A quality detection method for auspicious herbal medicine

Abstract

The present invention discloses a quality detection method for auspicious herbal medicine, including identification and / or content determination items; wherein the identification is thin layer chromatography identification of the auspicious herbal medicine; and the content determination is to determine the content of saponin A in the auspicious herbal medicine by high performance liquid chromatography. The quality detection method can accurately identify the authenticity of the medicinal materials, effectively distinguish the auspicious herbal medicine from its counterfeits and confusion products; and can accurately and quickly determine the content of saponin A in the auspicious herbal medicine, which is beneficial to the quality control of the auspicious herbal medicine. The quality detection method of the auspicious herbal medicine can effectively control the overall quality of the auspicious herbal medicine and ensure the safety and effectiveness of the clinical use of the auspicious herbal medicine.

Description

Technical Field

[0001] The invention relates to the field of auspicious herb material detection, in particular to a quality detection method for auspicious herb materials. Background Art

[0002] Reineckia carnea (Andr.) Kunth is the dried whole herb of Reineckia carnea (Andr.) Kunth, a plant of the Liliaceae family. In the relevant monographs and standards, the name of Reineckia carnea is mostly "Reineckia carnea". Only the Shaanxi Provincial Medicinal Material Standard (2015 edition) uses the name "Xiaozhugenqi (Reineckia carnea)", which is a local common name. This standard uses the name "Reineckia carnea".

[0003] Reineckia carnea (Andr.) Kunth is a dried whole herb of the Liliaceae plant Reineckia carnea (Andr.) Kunth. Reineckia carnea is mainly distributed in the provinces and regions of East China, Central South, and Southwest China, and is one of the traditional herbal medicines commonly used by the people and ethnic minority areas. The Jixiang Grassland Standard is included in the "Guangxi Zhuang Autonomous Region Traditional Chinese Medicine Standard" (1990 edition), "Jiangxi Province Traditional Chinese Medicine Standard" (1996 edition), "Guizhou Province Traditional Chinese Medicine, National Medicinal Materials Quality Standard" (2003 edition), "Yunnan Province Traditional Chinese Medicine Standard" (2005 edition, Volume 1: Yi Medicine), "Hunan Province Traditional Chinese Medicine Standard" (2009 edition), "Shaanxi Province Medicinal Materials Standard" [2015 edition, Xiaozhugenqi (Jixiang Grass)], which stipulates the properties, microscopic identification (cross section, powder), thin layer identification of reference medicinal materials, magazines, total ash, acid insoluble ash, 70% alcohol extract (hot leaching method), water-soluble extract, etc., but there is no provision for content determination, and the standard is not perfect. Therefore, it is urgent to develop and establish a quality detection method for Jixiang Grass, which is of great significance to control the overall quality of Jixiang Grass materials and ensure the safety and effectiveness of Jixiang Grass materials in clinical use. Summary of the invention

[0004] The purpose of the present invention is to provide a quality detection method for Jixiang herb materials. The present invention has the characteristics of being able to effectively control the overall quality of Jixiang herb materials and ensure the safety and effectiveness of the clinical use of Jixiang herb materials.

[0005] The technical solution of the present invention is a quality detection method for auspicious herb materials, including identification and / or content determination items; wherein the identification is a thin layer chromatography identification of the auspicious herb materials; and the content determination is to determine the content of saponin A in the auspicious herb materials by high performance liquid chromatography.

[0006] In the aforementioned quality detection method of the auspicious herb material, the thin layer chromatography identification of the auspicious herb material comprises the following steps:

[0007] (1) Take 1 g of the test sample powder of the herb, add 20 ml of methanol, heat and reflux for 30 minutes, filter, and take the supernatant as the test solution; take 1 g of the herb ...

[0008] (2) According to the thin layer chromatography test, 10 μl of each of the above two solutions were taken and spotted on the same silica gel G thin layer plate, and the lower layer of the mixture of chloroform: ethyl acetate: methanol: water = 15:40:22:10 was used as the developing solvent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and then inspected under sunlight;

[0009] If a spot of the same color appears in the chromatogram of the test sample at the corresponding position in the chromatogram of the control medicinal material, it is an auspicious herbal material.

[0010] In the aforementioned quality detection method of the auspicious herb, the high performance liquid chromatography determination method comprises the following steps:

[0011] 1) Preparation of standard curve

[0012] Accurately measure 0.04ml, 0.08ml, 0.1ml, 0.2ml, 0.4ml and 0.6ml of the reference solution and place them in 10ml stoppered test tubes respectively. Treat the reference solution according to the vanillin-ethanol-perchloric acid method and measure the absorbance at 452nm. Draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0013] 2) Preparation of test solution for content determination

[0014] Take 0.5g of medicinal powder and place it in a 50ml conical flask with a stopper, accurately add 20ml of methanol, weigh, heat and reflux for 1.5 hours, cool, make up the lost weight with methanol, filter, and extract the residue twice with methanol in the same way. Combine the three filtrates, recover the solvent to dryness, dissolve the residue in methanol, transfer it to a 50ml volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate to obtain the test solution for content determination.

[0015] 3) Determination

[0016] Accurately measure 0.15 ml of the test solution for content determination and place it in a 10 ml stoppered test tube. According to the method under the preparation of the standard curve, determine the absorbance according to the law, read the weight of saponin A in the test solution for content determination from the standard curve, and calculate the content of total saponins.

[0017] In the aforementioned method for testing the quality of auspicious herbal medicines, in step 2), the powder of the medicinal material needs to be passed through a No. 4 sieve.

[0018] In the aforementioned method for quality testing of auspicious herbal medicines, in step 1), the preparation process of the reference solution is: take an appropriate amount of saponin A reference substance, dissolve it with methanol, transfer it to a 25 ml volumetric flask, dilute it to the scale, and shake it well to obtain a reference solution with a concentration of 0.85 mg / ml.

[0019] In the aforementioned quality detection method for auspicious herbal medicine, in step 1), the specific method of treating the reference solution with the vanillin-ethanol-perchloric acid method is as follows: evaporate the solvent of each reference solution in a water bath, accurately add 0.2 ml of 8% vanillin ethanol solution and 0.1 ml of perchloric acid solution in sequence, seal, shake well, and heat at a constant temperature of 60°C in a water bath for 15 minutes, take out, cool with ice water for 5 minutes, accurately add 5 ml of glacial acetic acid, shake well, use the corresponding reagent as a blank reference, and measure the absorbance at a wavelength of 452 nm by UV-visible spectrophotometry.

[0020] In the aforementioned quality detection method of the auspicious herb, the regression equation for drawing the standard curve is Y=11.0109X-0.0120.

[0021] In the aforementioned method for detecting the quality of the herb, the saponin A is saponin (1β,3β,16 β,22S)-cholest-5-ene-1,3,16,22-tetrol 1,16-di(β-D-glucopyranoside).

[0022] Compared with the prior art, the quality inspection method of the present invention includes a thin layer identification method of auspicious herb medicinal materials and a method for determining the content of saponin A in auspicious herb medicinal materials. The quality inspection method can accurately identify the authenticity of medicinal materials and effectively distinguish auspicious herb medicinal materials from their counterfeits and mixed products; and can accurately and quickly determine the content of saponin A in auspicious herb medicinal materials, thereby facilitating the quality control of auspicious herb medicinal materials. The quality inspection method of auspicious herb medicinal materials can effectively control the overall quality of auspicious herb medicinal materials and ensure the safety and effectiveness of auspicious herb medicinal materials in clinical use.

[0023] Specifically, the thin-layer identification method of the auspicious herbal medicine of the present application has the characteristics of high sensitivity, strong specificity and good separation effect, is simple and feasible, has strong reproducibility, can accurately identify the authenticity of the medicinal materials, and effectively distinguish the auspicious herbal medicine from its counterfeits and mixed products.

[0024] The method for determining the content of saponin A in the herb of the present invention can accurately and quickly determine the content of the herb, providing an effective qualitative and quantitative analysis method for the herb, thereby facilitating the quality control of the herb. The quality detection method of the herb provided by the present invention can effectively control the overall quality of the herb and ensure the safety and effectiveness of the herb in clinical use.

[0025] The quality detection method of the auspicious herbal medicine provided by the present invention helps to make up for the "Guangxi Zhuang Autonomous Region Chinese Medicinal Materials Standard" (1990 edition), "Jiangxi Province Chinese Medicinal Materials Standard" (1996 edition), "Guizhou Province Chinese Medicinal Materials, National Medicinal Materials Quality Standard" (2003 edition), "Yunnan Province Chinese Medicinal Materials Standard" (2005 edition, Volume 1: Yi Medicine), "Hunan Province Chinese Medicinal Materials Standard" (2009 edition), "Shaanxi Province Medicinal Materials Standard" [2015 edition, Xiaozhugenqi (auspicious grass)], which stipulates that there are provisions for properties, microscopic identification (cross section, powder), thin layer identification of reference medicinal materials, magazines, total ash, acid-insoluble ash, 70% alcohol extract (hot immersion method), water-soluble extract, etc., but no provisions for measurement items, and the standards are not perfect. The method of the present invention is of great significance for the further quality control of auspicious herbal medicines, and can provide a scientific basis for the formulation of auspicious herbal medicine quality tables.

[0026] In summary, the present invention has the characteristics of being able to effectively control the overall quality of the Jixiang herbal medicine and ensure the safety and effectiveness of the clinical use of the Jixiang herbal medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a thin layer chromatogram developed with chloroform-methanol-water (65:35:10, lower layer);

[0028] Figure 2 is a thin layer chromatogram developed with chloroform-ethyl acetate-methanol-water (15:40:22:10, lower layer);

[0029] Figure 3 It is a thin layer chromatogram developed with chloroform-ethyl acetate-methanol-water (1:2:1:0.2);

[0030] Figure 4 It is a thin layer chromatogram developed with chloroform-ethyl acetate-methanol-water (1.5:4:2.7:1);

[0031] ( Figure 1-4 In the figure, 1 is saponin A reference substance 6 μL; 2 is lucky grass (S14) 10 μL; 3 is lucky grass (S14) 10 μL; 4 is lucky grass (S14) 10 μL);

[0032] Figure 5 It is the thin layer chromatogram investigated with different extraction solvents;

[0033] ( Figure 5 In the mixture, 1-saponin A reference substance 6 μL; 2-lucky grass (S14) 10 μL (methanol); 3-lucky grass (S14) 10 μL (ethanol); 4-lucky grass (S14) 10 μL (water);

[0034] Figure 6 It is the thin layer chromatogram of different extraction methods;

[0035] ( Figure 6 1-Saponin A reference substance 6 μL; 2-School of Medicinal Herbs (S14) 10 μL (heating reflux extraction for 30 min); 3-School of Medicinal Herbs (S14) 10 μL (ultrasonic extraction for 30 min); 4-School of Medicinal Herbs (S14) 10 μL (cold soaking for 1 h and ultrasonic extraction for 30 min);

[0036] Figure 7 It is a thin layer chromatogram of spot sample quantity investigation;

[0037] Figure 8 This is a thin layer chromatogram developed under a humidity of 25%;

[0038] Fig. 9 This is a thin layer chromatogram developed under a humidity of 75%;

[0039] Fig.10 It is a thin layer chromatogram developed at a temperature of 25°C;

[0040] Fig.11 It is a thin layer chromatogram developed at a temperature of 4°C;

[0041] ( Figure 7-11 In the mixture, 1-saponin A reference substance 6 μL; 2-lucky grass (S14) 6 μL; 3-lucky grass (S14) 8 μL; 4-lucky grass (S14) 10 μL);

[0042] Fig.12 It is the reference material for 15 batches of auspicious herbal medicines. Figure 1 ;

[0043] ( Fig.12 In the table, s is saponin A reference substance 6 μL, S1 to S7 are scutellaria baicalensis 10 μL);

[0044] Fig.13 It is the reference material for 15 batches of auspicious herbal medicines. Figure 2 ;

[0045] ( Fig.13 In the sample, s is saponin A reference substance 6 μL, S8-S15 are scutellaria baicalensis 10 μL);

[0046] Fig.14 is the absorption spectra of the reference solution and the test solution (vanillin-ethanol-perchloric acid method); ( Fig.14 In the figure, a is the reference substance; b is the test substance; c is the reference substance and the test substance);

[0047] Fig.15 It is the absorption spectrum of reference solution and test solution (perchloric acid method);

[0048] ( Fig.15 In the figure, d: control substance; e: test substance; f: control substance and test substance);

[0049] Fig.16 is the absorption spectrum of the reference solution and the test solution (vanillin-glacial acetic acid-perchloric acid method); ( Fig.16 In the figure, g: control substance; h: test substance; i: control substance and test substance);

[0050] Fig.17 This is the linear relationship diagram of saponin A. DETAILED DESCRIPTION

[0051] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0052] Embodiment. A method for testing the quality of auspicious herbal medicines,

[0053] instrument:

[0054] Agilent 1260 liquid chromatograph; evaporative light scattering detector (Alltech ELSD-2000); AUW220D electronic balance (1 / 100,000, Shimadzu Corporation, Japan); BS224S electronic balance (1 / 10,000, Sartorius); KQ-500DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); DHG-9140A electric constant temperature blast drying oven (Gongyi Yuhua Instrument Co., Ltd.); 8-10 box-type resistance furnace (manufactured by Shenyang Energy Saving Electric Furnace Factory, China).

[0055] Reagents and test drugs

[0056] 1. Reference

[0057] (1β,3β,16β,22S)-cholest-5-ene-1,3,16,22-tetrol1,16-di(β-D-glucopyranoside) (hereinafter referred to as "saponin A") is produced by the National Engineering Research Center for the Manufacturing Technology of Traditional Chinese Medicine Solid Preparations with a purity of >98%.

[0058] 2. Reagents

[0059] Acetonitrile (chromatographic grade, Fisher Scientific, Fairlawn, NJ, USA); formic acid (chromatographic grade, Sigma - Aldrich Co., Ltd., St Louis, MO, USA); methanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); chloroform (analytical grade, Shanghai Experiment Reagent Co., Ltd.); ethyl acetate (analytical grade, Guangdong Guanghua Sci - Tech Co., Ltd.); absolute ethanol (analytical grade, Shanghai Zhenxing Chemical Factory No. 1); silica gel plate GF254 (Qingdao Ocean Chemical Factory); sulfuric acid (analytical grade, Xilong Scientific Co., Ltd.); water was ultrapure water.

[0060] 3. Medicinal materials

[0061] The medicinal materials used in this study were purchased from Guizhou Province and identified by Professor Zhong Guoyue of the Ethnic Medicine Resource Center of Jiangxi University of Traditional Chinese Medicine as the whole herb of Reineckia carnea (Andr.) Kunth, a plant of the genus Reineckia in the family Liliaceae. The specimen (Z - 140310 - 01) was preserved in the Ethnic Medicine Resource Center of Jiangxi University of Traditional Chinese Medicine. (Table 1)

[0062] Table 1 Batches of Reineckia carnea medicinal materials

[0063]

[0064]

[0065]

Medicinal material name

[0066] The name of Reineckia carnea medicinal materials is mostly "Reineckia carnea" in relevant monographs and standards. Only in the "Shaanxi Provincial Medicinal Material Standard" (2015 edition), the name "Xiaozhugengenqi (Reineckia carnea)" is used, which is a local common name. In this standard, the name "Reineckia carnea" is adopted.

[0067] Reineckia carnea was first recorded in "Supplements to Newly Revised Materia Medica" by Chen Cangqi in the Sui and Tang dynasties. "Compendium of Materia Medica" states: "Reineckia carnea has leaves like Zhanglan, evergreen throughout the four seasons, with purple flowers in spikes in summer and is easy to propagate." "Illustrated Classics of Plants" says: "Songshoulan has slightly wider leaves, six - petaled flowers that are slightly larger, blooming in winter, planted in pots. In autumn, it bears fruits like Asparagus cochinchinensis, and the fruits are red - purple with tips." Combining the above descriptions with the attached pictures, it can be confirmed that its original plant conforms to the current Reineckia carnea (Andr.) Kunth of the family Liliaceae. Also, the "Rohdea japonica" described in "Outline of Medicinal Herbs" mentions that it "looks like orchid leaves", and its characteristics do not match those of Rohdea japonica, but should be Reineckia carnea. "Supplements to Compendium of Materia Medica" calls it Jieyuncao. "Classified Treatises on Febrile Diseases" also names it Yangjixiangcao. "Flora of Chinese Medicinal Plants" names it Zhuyeqing. "Sichuan Traditional Chinese Medicine Standards" names it Jiujielian. "Guizhou Herbal Medicine" names it Xiaojiulongpan. "Guizhou Chinese Herbal Medicine Directory" names it Guanyincao, Diyuyi, Qianlima. [2]Reineckia carnea is mainly distributed in the provinces of East China, Central South and Southwest China, and is widely used in folk medicine in various places.

[0068] In relevant monographs and original standards, the source of Reineckia carnea is stipulated as the whole plant of Reineckia carnea (Andr.) Kunth of the Liliaceae family, and the medicinal part is "dry whole herb" or "dry whole herb with roots". In this draft standard, it is stipulated as "dry whole herb of Reineckia carnea (Andr.) Kunth of the Liliaceae family".

[0069] The name of Reineckia carnea medicinal material is mostly "Reineckia carnea" in relevant monographs and standards. Only in the "Shaanxi Provincial Medicinal Material Standard" (2015 edition), the name "Xiaozhugenqi (Reineckia carnea)" is used, which is a local common name. In this standard, the name "Reineckia carnea" is adopted.

[0070] Perennial herb, with stems creeping on the ground, resembling rhizomes, green, multi-jointed, with fibrous roots growing at the joints. Leaves are clustered at the top of the stem or at the stem nodes, 3 - 8 leaves in each cluster, the leaf blades are linear to lanceolate, 10 - 38 cm long, 0.5 - 3.5 cm wide, gradually tapering at the apex, and gradually narrowing downward into a petiole. The scape is 5 - 15 cm long, the spike inflorescence is 2 - 6.5 cm long, and the upper flowers sometimes only have stamens; the bracts are ovate-triangular, membranous, light brown, or purplish; the perianth segments are united into a short tube, with 6 lobes at the upper part, the lobes are oblong, 5 - 7 mm long, slightly fleshy, reflexed when flowering, pink, and the flowers are fragrant; there are 6 stamens, the filaments are filiform, the anthers are nearly oblong, slightly concave at both ends; the ovary is flask-shaped, 3-locular, shorter than the style, the stigma is capitate, 3-lobed. The berry is spherical, 6 - 10 mm in diameter, bright red when ripe. The flowering and fruiting period is from July to November. It grows in shady and humid slopes, valleys or under dense forests at an altitude of 170 - 3200 m or is cultivated. Distributed in Jiangsu, Zhejiang, Anhui, Jiangxi, Hubei, Hunan, Guizhou, Yunnan, Sichuan, Guangxi, Guangdong, Henan, Shaanxi (south of the Qinling Mountains) and other places.

[0071]

Character

[0072] The rhizome of this product is cylindrical, of varying lengths, 0.2 - 0.5 cm in diameter, with a yellowish-brown or yellowish-green surface; the nodes are obvious, slightly swollen, often with residual membranous scale leaves and curly fibrous roots, and the roots are densely covered with white hairs; the internodes are short and contracted, with longitudinal wrinkles. The leaves are clustered at the top of the stem or at the nodes, the leaf blades are greenish-brown or brownish, mostly wrinkled, and when fully moistened and unfolded, they are lanceolate in shape, entire, sessile, with parallel veins, and the midvein is obvious. It has a slight odor and tastes bitter and slightly bitter.

[0073] Thin-layer identification:

[0074] The original TLC identification methods for reference medicinal materials in the standards of Guizhou, Hunan and Shaanxi provinces were different. The developing agents were: 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). After the three developing agents were re-tested, the development effect was not good. Therefore, a new TLC identification method was established. In the study, a new TLC identification method was established based on the main saponin component (1β,3β,16β,22S)-cholest-5-ene-1,3,16,22-tetrol 1,16-di(β-D-glucopyranoside, C 39 H 66 O 14 , hereinafter referred to as saponin A as the control thin layer identification, so in the methodological investigation of control medicinal materials, saponin A is used as the control.

[0075] Take 1g of the powder of this product, add 20ml of methanol, heat and reflux for 30 minutes, filter, and take the supernatant as the test solution. Take another 1g of the reference medicinal material of Jixiangcao, and prepare the reference medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 10μl of each of the above two solutions, and spot them on the same silica gel G thin layer plate, use chloroform-ethyl acetate-methanol-water (15:40:22:10) as the lower layer as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under sunlight. In the chromatogram of the test product, spots of the same color appear at the corresponding position of the chromatogram of the reference medicinal material.

[0076] 1) Investigation of developing agent

[0077] Take 3 portions of Jixiang herbal powder and prepare the test solution according to the above method. With saponin A as the reference substance, 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 plate: silica gel GF254 plate (50×100mm), development method and development distance: upward development 8cm, color development method: 10% sulfuric acid ethanol solution heated at 105℃ for 5min, placed under sunlight for inspection.

[0078] The results showed that the spots were well separated and clear in the chromatogram developed with chloroform-ethyl acetate-methanol-water (15:40:22:10, lower layer) as the developing solvent. Therefore, chloroform-ethyl acetate-methanol-water (15:40:22:10, lower layer) was finally selected as the developing solvent. (See Figure 1 to Figure 4 )

[0079] 2) Investigation of extraction solvent

[0080] Take 1g of the powder of the herb, add 20ml of methanol, ethanol and water respectively, heat and reflux for 30min, cool, filter and take the filtrate as the test solution. Spot the sample and develop.

[0081] When methanol is used as the extraction solvent, the extraction effect is better and the spots are clearer, so methanol is selected as the extraction solvent (see Figure 5 ).

[0082] 3) Investigation of extraction methods

[0083] Take 1g of the powder of the herb, add 20ml of methanol, and extract by heating reflux extraction for 30min, ultrasonic extraction for 30min, and ultrasonic extraction for 30min after cold soaking for 1h, cool, filter, and take the filtrate as the test solution. Spot the sample and develop it.

[0084] The results showed that the extraction effect of heating reflux extraction for 30 minutes was better, and the spots were clearer. Therefore, heating reflux extraction for 30 minutes was selected as the extraction method for the identification of the herb of Jixiangcao. (See Figure 6 )

[0085] 4) Sample quantity inspection

[0086] Take 1g of the powder of the auspicious herb (passed through a No. 4 sieve) in triplicate, prepare the test solution according to the above method, apply the sample, and develop it.

[0087] The results show that when the sample volume is 10 μl, the spots are clearer. Therefore, the sample volume is determined to be 10 μl. (See Figure 7 ).

[0088] 5) Conduct durability test under environmental conditions

[0089] Take 1g of the powder of the auspicious herb (passed through a No. 4 sieve) in triplicate, prepare the test solution according to the above method, apply the sample, and develop it.

[0090] The results showed that the selected developing agent was tested at 25% and 75% relative humidity and 4°C (refrigerator) and room temperature (25°C), and good separation results were obtained (see Figure 8 to Figure 11 ).

[0091] 6) Identification results of 15 batches of samples

[0092] According to the above method, 15 batches of Herba Lycopodii samples were subjected to TLC identification as reference medicinal materials.

[0093] The final determined thin-layer identification method for the reference medicinal material of the herb is: take 1g of the powder of this product, add 20ml of methanol, heat and reflux for 30 minutes, filter, and take the supernatant as the test solution. Take another 1g of the reference medicinal material of the herb, and prepare the reference medicinal solution in the same way. According to the thin-layer chromatography method (General Rule 0502), take 10μl of each of the above two solutions, spot them on the same silica gel G thin-layer plate, and use chloroform-ethyl acetate-methanol-water (15:40:22:10) as the lower layer as the developing agent. Develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under sunlight. In the chromatogram of the test product, spots of the same color appear at the corresponding position of the chromatogram of the reference medicinal material. (See Figure 12-13 )

[0094] [Content determination] There is no "content determination" provision in the original Jixiang Grassland standards.

[0095] Literature reports show that the types of compounds found in the herb include steroidal saponins, flavonoids, lignans, terpenes, etc. Among them, steroidal saponins have been studied more. The more common steroidal saponins and steroidal sapogenins are: proto-aspidosporin, neo-aspidosporin, katisaponin, iso-aspidosporin, (1β,3β,16β,22S)-cholest-5-ene-1,3,16,22-tetrol1,16-di(β-D-glucopyranoside), (1β,3β,16β,22S)-cholest-5-ene-1,3,16,22-tetrol1-[O-α-L-rhamnopyranosyl-(1→2)-β-D -glucopyranoside]16-(β-D-glucopyranoside), sapogenin, diosgenin, aspidistanin, isokariagenin, β-sitosterol, β-sitosterol-β-D-glucoside, stigmasterol-3-O-β-D-pyranoglucoside, β-amyrin, α-spinasterol-3-O-glucoside, etc.; flavonoids include: 7-methoxy-8-methyl-4'-hydroxy-flavone, 1,6-dihydroxy-8-methyl-xanthane, quercetin-3-O-α-L-pyranorhamnosyl-(1-6)-β-D-pyranoglucoside, broad bean root flavonoid glycoside B, 6,8-dimethyl-5,7,4'-trihydroxyflavone, (R)-8- Methylnaringenin, naringenin, daidzein, 3'-hydroxydaidzein, etc.; terpenes include ursolic acid squalene, etc.; lignans include syringaresinol-1-β-D-glucoside, syringaresinol, etc.; others include 1O-hexadecanoic acid glyceride, palmitic acid, tetradecanoic acid, inositol, Np-coumaroyltyramine, methyl linoleate, n-triacontane, daidzein N-benzoyl-L-phenylalaninol, 2-ethylhexyl phthalate, dibutyl phthalate, eleutheroside, etc.[5-11] . Studies have shown that the saponin components of scutellaria baicalensis have anti-tumor activity. Regarding the quality evaluation of scutellaria baicalensis, there are literature reports that the total saponin content of 13 samples in Guizhou was measured, and the average value was 1.28% (0.86-1.73%), and it is recommended that the total saponin content be no less than 1.0%; the results of the measurement of 3 batches of scutellaria baicalensis materials showed that the ferulic acid content was 0.011-0.013%, and the rutin content was 0.047-0.054%.

[0096] In this study, we first conducted a preliminary investigation on the components of the herb reported in the literature, and the results showed that saponins were its main components, while the contents of monomer compounds of other components were all low. At the same time, the antitussive and expectorant activity tests were conducted on the various extracted parts of the herb, and the results showed that the 80% ethanol extract and its ethyl acetate and n-butanol parts had good antitussive and expectorant effects; further separation, purification and identification of 27 compounds (including 4 new compounds, 1 new natural product, and 13 first-time discoveries in this genus) from the ethyl acetate and n-butanol parts of the 80% ethanol extract were obtained, mainly saponins; the anti-complement activity of the 27 components was then determined, and the results showed that most saponin components showed anti-complement activity after hydrolysis.

[0097] The above research results show that saponins are the main biologically active components of the herb, so the main saponin (1β,3β,16β,22S)-cholest-5-ene-1,3,16,22-tetrol 1,16-di(β-D-glucopyranoside) (hereinafter referred to as saponin A, C 39 H 66 O 14 ) and prepare the compound monomer (purity greater than 98%). Referring to the literature reports on the content determination of total saponins, ferulic acid, rutin, etc. of the herb, using total saponins as the content measurement index should be more in line with the actual situation of the medicinal materials, so total saponins are selected as the content measurement item limit regulation.

[0098] Determine according to the UV-Vis spectrophotometric chromatography method (General Rule 0401 of Part 4 of the 2020 edition of the Chinese Pharmacopoeia). The determination method is as follows:

[0099] Preparation of reference solution: Take 21.22 mg of saponin A reference substance, dissolve it in methanol, transfer it to a 25 ml volumetric flask, dilute to the scale, and shake well to obtain a reference solution with a concentration of 0.8488 mg / ml.

[0100] 1. Selection of color development system and detection wavelength

[0101] Weigh 0.5 g of the powder of the auspicious herb (passed through a No. 4 sieve), accurately weigh it, place it in a 50 ml stoppered conical flask, add 20 ml of methanol, weigh it, heat and reflux to extract for 30 minutes, cool it, make up the loss with methanol, filter it, and take the filtrate to obtain the product.

[0102] (1) Vanillin-ethanol-perchloric acid method: Accurately pipette 0.5 ml of the reference solution and 0.15 ml of the test solution into 10 ml stoppered test tubes, evaporate the solvent in a water bath, add 0.2 ml of 8% vanillin ethanol solution and 0.1 ml of perchloric acid solution in turn, seal the tubes, shake well, and heat in a 60°C water bath for 15 min. Immediately cool with ice water for 5 min, add 5 ml of glacial acetic acid, and shake well. Use the colorimetric agent as the blank reference.

[0103] (2) Perchloric acid method: Accurately pipette 0.4 ml of the reference solution and 0.15 ml of the test solution into 10 ml stoppered test tubes, evaporate the solvent in a water bath, accurately add 5 ml of perchloric acid, shake well, and react at room temperature for 25 min. Use the colorimetric reagent as a blank reference.

[0104] (3) Vanillin-acetic acid-perchloric acid method: Accurately pipette 0.2 ml of the reference solution and 40 μl of the test solution into 10 ml stoppered test tubes, evaporate the solvent in a water bath, add 0.2 ml of 5% vanillin-acetic acid solution and 0.8 ml of perchloric acid in turn, seal the tubes, shake well, and heat in a 60°C water bath for 20 min. Immediately cool with ice water for 5 min, add 5 ml of acetic acid, and shake well. Use the colorimetric agent as the blank reference.

[0105] The results of scanning with an ultraviolet spectrophotometer within 350-800 nm showed that when the vanillin-ethanol-perchloric acid method was used as the color developer, the absorption spectra of the reference solution and the test solution in the visible light region were consistent, so the maximum absorption wavelength was determined to be 452 nm. The results are shown in Table 2. Figure 14 to Figure 16

[0106] Table 2 Maximum absorption wavelengths of reference solution and test solution

[0107]

[0108] 2. Investigation of extraction methods

[0109] 2.1 Investigation of extraction solvents

[0110] Weigh 0.5g of medicinal material powder (passed through a No. 4 sieve), weigh six portions accurately, place in a 50ml stoppered conical flask, add 20ml of three different solvents, methanol, ethanol, and water, weigh, heat and reflux for 30 minutes, cool, make up the lost weight, filter, take the filtrate, and determine the total saponin content. (Table 3) Table 3 Results of investigation of different extraction solvents

[0111]

[0112] It can be seen from the above table that when methanol is used as the extraction solvent, the total saponin content is the highest, so methanol is selected as the extraction solvent.

[0113] 2.2 Investigation of extraction solvents with different concentrations

[0114] Weigh 0.5 g of medicinal powder (passed through No. 4 sieve) and take eight portions, accurately weigh them, place them in 50 ml stoppered conical flasks, add 30% methanol, 50% methanol, 70% methanol, and 20 ml of methanol respectively, weigh them, heat and reflux to extract for 30 minutes, cool, make up the lost weight, filter, take the filtrate, and determine the total saponin content respectively.

[0115] (Table 4)

[0116] Table 4 Results of investigation on different concentrations of extraction solvents

[0117]

[0118] It can be seen from the above table that when anhydrous methanol is used as the extraction solvent, the total saponin content is the highest, so methanol is selected as the extraction solvent.

[0119] 2.3 Extraction time investigation

[0120] Weigh 0.5g of medicinal material powder (passed through No. 4 sieve), weigh eight portions accurately, place in 50ml stoppered conical flasks, add 20ml of methanol respectively, weigh, heat and reflux to extract for 0.5h, 1h, 1.5h, 2h respectively, cool, make up the lost weight, filter, take the filtrate, and determine the total saponin content respectively. (Table 5)

[0121] Table 5 Results of investigation at different extraction times

[0122]

[0123] It can be seen from the above table that the total saponin content is slightly different between the heating reflux extraction for 1.5 h and the heating reflux extraction for 2 h. In order to save time, the heating reflux extraction for 1.5 h is selected.

[0124] 2.4 Investigation of extraction times

[0125] Weigh 0.5g of medicinal material powder (passed through No. 4 sieve), weigh six portions accurately, place in 50ml stoppered conical flasks, add 20ml of methanol respectively, weigh, heat and reflux extraction 1, 2, 3 times, each time for 1.5h, cool, make up the lost weight, filter, take the filtrate, and determine the total saponin content respectively. (Table 6) Table 6 Results of different extraction times

[0126]

[0127] It can be seen from the above table that the total saponin content is the highest when the extraction is carried out under heating reflux for 3 times, so the extraction is carried out under heating reflux for 3 times.

[0128] The final method for preparing the test solution for the determination of the total saponin content of Herba Lycopodii was as follows: 0.5 g of medicinal material powder (passed through a No. 4 sieve) was accurately weighed, placed in a 50 ml conical flask with a stopper, 20 ml of methanol was accurately added, weighed, heated under reflux for 1.5 hours, cooled, and the lost weight was made up with methanol, filtered, and the residue was extracted twice with methanol in the same way, the three filtrates were combined, the solvent was recovered to dryness, the residue was dissolved in methanol, transferred to a 50 ml volumetric flask, methanol was added to the scale, shaken well, filtered, and the filtrate was obtained.

[0129] 3 Methodological validation

[0130] 3.1 Linearity experiment

[0131] Accurately pipette 0.04, 0.08, 0.1, 0.2, 0.4, and 0.6 ml of the reference solution into 10 ml stoppered test tubes, treat the samples according to the vanillin-ethanol-perchloric acid method, and measure the absorbance at 452 nm. Draw a standard curve with absorbance (A) as the ordinate and concentration (C) as the abscissa. The regression equation is Y=11.0109X-0.0120(r 2 =0.9996), saponin A has good linearity with absorbance in the range of 33.95-509.28 μg. Fig.17 )

[0132] 3.2 Stability test

[0133] Take 0.15 ml of the same auspicious herbal medicine sample solution, treat the sample according to the vanillin-ethanol-perchloric acid method, and measure the absorbance at 452 nm after 0, 10, 20, 30, 40, 50, and 60 minutes, and calculate the RSD value. (Table 7)

[0134] Table 7 Stability test results

[0135]

[0136]

[0137] The results showed that the RSD value of the same sample solution of Herba Lysimachiae was 2.02% when measured after 0, 10, 20, 30, 40, 50 and 60 min, indicating that the solution stability of the method was good.

[0138] 3.3 Precision experiment

[0139] Take 0.15 ml of the same sample solution of the herbal medicine, treat the sample according to the vanillin-ethanol-perchloric acid method, and measure the absorbance at 452 nm for 6 consecutive times to calculate the RSD value (Table 8).

[0140] Table 8 Precision test results

[0141]

[0142] The results showed that the RSD value was 1.45% after six consecutive measurements, indicating that the instrument had good precision.

[0143] 3.4 Repeatability Experiment

[0144] Take 6 portions of the same batch of Herba Lucidae, prepare the test solution according to the above Herba Lucidae total saponin extraction method, treat the sample according to the vanillin-ethanol-perchloric acid method, measure the absorbance at 452nm, and calculate the RSD value. (Table 9)

[0145] Table 9 Results of repeatability test

[0146]

[0147]

[0148] The results showed that 6 samples of the test solution of the herb were prepared in parallel and the RSD value was 1.43%, indicating that the method had good repeatability.

[0149] 3.5 Sample recovery experiment

[0150] Accurately weigh 6 portions of 0.25g of Herba Lycopodiellae with known total saponin content, add a certain amount of saponin A reference substance, prepare the test solution according to the above-mentioned Herba Lycopodiellae total saponin extraction method, treat the sample according to the vanillin-ethanol-perchloric acid method, measure the absorbance at 452nm, calculate the total saponin content and the recovery rate.

[0151] (Table 10)

[0152] Table 10 Results of sample recovery experiment

[0153]

[0154] The average recovery rate of the sample was 99.24% (RSD = 2.43%), indicating that the recovery rate of the method was good.

[0155] 4. Determination of total saponins in herbal medicine

[0156] Take 0.5g of each batch of Jixiang herb materials, prepare according to the preparation method of the test solution, take 0.15ml of the test solution and treat the sample according to the vanillin-ethanol-perchloric acid method, measure the absorbance at 452nm, read the weight (mg) of saponin A in the test solution from the standard curve, and calculate the content of total saponins. (Table 11)

[0157] Table 11 Determination results of total saponin content in 28 batches of Jixiang herbal medicine

[0158]

[0159]

[0160] The results showed that the total saponin content of 28 batches of Jixiangcao medicinal materials ranged from 3.00% to 9.49%, with an average of 5.98%. Taking the average value down by 20% to 4.78%, 6 batches of the 28 batches of medicinal materials failed, and the qualified rate was 79%. Based on this, it was determined that the total saponin content of Jixiangcao should not be less than 4.5%, and the qualified rate was 86%.

Claims

1. A method for testing the quality of auspicious herbal medicine, characterized in that: It includes identification and content determination items; the identification is the thin layer chromatography identification of the auspicious herb materials; the content determination is the determination of the content of saponin A in the auspicious herb materials by high performance liquid chromatography; The thin layer chromatography identification of auspicious herbal medicine includes the following steps: (1) Take 1 g of the test sample powder of the herb, add 20 ml of methanol, heat and reflux for 30 minutes, filter, and take the supernatant as the test solution; take 1 g of the herb ... (2) According to the thin layer chromatography test, take 10 μl of each of the two solutions in step (1) and spot them on the same silica gel G thin layer plate, use the lower layer of the mixture of chloroform: ethyl acetate: methanol: water = 15:40:22:10 as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under sunlight; If the test sample chromatogram shows spots of the same color at the corresponding positions of the control herbal material chromatogram, it is an auspicious herbal material; The high performance liquid chromatography method comprises the following steps: 1) Preparation of standard curve Accurately measure 0.04 ml, 0.08 ml, 0.1 ml, 0.2 ml, 0.4 ml, and 0.6 ml of the reference solution and place them in 10 ml stoppered test tubes respectively. Treat the reference solution according to the vanillin-ethanol-perchloric acid method and measure the absorbance at 452 nm. Draw a standard curve with absorbance as the ordinate and concentration as the abscissa. 2) Preparation of test solution for content determination Take 0.5 g of medicinal material powder, put it in a 50 ml conical flask with a stopper, accurately add 20 ml of methanol, weigh it, heat and reflux for 1.5 hours, cool it, make up the lost weight with methanol, filter it, extract the residue with methanol twice in the same way, combine the three filtrates, recover the solvent to dryness, dissolve the residue in methanol, transfer it to a 50 ml volumetric flask, add methanol to the scale, shake it well, filter it, and take the filtrate to obtain the test solution for content determination; 3) Determination Accurately measure 0.15 ml of the test solution for content determination and place it in a 10 ml stoppered test tube. According to the method under the preparation of the standard curve, measure the absorbance according to the law, read the weight of saponin A in the test solution for content determination from the standard curve, and calculate the content of total saponins; The saponin A is saponin (1β,3β,16β,22S)-cholest-5-ene-1,3,16,22-tetrol 1,16-di(β-D-glucopyranoside).

2. The quality inspection method of auspicious herbal medicine according to claim 1, characterized in that: In step 2), the medicinal powder needs to be passed through a No. 4 sieve.

3. The quality inspection method of auspicious herbal medicine according to claim 1, characterized in that: In step 1), the preparation process of the reference solution is as follows: take an appropriate amount of saponin A reference, dissolve it in methanol, transfer it to a 25 ml volumetric flask, dilute it to the scale, and shake well to obtain a reference solution with a concentration of 0.85 mg / ml.

4. The quality inspection method of auspicious herb materials according to claim 1, characterized in that: In step 1), the specific method for treating the reference solution by the vanillin-ethanol-perchloric acid method is as follows: evaporate the solvent of each reference solution in a water bath, accurately add 0.2 ml of 8% vanillin ethanol solution and 0.1 ml of perchloric acid solution in sequence, seal, shake well, and heat at a constant temperature of 60°C in a water bath for 15 minutes, take out, cool with ice water for 5 minutes, accurately add 5 ml of glacial acetic acid, shake well, use the corresponding reagent as a blank reference, and measure the absorbance at a wavelength of 452 nm by UV-visible spectrophotometry.

5. The quality inspection method of auspicious herbal medicine according to claim 1, characterized in that: The regression equation for drawing the standard curve is Y=11.0109X-0.0120.

Images