Pretreatment method for detecting active substance epicafucatechin in ephedra root formula granules

Abstract

The invention discloses a pretreatment method for detecting an active substance epicafucatechin in radix ephedrae formula granules. The pretreatment method comprises the following steps: (1) crushing and sieving the radix ephedrae formula granules, and collecting screen underflow; (2) putting a proper amount of crushed ephedra root formula granules into a conical flask with a plug, adding ultrapure water, performing ultrasonic extraction in an ultrasonic machine, standing to room temperature, adding water to complement the weight, and filtering to obtain a filtrate; (3) extracting a proper amount of filtrate with water-saturated n-butyl alcohol, and combining extract liquor; and (4) evaporating the n-butyl alcohol extract to dryness, dissolving residues with methanol, metering the volume in a 10mL volumetric flask, and filtering to obtain a sample to be detected. The method can effectively guarantee the detection stability and accuracy of the active substance epialfallocatechin in the Chinese ephedra root formula granules.

Description

Technical Field

[0001] This invention relates to the field of active ingredient detection technology in traditional Chinese medicine formula granules, specifically to a pretreatment method for detecting the active substance epi-aureoside in ephedra root formula granules. Background Technology

[0002] Ephedra root, a traditional medicinal part, is recorded in ancient herbal classics such as the *Compendium of Materia Medica*, and is known for its antiperspirant and lung-clearing effects. Modern pharmacological studies have shown that ephedra root contains various bioactive components, among which epigallocatechin, as a key active substance, is considered to play an important role in its pharmacological effects. However, this component is easily affected by external environmental and process conditions during detection, exhibiting poor stability and significant fluctuations in detection results, which severely restricts the development and standardized evaluation of related formulations using it as a quality control marker. Therefore, establishing a stable and reliable detection method for epigallocatechin has become an urgent technical challenge in this field.

[0003] The article "Determination of Three Flavonoids in Different Parts of Ficus microcarpa by UPLC" discloses a method for simultaneously determining the contents of epiaurocin, vitexin, and isovitexin in samples from different parts of Ficus microcarpa. This method involves sieving Ficus microcarpa leaf powder, ultrasonically treating it with 50% methanol, centrifuging at 5000 r / min for 15 min, and filtering the supernatant through a 0.22 μm filter membrane to obtain the sample solution. This method uses samples from different parts of Ficus microcarpa leaves as research materials, which differs from the raw material used in this invention, namely, the ephedra root formulation granules. The centrifugation after 50% methanol extraction indicates the presence of interfering substances in the sample solution, affecting the stability and accuracy of the detection results.

[0004] The article "Analytical Method for Avramine Extract, an Effective Component of Ficus microcarpa Leaves" discloses a pretreatment method for avramine extract, an effective component of Ficus microcarpa leaves. The method involves sieving Ficus microcarpa leaf powder, ultrasonically extracting it with methanol, and filtering the sample solution through a 0.45 μm filter membrane. However, this method uses Ficus microcarpa leaves as the research material and employs ultrasonic extraction with methanol. The lack of subsequent extraction with water-saturated n-butanol may lead to fluctuations in the test results due to the potential influence of water-soluble macromolecular impurities.

[0005] The types and contents of active ingredients vary considerably among different medicinal herbs. Selecting a suitable pretreatment method based on the specific characteristics of the herb is crucial for obtaining accurate detection results. In this invention, the pretreatment of epiafercatechin in Ephedra root using existing literature-reported methods results in significant fluctuations in the detection results, leading to poor stability. Therefore, finding a stable and accurate pretreatment method for detecting epiafercatechin in Ephedra root formulation granules is an urgent problem to be solved. Summary of the Invention

[0006] In view of this, the present invention provides a pretreatment method for detecting the active substance epiarodine in ephedra root formulation granules, aiming to solve the above-mentioned problems in the background art, thereby ensuring the stability and accuracy of the detection of the active substance epiarodine in ephedra root formulation granules.

[0007] The technical solution adopted in this invention is:

[0008] A pretreatment method for detecting the active substance epidermalin in ephedra root formulation granules includes the following steps: Step 1: Crush the ephedra root formula granules, sieve them, and collect the sieve residue; Step 2: Take an appropriate amount of the crushed ephedra root formula granules into a stoppered conical flask, add ultrapure water, extract by ultrasonication in an ultrasonic machine, let stand to room temperature, add water to make up the weight, and filter to obtain the filtrate. Step 3: Take an appropriate amount of the filtrate and extract it with water-saturated n-butanol, then combine the extracts; Step 4: Evaporate the n-butanol extract to dryness, dissolve the residue in methanol, dilute to 10 mL in a volumetric flask, filter, and obtain the sample to be tested.

[0009] Preferably, the pharmacopoeia sieve used for filtering the ephedra root formula granules in step one has a mesh size of 24-65.

[0010] Preferably, the mass-to-volume ratio of the pulverized ephedra root formulation granules to ultrapure water in step two is 1:30-60 g / mL.

[0011] Preferably, in step two, the ultrasonic power is 200-300 W, the ultrasonic frequency is 20-60 kHz, and the ultrasonic time is 20-40 min.

[0012] Preferably, in step three, the volume ratio of the filtrate to water-saturated n-butanol is (1-1.5):1, and the extraction is performed 1-3 times.

[0013] Preferably, in step four, the evaporation time of the n-butanol solution is 60-80 min, and the evaporation temperature is 70-90℃.

[0014] Preferably, in step four, the residue is dissolved in a methanol aqueous solution with a volume percentage concentration of 30-50% and the volume is adjusted to 10 mL.

[0015] Preferably, in steps two and four, a 0.22 μm filter membrane is used for filtration.

[0016] This invention also provides the application of the test sample obtained by the pretreatment method in the detection of the active substance arbutin in Ephedra root formulation granules.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention studies the pretreatment factors affecting the detection stability of epi-aurein, an active substance in Ephedra root formulation granules. It was found that the amount of extractant, evaporation temperature, and evaporation time have a significant impact on the detection results. By optimizing the pretreatment factors, a pretreatment method for detecting epi-aurein, an active substance in Ephedra root formulation granules, with higher stability and stronger detection accuracy was obtained.

[0018] 2. The overall operation method of this invention is simple, and the detection results have high volatility and accuracy. It is particularly suitable for the detection of epi-aureochemical in ephedra root formula granules. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of this invention pertain. If any definition stated in this section is contrary to or otherwise inconsistent with a definition stated in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definitions listed here shall prevail over those incorporated herein by reference.

[0021] Unless otherwise specified, the methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0022] Example 1 This embodiment provides a pretreatment method for detecting the active substance epifurtine in ephedra root formulation granules, and the specific steps of the method are as follows: The granules of Ephedra root were pulverized and passed through a 24-mesh pharmacopoeia sieve. The sieve residue (1 g) was weighed into a stoppered conical flask, and 30 mL of ultrapure water was added. The flask was ultrasonically extracted for 20 min (ultrasonic power 200 W, ultrasonic frequency 20 kHz). After standing to room temperature, water was added to make up the weight, and the mixture was filtered to obtain the filtrate. 15 mL of the filtrate was extracted once with 15 mL of water-saturated n-butanol solution. Finally, the n-butanol extract was evaporated to dryness at 90 ℃ for 60 min. The residue was dissolved in 30% methanol aqueous solution and diluted to volume with 30% methanol aqueous solution in a 10 mL volumetric flask. The solution was filtered through a 0.22 μm filter membrane to obtain the sample to be tested. The sample was prepared in parallel six times using this method.

[0023] Example 2 This embodiment provides a pretreatment method for detecting the active substance epifurtine in ephedra root formulation granules, and the specific steps of the method are as follows: The granules of Ephedra root were pulverized and passed through a 50-mesh pharmacopoeia sieve. The sieve residue was collected, and 1 g was weighed into a stoppered conical flask. 50 mL of ultrapure water was added, and the mixture was ultrasonically extracted for 30 min (ultrasonic power 250 W, ultrasonic frequency 40 kHz). After standing to room temperature, water was added to make up the weight, and the mixture was filtered to obtain the filtrate. 25 mL of the filtrate was extracted twice with 20 mL of water-saturated n-butanol solution, and the extracts were combined. Finally, the n-butanol extract was evaporated to dryness at 80 ℃ for 70 min. The residue was dissolved in 40% methanol aqueous solution and diluted to volume with 40% methanol aqueous solution in a 10 mL volumetric flask. The solution was filtered through a 0.22 μm filter membrane to obtain the sample to be tested. The sample was prepared in parallel six times using this method.

[0024] Example 3 This embodiment discloses a pretreatment method for detecting the active substance epifurtine in ephedra root formulation granules, the specific steps of which are as follows: The granules of Ephedra root were pulverized and passed through a 65-mesh pharmacopoeia sieve. The sieve residue (1 g) was weighed into a stoppered conical flask, and 60 mL of ultrapure water was added. The flask was ultrasonically extracted for 40 min (300 W, 60 kHz). After standing at room temperature, water was added to make up the weight, and the mixture was filtered to obtain the filtrate. 30 mL of the filtrate was extracted three times with 20 mL of water-saturated n-butanol solution, and the extracts were combined. Finally, the n-butanol extract was evaporated to dryness at 70 °C for 80 min. The residue was dissolved in 50% methanol aqueous solution and diluted to volume with 50% methanol aqueous solution in a 10 mL volumetric flask. The solution was filtered through a 0.22 μm filter membrane to obtain the sample to be tested. The sample was prepared in parallel six times using this method.

[0025] Comparative Example 1 In this comparative example, after ultrasonic extraction, water-saturated n-butanol solution was not used for extraction, i.e., steps three and four were omitted, resulting in six parallel sample solutions.

[0026] Comparative Example 2 In this comparative example, the n-butanol extract was evaporated to dryness at 100 °C, and the other steps remained unchanged, resulting in 6 parallel sample solutions.

[0027] Comparative Example 3 In this comparative example, the n-butanol extract was evaporated to dryness for 90 min, while the other steps remained unchanged, resulting in 6 parallel sample solutions.

[0028] Comparative Example 4 In this comparative example, 2 mg of epidermal tea extract reference standard was used to replace the ephedra root formula granules in step one, while the other steps remained unchanged, resulting in 6 parallel sample solutions.

[0029] Experimental Example 1 1. Instruments: Agilent 1290Ⅱ High Performance Liquid Chromatograph (HPLC) from Agilent Technologies, Inc.; SK8200HP Ultrasonic Cleaner from Shanghai Kedao Ultrasonic Instrument Co., Ltd.; BSA224S-CW Analytical Balance from Sartorius Scientific Instruments (Beijing) Co., Ltd. 2. Reagents: n-Butanol Sinopharm Chemical Reagent Co., Ltd.; Thermo Fisher Scientific, Inc. Phosphoric acid, Shanghai Maclean Biochemical Technology Co., Ltd.; Table Afuer tea extract standard (purity 99.95%), National Institutes for Food and Drug Control (NIFDC).

[0030] 3. Preparation of reference standards and samples: 3.1 Preparation of reference solution Take an appropriate amount of epiaurantia extract reference standard, accurately weigh it, and add 30% methanol to prepare a 10 μg / mL reference solution.

[0031] 3.2 Preparation of sample solutions The sample solutions were prepared using the pretreatment methods described in the Examples, Comparative Examples and Experimental Example 3.1 of this invention.

[0032] 4. Chromatographic conditions: Chromatographic column: Waters Acquity BEH shield RP18 column (2.1 mm × 100 mm, 1.7 μm); mobile phase: acetonitrile-0.1% phosphoric acid solution (15:85); flow rate: 0.3 mL / min; column temperature: 25 ℃; detection wavelength: 210 nm. Injection volume: 1 μL.

[0033] 5. Testing methods: Accurately pipette 1 μL each of the reference solution and the sample solutions of the examples and comparative examples, inject them into the liquid chromatograph, and measure them to obtain the results.

[0034] The experimental results are shown in Table 1.

[0035] Table 1 shows the RSD values ​​for the detection of tea extract content in each example and comparative example.

[0036] As shown in Table 1, the RSD values ​​for the epiarodine content detection in Examples 1-3 are all less than 2%, indicating that the epiarodine content in the samples obtained using the pretreatment method of this invention is highly stable. In Comparative Example 1, the RSD value for epiarodine is significantly higher than 2%, indicating that the absence of water-saturated n-butanol solution extraction after ultrasonic extraction leads to large fluctuations in the epiarodine content detection results. The RSD values ​​for epiarodine in Comparative Examples 2-3 are also significantly higher than 2%, indicating that the evaporation temperature and time of n-butanol evaporation have a significant impact on the stability of the epiarodine content detection results in the Ephedra root formulation granules. In Comparative Example 4, the RSD value for epiarodine is significantly lower than 2%, indicating that the pretreatment method in this patent has strong stability and high accuracy.

[0037] To further highlight the inventiveness of this invention, the inventors conducted the following experiments to demonstrate the advantages of the method of this invention, as shown in Experiment 2, Experiment 3 and Experiment 4.

[0038] Experimental Example 2 The effect of different volume ratios of filtrate to water-saturated n-butanol (0.5:1, 1:1, 1.2:1, 1.5:1, 2:1) on the stability of the detection results was investigated. The volume ratios of filtrate to water-saturated n-butanol were varied, with the remaining steps the same as in Example 1. The detection results are shown in Table 2.

[0039] Table 2. Effect of different volume ratios of filtrate to water-saturated n-butanol on the stability of detection results.

[0040] As shown in Table 2, the volume ratio of the filtrate to water-saturated n-butanol used in this invention can ensure that the RSD value of the epiaurantchain content detection is less than 2%, thereby stabilizing the epiaurantchain content detection value.

[0041] Experimental Example 3 The effect of different evaporation temperatures (60, 70, 80, 90, 100 °C) on the stability of the detection results was investigated. The evaporation temperature of the n-butanol extract was varied, while the remaining steps were the same as in Example 1. The detection results are shown in Table 3.

[0042] Table 3. Effect of different n-butanol extract evaporation temperatures on the stability of detection results.

[0043] As shown in Table 3, the evaporation temperature of the n-butanol extract in this invention can ensure that the RSD value of the epiaurocin content detection is less than 2%, thereby stabilizing the epiaurocin content detection value. Temperatures higher or lower than the n-butanol evaporation temperature in this invention will cause large fluctuations in the epiaurocin content detection value.

[0044] Experiment Example 4 The effect of different evaporation times (50, 60, 70, 80, 90 min) of the n-butanol extract on the stability of the detection results was investigated. The evaporation time of the n-butanol extract was varied, while the remaining steps were the same as in Example 1. The detection results are shown in Table 4.

[0045] Table 4. Effect of different n-butanol extract evaporation times on the stability of detection results.

[0046] As shown in Table 4, the evaporation time of the n-butanol extract in this invention can ensure that the RSD value of the epiaurea extract content detection is less than 2%, thereby stabilizing the epiaurea extract content detection value. Evaporation times higher or lower than those in this invention will cause large fluctuations in the epiaurea extract content detection value.

[0047] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A pretreatment method for detecting the active substance epidermalin in ephedra root granules, characterized in that, Includes the following steps: Step 1: Crush the ephedra root formula granules, sieve them, and collect the sieve residue; Step 2: Take an appropriate amount of the crushed ephedra root formula granules into a stoppered conical flask, add ultrapure water, extract by ultrasonication in an ultrasonic machine, let stand to room temperature, add water to make up the weight, and filter to obtain the filtrate. Step 3: Take an appropriate amount of the filtrate and extract it with water-saturated n-butanol, then combine the extracts; Step 4: Evaporate the n-butanol extract to dryness, dissolve the residue in methanol, dilute to 10 mL in a volumetric flask, filter, and obtain the sample to be tested.

2. The pretreatment method according to claim 1, characterized in that, In step one, the pharmacopoeia sieve used for filtering the ephedra root formula granules is 24-65 mesh.

3. The pretreatment method according to claim 1, characterized in that, In step two, the mass-to-volume ratio of the pulverized ephedra root formulation granules to ultrapure water is 1:30-60 g / mL.

4. The pretreatment method according to claim 1, characterized in that, In step two, the ultrasonic power is 200-300 W, the ultrasonic frequency is 20-60 kHz, and the ultrasonic time is 20-40 min.

5. The pretreatment method according to claim 1, characterized in that, In step three, the volume ratio of the filtrate to water-saturated n-butanol is (1-1.5):1, and the extraction is performed 1-3 times.

6. The pretreatment method according to claim 1, characterized in that, In step four, the n-butanol solution is evaporated to dryness for 60-80 minutes at a temperature of 70-90℃.

7. The pretreatment method according to claim 1, characterized in that, In step four, the residue is dissolved in a 30-50% methanol aqueous solution and the volume is adjusted to 10 mL.

8. The pretreatment method according to claim 1, characterized in that, In steps two and four, a 0.22 μm filter membrane is used for filtration.

9. The use of a sample obtained by the pretreatment method according to any one of claims 1-8 in the detection of the active substance epidermal extract in ephedra root formulation granules.

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