CN115840018A - Quality detection method of reineckea carnea medicinal material - Google Patents

Abstract

The invention discloses a quality detection method of a pink reineckea herb medicinal material, which comprises identification and/or content determination items; wherein the identification is thin-layer chromatography identification of the reineckia carnea medicinal material; the content determination is performed by high performance liquid chromatography to determine the content of the saponin A of the lucky herbal materials. By adopting the quality detection method, the authenticity of the medicinal materials can be accurately identified, and the lucky herbal materials can be effectively distinguished from counterfeit and confused medicinal materials; and the content of the saponin A in the reineckia carnea medicinal material can be accurately and quickly determined, so that the quality control of the reineckia carnea medicinal material is facilitated. The quality detection method of the reineckia carnea medicinal material can effectively control the overall quality of the reineckia carnea medicinal material and ensure the safety and effectiveness of clinical medication of the reineckia carnea medicinal material.

Description

Quality detection method of reineckea carnea medicinal material

Technical Field

The invention relates to the field of auspicious herbal medicine detection, in particular to a quality detection method of an auspicious herbal medicine.

Background

The herba Reineckeae Carneae is dry whole plant of Reineckia carnea (Andr.) of Liliaceae. The title of auspicious herbs is "auspicious herbs" in the monograph and standard, and the title of "Xiaozhugen qin (auspicious herbs)" is used only in the standard of the medicine materials in Shaanxi province (2015 edition), which is a common name in local. The standard uses the name "luckiness grass".

The herba Reineckeae Carneae is dried whole plant of Reineckia carnea (Andr.) of Liliaceae. The reineckia carnea is mainly distributed in provinces of east China, south China and south China, and is one of the traditional herbal medicines commonly used in folk and minority regions. The reineckia carnea standard is collected in the traditional Chinese medicine standards of the autonomous region of the Zhuang nationality in Guangxi province (1990 edition), the traditional Chinese medicine standards in Jiangxi province (1996 edition), the quality standards of traditional Chinese medicines and national medicines in Guizhou province (2003 edition), the traditional Chinese medicine standards in Yunnan province (2005 edition, first book: yi medicine), the traditional Chinese medicine standards in Hunan province (2009 edition), and the medicine standards in Shaanxi province (2015 edition, small bamboo root seven (reineckia carnea)), wherein the regulations include properties, microscopic identification (transverse section and powder), thin-layer identification of reference medicinal materials, magazines, total ash, acid insoluble ash, 70% alcohol extract (hot dipping method), water soluble substances and the like, but the regulations of content determination items are not complete, and the standards are not complete. Therefore, the development and establishment of a quality detection method of reineckia carnea is urgently needed, and the method has important significance for controlling the overall quality of reineckia carnea medicinal materials and ensuring the safety and effectiveness of clinical medication of the reineckia carnea medicinal materials.

Disclosure of Invention

The invention aims to provide a quality detection method of a pink reineckea herb medicinal material. The invention has the characteristics of effectively controlling the overall quality of the reineckia carnea medicinal material and ensuring the safety and effectiveness of clinical medication of the reineckia carnea medicinal material.

The technical scheme of the invention is as follows: a quality detection method of herba Reineckeae Carneae medicinal material comprises identification and/or content determination items; wherein the identification is thin-layer chromatography identification of the reineckia carnea medicinal material; the content determination is performed by high performance liquid chromatography to determine the content of the saponin A of the lucky herbal materials.

In the quality detection method for the reineckia carnea medicinal material, the thin-layer chromatography identification of the reineckia carnea medicinal material comprises the following steps of:

(1) Taking 1g of reineckea carnea medicinal material sample powder, adding 20ml of methanol, heating and refluxing for 30 minutes, filtering, and taking supernate as a sample solution; preparing reference medicinal material 1g of herba Reineckeae Carneae, and making reference medicinal material solution by the same method;

(2) The two solutions were pipetted 10. Mu.l each for thin layer chromatography on the same silica gel G thin layer plate and mixed with chloroform: ethyl acetate: methanol: water =15:40:22: developing the lower layer of the 10 mixed solution with a developing agent, taking out, air drying, spraying with 10% sulfuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight;

in the chromatogram of the sample, spots of the same color appear at the positions corresponding to those in the chromatogram of the reference medicinal material, and the sample is herba Reineckeae Carneae medicinal material.

In the quality detection method of the reineckia carnea medicinal material, the high performance liquid chromatography determination method comprises the following steps:

1) Preparation of the Standard Curve

Precisely measuring reference substance solution 0.04ml, 0.08ml, 0.1ml, 0.2ml, 0.4ml and 0.6ml, respectively placing in 10ml test tubes with plugs, treating the reference substance solution according to vanillin-ethanol-perchloric acid method and measuring absorbance at 452 nm; drawing a standard curve by taking the absorbance as a vertical coordinate and the concentration as a horizontal coordinate;

2) Preparation of content determination test sample solution

Taking 0.5g of medicinal powder, placing the medicinal powder in a 50ml conical flask with a plug, precisely adding 20ml of methanol, weighing, heating and refluxing for 1.5 hours, cooling, complementing the lost weight with methanol, filtering, extracting filter residue twice with methanol in the same way, combining the three filtrates, recovering the solvent to dryness, dissolving the residue with methanol, transferring to a 50ml volumetric flask, adding methanol to scale, shaking up, filtering, and taking the subsequent filtrate to obtain the content measurement sample solution.

3) Measurement of

Precisely measuring 0.15ml of the content determination sample solution, placing in a 10ml test tube with a plug, measuring absorbance according to the method under the preparation item of the standard curve, reading the weight of saponin A in the content determination sample solution from the standard curve, and calculating to obtain the content of total saponin.

In the quality detection method for the reineckia carnea medicinal material, in the step 2), the medicinal material powder needs to be sieved by a fourth sieve.

In the quality detection method of the reineckea carnea medicinal material, in the step 1), the preparation process of the reference solution is as follows: taking a proper amount of saponin A reference substance, dissolving with methanol, transferring to a 25ml volumetric flask, diluting to scale, and shaking to obtain a reference substance solution with a concentration of 0.85 mg/ml.

In the quality detection method of the reineckea carnea medicinal material, in the step 1), the specific method for treating the reference solution by the vanillin-ethanol-perchloric acid method comprises the following steps: volatilizing the solvent of each reference substance solution in a water bath, sequentially and precisely adding 0.2ml of 8% vanillin ethanol solution and 0.1ml of perchloric acid solution, sealing, shaking up, heating in a water bath at the constant temperature of 60 ℃ for 15 minutes, taking out, cooling with ice water for 5min, precisely adding glacial acetic acid 5ml, shaking up, taking the corresponding reagent as a blank reference, and respectively measuring the absorbance at the wavelength of 452nm by an ultraviolet-visible spectrophotometry.

In the quality detection method for the reineckia carnea medicinal material, the regression equation for drawing the standard curve is Y =11.0109X-0.0120.

In the quality detection method for the reineckia carnea medicinal material, the saponin A is saponin (1 beta, 3 beta, 16 beta, 22S) -cholest-5-ene-1,3,16,22-tetrol1,16-di (beta-D-glucopyranoside).

Compared with the prior art, the quality detection method comprises a thin-layer identification method of the reineckia carnea medicinal material and a content determination method of the saponin A in the reineckia carnea medicinal material, and by adopting the quality detection method, the authenticity of the medicinal material can be accurately identified, and the reineckia carnea medicinal material is effectively distinguished from a fake product and a confused product thereof; and the content of the saponin A in the reineckia carnea medicinal material can be accurately and quickly determined, so that the quality control of the reineckia carnea medicinal material is facilitated. The quality detection method of the reineckia carnea medicinal material can effectively control the overall quality of the reineckia carnea medicinal material and ensure the safety and effectiveness of clinical medication of the reineckia carnea medicinal material.

The thin-layer identification method for the reineckia carnea medicinal materials has the advantages of being high in sensitivity, strong in specificity, good in separation effect and the like, simple, convenient and feasible, high in reproducibility, capable of accurately identifying the truth of the medicinal materials and effectively distinguishing the reineckia carnea medicinal materials from fake products and confused products of the reineckia carnea medicinal materials.

The method for measuring the content of the saponin A in the reineckia carnea medicinal material can accurately and quickly measure the content of the reineckia carnea medicinal material, and provides an effective qualitative and quantitative analysis means for the reineckia carnea medicinal material, so that the quality of the reineckia carnea medicinal material can be controlled. The quality detection method of the reineckia carnea medicinal material provided by the invention can effectively control the overall quality of the reineckia carnea medicinal material and ensure the safety and effectiveness of clinical medication of the reineckia carnea medicinal material.

The quality detection method of the reineckia carnea medicinal material provided by the invention is helpful for making up for the Chinese medicinal material standards of autonomous regions of Guangxi Zhuang nationality (1990 edition), the Chinese medicinal material standards of Jiangxi province (1996 edition), the Chinese medicinal material quality standards of Guizhou province and ethnic medicinal materials (2003 edition), the Chinese medicinal material standards of Yunnan province (2005 edition, first book: yi medicine), the Chinese medicinal material standards of Hunan province (2009 edition), the Chinese medicinal material standards of Shanxi province (2015 edition, root of small bamboo seven (reineckia carnea)), wherein the specifications of properties, microscopic identification (transverse section and powder), thin-layer identification of reference medicinal materials, magazines, total ash content, acid insoluble ash content, 70% alcohol extract (hot dipping method), water-soluble substances and the like are specified, and the specifications do not contain measurement items and are not perfect. The method has important significance for further quality control of the reineckia carnea medicinal material, and can provide scientific basis for formulation of reineckia carnea material quality scale mixing.

In conclusion, the invention has the characteristics of effectively controlling the overall quality of the pink reineckea herb medicine and ensuring the safety and the effectiveness of clinical medication of the pink reineckea herb medicine.

Drawings

FIG. 1 is a thin layer chromatogram developed with chloroform-methanol-water (65: 35: 10, lower layer);

FIG. 2 is a thin layer chromatogram developed from chloroform-ethyl acetate-methanol-water (15: 40:22:10, lower layer);

FIG. 3 is a thin layer chromatogram developed with chloroform-ethyl acetate-methanol-water (1: 2: 1: 0.2);

FIG. 4 is a thin layer chromatogram developed with chloroform-ethyl acetate-methanol-water (1.5: 4: 2.7: 1);

(in FIGS. 1-4, 1 is 6. Mu.L of saponin A reference substance, 2 is 10. Mu.L of Reineckia carnea (S14), 3 is 10. Mu.L of Reineckia carnea (S14), and 4 is 10. Mu.L of Reineckia carnea (S14));

FIG. 5 is a thin layer chromatogram for different extraction solvent studies;

(in FIG. 5, 6. Mu.L of 1-saponin A control; 10. Mu.L of 2-Reineckia carnea (S14) (methanol), 10. Mu.L of 3-Reineckia carnea (S14) (ethanol), and 10. Mu.L of 4-Reineckia carnea (S14) (water));

FIG. 6 is a thin layer chromatogram for different extraction mode studies;

(in FIG. 6, 6. Mu.L of 1-saponin A reference substance; 10. Mu.L of 2-Reineckia carnea (S14) (heating reflux extraction for 30 min), 10. Mu.L of 3-Reineckia carnea (S14) (ultrasonic extraction for 30 min), 10. Mu.L of 4-Reineckia carnea (S14) (ultrasonic extraction for 30min after cold soaking for 1 h));

FIG. 7 is a thin layer chromatogram for spot size investigation;

FIG. 8 is a thin layer chromatogram developed at a humidity of 25%;

FIG. 9 is a thin layer chromatogram developed at a humidity of 75%;

FIG. 10 is a thin layer chromatogram developed at a temperature of 25 ℃;

FIG. 11 is a thin layer chromatogram developed at a temperature of 4 ℃;

(in FIGS. 7-11, 6. Mu.L of 1-saponin A reference substance; 6. Mu.L of 2-Reineckea carnea (S14); 8. Mu.L of 3-Reineckea carnea (S14); 10. Mu.L of 4-Reineckea carnea (S14));

FIG. 12 is a thin-layer chromatogram for reference drug identification of 15 batches of Reineckia carnea (L.) Kuntze;

(in FIG. 12, S is 6. Mu.L of saponin A reference substance, and S1-S7 are 10. Mu.L of Reineckea carnea);

FIG. 13 is a thin-layer chromatogram for reference drug identification of 15 batches of Reineckia carnea (L.) Kuntze;

(in FIG. 13, S is 6. Mu.L of saponin A reference substance, and S8-S15 are 10. Mu.L of Reineckea carnea);

FIG. 14 is a graph of absorption spectra of a control solution and a test solution (vanillin-ethanol-perchloric acid method); (in FIG. 14, a is a reference substance, b is a test substance, and c is the reference substance and the test substance);

FIG. 15 is an absorption spectrum chart of a control solution and a sample solution (perchloric acid method);

(in FIG. 15, d is a reference substance; e is a test substance; f is a reference substance and a test substance);

FIG. 16 is an absorption spectrum (vanillin-glacial acetic acid-perchloric acid method) of a control solution and a test solution; (in FIG. 16, g: reference substance; h: test substance; i: reference substance and test substance);

FIG. 17 is a graph of the linear relationship of saponin A.

Detailed Description

The invention is further described with reference to the following figures and examples, which are not to be construed as limiting the invention.

Examples are given. A quality detection method of a reineckea carnea medicinal material,

the instrument comprises the following steps:

agilent model 1260 liquid chromatograph; an evaporative light scattering detector (Alltech ELSD-2000); an electronic balance of type AUW220D (one hundred thousand, shimadzu, japan); BS224 model S electronic balance (Sartorius); KQ-500DB type digital control ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); DHG-9140A electric heating constant temperature air blast drying oven (Steud City Instrument, inc.); 8-10 type box-type resistance furnace (manufactured by energy-saving electric furnace factory in Shenyang city, china).

Reagent and reagent

1. Reference substance

(1 beta, 3 beta, 16 beta, 22S) -cholest-5-ene-1,3,16,22-tetrol1,16-di (beta-D-glucopyranoside) (hereinafter referred to as saponin A) prepared by national engineering research center of manufacturing technology of Chinese medicinal solid preparation, and the purity is more than 98%.

2. Reagent

Acetonitrile (chromatographically pure, fisher Scientific, fairlawn, NJ, USA); formic acid (chromatographically pure, sigma-Aldrich co.ltd, st Louis, MO, USA); methanol (analytical grade, chemical reagents of national drug group, ltd); chloroform (analytical grade, shanghai laboratory reagents, inc.); ethyl acetate (analytical pure, guangdong Guanghua science and technology, inc.); absolute ethanol (analytically pure, shanghai gao xing chemical plant); GF254 silica gel plate (Qingdao oceanic factory); sulfuric acid (analytically pure, west longa science ltd); the water is ultrapure water.

3. Medicinal materials

The medicinal materials used in the research are purchased from Guizhou province, identified as the whole herb of Reineckia Reineckia carnea (Andr.) Kunth of Reineckia of Liliaceae by professor Zhong Guoyue of national medicine resource center of Jiangxi traditional Chinese medicine university, and the specimen (Z-140310-01) is stored in the national medicine resource center of Jiangxi traditional Chinese medicine university. (Table 1)

TABLE 1 lucky herbal batches

[ medicinal material name ] Reineckia carnea.

The title of auspicious herbs is "auspicious herbs" in the monograph and standard, and the title of "Xiaozhugen qin (auspicious herbs)" is used only in the standard of the medicine materials in Shaanxi province (2015 edition), which is a common name in local. The standard adopts the name of' lucky grass

Luck grass was recorded in Suitang Shi Chen Cangqi, bencao Shi Yi (supplement to materia Medica). Compendium of materia Medica cloud: the reineckia carnea, leaves like Zhanglan, green Sishi and Xia Kai are purple flowers and easy to propagate. "plant famous and practical drawings examination" is in fact: "Songhualan, she Weikuan, flower six is slightly bigger, opened in winter, planted in one flower pot. Autumn fruit like asparagus root, with red purpleAnd (4) a tip. "the above description in conjunction with the accompanying drawings confirmed that the original plant thereof is compatible with the Reineckia carnea (Andr.) Kunth, a plant of Liliaceae. The Wannian Qing Zhongyun recorded in the original materia of herb Property "is like orchid She Yang, and should be luckiness herb, with characteristics not consistent with those of Wannian Qing. The compendium of materia medica is called Jie Yu Cao. The book of classified diseases and livelihood is named as Yang Xiang Cao. Zhuyeqing from Zhong Guo Yao Zhi (Chinese medicinal plant record). Jieguian from Sichuan Chinese materia Medica (famous Jiuhuai lotus root). Guizhou herbal medicine named as Xiaojiulong (nine-Dragon disk). Guizhou Chinese herbal medicine famous book (famous records of Guizhou) famous kwan-yin herb, amusing ant and Musca puama ^[2] . The pink reineckea herbs are mainly distributed in provinces of east China, south China and south China, and are widely used in folks.

The reineckea carnea is specified in the monograph and the original standard to be from Reineckia carnea (andr.) Kunth of liliaceae, and the medicinal part is dry whole grass or dry whole grass with roots. The standard draft is defined as "dried whole plant of Reineckia carnea (Andr.) of the family Liliaceae" in Kunth.

The title of auspicious herbs is "auspicious herbs" in the monograph and standard, and the title of "Xiaozhugen qin (auspicious herbs)" is used only in the standard of the medicine materials in Shaanxi province (2015 edition), which is a common name in local. The standard uses the name "luckiness grass". .

Perennial herbs are planted in the ground in a meadow, are similar to rhizomes, green and multisection, and have fibrous roots on joints. The leaf clusters grow on the top of the stem or the stem node, each cluster has 3 to 8 leaves, the shape of the leaf is strip-shaped to be needle-shaped, the length is 10 to 38 centimeters, the width is 0.5 to 3.5 centimeters, the tip is gradually sharp, and the leaf gradually narrows downwards to form a handle. Scape is 5-15 cm long, spike-shaped inflorescence is 2-6.5 cm long, and upper flowers sometimes only have stamens; an oval, membranous, pale brown, or purple bud; the comforter slices are combined into a short tube shape, the upper part of the comforter slices is 6-split, the split slices are long round, the length of the comforter slices is 5-7 mm, the comforter slices are slightly fleshy, the comforter slices are curled reversely when the comforter slices bloom, and the comforter slices are pink and fragrant; 6 stamens, filaments, anthers are nearly oblong, and two ends are slightly concave; ovary bottle shape, 3 chambers, shorter than style, stigma head shape, 3 fissures. The berry is spherical, has the diameter of 6-10 mm, and is fresh red when cooked. The flower and fruit period is 7-11 months. Growing in the shady and wet hillside, valley or compact forest with the altitude of 170-3200 m or cultivating. Distributed in Jiangsu, zhejiang, anhui, jiangxi, hubei, hunan, guizhou, yunnan, sichuan, guangxi, guangdong, henan, shaanxi (south of Qinling mountains), etc.

[ PROPERTIES ] the original standard of the auspicious herbs is stipulated with sexual identification, through observation, the character description of the original standard basically conforms to the character of the medicinal materials, and the characters are modified according to actual observation.

The rootstock of the product is cylindrical, has different lengths, has the diameter of 0.2-0.5 cm, and has yellowish brown or yellowish green surface; obvious knots are slightly enlarged, residual membranous scaly leaves and bent and curled fibrous roots are frequent, and white wools are densely distributed on the roots; internodes were shortened with longitudinal wrinkles. The leaf cluster grows at the stem top or node, the leaves are green brown or tan, and are more wrinkled, and the intact one is in a strip shape like a needle after being wetted and unfolded, has complete edges and no handle, and has parallel veins and obvious middle veins. Light smell, bitter taste and slight bitter taste.

Thin-layer identification:

the thin layer identification method of the reference medicinal materials in the original Guizhou, hunan and Shaanxi province standards is different, and the developing agents are respectively as follows: chloroform-ethyl acetate-formic acid (8: 5: 0.2) (Guizhou), dichloromethane-methanol-water (8: 3: 0.4) (Hunan), chloroform-methanol-formic acid (36: 4: 0.5) (Shaanxi). By the rechecking test of the three developing agents, the developing effect is poor. Therefore, a new thin layer identification method is established. In the research, the main saponin component (1 beta, 3 beta, 16 beta, 22S) -cholest-5-ene-1,3,16,22-tetrol1,16-di (beta-D-glucopyranoside, C) in the saponin contained in the reineckia carnea is established ₃₉ H ₆₆ O ₁₄ The thin-layer identification using saponin A as a reference is used for methodology investigation of reference medicinal materials.

Taking 1g of the product powder, adding 20ml of methanol, heating and refluxing for 30 minutes, filtering, and taking supernatant as a test solution. Taking another herba Reineckeae Carneae reference medicinal material 1g, and making into reference medicinal material solution by the same method. Performing thin layer chromatography (general 0502) test, sucking 10 μ l of each of the above two solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-ethyl acetate-methanol-water (15: 40:22: 10) as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.

1) Examination of developing Agents

Taking 3 parts of reineckea carnea medicinal material powder, and preparing a test solution according to the method. With saponin a control, four developing agents were investigated: chloroform-methanol-water (65: 35: 10, lower layer); chloroform-ethyl acetate-methanol-water (15: 40:22:10, lower layer); chloroform-ethyl acetate-methanol-water (1: 2: 1: 0.2); chloroform-ethyl acetate-methanol-water (1.5: 4: 2.7: 1), thin layer plates: silica gel GF254 board (50X 100 mm), development mode and span: unfolding the materials upwards for 8cm, and developing the materials: heating 10% sulphuric acid ethanol solution at 105 deg.C for 5min, and inspecting in sunlight.

The results showed that in the chromatogram developed with chloroform-ethyl acetate-methanol-water (15: 40:22:10, lower layer) as developing agent, each spot was well separated and the spot was clear, and therefore, chloroform-ethyl acetate-methanol-water (15: 40:22, lower layer). (see FIG. 1 to FIG. 4)

2) Examination of extraction solvent

Adding 1g and three parts of herba Reineckeae Carneae powder into 20ml of methanol, ethanol and water, respectively, heating and reflux-extracting for 30min, cooling, filtering, and collecting filtrate as sample solution. Spotting and developing.

Methanol is selected as the extraction solvent because it has good extraction effect and clear spots (see FIG. 5).

3) Examination of extraction methods

Taking 1g and three parts of lucky herbal medicine powder, adding 20ml of methanol, extracting by three extraction modes of heating reflux extraction for 30min, ultrasonic extraction for 30min and ultrasonic extraction for 30min after cold soaking for 1h, cooling, filtering, and taking filtrate as a test solution. Spotting and developing.

The result shows that the heating reflux extraction for 30min has better extraction effect, and the spot is clearer, so the heating reflux extraction for 30min is selected as the extraction mode of the reineckia carnea thin-layer identification medicinal material. (see FIG. 6)

4) Examination of sample application amount

Taking 1g of herba Reineckeae Carneae medicinal material powder (sieved with a sieve of four numbers), and mixing three parts, preparing sample solution according to the above method, spotting, and developing.

The result shows that when the sample amount of the test sample is 10 mul, the spots are clearer. The amount of the sample applied was determined to be 10. Mu.l (see FIG. 7).

5) Durability test investigation of development environmental conditions

Taking 1g of herba Reineckeae Carneae medicinal material powder (sieved with a sieve of four numbers), and mixing three parts, preparing sample solution according to the above method, spotting, and developing.

The results showed that good separation was obtained for both the selected developing solvent at 25% and 75% relative humidity and at 4 ℃ (refrigerator) and room temperature (25 ℃) (see fig. 8-11).

6) Results of 15 batches of samples

Thin-layer identification of control materials was performed on 15 Reineckia carnea samples as above.

The finally determined reineckea carnea control medicinal material thin-layer identification method comprises the following steps: taking 1g of the product powder, adding 20ml of methanol, heating and refluxing for 30 minutes, filtering, and taking supernatant as a test solution. Taking another herba Reineckeae Carneae reference medicinal material 1g, and making into reference medicinal material solution by the same method. Performing thin layer chromatography (general 0502) test, sucking 10 μ l of each of the above two solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-ethyl acetate-methanol-water (15: 40:22: 10) as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution. (see FIGS. 12 to 13)

[ CONTENT DETERMINATION ] No "CONTENT DETERMINATION" is specified in each standard of the original luckiness grassland.

The types of compounds found in Reineckia carnea reported in the literature include steroidal saponins, flavonoids, lignans, terpenes, and the like. The steroid saponin components are researched more, and the common steroid saponins and the common steroid sapogenins comprise: original spider ampelopsin, new spider ampelopsin, kaitigenin, isovancomycin, (1 beta, 3 beta, 16 beta, 22S) -cholest-5-ene-1,3,16,22-tetrol1,16-di (beta-D-glucopyranoside))、 (1β,3β,16β,22S)-cholest-5-ene-1,3,16,22-tetrol1-[O-α-L-rhamnopyranosyl-(1→2)-β-D -glucopyranoside]16- (beta-D-glucopyranoside), reineckia carnea sapogenin, diosgenin, spicamarin, isocayitin, beta-sitosterol-beta-D-glucoside, stigmasterol-3-O-beta-D-glucopyranoside, beta-balsamic alcohol, alpha-bromosterol-3-O-glucoside and the like; the flavonoids comprise: 7-methoxy-8-methyl-4 ' -hydroxy-flavone, 1,6-dihydroxy-8-methyl-xanthane, quercetin-3-O-alpha-L-rhamnopyranose- (1-6) -beta-D-glucopyranoside, daidzein B, 6,8-dimethyl-5,7,4 ' -trihydroxyflavone, (R) -8-methylnaringenin, naringenin, daidzein, 3' -hydroxydaidzein, etc.; the terpenes are: squalene ursolate, etc.; lignans include: syringaresinol-1-beta-D-glucoside, syringaresinol, etc.; other classes are: L-O-hexadecanoic acid glyceride, palmitic acid, myristic acid, inositol, N-p-coumaroyl tyramine, methyl linoleate, N-triacontane, soyasaponin N-benzoyl-L-phenylalaninol, 2-ethylhexyl phthalate, dibutyl phthalate, eleutherobin, etc ^[5-11] . Studies show that the reineckia carnea saponin component has anti-tumor activity. As for the quality evaluation research of the reineckia carnea, the literature reports that the total saponin content is measured on 13 samples in Guizhou, the average value is 1.28 percent (0.86-1.73 percent), and the total saponin content is recommended to be defined to be not less than 1.0 percent; the test results of 3 batches of reineckea carnea medicinal materials show that the ferulic acid content is 0.011 to 0.013 percent, and the rutin content is 0.047 to 0.054 percent.

In the research, the components contained in the reineckia carnea reported in the literature are primarily investigated, and the result shows that the saponins are the main components of the reineckia carnea, and the content of the monomer compounds of other components is lower. Meanwhile, the test of cough-relieving and phlegm-eliminating activity is carried out on each extraction part of the pink reineckea herb, and the result shows that the 80% ethanol extract, the ethyl acetate part and the n-butyl alcohol part have good cough-relieving and phlegm-eliminating effects; further separating, purifying and identifying ethyl acetate part and n-butanol part of 80% ethanol extract to obtain 27 compounds (4 compounds, 1 new natural product, 13 compounds found in the plant for the first time) mainly containing saponins; and the anti-complement activity of 27 components is determined, and the result shows that most saponin components show the anti-complement activity after hydrolysis.

The above research results show that the saponin component is the main bioactive component of Reineckia carnea, so the main saponin (1 beta, 3 beta, 16 beta, 22S) -cholest-5-ene-1,3,16,22-tetrol1,16-di (beta-D-glucopyranoside) (hereinafter referred to as saponin A, C) ₃₉ H ₆₆ O ₁₄ ) The monomer of the compound is prepared and obtained (the purity is more than 98 percent), the total saponin is taken as the content measurement index to better accord with the actual condition of the medicinal materials by referring to the literature report about the content measurement of the pink reineckea herb total saponin, the ferulic acid, the rutin and the like, so the total saponin is taken as the content limit regulation.

Measuring by ultraviolet-visible spectrophotometry (in "Chinese pharmacopoeia" 2020 edition, fourth general rule 0401). The measurement method is as follows:

preparation of control solution 21.22mg of saponin A control was taken, dissolved in methanol and transferred to a 25ml volumetric flask, diluted to the scale and shaken up to obtain a control solution of 0.8488 mg/ml.

1 selection of the color system and the detection wavelength

Weighing 0.5g of lucky herbal medicine powder (sieved by a sieve IV), precisely weighing, placing in a conical flask with a plug of 50ml, adding 20ml of methanol, weighing, heating and refluxing for extraction for 30 minutes, cooling, supplementing methanol to reduce weight loss, filtering, and taking the subsequent filtrate.

(1) Vanillin-ethanol-perchloric acid method: precisely sucking 0.5ml of a reference substance solution and 0.15ml of a test solution, respectively placing into 10ml test tubes with plugs, volatilizing the solvent in a water bath, sequentially adding 0.2ml of 8% vanillin ethanol solution and 0.1ml of perchloric acid solution, sealing the plugs, shaking uniformly, then placing into a 60 ℃ water bath, heating at constant temperature for 15 min, taking out, immediately cooling with ice water for 5min, adding 5ml of glacial acetic acid, and shaking uniformly. Color developing agent is used as blank reference.

(2) Perchloric acid method: precisely sucking 0.4ml of the reference solution and 0.15ml of the test solution, respectively placing in a 10ml test tube with a plug, volatilizing the solvent in a water bath, precisely adding 5ml of perchloric acid, shaking up, and placing at room temperature for reaction for 25min. Color developing agent is used as blank reference.

(3) The vanillin-glacial acetic acid-perchloric acid method: precisely sucking 0.2ml of the reference solution and 40 μ l of the sample solution, respectively placing in 10ml test tubes with plugs, volatilizing the solvent in a water bath, sequentially adding 0.2ml of 5% vanillin-glacial acetic acid solution and 0.8ml perchloric acid, sealing the plugs, shaking uniformly, placing in a 60 ℃ water bath, heating at constant temperature for 20 min, taking out, immediately cooling with ice water for 5min, adding 5ml glacial acetic acid, and shaking uniformly. Color developing agent is used as blank reference.

Scanning with an ultraviolet spectrophotometer within 350-800 nm, and the result shows that when the vanillin-ethanol-perchloric acid method is used as a color developing agent, the reference solution and the test solution have good consistency of absorption spectra in a visible light region, so that the maximum absorption wavelength is determined to be 452nm. The results are shown in Table 2, FIGS. 14 to 16

TABLE 2 maximum absorption wavelength of control solution and test solution

2. Examination of extraction methods

2.1 Investigation of extraction solvent

Weighing 0.5g of medicinal powder (sieved by a sieve IV) and six parts of medicinal powder, precisely weighing, placing in a conical flask with a plug of 50ml, respectively adding 20ml of three different solvents of methanol, ethanol and water, weighing, heating and refluxing for 30 minutes, cooling, complementing the lost weight, filtering, taking the subsequent filtrate, and respectively measuring the content of the total saponin. (Table 3) results of investigation of different extraction solvents

As can be seen from the above table, methanol was selected as the extraction solvent because of the highest total saponin content when methanol was used as the extraction solvent.

2.2 investigation of extraction solvents at different concentrations

Weighing 0.5g and eight parts of medicinal powder (sieved by a sieve IV), precisely weighing, placing in a conical flask with a plug of 50ml, respectively adding 30% methanol, 50% methanol, 70% methanol and 20ml methanol, weighing, heating and refluxing for 30 minutes, cooling, complementing the lost weight, filtering, and taking the subsequent filtrate to respectively measure the content of the total saponin.

(Table 4)

TABLE 4 examination of different concentrations of extraction solvent

As can be seen from the above table, methanol was selected as the extraction solvent because anhydrous methanol had the highest total saponin content.

2.3 extraction time study

Weighing 0.5g of medicinal powder (sieved by a sieve IV) and eight parts of medicinal powder, precisely weighing, placing in a conical flask with a plug of 50ml, respectively adding 20ml of methanol, weighing, respectively heating and refluxing for extraction for 0.5h, 1h, 1.5h and 2h, cooling, complementing the loss weight, filtering, taking the subsequent filtrate, and respectively measuring the content of the total saponin. (Table 5)

TABLE 5 investigation results of different extraction times

As can be seen from the above table, the difference between the total saponin content in the heating reflux extraction for 1.5h and the heating reflux extraction for 2h is smaller, and the heating reflux extraction for 1.5h is selected from the viewpoint of time saving.

2.4 examination of extraction times

Weighing 0.5g of medicinal powder (sieved by a sieve IV) and six parts of medicinal powder, precisely weighing, placing in a conical flask with a plug of 50ml, respectively adding 20ml of methanol, weighing, respectively heating and refluxing for extraction for 1 time, 2 times and 3 times, cooling for 1.5h each time, complementing the lost weight, filtering, and taking the subsequent filtrate to respectively measure the content of the total saponin. (Table 6) results of examination of different extraction times in Table 6

As can be seen from the above table, the total saponin content was highest when the extract was heated under reflux for 3 times, so the extract was selected to be heated under reflux for 3 times.

The preparation method of the test solution for finally determining the content of the pink reineckea herb total saponin comprises the following steps: weighing 0.5g of medicinal powder (sieved by a sieve IV), accurately weighing, placing in a 50ml conical flask with a plug, accurately adding 20ml of methanol, weighing, heating and refluxing for 1.5 hours, cooling, complementing the loss weight with methanol, filtering, extracting the filter residue twice with methanol in the same way, combining the three filtrates, recovering the solvent to dryness, dissolving the residue with methanol, transferring to a 50ml volumetric flask, adding methanol to scale, shaking uniformly, filtering, and taking the subsequent filtrate.

3 methodological validation

3.1 Linear experiment

Precisely sucking 0.04ml, 0.08ml, 0.1ml, 0.2ml, 0.4ml and 0.6ml of reference substance solution, respectively placing into 10ml test tubes with plugs, treating the sample according to the vanillin-ethanol-perchloric acid method and measuring the absorbance at 452nm. Taking the absorbance (A) as the ordinate and the concentration (C) as the abscissa, drawing a standard curve, and taking a regression equation of Y =11.0109X-0.0120 (r) ² = 0.9996), saponin a has good linearity with absorbance at 33.95-509.28 μ g. (FIG. 17)

3.2 stability test

Taking 0.15ml of the same reineckia carnea medicinal material sample solution, processing the sample according to a vanillin-ethanol-perchloric acid method, respectively measuring absorbance at 452nm after 0, 10, 20, 30, 40, 50 and 60min, and calculating the RSD value. (Table 7)

Table 7 stability test investigation results

The result shows that the same reineckea carnea medicinal material test solution is measured after 0, 10, 20, 30, 40, 50 and 60min, the RSD value is 2.02 percent, and the method has good solution stability.

3.3 precision test

Taking 0.15ml of the same reineckia carnea medicinal material sample solution, treating the sample according to a vanillin-ethanol-perchloric acid method, measuring the absorbance at 452nm, continuously measuring for 6 times, and calculating the RSD value. (Table 8).

TABLE 8 results of precision test

The result shows that the RSD value is 1.45 percent after six times of continuous measurement, and the precision of the instrument is good.

3.4 repeatability experiments

Taking 6 parts of the pink reineckea herb medicinal material of the same batch, preparing a sample solution according to the pink reineckea herb total saponin extraction method, treating a sample according to a vanillin-ethanol-perchloric acid method, measuring absorbance at 452nm, and calculating an RSD value. (watch 9)

TABLE 9 results of repeatability tests

The result shows that the RSD value of the test solution of 6 reineckea carnea medicinal materials is 1.43 percent and the method has good repeatability when the test solution is prepared in parallel for determination.

3.5 sample application recovery test

Respectively and precisely weighing 6 parts of pink reineckea herb with the known total saponin content by 0.25g, adding a certain amount of saponin A reference substance, preparing a sample solution according to the pink reineckea herb total saponin extraction method, treating a sample according to a vanillin-ethanol-perchloric acid method, measuring absorbance at 452nm, calculating the content of the total saponin and calculating the recovery rate.

(watch 10)

TABLE 10 sample recovery test results

The average sample recovery was measured to be 99.24% (RSD = 2.43%), indicating good process recovery.

4. Method for measuring content of total saponins in reineckea carnea medicinal material

Taking 0.5g of reineckia carnea medicinal materials of different batches respectively, preparing according to a preparation method of a test solution, taking 0.15ml of the test solution respectively, treating a sample according to a vanillin-ethanol-perchloric acid method, measuring the absorbance at 452nm, reading the weight (mg) of saponin A in the test solution from a standard curve, and calculating to obtain the content of total saponin. (watch 11)

TABLE 11 Total saponins content determination results in Pink reineckea herb batches of 28

The results show that the total saponin content in 28 batches of reineckia carnea medicinal materials is between 3.00 and 9.49 percent, and the average content is 5.98 percent. Taking the mean value that 20% of the total saponins in the pink reineckea herb is 4.78%, 6 of the 28 medicinal materials are unqualified, and the percent of pass is 79%, thereby determining that the content of the total saponins in the pink reineckea herb is not lower than 4.5%, and the percent of pass is 86%.

Claims (8)

1. A quality detection method of a pink reineckea herb medicinal material is characterized by comprising the following steps: determination items including identification and/or content; wherein the identification is thin-layer chromatography identification of the reineckia carnea medicinal material; the content determination is performed by high performance liquid chromatography to determine the content of the saponin A of the lucky herbal materials.

2. The quality detection method of the reineckia carnea medicinal material according to claim 1, wherein the thin-layer chromatography identification of the reineckia carnea medicinal material comprises the following steps:

(1) Taking 1g of reineckea carnea medicinal material sample powder, adding 20ml of methanol, heating and refluxing for 30 minutes, filtering, and taking supernate as a sample solution; preparing reference medicinal material 1g of herba Reineckeae Carneae, and making reference medicinal material solution by the same method;

(2) The thin layer chromatography test was performed by pipetting 10. Mu.l of each of the above two solutions, spotting the solution on the same silica gel G thin layer plate, adding chloroform: ethyl acetate: methanol: water =15:40:22: developing the lower layer of the 10 mixed solution with a developing agent, taking out, air drying, spraying with 10% sulfuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight;

in the chromatogram of the test solution, spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material, and the sample is herba Reineckeae Carneae medicinal material.

3. The quality detection method of the reineckia carnea medicinal material according to claim 2, wherein the high performance liquid chromatography determination method comprises the following steps:

1) Preparation of the Standard Curve

Precisely measuring control solution 0.04ml, 0.08ml, 0.1ml, 0.2ml, 0.4ml and 0.6ml, respectively placing in 10ml test tube with plug, treating the control solution according to vanillin-ethanol-perchloric acid method and measuring absorbance at 452 nm; drawing a standard curve by taking the absorbance as a vertical coordinate and the concentration as a horizontal coordinate;

2) Preparation of content determination test sample solution

Taking 0.5g of medicinal powder, placing the medicinal powder in a 50ml conical flask with a plug, precisely adding 20ml of methanol, weighing, heating and refluxing for 1.5 hours, cooling, complementing the lost weight with methanol, filtering, extracting filter residue twice with methanol in the same way, combining the three filtrates, recovering the solvent to dryness, dissolving the residue with methanol, transferring to a 50ml volumetric flask, adding methanol to scale, shaking up, filtering, and taking the subsequent filtrate to obtain the content measurement sample solution.

3) Measurement of

Precisely measuring 0.15ml of the content determination sample solution, placing in a 10ml test tube with a plug, measuring absorbance according to the method under the preparation item of the standard curve, reading the weight of saponin A in the content determination sample solution from the standard curve, and calculating to obtain the content of total saponin.

4. The quality detection method of the reineckea carnea medicinal material according to claim 3, characterized in that: in the step 2), the medicinal powder needs to be sieved by a fourth sieve.

5. The quality detection method of the reineckea carnea medicinal material according to claim 3, wherein in the step 1), the preparation process of the reference solution is as follows: taking a proper amount of saponin A reference substance, dissolving with methanol, transferring to a 25ml volumetric flask, diluting to scale, and shaking to obtain a reference substance solution with a concentration of 0.85 mg/ml.

6. The method for detecting the quality of the reineckia carnea medicinal material according to claim 3, wherein in the step 1), the specific method for processing the reference solution by the vanillin-ethanol-perchloric acid method comprises the following steps: volatilizing the solvent of each reference substance solution in a water bath, sequentially and precisely adding 0.2ml of 8% vanillin ethanol solution and 0.1ml of perchloric acid solution, sealing, shaking up, heating in a water bath at 60 ℃ for 15 minutes at constant temperature, taking out, cooling with ice water for 5min, precisely adding 5ml of glacial acetic acid, shaking up, and respectively measuring the absorbance at the wavelength of 452nm by using a corresponding reagent as a blank reference according to an ultraviolet-visible spectrophotometry method.

7. The quality detection method of the reineckia carnea medicinal material according to claim 3, characterized by comprising the following steps: the regression equation to plot the standard curve was Y =11.0109X-0.0120.

8. The method for detecting the quality of the reineckia carnea medicinal material according to any one of claims 1 to 7, wherein the saponin A is saponin (1 beta, 3 beta, 16 beta, 22S) -cholest-5-ene-1,3,16,22-tetrol1,16-di (beta-D-glucopyranoside).

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