A scutellarin extract and its preparation method

Abstract

The present invention discloses a scutellarin extract and a preparation method thereof. By combining the aqueous solution obtained by removing ethanol from the water extract or ethanol extract of the dried whole herb of the plant of the genus Erigeron in the Compositae family with the water extract of the stem and / or leaf of Scutellaria baicalensis, a scutellarin extract can be obtained, where 60% ≤ the scutellarin content < 100 wt%. The plant of the genus Erigeron in the Compositae family is selected from any one or more of Erigeron breviscapus, Erigeron multiradiatus, Erigeron elongatus, Conyza canadensis, and Erigeron acer. The water extract of the stem and / or leaf of Scutellaria baicalensis can efficiently enzymatically convert scutellarin (baicalin) in the aqueous solution obtained by removing ethanol from the water extract or ethanol extract of the dried whole herb of the plant of the genus Erigeron in the Compositae family into scutellarin aglycone (scutellarin). The present invention uses the plant of the genus Erigeron in the Compositae family as the raw material for preparing scutellarin, and fully utilizes the stems and leaves of Scutellaria baicalensis, reducing the cost of producing scutellarin; the content of scutellarin in the product obtained by the preparation method is high, and the production process is simple and feasible.

Description

Technical Field

[0001] The present invention relates to an extract of effective components of traditional Chinese medicine, and particularly to an extract of scutellarein from Erigeron breviscapus (Vant.) Hand.-Mazz. and its preparation method. Background Art

[0002] Scutellarein is the aglycone of scutellarin. Scutellarin can be metabolized into scutellarein (active metabolite), that is, scutellarein, under the action of in vivo enzymes, acid hydrolysis or intestinal flora. Its biological activity is stronger, it is more easily absorbed orally than scutellarin, and it has better bioavailability (Zhang Peipei et al., Research status of physicochemical properties and in vitro and in vivo stability of scutellarin and its aglycone, Yunnan Journal of Traditional Chinese Medicine and Materia Medica, 2019, 40(10): 77). Literature reports that rats were intragastrically administered equal amounts of scutellarein aglycone and scutellarin. The results showed that scutellarein aglycone was easily absorbed orally. Compared with scutellarin, its relative bioavailability was 301.8% (Che Qingming et al., Comparison of pharmacokinetics of different doses of scutellarein aglycone in rats, Chinese Journal of New Drugs, 2006, 15(18): 1557). Pharmacodynamic experiments showed that: (1) Scutellarein aglycone has a protective effect on the experimental cerebral ischemia model in rats, and its curative effect is better than that of scutellarin (Song Yan et al., Protective effect of scutellarin aglycone on experimental cerebral ischemia, Chinese Journal of New Drugs, 2009, 18(21): 2061); (2) Scutellarein aglycone has the effects of anti-thrombosis, anti-platelet aggregation and improving hemorheological indexes, and its curative effect is better than that of scutellarin (Song Yan et al., Effects of scutellarin aglycone on thrombosis formation and hemorheology in rats, Chinese Journal of New Drugs, 2011, 20(15): 1446).

[0003] The natural source of scutellarein (scutellarin aglycone) is relatively scarce, and it only exists in a few plants with very low content, making it difficult to achieve large-scale production. At present, there are already chemical methods for the total synthesis of scutellarein, but the synthesis steps are long and the yield is low. Scutellarin exists in relatively high content in some plants, such as Erigeron breviscapus (Vant.) Hand.-Mazz., Erigeron multiradiatus (Lindl.) Benth., Erigeron elongatus Ledeb., Conyza canadensis (L.) Cronq., Erigeron acer L., etc. It can be obtained in large quantities from these plants. Scutellarin can be hydrolyzed or enzymatically hydrolyzed into scutellarein by chemical or biological methods, but there are also many defects and deficiencies. For example, acid hydrolysis often damages the aglycone structure, is difficult to separate and purify, and has a low conversion rate, resulting in acid emission pollution; the biological conversion method has problems such as low substrate concentration and low production yield. Summary of the Invention

[0004] In order to overcome the deficiencies of the existing methods for preparing scutellarein, the present invention provides an extract of scutellarein and its preparation method, which can satisfactorily solve the above problems.

[0005] Through in-depth research, the applicant unexpectedly found that the aqueous extract of the above-ground part of Scutellaria baicalensis Georgi, namely the aerial parts of Scutellaria baicalensis Georgi (Scutellaria baicalensis Georgi stems and leaves, Scutellaria baicalensis Georgi stems or Scutellaria baicalensis Georgi leaves), can efficiently enzymatically convert scutellarin (baicalin) in the aqueous solution obtained by removing ethanol from the aqueous extract or ethanol extract of the dried whole herb of Erigeron plants of the Asteraceae family into scutellarein (baicalein).

[0006] The applicant's research also found that during the growth process of Scutellaria baicalensis Georgi, the above-ground parts of Scutellaria baicalensis Georgi (Scutellaria baicalensis Georgi stems and leaves, Scutellaria baicalensis Georgi stems or Scutellaria baicalensis Georgi leaves) harvested at different harvesting periods (such as from June to December) have similar enzyme activities (enzymatically converting scutellarin into scutellarein), and moreover, harvesting the above-ground parts (Scutellaria baicalensis Georgi stems and leaves) has no obvious adverse effects on the underground part, Scutellaria baicalensis Georgi rhizome.

[0007] Accordingly, the present invention provides a method for preparing scutellarein extract, which comprises combining and reacting the aqueous solution obtained by removing ethanol from the aqueous extract or ethanol extract of the dried whole herb of Erigeron plants of the Asteraceae family with the aqueous extract of Scutellaria baicalensis Georgi stems and / or Scutellaria baicalensis Georgi leaves, collecting the precipitate, and obtaining the scutellarein extract.

[0008] The Erigeron plants of the Asteraceae family are selected from any one or more of Erigeron breviscapus (Vant.) Hand.-Mazz., Erigeron multiradiatus (Lindl.) Benth., Erigeron elongatus Ledeb., Conyza canadensis (L.) Cronq., and Erigeron acer L. Erigeron breviscapus (Vant.) Hand.-Mazz. is the dried whole herb of Erigeron breviscapus (Vant.) Hand.-Mazz., a plant of the Asteraceae family included in the Chinese Pharmacopoeia (2020 Edition). In the present invention, Erigeron breviscapus (Vant.) Hand.-Mazz. can be represented by Erigeron breviscapus (Vant.) Hand.-Mazz. Similar plants of the same genus as Erigeron breviscapus (Vant.) Hand.-Mazz., such as Erigeron multiradiatus (Lindl.) Benth., Erigeron elongatus Ledeb., Conyza canadensis (L.) Cronq., and Erigeron acer L., all contain relatively high levels of scutellarin and can be used as starting materials for extracting scutellarein.

[0009] The method for preparing the aqueous extract of the dried whole herb of the Erigeron plants of the Asteraceae family is: decocting the dried whole herb of the Erigeron plants of the Asteraceae family with water to obtain the aqueous extract; the method for preparing the aqueous solution obtained by removing ethanol from the ethanol extract is: refluxing and extracting the dried whole herb of the Erigeron plants of the Asteraceae family with an ethanol solution, then collecting the ethanol extract, removing ethanol, and diluting with water.

[0010] In the above method of the present invention, the conventional method is used to extract the scutellarin component from the Erigeron plants of the Asteraceae family with water or an ethanol solution containing water, including the aqueous extract of the Erigeron plants of the Asteraceae family or the aqueous solution obtained by removing ethanol from the ethanol extract. For example, in the current edition of the Chinese Pharmacopoeia, for "Erigeron breviscapus (Vant.) Hand.-Mazz. Injection", Erigeron breviscapus (Vant.) Hand.-Mazz. is extracted by decocting with water, and for "Erigeron breviscapus (Vant.) Hand.-Mazz. Granules", Erigeron breviscapus (Vant.) Hand.-Mazz. is extracted by refluxing with 75% ethanol.

[0011] According to the preferred technical solution of the present invention, the preparation method of the aqueous extract of the dried whole herb of the Erigeron plant of the Compositae family is as follows: Add 8 - 15 times the weight - volume ratio (g / ml) of water or boiling water to the dried whole herb of the Erigeron plant of the Compositae family, decoct and extract 2 - 3 times, with each extraction time being 1 - 2 hours, and collect the aqueous extract; the preparation method of the aqueous solution after removing ethanol from the ethanol extract is as follows: Add 8 - 15 times the weight - volume ratio of an ethanol solution with a concentration of 30% - 80% to the dried whole herb of the Erigeron plant of the Compositae family, reflux and extract 2 - 3 times, with each extraction time being 1 - 2 hours, collect the ethanol extract, remove ethanol, and then add water to adjust the concentration of the aqueous solution to 0.1 - 1 g of crude drug / ml. Here, the crude drug or raw material is calculated by weight based on the dry herb, and the ethanol solution mentioned is an ethanol - water solution (the same hereinafter).

[0012] The present invention generally recommends using the whole dry herb of the Erigeron plant of the Compositae family for extraction. However, under some special process requirements, it is also possible to select a part of the dry herb of the Erigeron plant of the Compositae family (such as the stem or leaf) for extraction, or use the fresh herb of the Erigeron plant of the Compositae family for extraction, which does not constitute a limitation to the present invention.

[0013] The preparation method of the aqueous extract (aqueous solution) of Scutellaria baicalensis Georgi stem and / or Scutellaria baicalensis Georgi leaf is as follows: Crush Scutellaria baicalensis Georgi stem and / or Scutellaria baicalensis Georgi leaf, add water and stir for extraction, and the collected aqueous extract is the aqueous solution of Scutellaria baicalensis Georgi stem and / or Scutellaria baicalensis Georgi leaf; or let the above - collected aqueous extract stand overnight, collect the precipitate, add water to dissolve and disperse it as the aqueous solution of Scutellaria baicalensis Georgi stem and / or Scutellaria baicalensis Georgi leaf; or adjust the pH of the above - collected aqueous extract to 3 - 5 after membrane concentration, let it stand overnight, collect the precipitate, add water to dissolve and disperse it as the aqueous solution of Scutellaria baicalensis Georgi stem and leaf. The Scutellaria baicalensis Georgi stem and / or Scutellaria baicalensis Georgi leaf mentioned in this specification refers to Scutellaria baicalensis Georgi stem and leaf (i.e., the above - ground part), Scutellaria baicalensis Georgi stem or Scutellaria baicalensis Georgi leaf, which have similar enzyme activities.

[0014] The aqueous solution of Scutellaria baicalensis Georgi stem and / or Scutellaria baicalensis Georgi leaf can be extracted with water under the condition of 0 - 50°C. The aqueous extract of the dried whole herb of the Erigeron plant of the Compositae family or the aqueous solution after removing ethanol from the ethanol extract is combined with the aqueous solution of Scutellaria baicalensis Georgi stem and / or Scutellaria baicalensis Georgi leaf, and heated to a temperature generally not higher than 50°C, as too high a temperature will affect the enzyme activity and enzymatic hydrolysis effect.

[0015] According to the preferred technical solution of the present invention, the preparation method of the water extract (aqueous solution) of Scutellaria baicalensis stem and / or Scutellaria baicalensis leaf is as follows: The Scutellaria baicalensis stem and / or Scutellaria baicalensis leaf are crushed into powder particles with a particle size ≤ 5-mesh sieve pores, added with 8-20 times the weight-volume ratio of water, and stirred and extracted 1-3 times under the condition of 0-50 °C, with each extraction time being 0.25-1 hour. The water extract is collected to obtain the aqueous solution of Scutellaria baicalensis stem and / or Scutellaria baicalensis leaf; or the above-collected water extract is left standing overnight, the precipitate is collected, and water is added to dissolve and disperse it as the aqueous solution of Scutellaria baicalensis stem and / or Scutellaria baicalensis leaf; or the above-collected water extract is membrane-concentrated, the pH is adjusted to 3-5, left standing overnight, the precipitate is collected, and water is added to dissolve and disperse it as the aqueous solution of Scutellaria baicalensis stem and / or Scutellaria baicalensis leaf.

[0016] The Scutellaria baicalensis stem and / or Scutellaria baicalensis leaf can be crushed into the coarsest powder, coarse powder, medium powder, etc. specified in the current Chinese Pharmacopoeia, which is more conducive to the extraction of Scutellaria baicalensis stem and leaf enzymes. At this time, a better extraction method is to carry out the extraction under stirring. Experiments show that although the smaller the particle size of the crushing, the more conducive to the efficiency and sufficiency of water extraction, but too small a particle size will also increase the difficulty of the separation operation after extraction, increase the production cost and reduce the production efficiency, which can be determined after comprehensive balance.

[0017] In the preparation method of the scutellarin extract, the dry weight of the dried whole herb of the plant of the genus Erigeron in the Compositae family and the Scutellaria baicalensis stem and / or Scutellaria baicalensis leaf is measured. The mass ratio of the raw material of the plant of the genus Erigeron in the Compositae family used in the aqueous solution after removing ethanol from the water extract or ethanol extract of the dried whole herb of the plant of the genus Erigeron in the Compositae family to the raw material of the Scutellaria baicalensis stem and / or Scutellaria baicalensis leaf used in the water extract of the Scutellaria baicalensis stem and / or Scutellaria baicalensis leaf is 10:(0.25-5).

[0018] The dried whole herb of the plant of the genus Erigeron in the Compositae family refers to being dried to a basically stable moisture content under conditions such as artificial drying, natural air drying, and sun drying. Generally, it should not exceed 12.0%, which is easy to achieve.

[0019] The dry weight of the Scutellaria baicalensis stem and / or Scutellaria baicalensis leaf can also be dried to a basically stable moisture content through natural air drying, sun drying or artificial drying, etc. Generally, it should not exceed 12.0%.

[0020] In the preparation method of the scutellarin extract, the conditions for the combined reaction are as follows: Maintaining for more than 5 hours under the condition of room temperature to ≤ 50 °C, collecting the precipitate, and obtaining the scutellarin extract, with 60% ≤ scutellarin content < 80%.

[0021] On the basis of the aforementioned scutellarin extract, the present invention can further obtain an extract with a higher scutellarin content. The method is as follows: The scutellarin extract is extracted with ethanol having a concentration ≥ 70%, the ethanol extract is collected, the ethanol is recovered, and precipitation or crystallization is carried out, or the operations of "extraction - collection - recovery - precipitation or crystallization" are further repeated to obtain a purified and refined scutellarin extract, with 80% ≤ scutellarin content < 100%. In this step, the time for ethanol reflux extraction can be selected from 30 minutes to 1 hour. After ethanol reflux extraction, activated carbon can also be used for decolorization and reflux extraction for 10 - 30 minutes; then filtration and vacuum recovery of ethanol are carried out.

[0022] In the preparation method of the scutellarin extract, before combining and reacting with the aqueous extract of Scutellaria baicalensis stems and / or leaves, 0.2 - 5% of sodium metabisulfite or sodium bisulfite, equivalent to the weight of the dried whole herb of the Compositae Erigeron plant, is added to the aqueous solution obtained by removing ethanol from the aqueous extract or ethanol extract of the dried whole herb of the Compositae Erigeron plant, and this is used as an antioxidant.

[0023] A further technical solution is to add 0.5 - 2% of sodium metabisulfite and / or sodium bisulfite, equivalent to the weight of the dried whole herb of the Compositae Erigeron plant, as an antioxidant, which can significantly improve the conversion rate of scutellarin.

[0024] On the basis of the above method of the present invention, the following process conditions can also be adopted alone or in any combination to obtain technical solutions, and in these solutions, Erigeron breviscapus can be replaced by any one or more of Erigeron multiradiatus, Erigeron elongatus, Conyza canadensis, and Erigeron acer. As a preferred method, these process conditions include:

[0025] The preferred weight part ratio of the raw materials of Erigeron breviscapus, Scutellaria baicalensis stems and / or leaves is 10:(2 - 0.5). Since the enzyme activity of Scutellaria baicalensis stems and leaves is relatively strong, appropriately extending the enzymolysis time can reduce the dosage of Scutellaria baicalensis stems and / or leaves and lower the production cost.

[0026] The aqueous solution obtained by removing ethanol from the aqueous extract or ethanol extract of Erigeron breviscapus is combined with the aqueous solution of Scutellaria baicalensis stems and leaves and heated at 30 - 50°C for 10 - 25 hours.

[0027] The aqueous solution obtained by removing ethanol from the aqueous extract or ethanol extract of Erigeron breviscapus is combined with the aqueous solution of Scutellaria baicalensis stems and / or leaves and heated and maintained at a pH value of 5 - 7 for 10 - 25 hours.

[0028] The aqueous solution obtained by removing ethanol from the aqueous extract or ethanol extract of Erigeron breviscapus is combined with the aqueous solution of Scutellaria baicalensis stems and / or leaves and maintained at a pH value of 5.5 - 6.5 and 40 - 50°C for 10 - 25 hours.

[0029] The described scutellarin extract is further extracted with 70 - 95% ethanol by a conventional method. The preferred method is stirring and / or reflux extraction. The ethanol extract is collected, the ethanol is recovered under reduced pressure and concentrated, then left to stand and precipitate (crystallize), or this operation is further repeated (recrystallized) to obtain a purified and refined scutellarin extract with a high content (refined product), where 80% ≤ scutellarin content < 100%.

[0030] In the present invention, the ethanol concentration mentioned, according to common understanding, refers to the volume percentage of the ethanol solution. Other percentages mentioned in the present invention, unless otherwise specified, refer to weight percentages.

[0031] The present invention also provides a scutellarin extract, which is characterized in that the scutellarin extract is prepared by the preparation method described above, wherein 60% ≤ scutellarin content < 100% wt%.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] The present invention uses plants of the genus Erigeron in the Compositae family as raw materials for preparing scutellarin, and makes full use of the stems and leaves of Scutellaria baicalensis, reducing the cost of producing scutellarin;

[0034] The product obtained by the above preparation method of the present invention has a high content of scutellarin, and the production process is simple and feasible. Description of the Drawings

[0035] Figure 1 HPLC chart of the mixed reference substances.

[0036] Figure 2 HPLC chart of the test sample of the Erigeron breviscapus decoction.

[0037] Figure 3 HPLC chart of the test sample of the enzymatic hydrolysis product.

[0038] In the figure, peak 1 is scutellarin glycoside and peak 2 is scutellarin. Detailed Embodiments

[0039] The above content of the present invention will be further described in detail below through the specific embodiments of the examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. Without departing from the above technical idea of the present invention, various substitutions or changes made according to ordinary technical knowledge and common means in the art should be included within the scope of the present invention.

[0040] Example 1

[0041] Take 10 Kg of Erigeron breviscapus, add 10 times the amount of water (weight - volume ratio Kg / L, the same below), and perform decoction and reflux extraction 3 times, each time for 1.5 hours. Collect the aqueous extract, add 0.1 Kg of sodium metabisulfite, dissolve it, and set aside (I). Crush the stems and leaves of Scutellaria baicalensis Georgi through a 40 - mesh sieve, weigh 1 Kg, add 10 times the amount of water, and stir - extract at room temperature 2 times, each time for 0.5 hours. Collect the aqueous extract (aqueous solution) and set aside (II). Combine I and II, adjust the pH value to 6, heat to 45 °C and keep warm for 20 hours to precipitate. Filter, wash with water, and dry to obtain the scutellarin extract, with a yield of 2.5% and a scutellarin content of 66.1%. Crush the scutellarin extract through an 80 - mesh sieve, weigh 100 g, add 80% ethanol with a volume equivalent to 0.15 times the mass of scutellarin (mg / ml), stir and reflux - extract for 30 minutes, filter, add an appropriate amount of activated carbon to the ethanol extract, heat and reflux for 25 minutes, filter, recover ethanol under reduced pressure to about 1 / 3 of the volume, let it stand overnight to precipitate or crystallize, filter, and dry to obtain 52.1 g of the refined scutellarin extract, with a scutellarin content of 90.5%.

[0042] Example 2

[0043] Take 10 Kg of Erigeron breviscapus, add 15 times the amount of water, and perform decoction and reflux extraction 2 times, each time for 2 hours. Collect the aqueous extract, add 0.5 Kg of sodium metabisulfite, dissolve it, and set aside (I). Crush the stems and leaves of Scutellaria baicalensis Georgi through a 5 - mesh sieve, weigh 5 Kg, add 20 times the amount of ice - water, and stir - extract at 0 - 5 °C for 1 hour. Collect the aqueous extract (aqueous solution) and set aside (II). Combine I and II, adjust the pH value to 5, heat to 50 °C and keep warm for 5 hours to precipitate. Filter, wash with water, and dry to obtain the scutellarin extract, with a yield of 2.2% and a scutellarin content of 64.3%. Crush the scutellarin extract through a 60 - mesh sieve, weigh 100 g, add 95% ethanol with a volume equivalent to 0.3 times the mass of scutellarin (mg / ml), stir and reflux - extract for 1 hour, filter, add an appropriate amount of activated carbon to the ethanol extract, heat and reflux for 15 minutes, filter, recover ethanol under reduced pressure to about 1 / 4 of the volume, let it stand overnight to precipitate or crystallize, filter, and dry to obtain 48.7 g of the refined scutellarin extract, with a scutellarin content of 91.5%.

[0044] Example 3

[0045] Take 10 Kg of Erigeron breviscapus, add 8 times the amount of water, and perform reflux extraction by decoction 3 times, each time for 1 hour. Collect the aqueous extract, add 0.02 Kg of sodium metabisulfite, dissolve it, and set aside (I). Crush the stems and leaves of Scutellaria baicalensis Georgi through an 80-mesh sieve, weigh 0.25 Kg, add 8 times the amount of water, and perform stirring extraction at room temperature 3 times, each time for 0.25 hour. Collect the aqueous extract (aqueous solution) and set aside (II). Combine I and II, adjust the pH value to 7, heat to 50 °C and keep warm for 30 hours to precipitate; filter, wash with water, and dry to obtain the scutellarin extract, with a yield of 2.1% and a scutellarin content of 62.5%. Crush the scutellarin extract through a 40-mesh sieve, weigh 100 g, add 70% ethanol in an amount equivalent to 0.2 times the mass of scutellarin (mg / ml), perform stirring reflux extraction for 30 minutes, filter, add an appropriate amount of activated carbon to the ethanol extract, heat and reflux for about 30 minutes, filter, recover ethanol under reduced pressure to about 1 / 3 of the volume, let stand overnight to precipitate or crystallize, filter, and dry to obtain 55.4 g of the refined scutellarin extract, with a scutellarin content of 88.1%.

[0046] Example 4

[0047] Take 10 Kg of Erigeron breviscapus, add 15 times the amount of 30% ethanol, perform reflux extraction 2 times, each time for 2 hours. Collect the ethanol extract, recover ethanol under reduced pressure and remove it, add water to 100 L, add 0.05 Kg of sodium metabisulfite, dissolve it, and set aside (I). Crush the stems and leaves of Scutellaria baicalensis Georgi through a 60-mesh sieve, weigh 3 Kg, add 15 times the amount of water, and perform stirring extraction at 30 °C 2 times, each time for 0.5 hour. Collect the aqueous extract (aqueous solution) and set aside (II). Combine I and II, adjust the pH value to 6, heat to 30 °C and keep warm for 25 hours to precipitate; filter, wash with water, and dry to obtain the scutellarin extract, with a yield of 2.4% and a scutellarin content of 64.7%. Crush the scutellarin extract through a 100-mesh sieve, weigh 100 g, add 80% ethanol in an amount equivalent to 0.1 times the mass of scutellarin (mg / ml), perform stirring reflux extraction for 30 minutes, filter, add an appropriate amount of activated carbon to the ethanol extract, heat and reflux for about 20 minutes, filter, recover ethanol under reduced pressure to about 1 / 3 of the volume, let stand overnight to precipitate or crystallize, filter, and dry to obtain 55.7 g of the refined scutellarin extract, with a scutellarin content of 87.6%.

[0048] Take 50 g of the refined scutellarin extract, add 80% ethanol in an amount equivalent to 0.15 times the mass of scutellarin (mg / ml), perform stirring reflux extraction for 30 minutes, filter, recover ethanol under reduced pressure to about 1 / 3 of the volume, let stand overnight to precipitate or crystallize, filter, and dry to obtain 25.3 g of the refined scutellarin extract with high purity, with a scutellarin content of 95.6%.

[0049] Example 5

[0050] Take 10 Kg of Erigeron breviscapus, add 8 times the amount of 60% ethanol, reflux extract 3 times, 1 hour each time, collect the ethanol extract, remove ethanol by reduced pressure recovery, add water to 20 L, add 0.2 Kg of sodium metabisulfite, dissolve, and set aside (I). Crush the stems and leaves of Scutellaria baicalensis Georgi through an 80-mesh sieve, weigh 0.5 Kg, add 12 times the amount of water, stir and extract at 40 °C for 3 times, 0.25 hour each time, collect the water extract (aqueous solution), and set aside (II). Combine I and II, adjust the pH value to 5.5, heat to 40 °C and keep warm for 20 hours to precipitate; filter, wash with water, and dry to obtain the scutellarin extract, with a yield of 2.3% and a scutellarin content of 63.9%.

[0051] Example 6

[0052] Take 10 Kg of Erigeron breviscapus, add 10 times the amount of 80% ethanol, reflux extract 2 times, 1.5 hours each time, collect the ethanol extract, remove ethanol by reduced pressure recovery, add water to 10 L, add 0.2 Kg of sodium bisulfite, dissolve, and set aside (I). Crush the stems and leaves of Scutellaria baicalensis Georgi through a 40-mesh sieve, weigh 4 Kg, add 15 times the amount of water, stir and extract at 50 °C for 2 times, 0.25 hour each time, collect the water extract (aqueous solution), and set aside (II). Combine I and II, adjust the pH value to 6.5, let stand at room temperature for 30 hours to precipitate; filter, wash with water, and dry to obtain the scutellarin extract, with a yield of 2.0% and a scutellarin content of 62.8%.

[0053] Example 7

[0054] Take 10 Kg of Erigeron breviscapus, add 15 times the amount of boiling water, decoct and reflux extract 2 times, 2 hours each time, collect the water extract, add 0.1 Kg of sodium bisulfite, dissolve, and set aside (I). Crush the stems and leaves of Scutellaria baicalensis Georgi through a 40-mesh sieve, weigh 2 Kg, add 10 times the amount of water, stir and extract at room temperature for 2 times, 0.5 hour each time, collect the water extract, let stand overnight, collect the precipitate, add water to dissolve and disperse to obtain the aqueous solution of the stems and leaves of Scutellaria baicalensis Georgi, and set aside (II). Combine I and II, adjust the pH value to 5, heat to 50 °C and keep warm for 10 hours to precipitate; filter, wash with water, and dry to obtain the scutellarin extract, with a yield of 2.1% and a scutellarin content of 68.7%.

[0055] Example 8

[0056] Take 10 kg of Erigeron breviscapus, add 8 times of boiling water, decoct and reflux for 3 times, each time for 1 hour, collect the water extract, add 0.3 kg of sodium bisulfite, dissolve, and set aside (I). Scutellaria baicalensis stems and leaves are crushed and passed through a 60-mesh sieve, weigh 1 kg, add 10 times of water, stir and extract at room temperature for 3 times, each time for 0.25 hours, collect the water extract, concentrate with a nanofiltration membrane, remove small molecules such as water and inorganic salts, intercept biological enzymes, adjust the pH value to 4, stand overnight, collect the precipitate, add water to dissolve and disperse to obtain an aqueous solution of Scutellaria baicalensis stems and leaves, and set aside (II). I and II are combined, the pH value is adjusted to 5.5, heated to 45°C and kept warm for 25 hours, and the precipitate is precipitated; filtered, washed with water, and dried to obtain a scutellarin extract with a yield of 2.0% and a scutellarin content of 70.6%.

[0057] The applicant provides some specific research results below to demonstrate the beneficial effects of the present invention.

[0058] The enzymatic hydrolysis conditions were investigated with the enzymatic conversion rate of baicalin as the evaluation index.

[0059] Scutellarin enzymatic conversion rate = m 2 ×M 1 / (m 1 ×M 2 )

[0060] In the formula, m 1 is the mass of scutellariae scutellariae, m 2 is the actual measured amount of scutellarin, M 1 is the relative molecular mass of scutellariae scutellariae, M 2 is the relative molecular mass of baicalein.

[0061] Scutellaria stem and leaf were crushed and passed through a 40-mesh sieve, 0.2 kg was weighed, 10 times of water was added (4 times more water was added for the first time, i.e. 14 times of water was added for the first time and 10 times of water was added for the second time, the same below), and the water extract was collected and adjusted to 4 L (0.05 g crude drug / ml) by adding water to obtain the scutellaria stem and leaf extract (containing scutellaria stem and leaf enzyme) for later use.

[0062] Take another 1 kg of Erigeron breviscapus, add 10 times of water (add 5 times more water for the first time), decoct and reflux for extraction twice, each time for 1.5 hours, collect the water extract, add water to adjust the volume to 20 L (0.05 g crude drug / ml), and you will get the Erigeron breviscapus water decoction (scutellaria baicalin extract), and set aside.

[0063] Content determination:

[0064] 1. HPLC conditions

[0065] Chromatographic column: Agilent Eclipse XDB-C 18(4.6×250 mm, 5 μm); Column temperature: 30 °C; Mobile phase: 0.1% formic acid aqueous solution (A) - methanol (B), gradient elution (0 - 12 min, 40% B; 12 - 30 min, 40% - 55% B); Flow rate: 1.0 mL·min -1 ; Detection wavelength was 278 nm; Injection volume: 5 - 10 μL. The number of theoretical plates calculated by scutellarin peak should be not less than 3000. The chromatogram is shown in Figures 1 to 3 .

[0066] 2. Preparation of reference substance solution

[0067] Accurately weigh 8.16 mg of scutellarin reference substance and 9.05 mg of scutellarein reference substance, place them in a 50 mL volumetric flask, dissolve with 70% methanol and dilute to the scale, then obtain a mixed reference substance solution of 163.2 μg·mL -1 scutellarin and 181 μg·mL -1 scutellarein.

[0068] 3. Preparation of test solution

[0069] Preparation of test solution of Erigeron breviscapus water decoction: Take 1 mL of Erigeron breviscapus water decoction into a 10 mL volumetric flask, add 70% ethanol to the scale, shake well, filter, and take the subsequent filtrate to obtain (for determination of scutellarin).

[0070] Preparation of test solution of enzymatic hydrolysis product: Combine the extract of Scutellaria baicalensis Georgi stems and leaves and Erigeron breviscapus water decoction for enzymatic hydrolysis, add ethanol with a volume three times that of the enzymatic hydrolysis system to terminate the reaction, transfer to a 50 mL or 100 mL volumetric flask, perform ultrasonic treatment (300 W, 40 kHz) for 20 min, cool, add 70% ethanol to the scale, shake well, filter, and take the subsequent filtrate to obtain (for determination of scutellarein).

[0071] From Figures 1 to 3 it can be seen that the test solution of Erigeron breviscapus water decoction contains scutellarin, while it is converted into scutellarein in the test solution of enzymatic hydrolysis product, indicating that enzymatic hydrolysis reaction occurs after combining the extract of Scutellaria baicalensis Georgi stems and leaves and Erigeron breviscapus water decoction, and scutellarin is fully converted into scutellarein.

[0072] Experiment 1 Effect of antioxidant types on conversion rate

[0073] Take 10 mL of Erigeron breviscapus water decoction, add no antioxidant, 1% sodium sulfite, 1% sodium metabisulfite, 1% sodium bisulfite (calculated based on the weight of Erigeron breviscapus, the same below) respectively, add 1 mL of the extract of Scutellaria baicalensis Georgi stems and leaves, and perform enzymatic hydrolysis in a 45 °C water bath for 20 h (pH is about 6, the same below). Determine the conversion rate of scutellarin in each sample according to the method under the above content determination item, and the results are shown in Table 1.

[0074] Table 1 Effect of antioxidants on conversion rate

[0075]

[0076] Experiment 2: Effect of pH on Conversion Rate

[0077] Take 10 mL of the water decoction of Erigeron breviscapus, add 1% sodium metabisulfite, add 1 mL of the extract of Scutellaria baicalensis Georgi stems and leaves, and adjust the pH to make the pH of the enzymatic hydrolysis system 5.0, 5.5, 6.0, 6.5, and 7.0 respectively. Carry out enzymatic hydrolysis in a water bath at 45°C for 20 h. Measure the conversion rate of scutellarin in each sample as above, and the results are shown in Table 2.

[0078] Table 2: Effect of pH on Conversion Rate

[0079]

[0080] Experiment 3: Effect of Enzymatic Hydrolysis Temperature on Conversion Rate

[0081] Take 10 mL of the water decoction of Erigeron breviscapus, add 1% sodium metabisulfite, add 1 mL of the extract of Scutellaria baicalensis Georgi stems and leaves, and carry out enzymatic hydrolysis in a water bath at room temperature, 40, 45, 50, 55, and 60°C for 20 h respectively. Measure the conversion rate of scutellarin in each sample as above, and the results are shown in Table 3.

[0082] Table 3: Effect of Temperature on Conversion Rate

[0083]

[0084] Experiment 4: Effect of Enzymatic Hydrolysis Time on Conversion Rate

[0085] Take 10 mL of the water decoction of Erigeron breviscapus, add 1% sodium metabisulfite, add 1 mL of the extract of Scutellaria baicalensis Georgi stems and leaves, and carry out enzymatic hydrolysis in a water bath at 45°C for 5, 10, 15, 20, and 25 h respectively. Measure the conversion rate of scutellarin in each sample as above, and the results are shown in Table 4.

[0086] Table 4: Effect of Enzymatic Hydrolysis Time on Conversion Rate

[0087]

[0088] Experiment 5: Effect of Antioxidant Dosage on Conversion Rate

[0089] Take 10 mL of the water decoction of Erigeron breviscapus, add 0.1%, 0.2%, 0.5%, 1%, 2%, and 5% sodium metabisulfite respectively, add 1 mL of the extract of Scutellaria baicalensis Georgi stems and leaves, and carry out enzymatic hydrolysis in a water bath at 45°C for 20 h. Measure the conversion rate of scutellarin in each sample as above, and the results are shown in Table 5.

[0090] Table 5: Effect of Antioxidant Dosage on Conversion Rate

[0091]

[0092]

[0093] Experiment 6: Effect of Scutellaria baicalensis Stem and Leaf Dosage on Conversion Rate

[0094] Take 10 mL of Erigeron breviscapus water decoction, add 1% sodium metabisulfite, and respectively add 0.1 mL, 0.25 mL, 0.5 mL, 1 mL, 2 mL, and 5 mL of Scutellaria baicalensis stem and leaf extract. Enzymatic hydrolysis is carried out in a water bath at 45°C for 20 h. The conversion rate of scutellarin in each sample is measured as above, and the results are shown in Table 6.

[0095] Table 6: Effect of Scutellaria baicalensis Stem and Leaf Dosage on Conversion Rate

[0096]

[0097] Experiment 7: Extraction of Scutellarin from Erigeron multiradiatus, Erigeron elongatus, Conyza canadensis, and Erigeron acer

[0098] The Scutellaria baicalensis stem and leaf is crushed and passed through a 40-mesh sieve. Weigh 0.4 Kg, add 10 times the amount of water (add 4 times the water absorption rate more for the first time), stir and extract at room temperature for 2 times, 0.25 hours each time. Collect the water extract and divide it into 4 equal parts for standby (I).

[0099] Respectively take 1 Kg of Erigeron multiradiatus, Erigeron elongatus, Conyza canadensis, and Erigeron acer, add 10 times the amount of water (add 5 times the water absorption rate more for the first time), decoct and reflux for 2 times, 1.5 hours each time. Collect the water extract, add 0.01 Kg of sodium metabisulfite, dissolve it, and keep it for standby (II).

[0100] Combine I and II, heat to about 45°C and keep warm for 20 hours to precipitate; filter, wash with water, and dry to obtain the scutellarin extract. Measure the scutellarin content, and the results are shown in Table 7.

[0101] Table 7: Experiment on Extraction of Scutellarin from Erigeron multiradiatus, Erigeron elongatus, Conyza canadensis, and Erigeron acer

[0102]

[0103] The above embodiments and experimental results show that the aqueous solution extracted from Scutellaria baicalensis Georgi stems and leaves is rich in Scutellaria baicalensis Georgi stems and leaves enzyme, which can efficiently enzymatically hydrolyze the aqueous solution of Erigeron breviscapus water extract or ethanol extract after removing ethanol, convert scutellarin into scutellarein. Scutellarein is poorly soluble in water and forms a precipitate. After filtration, washing with water, and drying, a scutellarein extract (crude extract) is obtained, with 60% ≤ scutellarein content < 80%; further extraction with ethanol with a concentration ≥ 70% is carried out, the ethanol extract is collected, ethanol is recovered, a precipitate or crystal is precipitated, or this operation is further repeated (recrystallization) to obtain a purified and refined scutellarein extract (refined product), with 80% ≤ scutellarein content < 100%. The mother liquor or filtrate after purification and refinement can be further concentrated, allowed to stand and precipitate to obtain scutellarein; according to the purity requirements of the target product, multiple purifications / recrystallizations can be carried out to obtain a high-purity scutellarein extract. The above experimental results also found that the conversion rate of scutellarin is closely related to the dosage of Scutellaria baicalensis Georgi stems and leaves, enzymatic hydrolysis temperature, enzymatic hydrolysis time, etc. For example, when the weight ratio of Erigeron breviscapus to Scutellaria baicalensis Georgi stems and leaves is 10:0.25, the enzymatic hydrolysis time can be appropriately extended (≥ 30 h), and when the weight ratio is 10:5, the enzymatic hydrolysis time can be appropriately reduced (such as 5 h). The optimal enzymatic hydrolysis temperature of Scutellaria baicalensis Georgi stems and leaves enzyme is about 40 - 50 °C. If enzymatic hydrolysis is carried out at room temperature, the enzymatic hydrolysis time needs to be appropriately extended or the dosage of Scutellaria baicalensis Georgi stems and leaves needs to be increased. The optimal enzymatic hydrolysis pH value of Scutellaria baicalensis Georgi stems and leaves enzyme is 5.5 - 6.5 (Table 2). After the aqueous solution of Erigeron breviscapus water extract or ethanol extract after removing ethanol is combined with the aqueous solution of Scutellaria baicalensis Georgi stems and leaves, its pH value is about 6. Therefore, generally, there is no need to adjust the pH value. Raw materials of Scutellaria baicalensis Georgi stems and leaves and Erigeron breviscapus from different sources have certain effects on the conversion rate, yield, and content. Therefore, it is recommended that in actual production, requirements such as raw material source, origin, harvesting and processing, enzyme activity, and content should be further fixed.

[0104] Although the present invention has been described herein with reference to illustrative embodiments thereof, it should be understood that those skilled in the art can devise many other modifications and embodiments that will fall within the scope and spirit of the principles disclosed in this application. More specifically, within the scope of the disclosure of this application, various variations and improvements can be made to the components and / or layout of the subject combination layout. In addition to the variations and improvements made to the components and / or layout, other uses will also be apparent to those skilled in the art.

Claims

1. A preparation method of scutellarin extract, characterized in that, the aqueous solution after removing ethanol from the water extract or ethanol extract of the dried whole herb of the plant of the genus Erigeron in the Compositae family is combined and reacted with the water extract of Scutellaria baicalensis stems and / or leaves. The conditions for the combined reaction are: maintaining at room temperature to ≤50 °C for more than 5 hours, collecting the precipitate to obtain the scutellarin extract; wherein, the plant of the genus Erigeron in the Compositae family is selected from any one or more of Erigeron breviscapus, Erigeron multiradiatus, Erigeron elongatus, Conyza canadensis, and Erigeron acer.

2. The preparation method of scutellarin extract according to claim 1, characterized in that, the preparation method of the water extract of the dried whole herb of the plant of the genus Erigeron in the Compositae family is: decocting the dried whole herb of the plant of the genus Erigeron in the Compositae family with water to obtain the water extract; the preparation method of the aqueous solution after removing ethanol from the ethanol extract is: refluxing and extracting the dried whole herb of the plant of the genus Erigeron in the Compositae family with an ethanol solution, then collecting the ethanol extract, removing ethanol and diluting with water.

3. The preparation method of scutellarin extract according to claim 2, characterized in that, the preparation method of the water extract of the dried whole herb of the plant of the genus Erigeron in the Compositae family is: adding 8 - 15 times the weight - volume ratio of water to the dried whole herb of the plant of the genus Erigeron in the Compositae family, decocting and extracting 2 - 3 times, with each extraction time of 1 - 2 hours, collecting the water extract; the preparation method of the aqueous solution after removing ethanol from the ethanol extract is: adding 8 - 15 times the weight - volume ratio of an ethanol solution with a concentration of 30% - 80% to the dried whole herb of the plant of the genus Erigeron in the Compositae family, refluxing and extracting 2 - 3 times, with each extraction time of 1 - 2 hours, collecting the ethanol extract, removing ethanol and adjusting the concentration of the aqueous solution to 0.1 - 1 g of crude drug / ml with water.

4. The preparation method of scutellarin extract according to claim 3, characterized in that, the preparation method of the water extract of the dried whole herb of the plant of the genus Erigeron in the Compositae family is: adding 8 - 15 times the weight - volume ratio of boiling water to the dried whole herb of the plant of the genus Erigeron in the Compositae family, decocting and extracting 2 - 3 times, with each extraction time of 1 - 2 hours, collecting the water extract.

5. The preparation method of scutellarin extract according to claim 1, characterized in that, the preparation method of the water extract of Scutellaria baicalensis stems and / or leaves is: crushing Scutellaria baicalensis stems and / or leaves, stirring and extracting with water, and collecting the water extract as the water extract of Scutellaria baicalensis stems and / or leaves; or allowing the water extract collected after the above - mentioned stirring and extracting with water to stand overnight, collecting the precipitate, dissolving and dispersing it with water as the water extract of Scutellaria baicalensis stems and / or leaves; or subjecting the water extract collected after the above - mentioned stirring and extracting with water to membrane concentration, adjusting the pH to 3 - 5, standing overnight, collecting the precipitate, dissolving and dispersing it with water as the water extract of Scutellaria baicalensis stems and / or leaves.

6. The preparation method of scutellarin extract according to claim 5, characterized in that, The preparation method of the aqueous extract of Scutellaria baicalensis stems and / or leaves is as follows: The Scutellaria baicalensis stems and / or leaves are crushed into powder particles with a particle size ≤ 5 mesh sieve pores, added with 8 - 20 times the weight - volume ratio of water, and stirred and extracted 1 - 3 times under the condition of 0 - 50 °C, with each extraction time of 0.25 - 1 hour. The collected aqueous extract is the aqueous extract of Scutellaria baicalensis stems and / or leaves; or the aqueous extract collected after the above stirring extraction is left standing overnight, the precipitate is collected, and dissolved and dispersed in water to be used as the aqueous extract of Scutellaria baicalensis stems and / or leaves; or the aqueous extract collected after the above stirring extraction is membrane - concentrated, the pH is adjusted to 3 - 5, left standing overnight, the precipitate is collected, and dissolved and dispersed in water to be used as the aqueous extract of Scutellaria baicalensis stems and / or leaves.

7. The preparation method of the scutellarin extract according to any one of claims 1 - 6, characterized in that, Based on the dry weight of the dried whole herb of the plant of the genus Erigeron in the Compositae family, Scutellaria baicalensis stems and / or leaves, the mass ratio of the raw material of the plant of the genus Erigeron in the Compositae family used in the aqueous extract or the aqueous solution after removing ethanol from the ethanol extract of the dried whole herb of the plant of the genus Erigeron in the Compositae family to the raw material of Scutellaria baicalensis stems and / or leaves used in the aqueous extract of Scutellaria baicalensis stems and / or leaves is 10:(0.25 - 5).

8. The preparation method of the scutellarin extract according to any one of claims 1 - 6, characterized in that, The scutellarin extract satisfies: 60% ≤ scutellarin content < 80%.

9. The preparation method of the scutellarin extract according to claim 8, characterized in that, For the scutellarin extract, it is extracted with ethanol with a concentration ≥ 70%, the ethanol extract is collected, the ethanol is recovered, and precipitated or crystallized, or further repeat "extraction - collection - recovery - precipitation or crystallization" to obtain a purified and refined scutellarin extract, with 80% ≤ scutellarin content < 100%.

10. The preparation method of the scutellarin extract according to claim 1, characterized in that, Before combining and reacting with the aqueous extract of Scutellaria baicalensis stems and / or leaves, 0.2 - 5% of sodium metabisulfite or sodium bisulfite equivalent to the weight of the dried whole herb of the plant of the genus Erigeron in the Compositae family is added to the aqueous extract or the aqueous solution after removing ethanol from the ethanol extract of the dried whole herb of the plant of the genus Erigeron in the Compositae family.

11. A scutellarin extract, characterized in that, The scutellarin extract is prepared by using the preparation method according to any one of claims 1 - 6, 9 - 10, wherein 60% ≤ scutellarin content < 100%.

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