Construction method of characteristic chromatogram of volatile oil in rhizoma nardostachyos aqueous extract and preparation of rhizoma nardostachyos aqueous extract and content determination method

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, and particularly provides a construction method of a specific chromatogram of volatile oil in a rhizoma nardostachyos aqueous extract and a preparation thereof and a content determination method. Comprising the following steps: by taking octadecylsilane chemically bonded silica as a filler and a mobile phase comprising acetonitrile and an acid-containing aqueous solution, carrying out gradient elution, screening through a large number of experiments to obtain a specific elution procedure, so as to obtain 10 or more common characteristic peaks, and realizing effective separation of the characteristic peaks containing the nardosinone H, the aristolochin and the nardosinone, so that the method is good in peak shape, stable in base line and high in accuracy. The method has the advantages of simple operation, realization of complete separation of the measured components, short detection time, provision of a basis for quality detection and control of the rhizoma nardostachyos aqueous extract and the preparation thereof, full reflection of the integrity and characteristics of the rhizoma nardostachyos aqueous extract and the preparation thereof, simple operation, and suitableness for quality control of different types of rhizoma nardostachyos preparations.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for constructing a characteristic spectrum of volatile oil in a nard water extract and a preparation thereof and a method for determining the content thereof. Background Art

[0002] Nardostachys jatamansi DC. is a plant of the family Patrinaceae. The product was first recorded in the Four Medical Classics and its Tibetan name is Bangbei. This product is pungent, sweet and warm in nature. It enters the spleen and stomach meridians and has the effects of regulating qi and relieving pain, relieving depression and invigorating the spleen. It can be used for abdominal distension, loss of appetite, and vomiting. It can also be used externally to remove dampness and reduce swelling, treat toothache, and beriberi swelling and poison. The chemical components of Nardostachys jatamansi DC. mainly include terpenes, flavonoids, etc. Terpenoid compounds are the main active ingredients of Nardostachys jatamansi and have a high content. They are a type of volatile oil.

[0003] Common dosage forms of Nardostachys galanga preparations include decoctions, powders, and formula granules. For example, standard decoctions are single-flavor Chinese herbal medicine decoctions prepared through standardized processes under the guidance of traditional Chinese medicine theory and based on clinical applications, with reference to hospital preparation extraction methods. As a standard substance, the properties of standard decoctions are closer to clinical medications and decoction drugs taken by patients. Formula granules are formulated granules obtained from Nardostachys galanga slices through water extraction, concentration, drying, preparation, and other steps. Their material basis is quite different from that of medicinal materials.

[0004] At present, there is no research report on the volatile oil in the water extract of Nardostachys grandiflora and its preparations. The existing literature mainly focuses on the research of water-soluble components in the water extract of Nardostachys grandiflora and its preparations. However, due to the low content of volatile oil in the water extract of Nardostachys grandiflora and its preparations, the existing method is not suitable for the quality detection of volatile oil in the water extract of Nardostachys grandiflora and its preparations, and there are problems such as few characteristic peaks and poor separation effect.

[0005] There is currently no such a problem in the qualitative detection of characteristic spectra of volatile oils in water extracts of Nardostachys grandiflora and its preparations by high performance liquid chromatography. How to improve the detection standard of volatile oils in water extracts of Nardostachys grandiflora and its preparations and comprehensively control the quality of finished products is a technical problem to be solved by the present invention. Summary of the invention

[0006] The object of the present invention is to provide a method for constructing a characteristic spectrum of volatile oil in a water extract of nard and a preparation thereof and a method for determining the content thereof. By adopting the method of the present invention, effective separation of volatile oil components in a water extract of nard and a preparation thereof is achieved, the number of characteristic peaks is significantly increased, the separation effect is good, the detection time is short, and the method is used for quality detection, and the quality of the finished product can be comprehensively controlled.

[0007] To this end, the present invention discloses a method for constructing a characteristic spectrum of volatile oil in a water extract of nard and a preparation thereof, comprising the following steps:

[0008] (1) Preparation of test solution;

[0009] (2) The sample solution is tested by high performance liquid chromatography, the filler is octadecylsilane bonded silica gel, and the mobile phase includes acetonitrile and acid-containing aqueous solution;

[0010] Gradient elution: the gradient elution program includes: 0→30 minutes→35 minutes, and the volume percentage of acetonitrile in the mobile phase is 40%→50%→55%.

[0011] Further, step (1) comprises:

[0012] 1) Take the test sample and extract the volatile oil;

[0013] 2) dissolving the volatile oil in an organic solvent and collecting the organic solvent layer to obtain the test solution;

[0014] Preferably, step (1) also satisfies any one or more of the following AD:

[0015] A. In step 1), volatile oil is extracted by hot reflux extraction;

[0016] B. In step 2), the organic solvent is selected from at least one of petroleum ether, cyclohexane and dichloromethane;

[0017] C. In step 1), the test sample is selected from an aqueous extract of nardus serrata or a preparation made from an aqueous extract of nardus serrata;

[0018] D. The volume ratio of the raw material nard used in step 1) to the test solution prepared in step 2) is 5-30 g:10-30 ml.

[0019] Furthermore, the volume percentage of phosphoric acid in the acid-containing aqueous solution is 0.18-0.25%; and / or the column length of the chromatographic column is 100 mm, the inner diameter is 2.1 mm, and the particle size is 1.8-1.9 μm; and / or the acid in the acid-containing aqueous solution is selected from one or more of phosphoric acid, acetic acid, and formic acid.

[0020] Furthermore, the chromatographic conditions of the high performance liquid chromatography in step (2) also include: a detection wavelength of 220-280 nm, a flow rate of 0.18-0.27 ml / min, a column temperature of 20-30° C., and an injection volume of 1 μl-5 μl; preferably, the detection wavelength is 230-270 nm.

[0021] Furthermore, the construction method also includes the step of preparing a reference solution using at least one reference substance selected from the group consisting of galantol H, aristolochicone, and galantol, and the step of detecting the reference solution by high performance liquid chromatography according to the above construction method to obtain a reference spectrum of the reference substance; preferably, the concentration of the reference solution is 10-100 μg / mL.

[0022] Furthermore, the characteristic spectrum of the volatile oil in the naringa water extract and its preparation has 10 characteristic peaks, the peak corresponding to the peak of the naringa ketone H reference substance is peak No. 5, peak No. 5 is taken as the reference peak, and the relative retention time of the characteristic peaks 1-4 and peaks 6-10 and the reference peak is within the range of ±10% of the specified value; the specified value is: 0.32 (peak 1), 0.70 (peak 2), 0.84 (peak 3), 0.88 (peak 4), 1.17 (peak 6), 1.31 (peak 7), 1.45 (peak 8), 1.66 (peak 9), 1.90 (peak 10).

[0023] The present invention also provides a method for determining the content of aristolochone H and / or aristolochone in a water extract of naringa and a preparation thereof, comprising the following steps:

[0024] Preparation of test solution;

[0025] Preparation of aristolochone H and / or aristolochone reference solution;

[0026] Test steps: Take the test solution and the reference solution and detect them respectively by high performance liquid chromatography. The filler is octadecylsilane bonded silica gel. The mobile phase includes acetonitrile and acid-containing aqueous solution. Gradient elution is performed. The gradient elution program includes: 0→30 minutes→35 minutes. The volume percentage of acetonitrile in the mobile phase is 40%→50%→55%. Calculate the content of aristolochone H and / or aristolochone reference in the water extract of naringa and its preparations.

[0027] Further, step (1) comprises:

[0028] 1) Take the test sample and extract the volatile oil;

[0029] 2) dissolving the volatile oil in an organic solvent and collecting the organic solvent layer to obtain the test solution;

[0030] Preferably, step (1) also satisfies any one or more of the following AD:

[0031] A. In step 1), volatile oil is extracted by hot reflux extraction;

[0032] B. In step 2), the organic solvent is selected from at least one of petroleum ether, cyclohexane and dichloromethane;

[0033] C. In step 1), the test sample is selected from an aqueous extract of nardus serrata or a preparation made from an aqueous extract of nardus serrata;

[0034] D. The volume ratio of the raw material nard used in step 1) to the test solution prepared in step 2) is 5-30 g:10-30 ml.

[0035] Furthermore, the volume percentage of phosphoric acid in the acid-containing aqueous solution is 0.18-0.25%; and / or the column length of the chromatographic column is 100 mm, the inner diameter is 2.1 mm, and the particle size is 1.8-1.9 μm; and / or the acid in the acid-containing aqueous solution is selected from one or more of phosphoric acid, acetic acid, and formic acid; and / or the chromatographic conditions of the high performance liquid chromatography in step (2) also include: a detection wavelength of 220-280 nm, a flow rate of 0.18-0.27 ml / min, a column temperature of 20-30° C., and an injection volume of 1 μl-5 μl.

[0036] The present invention also provides a quality control method for a water extract of nardostachys grandiflora and a preparation thereof, comprising the steps of taking a nardostachys grandiflora product to be tested and constructing a characteristic spectrum according to any of the construction methods described above and / or the step of taking a nardostachys grandiflora product to be tested and determining the content of naringenone H and / or aristolochone in the nardostachys grandiflora product to be tested according to any of the content determination methods described above.

[0037] Furthermore, the quality control method of the water extract of Nardostachys grandiflora and its preparation also includes a step of comparing the characteristic spectrum of the Nardostachys grandiflora product to be tested with the characteristic spectrum of volatile oil in the water extract of Nardostachys grandiflora and its preparation; the characteristic spectrum of the Nardostachys grandiflora product to be tested is obtained using the Nardostachys grandiflora product to be tested according to the above-mentioned construction method.

[0038] In the present invention, the aqueous extract of naringa and its preparation are selected from the aqueous extract of naringa or the preparation made from the aqueous extract of naringa. Among them, the naringa preparation includes the aqueous extract of naringa and also includes pharmaceutically acceptable excipients; preferably, the preparation is selected from one or more of decoction (such as standard decoction of naringa slices), powder (such as freeze-dried powder of standard decoction of naringa slices), tablets, capsules, pills, granules (such as naringa formula granules), and injections.

[0039] Further, the pharmaceutically acceptable excipient is selected from one or more of fillers, disintegrants, lubricants, suspending agents, adhesives, flavoring agents, preservatives, and bases; preferably, the filler is selected from one or more of starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, and sucrose; preferably, the disintegrant is selected from one or more of starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, and cross-linked sodium carboxymethyl cellulose; preferably, the lubricant is selected from one or more of magnesium stearate, sodium lauryl sulfate, talc, and silicon dioxide; preferably, the suspending agent is selected from polyvinyl pyrrolidone. Preferably, the adhesive is selected from one or more of starch slurry, polyvinyl pyrrolidone, hydroxypropyl methylcellulose; preferably, the flavoring agent is selected from one or more of saccharin sodium, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid; preferably, the preservative is selected from one or more of parabens, benzoic acid, sodium benzoate, sorbic acid and its salt preservatives, benzalkonium bromide, chloroethidine acetate, eucalyptus oil; preferably, the matrix is ​​selected from one or more of PEG6000 (polyethylene glycol 6000), PEG4000 (polyethylene glycol 4000), and insect wax.

[0040] The standard decoction of Nardostachys radix slices can be prepared by filtering the water extract. The freeze-dried powder of the standard decoction of Nardostachys radix slices can be prepared by freeze-drying the standard decoction of Nardostachys radix slices. The formula granules of Nardostachys radix can be prepared by filtering and concentrating the water extract and then adding a volatile oil inclusion compound, with or without excipients. Preferably, the relative density of the concentrated extract at 58-62° C. is 1.05-1.10 g / m.

[0041] In the present invention, the water extract of Nardostachys grandiflora can be prepared by an existing conventional method, for example, after extracting Nardostachys grandiflora with water, the water extract of Nardostachys grandiflora can be obtained. Preferably, the extraction method is heating reflux extraction, decoction extraction or ultrasonic extraction; preferably, the number of extractions is 1-3 times, and the time of each extraction is 15-60 minutes; preferably, the mass of water added for each extraction is 6-12 times the mass of Nardostachys grandiflora.

[0042] The technical solution of the present invention has the following advantages:

[0043] 1. The method for constructing a characteristic spectrum of volatile oil in an aqueous extract of naringa and a preparation thereof provided by the present invention uses octadecylsilane bonded silica gel as a filler, a mobile phase comprising acetonitrile and an acid-containing aqueous solution, and gradient elution. A specific elution program is obtained through a large number of experimental screenings, 10 or more common characteristic peaks are obtained, and effective separation of characteristic peaks including naringa ketone H, aristolochicone, and naringa ketone is achieved. The peak shape is good, the baseline is stable, the measured components can be completely separated, and the detection time is short. The method provides a basis for the quality detection and control of the aqueous extract of naringa and the preparation thereof, can fully reflect the integrity and characteristic of the aqueous extract of naringa and the preparation thereof, the method is simple to operate, and can be applicable to the quality control of different types of naringa preparations.

[0044] 2. The method for constructing the characteristic spectrum of the volatile oil in the water extract of Nardostachys grandiflora and its preparation provided by the present invention, after identification by the reference substance, peaks 5, 7, and 8 are respectively Nardostachys grandiflora H, aristolochicone, and Nardostachys grandiflora, which are well separated and have moderate areas. The accuracy of the quality control of Nardostachys grandiflora can be achieved by qualitative means with one or more of Nardostachys grandiflora H, aristolochicone, and Nardostachys grandiflora as references. The 10 or more characteristic peaks determined in the characteristic spectrum can well guarantee the accuracy of the method in the quality control of Nardostachys grandiflora.

[0045] 3. The method for determining the content of naringenone H and / or aristolochone in the water extract of naringensis and its preparations provided by the present invention can quickly and accurately determine the content of naringenone H, aristolochone, and the reference substance naringenone H and aristolochone in the water extract of naringensis and its preparations.

[0046] 4. The quality control method of the aqueous extract of Nardostachys grandiflora and its preparation provided by the present invention, by studying the characteristic spectrum and component content of the volatile oil in the aqueous extract of Nardostachys grandiflora and its preparation, can more clearly know the effective substance benchmark and biological benchmark of the decoction medicine taken by the patient. On the one hand, by controlling the quality of the components of the volatile oil in the aqueous extract of Nardostachys grandiflora and its preparation, the accuracy of medication and the consistency of dosage can be guaranteed. On the other hand, by establishing the characteristic spectrum and content determination method of the volatile oil in the aqueous extract of Nardostachys grandiflora and its preparation, it has a wide range of applications and can be widely used in production practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0048] Figure 1 is a chromatogram overlay of the test solution in Example 2 of the present invention;

[0049] Figure 2 is the reference characteristic spectrum in Example 2 of the present invention;

[0050] Figure 3 is the chromatographic peak identification diagram in Example 2 of the present invention;

[0051] Figure 4 It is the chromatogram of the negative control solution in Experimental Example 3 of the present invention - specificity test;

[0052] Figure 5 It is the chromatogram-specificity test of the mixed reference solution in Experimental Example 3 of the present invention;

[0053] Figure 6 It is the chromatogram-specificity test of the test solution in Experimental Example 3 of the present invention;

[0054] Figure 7 is a linear relationship diagram of the naringenone H reference substance in Experimental Example 3 of the present invention;

[0055] Figure 8 is a linear relationship diagram of the aristolochicone reference substance in Example 3 of the present invention;

[0056] Reference numerals: Peak 5: aristolochicone H; Peak 7: aristolochicone; Peak 8: aristolochicone.

[0057] Fig. 9 This is a comparison of chromatograms at different detection wavelengths in Experimental Example 1;

[0058] Fig.10 Chromatograms obtained with different mobile phase systems in Experimental Example 1;

[0059] Fig.11 The chromatograms obtained from different chromatographic columns in Experimental Example 1;

[0060] Fig.12 The chromatograms obtained with different extraction solvents in Experimental Example 2;

[0061] Fig.13 The chromatograms obtained with different injection volumes in Experimental Example 2;

[0062] Fig.14 This is the chromatogram of the reference medicinal material in Example 2. DETAILED DESCRIPTION

[0063] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0064] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0065] The instruments and reagents used in the present invention are as follows:

[0066] 1. Chromatograph: Waters Hclass chromatography system, including Quaternary Solvent Manager quaternary pump, FTN-H autosampler, PDA diode array detector, and Empower chromatography management system.

[0067] 2. Chromatographic column: Agilent InfinityLab Poroshell 120SB-C18, 2.1×100 mm, 1.9 μm.

[0068] 3. Other instruments: high-power CNC ultrasonic instrument (KQ-400KDB, Kunshan Ultrasonic Instrument Co., Ltd.), one-tenth electronic balance (ShimadzμAY120, Shimadzu Corporation, Japan), one-hundred-thousandth electronic balance (Sartoriμs SQP SECΜRA225D-1CN, Sartorius Scientific Instruments (Beijing) Co., Ltd.), digital display constant temperature water bath (HH-S6, Jiangsu Jinyi Instrument Technology Co., Ltd.).

[0069] 4. Reagents: Methanol and acetonitrile were of chromatographic grade (Merck); phosphoric acid (chromatographic grade, 85-90%, Aladdin); glacial acetic acid (HPLC, >99.9%, Aladdin); formic acid (chromatographic grade, ≥98%, Aladdin); water (Milli-Q).

[0070] 5. Test drug: Nardostachys grandiflora control medicinal material (purchased from Shanghai Hongyong Biotechnology Co., Ltd., batch number 290134-202206).

[0071] Gastrosinone H reference substance (purchased from Shanghai Hongyong Biotechnology Co., Ltd., batch number 070101-202405, purity ≥98%);

[0072] Aristolochicone reference substance (purchased from Shanghai Hongyong Biotechnology Co., Ltd., batch number 130157-202212, purity ≥98%);

[0073] Gastrosinone reference substance (purchased from Jiangxi Baicaoyuan Biotechnology Co., Ltd., batch number 002077-202401);

[0074] The information of Gansong decoction pieces is as follows:

[0075] Table 1 Information of Nardostachys grandiflora slices

[0076]

[0077] Nardostachys radix formula granules: take about 1000g of Nardostachys radix slices, weigh accurately, soak in water 10 times the weight of the Nardostachys radix slices for 30 minutes, heat and reflux for extraction for 30 minutes, collect the water extract, extract the volatile oil at the same time, add 8 times the amount of water to the filtered residue and boil for 25 minutes, filter while hot, combine the filtered decoction according to the mass ratio of volatile oil to β-cyclodextrin of 1:8, add β-cyclodextrin for grinding and inclusion, and prepare volatile oil inclusion compound for standby use. The water extract is filtered, the filtrate is concentrated into a clear paste, the volatile oil inclusion compound is added, dried, and an appropriate amount of auxiliary materials is added (the amount of auxiliary materials added accounts for 2% to 7% of the total mass of the particles), mixed, and made into granules.

[0078] Standard decoction of Nardostachys galanga slices: Take about 25g of Nardostachys galanga slices, weigh accurately, add water that is 10 times the weight of the Nardostachys galanga slices, soak for 30 minutes, boil for 30 minutes, and filter while hot; add 8 times the amount of water to the filtered residue, boil for 25 minutes, filter while hot, and combine the filtered decoctions to obtain the standard decoction of Nardostachys galanga slices.

[0079] Example 1

[0080] This embodiment provides a method for constructing a characteristic spectrum of volatile oil in a water extract of nard and its preparation, and a method for determining the content of aristolochone H and / or aristolochone in a water extract of nard and its preparation, comprising the following steps:

[0081] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.9 μm); acetonitrile was used as mobile phase A, 0.2% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 2 below; detection wavelength was 254 nm; column temperature was 25°C; flow rate was 0.2 ml per minute. The theoretical plate number calculated based on the galactone H peak should not be less than 3000.

[0082] Table 2 Elution gradient

[0083]

[0084] Preparation of reference solution Take 6g of naringa pine reference medicinal material, add 200ml of water, and keep the decoction slightly boiling for 5 hours to extract volatile oil according to the volatile oil determination method (Chinese Pharmacopoeia 2020 General Rules 2204 A method), collect the volatile oil, dissolve it in a 20ml volumetric flask with petroleum ether, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of naringa pine ketone H and aristolochic ketone reference substances, accurately weigh them, add petroleum ether to make a solution containing 50μg per 1ml, and use it as a reference substance reference mixed solution.

[0085] Take appropriate amounts of gabentone H, aristolochicone, and gabentone reference substance, accurately weigh them, and add petroleum ether to make solutions containing 50 μg per 1 ml, which serve as single reference substance solutions.

[0086] Preparation of the test solution Take about 25g of Nardostachys radix slices, weigh accurately, soak in water 10 times the weight of Nardostachys radix slices for 30 minutes, decoct for 30 minutes, and filter while hot; add 8 times the amount of water to the filtered residue and decoct for 25 minutes, filter while hot, and combine the filtered decoction to obtain the standard decoction of Nardostachys radix slices. The standard decoction of Nardostachys radix slices is kept slightly boiling for 5 hours to extract the volatile oil according to the volatile oil determination method (Method 2204A of the General Rules of the 2020 Edition of the Chinese Pharmacopoeia, no water is added), dissolve the volatile oil with petroleum ether, collect the petroleum ether layer, add petroleum ether to a 20ml volumetric flask, shake well, filter, and take the filtrate.

[0087] The determination method is to accurately pipette 2 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0088] The test results are shown in Table 3. There are 10 characteristic peaks in the chromatogram corresponding to the test solution, among which peak 5 corresponds to the retention time of the reference peak of galactone H, peak 7 corresponds to the retention time of the reference peak of aristolochicone, and peak 8 corresponds to the retention time of the reference peak of galactone; the peak corresponding to the peak of galactone H is the S peak, and the relative retention times of peaks 1 to 4 and peaks 6 to 10 are calculated, and their relative retention times are within the range of ±10% of the specified value. The specified values ​​are: 0.32 (peak 1), 0.70 (peak 2), 0.84 (peak 3), 0.88 (peak 4), 1.17 (peak 6), 1.31 (peak 7), 1.45 (peak 8), 1.66 (peak 9), and 1.90 (peak 10). The system adaptability parameters show that the method is feasible, the chromatographic peaks of the test sample can correspond to the reference, and the separation of each chromatographic peak is good.

[0089] Table 3 System adaptability parameters

[0090]

[0091]

[0092] Example 2

[0093] 18 batches of Nardostachys grandiflora slices were taken and the characteristic spectra of 18 batches of test solution were constructed according to the method of Example 1. The results are shown in Table 4. Figure 1 shown.

[0094] Table 4 Relative retention time results of characteristic spectra of 18 batches of Nardostachys grandiflora slices

[0095]

[0096]

[0097] The fingerprint similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate the reference characteristic spectrum, such as Figure 2 , as shown in Table 5-6. Figure 2 The control characteristic spectrum shown can be used to analyze and compare the test results of the characteristic spectrum of Nardostachys grandiflora slices, and is used for quality control of Nardostachys grandiflora slices. The specific method of using the control characteristic spectrum for quality control is as follows:

[0098] pass Figure 2-3 , Fig.14 It can be seen that there are 10 characteristic peaks in the characteristic spectrum of the Nardostachys radix slices, which correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material, among which peak 5 is the reference peak of the reference substance ...

[0099] The characteristic spectra of 18 batches of Nardostachys grandiflora slices were compared with the control characteristic spectra, and the similarity was between 0.90-1. The results are shown in Table 7.

[0100] Table 5 Relative retention time of the comparison chart of standard decoction of Nardostachys grandiflora slices

[0101]

[0102] Table 6 Common pattern matching data of Gansuo decoction pieces

[0103]

[0104] Table 7 Similarity results of characteristic spectra of 18 batches of Nardostachys grandiflora slices

[0105]

[0106]

[0107] Example 3

[0108] 18 batches of Nardostachys grandiflora slices were taken to detect the contents of naringenone H and aristolochone according to the method of Example 1, and the contents of naringenone H and / or aristolochone reference substances in the Nardostachys grandiflora aqueous extract and its preparations were calculated.

[0109] The results are shown in Table 8 below. From the results, it can be seen that the content of naringenone H in the volatile oil of multiple batches of naringensis formula granules ranges from 0.7% to 1.7%, and the content of aristolochone ranges from 0.7% to 1.6%. Through multiple batches of test data, the proportion of the two components in the naringensis formula granules can be obtained, providing a basis for the formulation of the quality standard of the naringensis formula granules.

[0110] Table 8 Determination results of naringenone H and aristolochone in standard decoction of naringensis slices

[0111]

[0112]

[0113] Experimental Example 1 Establishment of Liquid Chromatography Conditions

[0114] 1. Selection of detection wavelength

[0115] Take the test solution prepared according to the method of Example 1, inject it into the liquid chromatograph, select and compare the chromatograms under different absorption wavelengths commonly used in the study of nard, and the other chromatographic conditions are as shown in Example 1. The results are shown in Fig. 9 As shown in the table below, the optimal absorption wavelength is selected based on the detected chromatographic peak information and system adaptability parameters as the selection index, and it is determined that the difference in the detection results at 230nm~270nm is small. The peak separation is better at 230nm~270nm, so the detection at 230nm~270nm can be selected.

[0116] Table 9 Investigation results of different detection wavelengths

[0117]

[0118]

[0119] 2. Selection of organic phase components of mobile phase

[0120] Take 5g of Gansong formula granules, add 200ml of water, and keep the decoction at a slight boil for 5 hours to extract the volatile oil according to the volatile oil determination method (Method 2204A of the General Rules of the 2020 Edition of the Chinese Pharmacopoeia). Collect the volatile oil, dissolve it in a 20ml volumetric flask with petroleum ether, shake well, filter, and take the filtrate to prepare the test solution.

[0121] The mixture was injected into a liquid chromatograph to compare the effects of different mobile phase components, 0.2% phosphoric acid (mobile phase B)-methanol (mobile phase A), 0.2% acetic acid (mobile phase B)-acetonitrile (mobile phase A), and 0.2% formic acid (mobile phase B)-acetonitrile (mobile phase A), on the chromatographic peak separation of the volatile oil components in the Nardostachys grandiflora granules. The other chromatographic conditions are shown in Example 1. The results are shown in Fig.10As shown in the table below. It is determined that the difference can be detected under phosphoric acid, formic acid, and acetic acid. Therefore, this method can be detected under 0.2% phosphoric acid, 0.2% formic acid, and 0.2% acetic acid.

[0122] Table 10 Results of investigation of different mobile phases

[0123]

[0124]

[0125] 3. Selection of different chromatographic columns

[0126] The test solution prepared in Item 2 of this Experimental Example was injected into a liquid chromatograph and measured using different brands of octadecylsilane bonded silica gel chromatographic columns (chromatographic column 1: Agilent InfinityLab Poroshell 120SB-C18, 2.1×100mm, 1.9μm; chromatographic column 2: Waters HSS T3 (1.8μm, 2.1×100mm); chromatographic column 3: Shim-pack GIST-HPC18-AQ (1.9μm, 2.1×100mm), and the other chromatographic conditions were the same as those in Example 1. The results are shown in Table 1. Fig.11 As shown in the table below. Through testing, it can be concluded that different chromatographic columns have large differences in determination. The Agilent InfinityLab Poroshell120SB-C18 chromatographic column has the best detection effect and good separation of each chromatographic peak, so this column is preferred for detection.

[0127] Table 11 Results of investigation on different chromatographic columns

[0128]

[0129]

[0130] 4. Determination of chromatographic conditions

[0131] According to the above optimization results, the chromatographic conditions of the characteristic spectrum are determined as follows:

[0132] Octadecylsilane bonded silica gel was used as the filler (column length was 100 mm, inner diameter was 2.1 mm, particle size was 1.9 μm); acetonitrile was used as mobile phase A, 0.2% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 12 below; the flow rate was 0.2 ml per minute, the column temperature was 25°C, and the detection wavelength was 254 nm.

[0133] Table 12 Gradient elution program

[0134]

[0135] Experimental Example 2 Establishment of the preparation method of the test solution

[0136] 1. Selection of extraction solvent

[0137] The extraction effects of different extraction solvents (petroleum ether, cyclohexane, and dichloromethane) on the volatile oil of Nardostachys grandiflora granules were investigated. The test solution was prepared by the method prepared in Item 2 of Experimental Example 1 and tested by high performance liquid chromatography. The only difference was the extraction solvent. The appropriate extraction solvent was selected by comparing the chromatograms of the test samples obtained with different extraction solvents and the system adaptability parameters. The results are shown in Fig.12 As shown in the table below, the three extraction solvents can extract characteristic spectra, and the characteristic spectra are not much different. However, petroleum ether has the best extraction effect on the test sample, and the system adaptability parameters are relatively good, so petroleum ether (60-90℃) is selected as the extraction solvent.

[0138] Table 13 Results of investigation on different extraction solvents

[0139]

[0140] 2. Investigation of injection volume

[0141] The effects of different injection volumes (1 μl, 2 μl, 3 μl, 5 μl) on the chromatogram of the test sample and the system suitability parameters were investigated. The test sample solution prepared in Item 2 of Experimental Example 1 was used for HPLC detection. The only difference was the injection volume. The results are shown in Fig.13 As shown in the table below, after comparative analysis, it was found that the system adaptability parameters of the test sample were relatively good when the injection volume was 1μl to 5μl, and the injection volume can be temporarily set to 2μl as the method detection injection volume.

[0142] Table 14 Injection volume investigation results

[0143]

[0144]

[0145] 3. Determination of the preparation method of the test solution

[0146] According to the above optimization results, the preparation method of the test solution was determined as follows: take about 25 g of Nardostachys galanga slices, accurately weigh, add 10 times the water, soak for 30 minutes, decoct for 30 minutes, and filter while hot; add 8 times the water to the residue, decoct for 25 minutes, combine the decoctions, and keep the decoctions slightly boiling for 5 hours to extract the volatile oil according to the volatile oil determination method (Method 2204A of the General Rules of the 2020 Edition of the Chinese Pharmacopoeia). Collect the volatile oil, dissolve it in a 20 ml volumetric flask with petroleum ether, shake well, filter, and take the filtrate.

[0147] Experimental Example 3 Methodological Verification

[0148] 1. Precision test

[0149] The test solution was prepared according to the preparation method of the test solution in Example 1, and the test solution was repeatedly injected 6 times for detection according to the chromatographic conditions in Example 1, and the retention time and peak area were recorded, and the relative retention time (t / ts) and relative peak area of ​​the common peak were calculated.

[0150] Taking peak 5 (galbanum H) as the S peak, the relative retention times and relative peak areas of peaks 1-4 and 6-10 were calculated.

[0151] The results are shown in Tables 15-16 below. From the results, it can be seen that the RSD of the relative retention time of each characteristic peak and the reference S peak is less than 2%, and the RSD of the peak area corresponding to galactone H and aristolochicone is less than 2.0%, indicating that the precision of the instrument is relatively good.

[0152] Table 15 Instrument precision relative retention time test results

[0153]

[0154] Table 16 Precision test results

[0155]

[0156] 2. Method repeatability test

[0157] Take the same batch of standard decoction of Nardostachys chinensis slices and prepare 6 test solutions according to the preparation method of the test solution in Example 1. The test was carried out according to the liquid phase conditions in Example 1, and the retention time and peak area were recorded, and the relative retention time (t / ts) and relative peak area of ​​the common peak were calculated.

[0158] The results are shown in Tables 17-18 below. From the results, it can be seen that the RSD of the relative retention time of each characteristic peak and the reference S peak is less than 2%, and the RSD of the peak area corresponding to naringenone H and aristolochicone is less than 2%, indicating that the method has good repeatability.

[0159] Table 17 Method repeatability relative retention time test results

[0160]

[0161] Table 18 Method repeatability test results

[0162]

[0163] 3. Intermediate precision test (different operators)

[0164] Three inspectors, at different times, took the same batch of standard decoction of Nardostachys chinensis slices to prepare test solutions according to the preparation method of the test solution in Example 1, and used the same equipment to determine the relative retention time and peak area of ​​each common peak of the prepared test solutions, and calculated the relative retention time and relative peak area.

[0165] The results are shown in Table 19. From the results, it can be seen that the RSD of the relative retention time of each characteristic peak and the reference S peak is less than 2%, indicating that the intermediate precision of this method is good.

[0166] Table 19 Intermediate precision relative retention time test results (different operators)

[0167]

[0168] 4. Specificity test

[0169] Select blank solvent as negative control solution (petroleum ether), prepare test solution according to the preparation method of test solution in Example 1, and detect negative control solution and test solution according to the chromatographic conditions under Example 1.

[0170] Test results see Figure 4-6 As can be seen from the figure, there is no interference in the negative, and the method has good specificity.

[0171] 5. Stability test

[0172] The test solution was prepared according to the preparation method of the test solution in Example 1 with the same batch of standard decoction of Nardostachys chinensis slices and tested according to the liquid phase conditions in Example 1. The test solution was injected 0, 2, 4, 8, 12, and 24 hours after preparation, and the retention time and peak area of ​​the common peak were recorded, and the relative retention time and relative peak area were calculated.

[0173] The results are shown in Tables 20-21 below. From the results, it can be seen that the RSD value of the relative retention time of each characteristic peak and the reference S peak is less than 2.0%, and the RSD value of the peak area is less than 2.0%, indicating that the test solution is stable within 24 hours and meets the measurement requirements.

[0174] Table 20 Stability relative retention time test results

[0175]

[0176]

[0177] Table 21 Stability test results

[0178]

[0179] 6. Durability test

[0180] A. Investigation of different flow rates

[0181] The test solution was prepared according to the preparation method of the test solution in Example 1, and the test solution was sampled and tested basically according to the chromatographic conditions in Example 1, with the only difference that the measurement was carried out at flow rates of 0.22 ml / min, 0.2 ml / min, and 0.18 ml / min to examine the separation effect of each characteristic peak of this product when the flow rate changes.

[0182] The results are shown in Tables 22-23 below. From the results, it can be seen that at different flow rates, each chromatographic peak was detected, the separation effect was good, and the peak area RSD value was less than 2.0%, indicating that the method is durable at different flow rates.

[0183] Table 22 Relative retention time results at different flow rates

[0184]

[0185] Table 23 Determination results of naringenone H and aristolochicone at different flow rates

[0186]

[0187] B. Investigation of different column temperatures

[0188] The test solution was prepared according to the preparation method of the test solution in Example 1, and the test solution was sampled and tested basically according to the chromatographic conditions in Example 1, with the only difference that the measurement was carried out at column temperatures of 20°C, 25°C, and 30°C to examine the separation effect of each characteristic peak of this product when the column temperature changes.

[0189] The results are shown in Tables 24-25 below. From the results, it can be seen that at different column temperatures, all chromatographic peaks were detected, the separation effect was good, and the peak area RSD value was less than 2.0%, indicating that the method is durable at different column temperatures.

[0190] Table 24 Comparison of relative retention time determination results at different column temperatures

[0191]

[0192] Table 25 Determination results of different column temperatures

[0193]

[0194] 7. Linear relationship investigation

[0195] Study on the linear relationship of naringenone H control concentration Take an appropriate amount of naringenone H reference substance, accurately weigh it, add petroleum ether to make solutions containing 313.7μg, 125.5μg, 62.7μg, 31.4μg, 12.5μg, 6.27μg, and 3.14μg per 1mL, respectively, accurately draw 2μL of the above naringenone H reference substance solutions of different concentrations and inject them into the liquid chromatograph to determine the peak area, and draw a standard curve with the injection amount of naringenone H as the horizontal axis and the peak area integral value as the vertical axis. The results are shown in Table 26 and Figure 7 , the regression equation is: y = 25003x + 21023, R 2 =0.9997. The experimental results show that the linear relationship is good in the range of injection concentration of naringenone H from 3.14 μg / ml to 313.7 μg / ml.

[0196] Table 26 Results of investigation on linear relationship of different concentrations of naringenone H

[0197]

[0198] Study on the linear relationship of aristolochicone control concentration Take an appropriate amount of aristolochicone reference substance, accurately weigh it, add petroleum ether to make solutions containing 142.4μg, 56.9μg, 28.5μg, 14.2μg, 5.7μg, 2.85μg, and 1.42μg per 1mL, accurately draw 2μL of the above aristolochicone reference substance solutions of different concentrations and inject them into the liquid chromatograph to determine the peak area, and draw a standard curve with the injection amount of aristolochicone as the horizontal axis and the peak area integral value as the vertical axis. The results are shown in Table 27 and Figure 8 , the regression equation is: y = 82738x + 35519, R 2 =0.9998. The experimental results show that the linear relationship of aristolochicone is good in the range of 1.42μg / ml to 142.4μg / ml.

[0199] Table 27 Results of investigation on linear relationship of different concentrations of aristolochicone

[0200]

[0201] 8. Accuracy test

[0202] Accurately weigh about 12.5g of 6 portions of standard decoction of Nardostachys odorifera (the content of Nardostachys odorifera H is 0.0126mg / g, and the content of Aristolochicone is 0.0115mg / g), respectively extract the volatile oil according to the preparation method of the test solution in Example 1, accurately add 10ml of the mixed reference solution (the concentration of Nardostachys odorifera H is 0.0152mg / g, and the concentration of Aristolochicone is 0.0136mg / g) to the volatile oil, and then dilute to a 20ml volumetric flask with petroleum ether, shake well, filter, and take the subsequent filtrate. Determine and calculate the recovery rate according to the liquid phase method in Example 1.

[0203] The results are shown in Tables 28-29 below. From the results in the table, it can be seen that the average recovery rate of aristolochicone H is 99.2%, the RSD value is 1.9%, and the average recovery rate of aristolochicone is 100.2%, the RSD value is 0.6%. Therefore, this method has good accuracy.

[0204] Table 28 Naphtalol H accuracy test results

[0205]

[0206] Table 29 Aristolochicone accuracy test results

[0207]

[0208] From the above methodological investigation results, it can be seen that among the 10 common peaks in the characteristic spectrum of the standard decoction of Nardostachys chinensis established in Example 1, the peak height of each characteristic peak is affected to a certain extent under different HPLC conditions, so the specified value range of the relative retention time is controlled within ±10%.

[0209] The above methodological research results show that this method meets the requirements of content determination and can be used to determine the content of naringenone H in the standard decoction of Nardostachys galanga slices.

[0210] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for constructing a characteristic spectrum of volatile oil in a water extract of nard and its preparation, characterized in that: The following steps are involved: (1) Preparation of test solution; (2) The sample solution is tested by high performance liquid chromatography, the filler is octadecylsilane bonded silica gel, and the mobile phase includes acetonitrile and acid-containing aqueous solution; Gradient elution: the gradient elution program includes: 0→30 minutes→35 minutes, and the volume percentage of acetonitrile in the mobile phase is 40%→50%→55%.

2. The construction method according to claim 1, characterized in that: Step (1) comprises: 1) Take the test sample and extract the volatile oil; 2) dissolving the volatile oil in an organic solvent and collecting the organic solvent layer to obtain the test solution; Preferably, step (1) also satisfies any one or more of the following AD: A. In step 1), volatile oil is extracted by hot reflux extraction; B. In step 2), the organic solvent is selected from at least one of petroleum ether, cyclohexane and dichloromethane; C. In step 1), the test sample is selected from an aqueous extract of nardostachys grandiflora or a preparation made from an aqueous extract of nardostachys grandiflora; D. The volume ratio of the raw material nard used in step 1) to the test solution prepared in step 2) is 5-30 g:10-30 ml.

3. The construction method according to claim 1, characterized in that: The volume percentage of phosphoric acid in the acid-containing aqueous solution is 0.18-0.25%; and / or the column length of the chromatographic column is 100 mm, the inner diameter is 2.1 mm, and the particle size is 1.8-1.9 μm; and / or the acid in the acid-containing aqueous solution is selected from one or more of phosphoric acid, acetic acid, and formic acid.

4. The construction method according to any one of claims 1 to 3, characterized in that: The chromatographic conditions of the high performance liquid chromatography in step (2) also include: a detection wavelength of 220-280 nm, a flow rate of 0.18-0.27 ml / min, a column temperature of 20-30° C., and an injection volume of 1 μl-5 μl; preferably, the detection wavelength is 230-270 nm.

5. The construction method according to any one of claims 1 to 4, characterized in that: The construction method also includes the step of preparing a reference solution using at least one reference substance selected from the group consisting of galantol H, aristolochicone, and galantol, and the step of detecting the reference solution by high performance liquid chromatography according to the construction method described in any one of claims 1 to 4 to obtain a reference spectrum of the reference substance; preferably, the concentration of the reference solution is 10-100 μg / mL.

6. The construction method according to any one of claims 1 to 5, characterized in that: The characteristic spectrum of the volatile oil in the nardostachys water extract and the preparation thereof has 10 characteristic peaks, the peak corresponding to the peak of the nardostachys H reference substance is peak No. 5, peak No. 5 is taken as the reference peak, and the relative retention time of characteristic peaks 1-4 and peaks 6-10 and the reference peak is within the range of ±10% of the specified value; the specified value is: 0.32 (peak 1), 0.70 (peak 2), 0.84 (peak 3), 0.88 (peak 4), 1.17 (peak 6), 1.31 (peak 7), 1.45 (peak 8), 1.66 (peak 9), 1.90 (peak 10).

7. A method for determining the content of aristolochone H and / or aristolochone in a water extract of nard and its preparation, characterized in that: The steps include: Preparation of test solution; Preparation of aristolochone H and / or aristolochone reference solution; Test steps: Take the test solution and the reference solution and detect them respectively by high performance liquid chromatography. The filler is octadecylsilane bonded silica gel. The mobile phase includes acetonitrile and acid-containing aqueous solution. Gradient elution is performed. The gradient elution program includes: 0→30 minutes→35 minutes. The volume percentage of acetonitrile in the mobile phase is 40%→50%→55%. Calculate the content of aristolochone H and / or aristolochone reference in the water extract of naringa and its preparations.

8. The content determination method according to claim 7, characterized in that: Step (1) comprises: 1) Take the test sample and extract the volatile oil; 2) dissolving the volatile oil in an organic solvent and collecting the organic solvent layer to obtain the test solution; Preferably, step (1) also satisfies any one or more of the following AD: A. In step 1), volatile oil is extracted by hot reflux extraction; B. In step 2), the organic solvent is selected from at least one of petroleum ether, cyclohexane and dichloromethane; C. In step 1), the test sample is selected from an aqueous extract of nardostachys grandiflora or a preparation made from an aqueous extract of nardostachys grandiflora; D. The volume ratio of the raw material nard used in step 1) to the test solution prepared in step 2) is 5-30 g:10-30 ml.

9. The content determination method according to claim 7 or 8, characterized in that: The volume percentage of phosphoric acid in the acid-containing aqueous solution is 0.18-0.25%; and / or, the column length of the chromatographic column is 100 mm, the inner diameter is 2.1 mm, and the particle size is 1.8-1.9 μm; and / or, the acid in the acid-containing aqueous solution is selected from one or more of phosphoric acid, acetic acid, and formic acid; and / or, the chromatographic conditions of the high performance liquid chromatography in step (2) also include: a detection wavelength of 220-280 nm, a flow rate of 0.18-0.27 ml / min, a column temperature of 20-30° C., and an injection volume of 1 μl-5 μl.

10. A quality control method for a water extract of nard and a preparation thereof, characterized in that: The method comprises the steps of obtaining a characteristic spectrum from a tested nard product by the construction method described in any one of claims 1 to 6 and / or the steps of determining the content of aristolochone H and / or aristolochone in the tested nard product by the content determination method described in any one of claims 7 to 9.

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