CN102617693B - Method utilizing subcritical water extraction technology to extract and prepare ursolic acid from loquat leaves - Google Patents

Abstract

The invention provides a method for extracting and preparing ursolic acid from loquat leaves by using subcritical water extraction technology. The method mainly uses the pretreated loquat leaves as raw materials, uses water as the solvent, and extracts the loquat leaves sequentially by adjusting the temperature, pressure and extraction time of the water when the extraction pressure is greater than 0 and less than or equal to 10 MPa and the extraction temperature is 100-300°C. Active ingredients such as polysaccharides, organic acids, flavonoids, saponins, triterpenoids, and volatile oils. On the basis of obtaining all kinds of extracts, the ursolic acid product with high purity (>95%) can be obtained through column chromatography separation and further purification. The extraction process does not use organic solvents, is environmentally friendly, natural, non-toxic, has high extraction efficiency, and can realize continuous separation and enrichment of various active ingredients at the same time, and has a good industrial application prospect.

Description

A method for extracting and preparing ursolic acid from loquat leaves using subcritical water extraction technology

technical field

The invention relates to a method for extracting ursolic acid in loquat leaves by using a subcritical water extraction technology, and belongs to the technical field of extraction of natural active ingredients.

Background technique

Loquat leaves are the dried leaves of the Rosaceae Loquat plant Loquat, which are widely distributed in my country. It is a kind of traditional Chinese medicine widely used in our country. It has a wide range of pharmacological effects, mainly in anti-inflammatory and cough-relieving, hypoglycemic, anti-virus, anti-oxidation and many other aspects. The chemical composition of loquat leaves is diverse, mainly including volatile oils, triterpenoids, sesquiterpenes, flavonoids, polyphenols, and glycosides. At present, the main use of loquat leaves is to produce anti-inflammatory cough medicine loquat dew and loquat extract by water extraction. The pure product of ursolic acid in triterpenoids is white needle-like crystals with a melting point of 277 ° C. It is insoluble in water and petroleum ether. Soluble in ethanol and methanol, soluble in benzene and chloroform. Ursolic acid is a pentacyclic triterpene compound, which has anti-carcinogenic, anti-cancer, induction of F9 teratoma cell differentiation and anti-angiogenesis effects. It is a candidate new drug with great potential in anti-cancer, and has a huge market and high economic benefits as an anti-cancer drug.

The extraction of active ingredients in loquat leaves mainly includes solvent extraction, solvent extraction after enzymatic treatment, ultrasonic-assisted solvent extraction, microwave-assisted solvent extraction and supercritical fluid extraction. Whether it is auxiliary treatment or supercritical fluid extraction, it mainly focuses on the adjustment and control of solvent extraction. Solvent extraction has problems such as long process, time-consuming, high impurity content in the extract, and difficulty in purification. At the same time, due to the use of a large amount of organic solvents, there are also problems such as residue problems and environmental pollution. In order to solve these problems, in recent years, some people have applied supercritical _CO2 extraction technology to the extraction of active ingredients of natural products. This technology is environmentally friendly, the resulting product is of high quality, and the production process is easy to control, but the _CO2 fluid is a non-polar substance. And the relative molecular mass is low, this technology can only be used for the extraction of non-polar compounds, and it is difficult to achieve the ideal extraction effect due to the lack of sufficient solubility for polar and large relative molecular mass substances.

Subcritical fluid technology is a new technology that just emerged in the 1990s and is rapidly penetrating into various fields. Among them, the research on subcritical water is the most extensive. Subcritical water can be used not only as a solvent, but also as a reactant and a catalyst. Eye-catching technology. Subcritical water (Sub-critical water, SCW) is also called high-temperature water, super-heated water, high-pressure hot water or hot liquid water. The body of water remains in a liquid state. It has been found to be a possible solvent for many traditional oxidation reactions, including carbon-carbon bond formation, isomerization, dehydrogenation, hydrolysis, partial oxidation, and hydrogen and tritium exchange reactions, among others. The polarity, surface tension and viscosity of water can be changed by controlling the temperature and pressure of subcritical water. As the temperature rises, the polarity decreases, and the subcritical water's ability to dissolve organic matter increases greatly. Using subcritical water as an environment-friendly solvent can change the phase behavior of the extraction system, accelerate the solvation effect, change the multi-phase reaction under traditional solvent conditions into a single reaction, increase the diffusion coefficient, lower mass transfer and heat transfer resistance, The extraction equilibrium can be reached in a shorter time, the processing time can be shortened, and the distribution of the extracted product can be controlled by the temperature and pressure of the subcritical system.

However, there is no report on the application of subcritical water extraction technology to the effective components of loquat leaves. Therefore, in order to overcome the shortcomings of the existing loquat leaf medicinal components extraction technology, subcritical water extraction technology, a new type of green and environmentally friendly extraction technology, was introduced into the study of loquat leaf medicinal components. By changing the subcritical water extraction temperature and pressure Furthermore, the polarity of the water is changed to selectively extract active ingredients with different polarities, realizing one-time charging and continuous extraction operations. The extract can be concentrated under reduced pressure to become loquat extract, and can also be further separated and purified to obtain high-purity ursolic acid products, which are widely used in the fields of medicines, health products and cosmetics as functional factors.

Contents of the invention

The invention provides a method for extracting ursolic acid in loquat leaves by using subcritical water extraction technology. The method mainly uses pretreated loquat leaves as raw materials, subcritical water as a solvent, extracting polysaccharides, polysaccharides, Active ingredients such as organic acids, flavonoids, saponins, triterpenes and volatile oils. At the same time, on the basis of obtaining various extracts with different properties, further separation and purification can obtain high-purity ursolic acid.

Technical scheme of the present invention: with pretreated loquat leaves, subcritical water is used as the extractant, and the extraction temperature is gradually increased within the range of 100-300°C under 10Mpa, and these extracts can be further purified after being collected step by step The ursolic acid in it can also be directly rotary steamed to obtain extract as loquat leaf extract. The specific steps are:

1. Raw material pretreatment: Weigh a certain amount of loquat leaf raw material, wash with water, remove hair, dry and pulverize, and classify with a sample sieve. The particle size of the sample is between 0.4 and 0.8 mm. The deionized water that has removed the dissolved oxygen is transported to the pre-heater and rapidly heated to a certain temperature.

2. Subcritical water extraction: Put the treated raw materials in the extraction kettle, inject deoxygenated deionized water into the kettle at a flow rate of less than 10mL/min by using a liquid phase pump, extract the temperature at 100-300°C, and press at 0-10Mpa. The extraction time is 0.5-3h, and the loquat leaves are extracted. After the extraction, the pressure relief valve is opened, and the extract flows into the collection tank to cool down to room temperature, and is separated at 100-150°C, 200-250°C, and 250-300°C under 10Mpa. Collected step by step according to polarity to obtain three parts of extract (extract I, II, III).

3. Separation and purification: The extracts collected step by step are concentrated by rotary evaporation, and the following three different processes are carried out respectively, and different products can be obtained in turn: (a) add absolute ethanol to the mixture to make the ethanol concentration reach 70% Finally, static precipitation, filtration to obtain insoluble matter, drying and crushing, can obtain loquat leaf polysaccharides with a purity of more than 50%; (b) add water and settle for 3 times, dissolve the precipitate with ethanol, adjust the pH to 9-10 with NaOH, the activity Carbon decolorization and filtration, and acidification of the filtrate, drying and crushing, can obtain flavonoid active substances with a purity of more than 40%; (c) add water and settle for 3 times, dissolve the precipitate with ethanol, adjust the pH to 9-10 with NaOH, and use activated carbon Decolorize and filter, and acidify the filtrate, and use non-polar macroporous resin to separate the ursolic acid in the enriched solution. After recrystallization from ethanol or methanol, the ursolic acid product is obtained with a purity of over 95%.

Beneficial effects of the present invention: the present invention utilizes the subcritical water extraction technology to simultaneously obtain a series of high-purity functional factors with different functional characteristics, and the whole process is green and pollution-free. This invention has developed a series of products, forming a series of functional factor ingredients such as loquat extract, loquat polysaccharide, triterpenoids, flavonoids, ursolic acid, etc., and the added value of the product has been greatly improved.

Description of drawings

Fig. 1 is a schematic structural view of the subcritical water extraction device used in the present invention.

Detailed ways

Example 1:

A certain amount of loquat leaf raw material is weighed, washed with water, dehaired, dried and crushed, and classified by a sample sieve, and the particle size is between 0.4 and 0.8 mm. Put 1000g of the processed raw material in the extraction kettle, use a liquid phase pump to inject deoxygenated deionized water into the kettle at a flow rate of less than 10mL/min, adjust the pressure and temperature, when the material pressure in the extraction tank reaches 10Mpa, within 1h, The extraction temperature is gradually increased from 100°C to 250°C, and 1800mL of the extract is collected and concentrated after rotary evaporation to obtain the extract of loquat leaf extract, which can also be further dried to obtain 42.4g of loquat leaf extract, which contains loquat polysaccharides, flavonoids , triterpenoids and ursolic acid and other biologically active ingredients.

Example 2:

A certain amount of loquat leaf raw material is weighed, washed with water, dehaired, dried and crushed, and classified by a sample sieve, and the particle size is between 0.4 and 0.8 mm. Put 1000g of the processed raw material in the extraction kettle, use a liquid phase pump to inject deoxygenated deionized water into the kettle at a flow rate of less than 10mL/min, adjust the pressure and temperature, when the material pressure in the extraction tank reaches 10Mpa, control the extraction tank Extract at 100°C for 1.5h, use the regulating valve to release the pressure, the extract flows into the collection tank, take out the extract I after cooling to room temperature; keep the pressure of the extraction tank at 10MPa, raise the temperature to 200°C and extract for 1h, release the pressure , the extract flows into the collection tank, and after cooling, take out the extract II; keep the pressure at 10MPa, raise the temperature to 250°C and extract for 1 hour, after the pressure is released, the extract flows into the collection tank, cool down, and take out the extract III.

Rotary evaporation of the extract I, concentrated to a paste, adding absolute ethanol until the ethanol concentration in the mixture reaches 70%, static precipitation, filtering to obtain insoluble matter, drying and crushing, can reach the loquat leaf polysaccharide with a purity of 58% about 17.5 g.

The extract II was rotary evaporated, concentrated to the extract, settled in water for 3 times, dissolved in ethanol, adjusted to pH 9-10 with NaOH, decolorized and filtered with activated carbon, and the filtrate was acidified, dried and crushed to obtain a purity of 45%. 14.2g of flavonoids.

Add water and settle the extract III three times, dissolve the precipitate with ethanol, adjust the pH to 9-10 with NaOH, decolorize and filter with activated carbon, and acidify the filtrate to obtain 108 g of triterpenoid active substances with a purity of 40%. Macroporous tree adsorption resin XDA-200 separates and enriches ursolic acid in the solution. After recrystallization from ethanol or methanol, the ursolic acid product is obtained with a purity of 95.0% and a recovery of ursolic acid of 31.5%.

Example 3:

A certain amount of loquat leaf raw material is weighed, washed with water, dehaired, dried and crushed, and classified by a sample sieve, and the particle size is between 0.4 and 0.8 mm. Put 1000g of the processed raw material in the extraction kettle, use a liquid phase pump to inject deoxygenated deionized water into the kettle at a flow rate of less than 10mL/min, adjust the pressure and temperature, when the material pressure in the extraction tank reaches 10Mpa, control the extraction tank Extract at 120°C for 1 hour, use the regulating valve to release the pressure, the extract flows into the collection tank, take out the extract I after cooling to room temperature; keep the pressure of the extraction tank at 10MPa, raise the temperature to 230°C for 1 hour, release the pressure, The extract flows into the collection tank, and extract II is taken out after cooling; the pressure is maintained at 10 MPa, and the temperature is raised to 280°C for extraction for 1 hour. After the pressure is released, the extract flows into the collection tank, cooled, and extract III is taken out.

Rotary evaporation of the extract I, concentrated to a paste, adding absolute ethanol until the ethanol concentration in the mixture reaches 70%, static precipitation, filtering to obtain insoluble matter, drying and crushing, can reach about 24.6% of the loquat leaf polysaccharide with a purity of 52%. g.

The extract II was rotary evaporated, concentrated to the extract, settled in water for 3 times, dissolved in ethanol, adjusted to pH 9-10 with NaOH, decolorized and filtered with activated carbon, and the filtrate was acidified, dried and crushed to obtain a purity of 48%. 17.0g of flavonoids.

Add water to the extract III and settle for 3 times, dissolve the precipitate with ethanol, adjust the pH to 9-10 with NaOH, decolorize and filter with activated carbon, and acidify the filtrate to obtain 15.7 g of triterpenoid active substances with a purity of 42%. Separation and enrichment of ursolic acid in the solution by XDA-200 adsorption resin of macroporous tree. After recrystallization from ethanol or methanol, the ursolic acid product is obtained with a purity of 96.4% and a recovery of ursolic acid of 38.2%.

Example 4:

A certain amount of loquat leaf raw material is weighed, washed with water, dehaired, dried and crushed, and classified by a sample sieve, and the particle size is between 0.4 and 0.8 mm. Put 1000g of the processed raw material in the extraction kettle, use a liquid phase pump to inject deoxygenated deionized water into the kettle at a flow rate of less than 10mL/min, adjust the pressure and temperature, when the material pressure in the extraction tank reaches 10Mpa, control the extraction tank Extract at 150°C for 2 hours, use the regulating valve to release the pressure, the extract flows into the collection tank, take out the extract I after cooling to room temperature; keep the pressure of the extraction tank at 10MPa, raise the temperature to 250°C for 1.5 hours, and release the pressure , the extract flows into the collection tank, and after cooling, take out the extract II; keep the pressure at 10MPa, raise the temperature to 300°C for extraction for 1 hour, after the pressure is released, the extract flows into the collection tank, cool down, and take out the extract III.

Rotary evaporation of the extract I, concentrated to a paste, adding absolute ethanol until the ethanol concentration in the mixture reaches 70%, static precipitation, filtering to obtain insoluble matter, drying and crushing, can reach about 20.3% of the loquat leaf polysaccharide with a purity of 50%. g.

The extract II was rotary evaporated, concentrated to the extract, settled in water for 3 times, dissolved in ethanol, adjusted to pH 9-10 with NaOH, decolorized and filtered with activated carbon, and the filtrate was acidified, dried and crushed to obtain a purity of 41%. 19.0g of flavonoids.

Add water to the extract III and settle for 3 times, dissolve the precipitate with ethanol, adjust the pH to 9-10 with NaOH, decolorize and filter with activated carbon, and acidify the filtrate to obtain 22.4 g of triterpenoid active substances with a purity of 47%. Separation and enrichment of ursolic acid in the solution by XDA-200 adsorption resin of macroporous tree. After recrystallization from ethanol or methanol, the ursolic acid product is obtained with a purity of 95.8% and a recovery rate of 40.5%.

Claims (1)

1. A method utilizing subcritical water extraction technology to extract and prepare ursolic acid from loquat leaves, characterized in that using loquat leaves as raw material, raw material is pretreated, and utilizing subcritical water extraction technology to extract the flavonoids in loquat leaves Compounds, saponins, and triterpenoids active ingredients, and further separation and purification to obtain high-purity ursolic acid, the specific steps are as follows: (1) Raw material pretreatment: the loquat leaf raw material is washed with water, dehaired, dried and crushed, and classified by a sample sieve, and the particle size of the sample is between 0.4 and 0.8 mm; (2) Subcritical water extraction: put the raw materials in the extraction kettle, use a liquid phase pump to inject deoxygenated deionized water into the kettle at a flow rate of less than 10mL/min, the extraction temperature is 100-300°C, and the pressure is greater than 0 and less than or equal to 10Mpa. The extraction time is 0.5 to 3 hours, and the loquat leaves are extracted. After the extraction, the pressure relief valve is opened, and the extract flows into the collection tank to cool to room temperature, and at 10Mpa, the temperature is greater than or equal to 100°C and less than or equal to 150°C, greater than or equal to 200°C and less than or equal to At 250°C, greater than 250°C and less than or equal to 300°C, three parts of extract are collected step by step according to polarity; (3) Separation and purification: the extracts collected step by step are concentrated by rotary evaporation respectively, and the following three different treatment processes are respectively carried out to obtain different products in turn: (a) adding absolute ethanol to the mixture to make the ethanol concentration reach 70% Afterwards, static precipitation, filtration to obtain insoluble matter, drying and crushing to obtain loquat leaf polysaccharides with a purity of more than 50%; (b) add water and settle for 3 times, dissolve the precipitate with ethanol, adjust the pH to 9-10 with NaOH, and use activated carbon Decolorize and filter, and acidify the filtrate, dry and pulverize to obtain flavonoid active substances with a purity of more than 40%; (c) add water and settle for 3 times, dissolve the precipitate with ethanol, adjust the pH to 9-10 with NaOH, decolorize and filter with activated carbon , and acidify the filtrate, use non-polar macroporous adsorption resin to separate and enrich the ursolic acid in the enriched solution, and obtain the ursolic acid product after recrystallization from ethanol or methanol, with a purity of more than 95%.