CN116370536A

CN116370536A - Substituted flower flavone, preparation method thereof and application thereof in preparation of weight-losing medicine

Info

Publication number: CN116370536A
Application number: CN202211595719.5A
Authority: CN
Inventors: 申春燕; 马攀宇; 李晓怡; 王亚莉; 郎登琴
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2022-12-13
Filing date: 2022-12-13
Publication date: 2023-07-04

Abstract

The invention relates to a substituted flower flavone, a preparation method and application thereof in preparing weight-losing medicaments, wherein the substituted flower flavone (CAVAF) is prepared by extracting, separating, gradient eluting and screening the pollen of substituted flowers, and is the component with the highest flavone content of 30 percent. The invention discovers that CAVAF has certain effects of inhibiting weight gain, improving insulin resistance, inhibiting white fat cell growth and the like on fat mice induced by high-fat diet for the first time, and has lower dosage and lower toxicity in the effective dosage range.

Description

Substituted flower flavone, preparation method thereof and application thereof in preparation of weight-losing medicine

Technical Field

The invention relates to the technical field of extraction and separation of natural products, in particular to a substituted flower flavone, a preparation method thereof and application thereof in preparing a weight-losing medicament.

Background

Obesity is a chronic disease that is a health-damaging, abnormal or excessive accumulation of fat. Over the past three decades, the number of obese people has doubled. There are currently 20 hundred million overweight people in the world, and this number is still growing rapidly. Concerns about obesity are growing worldwide, not only because obesity affects one's appearance, but also causes various complications associated with obesity, including type 2 diabetes (T2 DM), hypertension, coronary atherosclerotic heart disease, neurodegenerative diseases, cancer, etc., with high incidence. Obesity is therefore recognized by organizations such as the united states medical community as a disease state and has become a global health problem. Clinical management of obesity has three main aspects: lifestyle intervention, weight-reducing medicine and weight-reducing operation. In clinical applications, viable therapeutic drugs such as orlistat, lorcaserin, phentermine and naltrexone are used for weight management as appetite suppressants, fat absorption inhibitors, thermogenic stimulants, and the like. However, the clinical therapeutic effect is not satisfactory due to side effects or serious complications (e.g., kidney injury). For example, long-term consumption of orlistat can have adverse effects on the intestines and stomach, such as diarrhea, flatulence, acute kidney injury, liver injury, urinary incontinence, etc.

The filth generation (Citrus aurantium L.var. Amara Engl) is a variety of Citrus plant lime, the filth generation flower is a dry product of flower buds, has good pharmacological action, contains more glycoside compounds such as cardiac glycoside and non-cardiac glycoside, is rich in vitamins, amino acids, cellulose, minerals, coumarin and the like, and is commonly used for treating qi depression, abdominal distention and pain, indigestion, nausea and vomiting and the like. The chemical components of the substituted flowers are various and mainly comprise flavonoids, alkaloids and volatile oil. The substituted flowers have the value of both medicine and food, are natural, safe, high-efficiency and small in side effect, are very likely to be developed into novel weight-losing medicaments, but the research on the substituted flowers at home and abroad at present mainly uses volatile oil compounds as main components, and the research reports on other effective components of the substituted flowers are almost absent. The seville orange flower flavone has rich content, mainly comprises hesperidin, neohesperidin, limonin and the like, but at present, the extraction methods of seville orange flower flavone are different, the extraction components are different, and the yield is low.

Disclosure of Invention

In order to overcome the defects and the shortcomings of the prior art, the invention aims to provide the substituted flower flavone, which is prepared by extracting, separating and gradient eluting the pollen of the substituted flower, wherein 30 percent of the eluting component with the highest flavone content is the substituted flower flavone.

The invention also provides a preparation method of the substituted flower flavone, which is characterized by comprising the following steps:

(1) Extracting:

pulverizing pollen, sieving, extracting with 80% ethanol under reflux, centrifuging, concentrating the supernatant under reduced pressure, and drying to obtain extract;

(2) Purifying:

dissolving the extract in water, standing, absorbing supernatant, adding into AB-8 macroporous resin column, sequentially washing AB-8 macroporous resin column with water, 30%, 50%, 70% and 95% ethanol solution, collecting and mixing the components, concentrating under reduced pressure, vacuum drying to obtain eluted component, and determining flavone content of the eluted component, wherein 30% of the eluted component with highest flavone content is substituted flower flavone.

Preferably, in the step (1), the heating reflux temperature is 70-80 ℃, the heating reflux time is 2-4 hours, and the heating reflux times are 2-4 times.

Preferably, in the step (1), the mass ratio of pollen to 80% ethanol is 1:15-1:30.

Preferably, in the step (1), the centrifugal speed is 4000-5000 r/min, the centrifugal time is 8-12 min, the drying temperature is 40-60 ℃, and the reduced pressure concentration temperature is 40-50 ℃.

Preferably, in the step (2), the concentration temperature under reduced pressure is 40-60 ℃, and the method for measuring the flavone content is NaNO ₂ -Al(NO ₃ ) ₃ NaOH colorimetry.

The invention also provides application of the daidzein prepared by the preparation method in preparation of weight-losing medicaments.

The invention also provides the application of the daidzein prepared by the preparation method in preparing medicines for inhibiting weight gain.

The invention also provides application of the substituted flower flavone prepared by the preparation method in preparation of the medicine for improving insulin resistance.

The invention also provides application of the daidzein prepared by the preparation method in preparation of medicines for inhibiting growth of white fat cells.

Compared with the prior art, the invention has the following advantages and effects:

(1) The invention obtains 30 percent of the component with the highest flavone content as the seville orange flavone (CAVAF) capable of effectively preventing and treating obesity through heating and reflux extraction of seville orange pollen, macroporous resin separation and gradient elution.

(2) The invention discovers that the CAVAF has a certain weight increase inhibiting effect on fat mice induced by high-fat diet for the first time;

(3) The invention discovers that CAVAF has a certain effect of improving insulin resistance on fat mice induced by high-fat diet for the first time;

(4) The invention discovers that CAVAF can obviously reduce the white fat specific gravity of fat mice induced by high-fat diet and inhibit the growth of white fat cells;

(5) The CAVAF dosage is relatively low and is less toxic over the effective dosage range.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention, wherein:

FIG. 1 is a LC-MS spectrum of daidzein (CAVAF) provided in example 1 of the present invention;

FIG. 2 is a diagram showing the analysis of the main components of the seville orange flavone provided in example 1 by LC-MS technique;

FIG. 3 is an improvement of weight gain of high fat diet induced obese mice by CAVAF;

FIG. 4 shows the effect of CAVAF on improving insulin resistance in obese mice induced by high fat diet, wherein FIG. 4a shows the trend of blood glucose in mice after insulin injection over time, and FIG. 4b shows the AUC of blood glucose in mice after insulin injection;

FIG. 5 shows the reduction of white adipose tissue weight to specific gravity of high fat diet induced obese mice by CAVAF, wherein FIG. 5a is the weight of white adipose tissue of each group of mice and FIG. 5b is the ratio of the weight of white adipose tissue to the weight of each group of mice;

figure 6 is a graph showing that CAVAF inhibits white adipocyte growth in high fat diet induced obese mice.

Detailed Description

The following disclosure provides many different embodiments, or examples, for implementing different features of the invention. In order to simplify the present disclosure, components and arrangements of specific examples are described below. They are, of course, merely examples and are not intended to limit the invention. Furthermore, the present invention may repeat reference numerals and/or letters in the various examples, which are for the purpose of brevity and clarity, and which do not themselves indicate the relationship between the various embodiments and/or arrangements discussed.

The principle of the invention is as follows: the flavone from plant sources has various pharmacological activities such as antioxidation, antivirus, anti-inflammatory, liver protection and the like, and is a research hot spot for utilizing natural medicines and developing health care products. The invention discovers and confirms that the plant flavone (seville orange flavone) has antioxidant, anti-tumor and immunity enhancing activities through in vivo experiments, and has low dosage, no acute toxic or side effect and no obvious chronic toxic or side effect.

1. Extracting:

example 1

Pulverizing flowers of Citrus aurantium L.var. Amara Engl, and sieving to obtain coarse powder of Citrus aurantium; extracting the substituted flower coarse powder by using 80% ethanol under heating and refluxing for 2 hours at the temperature of 80% and the times of 2 times, wherein the material-liquid ratio is 1:15 (mass ratio); and then 110 centrifuging the extracting solution, wherein the rotating speed is 4500r/min, the time is 12min, and the filtering times are 2 times.

Concentrating the supernatant under reduced pressure at 50deg.C, and drying to obtain 80% ethanol extract of herba Cynanchi Paniculati.

Example 2

Pulverizing flowers of the generation (Citrus aurantium L.var. Amara Engl), and sieving to obtain

To meal of seville orange flower; extracting the substituted flower coarse powder by using 80% ethanol under heating and refluxing at the temperature of 115 ℃ for 4 hours at the temperature of 70 ℃ for 3 times, wherein the material-liquid ratio is 1:30 (mass ratio); however, the method is that

And centrifuging the extract at a rotation speed of 5000r/min for 8min, and filtering for 4 times. Concentrating the supernatant under reduced pressure at 40deg.C, and drying to obtain 80% ethanol extract of herba Cynanchi Paniculati.

Example 3

120 pulverizing flowers of the generation (Citrus aurantium L.var. Amara Engl), and sieving to obtain

To meal of seville orange flower; extracting the substituted flower coarse powder by using 80% ethanol under heating and refluxing for 3 hours at a temperature of 75% and for 4 times, wherein the material-liquid ratio is 1:20 (mass ratio); and centrifuging the extract at 4000r/min for 10min for 3 times.

Concentrating the supernatant at 45deg.C under reduced pressure, and drying to obtain 80% ethanol extract of herba Cynanchi Paniculati. 125 Secondly, separating and purifying:

the 80% ethanol extract of seville orange flower in example 1 was dissolved in a proper amount of distilled water and left to stand,

adding the supernatant into pretreated macroporous resin, sequentially washing resin column with distilled water, 30%, 50%, 70% and 95% ethanol solution for 5 column volumes, and collecting respectively

Collecting and combining the components, concentrating under reduced pressure, and vacuum drying to obtain 30%, 50%, 70% and 95% 130% ethanol elution components. Measuring the flavone content of each component, wherein the flavone content is 30% of the highest

The component was substituted floral flavone (CAVAF) as provided in example 1.

The pretreatment method of the macroporous adsorption resin comprises the following steps: cleaning the chromatographic column, adding a clean funnel above the chromatographic column, slowly pouring a proper amount of AB-8 macroporous resin suspension into the chromatographic column, knocking the column body with an ear-washing ball, and discharging air bubbles. And (5) soaking the resin for 24 hours. The resin column was washed with 95% ethanol solution at a flow rate of 2BV/h until the effluent was not white cloudy by mixing with distilled water, and then the resin column was washed with distilled water until the effluent had no alcoholic smell. And (3) flushing the resin column for 5 column volumes by using a hydrochloric acid solution with concentration of 3-5%, soaking the resin for 4 hours, and flushing the resin column by using distilled water until effluent liquid is neutral. And (3) washing the resin column for 5 column volumes by using NaOH solution of 3-5% sodium hydroxide, soaking the resin for 4 hours, and washing the resin column by using distilled water until effluent liquid is neutral.

3. Measuring the content of the flavone component:

with NaNO ₂ -Al(NO ₃ ) ₃ The principle of the method is that the complex formed by coordination of the ortho-diphenol hydroxyl and aluminum ions under alkaline condition is developed in NaOH solution, and the absorbance value is determined by utilizing the generated red substance. Firstly, rutin standard substances with different concentrations are used for reaction, absorbance is measured at 510nm, and a standard curve chart is drawn. And calculating the flavone content in the sample according to a regression equation.

4. The components were analyzed by liquid chromatography-mass spectrometry (LC-MS):

the UPLC/MS technology is utilized to separate and identify the chemical components of the total flavone of the seville orange flowers, potential chemical component databases in the seville orange flowers are collected and arranged through the component databases, the Xcalibur software is adopted to process mass spectrum data, the standard substance and the literature are compared, the components are identified, and a material basis is provided for the follow-up study of the effect and mechanism of the monomer compound on the brown coloration of white fat.

Wherein UPLC/MS chromatographic conditions and mass spectrometry conditions are as follows:

chromatographic conditions: chromatographic column Hypersil Gold (100 x 2.1mm,1.9 μm), mobile phase: (A) 0.1% formic acid water, (B) acetonitrile, 2. Mu.L of sample injection amount and 0.3ml/min of flow rate. Elution was performed under the gradient elution conditions shown in Table 1.

HPLC analysis gradient elution conditions

Mass spectrometry conditions: the scanning range of mass spectrum ions is 100-1000 (m/z); electrospray ion source (ESI), 3500V in positive ion mode, and-2500V in negative ion mode. Sheath gas 40Arb, auxiliary gas 10Arb, ion transport tube temperature 320 ℃ and evaporator temperature 350 ℃. And respectively adopting positive and negative ion modes for detection.

The chemical components in the plant are searched by CNKI, pubMed, scifinder database and the like, and the potential chemical component database in the finished substituted flower flavone is collected. Mass spectrum data of the seville orange flavone extract were processed using Xcalibur software and found in table 2.

TABLE 2 analysis of substituted flower flavone compositions by liquid chromatography-Mass Spectrometry (LC-MS)

5. The substituted flower flavone has a certain obesity inhibiting effect on high-fat diet induced obese mice:

5.1 animal model and dosing:

c57BL/6J male mice were randomized, feed on a group basis, and daily gavaged. 8 animals are fed into the environment with alternating darkness and illumination for 12 hours at 20-26 ℃ and humidity of 40-70% in each group. Body weight was measured every two or three days and dosed according to the average mass of each group of mice. The culture was continued for 16 weeks. Mice body weight and food intake were recorded weekly. During the experiment, the physiological state of the mice is closely taken into consideration, and whether the sample solution has toxicity or side effect on the growth of the mice is judged.

TABLE 3 grouping and administration of experimental animals

5.2 weekly recordings of mouse body weights

As shown in fig. 3, from the trend of weight gain of each group of mice, the weight of the mice induced by the high fat diet was higher than that of the mice fed with the normal feed, and from the 10 th week, the weight of the mice of the model group was higher than that of the mice of the blank group and the mice of the daidzein group. At week 21, the weight of each dosing group was lower than the model group. Experimental results prove that the CAVAF has a certain improvement effect on the weight gain of the obese mice induced by the high-fat diet.

5.3 insulin resistance test

Mice were fasted for 12h, insulin was intraperitoneally injected (0.5U/kg body weight) after 6h fasting blood glucose, and blood glucose levels of 0, 15, 30, 60, 120min after insulin injection were measured using a blood glucose meter and recorded.

As shown in fig. 4, the blood glucose values of the time points of the substituted flower flavone group were lower than those of the model group, and the AUC values were between the blank group and the model group. Experimental results prove that the CAVAF has a certain effect of improving the insulin resistance of the obese mice induced by the high-fat diet.

5.4 results of HE staining of white adipose tissue pathological sections of mice

White adipose tissues are fixed, embedded and sliced, then hematoxylin-eosin (HE) stained, placed under an inverted microscope, and randomly selected for 5 fields of view, and white adipose morphology and pathological changes are observed.

As shown in fig. 5 and 6, the white adipocytes of the blank group have smaller volume, the adipocytes of the high-fat model group have increased volume, irregular arrangement and evacuation, and new adipocytes are arranged among cells, so that inflammatory cells infiltrate into a focus, the number of cells in the same field is remarkably reduced, while the white cells of the substituted flower flavone group have improved morphology to a certain extent, the cell volume is reduced, and the number of cells in the same field is between the two, which indicates that the white adipocytes can be reduced. Experimental results show that CAVAF can remarkably reduce the white fat specific gravity of fat mice induced by high-fat diet and inhibit the growth of white fat cells.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention herein. This invention is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

It is to be understood that the invention is not limited to the precise arrangements and instrumentalities shown in the drawings, which have been described above, and that various modifications and changes may be effected without departing from the scope thereof. The scope of the invention is limited only by the appended claims.

Claims

1. The seville orange flower flavone is characterized in that the seville orange flower pollen is extracted, separated and eluted in a gradient way to prepare an elution component, and 30% of the elution component with the highest flavone content is the seville orange flower flavone.

2. The method for preparing a substituted flower flavone according to claim 1, comprising the steps of:

(1) Extracting:

pulverizing pollen, sieving, extracting with 80% ethanol under reflux, centrifuging, concentrating supernatant under reduced pressure, and drying to obtain extract;

(2) Purifying:

dissolving the extract in water, standing, absorbing supernatant, adding the supernatant into an AB-8 macroporous resin column, sequentially flushing the AB-8 macroporous resin column with water, 30%, 50%, 70% and 95% ethanol solution, collecting and combining the components, concentrating under reduced pressure, and vacuum drying to obtain the eluted component, and determining the flavone content of the eluted component, wherein the eluted component with the highest flavone content of 30% is the substituted flower flavone.

3. The method for producing a substituted flower flavone according to claim 2, wherein in the step (1), the heating reflux temperature is 70 to 80 ℃, the heating reflux time is 2 to 4 hours, and the heating reflux times are 2 to 4 times.

4. The method for producing a substituted flower flavone according to claim 2, wherein in the step (1), the mass ratio of the pollen to the 80% ethanol is 1:15 to 1:30.

5. The method for producing a substituted flower flavone according to claim 2, wherein in the step (1), the centrifugal speed is 4000 to 5000r/min, the centrifugal time is 8 to 12min, the drying temperature is 40 to 60 ℃, and the reduced pressure concentration temperature is 40 to 50 ℃.

6. The method for producing a substituted flower flavone according to claim 2, wherein in the step (2), the concentration temperature under reduced pressure is 40 to 60 ℃, and the method for measuring the flavone content is NaNO ₂ -Al(NO ₃ ) ₃ NaOH colorimetry.

7. Use of the daidzein prepared by the preparation method according to any one of claims 2 to 6 in the preparation of weight-losing medicaments.

8. Use of the daidzein prepared by the preparation method according to any one of claims 2 to 6 in the preparation of a medicament for inhibiting weight gain.

9. Use of the daidzein prepared by the preparation method according to any one of claims 2 to 6 in the preparation of a medicament for improving insulin resistance.

10. Use of the daidzein prepared by the preparation method according to any one of claims 2 to 6 in the preparation of a medicament for inhibiting white adipocyte growth.