CN111333693A - Method for extracting betulinic acid - Google Patents

Abstract

The invention relates to the technical field of traditional Chinese medicine extraction, in particular to a method for extracting betulinic acid. The method for extracting betulinic acid in the present invention comprises the following steps: ultrasonically treating the mixture of bark of Sycamore tree and water, leaching the ultrasonically treated bark with a composite solvent, and filtering the obtained leaching solution in turn , concentration and drying treatment; in the mixture of sycamore bark and water, the temperature of the water is 10-30 ° C; the composite solvent includes an organic solvent and water; the organic solvent includes methanol, ethanol, At least one of alcohol and acetone; the volume percentage of water in the composite solvent is 10% to 20%. In the present invention, the bark of the Sycamore family is mixed with water at a specific temperature to carry out ultrasonic treatment, and then the specific compound solvent is used for leaching, so that the obtained betulinic acid has higher yield and purity.

Description

A kind of extraction method of betulinic acid

technical field

The invention relates to the technical field of traditional Chinese medicine extraction, in particular to a method for extracting betulinic acid.

Background technique

Betulinic acid can be extracted from cattail leaves, birch bark, and jujube kernels, and can also be obtained by chemical synthesis using betulin as raw material. Studies have found that betulinic acid can selectively kill human melanoma cells without killing healthy cells; betulinic acid has an inhibitory effect on HIV-1 infection. Malignant cells such as germ layer tumors and leukemia also have inhibitory effects.

At present, the method for extracting and separating betulinic acid from natural plants has the following disadvantages: a large amount of solvent is consumed, the yield is low, impurities are not easily separated, and the purity is low.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, the present invention relates to a kind of extraction method of betulinic acid, comprising the following steps:

The mixture of the bark of Sycamore tree and water is subjected to ultrasonic treatment, the bark after the ultrasonic treatment is leached with a composite solvent, and the obtained leaching solution is successively filtered, concentrated and dried;

In the mixture of sycamore bark and water, the temperature of the water is 10-30°C;

The composite solvent includes a mixed solution of at least two of methanol, ethanol, butanol and acetone;

The volume percentage of water in the composite solvent is 5% to 10%.

In the present invention, the bark of the sycamore family is mixed with water at a specific temperature to carry out ultrasonic treatment, and then a specific compound solvent is used for extraction, so that the obtained betulinic acid has higher yield and purity.

Compared with the prior art, the beneficial effects of the present invention are:

In the present invention, the bark of the Sycamore family is mixed with water at a specific temperature to carry out ultrasonic treatment, and then a specific compound solvent is used for extraction, and the obtained betulinic acid has higher yield and purity, and the purity is higher than 96.9%. Above 6.11% (relative to the mass percentage of dry bark), the amount of betulinic acid in the bark is already close to 100%.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

According to one aspect of the present invention, the present invention relates to a kind of extraction method of betulinic acid, comprising the following steps:

The mixture of the bark of Sycamore tree and water is subjected to ultrasonic treatment, the bark after the ultrasonic treatment is leached with a composite solvent, and the obtained leaching solution is successively filtered, concentrated and dried;

In the mixture of sycamore bark and water, the temperature of the water is 10-30°C;

The composite solvent includes an organic solvent and water;

The organic solvent includes at least one of methanol, ethanol, butanol and acetone;

The volume percentage of water in the composite solvent is 10% to 20%.

The Sycamore family described in the present invention includes Platanus orientalis, Platanus occidentalis, and Platanus acerifolia.

The bark of the sycamore family used in the present invention has a length of 2 to 5 cm and a thickness of 2 to 6 mm.

Preferably, the bark of the sycamore family used has a length of 3-4 cm and a thickness of 2-4 mm.

Hot water extraction is energy-intensive for industrial processing, especially in winter when the outdoor temperature is relatively low. Cold water (temperature is 10-30 ℃) plus ultrasonic treatment can achieve the effect of transforming and transporting the plant cell wall of the extracted sycamore bark in a short time, which is the most simple and effective method for improving the efficiency of compound solvent extraction. preprocessing.

Ultrasonic treatment has two functions: one is to promote the dissolution of other water-soluble extracts in the bark. Considering industrial scale production, cold water consumes less energy than hot water. Although ultrasound also consumes energy, adding a short time of ultrasonic treatment, It has high timeliness and can replace the pretreatment effect of hot water boiling; the second effect is that through ultrasonic treatment, the structure of the bark cell wall becomes loose and the pores become larger, which is more conducive to the penetration of the solvent and the subsequent treatment with the solvent. Improve the overall extraction efficiency of betulinic acid.

In the present invention, the bark of the sycamore family is mixed with water at a specific temperature to carry out ultrasonic treatment, and then a specific compound solvent is used for extraction, so that the obtained betulinic acid has higher yield and purity.

In one embodiment, the temperature of the water is 10-30°C, and 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C can also be selected. , 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C or 29°C.

In one embodiment, the volume percentage of water in the composite solvent is 10% to 20%, and 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% or 19%.

More preferably, the volume percentage of water in the composite solvent is 13-18%.

More preferably, the volume percentage of water in the composite solvent is 15%.

Preferably, the step of carrying out ultrasonic treatment to the mixture of Sycamore bark and water is specifically as follows: adding water to Sycamore bark,

The frequency of adding the water in every 24h is 1-3 times, and the amount of water added each time is 10-15mL/g bark of Sycamore;

Preferably, the number of times of adding the water in every 24h is 3 times, and the amount of water added each time is 12 mL/g bark of Sycamore tree.

In the present invention, a specific amount of water is used to soak the bark of Sycamore, that is, the number of times of adding the water is 1 to 3 times per 24 hours, and the amount of water added per gram of the bark of Sycamore is 10 to 15 mL. , which is more conducive to the loosening of the bark cell wall structure and the larger pores, which is more conducive to the penetration of the solvent during subsequent solvent treatment and improves the overall extraction efficiency of betulinic acid.

Preferably, the organic solvent includes at least two of methanol, ethanol, butanol and acetone.

Preferably, the organic solvent includes methanol, ethanol, butanol and acetone;

The mass ratio of methanol, ethanol, butanol and acetone is (3-5):(2-4):(1-2):1.

More preferably, the mass ratio of methanol, ethanol, butanol and acetone is (4):(3):(2):1.

In one embodiment, the mass ratio of methanol, ethanol, butanol and acetone is (3-5):(2-4):(1-2):1, and 3:2:1:1 can also be selected , 3:4:2:1, 5:2:1:1, 5:4:2:1, or 4:3:2:1.

Preferably, the power of the ultrasonic wave is 1100-3500W, the frequency is 20-40kHz, the ultrasonic wave is processed for 1-3 times per 24h, and the time for each processing is 4-8min.

In one embodiment, the power of the ultrasonic wave is 1100-3500W, and 1200W, 1300W, 1400W, 1500W, 1600W, 1700W, 1800W, 1900W, 2000W, 2100W, 2200W, 2300W, 2400W, 2500W, 2600W, 2700W, 2800W, 2900W, 3000W, 3100W, 3200W, 3300W or 3400W.

In one embodiment, the frequency of the ultrasonic wave is 20-40kHz, and 21kHz, 22kHz, 23kHz, 24kHz, 25kHz, 26kHz, 27kHz, 28kHz, 29kHz, 30kHz, 31kHz, 32kHz, 33kHz, 34kHz, 35kHz, 36kHz, 37kHz, 38kHz, 39kHz.

In an embodiment, the number of times of the ultrasonic treatment per 24h is 1 to 3 times, and 2 times can also be selected.

In one embodiment, the time for each ultrasonic treatment is 4-8 minutes, and 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, 6.5 minutes, 7 minutes, or 7.5 minutes can also be selected.

Ultrasonic treatment and soaking treatment time have a great influence on the extraction efficiency. If the time is too long, the mucus in the bark tissue will diffuse out and adhere to the surface of the bark to hinder the entry of the solvent, thereby affecting the extraction efficiency. If the time is too short, and good treatment effect cannot be achieved, the present invention obtains suitable ultrasonic treatment time and soaking time through a large number of experiments.

According to the characteristics of the bark and betulinic acid of the sycamore family, the present application adopts a specific ultrasonic frequency and power, which is more conducive to improving the yield and purity of the betulinic acid leaching in the later stage.

Preferably, the power of the ultrasonic wave is 1600-3000W, the frequency is 30-40kHz, the ultrasonic wave is processed three times per 24h, and the time of each processing is 7min.

In the present invention, by further optimizing the ultrasonic cold water treatment conditions, the power of the ultrasonic wave is 1600-3000W, the frequency is 30-40kHz, the number of ultrasonic treatment is 3 times per 24h, and the time of each treatment is 7min, which can further improve the extraction efficiency of betulin acid. Yield and Purity.

Preferably, the volume of the compound solvent added to each gram of the bark is 1-2.5 mL.

In one embodiment, the volume of the compound solvent added to each gram of the bark is 1.1mL, 1.2mL, 1.3mL, 1.4mL, 1.5mL, 1.6mL, 1.7mL, 1.8mL, 1.9mL, 2.0mL mL, 2.1 mL, 2.2 mL, 2.3 mL, or 2.4 mL.

Preferably, the temperature of the leaching is 21-26°C;

Preferably, the number of times of the leaching is 1 to 4 times, and the time of each leaching is 10 to 30 hours.

In one embodiment, the number of times of the leaching is 1 to 4 times, and 2 or 3 times can also be selected.

In one embodiment, the leaching temperature is 21-26°C, and 21.5°C, 22°C, 22.5°C, 23°C, 23.5°C, 24°C, 24.5°C, 25°C or 25.5°C can also be selected.

In one embodiment, the time of each extraction is 10-30h, and 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h can also be selected , 26h, 27h, 28h or 29h.

Preferably, the concentration includes concentration under reduced pressure, the temperature of the concentration under reduced pressure is 25-50° C., and the pressure is 280-450 KPa.

By concentrating under reduced pressure at a temperature of 25-50°C and a pressure of 280-450Kpa, it can ensure that the compound solvent is evaporated without destroying the betulinic acid, thereby further improving the purity.

In one embodiment, the decompression concentration temperature is 25-50°C, and 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C can also be selected. °C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C or 49°C.

More preferably, the decompression concentration temperature is 41-45°C, and the pressure is 360-400KPa.

By further controlling the decompression concentration temperature to be 41-45°C and the pressure to be 360-400Kpa, it is more beneficial to volatilize the composite solvent.

Preferably, the drying temperature is 5˜45° C., and the pressure is 3.5˜9 mmHg.

In one embodiment, the drying temperature is 5 to 45°C, and 6°C, 8°C, 10°C, 12°C, 15°C, 18°C, 21°C, 25°C, 28°C, 31°C can also be selected. , 35°C, 37°C, 39°C, 41°C, 43°C or 44°C.

In one embodiment, the drying pressure is 3.5-9 mmHg, and 4 mmHg, 4.5 mmHg, 5 mmHg, 5.5 mmHg, 6.5 mmHg, 7 mmHg, 7.5 mmHg, 8 mmHg or 8.5 mmHg can also be selected.

In a preferred embodiment, the extraction method of described betulinic acid, comprises the following steps:

The water is added to the bark of the Sycamore family for ultrasonic treatment, the number of times of adding the water is 1 to 3 times per 24h, and the amount of water added each time is 10 to 15 mL/g of the bark of the Sycamore family; the ultrasonic treatment is performed by using a composite solvent. The latter bark is leached, and the composite solvent includes a mixed solution of methanol, ethanol, butanol and acetone, and the mass ratio of methanol, ethanol, butanol and acetone in the composite solvent is (3-5): (2-4 ): (1～2): 1, the volume percentage of water in the composite solvent is 10% to 20%, and the leaching solution is filtered, concentrated and dried successively; Among them, the temperature of the water is 10-30°C; the power of the ultrasonic wave is 1100-3500W, the frequency is 20-40kHz, and the number of ultrasonic treatments per 24h is 1-3 times, and the time of each treatment is 4 ～8min; the temperature of the leaching is 21～26℃; the times of the leaching are 1～4 times, the time of each leaching is 10～30h, and the compound solvent is added to each gram of the bark The volume of the drying is 1-2.5mL; the concentration includes concentration under reduced pressure, the temperature of the concentration under reduced pressure is 25-50°C, and the pressure is 280-450Kpa; the drying temperature is 5-45°C, and the pressure is 3.5-9mmHg .

The above-mentioned specific extraction method of the present invention further improves the yield and purity of betulinic acid.

The present invention will be further explained below in conjunction with specific examples and comparative examples.

Example 1

A method for extracting betulinic acid, comprising the following steps:

Water is added to 500g of Sycamore bark for ultrasonic treatment, the number of times of adding the water is 3 times per 24h, the amount of water added each time is 12mL/g Sycamore bark, the temperature of the water is 22 ° C, the ultrasonic The power is 2500W, the frequency is 35kHz, the number of times of the ultrasonic treatment is 3 times per 24h, and the time of each treatment is 7min; the bark after the ultrasonic treatment is leached with a composite solvent, and the bark in each gram of the bark is extracted. The volume of the composite solvent added is 1-2.5 mL, the composite solvent is a mixed solution of methanol, ethanol, butanol and acetone, and the mass ratio of methanol, ethanol, butanol and acetone in the composite solvent is 4:3:2 : 1. The temperature of the leaching is 23°C, the number of times of the leaching is 4 times, the time of each leaching is 25h, the volume percentage of water in the composite solvent is 15%, and then the leaching solution Filtration and collection of the filtrate were carried out, and the filtrate was further concentrated and dried under reduced pressure. The temperature for concentration under reduced pressure was 42°C, the pressure was 400KPa, the drying temperature was 28°C, and the pressure was 5 mmHg.

Example 2

A method for extracting betulinic acid, comprising the following steps:

Water was added to 420 g of Sycamore bark for ultrasonic treatment, the number of times of adding the water was 1 time per 24h, the amount of water added each time was 10 mL/g Sycamore bark, the temperature of the water was 10 ° C, and the ultrasonic wave was used. The power of the ultrasonic wave is 1100W, the frequency is 40kHz, the number of times of the ultrasonic treatment is 1 time per 24h, and the time of each treatment is 8min; the bark after the ultrasonic treatment is leached with a composite solvent, and the composite solvent is methanol, A mixed solution of ethanol, butanol and acetone, the volume of the composite solvent added to each gram of the bark is 2.5 mL, and the mass ratio of methanol, ethanol, butanol and acetone in the composite solvent is 4:4:1:1 , the temperature of the leaching is 21°C, the number of times of the leaching is 3 times, the time of each leaching is 10h, the volume percentage of water in the composite solvent is 10%, and then the leaching solution is filtered. The filtrate was collected, and the filtrate was further concentrated and dried under reduced pressure. The temperature for concentration under reduced pressure was 25°C, the pressure was 280Kpa, the drying temperature was 5°C, and the pressure was 9mmHg.

Example 3

A method for extracting betulinic acid, comprising the following steps:

Water was added to 470 g of Sycamore bark for ultrasonic treatment, and the number of times of adding the water in every 24h was 2 times, and the amount of water added each time was 15mL/g Sycamore bark, and the temperature of the water was 30 ° C. The power is 3500W, the frequency is 20kHz, the number of times of the ultrasonic treatment is 2 times per 24h, and the time of each treatment is 4min; the bark after the ultrasonic treatment is leached with a composite solvent, and the composite solvent is methanol, A mixed solution of ethanol, butanol and acetone, the volume of the composite solvent added to each gram of the bark is 1 mL, and the mass ratio of methanol, ethanol, butanol and acetone in the composite solvent is 3:4:1:2, The temperature of the leaching is 26°C, the number of times of leaching is 1 time, the time of each leaching is 30h, the volume percentage of water in the composite solvent is 20%, and then the leaching solution is filtered and collected. The filtrate was collected, and the filtrate was further concentrated and dried under reduced pressure. The temperature for concentration under reduced pressure was 50°C, the pressure was 450Kpa, the drying temperature was 45°C, and the pressure was 3.5 mmHg.

Example 4

A method for extracting betulinic acid, except that the organic solvent is methanol solution, other operating conditions are the same as in Example 1.

Example 5

A method for extracting betulinic acid, except that the composite solvent is a mixed solution of methanol and acetone, and the mass ratio is 4:1, and other operation steps are the same as those in Example 1.

Example 6

A method for extracting betulinic acid, except that the composite solvent is methanol, a mixed solution of ethanol and acetone, and the mass ratio is 2:2:1, other operation steps are the same as those in Example 1.

Example 7

A method for extracting betulinic acid, except that the power of the ultrasonic wave is 1200W, the frequency is 38kHz, and the time of each ultrasonic treatment is 8min, and other operation steps are the same as those in Example 1.

Example 8

A method for extracting betulinic acid, except that the power of the ultrasonic wave is 3400W, the frequency is 22kHz, and the time of each ultrasonic treatment is 5min, and other operation steps are the same as those in Example 1.

Comparative Example 1

A method for extracting betulinic acid, except that the power of the ultrasonic wave is 900W, the frequency is 18kHz, and the time for each treatment is 2min, and other operating conditions are the same as in Example 1.

Comparative Example 2

A method for extracting betulinic acid, except that the temperature of the water in the ultrasonic treatment process is 8°C, and other operating conditions are the same as in Example 1.

Comparative Example 3

A method for extracting betulinic acid, except that the volume percentage of water in the composite solvent is 1%, and other operating conditions are the same as in Example 1.

Comparative Example 4

A method for extracting betulinic acid, the bark of Sycae family and Example 1 are taken from the same tree, and the patent application number is CN201110083158.6 (a kind of method for extracting betulinic acid from bark of Sycae family) Method 1 for extracting betulinic acid.

Experimental example

The yield calculation and purity detection of the betulinic acid obtained in the present invention, the examples and the comparative examples are carried out. The betulinic acid yield results are shown in Table 1, and the betulinic acid purity detection is shown in Table 2.

Table 1 Yield of betulinic acid

Table 2 Betulinic acid purity

Examples and Comparative Examples Betulinic acid purity (%) Example 1 97.5 Example 2 97.2 Example 3 97.1 Example 4 97.3 Example 5 97.2 Example 6 97.0 Example 7 97.3 Example 8 96.9 Comparative Example 1 97.1 Comparative Example 2 96.8 Comparative Example 3 97.1 Comparative Example 4 95.2

As can be seen from Table 1 and Table 2, the betulinic acid obtained by the specific purification method of the present invention has higher yield and purity, the purity is higher than 96.9%, and the yield is higher than 6.11% (relative to the absolute dry bark). mass percent).

The ultrasonic treatment conditions of comparative example 1 are not within the scope of protection of the present invention, the temperature of the ultrasonic treatment process of comparative example 2 is not within the scope of protection of the present invention, and the volume percent of water in the composite solvent in comparative example 3 is not within the scope of protection of the present invention The betulinic acid obtained in Comparative Example 1, Comparative Example 2, and Comparative Example 3 is inferior to the betulinic acid obtained by the specific purification method of the present application in terms of purity and yield.

Comparative example 4 is that the bark of Sycamore and Example 1 are taken from the same tree, and the method of Example 1 that the patent application number is CN201110083158.6 (a kind of method for extracting betulinic acid from the bark of Sycamore) is extracted. Betulinic acid. The yield of betulinic acid was 5.71% (relative to the mass percentage of dry bark), and the purity was 95.2%, which was far worse than the betulinic acid obtained by the purification method specified in the present application.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand: It is still possible to modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements to some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. range.

Claims (10)

1. The method for extracting betulinic acid is characterized by comprising the following steps of:

performing ultrasonic treatment on the mixture of the barks of the firmiana and water, leaching the ultrasonically treated barks by adopting a composite solvent, and sequentially filtering, concentrating and drying the obtained leaching liquor;

in the mixture of the tree bark of the firmiana family and water, the temperature of the water is 10-30 ℃;

the composite solvent comprises an organic solvent and water;

the organic solvent comprises at least one of methanol, ethanol, butanol and acetone;

the volume percentage of water in the composite solvent is 10-20%.

2. The method for extracting betulinic acid according to claim 1, wherein the step of subjecting the mixture of the bark of firmianaceae and water to ultrasonic treatment is specifically: adding water into the tree bark of the firmiana family, wherein the adding time of the water is 1-3 times every 24 hours, and the adding amount of water is 10-15 mL/g each time;

preferably, the water is added 3 times per 24h, each time with an amount of 12mL/g of the bark of Sterculiaceae family.

3. The method of extracting betulinic acid according to claim 1, wherein the organic solvent includes at least two of methanol, ethanol, butanol and acetone.

4. The method for extracting betulinic acid according to claim 1, wherein the organic solvent comprises methanol, ethanol, butanol and acetone;

the mass ratio of the methanol to the ethanol to the butanol to the acetone is (3-5) to (2-4): (1-2): 1.

5. the method for extracting betulinic acid as claimed in claim 1, wherein the power of the ultrasonic wave is 1100-3500W, the frequency is 20-40kHz, the number of times of treatment per 24h of the ultrasonic wave is 1-3, and the time of each treatment is 4-8 min.

6. The method for extracting betulinic acid as claimed in claim 5, wherein the power of the ultrasonic wave is 1600-3000W, the frequency is 30-40kHz, the number of times of treatment by the ultrasonic wave per 24h is 3, and the time of each treatment is 7 min.

7. The method for extracting betulinic acid according to claim 1, wherein the volume of the composite solvent added to each gram of the bark is 1-2.5 mL.

8. The method for extracting betulinic acid according to claim 1, wherein the leaching temperature is 21-26 ℃;

preferably, the leaching times are 1-4 times, and the leaching time is 10-30 hours each time.

9. The method for extracting betulinic acid according to claim 1, wherein the concentration comprises concentration under reduced pressure, wherein the temperature of the concentration under reduced pressure is 25-50 ℃ and the pressure is 280-450 KPa.

10. The method for extracting betulinic acid according to claim 1, wherein the drying temperature is 5 to 45 ℃ and the pressure is 3.5 to 9 mmHg.