A method for detecting the quality of Psoralea corylifolia medicinal material

Abstract

The present invention discloses a quality detection method for Caulis dasyphylla medicinal materials, including using thin layer chromatography to identify Caulis dasyphylla medicinal materials, including the following operating steps: (1) taking Caulis dasyphylla medicinal materials, adding methanol, heating and refluxing extraction, cooling to room temperature, supplementing the amount lost with methanol, filtering, and taking the filtrate as a test solution; (2) taking Caulis dasyphylla control medicinal materials, adding methanol, heating and refluxing extraction, cooling to room temperature, supplementing the amount lost with methanol, filtering, and taking the filtrate as a control medicinal material solution; (3) taking the test solution and the control medicinal material solution, respectively, spotting on the same silica gel thin layer plate, using acetone-ethanol-acetic acid-ammonia water with a volume ratio of 3:1.5:0.2:0.5 as a developing agent, developing, taking out, drying, and placing under ultraviolet light for inspection. The thin layer chromatography used in the present invention can accurately identify the authenticity of Caulis dasyphylla medicinal materials.

Description

Technical Field

[0001] The invention relates to a method for detecting the quality of traditional Chinese medicine, in particular to a method for detecting the quality of Paridis parviflora medicinal material. Background Art

[0002] SabiaparvifloraWall.ex Roxb. is a plant of the family Sabiaparviaceae. It is an evergreen woody climbing vine, 2-4m long. The bud scales are ovate, with long pointed tips, midribs on the back, and ciliate margins. The leaves are simple and alternate; the petiole is 0.5-2cm long; the leaves are papery or nearly thin leathery, ovate-lanceolate, narrowly oblong or oblong-elliptic, 5-12cm long, 1-3cm wide, with acuminate tips and rounded or broadly cuneate bases; there are 5-8 lateral veins on each side. Flowers are small, bisexual; cymes are integrated into panicles, with 10 to 20 (~25) flowers, peduncle 2 to 6 cm long, pedicel 3 to 6 mm long; flowers are green or yellow-green; sepals 5, about 0.8 mm long, with cilia; petals 5, oblong or oblong-lanceolate, 2 to 3 mm long, acute or obtuse at the apex, with red veins; stamens 5, filaments thick and flat; disc cup-shaped, margin 5 deeply lobed; ovary glabrous. Segmented fruit valves are nearly circular, 5 to 7 mm in diameter; ribs in the core are not obvious. Flowering period is March to May, fruiting period is July to September. Grown in ravines, streamside forests or hillside shrub forests at an altitude of 800 to 2800 m. Distributed in Guangxi, Guizhou and Yunnan.

[0003] The dried stems and leaves of Sabiaparviflora Wall.ex Roxb. are used as medicines. They have the effects of clearing away heat and dampness, promoting bile secretion, and stopping bleeding. They are used for jaundice and damp-heat syndrome, and traumatic bleeding. They are used as ethnic folk medicines. Currently, only the Quality Standards for Traditional Chinese and Ethnic Medicinal Materials in Guizhou Province (2003 edition) contains "Sabiaparviflora Wall.ex Roxb." Its origin is only Sabiaparviflora Wall.ex Roxb., and it is recorded as a "medicine for ethnic minorities" with stems and leaves as medicine. The original standard only has provisions for [properties] and [identification] (stem and leaf cross-section, physical and chemical identification), but the original identification method is not accurate enough, and some components cannot be well identified, resulting in the inability to effectively control the overall quality of Sabiaparviflora Wall.ex Roxb. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting the quality of the medicinal material of Psoralea corylifolia. The present invention has the characteristic of being able to accurately identify the authenticity of the medicinal material of Psoralea corylifolia.

[0005] The technical solution of the present invention is a method for detecting the quality of the medicinal material of Psoralea corylifolia, comprising using thin layer chromatography to identify the medicinal material of Psoralea corylifolia, and comprising the following steps:

[0006] (1) Take the medicinal material of Psoralea corylifolia, add methanol, heat and reflux to extract, cool to room temperature, make up the lost amount with methanol, filter, and take the filtrate as the test solution;

[0007] (2) Take the control medicinal material of Psoralea corylifolia, add methanol, heat and reflux to extract, cool to room temperature, make up the lost amount with methanol, filter, and take the filtrate as the control medicinal material solution;

[0008] (3) Take the test sample solution and the control drug solution and spot them on the same silica gel thin layer plate respectively. Use acetone-ethanol-acetic acid-ammonia water with a volume ratio of 3:1.5:0.2:0.5 as the developing agent, develop, take out, dry, and examine under ultraviolet light.

[0009] In the aforementioned method for detecting the quality of the medicinal material of Pleurotus eryngii, in step (1) and step (2), the solid-liquid ratio between the medicinal material and methanol is 1 g:20 ml, and the heating reflux extraction time is 30 min.

[0010] In the aforementioned method for detecting the quality of the medicinal material of Paridis parviflora, in the thin layer chromatogram of the test solution, fluorescent spots of the same color appear at corresponding positions in the thin layer chromatogram of the control medicinal material solution.

[0011] In the aforementioned method for detecting the quality of the medicinal material of Pleurotus eryngii, in step (3), the wavelength of the ultraviolet lamp is selected to be 365nm, and the spot volume of the test solution and the control medicinal material solution is 10μl.

[0012] In the aforementioned quality detection method for the medicinal material of Caulis Spatholobi, the quality detection method also includes using high performance liquid chromatography to determine the content of magnolamine and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside in the medicinal material of Caulis Spatholobi, comprising the following steps:

[0013] (1) Take magnolia alkaloids and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside reference substances and add 20% methanol to prepare reference substance solutions;

[0014] (2) Take the medicinal material of Psoralea corylifolia, add 70% methanol, weigh it, ultrasonicate it for 30 minutes, cool it to room temperature, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution;

[0015] (3) Take the reference solution and the test solution, inject them into the liquid chromatograph, and measure the content of magnolamine and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside.

[0016] In the aforementioned method for testing the quality of the medicinal material of Pleurotus eryngii, in the step (1), every 1 ml of the reference solution contains 0.1 mg of magnolamine and 0.2 mg of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside.

[0017] In the aforementioned method for detecting the quality of the medicinal material of Pleurotus eryngii, in the step (2), the solid-liquid ratio of the medicinal material of Pleurotus eryngii to 70% methanol is 1 g:25 ml.

[0018] In the aforementioned method for detecting the quality of the medicinal material of Pleurotus eryngii, in the step (3), the injection volume of the reference solution and the test solution is 5 μl.

[0019] In the aforementioned method for detecting the quality of the medicinal material of Psoralea corylifolia, in the step (3), the chromatographic conditions of the liquid chromatograph are: octadecylsilane bonded silica gel as the filler; methanol-0.2% triethylamine with a volume ratio of 20:80 as the mobile phase, wherein phosphoric acid is added to the 0.2% triethylamine to adjust the pH to 3.0; and the detection wavelength is 260nm.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The thin-layer identification method of the small-flowered Qingfengteng medicinal material of the present invention has the characteristics of high sensitivity, strong specificity and good separation effect. It is simple and feasible, with strong reproducibility. It can accurately identify the authenticity of the medicinal material and effectively distinguish the small-flowered Qingfengteng medicinal material from its counterfeits and confused products.

[0022] 2. The method for determining the content of magnolamine and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrenediol-4-O-β-D-glucoside in the medicinal material of Caulis Sinensis can accurately and quickly determine the content of Caulis Sinensis, providing an effective qualitative and quantitative analysis method for the medicinal material of Caulis Sinensis, which is beneficial to the quality control of the medicinal material of Caulis Sinensis. The quality detection method of Caulis Sinensis provided by the present invention can effectively control the overall quality of the medicinal material of Caulis Sinensis and ensure the safety and effectiveness of the clinical use of the medicinal material of Caulis Sinensis.

[0023] 3. The quality detection method of the medicinal material of the small flowered Pleurotus eryngii provided by the present invention helps to make up for the deficiencies of the quality detection method in the "Quality Standards of Traditional Chinese and Ethnic Medicinal Materials in Guizhou Province" (2003 edition), and has strong practicality. The method of the present invention is of great significance for the further quality control of the medicinal material of the small flowered Pleurotus eryngii, and can provide a scientific basis for the formulation of the quality table of the medicinal material of the small flowered Pleurotus eryngii. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the TLC of three developing agents in the identification of comparative medicinal materials;

[0025] Figure 2 It is the thin layer chromatogram of different extraction solvents in the identification of comparative medicinal materials;

[0026] Figure 3 It is a thin layer chromatogram of three extraction methods in the identification of comparative medicinal materials;

[0027] Figure 4 It is a thin layer chromatogram of different sample amounts in the identification of reference medicinal materials;

[0028] Figure 5 It is the thin layer chromatogram of the control medicinal material of Caulis Spatholobi and 18 batches of Caulis Spatholobi medicinal material samples;

[0029] Figure 6 It is the thin layer chromatogram of different extraction methods in the identification of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrenediol-4-O-β-D-glucoside;

[0030] Figure 7 It is the thin layer chromatogram of different extraction solvents in the identification of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrenediol-4-O-β-D-glucoside;

[0031] Figure 8 This is a thin layer chromatogram of different sample amounts in the identification of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrenediol-4-O-β-D-glucoside;

[0032] Fig. 9 These are the thin layer chromatograms of three different developing agents in the identification of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrenediol-4-O-β-D-glucoside;

[0033] Fig.10 This is a thin layer chromatogram developed under different humidity conditions in the identification of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrenediol-4-O-β-D-glucoside;

[0034] Fig.11 It is a thin layer chromatogram developed under different temperature conditions in the identification of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrenediol-4-O-β-D-glucoside;

[0035] Fig.12 It is the thin layer chromatogram of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside of 18 batches of Pleurotus eryngii medicinal materials samples;

[0036] Fig.13 This is the UV absorption spectrum of the magnolia alkaloid reference substance;

[0037] Fig.14It is the ultraviolet absorption spectrum of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrenediol-4-O-β-D-glucoside reference substance;

[0038] Fig.15 It is the liquid chromatogram of mobile phase systems 1 to 6;

[0039] Fig.16 It is the liquid chromatogram of different extraction solvents;

[0040] Fig.17 These are liquid chromatograms of four different solvent concentrations;

[0041] Fig.18 It is the liquid chromatogram of different extraction methods;

[0042] Fig.19 is the liquid chromatogram at different extraction times;

[0043] Fig. 20 It is the liquid chromatogram of different solvent dosage;

[0044] Fig.21 is the linear relationship diagram of magnolamine and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside;

[0045] Fig. 22 It is the spss stem and leaf diagram of magnolia alkaloid content determination;

[0046] Fig.23 It is the spss stem-leaf diagram for the determination of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrenediol-4-O-β-D-glucoside content. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0048] Example:

[0049] instrument:

[0050] Agilent 1260 liquid chromatograph with autosampler; chromatographic column: YMC C 18 (5μm, 4.6mm×250mm); AUW220D electronic balance: 1 / 100,000, Shimadzu Corporation, Japan; BS224S electronic balance: 1 / 10,000, Sartorius; CAMAG thin layer imager; KQ250DB CNC ultrasonic cleaner: Gongyi Yuhua Instrument Co., Ltd.;

[0051] KQ5200 water bath: Kunshan Ultrasonic Instrument Co., Ltd.; DHG-9140A electric constant temperature blast drying oven: Gongyi Yuhua Instrument Co., Ltd.; 8-10 box-type resistance furnace: Shenyang Energy Saving Electric Furnace Factory, China; Sony 717 digital camera: Sony Corporation, Japan.

[0052] Reference substances, medicines and medicinal materials:

[0053] Reference substances: Magnolia alkaloids reference substance (batch number: 0735-170801, National Engineering Research Center for the Manufacturing Technology of Traditional Chinese Medicine Solid Preparations); 1-[2-(Dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside reference substance (batch number: 0734-170801, National Engineering Research Center for the Manufacturing Technology of Traditional Chinese Medicine Solid Preparations).

[0054]

[0055] 1-[2-(dimethylamino)ethyl]-3,4-phenanthrenediol-4-O-β-D-glucoside, C 24 H 29 NO7.

[0056] Reagents: acetonitrile (chromatographic grade): Yucheng Chemical Plant of Shandong Yuwang Industrial Co., Ltd.; methanol (analytical grade): Shanghai Zhenxing Chemical Plant No. 1; dichloromethane, chloroform (analytical grade): Yucheng Chemical Plant of Shandong Yuwang Industrial Co., Ltd.; triethylamine, phosphoric acid, formic acid (analytical grade): Shanghai Zhenxing Chemical Plant No. 1; ethyl acetate (analytical grade): Shanghai Sinopharm Chemical Reagent Co., Ltd.; silica gel GF 254 Thin layer plate: Qingdao Ocean Chemical Plant; methanol (chromatographic grade): Yucheng Chemical Plant of Shandong Yuwang Industrial Co., Ltd.

[0057] Medicinal materials: Existing classification documents record that Sabiaparviflora Wall.ex Roxb. is distributed in Guizhou, Anhui, Henan, Yunnan and other places in my country. The medicinal materials used in this invention were purchased from Guizhou Province and identified as Sabiaparviflora Wall.ex Roxb. of the family Sabiaparviflora by Professor Zhong Guoyue of the Ethnic Medicine Resource Center of Jiangxi University of Traditional Chinese Medicine. The information of the purchased medicinal material samples is shown in Table 1.

[0058] Table 1 List of medicinal materials information of Psoralea corylifolia

[0059]

[0060] The stems and leaves of Sabiaparviflora Wall.ex Roxb. contain alkaloids, among which alkaloids are the main active ingredients of Sabiaparviflora Wall.ex Roxb. The researchers used a variety of separation techniques (silica gel column chromatography, preparative HPLC chromatography, Sephadex LH-20 gel column chromatography, ODS medium and low pressure column chromatography, preparative HPLC) to separate the ethanol extract of Sabiaparviflora Wall.ex Roxb. and used a variety of spectral techniques to identify the structures of the obtained compounds. 40 monomer compounds were separated and identified, including 11 alkaloids: grossamide (9), magnoflorine (24), lyciumamide C (25), cannabisin D (26), cannabisin E (27), iso-cannabisin E (28), cis-grossamide K(29), dihydroisoflavipucine(30), N-trans-feruloyloctopamine(31), N-trans-feruloyltyramine(32), 1-[2-(dimethylamino)ethyl]-3,4-phenanthrenediol-4-β-D-glucose(40); 8 lignans: ervatamisin(10), cleomiscosin C(7), (+)-lyoniresinol(8), (+)-syringaresinol(11), (-)-simulanol(14), (-)-7R,8S-dehydrodiconiferyl alcohol(15), Seslignanoccidentaliol A(12), (+)-syringaresinol-O-β-D-glucopyranoside (13);9 terpenes: darutigenol (3), saikosaponin A (16), saikosaponin B2 (17), 6”-O-acetyl-saikosaponin b2 (18), saikosaponin B1 (19), prosaikogenin D (20), 6”-O-acetyl-saikosaponin B1 (21), 1α,3β-di hydroxyl-olean-12-en-28-oic acid (22), 2α-hydroxyoleanolic acid (23). 12 other categories: Di(2-ethylhexyl) phthalate (DEHP, 1), Dibutyl phthalate (DBP, 2), Sucrose (D(+)-Sucrose, 4), 3,5-Dimethoxy-4-hydroxybenzaldehyde (5), Vanillin (Vanillin, 6), Sabianin A (sabianin A, 33), Sabianin B (sabianin B, 34), Sabianin C (sabianin C, 35), Sabianin D (sabianin D, 36), celastrol A-4-O-glucoside (37), 3,5,6-trihydroxy-7-(3-hydroxy-3-methylbut-1-en-1-yl)-2-(hydroxymethyl)-2-methyl-3,4-dihydronaphthalen-1-(2H)-one-5-O-glucoside (38), 2,2,7,7-tetramethyl-8,10-dihydroxy-2,7,8,9-tetrahydro-2H-naphtho(2,3-b)pyran-6-one-10-O-glucoside (39). Among them, 8 were new compounds (33-40), and 30 were isolated from the genus Celastrol for the first time (1-21, 23-32). After further in vitro screening of anti-lipid accumulation activity, the results showed that alkaloid components such as magnolamine and [2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside can inhibit FFAs-induced lipid accumulation in LO2 cells to varying degrees, and have certain anti-lipid accumulation activity, which is related to the function of Psoralea parviflora in promoting choleretic function and being used for jaundice and damp-heat syndrome. ;

[0061] In the thin-layer identification, this invention established a thin-layer identification of reference medicinal materials, and based on the results of this study on the chemistry and pharmacology of Psoralea corylifolia, established a thin-layer identification method with 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene glycol-4-O-β-D-glucoside as a reference. In addition, 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene glycol-4-O-β-D-glucoside was used as one of the indicator components of the [content determination] item.

[0062] A method for detecting the quality of a Caulis Spatholobi var. parviflora medicinal material comprises identifying the Caulis Spatholobi var. parviflora medicinal material by thin layer chromatography, and comprises the following steps:

[0063] (1) Take 1 g of Psoralea corylifolia powder (passed through a No. 4 sieve), add 20 ml of methanol at a solid-liquid ratio of 1 g:20 ml, heat and reflux for 30 min, cool to room temperature, make up the loss with methanol, filter, and take the filtrate as the test solution;

[0064] (2) Take 1 g of Psoralea corylifolia reference medicinal material powder (passed through a No. 4 sieve), add 20 ml of methanol according to a solid-liquid ratio of 1 g:20 ml, heat and reflux for 30 min, cool to room temperature, make up the loss with methanol, filter, and take the filtrate as the reference medicinal material solution;

[0065] (3) Take 10 μl of the test solution and the control medicinal material solution, respectively, and spot them on the same silica gel thin layer plate (silica gel G plate, 50×100 mm), use acetone-ethanol-acetic acid-ammonia water with a volume ratio of 3:1.5:0.2:0.5 as the developing solvent, develop 8 cm upward, take out, dry, and examine under a 365 nm ultraviolet lamp. In the thin layer chromatogram of the test solution, fluorescent spots of the same color appear at the corresponding positions of the thin layer chromatogram of the control medicinal material solution.

[0066] 1. Thin layer identification of reference medicinal materials:

[0067] 1.1 Developing agent:

[0068] Take 1g of the powder of the medicinal material of Caulis Agrimoniae Parviflora (passed through a No. 4 sieve), add 20ml of methanol, heat and reflux to extract for 30min, cool to room temperature, add methanol to make up to reduce the weight, filter, and take the filtrate as the test solution. The Caulis Agrimoniae Parviflora control medicinal material was used for comparison. In the experiment, the effects of three developing agents on the identification effect were investigated: acetone-ethanol-ammonia water (3:5:0.5), ethyl acetate-ethanol-ammonia water (3:4:0.5), and acetone-ethanol-acetic acid-ammonia water (3:1.5:0.2:0.5). Take 10μl of the test solution and 6μl of the control medicinal material solution, respectively, and spot them on the same silica gel thin layer plate (silica gel G plate, 50×100mm), develop 8cm upward, and inspect under a 365nm ultraviolet lamp. The chromatogram results are shown in the figure. Figure 1The results showed that the spots were better separated and clear when acetone-ethanol-acetic acid-ammonia water (3:1.5:0.2:0.5) was used as the developing solvent.

[0069] 1.2 Extraction solvent:

[0070] Take three portions of the powder of the medicinal material of the small flowered Qingfengteng (passed through a No. 4 sieve), 1g of each portion, add 20ml of methanol, ethanol and water respectively, heat and reflux for extraction for 30min, cool, make up the weight loss with the extraction solvent, filter, and take the filtrate as the test solution. Take 1g of the small flowered Qingfengteng control medicinal material powder, according to the solid-liquid ratio of 1g:20ml, add 20ml of methanol, heat and reflux for extraction for 30min, cool to room temperature, make up the loss with methanol, filter, and take the filtrate as the control medicinal material solution; the rest are tested by thin layer chromatography according to the above method, and the test results are shown in Figure 2 The results of thin layer chromatography showed that methanol had a better extraction effect when used as the extraction solvent. Therefore, methanol was selected as the extraction solvent.

[0071] 1.3 Extraction method:

[0072] Take three portions of the powder of the medicinal material of Psoralea corylifolia (passed through a No. 4 sieve), 1 g each, add 20 ml of methanol, and extract by ultrasonic extraction for 30 min, ultrasonic extraction for 30 min after cold soaking for 1 h, and heating reflux extraction for 30 min. Cool to room temperature, add methanol to reduce the weight, filter, and take the filtrate as the test solution. The rest is tested by thin layer chromatography according to the above method. The test results are shown in Figure 3 The results of thin layer chromatography showed that the effect of heating reflux extraction for 30 minutes was better.

[0073] 1.4 Sample volume:

[0074] Take three portions of the powder of the medicinal material of Psoralea corylifolia (passed through a No. 4 sieve), 1g each, add 20ml of methanol, heat and reflux for extraction for 30min, add methanol to reduce the weight, filter, and take the filtrate as the test solution. The test was performed with the sample volume of 6μl, 8μl and 10μl respectively. The test results are as follows Figure 4 The results show that when the sample volume is 10 μl, the spots are clearer.

[0075] According to the test results, the thin layer identification method is determined as follows: take 1g of the powder of the medicinal material of the small flower Qingfengteng (passed through a No. 4 sieve), add 20ml of methanol, heat and reflux to extract for 30min, cool to room temperature, use methanol to make up for the loss, filter, and take the filtrate as the test solution. Take another 1g of the small flower Qingfengteng reference medicinal material and prepare the reference medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 10μl of the test solution and the reference medicinal material solution, respectively, and spot them on the same silica gel G thin layer plate, use acetone-ethanol-acetic acid-ammonia water (3:1.5:0.2:0.5) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference medicinal material, a fluorescent spot of the same color appears.

[0076] Thin layer identification of medicinal material samples (18 batches):

[0077] According to the above method, 18 batches of samples of Psoralea corylifolia were tested. The results are as follows: Figure 5 As shown, the results show that the development effect is good and the thin layer chromatography of each sample Figure 1 Yes, the method is feasible.

[0078] 2. Thin layer identification of alkaloid compound 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside: 2.1 Preparation method of test solution:

[0079] During the experiment, three portions of powder of the medicinal material of Psoralea corylifolia (2#) (passed through a No. 4 sieve) were taken, 1g each, and 20ml of methanol was added. The three extraction methods of methanol ultrasonic (30 minutes), cold soaking (1 hour) and cold soaking (1 hour) plus ultrasonic (30 minutes) were compared, cooled to room temperature, supplemented with methanol to reduce the weight, filtered, and the filtrate was taken as the test solution. The 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene glycol-4-O-β-D-glucoside reference substance was used for comparison. 1.0g of the 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene glycol-4-O-β-D-glucoside reference substance was taken, 10ml of methanol was added, ultrasonic treatment was performed for 15 minutes, filtered, and used as the reference solution.

[0080] Take 4 μl of the test solution and 2 μl of the reference solution and spot them on the same silica gel thin layer plate (GF 254 Plate, 50×100 mm), with acetone-ethanol-acetic acid-ammonia water in a volume ratio of 3:8:0.2:0.5 as the developing agent, 8 cm upward, and examined under ultraviolet light (254 nm). The results are shown in Figure 6 The results showed that there was no significant difference between the different extraction methods. Since ultrasonic extraction is simple and easy to operate, direct ultrasonic extraction was selected.

[0081] 2.2 Extraction solvent and sample volume of the test solution:

[0082] The direct ultrasonic extraction method was used to investigate the extraction solvents. Methanol, ethanol and water were selected for ultrasonic extraction for 30 min. The sample volumes were 2 μl, 4 μl and 5 μl, respectively, and were spotted on the same silica gel thin layer plate (GF 254 Plate, 50×100 mm), with acetone-ethanol-acetic acid-ammonia water in a volume ratio of 3:8:0.2:0.5 as the developing agent, 8 cm upward, and examined under ultraviolet light (254 nm). The results are shown in Figure 7 , Figure 8 The results showed that different extraction solvents and sample volumes had certain differences. The spots were clear when methanol was used as the solvent and the sample volume was 5μl. Therefore, methanol was selected as the extraction solvent and the sample volume was 5μl.

[0083] According to the above experimental results, the preparation method of the test solution is determined as follows: take 2.0 g of the product powder, add 25 ml of methanol, ultrasonically treat for 30 minutes, filter, and use as the test solution. The test sample spot volume is 5 μl.

[0084] 2.3 Developing agent:

[0085] The reference substance 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-β-D-glucoside was used for comparison. The test solution and the reference substance solution were prepared according to the above method, and three thin layer identification developing agents were compared: acetone-ethanol-acetic acid-ammonia water (3:8:0.2:0.5), chloroform-methanol-acetic acid-ammonia water (5:5:0.2:0.5), and dichloromethane-methanol-acetic acid-ammonia water (5:5:0.2:0.5). 5 μl of the test solution and 2 μl of the reference substance solution were taken respectively and spotted on the same silica gel thin layer plate (GF 254 Plate, 50×100mm), expand upward 8cm, and inspect under ultraviolet light (254nm). The experimental results are as follows Fig. 9 According to the experimental results, acetone-ethanol-acetic acid-ammonia water (3:8:0.2:0.5) was selected as the final developing agent.

[0086] 2.4 Durability under unfolding environmental conditions:

[0087] The above selected developing agents were tested at 25% and 72% relative humidity and at temperatures of 4°C to 10°C (refrigerator) and room temperature (25°C). The experimental results are shown in Fig.10 and Fig.11 The results show that good separation effects were achieved.

[0088] Thin layer identification of medicinal materials samples of Psoralea corylifolia (18 batches):

[0089] According to the above method, 18 batches of small flowered gentian herbal medicine were tested. Fig.12 As shown, the results showed that all 18 batches of Pleurotus eryngii medicinal materials contained 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside, which can be used as one of the indicator components.

[0090] There is no content determination item in the Guizhou Provincial Standard of P. parviflora, so the inventors have studied this, selected magnolamine and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside (characteristic components of P. parviflora) as indicator components, and established a method for simultaneous determination of the two components by HPLC. The content of 18 batches of medicinal materials was determined and the limit was specified.

[0091] The quality detection method also includes using high performance liquid chromatography (General Rule 0512) to determine the content of magnolamine and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside in the medicinal material of Psoralea corylifolia, including the following steps:

[0092] (1) Take magnolia alkaloids and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene glycol-4-O-β-D-glucoside reference substances, add 20% methanol to prepare a reference substance solution containing 0.1 mg of magnolia alkaloids and 0.2 mg of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene glycol-4-O-β-D-glucoside per 1 ml;

[0093] (2) Take 1.0 g of Pleurotus eryngii herb, add 25 ml of 70% methanol according to the material-liquid ratio of 1 g:25 ml, weigh the weight, and ultrasonicate for 30 min at a power of 300 W and a frequency of 40 kHz. Cool to room temperature, weigh the weight again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution;

[0094] (3) Take 5 μl of the reference solution and the test solution, inject them into the liquid chromatograph, and measure the content of magnolamine and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside.

[0095] The chromatographic conditions of the liquid chromatograph were: YMC C 18 The column (250mm×4.6mm, 5μm) was used as the chromatographic column; octadecylsilane bonded silica gel was used as the filler; methanol-0.2% triethylamine (0.2% triethylamine plus phosphoric acid adjusted to pH 3.0) with a volume ratio of 20:80 was used as the mobile phase; the detection wavelength was 260nm; the flow rate was 1.0ml / min; the column temperature was 40℃; and the injection volume was 5μl.

[0096] 1. System adaptability test:

[0097] 1.1 Detection wavelength test:

[0098] Using the above method, the wavelength scanning function of the diode array detector in the high performance liquid chromatograph was used to perform full wavelength scanning to obtain magnolia alkaloids ( Fig. 20 ) and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside reference substances, such as Fig.13 , Fig.14 As shown in the figure, magnolia alkaloids have a maximum absorption at 268nm, and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside has a maximum absorption at 254nm, so the middle wavelength of the two, 260nm, is selected as the detection wavelength.

[0099] 1.2 Comparative test of different mobile phase systems:

[0100] Six different mobile phase systems and gradients were tested, and the test conditions are shown in Tables 2 to 7. The test results are shown in Fig.15 .

[0101] Table 2 Mobile phase system 1 and its gradient

[0102]

[0103] Table 3 Mobile phase system 2 and its gradient table

[0104]

[0105] Table 4 Mobile phase system 3 and its gradient table

[0106]

[0107] Table 5 Mobile phase system 4 and its gradient table

[0108]

[0109] Table 6 Mobile phase system 5 and its gradient

[0110]

[0111] Table 7 Mobile phase system 6 and its gradient table

[0112]

[0113] According to the chromatographic results, mobile phase system 6 was selected as the mobile phase, namely methanol-0.2% triethylamine (adjusting the pH to 3.0 with phosphoric acid) (20:80); flow rate: 1 ml / min.

[0114] 1.3 Comparative test of different extraction solvent types:

[0115] Weigh six portions of Pleurotus eryngii powder, 1.0 g each, accurately weigh, place in a stoppered conical flask, add 25 ml of three different solvents, methanol, ethanol, and water, respectively, weigh, ultrasonically treat for 30 minutes, cool to room temperature, make up for the lost weight, shake well, filter through a 0.45 μm filter membrane, and use as the test solution. Accurately pipette 5 μl of each of the three test solutions mentioned above, inject into the liquid chromatograph, and measure. Calculate the size of the specific peak area (the ratio of the peak area to the sampling amount is used as an experimental investigation indicator, and the larger the ratio, the more complete the extraction of the component). The measurement results are shown in Tables 8 and Fig.16 .

[0116] Table 8 Comparison results of different extraction solvent types

[0117]

[0118] As can be seen from Table 8, methanol and water have higher extraction efficiency, while ethanol has the worst efficiency. Therefore, we continue to examine different concentrations of methanol-water to determine the extraction solvent.

[0119] 1.4 Comparative test of different solvent concentrations:

[0120] Weigh four portions of Pleurotus eryngii powder, 1.0 g of each sample and 2 parallel portions, accurately weigh, place in a stoppered conical flask, add 25 ml of 90% methanol-water, 70% methanol-water, 50% methanol-water, and 30% methanol-water respectively, weigh, ultrasonically treat for 30 minutes, cool, make up for the lost weight, shake well, filter through a 0.45μm filter membrane, and use as the test solution. Accurately pipette 5μl of each of the three test solutions mentioned above, inject into the liquid chromatograph, and measure. Calculate the size of the specific peak area (the ratio of peak area to sampling amount is used as an experimental investigation indicator, and the larger the ratio, the more complete the extraction of the component). The measurement results are shown in Tables 9 and Fig.17 .

[0121] Table 9 Comparison results of different methanol concentrations

[0122]

[0123] As shown in Table 3, 70% methanol has the highest efficiency, so 70% methanol is determined as the extraction solvent.

[0124] 1.5 Comparative test of different extraction methods:

[0125] Weigh 1.0g of the powder of the medicinal material of Paridis parviflora, and make 2 parallel portions for each sample, accurately weigh them, place them in a stoppered conical flask, add 25ml of 70% methanol respectively, weigh them, and examine three extraction methods: reflux for 30 minutes, ultrasound for 30 minutes, and cold soaking for 1 hour. Let it cool, make up for the lost weight, shake it well, and filter it through a 0.45μm filter membrane to prepare the test solution. Accurately pipette 5μl of each of the three test solutions mentioned above, inject them into the liquid chromatograph, and measure them. Calculate the specific peak area (the ratio of the peak area to the sampling amount is used as an experimental investigation indicator, and the larger the ratio, the more complete the extraction of the component). The measurement results are shown in Tables 10 and Fig.18 .

[0126] Table 10 Comparison results of different extraction methods

[0127]

[0128] As can be seen from Table 10, there is little difference between the content results of reflux and ultrasonic extraction. Considering that ultrasonic extraction is simple and energy-saving, ultrasonic extraction was selected in this experiment.

[0129] 1.6 Comparative test of different extraction times:

[0130] Weigh 1.0g of the powder of the medicinal material of Paridis parviflora, and make 2 parallel portions for each sample, accurately weigh them, place them in a stoppered conical flask, add 25ml of 70% methanol respectively, weigh them, ultrasonicate for 15, 30, and 60 minutes respectively, let them cool, make up for the lost weight, shake them well, and filter them through a 0.45μm filter membrane as the test solution. Accurately pipette 5μl of each of the three test solutions mentioned above, inject them into the liquid chromatograph, and measure them. Calculate the size of the specific peak area (the ratio of the peak area to the sampling amount is used as an experimental investigation indicator, and the larger the ratio, the more complete the extraction of the component). The measurement results are shown in Tables 11 and Fig.19 .

[0131] Table 11 Comparison results of different extraction times

[0132]

[0133] As shown in Table 11, ultrasonic extraction can be completely extracted in 30 minutes, but it decreases in 60 minutes, so the extraction time is selected as 30 minutes. 1.7 Comparative test of different solvent dosages:

[0134] Weigh six portions of Pleurotus eryngii powder, 1.0 g each, accurately weigh, place in a round-bottom flask, add 70% methanol 15, 25, 50 ml respectively, weigh, ultrasonicate for 30 minutes respectively, cool, make up for the lost weight, shake well, filter through 0.45 μm filter membrane, and use as test solution. Accurately pipette 5 μl of each of the three test solutions mentioned above, inject into liquid chromatograph, and measure. Calculate the size of the specific peak area (the ratio of peak area to sampling amount is used as an experimental investigation indicator, and the larger the ratio, the more complete the extraction of the component). The measurement results are shown in Tables 12 and Fig. 20 .

[0135] Table 12 Comparison results of different solvent dosages

[0136]

[0137] As shown in Table 6, when the solvent dosage is 15, 25, and 50 ml, the extraction can be basically complete. Considering that the medicinal powder is relatively fluffy, from the perspective of reducing experimental errors and saving environmental protection, the solvent dosage is determined to be 25 ml.

[0138] Methodological validation:

[0139] Verification of linear relationship: 5 μL of mixed reference solution of different concentrations was injected into the HPLC spectrometer, and the peak areas of magnolia alkaloids and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene glycol-4-O-β-D-glucoside were recorded. The standard curves were drawn with the concentration X (μg / ml) as the horizontal coordinate and the peak area as the vertical coordinate. The regression equations of magnolia alkaloids and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene glycol-4-O-β-D-glucoside were: Y=7.061X+13.67R 2 =0.9998; Y = 16.17X + 88.98R 2 =0.9997. This indicates that the concentrations of magnolamine and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside in the range of 5-250 μg / ml and 16-800 μg / ml, respectively, show good linear relationships with the corresponding peak areas. The determination results are shown in Tables 13 and Fig.21 .

[0140] Table 13 Linear relationship investigation results

[0141]

[0142] Precision experiment: Accurately pipette the same mixed reference solution, inject it 6 times in a row, record the peak area, calculate the RSD value, and the RSD values ​​of the peak area of ​​magnolia alkaloids and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene diol-4-O-β-D-glucoside are 0.67% and 0.60%, respectively. The test results are shown in Table 14, which shows that the precision of the instrument is good.

[0143] Repeatability experiment: Weigh an appropriate amount of the same sample of Psoralea corylifolia, weigh it accurately, prepare 6 test sample solutions according to the test sample preparation method, inject 5 μL of each sample, calculate the content, and use the obtained content to calculate the RSD values ​​of magnolamine and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside to be 2.7% and 2.8%, respectively. The test results are shown in Table 14, indicating that the method has good repeatability.

[0144] Stability test: Weigh an appropriate amount of the sample of Psoralea corylifolia, weigh it accurately, prepare the test solution according to the test sample preparation method, and inject the sample at 0, 3, 6, 9, and 12 hours. The peak area RSD values ​​of magnolamine and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside were 0.71% and 0.61%, respectively. The test results are shown in Table 14, indicating that the test solution is stable after being placed at room temperature for 12 hours.

[0145] Table 14 Precision, repeatability and stability test results

[0146]

[0147] Sample recovery rate: Accurately weigh 6 portions of the same batch of small flowered gentiana medicinal materials with known content, add appropriate amounts of magnolia alkaloids and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene diol-4-O-β-D-glucoside reference substances, prepare the test solution according to the preparation method of the test solution, and inject the sample according to the above chromatographic conditions, calculate the sample recovery rate, and the sample recovery rates of magnolia alkaloids and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene diol-4-O-β-D-glucoside are 95.05% and 95.845, respectively, with RSDs of 2.17% and 2.04%. The test results are shown in Table 15, indicating that the method has a good recovery rate.

[0148] Table 15 Dissolution and sample recovery experimental results

[0149]

[0150] Sample content determination: Take 1.0g of each of 18 batches of Caulis Scutellariae, prepare the test solution according to the preparation method of the test solution, inject according to the above chromatographic conditions, inject each sample twice, determine the peak area, and calculate the content of magnolamine and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside in each batch of Caulis Scutellariae by regression equation. The determination results are shown in Table 16.

[0151] Table 16 Results of determination of the content of Psoralea corylifolia

[0152]

[0153]

[0154] The results of content determination showed that the contents of magnolamine and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside ranged from 0.19-0.51% and 0.40-1.05%, respectively, with average values ​​of 0.29% and 0.60%, respectively.

[0155] In order to prevent the above-mentioned measurement results from having extreme values ​​far from the mean, SPSS 21.0 in the statistical method was used to analyze the measurement data. The analysis results are shown in Tables 17 and 18; explore was used to draw the stem-and-leaf plot with a confidence interval of 95%.

[0156] Table 17 Analysis results of magnolia alkaloids

[0157]

[0158] Table 18 Analysis results of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene glycol-4-O-β-D-glucoside

[0159]

[0160]

[0161] For indicator A (magnolia alkaloids), Fig. 22 As shown in the figure, Z-160423-01 and Z-170313-01 are outlier data. After removing the outlier data, the average value of the content in the samples of Psoralea corylifolia was 0.26%, and the content limit value was determined to be 0.21% by taking the average value down by 20%. Two batches of the 18 batches of medicinal materials failed, and the qualified rate was 89%.

[0162] For indicator B (1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside), Fig.23As shown in the figure, Z-160423-01 and Z-170313-01 are outlier data. After removing the outlier data, the average value of the content in the samples of Psoralea corylifolia was 0.54%, and the content limit value was determined to be 0.43% by taking the average value down by 20%. Among the 18 batches of medicinal materials, 3 batches failed, and the qualified rate was 83%.

[0163] Taking all factors into consideration, the content limit of magnolia alkaloids in the dry product of Psoralea corylifolia was determined to be "not less than 0.21%" and the content limit of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside was determined to be "not less than 0.43%". Combining the two indicators, the qualified rate of samples was 83%.

Claims

1. A method for detecting the quality of Psoralea corylifolia medicinal materials, characterized in that: The method comprises the following steps: (1) Take the medicinal material of Psoralea corylifolia, add methanol, heat and reflux to extract, cool to room temperature, make up the lost amount with methanol, filter, and take the filtrate as the test solution; (2) Take the control medicinal material of Psoralea corylifolia, add methanol, heat and reflux to extract, cool to room temperature, make up the lost amount with methanol, filter, and take the filtrate as the control medicinal material solution; (3) Take the test sample solution and the control drug solution, spot them on the same silica gel thin layer plate, use acetone-ethanol-acetic acid-ammonia water with a volume ratio of 3:1.5:0.2:0.5 as the developing agent, develop, take out, dry, and inspect under ultraviolet light; The quality detection method also includes using high performance liquid chromatography to determine the content of magnolamine and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside in the medicinal material of Psoralea corylifolia, including the following steps: (4) Take magnolia alkaloids and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside reference substances and add 20% methanol to prepare reference substance solutions; (5) Take the medicinal material of Psoralea corylifolia, add 70% methanol, weigh it, ultrasonicate it for 30 min, cool it to room temperature, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution; (6) Take the reference solution and the test solution, inject them into the liquid chromatograph, and determine the content of magnolia alkaloids and 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside; The chromatographic conditions of the liquid chromatograph are as follows: octadecylsilane bonded silica gel as filler; methanol-0.2% triethylamine with a volume ratio of 20:80 as mobile phase, wherein phosphoric acid is added to 0.2% triethylamine to adjust the pH to 3.0; and the detection wavelength is 260 nm.

2. The method for detecting the quality of the medicinal material of Psoralea corylifolia according to claim 1, characterized in that: In step (1) and step (2), the material-liquid ratio between the medicinal material and methanol is 1 g:20 ml, and the heating reflux extraction time is 30 min.

3. The method for detecting the quality of the medicinal material of Psoralea corylifolia according to claim 1, characterized in that: In the thin layer chromatography of the test solution, fluorescent spots of the same color appear at the corresponding positions in the thin layer chromatography of the control medicinal material solution.

4. The method for detecting the quality of the medicinal material of Psoralea corylifolia according to claim 1, characterized in that: In step (3), the wavelength of the ultraviolet lamp is selected to be 365 nm, and the spot volume of the test solution and the control medicinal material solution is 10 μl.

5. The method for detecting the quality of the medicinal material of Psoralea corylifolia according to claim 1, characterized in that: In the step (4), each 1 ml of the reference solution contains 0.1 mg of magnolia alkaloids and 0.2 mg of 1-[2-(dimethylamino)ethyl]-3,4-phenanthrene-diol-4-O-β-D-glucoside.

6. The method for detecting the quality of the medicinal material of Psoralea corylifolia according to claim 1, characterized in that: In the step (5), the solid-liquid ratio of the medicinal material of Psoralea corylifolia to 70% methanol is 1 g:25 ml.

7. The method for detecting the quality of the medicinal material of Psoralea corylifolia according to claim 1, characterized in that: In step (6), the injection volume of the reference solution and the test solution is 5 μl.

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