A quality detection method for wild tobacco medicinal material

Abstract

The present invention discloses a quality detection method for wild tobacco medicinal materials, the detection method includes identification and / or content determination items; wherein the identification is a microscopic identification method for wild tobacco medicinal materials; the content determination is to determine the content of 1,5-dicaffeoylquinic acid in wild tobacco medicinal materials by high performance liquid chromatography. The microscopic identification method of wild tobacco medicinal materials is simple and feasible, and can accurately identify the authenticity of medicinal materials. The content determination method of 1,5-dicaffeoylquinic acid in wild tobacco medicinal materials can accurately and quickly determine the content of wild tobacco medicinal materials, provide effective qualitative and quantitative analysis means for wild tobacco medicinal materials, and thus facilitate the quality control of wild tobacco medicinal materials. The quality detection method of wild tobacco medicinal materials provided by the present invention can effectively control the overall quality of wild tobacco medicinal materials and ensure the safety and effectiveness of clinical use of wild tobacco medicinal materials.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine analysis, in particular to a quality detection method for wild tobacco leaf medicinal materials. Background Art

[0002] Wild tobacco leaf medicinal material is the dried whole herb of CarpesiumcernuumL. or Carpesiumdivaricatum Sieb.etZucc. of Asteraceae. It is harvested before fruiting in autumn, impurities are removed and dried.

[0003] The stem is 50-100 cm tall, densely covered with long white hairs and curly short hairs at the bottom, especially at the base and leaf axils, often in a cottony state, and sparsely covered with hairs at the top, which gradually fall off and become sparse, with obvious vertical stripes and many branches. The basal leaves wither before flowering and rarely persist; the leaves at the bottom of the stem are larger, with long petioles, which are about 2 / 3 of the length of the leaf blade or nearly the same length, with narrow wings at the bottom, gradually widening towards the leaf base, the leaf blade is oblong or spoon-shaped oblong, 6-12 cm long, 4-6 cm wide, with acute or obtuse apex, and the base is long and gradually narrowed and decurrent, green above, with slightly dense obcuminate hairs, light green below, with white long soft hairs, denser along the veins, often velvety on the midrib and petiole, with glandular dots on both sides, and slightly irregular serrations with callose edges; the leaves in the middle are elliptic to oblong, 8-11 cm long, 3-4 cm wide, with acuminate or acute apex, cuneate base, and short petioles; the leaves at the top are gradually smaller, elliptic to elliptic-lanceolate, and nearly entire. The capitula is solitary at the stem and branch ends, drooping when flowering; there are many bracts of varying sizes, of which 2-3 are larger, elliptic-lanceolate, 2-5 cm long, gradually narrowed at both ends, with short petioles, densely covered with soft hairs and glandular dots, and the rest are smaller, linear-lanceolate or linear-spatulate, slightly longer than the involucre. The involucre is bucket-shaped, 1-2 cm in diameter, 7-8 mm long; there are 4 layers of bracts, the outer bracts are leaf-shaped, lanceolate, equal to or slightly longer than the inner bracts, herbaceous or membranous at the base, densely covered with long soft hairs, obtuse at the apex, usually reflexed, the middle and inner layers are membranous, narrowly oblong to linear, obtuse at the apex, with irregular micro-teeth. The female flowers are narrowly tubular, about 1.5 mm long, wider in the middle, slightly contracted at both ends, and the bisexual flowers are tubular, widening upwards, with 5 tooth cracks on the tube eaves. The achenes are 4-4.5 mm long. It is distributed in provinces and regions in Northeast, North China, Central China, East China, South China and Southwest China, as well as Shaanxi and Gansu in Northwest China.

[0004] Golden ear is a perennial herb with an erect stem, 25-150 cm high, covered with white soft hairs, dense at first, then gradually sparse, branched above the middle, and the branches are usually nearly flat. The basal leaves wither before flowering. The lower leaves are ovate or ovate-oblong, 5-12 cm long and 3-7 cm wide, with acute or obtuse apex, rounded or slightly cordate base, sometimes broadly cuneate, with coarse callose teeth on the margin, dark green above, covered with soft hairs with a spherical swollen base, which fall off sparsely when old, leaving the swollen base. The leaf surface is slightly rough, light green below, covered with white short soft hairs and mixed with sparse long soft hairs, denser along the midrib; the petiole is shorter than or nearly as long as the leaf blade, with narrow wings at the junction with the leaf blade, and wingless at the lower part; the middle leaves are oblong, with acuminate apex, cuneate base, short petiole and no wings; the upper leaves gradually become smaller, oblong or oblong-lanceolate, gradually narrow at both ends, and almost sessile. Inflorescence solitary at stem and branch ends; 3-5 bracts, lanceolate to elliptic, 2 of which are larger, 2-5 times longer than the involucre, densely covered with soft hairs and glandular dots. Involucre ovate-globose, wide at base, less contracted at top, 5-6 mm long, 6-10 mm in diameter, 4 layers of bracts, imbricate, outer layer short (increases layer by layer inward), broadly ovate, membranous or slightly grassy at apex, pubescent on back, middle layer narrowly elliptic, membranous, obtuse at apex, inner layer strip-shaped. Female flowers narrowly tubular, 1.5-2 mm long, tube eaves 4-5 tooth-cleft, bisexual flowers tubular, 3-3.5 mm long, slightly wider upward, tube eaves 5 tooth-cleft, very few soft hairs can be seen under a magnifying glass. Achenes 3-3.5 mm long. Distributed in East China, South China, Central China, Southwest China, Northeast China provinces.

[0005] This product is also a traditional medicinal material used by the Miao people. It is the dried whole herb of Carpesium cernuum L. or Carpesium divaricatum Sieb.et Zucc. of the Asteraceae family. It is dug before fruiting in autumn, removed of impurities, and dried. (Same as the Quality Standards of Traditional Chinese and Ethnic Chinese Medicinal Materials in Guizhou Province)

[0006] The original standard of wild tobacco leaves stipulates that its origin is "the dried whole herb of Carpesium cernuumL. or Carpesium divaricatum Sieb.etZucc." The name "Golden Ear" is recorded in the Qing Dynasty's "Plant Names and Realities Illustrated Records" and the herbal name "Chuzhou Heshi" in the "Daguan Materia Medica"; the alias of Carpesium divaricatum is also called "Golden Ear". The two species are widely distributed in my country, and their distribution areas are mostly the same, and the resources are abundant. After consulting the "Chinese Traditional Medicine Resources", "National Compilation of Chinese Herbal Medicines" and the literature on Chinese herbal medicines in Guizhou, Yunnan, Sichuan, Chongqing, Guangxi and other places, most of the places use the two species as medicines, and their functions and indications are also similar, so the original plants specified in the original standard are used. At the same time, the Miao (Woying, Wild Tobacco), Dong (Jinqueka), Tujia (Waer Grass, Wild Tobacco), Yi (Walebo), Yao (Gutanmi) and other folks also use it for medicine, so it is noted as a common medicinal material used by the Miao people.

[0007] The plants Carpesium cernuum L. and Carpesium divaricatum Sieb.et Zucc. of the Asteraceae family are used as medicines by the people in Guizhou, Sichuan, Chongqing, Yunnan, Anhui, Guangxi, etc. The name of the medicinal material "Wild Tobacco Leaf" is found in the Quality Standards of Traditional Chinese and Ethnic Medicinal Materials in Guizhou Province. It is also called "Wa'er Grass" in Guizhou, Yunnan, Chongqing, etc., "Tobacco Bag Grass" in Anhui and Yunnan, etc., and "Wild Tobacco" by the Miao people. Therefore, this standard refers to the original standard and continues to use the name "Wild Tobacco Leaf".

[0008] Wild tobacco leaves belong to the genus Asteraceae, which contains various types of compounds such as terpenes, steroids, flavonoids, coumarins, lignans, fatty acids, etc. However, most of the current research on its chemical composition focuses on weak polar compounds, especially sesquiterpenoids, which have antibacterial, cytotoxic and herbicidal activities, while there are fewer studies on its polar compounds, and there is no content determination regulation in the original standard. Therefore, it is urgent to develop a quality detection method for wild tobacco leaves, so as to effectively control the overall quality of wild tobacco leaves and ensure the safety and effectiveness of wild tobacco leaves in clinical use. Summary of the invention

[0009] The purpose of the present invention is to provide a quality detection method for wild tobacco medicinal materials, which includes microscopic identification characteristics of wild tobacco medicinal materials and a method for determining the content of 1,5-dicaffeoylquinic acid in wild tobacco medicinal materials. The quality detection method can accurately identify the authenticity of medicinal materials; and can accurately and quickly determine the content of 1,5-dicaffeoylquinic acid in wild tobacco medicinal materials, thereby facilitating the quality control of wild tobacco medicinal materials. The quality detection method for wild tobacco medicinal materials can effectively control the overall quality of wild tobacco medicinal materials and ensure the safety and effectiveness of clinical use of wild tobacco medicinal materials.

[0010] To achieve the above object, the present invention adopts the following technical solution:

[0011] A quality detection method for wild tobacco leaf medicinal materials includes identification and / or content determination items; wherein the identification is a microscopic identification method for wild tobacco leaf medicinal materials; and the content determination is to determine the content of 1,5-dicaffeoylquinic acid in the wild tobacco leaf medicinal materials by high performance liquid chromatography.

[0012] In the aforementioned quality inspection method for wild tobacco leaf medicinal materials, the microscopic identification method for wild tobacco leaf medicinal materials includes microscopic identification of root cross section, stem cross section, leaf cross section, leaf surface and powder.

[0013] (1) Root cross section: Subcircular. The epidermis is composed of one layer of thin-walled cells, with irregular shapes, mostly broken or falling off, and occasionally root hairs. The cortex is broad, the cells are subrectangular, the endodermis is not obvious, and there are secretory cavities arranged in a ring near the phloem. The vascular bundles are externally tough and annular. The cambium between the bundles is not obvious, the phloem cells are arranged closely, and there are intermittent annular cracks between the xylem and the xylem. The xylem cells are all lignified, and the vessels are mostly single and scattered. The xylem ray cells are composed of 1 to 3 rows of cells.

[0014] (2) Stem cross section:

[0015] The epidermis of the tobacco pipe grass is composed of a row of circular and closely arranged cells, and non-glandular hairs and a small amount of glandular hairs can be seen on the epidermis. The cortex is relatively wide, composed of thin-walled cells, and there are 2 to 3 rows of closely arranged thick-walled tissues on the outside. The vascular bundles are exo-tough, with about 20 to 30 bundles arranged in a ring; the cambium is not obvious; the phloem is narrow, with fiber bundles on the outside, which are semi-lunar; the xylem is composed of wood fibers and vessels, and the cell walls are lignified. The pith is well developed, and the medullary rays are narrow, composed of 2 to 8 rows of radially elongated thin-walled cells;

[0016] The hairs on the surface of the golden ear are rare; the cortex is narrow, the outer phloem fiber bundles are well developed, and the medulla is wide;

[0017] (3) Leaf cross section: The upper epidermal cells are composed of one row of irregularly arranged thin-walled cells. Both the upper and lower epidermis are covered with glandular hairs and non-glandular hairs. The non-glandular hairs are mostly composed of 2 to 6 cells, and the glandular hairs are mostly single-petioled. The mesophyll tissue is not clearly differentiated from the lower epidermal cells, and there is no distinction between palisade tissue and spongy tissue. There are several rows of thick-cornered tissue in the upper and lower epidermis of the main vein. The vascular bundles are double-tough. The phloem is in two half-moons surrounding the xylem, and the xylem vessels are radially arranged or scattered.

[0018] (4) Leaf surface: The upper epidermal cells are polygonal, with glandular and non-glandular hairs visible. The lower epidermal cells are polygonal, with slightly wavy walls, many stomata in an indefinite pattern, and densely covered with glandular and non-glandular hairs.

[0019] (5) Powder microscopy: The powder of this product is yellow-green to gray-green. Vessels are common, mostly spiral and reticular vessels, occasionally ladder-shaped and pit-shaped vessels, with a diameter of 20-75μm. Non-glandular hairs are common, composed of 2-4 cells, slightly curved, and often broken; glandular hairs are rare, mostly single-cell glandular hairs. Wood fibers are mostly bundled, long and narrow, with flat ends and slightly thickened walls. Pollen grains are rare, spherical, 18-25μm in diameter, with sharp spines on the surface and 3 germination pores.

[0020] In the above-mentioned method for testing the quality of wild tobacco medicinal materials, the method for determining the content of 1,5-dicaffeoylquinic acid in wild tobacco medicinal materials is determined by high performance liquid chromatography, comprising the following steps:

[0021] (1) Chromatographic conditions and system suitability test: Intersil ODS-SP (4.6×150 mm, 5 μm) was used as the chromatographic column; acetonitrile-0.4% formic acid solution (14:86) was used as the mobile phase, and the detection wavelength was 325 nm. The theoretical plate number calculated based on the 1,5-dicaffeoylquinic acid peak should be no less than 2500.

[0022] (2) Preparation of reference solution: Take an appropriate amount of 1,5-dicaffeoylquinic acid reference substance, accurately weigh it, and add 70% methanol to make a solution containing 24 μg per 1 ml.

[0023] (3) Preparation of test solution: Take about 1 g of the product powder (passed through a No. 4 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 70% methanol, weigh it, ultrasonicate it for 30 minutes, cool it, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0024] (4) Determination method: Accurately pipette 20 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine;

[0025] (5) This product contains 1,5-dicaffeoylquinic acid (C 25 H 24 O 12 ) shall not be less than 0.045%.

[0026] In the above-mentioned method for quality detection of wild tobacco medicinal materials, the power of ultrasonic treatment is 250W and the frequency is 40KHz.

[0027] The beneficial effects of the present invention are:

[0028] 1. The present invention provides a quality detection method for wild tobacco medicinal materials, which includes a microscopic identification method for wild tobacco medicinal materials and a method for determining the content of 1,5-dicaffeoylquinic acid in wild tobacco medicinal materials. The microscopic identification method for wild tobacco medicinal materials is simple and feasible, and can accurately identify the authenticity of medicinal materials. The method for determining the content of 1,5-dicaffeoylquinic acid in wild tobacco medicinal materials can accurately and quickly determine the content of wild tobacco medicinal materials, providing an effective qualitative and quantitative analysis method for wild tobacco medicinal materials, thereby facilitating the quality control of wild tobacco medicinal materials. The quality detection method for wild tobacco medicinal materials provided by the present invention can effectively control the overall quality of wild tobacco medicinal materials and ensure the safety and effectiveness of clinical use of wild tobacco medicinal materials.

[0029] The quality detection method of the Herba Blumeae Balsamiferae provided by the present invention is helpful to make up for the deficiencies of the quality detection method in the "Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province" (2003 Edition: Wild Tobacco Leaves), and has strong practicality. The method of the present invention is of great significance for further quality control of wild tobacco leaves, and can provide a scientific basis for the formulation of quality tables for wild tobacco leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the cross-section tissue of the root of wild tobacco leaf (in the figure, A: cross-section of the root of tobacco pipe head grass; B: cross-section of the root of golden ear);

[0031] Figure 2 It is the cross-section tissue of the stem of wild tobacco leaves (in the figure, A: cross-section of the stem of tobacco tube grass; B: cross-section of the stem of golden ear);

[0032] Figure 3 It is the cross-section tissue of wild tobacco leaves (in the figure, A: cross-section of tobacco tube grass leaves; B: cross-section of golden ear leaves);

[0033] Figure 4 This is the surface view of a wild tobacco leaf (in the figure, A: the surface view of a tobacco leaf; B: the surface view of a golden ear leaf);

[0034] Figure 5 It is the characteristic of wild tobacco leaf powder (in the figure, A: duct, B: sieve tube, wood fiber, non-glandular hair);

[0035] Figure 6 It is the mass spectrum of 1,5-dicaffeoylquinic acid (in the figure, A is the chromatogram and TIC diagram of wild tobacco leaves; B is the identification result diagram of the main chemical components of wild tobacco leaves; C is the main ion fragment attribution diagram of the main chemical components of wild tobacco leaves);

[0036] Figure 7 It is the UV absorption spectrum of the sample (A: 1,5-dicaffeoylquinic acid reference substance; B: wild tobacco leaf medicinal material);

[0037] Figure 8 This is the sample purity view (A: 1,5-dicaffeoylquinic acid reference substance; B: wild tobacco leaf medicinal material);

[0038] Fig. 9 It is the NMR diagram of the reference substance;

[0039] Fig.10 This is the standard curve of 1,5-dicaffeoylquinic acid;

[0040] Fig.11 It is the HPLC spectrum of the sample (in the figure, A: 1,5-dicaffeoylquinic acid reference substance; B: wild tobacco leaf medicinal material);

[0041] Fig.12It is the spectrum of Intersil ODS-SP (4.6×150mm, 5μm) reference product;

[0042] Fig.13 It is the spectrum of Intersil ODS-SP (4.6×150mm, 5μm) sample;

[0043] Fig.14 It is the Agilent Eclipse XDB-C18 (4.6×150mm, 5μm) reference spectrum;

[0044] Fig.15 It is the sample spectrum of Agilent Eclipse XDB-C18 (4.6×150mm, 5μm);

[0045] Fig.16 It is the spectrum of the reference substance test report at a column temperature of 28°C;

[0046] Fig.17 It is the HPLC spectrum of the reference substance at a column temperature of 28°C;

[0047] Fig.18 It is the HPLC spectrum of the sample at a column temperature of 28°C;

[0048] Fig.19 It is the HPLC spectrum of the reference substance at a column temperature of 32°C;

[0049] Fig. 20 This is the HPLC spectrum of the sample at a column temperature of 32°C;

[0050] Fig.21 It is the HPLC spectrum of the reference substance at a flow rate of 0.8 ml / min;

[0051] Fig. 22 This is the HPLC profile of the sample at a flow rate of 0.8 ml / min;

[0052] Fig.23 It is the HPLC spectrum of the reference substance at a flow rate of 0.6 ml / min-;

[0053] Fig.24 This is the HPLC profile of the sample at a flow rate of 0.6 ml / min;

[0054] Fig.25 It is the HPLC spectrum of the reference substance at a wavelength of 323 nm;

[0055] Fig.26 It is the HPLC spectrum of the sample at a wavelength of 323 nm;

[0056] Fig. 27 It is the HPLC spectrum of the reference substance at a wavelength of 327nm;

[0057] Fig.28 It is the HPLC spectrum of the sample at a wavelength of 327 nm;

[0058] Fig.29 It is the HPLC spectrum of the reference substance of acetonitrile:0.4% formic acid solution 15:85;

[0059] Fig.30 It is the HPLC spectrum of the sample of acetonitrile:0.4% formic acid solution 15:85;

[0060] Fig.31 It is the HPLC spectrum of the reference substance of acetonitrile:0.4% formic acid solution 13:87;

[0061] Fig.32 It is the HPLC spectrum of the sample of acetonitrile:0.4% formic acid solution 13:87;

[0062] Fig.33 This is the HPLC spectrum of the reference substance of Shimadzu LC-20A;

[0063] Fig.34 This is the sample HPLC spectrum of Shimadzu LC-20A;

[0064] Fig.35 This is the HPLC spectrum of the reference substance of Shimadzu LC-2010CHT;

[0065] Fig.36 This is the sample HPLC spectrum of Shimadzu LC-2010CHT;

[0066] Fig.37 It is the HPLC spectrum of the reference substance of Waters e2695;

[0067] Fig.38 is the sample HPLC spectrum of Waters e2695;

[0068] Fig.39 This is the HPLC spectrum of the reference substance of the chromatographic column Inertsil ODS-SP S / N 2AI92266;

[0069] Fig.40 This is the sample HPLC spectrum of the chromatographic column Inertsil ODS-SP S / N 2AI92266. DETAILED DESCRIPTION

[0070] 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.

[0071] Embodiments. The method for detecting the quality of wild tobacco leaves described in the present invention is not limited to the following embodiments.

[0072] instrument:

[0073] Waters e2695 high performance liquid chromatograph, Shimadzu LC-20A high performance liquid chromatograph, Shimadzu LC-2010CHT high performance liquid chromatograph;

[0074] CPA225D electronic balance: 1 / 100,000, Sartorius;

[0075] BS224S electronic balance: 1 / 10,000, Sartorius;

[0076] KQ5200 ultrasonic cleaner: Kunshan Ultrasonic Instrument Co., Ltd.

[0077] HH-4 digital constant temperature water bath: Jintan Huaou Experimental Instrument Factory;

[0078] CS101-2EBN electric constant temperature blast drying oven: Chongqing Yongsheng Experimental Instrument Factory;

[0079] 8-10 type box-type resistance furnace: manufactured by Shenyang Energy Saving Electric Furnace Factory, China;

[0080] ZF-20D dark box UV analyzer: Gongyi Yuhua Instrument Co., Ltd.

[0081] Reagents and test drugs

[0082] 1,5-dicaffeoylquinic acid reference substance (Lot: PRF10041043, Chengdu PRF Technology Development Co., Ltd.).

[0083] Medicinal Materials

[0084] A total of 15 batches of wild tobacco leaf samples were provided by Guizhou Bailing Group. They were identified by Zhong Guoyue, a researcher at Jiangxi University of Traditional Chinese Medicine, as the dried whole herbs of Carpesiumcernuum L. and Carpesiumdivaricatum Sieb.et Zucc. of the Asteraceae family. Sample information is shown in Table 1.

[0085] Table 1 List of wild tobacco medicinal material sample information

[0086]

[0087]

[0088] The microscopic identification methods for wild tobacco leaf medicinal materials include root cross section, stem cross section, leaf cross section, leaf surface and powder microscopic identification.

[0089] (1) Root cross section: Subcircular. The epidermis is composed of a layer of thin-walled cells, which are irregular in shape, often broken or falling off, with occasional root hairs. The cortex is broad, the cells are subrectangular, the endodermis is not obvious, and there are secretory cavities arranged in rings near the phloem. The vascular bundles are extra-tough and annular. The cambium between bundles is not obvious, the phloem cells are arranged closely, and there are intermittent annular cracks between the xylem and the xylem. The xylem cells are all lignified, the vessels are mostly single and scattered, and the xylem ray cells are composed of 1 to 3 rows of cells. Figure 1 shown.

[0090] (2) Stem cross section:

[0091] The epidermis of the tobacco pipe grass is composed of a row of circular and closely arranged cells, and non-glandular hairs and a small amount of glandular hairs can be seen on the epidermis. The cortex is relatively wide, composed of thin-walled cells, and there are 2 to 3 rows of closely arranged thick-walled tissues on the outside. The vascular bundles are exo-tough, with about 20 to 30 bundles arranged in a ring; the cambium is not obvious; the phloem is narrow, with fiber bundles on the outside, which are semi-lunar; the xylem is composed of wood fibers and vessels, and the cell walls are lignified. The pith is well developed, and the medullary rays are narrow, composed of 2 to 8 rows of radially elongated thin-walled cells; such as Figure 2 shown.

[0092] The hairs on the surface of the golden ear are rare; the cortex is narrow, the outer phloem fiber bundles are well developed, and the medulla is wide;

[0093] (3) Leaf cross section: The upper epidermal cells are composed of one row of irregularly arranged thin-walled cells. Both the upper and lower epidermis are covered with glandular hairs and non-glandular hairs. The non-glandular hairs are mostly composed of 2 to 6 cells, and the glandular hairs are mostly single-petioled. The mesophyll tissue is not clearly differentiated from the lower epidermal cells, and there is no distinction between palisade tissue and spongy tissue. There are several rows of thick-cornered tissue in the upper and lower epidermis of the main vein. The vascular bundles are double-tough. The phloem is in two half-moons surrounding the xylem, and the xylem vessels are radially arranged or scattered. Figure 3 shown.

[0094] (4) Leaf surface: The upper epidermal cells are polygonal, with glandular and non-glandular hairs visible. The lower epidermal cells are polygonal, with slightly wavy walls, many stomata in an indefinite pattern, and densely covered with glandular and non-glandular hairs. Figure 4 shown.

[0095] (5) Powder microscopy: The powder of this product is yellow-green to gray-green. Vessels are common, mostly spiral and reticular vessels, occasionally ladder-shaped and pitted vessels, with a diameter of 20 to 75 μm. Non-glandular hairs are common, composed of 2 to 4 cells, slightly curved, and often broken; glandular hairs are rare, mostly single-cell glandular hairs. Wood fibers are mostly bundled, long and narrow, with flat ends and slightly thickened walls. Pollen grains are rare, spherical, 18 to 25 μm in diameter, with sharp spines on the surface and 3 germination pores. Figure 5 shown.

[0096] In this study, 50% ethanol was used for extraction, followed by ethyl acetate and n-butanol extraction, and HPLC-MS was used to analyze the characteristic compound components of the n-butanol part. It was found that caffeoylquinic acid was the main component, one of which was 1,5-dicaffeoylquinic acid ( Figure 6-10 ).

[0097]

Content Determination

[0098] Studies have shown that caffeoylquinic acid compounds have anti-inflammatory, antioxidant, cell damage protection, anti-fibrosis and other pharmacological activities, and 1,5-dicaffeoylquinic acid has a strong cell protection effect. Therefore, in this standard revision, 1,5-dicaffeoylquinic acid was selected as the indicator component, and its content determination method was established and its content limit was specified.

[0099] Determine by high performance liquid chromatography (General Rule 0512 of Part IV of the 2015 edition of the Chinese Pharmacopoeia). The determination method is as follows:

[0100] Test solution: Take about 1 g of the powder of the product (passed through No. 4 sieve), weigh it accurately, put it in a stoppered conical flask, add 25 ml of 70% methanol accurately, weigh it, treat it with ultrasound for 30 minutes, cool it, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0101] Preparation of reference solution: Take an appropriate amount of 1,5-dicaffeoylquinic acid reference substance, weigh it accurately, and add 70% methanol to make a solution containing 24 μg per 1 ml.

[0102] The chromatographic conditions were Intersil ODS-SP (4.6×150 mm, 5 μm) as the chromatographic column, acetonitrile-0.4% formic acid solution (14:86) as the mobile phase, and the detection wavelength was 325 nm. The theoretical plate number calculated based on the 1,5-dicaffeoylquinic acid peak should not be less than 2500.

[0103] 1. Selection of detection wavelength

[0104] Shimadzu LC-20A high performance liquid chromatograph, chromatographic column: Inertsil ODS-SP (4.6mm×150mm, 5μm), mobile phase: acetonitrile-0.4% formic acid solution (14:86), column temperature: 30℃, flow rate: 0.7ml / min, detection wavelength: 325nm. Under the above chromatographic conditions, 1,5-dicaffeoylquinic acid was well separated, and the theoretical plate number was >2500.

[0105] This product contains a large number of caffeoylquinic acid compounds and has a high selectivity for chromatographic columns. Among them, the Intersil ODS-SP (4.6×150mm, 5μm) chromatographic column can achieve baseline separation for 1,5-dicaffeoylquinic acid, while other chromatographic columns such as Agilent Eclipse XDB-C18 (4.6×150mm, 5μm) cannot achieve baseline separation, and the target peak is mixed with the previous impurity peak, indicating that this type of compound has high requirements for the chromatographic column. In this study, the Intersil ODS-SP (4.6×150mm, 5μm) chromatographic column was selected, which is commercially available and moderately priced.

[0106] The China Food and Drug Inspection Institute does not have 1,5-dicaffeoylquinic acid reference substances for sale. The reference substance used in this study is a commercially available reference substance purchased from Chengdu Purifa Technology Development Co., Ltd. The HPLC normalization method detected its purity to be 98.80% (not Intersil ODS-SP (4.6×150mm, 5μm) chromatographic column); the purity was tested using the chromatographic conditions of this article, and the result was 93.33%, which once again verified that this compound has strict requirements on the chromatographic column. This experiment is based on a content of 93.33%. Figure 10-15 shown.

[0107] 2. Preparation of reference solution

[0108] Accurately weigh an appropriate amount of 1,5-dicaffeoylquinic acid reference substance, and use 70% methanol to prepare a stock solution containing 194.1μg of 1,5-dicaffeoylquinic acid per 1ml, and store it in a 4℃ refrigerator away from light for later use. Dilute the reference substance stock solution 8 times to prepare a solution containing 24.3μg of 1,5-dicaffeoylquinic acid per 1ml as the reference substance solution.

[0109] 3 Investigation of the preparation method of test solution

[0110] 3.1 Investigation of extraction solvent

[0111] Take about 1g of coarse powder (pass through No. 4 sieve) of wild tobacco leaf medicinal material (batch number: 20161011), accurately weigh it, put it in a stoppered conical bottle, accurately add 25ml of methanol, 70% methanol, 50% methanol, and 30% methanol, weigh it, ultrasonically treat it for 30 minutes, cool it, use each solvent to make up the lost weight, shake it well, centrifuge it, and filter it. Take 20μl of each sample for injection, calculate the content of 1,5-dicaffeoylquinic acid, and the results are shown in Table 2. From the results, it can be seen that the content of 1,5-dicaffeoylquinic acid in the extract with 70% methanol as the solvent is higher, so 70% methanol is selected as the solvent.

[0112] Table 2 Solvent investigation results

[0113]

[0114] 3.2 Examination of solvent dosage

[0115] Take about 1g of coarse powder (pass through No. 4 sieve) of wild tobacco leaf medicinal material (batch number: 20161011), accurately weigh it, put it in a stoppered conical bottle, accurately add 25ml, 50ml, and 100ml of 70% methanol, weigh it, ultrasonically treat it for 30 minutes, let it cool, make up the lost weight with 70% methanol, shake it well, centrifuge it, and filter it. Take 20μl of each sample for injection, and calculate the content of 1,5-dicaffeoylquinic acid. The results are shown in Table 3. From the results, it can be seen that the amount of solvent has little effect on the extraction rate of 1,5-dicaffeoylquinic acid. In order to save solvent, the amount of solvent is selected as 25ml.

[0116] Table 3 Results of investigation on solvent dosage

[0117]

[0118]

[0119] 3.3 Ultrasonic time investigation

[0120] Take about 1g of coarse powder (pass through No. 4 sieve) of wild tobacco leaf medicinal material (batch number: 20161011), accurately weigh it, put it in a stoppered conical bottle, accurately add 25ml of 70% methanol, weigh it, and ultrasonically treat it for 15, 30, and 40 minutes respectively, let it cool, use 70% methanol to make up the lost weight, shake it well, centrifuge it, and filter it. Take 20μl of each sample for injection, and calculate the content of 1,5-dicaffeoylquinic acid. The results are shown in Table 4. It can be seen from the results that as the ultrasonic time increases, the content of 1,5-dicaffeoylquinic acid also increases accordingly. The content of 1,5-dicaffeoylquinic acid is not much different after ultrasonic treatment for 30 minutes and 45 minutes, so the ultrasonic time is determined to be 30 minutes.

[0121] Table 4 Ultrasonic time investigation results

[0122]

[0123] According to the above test results, the method for preparing the test solution is as follows: take about 1 g of the powder of the product (passed through No. 4 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 70% methanol, weigh the weight, ultrasonically treat it for 30 minutes, cool it, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0124] 4 Methodological Validation

[0125] 4.1 Investigation of linear relationship

[0126] Take the mixed reference stock and dilute it a certain multiple, take the mixed reference and its dilutions for injection. Perform linear regression processing on the peak area integral value with the injection volume to obtain the regression equation of 1,5-dicaffeoylquinic acid. The results show that 1,5-dicaffeoylquinic acid has good linearity under this condition. The results are shown in Table 5 and the standard curve is shown in Fig.10 .

[0127] Table 5 Regression equation and linear range of 1,5-dicaffeoylquinic acid

[0128]

[0129] Based on the above methodological investigation results, the method for preparing the test solution is determined as follows: weigh about 0.5 g of medicinal powder, accurately weigh it, place it in a stoppered conical flask, add 25 ml of 70% ethanol, weigh it, ultrasonicate for 30 minutes, cool it, make up the lost weight with 70% ethanol, filter it, and take the filtrate.

[0130] 4.2 Durability test

[0131] Wild tobacco leaf powder (20161106) was used to prepare the test solution according to the “Test Sample Preparation Method”, and the effects of column temperature, flow rate, mobile phase ratio, wavelength, chromatograph, and chromatographic column on the content determination method were investigated. The results are shown in Table 6. It can be seen from the results that fine-tuning the column temperature, flow rate, mobile phase ratio, wavelength, different chromatographs, and the same type of chromatographic columns have little effect on the content determination results.

[0132] Table 6 Durability test results

[0133]

[0134]

[0135] 4.3 Determination of detection limit and quantification limit

[0136] Prepare a lower concentration of reference solution, inject it into the liquid chromatograph, and observe how many times its peak height is higher than the baseline noise (i.e. S / N). When S / N is approximately equal to 3, the concentration at this time is the detection limit. When S / N is approximately equal to 10, the concentration at this time is the quantification limit. The results are shown in Table 7.

[0137] Table 7 Detection limit and quantification limit of 1,5-dicaffeoylquinic acid

[0138]

[0139] 4.4 Precision test

[0140] The mixed reference solution was taken and injected 6 times in succession, 20 μL each time, and the peak area of ​​1,5-dicaffeoylquinic acid was recorded. The results are shown in Table 8. The results show that the precision of 1,5-dicaffeoylquinic acid is good under this condition.

[0141] Table 8 Precision determination results (measured by peak area integral value)

[0142]

[0143] 4.5 Repeatability test

[0144] Take wild tobacco leaf medicinal material (batch number: 20150121), prepare 6 test solutions according to the "Test solution preparation method", inject 20 μL, and determine the content of 1,5-dicaffeoylquinic acid. The results are shown in Table 9. The results show that wild tobacco leaves have good repeatability under this condition.

[0145] Table 9 Repeatability test results (average content, mg / g)

[0146]

[0147] 4.6 Solution stability test

[0148] Take wild tobacco leaf medicinal material (batch number: 20150121), prepare the test solution according to the "Test solution preparation method", inject 20 μL at 0, 4, 8, 12, and 24 hours, and record the peak area of ​​1,5-dicaffeoylquinic acid. The results are shown in Table 10. The results show that the treated sample is stable within 1 day.

[0149] Table 10 Stability test results (measured by peak area integral value)

[0150]

[0151] 4.7 Sample recovery rate

[0152] Weigh about 0.5g of wild tobacco leaf medicinal material (batch number: 20150121), add 2ml of stock solution, prepare 6 samples according to the "Test solution preparation method", determine the content of 1,5-dicaffeoylquinic acid, and calculate the recovery rate of the sample. The average recovery rate and RSD% of 1,5-dicaffeoylquinic acid are shown in Table 11. RSD%≤2.0%, indicating that the determination result is accurate. The data meets the methodological validation requirements under Item 9101 of Part IV of the 2015 edition of the Chinese Pharmacopoeia (the content of the component to be tested is 0.1%, and the corresponding recovery limit is 90%-108%).

[0153] Table 11: Results of sample recovery of wild tobacco leaves (n=6)

[0154]

[0155] 5. Sample content determination

[0156] Fifteen batches of wild tobacco leaf medicinal materials were taken, and the test solution was prepared according to the "Test solution preparation method". The content of 1,5-dicaffeoylquinic acid in each sample was determined according to the "Chromatographic conditions". The results are shown in Table 12.

[0157] Table 12 Determination results of content in wild tobacco medicinal materials

[0158]

[0159] * indicates outliers.

[0160] In order to prevent the measurement results from showing extreme values ​​far from the mean, the G test method in statistical methods was used to test the measured values, and the outlier value (sample No. 6) was eliminated. The average value of the remaining samples was calculated to be 0.706 mg / g.

[0161] The results showed that the content of 1,5-dicaffeoylquinic acid in 15 batches of medicinal samples was between 0.346 and 3.475 mg / g. The average value after removing outliers was 0.706 mg / g, and the average value was 0.4942 mg / g after 30% of the average was lowered. Four of the 15 batches of samples were unqualified, and the qualified rate was 74%. The content of 1,5-dicaffeoylquinic acid in each sample varied widely, which may be related to the conversion between various dicaffeoylquinic acid components in the samples. Taking all factors into consideration, the content limit of 1,5-dicaffeoylquinic acid was determined to be no less than 0.045%.

[0162] Fig.12 The readings are shown in Table 13.

[0163] Table 13

[0164]

[0165] Fig.13 The readings in are shown in Table 14.

[0166] Table 14

[0167]

[0168] Fig.14 The readings are shown in Table 15.

[0169] Table 15

[0170]

[0171] Fig.15 The readings in are shown in Table 16.

[0172] Table 16

[0173]

[0174] Fig.17 The readings in are shown in Table 17.

[0175] Table 17

[0176]

[0177] Fig.18 The readings in are shown in Table 18.

[0178] Table 18

[0179]

[0180] Fig.19 The readings in are shown in Table 19.

[0181] Table 19

[0182]

[0183] Fig. 20 The readings are shown in Table 20.

[0184] Table 20

[0185]

[0186] Fig.21 The readings in are shown in Table 21.

[0187] Table 21

[0188]

[0189] Fig. 22 The readings in are shown in Table 22.

[0190] Table 22

[0191]

[0192] Fig.23 The readings in are shown in Table 23.

[0193] Table 23

[0194]

[0195] Fig.24 The readings in are shown in Table 24.

[0196] Table 24

[0197]

[0198] Fig.25 The readings in are shown in Table 25.

[0199] Table 25

[0200]

[0201] Fig.26 The readings in are shown in Table 26.

[0202] Table 26

[0203]

[0204] Fig. 27 The readings in are shown in Table 27.

[0205] Table 27

[0206]

[0207] Fig.28 The readings in are shown in Table 28.

[0208] Table 28

[0209]

[0210] Fig.29 The readings in are shown in Table 29.

[0211] Table 29

[0212]

[0213] Fig.30 The readings in are shown in Table 30.

[0214] Table 30

[0215]

[0216] Fig.31 The readings in are shown in Table 31.

[0217] Table 31

[0218]

[0219] Fig.32 The readings in are shown in Table 32.

[0220] Table 32

[0221]

[0222] Fig.33 The readings in are shown in Table 33.

[0223] Table 33

[0224]

[0225] Fig.34 The readings in are shown in Table 34.

[0226] Table 34

[0227]

[0228] Fig.35 The readings in are shown in Table 35.

[0229] Table 35

[0230]

[0231] Fig.36 The readings in are shown in Table 36.

[0232] Table 36

[0233]

[0234] Fig.37 The readings in are shown in Table 37.

[0235] Table 37

[0236] Chromatographic peak results

[0237]

[0238] Fig.38 The readings in are shown in Table 38.

[0239] Table 38

[0240] Chromatographic peak results

[0241] Retention time area Peak height %area %high Separation USP Theoretical Plate Number JP theoretical increase in number of boards EP theoretical plates 1 35.115 493548 7925 3.80 4.99 7.276199e+003 7.414394e+003 7.414394e+003 2 38.96 1441047 21670 11.10 13.65 2006625e+000 7.677666e+003 7.763986e+003 7.763966e+003 3 42.849 11043316 129186 86.09 81.36 2.203720e+000 5.912873e+003 5.996676e+003 5.996676e+003

[0242] Fig.39 The readings in are shown in Table 39.

[0243] Table 39

[0244] Chromatographic peak results

[0245] Retention time area Peak height %area %high Height USP Theoretical Plate Number JP theoretical plate number EP on the number of plates 1 38.036 1282316 19189 5.71 6.30 2 41.367 21182589 286609 94.29 93.70

[0246] Fig.40 The readings in are shown in Table 40.

[0247] Table 40

[0248] Chromatographic peak results

[0249] Retention time area Peak height %area %high Separation USP Theoretical Plate Number JP theoretical plate number EP theoretical plates 1 39.703 586138 7372 4.38 4.93 2 43.260 1458397 19447 10.90 13.01 3 48.269 11331805 122637 84.72 82.06

Claims

1. A method for detecting the quality of wild tobacco medicinal materials, characterized in that: The detection method includes identification and / or content determination items; wherein the identification is a microscopic identification method for wild tobacco medicinal materials; the content determination is to determine the content of 1,5-dicaffeoylquinic acid in wild tobacco medicinal materials by high performance liquid chromatography; Microscopic identification methods for wild tobacco leaf medicinal materials include root cross section, stem cross section, leaf cross section, leaf surface and powder microscopic identification; The method for determining the content of 1,5-dicaffeoylquinic acid in the wild tobacco medicinal material is determined by high performance liquid chromatography, comprising the following steps: (1) Chromatographic conditions Intersil ODS-SP was used as the chromatographic column; a mixture of acetonitrile and 0.4% formic acid solution = 14:86 was used as the mobile phase, and the detection wavelength was 325 nm; (2) Preparation of reference solution: Take an appropriate amount of 1,5-dicaffeoylquinic acid reference substance, weigh it accurately, and add 70% methanol to make a solution containing 24 µg per 1 ml to obtain the reference solution. (3) Preparation of test solution: Take 1 g of the powder of the product, weigh it accurately, place it in a stoppered conical flask, add 25 ml of 70% methanol accurately, weigh it, ultrasonicate it for 30 minutes, let it cool, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution; (4) Determination: Accurately pipette 20 µl of the reference solution and the test solution respectively and inject them into the liquid chromatograph for determination.

2. The method for detecting the quality of wild tobacco leaf medicinal material according to claim 1, characterized in that: The wild tobacco leaf medicinal material is sliced ​​according to the microscopic identification method of the wild tobacco leaf medicinal material, and observed under a microscope. The microscopic characteristics should be consistent with the standard.

3. The method for detecting the quality of wild tobacco leaf medicinal material according to claim 1, characterized in that: This product contains 1,5-dicaffeoylquinic acid C calculated on the basis of dry product. 25 H 24 O 12 Not less than 0.045%.

4. The method for detecting the quality of wild tobacco leaf medicinal material according to claim 1, characterized in that: In step (3) of the method for determining the content of 1,5-dicaffeoylquinic acid in wild tobacco medicinal materials, the power of ultrasonic treatment is 250 W and the frequency is 40 KHz.

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