Multi-index quality analysis method of dendrobium nobile and application of multi-index quality analysis method

Abstract

The invention discloses a multi-index quality analysis method of dendrobium nobile and application of the multi-index quality analysis method. According to the invention, a high performance liquid chromatography (HPLC) method is adopted to establish a method for determining the content of Dendrobin A, xanthophyll, New Zealand vitexin and isophorone in stems and flowers of Dendrobium nobile; a method for determining the content of dendrobine is established by adopting a gas chromatography (GC) method, and a method for determining the content of large components including total polyphenols, total flavonoids, total anthocyanin, total polysaccharides and total alkaloids in stems and flowers of dendrobium nobile lindl is established by adopting an ultraviolet-visible (UV-Vis) spectrophotometric method. According to the quality standard disclosed by the invention, systematic and comprehensive quality control is carried out on the dendrobium nobile for the first time, and the quality standard not only has an important guiding significance on establishing the quality standard of the dendrobium nobile medicinal material red water in Guizhou, but also has an important guiding effect on research of a dendrobium nobile extraction process and development and quality control of related products.

Description

Technical Field

[0001] The present invention relates to the technical field of quality analysis of traditional Chinese medicines, and in particular to a multi-index quality analysis method for Dendrobium nobile and an application thereof. Background Art

[0002] Dendrobium nobile Lindl., a plant of the Orchidaceae family, is sweet, slightly cold, and bitter in nature. It enters the stomach and kidney meridians. It is commonly used clinically to treat mouth and tongue sores, restlessness and thirst due to deficiency, stomach yin deficiency, liver-stomach disharmony, yin deficiency and excessive fire, bone steaming and fever, and soreness and weakness in the waist and knees. The Compendium of Materia Medica describes Dendrobium nobile as follows: "Its stem is shaped like the strands of a golden hairpin, hence the ancient name 'Golden Hairpin Dendrobium.' Today, people in Sichuan cultivate it and call it 'Golden Hairpin Flower.' Growing in clusters on rocks, its roots are densely tangled, and its stems are soft white. Its stems and leaves are green when young, then yellow when dry. It blooms red flowers. With frequent watering, it endures year after year, earning it the name 'Thousand-Year Moisture.' Dendrobium spp. is short and solid in the middle, while Dendrobium spp. is long and hollow in the middle, making it easy to distinguish. They are found everywhere, but those in Sichuan are considered the best." The Compendium of Materia Medica describes the optimal growing environment for Dendrobium nobile as Sichuan, which is today's Chishui, Guizhou. Volume 10 of the Xuanping County Chronicle, published in the 20th year of the Daoguang reign, lists "Dendrobium, commonly known as 'Chlorophytum,' grows in clusters on high mountain cliffs," indicating that Dendrobium often grows on cliffs in valleys. Today, resources of Dendrobium nobile are primarily distributed in my country's subtropical regions south of the Yangtze River, including Guizhou, Yunnan, Sichuan, Guangxi, Hubei, Guangdong, and Taiwan. Chishui and Xishui in Guizhou boast significant production. Traditionally, Guizhou is the origin and primary production area of ​​wild Dendrobium nobile. The primary source of Guizhou's Dendrobium nobile seeds comes from Chishui City, traditionally a renowned authentic production area.

[0003] Numerous factors influence the quality of Traditional Chinese Medicine (TCM), closely related to its variety, origin, ecological environment, growth cycle, and harvesting season. Different varieties, origins, soil conditions, harvesting times, transportation and storage, and processing methods all directly impact the quality and active ingredient content of TCM. TCM quality control is fundamental to ensuring the stability of its efficacy and safety. Its standardization, standardization, and modernization are crucial prerequisites for promoting the internationalization of TCM.

[0004] Currently, the quality analysis methods for Dendrobium officinale mostly use ultraviolet spectrophotometry (UV-Vis) and gas chromatography (GC), focusing on the determination of total alkaloids, total polysaccharides, or dendrobine content. For example, Lu Anjing et al. compared the alkaloid content within a population of Dendrobium nobile and found that age was the primary factor affecting dendrobium content in different branches within the same clump, with dendrobium content increasing in 1-year-old plants > 2-year-old plants > 3-year-old plants. Topography was also a secondary factor influencing dendrobium content, with higher dendrobium content in Dendrobium nobile plants grown at higher elevations for the same age. Wang Qin et al. studied the accumulation of secondary metabolites in Dendrobium nobile plants grown at different ages in wild biomimetic stone cultivation and found that with increasing age, levels of secondary metabolites such as flavonoids, phenolic acids, alkaloids, and terpenes increased significantly. Xu Delin et al. analyzed the differences in the content of Dendrobium nobile and Dendrobium officinale from different production areas. The content of dendrobium alkaloids in the dried stems of Dendrobium nobile was higher than 0.6%, while the content of dendrobium alkaloids in the dried stems of Dendrobium officinale was 0.09-0.28%, which was only 12%-46% of that of Dendrobium nobile; but the total polysaccharide content in the dried stems of Dendrobium officinale was 13.49-22.94%, which was significantly higher than the total polysaccharide content of 2.67-7.59% in the dried stems of Dendrobium nobile.

[0005] However, with the advancement of science and technology, bibenzyls and flavonoids have been gradually discovered in Dendrobium. Due to their promising antitumor activity, they have become a hot topic of research in recent years. With the continuous advancement of quality inspection technologies, new quality control methods for Dendrobium species have emerged. Wei Gang's research group at Guangzhou University of Chinese Medicine systematically analyzed and identified C-glycosylated flavonoids in Dendrobium nobile from different origins using liquid chromatography-mass spectrometry (LC-MS). Chemometric analysis revealed that foleyroside and isofleyroside are primarily present in Dendrobium nobile from Chishui, Guizhou, while absent in D. nobile from Yunnan. These two compounds could serve as quality control markers for D. nobile from Guizhou. Furthermore, the C-glycosylated flavonoids vitexin and isofleyroside can reverse TGF-β1-mediated epithelial-mesenchymal transition (EMT), suggesting they may be potential anticancer agents. New Zealand vitexin is a relatively common component in Dendrobium nobile, having been detected in samples from Guizhou, Yunnan, Guangxi, and Sichuan, and can be used as a quality control indicator. Dendrobin A, a typical active ingredient in Dendrobium, has been reported to significantly inhibit the proliferation, migration, invasion, and epithelial-mesenchymal transition of liver cancer cells by blocking cell cycle progression and inducing apoptosis, thereby mediating its anti-liver cancer effects. Its mechanism of action is related to the NF-κB signaling pathway.

[0006] However, the quality control system for Dendrobium plants still has the problem of single indicators, which cannot fully reflect the quality of medicinal materials and products. Taking the 2020 edition of the "Chinese Pharmacopoeia" as an example, the content determination item stipulates that the content of dendrobium (alkaloids) in Dendrobium nobile shall not be less than 0.40%, the content of eryngol (bibenzyl) in Dendrobium chrysotoxum shall not be less than 0.03%, the content of mannose in Dendrobium officinale shall be 13.0% to 38.0%, and the total polysaccharide content of Dendrobium officinale and Dendrobium Huoshanense shall not be less than 25% and 17%, respectively; Dendrobium Huoshanense shall use schaftoside (flavonoids) as a reference for the characteristic spectrum.

[0007] As we all know, Traditional Chinese Medicine (TCM) possesses the characteristics of multi-component synergy, multi-target effects, and overall regulation. Its active substances are the core components and foundation for its therapeutic effects. The multiple active ingredients contained in Dendrobium plants, such as alkaloids, polysaccharides, flavonoids, and bibenzyls, and their respective ratios, may directly influence their efficacy. Consequently, the current quantitative analysis model based on a single component is unable to fully reflect the overall quality of the medicinal material and the synergistic effects of the active ingredients, highlighting the limitations of the current quality control system. Therefore, it is necessary to break away from the traditional single-indicator evaluation model and promote a multi-indicator evaluation model to promote the scientific development of quality evaluation standards for Dendrobium medicinal materials. Summary of the Invention

[0008] In order to overcome the problems existing in the prior art, one of the purposes of the present invention is to provide a multi-index quality analysis method for Dendrobium nobile.

[0009] A second object of the present invention is to provide an application of the quality analysis method of the Dendrobium nobile.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] The invention discloses a multi-index quality analysis method for Dendrobium nobile, which uses one or more of the following substances as detection indicators to analyze the quality of Dendrobium nobile: lutein, dendrobin A, dendrobine, vitexin, isoflavone glycosides, total polysaccharides, total polyphenols, total flavonoids, total anthocyanins, and total alkaloids.

[0012] Furthermore, the multi-index quality analysis method includes:

[0013] (1) The contents of lutein, dendrobin A, vitexin, and isoflavone in Dendrobium nobile were determined by high performance liquid chromatography (HPLC);

[0014] (2) The content of dendrobine in Dendrobium nobile was determined by gas chromatography (GC);

[0015] (3) The contents of total polyphenols, total flavonoids, total anthocyanins, total polysaccharides and total alkaloids in Dendrobium nobile were determined by ultraviolet-visible (UV-Vis) spectrophotometry.

[0016] Furthermore, the (1) includes:

[0017] 1) Take the sample of Dendrobium nobile to be tested, add methanol, sonicate, centrifuge the extract, and filter the supernatant through a filter membrane to obtain the test solution;

[0018] 2) Perform HPLC on the test solution and determine the content of lutein and Dendrobin A using the standard curve method;

[0019] Chromatographic conditions were as follows: a Phenomenex Luna C18 column was used, the mobile phase was water (A)-acetonitrile (B), gradient elution, the elution program was shown in the table below, the flow rate was 1.0 mL / min, the column temperature was 30°C, and the detection wavelengths were 210 nm and 450 nm;

[0020]

[0021] 3) Take the sample of Dendrobium nobile to be tested, add ethanol, sonicate, centrifuge the extract, and filter the supernatant through a filter membrane to obtain the test solution;

[0022] 4) Performing high performance liquid chromatography on the test solution and determining the contents of vitexin and isoflavone by standard curve method;

[0023] Chromatographic conditions were as follows: a Phenomenex Luna C18 column was used, the mobile phase was water (A)-acetonitrile (B), gradient elution, the elution program was shown in the table below, the flow rate was 1.0 mL / min, the column temperature was 30°C, and the detection wavelength was 340 nm.

[0024]

[0025]

[0026] Furthermore, the Dendrobium nobile sample to be tested needs to be pretreated as follows:

[0027] The crushed drug powder was weighed and put into the extraction kettle, and the extraction conditions were set as follows: extraction temperature 65℃, extraction pressure 35MPa, carrier agent anhydrous ethanol, carrier agent ratio 1:3, and cooling mechanism was turned on. After the condenser temperature dropped to near the set temperature, the CO2 cylinder was turned on, and the extraction kettle was pressurized by the high-pressure pump. At the same time, the automatic control valve of the extraction kettle was opened, and the experimental pressure was set. When the experimental design pressure and temperature were reached, the CO2 gas source was turned off, the carrier agent pump was turned on to adjust the carrier agent injection speed, and the cyclic extraction was started. The extraction was stopped when the extraction time was reached. Among them, the pressure of the separation kettle was fixed at 5-6MPa, the temperature was 35℃, the extraction flow rate was 35L / h, the carrier agent addition rate was 10mL / min, and the extraction time was 120min. The extract of the separation kettle was collected, centrifuged at 4℃ and 10000r / min for 20min, the supernatant was filtered, and the filtrate was recovered by rotary evaporation to dryness.

[0028] Furthermore, the ratio of the Dendrobium nobile sample to be tested and methanol described in 1) is 0.5 g:20 mL.

[0029] Furthermore, the ratio of the Dendrobium nobile sample to be tested and ethanol described in 3) is 1 g:20 mL.

[0030] Furthermore, the ultrasonic conditions described in 1) and 3) are: 30 min, the centrifugal conditions are: 4500 r / min, 15 min, and the pore size of the filter membrane is 0.45 μm.

[0031] Furthermore, the reference substance used in the construction of the Dendrobin A standard curve is constructed by the following steps:

[0032] Take the supercritical CO2 extract of Dendrobium nobile stem, add an appropriate amount of ethyl acetate, and sonicate to completely dissolve it; add 80 mesh silica gel powder in a mass ratio of 1:1 and mix evenly, and recover the solvent in a rotary evaporator until the silica gel becomes mobile; use 200-300 mesh normal phase silica gel for column chromatography separation, use petroleum ether-ethyl acetate as the solvent system, and perform gradient elution in the ratio of 30:1, 15:1, 8:1, 4:1, 2:1, 1:1, and 0:1 by volume, each gradient elution is 5 column volumes, the elution solvent is detected by thin layer chromatography, and the solvents with the same spots are combined; take the fractions obtained by 15:1 combination, perform column chromatography separation on 200-300 mesh normal phase silica gel, perform isocratic elution with petroleum ether-ethyl acetate in a volume ratio of 6:1, detect by thin layer chromatography and combine the elution solvents with the same spots, recover the solvent and enter semi-preparative liquid chromatography, the mobile phase is acetonitrile: water = 35%:65%, the flow rate is 4 mL / min, t R = 20 min, recover the solvent, and obtain the reference substance Dendrobin A.

[0033] Furthermore, the (2) includes:

[0034] 1) Take a sample of Dendrobium nobile to be tested, add methanol, sonicate, centrifuge the extract, filter the supernatant through a filter membrane, take the filtrate and add an internal standard solution containing naphthalene, and then add methanol to obtain a test solution;

[0035] 2) The test solution was subjected to gas chromatography and the content of dendrobine was determined by the standard curve method;

[0036] The chromatographic conditions were as follows: inlet temperature of 250°C, split ratio of 30:1, carrier gas of N2, flow rate of 1.0 mL / min; a TR-1MS column was used, temperature program: initial temperature of 80°C, heating to 250°C at a rate of 10°C per minute, and holding for 16 min; FID detector temperature of 250°C, air: gas = 10:1.

[0037] Furthermore, the naphthalene concentration in the naphthalene-containing internal standard solution described in 1) is 0.1340 mg / mL.

[0038] Furthermore, the ratio of the Dendrobium nobile sample to be tested, the methanol added for the first time, the internal standard solution containing naphthalene, and the methanol added for the second time described in 1) is 0.5 g: 20 mL: 0.5 mL: 0.5 mL.

[0039] Furthermore, the ultrasonic conditions described in 1) are: 30 min, the centrifugal conditions are: 4500 r / min, 15 min, and the pore size of the filter membrane is 0.45 μm.

[0040] Furthermore, the (3) includes:

[0041] 1) Take a sample of Dendrobium nobile to be tested, add water / ethanol, sonicate, and filter the supernatant through a filter membrane to obtain a test solution; based on ultraviolet spectrophotometry and a standard curve method, the total polyphenol content in the test solution is determined by a folin-phenol colorimetric method; the total flavonoid content in the test solution is calculated as rutin, and the total flavonoid content in the test solution is determined by an aluminum salt colorimetric method; the total anthocyanin content in the test solution is determined by an iron salt catalytic colorimetric method;

[0042] 2) Take the Dendrobium nobile sample to be tested, add ethanol solution, sonicate, centrifuge and discard the supernatant, add appropriate amount of water to the precipitate, sonicate, and take the supernatant to obtain the test solution; based on ultraviolet spectrophotometry and standard curve method, use phenol-sulfuric acid method to determine the total polysaccharide content in the test solution;

[0043] 3) Taking a sample of Dendrobium nobile to be tested, adding chloroform to obtain a test solution; based on ultraviolet spectrophotometry and a standard curve method, the amount of total alkaloids contained is calculated as dendrobine, and the total alkaloid content in the test solution is determined by bromocresol green acid dye colorimetry.

[0044] Furthermore, the Dendrobium nobile sample to be tested described in 1) needs to be pretreated as follows:

[0045] Take 5 g of the medicinal material of Dendrobium nobile stem or flower crushed through an 80-mesh sieve, add water / ethanol according to a solid-liquid ratio of 1:30, reflux extraction, high-speed centrifugation, filter the supernatant, and freeze-dry the filtrate in a freeze dryer to obtain the water extract / alcohol extract of Dendrobium nobile stem and Dendrobium flower.

[0046] Furthermore, the ratio of the Dendrobium nobile sample to be tested and water / ethanol described in 1) is 10 mg:5 mL.

[0047] Furthermore, the ultrasonic conditions described in 1) are: 30 min, and the pore size of the filter membrane is 0.45 μm.

[0048] Furthermore, the ethanol solution described in 2) is an 80% ethanol solution, and the ratio of the Dendrobium nobile sample to be tested to the ethanol solution is a mass-to-volume ratio of 1:8.

[0049] Furthermore, in 2), the conditions for the first ultrasonication are: 20 min, the conditions for the centrifugation are: 4500 r / min, 15 min, the pore size of the filter membrane is 0.45 μm, and the conditions for the second ultrasonication are: 15 min.

[0050] Furthermore, the Dendrobium nobile sample to be tested described in 3) needs to be pretreated as follows:

[0051] The crushed drug powder was weighed and put into the extraction kettle, and the extraction conditions were set as follows: extraction temperature 65℃, extraction pressure 35MPa, carrier agent anhydrous ethanol, carrier agent ratio 1:3, and cooling mechanism was turned on. After the condenser temperature dropped to near the set temperature, the CO2 cylinder was turned on, and the extraction kettle was pressurized by the high-pressure pump. At the same time, the automatic control valve of the extraction kettle was opened, and the experimental pressure was set. When the experimental design pressure and temperature were reached, the CO2 gas source was turned off, the carrier agent pump was turned on to adjust the carrier agent injection speed, and the cyclic extraction was started. The extraction was stopped when the extraction time was reached. Among them, the pressure of the separation kettle was fixed at 5-6MPa, the temperature was 35℃, the extraction flow rate was 35L / h, the carrier agent addition rate was 10mL / min, and the extraction time was 120min. The extract of the separation kettle was collected, centrifuged at 4℃ and 10000r / min for 20min, the supernatant was filtered, and the filtrate was recovered by rotary evaporation to dryness.

[0052] Furthermore, the amount of chloroform added in 3) is calculated based on the concentration of the sample to be tested being 8 mg / mL.

[0053] Furthermore, the Dendrobium nobile sample to be tested is a stem or flower of Dendrobium nobile.

[0054] The application of the above-mentioned multi-index quality analysis method of Dendrobium nobile in the quality control of Dendrobium nobile medicinal materials.

[0055] The present invention has the following advantages and effects compared to the prior art:

[0056] The present invention is based on the previous research on Dendrobium and prepares Dendrobin A reference substance for quality control. In view of the current situation that the quality control standard of Dendrobium nobile is single, ultraviolet spectrophotometry (UV-Vis), gas chromatography (GC) and high performance liquid chromatography (HPLC) are combined to establish an overall evaluation system for the stems, flowers and extracts of Dendrobium nobile. The Dendrobium nobile is evaluated overall based on five major components (total polysaccharides, total polyphenols, total flavonoids, total anthocyanins, total alkaloids) and five index components (dendrobin, Dendrobin A, lutein, New Zealand vitexin, isoflavone glycosides). For the first time, the contents of four index components, Dendrobin A, lutein, New Zealand vitexin and isoflavone glycosides, in the stems of Dendrobium nobile are determined simultaneously. The results show that the contents of the four components in the stems of Dendrobium nobile are Dendrobin A, Lutein, New Zealand vitexin and isoflavone glycosides, respectively. A0.13%, lutein 0.23%, New Zealand vitexin 0.02% and isoflavone glycoside 0.13%; while the above four components were not detected in Dendrobium nobile flowers.

[0057] The quality standard of the present invention is the first to carry out systematic and comprehensive quality control on Dendrobium nobile. It not only has important guiding significance for establishing the quality standard of Dendrobium nobile medicinal materials in Chishui, Guizhou, but also plays an important guiding role in the research of Dendrobium nobile extraction technology and the development and quality control of related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is the structural formula of Dendrobin A.

[0059] Figure 2 The chromatograms of the test and reference solutions at different wavelengths are shown in Figure 2. (A) (B) are the chromatograms of the reference Dendrobin A and the test solution at 210 nm, and (C) (D) are the chromatograms of the reference lutein and the test solution at 450 nm.

[0060] Figure 3 Standard curves of Dendrobin A (left) and lutein (right).

[0061] Figure 4 Chromatograms of the mixed reference substance (top) and test substance (bottom); 1. New Zealand vitexin; 2. Isophora glycoside.

[0062] Figure 5 This is the standard curve of isoflavone glycoside (left) and New Zealand vitexin (right).

[0063] Figure 6 GC chromatograms of the reference substance (top) and the test substance (bottom) (naphthalene is the internal standard).

[0064] Figure 7 A standard curve was established for GC chromatography detection of dendrobium alkaloids.

[0065] Figure 8 This is the standard curve of gallic acid.

[0066] Figure 9 This is the standard curve of rutin.

[0067] Figure 10 This is the standard curve of proanthocyanidins.

[0068] Figure 11 This is the standard curve of glucose.

[0069] Figure 12 This is the standard curve of dendrobium alkaloids. DETAILED DESCRIPTION

[0070] The following will clearly and completely describe the technical solutions of the specific embodiments of the present invention in combination with various specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, can be obtained through ordinary commercial channels.

[0071] The present invention uses high performance liquid chromatography (HPLC) to establish a quantitative analytical method for Dendrobin A, lutein, vitexin, and isoflavone in Dendrobium nobile. Gas chromatography (GC) is used to optimize the method for determining the content of dendrobinine in Dendrobium nobile. Ultraviolet-visible (UV-Vis) spectrophotometry is used to establish a method for determining the content of major components such as total polyphenols, total flavonoids, total anthocyanins, total polysaccharides, and total alkaloids in the stems and flowers of Dendrobium nobile. The established HPLC, GC, and UV-Vis spectrophotometry methods are used to qualitatively and quantitatively analyze the index components of the stems and flowers of Dendrobium nobile.

[0072] The instruments and equipment used in the following examples are:

[0073] High-performance liquid chromatograph (SHIMHDZU LC-20A, Shimadzu Instrument Co., Ltd., Japan); gas chromatograph (7890B, Agilent Technologies, Inc.); UV-visible spectrophotometer (UV-2600, Shimadzu Corporation, Japan); electronic analytical balance (ME204, Mettler-Toledo Instrument Co., Ltd.); electronic analytical balance (BSA224S-CW, Beijing Sartorius Balance Co., Ltd.); ultrasonic cleaner (JP-060S, Guangdong Jiemeng Ultrasonic Industrial Co., Ltd.); ultrasonic cleaner (PS-60A, Shenzhen Jiekang Intelligent Technology Co., Ltd.); rotary evaporator (XD2000A, Shanghai Xiande Experimental Instrument Co., Ltd.); desktop low-speed automatic balancing centrifuge (L400, Xiangyi Centrifuge Instrument Co., Ltd.); vacuum freeze dryer (SCIENTZ-10N, Ningbo Xinzhi Biotechnology Co., Ltd.); and drum juice press (RH311, Midea Group Co., Ltd.).

[0074] Materials and reagents used in the following examples:

[0075] The stems and flowers of Dendrobium nobile were provided by Chishui Zhilu Dendrobium nobile Ecological Park Development Co., Ltd. (the method of the present invention can be widely applied to the authentic production areas of Dendrobium nobile in Guizhou). Dendrobin A (≥96%) was prepared in the laboratory; lutein (97.6%, Lot No. 24070812) was purchased from Tanmo Quality Inspection Technology Co., Ltd.; dendrobin (≥98%, Lot No. M02IB208625), vitexin (≥98%, Lot No. D09GB170690), isophrynoside (≥95%, Lot No. A11HB190322), rutin (purity: ≥97%, Lot No. H24N9Z75966), and folin phenol reagent (Lot No. J24GS142729) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; proanthocyanidins, naphthalene (Lot No. C15008371), gallic acid (purity: ≥99%, Lot No. C11987274), aluminum nitrate (Lot No. No. C10094520) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; sodium nitrite (Lot No. L1904014) was purchased from Aladdin China Technology Co., Ltd.; sodium hydroxide (Lot No. G2031120) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; acetonitrile and methanol were both chromatographically grade and purchased from BCR Reagents, Inc.; methanol was chromatographically grade and purchased from Merck Chemical Technology Shanghai Co., Ltd.; chloroform, bromocresol green, sulfuric acid, and phenol were all analytically grade and purchased from Guangdong Guangshi Reagent Technology Co., Ltd.; anhydrous sodium carbonate was analytically grade and purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.; anhydrous ethanol was analytically grade and purchased from Shanghai Zhanyun Chemical Co., Ltd.; and distilled water was purchased from China Resources C'estbon Beverages China Co., Ltd. All other reagents were analytically grade.

[0076] Example 1: Method for determining the content of multiple components of Dendrobium nobile based on high performance liquid chromatography (HPLC)

[0077] 1.1 Establishment of HPLC-based method for determination of Dendrobin A and lutein

[0078] 1.1.1 Preparation of Dendrobin A

[0079] Weigh 80.0 g of crushed Dendrobium nobile stem powder and put it into the extraction kettle. Set the extraction conditions: extraction temperature 65℃, extraction pressure 35 MPa, carrier is anhydrous ethanol, carrier ratio 1:3, turn on the refrigeration machine for cooling, and turn on the CO2 gas cylinder after the condenser temperature drops to near the set temperature. Pressurize the extraction kettle through the high-pressure pump, and at the same time, open the automatic control valve of the extraction kettle, and set the experimental pressure. When the experimental design pressure and temperature are reached, turn off the CO2 gas source, turn on the carrier pump to adjust the carrier injection speed, and enter the circulation extraction. Stop the extraction when the extraction time is reached. To ensure the normal circulation state and the extract is collected from the collection kettle as much as possible, the pressure of the separation kettle is fixed at 5-6 MPa, the temperature is 35℃, the extraction flow rate is 35 L / h, the carrier addition rate is 10 mL / min, and the extraction time is 120 min. The extract in the separation kettle was collected, centrifuged at 4°C and 10,000 r / min for 20 min, the supernatant was filtered, and the filtrate was subjected to a rotary evaporator to recover the solvent until dryness to obtain a supercritical CO2 extract of the stems and flowers of Dendrobium nobile.

[0080] Take the supercritical CO2 extract of the stem of Dendrobium nobile, add an appropriate amount of ethyl acetate, and sonicate until completely dissolved. Add 80-mesh silica gel powder in a 1:1 mass ratio and mix thoroughly. Recover the solvent on a rotary evaporator until the silica gel becomes fluid. Column chromatography separation is performed using normal-phase silica gel (200-300 mesh). Using petroleum ether and ethyl acetate as the solvent system, gradient elution is performed in the following ratios: 30:1, 15:1, 8:1, 4:1, 2:1, 1:1, and 0:1 by volume, with each gradient elution lasting five column volumes. Elution solvents are detected by thin-layer chromatography (TLC), and solvents with identical spots are combined. The fractions obtained by combining the ratio of 15:1 were separated by column chromatography on normal phase silica gel (200-300 mesh) and eluted isocratically with petroleum ether-ethyl acetate (volume ratio 6:1). The elution solvents with the same spots were detected by TLC and combined. After the solvent was recovered, the mixture was subjected to semi-preparative liquid chromatography with a mobile phase of acetonitrile: water = 35%:65% at a flow rate of 4 mL / min. R = 20min, and the solvent was recovered. The compound was analyzed by nuclear magnetic resonance 1 H NMR, 13 C NMR and mass spectrometry identification showed that it was Dendrobin A (Dendrobin A, 4,5-dihydroxy-3,3′-dimetheoxybibenzyl), with a molecular formula of C 16 H 18 O4, purity PDA ≥ 95%, structural formula Figure 1 shown.

[0081] 1.1.2 Preparation of reference solution

[0082] Accurately weigh appropriate amounts of reference substances Dendrobin A and lutein, place them in a 10 mL volumetric flask, add acetonitrile to completely dissolve them, and then dilute to 10 mL to prepare reference substance solutions with concentrations of 0.0979 mg / mL for Dendrobin A and 0.0134 mg / mL for lutein, respectively.

[0083] 1.1.3 Preparation of test solution

[0084] Take 0.5 g of Dendrobium nobile stems or flowers, add 20 mL of methanol and ultrasonicate for 30 min. Centrifuge the extract at 4500 r / min for 15 min, and filter the supernatant through a 0.45 μm filter membrane to obtain the product.

[0085] 1.1.4HPLC chromatographic conditions

[0086] A Phenomenex Luna C18 column (250 mm × 4.6 mm, 5 μm) was used with a mobile phase of water (A)-acetonitrile (B) in a gradient elution process. The elution program is shown in Table 1. The flow rate was 1.0 mL / min, the column temperature was 30°C, and the detection wavelengths were 210 nm and 450 nm. The chromatograms of the reference and test samples at different wavelengths are shown in Figure 1. Figure 2 shown.

[0087] Table 1 HPLC mobile phase elution program

[0088]

[0089] 1.1.5 Drawing of the standard curve

[0090] Inject different volumes of reference solution into the liquid chromatograph, record the peak area, and calculate the standard curve. Figure 3 As shown in the figure, Dendrobin A and lutein have good linear relationships in the range of 0.1957-1.1742 μg and 0.0268-0.3350 μg, respectively. The standard curve equations are y=8×10 6 x-212563(R 2 =0.9998) and y = 3 × 10 6 x-18876(R 2 =0.9994).

[0091] 1.2 Establishment of an HPLC-based method for the determination of vitexin and isoflavone

[0092] 1.2.1 Preparation of reference solution

[0093] Accurately weigh appropriate amounts of New Zealand vitexin and isoflavone glycoside reference substances, place them in a 10 mL volumetric flask, add methanol to completely dissolve them, and then dilute the volume to 10 mL to prepare reference substance solutions with New Zealand vitexin and isoflavone glycoside concentrations of 0.0616 mg / mL and 0.0088 mg / mL, respectively.

[0094] 1.2.2 Preparation of test solution

[0095] Take an appropriate amount of Dendrobium nobile stems or flowers, add ethanol at a mass-to-volume ratio of 1:20, ultrasonicate for 30 minutes, centrifuge the extract at 4500 r / min for 15 minutes, and filter the supernatant through a 0.45 μm filter membrane to obtain the extract.

[0096] 1.2.3 HPLC chromatographic conditions

[0097] A Phenomenex Luna C18 column (250 mm × 4.6 mm, 5 μm) was used with a mobile phase of water (A)-acetonitrile (B) in a gradient elution process. The elution program is shown in Table 2. The flow rate was 1.0 mL / min, the column temperature was 30°C, and the detection wavelength was 340 nm. Figure 4 shown.

[0098] Table 2 HPLC mobile phase elution program

[0099]

[0100] 1.2.4 Drawing of the standard curve

[0101] Inject different volumes of reference solution into the liquid chromatograph, record the peak area, and calculate the standard curve. Figure 5 As shown in the figure, New Zealand vitexin and isoflavone glycoside have good linear relationships in the range of 0.0176-0.1056 μg and 0.1232-0.7392 μg, respectively. The equations of their standard curves are y=2×10 6 x-1831.3(R 2 =0.9997) and y = 2 × 10 6 x-962.13(R 2 =0.9999).

[0102] 1.3. Determination of dendrobine content in Dendrobium nobile based on gas chromatography (GC)

[0103] 1.3.1 Preparation of reference solution

[0104] Accurately weigh appropriate amounts of the reference substance dendrobium daptomycin and the internal standard naphthalene, add methanol to completely dissolve them, and then prepare a reference solution containing the internal standard with a naphthalene concentration of 0.1340 mg / mL and a dendrobium daptomycin concentration of 0.6550 mg / mL. Filter through a 0.45 μm filter membrane to obtain the solution.

[0105] 1.3.2 Preparation of test solution

[0106] Take 0.5 g of Dendrobium nobile stems or flowers, add 20 mL of methanol, sonicate for 30 minutes, centrifuge the extract at 4500 rpm for 15 minutes, and filter the supernatant through a 0.45 μm filter. Take 0.5 mL of the filtrate, add 0.5 mL of an internal standard solution containing 0.1340 mg / mL naphthalene, and then add 0.5 mL of methanol to obtain the product.

[0107] 1.3.3GC Chromatographic Conditions

[0108] With reference to the method for determining the content of Dendrobium nobile under Dendrobium in the 2020 edition of the Chinese Pharmacopoeia, with slight adjustments, the GC chromatographic conditions are as follows: inlet temperature of 250°C, split ratio of 30:1, carrier gas of N2, flow rate of 1.0 mL / min; TR-1MS column (30 m × 250 μm × 0.25 μm) is used, programmed temperature: initial temperature of 80°C, temperature increased to 250°C at a rate of 10°C per minute, and maintained for 16 min; inlet temperature of 250°C, FID detector temperature of 250°C, air: gas = 10:1, and the sample spectra of the reference and test products are as follows: Figure 6 shown.

[0109] 1.3.4 Drawing of the standard curve

[0110] Prepare different concentrations of dendrobium alkaloid reference solution containing internal standard, inject into gas chromatograph, record its peak area. Fit the standard curve with the dendrobium alkaloid / naphthalene mass ratio as the horizontal axis and the dendrobium alkaloid / naphthalene peak area ratio as the vertical axis. Figure 7 As shown in Figure 2, the linear relationship of dendrobium alkaloids was good in the range of 32.75 to 196.50 μg, and the standard curve equation was y = 0.7171x - 0.0719 (R 2 =0.9997).

[0111] 1.4. Determination of major components in Dendrobium nobile based on UV-Vis spectrophotometry

[0112] 1.4.1 Establishment of the total polyphenol determination method

[0113] 1.4.1.1 Preparation of reference solution

[0114] Accurately weigh an appropriate amount of gallic acid reference substance, add water to prepare a stock solution containing 0.201 mg of gallic acid per 1 mL, and dilute it into a series of reference substance solutions with a concentration of 0.02-0.20 mg / mL.

[0115] 1.4.1.2 Preparation of test solution

[0116] Take 5 g of the medicinal material of Dendrobium nobile stem or flower crushed through an 80-mesh sieve, add water / ethanol according to a solid-liquid ratio of 1:30, reflux extraction for 2 hours, high-speed centrifugation, filter the supernatant, and freeze-dry the filtrate in a freeze dryer to obtain the water extract / alcohol extract of Dendrobium nobile stem and Dendrobium flower.

[0117] Weigh 10 mg of the water extract / alcohol extract of the above-mentioned Dendrobium nobile stem and Dendrobium flower, add 5 mL of water / ethanol accordingly, sonicate for 30 min, and filter through a 0.45 μm microporous membrane to obtain the test solution.

[0118] 1.4.1.3 Total polyphenols determination method

[0119] Accurately pipette 0.5 mL of each reference solution and test solution into 10 mL volumetric flasks, add 2.5 mL of 10-fold diluted Folin phenol reagent, react for 5 minutes, then add 2 mL of 10% Na2CO3 solution, add water to the scale, mix thoroughly, react at room temperature in the dark for 1 hour, and measure the absorbance at a wavelength of 760 nm. Draw a standard curve with absorbance as the ordinate and concentration as the abscissa, as shown in the following example: Figure 8 As shown. The linear regression equation y=1.5851x+0.0424, R 2 =0.999 (n=3), with good linearity in the range of 0-0.5 mg / mL. Gallic acid was calculated from the standard curve and regression equation based on the absorbance of the test solution. The total polyphenol content of the Dendrobium nobile sample was calculated as gallic acid based on the dry product.

[0120] 1.4.2 Establishment of the total flavonoids determination method

[0121] 1.4.2.1 Preparation of reference solution

[0122] Accurately weigh an appropriate amount of rutin reference substance, add water to prepare a stock solution containing 5.54 mg of rutin per 1 mL, and dilute it into a series of reference substance solutions with a concentration of 0.11-0.77 mg / mL.

[0123] 1.4.2.2 Preparation of test solution

[0124] Take 5 g of the medicinal material of Dendrobium nobile stem or flower crushed through an 80-mesh sieve, add water / ethanol according to a solid-liquid ratio of 1:30, reflux extraction for 2 hours, high-speed centrifugation, filter the supernatant, and freeze-dry the filtrate in a freeze dryer to obtain the water extract / alcohol extract of Dendrobium nobile stem and Dendrobium flower.

[0125] Weigh 10 mg of the water extract / alcohol extract of the above-mentioned Dendrobium nobile stem and Dendrobium flower, add 5 mL of water / ethanol accordingly, sonicate for 30 min, and filter the supernatant through a 0.45 μm microporous membrane to obtain the test solution.

[0126] 1.4.2.3 Determination of total flavonoids

[0127] Accurately pipette 1 mL each of the reference solution and the test solution into 10 mL volumetric flasks, add 0.3 mL of 5% sodium nitrite solution, shake well, and let it stand for 6 minutes; then add 0.3 mL of 10% aluminum nitrate solution, shake well, and let it stand for 6 minutes; finally, add 4 mL of 4% sodium hydroxide solution, dilute to the mark with water, and let it stand for 15 minutes. Measure the absorbance at a wavelength of 499 nm. Use absorbance as the ordinate and concentration as the abscissa to draw a standard curve, as shown in the following example: Figure 9 As shown. The linear regression equation y=1.2646x-0.0054, R 2 =0.998 (n=3), with good linearity in the range of 0.11-0.77 mg / mL. The amount of rutin was calculated from the standard curve and regression equation based on the absorbance of the test solution. The total flavonoid content of the Dendrobium nobile sample was calculated as rutin based on the dry product.

[0128] 1.4.3 Establishment of the total anthocyanin determination method

[0129] 1.4.3.1 Preparation of reference solution

[0130] Accurately weigh an appropriate amount of proanthocyanidin reference substance and use methanol to accurately prepare a series of working solutions with mass concentrations of 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, and 0.25 mg / mL.

[0131] 1.4.3.2 Preparation of test solution

[0132] Take 5 g of the medicinal material of Dendrobium nobile stem or flower crushed through an 80-mesh sieve, add water / ethanol according to a solid-liquid ratio of 1:30, reflux extraction for 2 hours, high-speed centrifugation, filter the supernatant, and freeze-dry the filtrate in a freeze dryer to obtain the water extract / alcohol extract of Dendrobium nobile stem and Dendrobium flower.

[0133] Weigh 10 mg of the water extract / alcohol extract of the above-mentioned Dendrobium nobile stem and Dendrobium flower, add 5 mL of water / ethanol accordingly, sonicate for 30 min, and filter the supernatant through a 0.45 μm microporous membrane to obtain the test solution.

[0134] 1.4.3.3 Establishment of the total anthocyanin determination method

[0135] Using the iron salt catalysis method, 1.0 mL of reference solution and test solution of different concentrations were taken respectively and placed in a 10 mL cuvette with a stopper. Then, 6.0 mL of n-butanol-hydrochloric acid solution (95:5) and 0.2 mL of ammonium ferric sulfate solution (prepared immediately) were added in sequence. After mixing, the mixture was heated in a boiling water bath for 40 minutes. After rapid cooling, the maximum absorption wavelength was scanned in the range of 200-800 nm of ultraviolet-visible light, and the absorbance of the total anthocyanin standard solution was measured at the maximum absorption wavelength. With absorbance as the ordinate and concentration as the abscissa, a standard curve was drawn, as shown below. Figure 10 As shown. The linear regression equation y=2.7032x+0.002, R 2 =0.9987 (n=3), with good linearity in the range of 0.11-0.26 mg / mL. The amount of proanthocyanidins was calculated from the standard curve and regression equation based on the absorbance of the test solution. The total anthocyanidin content of the Dendrobium nobile sample was calculated as proanthocyanidins based on the dry product.

[0136] 1.4.4 Establishment of total polysaccharide determination method

[0137] 1.4.4.1 Preparation of reference solution

[0138] Accurately weigh 10 mg of D-anhydrous glucose and place it in a 100 mL volumetric flask. Dissolve it in water and dilute to the mark to prepare a 0.11 mg / mL D-anhydrous glucose reference stock solution. Accurately measure 1 mL, 2 mL, 4 mL, 6 mL, 8 mL, and 10 mL of the D-anhydrous glucose reference stock solution and place them in 10 mL stoppered test tubes, dilute to the mark with water, and mix thoroughly.

[0139] 1.4.4.2 Preparation of test solution

[0140] Weigh a certain mass of Dendrobium nobile stem water extract, add 80% ethanol solution according to the mass-to-volume ratio of 1:8, ultrasonicate for 20 minutes, centrifuge the above solution at 4500r / min for 15 minutes, discard the supernatant, add appropriate amount of water to the precipitate, ultrasonicate for 15 minutes, and take the supernatant as the test solution.

[0141] 1.4.4.3 Total polysaccharide determination method

[0142] The detection method of total polysaccharides refers to the phenol-sulfuric acid method adopted in the Chinese Pharmacopoeia. Take 1.0 mL of each of the reference solution and the test solution, place them in 10 mL stoppered test tubes, accurately add 1 mL of 5% phenol aqueous solution (prepared before use), shake well, then accurately add 5 mL of concentrated sulfuric acid, shake well, heat in a boiling water bath for 20 minutes, take out and cool in an ice bath for 5 minutes. Use the corresponding reagent as a blank control and measure the absorbance at a wavelength of 488 nm. Use absorbance as the vertical axis and concentration as the horizontal axis to draw a standard curve, such as Figure 11 As shown. The linear regression equation y=9.5428x+0.1477, R 2 = 0.9978 (n = 3), with good linearity in the range of 0.01 to 0.11 mg / mL. The amount of glucose was calculated from the standard curve and regression equation based on the absorbance of the test solution. The total polysaccharide content of the Dendrobium nobile sample was calculated as glucose based on the dry product.

[0143] 1.4.5 Establishment of the total alkaloid determination method

[0144] 1.4.5.1 Preparation of reference solution

[0145] Accurately measure an appropriate amount of the reference substance dendrobium daptomycin, add chloroform to prepare a stock solution with a concentration of 83.60 μg / mL, and then dilute it with chloroform to a series of working solutions with concentrations of 37.62, 29.26, 20.90, 16.70, 12.54, and 8.36 μg / mL.

[0146] 1.4.5.2 Preparation of test solution

[0147] Weigh a certain amount of supercritical extract of Dendrobium stem and add chloroform to prepare a test solution with a concentration of 8 mg / mL.

[0148] 1.4.5.3 Total alkaloid determination method

[0149] Take 2.0mL of working solution or test solution, place it in a 10mL stoppered test tube, and add 0.2mL of pH 3 citric acid-sodium citrate buffer solution and 0.3mL of bromocresol green acid dye solution in sequence, shake vigorously for 3min, and let it stand for 30min. Discard the supernatant, take the lower organic layer, use chloroform as a blank control, and measure the absorbance value at a wavelength of 416nm. Use absorbance as the vertical axis and the mass of dendrobium alkaloids as the horizontal axis to draw a standard curve, as shown in Figure 2. Figure 12 As shown. The linear regression equation is y = 0.0406x-0.1491, R 2=0.9942 (n=3), with good linearity in the range of 8.36-37.62 μg / mL. The amount of dendrobine was calculated from the standard curve and regression equation based on the absorbance of the test solution. The total alkaloid content of the Dendrobium nobile sample was calculated as dendrobine based on the dry product.

[0150] Example 2: Results of multi-index content determination of Dendrobium nobile stems and flowers

[0151] The multi-index content determination method based on HPLC and GC established in Example 1 was used to determine the contents of the five index components in the stems and flowers of Dendrobium nobile. The determination results are shown in Table 3.

[0152] Table 3 Determination of medicinal material content of Dendrobium nobile stems and flowers

[0153]

[0154]

[0155] Note: Each sample was tested three times and the average value was taken. "-" means the sample was below the detection limit and not detected.

[0156] The content of dendrobine in the stem of Dendrobium nobile was 0.56%, exceeding the 0.40% limit stipulated in the Chinese Pharmacopoeia. Dendrobin A, lutein, vitexin, and isophora indigofera were found at 0.13%, 0.02%, 0.02%, and 0.13%, respectively. This is the first time that the contents of these four components have been simultaneously determined in Dendrobium nobile.

[0157] The dendrobinoid content in Dendrobium nobile flowers was determined to be 1.81%, significantly higher than the 0.4% specified in the "Local Food Safety Standard for Dendrobium nobile Flowers (Dried Products)" (DBS52 / 049-2021), as well as the average content of 1.35% in the "Local Food Safety Standard for Dendrobium nobile Flowers (Compilation Instructions)" and close to the maximum value of 1.94%. This study is the first to simultaneously determine the contents of four components in Dendrobium nobile flowers: dendrobin A, lutein, vitexin, and isoflavone. However, none of these components were detected in the flowers.

[0158] Example 3: Methodological Investigation of Multi-Indicator Quality Analysis Method for Dendrobium nobile

[0159] 1. Investigation of DA and Lutein HPLC Methodology

[0160] (1) Repeatability study

[0161] Referring to Example 1, six replicates of the test solution were prepared. The samples were analyzed using the chromatographic method. The standard curve was substituted and the contents and relative standard deviations (RSDs) were calculated. The results showed that the RSDs for DA and lutein content in all six test solutions were less than 5%, demonstrating good reproducibility.

[0162] (2) Precision inspection

[0163] Referring to Example 1, a test solution was prepared and injected six times. Chromatographic analysis was performed, and the relative standard deviation (RSD) was calculated based on the peak area. The results showed that the RSDs for the peak areas of DA and lutein for the six repeated injections were all less than 5%, indicating good precision of the method.

[0164] (3) Stability investigation

[0165] Referring to Example 1, the chromatographic method was used to detect the lutein content at 0, 4, 8, 12, 16, 20, and 24 hours, and the content and relative standard deviation (RSD) were calculated by substituting the standard curve into the test solution. The results showed that the RSDs of DA and lutein content in the test solution from 0 to 24 hours were less than 5%, indicating that the test solution had good stability within 24 hours.

[0166] (4) Sample recovery rate investigation

[0167] Referring to Example 1, reference and test solutions were prepared, and the mass of the target component in the test solution was measured. An equal volume of reference solution was added to the test solution, mixed thoroughly, and the mass of the target component was analyzed. The results showed that the average recovery rates of the target components were less than 5% RSD, indicating that the analytical method is accurate.

[0168] 2. Investigation of HPLC Methodology of Vitexin and Isophoroside in New Zealand

[0169] (1) Repeatability study

[0170] Referring to Example 1, six replicates of the test solution were prepared. The samples were analyzed using the chromatographic method. The standard curve was substituted and the contents and relative standard deviations (RSDs) were calculated. The results showed that the RSDs for DA and lutein content in all six test solutions were less than 5%, demonstrating good reproducibility.

[0171] (2) Precision inspection

[0172] Referring to Example 1, a test solution was prepared and injected six times. The solution was detected by chromatography, and the relative standard deviation (RSD) was calculated based on the peak area. The results showed that the peak area RSD values ​​of New Zealand peony glycoside and isoflavone glycoside for six repeated injections were all less than 5%, indicating good precision of the method.

[0173] (3) Stability investigation

[0174] Referring to Example 1, the test was performed at 0, 4, 8, 12, 16, 20, and 24 hours, respectively, and the content and relative standard deviation (RSD) were calculated by substituting the standard curve. The results showed that the RSDs of the New Zealand peony glycoside and isophora glycoside contents in the test solution within 0 to 24 hours were less than 5%, indicating that the test sample had good stability within 24 hours.

[0175] (4) Sample recovery rate investigation

[0176] Referring to Example 1, reference and test solutions were prepared, and the mass of the target component in the test solution was measured. An equal volume of reference solution was added to the test solution, mixed thoroughly, and the mass of the target component was analyzed. The results showed that the average recovery rates of the target components were less than 5% RSD, indicating that the analytical method is accurate.

[0177] 3. Investigation of GC Methodology of Dendrobium

[0178] (1) Repeatability study

[0179] With reference to Example 1, a test solution was prepared, and 6 portions were prepared in parallel. The test was performed according to the chromatographic method, and the standard curve was substituted to calculate the content and relative standard deviation (RSD). The results showed that the RSD values ​​of the dendrobine content in the 6 test solutions were all less than 5%, indicating that the method had good reproducibility.

[0180] (2) Precision inspection

[0181] With reference to Example 1, preparation need testing solution, repeated sample introduction 6 times, detected by chromatographic method, with peak area calculation of dendrobium dodecanoides peak area / naphthalene peak area calculation its relative standard deviation (RSD). Result show, the peak area RSD value of dendrobium dodecanoides peak area / naphthalene peak area of ​​6 repeated sample introductions is all less than 5%, illustrate that method precision is good.

[0182] (3) Stability investigation

[0183] With reference to Example 1, the test was performed by chromatography at 0, 4, 8, 12, 16, 20, and 24 hours, and the standard curve was substituted to calculate the content and relative standard deviation (RSD). The results showed that the RSD of the dendrobine content of the test solution within 0 to 24 hours was less than 5%, indicating that the test sample had good stability within 24 hours.

[0184] (4) Sample recovery rate investigation

[0185] Referring to Example 1, reference and test solutions were prepared, and the mass of the target component in the test solution was measured. An equal volume of reference solution was added to the test solution, mixed thoroughly, and the mass of the target component was analyzed. The results showed that the average recovery rates of the target components were less than 5% RSD, indicating that the analytical method is accurate.

[0186] Comparative Example 1: Optimization of pretreatment process of Dendrobium nobile stems

[0187] Ethyl acetate reflux extraction of Dendrobium nobile stem: Weigh 5.00 g of Dendrobium nobile powder and place it in a round-bottom flask, add 50 times the amount of ethyl acetate, mix well, and place it in an 85°C water bath for reflux extraction for 2 hours. After the extraction is completed, cool it, centrifuge it at 4°C and 10,000 r / min for 20 minutes, take the supernatant and filter it. The filtrate is passed through a rotary evaporator to recover the solvent and dry it to obtain the ethyl acetate extract of Dendrobium nobile stem.

[0188] The content determination results are shown in Table 4. The contents of Dendrobin A, lutein, dendrobine and total alkaloids in the supercritical CO2 extract were significantly higher than those in the ethyl acetate extract, and the contents were increased by more than 1 times. The supercritical CO2 extraction process can effectively target and enrich the active ingredients in the stems of Dendrobium nobile and improve the sensitivity of the analysis.

[0189] Table 4 Comparative results of content determination of extracts from Dendrobium nobile stems using different processes

[0190]

[0191] Comparative Example 2: Optimization of HPLC analysis parameters for Dendrobin A and lutein

[0192] (1) Influence of column temperature

[0193] Referring to Example 1, other chromatographic conditions remained unchanged, and the effects of different column temperatures (30°C, 35°C, and 40°C) on the separation of the same test solution were investigated. The results showed that as the column temperature increased, the retention time of each component shortened. At 40°C, the target component peaks were not separated, while the separation effects of the peaks at 30 and 35°C were similar.

[0194] (2) Influence of flow rate

[0195] With reference to Example 1, other chromatographic conditions remained unchanged, and the effects of different flow rates (0.6 mL / min, 0.8 mL / min, and 1.0 mL / min) on the separation of the same test solution were investigated. The results showed that with increasing flow rate, the retention time of each component shortened. At 0.6 mL / min and 0.8 mL / min, the target component peaks were not separated and had longer retention times.

[0196] Comparative Example 3: Optimization of HPLC analysis parameters for New Zealand peony glycosides and isoflavone glycosides

[0197] (1) Influence of column temperature

[0198] Referring to Example 1, other chromatographic conditions remained unchanged, and the effects of different column temperatures (30°C, 35°C, and 40°C) on the separation of the same test solution were investigated. The results showed that as the column temperature increased, the retention time of each component shortened, and the peak shapes of the target components were more symmetrical.

[0199] (2) Influence of flow rate

[0200] With reference to Example 1, other chromatographic conditions remained unchanged, and the effects of different flow rates (0.6 mL / min, 0.8 mL / min, and 1.0 mL / min) on the separation of the same test solution were investigated. The results showed that as the flow rate increased, the retention time of each component shortened, and the separation of the target components was better at different flow rates.

[0201] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A multi-index quality analysis method for Dendrobium nobile, characterized in that: The following substances were used as detection indicators for the quality analysis of Dendrobium nobile: lutein, Dendrobin A, dendrobine, vitexin, isoflavone glycosides, total polysaccharides, total polyphenols, total flavonoids, total anthocyanins, and total alkaloids.

2. The multi-index quality analysis method for Dendrobium nobile according to claim 1, wherein: The multi-index quality analysis method includes: (1) The contents of lutein, dendrobin A, vitexin, and isoflavone in Dendrobium nobile were determined by HPLC; (2) The content of dendrobine in Dendrobium nobile was determined by gas chromatography; (3) The contents of total polyphenols, total flavonoids, total anthocyanins, total polysaccharides and total alkaloids in Dendrobium nobile were determined by UV-visible spectrophotometry.

3. The multi-index quality analysis method of Dendrobium nobile according to claim 2, wherein: 1) Take the sample of Dendrobium nobile to be tested, add methanol, sonicate, centrifuge the extract, and filter the supernatant through a filter membrane to obtain the test solution; 2) Perform HPLC on the test solution and determine the content of lutein and Dendrobin A using the standard curve method; Chromatographic conditions were as follows: a Phenomenex Luna C18 column was used, the mobile phase was water (A)-acetonitrile (B), gradient elution, the elution program was shown in the table below, the flow rate was 1.0 mL / min, the column temperature was 30°C, and the detection wavelengths were 210 nm and 450 nm; 3) Take the sample of Dendrobium nobile to be tested, add ethanol, sonicate, centrifuge the extract, and filter the supernatant through a filter membrane to obtain the test solution; 4) Performing high performance liquid chromatography on the test solution and determining the contents of vitexin and isoflavone by standard curve method; Chromatographic conditions were as follows: a Phenomenex Luna C18 column with a mobile phase of water (A)-acetonitrile (B), gradient elution (elution program shown in the table below), a flow rate of 1.0 mL / min, a column temperature of 30°C, and a detection wavelength of 340 nm; 4. The multi-index quality analysis method for Dendrobium nobile according to claim 3, wherein: The sample to be tested of Dendrobium nobile needs to be pretreated as follows: The crushed drug powder was weighed and put into the extraction kettle, and the extraction conditions were set as follows: extraction temperature 65°C, extraction pressure 35MPa, carrier agent anhydrous ethanol, carrier agent ratio 1:3, and cooling mechanism was turned on. After the condenser temperature dropped to near the set temperature, the CO2 cylinder was turned on, and the extraction kettle was pressurized by the high-pressure pump. At the same time, the automatic control valve of the extraction kettle was opened, and the experimental pressure was set. When the experimental design pressure and temperature were reached, the CO2 gas source was turned off, the carrier agent pump was turned on to adjust the carrier agent injection speed, and the cyclic extraction was started. The extraction was stopped when the extraction time was reached. Among them, the pressure of the separation kettle was fixed at 5-6MPa, the temperature was 35°C, the extraction flow rate was 35L / h, the carrier agent addition rate was 10mL / min, and the extraction time was 120min. The extract of the separation kettle was collected, centrifuged at 4°C and 10000r / min for 20min, the supernatant was filtered, and the filtrate was recovered by rotary evaporation to dryness. 1) The ratio of the Dendrobium nobile sample to be tested and methanol is 0.5 g: 20 mL; 3) The ratio of the Dendrobium nobile sample to be tested and ethanol is 1 g: 20 mL; 1) and 3) the ultrasonic conditions are: 30 min, the centrifugal conditions are: 4500 r / min, 15 min, and the pore size of the filter membrane is 0.45 μm; The reference substance used in the construction of the standard curve of Dendrobin A was constructed by the following steps: Take the supercritical CO2 extract of Dendrobium nobile stem, add an appropriate amount of ethyl acetate, and sonicate to completely dissolve it; add 80 mesh silica gel powder in a mass ratio of 1:1 and mix evenly, and recover the solvent in a rotary evaporator until the silica gel becomes mobile; use 200-300 mesh normal phase silica gel for column chromatography separation, use petroleum ether-ethyl acetate as the solvent system, and perform gradient elution in the ratio of 30:1, 15:1, 8:1, 4:1, 2:1, 1:1, and 0:1 by volume, each gradient elution is 5 column volumes, the elution solvent is detected by thin layer chromatography, and the solvents with the same spots are combined; take the fractions obtained by 15:1 combination, perform column chromatography separation on 200-300 mesh normal phase silica gel, perform isocratic elution with petroleum ether-ethyl acetate in a volume ratio of 6:1, detect by thin layer chromatography and combine the elution solvents with the same spots, recover the solvent and enter semi-preparative liquid chromatography, the mobile phase is acetonitrile: water = 35%:65%, the flow rate is 4 mL / min, t R = 20 min, recover the solvent, and obtain the reference substance Dendrobin A.

5. The multi-index quality analysis method for Dendrobium nobile according to claim 2, wherein: Said (2) includes: 1) Take a sample of Dendrobium nobile to be tested, add methanol, sonicate, centrifuge the extract, filter the supernatant through a filter membrane, take the filtrate and add an internal standard solution containing naphthalene, and then add methanol to obtain a test solution; 2) The test solution was subjected to gas chromatography and the content of dendrobine was determined by the standard curve method; The chromatographic conditions were as follows: inlet temperature of 250°C, split ratio of 30:1, carrier gas of N2, flow rate of 1.0 mL / min; a TR-1MS column was used, temperature program: initial temperature of 80°C, heating to 250°C at a rate of 10°C per minute, and holding for 16 min; FID detector temperature of 250°C, air: gas = 10:

1.

6. The multi-index quality analysis method for Dendrobium nobile according to claim 5, characterized in that: 1) The naphthalene-containing internal standard solution contains naphthalene at a concentration of 0.1340 mg / mL; 1) The ratio of the Dendrobium nobile sample to be tested, the methanol added for the first time, the internal standard solution containing naphthalene, and the methanol added for the second time is 0.5 g: 20 mL: 0.5 mL: 0.5 mL; The ultrasonic conditions described in 1) are: 30 min, the centrifugal conditions are: 4500 r / min, 15 min, and the pore size of the filter membrane is 0.45 μm.

7. The multi-index quality analysis method for Dendrobium nobile according to claim 2, wherein: Said (3) includes: 1) Take a sample of Dendrobium nobile to be tested, add water / ethanol, sonicate, and filter the supernatant through a filter membrane to obtain a test solution; based on ultraviolet spectrophotometry and a standard curve method, the total polyphenol content in the test solution is determined by a folin-phenol colorimetric method; the total flavonoid content in the test solution is calculated as rutin, and the total flavonoid content in the test solution is determined by an aluminum salt colorimetric method; the total anthocyanin content in the test solution is determined by an iron salt catalytic colorimetric method; 2) Take the Dendrobium nobile sample to be tested, add ethanol solution, sonicate, centrifuge and discard the supernatant, add appropriate amount of water to the precipitate, sonicate, and take the supernatant to obtain the test solution; based on ultraviolet spectrophotometry and standard curve method, use phenol-sulfuric acid method to determine the total polysaccharide content in the test solution; 3) Taking a sample of Dendrobium nobile to be tested, adding chloroform to obtain a test solution; based on ultraviolet spectrophotometry and a standard curve method, the amount of total alkaloids contained is calculated as dendrobine, and the total alkaloid content in the test solution is determined by bromocresol green acid dye colorimetry.

8. The multi-index quality analysis method for Dendrobium nobile according to claim 7, characterized in that: The Dendrobium nobile sample to be tested described in 1) needs to be pretreated as follows: 5 g of the medicinal material of Dendrobium nobile stem or flower crushed through an 80-mesh sieve was taken, water / ethanol was added according to a material-liquid ratio of 1:30, and after reflux extraction and high-speed centrifugation, the supernatant was filtered and the filtrate was freeze-dried in a freeze dryer to obtain the water extract / alcohol extract of Dendrobium nobile stem and Dendrobium flower; 1) The ratio of the Dendrobium nobile sample to be tested and water / ethanol is 10 mg:5 mL; The ultrasonic conditions described in 1) are: 30 min, the pore size of the filter membrane is 0.45 μm; 2) The ethanol solution described in the above is an 80% ethanol solution, and the ratio of the Dendrobium nobile sample to be tested to the ethanol solution is a mass-to-volume ratio of 1:8; 2) The first ultrasonication was performed for 20 min, the centrifugation was performed at 4500 rpm for 15 min, the pore size of the filter membrane was 0.45 μm, and the second ultrasonication was performed for 15 min; 3) The Dendrobium nobile sample to be tested needs to be pretreated as follows: The crushed drug powder was weighed and put into the extraction kettle, and the extraction conditions were set as follows: extraction temperature 65°C, extraction pressure 35MPa, carrier agent anhydrous ethanol, carrier agent ratio 1:3, and cooling mechanism was turned on. After the condenser temperature dropped to near the set temperature, the CO2 cylinder was turned on, and the extraction kettle was pressurized by the high-pressure pump. At the same time, the automatic control valve of the extraction kettle was opened, and the experimental pressure was set. When the experimental design pressure and temperature were reached, the CO2 gas source was turned off, the carrier agent pump was turned on to adjust the carrier agent injection speed, and the cyclic extraction was started. The extraction was stopped when the extraction time was reached. Among them, the pressure of the separation kettle was fixed at 5-6MPa, the temperature was 35°C, the extraction flow rate was 35L / h, the carrier agent addition rate was 10mL / min, and the extraction time was 120min. The extract of the separation kettle was collected, centrifuged at 4°C and 10000r / min for 20min, the supernatant was filtered, and the filtrate was recovered by rotary evaporation to dryness. The amount of chloroform added described in 3) is calculated based on the concentration of the sample to be tested being 8 mg / mL.

9. The multi-index quality analysis method for Dendrobium nobile according to any one of claims 2 to 8, characterized in that: The Dendrobium nobile sample to be tested is the stem or flower of Dendrobium nobile.

10. Application of the multi-index quality analysis method for Dendrobium nobile described in any one of claims 1 to 9 in quality control of Dendrobium nobile medicinal materials.

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