A method for constructing a fingerprint of Cordyceps sinensis and its application

Abstract

The present invention discloses a method for constructing a fingerprint spectrum of Cordyceps sinensis and its application. The method includes preparation of a test solution, preparation of a reference solution, preparation of a reference solution and high performance liquid chromatography detection, and the formulation of a fingerprint spectrum of Cordyceps sinensis sterol components. The liquid chromatography conditions of the present invention can effectively separate and detect the sterol components in Cordyceps sinensis, and the separation effect is good. This is the first HPLC fingerprint spectrum detection and identification method for sterol components in Cordyceps sinensis; the method provides favorable data support for the authenticity identification and / or quality control of Cordyceps sinensis and its products.

Description

Technical field:

[0001] The invention relates to a method for constructing a fingerprint spectrum of sterol components of Cordyceps sinensis and application thereof, belonging to the technical field of traditional Chinese medicine detection. Background technology:

[0002] Cordyceps sinensis refers to the complex of larval corpses and fungal stroma formed by the infection of Hepialidae insect larvae by Ophiocordyceps sinensis (Berk.) Sung, Hywel-Jones & Spatafora. It is a biological resource unique to the Qinghai-Tibet Plateau. Modern medicine shows that Cordyceps sinensis has a wide range of pharmacological effects, including immunomodulation, anti-oxidation, anti-aging, anti-fatigue, anti-bacterial, anti-tumor, and liver and kidney protection. It is known as the "three treasures of Chinese medicine" together with ginseng and deer antlers. It has a long history of use and is a precious tonic Chinese herbal medicine. Due to the scarcity of Cordyceps sinensis resources and its high price, the quality of Cordyceps sinensis materials and products on the market varies greatly, and the phenomenon of inferior products being sold as good ones and fake products being sold as real ones is common. The quality control of Cordyceps sinensis in the Chinese Pharmacopoeia only uses the content of adenosine to evaluate the quality. Although it can effectively guarantee the quality of Cordyceps sinensis as a raw material to a certain extent, it is not enough to guarantee the authenticity and quality of Cordyceps sinensis, especially for Cordyceps sinensis products that do not have the appearance of Cordyceps sinensis, it is even more difficult to guarantee their authenticity and quality.

[0003] Traditional Chinese medicine fingerprint technology is a quality control model that comprehensively reflects the characteristics of multiple components of traditional Chinese medicine. It can accurately evaluate the uniformity, efficacy and stability of the quality of traditional Chinese medicine materials. It has the characteristics of integrity and fuzziness. It is one of the traditional Chinese medicine quality evaluation systems recognized at home and abroad. At present, there have been many studies on the fingerprint of Cordyceps sinensis, and most of the professional literature has been published on the fingerprint construction method of Cordyceps sinensis water extract or alcohol extract. For example, the literature (Qian Zhengming et al. HPLC fingerprint analysis of Cordyceps sinensis water extract and alcohol extract [J]. World Traditional Chinese Medicine, December 2017, Vol. 12, No. 12, 3133-3137.) constructed the fingerprint of Cordyceps sinensis water extract and alcohol extract. The literature (Qian Zhengming et al. Water-soluble and alcohol-soluble substances in Cordyceps sinensis) Simultaneous analysis of fat-soluble components by HPLC [J]. Lishizhen Traditional Chinese Medicine, Vol. 31, No. 7, 2020) simultaneously determined the fingerprints of water-soluble and fat-soluble components. There are many similar public documents, but the fingerprints of Cordyceps sinensis reported in these documents are complex, containing multiple chemical components such as nucleosides, adenosines, sterols, etc., lacking obvious characteristic features. The fingerprints of active sites containing a single type of component are more conducive to the authenticity and / or quality identification of Chinese medicinal materials.

[0004] Cordyceps sinensis contains a variety of chemical components, and the recognized active ingredients are mainly nucleosides, sterols, polysaccharides, and also organic acids, vitamins, inorganic elements and other compounds. Modern pharmacological studies have shown that sterol compounds have a wide range of pharmacological activities, such as anti-tumor, cholesterol-lowering and cardiovascular disease prevention. They are recommended by the International Nutrition Society as one of the top ten functional nutrients for humans in the future, and are now widely used in medicine, chemical industry and food. Sterol compounds, as the main active ingredients in Cordyceps sinensis, should be used as a signature component for quality control and authenticity identification. At present, there is no method for constructing a fingerprint for sterol compounds in Cordyceps sinensis and applying it to the authenticity and quality identification of Cordyceps sinensis. Patent CN201810236777 constructs a sterol HPLC fingerprint of Cordyceps sinensis powder and preparations. Cordyceps sinensis powder and preparations are products obtained by fermenting a strain isolated from Cordyceps sinensis, which are two completely different products from Cordyceps sinensis medicinal materials and products. The present invention constructs the sterol component fingerprint of Cordyceps sinensis for the first time, which is mainly used for authenticity and / or quality identification of Cordyceps sinensis and its products. Summary of the invention:

[0005] The purpose of the present invention is to construct a fingerprint of sterol components of Cordyceps sinensis, so as to provide a scientific basis for the authenticity identification and / or quality control of Cordyceps sinensis and its products. The liquid chromatography conditions of the present invention can effectively separate and detect the sterol components in Cordyceps sinensis, with good separation effect. It is the first HPLC fingerprint detection and identification method for sterol components in Cordyceps sinensis; the method provides favorable data support for the authenticity identification and / or quality control of Cordyceps sinensis and its products.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for constructing a fingerprint of Cordyceps sinensis, comprising the following steps:

[0007] (1) Preparation of test solution: Take Cordyceps sinensis medicinal materials, grind them, add n-hexane for ultrasonic extraction, extract the n-hexane extract with acidic and alkaline inorganic salt solutions, concentrate the n-hexane extract after extraction, adsorb it with a solid phase extraction column and then elute it to separate the sterol components, collect and combine the eluate, recover the solvent from the eluate, dissolve it with a methanol-isopropanol mixed solvent, filter it with a microporous filter membrane for sterilization, and obtain the test solution;

[0008] (2) Preparation of reference substance solution: Take reference substances ergosterol, cholesterol and sitosterol, add methanol-isopropanol mixed solvent respectively to dissolve them to obtain reference substance stock solutions, mix the three reference substance stock solutions in a certain volume, filter and sterilize them with a microporous filter membrane to obtain a reference substance mixed solution;

[0009] (3) Preparation of reference solution: Take Cordyceps sinensis as a reference medicinal material, grind it, add anhydrous ethanol to ultrasonically extract it, concentrate the extract, add methanol-isopropanol mixed solvent to dissolve it, and filter it with a microporous filter membrane to sterilize it to obtain a reference solution;

[0010] (4) High performance liquid chromatography detection to develop a fingerprint of Cordyceps sinensis sterol components.

[0011] Preferably, the chromatographic conditions for high performance liquid chromatography detection in step (4) are:

[0012] Chromatographic column: C18 column;

[0013] Mobile phase: ultrapure water is used as mobile phase A, and methanol is used as mobile phase B for gradient elution, and the mobile phase gradient elution conditions are: 0-7 min, mobile phase A (65%→20%), mobile phase B (35%→80%); 7-15 min, mobile phase A (20%→0%), mobile phase B (80%→100%); 15-30 min, mobile phase A (0%→0%), mobile phase B (100%→100%); 30-35 min, mobile phase A (0%→35%), mobile phase B (100%→65%); 35-40 min, mobile phase A (35%→35%), mobile phase B (65%→65%);

[0014] Detection wavelength: 216nm;

[0015] Flow rate: 0.5ml / min;

[0016] Column temperature: 30°C;

[0017] Accurately pipette 5 μl of the reference substance mixed solution, 15 μl of the reference substance solution and 15 μl of the test substance solution respectively, inject into the high performance liquid chromatograph for determination.

[0018] Preferably, in step (4), the C18 chromatographic column is InfinityLab Poroshell 120EC-C18, specification: 4.6×100 mm, 2.7 Micron.

[0019] Preferably, the method for formulating the fingerprint of Cordyceps sinensis sterol components in step (4) is: by comparing the peak appearance time and the existence of the chromatographic peaks in the chromatograms of the reference solution, the reference solution and the test solution, the common characteristic peaks in the test solution are obtained.

[0020] Preferably, the Cordyceps sinensis sterol component fingerprint includes 12 common characteristic peaks, among which peak No. 9 is ergosterol, peak No. 11 is cholesterol, peak No. 12 is sitosterol, and the others are unknown component peaks;

[0021] Taking ergosterol peak No. 9 as the reference peak, the relative retention times of the characteristic peaks are: peak 1: 0.579±0.029; peak 2: 0.672±0.034; peak 3: 0.756±0.038; peak 4: 0.794±0.040; peak 5: 0.814±0.041; peak 6: 0.850±0.042; peak 7: 0.936±0.047; peak 8: 0.949±0.047; peak 9: 1.000±0.05; peak 10: 1.016±0.051; peak 11: 1.063±0.053; peak 12: 1.169±0.058.

[0022] Preferably, in the step (1), the Cordyceps sinensis material is crushed to about 10 meshes, and each gram of Cordyceps sinensis is processed according to the following method: the mass volume ratio of the Cordyceps sinensis material to n-hexane is 1.0g:100ml, the power used for ultrasonic extraction is 400W, the working frequency is 40KHz, and the extraction time is 30min; the acidic inorganic salt solution is a sodium dihydrogen phosphate aqueous solution with a mass volume ratio of 1%g / ml, and the alkaline inorganic salt solution is a sodium hydrogen phosphate aqueous solution with a mass volume ratio of 1%g / ml, and the acidic and alkaline inorganic salt solutions are extracted once respectively; after acid The n-hexane extract extracted with an alkaline inorganic salt solution was concentrated to 2 ml. The solid phase extraction column was a C18 column. The solid phase extraction column was first activated with 3 ml of n-hexane, and then eluted with 3 ml of n-hexane (Ⅰ), n-hexane-dichloromethane (Ⅱ, 1:1 v / v), dichloromethane (Ⅲ), dichloromethane-methanol (Ⅳ, 1:1 v / v), and methanol (Ⅴ) in sequence. The eluents Ⅱ and Ⅲ were collected, and after the solvent was recovered, it was dissolved with 2 ml of a methanol-isopropanol mixed solvent. The volume ratio of the methanol-isopropanol mixed solvent was 1:1. The sample was filtered with a microporous filter membrane to obtain a test solution.

[0023] The present invention uses sodium dihydrogen phosphate aqueous solution and disodium hydrogen phosphate aqueous solution to extract cordyceps sinensis n-hexane extract, removes the interference of acidic and alkaline impurities, uses a C18 solid phase extraction column, successfully separates and enriches the sterol components of cordyceps sinensis, and removes most of the interference of weak polar impurities, which is conducive to the subsequent use of high performance liquid chromatography to detect and formulate a fingerprint.

[0024] Preferably, the specifications of the C18 solid phase extraction column are: 500 mg of filler and 3 ml of space volume above the filler.

[0025] Preferably, in the step (2), the volume ratio of the methanol-isopropanol mixed solvent is 1:1, the mass volume ratio of ergosterol, cholesterol and sitosterol in the reference substance stock solution to the methanol-isopropanol mixed solvent is 2 mg:2 ml, and the volume ratio of the ergosterol, cholesterol and sitosterol reference substance stock solutions mixed is 1:2:2.

[0026] Preferably, in the step (3), the Cordyceps sinensis reference medicinal material is crushed to about 10 meshes, the mass volume ratio of the Cordyceps sinensis reference medicinal material to n-hexane is 1.0 g:100 ml, the power used for ultrasonic extraction is 400 W, the operating frequency is 40 KHz, the extraction time is 30 min, and the extract of each gram of medicinal material is concentrated and dissolved with 3 ml of methanol-isopropanol mixed solvent, the volume ratio of methanol-isopropanol mixed solvent is 1:1, and filtered with a microporous filter membrane to obtain a reference solution.

[0027] The present invention also provides the application of the fingerprint spectrum of Cordyceps sinensis sterol components constructed according to the above construction method in the detection of Cordyceps sinensis and its products.

[0028] The fingerprint of Cordyceps sinensis sterol components constructed according to the construction method of the present invention can be used to detect the authenticity and / or quality of Cordyceps sinensis and its products.

[0029] The present invention also provides a method for detecting Cordyceps sinensis and its products, comprising comparing the characteristic spectrum of the Cordyceps sinensis and its products to be detected with the fingerprint spectrum of Cordyceps sinensis sterol components constructed by the above construction method.

[0030] Preferably, the detection method comprises the following steps:

[0031] (1) Take Cordyceps sinensis or Cordyceps sinensis products, crush them, add n-hexane for ultrasonic extraction, extract the n-hexane extract with acidic and alkaline inorganic salt solutions, concentrate the extracted n-hexane extract, adsorb it with a solid phase extraction column and then elute it to separate the sterol components, collect and combine the eluate, recover the solvent from the eluate, dissolve it with a methanol-isopropanol mixed solvent, filter it with a microporous filter membrane for sterilization, and obtain a test solution;

[0032] The characteristic spectrum of Cordyceps sinensis or Cordyceps sinensis products is detected by high performance liquid chromatography, and the chromatographic conditions are:

[0033] Chromatographic column: C18 column;

[0034] Mobile phase: ultrapure water is used as mobile phase A, and methanol is used as mobile phase B for gradient elution, and the mobile phase gradient elution conditions are: 0-7 min, mobile phase A (65%→20%), mobile phase B (35%→80%); 7-15 min, mobile phase A (20%→0%), mobile phase B (80%→100%); 15-30 min, mobile phase A (0%→0%), mobile phase B (100%→100%); 30-35 min, mobile phase A (0%→35%), mobile phase B (100%→65%); 35-40 min, mobile phase A (35%→35%), mobile phase B (65%→65%);

[0035] Detection wavelength: 216nm;

[0036] Flow rate: 0.5ml / min;

[0037] Column temperature: 30°C;

[0038] Accurately pipette 5 μl of the reference substance mixed solution, 15 μl of the reference substance solution and the test substance solution respectively, inject into the HPLC and measure;

[0039] (2) comparing the characteristic spectrum of the Cordyceps sinensis or the Cordyceps sinensis product obtained in step (1) with the fingerprint spectrum of the Cordyceps sinensis sterol components;

[0040] The fingerprint of the Cordyceps sinensis sterol components includes 12 common characteristic peaks, among which peak No. 9 is ergosterol, peak No. 11 is cholesterol, peak No. 12 is sitosterol, and the others are unknown component peaks;

[0041] Taking ergosterol peak 9 as the reference peak, the relative retention times of the characteristic peaks are: peak 1: 0.579±0.029; peak 2: 0.672±0.034; peak 3: 0.756±0.038; peak 4: 0.794±0.040; peak 5: 0.814±0.041; peak 6: 0.850±0.042; peak 7: 0.936±0.047; peak 8: 0.949±0.047; peak 9: 1.000±0.05; peak 10: 1.016±0.051; peak 11: 1.063±0.053; peak 12: 1.169±0.058;

[0042] (3) Determining the authenticity and / or quality of Cordyceps sinensis or Cordyceps sinensis products based on the comparison results of step (2).

[0043] Preferably, the judgment standard in step (3) is: if the characteristic spectrum of the test solution of the Cordyceps sinensis or Cordyceps sinensis product to be tested conforms to the fingerprint spectrum of the Cordyceps sinensis sterol components, the Cordyceps sinensis or Cordyceps sinensis product to be tested can be judged as a qualified sample; otherwise, the Cordyceps sinensis or Cordyceps sinensis product to be tested can be judged as an unqualified sample.

[0044] Compared with the prior art, the beneficial effects of the present invention are:

[0045] (1) The liquid chromatography conditions of the present invention can effectively separate and detect the sterol components in Cordyceps sinensis, with good separation effect, low analysis and detection cost, simple and easy operation of mobile phase preparation, and is the first HPLC fingerprint detection and identification method for Cordyceps sinensis sterol components;

[0046] (2) The present invention uses three reference substances, namely, ergosterol, cholesterol and sitosterol, which are representative sterol components in Cordyceps sinensis, as reference substances, and also uses Cordyceps sinensis reference medicinal material extract as medicinal material reference substance, and conducts qualitative comparison respectively, and the method is reliable and effective;

[0047] (3) The present invention can provide favorable data support for the quality control and formulation of quality standards of Cordyceps sinensis products, and play an important guiding role in the process development of Cordyceps sinensis products;

[0048] (4) The present invention has the advantages of convenience, speed, stability, high precision, good reproducibility, etc., and can accurately and reliably detect and control the quality of Cordyceps sinensis and Cordyceps sinensis products. Description of the drawings:

[0049] Figure 1 The characteristic spectrum of the Cordyceps sinensis test solution obtained by the fingerprint spectrum construction method of the present invention;

[0050] Figure 2 The characteristic spectrum of the reference solution of Cordyceps sinensis reference medicinal material measured by the liquid chromatography conditions of the present invention;

[0051] Figure 3 A characteristic spectrum of a reference solution measured using the liquid chromatography conditions of the present invention;

[0052] Figure 1 , Figure 2 and Figure 3 Among them, the chromatographic peak No. 9 is ergosterol, the chromatographic peak No. 11 is cholesterol, the chromatographic peak No. 12 is sitosterol, and the other chromatographic peaks are unknown substances.

[0053] Figure 4 is the analytical spectrum of thin layer chromatography (TLC) of the extract (from left to right are petroleum ether, n-hexane, ethyl acetate and 95% ethanol extracts, ergosterol, cholesterol and sitosterol reference substances);

[0054] Figure 5 is the HPLC spectrum of the extract not treated with the inorganic salt solution;

[0055] Figure 6 It is the HPLC spectrum of the extract treated with an inorganic salt solution. Specific implementation method:

[0056] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0057] Example 1 Selection of HPLC Conditions and System Suitability

[0058] At present, there is no literature related to the high performance liquid chromatography conditions for the determination of sterol components in Cordyceps sinensis. Referring to the determination methods of sterol components in other medicinal materials, it is found that the separation effect of the reported chromatographic conditions is not ideal. After trying various chromatographic conditions, the inventors found that gradient elution with a methanol-water system has a better separation degree for the sterol components in Cordyceps sinensis, and the chromatographic conditions of the present invention do not require the use of an acidic system and are easy to operate.

[0059] The present invention adopts two reference control substances for comparative research. One is ergosterol, cholesterol and sitosterol, which are representative components of sterols. A total of three reference substances are used as reference control substances, which are prepared into reference substance solutions for qualitative comparison of reference substances. The second is the use of anhydrous ethanol extract of Cordyceps sinensis reference medicinal material as a medicinal material extract reference, which represents the alcohol-soluble components such as Cordyceps sinensis sterols and is prepared into a reference solution for qualitative comparison of medicinal material extract reference substances. Therefore, the effectiveness, accuracy and reliability of the present invention can be fully explained.

[0060] The method for constructing the fingerprint spectrum of the sterol components of Cordyceps sinensis of the present invention is used for detection, and comprises the following steps:

[0061] (1) Crush the Cordyceps sinensis into about 10 meshes, take 1 g, add 100 ml of n-hexane, and perform ultrasonic extraction for 30 min (power is 400 W, working frequency is 40 kHz). The n-hexane extract is first extracted with 100 ml of 1% g / ml sodium dihydrogen phosphate aqueous solution, and then extracted with 100 ml of 1% g / ml disodium hydrogen phosphate solution. The n-hexane extract after extraction is concentrated to 2 ml, and 3 ml of n-hexane is used to activate the C18 solid phase extraction column. 2 ml of the n-hexane concentrate is added to the C18 solid phase extraction column. Adsorb in the column, and elute with 3 ml of n-hexane (Ⅰ), n-hexane-dichloromethane (Ⅱ, 1:1 v / v), dichloromethane (Ⅲ), dichloromethane-methanol (Ⅳ, 1:1 v / v), and methanol (Ⅴ) in sequence to separate the sterol components, collect the eluents Ⅱ and Ⅲ, recover the solvent, dissolve it with 2 ml of a methanol-isopropanol mixed solvent with a volume ratio of 1:1, and filter it with a 0.22 μm microporous filter membrane to obtain the test solution; the specifications of the C18 solid phase extraction column are: 500 mg of filler material and 3 ml of the space volume above the filler material.

[0062] (2) Preparation of reference substance solution: 2 mg of each of the reference substances ergosterol, cholesterol and sitosterol were taken and dissolved in 2 ml of a methanol-isopropanol mixed solvent at a volume ratio of 1:1 to obtain a reference substance stock solution. The ergosterol, cholesterol and sitosterol reference substance stock solutions were mixed evenly at a volume ratio of 1:2:2, and filtered through a 0.22 μm microporous filter membrane to obtain a reference substance mixed solution;

[0063] (3) Preparation of reference solution: Grind the Cordyceps sinensis reference medicinal material into about 10 meshes, take 1 g, then add 100 ml of anhydrous ethanol and ultrasonically extract for 30 min. After the extract solvent is recovered, 3 ml of a methanol-isopropanol mixed solvent with a volume ratio of 1:1 is added to dissolve, and filtered with a 0.22 μm microporous filter membrane to obtain a reference solution;

[0064] High performance liquid chromatography detection, the development of Cordyceps sinensis sterol component fingerprint:

[0065] Chromatographic conditions:

[0066] Chromatographic column: C18 chromatographic column, the chromatographic column is InfinityLab Poroshell 120EC-C18, specification: 4.6×100mm, 2.7Micron.

[0067] Mobile phase: ultrapure water is used as mobile phase A, and methanol is used as mobile phase B for gradient elution, and the mobile phase gradient elution conditions are: 0-7 min, mobile phase A (65%→20%), mobile phase B (35%→80%); 7-15 min, mobile phase A (20%→0%), mobile phase B (80%→100%); 15-30 min, mobile phase A (0%→0%), mobile phase B (100%→100%); 30-35 min, mobile phase A (0%→35%), mobile phase B (100%→65%); 35-40 min, mobile phase A (35%→35%), mobile phase B (65%→65%);

[0068] Detection wavelength: 216nm;

[0069] Flow rate: 0.5ml / min;

[0070] Column temperature: 30°C;

[0071] Accurately pipette 5 μl of the reference substance mixed solution, 15 μl of the reference substance solution and 15 μl of the test substance solution respectively, inject into the high performance liquid chromatograph for determination.

[0072] The results of the test are as follows Figure 1 , Figure 2 and Figure 3 As shown, it can be seen that the sterol components of Cordyceps sinensis can be completely separated and detected under the chromatographic conditions of the present invention, and the separation effect is good.

[0073] The reference substances ergosterol, cholesterol and sitosterol were taken respectively, diluted with methanol-isopropanol (1:1 v / v), and a detection limit test solution of ergosterol containing 5.15 μg per 1 ml was prepared, a detection limit test solution of cholesterol containing 7.23 μg per 1 ml was prepared, and a detection limit test solution of sitosterol containing 9.32 μg per 1 ml was prepared, and a mixed reference solution of 1 ml was prepared, and 1 μl was respectively injected into the high performance liquid chromatograph for determination, and the chromatogram was recorded, and the retention time, detection limit, theoretical plate number and separation degree were determined. The results are shown in Table 1. The theoretical plate number of ergosterol is greater than 300,000, and the theoretical plate number of cholesterol and sitosterol is greater than 200,000. The detection limits of the three components of ergosterol, cholesterol and sitosterol are 5.15 ng, 7.23 ng, and 9.32 ng, respectively, all of which are nanogram level, indicating that the detection method has high sensitivity and good separation effect.

[0074] Table 1 System suitability test results

[0075]

[0076] Example 2 Development and research of sample pretreatment method

[0077] The inventors investigated the extraction of Cordyceps sinensis medicinal materials with different solvents. 1g of Cordyceps sinensis medicinal material powder was extracted once for 30min using 100ml of petroleum ether, n-hexane, ethyl acetate and 95% ethanol ultrasonically (power was 400W, working frequency was 40KHz). The extracts were analyzed by thin layer chromatography (TLC, developing solvent petroleum ether: ethyl acetate = 5: 1 v / v) to compare the contents of sterol compounds in the extracts obtained with different extraction solvents ( Figure 4 ) and found that the components of petroleum ether and n-hexane extracts were relatively simple, and the content of sterols was relatively high. Considering the stable nature of n-hexane, it was finally decided to use n-hexane as the extraction solvent.

[0078] In addition, the inventors also considered that the extract may contain acidic and alkaline components. In order to remove the interference of these components and make the chromatographic peak simpler and more beautiful, the extract was first extracted once with 1% g / ml sodium dihydrogen phosphate solution in a separatory funnel at a volume ratio of 1:1, and the sodium dihydrogen phosphate solution was removed. The extract was extracted once again with 1% g / ml disodium hydrogen phosphate solution at a volume ratio of 1:1, and the disodium hydrogen phosphate solution was removed to obtain the treated extract. The specific treatment procedure is the same as that of the test solution in Example 1 (including high performance liquid chromatography), and the solution that was not treated with 1% g / ml sodium dihydrogen phosphate solution and 1% g / ml disodium hydrogen phosphate solution was used as a control. For comparison, ( Figure 6 ), no inorganic salts ( Figure 5 ) processed HPLC spectrum, and found that the processed spectrum is more simplified, the peak shape of the chromatographic peak is more beautiful, and the interference of some impurity peaks is eliminated ( Figure 5 and 6 The sample pretreatment method adopted by the present invention does not require a saponification process, is simple and convenient to operate, and obtains native sterol components, which is more conducive to component analysis.

[0079] Example 3 Identification study of ten batches of Cordyceps sinensis sterol components fingerprint

[0080] Take 10 batches of Cordyceps sinensis materials, prepare the test solution and detect it according to the operating method of Example 1 of the present invention, and record the chromatogram. Mark the 12 main chromatographic peaks in the fingerprint, among which the peak No. 9 is consistent with the peak time of the ergosterol reference substance, the peak No. 11 is consistent with the peak time of the cholesterol reference substance, and the peak No. 12 is consistent with the peak time of the sitosterol reference substance. Record the ratio of the retention time of each component in the 10 batches of samples to the retention time of the peak No. 9 (relative retention time), and the relative retention time average value ± 5%, calculate the qualitative identification limit of each component peak, and the results are shown in Table 2. The RSD of the relative retention time of the chromatographic peaks between the batches does not exceed 0.5%, and the difference is not large. This shows that the method of the present invention has good repeatability.

[0081] Table 2 Relative retention time of each peak and qualitative identification limit range of ten batches of Cordyceps sinensis sterol components fingerprint

[0082]

[0083]

Claims

1. A method for constructing a fingerprint of Cordyceps sinensis, characterized in that: The following steps are involved: (1) Preparation of test solution: Take Cordyceps sinensis medicinal materials, grind them, add n-hexane for ultrasonic extraction, extract the n-hexane extract with acidic and alkaline inorganic salt solutions, concentrate the n-hexane extract after extraction, adsorb it with a solid phase extraction column and then elute it to separate the sterol components, collect and combine the eluate, recover the solvent from the eluate, dissolve it with a methanol-isopropanol mixed solvent, filter it with a microporous filter membrane for sterilization, and obtain the test solution; (2) Preparation of reference substance solution: Take reference substances ergosterol, cholesterol and sitosterol, add methanol-isopropanol mixed solvent respectively to dissolve them to obtain reference substance stock solution, mix the three reference substance stock solutions in a certain volume, filter and sterilize them with a microporous filter membrane to obtain a reference substance mixed solution; (3) Preparation of reference solution: Take Cordyceps sinensis as a reference medicinal material, grind it, add anhydrous ethanol to perform ultrasonic extraction, concentrate the extract, add methanol-isopropanol mixed solvent to dissolve it, filter it with a microporous filter membrane to sterilize it, and obtain a reference solution; (4) HPLC detection to develop a fingerprint of Cordyceps sinensis sterol components; In the step (1), the cordyceps sinensis material is crushed to about 10 meshes, and each gram of cordyceps sinensis is processed according to the following method: the mass volume ratio of the cordyceps sinensis material to n-hexane is 1.0g:100ml, the power used for ultrasonic extraction is 400W, the operating frequency is 40KHz, and the extraction time is 30min; the acidic inorganic salt solution is a sodium dihydrogen phosphate aqueous solution with a mass volume ratio of 1% g / ml, and the alkaline inorganic salt solution is a sodium hydrogen phosphate aqueous solution with a mass volume ratio of 1% g / ml, and the acidic and alkaline inorganic salt solutions are extracted once respectively; the n-hexane extract extracted with the acidic and alkaline inorganic salt solutions is concentrated to 2ml, the solid phase extraction column is a C18 column, the solid phase extraction column is first activated with 3ml of n-hexane, and then activated with 3ml of n-hexane, n-hexane: dichloromethane = 1:1 v / v, dichloromethane, and dichloromethane: methanol = 1:1 v / v. v / v, eluted with methanol to obtain eluent I, eluent II, eluent III, eluent IV and eluent V respectively, collected eluent II and eluent III, recovered the solvent and dissolved it with 2 ml of methanol-isopropanol mixed solvent, the volume ratio of methanol-isopropanol mixed solvent was 1:1, filtered with a microporous filter membrane to obtain a test solution; The chromatographic conditions for high performance liquid chromatography detection in step (4) are: Chromatographic column: C18 column; Mobile phase: ultrapure water was used as mobile phase A, and methanol was used as mobile phase B for gradient elution. The mobile phase gradient elution conditions were as follows: 0-7 min, mobile phase A 65%→20%, mobile phase B 35%→80%; 7-15 min, mobile phase A 20%→0%, mobile phase B 80%→100%; 15-30 min, mobile phase A 0%→0%, mobile phase B 100%→100%; 30-35 min, mobile phase A 0%→35%, mobile phase B 100%→65%; 35-40 min, mobile phase A 35%→35%, mobile phase B 65%→65%; Detection wavelength: 216nm; Flow rate: 0.5ml / min; Column temperature: 30°C; Accurately pipette 5 μl of the reference substance mixed solution, 15 μl of the reference substance solution and 15 μl of the test substance solution respectively, inject into the high performance liquid chromatograph for determination.

2. The method for constructing the fingerprint of Cordyceps sinensis according to claim 1, characterized in that: The method for preparing the fingerprint of Cordyceps sinensis sterol components in step (4) is: by comparing the chromatographic peak appearance time and the existence of the chromatographic peak in the chromatograms of the reference solution, the reference solution and the test solution, the common characteristic peak in the test solution is obtained.

3. The method for constructing the fingerprint of Cordyceps sinensis according to claim 2, characterized in that: The fingerprint spectrum of the Cordyceps sinensis sterol components includes 12 common characteristic peaks, among which peak No. 9 is ergosterol, peak No. 11 is cholesterol, peak No. 12 is sitosterol, and the others are unknown component peaks; Taking ergosterol peak No. 9 as the reference peak, the relative retention times of the characteristic peaks are: peak 1: 0.579±0.029; peak 2: 0.672±0.034; peak 3: 0.756±0.038; peak 4: 0.794±0.040; peak 5: 0.814±0.041; peak 6: 0.850±0.042; peak 7: 0.936±0.047; peak 8: 0.949±0.047; peak 9: 1.000±0.05; peak 10: 1.016±0.051; peak 11: 1.063±0.053; peak 12: 1.169±0.

058.

4. The method for constructing the fingerprint of Cordyceps sinensis according to claim 1, characterized in that: In the step (2), the volume ratio of the methanol-isopropanol mixed solvent is 1:1, the mass volume ratio of ergosterol, cholesterol and sitosterol in the reference stock solution to the methanol-isopropanol mixed solvent is 2 mg:2 ml, and the volume ratio of the ergosterol, cholesterol and sitosterol reference stock solutions is 1:2:2; in the step (3), the Cordyceps sinensis reference medicinal material is crushed to about 10 mesh, the mass volume ratio of the Cordyceps sinensis reference medicinal material to n-hexane is 1.0 g:100 ml, the power used for ultrasonic extraction is 400 W, the operating frequency is 40 kHz, the extraction time is 30 min, and each gram of the extract of the medicinal material is concentrated and dissolved with 3 ml of methanol-isopropanol mixed solvent, the volume ratio of methanol-isopropanol mixed solvent is 1:1, and filtered with a microporous filter membrane to obtain a reference solution.

5. Application of the fingerprint of Cordyceps sinensis sterol components constructed according to the method for constructing the fingerprint of Cordyceps sinensis according to claim 1 in the detection of Cordyceps sinensis and its products.

6. A method for detecting Cordyceps sinensis and its products, characterized in that: The method comprises comparing the characteristic spectrum of the Cordyceps sinensis and its products to be tested with the fingerprint spectrum of the Cordyceps sinensis sterol components constructed by the construction method according to claim 1.

7. The detection method according to claim 6, characterized in that: The detection method comprises the following steps: (1) Take Cordyceps sinensis or Cordyceps sinensis products, crush them, add n-hexane for ultrasonic extraction, extract the n-hexane extract with acidic and alkaline inorganic salt solutions, concentrate the n-hexane extract after extraction, adsorb it with a solid phase extraction column and then elute it to separate the sterol components, collect and combine the eluate, recover the solvent from the eluate, dissolve it with a methanol-isopropanol mixed solvent, filter it with a microporous filter membrane for sterilization, and obtain a test solution; The characteristic spectrum of Cordyceps sinensis or Cordyceps sinensis products is detected by high performance liquid chromatography, and the chromatographic conditions are: Chromatographic column: C18 column; Mobile phase: ultrapure water was used as mobile phase A, and methanol was used as mobile phase B for gradient elution. The mobile phase gradient elution conditions were as follows: 0-7 min, mobile phase A 65%→20%, mobile phase B 35%→80%; 7-15 min, mobile phase A 20%→0%, mobile phase B 80%→100%; 15-30 min, mobile phase A 0%→0%, mobile phase B 100%→100%; 30-35 min, mobile phase A 0%→35%, mobile phase B 100%→65%; 35-40 min, mobile phase A 35%→35%, mobile phase B 65%→65%; Detection wavelength: 216nm; Flow rate: 0.5ml / min; Column temperature: 30°C; Accurately pipette 5 μl of the reference substance mixed solution, 15 μl of the reference substance solution and the test solution respectively, inject into the HPLC and measure; (2) comparing the characteristic spectrum of the Cordyceps sinensis or the Cordyceps sinensis product obtained in step (1) with the fingerprint spectrum of the Cordyceps sinensis sterol components; The fingerprint of the Cordyceps sinensis sterol components includes 12 common characteristic peaks, among which peak No. 9 is ergosterol, peak No. 11 is cholesterol, peak No. 12 is sitosterol, and the others are unknown component peaks; Taking ergosterol peak 9 as the reference peak, the relative retention times of the characteristic peaks are: peak 1: 0.579±0.029; peak 2: 0.672±0.034; peak 3: 0.756±0.038; peak 4: 0.794±0.040; peak 5: 0.814±0.041; peak 6: 0.850±0.042; peak 7: 0.936±0.047; peak 8: 0.949±0.047; peak 9: 1.000±0.05; peak 10: 1.016±0.051; peak 11: 1.063±0.053; peak 12: 1.169±0.058; (3) Determine the authenticity and / or quality of Cordyceps sinensis or Cordyceps sinensis products based on the comparison results of step (2).

8. The detection method according to claim 7, characterized in that: The judgment standard in step (3) is: if the characteristic spectrum of the test solution of the Cordyceps sinensis or Cordyceps sinensis product to be tested conforms to the fingerprint spectrum of the Cordyceps sinensis sterol components, then the Cordyceps sinensis or Cordyceps sinensis product to be tested can be judged as a qualified sample; Otherwise, the tested Cordyceps sinensis or Cordyceps sinensis products can be determined as unqualified samples.

Images