Quality standard detection method of herba patriniae decoction pieces

Abstract

The invention discloses a quality standard detection method of a patrinia villosa decoction piece, which researches the character, microscopy, thin layer, moisture, acid-insoluble ash content, extract and content determination of the patrinia villosa decoction piece, and the content determination adopts high performance liquid chromatography to determine the content of chlorogenic acid in the patrinia villosa decoction piece. The method for detecting the quality standard of the herba patriniae decoction pieces has the advantages of being good in reproducibility and high in specificity, and reliable scientific basis is provided for development and utilization of the herba patriniae decoction pieces.

Description

Technical Field

[0001] The invention relates to the technical field of quality standards for Chinese medicinal material pieces, and in particular to a method for detecting the quality standards of Patrinia scabra pieces. Background Art

[0002] Patrinia scabra is the dried whole herb of Sonchus transcaspicus Nevski., a plant of the Asteraceae family. It is also known as wild bitter vegetable and bitter bitter vegetable. There are also records about Patrinia scabra in ancient books. "Shennong's Herbal Classic" says: "Bitter vegetable" is listed as a top-grade product, saying that "it tastes bitter and cold, and can cure evil spirits in the five internal organs, aversion to food, and stomach numbness. Long-term use can calm the mind and replenish qi, make people smarter and less prone to sleep, and make the body lighter and more resistant to aging." Patrinia scabra can clear away heat and detoxify, clear swelling and discharge pus, and promote blood circulation and remove blood stasis. It is used for painful swelling caused by herpes, dysentery caused by lung abscess and intestinal abscess, postpartum blood stasis, abdominal pain, etc.

[0003] Related studies have found that the chemical components of the extract of Patrinia scabra are mainly flavonoids, phenylpropanoids, steroids, etc. Flavonoids mainly contain luteolin, luteolin, quercetin, etc. Phenylpropanoids contain caffeic acid, chlorogenic acid, etc. Chlorogenic acid is abundant in many plants and has good anti-inflammatory and antioxidant activities. It is consistent with the efficacy of Patrinia scabra and is suitable as a substance for qualitative and quantitative control of the standard of Patrinia scabra. At present, the Chinese Pharmacopoeia has not yet included the medicinal materials and pieces of Patrinia scabra in Chinese medicine. The standards included in the local medicinal materials standards and preparation specifications of pieces of pieces in some provinces and cities are not perfect, with only properties or microscopic identification items, and the degree of control is low. In order to further improve the quality standards of this variety, this study collected 14 batches of Patrinia scabra pieces from different origins. After determining the origin, the properties, microscopic identification, thin layer identification, moisture, acid-insoluble ash and extracts of 14 batches of Patrinia scabra samples from different origins were systematically studied. A new thin-layer identification method was proposed, the limits of each inspection item were determined, and the content of chlorogenic acid was determined by liquid chromatography to improve and enhance the quality standards of existing Patrinia dasyphylla slices. Summary of the invention

[0004] The invention aims to provide a quality standard detection method for Patrinia scabra decoction pieces, which can scientifically and effectively evaluate and control the intrinsic quality and medication quality of the medicinal material.

[0005] The technical solution adopted by the present invention to solve the problem is: a quality standard detection method for Patrinia scabra slices, establishing property identification, microscopic identification, thin layer identification, moisture, acid-insoluble ash, extract and content determination, and the content determination adopts high performance liquid chromatography to determine the content of chlorogenic acid in Patrinia scabra slices.

[0006] The source is: this product is a processed product of the dried whole herb of Sonchus transcaspicus Nevski., a plant of the Asteraceae family.

[0007] The properties are as follows: This product is segmented. The stem is cylindrical, the surface is yellow-brown, the longitudinal ridges are obvious, and most of them are hollow. The leaves are curled or broken, gray-green, and have small sharp teeth on the edges. The texture is brittle. The smell is slight and the taste is slightly bitter.

[0008] The microscopic identification is the microscopic identification of the surface of the medicinal leaf, and the characteristics are as follows: the upper and lower epidermal cells are polygonal, the anticlimax wall is straight or slightly curved; the upper epidermal stomata are relatively few, the lower epidermal stomata are numerous, and some are connected in pairs. The stomata are unequal or indefinite, and there are 3 to 5 accessory cells. The thin layer chromatography identification steps are as follows: taking Patrinia chinensis powder, adding ethanol, reflux treatment, filtering, evaporating the filtrate to dryness, adding ethyl acetate to the residue to dissolve it, and using it as a test solution; taking another Patrinia chinensis control medicinal material, and preparing a control medicinal material solution in the same way; according to the thin layer chromatography test, absorbing the above solutions, spotting them on the same silica gel G plate respectively, using dichloromethane-ethyl acetate-formic acid-water as a developing agent, developing, taking out, drying, spraying with an ethanol solution of sulfuric acid with a mass concentration of 10%, heating at 105°C until the spots are clearly colored, and inspecting under ultraviolet light, comparing the states of the test sample and the control medicinal material solutions at the corresponding positions, and determining the quality of the Patrinia chinensis slices.

[0009] The specific identification steps of the thin layer chromatography are as follows: take 0.5g of the powder of this product, add 20mL of ethanol, heat and reflux for 1 hour, filter, evaporate the filtrate, and add 1mL of ethyl acetate to the residue to dissolve it as the test solution. Take 0.5g of the control medicinal material of Patrinia scabra and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 2μL of each of the above two solutions are taken and spotted on the same silica gel G thin layer plate, respectively, with dichloromethane-ethyl acetate-formic acid-water volume ratio (25:1:0.1:0.1) as the developing agent, develop, take out, dry, spray with ethanol solution of 10% sulfuric acid by mass concentration, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365nm). In the chromatogram of the test product, fluorescent spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material.

[0010] The general inspection items include moisture, acid-insoluble ash, and water-soluble extracts: (1) Moisture inspection: Determine the moisture content in accordance with the second method of the general rule 0832 Moisture Determination Method of the 2020 edition of the Chinese Pharmacopoeia (Volume IV). The mass fraction of moisture shall not exceed 12.0%. (2) Acid-insoluble ash inspection: Determine the acid-insoluble ash content in accordance with the ash determination method of the general rule 2302 of the 2020 edition of the Chinese Pharmacopoeia (Volume IV). The mass fraction of acid-insoluble ash shall not exceed 2.0%. (3) Extract inspection: Determine the water-soluble extract of the Patrinia dasyphylla slices in accordance with the hot leaching method under the general rule 2201 of the 2020 edition of the Chinese Pharmacopoeia (Volume IV), using water as the solvent. The mass fraction of the extract shall not be less than 23.0%.

[0011] The content determination method is as follows.

[0012] (1) Chromatographic conditions and system suitability test

[0013] High performance liquid chromatography was used to quantitatively analyze chlorogenic acid. The chromatographic conditions and system suitability test for determining the peak area were as follows: octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as the mobile phase A, and 0.3% mass concentration of phosphoric acid was used as the mobile phase B; the detection wavelength was 327 nm; the flow rate was 1 mL / min, and the injection volume was 10 μL; the theoretical plate number calculated based on the chlorogenic acid peak should be no less than 20,000; the gradient elution program was: time 0 to 25 minutes, mobile phase A volume fraction 8→15%, mobile phase B volume fraction 92→85%; Time 25-35 minutes, mobile phase A volume fraction 15→60%, mobile phase B volume fraction 85→40%;

[0014] (2) Preparation of reference solution

[0015] Accurately weigh an appropriate amount of chlorogenic acid and add 50% methanol to prepare a reference solution containing 20 μg per 1 mL.

[0016] (3) Preparation of test solution

[0017] Accurately weigh 1.0 g of the decoction powder (passed through No. 3 sieve), accurately add 50 mL of 50% methanol, weigh the weight, heat and reflux for 1 hour, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, pass through a 0.45 μm microporous filter membrane, and obtain the filtrate.

[0018] (4) Determination method

[0019] Accurately pipette 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0020] This product is calculated on the basis of dry product. 16 H 18 O9) The mass fraction shall not be less than 0.040%.

[0021] Beneficial effects of the present invention.

[0022] The identification method of the medicinal pieces established in the present invention has good reproducibility and strong specificity. The content determination method can separate the chlorogenic acid content in a short time, and has good peak shape and separation degree, and can effectively evaluate and control the intrinsic quality and medication quality of the medicinal pieces. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram of the properties of the medicinal slices of Patrinia dasyphylla.

[0024] Figure 2 This is a microscopic image of the leaf surface of the Patrinia dasyphylla medicinal piece.

[0025] Figure 3 This is the TLC diagram for the identification of Patrinia dasyphylla slices (1 and 16 are control herbs, 2-15 are 14 batches of test samples).

[0026] Figure 4-1 HPLC chart of the reference substance. Figure 4-2 This is the HPLC chart of the test sample of Patrinia dasyphylla decoction pieces.

[0027] Figure 5 This is the linear curve of chlorogenic acid. DETAILED DESCRIPTION

[0028] According to the method and standard provided by the present invention, quality inspection is performed on different batches of Patrinia dasyphylla decoction pieces.

[0029] Embodiments of the present invention.

[0030] Instruments used: Flow-type Chinese medicine pulverizer (DLF-18, Wenzhou Dingli Medical Instrument Co., Ltd.); electronic analytical balance (AL-204, Shanghai Mettler-Toledo Instrument Co., Ltd.); SOP 1 / 100,000 microanalytical balance (Sartorius Scientific Instrument Co., Ltd.); blast drying oven (101-2AB, Tianjin Test Instrument Co., Ltd.); double-beam UV-visible spectrophotometer (Shanghai Yuanxi Instrument Co., Ltd.); constant temperature water bath (DZKW-S-4, Beijing Yongguangming Medical Instrument Co., Ltd.); muffle furnace (SX2-4-10, Shenyang Energy Saving Instrument Factory); silica gel G thin layer plate (Yantai Chemical Industry Research Institute); LC-10ATVP high performance liquid chromatograph (Shimadzu Corporation, Japan); biological microscope (CX22RFS1, Olympus Corporation).

[0031] Test drugs used: Chlorogenic acid reference substance (batch number 110753-202119, content 96.3%), purchased from China Food and Drug Inspection Institute; analytical grade formic acid, ethanol, ethyl acetate, dichloromethane (Tianjin Damao Chemical Reagent Factory); chromatographic grade phosphoric acid (Tianjin Damao Chemical Reagent Factory); chromatographic grade acetonitrile (Sunrise Chem, USA); Wahaha purified water (Hangzhou Wahaha Group Co., Ltd.).

[0032] [Source] This product is a processed product of the dried whole herb of Sonchus transcaspicus Nevski., a plant of the Asteraceae family.

[0033] The 14 batches of Patrinia chinensis slices originated from slice production enterprises in Hebei Province, Anguo Chinese medicinal materials market, etc. The information of Patrinia chinensis slices is shown in Table 1.

[0034] Table 1 Source information of Patrinia dasyphylla decoction pieces.

[0035]

[0036]

Properties

[0037]

Identification

[0038] Microscopic identification steps of leaf surface: pinch the Patrinia dasyphylla slices between the thumbs and index fingers of both hands, tear them apart diagonally, tear off a small piece of the upper and lower epidermis of the leaf with tweezers, immerse them in water droplets on the slide and flatten them, and observe them under a microscope.

[0039] The samples that meet the following characteristics are the Northern Patrinia scabra slices: the upper and lower epidermal cells are polygonal, the vertical wall is straight or slightly curved; the upper epidermal stomata are few, the lower epidermal stomata are numerous, and some are connected in pairs. The stomata are unequal or indefinite, and there are 3 to 5 accessory cells. Figure 2 .

[0040]

Identification

[0041] The steps are as follows: take 0.5g of the powder of this product, add 20mL of ethanol, heat and reflux for 1 hour, filter, evaporate the filtrate, add 1mL of ethyl acetate to the residue to dissolve it, and use it as the test solution. Take another 0.5g of the control medicinal material of Patrinia dasyphylla and prepare the control medicinal solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the "Chinese Pharmacopoeia"), take 2μL of each of the above two solutions and spot them on the same silica gel G thin layer plate, use dichloromethane-ethyl acetate-formic acid-water (25:1:0.1:0.1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365nm). See the results. Figure 3 In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material, which meets the quality requirements.

[0042] [Inspection] General inspections include moisture, acid-insoluble ash, and extractables. The following inspections must meet the quality requirements.

[0043] (1) Moisture inspection: The detection method is determined in accordance with the second method of General Chapter 0832 of the 2020 edition of the "Chinese Pharmacopoeia" (Part IV). The moisture mass fraction shall not exceed 12.0%. Each batch of samples is measured in parallel 3 times and the average value is taken. As a result, the moisture mass fraction of the samples in this embodiment is 7.3-9.9%, with an average of 8.3%. See Table 2 for details.

[0044] (2) Acid-insoluble ash inspection: The acid-insoluble ash inspection is carried out in accordance with General Rule 2302 of the 2020 edition of the Chinese Pharmacopoeia (Part IV). The mass fraction of the acid-insoluble ash shall not exceed 2.0%. Each batch of samples shall be measured in parallel 3 times. In this embodiment, the mass fraction of the acid-insoluble ash of the samples is 0.8% to 2.3%, with an average value of 1.4%. The acid-insoluble ash is detailed in Table 2.

[0045] (3) Extract inspection: According to the hot infusion method under Item 2201 of the General Rules of the 2020 edition of the Chinese Pharmacopoeia (Volume 4), water was used as the solvent to determine the water-soluble extract of Patrinia dasyphylla decoction pieces, and the mass fraction shall not be less than 23.0%. Each batch of samples was measured three times in parallel and the average value was taken. The mass fraction of water-soluble extract ranged from 27.2% to 38.1%, and the average mass fraction was 33.3%, as shown in Table 2.

[0046] Table 2 Determination results of moisture content, acid-insoluble ash and extract of Patrinia dasyphylla slices.

[0047]

[0048] [Content Determination] After literature research and based on the chemical components contained in the Patrinia scabra slices, chlorogenic acid was selected as the content determination component of the Patrinia scabra slices from the perspective of the characteristics of the components, the source and content of the reference substances, and an HPLC method was developed according to the high performance liquid chromatography method (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia) to determine the content of chlorogenic acid in the Patrinia scabra slices. The specific steps are as follows.

[0049] 1. The chromatographic conditions for determining the peak area are: Chromatographic conditions are: InertsiL ODS-SP C18 chromatographic column (250×4.6 mm, 5 μm); mobile phase acetonitrile (A)-0.3% phosphoric acid (B), flow rate 1.0 mL / min; column temperature 30°C; injection volume 10 μL; detection wavelength 327 nm; gradient elution program: Time 0-25 minutes, mobile phase A volume fraction 8→15%, mobile phase B volume fraction 92→85%; Time 25-35 minutes, mobile phase A volume fraction 15→60%, mobile phase B volume fraction 85→40%;

[0050] 2. Solution Preparation

[0051] 2.1 Preparation of reference solution

[0052] Accurately weigh an appropriate amount of chlorogenic acid and add 50% methanol to make a solution containing 20 μg per 1 mL.

[0053] 2.2 Preparation of test solution

[0054] 2.2.1 Selection of extraction method

[0055] Take 2 portions of the same batch of samples (No. BBJC-004), each 1g, accurately weighed, placed in a stoppered conical flask, use 50% methanol by volume as the extraction solvent, the extraction time is 60 minutes, and use ultrasonic and reflux extraction methods to test respectively, and inject and measure under the chromatographic conditions of "1". The results show that the reflux extraction method has a higher determination result, and reflux is selected as the extraction method of this experiment (results are shown in Table 3).

[0056] Table 3 Effect of extraction method on chlorogenic acid

[0057]

[0058] 2.2.2 Selection of extraction solvent

[0059] Take the same batch of samples (No. BBJC-004), 5 portions, 1g each, accurately weighed, placed in a stoppered conical flask, and used 30% methanol by volume, 40% methanol by volume, 50% methanol by volume, 60% methanol by volume, and 70% methanol by volume as extraction solvents, respectively, reflux extraction for 60 minutes, prepare the test solution, and inject and measure under the chromatographic conditions of "1". The results show that the content determination result is the highest when using 50% methanol by volume as the extraction solvent, so 50% methanol by volume is selected as the extraction solvent (results are shown in Table 4).

[0060] Table 4 Effect of extraction solvent on chlorogenic acid extraction rate

[0061]

[0062] 2.2.3 Selection of extraction time

[0063] Take the same batch of samples (No. BBJC-004), 4 portions, 1g each, accurately weighed, placed in a stoppered conical flask, and refluxed for 30min, 45min, 60min, and 75min respectively, and determined according to the proposed chromatographic conditions. The results showed that the content determination result of the extraction time of 60 minutes was higher, so the extraction time of 60 minutes was selected (the results are shown in Table 5).

[0064] Table 5 Effect of extraction time on chlorogenic acid extraction rate

[0065]

[0066] The final method for preparing the test solution of Patrinia dasyphylla decoction pieces was determined as follows: accurately weigh 1.0 g of decoction piece powder (passed through No. 3 sieve), accurately add 50 mL of 50% by volume methanol, weigh the weight, heat and reflux for 1 hour, cool, weigh again, make up the lost weight with 50% by volume methanol, shake well, filter, and pass through a 0.45 μm microporous filter membrane to obtain the filtrate.

[0067] 3. System specificity test: Inject the test sample and standard sample according to the above chromatographic conditions and record the chromatogram. Figure 4-1 and Figure 4-2 The number of theoretical plates calculated based on chlorogenic acid is not less than 20,000, and the blank solution has no interference with the determination.

[0068] 4. Investigation of the methodology of content determination by high performance liquid chromatography.

[0069] (1) Linear relationship investigation: Take the reference solution under "2.1" and use the injection volume as the horizontal axis X, and inject 1μL, 4μL, 8μL, 12μL, and 16μL respectively. Perform the injection measurement under the chromatographic conditions of "1". Record the peak area and use the peak area as the vertical axis Y to draw the standard curve. Calculate the linear regression equation, linear range, and correlation coefficient of chlorogenic acid. The concentration of chlorogenic acid shows a good linear relationship between 0.04105 and 0.8210μg (R 2 =0.9989), the average recovery rate was 94.84%, the results are shown in Figure 5 ,It can be seen from the figure that each component has a good linear relationship within its respective concentration range.

[0070] (2) Precision test: Accurately pipette the reference solution of item "2.1" and inject it 6 times continuously under the chromatographic conditions of item "1". The RSD of the chlorogenic acid peak area was measured to be 0.18%, indicating that the instrument has good precision. The results are shown in Table 6.

[0071] Table 6 Precision test results.

[0072]

[0073] (3) Stability test: The same sample solution was accurately aspirated and measured under the chromatographic conditions of item "1". The samples were injected at 0, 2, 6, 10, 16, and 24 hours. The RSD of the chlorogenic acid peak area was 0.43%, indicating that the sample solution had good stability within 24 hours. The results are shown in Table 7.

[0074] Table 7 Stability test results.

[0075]

[0076] (4) Repeatability test: 6 portions of the same batch of decoction powder were accurately weighed, and the test solution was prepared according to the conditions in item "2.2". The chlorogenic acid content was measured under the chromatographic conditions in item "1". The RSD of chlorogenic acid content was 0.93%, indicating that the method had good repeatability. The results are shown in Table 8.

[0077] Table 8 Repeatability test results.

[0078]

[0079] (5) Sample addition recovery test: Accurately weigh 6 portions of herbal medicine powder, add 1 mL of reference solution to each portion, prepare the test solution according to the conditions in item "2.2", and measure under the chromatographic conditions in item "1". Calculate the recovery rate. The results show that this method has a good recovery rate. The results are shown in Table 9.

[0080] Table 9 The recovery test results of chlorogenic acid in Patrinia dasyphylla slices.

[0081]

[0082] (6) Sample content determination: Take an appropriate amount of samples from each batch and prepare the test solution according to the conditions in item “2.2”. Determine the solution under the chromatographic conditions in item “1” and calculate the average content of chlorogenic acid. The results are shown in Table 10.

[0083] Table 10 Results of content determination of Patrinia dasyphylla decoction pieces.

[0084]

[0085] By adopting the quality detection method of the Patrinia chinensis slices of the present invention, the quality detection method of the Patrinia chinensis slices is improved, the quality control of the medicinal materials is achieved in an all-round and systematic manner, and a reliable scientific basis is provided for the development and utilization of Patrinia chinensis.

[0086] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A quality standard detection method for Patrinia dasyphylla decoction pieces, comprising establishing property identification, microscopic identification, thin layer identification, water content, acid-insoluble ash, extract and content determination, characterized in that: The content determination adopts high performance liquid chromatography to determine the content of chlorogenic acid in the Patrinia scabra slices.

2. The quality standard detection method of a Patrinia dasyphylla decoction piece according to claim 1, characterized in that: The properties are as follows: segment-shaped, cylindrical stem, yellow-brown surface, obvious longitudinal ridges, hollow, curled or broken leaves, gray-green, with small sharp teeth on the edges, crisp texture, slight odor, and slightly bitter taste.

3. The quality standard detection method of a Patrinia dasyphylla decoction piece according to claim 1, characterized in that: The microscopic identification is a microscopic identification of the surface of the medicinal leaf. The upper and lower epidermal cells are polygonal, and the anticlimax walls are straight or slightly curved. The number of stomata in the upper epidermis is less than that in the lower epidermis, the stomata are unequal or indefinite, and there are 3 to 5 accessory cells.

4. The quality standard detection method of a Patrinia dasyphylla decoction piece according to claim 1, characterized in that: The thin layer chromatography identification steps are as follows: taking Patrinia chinensis powder, adding ethanol, reflux treatment, filtering, evaporating the filtrate to dryness, adding ethyl acetate to the residue to dissolve it, and using it as a test solution; taking another Patrinia chinensis control medicinal material, and preparing a control medicinal material solution in the same way; according to the thin layer chromatography test, absorbing the above solutions, spotting them on the same silica gel G plate respectively, using dichloromethane-ethyl acetate-formic acid-water as a developing agent, developing, taking out, drying, spraying with an ethanol solution of sulfuric acid with a mass concentration of 10%, heating at 105°C until the spots are clearly colored, and inspecting under ultraviolet light, comparing the states of the test sample and the control medicinal material solutions at the corresponding positions, and determining the quality of the Patrinia chinensis slices.

5. The quality standard detection method of a Patrinia dasyphylla decoction piece according to claim 1, characterized in that: The volume ratio of dichloromethane:ethyl acetate:formic acid:water is 25:1:0.1:0.

1.

6. The quality standard detection method of a Patrinia dasyphylla decoction piece according to claim 1, characterized in that: The content determination method is as follows: HPLC is used to quantitatively analyze chlorogenic acid, and the chromatographic conditions and system suitability test for determining the peak area are as follows: octadecylsilane bonded silica gel is used as filler; acetonitrile is used as mobile phase A, and 0.3% mass concentration of phosphoric acid is used as mobile phase B; the detection wavelength is 327nm; the flow rate is 1mL / min, and the injection volume is 10μL; the theoretical plate number calculated according to the chlorogenic acid peak should be not less than 20000; the gradient elution program is: Time 0-25 minutes, mobile phase A volume fraction 8→15%, mobile phase B volume fraction 92→85%; The time is 25 to 35 minutes, the volume fraction of mobile phase A is 15→60%, and the volume fraction of mobile phase B is 85→40%.

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