing with 60% isopropanol for 2min, dyeing with oil red O working solution for 5min, toning with 60% isopropanol, washing with distilled water, counterstaining with hematoxylin, rewarding with running water, sealing with glycerol gelatin, and observing under a 400-fold microscope, wherein the oil red O staining of mice in DBM group, DB-QFA group, DB-QFAE group and DB-QFAW group is shown in figure 1; oil red O staining of mice in CON, HFD-QFA, HFD-QFAE and HFD-QFAW groups is shown in FIG. 2.

6. Data processing

All data were processed using SPSS 17.0 statistical software, with data as mean. + -. standard deviation

Showing that differences between groups were examined by ANOVA and were compared pairwise by least significant difference examination, as P<0.05 is statistically significant.

7. Results

(1) Effects on body weight and liver weight in mice

The results of the changes in body weight and liver weight in db/db mice with type 2 diabetes are shown in Table 1(

n＝9)。

TABLE 1

Note: in contrast to the set of DBMs,^*P<0.05; in contrast to the DB group,^#P<0.05。

as can be seen from Table 1, the body weight and liver weight of db/db mice with type 2 diabetes were significantly increased compared to the normal control group (i.e., DBM group); compared with DB group, thoroughfare bitter orange dry powder (DB-QFA group), thoroughfare bitter orange alcohol extract (DB-QFAE group) and thoroughfare bitter orange water extract (DB-QAW group) can obviously reduce the liver weight of DB/DB mice, but the effect of reducing the weight is not obvious. In contrast, the alcohol extract of mature fruit of grapefruit (DB-HYE group) and the alcohol extract of pericarp of grapefruit (DB-HYPE group) had no significant effect on reducing body weight and liver weight in DB/DB mice with type 2 diabetes.

Results of the changes in body weight and liver weight in high fat diet-induced obese mice are shown in Table 2(

n＝12)。

TABLE 2

Group of	Body weight (g)	Liver weight (g)
			CON group	28.83±1.65	1.12±0.08
HFD group	42.70±1.77^*	1.69±0.13^*
			HFD-QFA group	35.17±1.55^#	1.23±0.17^#
HFD-QFAE group	32.27±3.41^#	1.20±0.09^#
			HFD-QFAW group	32.00±3.60^#	1.20±0.10^#
HFD-HYE group	39.11±3.21^*	1.61±0.05^*
			HFD-HYPE group	38.78±3.01^*	1.57±0.19^*

Note: in contrast to the CON group,^*P<0.05; in contrast to the HFD group,^#P<0.05。

as can be seen from table 2, the high fat diet induced significant increase in body weight and liver weight in obese mice compared to the normal control group (i.e., CON group); compared with HFD group, thoroughfare bitter orange dry powder (HFD-QFA group), thoroughfare bitter orange alcohol extract (HFD-QFAAE group) and thoroughfare bitter orange water extract (HFD-QFAW group) can significantly reduce the weight and liver weight of fat mice induced by high fat diet. In contrast, alcohol extracts of mature grapefruit fruit (HFD-HYE group) and alcohol extracts of grapefruit pericarp (HFD-HYPE group) were not effective in reducing body weight and liver weight in high-fat diet-induced obese mice.

(2) Effect on serum ALT, AST, NEFA and TG levels in mice

The results of the changes in serum ALT, AST, NEFA and TG levels in db/db type 2 diabetic mice are shown in Table 3: (

n＝9)。

TABLE 3

Group of	ALT(IU/L)	AST(IU/L)	NEFA(mmol/L)	TG(mmol/L)
					DBM group	51.08±4.05	116.40±9.56	3.37±0.49	0.90±0.09
DB group	68.12±3.97^*	205.27±22.46^*	9.20±1.09^*	6.10±0.96^*
					DB-QFA group	53.74±2.17^#	138.88±21.43^#	5.83±0.99^*#	4.53±0.20^*#
DB-QFAE group	60.78±3.46^#	139.63±22.48^#	5.80±0.99^*#	3.38±0.78^*#
					DB-QFAW group	53.08±5.26^#	133.10±13.48^#	3.75±1.12^*#	3.29±0.56^*#
DB-HYE group	63.87±5.10	163.98±26.37	7.89±1.01^*	5.71±0.36
					DB-HYPE group	65.27±3.98	159.77±21.89	6.98±0.73^*	5.22±0.77^*

as can be seen from Table 3, the serum ALT, AST, NEFA and TG levels of db/db mice with type 2 diabetes were significantly increased as compared with the normal control group (i.e., DBM group); compared with DB group, thoroughfare bitter orange dry powder (DB-QFA group), thoroughfare bitter orange alcohol extract (DB-QFAE group) and thoroughfare bitter orange water extract (DB-QFAW group) can obviously reduce serum ALT, AST, NEFA and TG levels of DB/DB mice. In contrast, the alcohol extract of mature fruit of grapefruit (DB-HYE group) and the alcohol extract of pericarp of grapefruit (DB-HYPE group) had no significant effect on reducing serum ALT, AST, NEFA and TG levels in DB/DB mice.

Results of changes in serum ALT, AST, NEFA and TG levels in high fat diet-induced obese mice are shown in Table 4: (

n＝12)。

TABLE 4

Group of	ALT(IU/L)	AST(IU/L)	NEFA(mmol/L)	TG(mmol/L)
					CON group	37.10±7.02	101.07±10.30	3.25±0.87	0.86±0.14
HFD group	78.50±7.34^*	460.50±62.60^*	6.51±1.05^*	1.98±0.20^*
					HFD-QFA group	48.90±4.69^#	246.05±74.10^*#	5.48±0.51^#	1.51±0.08^*#
HFD-QFAE group	52.87±7.39^#	246.20±16.10^*#	5.39±0.63^#	1.17±0.21^*#
					HFD-QFAW group	54.77±2.78^#	266.90±24.06^*#	5.18±0.45^#	1.19±0.11^*#
HFD-HYE group	67.59±5.67^*	397.29±31.67^*	6.37±0.71	1.71±0.21^*
					HFD-HYPE group	70.14±1.98^*	377.99±58.10^*	6.80±1.03	1.73±0.14^*

Note: in contrast to the CON group,^*P<0.05; and HThe comparison of the FD group is carried out,^#P<0.05。

as can be seen from table 4, serum ALT, AST, NEFA and TG were significantly increased in the high fat diet-induced obese mice compared to the normal control group (i.e., CON group); compared with HFD group, thoroughfare bitter orange dry powder (HFD-QFA group), thoroughfare bitter orange alcohol extract (HFD-QFAAE group) and thoroughfare bitter orange water extract (HFD-QFAW group) can significantly reduce serum ALT, AST, NEFA and TG levels of fat mice induced by high fat diet. In contrast, alcohol extracts of mature grapefruit fruits (HFD-HYE group) and grapefruit pericarp (HFD-HYPE group) had no significant effect on reducing serum ALT, AST, NEFA, and TG levels in high-fat diet-induced obese mice.

(3) Effect on mouse liver TG levels

Results of changes in hepatic TG levels in db/db mice with type 2 diabetes mellitus: (

n-9) and the results of changes in hepatic TG levels in obese mice resulting from high fat diet (

n-12) is shown in table 5.

TABLE 5

Note: in contrast to the set of DBMs,^*P<0.05; in contrast to the DB group,^#P<0.05. in contrast to the CON group,^△P<0.05; in contrast to the HFD group,^▲P<0.05。

as can be seen from table 5, liver TG levels of db/db mice with type 2 diabetes and obese mice induced by high fat diet were significantly increased compared to the normal control group (i.e., DBM group or CON group); compared with DB group or HFD group, the thoroughfare bitter orange dry powder, the thoroughfare bitter orange alcohol extract and the thoroughfare bitter orange water extract can obviously reduce the liver TG level of DB/DB mice or fat mice induced by high fat diet. In contrast, alcohol extracts of mature grapefruit fruit and alcohol extracts of grapefruit pericarp were not effective in reducing hepatic TG levels in db/db type 2 diabetic mice or in obese mice induced by high fat diet.

(4) Influence on pathological morphology of liver tissue and quantity of red fat drops of liver of mouse

As shown in the results of fig. 1 and 2, the hepatocytes of the mice in the normal control group (DBM group or CON group) were intact, well-arranged, had clear liver lobules, and had no red lipid droplets; the liver tissue structure of a DB/DB type 2 diabetes mouse (DB group) or an obese mouse (HFD group) caused by high-fat diet is disordered, hepatic lobules are not clearly demarcated, the volume of hepatic cells is increased and the hepatic cells swell, and a large amount of large vacuolar vacuole-like changes appear in the hepatic cells and contain a large amount of red lipid drops, so that the liver fatty pathological changes of the obese mouse caused by the DB/DB type 2 diabetes mouse and the high-fat diet are shown, and the lipid accumulation is remarkably increased; and the symptoms of mouse liver structural disorder, hepatocyte volume increase, hepatocyte vacuole sample change and the like are obviously improved or relieved, and the liver lipid accumulation is obviously reduced. In contrast, the effect of alcohol extracts of mature grapefruit fruit and of grapefruit pericarp was not significant in improving liver cell morphology in both model mice (results not shown).

From the above results, the thoroughfare type fructus aurantii dry powder, the thoroughfare type fructus aurantii alcohol extract and the thoroughfare type fructus aurantii water extract have the effects of reducing liver lipid accumulation of obese mice caused by spontaneous type 2 diabetes mellitus mice and high fat diet, or improving and/or relieving liver fatty lesion. The specific expression is in the aspects of reducing weight and liver weight, reducing ALT, AST, NEFA and TG levels of serum, reducing TG level of liver, improving hepatocyte morphology, reducing the number of red lipid drops and the like.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

1. The application of thoroughfare bitter orange, thoroughfare bitter orange extract or compositions containing the thoroughfare bitter orange extract in preparing products for preventing and/or treating metabolic liver diseases, particularly non-alcoholic fatty liver diseases.

2. The application of thoroughfare bitter orange, thoroughfare bitter orange extract or a composition containing thoroughfare bitter orange in preparing products for reducing liver lipid accumulation or improving and/or relieving fatty lesions of the liver.

3. The use of claim 1 or 2, wherein the thoroughfare bitter orange extract is an alcohol extract of thoroughfare bitter orange or a water extract of thoroughfare bitter orange.

4. Use according to claim 3, wherein the alcohol is ethanol.

5. The use of any one of claims 1 to 4, wherein the thoroughfare bitter orange extract is prepared by any one of the extraction methods of heating reflux extraction, ultrasonic extraction, supercritical fluid extraction and macroporous resin adsorption.

6. The use of claim 5 wherein the thoroughfare bitter orange extract is thoroughfare bitter orange alcohol extract prepared by heating reflux extraction, the extraction method comprises:

7. The use of claim 6, wherein the alcohol solvent is used in an amount of 10 to 20 times per kilogram of thoroughfare bitter orange;

preferably, the concentration of the alcohol solvent is 70 to 90% by weight.

8. The use as claimed in claim 6 or 7, wherein, in step (1), the thoroughfare bitter orange pulverized product is immersed in the alcohol solvent for 0.5-2 hours before the reflux extraction.

9. Use according to claim 1 or 2, wherein the product is a medicament, a nutraceutical or a food product.

10. The use according to claim 9, wherein the pharmaceutical or nutraceutical is a tablet, capsule, powder, oral liquid, granule or pill.