Extraction method for improving transfer rate of kaempferol-3-O-beta-D-sophoroside in equisetum hyemale medicinal material
By using water, ascorbic acid and polyethylene glycol compound solution for low-temperature immersion and mechanical stirring in the extraction of pike medicinal materials, the transfer rate of yamphe-3-O-β-D-sogoside was improved, and the solution was solved The problem of low extraction rate in the prior art reduces the content requirements in medicinal materials and avoids difficulties in purchasing medicinal materials and waste of resources.
Patent Information
- Application Number
- CN202411306107.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-19
- Publication Date
- 2024-12-20
AI Technical Summary
In the prior art, the extraction rate of Zhongshan thief medicinal material Zhongshan thief-3-O-β-D-sogoside is low, resulting in difficulty in purchasing medicinal materials and waste of resources, making it difficult to meet the content requirements in drug standards.
The extraction method of water, ascorbic acid and polyethylene glycol is used to perform low-temperature irrigation combined with mechanical stirring to improve enzyme activity and solubility and improve the transfer rate of yamphenol-3-O-β-D-sogoside.
It significantly improved the content transfer rate of Shanxi Phenol-3-O-β-D-sogoside in the trefoil extract, reduced the content requirements in raw materials, increased from 20% to 230%, and solved the difficulties in purchasing medicinal materials and waste of resources. The problem.
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Abstract
Description
Technical Field
[0001] The invention relates to an extraction method for improving the transfer rate of kaempferol-3-O-β-D-sophoroside in horsetail medicinal materials, and belongs to the technical field of traditional Chinese medicine extraction. Background Art
[0002] The horsetail formula granules YBZ-PFKL-2022041 have been published as a national drug standard. Water is used as the solvent in the standard, and the specification is that 1g of the formula granules is equivalent to 5.5g of the decoction pieces. It is stipulated that the content of kaempferol-3-O-β-D-sophoroside in each 1g of the finished product should be 2.0mg~6.0mg.
[0003] Equisetum is the dried aerial part of the plant Equisetum equisetum of the family Equisetaceae. It is a species included in the pharmacopoeia. Currently, there is no control standard for the content of kaempferol-3-O-β-D-sophoroside. Lai Zhengquan et al. found that the content of kaempferol-3-O-β-D-sophoroside in Equisetum is mainly concentrated in 0.06% to 0.54%, with an average value of about 0.22% and a median value of about 0.20%. 23 batches of Equisetum medicinal materials were purchased from the market, and the content of kaempferol-3-O-β-D-sophoroside ranged from 0.10% to 0.28%, with an average value of about 0.20% and a median value of about 0.21%.
[0004] Kaempferol-3-O-β-D-sophoroside belongs to the flavonol class of compounds, and the experimental results show that it has poor thermal stability. In the prior art, when horsetail is cut into 5mm segments, decocted twice, and the total amount of water added is more than 20 times, the transfer rate of kaempferol-3-O-β-D-sophoroside content is about 20%. In fact, in the industrial production process of horsetail formula granules, it is also necessary to undergo concentration and drying processes, so the transfer rate of kaempferol-3-O-β-D-sophoroside content in horsetail extract is generally about 15%. If the lower limit of the kaempferol-3-O-β-D-sophoroside content requirement of horsetail formula granules is 2.0 mg / g, and the transfer rate of kaempferol-3-O-β-D-sophoroside content in the prior art is calculated as 15%, the content of kaempferol-3-O-β-D-sophoroside in horsetail raw materials should not be less than 0.243%. In reality, the content of kaempferol-3-O-β-D-sophoroside in most horsetail raw materials is difficult to meet the theoretical requirements. For example, according to the research data of Lai Zhengquan et al., about 80% of the batches do not meet the requirements. According to the research data of self-purchased medicinal materials, about 65% of the batches do not meet the requirements, which leads to difficulties in purchasing medicinal materials and waste of medicinal resources. Summary of the invention
[0005] Purpose of the invention: The purpose of the present invention is to provide a method for extracting kaempferol-3-O-β-D-sophoroside from Equisetum arvense with a transfer rate of up to 230%.
[0006] Technical solution: The present invention provides an extraction method for improving the transfer rate of kaempferol-3-O-β-D-sophoroside in horsetail medicinal materials, comprising the following steps: pre-treating the horsetail medicinal materials, adding water and stirring, then adding ascorbic acid and polyethylene glycol, soaking at low temperature, filtering, and obtaining the horsetail medicinal material extract.
[0007] Furthermore, the weight ratio of the horsetail herb to ascorbic acid is 100:0.5-2.
[0008] Furthermore, the weight ratio of the horsetail herb to ascorbic acid is 100:1-2.
[0009] Furthermore, the weight ratio of the horsetail herb to polyethylene glycol is 100:0.5-1.
[0010] Furthermore, the weight ratio of the horsetail herb to polyethylene glycol is 100:0.8-1.
[0011] Furthermore, the polyethylene glycol includes one of polyethylene glycol 1500, polyethylene glycol 4000 or polyethylene glycol 6000.
[0012] Furthermore, the temperature of the low-temperature warm immersion is 40-65°C.
[0013] Furthermore, the temperature of the low-temperature warm immersion is 40-50°C.
[0014] Furthermore, the low-temperature soaking time is 2 to 6 hours.
[0015] Furthermore, the low-temperature soaking time is 4 to 6 hours.
[0016] In the technical solution of the present application, a composite solution of water, ascorbic acid and polyethylene glycol is used as the extraction solvent, wherein ascorbic acid, as an enzyme activator, can directly increase the enzyme activity and increase the conversion of precursor substances to kaempferol-3-O-β-D-sophoroside; on the other hand, as a pH regulator, the pH value of the extract can be maintained within a suitable acidic range, thereby indirectly increasing the enzyme activity and increasing the conversion of precursor substances to kaempferol-3-O-β-D-sophoroside; polyethylene glycol (model 1500 or 4000 or 6000), as an enzyme activator, can directly increase the enzyme activity and increase the conversion of precursor substances to kaempferol-3-O-β-D-sophoroside; on the other hand, as a solubilizer, it can increase the solubility of kaempferol-3-O-β-D-sophoroside in the extract. In addition, the technical solution of the present application adopts an extraction method of low-temperature warm immersion combined with mechanical stirring to provide a suitable conversion temperature for the enzyme while avoiding the loss of kaempferol-3-O-β-D-sophoroside due to thermal instability at high temperature, thereby greatly improving the transfer rate of kaempferol-3-O-β-D-sophoroside content in the horsetail extract, thereby greatly reducing the content requirement of kaempferol-3-O-β-D-sophoroside in the raw material.
[0017] Beneficial effects: Compared with the prior art, the present invention has the following outstanding significant advantages: the extraction method of the present invention is based on the thermal stability of kaempferol-3-O-β-D-sophoroside in horsetail raw materials and the transformation law of its precursor substances, selects a composite solution of water, ascorbic acid and polyethylene glycol as an extraction solvent, and adopts a low-temperature warm immersion combined with a mechanical stirring extraction method, so that the extraction transfer rate of kaempferol-3-O-β-D-sophoroside in horsetail can be increased to 230% at the highest, so that the content requirement of kaempferol-3-O-β-D-sophoroside in horsetail raw materials can be reduced to 0.016% at most, which solves the problems of difficulty in purchasing horsetail medicinal materials and waste of medicinal material resources, and reduces the difficulty of material selection. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 is the liquid chromatogram of kaempferol-3-O-β-D-sophoroside reference solution;
[0019] Figure 2 The liquid chromatogram of the extract solution of Example 1 is shown;
[0020] Figure 3 The liquid chromatogram of the extract solution of Example 2 is shown in FIG.
[0021] Figure 4 The liquid chromatogram of the extract solution of Example 4 is shown in FIG.
[0022] Figure 5 The liquid chromatogram of the extract for testing solution of Example 5;
[0023] Figure 6 The liquid chromatogram of the extract of comparative example 1 and the test solution. DETAILED DESCRIPTION
[0024] The technical solution of the present invention is further described below in conjunction with specific technical solutions.
[0025] Equisetum arvense (batch number: 1022302088) was purchased from Bozhou Jiyang Zhenyuan Chinese Medicine Pieces Co., Ltd.
[0026] Example 1
[0027] 1. Pre-treatment
[0028] Remove the dead stems and roots of the horsetail herb and cut into 5mm segments.
[0029] 2. Extraction
[0030] Add 20 times the weight of water to the horsetail medicinal material, turn on the mechanical stirring device (50r / min), and then add ascorbic acid and polyethylene glycol 6000, wherein the weight ratio of horsetail medicinal material, ascorbic acid and polyethylene glycol 6000 is 100:1:0.8. Raise the temperature to 50°C, keep warm at 50°C for 6 hours, and filter the extract through a 200-mesh sieve to obtain the horsetail medicinal material extract (pH 4.18).
[0031] Example 2
[0032] 1. Pre-treatment
[0033] Remove the dead stems and roots of the horsetail herb and cut into 5mm segments.
[0034] 2. Extraction
[0035] Add 20 times the weight of water to the horsetail medicinal material, turn on the mechanical stirring device (50r / min), and then add ascorbic acid and polyethylene glycol 4000, wherein the weight ratio of horsetail medicinal material, ascorbic acid and polyethylene glycol 4000 is 100:2:1. Raise the temperature to 50°C, keep warm at 50°C for 4 hours, and filter the extract through a 200-mesh sieve to obtain the horsetail medicinal material extract (pH 4.06).
[0036] Example 3
[0037] 1. Pre-treatment
[0038] Remove the dead stems and roots of the horsetail herb and cut into 5mm segments.
[0039] 2. Extraction
[0040] Add 20 times the weight of water to the horsetail medicinal material, turn on the mechanical stirring device (50r / min), and then add ascorbic acid and polyethylene glycol 1500, wherein the weight ratio of horsetail medicinal material, ascorbic acid and polyethylene glycol 1500 is 100:1.5:1. Raise the temperature to 65°C, keep warm at 65°C for 4 hours, and filter the extract through a 200-mesh sieve to obtain the horsetail medicinal material extract (pH 4.09).
[0041] Example 4
[0042] 1. Pre-treatment
[0043] Remove the dead stems and roots of the horsetail herb and cut into 5mm segments.
[0044] 2. Extraction
[0045] Add 20 times the weight of water to the horsetail medicinal material, turn on the mechanical stirring device (50r / min), and then add ascorbic acid and polyethylene glycol 6000, wherein the weight ratio of horsetail medicinal material, ascorbic acid and polyethylene glycol 6000 is 100:1:0.8. Raise the temperature to 40°C, keep warm at 40°C for 6 hours, and filter the extract through a 200-mesh sieve to obtain the horsetail medicinal material extract (pH 4.20).
[0046] Example 5
[0047] 1. Pre-treatment
[0048] Remove the dead stems and roots of the horsetail herb and cut into 5mm segments.
[0049] 2. Extraction
[0050] Add 20 times the weight of water to the horsetail medicinal material, turn on the mechanical stirring device (50r / min), and then add ascorbic acid and polyethylene glycol 4000, wherein the weight ratio of horsetail medicinal material, ascorbic acid and polyethylene glycol 4000 is 100:0.5:0.5. Raise the temperature to 50°C, keep warm at 50°C for 2 hours, and filter the extract through a 200-mesh sieve to obtain the horsetail medicinal material extract (pH 4.35).
[0051] Comparative Example 1
[0052] 1. Pre-treatment
[0053] Remove the dead stems and roots of the horsetail herb and cut into 5mm segments.
[0054] 2. Water Extraction
[0055] First decoction: add 10 times the weight of water to the horsetail medicinal material, heat to boiling, extract with slight boiling for 0.5 hour, filter the extract through a 200-mesh sieve to obtain the first decoction; after the first decoction, add 10 times the weight of water of the horsetail medicinal material to the medicinal residue, heat to boiling, extract with slight boiling for 0.5 hour, filter the extract through a 200-mesh sieve to obtain the second decoction; combine the first and second decoctions to obtain the extract.
[0056] Comparative Example 2
[0057] 1. Pre-treatment
[0058] Remove the dead stems and roots of the horsetail herb and cut into 5mm segments.
[0059] 2. Water Extraction
[0060] First decoction: add 10 times the weight of water to the horsetail medicinal material, soak it at room temperature (25°C) for 2 hours, then heat it to boiling, extract it with a slight boiling for 0.5 hour, filter the extract through a 200-mesh sieve to obtain a first decoction; after the first decoction, add 10 times the weight of water of the medicinal piece to the residue, heat it to boiling, extract it with a slight boiling for 0.5 hour, filter the extract through a 200-mesh sieve to obtain a second decoction; combine the first and second decoctions to obtain an extract.
[0061] Comparative Example 3
[0062] 1. Pre-treatment
[0063] Remove the dead stems and roots of the horsetail herb and cut into 5mm segments.
[0064] 2. Water Extraction
[0065] First decoction: add 10 times the weight of water to the horsetail medicinal material, heat to boiling, extract with slight boiling for 2.5 hours, and filter the extract through a 200-mesh sieve to obtain a first decoction; after the first decoction, add 10 times the weight of water of the horsetail medicinal material to the medicinal residue, heat to boiling, extract with slight boiling for 0.5 hour, and filter the extract through a 200-mesh sieve to obtain a second decoction; combine the first and second decoctions to obtain an extract.
[0066] The Equisetum equisetum extract prepared in Examples 1 to 5 and Comparative Examples 1 to 3 was subjected to pre-test treatment to prepare an extract test solution. Preparation method: Accurately pipette 2 mL of Equisetum equisetum extract into a 10 mL volumetric flask, add ethanol to the scale, shake well, filter, and take the filtrate to obtain.
[0067] Preparation of reference solution: Take an appropriate amount of kaempferol-3-O-β-D-sophoroside reference substance, accurately weigh it, and add 10% methanol to make a solution containing 10 μg per 1 ml.
[0068] Preparation method of medicinal material test sample solution: Take about 0.75g of the powder of this product (passed through No. 3 sieve), weigh it accurately, put it in a stoppered conical flask, accurately add 50ml of 10% methanol, stopper it tightly, weigh it, soak it for 24h, ultrasonically treat it for 30min, cool it, make up for the weight loss, shake it well, filter it, and obtain it.
[0069] Take the extract test solution, reference solution and medicinal material test solution, determine the content of kaempferol-3-O-β-D-sophoroside according to the high performance liquid chromatography method (General Rule 0512 of the 2020 edition of the Chinese Pharmacopoeia), and calculate the transfer rate.
[0070] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the provisions in Table 1, flow rate 0.30 ml per minute; column temperature 30°C; detection wavelength 265 nm. Injection volume 1 μl.
[0071] Table 1 Elution conditions
[0072] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～20 7→25 93→75
[0073] Kaempferol-3-O-β-D-sophoroside content transfer rate = (extract content μg·mL -1 × extract volume mL) ÷ (Equisetum content % g / g × Equisetum weight g) × 100%.
[0074] The test results of each embodiment and comparative example are shown in Table 2.
[0075] Table 2 Transfer rate of kaempferol-3-O-β-D-sophoroside content in Equisetum arvense under different extraction methods
[0076]
[0077] As can be seen from Table 2, under the extraction method provided by the embodiment of the present invention, the transfer rate of kaempferol-3-O-β-D-sophoroside content in the extract is much higher than that of the conventional extraction method, and can be increased from about 20% in the prior art to 230% at the highest, indicating that the present method can convert a large amount of kaempferol-3-O-β-D-sophoroside precursors in horsetail into kaempferol-3-O-β-D-sophoroside. Compared with the existing extraction technology, the requirement for the content of kaempferol-3-O-β-D-sophoroside in horsetail raw materials is greatly reduced, from 0.243% to 0.016%.
Claims
1. A method for improving the transfer rate of kaempferol-3-O-β-D-sophoroside in horsetail medicinal materials, characterized in that: The following steps are involved: After pre-treatment of the horsetail medicinal material, water is added and stirred, and then ascorbic acid and polyethylene glycol are added, and the mixture is soaked at low temperature and filtered to obtain the horsetail medicinal material extract.
2. The extraction method according to claim 1, characterized in that The weight ratio of the horsetail herb to ascorbic acid is 100:0.5-2.
3. The extraction method according to claim 1, characterized in that The weight ratio of the horsetail medicinal material to ascorbic acid is 100:1-2.
4. The extraction method according to claim 1, characterized in that The weight ratio of the horsetail medicinal material to polyethylene glycol is 100:0.5-1.
5. The extraction method according to claim 1, characterized in that The weight ratio of the horsetail medicinal material to polyethylene glycol is 100:0.8-1.
6. The extraction method according to claim 1, characterized in that The polyethylene glycol includes one of polyethylene glycol 1500, polyethylene glycol 4000 or polyethylene glycol 6000.
7. The extraction method according to claim 1, characterized in that The temperature of the low temperature soaking is 40-65°C.
8. The extraction method according to claim 1, characterized in that The temperature of the low temperature soaking is 40-50°C.
9. The extraction method according to claim 1, characterized in that The low temperature soaking time is 2 to 6 hours.
10. The extraction method according to claim 1, characterized in that The low temperature soaking time is 4 to 6 hours.