A quality detection method for Malan herb

Abstract

The invention discloses a quality detection method of amaranth herb, including identification and / or content determination items; the identification is a thin layer chromatography identification method for α-bolsterol-3-O-β-glucoside control; the content determination is to determine the content of 3,5-dicaffeoylquinic acid in amaranth herb by high performance liquid chromatography. The invention can effectively control the overall quality of amaranth herb and ensure the safety and effectiveness of clinical use of amaranth herb.

Description

Technical Field

[0001] The invention relates to a method for detecting the quality of Chinese medicinal materials, in particular to a method for detecting the quality of Amaranthus chinensis medicinal materials. Background Art

[0002] Kalimeris indica (L.) Sch.-Bip is a perennial herbaceous plant of the genus Kalimeris. The rhizome has creeping branches, the stem is upright, up to 70 cm high, with short hairs on the upper part, and the leaves at the base wither during the flowering period; the leaves on the stem are oblanceolate or obovate-rectangular, all the leaves are slightly thin, and the head inflorescence is solitary at the end of the branch and arranged in a sparse corymb. The involucre is hemispherical, and the involucre bracts are arranged in a shingle-like manner; the outer layer is oblanceolate, the inner layer is oblanceolate-rectangular, the upper part is herbaceous, with sparse short hairs, the edge is membranous, and the receptacle is conical. The ligulate flowers, the ligules are light purple, the achenes are obovate-rectangular, very flat, flowering from May to September, and fruiting from August to October. It is distributed in western, central, southern, and eastern China. It is also distributed in Korea, Japan, Indochina Peninsula to India. It grows on the edge of the forest, in the grass, on the bank of the stream, and on the roadside.

[0003] The whole herb of Malan is used for medicine, which has the effects of clearing away heat and detoxifying, eliminating food accumulation, promoting urination, dispersing blood stasis and stopping bleeding. The young leaves are usually eaten as vegetables, commonly known as "Malan head". At the same time, it is recorded in various documents. "Rihuazi Materia Medica" says: "Roots and leaves can break old blood, nourish new blood, stop nosebleeds, vomit blood, combine with alloy sores, stop bloody dysentery, relieve alcohol jaundice and various bacteria and toxins; apply raw to snake bites." "Bencao Zhengyi" says: Malan is the best heat-removing and toxin-removing, can specifically enter the blood, stop bleeding and cool blood, and is particularly good. For all warm and hot evils that penetrate into the nutrient division, as well as carbuncle, blood heat, rot and other symptoms, it is a special medicine. It is widely used for internal and external application, and is also an important product for clearing heat and detoxification. If it is said that it breaks old blood and produces new blood, it is an exaggeration; "Bencao Shiyi" says: Malan grows beside the marsh. Like the marsh orchid, it smells bad. Northerners call it purple chrysanthemum when they see its flowers, because it resembles a single-petal chrysanthemum but is purple. There is also a mountain orchid, which grows on the side of the mountain, resembles Liu Ji Nu leaves, has no twigs, grows oppositely, and has a slightly yellow and red flower heart. It also breaks blood and can be used. It is also used clinically to treat chronic tracheitis: 4 liang of fresh Malan grass or 2 liang of dry product, add 200 ml of water after washing, decoct and filter, concentrate to 45 ml, add sugar and preservatives. Take 3 times a day, 15 ml each time, and 6 days as a course of treatment. 328 cases were treated, 5 cases were cured, 67 cases were effective, 167 cases were improved, 89 cases were invalid, and the total effective rate was 72.9%. Malan grass has a good antitussive effect, and it also has certain effects on expectoration, antiasthma, and anti-inflammatory. Only a very small number of side effects such as upper abdominal discomfort, chest tightness, and vomiting occurred within 1 to 2 days after taking the medicine.

[0004] The quality standards of original medicinal materials related to "Malan grass" are included in the "Chinese Pharmacopoeia" (1977 edition), "Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province" (2004), "Fujian Chinese Medicinal Materials Standards" (2006), "Hunan Chinese Medicinal Materials Standards" (2009 edition), and "Sichuan Chinese Medicinal Materials Standards" (2010). The original standards summarize the provisions of property identification, powder identification, thin layer identification with reference medicinal materials, moisture, total ash, acid-insoluble ash, etc. The original standards did not establish content determination items and could not effectively control the overall quality of Malan grass materials. Therefore, it is urgent to develop and establish a quality detection method for Malan grass to control the overall quality of Malan grass materials and ensure the safety and effectiveness of Malan grass materials in clinical use. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting the quality of Amaranthus chinensis. The present invention can effectively control the overall quality of Amaranthus chinensis and ensure the safety and effectiveness of clinical use of Amaranthus chinensis.

[0006] The technical solution of the present invention is a quality detection method for Malan herb, including identification and / or content determination items; the identification is a thin layer chromatography identification method for α-bolsterol-3-O-β-glucoside control; the content determination is to determine the content of 3,5-dicaffeoylquinic acid in Malan herb by high performance liquid chromatography.

[0007] In the aforementioned quality detection method of Amaranthus herba, the thin layer chromatography identification method for α-sterol-3-O-β-glucoside comprises the following steps:

[0008] 1) Take 0.5 g of amaranth powder (passed through a No. 4 sieve), add 20 mL of methanol, ultrasonicate for 30 minutes, filter, evaporate the filtrate to dryness, add methanol to the residue to dissolve to 1 mL, as the test solution; take 2 mg of α-spinasterol-3-O-β-D-glucoside, add methanol to make a solution containing 0.1 mg of α-spinasterol-3-O-β-D-glucoside per 1 mL, as the reference solution;

[0009] 2) According to the thin layer chromatography test, take 6 μl of each of the above solutions and spot them on the same silica gel G thin layer plate, use chloroform-methanol (9:1) as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol test solution, heat at 105℃ until the spots are clearly colored, and examine under visible light. In the chromatogram of the test sample, a spot of the same color appears at the corresponding position of the chromatogram of the reference sample.

[0010] In the aforementioned quality detection method of Amaranthus herbaceus, the method for determining the content of 3,5-dicaffeoylquinic acid in Amaranthus herbaceus is determined by high performance liquid chromatography, comprising the following steps:

[0011] (1) Preparation of reference solution: Take an appropriate amount of 3,5-dicaffeoylquinic acid reference substance, weigh accurately, and add methanol to make a solution containing 30 μg per 1 ml to obtain the reference solution;

[0012] (2) Preparation of test solution: Weigh 0.5 g of medicinal material powder, accurately weigh it, place it in a stoppered conical flask, add 25 ml of 70% methanol, weigh it, heat and reflux it for 30 minutes, cool it, make up the lost weight with 70% methanol, filter it, and take the filtrate to obtain the test solution;

[0013] (3) Determination method: Accurately pipette 10 μl of the reference solution and 10 μl of the test solution respectively, inject into the liquid chromatograph, and determine.

[0014] In the aforementioned quality testing method for Amaranthus herb, the product, calculated as a dry product, contains no less than 0.05% of 3,5-dicaffeoylquinic acid.

[0015] In the aforementioned quality detection method for Amaranthus herb, the chromatographic conditions of the liquid chromatograph are as follows: the chromatographic column is a Cosmosil C18 column, the wavelength is 327nm, the flow rate is 0.6ml / min, and the column temperature is 35°C; methanol is used as mobile phase A, and 0.1% glacial acetic acid water is used as mobile phase B, and gradient elution is performed as specified.

[0016] Compared with the prior art, the quality detection method of the present invention includes a thin layer identification method of Malan herbal medicine and a method for determining the content of 3,5-dicaffeoylquinic acid (C25H24O12) in Malan herbal medicine. Among them, the thin layer identification method of Malan herbal medicine has the characteristics of high sensitivity, strong specificity and good separation effect, is simple and feasible, has strong reproducibility, and can accurately identify the authenticity of medicinal materials; the method for determining the content of 3,5-dicaffeoylquinic acid (C25H24O12) in Malan herbal medicine can accurately and quickly determine the content of Malan herbal medicine, and provide effective qualitative and quantitative analysis means for Malan herbal medicine, which is conducive to controlling the quality of Malan herbal medicine. The quality detection method of Malan herbal medicine provided by the present invention can effectively control the overall quality of Malan herbal medicine and ensure the safety and effectiveness of Malan herbal medicine in clinical use.

[0017] At the same time, the quality detection method of the herbaceous amaranth provided by the present invention helps to make up for the deficiencies of the quality detection methods in the Chinese Pharmacopoeia (1977 edition), the Quality Standards of Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province (2004), the Standards of Chinese Medicinal Materials in Fujian Province (2006), the Standards of Chinese Medicinal Materials in Hunan Province (2009 edition), and the Standards of Chinese Medicinal Materials in Sichuan Province (2010), and has strong practicality. The method of the present invention is of great significance for the further quality control of herbaceous amaranth materials, and can provide a scientific basis for the formulation of the quality table of herbaceous amaranth materials.

[0018] In summary, the present invention can effectively control the overall quality of Amaranthus herbaceus medicinal materials and ensure the safety and effectiveness of clinical use of Amaranthus herbaceus medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the investigation thin layer chromatogram of the test solution; ( Figure 1 In the figure, 1 is the α-bosterol-3-O-β-glucoside reference substance, 2 is the sample left to stand for 2 hours, 3 is the sample ultrasonicated for 30 minutes, and 4 is the sample refluxed for 30 minutes);

[0020] Figure 2 Thin layer chromatograms of different solvents; ( Figure 2 In the figure, 1 is the reference substance of α-bosterol-3-O-β-glucoside, 2 is the sample prepared with water solvent, 3 is the sample prepared with ethanol solvent, and 4 is the sample prepared with methanol solvent);

[0021] Figure 3 is a thin layer chromatogram with different sample amounts; ( Figure 3 In the table, 1 is the α-bosterol-3-O-β-glucoside reference substance, 2 is the test solution 2 μL, 3 is the test solution 4 μL, and 4 is the test solution 2 μL);

[0022] Figure 4 This is the thin layer chromatogram when the developing solvent is chloroform-methanol (9:1), ( Figure 4 Among them, 1 is the α-bosterol-3-O-β-glucoside reference substance, and 2 is the amaranth sample);

[0023] Figure 5 This is a thin layer chromatogram with the developing solvent being dichloromethane-methanol-formic acid (7:2). Figure 5 Among them, 1 is the α-bosterol-3-O-β-glucoside reference substance, and 2 is the amaranth sample);

[0024] Figure 6 This is a thin layer chromatogram developed under a humidity of 25%. Figure 6 Among them, 1 is the α-bosterol-3-O-β-glucoside reference substance, and 2 is the amaranth sample);

[0025] Figure 7 This is a thin layer chromatogram developed under a humidity of 75%. Figure 7 Among them, 1 is the α-bosterol-3-O-β-glucoside reference substance, and 2 is the amaranth sample);

[0026] Figure 8 It is a thin layer chromatogram developed at 4℃~10℃ (refrigerator), ( Figure 8 In the table, 1 is the α-sterol-3-O-β-glucoside reference substance, and 2 is the amaranth sample)

[0027] Fig. 9 It is a thin layer chromatogram developed at room temperature (25°C). Fig. 9 Among them, 1 is the α-bosterol-3-O-β-glucoside reference substance, and 2 is the amaranth sample);

[0028] Fig.10 This is the thin layer identification diagram of the M1-M7 batches of Malan grass samples ( Fig.10 Among them, 1 is the α-bosterol-3-O-β-glucoside reference substance, and 2-8 are the samples of Malan grass from batches M1-M7);

[0029] Fig.11 This is the thin layer identification diagram of the M8-M13 batch of Malan grass samples ( Fig.10 Among them, 1 is the α-bosterol-3-O-β-glucoside reference substance, and 9-14 are the Malan grass samples from batches M8-M13);

[0030] Fig.12 This is the chromatogram of the 3,5-dicaffeoylquinic acid reference substance;

[0031] Fig.13 It is the chromatogram of the herbal medicine of Malan;

[0032] Fig.14 It is the linear relationship diagram of 3,5-dicaffeoylquinic acid;

[0033] Fig.15 It is the extraction solvent (methanol) investigation diagram;

[0034] Fig.16 It is the extraction solvent (ethanol) investigation diagram;

[0035] Fig.17 It is the extraction solvent (water) investigation diagram;

[0036] Fig.18 This is a graph of investigation of different solvent concentrations (30% methanol);

[0037] Fig.19 This is a graph of investigation of different solvent concentrations (50% methanol);

[0038] Fig. 20 This is a graph of investigation of different solvent concentrations (70% methanol);

[0039] Fig.21 This is a survey chart of different extraction methods (cold soaking overnight);

[0040] Fig. 22 This is a survey diagram of different extraction methods (reflux for 30 minutes);

[0041] Fig.23 This is a survey diagram of different extraction methods (ultrasound for 30 minutes);

[0042] Fig.24 This is the investigation graph of extraction time (reflux for 15 minutes);

[0043] Fig.25 This is a graph examining the extraction time (reflux for 45 minutes). DETAILED DESCRIPTION

[0044] 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.

[0045] Embodiment. A method for detecting the quality of Amaranthus herba,

[0046] instrument:

[0047] Shimadzu LC-20AT high performance liquid chromatography; 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).

[0048] Reagents and test drugs

[0049] 1. Reference

[0050] α-Bosterol-3-O-β-glucoside and 3,5-dicaffeoylquinic acid are both produced by the National Engineering Research Center for Traditional Chinese Medicine Solid Preparation Manufacturing Technology, with a purity of >98%.

[0051] 2. Reagents

[0052] 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 Experimental Reagent Co., Ltd.); ethyl acetate (analytical grade, Guangdong Guanghua Technology Co., Ltd.); anhydrous ethanol (analytical grade, Shanghai Zhenxing Chemical Plant No. 1); GF254 silica gel plate (Qingdao Ocean Chemical Plant); sulfuric acid (analytical grade, Xilong Scientific Co., Ltd.); water was ultrapure water.

[0053] Medicinal Materials

[0054] The medicinal materials used in the experiment were provided by Guizhou Bailing Pharmaceutical Co., Ltd. and identified by Zhong Guoyue, a researcher at the Ethnic Medicine and Chinese Medicine Germplasm Resources Center of Jiangxi University of Traditional Chinese Medicine, as the whole herb of Kalimeris indica (L.) Sch. Bip. of the Asteraceae family. The specimen (Z-140310-01) is preserved in the Ethnic Medicine Resources Center of Jiangxi University of Traditional Chinese Medicine. The medicinal material information is shown in Table 1.

[0055] Table 1 Batch of Malan herbs

[0056]

[0057]

Medicinal material name

[0058] Synonyms: Purple Chrysanthemum (Supplement to Materia Medica), Step Chrysanthemum (Lueyan Materia Medica), Chicken Intestine, Malantou (Jiuhuang Materia Medica), Bamboo Grass (Zhaiyuan Fang), Malanchrysanthemum (Yilin Zuanyao. Yaoxing), Wedeli Chrysanthemum (Zhiwen Materia Medica), Fish Loach (Caomu Bianfang), Red Stem Vegetable (Bencao Zhengyi), Field Chrysanthemum (Guangzhou Flora), Field Chrysanthemum (Fujian Folk Herbs), Hairy Crab Vegetable (Southern Fujian Folk Herbs), Red Malan, Malanqing (Common Folk Herbs in Zhejiang), Roadside Chrysanthemum, Crab Head Grass (Common Chinese Herbs in Shanghai), Chicken Oil (Hunan Pharmacopoeia), Loach String (Hunan, Sichuan, Jiangxi, Yunnan) [3], etc. Combined with the family and genus name, it was finally named "Malancao".

[0059] [Origin] This product is the dried whole herb of Kalimeris indica (L.) Sch.Bip. of the Asteraceae family.

[0060] Amaranthus is mainly distributed in southeastern Asia[1]. It grows in forest edges, grass, stream banks, roadsides, etc.[2] It is a traditional herbal medicine commonly used by the people in southwestern my country and by ethnic minorities such as the Miao, Yi, Dong, and Lisu, and has a long history of medicinal use.

[0061] [Compendium of Materia Medica] It was first recorded in the Supplement to Compendium of Materia Medica (edition in 741 AD), which says: "Malan grows beside marshes. It is like marsh orchid but has a smelly smell. Northerners call its flowers "purple chrysanthemum" because they resemble single-petal chrysanthemums but are purple. [4]" The Compendium of Materia Medica records: "Malan is abundant in low-lying and wet places in lakes and marshes. It sprouts in February, has red stems and white roots, and long leaves with serrated teeth. It looks like marsh orchid, but is not as fragrant. Southerners often pick it and dry it for vegetables and steamed bun fillings. It is three feet tall in summer, blooms purple flowers, and has fine seeds after the flowers have bloomed." The attached picture and the description and attached picture of Malan in the Plant Names and Realities Illustrated Records are consistent with the Malan of today.

[0062] [Original plant] Perennial herb, 30-70cm high. The rhizome has creeping branches. The stem is erect, with short hairs on the upper part, and branches on the upper part or from the lower part. The leaves are alternate; the base gradually narrows into a long petiole with wings; the leaf blade is obovate or obovate-oblong, 3-6cm long, rarely up to 10cm, 0.8-2cm wide, rarely up to 5cm, with obtuse or pointed tips, and the margin has obtuse or pointed teeth with small tips from the middle or above, or pinnate lobes, with sparse hairs on both sides or the upper side or nearly hairless, thin; the upper leaves are small, sessile, and entire. The capitula is solitary at the end of the branch and arranged in a sparse corymbose shape; the involucre is hemispherical, 6-9mm in diameter and 4-5mm long; the involucre is 2-3 layers, arranged in a shingle-like manner, the outer layer is oblanceolate, about 2mm long, the inner layer is oblanceolate-oblong, up to 4mm, the apex is obtuse or slightly pointed, the upper part is herbaceous, with sparse short hairs, the edge is membranous and has cilia; the ligulate flowers are 1 layer, 15-20, and the tube length is 1.5-1.7mm; the tongue is light purple, up to 10mm long and 1.5-2mm wide; the tubular flowers are 1.5-2mm long, about 1.5mm long, and covered with short hairs. The achenes are obovate-oblong, extremely flat, 1.5-2mm long, about 1mm wide, brown, with light-colored edges and thick ribs, the upper part is covered with glandular hairs and short soft hairs, and the pappus is 0.1-0.8mm long, easy to fall off, and of unequal length. The flowering period is from May to September, and the fruiting period is from August to October.

[0063] [Properties] Based on the description of the properties of the herb Amaranthus chinensis. It is consistent with the Quality Standards for Traditional Chinese and Ethnic Medicinal Materials in Guizhou Province (2003 edition).

[0064] Thin layer identification: The Quality Standards of Traditional Chinese Medicines and Ethnic Medicinal Materials in Guizhou Province and the Standards of Traditional Chinese Medicines in Hunan Province have regulations on thin layer identification of reference medicinal materials. The method has been verified to be feasible, so it is retained. Literature reports that the content of α-spinasterol-3-O-β-D-glucoside in Amaranthus is relatively high. In order to improve the quality control of Amaranthus, a thin layer identification of α-spinasterol-3-O-β-glucoside reference was established.

[0065] Comparative medicinal material thin layer identification:

[0066] Take 2g of this product powder, add 30ml of petroleum ether (30-60℃), heat and reflux for 30 minutes, filter, and concentrate the filtrate to about 0.5ml as the test solution. Take another 2g of Malan reference medicinal material and prepare the reference medicinal material solution in the same way.

[0067] According to the thin layer chromatography method (General Rule 0502), 5 μl of the test solution and the control medicinal material solution were taken and spotted on the same silica gel G thin layer plate, cyclohexane-ethyl acetate (9:1) was used as the developing solvent, developed, taken out, dried, sprayed with vanillin sulfuric acid test solution, and heated at 105°C until the spots were clear. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatogram of the control medicinal material.

[0068] Thin layer identification of α-Bosterol-3-O-β-glucoside control:

[0069] 1 Investigation of the preparation method of the test solution

[0070] Take 0.5g of amaranth powder (passed through a No. 4 sieve) (batch number: Z-161203-01, the same below), add 20mL of methanol, perform ultrasonic extraction for 30 minutes, cold soak for 2 hours and reflux for 30 minutes, filter respectively, evaporate the filtrate in a water bath, add methanol to the residue to 1mL, as the test solution. Take another 2mg of α-spinasterol-3-O-β-D-glucoside, add methanol to make a solution containing 0.1mg of α-spinasterol-3-O-β-D-glucoside per 1mL, as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 6μl of each of the above solutions, spot on the same silica gel G thin layer plate, use chloroform-methanol (9:1) as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol test solution, heat at 105℃ until the spots are clearly colored, and inspect under visible light. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatogram of the reference sample. The results showed that there was no significant difference between the different extraction methods, so ultrasonic extraction was selected. Figure 1 )

[0071] 2 Investigation of solvents for preparing test samples

[0072] Take 0.5g of amaranth powder (passed through a No. 4 sieve), add 30mL of methanol, ethanol and water respectively, treat with ultrasound for 30 minutes, filter respectively, evaporate the filtrate to dryness, and add methanol to the residue to 1mL. Perform a thin layer test according to the method under "1". The results show that different extraction solvents have significant differences. When methanol is used as the solvent, the extraction is more complete, so methanol is selected as the extraction solvent. ( Figure 2 )

[0073] 3. Investigation of different spotting amounts

[0074] Take 0.5g of amaranth powder (passed through a No. 4 sieve), add 30mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, and add methanol to the residue to 1mL. Perform thin layer test according to the method under "1". The sample spot volume is 1μl, 3μl, 5μl, and 7μl respectively. The results show that there are significant differences in different spot volumes. When 5μl is used, the spots are clearly visible. Figure 3 )

[0075] 4. Investigation of developing agent

[0076] The α-spinasterol-3-O-β-D-glucoside reference substance was used for comparison. The preparation methods of the test and reference solutions are shown in the main text. The systems for thin layer identification of α-spinasterol-3-O-β-D-glucoside were investigated: chloroform-methanol (9:1); dichloromethane-methanol-formic acid (7:2).

[0077] According to the experimental results, chloroform-methanol (9:1) was selected as the developing solvent. Figure 4 , 5 )

[0078] 5. Conduct durability test under environmental conditions

[0079] The selected developing agent was tested at 25% and 75% relative humidity and 4°C to 10°C (refrigerator) and room temperature (25°C), and good separation effects were achieved.

[0080] ( Figures 6 to 9 )

[0081] 6 13 batches of Malan grass thin layer chromatography

[0082] The final thin layer identification method for the α-spinasterol-3-O-β-D-glucoside control is as follows: take 0.5g of amaranth powder (passed through a No. 4 sieve), add 20mL of methanol, ultrasonicate for 30 minutes, filter, evaporate the filtrate, and add methanol to the residue to dissolve it to 1mL as the test solution. Take another 2mg of α-spinasterol-3-O-β-D-glucoside, add methanol to make a solution containing 0.1mg of α-spinasterol-3-O-β-D-glucoside per 1mL, as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 6μl of each of the above solutions, spot on the same silica gel G thin layer plate, use chloroform-methanol (9:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol test solution, heat at 105℃ until the spots are clearly colored, and examine under visible light. In the chromatogram of the test product, spots of the same color appear at the corresponding position of the chromatogram of the reference product. Thin layer identification of 13 batches of Malan grass samples Fig.10 , 11 .

[0083] [Content Determination] There is no provision for content determination in the standards of Malan Grassland.

[0084] Literature reports that Malan contains flavonoids, alkaloids, phytosterols, triterpenes, phenolic acids, volatile oils and polysaccharides. The results show that the flavonoids contained in Malan include apigenin, quercetin, kaempferol, wogonin, apigenin-7-O-β-D-glucoside, genkwain, rutin, etc.; the phenolic acids include ferulic acid, vanillic acid, syringic acid, 3,5-dicaffeoylquinic acid, protocatechuic acid, etc.; the anthraquinones include chrysophanol, rhein, rhein, etc.; the triterpenes include oleanolic acid, ergodiol, lupinone, etc.; the steroids include α-spinasterol-3-O-β-D-pyranoglucoside, stigmasterol, α-bosterol, etc. Literature reports that Amaranth has anti-inflammatory, analgesic, anti-aging, and antioxidant pharmacological effects; its phenolic acids are one of the active sites of Amaranth's antibacterial and anti-inflammatory effects; 3,5-dicaffeoylquinic acid, chrysophanol, 2α,3β,19α,23-tetrahydroxy-oleanane-12-ene-28-acid, etc. have a protective effect on CCl4-induced liver damage.

[0085] In this study, the UHPLC-QTOF-MS / MS method was used to identify the components of the ethyl acetate part of the effective antitussive and anticomplementary part of Amaranthus chinensis. A total of 18 components were identified. The component types were complex, mainly flavonoids and phenolic acids, which were speculated to be the main active substances of Amaranthus chinensis for antitussive and anticomplementary effects. However, the content of the monomer components was low. After comprehensive consideration, 3,5-dicaffeoylquinic acid, which has anti-inflammatory and hepatoprotective effects, was selected as the indicator component for [content determination]. The HPLC determination method for the content of this component was established, and 13 batches of samples were determined, and the content limit was specified.

[0086] Determined by high performance liquid chromatography (General Rule 0512).

[0087] Test solution: weigh 0.5 g of medicinal powder (passed through No. 4 sieve), accurately weigh, place in a stoppered conical flask, add 25 ml of 70% methanol, weigh, heat and reflux to extract for 30 minutes, cool, make up the lost weight with 70% methanol, filter, and obtain the filtrate.

[0088] Reference solution: Take an appropriate amount of 3,5-dicaffeoylquinic acid reference substance, accurately weigh (1.62 mg), dissolve it in methanol, transfer it to a 50 ml volumetric flask, dilute to the scale, shake well, and obtain a reference solution with a concentration of 0.0324 mg / ml.

[0089] Chromatographic conditions: Chromatographic column: Elite Hypersil ODS2 (5μm, 4.6mm×250mm); Mobile phase: methanol (A)-0.1% glacial acetic acid water (B), gradient elution as specified in the following table; Column temperature 35°C, flow rate 0.6ml / min, detection wavelength 327nm. The theoretical plate number calculated based on the isochlorogenic acid A peak should not be less than 3000.

[0090]

[0091] 1 Preparation of test solution

[0092] 1.1 Investigation of extraction solvent

[0093] Weigh 0.5g of medicinal material powder (passed through No. 4 sieve), a total of 12 portions, accurately weighed, placed in a 50ml stoppered conical flask, and added 25ml of three different solvents: methanol, ethanol, water, 30% methanol, 50% methanol and 70% methanol, respectively, weighed, ultrasonicated for half an hour, cooled, supplemented the lost weight, filtered, and the filtrate was obtained. Samples were injected and analyzed according to the above chromatographic conditions. The results are shown in Table 2 and Attached Figure 15-20 .

[0094] Table 2 Extraction solvent investigation results

[0095]

[0096]

[0097] It can be seen from the above table that 70% methanol has the highest extraction efficiency among the 6 extraction solvents, so 70% methanol was determined as the extraction solvent.

[0098] 1.2 Investigation of different extraction methods

[0099] Weigh 0.5g of medicinal material powder (passed through No. 4 sieve), a total of 6 portions, accurately weighed, placed in a 50ml stoppered conical bottle, added 25ml of 70% methanol respectively, weighed, cold soaked overnight, heated under reflux extraction and ultrasonic treatment for 30 minutes, cooled, supplemented the lost weight, filtered, and took the filtrate to obtain. Sample injection and analysis were carried out under the above chromatographic conditions. The results are shown in Table 3 and Appendix. Figures 21 to 23 .

[0100] Table 3 Results of different extraction methods

[0101]

[0102] It can be seen from the above table that among the three extraction methods, reflux extraction has the highest extraction efficiency, so heating reflux extraction was determined.

[0103] 1.3 Extraction time investigation

[0104] Weigh 0.5g of medicinal material powder (passed through No. 4 sieve), a total of 4 portions, accurately weighed, placed in 50ml stoppered conical flasks, added 25ml of 70% methanol, weighed, heated under reflux for 15 minutes and 45 minutes respectively, cooled, made up for the lost weight, filtered, and took the filtrate to obtain. Injected and analyzed according to the above chromatographic conditions, the results are shown in Table 4, attached Figures 24-25

[0105] Table 4 Results of investigation at different extraction times

[0106]

[0107]

[0108] It can be seen from the above table that there is little difference in extraction efficiency between the heating reflux extraction for 30 minutes and 45 minutes. Considering time and energy saving, the heating reflux extraction time is determined to be 30 minutes.

[0109] The final method for preparing the test solution was determined as follows: weigh 0.5 g of medicinal powder (passed through a No. 4 sieve), accurately weigh, place in a 50 ml stoppered conical flask, add 25 ml of 70% methanol, weigh, heat and reflux to extract for 30 minutes, cool, make up the lost weight with 70% methanol, filter, and obtain the filtrate.

[0110] 2 HPLC method content determination method validation

[0111] 2.1 System adaptability

[0112] Chromatograms of the reference and test solutions injected under the above chromatographic conditions. Fig.12 , 13 )

[0113] The results showed that the chromatogram of Malan herb could achieve good separation under these conditions, and the peaks were sharp.

[0114] 2.2 Linear Relationship

[0115] Take the reference solution (0.0324 mg / ml) and inject it according to the above liquid phase conditions. The injection volumes are 1, 4, 7, 10, 13, and 16 μl respectively. Record the chromatographic peak area and plot with the injection volume μg as the abscissa and the peak area as the ordinate. The regression equation is y=6,157,040.5644x-44,086.6381, R 2 =0.9999. (Table 5, Fig.14 )

[0116] Table 5 Linear relationship investigation results

[0117]

[0118]

[0119] The results showed that there was a good linear relationship between the injection amount and the peak area of ​​3,5-dicaffeoylquinic acid in the range of 0.0324 to 0.5184 μg.

[0120] 2.3 Precision test

[0121] Take the same portion of 3,5-dicaffeoylquinic acid reference solution and inject it according to the above liquid phase conditions for 6 consecutive times. Calculate its RSD value. The results are shown in Table 6.

[0122] Table 6 Precision test results

[0123]

[0124] The results showed that the RSD value of 6 consecutive injections was 0.37%, indicating that the instrument had good precision.

[0125] 2.4 Repeatability test

[0126] Six samples of Amaranthus herb test solutions were prepared in parallel and injected and tested according to the aforementioned liquid phase conditions. The RSD values ​​were calculated and the results are shown in Table 7.

[0127] Table 7 Repeatability test results

[0128]

[0129]

[0130] 2.5 Stability test

[0131] The same Malan herb test solution was taken and measured after 0, 2, 4, 8, 12, 18 and 24 hours, respectively. The peak area was recorded and the RSD value was calculated. The results are shown in Table 8.

[0132] Table 8 Stability test results

[0133]

[0134] The results showed that the RSD value of the same Amaranthus herba test solution was 0.57% when measured within 24 hours, indicating that the solution had good stability.

[0135] 2.6 Sample recovery test

[0136] Weigh 6 samples of the same batch with known content accurately, add 2.1824 of 3,5-dicaffeoylquinic acid reference substance accurately, prepare according to the test solution preparation method, inject 10μl of sample according to the above liquid phase conditions, and calculate the sample recovery rate and RSD value. The results are shown in Table 9.

[0137] Table 9 Sample recovery determination results

[0138]

[0139]

[0140] The results showed that the average recovery rate of the sample was 98.82% (RSD = 2.40%), indicating that the recovery rate of the method was good.

[0141] 2.7 Sample content determination

[0142] Thirteen batches of Amaranthus herba samples were taken, and the test solutions were prepared according to the test solution preparation method. The samples were injected and measured according to the above-mentioned chromatographic conditions, and the peak areas were recorded and substituted into the linear equation to calculate the content of 3,5-dicaffeoylquinic acid in each sample. The results are shown in Table 10.

[0143] Table 10 Determination of 3,5-dicaffeoylquinic acid content in samples

[0144]

[0145] The results showed that the content of 3,5-dicaffeoylquinic acid in 13 batches of Amaranthus herb samples ranged from 0.04% to 0.30%, with an average of 0.11%. The average value was reduced by 20% to 0.088%, and 5 of the 13 batches of herb samples were unqualified (the unqualified rate was 38%). After observing the 13 batches of herb samples, the plant sizes of the herb varied greatly. Amaranthus is used as medicine in its entirety. Currently, all herb production is wild-collected. The content of 3,5-dicaffeoylquinic acid in each sample varies greatly. The reason for this may be that flowering plants (larger) and tenderer plants (smaller) were collected at the same time when collecting herb (collected in summer and autumn, the flowering period of Amaranthus is from May to September), which led to differences in the content of the ingredients. After comprehensive consideration (avoiding the collection of young plants and ensuring the quality of medicinal materials), the content limit of 3,5-dicaffeoylquinic acid was determined: based on the dry product, the content of 3,5-dicaffeoylquinic acid (C25H24O12) must not be less than 0.05% (the qualified rate of 13 batches of medicinal materials is 85%).

Claims

1. A method for detecting the quality of Amaranthus chinensis, characterized in that: It includes identification and / or content determination items; identification is a thin layer chromatography identification method for α-bosterol-3-O-β-glucoside; content determination is to determine the content of 3,5-dicaffeoylquinic acid in Malan herb by high performance liquid chromatography; The thin layer chromatography identification method for α-sterol-3-O-β-glucoside comprises the following steps: 1) Take 0.5g of Amaranthus powder, add 20mL of methanol, ultrasonicate for 30 minutes, filter, evaporate the filtrate to dryness, add methanol to the residue to dissolve to 1mL, as the test solution; take 2mg of α-spinasterol-3-O-β-D-glucoside, add methanol to make a solution containing 0.1mg of α-spinasterol-3-O-β-D-glucoside per 1mL, as the reference solution; 2) According to the thin layer chromatography test, take 6 μl of each of the above solutions and spot them on the same silica gel G thin layer plate, use a mixture of chloroform and methanol = 9:1 as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol test solution, heat at 105°C until the spots are clearly colored, and examine under visible light; in the chromatogram of the test sample, a spot of the same color should appear at the corresponding position in the chromatogram of the reference sample; The method for determining the content of 3,5-dicaffeoylquinic acid in Malan herb is determined by high performance liquid chromatography, comprising the following steps: (1) Preparation of reference solution: Take an appropriate amount of 3,5-dicaffeoylquinic acid reference substance, weigh accurately, and add methanol to make a solution containing 30 μg per 1 ml to obtain the reference solution; (2) Preparation of test solution: Weigh 0.5 g of medicinal material powder, accurately weigh it, place it in a stoppered conical flask, add 25 ml of 70% methanol, weigh it, heat and reflux it for 30 minutes, cool it, make up the lost weight with 70% methanol, filter it, and take the filtrate to obtain the test solution; (3) Determination method: Accurately pipette 10 μl of the reference solution and 10 μl of the test solution respectively, inject into the liquid chromatograph, and determine.

2. The method for quality inspection of Amaranthus herba according to claim 1, characterized in that: This product, calculated on the basis of dry product, contains not less than 0.05% 3,5-dicaffeoylquinic acid.

3. The method for quality inspection of Amaranthus herba according to claim 1, characterized in that: The chromatographic conditions of the liquid chromatograph are as follows: a Cosmosil C18 column, a wavelength of 327 nm, a flow rate of 0.6 ml / min, and a column temperature of 35° C.; methanol is used as mobile phase A, 0.1% glacial acetic acid water is used as mobile phase B, and gradient elution is performed as specified.

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