CN103304490B - Method for separating and purifying five purine and pyrimidine bases from trichosanthes bark - Google Patents

Abstract

The present invention relates to a method for separating and purifying 5 kinds of purines and pyrimidine bases (cytosine, uracil, hypoxanthine, guanine and xanthine) from the skin of Trichosanthes quinces, using the skins of Trichosanthes as raw materials, through the following steps: ( 1) Preparation of crude extract of Trichosanthes barbatus; (2) Coarse separation with macroporous adsorption resin column; (3) Separation and purification by semi-preparative high-performance liquid chromatography: Sample 1 and sample 2 were separated by semi-preparative high-performance liquid chromatography Separation and purification are carried out, the mobile phase is methanol-water, and five monomer compounds of cytosine, uracil, hypoxanthine, guanine and xanthine are obtained with high purity. The technological process is green and environmentally friendly, has no serious harm to the environment, and has low overall cost.

Description

A method for separating and purifying 5 kinds of purine and pyrimidine bases from Trichosanthes rind

technical field

The invention belongs to the field of chemical industry, and in particular relates to a method for separating and purifying five kinds of purine and pyrimidine bases (cytosine, uracil, hypoxanthine, guanine and xanthine) from the skin of the traditional Chinese medicine Trichosanthes quince.

Background technique

Trichosanthes kirilowii Marim. or Trichosanthes rosthornii Harms. is the dried and mature peel of Trichosanthes kirilowii Marim. or Trichosanthes rosthornii Harms. Clinically, it is widely used in the treatment of phlegm-heat cough, phlegm turbid yellow thick, sore throat, chest pain, hematemesis, epistaxis, constipation, carbuncle sores and swollen toxins. Modern pharmacological experiments and clinical practice have shown that Trichosanthes peel has various pharmacological effects such as expanding coronary arteries, increasing coronary blood flow, improving myocardial hypoxia resistance, lowering serum cholesterol, antibacterial, and anticancer, especially for cardiovascular diseases. Good therapeutic effect.

Nucleic acid contains biological genetic information. Purine and pyrimidine alkaloids are important components of nucleic acid, and they are widely involved in various life activities such as heredity and metabolism. Their metabolic abnormalities often cause many serious diseases, such as kidney failure, gout and Male infertility etc. Hypoxanthine is a very important biological purine base, which has pharmacological activities such as lowering blood pressure, relieving asthma, and treating gout. It is widely distributed in the human body and can participate in the regulation of some important physiological functions in the human body. It can also be used to treat various Leukopenia, thrombocytopenia and other diseases caused by various causes, foreign countries have also reported that xanthine and hypoxanthine are closely related to the growth and metabolism of tumor cells.

A method for extracting and purifying purine and pyrimidine alkaloids from Trichosanthes rind has been reported in the literature. Teng Yongrong [Study on the chemical composition of Gualoupi and Gualoupi injection, master's degree thesis of Shandong University of Traditional Chinese Medicine, 2011] using repeated silica gel column chromatography, macroporous adsorption resin column chromatography and preparative high performance liquid chromatography to separate the The chemical constituents of the skin of Trichosanthes L. were studied, and a total of 7 compounds were obtained, including 3 nucleoside components (adenine, guanosine and 6-isosinosine). Shi Yanpeng [Research on the chemical composition of Trichosanthes and the determination of α-spinasterol (I), Chinese herbal medicine, 2002, No. 1] using repeated silica gel column chromatography to study the chemical composition of Trichosanthes, from the petroleum ether part , chloroform, and n-butanol were separated to obtain 12 compounds, 10 of which were identified, including 1 nucleoside component—uracil. Liu Dailin [Study on the anti-platelet aggregation active ingredients of Trichosanthes trichosanthes. Chinese herbal medicine, 2004, No. 12] Using the anti-platelet aggregation activity tracking method, using Sephadex LH-20 column chromatography and repeated silica gel column chromatography, 7 compounds were isolated from Trichosanthes trichosanthes. Compounds, including a nucleoside component - adenosine.

The main problem of the above-mentioned method is: the separation process step is comparatively loaded down with trivial details, needs to use chloroform sometimes as extraction agent or

The eluent has serious harm to the human body and the environment, and the types of purine and pyrimidine bases obtained are few, the production cycle is long, and the production cost is high.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a method for quickly separating and purifying purine and pyrimidine bases from the traditional Chinese medicine Trichosanthes quince, which is easy to operate, environmentally friendly, low in overall cost, and short in production cycle.

The scheme of the present invention is as follows:

The method for separating and purifying purine and pyrimidine bases from the peel of the traditional Chinese medicine Trichosanthes quince, the steps are:

(1) Preparation of the crude extract of Trichosanthes rind: Take the crushed medicinal material of Trichosanthes piscifolia, heat and reflux with water to extract, the extract is concentrated under reduced pressure to a relative density of about 1.2, and 95% ethanol to the concentration of ethanol is added to the concentrated solution. The concentration is about 70%, let it stand for 72 hours, filter, take the supernatant, and concentrate under reduced pressure to obtain the crude extract;

(2) Coarse separation with macroporous adsorption resin column: Dissolve the crude extract of Trichosanthes piperi in water, apply HPD826 macroporous adsorption resin column for chromatography, first elute with water, and then use ethanol with a concentration of about 5%- The aqueous solution was used for elution, and water and about 5% ethanol-water eluent were collected respectively. The former was concentrated under reduced pressure to obtain sample 1, and the latter was concentrated under reduced pressure to obtain sample 2;

(3) Further separation and purification of sample 1: The sample 1 was separated and purified by semi-preparative high performance liquid chromatography, the chromatographic column was YMC ODS-AQ column, the mobile phase was methanol-water, and the detection wavelength was 254 nm;

(4) Further separation and purification of sample 2: The sample 2 was separated and purified by semi-preparative high performance liquid chromatography, the chromatographic column was YMC ODS-AQ column, the mobile phase was methanol-water, and the detection wavelength was 254 nm;

The fractions of the target components are collected, and the obtained fractions are concentrated under reduced pressure to obtain the monomer compound to be separated.

In the above-mentioned method, the preferred solution is that the extraction time in step (1) heating under reflux extraction is 1-3 hours, and the number of extractions is 2-6 times. More preferably, the extraction time is 1.5-2.5 hours (preferably 2 hours), and the extraction frequency is 3-5 times (preferably 4 times).

The method described above, the preferred scheme is that the mobile phase of step (3) is 0-5% methanol-water eluent (preferably 2%), and the eluent flow rate is 2.5-4.5 mL/min (preferably 3.5 mL /min).

In the aforementioned method, a preferred scheme is that when step (4) is eluted with methanol-water eluent, the mass concentration of methanol used is 2%-6% (preferably 4%). The eluent flow rate is 2.5-4.5 mL/min (preferably 3.5 mL/min).

The method described above, the preferred solution is that the elution method of step (3) is methanol-water gradient elution/2% methanol-water isocratic elution/100% water isocratic elution/5% methanol-water, etc. Speed elution, preferably, the eluent flow rate is 2.5-4.5 mL/min (preferably 3.5 mL/min).

For the method described above, the preferred scheme is that the elution method during the separation and purification of step (4) is 6% methanol-water isocratic elution/4% methanol-water isocratic elution/2% methanol-water isocratic elution Elution/methanol-water gradient elution, preferably, the eluent flow rate is 2.5-4.5 mL/min (preferably 3.5 mL/min).

The invention relates to a method for separating and purifying purine and pyrimidine bases from the traditional Chinese medicine Trichosanthes peel, the steps are as follows: (1) Preparation of the crude extract of Trichosanthes peel: Take the crushed medicinal material of Trichosanthes peel, heat and reflux with water to extract, and extract Concentrate under reduced pressure to a relative density of about 1.2, add 95% ethanol to the concentrated solution until the concentration of ethanol is about 70%, let it stand for 72 hours, filter, take the supernatant, and concentrate under reduced pressure to obtain a crude extract. (2) Coarse separation with macroporous adsorption resin column: Dissolve the crude extract of Trichosanthes piperi in water, apply HPD826 macroporous adsorption resin column for chromatography, first elute with water, and then use ethanol with a concentration of about 5%- Water was used as the eluent for elution, and water and about 5% ethanol-water eluent were collected respectively. The former was concentrated under reduced pressure to obtain sample 1, and the latter was concentrated under reduced pressure to obtain sample 2. (3) Further separation and purification of sample 1: The sample 1 was separated and purified by semi-preparative high performance liquid chromatography, and the chromatographic column was a YMC ODS-AQ column (250 mm×10 mm I. D., 10 μm, Dalian Jiangshen Separation Technology Co., Ltd.), the mobile phase was methanol-water, and the detection wavelength was 254 nm. (4) Further separation and purification of sample 2: The sample 2 was separated and purified by semi-preparative high-performance liquid chromatography, and the chromatographic column was YMC ODS-AQ column (250 mm×10 mm I. D., 10 μm, Dalian Jiangshen Separation Technology Co., Ltd.), the mobile phase was methanol-water, and the detection wavelength was 254 nm.

The technological process is green and environmentally friendly, has no serious harm to the environment, and has low overall cost.

The method of the present invention for separating and purifying five kinds of purine and pyrimidine bases from the rind of the traditional Chinese medicine Trichosanthes quinquefolium, first adopts a macroporous adsorption resin column for rough separation, and then uses semi-preparative high-performance liquid chromatography for separation and purification, and the obtained target compound has high purity, The impurity content is extremely low, which can be seen from Figures 5-9. In addition, it also has the following advantages:

(1) After the extract is roughly separated by a macroporous adsorption resin column, most of the impurities can be removed, the composition of the mixture is simplified, the difficulty of subsequent separation can be reduced, and the semi-preparative high-performance liquid chromatography column can be protected from being Contamination effectively prolongs the service life of the chromatographic column.

(2) Five high-purity monomeric compounds of cytosine, uracil, hypoxanthine, guanine and xanthine can be obtained at the same time by using semi-preparative high-performance liquid chromatography for two separations and purifications. The method is simple to operate and high in efficiency. The process cycle is short, reagents are saved, and production costs are reduced.

(3) Organic solvents such as chloroform, which are harmful to the environment and human body, are not used in the purification process. The macroporous adsorption resin can be reused many times after regeneration, which is green and environmentally friendly.

(4) The conditions of the chromatographic method (composition and flow rate of the eluent) were optimized, so that the purity and efficiency of the compound were greatly improved.

Description of drawings

Figure 1 is a high performance liquid chromatogram of sample 1.

Figure 2 is a high performance liquid chromatogram of sample 2.

Figure 3 is a semi-preparative high performance liquid chromatogram of sample 1.

Figure 4 is a semi-preparative high performance liquid chromatogram of sample 2.

Fig. 5 is the high performance liquid chromatogram and the ultraviolet spectrogram of cytosine.

Fig. 6 is the high-performance liquid chromatogram and the ultraviolet spectrogram of uracil.

Figure 7 is a high performance liquid chromatogram and an ultraviolet spectrogram of hypoxanthine.

Figure 8 is a high performance liquid chromatogram and an ultraviolet spectrogram of guanine.

Fig. 9 is a high-performance liquid chromatogram and an ultraviolet spectrogram of xanthine.

Detailed ways

The technical solutions of the present invention will be described in detail below in conjunction with the embodiments and drawings, but the scope of protection is not limited thereto. The equipment or raw materials used in the examples are all available from the market. All reagents used were purchased from Jinan General Reagent Factory, and the water used was deionized water.

Embodiment: the method for separating and purifying purine and pyrimidine bases from the peel of the traditional Chinese medicine Trichosanthes quince, the steps are:

(1) Preparation of the crude extract of Trichosanthes rind: after crushing the medicinal material of Trichosanthes trichosanthes, heat and reflux extraction with water for 4 times, combine the extracts, concentrate under reduced pressure to a relative density of about 1.2, and add 95 % ethanol until the concentration of ethanol is about 70%, let it stand for 72 h, filter, take the supernatant, and concentrate under reduced pressure to obtain the crude extract.

(2) Coarse separation with macroporous adsorption resin column: Dissolve the crude extract of Trichosanthes piperi in water, apply HPD826 macroporous adsorption resin column for chromatography, first elute with water, and then use ethanol with a concentration of about 5%- The aqueous solution was eluted, and water and about 5% ethanol-water eluate were collected respectively. The former was concentrated under reduced pressure to obtain sample 1, and the latter was concentrated under reduced pressure to obtain sample 2.

(3) Further separation and purification of sample 1: The sample 1 was separated and purified by semi-preparative high performance liquid chromatography, and the chromatographic column was a YMC ODS-AQ column (250 mm×10 mm I. D., 10 μm, Dalian Jiangshen Separation Technology Co., Ltd.), the mobile phase was methanol-water, and the detection wavelength was 254 nm. the

(4) Further separation and purification of sample 2: The sample 2 was separated and purified by semi-preparative high-performance liquid chromatography, and the chromatographic column was YMC ODS-AQ column (250 mm×10 mm I. D., 10 μm, Dalian Jiangshen Separation Technology Co., Ltd.), the mobile phase was methanol-water, and the detection wavelength was 254 nm. the

The fractions of the target components are collected, and the obtained fractions are concentrated under reduced pressure to obtain the monomer compound to be separated.

The inventor used different concentrations of methanol as the mobile phase, adopted different elution methods, and controlled the flow rate of the eluent to be 2.5-4.5 mL/min (preferably 3.5 mL/min), and optimized the purification conditions to achieve the purpose of the present invention , the relevant experimental results are as follows:

Table 1 Semi-preparative HPLC separation conditions of sample 1

the eluent condition Example 1 5% methanol-water isocratic elution Example 2 2% methanol-water isocratic elution Example 3 100% water isocratic elution Example 4 Methanol-water gradient elution

In Example 1, 5% methanol-water was used as the eluent for isocratic elution, and the elution time of each component was shorter, but the separation effect between them was not ideal, and the purity of the obtained components was low. In Example 2, 2% methanol-water was used as the eluent for isocratic elution, and the components were separated well, and the separation time was also relatively appropriate. In Example 3, 100% water was used as the eluent for isocratic elution, and the components were separated well, but the separation time was too long. Example 4 adopts methanol-water gradient elution, the components are separated well, and the separation time is also acceptable, but the recovery and reuse of the eluate is difficult due to concentration changes.

Fig. 3 is the chromatogram when the system of Example 2 is selected for use, as can be seen from Fig. 3, each component is well separated, and the separation time is also comparatively suitable. The peak components are collected according to the chromatogram, and the corresponding high-purity compound can be obtained after recovering the solvent. After analysis and testing by the high performance liquid chromatography area normalization method, the purity of each component obtained in Example 2 is very high, all above 98%, which can be seen from Figures 5-6.

Table 2 The semi-preparative high performance liquid chromatography separation conditions of sample 2

the eluent condition Example 5 6% methanol-water isocratic elution Example 6 4% methanol-water isocratic elution Example 7 2% methanol-water isocratic elution Example 8 Methanol-water gradient elution

In Example 5, 6% methanol-water was used as the eluent for isocratic elution, and the elution time of each component was shorter, but the separation effect between them was not ideal, and the purity of the obtained components was low. In Example 6, 4% methanol-water was used as the eluent for isocratic elution, and the components were separated well, and the separation time was also relatively appropriate. In Example 7, 2% methanol-water was used as the eluent for isocratic elution, and the components were separated well, but the separation time was too long. Example 8 adopts methanol-water gradient elution, the components are separated well, and the separation time is also acceptable, but the recovery and reuse of the eluate is difficult due to concentration changes.

Fig. 4 is the chromatogram when selecting embodiment 6 system for use, as can be seen from Fig. 4, each component is separated well, and separation time is also comparatively suitable. The peak components are collected according to the chromatogram, and the corresponding high-purity compound can be obtained after recovering the solvent. After analysis and testing by the high performance liquid chromatography area normalization method, the purity of each component obtained in Example 6 is very high, all above 98%, which can be seen from Figures 7-9.

According to the test of H NMR spectrum and C NMR spectrum, the obtained peak components were identified, and cytosine, uracil, hypoxanthine, guanine and xanthine were obtained. The structural formulas of the extracted and purified five compounds confirmed by modern spectral data are as follows:

The identification results of the five compounds are as follows:

Compound I (cytosine): ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ ppm: 10.47 (1H, br s, 1-H), 7.32 (1H, d, J = 6.8 Hz, 6-H) , 7.04 (2H, br s, 4-NH ₂ ), 5.57 (1H, d, J =6.8 Hz, 5-H). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ ppm: 166.6 (C-4), 156.8 (C-2), 142.5 (C-6), 92.4 (C-5).

Compound II (uracil): ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ ppm: 10.48 (2H, br s, 1-H, 3-H), 7.39 (1H, d, J = 7.6 Hz, H-6), 5.44 (1H,d, J =7.6 Hz, H-5). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ ppm: 164.1 (C-4), 151.4 (C-2), 142.1 (C-6), 100.0 (C-5).

Compound III (hypoxanthine): ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ ppm: 13.20 (1H, br s, 9-H), 12.29 (1H, br s, 1-H), 8.18 ( 1H, s, 2-H), 8.11 (1H, s, H-8). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ ppm: 155.6 (C-6), 153.7 (C-4), 152.7 (C-8), 144.1 (C-2), 119.5 (C-5) .

Compound IV (guanine): ¹ H-NMR (400 MHz, D ₂ O) δ ppm: 7.34 (1H, s, 8-H). ¹³ C-NMR (100 MHz, D ₂ O) δ ppm: 155.8 (C-6), 149.1 (C-4), 147.2 (C-2), 136.7 (C-8), 106.5 (C-5).

Compound V (xanthine): ¹ H-NMR (400 MHz, D ₂ O) δ ppm: 7.35 (1H, s, 8-H). ¹³ C-NMR (100 MHz, D ₂ O) δ ppm: 155.4 (C-6), 152.2 (C-2), 150.4 (C-4), 141.6 (C-8), 106.9 (C-5).

It should be noted that the specific embodiments are only representative examples of the present invention, and obviously the technical solution of the present invention is not limited to the above-mentioned embodiments. Many variants are also possible. Those of ordinary skill in the art should consider the disclosure, mention or association of this document as the scope of protection of this patent.

Claims (13)

1. the method for separation and purification purine and pyrimidine bases from Chinese medicine Snakegourd Peel, it is characterized in that, step is:

(1) preparation of Snakegourd Peel crude extract: get the Snakegourd Peel medicinal material crushed, use water heating and refluxing extraction, extracting solution is 1.2 through being evaporated to relative density, in concentrated solution, add 95% ethanol to the concentration of ethanol is 70%, leave standstill 72h, filter, get supernatant liquid, concentrating under reduced pressure obtains crude extract;

(2) macroporous adsorptive resins roughing out: by soluble in water for Snakegourd Peel crude extract, upper HPD826 type macroporous adsorptive resins carries out chromatography, first carry out wash-out with water, wash-out is carried out again with the ethanol-water solution that concentration is 5%, collect the alcohol-water elutriant of water and 5% respectively, the former concentrating under reduced pressure, obtains sample 1, the latter's concentrating under reduced pressure, obtains sample 2;

(3) the further separation and purification of sample 1: sample 1 Semipreparative chromatography is carried out separation and purification, chromatographic column is YMC ODS-AQ post, and moving phase is methanol-water, and determined wavelength is 254nm;

(4) the further separation and purification of sample 2: sample 2 Semipreparative chromatography is carried out separation and purification, chromatographic column is YMC ODS-AQ post, and moving phase is methanol-water, and determined wavelength is 254nm;

Collect target components cut, by the cut concentrating under reduced pressure obtained, namely obtain cytosine(Cyt), uridylic, xanthoglobulin, guanine and xanthine monomeric compound.

2. method according to claim 1, is characterized in that, during step (1) reflux, extraction time is 1-3 hour.

3. method according to claim 2, is characterized in that, step (1) extraction time is 1.5-2.5 hour.

4. method according to claim 3, is characterized in that, step (1) extraction time is 2 hours.

5. method according to claim 1, is characterized in that, step (1) extraction time is 2-6 time.

6. method according to claim 1, is characterized in that, step (1) extraction time is 3-5 time.

7. method according to claim 1, is characterized in that, step (1) extraction time is 4 times.

8. method according to claim 1, is characterized in that, step (3) type of elution is methanol-water gradient elution/2% methanol-water isocratic elution/100% water isocratic elution/5% methanol-water isocratic elution.

9. method according to claim 8, is characterized in that, eluent flow rate is 2.5-4.5mL/min.

10. method according to claim 8, is characterized in that, eluent flow rate is 3.5mL/min.

11. methods according to claim 1, is characterized in that, type of elution during step (4) separation and purification is 6% methanol-water isocratic elution/4% methanol-water isocratic elution/2% methanol-water isocratic elution/methanol-water gradient elution.

12. methods according to claim 11, is characterized in that, eluent flow rate is 2.5-4.5mL/min.

13. methods according to claim 12, is characterized in that, eluent flow rate is for selecting 3.5mL/min.