CN104744602B - Radix Scrophulariae polysaccharide and the method for microwave extraction thereof - Google Patents

Abstract

The invention discloses a polysaccharide from Scrophulariaceae and a microwave extraction method thereof. (1) Beat or pulverize raw medicinal materials, and then enzymolyze them with a compound enzyme preparation; (2) The ratio of solid to water for Scrophulariaceae in water is 1:10-1:100 Microwave-assisted extraction is carried out, and the extract is obtained after centrifugal filtration. During the extraction process, the extraction tank is intermittently evacuated and vacuum-broken; (3) The Scrophulariaceae polysaccharide extract is loaded onto a macroporous adsorption resin chromatographic column, and the degassing method is used to remove Elute with deionized water and collect the eluent; (4) After concentrating the eluent under reduced pressure, add ethanol to fully stir and mix, and let stand; (5) Freeze-dry. The invention effectively integrates multiple technologies such as enzymolysis of compound enzyme preparation, vacuum pulsation, microwave assistance, resin adsorption, etc., which significantly improves the extraction rate and purity of Scrophulariaceae polysaccharides, shortens the extraction time, has strong market competitiveness, and has great application prospects. Broadly, the obtained Scrophulariaceae polysaccharide has obvious inhibitory effect on cancer cells.

Description

Scrophulariaceae polysaccharide and its microwave extraction method

technical field

The invention relates to polysaccharides from Scrophulariaceae Scrophulariae and a method for extracting polysaccharides from Scrophulariaceae Scrophulariae, a Chinese medicinal material, and a method for extracting polysaccharides from Scrophulariaceae Scrophulariae.

Background technique

Plant polysaccharides are an important topic in the field of biochemistry, and have become a new research hotspot at home and abroad, among which water-soluble polysaccharides extracted from Chinese herbal medicines are particularly popular. Polysaccharides are polymers that are linked by monosaccharides and have a polymerization degree of more than 100,000. Plant polysaccharides are a component commonly found in plant cell walls and cells. At present, hundreds of polysaccharides have been isolated and extracted from nature. A large number of studies have proved that polysaccharides have unique biological activities, and are the main active substances of many Chinese herbal medicines. . According to the statistics of the China Chamber of Commerce for Import and Export of Medicines and Health Products, driven by the growth of demand in the world's plant drug market, my country's plant extract exports showed a good momentum of both volume and price in 2011, especially the average export price has increased significantly compared with 2010. Improvement, the market prospect is very broad.

Scrophulariaceae is also known as Yuanshen, Black Ginseng, Chongtai, Guizang, Luchang, Xuantai, etc. Scrophulariaceae is a plant of Scrophulariaceae (Scrophularia ningpoensis The dried root of Hems1. ) is a traditional Chinese medicine in my country. Scrophulariaceae is slightly cold in nature, sweet, bitter, and salty in taste, and returns to the lung, stomach, and kidney meridians. It has the effects of cooling blood, nourishing yin, purging fire and detoxifying. Modern pharmacological studies have shown that the main functions of Scrophulariaceae are anti-inflammatory, enhancing immune function, lowering blood sugar, protecting the liver, anti-tumor, and anti-fatigue. In addition, Scrophulariaceae also has certain effects on the nervous system, such as protecting the nerves, preventing depression, Improve memory, etc.

As an important extract of Scrophulariae Scrophulariae, polysaccharides also have great pharmacological effects. Scrophulariae polysaccharides have different degrees of scavenging capabilities for hydroxyl radicals, superoxide anion radicals, and nitrite. Wang Hui et al.’s research on the water-soluble components of Scrophulariaceae showed that Scrophulariaceae polysaccharides can prolong the swimming time of mice, reduce the serum urine nitrogen content of mice after exercise, reduce the blood lactic acid content of mice after exercise and increase liver glycogen in mice. Scrophulariaceae polysaccharides can increase the activity of SOD and GSH-Px in aging mice and reduce the content of MDA in aging mice.

As a medical and health product, Scrophulariaceae polysaccharide has great functions, but its extraction process needs to be improved. At present, the extraction of polysaccharides from Scrophulariaceae Scrophulariae is mostly carried out in a single batch of cans, and the yield is low, the extraction time is long, the extraction solvent consumes a lot, the operation is cumbersome, and the environmental pollution is serious. Therefore, it is of great significance to develop the extraction method of Scrophulariaceae polysaccharides, simplify the operation, reduce the loss of active substances of polysaccharides, shorten the extraction cycle, and improve the yield and purity of polysaccharides.

Contents of the invention

In order to overcome the deficiencies in the prior art, the inventor explored and summed up a microwave extraction method of polysaccharides from Scrophulariaceae through a large number of experiments. Using this method, the extraction rate and purity of polysaccharides from Scrophulariaceae can be significantly improved, the extraction time can be shortened, and the extraction time can be reduced. cost and improve economic efficiency.

In order to overcome the deficiencies in the prior art, the present invention takes the following technical solutions:

(1) Processing of raw materials: Radix Scrophulariae can be dry or wet. If it is wet, it should be beaten. If it is dry, it should be crushed to 20-50 mesh. Add water and a compound enzyme preparation of 2-8% of the mass of medicinal materials. Carry out enzymatic hydrolysis, the enzymatic hydrolysis time is 4-10 hours, and the enzymatic hydrolysis temperature is 40-50°C; it has been proved by experiments that the extraction rate of polysaccharides can be significantly improved after treatment with enzymes.

The compounding ratio of the compound enzyme preparation is 50-70% of the mass fraction of cellulase, 15-40% of the mass fraction of pectinase, and the rest is xylanase, and the average enzyme activity of the compound enzyme preparation is 10000-30000 units;

(2) Microwave extraction: using water as solvent, the solid-liquid ratio of scrophulariaceae to water is 1:10-1:100, extracting polysaccharides from scrophulariaceae by microwave-assisted method, and obtaining the extract after centrifugal filtration;

During the above extraction process, the extraction tank is vacuumed and broken intermittently, and vacuum pulses are used to enhance mass transfer. The mechanism of vacuum pulsation to enhance mass transfer is: when the vacuum is pumped, the water in the cell tissue of Scrophulariaceae will be vaporized due to the decompression, causing the pressure inside the cell tissue of Scrophulariaceae to be greater than that outside the cell tissue of Scrophulariae scrophulariae. The lower energy can be forced to pass through the cell tissue of Scrophulariae; when the vacuum is broken, the external pressure of the cells of Scrophulariae is greater than the internal pressure of cells of Scrophulariae, and the water outside the cells of Scrophulariae quickly enters the cells of Scrophulariae tissue to dissolve due to the pressure difference Polysaccharides, the alternation of pressure inside and outside the cells of Scrophulariaceae Scrophulariae strengthens the mass transfer of water and polysaccharides dissolved in water, so that the extraction efficiency of polysaccharides is improved and the residual rate is reduced.

(3) Chromatography: load the Scrophulariaceae polysaccharide extract onto a macroporous adsorption resin chromatography column, elute with deionized water, and collect the eluate;

(4) Alcohol precipitation: After concentrating the eluate under reduced pressure, add ethanol to fully stir and mix, let stand, and collect the precipitate;

(5) Freeze-drying: reconstitute the precipitate obtained by alcohol precipitation with water, and freeze-dry to obtain pure Scrophulariaceae polysaccharide.

Among the above technical solutions, in step (2), the preferred technical solution for microwave extraction is as follows: the microwave power is 800-1500W, the extraction temperature is 60°C-100°C, and the extraction time is 10-60 minutes.

In the above technical scheme, in step (3), the chromatography step is preferably: load the polysaccharide extract to the ADS-17 macroporous adsorption resin column, elute with deionized water at a speed of 1.5-2.5ml/min, collect eluent. ADS-17 macroporous adsorption resin column has a high adsorption rate for Scrophulariaceae polysaccharides, and is easy to elute and easy to regenerate.

In the above technical solution, in step (4), the alcohol precipitation step is preferably: concentrating the eluate obtained from chromatography to 4-6 mg/ml under reduced pressure, adding 3-6 times 95% ethanol, and standing at 3-5°C After 12-72 hours, the precipitate was collected by centrifugation.

The Scrophulariaceae polysaccharide obtained in the present invention is characterized by infrared spectroscopy, and a broad peak appears at 3415.31cm ^-1 , which is O—H stretching vibration; the absorption peak in the region of 3000-2800cm ^-1 is a characteristic peak of carbohydrates, The absorption peak of Scrophulariae polysaccharide at 2924.52cm ^-1 is the C—H stretching vibration of the polysaccharide molecule; the absorption peak at 1617.02cm ^-1 is the C=0 stretching vibration of the acetyl amino group in Scrophulariaceae polysaccharides, indicating that Scrophulariaceae polysaccharides contain Uronic acid; absorption bands of C—O—C asymmetric and symmetric stretching vibrations with ester groups at 1265.07 and 1142.62 cm ^-1 .

The functional test proves that the crocodile polysaccharide obtained by the invention has obvious inhibitory effect on cancer cells.

Compared with prior art, the beneficial effect of the present invention also lies in:

The compound enzyme preparation is used for enzymolysis before the extraction of the raw materials, so as to destroy the cell wall of Scrophulariaceae Scrophulariae, so that the polysaccharide components can be dissolved quickly, and the extraction rate of polysaccharides is improved.

The compound enzyme preparation of the present invention is an optimized combination. According to the characteristics of the main components of the cell wall of Scrophulariaceae, the mass fraction of cellulase is designed to be 50-70%, the mass fraction of pectinase is 15-40%, and the rest is xylanase. Enzymatic hydrolysis works best.

Using water as a solvent, microwave-assisted extraction was used to extract the polysaccharide components of Scrophulariaceae, which effectively shortened the extraction time and improved the extraction rate of active components; Strong, easy to elute, easy to regenerate.

During the extraction process of polysaccharides from Scrophulariaceae Scrophulariae, vacuuming and vacuum breaking are carried out, and vacuum pulses are used to enhance mass transfer, further shortening the extraction time and the extraction rate of polysaccharides.

In summary, the present invention significantly improves the extraction rate and purity of Scrophulariaceae polysaccharides, shortens the extraction time, reduces extraction costs, improves economic benefits, has strong market competitiveness, and has broad application prospects by effectively integrating the above multiple technologies. .

Description of drawings

Fig. 1 is the infrared absorption spectrogram of polysaccharides of Scrophulariaceae obtained by the present invention.

detailed description

Now with reference to the accompanying drawings, the microwave extraction method of Scrophulariaceae polysaccharides is further specifically described.

Example 1:

(1) Processing of raw materials: Radix Scrophulariae can be wet or dry. If it is wet, it will be beaten to 25 mesh. If it is dry, it will be crushed to 25 mesh. Water and a compound enzyme preparation of 3/1000 of the medicinal material will be added for enzymolysis. The enzymatic hydrolysis time 9 hours, enzymatic hydrolysis temperature 45°C;

The proportioning of the compound enzyme preparation is 52% of the mass fraction of cellulase, 30% of the mass fraction of pectinase, and the rest is xylanase. The average enzyme activity of the compound enzyme preparation is 18000 units;

(2) Microwave extraction: using water as solvent, the solid-liquid ratio of scrophulariaceae to water is 1:12, extracting polysaccharides from scrophulariaceae by microwave-assisted method, and obtaining the extract after centrifugal filtration;

During the above extraction process, the extraction tank is vacuumed and broken intermittently, and vacuum pulses are used to enhance mass transfer.

(3) Chromatography: load the Scrophulariaceae polysaccharide extract onto a macroporous adsorption resin chromatography column, elute with deionized water, and collect the eluate;

(4) Alcohol precipitation: After concentrating the eluate under reduced pressure, add ethanol to fully stir and mix, let stand, and collect the precipitate;

(5) Freeze-drying: reconstitute the precipitate obtained by alcohol precipitation with water, and freeze-dry to obtain pure Scrophulariaceae polysaccharide.

In step (2), the microwave power is 850W, the extraction temperature is 65°C, and the extraction time is 30 minutes.

In step (3), the polysaccharide extract was loaded onto the ADS-17 macroporous adsorption resin column, eluted with deionized water at a rate of 1.6ml/min, and the eluate was collected.

In step (4), the eluate obtained from chromatography was concentrated to 4.2 mg/ml under reduced pressure, 4 times of 95% ethanol was added, left standing at 4°C for 15 hours, and the precipitate was collected by centrifugation.

The Scrophulariaceae polysaccharide obtained in the present invention is characterized by infrared spectroscopy, and a broad peak appears at 3415.31cm ^-1 , which is O—H stretching vibration; the absorption peak in the region of 3000-2800cm ^-1 is a characteristic peak of carbohydrates, The absorption peak of Scrophulariae polysaccharide at 2924.52cm ^-1 is the C—H stretching vibration of the polysaccharide molecule; the absorption peak at 1617.02cm ^-1 is the C=0 stretching vibration of the acetyl amino group in Scrophulariaceae polysaccharides, indicating that Scrophulariaceae polysaccharides contain Uronic acid; absorption bands of C—O—C asymmetric and symmetric stretching vibrations with ester groups at 1265.07 and 1142.62 cm ^-1 .

The functional test proves that the crocodile polysaccharide obtained by the invention has obvious inhibitory effect on cancer cells.

Example 2:

(1) Processing of raw materials: Radix Scrophulariae can be dry or wet. If it is wet, it will be beaten to a pulp. If it is dry, it will be crushed to 45 mesh. Add water and a compound enzyme preparation of 7/1000 of the medicinal material to carry out enzymatic hydrolysis. The enzymatic hydrolysis time 4.5 hours, enzymatic hydrolysis temperature 48°C;

The proportioning of the compound enzyme preparation is 67% of the mass fraction of cellulase, 18% of the mass fraction of pectinase, and the rest is xylanase. The average enzyme activity of the compound enzyme preparation is 27000 units;

(2) Microwave extraction: using water as solvent, the solid-liquid ratio of scrophulariaceae to water is 1:91, the microwave-assisted method is used to extract the polysaccharides of scrophulariaceae, and the extract is obtained after centrifugal filtration;

During the above extraction process, the extraction tank is vacuumed and broken intermittently, and vacuum pulses are used to enhance mass transfer.

(3) Chromatography: load the Scrophulariaceae polysaccharide extract onto a macroporous adsorption resin chromatography column, elute with deionized water, and collect the eluate;

(4) Alcohol precipitation: After concentrating the eluate under reduced pressure, add ethanol to fully stir and mix, let stand, and collect the precipitate;

(5) Freeze-drying: reconstitute the precipitate obtained by alcohol precipitation with water, and freeze-dry to obtain pure Scrophulariaceae polysaccharide.

In step (2), the microwave power is 1400W, the extraction temperature is 90°C, and the extraction time is 50 minutes.

In step (3), the polysaccharide extract was loaded onto the ADS-17 macroporous adsorption resin column, eluted with deionized water at a rate of 2.3ml/min, and the eluate was collected.

In step (4), the eluate obtained from chromatography was concentrated to 5.8 mg/ml under reduced pressure, 4.8 times of 95% ethanol was added, left standing at 4.7°C for 15 hours, and the precipitate was collected by centrifugation.

The Scrophulariaceae polysaccharide obtained in the present invention is characterized by infrared spectroscopy, and a broad peak appears at 3415.31cm ^-1 , which is O—H stretching vibration; the absorption peak in the region of 3000-2800cm ^-1 is a characteristic peak of carbohydrates, The absorption peak of Scrophulariae polysaccharide at 2924.52cm ^-1 is the C—H stretching vibration of the polysaccharide molecule; the absorption peak at 1617.02cm ^-1 is the C=0 stretching vibration of the acetyl amino group in Scrophulariaceae polysaccharides, indicating that Scrophulariaceae polysaccharides contain Uronic acid; absorption bands of C—O—C asymmetric and symmetric stretching vibrations with ester groups at 1265.07 and 1142.62 cm ^-1 .

The functional test proves that the crocodile polysaccharide obtained by the invention has obvious inhibitory effect on cancer cells.

Claims (5)

1. a microwave extraction method of polysaccharides from Scrophulariaceae Scrophulariae, characterized in that it comprises the following steps: (1) Processing of raw materials: Radix Scrophulariae can be dry or wet. If it is wet, it should be beaten. If it is dry, it should be crushed to 20-50 mesh. Add water and a compound enzyme preparation of 2-8% of the mass of medicinal materials. Carry out enzymatic hydrolysis, the enzymatic hydrolysis time is 4-10 hours, and the enzymatic hydrolysis temperature is 40-50°C; The compounding ratio of the compound enzyme preparation is 50-70% of the mass fraction of cellulase, 15-40% of the mass fraction of pectinase, and the rest is xylanase, and the average enzyme activity of the compound enzyme preparation is 10000-30000 units; (2) Microwave extraction: using water as the solvent, the solid-liquid ratio of scrophulariaceae to water is 1:10-1:100, the microwave-assisted method is used to extract the polysaccharides of scrophulariae scrophulariae, the extract is obtained after centrifugal filtration, and the extraction tank is cleaned during the extraction process. Intermittent vacuuming and vacuum breaking, using vacuum pulsation to enhance mass transfer; (3) Chromatography: load the Scrophulariaceae polysaccharide extract onto a macroporous adsorption resin chromatography column, elute with deionized water, and collect the eluate; (4) Alcohol precipitation: After concentrating the eluate under reduced pressure, add ethanol to fully stir and mix, let stand, and collect the precipitate; (5) Freeze-drying: reconstitute the precipitate obtained by alcohol precipitation with water, and freeze-dry to obtain pure Scrophulariaceae polysaccharide. 2. The microwave extraction method of Scrophulariaceae polysaccharide according to claim 1, characterized in that: in the microwave extraction step, the microwave power is 800-1500W, the extraction temperature is 60°C-100°C, and the extraction time is 10-60 minutes. 3. The microwave extraction method of polysaccharides from Scrophulariaceae Scrophulariae according to claim 1, characterized in that: in the chromatography step, the polysaccharide extract is loaded to the ADS-17 macroporous adsorption resin column at a speed of 1.5-2.5mL/min Elute with deionized water and collect the eluate. 4. The microwave extraction method of Scrophulariaceae polysaccharide according to claim 1, characterized in that: in the alcohol precipitation step, the eluate obtained from chromatography is concentrated to 4-6 mg/mL under reduced pressure, and 3-6 times of 95% Ethanol, let stand at 3-5°C for 12-72 hours, and collect the precipitate by centrifugation. 5. The pure Scrophulariaceae polysaccharide obtained by the method according to claim 1 or 2 or 3 or 4, characterized in that: Scrophulariaceae polysaccharide has obvious inhibitory effect on cancer cells.