A method for identifying Nobilis soft purple gromwell

Abstract

This invention discloses a method for identifying *Lithospermum oryzae*, which includes morphological observation, microscopic identification, thin-layer chromatography identification, and high-performance liquid chromatography identification. Experiments show that the root head of *Lithospermum oryzae* is densely covered with hairs, some of which grow in the bark. The bark is brittle and easily broken, and the woody core is large, sometimes reaching several centimeters. In component identification, *Lithospermum oryzae* lacks β-acetoxyethylcarboxylic acid, which can serve as a marker component for identifying *Lithospermum oryzae*. The method established in this invention can accurately distinguish *Lithospermum oryzae* from *Lithospermum erythrorhizon*. The identification method provided by this invention has the advantages of being fast, simple, and accurate.

Description

Technical Field

[0001] This invention relates to a method for identifying Chinese medicinal materials, and particularly to a method for identifying Arnebia nobilis Rech. fil. Background Technology

[0002] The traditional Chinese medicine Arnebiaeuchroma is the dried root of a plant in the Boraginaceae family, primarily sourced from Xinjiang. Its properties and meridian tropism are: sweet, salty, and cold; it enters the heart and liver meridians. It has the effects of clearing heat and cooling blood, promoting blood circulation and detoxifying, and promoting rash eruption and eliminating spots. Clinically, it is used to treat conditions such as blood heat and toxicity, sores, eczema, vulvar itching, and burns. However... Xinjiang gromwell is mainly a wild resource, and although it is still included in the pharmacopoeia, the resources are nearly exhausted. Currently, the mainstream gromwell products on the market are "imported gromwell," which mostly originate from Arnebia nobilis Rech.fil., produced in Pakistan and other regions. Due to its large yield, low price, and similar characteristics to Xinjiang gromwell, it circulates in the market.

[0003] There is currently no reliable and accurate method for distinguishing between Xinjiang Lithospermum and Nobilis soft Lithospermum. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for identifying Arnebia nobilis Rech.fil. This method can quickly and accurately identify Arnebia nobilis and can be applied in pharmaceutical testing. It is of great significance to ensure the quality of Arnebia and control its efficacy.

[0005] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows: A method for identifying Nobilis soft purple gromwell, characterized by including morphological observation, microscopic identification, thin-layer chromatography identification, and high-performance liquid chromatography identification.

[0006] As a preferred method, the identification method for *Lithospermum erythrorhizon* described above includes the following morphological observations: It is conical or cylindrical, twisted, 10–50 cm long, and 1–20 cm in diameter, with several large, thick root heads. The inner surface of the root head is smooth, while the outer surface is densely covered with fine white hairs. The surface is purplish-red or dark purple, the bark is thin, often with multiple overlapping layers, easily peeled off, hard and brittle, easily broken, with a relatively neat cross-section. The bark is purplish-red, the xylem is large, yellowish-white, solid, 0.5–2 cm in diameter, has a peculiar odor, and an astringent taste.

[0007] As a preferred option, the microscopic identification characteristics of the above-described method for identifying *Lithospermum erythrorhizon* are as follows: skin non-glandular hairs: thick-walled, straight, with flat or rough walls; leaves non-glandular hairs: thin-walled, soft.

[0008] As a preferred embodiment, the identification method for Nobilis soft comfrey described above, wherein the component identification is that Nobilis soft comfrey does not contain β-acetoxyethyl valerate acetonitrile.

[0009] As a preferred embodiment, the identification method for Nobilis soft comfrey described above, wherein the component identification method is as follows: take Nobilis soft comfrey powder, add cyclohexane, sonicate, filter, concentrate the filtrate, and use it as Nobilis soft comfrey test solution. Xinjiang soft gromwell was used as a reference herb. Cyclohexane was added, the mixture was ultrasonically treated, filtered, and the filtrate was concentrated to obtain the Xinjiang soft gromwell reference herb solution. Separately prepare reference solutions of deoxyshikonin, β,β-dimethylacryloylshikonin, acetylshikonin, β-acetoxyvalerate acarine and shikonin reference standards; Take the test solution of Nobilis soft gromwell, the reference herb solution of Xinjiang soft gromwell, and the solutions of each reference standard, and spot them separately on the same silica gel G thin layer plate. Use petroleum ether-benzene-acetone-formic acid (volume ratio 4.5:5.5:1:0.1) as the developing solvent, develop, remove, and air dry. In the chromatogram of Nobilis soft gromwell test sample, except for β-acetoxyethyl valerate acarine, the other reference standards appear as purple-red spots. The solution of Xinjiang soft purple gromwell reference material showed purplish-red spots of all reference components.

[0010] As a preferred embodiment, the high-performance liquid chromatography (HPLC) method for identifying Noberrine comfrey described above is as follows: (1) Preparation of reference solution: Take an appropriate amount of β-acetoxyvalerate acarine reference standard, add ethanol to prepare a solution containing 0.1 mg per ml; (2) Preparation of test solution: Take the medicinal powder, pass it through a No. 4 sieve, weigh it accurately, place it in a stoppered conical flask, add cyclohexane accurately, weigh it, sonicate it, cool it, weigh it again, make up the weight loss with cyclohexane, shake it well, and filter it; accurately measure the filtrate, evaporate it to dryness, dissolve the residue in the mobile phase, transfer it to a volumetric flask, add the mobile phase to the mark, shake it well, filter it, and take the filtrate to obtain the test solution; (3) Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine that the limonite root does not contain β-acetoxyvalerate acetonitrile.

[0011] As a preferred option, in the identification method of Noberrin soft purple gromwell described above, step (3) uses acetonitrile-water-trifluoroacetic acid with a volume ratio of 75:25:0.1 as the mobile phase and the detection wavelength is 275nm.

[0012] Application of β-acetoxyvalerate as a marker for distinguishing between Lithospermum erythrorhizon and Lithospermum praecox.

[0013] As another preferred option, β-acetoxyvalerate can be used to identify Arnebianobilis Rech.fil. and its extracts and prepared traditional Chinese medicine preparations. Beneficial effects

[0014] This invention discloses a method for identifying the root of Arnebia nobilis Rech.fil. Based on traditional empirical and physicochemical identification methods, this invention identifies Arnebia nobilis and Xinjiang Lithospermum. Experiments show that the root head of Arnebia nobilis is densely covered with hairs, some of which grow on the bark. The bark is brittle and easily broken, and the woody core is large, sometimes reaching several centimeters. In component identification, Arnebia nobilis lacks the β-acetoxyethylcarboxylamine spot. The identification method provided by this invention has the advantages of being fast, simple, and accurate. Attached Figure Description

[0015] Figure 1 These are morphological diagrams of Lithospermum erythrorhizon. A. Whole Lithospermum erythrorhizon (without root head), B. Whole Lithospermum erythrorhizon (with root head) and root head, C. Top view of root head, D. Side view of root head, E. Root head hairs, F. Cross-section, G. Lithospermum erythrorhizon bark.

[0016] Figure 2 The image shows the non-glandular hairs of *Lithospermum erythrorhizon*. A. *Lithospermum erythrorhizon* bark, non-glandular hairs, under natural light; B. *Lithospermum erythrorhizon* bark, non-glandular hairs, under polarized light; C. *Lithospermum erythrorhizon* bark, non-glandular hairs, under natural light; D. *Lithospermum erythrorhizon* bark, non-glandular hairs, under polarized light.

[0017] Figure 3 These are thin-layer chromatography (TLC) images of the components of *Lithospermum erythrorhizon* and *Lithospermum xanthina*. A. *Lithospermum erythrorhizon*, B. *Lithospermum erythrorhizon*, C. *Lithospermum xanthina*, D. *Lithospermum xanthina*.

[0018] Figure 4 The results are HPLC chromatograms. A represents mixed standard, B represents Nobilis alpinia oxyphylla, and C represents Xinjiang alpinia oxyphylla. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0020] Example 1

[0021] A method for identifying Nobilis soft purple gromwell, which includes morphological observation, microscopic identification and component identification.

[0022] 1. Experimental Materials. 1.1 Observation of morphological characteristics Method: The samples were placed under natural light for morphological observation.

[0023] Results: The root is conical or cylindrical, twisted, 10–50 cm long and 1–20 cm in diameter. It has several large root heads, the inner part of which is smooth, while the outer part is densely covered with fine white hairs. The surface is purplish-red or dark purple, the bark is thin, often in overlapping layers, and easily peeled off. It is hard and brittle, easily broken, with a relatively neat cross-section. The bark is purplish-red, the xylem is large, yellowish-white, solid, and 0.5–2 cm in diameter. It has a peculiar odor and an astringent taste.

[0024] 1.2 Microscopic observation Pass the sample powder through a No. 5 sieve, take a small amount and place it on a glass slide, add 1-2 drops of chloral hydrate test solution, gently heat over an alcohol lamp to allow it to bleed through, and cover with a coverslip. After cooling, observe and photograph the characteristics under a microscope.

[0025] Results: Skin non-glandular hairs: thick-walled, straight, with flat or rough walls; Leaf non-glandular hairs: thin-walled, soft.

[0026] 1.3 Thin-layer chromatography (TLC) identification Take 1g of this product powder, add 10ml of cyclohexane, sonicate for 15 minutes, filter, and concentrate the filtrate to 1ml to obtain the test solution. Take 1g of newly prepared *Lithospermum erythrorhizon* reference material and prepare a reference material solution using the same method. Separately, prepare 1mg / mL reference solutions of deoxyshikonin, β,β-dimethylacryloylshikonin, acetylshikonin, β-acetoxyvalerate, and shikonin. Apply 5μl of each solution separately to the same silica gel G thin-layer plate, develop using petroleum ether-benzene-acetone-formic acid (4.5:5.5:1:0.1) as the developing solvent, remove, and air dry. In the chromatogram of the test sample, purple-red spots appear at the corresponding positions as in the chromatogram of the reference material.

[0027] like Figure 3 Experimental results: Nobilis soft comfrey lacks β-acetoxyethyl valerate acarine.

[0028] 1.4 The high-performance liquid chromatography (HPLC) identification method is as follows: Preparation of reference solution Take an appropriate amount of β-acetoxyvalerate acarinin reference standard, add ethanol to prepare a solution containing 0.1 mg per 1 ml; Preparation of test solution Accurately weigh approximately 1g of the herbal powder (passed through a No. 4 sieve), place it in a stoppered conical flask, accurately add 50ml of cyclohexane, weigh again, sonicate (250W power, 33kHz frequency) for 30 minutes, cool, weigh again, replenish the lost weight with cyclohexane, shake well, and filter. Accurately measure 10ml of the subsequent filtrate, evaporate to dryness, dissolve the residue in the mobile phase, transfer to a 10ml volumetric flask, add the mobile phase to the mark, shake well, filter, and collect the subsequent filtrate.

[0029] (3) Accurately pipette 10 μl of the reference solution and the test solution respectively, and inject them into the liquid chromatograph (chromatographic conditions: acetonitrile-water-trifluoroacetic acid with a volume ratio of 75:25:0.1 as the mobile phase, detection wavelength of 275 nm, C 18 Chromatographic column, column temperature 30℃, flow rate 1mL / min, determination, such as Figure 4 As shown, Nobilis soft purple gromwell does not contain β-acetoxyvalerate acarine, while Xinjiang soft purple gromwell does contain β-acetoxyvalerate acarine.

[0030] 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 method for identifying Nobilis soft purple gromwell, characterized in that, This includes morphological observation, microscopic identification, thin-layer chromatography identification, and high-performance liquid chromatography identification.

2. The method for identifying Nobilis Soft Lithospermum according to claim 1, characterized in that, The observed characteristics include: conical or cylindrical shape, twisted, 10–50 cm long, 1–20 cm in diameter, with several thick root heads, the inner side of the upper part of the root head is smooth, and the outer side is densely covered with fine white hairs. The surface is purplish-red or dark purple, the bark is thin, often with multiple overlapping layers, easily peeled off, hard and brittle, easily broken, with a relatively neat cross-section, the bark is purplish-red, the xylem is large, yellowish-white, solid, 0.5–2 cm in diameter, with a peculiar odor and astringent taste.

3. The method for identifying Nobilis Soft Lithospermum according to claim 1, characterized in that, The microscopic identification features are as follows: for non-glandular hairs on the skin: thick-walled, straight, with flat or rough walls; for non-glandular hairs on the leaves: thin-walled, soft.

4. The method for identifying Nobilis Soft Lithospermum according to claim 1, characterized in that, The method for identifying the components is as follows: take Nobilis soft purple gromwell powder, add cyclohexane, sonicate, filter, concentrate the filtrate, and use it as the Nobilis soft purple gromwell test solution; Xinjiang soft gromwell was used as a reference herb. Cyclohexane was added, the mixture was ultrasonically treated, filtered, and the filtrate was concentrated to obtain the Xinjiang soft gromwell reference herb solution. Separately prepare reference solutions of deoxyshikonin, β,β-dimethylacryloylshikonin, acetylshikonin, β-acetoxyvalerate acarine and shikonin reference standards; Take the test solution of Nobilis soft gromwell, the reference herb solution of Xinjiang soft gromwell, and the solutions of each reference standard, and spot them separately on the same silica gel G thin layer plate. Use petroleum ether-benzene-acetone-formic acid (volume ratio 4.5:5.5:1:0.1) as the developing solvent, develop, remove, and air dry. In the chromatogram of Nobilis soft gromwell test sample, except for β-acetoxyethyl valerate acarine, the other reference standards appear as purple-red spots. The solution of Xinjiang soft purple gromwell reference material showed purplish-red spots of all reference components.

5. The method for identifying Nobilis Soft Lithospermum according to claim 1, characterized in that, The high-performance liquid chromatography (HPLC) identification method is as follows: (1) Preparation of reference solution: Take an appropriate amount of β-acetoxyvalerate acarine reference standard, add ethanol to prepare a solution containing 0.1 mg per ml; (2) Preparation of test solution: Take the medicinal powder, pass it through a No. 4 sieve, weigh it accurately, place it in a stoppered conical flask, add cyclohexane accurately, weigh it, sonicate it, cool it, weigh it again, make up the weight loss with cyclohexane, shake it well, and filter it; accurately measure the filtrate, evaporate it to dryness, dissolve the residue in the mobile phase, transfer it to a volumetric flask, add the mobile phase to the mark, shake it well, filter it, and take the filtrate to obtain the test solution; (3) Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine that the limonite root does not contain β-acetoxyvalerate acetonitrile.

6. The method for identifying Nobilis Soft Lithospermum according to claim 4, characterized in that, Step (3) uses acetonitrile-water-trifluoroacetic acid with a volume ratio of 75:25:0.1 as the mobile phase, and the detection wavelength is 275 nm. 18 Chromatographic column, column temperature 30℃, flow rate 1mL / min.

7. Application of β-acetoxyvalerate as a marker for distinguishing between Lithospermum erythrorhizon and Lithospermum praecox.

8. Application of β-acetoxyvalerate as a marker for identifying Lithospermum erythrorhizon and its extracts and prepared traditional Chinese medicine preparations.

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