CN101549010B - A preparing method and application of malaytea scurfpea fruit total glycosides extract - Google Patents

Abstract

The invention provides a preparation method of total psoralen glycosides extract, which comprises extracting psoralen with water, concentrating the extract and then ethanol precipitation, extracting the supernatant with a low-polarity organic reagent, and purifying the water layer with a macroporous resin after extraction , to obtain the total psoralen extract, wherein the total psoralen content is more than 80%, and through HPLC content determination, it contains 40-50% psoralen and 40-50% isopsoralen. The total psoralen glycosides extract provided by the present invention has been proved by pharmacological experiments to resist leukopenia caused by cyclophosphamide, has a significant immune enhancement effect, and can be used in the preparation of medicines for increasing leukocytes. The method of the invention adopts a simple flow process to obtain total psoralen glycosides from the traditional Chinese medicine psoralen, and the method is simple, economical, stable and easy to operate, and is beneficial to large-scale industrial production.

Description

A kind of preparation method and application of total psoralen glycosides extract

technical field

The invention belongs to the field of medicine, and in particular relates to a method for obtaining total psoralen extract from the traditional Chinese medicine psoraleae through extraction and refining, and the medicinal use of the extract.

Background technique

At present, Chinese herbal medicine has a certain market all over the world. With the increase of people's awareness of health requirements and the aging of the population, sub-health status, people are more eager to return to nature, and use pure natural medicine with high degree of treatment, To prevent problems that cannot be solved by some chemically synthesized drugs, the application of natural herbal medicines is beyond the background of its original national traditional culture. It has become the goal pursued by pharmaceutical companies all over the world to seek medicines with low side effects and high quality and low price from natural medicines. The European Community has carried out unified legislation on herbal medicines, and the status of herbal medicines in countries such as Canada and Australia has been legalized. The US government has also drafted a management method for herbal medicines, and began to accept compound mixtures of natural medicines as therapeutic medicines. These are traditional Chinese medicines as therapeutic medicines. Entering the international pharmaceutical market provides a good international environment. On the other hand, with the acceleration of the global economic integration process, especially my country's formal accession to the WTO, the breadth and depth of the Chinese pharmaceutical market's integration into the international pharmaceutical market will be further intensified. Faced with fierce competition from powerful multinational pharmaceutical groups and the huge impact of traditional medicine products from Asian countries such as Japan, South Korea, India, and Thailand, and herbal medicines from European countries such as Germany and France, many products produced by traditional Chinese medicine in my country cannot meet the requirements of the international pharmaceutical market. rejected by standards and requirements.

The traditional Chinese medicine Psoralen is the fruit of the leguminous plant Psoralea corylifolia L. It is warm in nature, pungent in taste, and has the effect of tonifying the kidney and yang. Indications for cold diarrhea due to kidney deficiency, frequent urination, impotence, cold pain in the waist and knees, dyspnea due to deficiency and cold, and vitiligo for external use. Modern studies have shown that psoralen has physiological activities such as dilating blood vessels, increasing myocardial contractility, antibacterial, estrogen-like effects, and treating vitiligo. Psoralen contains a variety of active ingredients, and glycosides represented by psoralen and isopsoralen are one of its important active ingredients. However, there is no report of obtaining psoralen in the current literature and patents.

Contents of the invention

The purpose of the present invention is to provide a low-cost, easy-to-operate, stable and easy-to-industrialize preparation method of total psoralen glycosides extract, and to measure the content of representative components therein.

The preparation method of the present invention is realized through the following technical solutions:

(1) Psoralen is extracted with water as a solvent;

(2) Alcohol precipitation after the extract is concentrated;

(3) The alcohol precipitation supernatant is extracted with a small polar organic reagent;

(4) After extraction, the aqueous layer is purified with a macroporous resin to obtain a total psoralen extract, wherein the total psoralen content is more than 80%;

Wherein: in step (1), the amount of solvent used is 8-15 times the amount, and the heating extraction is performed 1-3 times, each time for 0.5-2 hours. The optimal amount of solvent is 10 times the amount, heating and extracting twice for 2 hours each time.

In step (2), the ethanol concentration is 70-95%, and the optimum ethanol concentration is 80%.

In step (3), the small polar organic reagent is selected petroleum ether, chloroform or ethyl acetate.

In step (4), after the extraction, the water layer passes through the macroporous adsorption resin chromatography column, rinses with water first, and then rinses with ethanol aqueous solution, collects the eluent, concentrates the eluent under reduced pressure to no alcohol, and freeze-dries to obtain the total psoralen. Wherein the alcohol concentration of the ethanol aqueous solution is 30-100%. Content determination is carried out by HPLC, and the content of psoralenoside (Psoralenoside) in the total psoralen glycosides is 40-50%, and the content of isopsoralenoside (Isopsoralenoside) is 40-50%.

Another object of the present invention is to provide the application of the total psoralen glycosides extract in the preparation of medicines for increasing white blood cells.

The total psoralen glycosides extract prepared by the method of the present invention is prepared into any pharmaceutical dosage form with dressings or excipients allowed by the preparation, and can also be prepared into pharmaceutical preparations together with other drugs or components.

The pharmaceutical dosage forms can be tablets, capsules, granules, oral liquids, sustained-release preparations, controlled-release preparations, gels, ointments, ointments, creams, suppositories, injections, powder injections, patches, dripping pills, mixed Suspension, etc.

The method of the invention adopts a simple process to obtain total psoralen glycosides from the traditional Chinese medicine psoralen, and measures the content of psoralen and isopsoralen. The method is simple and economical, and is beneficial to large-scale industrial production. It has been proved by pharmacological experiments that the total psoralen glycosides extract and the psoralen and isopsoralen contained in it can resist the leukopenia caused by cyclophosphamide, and have a significant immune enhancement effect, which can be used in the preparation of It is used in drugs with high white blood cells.

Description of drawings

Figure 1 Chromatogram of total psoralen glycosides.

Detailed ways

The present invention is described further in conjunction with embodiment. The embodiments of the present invention are only used for illustration rather than limitation of the present invention.

The HPLC analysis and content determination method of embodiment 1 psoralen and isopsoralen

Chromatographic conditions Chromatographic column Agilent Zorbax SB-C18 column (4.6mm × 250mm, 5 μ m); Adopt gradient elution, mobile phase A phase is 0.1% glacial acetic acid aqueous solution, mobile phase B phase is the acetonitrile solution containing 0.1% glacial acetic acid; Gradient The elution procedure is as follows: at 0 minutes, the mobile phase is 95% A, 5% B; at 20 minutes, the mobile phase is 70% A, 30% B; at 25 minutes, the mobile phase is 50% A , 50% B; at 30 minutes, the mobile phase was 5% A, 95% B. The solution flow rate is 1.0mL·min ^-1 ; the detection wavelength is 246nm; the column temperature is 30°C, and the content of psoralen and isopsoralen can be determined. Referring to Fig. 1, among the figure 1 is isopsoralen, 2 is psoralen.

Embodiment two

Take 1 kg of psoraleae, dry at 50°C for 6 hours, add 8 times the amount of water, heat and reflux for 2 hours in a slightly boiling state, extract the extract, filter it, concentrate under reduced pressure, and precipitate with alcohol, adjust the alcohol concentration to 80%, take the supernatant, and concentrate Add ethyl acetate to extract after reaching no alcohol, separate the water layer, apply macroporous resin, wash with water, discard, and continue to elute with 30% ethanol aqueous solution. The eluate was collected, concentrated under reduced pressure until alcohol-free, and freeze-dried to obtain total psoralen glycosides. As determined by the method of Example 1, the content of psoralen in the total psoralen was 38%, and the content of isopsoralen was 42%.

Embodiment Three

Take 1 kg of psoraleae, dry at 50°C for 6 hours, add 8 times the amount of water and heat to reflux for 2 hours, then filter the extract, concentrate under reduced pressure, and precipitate with alcohol, adjust the alcohol concentration to 70%, take the supernatant, and concentrate Add ethyl acetate to extract after reaching no alcohol, separate the water layer, apply macroporous resin, wash with water, discard, and continue to elute with 50% ethanol aqueous solution. The eluate was collected, concentrated under reduced pressure until alcohol-free, and freeze-dried to obtain total psoralen glycosides. Measured according to the method of Example 1, the content of psoralen in the total psoralen is 40%, and the content of isopsoralen is 42%.

Embodiment Four

Take 1 kg of psoraleae, dry at 50°C for 6 hours, add 12 times the amount of water and heat under reflux for 2 hours, then filter the extract, concentrate under reduced pressure, and precipitate with alcohol, adjust the alcohol concentration to 70%, take the supernatant, and concentrate Add ethyl acetate to extract after the alcohol is gone, separate the water layer, apply macroporous resin, wash with water, discard, and continue to elute with 70% ethanol aqueous solution. The eluate was collected, concentrated under reduced pressure until alcohol-free, and freeze-dried to obtain total psoralen glycosides. Measured according to the method of Example 1, the content of psoralen in the total psoralen was 41%, and the content of isopsoralen was 43%.

Embodiment five

Take 1 kg of psoraleae, dry at 50°C for 6 hours, add 10 times the amount of water and heat it under reflux to extract twice, 1 hour each time, filter the extract, concentrate under reduced pressure, alcohol precipitation, adjust the alcohol concentration to 80%, and take The supernatant was concentrated to no alcohol, then extracted twice with chloroform, the water layer was separated, applied to a macroporous resin, washed with water, discarded, and continued to be eluted with 80% aqueous ethanol. The eluate was collected, concentrated under reduced pressure until alcohol-free, and freeze-dried to obtain total psoralen glycosides. Measured according to the method of Example 1, the content of psoralen in the total psoralen was 43%, and the content of isopsoralen was 41%.

Embodiment six

Take 1 kg of psoraleae, dry at 50°C for 6 hours, add 10 times the amount of water and heat and reflux to extract twice, each time for 2 hours, filter the extract, concentrate under reduced pressure, alcohol precipitation, adjust the alcohol concentration to 70%, take The supernatant was concentrated to no alcohol and then extracted three times with ethyl acetate. The aqueous layer was separated, applied to macroporous resin, washed with water until sugar-free (molish reaction), discarded, and continued to elute with 70% aqueous ethanol. The eluate was collected, concentrated under reduced pressure until alcohol-free, and freeze-dried to obtain total psoralen glycosides. Measured according to the method of Example 1, the content of psoralen in the total psoralen is 46%, and the content of isopsoralen is 45%.

The preparation of embodiment seven drop pills

Take 0.5 g of total psoralen glycosides and 10.5 g of polyethylene glycol-6000, mix evenly, heat and melt, transfer the material to drip irrigation, drop the drug solution into liquid paraffin at 6-8°C, remove oil, and obtain 300 drop pills.

The preparation of embodiment eight freeze-dried powder injections

Take 1.5 g of balm balm oil, add it to 13 ml of saturated hydroxypropyl β-cyclodextrin, stir to dissolve, filter, and dry the filtrate at low temperature to obtain an inclusion compound powder of balm balm oil and hydroxypropyl β-cyclodextrin. In addition to the above-mentioned clathrate powder of balsamic oil and hydroxypropyl β-cyclodextrin, take 0.5g of psoralen, 5.5g of mannitol, 0.9g of calcium sodium edetate and 2ml of distilled water, mix the above components After homogenization, freeze-dry and pack into 350 tubes, that is to say.

The preparation of embodiment nine tablet

Take 100 grams of total psoralen glucosides and mix them evenly with microcrystalline cellulose, add 3% povidone ethanol solution to make soft materials, pass through a 18-mesh sieve to make granules, dry at 60°C for 1 hour, granulate, add appropriate amount of talcum powder, mix Evenly, pressed into tablets, ready to serve.

The preparation of embodiment ten capsules

Take psoralen glucosides and peanut oil in a water-soluble stainless steel mixing tank in a ratio of 2:18, add an appropriate amount of gelatin and glycerin to mix, release and then grind with a colloid, and stir the ground mixed solution to make a medicinal solution; Glycerin and distilled water are stirred and miscible at 40-50°C, then warm the glycerin solution to 80-90°C, add gelatin to it and mix and stir until it is completely melted into a glue, and then let the glue stand at a temperature of 60°C After 4 hours, use a plate making machine to make a rubber plate for the pill making process; send the above-mentioned medicinal liquid and rubber plate into the pill pressing machine, and then blow it at a temperature of 22-25°C for 4 hours to make a roller shape, and then Air-dry at 25-30°C for 16-20 hours, check and select qualified capsules, wash with 95% ethanol, and air-dry at 25-30°C for 4 hours to obtain.

The preparation of embodiment eleven injection

Take 100 grams of total psoralen glycosides and dissolve them in water for injection at 40°C, add an appropriate amount of pharmaceutical carrier isotonic agent, adjust the pH of the solution to 7.0-8.0, add water for injection to the full amount, and use an ultrafilter to remove the heat source, and measure After the pH value is adjusted, it is filtered with a membrane, subpackaged, sterilized under high pressure, inspected and packaged, and the product is obtained.

Example 12 Effect of total psoralen glycosides on reduction of white blood cell count in normal mice caused by cyclophosphamide

1. Purpose of the experiment: To investigate the effect of total psoralen glycosides on leukopenia induced by cyclophosphamide.

2. Experimental materials

2.1 Drugs and reagents: donkey-hide gelatin, Shandong Dong'e Ejiao Co., Ltd., batch number: 0311101; cyclophosphamide for injection (CTX), Jiangsu Hengrui Medicine Co., Ltd., batch number: 7063021; white blood cell diluent: glacial acetic acid 2ml, 1% Gentian violet 1ml, add distilled water to 100ml.

2.2 Experimental equipment: cell counting plate, microscope, 1000μl, 100μl micropipette.

2.3 Experimental animals: ICR mice, male and female, weighing 18-22 g, provided by the Animal Center of Zhejiang Academy of Medical Sciences.

3. Methods and steps

3.1 Take healthy 18-22g ICR mice, feed them for 2 days under the conditions of this laboratory, and randomly divide them into groups:

Normal control group: equal volume of normal saline

Model group: equal volume of normal saline

Donkey-hide gelatin group: 1.5g Kg ^-1 ;

Experimental group: low and high dose groups (crude drug amount 2g·Kg ^-1 , 4g·Kg ^-1 );

10 in each group. The mice in each group were administered continuously for 8 days.

3.2 On the third day of administration, each mouse was intraperitoneally injected with cyclophosphamide 100mg/kg (0.2ml/10g).

3.3 On the eighth day (fifth day after injection of cyclophosphamide), 2 hours after administration, blood was collected from the orbital vein, 20 μl of blood was drawn, immediately transferred to 380 μl of white blood cell diluent, shaken gently for 1 to 2 minutes, fully mixed and then counted.

4. Experimental results

The experimental results showed that on the fifth day after cyclophosphamide injection, the white blood cell count in the model group was (3.33±0.98)×10 ⁶ /ml, which was significantly lower than that in the normal group (7.24±1.62)×10 ⁶ /ml, and the difference was significant ( P<0.01). The white blood cell counts in the low-dose group and the high-dose group were (4.05±0.80)×10 ⁶ /ml, (4.29±1.37)×10 ⁶ /ml, which were significantly higher than those in the model group, and the difference was significant (P<0.05, P< 0.05). The white blood cell count in the positive control group (donkey-hide gelatin group) was (4.05±0.83)×10 ⁶ /ml, which was significantly higher than that in the model group, and the difference was significant (P<0.05).

Table 1 The effect of total psoralen glycosides on the reduction of white blood cell count in normal mice caused by cyclophosphamide (x±s, n=10)

Note: Compared with the normal group, ^* P<0.05, ^** P<0.01;

Compared with the model group, ^△ P<0.05, ^△△ P<0.01.

5 Conclusion

Both low and high doses of psoralen glycosides can counteract the leukopenia induced by cyclophosphamide.

Example 13 Effect of psoralen on leukocyte count reduction in normal mice caused by cyclophosphamide

1. Purpose of the experiment: To investigate the effect of psoralen on leukopenia induced by cyclophosphamide.

2. Experimental materials

2.1 Drugs and reagents

Ejiao, Shandong Dong'e Ejiao Co., Ltd., batch number: 0311101;

Cyclophosphamide for injection (CTX), Jiangsu Hengrui Medicine Co., Ltd., batch number: 07063021;

Leukocyte diluent: glacial acetic acid 2ml, 1% gentian violet 1ml, distilled water to 100ml.

2.2 Experimental equipment: cell counting plate, microscope, 1000μl, 100μl micropipette.

2.3 Experimental animals

ICR mice, half male and half female, weighing 18-22 g, were provided by the Animal Center of Zhejiang Academy of Medical Sciences.

3. Methods and steps

3.1 Take healthy 18-22g ICR mice, feed them for 2 days under the conditions of this laboratory, and randomly divide them into groups:

Normal control group: equal volume of normal saline

Model group: equal volume of normal saline

Donkey-hide gelatin group: 1.5g Kg ^-1 ;

Experimental group: low and high dose groups (0.1g·Kg ^-1 , 0.3g·Kg ^-1 );

10 in each group. The mice in each group were administered continuously for 8 days.

3.2 On the third day of administration, each mouse was intraperitoneally injected with cyclophosphamide 100mg/kg (0.2ml/10g).

3.3 On the eighth day (fifth day after injection of cyclophosphamide), 2 hours after administration, blood was collected from the orbital vein, 20 μl of blood was drawn, immediately transferred to 380 μl of white blood cell diluent, shaken gently for 1 to 2 minutes, fully mixed and then counted.

4. Experimental results

The experimental results showed that on the fifth day after cyclophosphamide injection, the white blood cell count in the model group was (3.33±0.98)×10 ⁶ /ml, which was significantly lower than that in the normal group (7.24±1.62)×10 ⁶ /ml, and the difference was significant ( P<0.01). The white blood cell counts in the low-dose group and the high-dose group were (3.82±0.66)×10 ⁶ /ml, (3.99±1.12)×10 ⁶ /ml, which were significantly higher than those in the model group, and the difference was significant (P＜0.05, P＜ 0.05). The white blood cell count in the positive control group (donkey-hide gelatin group) was (4.05±0.83)×10 ⁶ /ml, which was significantly higher than that in the model group, and the difference was significant (P<0.05).

Table 2 Effect of psoralen on leukocyte count reduction in normal mice caused by cyclophosphamide (x±s, n=10)

Note: Compared with the normal group, ^* P<0.05, ^** P<0.01;

Compared with the model group, ^△ P<0.05, ^△△ P<0.01.

5 Conclusion

Both low and high doses of psoralen can counteract cyclophosphamide-induced leukopenia.

Example 14 Effect of Isopsoralen on the Reduced White Blood Cell Count in Normal Mice Caused by Cyclophosphamide

1. Purpose of the experiment: To investigate the effect of isopsoralen on leukopenia induced by cyclophosphamide.

2. Experimental materials

2.1 Drugs and reagents: donkey-hide gelatin, Shandong Dong'e Ejiao Co., Ltd., batch number: 0311101; cyclophosphamide for injection (CTX), Jiangsu Hengrui Medicine Co., Ltd., batch number: 07063021; white blood cell diluent: glacial acetic acid 2ml, 1% Gentian violet 1ml, add distilled water to 100ml.

2.2 Experimental equipment: cell counting plate, microscope, 1000μl, 100μl micropipette.

2.3 Experimental animals

ICR mice, half male and half female, weighing 18-22 g, were provided by the Animal Center of Zhejiang Academy of Medical Sciences.

3. Methods and steps

3.1 Take healthy 18-22g ICR mice, feed them for 2 days under the conditions of this laboratory, and randomly divide them into groups:

Normal control group: equal volume of normal saline

Model group: equal volume of normal saline

Donkey-hide gelatin group: 1.5g Kg ^-1 ;

Experimental group: low and high dose groups (0.1g·Kg ^-1 , 0.3g·Kg ^-1 );

10 in each group. The mice in each group were administered continuously for 8 days.

3.2 On the third day of administration, each mouse was intraperitoneally injected with cyclophosphamide 100mg/kg (0.2ml/10g).

3.3 On the eighth day (fifth day after injection of cyclophosphamide), 2 hours after administration, blood was collected from the orbital vein, 20 μl of blood was drawn, immediately transferred to 380 μl of white blood cell diluent, shaken gently for 1 to 2 minutes, fully mixed and then counted.

4. Experimental results

The experimental results showed that on the fifth day after cyclophosphamide injection, the white blood cell count in the model group was (3.33±0.98)×10 ⁶ /ml, which was significantly lower than that in the normal group (7.24±1.62)×10 ⁶ /ml, and the difference was significant ( P<0.01). The white blood cell counts in the low-dose group and the high-dose group were (4.01±1.02)×10 ⁶ /ml, (3.89±0.65)×10 ⁶ /ml, which were significantly higher than those in the model group, and the difference was significant (P＜0.05, P＜ 0.05). The white blood cell count in the positive control group (donkey-hide gelatin group) was (4.05±0.83)×10 ⁶ /ml, which was significantly higher than that in the model group, and the difference was significant (P<0.05).

The effect of table 3 isopsoralen on the reduction of white blood cell count in normal mice caused by cyclophosphamide (x±s, n=10)

Note: Compared with the normal group, ^* P<0.05, ^** P<0.01;

Compared with the model group, ^△ P<0.05, ^△△ P<0.01.

5 Conclusion

Both low and high doses of isopsoralen can counteract cyclophosphamide-induced leukopenia.

Claims (6)

1. the preparation method of a malaytea scurfpea fruit total glycosides extract is characterized in that being achieved through the following technical solutions:

(1) Fructus Psoraleae is solvent extraction with water,

(2) extracting solution concentrates the back precipitate with ethanol,

(3) the precipitate with ethanol supernatant extracts with little polarity organic reagent,

(4) extraction back water layer purification by macroporous resin, first water flushing, eluent is collected in the flushing of reuse ethanol water, and eluent is evaporated to does not have alcohol, and lyophilization promptly gets malaytea scurfpea fruit total glycosides extract;

In the described step (1), solvent load 8-15 doubly measures, heating extraction 1-3 time, each 0.5-2 hour;

The used concentration of alcohol of precipitate with ethanol is 70-95% in the described step (2);

The medium and small polarity organic reagent of described step (3) is selected a kind of in chloroform or the ethyl acetate for use;

The determining alcohol of ethanol water is 30-100% in the described step (4), carries out assay by HPLC, and containing percentage by weight in the malaytea scurfpea fruit total glycosides is the Fructus Psoraleae glycosides of 40-50% and the different Fructus Psoraleae glycosides of 40-50%.

2. the preparation method of a kind of malaytea scurfpea fruit total glycosides extract according to claim 1, it is characterized in that: the described solvent load of step (1) is 10 times of amounts, heating extraction 2 times, each 2 hours.

3. the preparation method of a kind of malaytea scurfpea fruit total glycosides extract according to claim 1, it is characterized in that: the described precipitate with ethanol concentration of alcohol of step (2) is 80%.

4. the application of malaytea scurfpea fruit total glycosides extract in the medicine of preparation leukocyte increasing for preparing according to the described method of claim 1.

5. the application of a kind of malaytea scurfpea fruit total glycosides extract according to claim 4 is characterized in that: drug prepared also contains preparation allowable pharmaceutical excipients or dressing.

6. the application of a kind of malaytea scurfpea fruit total glycosides extract according to claim 5 is characterized in that: described pharmaceutical dosage forms comprises tablet, capsule, granule, oral liquid, slow releasing preparation, controlled release preparation, gel, ointment, ointment, cream, suppository, injection, injectable powder, patch, drop pill or suspensoid.

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