Syzygium jambos extract, preparation method thereof and application of syzygium jambos extract in obesity combined with fatty liver

Abstract

The invention provides a syzygium jambos extract, a preparation method thereof and application of the syzygium jambos extract in obesity combined with fatty liver. The preparation method comprises the following steps: (1) cutting the syzygium jambos into slices, and freeze-drying to obtain freeze-dried syzygium jambos; and (2) adding the freeze-dried syzygium jambos prepared in the step (1) into ethanol for soaking, performing ultrasonic-assisted extraction, soaking overnight, performing suction filtration and extraction next day, repeating the suction filtration and extraction operations for three times, combining extracting solutions, and performing vacuum concentration to obtain the syzygium jambos extract. The syzygium jambos extract prepared by the invention can obviously reduce the relative weight growth rate and liver weight of mice so as to achieve the purpose of effectively losing weight, can be used in various fields of medicines, health care products and the like, and has great economic and social values.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical chemistry, and in particular to a Syzygium wilfordii extract, a preparation method thereof and application thereof in obesity combined with fatty liver. Background Art

[0002] Obesity is a chronic metabolic disease. As changes in lifestyle and age bring about changes in hormone levels in the body, the probability of an individual becoming obese increases. According to global health statistics, the proportion of obese people is increasing year by year. The obesity problem has a huge impact on national health. Obesity not only increases people's risk of cardiovascular and cerebrovascular diseases, diabetes and various cancers, but is also one of the top five risk factors for death worldwide. Every year, tens of thousands of people die from obesity and its complications.

[0003] Obesity significantly affects people's health. Related studies have shown that obese people have abnormal oxygen metabolism, increased maximum oxygen consumption, decreased blood oxygen utilization, reduced maximum tolerance time and vital capacity per kilogram of body weight, and impaired cardiopulmonary reserve function. With the increase in obesity and the extension of the course of the disease, glucose tolerance may decrease and insulin levels may increase. In the early stage, it is often manifested as increased blood sugar after meals, and in the later stage, it may affect fasting blood sugar and is often accompanied by insulin resistance, which significantly increases the incidence of diabetes. Obese patients are prone to significant lipid metabolism disorders, which are often manifested as increased triglycerides, cholesterol, and very low-density lipoprotein cholesterol, leading to fatty liver, hyperlipidemia, etc.

[0004] It is worth noting that there is a close connection between obesity and metabolic dysfunction-associated fatty liver disease (MASLD, formerly known as non-alcoholic fatty liver disease NAFLD). Due to excessive energy intake and insufficient energy expenditure, the excess energy in obese patients will be converted into fat. As an important organ of metabolism, the liver can easily become a place for fat accumulation, thus forming metabolic dysfunction-associated fatty liver disease (MASLD). MASLD has become a serious public health problem worldwide, and its prevalence continues to rise, from 25% in 2016 to more than 30% at present. Some studies even show that the global incidence of MASLD is as high as 32.4%. This disease not only affects nearly a quarter to a third of the world's population, but there are still a large number of potential cases that have not been diagnosed.

[0005] The treatment and management of MASLD mainly include lifestyle intervention and drug therapy. Lifestyle intervention includes weight loss, diet adjustment, increased exercise and abstinence from alcohol, which is the basis for the prevention and treatment of MASLD. In terms of drug treatment, some drugs for MASLD have shown efficacy in clinical trials, such as thyroid hormone receptor β agonist Resmetirom (Rezdiffra), farnesoid X receptor (FXR) agonist, peroxisome proliferator-activated receptor (PPAR) agonist, etc. However, the overall treatment results are still unsatisfactory, mainly due to the heterogeneity of MASLD disease and the challenges of new drug development.

[0006] The main characteristic of obesity is excessive fat tissue in the body, while fatty liver is caused by abnormal accumulation of fat in the liver. Although traditional treatments, such as diet control and exercise, can reduce weight and body fat to a certain extent, these methods may be difficult to adhere to or ineffective for some patients. Therefore, it is particularly important to find a safe and effective alternative treatment. Summary of the invention

[0007] Based on the above, the object of the present invention is to provide a Syzygium wilfordii extract, a preparation method thereof and an application thereof in obesity combined with fatty liver.

[0008] The technical solution adopted by the present invention to achieve the technical purpose is:

[0009] In a first aspect of the present invention, a method for preparing a Syzygium wilfordii extract is provided, comprising the following steps:

[0010] (1) cutting the syzygium wilfordii into thin slices and then freeze-drying them to obtain freeze-dried syzygium wilfordii;

[0011] (2) The lyophilized Syzygium wilfordii prepared in step (1) is soaked in ethanol, subjected to ultrasonic-assisted extraction and soaked overnight, and subjected to suction filtration and extraction the next day. The above suction filtration and extraction operation is repeated 3 times, the extracts are combined, and the Syzygium wilfordii extract is obtained after reduced pressure concentration.

[0012] Preferably, the freeze-drying conditions in step (1) are: temperature of -50°C, vacuum degree of 10 Pa, and time of 72 hours.

[0013] Preferably, step (1) further comprises cutting the syzygium wilfordii into thin slices, placing the slices in a sealed bag and pre-freezing the bag in a -80°C refrigerator for 24 hours, and then taking the slices out and placing the slices in a freeze dryer for freeze drying.

[0014] Preferably, in step (2), the liquid ratio (g / mL) of lyophilized Syzygium wilfordii to ethanol is 1:8-1:15; and the ethanol used is 75% ethanol.

[0015] More preferably, in step (2), the solid-liquid ratio (g / mL) of lyophilized Syzygium wilfordii to ethanol is 1:10.

[0016] Preferably, the ultrasonic frequency of the ultrasonic-assisted extraction in step (2) is 35 kHz and the time is 20 min.

[0017] In the second aspect of the present invention, a Syzygium wilfordii extract is provided, which is prepared by the above-mentioned preparation method.

[0018] In the third aspect of the present invention, there is provided use of the Syzygium wilfordii extract in the preparation of a medicament for treating obesity combined with fatty liver.

[0019] Preferably, the Syzygium wilfordii extract can reduce body weight and subcutaneous fat weight.

[0020] Preferably, the Syzygium wilfordii extract can significantly reduce liver tissue weight.

[0021] Preferably, the Syzygium wilfordii extract can improve liver steatosis, vacuolar degeneration and lipid accumulation, and reduce the size of adipocytes.

[0022] The present invention has the following advantages:

[0023] (1) The preparation method of the present invention is simple and easy to operate, and has low operating costs. The extraction solvents used in the preparation method of the present invention are only ethanol and water, which can be recycled and reused, and there is no risk of organic reagent residues. The natural quality of the syzygium wilfordii extract is ensured while being environmentally friendly, and belongs to a green manufacturing technology.

[0024] (2) The Syzygium wilfordii extract of the present invention can significantly reduce the relative weight growth rate and liver weight of mice, thereby achieving the purpose of effective weight loss. It can be used in various fields such as medicines and health products, and has great economic and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph showing the effect of the Syzygium wilfordii extract of the present invention on the body weight of HFC mice;

[0026] Figure 2 This is a graph showing the effect of the Syzygium wilfordii extract of the present invention on subcutaneous adipose tissue in HFC mice;

[0027] Figure 3 This is a graph showing the effect of the Syzygium wilfordii extract of the present invention on the liver weight of HFC mice;

[0028] Figure 4 The figure shows the effect of the Syzygium wilfordii extract of the present invention on the liver tissue of HFC mice, and the figure shows the changes in the lipid level and histopathology of the liver tissue observed by HE staining and Oil Red O staining under an optical microscope. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0030] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0031] Example

[0032] 1. Preparation of freeze-dried Syzygium wilfordii

[0033] 3 kg of Syzygium wilfordii slices were placed in a sealed bag and pre-frozen in a -80°C refrigerator for 24 hours, then taken out and placed in a freeze dryer for freeze drying at a temperature of -50°C and a vacuum degree of 10 Pa for 72 hours to obtain Syzygium wilfordii freeze-dried product.

[0034] 2. Preparation of 75% ethanol extract of Syzygium wilfordii

[0035] Weigh 400.0 g of freeze-dried Syzygium wilfordii, add 4 liters of 75% ethanol to fully soak the freeze-dried Syzygium wilfordii, set the ultrasonic frequency to 35 kHz, and perform assisted extraction for 20 minutes. Then, soak it at room temperature (about 25°C) overnight. The next day, use a 1L suction funnel device to perform filtration and extraction. Repeat the above operation three times, then combine the extracts, and use a rotary evaporator to reduce the pressure and concentrate the extracts to obtain a 75% ethanol extract of Syzygium wilfordii. The temperature is 45°C, and the vacuum degree is gradually reduced from 200 hPa to 5 hPa.

[0036] 3. Effect of 75% ethanol extract of Syzygium wilfordii on obesity combined with fatty liver disease

[0037] In this example, 75% ethanol extract of Syzygium wilfordii was used as a test object, and an in vivo validation experiment was conducted on a MASLD mouse model induced by a high-fat, high-cholesterol diet.

[0038] 1. Experimental methods:

[0039] 1.1 Preparation of dosing solution

[0040] The 75% ethanol extract of Syzygium wilfordii prepared in the above step 2 was dissolved in pure water to prepare Syzygium wilfordii extract solutions with concentrations of 20 mg / mL and 40 mg / mL, and the solutions were thoroughly mixed and dissolved.

[0041] A 1 mg / mL positive drug rosiglitazone solution was prepared with a physiological saline solution containing 2% DMSO and 5% Tween 80, and the solution was thoroughly mixed and dissolved.

[0042] 1.2 Grouping and establishment of MAFLD model

[0043] After the animals were purchased and adapted for one week, they entered the experimental state. 6-8 week old male C57BL / 6J mice (weight 18-20 grams) were used as the subjects, and 40 mice were randomly divided into 5 groups, normal control group, HFC model control group (high fat and high cholesterol group), positive drug rosiglitazone control group (10mg / kg), Syzygium wilfordii high-dose group (400mg / kg), Syzygium wilfordii low-dose group (200mg / kg), 8 mice in each group. The mice fed with normal feed continued to be fed with ordinary feed, and the mice in the HFC model control group, Syzygium wilfordii high / low-dose group and positive drug rosiglitazone control group continued to be fed with high fat and high cholesterol feed (HFC, containing 60% fat and 1% cholesterol).

[0044] 1.3 Grouped medication

[0045] The drug administration was started at the same time as the experimental modeling. The Syzygium syzygium extract solution prepared in step (2) was administered to the Syzygium syzygium administration group at a dose of 200 mg / kg and 400 mg / kg respectively by gavage, and the positive drug rosiglitazone control group was administered with a positive drug solution prepared in step (2) at a dose of 10 mg / kg by intraperitoneal injection, and the control group was treated with a solvent. The treatment was performed once a day, and the food intake and body weight of the mice were observed and recorded. The dosage was adjusted according to the change in body weight, and the drug administration was continued for 8 weeks. During the drug administration period, except for the normal group, the mice in each group continued to be modeled.

[0046] 1.4 Experimental Animal Treatment

[0047] At the end of the 8th week, the animals were fasted but not watered for 12 hours after the administration and weighed. All mice were anesthetized for blood sampling, killed by cervical dislocation and dissected. The body weight and liver weight of mice in each group were recorded, and adipose tissue and liver tissue were collected and frozen in a -80℃ refrigerator for subsequent analysis and detection.

[0048] 1.5 Analysis of mouse liver tissue samples:

[0049] A certain amount of mouse liver tissue samples collected in 1.4 were weighed for histopathological analysis (H&E staining and Oil Red O staining) to evaluate the therapeutic effect of the extract on MASLD.

[0050] 2. Experimental results:

[0051] 2.1 Analysis of mouse body weight changes

[0052] like Figure 1 As shown, the body weight of mice in the HFC group was significantly higher than that in the normal control group (p<0.001); compared with the HFC group, treatment with Syzygium wilfordii extract significantly reduced the body weight of mice (p<0.01), and the reduction effect was dose-dependent. The above experimental results also showed that Syzygium wilfordii extract had a therapeutic effect on obesity in mice induced by a high-fat diet.

[0053] 2.2 Effect of Syzygium wilfordii extract on subcutaneous adipose tissue weight in mice

[0054] Results Figure 2 It can be seen that compared with the normal control group, the subcutaneous fat tissue in the HFC group increased significantly (P<0.001), and compared with the HFC group, the weight of subcutaneous fat tissue was significantly reduced after oral administration of Syzygium wilfordii extract. In addition, Syzygium wilfordii 400 mg / kg significantly reduced the weight of subcutaneous fat tissue (P<0.01), and the effect was dose-dependent. This shows that Syzygium wilfordii extract can significantly reduce the subcutaneous fat weight of mice.

[0055] 2.3 Effect of Syzygium wilfordii extract on liver weight in mice

[0056] Effects of Syzygium wilfordii extract on liver weight in mice Figure 3 As shown, compared with the normal control group, the liver weight of mice in the HFC group increased significantly (P<0.001). Compared with the HFC group, the liver weight of Syzygium wilfordii extract was significantly reduced after oral administration, and the content of adipose tissue in the liver was significantly reduced. In addition, Syzygium wilfordii 400 mg / kg significantly reduced the weight of liver tissue (P<0.05). The above experimental results also show that the inhibitory effect of Syzygium wilfordii extract on the liver weight of mice is significantly better than that of the positive drug rosiglitazone.

[0057] The liver weight of mice in the MASLD model group was greater than that in the Syzygium wilfordii extract group and the rosiglitazone group. Figure 4 As shown, the liver showed different degrees of milky white. In addition, the positive drug rosiglitazone had no therapeutic effect on the liver tissue of HFC mice; while the Syzygium wilfordii extract could effectively reduce the liver weight of HFC mice.

[0058] 2.4 Mouse liver section staining analysis

[0059] Results Figure 4 H&E staining and Oil Red O staining showed that the MASLD model mice induced by high fat had different degrees of fatty degeneration and vacuolar changes, and the vacuolar changes in the liver of mice treated with rosiglitazone were more serious. The results of Oil Red O staining showed that the fat content in the liver of the MASLD model mice induced by high fat increased significantly. The Syzygium wilfordii extract significantly improved the fatty degeneration, vacuolar changes and lipid accumulation in the liver of mice after treatment, and reduced the size of adipocytes, and the inhibitory effect on liver fat was better than that of the positive drug rosiglitazone.

[0060] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a Syzygium wilfordii extract, comprising the following steps: (1) cutting the syzygium wilfordii into thin slices and then freeze-drying them to obtain freeze-dried syzygium wilfordii; (2) soaking the lyophilized Syzygium wilfordii prepared in step (1) in ethanol, and soaking overnight after ultrasonic-assisted extraction. The next day, extraction was performed by filtration, and the above extraction operation was repeated 3 times. The extracts were combined and concentrated under reduced pressure to obtain the Syzygium wilfordii extract.

2. The method for preparing a Syzygium wilfordii extract according to claim 1, characterized in that: The freeze-drying conditions in step (1) are: temperature of -50°C, vacuum degree of 10Pa, and time of 72h.

3. The method for preparing a Syzygium wilfordii extract according to claim 1, characterized in that: Step (1) also includes cutting the syzygium wilfordii into thin slices, placing the slices in a sealed bag and pre-freezing the slices in a -80°C refrigerator for 24 hours, and then taking the slices out and placing the slices in a freeze dryer for freeze drying.

4. The method for preparing a Syzygium wilfordii extract according to claim 1, characterized in that: In step (2), the material-liquid ratio of lyophilized Syzygium wilfordii to ethanol is 1:8-1:15; the ethanol used is 75% ethanol.

5. The method for preparing a Syzygium wilfordii extract according to claim 1, characterized in that: The ultrasonic frequency of the ultrasonic-assisted extraction in step (2) is 35 kHz and the time is 20 min.

6. A Syzygium wilfordii extract prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the Syzygium wilfordii extract according to claim 6 in the preparation of a medicament for treating obesity combined with fatty liver.

8. The use according to claim 7, characterized in that: The Syzygium wilfordii extract can reduce body weight and subcutaneous fat weight.

9. The use according to claim 7, characterized in that: The Syzygium wilfordii extract can significantly reduce liver tissue weight.

10. The use according to claim 7, characterized in that: The Syzygium wilfordii extract can improve liver fatty degeneration, vacuolar degeneration and lipid accumulation, and reduce the size of fat cells.

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