CN112876366B - Broom-like isoesterasum, preparation method and application thereof, and pharmaceutical composition - Google Patents

Abstract

A broom-shaped isoester, its preparation method and application relate to the technical field of medicine, the broom-shaped isoester has novel structure, has inhibition effect on T and B lymphocyte proliferation, has selectivity on the inhibition effect on T lymphocyte, and can be widely applied to preparation of immunosuppressive drugs. The preparation method of the broom-shaped isoesteridin is simple to operate, and the broom-shaped isoesteridin can be quickly and efficiently extracted from rabdosia rubescens. A pharmaceutical composition, which comprises the broom-shaped isoester and pharmaceutically acceptable auxiliary materials. It has good immunosuppressive activity, and can be used as immunosuppressant.

Description

A kind of brush-like isoesterin, its preparation method and application, and pharmaceutical composition

technical field

The present invention relates to the technical field of extraction and separation of natural compounds, in particular, to a fucoidin, a preparation method and application thereof, and a pharmaceutical composition.

Background technique

Caraway is one of the important medicinal plant resources in my country. Among them, Rubescens has been included in the Chinese Pharmacopoeia. At the same time, various plants of this genus are widely used as herbal medicines in my country for clearing heat and detoxifying, anti-inflammatory and antibacterial. , anti-tumor and so on. Plants of this genus are rich in diterpenoids, which have diverse structures and are active in anti-tumor, antibacterial and immunosuppressive aspects, such as Rubescensine A, calyxine B, and calyxine B have significant antitumor activity . Isodonscoparius C.Y.Wu et H.W.Li(Dunn) Kudo belongs to the genus Lamiaceae, mainly distributed in Yunnan Province, China. It is used in folk to treat inflammation of the digestive system of humans and livestock and external infections, etc. . Studies have shown that this plant contains novel diterpenoids, with dicyclic diterpenes as the main structural type. At present, only three heteroditerpenoids containing asymmetric four-membered rings have been found, namely broom tea. Vegetable lactones A and B (scopariusicides A and B), scopariusicacid. However, in the prior art, there is not yet a better acquisition channel for the above-mentioned heteroditerpenoids, and the technology of extraction from natural products or the technology of artificial synthesis still needs to be further developed.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a fucoidin, which has a novel structure, a wide range of sources and great medicinal value.

The second object of the present invention is to provide a preparation method of the above-mentioned lepidopteran, which is simple and convenient to operate, and can quickly and efficiently extract the above-mentioned lepidopteran from the broccoli.

The third object of the present invention is to provide an application of the above-mentioned spirochete, which has a certain immunosuppressive activity and can be widely used in the preparation of immunosuppressive drugs.

The fourth object of the present invention is to provide a pharmaceutical composition, which comprises the above-mentioned strobilurin, which has better immunosuppressive activity.

Embodiments of the present invention are implemented as follows:

A kind of brush-shaped isoesterin, the structural formula of brush-shaped isoesterin is

A kind of preparation method of above-mentioned brush-shaped isoesterin, it comprises:

Pulverizing the stems and/or leaves of C. brussel sprouts, leaching with an extraction solvent, and then desolventizing, to obtain an extract of C. brussibilis; using silica gel column chromatography, reverse phase phase Column chromatography and semi-preparative HPLC were used to separate the isoesterin.

An application of the above spicules in the preparation of immunosuppressive drugs.

A pharmaceutical composition, which comprises the above-mentioned Isoesterin and pharmaceutically acceptable excipients.

The beneficial effects of the embodiments of the present invention are:

The embodiments of the present invention provide a broomlike isoesterin, a preparation method and an application thereof. The broomlike isoesterin has a novel structure, has an inhibitory effect on the proliferation of T lymphocytes, and can be widely used in the preparation of immunosuppressive drugs. The preparation method of the broom-shaped isoesterin is not only simple to operate, but also can be extracted from the broom-shaped isoesterin with high efficiency and high purity from the broom-shaped isoesterin.

The embodiment of the present invention also provides a pharmaceutical composition, which comprises the above-mentioned succinyl isoesterin, and pharmaceutically acceptable excipients. It has good immunosuppressive activity and can be used as an immunosuppressant.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is the ¹³ C NMR spectrogram of the fucoidin provided by the embodiment of the present invention;

Fig. 2 is the ¹ H NMR spectrogram of the fucoidin provided by the embodiment of the present invention;

Fig. 3 is the HSQC spectrogram of the brush-like isoesterin provided in the embodiment of the present invention;

Fig. 4 is the HMBC spectrogram of the brush-like isoesterin provided in the embodiment of the present invention;

FIG. 5 is an enlarged view of the HMBC spectrum of the broom-like isoesterin provided in the embodiment of the present invention at a position where the chemical shift of the horizontal axis is 0-2.1 ppm.

Fig. 6 is the ¹ H- ¹ H COSY spectrogram of the brush-like isoesterin provided in the embodiment of the present invention;

FIG. 7 is an enlarged view of the ¹ H- ¹ H COSY spectrum of the fucoidin provided in the embodiment of the present invention where the chemical shift of the horizontal axis is 0-2.7 ppm.

Fig. 8 is the ROESY spectrogram of the brush-like isoesterin provided in the embodiment of the present invention;

FIG. 9 is an enlarged view of the ROESY spectrum of the broom-like isoesterin provided in the embodiment of the present invention where the chemical shift of the horizontal axis is 0-2.2 ppm;

Fig. 10 is a schematic diagram showing the correlation of ¹ H- ¹ H COSY (indicated by bold keys) and HMBC (indicated by arrows) of the styloplasts provided in the examples of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. 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 purchased from the market.

The following will specifically describe a kind of broom-like isoesterin, the preparation method and application thereof, and the pharmaceutical composition of the embodiment of the present invention.

The embodiment of the present invention provides a kind of broom-like isoesterin, and the structural formula of the broom-like isoesterin is:

The broom-like isoesterin is extracted from the stems and/or leaves of the broom-like caraway, has a novel structure, and has biological activity, exhibits inhibitory activity on T and B cell lines, and is selective for T cell inhibitory effect. As the lead compound of immunosuppressant, it has good application potential.

The embodiment of the present invention also provides a kind of preparation method of the above-mentioned brush-like isoesterin, and it comprises:

Pulverizing the stems and/or leaves of C. brussel sprouts, leaching with an extraction solvent, and then desolventizing, to obtain an extract of C. brussibilis; using silica gel column chromatography, reverse phase phase Column chromatography and semi-preparative HPLC were used to separate the isoesterin.

Wherein, the extraction solvent includes at least one of ethanol, acetone, methanol, ethyl acetate and water. Preferably, the extraction solvent includes a solution formed by mixing at least one of ethanol, acetone, methanol and ethyl acetate with water. More preferably, the extraction solvent includes any one of 70%-100% ethanol, 70%-100% acetone, 70%-100% methanol or ethyl acetate by volume fraction.

Further, the above-mentioned preparation method comprises:

Pulverize the stems and/or leaves of C. brussibilis, use an extraction solvent for leaching and then desolubilize to obtain a crude extract; disperse the crude extract in water, extract with an organic solvent, and take the organic layer to concentrate and desolubilize. Obtained the extract of Chamomile cabbage. Before the silica gel column chromatography, the impurities in the crude extract can be effectively removed by an extraction, so that the subsequent separation and purification efficiency is higher, and the obtained product has a higher purity. The organic solvent used for extraction can be ethyl acetate. Ultrasound can be used to assist in the extraction process, thereby improving the extraction efficiency.

Further, above-mentioned preparation method also comprises:

S1. Separation by silica gel column chromatography is adopted for the extract of Chamomile tea coriander, detected according to thin-layer chromatography, and eight parts of A, B, C, D, E, F, G and H are obtained by segmental collection;

S2. The B part collected in the S1 step is separated by a reversed-phase chromatographic column, detected according to thin-layer chromatography, and collected in stages to obtain four parts B1, B2, B3 and B4;

S3. The B2 part collected in the S2 step is separated by semi-preparative HPLC to obtain the broom-like isoesterin.

Preferably, in step S1, a mixed organic solvent system composed of solvent A and solvent B is used to carry out the gradient system, wherein solvent A includes any one of dichloromethane, chloroform and petroleum ether, and solvent B includes methanol, ethyl acetate and acetone. During gradient elution, the volume ratio of solvent A and solvent B was started from 19:1, and the ratio of solvent A was gradually reduced until finally pure solvent B was used for elution. Optionally, the volume ratio of solvent A and solvent B can be set to eight gradients of 19:1, 9:1, 8:2, 7:3, 6:4, 5:5, 1:2 and 0:1, to Corresponding to the eight parts A, B, C, D, E, F, G and H, the volume ratio of solvent A and solvent B was changed after each part was collected. Under the above separation conditions, not only the separation efficiency is high, but also the separation effect between the parts is better.

Further, in step S2, an aqueous methanol solution is used for gradient elution. Optionally, the reversed-phase material of the reversed-phase chromatography column is any one of C-8, C-18 or MCI. During gradient elution, 20 vol% methanol aqueous solution was used first, and then the methanol content was gradually increased until the final elution was performed with pure methanol.

Further, an aqueous methanol solution or an aqueous acetonitrile solution is used as the mobile phase in step S3. The volume fraction of the methanol aqueous solution is 75% to 95%, and the volume fraction of the acetonitrile aqueous solution is 60% to 90%. In order to improve the separation effect, gradient elution can also be used in the mobile phase, and the volume fraction of the mobile phase can be gradually increased. In addition, during elution, the flow rate of the mobile phase was 3mL/min, and a 9.4×250mm, 5μm Zorbax SB-C18 reversed-phase semi-preparative column could be selected as the stationary phase, and the detection wavelength of the UV detector was 212nm.

Further, the embodiments of the present invention also provide an application of the above-mentioned spicules in the preparation of immunosuppressive drugs. It has inhibitory effect on T and B lymphocyte proliferation, and has selective inhibitory effect on T cells, with an IC ₅₀ value of 0.68 μm.

Further, the embodiment of the present invention also provides a pharmaceutical composition, which comprises the above-mentioned succinyl isoesterin, and pharmaceutically acceptable excipients. It has better immunosuppressive activity. Has better application prospects.

The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

Example 1

The present embodiment provides a kind of brush-shaped isoesterin, and its structural formula is

Its preparation method includes:

S1. Crush the stems and leaves of 4 kg of Fructus esculenta into powder, add it to the extraction tank, add 20 L of 70% ethanol aqueous solution by volume, soak at 25°C for 3 days, soak 3 times in total, filter the combined filtrate, reduce Concentrate under pressure to obtain crude extract.

S2. Disperse the above crude extract in 2L of water, extract 3 times with 2L of ethyl acetate respectively, combine the ethyl acetate layers, and concentrate under reduced pressure to obtain 250g of 250g of tea coriander extract.

S3. Mix and dissolve the above extract of Coriander sp. with 600 mL of methanol, mix the sample with 400 g of 80-100 mesh silica gel, and pack the column with 4 kg of 200-300 mesh silica gel, and the volume ratios are 1:0, 19:1, 9 :1, 8:2, 3:2, 1:1, 1:2, 0:1 chloroform-methanol mixed organic solvent gradient elution, the gradient eluent was collected and concentrated under reduced pressure, monitored by TLC, the fractions were combined The same eluent yielded eight fractions A, B, C, D, E, F, G and H.

S4. Collect part B (78 g) in S3 and carry out reverse phase C-18 column chromatography separation, carry out gradient elution with methanol aqueous solution with a volume content of 40 to 90%, collect and concentrate each part of the eluent, and monitor by TLC , get four parts B1, B2, B3 and B4.

S5. Collect the B2 part (600 mg) in S4, and then use 90% methanol aqueous solution (0.5% formic acid) as the mobile phase, flow rate 3 mL/min, and use 9.4 × 250 mm, 5 μm Zorbax SB-C18 reverse phase half The preparative column is a stationary phase, and the detection wavelength of the ultraviolet detector is 212 nm. The chromatographic peaks of 18.0 min are collected respectively, and the required succinyl isoesterin is obtained by evaporating to dryness after accumulating for many times.

Example 2

The present embodiment provides a kind of brush-shaped isoesterin, and its structural formula is

Its preparation method includes:

S1. Crush the stems and leaves of 4 kg of Fructus esculenta into powder, add it to an extraction tank, add 20 L of methanol aqueous solution with a volume content of 70%, soak at 25° C. for 3 days, soak for 3 times in total, filter the combined filtrate, reduce Concentrate under pressure to obtain crude extract.

S2. Disperse the above crude extract in 2.5L of water, extract 3 times with 2.5L of ethyl acetate respectively, combine the ethyl acetate layers, and concentrate under reduced pressure to obtain 335g of the extract of Chamomile tea coriander.

S3. Mix and dissolve the above extract of Coriander sp. with 750 mL of methanol, mix the sample with 500 g of 80-100 mesh silica gel, and pack the column with 4.5 kg of 200-300 mesh silica gel. The volume ratios are 1:0, 19:1, 9:1, 8:2, 3:2, 1:1, 1:2, 0:1 petroleum ether-ethyl acetate mixed organic solvent gradient elution, the gradient eluent was collected and concentrated under reduced pressure, monitored by TLC , the eluates with the same components were combined to obtain eight fractions A, B, C, D, E, F, G and H.

S4. Collect part B (64 g) in S3 and carry out reverse-phase C-18 column chromatography separation, carry out gradient elution with methanol aqueous solution with a volume content of 40 to 95%, collect and concentrate each part of the eluent, and monitor by TLC , get four parts B1, B2, B3 and B4.

S5. Collect part B2 (710 mg) in S4, and then use 90% acetonitrile aqueous solution as mobile phase, flow rate 3 mL/min, and use 9.4×250 mm, 5 μm Zorbax SB-C18 reversed-phase semi-preparative column as stationary phase , the detection wavelength of the ultraviolet detector is 212nm, and the chromatographic peaks of 18.0min are collected respectively, and after being accumulated for many times, evaporated to dryness to obtain the required broom-like isoesterin.

Example 3

The present embodiment provides a kind of brush-shaped isoesterin, and its structural formula is

Its preparation method includes:

S1. Crush the stems and leaves of 4kg of Radix Papillae to powder, add it to the extraction tank, add 20L of acetone aqueous solution with a volume content of 70%, soak at 25°C for 3 days, soak 3 times in total, filter the combined filtrate, reduce Concentrate under pressure to obtain crude extract.

S2. Disperse the above crude extract in 2L of water, extract 3 times with 2L of ethyl acetate respectively, combine the ethyl acetate layers, and concentrate under reduced pressure to obtain 220g of the extract of broom-shaped tea coriander.

S3. Mix and dissolve the above-mentioned extract of coriander tea and coriander with 450 mL of methanol, mix the sample with 300 g of 80-100 mesh silica gel, and pack the column with 4 kg of 200-300 mesh silica gel, and the volume ratios are respectively 1:0, 19:1, and 9:0. 1, 8:2, 3:2, 1:1, 1:2, 0:1 chloroform-acetone mixed organic solvent gradient elution, the gradient eluent was collected and concentrated under reduced pressure, monitored by TLC, the combined components were the same The eluates obtained were A, B, C, D, E, F, G and H in eight fractions.

S4. Collect part B (75g) in S3 and carry out reverse-phase C-18 column chromatography separation, carry out gradient elution with methanol aqueous solution with a volume content of 30 to 95%, collect and concentrate each part of the eluent, and monitor by TLC , get four parts B1, B2, B3 and B4.

S5. Collect part B2 (600 mg) in S4, and then use 90% acetonitrile aqueous solution as mobile phase, flow rate 3 mL/min, and use 9.4×250 mm, 5 μm Zorbax SB-C18 reversed-phase semi-preparative column as stationary phase , the detection wavelength of the ultraviolet detector is 212nm, and the chromatographic peaks of 18.0min are collected respectively, and after being accumulated for many times, evaporated to dryness to obtain the required broom-like isoesterin.

Example 4

The present embodiment provides a tablet of a pharmaceutical composition, which takes the fucoidin prepared in Example 3, adds excipients in a ratio of 1:1 by weight to excipients, and granules and compresses the tablets.

Example 5

The present embodiment provides a capsule of a pharmaceutical composition, and takes the fucoidin prepared in Example 3 and prepares a capsule according to a conventional capsule preparation method.

Example 6

The present embodiment provides a tablet of a pharmaceutical composition, which is obtained by mixing the fucoidin prepared in Example 3 with an appropriate amount of starch, corn steep liquor and magnesium stearate, and compressing it into a tablet.

Example 7

The present embodiment provides a capsule of a pharmaceutical composition. Take 100 mg of the fucoidin prepared in Example 3, mix it with an appropriate amount of starch and magnesium stearate, sieve it, and uniformly mix it in a suitable container, and the obtained The mixture is filled into hard gelatin capsules.

Example 8

The present example provides a nasal spray of a pharmaceutical composition, which is prepared in Example 3 by taking 80 mg of Isoesterin, 8 mg of sodium oxide, 1 mg of EDTA, 1 mg of uranyl phosphate buffer solution (pH 6.5), and 1 mg of polyethoxylate. ether 10mg, redistilled water 2mL. Stir and mix until completely dissolved to obtain a solution. The solution is filtered on a sterile filter, bottled and divided into appropriate doses.

Example 9

The present embodiment provides a dripping pill of a pharmaceutical composition, which is 1 g of the strobilurin prepared in Example 9 and 9 g of polyethylene glycol 6000. After slightly thermally dissolving, add the polyethylene glycol melt in the recipe amount (60°C water bath heat preservation), stir and mix evenly until the ethanol evaporates, leave it to stand in a 60°C water bath and keep warm for 30 minutes. The above-mentioned mixed molten liquid with all the bubbles removed is transferred into the liquid storage cylinder. Under the temperature preservation condition of 80-85 °C, the dripping speed is controlled, and it is dropped into the condensed liquid drop by drop. After the condensation is complete, the condensed liquid is poured out and the dripping pills are collected. Drain and remove the condensate on the pellets with filter paper, then place in a silica gel desiccator or dry naturally.

Test Example 1

The compounds prepared in Examples 1-3 were white solids; the obtained products were identified by ¹ H NMR, ¹³ CNMR, ¹ H- ¹ H COSY, ROESY, HSQC, IR and HRESIMS respectively.

The HREIMS of the compound Isoesterin shows the compound quasi-molecular ion peak m/z 578.3941[M]+(calcd for C ₃₇ H ₅₄ O ₅ , 578.3971), combined with ¹ H and ¹³ C NMR spectra and two-dimensional nuclear magnetic resonance spectra (Fig. 1-Fig. 6), the NMR data were assigned, the results are shown in Table 1, the molecular formula was deduced to be C ₃₇ H ₅₄ O ₅ , and its chemical structure was determined.

Table 1 Assignment of ¹ H and ¹³ C NMR data of Isoesterin A (solvent is pyridine-d ₅ )

Combined with all the spectral data, it is confirmed that the chemical structural formula of the purified succinyl isoesterin is as follows:

Test Example 2

The immunosuppressive activity test was carried out by taking the fucoidin prepared in Examples 1-3, and the specific method was as follows:

Detection of cytotoxicity of compounds on mouse spleen lymphocytes by MTT method: Take the isolated Balb/c mouse spleen, prepare a single cell suspension, and inoculate 8×10 ⁵ /well of mouse spleen lymphocyte suspension in a 96-well plate , after adding different concentrations of screening drugs at the same time, the immunosuppressant cyclosporin A (cyclosporin A, CsA) was used as the positive control drug, and the corresponding vehicle control (cell control) and culture medium background control (blank control) were also set. The total volume is 200 μL. Incubate for 48 hours in a 37°C, 5% CO ₂ incubator. MTT solution at a concentration of 5 mg/mL was added 4 hours before the end of the incubation. When the culture was over, the supernatant was discarded, 200 μL of DMSO was added to each well to dissolve the purple crystals, and the OD value was measured at 570 nM on a microplate reader.

³ H-TdR incorporation method to detect the proliferation inhibitory activity of compounds on mouse spleen T| and B lymphocytes: mouse spleen lymphocyte suspension 5×10 ⁵ /well was inoculated in 96-well plate, and ConA (final concentration 1μg/well) was added. ml) or LPS (final concentration 10 μg/ml) and compounds of different concentrations, the immunosuppressant cyclosporin A (CsA) was used as the positive control drug, and the corresponding control wells without ConA, LPS cells and stimulation without Drug control wells with a total volume of 200 μL. Incubate for 48 hours in a 37°C, 5% CO ₂ incubator. Eight hours before the end of the culture, 25 μL of ³ H-thymidine nucleotides (10 μCi/ml) were added to each well. Continue to cultivate until the end of the experiment. The cells were collected on a glass fiber membrane with a cell harvester, and after adding scintillation fluid, the amount of ³ H-TdR incorporated into cellular DNA was read in a Beta counter (MicroBetaTrilux, PerkinElmer), and the cpm value represented the cell proliferation.

The experimental results found that the compound succinyl _isoesterin showed a certain immunosuppressive activity, and at a concentration without cytotoxic activity, it had an inhibitory effect on the proliferation of T lymphocytes. It has inhibitory effect on lymphocyte proliferation with IC ₅₀ of 13.81μm (CsA is the positive control), which is selective for the inhibitory effect on T lymphocyte proliferation.

To sum up, the embodiments of the present invention provide a broomlike isoesterin, a preparation method and application thereof, the broomlike isoesterin has a novel structure, has an inhibitory effect on the proliferation of T and B lymphocytes, and has an inhibitory effect on the T lymphocyte proliferation. The inhibitory effect on cell proliferation is selective and can be widely used in the preparation of immunosuppressive drugs. The preparation method of the broom-shaped isoesterin is not only simple to operate, but also can be extracted from the broom-shaped isoesterin with high efficiency and high purity from the broom-shaped isoesterin.

The embodiment of the present invention also provides a pharmaceutical composition, which comprises the above-mentioned succinyl isoesterin, and pharmaceutically acceptable excipients. It has good immunosuppressive activity and can be used as an immunosuppressant.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (3)

1. A preparation method of broom-like isoesterasum is characterized by comprising the following steps:

s1, smashing stems and/or leaves of rabdosia broom-like tea vegetables, leaching by using an extraction solvent, and then desolventizing to obtain a crude extraction solution;

s2, dispersing the crude extract in water, extracting with an organic solvent, and concentrating and desolventizing an organic layer to obtain a rabdosia broom-shaped rabdosia extract;

s3, separating the rabdosia broom extract by silica gel column chromatography, detecting by thin-layer chromatography, and collecting eight parts A, B, C, D, E, F, G and H in sections; in the step S3, a mixed organic solvent system consisting of a solvent A and a solvent B is adopted for gradient elution, wherein the solvent A comprises any one of dichloromethane, chloroform and petroleum ether, and the solvent B comprises any one of methanol, ethyl acetate and acetone; the volume ratio of the solvent a and the solvent B is set at 19;

s4, separating the part B collected in the step S3 by adopting a reverse phase chromatographic column, detecting according to a thin-layer chromatography, and collecting the parts B1, B2, B3 and B4 in a segmented manner; in the step S4, methanol aqueous solution is adopted for gradient elution, 20vol% methanol aqueous solution is adopted, and then the content of methanol is gradually increased until pure methanol is used for elution;

s5, separating the part B2 collected in the step S4 by adopting semi-preparative HPLC to obtain the broom-like isoesteridin; in the step S5, a methanol water solution or an acetonitrile water solution is used as a mobile phase; the volume fraction of the methanol aqueous solution is 75-95%, and the volume fraction of the acetonitrile aqueous solution is 60-90%;

wherein the broom-shaped isoesterol has a structural formula

。

2. The method of claim 1, wherein the extraction solvent comprises at least one of ethanol, acetone, methanol, ethyl acetate, and water.

3. The method of claim 2, wherein the extraction solvent comprises a solution of at least one of ethanol, acetone, methanol, and ethyl acetate mixed with water.

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