Prunella vulgaris polysaccharide as well as extraction method and application thereof

Abstract

The invention relates to the technical field of spica prunellae polysaccharide extraction, and discloses a spica prunellae polysaccharide and an extraction method and application thereof, the method comprises the following steps: (1) crushing spica prunellae clusters, sieving, and degreasing with an organic solvent; (2) mixing the degreased prunella vulgaris powder with water, stirring at 20-28 DEG C, and repeating for 2-4 times to obtain first medicinal material residues; (3) stirring the first medicinal material residues in hot water at 75-85 DEG C, repeating for 1-3 times, and stirring the obtained second medicinal material residues with acidified water; and (4) extracting the third medicinal material residue by using an ammonium oxalate solution, repeatedly extracting for 1-3 times, and decoloring and deproteinizing the collected extracting solution. According to the method, cold water, hot water and acidified water are sequentially adopted for pre-treatment of selfheal, then an ammonium oxalate solution is utilized for polysaccharide extraction, the selfheal polysaccharide is obtained, the purity and the yield of the selfheal polysaccharide are high, and the selfheal polysaccharide is identified as pectic polysaccharide; the prunella vulgaris pectic polysaccharide has good prevention and treatment effects on alcoholic liver injury.

Description

Technical Field

[0001] The invention relates to the technical field of extracting Prunella vulgaris polysaccharides, and in particular to Prunella vulgaris polysaccharides and an extraction method and application thereof. Background Art

[0002] The Chinese herbal medicine Prunella vulgaris is the dried fruit spike of the Lamiaceae plant Prunella vulgaris. It tastes pungent and bitter, has the effects of clearing away heat and purging fire, improving eyesight and reducing swelling, and is used to treat symptoms such as dizziness, breast pain, etc. Prunella vulgaris has a complex composition, including flavonoids, triterpenes, phenolic acids, volatile oils, etc., among which the polysaccharide content accounts for about 10% of the Prunella vulgaris medicinal material. At present, most of the research on it is focused on triterpenes and phenolic acid components, and there are few reports on polysaccharide components. However, modern pharmacological studies have shown that Prunella vulgaris polysaccharides have multiple biological activities such as anti-inflammatory, antioxidant, antiviral, and anti-tumor.

[0003] The extraction methods of polysaccharides include hot water extraction, ultrasonic extraction, heating reflux, ultrasonic-assisted extraction, enzymatic-assisted extraction, etc. At present, the extraction of Prunella vulgaris polysaccharides is usually carried out by hot water extraction, which is simple to operate and has stable conditions. However, the structure of Prunella vulgaris polysaccharides obtained by hot water extraction is complicated, the purity is low, and the activity is unclear, which limits its application. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems in the prior art that the extracted selfheal polysaccharides have a complex structure, low purity, low activity, are difficult to apply or require complex processing before application, and to provide selfheal polysaccharides and an extraction method and application thereof.

[0005] In order to achieve the above object, the present invention provides a method for extracting Prunella vulgaris polysaccharides, which comprises the following steps:

[0006] (1) crushing and sieving the fruit ears of Prunella vulgaris, and then performing a degreasing treatment with an organic solvent;

[0007] (2) mixing the defatted Prunella vulgaris powder obtained in step (1) with water, stirring and mixing at 20 to 28° C. for 1 to 4 hours, repeating 2 to 4 times to obtain a first medicinal material residue;

[0008] (3) stirring and mixing the first medicinal material residue in hot water at 75 to 85° C. for 1 to 4 hours, repeating 1 to 3 times to obtain a second medicinal material residue, and then stirring and mixing with acidified water for 1 to 4 hours to obtain a third medicinal material residue;

[0009] (4) extracting polysaccharides from the third medicinal material residue with an ammonium oxalate solution, repeating the extraction 1 to 3 times, and decolorizing and deproteinizing the collected extract to obtain Prunella vulgaris polysaccharides.

[0010] Preferably, in step (4), the concentration of the ammonium oxalate solution is 0.5-1%.

[0011] Preferably, in step (4), the extraction time is 5 to 7 hours.

[0012] Preferably, in step (4), the extraction is repeated 1 to 2 times.

[0013] Preferably, in step (1), the sieving is through a 30-50 mesh sieve;

[0014] Preferably, in step (1), the organic solvent is anhydrous ethanol and ethyl acetate.

[0015] Preferably, in step (2), the ratio of the herbaceous spring grass powder to water is 50-150 g:1 L.

[0016] Preferably, in step (3), the pH of the acidified water is 1.5 to 2.5.

[0017] Preferably, during the stirring with acidified water, the stirring and mixing temperature is 40-60°C.

[0018] The second aspect of the present invention provides Prunella vulgaris polysaccharide extracted by the method as described above.

[0019] Preferably, the Prunella vulgaris polysaccharide comprises at least one of the following polysaccharides:

[0020] Prunella vulgaris polysaccharide OK1: mainly composed of mannose, glucuronic acid, rhamnose, galacturonic acid, galactose, xylose and arabinose, and the content of galacturonic acid is more than 80 mol%;

[0021] Prunella vulgaris polysaccharide OK2: mainly composed of rhamnose, galacturonic acid, galactose, xylose and arabinose, and the content of galacturonic acid is more than 80 mol%.

[0022] Preferably, the Prunella Vulgaris polysaccharide is an HG-type pectin polysaccharide with a galacturonic acid structure linked by α-1,4 glycosidic bonds.

[0023] Preferably, the molecular weight of the Prunella Vulgaris polysaccharide OK2 is 100-120 KDa.

[0024] The third aspect of the present invention provides the use of the above-mentioned Prunella Vulgaris polysaccharide in the preparation of a medicament for preventing and treating acute alcoholic liver injury.

[0025] In the extraction method provided by the present invention, cold water, hot water and acidified water are sequentially used for pretreatment to destroy the cell wall step by step, the obtained medicinal residue is then extracted with an ammonium oxalate solution, and the extract is subjected to conventional decolorization / deproteinization treatment to obtain the Prunella vulgaris polysaccharide; the extraction method can remove most of the heteropolysaccharides through the design of the pretreatment steps, so that the obtained extract does not need to be separated by a chromatographic column to obtain the Prunella vulgaris polysaccharide with high purity, the high-purity Prunella vulgaris polysaccharide can be directly used and used for molecular weight and structure characterization tests, and the operation is simple; the Prunella vulgaris polysaccharide has a high yield, and is identified as a pectin polysaccharide; and at the same time, animal experiments have proved that the Prunella vulgaris pectin polysaccharide has a good effect on preventing and treating alcoholic liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the HPSEC result diagram of Prunella vulgaris polysaccharide OK2 prepared in Example 1 of the present invention;

[0027] Figure 2 are SEM images of Prunella vulgaris polysaccharide prepared in Example 1 of the present invention at different magnifications;

[0028] Figure 3 The test results of enzyme and inflammation indicators in the serum of each group of mice after the experiment in Test Example 3 of the present invention are as follows;

[0029] Figure 4 The H&E staining results of the colon of each group of mice after the experiment in Test Example 3 of the present invention;

[0030] Figure 5 This is the oil red staining result of the liver of each group of mice after the experiment in Test Example 3 of the present invention. DETAILED DESCRIPTION

[0031] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0032] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0033] As a plant with high medicinal activity, most of the current research on Prunella vulgaris is focused on triterpenoids and phenolic acid components, and there are few reports on polysaccharide components. However, modern pharmacology has gradually discovered that Prunella vulgaris polysaccharides have multiple biological activities such as anti-tumor, antibacterial, antiviral, and immunomodulatory.

[0034] The extraction methods of polysaccharides include hot water extraction, ultrasonic extraction, heating reflux, ultrasonic-assisted extraction, enzymatic-assisted extraction, etc. At present, hot water extraction is commonly used for Prunella vulgaris polysaccharides, which is simple to operate and has stable conditions.

[0035] It is worth noting that the biological activity of polysaccharides is closely related to their structure. However, the structure-activity relationship of the structure and activity of Prunella vulgaris polysaccharides is not clear at present, which brings difficulties to the discovery of highly active polysaccharides from Prunella vulgaris. High-purity polysaccharides are the primary condition for characterizing their chemical structure. Currently, polysaccharides obtained by hot water extraction have complex structures, low purity and activity, and although high-purity polysaccharides can be obtained by multiple purifications with chromatographic columns, the operation is complicated and the yield is extremely low. In view of this, the present invention proposes a method for extracting Prunella vulgaris polysaccharides, which comprises the following steps:

[0036] (1) crushing and sieving the fruit ears of Prunella vulgaris, and then performing a degreasing treatment with an organic solvent;

[0037] (2) mixing the defatted Prunella vulgaris powder obtained in step (1) with water, stirring and mixing at 20 to 28° C. for 1 to 4 hours, repeating 2 to 4 times to obtain a first medicinal material residue;

[0038] (3) stirring and mixing the first medicinal material residue in hot water at 75 to 85° C. for 1 to 4 hours, repeating 1 to 3 times to obtain a second medicinal material residue, and then stirring and mixing with acidified water for 1 to 4 hours to obtain a third medicinal material residue;

[0039] (4) extracting polysaccharides from the third medicinal material residue with an ammonium oxalate solution, repeating the extraction 1 to 3 times, and decolorizing and deproteinizing the collected extract to obtain Prunella vulgaris polysaccharides.

[0040] At present, the conventional extraction methods of Prunella vulgaris polysaccharides are hot water extraction and ethanol precipitation. The obtained polysaccharides have complex structures, unclear activities, and are difficult to apply. Even if multiple chromatographic column separations are further performed to obtain uniform polysaccharides, the processing flow is complicated, the yield is extremely low, and it is difficult to transform and apply. In the present invention, cold water, hot water and acidified water are used in turn to pre-treat Prunella vulgaris materials, and the cell walls are destroyed step by step. The obtained medicinal residues are then extracted with ammonium oxalate solution for polysaccharide extraction, which can remove most of the heteropolysaccharides, and finally Prunella vulgaris polysaccharides with high purity and yield are obtained, which is convenient for application and detection. It is identified as pectin polysaccharide; the Prunella vulgaris pectin polysaccharide has a good preventive and therapeutic effect on alcoholic liver injury.

[0041] In a preferred embodiment, in step (1), the sieving is through a 30-50 mesh sieve, which facilitates subsequent extraction.

[0042] In some embodiments, in step (1), the organic solvent is anhydrous ethanol and ethyl acetate. In specific implementation, the sieved powder is treated with anhydrous ethanol and ethyl acetate respectively using a Soxhlet extractor to achieve the effect of depigmentation and degreasing.

[0043] In the method of the present invention, in step (2), the defatted Prunella Vulgaris powder is stirred and mixed in water (cold water) at 20 to 28°C. Specifically, the stirring and mixing temperature can be, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C or 28°C.

[0044] In a preferred embodiment, in step (2), the treatment is repeated three times, so that the heteropolysaccharides can be removed better.

[0045] In a preferred embodiment, in step (2), the ratio of the herbaceous earth powder to water is 50-150 g:1L, specifically, for example, 50 g:1L, 60 g:1L, 70 g:1L, 80 g:1L, 100 g:1L, 120 g:1L, 130 g:1L or 150 g:1L.

[0046] In a preferred embodiment, step (2) comprises: taking 50-150 g of defatted Prunella vulgaris powder into a container, adding 1 L of distilled water, stirring the resulting mixture in a mechanical stirrer at 20-28° C. for 1-4 h, centrifuging the mixture, and performing the above treatment on the residue of the medicinal material again, and repeating the process 3 times.

[0047] In a preferred embodiment, in step (3), the pH of the acidified water is 1.5 to 2.5. More preferably, the pH of the acidified water is 2.

[0048] In the method of the present invention, in step (3), the second medicinal material residue obtained by hot water treatment is stirred and mixed with acidified water at a temperature of 40 to 60°C. Specifically, the extraction temperature can be, for example, 40°C, 42°C, 45°C, 50°C, 52°C, 55°C or 60°C.

[0049] In a preferred embodiment, step (3) comprises: stirring and mixing the first medicinal material residue obtained in step (2) in hot water at 75 to 85° C. for 1 to 4 hours, centrifuging the obtained mixture, and subjecting the residue to the above treatment, repeating the process twice to obtain a second medicinal material residue; and then stirring and mixing the second medicinal material residue with acidified water having a pH of 1.5 to 2.5 at 40 to 60° C. for 1 to 4 hours.

[0050] In a preferred embodiment, in step (4), the concentration of the ammonium oxalate solution is 0.5-1%. Specifically, the ammonium oxalate solution with a concentration of 0.5-1% means that every 100 mL of the ammonium oxalate solution contains 0.5-1 g of ammonium oxalate.

[0051] In some embodiments, in step (4), the extraction time is 5 to 7 hours.

[0052] In some embodiments, the ratio of the amount of the Prunella Vulgaris powder in step (2) to the amount of the ammonium oxalate solution used in each extraction in step (4) is 50-150 g:1 L.

[0053] In some embodiments, the present invention does not limit the specific method of decolorization / deproteinization in step (4), and conventional decolorization / deproteinization methods in the art can be used. Specifically, for example, macroporous resin can be used for decolorization, and sevage reagent can be used for deproteinization.

[0054] In the method described in the present invention, in step (4), the yield of polysaccharides obtained by the first extraction and the second extraction using ammonium oxalate solution is relatively high. Therefore, based on comprehensive considerations of cost and yield, in a preferred embodiment, in step (4), the extraction is repeated 1 to 2 times.

[0055] Further preferably, step (4) comprises: extracting polysaccharides from the third medicinal material residue obtained in step (3) using an ammonium oxalate solution, repeating the extraction twice, and decolorizing and deproteinizing the obtained extracts to obtain Prunella vulgaris polysaccharide OK1 and Prunella vulgaris polysaccharide OK2, respectively.

[0056] The present invention also provides Prunella vulgaris polysaccharide extracted by the method as described above.

[0057] In some embodiments, the Prunella vulgaris polysaccharide comprises at least one of the following polysaccharides:

[0058] Prunella vulgaris polysaccharide OK1: mainly composed of mannose, glucuronic acid, rhamnose, galacturonic acid, galactose, xylose and arabinose, and the content of galacturonic acid is more than 80 mol%;

[0059] Prunella vulgaris polysaccharide OK2: mainly composed of rhamnose, galacturonic acid, galactose, xylose and arabinose, and the content of galacturonic acid is more than 80 mol%.

[0060] Specifically, the Prunella vulgaris polysaccharide OK1 is a polysaccharide obtained by decolorizing / deproteinizing the extract OK1 obtained by the first extraction using an ammonium oxalate solution, and the Prunella vulgaris polysaccharide OK2 is a polysaccharide obtained by decolorizing / deproteinizing the extract OK2 obtained by the second extraction using an ammonium oxalate solution.

[0061] In the present invention, the Prunella Vulgaris polysaccharide OK1 and Prunella Vulgaris polysaccharide OK2 have a high galacturonic acid content and are pectin polysaccharides. They not only have many activities in medicine, but also have greater value in food.

[0062] In the present invention, more specifically, based on the total molar amount of the Prunella Vulgaris OK1, the content of mannose is 1-1.5 mol%, the content of glucuronic acid is 0.1-1 mol%, the content of rhamnose is 1.5-2 mol%, the content of galacturonic acid is 85-90 mol%, the content of galactose is 1-2 mol%, the content of xylose is 4-5 mol%, and the content of arabinose is 2.4-3.5 mol%.

[0063] In the present invention, more specifically, based on the total molar amount of the Prunella Vulgaris OK2, the content of rhamnose is 2-3 mol%, the content of galacturonic acid is 85-88 mol%, the content of galactose is 1-2 mol%, the content of xylose is 6-7 mol%, and the content of arabinose is 3-4 mol%.

[0064] In the present invention, the Prunella Vulgaris polysaccharide has high yield, high purity, good antioxidant activity and good liver damage repair effect.

[0065] In the present invention, the Prunella vulgaris polysaccharide is an HG-type pectin polysaccharide with a galacturonic acid structure linked by α-1,4 glycosidic bonds. In the specific implementation, the Prunella vulgaris polysaccharide OK2 obtained by the second extraction with an ammonium oxalate solution was subjected to structural characterization, and the results showed that the Prunella vulgaris polysaccharide OK2 is an HG-type pectin polysaccharide with a galacturonic acid structure linked by α-1,4 glycosidic bonds.

[0066] The molecular weight of Prunella vulgaris polysaccharide OK2 was measured, and the result showed that the molecular weight of Prunella vulgaris polysaccharide OK2 was 100-120 KDa. In a specific embodiment, the molecular weight of Prunella vulgaris polysaccharide OK2 was 102.8 KDa.

[0067] The present invention also proposes the use of the above-mentioned Prunella Vulgaris polysaccharide in the preparation of medicines for preventing and treating acute alcoholic liver injury.

[0068] The present invention will be described in detail below by way of examples, but the protection scope of the present invention is not limited thereto. The experimental methods in the following examples, unless otherwise specified, are conventional methods in the art. The experimental materials used in the following examples, unless otherwise specified, are commercially available products.

[0069] In the following embodiments and test examples, the materials and reagents involved include:

[0070] Prunella vulgaris, produced in Henan, was purchased from a supplier in Wuhan, Hubei Province;

[0071] Anhydrous glucose was purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0072] DPPH, Shanghai MacLean Biochemical Technology Co., Ltd.;

[0073] DEAE-Sepharose fast flow, sephacryl S-300, cytiva company

[0074] Phenol, concentrated sulfuric acid, ferric chloride, ferrous sulfate, and TFA were purchased from Sinopharm Chemical Reagent Co., Ltd.; all of the above reagents were of analytical grade.

[0075] Ammonium oxalate monohydrate was purchased from Sinopharm Chemical Reagents;

[0076] Silymarin was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0077] In the following embodiments and test examples, the instruments and equipment involved include:

[0078] Analytical balance, Ohaus Instruments Changzhou Co., Ltd.;

[0079] UV-1800PC UV spectrophotometer, Aoyi Instrument Co., Ltd.;

[0080] Rotary evaporator, Shanghai Yarong Technology Co., Ltd.;

[0081] Vacuum freeze dryer, Qingdao Yonghe Chuangxin Electronic Technology Co., Ltd.

[0082] In the following embodiments and test examples, the solutions involved include:

[0083] Preparation of 0.7% (w / v) ammonium oxalate solution: Add 8.02 g of ammonium oxalate monohydrate to every 1 L of pure water and mix well.

[0084] Example 1

[0085] (1) Pulverizing the fruit spikes of Prunella vulgaris, passing through a 40-mesh sieve, taking an appropriate amount of the sieved powder and treating it with anhydrous ethanol and ethyl acetate respectively using a Soxhlet extractor to achieve the effects of depigmentation and degreasing;

[0086] (2) 100 g of defatted Prunella vulgaris powder was placed in a container, distilled water (1 L) was added, and the resulting mixture was stirred in a mechanical stirrer at 25° C. for 3 hours. The mixture was centrifuged, and the above treatment was repeated for 3 times to obtain the first medicinal residue and cold water extracts CW1, CW2 and CW3;

[0087] (3) the first medicinal material residue obtained after the cold water extraction in step (2) was stirred and mixed with 80° C. hot water for 3 h to extract polysaccharides, and the extraction was repeated twice (each time with 1 L of water added) to obtain two hot water extracts HW1 and HW2 and the second medicinal material residue, and then the second medicinal material residue was stirred and mixed with acidified water (pH = 2.0, 1 L) at 50° C. for 3 h to obtain extract AC and the third medicinal material residue;

[0088] (4) extracting polysaccharides from the third medicinal material residue obtained after the acidified water extraction in step (3) with an ammonium oxalate solution (0.7%, w / v) at 70° C. for 6 hours, repeating the extraction three times (adding 1 L of solution each time), and obtaining ammonium oxalate extracts OK1, OK2 and OK3, respectively;

[0089] (5) The ammonium oxalate extracts OK1, OK2 and OK3 were decolorized by macroporous resin and deproteinized by sevage reagent, respectively, to obtain Prunella vulgaris polysaccharides OK1, OK2 and OK3 (i.e., Prunella vulgaris polysaccharide OK1 was obtained by decolorizing and deproteinizing the extract OK1 obtained in the first extraction, Prunella vulgaris polysaccharide OK2 was obtained by decolorizing and deproteinizing the extract OK2 obtained in the second extraction, and Prunella vulgaris polysaccharide OK3 was obtained by decolorizing and deproteinizing the extract OK3 obtained in the third extraction).

[0090] Example 2

[0091] The method described in Example 1 was followed, except that in step (4), the ammonium oxalate solution was used for extraction once, and finally Prunella vulgaris polysaccharide OK1 was obtained.

[0092] Example 3

[0093] The method described in Example 1 was followed, except that in step (4), the ammonium oxalate solution was used for repeated extraction twice, and finally Prunella vulgaris polysaccharides OK1 and OK2 were obtained (Prunella vulgaris polysaccharide OK1 was obtained by decolorizing and deproteinizing the extract OK1 obtained in the first extraction, and Prunella vulgaris polysaccharide OK2 was obtained by decolorizing and deproteinizing the extract OK2 obtained in the second extraction).

[0094] Test Example 1 Yield, Purity, Monosaccharide Composition and Ratio Test

[0095] The various extracts obtained in Example 1 and Prunella vulgaris polysaccharides OK1, OK2 and OK3 were tested as follows:

[0096] 1. Protein content and yield test

[0097] The protein content was detected by the Coomassie Brilliant Blue method.

[0098] (1) Each extract obtained in Example 1 was first subjected to the same decolorization and deproteinization treatment as the ammonium oxalate solution (macroporous resin decolorization, sevage method deproteinization) to obtain a polysaccharide solution;

[0099] (2) The above polysaccharide solution and Prunella vulgaris polysaccharides OK1, OK2 and OK3 were respectively concentrated under reduced pressure in a rotary evaporator, and the polysaccharides were precipitated from the concentrate with 4 times the volume of anhydrous ethanol, centrifuged, washed twice with 95% ethanol, dissolved in distilled water, and dialyzed for 24 hours. The dialyzate was freeze-dried to obtain crude polysaccharides; the yield and protein content test results of each polysaccharide component are shown in Table 1.

[0100] 2. Determination of monosaccharide composition and ratio

[0101] The monosaccharide composition and ratio were determined by HPLC. Specifically, the test method is as follows:

[0102] 2.1 Acid hydrolysis

[0103] (1) Accurately weigh 1 mg of crude polysaccharide into an acid hydrolysis vial, add 1 mL of hydrochloric acid methanol solution, fill with N2, and react in a constant temperature metal bath at 80°C for 16 h.

[0104] (2) After the reaction, the methanol hydrochloride in the sample was blown dry with a nitrogen blower, and then 1 mL of 2 M trifluoroethanol (TFA) was added to react at 120° C. for 1 h. After the reaction was completed, the sample was blown dry again.

[0105] 2.2 Monosaccharide derivatization

[0106] (1) Add 500uL of 0.3M NaOH solution to the acid hydrolysis vial to completely dissolve the dried monosaccharide sample.

[0107] (2) Add 500uL of 0.5M PMP-methanol. PMP and NaOH can be seen to diffuse and mix rapidly. Use a pipette to blow the insoluble matter at the bottom to disperse it evenly.

[0108] (3) Take 200uL of the mixed solution into an EP tube and place the sample in a 70℃ water bath for 30min.

[0109] (4) Take out the sample after the reaction, add 100uL 0.3M hydrochloric acid, and then extract.

[0110] (5) Add 700uL of dichloromethane, shake to mix, centrifuge, and use a syringe to remove the lower organic phase, repeat twice. Use a syringe to remove the remaining aqueous phase, filter through a 0.22um microporous membrane, and load it into HPLC to detect the monosaccharide composition. The test results are shown in Table 1.

[0111] Table 1

[0112]

[0113] As can be seen from Table 1, the GalA (galacturonic acid) content in the Prunella vulgaris polysaccharides OK1, OK2 and OK3 extracted by the present invention is very high, among which OK1 and OK2 are both above 86%, which are much higher than other solvent extraction components, and the monosaccharide composition is uniform; at the same time, the protein content in Prunella vulgaris polysaccharides OK1, OK2 and OK3 is only about 1%, with high purity, which meets the application requirements; among them, the yield of Prunella vulgaris polysaccharide OK1 is the highest at 2.65%, the yield of Prunella vulgaris polysaccharide OK2 is 2.11%, and the yield of Prunella vulgaris polysaccharide OK3 is 0.61%, and there are more protein impurities, therefore, the preferred scheme is to repeat the extraction twice.

[0114] In addition, it can be seen from Table 1 that the monosaccharide composition of the three components CW1, CW 2 and CW3 obtained by cold water extraction is complex, and the main monosaccharide components are Xyl and Ara, among which the content of Xyl is greater than 40%. This may be due to the low melting point of xylan, which can be soaked out by cold water; the polysaccharides HW1 and HW2 obtained by hot water extraction have complex monosaccharide composition and uneven distribution, which is basically the same as the effect obtained by traditional direct hot water extraction; acidified water extracted Prunella vulgaris polysaccharide is also an acidic polysaccharide with a relatively high uronic acid content, but its yield is low.

[0115] Test Example 2 Molecular Weight Test and Structural Characterization

[0116] Taking Prunella vulgaris OK2 obtained in Example 1 as an example, molecular weight test and structural characterization of Prunella vulgaris polysaccharide are performed as follows:

[0117] 1. Molecular weight test

[0118] The molecular weight of Prunella vulgaris polysaccharide OK2 was tested by high performance gel permeation chromatography. The test method was as follows: LC-10Avp high performance liquid chromatography system produced by Shimadzu Company of Japan, RID-10A parallax refractometer, Shimadzu CLASS-Vp workstation, TSK-gel G-3000PWXL stainless steel chromatographic column (7.8×300mm), column temperature was 40°C. 2 mg of sample was dissolved in 0.2 M NaCl aqueous solution, the concentration was 5 mg / mL, and the sample volume was 20 μL. The mobile phase was 0.2 M NaCl aqueous solution, and the flow rate was 0.6 mL / min.

[0119] HPSEC results of Prunella vulgaris polysaccharide OK2 are as follows Figure 1 As shown by Figure 1 It can be seen that the Prunella Vulgaris polysaccharide OK2 obtained by extraction with ammonium oxalate solution has only one peak and is a uniform polysaccharide with a molecular weight of 102.8 KDa.

[0120] 2. Structural characterization

[0121] Experimental methods:

[0122] (1) Dissolution of sugar samples: Weigh 5 mg of dried sample and dissolve it in 0.5 mL of DMSO (dehydrated with 4A molecular sieves), fill with N2 and seal, stir magnetically until the sugar sample is fully dissolved. Add 0.5 mL of NaOH-DMSO suspension to the dissolved sugar sample, fill with N2 and seal, stir magnetically for 2 min, and mix well. Slowly add 1 mL of iodomethane in an ice bath, seal, protect from light, stir magnetically for 30 min, add 2 mL of distilled water to stop the reaction, dialyze with running water for 24 h, dialyze with distilled water for 24 h, concentrate to a small volume and freeze-dry.

[0123] (2) Repeat the above steps for the second methylation. After terminating the methylation reaction, add an equal volume of dichloromethane and stir to extract for 30 minutes. Let stand to separate the layers. Take the dichloromethane layer (lower layer) and repeat the extraction three times. Combine the extracts, add 5-7 mL of water and stir to extract the organic phase for 20 minutes. Discard the aqueous phase and repeat three times. Dry the organic phase with an air pump and add 1 mL of distilled water to freeze-dry.

[0124] (3) The methylated sample is subjected to infrared spectroscopy. If the IR spectrum is at 3400 cm -1 There is no absorption peak at 1000cm -1 If there is a high absorption peak around , it proves that the polysaccharide is completely methylated.

[0125] (4) Hydrolysis of methylated polysaccharides: Add 1 mL of mixed acid (HCOOH:H2O:TFA=3:2:1) to the dried methylated sugar sample, seal with N2, and hydrolyze at 100°C for 6 h. After the hydrolysis, add anhydrous ethanol repeatedly to evaporate the mixed acid until pH = 7 (temperature below 40°C).

[0126] Reduction: Add 1 mL of 30 mg / mL NaBH4 solution and stir at room temperature for 12 h, add about 100 uL of 50% glacial acetic acid to neutralize to neutrality, add an appropriate amount of strong acid cation exchange resin, stir magnetically for 20 min, filter (remove the resin), repeatedly add methanol to the filtrate, and evaporate the boric acid to neutrality (temperature below 40°C).

[0127] (5) Acetylation: Add 0.5 mL of acetic anhydride and anhydrous pyridine, seal with N2, and react at 100°C for 2 h. After the reaction is completed, quickly add 1 mL of distilled water in an ice bath to terminate the reaction, cover the bottle cap tightly, and cool in an ice bath for 5 min. Add 2 mL of dichloromethane and 2 mL of distilled water, and back-extract the organic phase 3 times. Remove the aqueous phase, blow dry the organic phase, dissolve it in 1 mL of chromatographically pure dichloromethane, filter it, and perform GC-MS analysis.

[0128] (6) GC-MS program: The chromatographic column model was Agilent DB-35ms, the injection port temperature was 300°C, the auxiliary heater temperature was 280°C, and the heating program was as follows: initial temperature 140°C, hold for 2 min, increase to 170°C at 5°C / min, hold for 3 min, increase to 180°C at 1°C / min, hold for 5 min, increase to 220°C at 3°C / min, hold for 1 min, increase to 295°C at 20°C / min, hold for 3 min.

[0129] Experimental results:

[0130] The results of methylation analysis of Prunella vulgaris polysaccharide OK2 are shown in Table 2.

[0131] Table 2

[0132]

[0133] It can be seen from Table 2 that Prunella vulgaris polysaccharide OK2 is a linear α-1,4-GalA-linked pectin polysaccharide. Combined with the monosaccharide composition and proportion results in the previous text, it can be seen that Prunella vulgaris polysaccharide OK2 is an HG-type pectin with a galacturonic acid structure linked by an α-1,4 glycosidic bond.

[0134] It is understandable that due to the same extraction method, the structures of Prunella Vulgaris polysaccharide OK2 and Prunella Vulgaris polysaccharide OK1 are similar, both of which are HG-type pectins with galacturonic acid structures connected by α-1,4 glycosidic bonds.

[0135] 2. Electron microscope detection

[0136] The Prunella Vulgaris polysaccharide OK2 obtained in Example 1 was subjected to scanning electron microscopy (SEM) analysis. Test method: Prunella Vulgaris polysaccharide OK2 was weighed and subjected to gold spraying using an ion plating machine. The appearance of the sample after gold spraying was detected using a scanning electron microscope. The results are as follows: Figure 2 shown.

[0137] Depend on Figure 2 It can be seen that the Prunella Vulgaris polysaccharide OK2 showed flake accumulation under a scanning electron microscope, with a dense and smooth surface and an overlapping structure, indicating that it has a certain flexibility.

[0138] Test Example 3: Animal Experiment on Acute Alcoholic Liver Injury

[0139] 70 male Balb / C mice, 18-20g, 6 weeks old. During the experiment, the mice were exposed to a temperature of 23±2°C, a humidity of 55±15%, and a light-dark cycle of 12h. Among them, the cold water Prunella vulgaris extract (CW) was obtained by mixing the cold water extracts CW1, CW2 and CW3 obtained in Example 1 after decolorization and deproteinization, and the ammonium oxalate Prunella vulgaris polysaccharide (OK) was obtained by mixing the Prunella vulgaris polysaccharides OK1, OK2 and OK3 obtained in Example 1.

[0140] The mice were randomly divided into 7 groups, 10 in each group, and adaptively fed for one week: normal control group (control), alcohol treatment model group (model), low-dose cold water Prunella vulgaris extract group (100 mg / kg, CW100), high-dose cold water Prunella vulgaris extract group (300 mg / kg, CW300), low-dose ammonium oxalate Prunella vulgaris polysaccharide group (100 mg / kg, OK100), high-dose ammonium oxalate Prunella vulgaris polysaccharide group (300 mg / kg, OK300), silymarin positive control (150 mg / kg, Sil). The control group and the model group were gavaged with normal saline every morning, the positive control group was gavaged with silymarin, and the other drug-administered groups were given different doses of drugs (specifically, the low-dose cold water Prunella vulgaris extract group was gavaged with 100 mg / kg of cold water extract, the high-dose cold water Prunella vulgaris extract group was gavaged with 300 mg / kg of cold water extract, the low-dose ammonium oxalate Prunella vulgaris extract group was gavaged with 100 mg / kg of ammonium oxalate Prunella vulgaris polysaccharide, and the high-dose ammonium oxalate Prunella vulgaris polysaccharide group was gavaged with 300 mg / kg of ammonium oxalate Prunella vulgaris polysaccharide). After 4 hours, except for the control group, the other groups were gavaged with 50% ethanol (10 mL / kg), and the control group was gavaged with normal saline. After the 28-day feeding period, the mice were euthanized by cervical dislocation, and the liver, fat, serum, ileum, colon and cecum contents of all mice were collected, and the organ index and fat index (liver index = liver weight / final weight, fat index = fat weight / final weight) were immediately weighed and calculated and stored at -80°C for subsequent detection and analysis.

[0141] After the experiment, the following tests were performed:

[0142] 1. After the experiment, the serum transaminase, inflammatory factors, lipoprotein, cholesterol and other indicators of each group of mice were tested using the kit assay method. The test results are as follows: Figure 3 shown.

[0143] Depend on Figure 3 It can be seen that the AST, ALT, TC, TG, LDL-C, TNF-α, IL-6, and IL-1β indicators of the model group mice were significantly increased, indicating that the liver of the mice was significantly damaged after alcohol administration; while the Prunella vulgaris polysaccharide extract group administered with Prunella vulgaris polysaccharide extract can effectively alleviate the inflammation and liver damage after the modeling of the mice. At the same time, the improvement effect of Prunella vulgaris polysaccharide extract was significantly better than that of cold water extract.

[0144] 2. Colon HE staining: Take out the paraffin sections of the mouse colon tissue and perform conventional HE staining. The staining results are as follows: Figure 4 shown.

[0145] Depend on Figure 4It can be seen that the colon villi in the model group were significantly shortened and the goblet cells became fewer, with obvious inflammatory effects. After administration of cold water extract and Prunella vulgaris polysaccharide extract, there was some improvement, and it was dose-dependent. At the same time, it can be seen from the high dose that the improvement effect of Prunella vulgaris polysaccharide extract was significantly better than that of cold water extract.

[0146] 3. Liver oil red staining, staining method: Take out the cut mouse liver tissue paraffin section and perform conventional oil red staining. The staining results are as follows Figure 5 shown.

[0147] Depend on Figure 5 It can be seen that the fat content in the liver of the model group was significantly higher than that of the normal group, indicating that after the administration of alcohol, the liver was damaged, resulting in fat accumulation in the liver. The administration of cold water extract and Prunella vulgaris polysaccharide extract can significantly reduce liver damage in a dose-dependent manner. At the same time, it can be seen from the high-concentration administration that the fat in the liver of the Prunella vulgaris polysaccharide extract was significantly less, and the improvement effect was significantly better than that of the cold water extract.

[0148] The above results show that in the extraction method described in the present invention, the solvent pretreatment method is adopted, and the high-purity Prunella vulgaris pectin polysaccharide obtained by final extraction with ammonium oxalate solution has a significant effect in preventing alcoholic liver damage, and can provide theoretical guidance for the clinical drug development of Prunella vulgaris polysaccharide.

[0149] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for extracting Prunella vulgaris polysaccharides, characterized in that: The method comprises the following steps: (1) crushing and sieving the fruit ears of Prunella vulgaris, and then performing a degreasing treatment with an organic solvent; (2) mixing the defatted Prunella vulgaris powder obtained in step (1) with water, stirring and mixing at 20 to 28° C. for 1 to 4 hours, repeating 2 to 4 times to obtain a first medicinal material residue; (3) stirring and mixing the first medicinal material residue in hot water at 75 to 85° C. for 1 to 4 hours, repeating 1 to 3 times to obtain a second medicinal material residue, and then stirring and mixing with acidified water for 1 to 4 hours to obtain a third medicinal material residue; (4) extracting polysaccharides from the third medicinal material residue with an ammonium oxalate solution, repeating the extraction 1 to 3 times, and decolorizing and deproteinizing the collected extract to obtain Prunella vulgaris polysaccharides.

2. The method according to claim 1, characterized in that: In step (4), the concentration of the ammonium oxalate solution is 0.5-1%.

3. The method according to claim 1 or 2, characterized in that: In step (4), the extraction time is 5 to 7 hours; and / or In step (4), the extraction is repeated 1 to 2 times.

4. The method according to any one of claims 1 to 3, characterized in that: In step (1), the sieving is through a 30-50 mesh sieve; and / or In step (1), the organic solvent is anhydrous ethanol and ethyl acetate.

5. The method according to any one of claims 1 to 4, characterized in that: In step (2), the ratio of the herbaceous spring grass powder to water is 50-150 g:1 L.

6. The method according to any one of claims 1 to 5, characterized in that: In step (3), the pH of the acidified water is 1.5 to 2.5; and / or In step (3), during the stirring with acidified water, the stirring and mixing temperature is 40 to 60°C.

7. Prunella vulgaris polysaccharide extracted by the method described in any one of claims 1 to 6.

8. The Prunella vulgaris polysaccharide according to claim 7, characterized in that The Prunella vulgaris polysaccharide comprises at least one of the following polysaccharides: Prunella vulgaris polysaccharide OK1: mainly composed of mannose, glucuronic acid, rhamnose, galacturonic acid, galactose, xylose and arabinose, and the content of galacturonic acid is more than 80 mol%; Prunella vulgaris polysaccharide OK2: mainly composed of rhamnose, galacturonic acid, galactose, xylose and arabinose, and the content of galacturonic acid is more than 80 mol%.

9. The Prunella vulgaris polysaccharide according to claim 8, characterized in that The Prunella Vulgaris polysaccharide is an HG type pectin polysaccharide with a galacturonic acid structure connected by α-1,4 glycosidic bonds; Preferably, the molecular weight of the Prunella Vulgaris polysaccharide OK2 is 100-120 KDa.

10. Use of the Prunella vulgaris polysaccharide according to any one of claims 7 to 9 in the preparation of a medicament for preventing and treating acute alcoholic liver injury.

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