Industrialized method for preparing shikonin and shikonin polysaccharide in one step

Abstract

The application discloses an industrialized method for preparing one-step preparation of shikonin and shikonin polysaccharide, which comprises the following steps: (1) after the shikonin is crushed, the shikonin is mixed with an alkaline solution, countercurrent extraction is carried out, and filtration is carried out, so as to obtain a shikonin extract; (2) the shikonin extract is adjusted to neutral by an acid solution, and then concentrated by an ultrafiltration membrane, so as to obtain a concentrated solution; (3) short-chain alcohol aqueous solution is added to the concentrated solution for alcohol precipitation, and the filter residue and the filtrate are collected respectively; (4) the pH of the filtrate in the step (3) is adjusted to be acidic by an acidic solution, then a low-polarity organic solvent is added for extraction, then an alkaline solution is added for reaction, standing, layer separation, liquid separation, and then the acidic solution is added to the lower shikonin alkali aqueous solution, so as to precipitate shikonin; (5) the filter residue in the step (3) is dissolved by water, and then adsorbed by a macroporous resin, washed by water, eluted by ethanol, concentrated and dried, so as to obtain a shikonin polysaccharide product. The application has higher extraction efficiency, is more beneficial to industrialized application, realizes production of shikonin and shikonin polysaccharide products at a lower cost, and makes the effective components of shikonin be fully utilized.

Description

TECHNICAL FIELD

[0001] The present application relates to the preparation of shikonin and shikonin polysaccharide, in particular to an industrialized method for preparing shikonin and shikonin polysaccharide in one step. BACKGROUND

[0002] Radix Lithospermi is a commonly used traditional Chinese medicine, and its roots are used as medicine. It is named after its roots are purple. Radix Lithospermi was first recorded in Shennong Bencao Jing, and it has the effects of cooling blood, clearing heat and detoxifying, and relieving inflammation and rash, and is used for blood heat and toxin, rash purple black, measles, sore, eczema, hematemesis and hematuria, and water and fire burns. There are three species of Radix Lithospermi in the Pharmacopoeia of China, namely, Radix Lithospermi Xinjiangense (commonly known as soft Radix Lithospermi), Radix Lithospermi and Radix Lithospermi Neimengguense. Because the chemical components of Radix Lithospermi and Radix Lithospermi Xinjiangense are very similar, Radix Lithospermi Xinjiangense is listed as the correct product in the 1995 edition of the Pharmacopoeia of the People's Republic of China, and Radix Lithospermi Xinjiangense is listed as the first in books such as Chinese Materia Medica, National Chinese Medicine Compilation and Chinese Medicine Dictionary. Pharmacological studies have shown that Radix Lithospermi contains a variety of physiologically active components, which have the effects of antibacterial, antioxidant, anticancer, antifertility, antithyroid, hypoglycemic, liver protection, immune enhancement, antiviral and antitumor.

[0003] Shikonin in Radix Lithospermi has the effects of anticancer, anti-inflammatory and antibacterial. Shikonin polysaccharide has the effect of immune regulation. At present, shikonin is mainly produced by solvent method, and the total naphthoquinone content in the product is less than 30%, but the solvent residue is high. Supercritical CO2 extraction has also been industrialized, but it is focused on the extraction of Radix Lithospermi oil, not shikonin. The shikonin polysaccharide in the filter residue after extraction of shikonin often cannot be effectively utilized.

[0004] There are a few existing technologies that can obtain shikonin and shikonin polysaccharide from Radix Lithospermi, but they are all extracted in two steps, that is, first, the shikonin components are extracted by using organic solvents or supercritical extraction method, and then the polysaccharides are extracted by using water or ethanol as solvent. For example, CN101721452A provides a new process for improving the utilization rate of Radix Lithospermi, which uses traditional Chinese medicine Radix Lithospermi as raw material, and after crushing treatment to obtain Radix Lithospermi powder, supercritical CO2 is used as extraction solvent, and the corresponding extraction temperature, pressure, CO2 flow and extraction time are controlled to separate out Radix Lithospermi extract containing fat-soluble naphthoquinone components. After the fat-soluble Radix Lithospermi extract is separated out, the remaining Radix Lithospermi powder residue after supercritical extraction is used as raw material, water or dilute ethanol is used as raw material, and the extraction temperature, time, solvent ratio and extraction times are controlled to extract the water-soluble polysaccharide components therein; the extraction liquid is concentrated, high-concentration ethanol solution is added in proportion, it is left to stand, and the precipitate is separated by filtration to obtain shikonin polysaccharide. The above method is complicated, the utilization rate of effective components is low, the equipment cost is high, and the production cost is high. SUMMARY

[0005] The application provides an industrialized method for preparing one-step preparation of shikonin and shikonin polysaccharide, which has high extraction efficiency and is more beneficial to industrial application, and realizes low-cost production of shikonin and shikonin polysaccharide.

[0006] The technical scheme provided by the application is as follows:

[0007] (1) After the shikonin is crushed, the shikonin is mixed with an alkaline solution, countercurrent extraction is carried out, filtration is carried out, and shikonin extract is obtained;

[0008] (2) The shikonin extract is adjusted to neutral by an acid solution, and then concentrated by an ultrafiltration membrane to remove salt and small-molecule substances such as monosaccharides, and a concentrated solution is obtained;

[0009] (3) The concentrated solution is added with a short-chain alcohol aqueous solution for alcohol precipitation, filtration is carried out, and the filter residue and the filtrate are collected respectively;

[0010] (4) The pH of the filtrate in step (3) is adjusted to be acidic by an acidic solution, a low-polarity organic solvent is added for extraction, an organic solvent extraction solution is obtained, then an alkaline solution is added for reaction, standing is carried out, layer separation is carried out, and the acidic solution is added to the lower shikonin alkali aqueous solution, shikonin is precipitated, filtration is carried out, and drying is carried out to obtain a shikonin product;

[0011] (5) The filter residue in step (3) is dissolved in water and then adsorbed by a macroporous resin, water washing is carried out, ethanol elution is carried out, and concentration and drying are carried out to obtain a shikonin polysaccharide product.

[0012] In step (1), the alkaline solution is one or a mixture of two of a sodium hydroxide solution, a potassium hydroxide solution, a sodium carbonate solution and an ammonia solution, the concentration is 0.01-1 mol / L, and the addition ratio is 6-50 times the weight of the shikonin raw material.

[0013] In step (1), the countercurrent extraction time is 2-3 hours, and the temperature is 20-30 DEG C.

[0014] In step (1), the obtained countercurrent extraction liquid is subjected to solid-liquid separation, the filtrate is collected, a shikonin crude extract is obtained, and the shikonin crude extract is subjected to ceramic membrane treatment to remove suspended matters, and a clear shikonin extract is obtained.

[0015] In step (2), the acid solution is sulfuric acid, hydrochloric acid, formic acid or acetic acid, the pH adjustment range is 6.0-8.0, the molecular weight of the ultrafiltration membrane is 100-200 daltons, and the concentration temperature is controlled to be 10-40 DEG C.

[0016] In step (3), the short-chain alcohol is one or any combination of several of methanol and ethanol, the volume fraction of the short-chain alcohol is 85-95%, the addition amount is 5-20 times the volume of the concentrated solution, and the filtration is plate-and-frame filtration or cloth bag centrifugation.

[0017] In step (4), the pH of the filtrate is adjusted to 1.0-3.0, the low-level organic solvent is one or a mixture of petroleum ether, n-hexane and n-heptane, the concentration is 0.1-1 mol / L, the volume of the low-level organic solvent added is 20-100%, the stirring time is 1-8 hours, and the drying temperature is ≤60℃.

[0018] In step (5), the macroporous resin is one or a mixture of AB-8, D101, T8100, NKA-9 and LS-315, the loading speed is 1-5 BV / h, and the concentration of the ethanol solution is 60-90%.

[0019] The present application has the following advantages:

[0020] 1. The present application uses alkaline water to extract shikonin and polysaccharide in one step, instead of the traditional step-by-step extraction process, which is more energy-saving, has higher extraction efficiency, and is more conducive to industrial application, thereby achieving the production of shikonin and shikonin polysaccharide products at a lower cost, and fully utilizing the effective components of shikonin. Finally, the purity of shikonin is ≥95.0%, and the yield is ≥2.8%; the purity of shikonin polysaccharide is ≥68.0%, and the yield is ≥5.5%.

[0021] 2. The naphthoquinone component in shikonin is a fat-soluble component. The present application uses the principle that shikonin and its derivatives can form salts with alkali, and uses an alkaline solution to react with shikonin and its derivatives to form a salt, thereby improving the solubility and facilitating the extraction of naphthoquinone components. At the same time, the alkaline solution can destroy the cell wall of the plant, thereby promoting the release of polysaccharide components and facilitating the dissolution of polysaccharide.

[0022] 3. In step (2), the pH of the shikonin extract is adjusted to neutral, and the shikonin extract is concentrated by ultrafiltration membrane. This can effectively remove salts generated by acid-base neutralization and small molecules such as monosaccharides, which is conducive to the separation of shikonin and polysaccharide. Shikonin can be dissolved in high-concentration short-chain alcohol, while polysaccharide will precipitate in high-concentration short-chain alcohol due to their different properties.

[0023] 4. Shikonin is insoluble in acidic short-chain alcohol. The low-level organic solvent used in the present application is immiscible with the short-chain alcohol containing water, and can extract shikonin from the acidic short-chain alcohol. This allows shikonin to enter the low-level organic solvent and remove some strong polar impurities. Then, dilute alkali is added to make shikonin enter the alkaline solution with higher solubility, and the shikonin-containing alkaline solution can be precipitated by adding acid. The addition of alkali not only removes impurities in the organic solvent, but also hydrolyzes various shikonin derivatives into shikonin, thereby improving the yield of shikonin.

[0024] 5. The present application selects a suitable macroporous resin, which can remove the protein of the pigment through ion exchange and physical adsorption, thereby improving the purity of polysaccharide.

[0025] 6. The shikonin structure has active groups such as phenolic hydroxyl, ester bond and double bond, and is heat unstable. In order to avoid degradation of shikonin, the present application adopts low-temperature process, including alkali water extraction at normal temperature and organic membrane concentration at low temperature. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The flow chart of the industrialized method for preparing shikonin and shikonin polysaccharide in one step according to the present application. DETAILED DESCRIPTION

[0027] The present application will be further described in combination with specific examples.

[0028] Example 1

[0029] (1) 500 kg of shikonin raw material was crushed and passed through a 40-mesh sieve to obtain shikonin powder, which was put into a countercurrent extraction device and added with 8000 L of 0.1 mol / L NaOH aqueous solution to perform countercurrent extraction at 20-30℃ for 2 hours. The extraction liquid was subjected to solid-liquid separation by horizontal screw centrifugation, and the filtrate was collected to obtain shikonin crude extract. The shikonin crude extract was subjected to ceramic membrane to remove suspended solids, and the clear shikonin extract was obtained.

[0030] (2) The shikonin extract was adjusted to pH 6.88 by hydrochloric acid, and then subjected to concentration and impurity removal by 200 dalton ultrafiltration membrane. The concentration temperature was controlled at 25-30℃, and the volume of the concentrated liquid was 280 L.

[0031] (3) 2000 L of 90% ethanol was added to the concentrated liquid to stir for 1 hour, and then plate and frame filtration was performed to collect the filtrate and the filter residue, respectively.

[0032] (4) Preparation of shikonin: the pH of the above filtrate was adjusted to 1.88 by hydrochloric acid solution, and then 3000 L of n-heptane was added to stir for 2 hours to obtain n-heptane extract. Then, 1000 L of 0.5 mol / L NaOH was added to the n-heptane extract to react for 3 hours, and then the mixture was allowed to stand for 1 hour. After the mixture was separated into two layers, the lower layer of shikonin NaOH solution was collected. Hydrochloric acid solution was added to the lower layer of shikonin alkali aqueous solution to adjust the pH to 1.52 to precipitate shikonin. The crystal was centrifuged and washed with water until the pH of the washing liquid was ≥6. The crystal was vacuum dried at 55℃, and finally 14.22 kg of shikonin product was obtained, with a purity of 96.88% detected by HPLC.

[0033] (5) Preparation of shikonin polysaccharide: the above filter residue was dissolved by stirring with 2000 L of water, and the dissolved liquid was subjected to AB-8 macroporous resin adsorption. The loading speed was 2 BV / h, and 70% ethanol was used for elution. The eluent was concentrated and dried, and finally 29.9 kg of shikonin polysaccharide product was obtained, with a purity of 70.2% detected by ultraviolet detection.

[0034] Example 2

[0035] (1) Take 500 kg of Lithospermum erythrorhizon raw material, crush it, pass it through a 40-mesh sieve to obtain Lithospermum erythrorhizon powder, put it into a countercurrent extraction device, add 8000 L of 0.1 mol / L ammonia water solution, and extract countercurrently at 30℃ for 2 hours. The extract is centrifuged by horizontal screw centrifugation to separate solids and liquids, and the filtrate is collected to obtain crude Lithospermum erythrorhizon extract. The crude Lithospermum erythrorhizon extract is passed through a ceramic membrane to remove suspended solids to obtain clear Lithospermum erythrorhizon extract.

[0036] (2) The pH of the purple gromwell extract was adjusted to 7.10 with hydrochloric acid, and then concentrated and purified by passing it through a 200 Dalton ultrafiltration membrane to remove small molecules such as salts and monosaccharides. The concentration temperature was controlled at 25-30℃ and the volume of the concentrated liquid was 320L.

[0037] (3) Add 2500L of 95% methanol to the concentrate and stir for 1.5 hours. Filter the solution using a plate and frame filter and collect the filtrate and residue separately.

[0038] (4) Preparation of Shikonin: The pH of the above filtrate was adjusted to 1.50 with acetic acid solution, and then 4000 L of petroleum ether was added and stirred for 2 hours to obtain petroleum ether extract. Then 2000 L of 0.5 mol / L KOH was added to the petroleum ether extract and reacted for 3 hours. After standing for 1 hour, the layers were separated, and the lower layer of shikonin KOH solution was collected. Then sulfuric acid solution was added to the lower layer of shikonin alkaline aqueous solution to adjust the pH to 1.52 to precipitate shikonin. The crystals were centrifuged and washed with water until the pH of the washing solution was ≥6. The crystals were dried under vacuum at 55℃ to finally obtain 14.88 kg of shikonin product. The purity was 95.22% as determined by HPLC.

[0039] (5) Preparation of Lithospermum erythrorhizon polysaccharide: The above filter residue was dissolved in 1500L of water by stirring. The solution was then introduced into LS-315 macroporous resin for adsorption at a loading rate of 3.5 BV / h. The solution was eluted with 70% ethanol, and the eluent was concentrated and dried to obtain 32.70 kg of Lithospermum erythrorhizon polysaccharide product. The purity of the Lithospermum erythrorhizon polysaccharide was 68.89% by UV measurement.

[0040] Example 3

[0041] (1) Take 500 kg of Lithospermum erythrorhizon raw material, crush it, pass it through a 40-mesh sieve to obtain Lithospermum erythrorhizon powder, put it into a countercurrent extraction device, add 10000 L of 0.1 mol / L KOH aqueous solution, and extract countercurrently at 25℃ for 2.5 hours. The extract is centrifuged by horizontal screw centrifugation to separate solids and liquids, and the filtrate is collected to obtain crude Lithospermum erythrorhizon extract. The crude Lithospermum erythrorhizon extract is passed through a ceramic membrane to remove suspended solids to obtain clear Lithospermum erythrorhizon extract.

[0042] (2) The pH of the purple gromwell extract was adjusted to 7.50 with hydrochloric acid, and then concentrated and purified by passing it through a 100 Dalton ultrafiltration membrane to remove small molecules such as salts and monosaccharides. The concentration temperature was controlled at 30℃ and the volume of the concentrated liquid was 350L.

[0043] (3) Concentrate 3200 L of 85% ethanol and stir for 1 hour. Filter using a plate frame and collect the filtrate and residue separately.

[0044] (4) Preparation of shikonin: Adjust the pH of the filtrate to 1.20 using acetic acid solution. Add 2800 L of n-hexane and stir for 2 hours to obtain n-heptane extract. Then add 1800 L of 0.5 mol / L NaOH solution to the n-heptane extract and react for 5 hours. Let it stand for 1 hour, separate the layers, collect the lower layer of shikonin NaOH solution. Add formic acid solution to the lower layer of shikonin alkaline solution to adjust the pH to 2.10 to precipitate shikonin. Centrifuge, wash the crystals with water until the pH of the washing solution is ≥6. Dry the crystals at 55°C under vacuum. Finally, 14.35 kg of shikonin product is obtained with a purity of 97.22% as determined by HPLC.

[0045] (5) Preparation of shikonin polysaccharide: Dissolve the residue in 1000 L of water and stir. Pass the solution through D101 macroporous resin adsorption at a speed of 4 BV / h. Elute with 70% ethanol, concentrate and dry the eluate. Finally, 29.30 kg of shikonin polysaccharide product is obtained with a purity of 69.80% as determined by UV.

Claims

1. An industrial method for one-step preparation of shikonin and shikonin polysaccharides, characterized in that, Includes the following steps: (1) Take the powdered Lithospermum erythrorhizon and mix it with an alkaline solution. Extract it countercurrently and filter it to obtain Lithospermum erythrorhizon extract. (2) The purple gromwell extract was adjusted to neutral with an acid solution and then concentrated through an ultrafiltration membrane to remove salts and small monosaccharide molecules, resulting in a concentrated solution; (3) Add a short-chain alcohol aqueous solution to the concentrate for alcohol precipitation, filter, and collect the filter residue and filtrate respectively; (4) Adjust the pH of the filtrate in step (3) to acidic with an acidic solution, then add a low-polarity organic solvent for extraction to obtain an organic solvent extract, then add an alkaline solution for reaction, let stand, separate the layers, separate the liquids, then add the acidic solution to the lower layer of shikonin alkaline aqueous solution to precipitate shikonin, filter, and dry to obtain the shikonin product. (5) Dissolve the filter residue in step (3) with water and then adsorb it into macroporous resin. Wash with water, elute with ethanol, concentrate and dry to obtain the Lithospermum erythrorhizon polysaccharide product.

2. The industrial method for preparing shikonin and shikonin polysaccharides in one step according to claim 1, characterized in that, In step (1), the alkaline solution is one or a mixture of two of the following: sodium hydroxide, potassium hydroxide, sodium carbonate and ammonia water, with a concentration of 0.01-1 mol / L and an addition ratio of 6-50 times the weight of the Lithospermum erythrorhizon raw material.

3. The industrial method for preparing shikonin and shikonin polysaccharides in one step according to claim 1, characterized in that, In step (1), the countercurrent extraction time is 2-3 hours and the temperature is 20-30℃.

4. The industrial method for preparing shikonin and shikonin polysaccharides in one step according to claim 1, characterized in that, In step (1), the countercurrent extract obtained is subjected to solid-liquid separation, the filtrate is collected to obtain crude extract of Lithospermum erythrorhizon, and the crude extract is then subjected to a ceramic membrane to remove suspended solids to obtain clear Lithospermum erythrorhizon extract.

5. The industrial method for preparing shikonin and shikonin polysaccharides in one step according to claim 1, characterized in that, In step (2), the acid solution is sulfuric acid, hydrochloric acid, formic acid or acetic acid, the pH adjustment range is 6.0-8.0, the molecular weight of the ultrafiltration membrane is 100-200 Daltons, and the concentration temperature is controlled at 10-40℃.

6. The industrial method for preparing shikonin and shikonin polysaccharides in one step according to claim 1, characterized in that, In step (3), the short-chain alcohol is one or more of methanol and ethanol in any combination, the volume fraction of the short-chain alcohol is 85-95%, the amount added is 5-20 times the volume of the concentrated liquid, and the filtration is plate and frame filtration or bag centrifugation.

7. The industrial method for preparing shikonin and shikonin polysaccharides in one step according to claim 1, characterized in that, In step (4), the pH of the filtrate is adjusted to a range of 1.0-3.0, the low-grade organic solvent is one or a mixture of petroleum ether, n-hexane and n-heptane, with a concentration of 0.1-1 mol / L, and the added volume is 20-100% of the low-grade organic solvent. The stirring time is 1-8 hours, and the drying temperature is ≤60℃.

8. The industrial method for preparing shikonin and shikonin polysaccharides in one step according to claim 1, characterized in that, In step (5), the macroporous resin is one or more of AB-8, D101, T8100, NKA-9 and LS-315, the loading speed is 1-5 BV / h, and the ethanol solution concentration is 60-90%.

Images