阅读说明：本技术 一种治疗肥胖及其代谢性疾病药物的提取方法及应用 (Extraction method and application of medicine for treating obesity and metabolic diseases thereof ) 是由 付明海 阿丽沙 巴根那 李慧芳 文杰 于 2021-09-27 设计创作，主要内容包括：本发明提供了一种治疗肥胖及其代谢性疾病药物的提取方法及应用；本发明以肋柱花为材料,建立獐牙菜苦苷提取工艺方法,作为判定苦味素提取率指标,从根本上解决肋柱花苦味素对于脂肪细胞的最佳疗效的问题。通过乙醇浓度,提取用量,提取时间,提取次数四个主要因素进行单因素实验分析,采用正交试验确定肋柱花提取工艺其最佳参数为：提取浓度60％,料液比1:10,提取时间2.0,提取次数1。(The invention provides an extraction method and application of a medicament for treating obesity and metabolic diseases thereof; the invention takes the lomatogonis as the material, establishes the swertiamarin extraction process method, and takes the swertiamarin extraction process method as the index for judging the extraction rate of the bitter principle, thereby fundamentally solving the problem of the best curative effect of the lomatogonis bitter principle on fat cells. The method comprises the following steps of carrying out single-factor experimental analysis on four main factors including ethanol concentration, extraction dosage, extraction time and extraction frequency, and determining the optimal parameters of the extraction process of the ribbonfilum by adopting an orthogonal test: the extraction concentration is 60%, the feed-liquid ratio is 1:10, the extraction time is 2.0, and the extraction times are 1.)

1. The extraction method of the medicine for treating obesity and metabolic diseases thereof is characterized by comprising the following steps:

step 1, crushing the lomatogonia carinata into powder, weighing 5g of lomatogonia carinata powder, adding 50-90% ethanol for reflux extraction to obtain extract, and freeze-drying;

and 2, dissolving the dried powder in methanol, filtering the solution by using a 0.45 mu L filter membrane, analyzing and identifying according to chromatographic conditions, and calculating the percentage content of the bitter principle.

2. The method for extracting drugs for treating obesity and metabolic diseases according to claim 1, wherein in the step 1, the ethanol concentration in the reflux extraction is 60%, and the ratio of the feed to the liquid is 1:10, the extraction time is 2.0 hours, and the extraction times is 1 time.

3. The application of the medicine for treating obesity and metabolic diseases thereof is characterized in that the medicine is used for preparing the medicine for treating obesity and metabolic diseases thereof.

Technical Field

The invention belongs to the field of extraction methods; in particular to an extraction method and application of a medicament for treating obesity and metabolic diseases thereof.

Background

The Mongolian medicine costalia flower is a traditional Mongolian medicine, has a long application history, and has a Mongolian medicine name of Harbidi and a Digeda. Is dried whole plant of Lomatogoniumrotatum (L.) Fries ex Nym. The Mongolian medicine costal flower is bitter in taste, cold in nature, dull, coarse, light and dry. Has effects of calming down the adverse-rising of the liver, clearing heat, invigorating stomach, and healing wound. Can be used for treating heat in synergistic period, pestilence, influenza, typhoid fever, sunstroke, headache, liver and gallbladder heat, jaundice, stomach heat in synergistic period, and traumatic heat, and is mainly used for treating diseases of liver and gallbladder system in clinical Mongolian medicine.

Costal column flower is widely distributed in inner Mongolia, Tibet, Yunnan, Sichuan, Qinghai, Gansu, Xinjiang, etc. Growing in hillside grass slopes and irrigation clusters below the elevation of 4200 m. The Gentianaceae plants have effects of purging excessive fire of liver and gallbladder, relieving cough, invigorating stomach, dispelling pathogenic wind and dampness, relaxing muscles and tendons, and relieving pain. Can be used for treating liver and gallbladder cystitis, arthritis and digestive system diseases. Therefore, the Mongolian medicine, namely the ribbing, is mainly or compatibly used in classical and modern Mongolian medicine prescriptions, such as ribbing four-ingredient decoction powder, cholagogic eight-ingredient powder, Sangeda sappan decoction, Yihe Hari twelve-ingredient decoction, elecampane ten-ingredient decoction and the like, and is mainly used for treating symptoms such as icteric hepatitis, acute enteritis, dysentery, cholecystitis, pneumonia and the like in Mongolian clinical practice.

The main pharmacological actions of the costal Styloides flower include liver protection, anti-inflammation, anti-tumor and fat reduction. The chemical components mainly comprise xanthones, flavonoids, triterpenes, iridoids and secoiridoids. The secoiridoid glycoside compounds are natural active molecules with the functions of protecting liver, reducing blood sugar, regulating blood fat, resisting inflammation, resisting tumor and protecting nerve. Bitter principle (bitter principles) is a general name of compounds with bitter taste, can be divided into a paste, sesquiterpenes, diterpenes and triterpenes, and has a great number of medicinal values; for example, rehmannia rich in bitter principle is a yin-nourishing medicine, and has certain effects on reducing blood sugar and promoting urination; the fruit of Gardenia jasminoides Ellis contains various bitter components, and has effects of clearing heat and purging pathogenic fire; the gentianaceae plants are mostly cold in nature and bitter in taste, and are regarded as bitter stomachic botanical drugs due to the effects of strengthening spleen and promoting digestion. Swertiamarin belongs to iridoid compounds, and modern pharmacological research shows that swertiamarin has multiple effects of protecting liver, regulating blood lipid, resisting diabetes, relieving pain, tranquilizing, resisting inflammation and the like. The content of the total substances of the costaria flowers is about 30.98 percent, and the method has the value of extracting bitter principles. The extraction and purification of the costaphylloside are important ways for improving the added value of the costaphylloside. Obesity has become the most common dystrophic disease in modern society in recent years. The basic mechanism that occurs is food intake over energy expenditure. Fat is the main form of energy storage of an organism and is closely related to the metabolism of the organism. Adipose tissue includes adipose precursor cells and adipocytes at various stages of differentiation, and obesity occurs due to the excessive proliferation and differentiation of adipocytes. The bitter taste receptor TAS2R38 is reported in the literature to be present at both the RNA and protein levels in human adipocytes, and that bitter taste receptor expression is higher in adipose tissue of obese humans than in lean humans. Bitter compounds can support weight control by several mechanisms involving expression of TAS2Rs in different parts of the body-including adipose tissue, obesity being closely related to type ii diabetes, cardiovascular diseases, hypertension and cancer.

Therefore, the research and control of the proliferation and differentiation mechanism of preadipocytes is very key to the prevention and treatment of the occurrence and the development of obesity and related diseases. Reducing the synthesis of fat in the body or accelerating its decomposition, etc., has become a difficult problem to overcome. At home and abroad, a plurality of scholars are dedicated to researching whether the compound separated and extracted from the plant raw material has the function of regulating the blood fat metabolism and fat differentiation. Pharmaceutical intervention is of great significance in preventing adipocyte differentiation and stimulating lipid absorption. The development of a safe, effective, side-effect free drug is critical to combating the obesity epidemic. Therefore, the method takes the stigmata miniata as a raw material, four main factors of ethanol concentration, extraction dosage, extraction time and extraction frequency in an orthogonal test are designed, the optimal extraction process of the stigmata stricta swertiamarin is researched, the content of the swertiamarin is used as an index for judging the extraction rate of the bitter principle, and a new thought is provided for solving the occurrence of obesity and metabolic diseases thereof.

Disclosure of Invention

The invention aims to provide an extraction method and application of a medicine for treating obesity and metabolic diseases thereof.

The invention is realized by the following technical scheme:

the invention relates to an extraction method of a medicine for treating obesity and metabolic diseases thereof, which comprises the following steps:

step 1, crushing the lomatogonia carinata into powder, weighing 5g of lomatogonia carinata powder, adding 50-90% ethanol for reflux extraction to obtain extract, and freeze-drying;

and 2, dissolving the dried powder in methanol, filtering the solution by using a 0.45 mu L filter membrane, analyzing and identifying according to chromatographic conditions, and calculating the percentage content of the bitter principle.

Preferably, in step 1, the ethanol concentration in the reflux extraction is 60%, and the ratio of the feed to the liquid is 1:10, the extraction time is 2.0 hours, and the extraction times is 1 time.

The invention also relates to the application of the medicament for treating the obesity and the metabolic diseases thereof, and the preparation of the medicament for treating the obesity and the metabolic diseases thereof.

The invention has the following advantages:

the method takes the content of the swertiamarin as an index, combines single factor investigation and orthogonal test, screens factors and conditions influencing the extraction of Mongolian swertiamarin, finally determines the optimal extraction process parameters, and lays a foundation for further comprehensive development of costcolumn flower medicinal materials.

Drawings

FIG. 1 is an intercostal HPLC chromatogram;

FIG. 2 is HPLC chromatogram of swertiamarin standard;

FIG. 3 is a graph of a swertiamarin standard curve;

FIG. 4 is a graph comparing the concentration of swertiamarin with different ethanol concentrations;

FIG. 5 is a graph showing the comparison of swertiamarin content with different extraction solvent amount;

FIG. 6 is a graph showing the comparison of swertiamarin content at different extraction times;

FIG. 7 is a graph showing the comparison of swertiamarin content with different extraction times.

Detailed Description

The present invention will be described in detail with reference to specific examples. It should be noted that the following examples are only illustrative of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

1. Main instrument of the embodiment

(1) Agilent 1260Infinity ii liquid chromatograph,

(2) one hundred thousand analytical balances (sidoris scientific instruments ltd) of the SQP type,

(3) a KQ-800E type ultrasonic cleaner,

(4) SHZ-95B model circulating water type multipurpose vacuum pump,

(5) KDM type temperature-adjusting electric heating jacket.

2. Major drugs and reagents

Lot number of intercostal flower (2020.9.25) was collected in the union of Cenline Guo and identified as whole grass of Lomatoconiumrotatum (L.) Fries ex Nym, Gentianaceae by professor Lomatoconiumgitatum, university of inner Mongolia. Swertiamarin standard (batch No. B21644 purity is more than or equal to 98%) is purchased from original leaf organism. Methanol, phosphoric acid and absolute ethyl alcohol are analytically pure, and water is purified water.

The embodiment relates to an extraction method of a medicine for treating obesity and metabolic diseases thereof, which comprises the following steps:

step 1, crushing the lomatogonia carinata into powder, weighing 5g of lomatogonia carinata powder, adding 50-90% ethanol for reflux extraction to obtain extract, and freeze-drying;

step 2, accurately weighing 2.18mg of swertiamarin, putting the swertiamarin into a 25ml measuring flask, adding methanol for dissolving, diluting until scales are marked, and shaking up. Degassing for 10min with ultrasonic wave, filtering with 0.45nm water system membrane to obtain 1ml of fluid containing 0.0872 mg, sampling amount of 10 μ L, and drawing standard curve with swertiamarin mass concentration as abscissa and peak area as ordinate.

As can be seen from FIGS. 1, 2 and 3, the retention time of swertiamarin is 6.775, and a regression equation is obtained: y is 641.66X +5.2603, the results show that the linear range R²0.9997; RSD in the precision experiment is 0.26%, which shows that the precision of the instrument is good; determination of swertiamarin content in 24 hours of sampleThe RSD of the result is 0.002%, which indicates that the test solution is stable within 24 h; RSD in a repeatability experiment is 0.026%, which shows that the repeatability is good; RSD in the sample recovery rate experiment is 0.084%. The method is proved to meet the requirement of content measurement.

Chromatographic conditions are as follows: high performance liquid chromatography is adopted. A chromatographic column: ZORBAX SB-C18 (4.6X 250mm) 5-Micron; mobile phase: water-0.1% phosphoric acid water solution gradient wash 0 → 70:30,15 → 65:35,25 → 50: 50; flow rate: 1.0 ml/min^－1(ii) a Detection wavelength: 254 nm; column temperature: at 30 ℃.

Comparative experiment

Single factor experiment in extraction process of costal column flower swertiamarin

The experiment takes Mongolian medicinal material, namely the whole herb of the costaliella as a material, and establishes an optimization method of the swertiamarin extraction process. The extract is used as an index for judging the extraction rate of the bitter principle. The optimal extraction process of the swertiamarin is obtained by an orthogonal experiment of four factors of ethanol concentration, extraction dosage, extraction time and extraction frequency after the medicinal materials are dried and crushed in the shade, and the data is shown in table 1.

TABLE 1

	A	B	C	D
						Ethanol concentration (%)	Ratio of material to liquid	Extraction time (h)	Number of times of extraction (times)
1	50％	1：10	0.5	1
					2	60％	1：20	1.0	2
3	70％	1：30	1.5	3
					4	80％	1：40	2.0	4
5	90％	1：50	2.5	5

(1) Selection of ethanol concentration

Precisely weighing 5 parts of costal anther powder, each 5g of costal anther powder, using 20 times of ethanol, extracting for 3 times, extracting for 2.0h, respectively setting the concentrations of 5 ethanol of 50%, 60%, 70%, 80% and 90% to extract, calculating the peak area content of the swertiamarin, and obtaining the result shown in figure 4, wherein the peak value of the content of the swertiamarin is reached when the ethanol concentration is 60%, so 60% ethanol is selected as an extraction solvent.

(2) Selection of extraction times

Precisely weighing 5 parts of costal column flower medicinal material powder, each 5g of costal column flower medicinal material powder, using 20 times of ethanol, extracting for 2.0h at the extraction concentration of 50%, respectively extracting for 1, 2, 3, 4 and 5 times, and calculating the peak content of the swertiamarin. As shown in FIG. 5, the content of swertiamarin reached the peak value at times 2, so 2 times were selected.

(3) Selection of extraction time

Precisely weighing 5 parts of costal column medicinal material powder, each 5g of costal column medicinal material powder, using 20 times of ethanol, extracting for 1 time, and respectively extracting for 5 times of 0.5h, 1.0h, 1.5h, 2.0h, 2.5h and the like to calculate the content of the swertiamarin. As shown in FIG. 6, the content of swertiamarin reaches the peak value at the time of 2.0h, so 2.0h is selected as the extraction time.

(4) Selection of the amount of extraction solvent

Accurately weighing 5 parts of costal column anther powder, each 5g, the extraction time is 2.0h, the extraction concentration is 50%, the extraction frequency is 1 time, and respectively setting 1: 10. 1: 20. 1: 30. 1: 40. 1: extracting with a material-liquid ratio of 50, and calculating the content of swertiamarin. As shown in fig. 7, the content of swertiamarin is in the range of multiple 1: the peak value is reached when the temperature is 10 ℃, and the temperature is selected from the following steps: extracting with 10 times of ethanol.

Orthogonal experiments and verifications

According to the results of the single-factor experiment, the orthogonal experiment is designed by selecting appropriate ethanol concentration, extraction dosage, extraction time and extraction times and tabulating, and the results are shown in Table 2.

TABLE 2

Test number	A	B	C	D
						Ethanol concentration (%)	Ratio of material to liquid	Extraction time (h)	Number of times of extraction (times)
1	50％	1：10	1.5	1
					2	60％	1：20	2.0	2
3	70％	1：30	2.5	3

Results of orthogonal experiments

The orthogonal experimental protocol and results are shown in Table 3, and the ANOVA is shown in Table 4. The results show that the time of extraction is known from the R value and the analysis of variance in Table 4The inter-effect is most pronounced. And the optimal extraction process is A₂B₁C₂D₁Namely, the extraction concentration is 60%, and the ratio of material to liquid is 1:10, extraction time 2.0, extraction times 1.

TABLE 3

TABLE 4

Factors of the fact	Sum of squares of deviation	Degree of freedom	F ratio	Critical value of F	Significance of
						Concentration of the extract	0.522	2	1.206	4.460	*
The amount of the extract is determined	0.222	2	0.513	4.460	-
						Extraction time	0.972	2	2.245	4.460	*
Number of times of extraction	0.016	2	0.037	4.460	-
						Error of the measurement	1.73	8

According to the invention, from a single-factor experiment, the peak areas are respectively in 60% ethanol, the extraction times are 2 times, the extraction time is 2.0h, the peak area reaches the peak when the feed-liquid ratio is 1:10, and the peak area shows a descending trend along with the increase of the concentration, times, time and specific gravity. The results of the orthogonal test conducted according to the above results are A₂B₁C₂D₁Because the ingredients of the medicinal materials are complex, the complexity and uncontrollable property of the reaction are increased, so that different medicinal materials are measured by the same method, and even different extracts of the same medicinal material are obtainedThe content of total iridoid glycoside in the product can be different. The iridoid and the glycoside thereof have unstable properties, and have long reflux heating time, which has certain influence on the chemical components of the costaphanidermatis. Accurate determination of the content of the extract and screening of a correct and specific method are yet to be further improved.

In the experiment, single-factor experimental analysis is carried out through four main factors of ethanol concentration, extraction dosage, extraction time and extraction frequency, a reflux extraction method is used as a basis, and an optimal extraction process obtained by adopting orthogonal experiment verification is that the extraction concentration is 60% ethanol, the ethanol dosage is 10 times, the extraction time is 2.0h, and the extraction frequency is 1 time.

Therefore, the experiment takes the content of the swertiamarin as an index, combines single factor investigation and orthogonal experiment, screens factors and conditions influencing the extraction of the picrorhiamarin of the bitter Mongolian medicine, namely the swertiamarin of the costcolumn flower, finally optimizes extraction process parameters, and lays a foundation for further comprehensive development of costcolumn flower medicinal materials.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.