CN115400165A - Eucalyptus leaf extract, preparation method and application thereof in preparation of weight-losing and lipid-lowering products - Google Patents

Abstract

The invention provides a eucalyptus leaf extract, a preparation method and its application in the preparation of weight-loss and lipid-lowering products, belonging to the technical field of plant extraction. The invention solves the problems of low extraction rate of effective components of eucalyptus leaves, poor activity of extraction products and the like in the prior art by adopting processes such as multi-solvent low-temperature continuous phase change extraction and column chromatography separation. The preparation method of the eucalyptus leaf extract of the present invention can realize high impurity removal rate, simple operation steps and high yield. The eucalyptus leaf extract obtained by the preparation method of the invention has higher polyphenol content and higher total yield, and has a good weight loss and fat-lowering effect, can effectively control body weight, regulate blood lipid levels, and effectively prevent and improve obesity. In addition, eucalyptus resources are abundant and cheap, and the preparation method is simple and low in cost, which meets the requirements of industrial production and has practical significance and application value for development as medicine or health care products. Therefore, the present invention also improves the utilization rate of eucalyptus resources.

Description

A kind of eucalyptus leaf extract, its preparation method and its application in the preparation of weight-loss and lipid-lowering products application

technical field

The invention relates to the technical field of plant extraction, in particular to a eucalyptus leaf extract, a preparation method and its application in the preparation of weight-loss and lipid-lowering products.

Background technique

In recent years, with the continuous improvement of living standards, the incidence of nutritional obesity caused by unhealthy work and rest and eating patterns is increasing day by day. According to reports, nutritional obesity will increase the risk of hypertension, type 2 diabetes, coronary heart disease and cancer, which will greatly affect the health of the body. Therefore, how to effectively prevent and treat obesity has become one of the public safety and health issues worldwide. Although weight-loss drugs can effectively control weight, long-term use may cause adverse effects on the body. Therefore, the market demand for developing safe, non-toxic and non-toxic functional natural products for weight loss and lipid reduction is gradually released, and the future development prospects of the industry are good.

Our country is rich in eucalyptus resources, which have the characteristics of large area, wide area and many varieties. As a by-product of eucalyptus, eucalyptus has been used as a traditional Chinese herbal medicine, and is often used for clearing away heat, detoxifying and treating colds. Existing studies have shown that eucalyptus leaf extract contains a large amount of polyphenols, which show strong antioxidant activity in vivo and in vitro, and a series of effects such as anti-aging, anti-tumor, anti-diabetes, and anti-inflammation have also been reported, but The existing methods such as chemical method and enzymatic method have low extraction rate of active components of eucalyptus leaves, and the activity of the extracted product is poor, which limits its application in industry. At present, there are no relevant reports on the prevention and treatment of obesity using eucalyptus leaf extract.

Contents of the invention

The object of the present invention is to provide a eucalyptus leaf extract, a preparation method and its application in the preparation of weight loss and lipid-lowering products, which solves the problems of low extraction rate of active ingredients of eucalyptus leaf and poor activity of the extracted product in the prior art. .

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a kind of preparation method of eucalyptus leaf extract, comprises the following steps:

Pulverizing eucalyptus leaves to obtain eucalyptus leaf powder;

The eucalyptus leaf powder is subjected to continuous phase change extraction to obtain an extract;

Concentrating and spray-drying the extract in sequence to obtain the crude extract of eucalyptus leaves;

purifying the crude extract of eucalyptus to obtain an extract of eucalyptus;

The extractant used in the continuous phase change extraction is butane, ethanol aqueous solution and water in sequence.

Preferably, the particle size of the eucalyptus leaf powder is below 550 μm.

Preferably, the extraction pressure of the continuous phase change extraction is 0.5-1.5 MPa, the extraction temperature is 55-65 °C, the analysis temperature is 65-75 °C, and the extraction time of each extractant is 80-120 min.

Preferably, the purification adopts silica gel column separation, and the mesh number of the silica gel column used for the silica gel column separation is 100-300 mesh.

Preferably, gradient elution is adopted in the separation process of the silica gel column, and the elution flow rate of the gradient elution is 30-50 ml/min.

Preferably, the eluent used for the silica gel column separation includes component A and component B;

The component A is ethanol, and the component B is ethyl acetate or petroleum ether.

Preferably, in the process of gradient elution, the volume ratio of component A and component B is 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60: 40, 70:30, 80:20, 90:10, 100:0, after the end of the gradient elution, collect the elution with the volume ratio of component A and component B at 60:40, 70:30, 80:20 product;

Each volume ratio of the eluent elutes 2 to 4 column volumes.

Preferably, the eluted product is concentrated and spray-dried to obtain eucalyptus leaf extract.

The present invention also provides the eucalyptus leaf extract prepared by the above preparation method, including 10-20% of evening primrose B, 1-2% of trimarin I, 1-1.5% of quercetin-3-O-glucuronide %, 0.8-1.2% gallic acid, 0.2-0.5% tremasin Ⅱ, 3-6% 1,2,3,4,6 pentagalloyl glucose, the total phenol content in the eucalyptus leaf extract is 50% ~60%.

The present invention also provides the application of the above eucalyptus leaf extract in the preparation of weight-loss and lipid-lowering products.

Technical effect and advantage of the present invention:

The preparation method of the eucalyptus leaf extract of the present invention can realize high impurity removal rate, simple operation steps and high yield. The eucalyptus leaf extract obtained by the preparation method of the invention has higher polyphenol content and higher total yield, and is suitable for practical industrial application. It is found through testing that the eucalyptus leaf extract obtained by the present invention has a good weight-loss and lipid-lowering effect, can effectively control body weight, regulate blood lipid levels, and effectively prevent and improve obesity. In addition, eucalyptus leaf resources are abundant and cheap, and the preparation method is simple and low-cost, which meets the requirements of industrial production. It has practical significance and application value for development as medicine or health care products, and also improves the utilization rate of eucalyptus leaf resources to a certain extent.

Description of drawings

Fig. 1 is liquid chromatography analysis result;

Fig. 2 is the measurement result of total triglyceride content in mouse serum;

Fig. 3 is the measurement result of total cholesterol content in mouse serum;

Fig. 4 is the determination result of low-density lipoprotein content in mouse serum;

Fig. 5 is the measurement result of high-density lipoprotein content in mouse serum.

Detailed ways

The present invention provides the present invention provides a kind of preparation method of eucalyptus leaf extract, comprises the following steps:

Pulverizing eucalyptus leaves to obtain eucalyptus leaf powder;

The eucalyptus leaf powder is subjected to continuous phase change extraction to obtain an extract;

Concentrating and spray-drying the extract in sequence to obtain the crude extract of eucalyptus leaves;

purifying the crude extract of eucalyptus to obtain an extract of eucalyptus;

The extractant used in the continuous phase change extraction is butane, ethanol aqueous solution and water.

In the present invention, the eucalyptus leaves are preferably pulverized after drying in the shade to obtain eucalyptus leaf powder, the particle size of the eucalyptus leaf powder is preferably below 550 μm, more preferably below 350 μm; the eucalyptus leaf powder is subjected to continuous phase change extraction, The continuous phase-change extraction is preferably carried out using a continuous phase-change extraction tank, the extraction pressure of the continuous phase-change extraction is preferably 0.5-1.5 MPa, more preferably 0.8-1.2 MPa, and the extraction temperature is preferably 55-65°C, and more preferably is 58～62°C, the analysis temperature is preferably 65～75°C, more preferably 68～72°C, and the extraction time is preferably 80～120min, more preferably 90～110min; the extraction used in the continuous phase change extraction The solvents are butane, ethanol aqueous solution and water, and the present invention has no requirement on the order of extraction, and the volume concentration of the ethanol aqueous solution is preferably 60-80%.

In the present invention, the concentration is preferably vacuum concentration, the temperature of the vacuum concentration is preferably 55-70°C, the vacuum degree is preferably 300-500Pa, and the air inlet temperature of the spray drying is preferably 200-70°C. 250°C, the feed rate is preferably 80-120 rpm, and the atomizer frequency is preferably 40-50 Hz.

In the present invention, the purification preferably adopts silica gel column separation, and the mesh number of the silica gel column used for the silica gel column separation is preferably 100-300 mesh, more preferably 150-250 mesh; Gradient elution, the elution flow rate of the gradient elution is preferably 30-50ml/min, more preferably 35-45ml/min; the eluent used for the silica gel column separation of the present invention preferably includes component A and component B, the component A is preferably ethanol, and the component B is preferably ethyl acetate or petroleum ether; in the process of gradient elution, the volume ratio of component A and component B is preferably sequentially 0:100, 10 :90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, after the gradient elution ends, collect fraction A and The volume ratio of component B is the elution product of 60:40, 70:30, 80:20; in the present invention, the eluent of each volume ratio is preferably eluted in 2 to 4 column volumes, after the gradient elution ends , preferably the eluted product is concentrated and spray-dried to obtain eucalyptus leaf extract, the concentration is preferably vacuum concentration, the temperature of the vacuum concentration is preferably 65-85°C, and the degree of vacuum is preferably 400-600Pa. The air inlet temperature of the spray drying is preferably 250-300° C., the feeding speed is preferably 100-150 rpm, and the atomizer frequency is preferably 50-60 Hz.

The present invention also provides the eucalyptus leaf extract prepared by the above preparation method.

The present invention also provides the application of the above eucalyptus leaf extract in the preparation of weight-loss and lipid-lowering products, and the weight-loss and lipid-lowering products are preferably health care products or medicines; the product preferably uses the eucalyptus leaf extract of the present invention as the only active ingredient; Said product preferably also includes adjuvant, the present invention has no special limitation to the kind of said adjuvant, just adopt the conventional product adjuvant in this field; The dosage form of the product described in the present invention is preferably powder, tablet, granule, capsule , solution, emulsion, suspension, injection, spray, aerosol or powder mist, etc. In the present invention, the product includes a carrier, and the carrier is preferably a binder, a lubricant, a disintegrant, a solubilizer, a diluent, a stabilizer, a suspending agent, a pigment, a flavoring agent, etc. for oral preparations; Preservatives, solubilizers, stabilizers, etc. for injectable preparations; bases, diluents, lubricants, preservatives, etc. for topical preparations.

The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.

Example 1

The eucalyptus leaf raw material of "Guanglin No. 9" was cooled and dried, and then mechanically crushed to 30 meshes. Weighed 20kg of eucalyptus leaf powder and put it into a continuous phase change extraction kettle for sealed extraction. The extraction pressure was 1.0 MPa and the extraction temperature was 65 °C, the analysis temperature is 75 °C, sequentially use butane, 70% ethanol, and water as the extractant, and the extraction time is 100 min each time to obtain the eucalyptus leaf extract. The eucalyptus leaf extract was concentrated under reduced pressure at 60°C and a vacuum of 400Pa, and then spray-dried under the conditions of an inlet air temperature of 200°C, a feed rate of 100rpm, and an atomizer frequency of 45Hz to obtain crude eucalyptus leaves. Extract. The crude extract of eucalyptus leaves was passed through a 100-mesh silica gel column, and the elution flow rate was 40ml/min. Gradient elution was carried out with ethanol and ethyl acetate. The volume ratio of ethanol and ethyl acetate was 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, each ratio eluted with 3 column volumes, at 70℃, Under the condition of 500Pa, vacuum concentrate the eluent with volume ratio of ethanol and ethyl acetate of 60:40, 70:30, 80:20, the inlet air temperature is 250℃, the feeding speed is 100rpm, and the nebulizer frequency is 50Hz. Spray-dry to obtain the eucalyptus extract.

Example 2

The "Guanglin No. 9" eucalyptus leaf raw material was cooled and dried, and then mechanically crushed to 40 mesh. Weighed 15kg of eucalyptus leaf powder and put it into a continuous phase change extraction kettle for sealed extraction. The extraction pressure was 1.2MPa and the extraction temperature was 60°C. °C, the analysis temperature is 70 °C, sequentially use butane, 65% ethanol, and water as the extractant, each extraction time is 120min, and collect the eucalyptus leaf extract. The eucalyptus leaf extract was concentrated under reduced pressure at 65° C. and a vacuum of 500 Pa, and then spray-dried at an inlet air temperature of 250° C., a feed rate of 150 rpm, and an atomizer frequency of 50 Hz. The crude extract of eucalyptus leaves was passed through a 200-mesh silica gel column, and the elution flow rate was 35ml/min. Gradient elution was carried out with ethanol and ethyl acetate. The volume ratio of ethanol and ethyl acetate was 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, each ratio eluted with 3 column volumes, at 70℃, Under the condition of 500Pa, vacuum concentrate the eluent with volume ratio of ethanol and ethyl acetate of 60:40, 70:30, 80:20, the inlet air temperature is 300℃, the feeding speed is 150rpm, and the nebulizer frequency is 60Hz. Spray-dry to get the eucalyptus extract.

Example 3

The eucalyptus leaf raw material of "Guanglin No. 9" was cooled and dried, and then mechanically crushed to 60 mesh. Weighed 15kg of eucalyptus leaf powder and put it into a continuous phase change extraction kettle for sealed extraction. The extraction pressure was 1.2MPa and the extraction temperature was 65 °C, the analysis temperature was 75 °C, butane, 80% ethanol, and water were used as extraction agents respectively, and the extraction time was 120 min each time to collect the eucalyptus leaf extract. The eucalyptus leaf extract was concentrated under reduced pressure at 60°C and a vacuum of 400Pa, and then spray-dried under the conditions of an inlet air temperature of 200°C, a feed rate of 100rpm, and an atomizer frequency of 45Hz to obtain crude eucalyptus leaves. Extract. The crude extract of eucalyptus leaves was passed through a 200-mesh silica gel column, and the elution flow rate was 35ml/min. Gradient elution was carried out with ethanol and petroleum ether. The volume ratio of ethanol and petroleum ether was 0:100, 10:90, 20: 80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, each ratio eluted with 3 column volumes, at 70℃, 500Pa Under the conditions of vacuum concentration ethanol and ethyl acetate volume ratio of 60:40, 70:30, 80:20 eluate, spray drying under the conditions of air inlet temperature 250 ℃, feed speed 100rpm, atomizer frequency 50Hz , that is, eucalyptus extract.

Experimental Example 1 Liquid Chromatography Analysis

The eucalyptus extract obtained in Example 1 is subjected to liquid chromatography analysis, and the liquid phase conditions are: Diamonsil C18 chromatographic column (250 × 4.6mm, 5 μm); detection wavelength 270nm; mobile phase is a gradient of 10%-90% methanol Elution was performed for 1 hour, and the flow rate was 1 mL/mim; the injection volume was 20 μL.

The results are shown in Figure 1, in which: 1. Gemin D; 2. Evening primrose B; 3. 5-methoxyfurfural; 4. Quercus ellagitannin; 5. Trimazin Ⅰ; 6. Chlorogenic acid; 7. Terimazol Ⅱ; 8. 1,2,3,6-tetragalloyl glucose; 9. Ethyl gallate; 10. 1,2,3,4,6-pentagalloyl glucose ; 11. Quercetin-3-O-glucuronide.

Experimental example 2 extraction rate, total phenol content determination

The eucalyptus leaf extract quality in embodiment 1 and embodiment 2 is measured, and the extraction rate is calculated (extraction rate=eucalyptus leaf essence extract quality/eucalyptus leaf powder quality×100%);

The total phenol content was detected by the Folin phenol method, and the results are shown in Table 1 below:

Table 1 Extraction Results of Eucalyptus Leaf Extract

Quality of eucalyptus extract Extraction rate Total phenol content Example 1 11.2kg 56% 55% Example 2 9.0kg 60% 50%

Experimental Example 3 Research on Weight Loss and Fat-lowering Effects

Experimental animals: male, SPF grade C57BL/6J mice, weighing about (23 ± 2g), purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.;

Test drugs and reagents: eucalyptus leaf extract, total phenol content about 55%; normal saline; etc. kits; total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-C), high-density lipoprotein Protein (HDL-C) and other kits;

Feed: 60% high-fat model feed and 10% low-fat control feed were purchased from Nantong Trophy Feed Technology Co., Ltd.;

Experimental method: After 7 days of adaptation, 32 C57BL/6J mice were divided into four groups, which were normal control group, model control group, low-dose eucalyptus extract group (50mg/kg) and high-dose eucalyptus extract group (100mg/kg). The normal control group was fed with 10% low-fat control feed, while the model group and low-dose and high-dose eucalyptus extract groups were fed with 60% high-fat model feed, and the feeding period was 10 weeks. From the 1st day, the mice in the normal control group and the model control group were given intragastric administration of normal saline, and the mice in the low-dose and high-dose eucalyptus extract groups were given intragastric administration of 50 mg/kg and 100 mg/kg eucalyptus extract. After the feeding period, the mice were sacrificed, and the serum, organs, and fat were collected and stored at -80°C for testing.

The body weight, food intake, organ coefficient, fat coefficient, serum lipid level, etc. of the mice were measured in the experiment, and the results are as follows. All experimental data were analyzed using SPSS 17.0 statistical software. One-way ANOVA was used for comparison among multiple groups, and Duncan's method was used for pairwise comparison. The results were expressed as mean ± standard deviation, and p<0.05 was considered a significant difference.

During the experiment, the body weight of the mice in each group was weighed and recorded every week, and the body weight and weight gain of the mice in each group are shown in Table 2.

Table 2 Mouse body weight gain (g)

Note: The data in the table are the sample mean ± standard deviation; compared with the normal control group, **p<0.01, compared with the model control group ##p<0.05.

It can be seen from Table 2 that the weight growth rate of the mice in the model control group was significantly higher than that in the normal control group, indicating that the mouse obesity model was successfully established. Compared with the model control group, the weight gain of the mice in the low-dose and high-dose groups of eucalyptus leaf extract was reduced, and the weight loss effect of the high-dose group was extremely significant. The results show that taking a certain dose of eucalyptus extract can effectively inhibit the body obesity caused by high-fat diet, and has a good weight loss effect.

In the experiment, the epididymal fat, inguinal fat, perirenal fat, brown fat and liver of mice in each group were weighed, and their indexes were calculated. The results are shown in Table 3.

Table 3 mouse liver coefficient and fat coefficient (%)

Note: The data in the table are the sample mean ± standard deviation; compared with the normal control group, **p<0.01, compared with the model control group ##p<0.05.

It can be seen from Table 3 that the liver coefficient of mice in low and high dose groups of eucalyptus leaf extract was significantly lower than that of the model control group, indicating that eucalyptus leaf extract can relieve liver congestion, swelling, hypertrophy and hyperplasia caused by obesity, and reduce the degree of fatty liver. In addition, compared with the model control group, the three white fat coefficients of the mice in the eucalyptus extract low- and high-dose groups, including inguinal fat, epididymis fat, and perirenal fat, were significantly reduced, reflecting that eucalyptus extract can effectively reduce the amount of fat in the body. The accumulation of fat plays a role in weight loss.

The contents of total triglyceride, total cholesterol, low-density lipoprotein and high-density lipoprotein in mouse serum were detected, and the results are shown in Figures 2-5 below.

It can be seen from Figures 2 to 5 that compared with the high-fat diet control group, the total triglyceride, total cholesterol, and low-density lipoprotein in the serum of the mice in the low- and high-dose eucalyptus extract groups were significantly reduced, and the serum HDL-C The level increased significantly (P<0.01), indicating that the eucalyptus leaf extract in the present invention can effectively improve the body lipid metabolism disorder induced by a high-fat diet, and has a good lipid-lowering effect.

It can be seen from the above examples that the eucalyptus leaf extraction method provided by the present invention has a high extraction rate, and the obtained eucalyptus leaf extract has a high total phenol content and a wide variety of active substances. And the eucalyptus leaf extract obtained by the preparation method of the present invention can effectively inhibit body obesity caused by high-fat diet, has a good weight loss effect, can relieve liver congestion and swelling caused by obesity, hypertrophy and hyperplasia, alleviate the degree of fatty liver, and can effectively reduce The accumulation of fat in the body has the effect of losing weight, and can also effectively improve the lipid metabolism disorder of the body induced by a high-fat diet, and has a good lipid-lowering effect.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (10)

1. a preparation method of Eucalyptus leaf extract, is characterized in that, comprises the steps: Pulverizing eucalyptus leaves to obtain eucalyptus leaf powder; The eucalyptus leaf powder is subjected to continuous phase change extraction to obtain an extract; Concentrating and spray-drying the extract in sequence to obtain the crude extract of eucalyptus leaves; purifying the crude extract of eucalyptus to obtain an extract of eucalyptus; The extractant used in the continuous phase change extraction is butane, ethanol aqueous solution and water in sequence. 2. the preparation method of eucalyptus leaf extract according to claim 1, is characterized in that, the particle diameter of described eucalyptus leaf powder is below 550 μm. 3. The preparation method of eucalyptus leaf extract according to claim 2, characterized in that the extraction pressure of the continuous phase change extraction is 0.5-1.5 MPa, the extraction temperature is 55-65 °C, and the analysis temperature is 65-75 °C. °C, the extraction time of each extractant is 80-120min. 4. The preparation method of eucalyptus leaf extract according to claim 3, characterized in that, said purification adopts silica gel column separation, and the mesh number of the silica gel column used for said silica gel column separation is 100-300 mesh. 5 . The preparation method of eucalyptus leaf extract according to claim 4 , characterized in that, gradient elution is adopted in the separation process of the silica gel column, and the elution flow rate of the gradient elution is 30˜50 ml/min. 6. the preparation method of Eucalyptus leaf extract according to claim 5, is characterized in that, the eluent that described silica gel column separation adopts comprises component A and component B; The component A is ethanol, and the component B is ethyl acetate or petroleum ether. 7. the preparation method of Eucalyptus leaf extract according to claim 6 is characterized in that, in the process of described gradient elution, the volume ratio of component A and component B is successively 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, after the end of gradient elution, collect fraction A and fraction B The volume ratio is the elution product of 60:40, 70:30, 80:20; Each volume ratio of the eluent elutes 2 to 4 column volumes. 8. The preparation method of the eucalyptus extract according to claim 7, characterized in that, the eluted product is concentrated and spray-dried to obtain the eucalyptus extract. 9. The eucalyptus leaf extract prepared by the preparation method described in any one of claims 1 to 8 is characterized in that it comprises 10-20% of evening primrose B, 1-2% of trimarin I, quercetin -3-O-glucuronide 1~1.5%, gallic acid 0.8~1.2%, tremasin II 0.2~0.5%, 1,2,3,4,6 pentagalloyl glucose 3~6%, all The total phenol content in the eucalyptus leaf extract is 50-60%. 10. the application of the eucalyptus leaf extract described in claim 9 in the preparation of fat-reducing products for weight loss.

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