阅读说明：本技术 一种药材胭脂花根的薄层鉴别方法及其应用 (Thin-layer identification method for medicinal material carmine flower root and application thereof ) 是由 张志杰 张海燕 单海涛 穆建国 甘惠仍 文丽 于 2021-02-07 设计创作，主要内容包括：本发明公开一种药材胭脂花根的薄层鉴别方法,其包括以下步骤：采用相同的方式分别将待鉴别的药材胭脂花根样品进行前处理得到供试品溶液、将对照药材样品进行前处理得到对照药材溶液；以及对该对供试品溶液和该对照药材溶液进行薄层色谱鉴别分析；其中,该薄层色谱鉴别分析的步骤中采用甲苯-乙酸乙酯-丙酮作为展开剂,甲苯、乙酸乙酯和丙酮的体积比为(1～5):(1～5):(1～5)。本发明的上述薄层鉴别方法还可应用于区分药材胭脂花根和药材喜马拉雅紫茉莉根以及鉴别包含药材胭脂花根的疗痔胶囊。本发明的上述鉴别方法展开剂选择适当、鉴别效率高、鉴别精度高并且操作方便。(The invention discloses a thin-layer identification method of a medicinal material common primrose root, which comprises the following steps: respectively pretreating a primula maximowiczii root sample to be identified in the same way to obtain a test solution, and pretreating a reference medicinal material sample to obtain a reference medicinal material solution; and carrying out thin-layer chromatography identification analysis on the test solution and the reference medicinal material solution; wherein, in the step of the thin-layer chromatography identification analysis, toluene-ethyl acetate-acetone is used as a developing solvent, and the volume ratio of toluene to ethyl acetate to acetone is (1-5) to (1-5). The thin-layer identification method can also be applied to distinguishing the medicinal material carmine root from the medicinal material himalayan Mirabilis root and identifying the hemorrhoid treatment capsule containing the medicinal material carmine root. The identification method has the advantages of proper selection of the developing solvent, high identification efficiency, high identification precision and convenient operation.)

1. A thin-layer identification method for medicinal material cochineal flower root comprises the following steps:

(1) respectively pretreating a primula maximowiczii root sample to be identified in the same way to obtain a test solution, and pretreating a reference medicinal material sample to obtain a reference medicinal material solution; and

(2) carrying out thin-layer chromatography identification analysis on the test solution and the reference medicinal material solution;

wherein, in the step of thin-layer chromatography identification and analysis, toluene-ethyl acetate-acetone is used as a developing solvent, and the volume ratio of toluene, ethyl acetate and acetone is (1-5): 1-5.

2. A thin-layer identification method for distinguishing a medicinal material carmine root from a medicinal material himalayan Mirabilis root comprises the following steps:

(1) respectively pretreating a medicinal material primula maximowiczii root sample to obtain a primula maximowiczii root sample solution, and pretreating a medicinal material himalayan mirabilis root sample to obtain a himalayan mirabilis root sample solution in the same mode; and

(2) carrying out thin-layer chromatography identification analysis on the primula maximowiczii root sample solution and the himalayan mirabilis root sample solution;

wherein, in the step of thin-layer chromatography identification and analysis, toluene-ethyl acetate-acetone is used as a developing solvent, and the volume ratio of toluene, ethyl acetate and acetone is (1-5): 1-5.

3. A thin layer identification method of a hemorrhoid treatment capsule containing a medicinal material nopaline root comprises the following steps:

(1) respectively pretreating a hemorrhoid treatment capsule sample to be identified in the same way to obtain a test solution, and pretreating a reference medicinal material or a reference substance to obtain a reference medicinal material solution or a reference substance solution; and

(2) carrying out thin-layer chromatography identification analysis on the test solution and the reference medicinal material solution or the reference solution;

wherein, in the step of thin-layer chromatography identification and analysis, toluene-ethyl acetate-acetone is used as a developing solvent, and the volume ratio of toluene, ethyl acetate and acetone is (1-5): 1-5.

4. The thin layer identification method of any one of claims 1 to 3, wherein the volume ratio of toluene, ethyl acetate and acetone is (1-4): (2-5); preferably, the developing agent further comprises an acid; more preferably, the acid is an organic acid; particularly preferably, the organic acid is formic acid and/or acetic acid; particularly preferably, the acid is present in a proportion of 2.5% to 3.5% by volume relative to the sum of the volumes of toluene, ethyl acetate and acetone.

5. The thin layer identification method as claimed in any one of claims 1 to 3, wherein the thin layer identification method further comprises a thin layer plate activation step; preferably, the thin layer plate activating step includes activating the thin layer plate at 100 to 120 ℃ for 20 to 60 minutes; more preferably, the lamella plates are selected from the group consisting of silica gel G lamella plates, silica gel GF₂₅₄Thin layer plate, silica gel H thin layer plate, silica gel HF₂₅₄At least one of a laminate sheet and a polyamide laminate sheet.

6. The thin layer identification method of any one of claims 1 to 3, wherein the inspection conditions in the step of thin layer chromatography identification analysis are: observing under visible light; preferably, the developed thin-layer plate is taken out during the inspection, dried and sprayed with 5 to 10 percent sulfuric acid ethanol solution or 5 to 25 percent phosphomolybdic acid ethanol solution for color development; more preferably, 5 to 10 percent of sulfuric acid ethanol solution is sprayed for color development.

7. The thin layer identification method according to any one of claims 1 to 3, wherein the pretreatment comprises adding an organic solvent to a sample powder or a reference powder for 5-50 minutes, filtering, evaporating the filtrate, and dissolving the residue with the organic solvent to obtain a corresponding sample solution or a corresponding reference solution; preferably, the sample solution is spotted in an amount of 3 to 10 μ L; more preferably, the control solution is spotted in an amount of 5 to 10 μ L; particularly preferably, the concentration of the control solution is 0.15mg/mL to 0.30 mg/mL; particularly preferably, the organic solvent is any one or a mixture of more than two of methanol, ethanol, n-butanol and ethyl acetate; particularly preferably, the treatment time is 10 to 45 minutes; particularly preferably, the treatment time is 20 to 30 minutes; most preferably, the treatment method is any one or more of ultrasound, reflux, decoction and immersion.

8. The thin-layer identification method as claimed in claim 1 or 3, wherein the control is daucosterol and/or β -sitosterol; preferably, the control medicinal material is a cochineal flower root control medicinal material.

9. Use of the thin-layer identification method according to any one of claims 1 to 8 for identifying the medicinal material nopaline root or for identifying a hemorrhoid treatment capsule comprising the medicinal material nopaline root.

10. A kit for identifying medicinal material primula maximowiczii or a hemorrhoid treatment capsule containing the medicinal material primula maximowiczii comprises a thin layer plate and a developing agent; preferably, toluene-ethyl acetate-acetone is used as the developing solvent; more preferably, the volume ratio of the toluene to the ethyl acetate to the acetone is (1-5) to (1-5); particularly preferably, the volume ratio of the toluene to the ethyl acetate to the acetone is (1-4) to (2-5); particularly preferably, the developing agent further comprises an acid; particularly more preferably, the acid is an organic acid; particularly more preferably, the organic acid is formic acid and/or acetic acid; most preferably, the acid is present in a volume fraction of 2.5% to 3.5% relative to the sum of the volumes of toluene, ethyl acetate and acetone.

Technical Field

The invention relates to a Chinese herbal medicine identification method, in particular to a thin-layer identification method of a medicinal material primula maximowiczii root, a thin-layer identification method for distinguishing the medicinal material primula maximowiczii root from a medicinal material himalayan mirabilis root, a thin-layer identification method of a hemorrhoid treatment capsule containing the medicinal material primula maximowiczii root and application of the thin-layer identification method.

Background

The nopal root is dried root of Mirabilis jalapa L.of Mirabilis of Mirabilis. Collected in autumn and winter, removed fibrous root, cleaned, and sliced or directly dried. The primula maximowiczii root is also used as a drug for minority nationality in Guizhou province.

Mirabilis jalapa is originally recorded in Dian nan Ben Cao (Dian nan Ben Cao) (1397-. The first name of Zi jasmine was used in Zhao Zhi Ming Dynasty, Qing Dynasty, Ben Cao gang mu Shi Yi. Mirabilis jalapa is used in folk, and has medical records in ShaanGangqing Chinese herbal medicine selection, Guizhou folk prescription medicine collection, Fujian medicine YangZhi, Tujian medicine collection and national Chinese herbal medicine compilation. Modern researches find that the mirabilis jalapa root has the effects of resisting virus, resisting cancer, resisting tumors, resisting bacteria and reducing blood sugar. At present, Mirabilis jalapa root is habitually called as carmine root in Guizhou province and is collected and carried by the quality standards of traditional Chinese medicinal materials and national medicinal materials in Guizhou province (2003 edition). Mirabilis jalapa medicinal material is also collected in the Standard of Chinese medicinal materials in Yunnan province (2005 edition) (fourth volume Yi nationality medicine).

The existing identification research of the annatto (Mirabilis asiatica Royle) root (Rayleigh et al, the identification research of the Mirabilis amara Royle root, Anhui agricultural science 2012,40(9): 5191-. Wherein, the thin layer identification only shows that the n-butanol extract on the thin layer plate has yellow brown spots separated out, and the chloroform extract and the petroleum ether extract have the same color spots at corresponding positions. However, the thin-layer identification method has the defects that a reference medicinal material, a reference substance and a specific developing agent are lacked, the identification precision is extremely low, the effective identification of the medicinal material of the carmine root (the Mirabilis javanica root) cannot be realized, and the effective identification of the medicinal material of the carmine root (the Mirabilis javanica root) and the similar medicinal material of the Mirabilis javanica root cannot be realized.

Based on the reasons, the thin-layer identification method of the medicinal material carmine root (the Mirabilis jalapa Linn.) is required to be further researched so as to solve the problems that the existing thin-layer identification method is lack of a reference medicinal material, a reference substance and specific developing agent conditions and is extremely low in identification precision.

Disclosure of Invention

Based on the above, the main objective of the present invention is to provide a thin-layer identification method for the medicinal material primula maximowiczii root, a thin-layer identification method for distinguishing the medicinal material primula maximowiczii root from the medicinal material himalayan mirabilis root, a thin-layer identification method for the hemorrhoid treatment capsule containing the medicinal material primula maximowiczii root, and an application of the thin-layer identification method, so as to solve the problems that the existing thin-layer identification method in the prior art is lack of a reference medicinal material, a reference substance, specific developing agent conditions and extremely low in identification precision.

In order to achieve the above object, according to one aspect of the present invention, there is provided a thin layer identification method for a medicinal material primula maximowiczii root, comprising the steps of:

(1) respectively pretreating a primula maximowiczii root sample to be identified in the same way to obtain a test solution, and pretreating a reference medicinal material sample to obtain a reference medicinal material solution; and

(2) carrying out thin-layer chromatography identification analysis on the test solution and the reference solution;

wherein, in the step of the thin-layer chromatography identification analysis, toluene-ethyl acetate-acetone is used as a developing solvent, and the volume ratio of toluene to ethyl acetate to acetone is (1-5) to (1-5).

According to another aspect of the present invention, there is provided a thin-layer identification method for distinguishing a medicinal material carmine root from a medicinal material heliotrope himalayan, comprising the steps of:

(1) respectively pretreating a medicinal material primula maximowiczii root sample to obtain a primula maximowiczii root sample solution, and pretreating a medicinal material himalayan mirabilis root sample to obtain a himalayan mirabilis root sample solution in the same mode; and

(2) carrying out thin-layer chromatography identification analysis on the primula maximowiczii root sample solution and the himalayan mirabilis root sample solution;

wherein, in the step of the thin-layer chromatography identification analysis, toluene-ethyl acetate-acetone is used as a developing solvent, and the volume ratio of toluene to ethyl acetate to acetone is (1-5) to (1-5).

According to another aspect of the invention, there is provided a thin layer identification method of a hemorrhoid treatment capsule containing the medicinal material nopaline root, comprising the following steps:

(1) respectively pretreating a hemorrhoid treatment capsule sample to be identified in the same way to obtain a test solution, and pretreating a reference medicinal material or a reference substance to obtain a reference medicinal material solution or a reference substance solution; and

(2) carrying out thin-layer chromatography identification analysis on the test solution and the reference medicinal material solution or the reference solution;

wherein, in the step of the thin-layer chromatography identification analysis, toluene-ethyl acetate-acetone is used as a developing solvent, and the volume ratio of toluene to ethyl acetate to acetone is (1-5) to (1-5).

Furthermore, the volume ratio of the toluene to the ethyl acetate to the acetone is (1-4) to (2-5).

Furthermore, the volume ratio of the toluene to the ethyl acetate to the acetone is (1-4) to (2-5).

Furthermore, the volume ratio of the toluene to the ethyl acetate to the acetone is (1-4) to (2-3).

Furthermore, the volume ratio of the toluene to the ethyl acetate to the acetone is (2-4) to (2-3).

Furthermore, the volume ratio of the toluene to the ethyl acetate to the acetone is 4 (2-4) to 2.

Further, the volume ratio of toluene, ethyl acetate and acetone was 4:4: 2.

Further, the developing agent further comprises an acid.

Further, the acid is an organic acid.

Further, the organic acid is formic acid and/or acetic acid.

Further, the acid is present in a volume fraction of 2.5% to 3.5% relative to the sum of the volumes of toluene, ethyl acetate and acetone.

Further, the thin layer identification method further comprises a thin layer plate activation step.

Further, the thin layer plate activating step includes activating the thin layer plate at 100 to 120 ℃ for 20 to 60 minutes.

Further, the thin layer plate is selected from silica gel G thin layer plate and silica gel GF₂₅₄Thin layer plate, silica gel H thin layer plate, silica gel HF₂₅₄At least one of a laminate sheet and a polyamide laminate sheet.

Further, the inspection conditions in the step of the thin layer chromatography differential analysis are: and (4) observing under visible light.

Further, taking out the developed thin-layer plate during inspection, airing, and spraying a 5-10% sulfuric acid ethanol solution or a 5-25% phosphomolybdic acid ethanol solution for color development.

Furthermore, the developed thin-layer plate is taken out during inspection, dried and sprayed with 5-10% sulfuric acid ethanol solution for color development.

Further, the pretreatment comprises the steps of adding an organic solvent into sample powder or reference powder for treatment for 5-50 minutes, filtering, evaporating filtrate, and dissolving residues by adding the organic solvent to obtain a corresponding sample solution or reference solution.

Further, the sample solution is spotted in an amount of 3. mu.L to 10. mu.L.

Further, the spotting amount of the control solution was 5. mu.L to 10. mu.L.

Further, the concentration of the control solution is 0.15mg/mL to 0.30 mg/mL.

Further, the organic solvent is any one or a mixture of more than two of methanol, ethanol, n-butanol and ethyl acetate.

Further, the treatment time is 10-45 minutes.

Further, the treatment time is 20-30 minutes.

Further, the treatment method comprises any one or more of ultrasound, reflux, decoction and immersion.

Further, the reference substance is daucosterol and/or beta-sitosterol.

Further, the reference medicinal material is a carmine root reference medicinal material.

According to another aspect of the invention, the thin-layer identification method is used for identifying the medicinal material nopaline root or identifying the hemorrhoid treatment capsule containing the medicinal material nopaline root.

According to another aspect of the present invention, there is provided a kit for identifying a medicinal material annatto or a hemorrhoid treatment capsule containing the medicinal material annatto, which includes a lamella plate and a developing agent.

Further, toluene-ethyl acetate-acetone was used as the developing solvent.

Furthermore, the volume ratio of the toluene to the ethyl acetate to the acetone is (1-5) to (1-5).

Furthermore, the volume ratio of the toluene to the ethyl acetate to the acetone is (1-4) to (2-5).

Furthermore, the volume ratio of the toluene to the ethyl acetate to the acetone is (1-4) to (2-5).

Furthermore, the volume ratio of the toluene to the ethyl acetate to the acetone is (1-4) to (2-3).

Furthermore, the volume ratio of the toluene to the ethyl acetate to the acetone is (2-4) to (2-3).

Furthermore, the volume ratio of the toluene to the ethyl acetate to the acetone is 4 (2-4) to 2.

Further, the volume ratio of toluene, ethyl acetate and acetone was 4:4: 2.

Further, the developing agent further comprises an acid.

Further, the acid is an organic acid.

Further, the organic acid is formic acid and/or acetic acid.

Further, the acid is present in a volume fraction of 2.5% to 3.5% relative to the sum of the volumes of toluene, ethyl acetate and acetone.

By applying the technical scheme of the invention, the thin-layer identification method has the advantages of proper selection of the developing solvent, high identification efficiency, high identification precision and convenient operation, thereby well solving the problems of lack of reference medicinal materials, lack of reference substances, lack of specific developing solvent conditions and extremely low identification precision of the existing thin-layer identification method.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without departing from the scope of the present invention as claimed.

FIG. 1 is a development of thin layer chromatography of example one (I), wherein number 1- -daucosterol; number 2- - -beta-sitosterol; number 3-reference drug (i.e. primula maximowiczii root reference drug sample) 1; number 4-control drug (i.e. primula maximowiczii root control drug sample) 2; number 5- -reference drug to be distinguished (i.e. Himalayan Mirabilis root reference sample); no. 6- -sample of test sample (i.e. Capsule for treating hemorrhoid).

FIG. 2 is a development of thin layer chromatography of example one (II), in which number 1- -daucosterol; number 2- - -beta-sitosterol; number 3-reference drug (i.e. primula maximowiczii root reference drug sample) 1; number 4-control drug (i.e. primula maximowiczii root control drug sample) 2; number 5- -reference drug to be distinguished (i.e. Himalayan Mirabilis root reference sample); no. 6- -sample of test sample (i.e. Capsule for treating hemorrhoid).

FIG. 3 is a developed view of TLC of example one (III), in which number 1- -daucosterol; number 2- - -beta-sitosterol; number 3-reference drug (i.e. primula maximowiczii root reference drug sample) 1; number 4-control drug (i.e. primula maximowiczii root control drug sample) 2; number 5- -reference drug to be distinguished (i.e. Himalayan Mirabilis root reference sample); no. 6- -sample of test sample (i.e. Capsule for treating hemorrhoid).

FIG. 4 is a development of thin layer chromatography of example two (one), in which number 1- -daucosterol; number 2- - -beta-sitosterol; number 3-reference drug (i.e. primula maximowiczii root reference drug sample) 1; number 4-control drug (i.e. primula maximowiczii root control drug sample) 2; number 5- -reference drug to be distinguished (i.e. Himalayan Mirabilis root reference sample); number 6- -test sample (i.e., Mediterranean Mirabilis root sample); no. 7- -test sample (i.e. Capsule sample for treating hemorrhoid).

FIG. 5 is a development of thin layer chromatography of example two (II), in which number 1- -daucosterol; number 2- - -beta-sitosterol; number 3-reference drug (i.e. primula maximowiczii root reference drug sample) 1; number 4-control drug (i.e. primula maximowiczii root control drug sample) 2; number 5- -reference drug to be distinguished (i.e. Himalayan Mirabilis root reference sample); number 6- -test sample (i.e., Mediterranean Mirabilis root sample); no. 7- -test sample (i.e. Capsule sample for treating hemorrhoid).

FIG. 6 is a development of thin layer chromatography of example two (III), in which number 1- -daucosterol; number 2- - -beta-sitosterol; number 3-reference drug (i.e. primula maximowiczii root reference drug sample) 1; number 4-control drug (i.e. primula maximowiczii root control drug sample) 2; number 5- -reference drug to be distinguished (i.e. Himalayan Mirabilis root reference sample); number 6- -test sample (i.e., Mediterranean Mirabilis root sample); no. 7- -test sample (i.e. Capsule sample for treating hemorrhoid).

FIG. 7 is a development of thin layer chromatography of example three (one), in which number 1- -daucosterol; number 2- - -beta-sitosterol; number 3-reference drug (i.e. primula maximowiczii root reference drug sample) 1; number 4-control drug (i.e. primula maximowiczii root control drug sample) 2; number 5- -reference drug to be distinguished (i.e. Himalayan Mirabilis root reference sample); no. 6- -sample of test sample (i.e. Capsule for treating hemorrhoid).

FIG. 8 is a development of thin layer chromatography of example III (II) in which the number 1- - -daucosterol; number 2- - -beta-sitosterol; number 3-reference drug (i.e. primula maximowiczii root reference drug sample) 1; number 4-control drug (i.e. primula maximowiczii root control drug sample) 2; number 5- -reference drug to be distinguished (i.e. Himalayan Mirabilis root reference sample); no. 6- -sample of test sample (i.e. Capsule for treating hemorrhoid).

FIG. 9 is a development of thin layer chromatography of example four (one), in which number 1- -daucosterol; number 2- - -beta-sitosterol; number 3-reference drug (i.e. primula maximowiczii root reference drug sample) 1; number 4-control drug (i.e. primula maximowiczii root control drug sample) 2; number 5- -reference drug to be distinguished (i.e. Himalayan Mirabilis root reference sample); number 6- -test sample (i.e., Mediterranean Mirabilis root sample); no. 7- -test sample (i.e. Capsule sample for treating hemorrhoid).

FIG. 10 is a development of thin layer chromatography of example four (II), in which the number 1- - -daucosterol; number 2- - -beta-sitosterol; number 3-reference drug (i.e. primula maximowiczii root reference drug sample) 1; number 4-control drug (i.e. primula maximowiczii root control drug sample) 2; number 5- -reference drug to be distinguished (i.e. Himalayan Mirabilis root reference sample); number 6- -test sample (i.e., Mediterranean Mirabilis root sample); no. 7- -test sample (i.e. Capsule sample for treating hemorrhoid).

FIG. 11 is a development of thin layer chromatography of example four (III), in which number 1- -daucosterol; number 2- - -beta-sitosterol; number 3-reference drug (i.e. primula maximowiczii root reference drug sample) 1; number 4-control drug (i.e. primula maximowiczii root control drug sample) 2; number 5- -reference drug to be distinguished (i.e. Himalayan Mirabilis root reference sample); number 6- -test sample (i.e., Mediterranean Mirabilis root sample); no. 7- -test sample (i.e. Capsule sample for treating hemorrhoid).

FIG. 12 is a development of thin layer chromatography of example five, in which number 1- -daucosterol; number 2- - -beta-sitosterol; number 3-reference drug (i.e. primula maximowiczii root reference drug sample) 1; number 4-control drug (i.e. primula maximowiczii root control drug sample) 2; number 5-control drug (i.e. primula maximowiczii root control drug sample) 3; number 6-sample (crude drug annatto root sample) 1; number 7- -sample to be tested (sample of primula maximowiczii root); number 8-sample (crude drug annatto root sample) 3; number 9- -sample to be tested (sample of primula maximowiczii root); number 10- -sample to be tested (sample of primula maximowiczii root); number 11- -sample to be tested (sample of primula maximowiczii root); number 12- -sample (sample of primula maximowiczii root) 7; no. 13- -sample to be tested (sample of Bixa orellana root).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

As described in the background art section, the existing thin layer identification methods lack the control medicinal materials, lack the control products, lack the specific developing agent conditions, and have extremely low identification accuracy. In order to solve the problems, the invention provides a thin-layer identification method of a medicinal material common primrose root, which comprises the following steps:

(1) respectively pretreating a primula maximowiczii root sample to be identified in the same way to obtain a test solution, and pretreating a reference medicinal material sample to obtain a reference medicinal material solution; and

(2) carrying out thin-layer chromatography identification analysis on the test solution and the reference solution;

wherein, in the step of the thin-layer chromatography identification analysis, toluene-ethyl acetate-acetone is used as a developing solvent, and the volume ratio of toluene to ethyl acetate to acetone is (1-5) to (1-5).

According to another aspect of the present invention, there is provided a thin-layer identification method for distinguishing a medicinal material carmine root from a medicinal material heliotrope himalayan, comprising the steps of:

(1) respectively pretreating a medicinal material primula maximowiczii root sample to obtain a primula maximowiczii root sample solution, and pretreating a medicinal material himalayan mirabilis root sample to obtain a himalayan mirabilis root sample solution in the same mode; and

(2) carrying out thin-layer chromatography identification analysis on the primula maximowiczii root sample solution and the himalayan mirabilis root sample solution;

wherein, in the step of the thin-layer chromatography identification analysis, toluene-ethyl acetate-acetone is used as a developing solvent, and the volume ratio of toluene to ethyl acetate to acetone is (1-5) to (1-5).

According to another aspect of the invention, there is provided a thin layer identification method of a hemorrhoid treatment capsule containing the medicinal material nopaline root, comprising the following steps:

(1) respectively pretreating a hemorrhoid treatment capsule sample to be identified in the same way to obtain a test solution, and pretreating a reference medicinal material or a reference substance to obtain a reference medicinal material solution or a reference substance solution; and

(2) carrying out thin-layer chromatography identification analysis on the test solution and the reference medicinal material solution or the reference solution;

wherein, in the step of the thin-layer chromatography identification analysis, toluene-ethyl acetate-acetone is used as a developing solvent, and the volume ratio of toluene to ethyl acetate to acetone is (1-5) to (1-5).

According to the method for simultaneously identifying the annatto root, the himalayan Mirabilis root and the hemorrhoid treatment capsule sample, the same pretreatment method is adopted for the annatto root, the himalayan Mirabilis root and the hemorrhoid treatment capsule sample, and a specific developing agent system is selected, so that various components in the annatto root, the himalayan Mirabilis root and the hemorrhoid treatment capsule sample can be well separated, and simultaneous identification is realized. In the chromatogram of each test sample, spots with the same color are displayed at the positions corresponding to the chromatogram of the reference substance or the chromatogram of the reference medicinal material, and strong specificity is displayed. Meanwhile, the method simplifies the operation process, reduces the time required by identification, improves the working efficiency and reduces the detection cost.

In order to achieve better separation between spots during thin layer development, in a preferred embodiment, the volume ratio of toluene, ethyl acetate and acetone is (1-4): 2-5.

In a preferred embodiment, the volume ratio of toluene, ethyl acetate and acetone is (1-4): (2-5).

In a preferred embodiment, the volume ratio of toluene, ethyl acetate and acetone is (1-4): (2-5).

In a preferred embodiment, the volume ratio of toluene, ethyl acetate and acetone is (1-4): (2-3).

In a preferred embodiment, the volume ratio of toluene, ethyl acetate and acetone is (2-4): (2-3).

In a preferred embodiment, the volume ratio of the toluene to the ethyl acetate to the acetone is 4 (2-4) to 2.

In a preferred embodiment, the volume ratio of toluene, ethyl acetate and acetone is 4:4: 2.

In order to prevent smearing of the spots during the spreading of the sheet, in a preferred embodiment, the spreading agent further comprises an acid.

In a preferred embodiment, the acid is an organic acid.

In a preferred embodiment, the organic acid is formic acid and/or acetic acid.

In a preferred embodiment, the acid is present in a volume fraction of 2.5% to 3.5% relative to the sum of the volumes of toluene, ethyl acetate and acetone.

In order to remove volatile impurities and moisture adsorbed on the laminate and improve separation efficiency, in a preferred embodiment, the laminate identification method further comprises a laminate activation step.

To further enhance the efficiency of the separation of spots on the lamina plate, in a preferred embodiment, the lamina plate activation step comprises activating the lamina plate at 100 ℃ to 120 ℃ for 20 minutes to 60 minutes.

In a preferred embodiment, the lamella plates are selected from the group consisting of silica gel G lamella plates, silica gel GF₂₅₄Thin layer plate, siliconGlue H thin layer plate, silica gel HF₂₅₄At least one of a laminate sheet and a polyamide laminate sheet.

In a preferred embodiment, the inspection conditions in the step of TLC differential analysis are: and (4) observing under visible light.

In a preferred embodiment, the developed thin-layer plate is taken out during the inspection, dried, sprayed with 5 to 10 percent sulfuric acid ethanol solution or 5 to 25 percent phosphomolybdic acid ethanol solution for color development.

In a preferred embodiment, the developed thin-layer plate is taken out during the inspection, dried and sprayed with 5-10% sulfuric acid ethanol solution for color development.

In a preferred embodiment, the pretreatment comprises adding an organic solvent into the sample powder or the reference powder for 5-50 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue with the organic solvent to obtain a corresponding sample solution or a corresponding reference solution.

In a preferred embodiment, the sample solution is spotted in an amount of 3. mu.L to 10. mu.L.

In order to allow the sample solution to show clear and non-streaking spots after thin layer development, in a preferred embodiment, the spots are most clear when the sample amount of the annatto root sample is 10 μ L; the hemorrhoid treating capsule (Miao medicine containing primula maximowiczii root) is most suitable when the sample amount is 3 μ L.

In order to allow the control solution to show clear and non-streaking spots after thin layer development, in a preferred embodiment, the control solution is spotted in an amount of 5 μ L to 10 μ L.

In a preferred embodiment, the concentration of the control solution is 0.15mg/mL to 0.30 mg/mL.

In a preferred embodiment, the organic solvent is any one or a mixture of two or more of methanol, ethanol, n-butanol and ethyl acetate.

In a preferred embodiment, the treatment time is 10 to 45 minutes.

In order to allow the sample solution or the control solution to show clear and moderate spots after the thin layer is spread, in a preferred embodiment, the treatment time is 30 to 40 minutes.

In a preferred embodiment, the treatment method is any one or more of ultrasound, reflux, decoction and immersion.

In a preferred embodiment, the control is daucosterol and/or beta-sitosterol.

In a preferred embodiment, the control drug is a cochineal root control drug.

According to another aspect of the invention, the thin-layer identification method is used for identifying the medicinal material nopaline root or identifying the hemorrhoid treatment capsule containing the medicinal material nopaline root.

According to another aspect of the present invention, there is provided a kit for identifying a medicinal material annatto or a hemorrhoid treatment capsule containing the medicinal material annatto, which includes a lamella plate and a developing agent.

In a preferred embodiment, toluene-ethyl acetate-acetone is used as the developing solvent.

In a preferred embodiment, the volume ratio of toluene, ethyl acetate and acetone is (1-5): (1-5).

In order to achieve better separation between spots during thin layer development, in a preferred embodiment, the volume ratio of toluene, ethyl acetate and acetone is (1-4): 2-5.

In a preferred embodiment, the volume ratio of toluene, ethyl acetate and acetone is (1-4): (2-5).

In a preferred embodiment, the volume ratio of toluene, ethyl acetate and acetone is (1-4): (2-3).

In a preferred embodiment, the volume ratio of toluene, ethyl acetate and acetone is (2-4): (2-3).

In a preferred embodiment, the volume ratio of the toluene to the ethyl acetate to the acetone is 4 (2-4) to 2.

In a preferred embodiment, the volume ratio of toluene, ethyl acetate and acetone is 4:4: 2.

In order to prevent smearing of the spots during the spreading of the sheet, in a preferred embodiment, the spreading agent further comprises an acid.

In a preferred embodiment, the acid is an organic acid.

In a preferred embodiment, the organic acid is formic acid and/or acetic acid.

In a preferred embodiment, the acid is present in a volume fraction of 2.5% to 3.5% relative to the sum of the volumes of toluene, ethyl acetate and acetone.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

The present invention is described in further detail below with reference to specific examples, which are not to be construed as limiting the scope of the invention as claimed herein.

The identification method of the invention comprises the following steps:

pretreating the test sample, the reference medicinal material and the reference substance respectively, and identifying by thin layer chromatography. Wherein, the pretreatment step can adopt the pretreatment means in the prior art.

Specifically, the identification method of the present invention specifically includes the steps of:

1. preparation of a test solution:

taking the powder of a test sample (i.e. a sample of medicinal material carmine roots or a sample of medicinal material himalayan Mirabilis roots or a sample of medicinal material hemorrhoid treating capsules), adding an organic solvent, treating for 5-50 minutes, filtering, evaporating the filtrate to dryness, and dissolving residues in the organic solvent to obtain a corresponding test sample solution.

2. Preparation of reference drug solution:

preparing reference medicinal material solution from powder of reference medicinal material (i.e. primula maximowiczii root reference medicinal material sample) according to the preparation method of test solution.

3. Preparation of reference medicinal material solution to be distinguished

Taking the powder of the reference medicinal material to be distinguished (i.e. the reference medicinal material sample of the himalayan Mirabilis jalapa) to prepare the solution of the reference medicinal material to be distinguished according to the preparation method of the solution of the sample.

4. Preparation of control solutions:

preparing reference substance (daucosterol and beta-sitosterol) powder into corresponding reference substance solution according to the preparation method of the test substance solution.

5. Performing thin-layer chromatography analysis on the test solution, the reference medicinal material solution, the solution to be distinguished and the reference solution by adopting a thin-layer chromatography identification method:

the thin layer conditions were as follows:

thin-layer plate: silica gel G thin layer plate;

sample application: sample application of the test solution, the reference solution and the reference solution is 1-10 μ l respectively;

developing agent: toluene at a volume ratio of (1-5) to (1-5): ethyl acetate: acetone;

the unfolding mode is as follows: unfolding the uplink;

and (6) inspection: observing under visible light;

and (3) chromatographic identification: and in the chromatogram of the test sample, if spots with the same color appear at the positions corresponding to the chromatograms of the reference medicinal materials or the reference substances, the test sample is a qualified product.

The following materials can be selected as the test materials used in the present invention, but the actual implementation is not limited to the following materials:

the instrument comprises the following steps: a rotary evaporator (EYELA, OSB-2100), a developer motor nebulizer (CAMAG, CH-4132MUTTENZ), an oven (Zhongxing Wei, 101-1AB) and an analytical balance (Yokov, YP 30002).

Reagent: silica gel G thin layer plate, produced by Qingdao Shenghai fine silica gel chemical Co., Ltd, with plate specification of 100X 100mm and thickness of 0.20-0.25 mm. Methanol, toluene, ethyl acetate, acetone, formic acid, concentrated sulfuric acid and absolute ethyl alcohol, all of which are analytically pure.

1-8 parts of test sample (medicinal material carmine flower root sample): the information is shown in table 1 below.

TABLE 1 Bixa orellana root sample information sheet

Test article (medicinal material himalayas Mirabilis jalapa sample): collected in 2019 from Tibetan Linzhi, lot number 2019001.

Test article (hemorrhoid treating capsule sample): the Miao medicine containing the primula maximowiczii roots is manufactured by Guizhou Fuhua pharmaceutical industry, namely, the Limited liability company, and the batch number is 20180504.

1-3 of a control medicinal material (a cochineal flower root control medicinal material sample): the information is shown in the following table 2, and is developed according to the first research and development guiding principles (trial) of national drug reference medicinal materials in the Zhongzhong college.

TABLE 2 Bixa orellana root sample information sheet

The reference medicinal materials to be distinguished (namely the himalayan Mirabilis jalapa root reference medicinal material sample) are as follows: purchased at the institute for testing and testing of Chinese food and drug, batch number: 121395-201303.

Comparison products: a daucosterol control substance (HPLC is more than or equal to 98 percent, purchased from Shanghai leaf Biotechnology Co., Ltd., batch number: P26F9F54684) and a beta-sitosterol control substance (the content is 97 percent, purchased from China food and drug testing institute, batch number: 110851-201608).

Example one

In this example, the effect of different developing agents on the results of the present technical solution is studied to further explain the present technical solution.

(1) Sample preparation:

number 1 — daucosterol: taking daucosterol control, adding methanol to make into solution containing 0.2mg per 1ml, and making into control solution. The concentration of the control in the test is 0.252 mg/mL.

Number 2- - -beta-sitosterol: taking beta-sitosterol reference substance, adding methanol to prepare a solution containing 0.2mg per 1ml, and using the solution as a reference substance solution. The control concentration in this experiment was 0.1956 mg/mL.

Number 3-control drug (i.e. primula maximowiczii root control drug sample) 1: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 4-control drug (i.e. primula maximowiczii root control drug sample) 2: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 5-reference medicinal material to be distinguished (i.e. himalayan Mirabilis jalapa reference medicinal material sample): taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

No. 6- -test sample (i.e. hemorrhoid treating capsule sample): taking 1 capsule for treating hemorrhoid, opening the capsule, weighing about 0.5g of powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

(2) Unfolding the system:

1. petroleum ether-ethyl acetate-acetone (5:2:3), 0.5ml formic acid;

2. toluene-ethyl acetate-acetone (1:2:5), 0.5ml formic acid;

3. toluene-ethyl acetate-acetone (5:2:3), 0.5ml formic acid.

(3) The color development method comprises the following steps: taking out, air drying, spraying 5% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed.

(4) And (3) test results:

in a developing system of petroleum ether-ethyl acetate-acetone (5:2:3) and 0.5ml of formic acid, the reference substance is clearer, the separation of the hemorrhoid treatment capsule sample is poorer, and the himalayan Mirabilis root and the annatto root are not obviously different, and the result is shown in figure 1; in a developing system of toluene-ethyl acetate-acetone (1:2:5) and 0.5ml formic acid, the control is clear, the himalayan Mirabilis root and annatto root are separated normally, the hemorrhoid treating capsule sample is separated normally, and the result is shown in fig. 2; in a developing system of toluene-ethyl acetate-acetone (5:2:3) and 0.5ml formic acid, the control product has clear spots, the himalayan Mirabilis root and the annatto root are separated normally, the hemorrhoid treating capsule sample is separated preferentially, and the result is shown in fig. 3.

Example two

In order to further optimize the toluene-ethyl acetate-acetone developer system, the present example further studies the effect of different developers on the results of the present technical solution, so as to further explain the present technical solution.

(1) Sample preparation:

number 1 — daucosterol: taking daucosterol control, adding methanol to make into solution containing 0.2mg per 1ml, and making into control solution. The concentration of the control in the test is 0.252 mg/mL.

Number 2- - -beta-sitosterol: taking beta-sitosterol reference substance, adding methanol to prepare a solution containing 0.2mg per 1ml, and using the solution as a reference substance solution. The control concentration in this experiment was 0.1956 mg/mL.

Number 3-control drug (i.e. primula maximowiczii root control drug sample) 1: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 4-control drug (i.e. primula maximowiczii root control drug sample) 2: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 5-reference medicinal material to be distinguished (i.e. himalayan Mirabilis jalapa reference medicinal material sample): taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 6- -test sample (i.e. medicinal material himalayas mirabilis root sample): taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

No. 7- -test sample (i.e. hemorrhoid treating capsule sample): taking 1 capsule for treating hemorrhoid, opening the capsule, weighing about 0.5g of powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

(2) Unfolding the system:

1. toluene-ethyl acetate-acetone (5:2:3), 0.5ml formic acid;

2. toluene-ethyl acetate-acetone (4:2:2), 0.5ml formic acid;

3. toluene-Ethyl acetate-acetone (4:4:2), 0.5ml formic acid.

(3) The color development method comprises the following steps: taking out, air drying, spraying 5% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed.

(4) And (3) test results:

in a developing system of toluene-ethyl acetate-acetone (5:2:3) and 0.5ml formic acid, the control product has clear spots, the himalayan Mirabilis root and the annatto root are separated normally, the separation of the hemorrhoid treating capsule sample is better, and the result is shown in figure 4; in a developing system of toluene-ethyl acetate-acetone (4:2:2) and 0.5ml formic acid, the control product has clear spots, the separation of Himalayan Mirabilis root and nopalin root is better, the separation of the capsule sample for treating hemorrhoid is better, and the result is shown in figure 5; separating the annatto root control sample in a developing system of toluene-ethyl acetate-acetone (4:4:2) and 0.5ml formic acid, wherein spots are clear and correspond to the daucosterol and beta-sitosterol control; the hemorrhoid treatment capsule sample is well separated, and has corresponding spots with the reference sample and the primula maximowiczii reference sample; furthermore, the primula maximowiczii root and the himalayan mirabilis root are clearly different, and the results are shown in fig. 6.

EXAMPLE III

In this embodiment, the influence of different color developers on the results of the technical scheme is studied to further explain the technical scheme.

(1) Sample preparation:

number 1 — daucosterol: taking daucosterol control, adding methanol to make into solution containing 0.2mg per 1ml, and making into control solution. The concentration of the control in the test is 0.252 mg/mL.

Number 2- - -beta-sitosterol: taking beta-sitosterol reference substance, adding methanol to prepare a solution containing 0.2mg per 1ml, and using the solution as a reference substance solution. The control concentration in this experiment was 0.1956 mg/mL.

Number 3-control drug (i.e. primula maximowiczii root control drug sample) 1: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 4-control drug (i.e. primula maximowiczii root control drug sample) 2: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 5-reference medicinal material to be distinguished (i.e. himalayan Mirabilis jalapa reference medicinal material sample): taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

No. 6- -test article (hemorrhoid treating capsule sample): taking 1 capsule for treating hemorrhoid, opening the capsule, weighing about 0.5g of powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

(2) Unfolding the system: petroleum ether-ethyl acetate-acetone (5:2:1), 0.5ml formic acid;

(3) the color development method comprises the following steps: taking out, air drying, spraying 5% sulphuric acid ethanol solution and 25% phosphomolybdic acid ethanol solution, respectively, and heating at 105 deg.C until the spots are clearly developed.

(4) And (3) test results: in the development of petroleum ether-ethyl acetate-acetone (5:2:1) with 0.5ml formic acid, 2 developers were compared with 5% ethanol sulfuric acid solution (results shown in FIG. 7) and 25% ethanol phosphomolybdic acid solution (results shown in FIG. 8), and the results showed that: 5% sulfuric acid ethanol solution is more convenient for identification.

Example four

In this example, the influence of different formic acid addition amounts on the results of the present technical solution is studied to further explain the present technical solution.

(1) Sample preparation:

number 1 — daucosterol: taking daucosterol control, adding methanol to make into solution containing 0.2mg per 1ml, and making into control solution. The concentration of the control in the test is 0.252 mg/mL.

Number 2- - -beta-sitosterol: taking beta-sitosterol reference substance, adding methanol to prepare a solution containing 0.2mg per 1ml, and using the solution as a reference substance solution. The control concentration in this experiment was 0.1956 mg/mL.

Number 3-control drug (i.e. primula maximowiczii root control drug sample) 1: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 4-control drug (i.e. primula maximowiczii root control drug sample) 2: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 5-reference medicinal material to be distinguished (i.e. himalayan Mirabilis jalapa reference medicinal material sample): taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 6- -test sample (i.e. medicinal material himalayas mirabilis root sample): taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

No. 7- -test sample (i.e. hemorrhoid treating capsule sample): taking 1 capsule for treating hemorrhoid, opening the capsule, weighing about 0.5g of powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

(2) Unfolding the system:

1. toluene-ethyl acetate-acetone (4:4:2), 60ml of the above three solvents added, and 1.5ml of formic acid (the volume of formic acid is 2.5% relative to the sum of the volumes of toluene, ethyl acetate and acetone);

2. toluene-ethyl acetate-acetone (4:4:2), 60ml of the above three solvents added, 0.5ml of formic acid (the volume of formic acid is about 0.83% relative to the sum of the volumes of toluene, ethyl acetate and acetone);

3. toluene-ethyl acetate-acetone (4:4:2), without addition of formic acid.

(3) The color development method comprises the following steps: taking out, air drying, spraying 5% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed.

(4) And (3) test results:

the content of formic acid has influence on the development result, the development system without formic acid has general separation result, the requirement of identifying medicinal materials can be basically realized, and the result is shown in figure 9; when the volume percentage of formic acid is about 0.83%, the separation effect is the same and general, the requirement of identifying the medicinal materials can be basically realized, and the result is shown in figure 10; when the volume ratio of formic acid is 2.5%, the separation effect is good, the spots are clear, the medicinal materials can be well identified, and the result is shown in fig. 11.

In the first, second and third examples, 0.5ml formic acid and 0.5ml formic acid developing agent are added into the prepared toluene-ethyl acetate-acetone, and the volume of the formic acid accounts for 2.44-3.45% of the sum of the volumes of the three solvents.

EXAMPLE five

The thin layer identification of the annatto roots in different producing areas is carried out by using the spreading system 1 in the fourth embodiment as the spreading agent condition, so as to further explain the technical scheme.

(1) Sample preparation:

number 1 — daucosterol: taking daucosterol control, adding methanol to make into solution containing 0.2mg per 1ml, and making into control solution. The concentration of the control in the test is 0.252 mg/mL.

Number 2- - -beta-sitosterol: taking beta-sitosterol reference substance, adding methanol to prepare a solution containing 0.2mg per 1ml, and using the solution as a reference substance solution. The control concentration in this experiment was 0.1956 mg/mL.

Number 3-control drug (i.e. primula maximowiczii root control drug sample) 1: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 4-control drug (i.e. primula maximowiczii root control drug sample) 2: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 5-control drug (i.e. primula maximowiczii root control drug sample) 3: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 6- -test sample (crude drug cochineal flower root sample) 1: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 7- -test sample (crude drug cochineal flower root sample) 2: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 8-sample (crude drug annatto root sample) 3: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 9- -sample to be tested (crude drug annatto root sample) 4: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 10- -test sample (crude drug annatto root sample) 5: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 11- -test sample (crude drug annatto root sample) 6: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 12- -test sample (crude drug annatto root sample) 7: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

Number 13- -sample to be tested (crude annatto root sample) 8: taking 1g of the powder, adding 10ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, and dissolving 1ml of methanol.

(2) Unfolding the system:

toluene-Ethyl acetate-acetone (4:4:2), 60ml of the above three solvent phases, and 1.5ml of formic acid (2.5% by volume formic acid relative to the sum of the volumes of toluene, ethyl acetate and acetone).

(3) The color development method comprises the following steps: taking out, air drying, spraying 5% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed.

(4) And (3) test results:

as shown in figure 12, the established thin-layer identification method comprises 2 reference products of daucosterol and beta-sitosterol and a cochineal flower root reference medicinal material, and has the advantages of good sample separation, proper Rf value and clear spots.

Corresponding spots are formed on the cochineal flower root medicinal material and the daucosterol and beta-sitosterol reference substances in all batches; the primula maximowiczii root medicinal material and the reference medicinal material in all batches have the same spots, and the thin-layer identification result shows that: the primula maximowiczii root herbs with numbers 6, 7, 8, 9, 10, 11, 12 and 13 are all genuine products.

The above description is only an example of the present invention, and common knowledge such as known technical means in the schemes is not described herein too much. It should be noted that, for those skilled in the art, without departing from the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

The present invention has been described in detail with reference to the embodiments and the accompanying drawings, wherein the principles and embodiments of the present invention are described in detail with reference to specific embodiments, which are provided to assist in understanding the methods and the core concepts of the present invention. Meanwhile, those skilled in the art should also be able to make modifications or variations to the embodiments and applications of the present invention based on the idea of the present invention. In view of the above, the present disclosure should not be construed as limiting the invention.