CN113087750A - Cyclocarya paliurus sweet glycoside, and preparation method and application thereof - Google Patents
Cyclocarya paliurus sweet glycoside, and preparation method and application thereof Download PDFInfo
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- CN113087750A CN113087750A CN202110310638.5A CN202110310638A CN113087750A CN 113087750 A CN113087750 A CN 113087750A CN 202110310638 A CN202110310638 A CN 202110310638A CN 113087750 A CN113087750 A CN 113087750A
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- Prior art keywords
- cyclocarya paliurus
- sweet
- leaves
- water
- glycoside
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Abstract
The invention relates to cyclocarya paliurus sweet glycoside, which is obtained by sequentially eluting cyclocarya paliurus leaves with water or alcohol, macroporous adsorption resin and water and ethanol solution. The preparation method comprises the following steps: adding water or ethanol into appropriate amount of cyclocarya paliurus leaves, heating and reflux-extracting, mixing extractive solutions, and concentrating under reduced pressure to obtain concentrated solution; passing the concentrated solution through macroporous adsorbent resin column, and sequentially eluting with water, 15-25% ethanol solution, and 75-85% ethanol solution; concentrating the 75-85% ethanol eluate under reduced pressure until no alcohol smell is present, adding water for suspension, extracting with ethyl acetate, and recovering solvent to obtain sweet component of folium cyclocarya paliurus; carrying out decoloring treatment on the sweet part of the cyclocarya paliurus leaves; drying to obtain cyclocarya paliurus sweet glycoside. The cyclocarya paliurus sweet glycoside or the cyclocarya paliurus sweet glycoside prepared by the method can be used for preparing a medicine with a lipid-lowering function, or used for common food or health food, or directly used as a food additive such as a sweetening agent, an aromatic agent and the like.
Description
Technical Field
The invention belongs to the technical field of natural products, and relates to cyclocarya paliurus sweet glycoside, and a preparation method and application thereof.
Background
Sweet taste is a taste that is most preferred by humans and is essentially the result of the action of certain compounds on taste receptors. The conventional sweeteners currently in wide use are almost all sugars and are considered to be of the high calorie type. Excessive intake of such high calorie sugar may cause not only obesity and caries but also diabetes, cardiovascular diseases, neurological dysfunction, and accelerated aging, resulting in various problems affecting physical health. Nowadays, due to the pursuit of health and deliciousness, people have higher and higher requirements on the inherent quality of ingested food, and food with low calorific value, good mouthfeel and high safety is pursued in many times. Among such foods, natural non-sugar sweeteners such as glycyrrhizin, stevioside, mogroside and the like are preferred by people because of their high sweetness and low calorie.
Among a plurality of sweet plant products, cyclocarya paliurus tea is known as "Shencha" and the third tree in medical science because of its strong and lingering sweet taste and its functions of reducing blood fat and blood sugar. At present, cyclocarya paliurus leaves are listed in a new resource food catalogue by the nation, and have great development potential.
Cyclocarya paliurus (Batal) Iljinskaja, also called Qingqian plum, Royal tree, sweet tea tree, etc., is a deciduous tree of Cyclocarya of Juglandaceae, is a unique single-species plant in China, and is mainly distributed in the provinces of south of Yangtze river. According to records of medical books such as Chinese materia medica and Chinese traditional medicine resource Zhi Yao, leaves, barks and roots of cyclocarya paliurus can be used as medicines, has the functions of clearing heat, reducing swelling and relieving pain, and can be used for treating intractable tinea. For a long time, most people adopt tender leaves of the tea leaves to prepare tea, tea soup is sweet and moist, and the tea soup is considered to have the effects of promoting the production of body fluid to quench thirst, clearing away summer heat, reducing blood pressure and strengthening heart, prolonging life and the like, and can effectively prevent and treat diseases such as diabetes, coronary heart disease, hyperlipidemia, neurasthenia and the like. Modern pharmacological studies show that cyclocarya paliurus leaves have physiological activities of reducing blood sugar and blood fat, resisting oxidation, resisting bacteria, improving immunity and the like. The research on the chemical components of the cyclocarya paliurus leaves shows that the cyclocarya paliurus leaves mainly contain flavone, terpenoids, polysaccharide, organic acid and other components. Of these chemical components, more than ten components with significant sweetness, such as cyclocarya paliurus glycoside I, have been found to be about 250 times as sweet as sucrose. From the chemical structure of the component with sweet taste separated from cyclocarya paliurus leaves, the triterpene mother nucleus mainly has dammarane or 2, 3-position split ring, usually contains multiple unsaturated double bonds, and is connected with one or more glycoside group(s) taking isorhamnese and/or arabinose as main type, as shown in figure 1. By literature investigations, in conjunction with pre-experimental analysis, we speculate that: besides the chemical components with sweet taste which are found at present, a plurality of chemical components with sweet taste which are not found yet exist in the cyclocarya paliurus leaves; structurally, such sweet ingredients should have triterpene dammarane or rhadammarane type glycosides that are structurally similar to those found in sweet ingredients; by type analysis, the sweet taste components are natural non-sugar compounds with low calorific value. Based on the analysis, referring to other natural non-sugar sweetener varieties such as stevioside, mogroside and the like, the extract which is refined by processes such as extraction, concentration, impurity removal, enrichment, purification, decoloration, drying and the like on cyclocarya paliurus leaves, has sweet taste and definite sweet index components and contains more chemical components with sweet index components and similar structures is named as cyclocarya paliurus sweet glycoside.
At present, more researches on the extraction process of the chemical components of cyclocarya paliurus are reported, the main focus is on cyclocarya paliurus polysaccharide, flavone and triterpene, and the research specially aiming at cyclocarya paliurus sweet glycosides is blank. Although cyclocarya paliurus sweet glycosides still belong to triterpenes in chemical structure, the cyclocarya paliurus sweet glycosides are obviously different from the total triterpenes of cyclocarya paliurus mentioned in general researches: (1) in terms of raw materials, the raw material of the total triterpenoids of cyclocarya paliurus in general research is a leaf blade which completely grows in 6-10 months, the cyclocarya paliurus leaf has obvious sweet taste in a tender leaf stage which does not completely grow in 3-5 months, and the cyclocarya paliurus sweet glycosides extracted from the leaf blade have a structure which is obviously different from the total triterpenoids of cyclocarya paliurus (represented by oleanolic acid and arjunolic acid without sweet taste). (2) In terms of preparation process, the extraction process of the total triterpenoids of cyclocarya paliurus generally comprises the steps of alcohol solvent extraction, organic solvent extraction and/or macroporous adsorption resin enrichment and purification, active carbon or ion exchange resin refining, drying and the like, and is an extraction scheme designed and developed according to the characteristics that a triterpenoid natural product is easily soluble in an alcohol solvent, has moderate molecular weight, has medium and small molecular polarity and the like; the cyclocarya paliurus sweet glycosides are sweet, have chemical structures obviously different from total triterpenes of cyclocarya paliurus, are particularly influenced by the picking time of raw materials, and are greatly changed in the growth and development process of cyclocarya paliurus leaves from tender leaves to old leaves. Examples that can be cited are the huge chemical composition differences of young leaves and old leaves of tea trees and cedrela sinensis trees. In view of this, it is not difficult to presume that: cyclocarya paliurus sweet glycosides contained in cyclocarya paliurus leaves obtained in different harvesting periods and coexisting impurities are different, so that the extraction, impurity removal, decoloration and other process operations of the cyclocarya paliurus sweet glycosides and the total triterpenes of the cyclocarya paliurus are different. (3) Evaluation indexes are as follows: a preparation process of total triterpenes of cyclocarya paliurus mostly takes oleanolic acid as a marker component and takes the content, purity, yield and the like of the total triterpenes as indexes; the preparation process of cyclocarya paliurus sweet glycosides takes the sweetness intensity as a main index. Therefore, the process links of extraction, screening separation, purification and refining of cyclocarya paliurus sweet glycosides are different from the process design of extraction of total triterpenes of cyclocarya paliurus, no matter from raw material screening or process design basis.
In addition, although a few researches on sweet components of cyclocarya paliurus exist at present, the conventional phytochemical separation means is mainly adopted to obtain component monomers, identify the structure and finally test the sweetness of the component monomers. A research report for carrying out systematic discovery and separation of sweet components based on cyclocarya paliurus sweet glycosides extracted from cyclocarya paliurus tender leaves is blank. The establishment of a quality control system based on cyclocarya paliurus sweet glycosides with sweet index components is still blank. The application of cyclocarya paliurus sweet glycosides in food and medicine industries is still blank.
Disclosure of Invention
In order to solve the technical problems, the cyclocarya paliurus sweet glycoside and the preparation method and the application thereof are provided, a crude extract with sweet taste of cyclocarya paliurus leaves is screened out through systematic research, and the cyclocarya paliurus sweet glycoside is obtained through separation, purification, refining and drying by further taking sweetness as an index; and then, separating and obtaining index components with obvious sweet taste from cyclocarya paliurus sweet glycosides, establishing an HPLC (high performance liquid chromatography) characteristic map of the index components, further investigating the lipid-lowering effect of the index components, and laying a foundation for trying to apply the index components to the production and processing of products in the food, beverage and pharmaceutical industries.
The technical scheme of the invention is as follows:
cyclocarya paliurus sweet glycoside is prepared by extracting cyclocarya paliurus leaves with water or alcohol, passing through macroporous adsorbent resin, and sequentially eluting with water and ethanol solution.
Preferably, the cyclocarya paliurus sweet glycosides comprise discoidin A or other triterpenoid saponins.
The preparation method of cyclocarya paliurus sweet glycoside comprises the following steps:
s1: adding water or ethanol into appropriate amount of cyclocarya paliurus leaves, heating and reflux-extracting, mixing extractive solutions, and concentrating under reduced pressure to obtain concentrated solution;
s2: passing the concentrated solution through macroporous adsorbent resin column, and sequentially eluting with water, 15-25% ethanol solution, and 75-85% ethanol solution;
s3: concentrating the 75-85% ethanol eluate under reduced pressure until no alcohol smell is present, adding water for suspension, extracting with ethyl acetate, and recovering solvent to obtain sweet component of folium cyclocarya paliurus;
s4: carrying out decoloring treatment on the sweet part of the cyclocarya paliurus leaves;
s5: drying to obtain cyclocarya paliurus sweet glycoside.
Preferably, 15-30 times of water or 70% ethanol is added in the step S1, and the heating reflux extraction is carried out for 3-4 times, wherein the single extraction time is 1.5-2.5 h.
Preferably, the concentrated solution obtained in step S2 is passed through a macroporous adsorbent resin column, the loaded material is nonpolar or low-polarity macroporous adsorbent resin, and the macroporous adsorbent resin is sequentially eluted with 5-15 times of column volume of water, 15-25% ethanol solution and 75-85% ethanol solution.
Further preferably, the concentrated solution of step S2 is passed through a macroporous adsorbent resin column, and the packed material is a nonpolar or low-polarity macroporous adsorbent resin, such as X-5 type macroporous adsorbent resin, which is sequentially eluted with 10 times of column volume of water, 20% ethanol solution, and 80% ethanol solution, respectively.
Preferably, the decoloring processing method in step S4 is to add water to the sweet part of cyclocarya paliurus leaves to prepare a stock solution to be decolored of the sweet part of cyclocarya paliurus, and add a decoloring agent for decoloring, wherein the decoloring agent is magnesium oxide, attapulgite, polyamide, alumina, activated carbon or a combination thereof, and the addition amount is 0.03-6%.
Preferably, the decoloring processing method in step S4 includes the steps of:
s4.1 preparation of a compound decolorizing agent: compounding magnesium oxide and an active carbon agent, and uniformly mixing to obtain a compound decolorizing agent;
s4.2 adding a compound decolorizing agent into the stock solution to be decolorized of the sweet part of the cyclocarya paliurus, decolorizing at constant temperature, shaking, filtering, and collecting the filtrate to obtain the decolorized sweet part of the cyclocarya paliurus.
Further preferably, the magnesium oxide and the active carbon agent in the step S4.1 are compounded according to the ratio of (75-85) to 1; s4.2 adding 2-5% of compound decoloring agent into the stock solution to be decolored at the sweet part of the cyclocarya paliurus, and decoloring at the constant temperature of 55-75 ℃ for 0.5-2 h.
Further preferably, the cyclocarya paliurus leaves are also preprocessed before the step S1, and the preprocessing method includes: adding lime water into cyclocarya paliurus leaves, soaking at room temperature for 0.5-1.5h, filtering, removing filtrate, quickly washing the leaves with water for 2-3 times, testing to show neutrality, and air drying for use.
The cyclocarya paliurus sweet glycoside or the cyclocarya paliurus sweet glycoside prepared by the method can be used for preparing a medicament with a lipid-lowering function, and is characterized by belonging to any one of oral liquid, capsules, tablets, pills, dripping pills, powder and injection, or being used as a food additive for processing and producing various types of wine, beverages and foods, or being used as a pharmaceutical adjuvant for producing pharmaceutical preparations.
The invention has the beneficial effects that:
1. cyclocarya paliurus sweet glycosides are extracts separated from cyclocarya paliurus leaves (especially tender leaves) and have sweet taste and no or little pigment and other impurities, and the sweet index components of the cyclocarya paliurus sweet glycosides are dammarane type triterpenes compounds with dammarane or 2, 3-position split rings represented by discoidin A (Pterocarposines A), usually contain a plurality of unsaturated double bonds, and are connected with one or a plurality of glycoside groups with isorhamnese and/or arabinose as main types.
2. Cyclocarya paliurus sweet glycoside is an extract which is obtained by taking sweetness as a main index through the steps of extraction, purification, decoloration, drying and the like, has the appearance of light brown yellow to white-like fine powder and has strong and lingering sweetness.
3. Based on the structural characteristics of the chemical composition of cyclocarya paliurus sweet glycoside, the preparation of the cyclocarya paliurus sweet glycoside is suitable for extracting, purifying, decoloring and other intermediate processes by using alcohol solution with higher concentration as an extraction solvent, and long-time high-temperature treatment is avoided; finally, the drying is suitable for adopting the modes of freeze drying with lower temperature or vacuum drying under reduced pressure and the like.
4. The cyclocarya paliurus sweet glycoside is prepared by adopting macroporous adsorption resin and/or organic solvent extraction method for enrichment and purification. Because a large amount of pigment impurities still exist in intermediate products such as sweet parts of cyclocarya paliurus which are obtained after extraction and purification and have obvious sweet taste, the intermediate products in the preparation process of cyclocarya paliurus sweet glycosides need to be decolored and refined.
5. The cyclocarya paliurus dulcoside is prepared by soaking in saturated or nearly saturated lime water in advance, so that part of pigment impurities can be effectively removed.
6. In order to effectively remove pigment impurities coexisting with cyclocarya paliurus sweet glycosides, various decolorizing treatment means are tried, and decolorizing agents such as magnesium oxide, attapulgite, polyamide, aluminum oxide, activated carbon and the like are preferably selected, so that the decolorizing agent has a good decolorizing effect. Furthermore, a composite decoloring agent is preferably selected, wherein magnesium oxide and active carbon are compounded in a weight ratio of 80:1, and a better decoloring effect can be obtained. The preferred magnesium oxide and active carbon composite decolorant is used for decoloring, and better process parameters are determined through experiments: the dosage of the composite decolorant is 1 to 5 percent, and the preferred dosage is 3 percent; the decoloring time is 0.5h-3h, preferably 1.5 h; the decolorizing temperature is 40-80 ℃, and the preferred decolorizing temperature is 60 ℃.
7. The cyclocarya paliurus sweet glycoside extract obtained by the steps of extraction, purification, decoloration, drying and the like has good lipid-lowering activity; and because the cyclocarya paliurus leaves are listed in a new resource food catalogue by the nation, and simultaneously, ethanol, ethyl acetate, magnesium oxide, activated carbon and the like adopted in the preparation process have good biological safety, the cyclocarya paliurus sweet glycoside extract has good application safety.
Drawings
FIG. 1 typical chemical structure of sweet component in cyclocarya paliurus leaves (source: CN108235686A 'novel triterpene glycoside as sweetener or sweetness enhancer');
FIG. 2 shows a typical HPLC chromatogram of a cyclocarya paliurus sample (S14: young leaves; S15: old leaves);
FIG. 3 shows a process of preparing sweet part of cyclocarya paliurus;
FIG. 4 the decolorizing effect of various decolorizing agents;
FIG. 5 the decolourisation efficacy and sweetness profile of the combined decolouriser;
FIG. 6 cyclocarya paliurus glycosides appearance;
FIG. 7 shows the chemical structure of discocarioside A (Pterocarposines A, D-1-2-1);
FIG. 8 is an HPLC characteristic spectrum of cyclocarya paliurus sweet glycoside sample (S6 is taken as a raw material);
FIG. 9 comparison of HPLC characteristic patterns of cyclocarya paliurus sweet glycoside samples of different raw materials (S14: tender leaves; S15: old leaves);
FIG. 103 is a typical photograph of cyclocarya paliurus sweet glycosides in 7 days after treatment of T3-L1 cells with oil red O staining;
FIG. 11 Effect of cyclocarya paliurus glycosides on Nrf2 translocation and HO-1 expression in 3T3-L1 cells.
Detailed Description
The invention is further illustrated by the following examples, but the scope of the invention as claimed is not limited to the scope of the examples.
We first performed HPLC analysis of chemical composition on cyclocarya paliurus leaves collected at different periods. Respectively pulverizing cyclocarya paliurus leaves, precisely weighing a proper amount of sample powder, placing into a conical flask, adding 70% methanol solution, weighing, ultrasonically treating for 1h, cooling to room temperature, weighing, dripping 70% methanol solution, supplementing lost mass, filtering with microporous membrane (0.45 μm), collecting filtrate, and injecting into HPLC for analysis. The chromatographic conditions employed were: a Dionex UltiMate 3000 HPLC system; ultimate XB-C18(4.6 mm. times.250 mm, 5 μm); column temperature: 30 ℃; detection wavelength: 205 nm; flow rate: 1.0 mL/min; mobile phase: a: 5mM potassium dihydrogen phosphate aqueous solution, B: acetonitrile; gradient elution: 0 min: 10% of B; 10 min: 20% of B; 30 min: 25% of B; and (4) 40 min: 75% of B; 50 min: 75% of B. The analysis result shows that: in a typical HPLC chromatogram of a cyclocarya paliurus sample (S14: tender leaves; S15: old leaves), the tender leaves and the old leaves of the cyclocarya paliurus mainly contain flavonoid components such as kaempferol-3-O-glucuronide and quercetin-3-O-glucuronide, and simultaneously contain a small amount of organic acid components such as chlorogenic acid and neochlorogenic acid; the content of flavone and organic acid contained in old leaves is far higher than that of corresponding components contained in young leaves; Quercetin-3-O-rhamnoside and kaempferol-3-O-glucoside contained in old leaves of cyclocarya paliurus are not obviously seen in tender leaves; however, the tender leaves of cyclocarya paliurus contain medium and small polar components (the retention time is 28-40 min), and are obviously more abundant than the old leaves, and the components are only absorbed by the ultraviolet end by ultraviolet spectrum analysis, which indicates that the components are probably triterpene components, and the result is shown in figure 2. It is known that flavonoid components such as kaempferol-3-O-glucuronide and quercetin-3-O-glucuronide, and organic acid components such as chlorogenic acid and neochlorogenic acid do not have sweet taste, and the above results suggest that: the cyclocarya paliurus leaves contain a large amount of flavonoids, organic acids, other pigments and other ingredients, and for cyclocarya paliurus sweet glycosides, the cyclocarya paliurus leaves belong to impuritiesMass components; the cyclocarya paliurus sweet glycosides are more suitable for being extracted from the cyclocarya paliurus tender leaves because the cyclocarya paliurus tender leaves contain less impurity components and are richer in sweetness after being tasted by mouth than the old leaves. Because the characteristics of the cyclocarya paliurus sweet glycosides are not completely understood, the types of the coexisting pigment impurities of the cyclocarya paliurus sweet glycosides are completely unknown, and the interference of the cyclocarya paliurus sweet glycosides in the extraction and refining processes is completely unknown, the sweetness and the decoloration rate are used as important indexes of the cyclocarya paliurus sweet glycosides in the process optimization. The ideal cyclocarya paliurus sweet glycoside extract has intense sweet taste and less impurities, is reflected in the appearance of the extract, and has the characteristics of light color, low hygroscopicity and the like.
Example 1
Cyclocarya paliurus sweet glycoside is prepared by extracting cyclocarya paliurus leaves with water or alcohol, passing through macroporous adsorbent resin, and sequentially eluting with water and ethanol solution.
Preferably, the cyclocarya paliurus sweet glycosides comprise discoidin A or other triterpenoid saponins.
The preparation method of cyclocarya paliurus sweet glycoside comprises the following steps:
s1: adding water or ethanol into appropriate amount of cyclocarya paliurus leaves, heating and reflux-extracting, mixing extractive solutions, and concentrating under reduced pressure to obtain concentrated solution;
s2: passing the concentrated solution through macroporous adsorbent resin column, and sequentially eluting with water, 15-25% ethanol solution, and 75-85% ethanol solution;
s3: concentrating the 75-85% ethanol eluate under reduced pressure until no alcohol smell is present, adding water for suspension, extracting with ethyl acetate, and recovering solvent to obtain sweet component of folium cyclocarya paliurus;
s4: carrying out decoloring treatment on the sweet part of the cyclocarya paliurus leaves;
s5: drying to obtain cyclocarya paliurus sweet glycoside.
Preferably, 15-30 times of water or 70% ethanol is added in the step S1, and the heating reflux extraction is carried out for 3-4 times, wherein the single extraction time is 1.5-2.5 h.
Preferably, the concentrated solution of step S2 is passed through X-5 type nonpolar macroporous adsorbent resin column, and eluted with 10 times column volume of water, 20% ethanol solution, and 80% ethanol solution, respectively.
Preferably, the decoloring processing method in step S4 is to add water to the sweet part of cyclocarya paliurus leaves to prepare a stock solution to be decolored of the sweet part of cyclocarya paliurus, and add a decoloring agent for decoloring, wherein the decoloring agent is magnesium oxide, attapulgite, polyamide, alumina, activated carbon or a combination thereof, and the addition amount is 0.03-6%.
Preferably, the decoloring processing method in step S4 includes the steps of:
s4.1 preparation of a compound decolorizing agent: compounding magnesium oxide and an active carbon agent, and uniformly mixing to obtain a compound decolorizing agent;
s4.2 adding a compound decolorizing agent into the stock solution to be decolorized of the sweet part of the cyclocarya paliurus, decolorizing at constant temperature, shaking, filtering, and collecting the filtrate to obtain the decolorized sweet part of the cyclocarya paliurus.
Further preferably, the magnesium oxide and the active carbon agent in the step S4.1 are compounded according to the ratio of (75-85) to 1; s4.2 adding 2-5% of compound decoloring agent into the stock solution to be decolored at the sweet part of the cyclocarya paliurus, and decoloring at the constant temperature of 55-75 ℃ for 0.5-2 h.
Further preferably, the cyclocarya paliurus leaves are also preprocessed before the step S1, and the preprocessing method includes: adding lime water into cyclocarya paliurus leaves, soaking at room temperature for 0.5-1.5h, filtering, removing filtrate, quickly washing the leaves with water for 2-3 times, testing to show neutrality, and air drying for use.
The cyclocarya paliurus sweet glycoside or the cyclocarya paliurus sweet glycoside prepared by the method can be used for preparing a medicament with a lipid-lowering function, and is characterized by belonging to any one of oral liquid, capsules, tablets, pills, dripping pills, powder and injection, or being used as a food additive for processing and producing various types of wine, beverages and foods, or being used as a pharmaceutical adjuvant for producing pharmaceutical preparations.
The detailed development process is as follows:
1. sources of drugs
Researching main distribution and cultivation areas of cyclocarya paliurus resources through literature research and field investigation, and collecting fresh plant materials; meanwhile, the cyclocarya paliurus leaf tea with different producing areas and different batches is purchased from various markets. All Cyclocarya paliurus plant material was identified as leaves of Cyclocarya paliurus (Batal.) lljinskaja, see Table 1. All experimental materials were used for the preparation of cyclocarya paliurus sweet glycosides in the present invention. As a research material, cyclocarya paliurus tender leaf S6 produced in the whole state of Guangxi is mainly selected as a research object.
Table 1 cyclocarya paliurus materials summary
2. Sweet taste testing method
All test solutions were prepared in distilled water at 25 + -5 deg.C. The taste panel consists of 5 men and 5 women, is interesting for sensory evaluation, is healthy, has good expression ability and good sensory resolution, and can objectively treat each sample to be tested. Between two samples, the mouth was rinsed several times with warm water until no aftertaste was perceived. Only one sample was evaluated per taste test.
The test methods were performed according to the methods reported in the literature [ A.T.Cameron. the relative fans of the vacuum compounds and of the air mix. Can J Res,1945,23: Sec.E.139-166], with minor adjustments. Tasters were asked to compare the sweetness of three randomly placed sucrose solutions of different concentrations prior to testing and in order, about 90% of the panelists could be in the correct order. Then, each member is assigned with a test solution with a certain concentration and six randomly placed sucrose solutions with different concentrations, and a taster is required to find out the sucrose solution with the same sweetness as the tested sample, namely the sucrose concentration (ES) with equal sweetness.
The tasting results of the samples are described in Relative Sweetness (RS). RS indicates that the sweetness of a test sample at a certain first-sweetness sucrose concentration (ES) is a multiple of the sweetness of sucrose (═ 1):
relative Sweetness (RS) ═ iso-sugar concentration (%)/sample solution concentration (%).
3. Preparation of cyclocarya paliurus tea soup and sweet taste test result
Cyclocarya paliurus leaves in table 1 are taken, dried under reduced pressure and vacuum and then crushed into coarse powder. And (3) putting 0.5g of cyclocarya paliurus leaf coarse powder into a corn fiber tea bag, and sealing. Respectively taking a bag of cyclocarya paliurus tea bag, adding 100ml of boiled water, soaking for 10 minutes, and filtering out tea soup. When the temperature of the tea soup is reduced to room temperature, the tea soup is tasted, and the sweet taste is evaluated and recorded. When the sweet taste test is carried out, sucrose solutions with the series of concentrations are respectively prepared to be 10%, 5%, 1%, 0.5%, 0.1% and 0.05% (m/m), and the concentrations of the sample solutions are all 0.5% (m/m). The determination of the ES value of the sample allows taking an intermediate value of each concentration of sucrose solution, for example, the sweet taste of a certain sample tea soup is slightly stronger than that of a 1% sucrose solution and weaker than that of a 2.5% sucrose solution, and the ES value of the sample solution is determined by the taster to be between 1% and 2.5%.
The sweet taste test result shows that the cyclocarya paliurus leaves in the table 1 have sweet taste except the tea soup of four cyclocarya paliurus old leaves of S1, S9, S16 and S19, and the change range of the RS value is 0.4-8. Wherein, the sweetness of the tea soup S6 (Guangxi Quanzhou) and S7 (Guizhou Reshan) is the highest, and the RS values are respectively 6 and 8. In the subsequent study, the tender leaf of cyclocarya paliurus S6 produced in the state of Guangxi with a relatively abundant stock is selected as a study material.
4. Screening and extracting method of sweet part of cyclocarya paliurus leaf
According to the mode of making cyclocarya paliurus tea with hot water for drinking, a proper amount of S6 cyclocarya paliurus leaf coarse powder is weighed, 20 times of drinking purified water is added, heating reflux extraction is carried out for 3 times, the single extraction time is 2 hours, extracting solutions are combined, and reduced pressure concentration is carried out. Extracting appropriate amount of concentrated solution with dichloromethane, ethyl acetate and n-butanol sequentially, recovering solvent, and freeze drying to obtain dichloromethane fraction, ethyl acetate fraction, n-butanol fraction and raffinate water fraction. And eluting with 10 times of water, 20% ethanol solution, 40% ethanol solution, 60% ethanol solution, 80% ethanol solution and 95% ethanol respectively. Recovering solvent from each eluate, and freeze drying to obtain water washing part, 20% ethanol eluting part, 40% ethanol eluting part, 60% ethanol eluting part, 80% ethanol eluting part and 95% ethanol eluting part.
Weighing appropriate amount of the above components respectively, adding hot water, and shaking to obtain 5 mg/ml solution-1The solution (a) of (b) is,the sweetness was evaluated and recorded. As a result, the dichloromethane part and the ethyl acetate part, as well as the 40% ethanol elution part, the 60% ethanol elution part and the 80% ethanol elution part have obvious sweet taste, and the RS value is all more than 6. The result indicates that both the solvent extraction method and the macroporous adsorption resin method have good enrichment effect on the sweet components in the cyclocarya paliurus leaves.
The dichloromethane and ethyl acetate in the solvent extraction method are eluted by 40 percent ethanol solution, 60 percent ethanol solution and 80 percent ethanol solution in a macroporous adsorption resin method, so that the aim of enriching sweet components in cyclocarya paliurus leaves can be achieved. Because dichloromethane and ethyl acetate both belong to medium-low polarity organic solvents, the polarity of ethyl acetate is slightly stronger than that of dichloromethane, and the principle that the dichloromethane and the ethyl acetate enrich sweet components in cyclocarya paliurus leaves is that the dichloromethane and the ethyl acetate are similar and soluble, the sweet components in the cyclocarya paliurus leaves can be enriched in a one-step extraction mode of ethyl acetate. The macroporous adsorption resin method is mainly realized according to the principle that sweet components are adsorbed by macroporous resin and the desorption effect of ethanol solutions with different concentrations is realized, the higher the concentration of the alcohol solution is, the stronger the desorption capacity is, so that the stepwise elution of water, 20% ethanol solution and 80% ethanol solution can be considered, and the elution part of 80% ethanol can be directly collected, namely almost all sweet components in the cyclocarya paliurus leaves can be effectively enriched. Further considering the difference of the working principles of the solvent extraction method and the macroporous adsorption resin method, the types and the capacities of removing impurities of the solvent extraction method and the macroporous adsorption resin method are different, and in order to obtain the sweet part of the cyclocarya paliurus leaves with less impurities, a mode of combining the solvent extraction method and the macroporous adsorption resin method is designed to extract the sweet part of the cyclocarya paliurus leaves. Through repeated tests, the following extraction process methods were determined:
weighing appropriate amount of cyclocarya paliurus leaves, adding 20 times of drinking purified water, heating and reflux-extracting for 3 times, extracting for 2h for one time, mixing extractive solutions, and concentrating under reduced pressure. The concentrated solution passes through pretreated X-5 type macroporous adsorption resin, and is sequentially eluted by 10 times of column volume of water, 20% ethanol solution and 80% ethanol solution. Concentrating the 80% ethanol eluted part under reduced pressure until no alcohol smell exists, adding a proper amount of water for suspension, extracting with ethyl acetate, recovering the solvent, and freeze-drying to obtain the sweet part of the cyclocarya paliurus leaves. The extraction process is shown in FIG. 3.
Taking appropriate amount of sweet part of folium cyclocarya paliurus, adding hot water, and shaking to obtain 5 mg/ml-1The obtained aqueous solution of (4) was found to have a strong and long-lasting sweet taste with an RS value of 16.
5. Screening for depigmenting agents
Through the research of the '4' mentioned above, the sweet part of the cyclocarya paliurus leaf has strong and lasting sweet feeling, but at the same time, the part is found to be semi-solid from brown to yellow green, which indicates that a large amount of impurities such as pigment coexist. For this purpose, it is necessary to subject it to a decolorizing treatment with a suitable decolorizing agent. The existing pigment removing method mainly comprises an adsorption method of activated carbon and the like, a chemical method of hydrogen peroxide and the like, an ion exchange resin, a macroporous resin adsorption method and the like. According to the test, 10 types of decolorizing materials including activated carbon, activated clay, polyamide, attapulgite, magnesium oxide, aluminum oxide, anion exchange resin, cation exchange resin, silica gel and diatomite are used for decolorizing sweet parts of cyclocarya paliurus, and the optimal decolorizing agent is screened out by taking the decolorizing rate and the sweet taste change as investigation indexes for subsequent decolorizing process research.
5.1 preparation of sweet part of cyclocarya paliurus adopts the process conditions of the step 4 to prepare the original liquid to be decolorized of the sweet part of cyclocarya paliurus with the concentration of 0.1g/ml for later use.
5.2 sweetness evaluation method the sweetness evaluation method described above under "2" was used. Here, due to the high concentration of the sample solution (10%, m/m), at this concentration, the sweetness of the oral test sample solution is extremely strong, exhibiting a pronounced aftertaste, while the sweetness duration exceeds 20 minutes, greatly affecting the efficiency of the sweetness test. Therefore, in order to compare the change of sweetness before and after the treatment of various decolorants more intuitively, each sample is repeatedly tasted for a plurality of times, the sweetness of the solution is quantitatively scored by recording the change of sweetness between the stock solution and the solution after the decoloration treatment, and the higher the score is, the higher the sweetness is, the closer to the stock solution is. Wherein the sweetness of the product is 5 minutes, slightly reduced to 4 minutes, obviously reduced to 2 minutes, only slightly reduced to 1 minute, and no obviously reduced to 0 minute.
5.3 determination of discoloration rate of sweet part of cyclocarya paliurus, a full-wavelength scanning absorption curve is carried out in the range of wavelength 200-. According to the related documents, wavelengths of 420, 520 and 620nm are selected as the wavelengths for measuring the decolorization rate of the sweet part of the cyclocarya paliurus. The decolorization ratio was calculated according to the following formula:
decolorization ratio [ [ (A)420+A520+A620)Before decolorization-(A420+A520+A620)After decolorization/(A420+A520+A620)Before decolorization]×100％
5.4 selection of decolorizing agent
5.4.1 determination of maximum appropriate amount of decolorizer A plurality of 10mL of stock solution are taken, respectively added with appropriate amount of 10 decolorizers of activated carbon, activated clay, polyamide, attapulgite, magnesium oxide, aluminum oxide, anion exchange resin, cation exchange resin, silica gel and diatomite, kept at 60 ℃ for 2h for decolorization, filtered to remove the decolorizer, and the filtrate is collected. The proper maximum dosage of each decoloring agent is selected by taking the fact that the sweet taste of the decoloring solution is basically consistent (more than 4 minutes) with the original solution as a judgment basis. The results show a suitable maximum amount of each decolorizing agent: 0.05 percent of activated carbon; activated clay 0.2%; 5% of polyamide; 5% of attapulgite; 4% of magnesium oxide; 2% of alumina; 2g of cation exchange resin; 5% of silica gel; 5 percent of diatomite. Wherein the anion exchange resin has obviously weakened sweet taste (less than 2 minutes) at the minimum dose (2g), and is not used for the research of the decolorization process.
5.4.2 the decolorization effect stock solution of each decolorizer is decolorized by activated carbon, activated clay, polyamide, attapulgite, magnesium oxide, aluminum oxide, cation exchange resin, silica gel and diatomite at the maximum suitable amount, the decolorization rate is calculated, and the decolorization effects are compared in parallel. The results are shown in FIG. 4. After the stock solution is decolorized by each decolorizing agent at the maximum suitable dosage, the decolorizing effect is as follows from good to bad: magnesium oxide, attapulgite, polyamide, alumina, activated carbon, cation exchange resin, silica gel, kieselguhr and activated clay; wherein, the magnesium oxide, the attapulgite, the polyamide, the alumina and the active carbon have better decoloration effect.
5.4.3 decolorizing agents are combined to consider that the decolorizing principle of each material is different, the types of pigment impurities removed by the decolorizing agents are different, and in order to obtain the best decolorizing effect, a composite decolorizing method is further adopted, namely, five decolorizing agents with better decolorizing effect are screened out: magnesium oxide (A), attapulgite (B), polyamide (C), alumina (D) and activated carbon (E) are combined in a pairwise proportion, the using dose is the maximum applicable dose of the decolorizing agent, the combined decolorizing effect is examined, and the result is shown in figure 5. The decolorizing effects of different combined decolorizing agents on the stock solution of the sweet part are sequenced: AE is AD > AC > AB > DE > > CE > BD > BE > BC > CD, wherein the AE combination (magnesium oxide + activated carbon) has the best decolorizing effect on stock solution, the decolorizing rate can reach 82.71%, the retention rate on sweet taste is relatively higher, and the sweet taste score is 4.5 (basically no reduction compared with the stock solution). The magnesium oxide and the activated carbon are used in a composite way, so that the decoloring operation is convenient and quick, the price is low, and the decoloring effect is excellent, so that the magnesium oxide and the activated carbon are selected to be used together as a decoloring agent for the sweet taste part solution of the cyclocarya paliurus.
6. Optimization of decoloring process of sweet part of cyclocarya paliurus leaves
6.1 preparation of Compound decolorizer magnesium oxide and active carbon are screened out according to the 5 to be used as decolorizer for sweet part of cyclocarya paliurus, a series of proportions are designed, the proportion of magnesium oxide and active carbon is determined by experiments to be 80:1, and the components are compounded and mixed uniformly to obtain the compound decolorizer.
6.2 determination of the amount of the decolorizing agent 10mL of stock solution was taken respectively in a conical flask, 1%, 2%, 3%, 4%, 5% of a compound decolorizing agent was added respectively, decolorized at constant temperature of 60 ℃ for 1.5h, shaken all the time, filtered, the filtrate was collected, the absorbance of the solution was measured at wavelengths of 420, 520, 620nm, the decolorization rate was calculated, and the sweetness was evaluated. The result shows that the larger the added compound decolorizing agent is, the better the decolorizing effect is; however, the sweetness of the sample to be tested is obviously reduced along with the increase of the dosage of the compound decolorant. The decolorizing effect and the sweet taste change are comprehensively considered, and 3 percent of the compound decolorizing agent is preferably selected.
6.3 determination of decolorization time 10mL of stock solution was taken respectively in a conical flask, 3% of compound decolorizer was added respectively, decolorization was carried out at 60 ℃ for 0.5h, 1h, 1.5h, 2h, 3h at constant temperature, shaking was carried out constantly, filtration was carried out, the filtrate was collected, the decolorization rate was calculated, and the sweetness was evaluated. The result shows that the prolongation of the decoloring time is beneficial to the contact adsorption of pigment molecules and the compound decoloring agent, and the decoloring rate is basically kept constant after the decoloring time is 1.5 h; after 1.5h, the sweetness of the solution was slightly reduced. Therefore, the decoloring time is preferably 1.5 hours.
6.4 determination of decolorizing temperature 10mL of stock solution was taken in conical flasks, 3% of compound decolorizing agent was added, decolorized at 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C for 1.5h, shaken from time to time, filtered, the filtrate was collected, the decolorizing rate was calculated, and the sweetness was evaluated. The result shows that the temperature has obvious influence on the decolorization of the stock solution, and the temperature is increased to be beneficial to the decolorization; however, when the temperature is raised to above 60 ℃, the decolorization rate tends to be flat, and the sweetness of the solution begins to decrease. Comprehensively considering, the decolorizing temperature is preferably 60 ℃.
6.5 summary through the above tests, the proper decolorization process conditions of the sweet part of cyclocarya paliurus are basically determined: magnesium oxide, activated carbon and other single or composite decolorants are adopted, the preferable decolorant is a compound decolorant obtained by combining more than two decolorant materials, the more preferable decolorant is a compound decolorant obtained by combining magnesium oxide and activated carbon according to the weight ratio of 80:1, the consumption of the decolorant is 1-5%, and the more preferable consumption is 3%; the decoloring time is 0.5h-3h, preferably 1.5 h; the decolorizing temperature is 40-80 ℃, and the preferred decolorizing temperature is 60 ℃. Under the condition of the process parameters, the discoloring effect of the sweet part of the cyclocarya paliurus is better, and the influence on the sweet is smaller.
7. Preparation of cyclocarya paliurus sweet glycoside
Through the tests from 4 to 6, the technological methods of enrichment, purification, decoloration and the like of the sweet part of cyclocarya paliurus are basically determined, but the problems of technological rationality such as production efficiency and the like, operation applicability and the like need to be considered for preparing cyclocarya paliurus sweet glycosides from cyclocarya paliurus leaves. Subsequent experimental research finds that the extraction mode of water decoction is adopted, the cyclocarya paliurus leaf extracting solution for the 4 th time or even the 5 th time still has obvious sweet taste, and the extraction efficiency of cyclocarya paliurus sweet glycosides is low by taking water as a solvent; meanwhile, the cyclocarya paliurus leaf extracting solution is heated (nearly 100 ℃) for a long time (more than 3 hours) for decoction and concentration, the sweet taste is obviously lost, and the process is suggested to possibly damage the cyclocarya paliurus sweet glycoside component. Further, through comparative research, it is found that the cyclocarya paliurus leaves are soaked in saturated or nearly saturated lime water (pH 10-13) at room temperature, so that part of pigment impurities can be effectively removed, and the influence on sweetness of the cyclocarya paliurus leaves is small. Therefore, the technological conditions for extracting cyclocarya paliurus sweet glycosides from cyclocarya paliurus leaves can consider that saturated or nearly saturated lime water is adopted to pretreat the cyclocarya paliurus leaves to remove part of pigment impurities; meanwhile, the more effective and safer extraction solvent is considered and selected, and the solution rich in cyclocarya paliurus sweet glycosides is prevented from being subjected to long-time high-temperature treatment. Through repeated tests, the suitable preparation process method of cyclocarya paliurus sweet glycoside is finally determined: taking a proper amount of cyclocarya paliurus leaves, adding a proper amount of saturated or nearly saturated lime water (pH value is about 13), soaking at room temperature for 1h, filtering, discarding filtrate, quickly washing the leaves with clear water for 3 times, testing pH test paper to be neutral, drying in the air, adding 70% ethanol solution with the material-liquid ratio of 1:20, heating, refluxing and extracting for 2h, filtering, and collecting filtrate. After repeated extraction for 4 times, the leaves had no sweet taste. Mixing extractive solutions, recovering ethanol under reduced pressure, concentrating, centrifuging and/or filtering to obtain cyclocarya paliurus leaf concentrated solution. Passing the concentrated solution through a pretreated X-5 macroporous adsorbent resin column, sequentially eluting with water, 20% ethanol and 80% ethanol, collecting 80% ethanol eluate, and recovering ethanol under reduced pressure to obtain folium cyclocarya paliurus 80% ethanol eluate. Dispersing the part with appropriate amount of water, extracting with ethyl acetate, and recovering solvent to obtain sweet part of cyclocarya paliurus leaf. And then, decoloring by adopting a method under the item 6.5, and finally, freeze-drying to obtain cyclocarya paliurus sweet glycosides.
The cyclocarya paliurus sweet glycoside extract (figure 6) is light brown yellow to white-like fine powder, slightly hygroscopic. The solubility in cold water is low, and the solvent is easy to dissolve in hot water and organic solvents such as ethanol and methanol. The water solution has strong and lasting sweet taste and is slightly bitter. By adopting the sweet taste testing method under item 2, the RS value of the cyclocarya paliurus sweet glycoside extract is 50-70.
8. Separation and identification of marker components in cyclocarya paliurus sweet glycosides
Taking a cyclocarya paliurus sweet glycoside sample of 0.5g, dissolving in methanol, filtering, carrying out Sephadex LH-20 gel column chromatography on filtrate, eluting with 60% methanol solution, collecting eluent every 8-10mL, and combining the eluates with similar components after HPLC and TLC detection to obtain fractions A-F. And treating the fraction D by using semi-preparative high performance liquid chromatography to obtain a sub-fraction D-1. The D-1 is subjected to Sephadex LH-20 gel column chromatography again and finally to semi-preparative high performance liquid chromatography to obtain a monomeric compound D-1-2-1(40 mg).
Taking appropriate amount of D-1-2-1, and substituting deuterated methanol (CD)3OD) after dissolution1H-NMR and13C-NMR measurement. The carbon spectrum data of the monomeric compound are as follows:13C NMR(100MHz，CD3OD) δ 37.5(C-1), 29.9(C-2), 179.2(C-3), 148.4(C-4), 49.5(C-5), 25.0(C-6), 31.0(C-7), 39.9(C-8), 40.5(C-9), 41.0(C-10), 33.8(C-11), 75.9(C-12), 44.4(C-13), 50.8(C-14), 34.9(C-15), 25.2(C-16), 52.1(C-17),16.1(C-18), 19.8(C-19), 75.0(C-20), 25.5(C-21), 44.1(C-22), 123.0(C-23), 141.2(C-24), 70.3(C-25), 29.1(C-26), 29.0(C-27), 29.2 (C-23), 29.7 (C-23), 16.2(C-30), 99.9(C-1 '), 74.7 (C-2'), 76.3(C-3 '), 77.1 (C-4'), 72.1(C-5 '), 17.2 (C-6'). According to the carbon spectrum data, combined with literature reports, the monomeric compound is identified as discoidin A (Pterocarposines A), and the structure is shown in figure 7.
9. Cyclocarya paliurus sweet glycoside HPLC characteristic map analysis
9.1 preparation of test solution A proper amount of cyclocarya paliurus sweet glycoside sample is taken, added with methanol and treated with ultrasound for 10min to be fully dissolved, filtered by a microporous filter membrane (0.45 mu m), and a subsequent filtrate is taken, thus obtaining the cyclocarya paliurus sweet glycoside.
9.2 preparation of control solution A proper amount of the self-made discoidin A control is precisely weighed and dissolved by adding methanol to prepare a control stock solution. Sucking appropriate amount of the stock solution, adding methanol for diluting, and shaking.
9.3 chromatographic conditions
Chromatographic conditions are as follows: a Dionex UltiMate 3000 HPLC system. Ultimate XB-C18(4.6 mm. times.250 mm, 5 μm); column temperature: 30 ℃; detection wavelength: 205 nm;flow rate: 1.0 mL/min; mobile phase: a: 0.1% aqueous phosphoric acid solution, B: acetonitrile; gradient elution: 0 min: 10% of B; 5 min: 10% of B; 20 min: 30% of B; 30 min: 45% of B; and (5) 60 min: 55% of B; 70 min: 80% of B; 80 min: 80% of B. Sample introduction amount: 10 μ l.
9.4 the acquisition time of the resulting atlas is 80 min. The retention time (Rt) is within the first 40 minutes, obvious chromatographic peaks appear except Rt 4-6min, and other chromatographic peaks are small. The smaller chromatographic peaks have the spectral characteristics of flavonoid components through ultraviolet spectral analysis, which indicates that a small amount of pigment and flavonoid components possibly exist in the cyclocarya paliurus sweet glycoside sample; in the Rt 40-65min range, obvious chromatographic peaks appear, and the chromatographic peaks are all shown as maximum absorption at the ultraviolet end through ultraviolet spectrum analysis, and meanwhile, according to the principle of reversed phase chromatographic separation, the molecular polarity of the corresponding chemical components of the chromatographic peaks appearing in the range is medium-low polarity, so that the components corresponding to the chromatographic peaks in the range are presumed to be triterpenoid components. Calibrating by a self-made reference substance of the discoidin A, wherein the component corresponding to the Rt 51min chromatographic peak is the discoidin A. The discoidin A is a schizodammarane type triterpene saponin with molecular weight of 636.8562, moderate polarity and strong sweet taste. Therefore, the chromatographic peak in the Rt 40-65min range is further proved to be mainly the schizodammarane type triterpene glycoside represented by the discoidin A, namely cyclocarya paliurus sweet glycoside compounds. The above results are shown in FIG. 8.
In addition, the cyclocarya paliurus sweet glycoside extract prepared by extracting and refining the cyclocarya paliurus tender leaves serving as the raw materials has strong and lingering sweet taste and is slightly bitter; the cyclocarya paliurus sweet glycoside extract extracted and refined from old cyclocarya paliurus leaves has slightly light sweet taste and obvious bitter taste. HPLC analysis shows that both of them contain the designated components of the discoidin A, but the content of the components in the extract obtained by taking old leaves as raw materials is obviously less, and meanwhile, an obvious impurity peak exists within the retention time of 8-30 min; in the extract obtained by using the tender leaves as the raw material, the content of the discoside A is high, and the chromatographic peak is rich within the retention time range (42-64min) which is similar to the index component of the discoside A, so that the extract can be used as an important characteristic of the chemical composition of cyclocarya paliurus sweet glycoside. The above results are shown in FIG. 9.
10. Lipid-lowering activity research of cyclocarya paliurus sweet glycosides
10.1 preadipocyte culture and differentiation after 3T3-L1 cells attached (Day 0), it was transferred to a medium containing differentiation Medium (MDI) [ formulation: 0.5mM 3-isobutyl-1-methylxanthine, 1. mu.M dexamethasone and 5. mu.g/mL insulin ] for 2 days (Day 2), followed by 2 days with DMEM high-sugar medium containing 5. mu.g/mL INS alone (Day 4) and finally 5 days with DMEM high-sugar medium (from Day 4 to Day 9). The time for intervention of the cyclocarya paliurus sweet glycoside extract tested is 7 days (from Day2 to Day 9); 3T3-L1 cells that were not treated to promote differentiation were used as a control group.
10.2 Effect on 3T3-L1 cell Activity after 5 and 7 days of culture of the cyclocarya paliurus glycoside extract, 3T3-L1 cells have 7.49 and 11.95% lower cell viability than the control group at the same time point, and do not show any toxicity to the cells within the concentration range of 0.1-100 mug/mL. Therefore, the experimental dose of the cyclocarya paliurus sweet glycoside extract is performed in a nontoxic concentration range of less than 100 μ g/mL.
10.3 effects on lipid accumulation and TG content during differentiation of 3T3-L1 cells after 2, 5 or 7 days of treatment of 3T3-L1 cells with cyclocarya paliurus sweet glycoside extract (50 and 100. mu.g/mL) and pioglitazone (100. mu.M), it can be seen that 3T3-L1 has significant lipid accumulation and TG content reduction, and has significant differences compared with the same time point model group, wherein the effect is most significant when the cells are treated for 7 days. See table 2 and fig. 10.
TABLE 2 influence of cyclocarya paliurus glycosides on lipid accumulation and TG content during differentiation of 3T3-L1 cells (Mean + -SD, n ═ 5)
Note: model group comparison with simultaneous points in time*P＜0.05，**P＜0.01
10.4 PPARgamma, C/EBP alpha, SREBP-1C, aP2 and adiponectin mRNA are obviously reduced after 3T3-L1 cells are treated by cyclocarya paliurus sweet glycoside extracts (50 and 100 mu g/mL) and pioglitazone (100 mu M) for regulating and controlling the expression of PPAR gamma/CEBP alpha signal channel related genes of 3T3-L1 cells; the expression of Nrf2 in nucleus and HO-1 protein in cell is obviously increased, and the expression of Nrf2 protein in cytoplasm is obviously reduced. See table 3, fig. 11.
TABLE 3 influence of cyclocarioside on PPAR γ/CEBP α signaling pathway-related gene expression controlling differentiation of 3T3-L1 cells (Mean + -SD, n ═ 5)
Note: comparison with model group of P < 0.05, P < 0.01
10.5 through interfering with the influence of HO-1 expression on the contents of lipid and TG in the differentiation process of 3T3-L1 cells, the contents of intracellular lipid and TG after treatment with the cyclocarya paliurus sweet glycoside extract are obviously reduced, and the contents of the reduced intracellular lipid and TG after treatment with the HO-1 inhibitor ZnPP alone and the cyclocarya paliurus sweet glycoside extract together are obviously reversed, and the results are shown in Table 4. In addition, after the treatment of the cyclocarya paliurus sweet glycoside extract, the mRNA expression of PPAR gamma, C/EBP alpha, SREBP-1C, aP2 and adiponectin in cells is obviously reduced, and after the HO-1 inhibitor ZnPP is singly used and the cyclocarya paliurus sweet glycoside terpene extract is used in combination, the mRNA expression is obviously reversed, and the result is shown in Table 5.
Table 4 influence of cyclocarya paliurus glycosides on lipid and TG contents in 3T3-L1 cell differentiation process by intervention of HO-1 expression (Mean ± SD, n ═ 5)
Note: comparison with model group*P＜0.05，**P is less than 0.01; comparing with salidroside groupΔP＜0.05，ΔΔP＜0.01
TABLE 5 influence of cyclocarya paliurus glycosides on PPAR γ/CEBP α signaling pathway-related genes during differentiation of 3T3-L1 cells by intervention of HO-1 expression (Mean + -SD, n ═ 5)
Note: comparison with model group*P＜0.05，**P is less than 0.01; comparing with salidroside groupΔP＜0.05，ΔΔP＜0.01
10.6 cyclocarya paliurus sweet glycoside lipid-lowering activity research shows that cyclocarya paliurus sweet glycoside extract has obvious inhibition effect on differentiation of 3T3-L1 preadipocytes and generation of fat; the action mechanism of the polypeptide may be related to the gene expression of the activation Nrf2/HO-1 signal channel; it is involved in inhibiting the expression of lipogenic transcription factors such as PPAR γ, C/EBP α, SREBP-1C, aP2, adiponectin, etc.
The experimental research from the '4' to the '10' proves that the cyclocarya paliurus sweet glycosides are really extracts with strong sweet taste which are extracted and refined from cyclocarya paliurus leaves; the index component of the extract is the chemical component of the dicarbarin A which is reported to have strong sweet taste; the structural characteristics of taking the discoidin A as a typical component are as follows: the dammarane type triterpene mother nucleus with 2, 3-position split ring contains 2 unsaturated double bonds and is connected with isorhamnetin glycoside group, and the structural characteristics are consistent with the characteristics of the main component contained in cyclocarya paliurus glycoside defined in the background technology. In addition, the cyclocarya paliurus sweet glycosides have definite lipid-lowering activity.
Compared with the research on the total flavone and total triterpene of cyclocarya paliurus, as well as other traditional Chinese medicines and natural product extracts, the cyclocarya paliurus sweet glycoside provided by the invention is obtained by taking sweetness as a process index and carrying out process screening and optimization. The sweetness index is not kept, and the common extraction, separation, refining and other process operations can cause the loss of sweet components, so the method is not suitable for preparing the cyclocarya paliurus sweet glycosides. After the sweet part of cyclocarya paliurus is obtained, although the sweet part is strong in sweetness, the bitter and astringent taste is obvious, and the part is in a semi-solid state from brown to yellow green, which shows that a large amount of impurities such as pigment coexist, so that further decoloration and refining are needed. The research on the decoloring process with sweetness and decoloring rate as process indexes shows that the type of pigment coexisting with cyclocarya paliurus sweet glycosides is complex, and conventional decoloring agents such as activated carbon, silica gel, macroporous adsorption resin and ion exchange resin cannot achieve a good decoloring effect under a front dike for effectively retaining the sweet components of cyclocarya paliurus. Through screening and comparison research, magnesium oxide as a decoloring agent can obtain a good decoloring effect under the front dike where sweet components of cyclocarya paliurus are reserved to a large extent; further research shows that although the decolorizing effect of the activated carbon is general when the activated carbon is used alone, the decolorizing effect can be greatly enhanced by combining the activated carbon with magnesium oxide, but the dosage and the proportion of the activated carbon and the magnesium oxide need to be controlled, otherwise, the common decolorizing degree or the serious loss of sweet components of the cyclocarya paliurus can be caused. Finally, determining that the dosage ratio of the magnesium oxide to the active carbon is 80:1 as a complexing agent, and under the condition of proper technological parameters, obtaining the cyclocarya paliurus sweet glycoside with good quality and high yield.
Although cyclocarya paliurus sweet glycosides contain triterpenoid saponins such as spilantoside A, the cyclocarya paliurus sweet glycosides are different from cyclocarya paliurus total triterpenes represented by oleanolic acid in general meanings, and are further different from extracts such as cyclocarya paliurus total flavonoids and cyclocarya paliurus total polysaccharides. The extraction of cyclocarya paliurus sweet glycosides needs to start with the raw materials and the treatment of the raw materials, retain sweet components as much as possible, prevent the sweet components from being damaged, and remove pigment impurities to the greatest extent, so that the quality of the cyclocarya paliurus sweet glycosides is improved. Particularly, the tender leaves of cyclocarya paliurus are the preferred raw materials, and lime water is adopted for pretreatment before extraction, so that part of impurities can be effectively removed, and the subsequent process operations such as enrichment, purification, decoloration and the like are particularly facilitated; because the sweet component represented by the discoidin A contains a plurality of unsaturated bonds and has chemical instability and thermal instability, the process parameters of extraction, refining and other operations of the cyclocarya paliurus sweet glycoside need to be carefully controlled, and the change of the sweetness of the material can be noticed at any time.
The foregoing detailed description is intended to illustrate and not limit the invention, which is intended to be within the spirit and scope of the appended claims, and any changes and modifications that fall within the true spirit and scope of the invention are intended to be covered by the following claims.
Claims (10)
1. Cyclocarya paliurus sweet glycoside is characterized in that cyclocarya paliurus sweet glycoside is obtained by sequentially eluting cyclocarya paliurus leaves with water or alcohol, macroporous adsorption resin and water and ethanol solution.
2. The cyclocarya paliurus sweet glycoside of claim 1, wherein the cyclocarya paliurus sweet glycoside comprises discoidin A or other triterpenoid saponins.
3. The preparation method of cyclocarya paliurus sweet glycoside according to any one of claims 1 to 2, which is characterized by comprising the following steps:
s1: adding water or ethanol into appropriate amount of cyclocarya paliurus leaves, heating and reflux-extracting, mixing extractive solutions, and concentrating under reduced pressure to obtain concentrated solution;
s2: passing the concentrated solution through macroporous adsorbent resin column, and sequentially eluting with water, 15-25% ethanol solution, and 75-85% ethanol solution;
s3: concentrating the 75-85% ethanol eluate under reduced pressure until no alcohol smell is present, adding water for suspension, extracting with ethyl acetate, and recovering solvent to obtain sweet component of folium cyclocarya paliurus;
s4: carrying out decoloring treatment on the sweet part of the cyclocarya paliurus leaves;
s5: drying to obtain cyclocarya paliurus sweet glycoside.
4. The production method according to claim 3, characterized in that: and step S1, adding 15-30 times of water or 70% ethanol, heating and refluxing for 3-4 times, wherein the single extraction time is 1.5-2.5 h.
5. The production method according to claim 3, characterized in that: and (3) allowing the concentrated solution obtained in the step (S2) to pass through a macroporous adsorption resin column, wherein the filled material is nonpolar or low-polarity macroporous adsorption resin, such as X-5 type macroporous adsorption resin, and is sequentially eluted by using water, 15-25% ethanol solution and 75-85% ethanol solution which are 5-15 times of the column volume respectively.
6. The production method according to claim 3, characterized in that: the step S4 decolorization treatment method comprises the steps of adding water into the sweet part of the cyclocarya paliurus leaves to prepare a stock solution to be decolorized of the sweet part of the cyclocarya paliurus, and adding a decolorizing agent for decolorization, wherein the decolorizing agent is magnesium oxide, attapulgite, polyamide, alumina, activated carbon or a combination thereof, and the adding amount is 0.03-6%.
7. The production method according to claim 3, characterized in that: the decoloring processing method of the step S4 includes the steps of:
s4.1 preparation of a compound decolorizing agent: compounding magnesium oxide and an active carbon agent, and uniformly mixing to obtain a compound decolorizing agent;
s4.2 adding a compound decolorizing agent into the stock solution to be decolorized of the sweet part of the cyclocarya paliurus, decolorizing at constant temperature, shaking, filtering, and collecting the filtrate to obtain the decolorized sweet part of the cyclocarya paliurus.
8. The method of claim 7, wherein: s4.1, compounding the magnesium oxide and the active carbon agent according to the ratio of (75-85) to 1;
s4.2 adding 2-5% of compound decoloring agent into the stock solution to be decolored at the sweet part of the cyclocarya paliurus, and decoloring at the constant temperature of 55-75 ℃ for 0.5-2 h.
9. The production method according to claim 3, characterized in that: the cyclocarya paliurus leaves are also preprocessed before the step S1, and the preprocessing method comprises the following steps: adding cyclocarya paliurus leaves into lime water solution with the pH value of 10-13, soaking at room temperature for 0.5-1.5h, filtering, removing filtrate, quickly washing the leaves with water for 2-3 times, testing to show neutrality, and air-drying for later use.
10. Cyclocarya paliurus sweet glycoside according to any one of claims 1 to 2 or cyclocarya paliurus sweet glycoside prepared by the method according to any one of claims 3 to 9, which can be used for preparing a medicament with a lipid-lowering function, belongs to any one of oral liquid, capsules, tablets, pills, dripping pills, powder and injection, or is used for common food or health food, or is directly used as a sweetener, an aromatic or other food additives.
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| CN202110310638.5A Pending CN113087750A (en) | 2021-03-23 | 2021-03-23 | Cyclocarya paliurus sweet glycoside, and preparation method and application thereof |
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Citations (3)
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| CN106511353A (en) * | 2016-09-08 | 2017-03-22 | 中国药科大学 | Preparation and application of triterpene drug for inhibiting synthesis and secretion of apoB48 in intestinal tracts |
| CN108235686A (en) * | 2015-06-24 | 2018-06-29 | 阿纳里蒂科探索有限责任公司 | Novel tetraterpene glycosides is as sweetener or sweetness enhancers |
| CN109364119A (en) * | 2018-12-26 | 2019-02-22 | 浙江大学 | Preparation has the method and application of the total triterpene of hypoglycemic effect from Qingqian Willow leaf |
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| CN108235686A (en) * | 2015-06-24 | 2018-06-29 | 阿纳里蒂科探索有限责任公司 | Novel tetraterpene glycosides is as sweetener or sweetness enhancers |
| CN106511353A (en) * | 2016-09-08 | 2017-03-22 | 中国药科大学 | Preparation and application of triterpene drug for inhibiting synthesis and secretion of apoB48 in intestinal tracts |
| CN109364119A (en) * | 2018-12-26 | 2019-02-22 | 浙江大学 | Preparation has the method and application of the total triterpene of hypoglycemic effect from Qingqian Willow leaf |
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| EDWARD J. KENNELLY ET AL.: "Novel Highly Sweet Secodammarane Glycosides from Pterocarya paliurust", 《J AGRIC FOOD CHEM》 * |
| YI WU ET AL.: "Chemical constituents from Cyclocarya paliurus (Batal.) Iljinsk", 《BIOCHEMICAL SYSTEMATICS AND ECOLOGY》 * |
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