Method for constructing characteristic chromatogram of volatile oil in water extract of glycyrrhiza glabra and its preparation and method for determining content of volatile oil

Abstract

The present application relates to the technical field of traditional Chinese medicine detection, and specifically provides a construction method and content determination method of a characteristic chromatogram of volatile oil in a mannsson water extract and preparation thereof. A specific elution procedure is obtained through a large number of experiments by using octadecylsilane bonded silica gel as a filler and a mobile phase including acetonitrile and an acid-containing aqueous solution for gradient elution, 10 or more common characteristic peaks are obtained, and effective separation of characteristic peaks containing mannsson ketone H, aristolane ketone and mannsson ketone is achieved, the peak shape is good, the baseline is smooth, the determined components can be completely separated, the detection time is short, and the method provides a basis for quality detection and control of the mannsson water extract and preparation thereof, can fully reflect the integrity and characteristics of the mannsson water extract and preparation thereof, and is simple to operate and applicable to quality control of different preparation types of mannsson.

Description

Technical Field

[0001] The present 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 water extract of spikenard and a preparation thereof and a method for determining the content of the volatile oil. Background Art

[0002] Nardostachys jatamansi DC. is the dried root and rhizome of Nardostachys jatamansi DC., a plant of the Patrinaceae family. This product was first recorded in the Four Medical Classics and is known as Bangbei in Tibetan. It is pungent, sweet, and warm in nature; it enters the spleen and stomach meridians, and has the effects of regulating qi, 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 treat beriberi. The chemical components of Nardostachys jatamansi primarily include terpenes and flavonoids. Terpenoid compounds are the main active ingredients of Nardostachys jatamansi and are present in high concentrations, making them a type of volatile oil.

[0003] Common dosage forms of ganjiang preparations include decoctions, powders, and formula granules. For example, standard decoctions are single-ingredient Chinese herbal medicine decoctions prepared using standardized processes, guided by Traditional Chinese Medicine theory and based on clinical application, with reference to hospital preparation extraction methods. As a standard substance, the properties of standard decoctions are closer to those of clinical medications and decoctions taken by patients. Formula granules are obtained from ganjiang preparations through water extraction, concentration, drying, and preparation. Their material basis differs significantly from that of the medicinal materials.

[0004] Currently, there are no reports on the volatile oils in Nardostachys japonica aqueous extracts and their preparations. Existing literature primarily focuses on the water-soluble components in these extracts. However, due to the low content of volatile oils in Nardostachys japonica aqueous extracts and their preparations, existing methods are not suitable for the quality detection of volatile oils in these extracts, resulting in a small number of characteristic peaks and poor separation.

[0005] There is currently no such a problem in the qualitative detection of characteristic patterns of volatile oils in Nardostachys grandiflora aqueous extracts and their preparations using high performance liquid chromatography. How to improve the detection standards of volatile oils in Nardostachys grandiflora aqueous extracts and their 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 present invention aims to provide a method for constructing a characteristic spectrum of volatile oil in an aqueous extract of naringa 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 the aqueous extract of naringa 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 can be used for quality detection to comprehensively control the quality of the finished product.

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

[0008] (1) Preparation of test solution;

[0009] (2) The test solution was tested by high performance liquid chromatography using octadecylsilane bonded silica gel as the filler and acetonitrile and acid-containing aqueous solution as the mobile phase;

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

[0011] Furthermore, step (1) includes:

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

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

[0014] Preferably, step (1) further 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 truncatus or a preparation made from an aqueous extract of nardus truncatus;

[0018] D. Preparation: The volume ratio of the raw material nardus used for the test sample in step 1) to the test sample 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 galbanum H, aristolochicone, and galbanum, 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 water extract of naringa and its preparation has 10 characteristic peaks, the peak corresponding to the peak of the naringa ketone H reference substance is peak No. 5, and peak No. 5 is used as the reference peak. The relative retention times of the characteristic peaks 1-4 and peaks 6-10 and the reference peak are 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), and 1.90 (peak 10).

[0023] The present invention also provides a method for determining the content of naringenone H and / or aristolochone in a water extract of naringensis 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, and the gradient elution is performed. The gradient elution program includes: 0→30 minutes→35 minutes, and the volume percentage of acetonitrile in the mobile phase is 40%→50%→55%. Calculate the content of naringenone H and / or aristolochicone reference substance in the aqueous extract of naringensis and its preparations.

[0027] Furthermore, step (1) includes:

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

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

[0030] Preferably, step (1) further 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 truncatus or a preparation made from an aqueous extract of nardus truncatus;

[0034] D. Preparation: The volume ratio of the raw material nardus used for the test sample in step 1) to the test sample 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 spikenard water extract and a preparation thereof, comprising the steps of taking a spikenard product to be tested and constructing a characteristic spectrum according to any of the construction methods described above and / or the steps of taking a spikenard product to be tested and determining the content of spikenardone H and / or aristolochone in the spikenard 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 naringa and its preparation also includes a step of comparing the characteristic spectrum of the naringa product to be tested with the characteristic spectrum of the volatile oil in the water extract of naringa and its preparation; the characteristic spectrum of the naringa product to be tested is obtained using the naringa 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. The naringa preparation comprises the aqueous extract of naringa and also includes pharmaceutically acceptable excipients; preferably, the preparation is selected from one or more of decoctions (e.g., standard decoction of naringa slices), powders (e.g., freeze-dried powder of standard decoction of naringa slices), tablets, capsules, pills, granules (e.g., naringa formula granules), and injections.

[0039] Furthermore, the pharmaceutically acceptable excipients are selected from one or more of fillers, disintegrants, lubricants, suspending agents, binders, flavoring agents, preservatives, and bases; preferably, the fillers are selected from one or more of starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, and sucrose; preferably, the disintegrants are 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 lubricants are selected from one or more of magnesium stearate, sodium lauryl sulfate, talc, and silicon dioxide; preferably, the suspending agents are selected from polyvinyl pyrrolidone. Preferably, the binder is selected from one or more of starch slurry, polyvinyl pyrrolidone, and hydroxypropyl methylcellulose; preferably, the flavoring agent is selected from one or more of saccharin sodium, aspartame, sucrose, sodium cyclamate, and glycyrrhetinic acid; preferably, the preservative is selected from one or more of parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate, and eucalyptus oil; preferably, the matrix is ​​selected from one or more of PEG6000 (polyethylene glycol 6000), PEG4000 (polyethylene glycol 4000), and insect wax.

[0040] A standard decoction of Nardostachys radix slices can be prepared by filtering the aqueous extract. A freeze-dried powder of the standard decoction of Nardostachys radix slices can be prepared by freeze-drying the standard decoction of Nardostachys radix slices. Nardostachys radix granules can be prepared by filtering and concentrating the aqueous extract, adding a volatile oil inclusion compound, and optionally adding excipients. Preferably, the concentrated extract has a relative density of 1.05-1.10 g / m3 at 58-62°C.

[0041] In the present invention, the water extract of nardus truncatus can be prepared by an existing conventional method, for example, by extracting nardus truncatus with water to obtain the water extract of nardus truncatus. 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 for each extraction is 15-60 minutes; preferably, the mass of water added for each extraction is 6-12 times the mass of nardus truncatus.

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

[0043] 1. The present invention provides a method for constructing a characteristic spectrum of volatile oil in an aqueous extract of jasmine and a preparation thereof, using octadecylsilane bonded silica gel as a filler, a mobile phase comprising acetonitrile and an acidic aqueous solution, and gradient elution. A specific elution procedure is obtained through a large number of experimental screenings, and 10 or more common characteristic peaks are obtained. Effective separation of characteristic peaks comprising jasmine ketone H, aristolochicone, and jasmine ketone is achieved, with good peak shape, a stable baseline, complete separation of the measured components, and a short detection time. This method provides a basis for quality inspection and control of the aqueous extract of jasmine and a preparation thereof, can fully reflect the integrity and characteristic properties of the aqueous extract of jasmine and a preparation thereof, is simple to operate, and can be applied to quality control of different types of jasmine preparations.

[0044] 2. The method for constructing a characteristic spectrum of the volatile oil in the aqueous extract of jasmine and its preparation provided by the present invention, wherein peaks 5, 7, and 8 are identified by reference substances as jasmine ketone H, aristolochicone, and jasmine ketone, respectively, and are well separated and have moderate areas. The accuracy of jasmine quality control can be achieved qualitatively by using one or more of jasmine ketone H, aristolochicone, and jasmine ketone as references. 10 or more characteristic peaks determined in the characteristic spectrum can well ensure the accuracy of the method in the quality control of jasmine.

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

[0046] 4. The quality control method of the aqueous extract of naringa and its preparation provided by the present invention, by studying the characteristic spectrum and the component content of volatile oil in the aqueous extract of naringa and its preparation, can more clearly know the effective substance benchmark and the biological benchmark of the patient taking decoction medicine. On the one hand, by controlling the quality of the component of volatile oil in the aqueous extract of naringa 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 the content assay method of volatile oil in the aqueous extract of naringa and its preparation, there is 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 embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

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

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

[0050] Figure 3 This 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 This 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 of the test solution in Experimental Example 3 of the present invention - specificity test;

[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 symbols: Peak 5: aristolochicone H; Peak 7: aristolochicone; Peak 8: aristolochicone.

[0057] Figure 9 Comparison of chromatograms at different detection wavelengths in Experimental Example 1;

[0058] Figure 10 The chromatograms obtained with different mobile phase systems in Experimental Example 1;

[0059] Figure 11 The chromatograms obtained with different chromatographic columns in Experimental Example 1;

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

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

[0062] Figure 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 and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection 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 this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[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 digitally controlled ultrasonic instrument (KQ-400KDB, Kunshan Ultrasonic Instrument Co., Ltd.), 1 / 10,000 electronic balance (ShimadzμAY120, Shimadzu Corporation, Japan), 1 / 100,000 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); and water (Milli-Q).

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

[0071] Nardone 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 slices is as follows:

[0075] Table 1 Information of Gansong decoction pieces

[0076]

[0077] Nardostachys radix granules: Take approximately 1000 g of Nardostachys radix slices, accurately weigh them, soak them in water 10 times the weight of the slices for 30 minutes, then heat and reflux for extraction for 30 minutes. Collect the water extract and simultaneously extract the volatile oil. Add 8 times the amount of water to the filtered medicinal residue and boil it for 25 minutes. Filter it while hot. Combine the filtered decoctions and grind and include β-cyclodextrin in a ratio of volatile oil to β-cyclodextrin of 1:8 to obtain a volatile oil inclusion compound for later use. Filter the water extract, concentrate the filtrate into a clear paste, add the volatile oil inclusion compound, dry it, add an appropriate amount of excipients (the amount of excipients added accounts for 2% to 7% of the total mass of the granules), mix well, and make granules.

[0078] Standard decoction of Nardostachys galanga slices: Take about 25g of Nardostachys galanga slices, weigh accurately, add 10 times the weight of water to soak for 30 minutes, then boil for 30 minutes and filter while hot; add 8 times the amount of water to the filtered residue and boil for 25 minutes, filter while hot, 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 an aqueous extract of nardus serrata and its preparation, and a method for determining the content of naringenone H and / or aristolochone in an aqueous extract of nardus serrata and its preparation, comprising the following steps:

[0081] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.9 μm); acetonitrile as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, with gradient elution as specified in Table 2 below; detection wavelength, 254 nm; column temperature, 25°C; flow rate, 0.2 ml / min. The number of theoretical plates, calculated based on the naringenone H peak, should be no less than 3000.

[0082] Table 2 Elution gradient

[0083]

[0084] Preparation of reference solution: Take 6g of the reference medicinal material Nardostachys grandiflora, add 200ml of water, and boil the decoction for 5 hours according to the volatile oil determination method (Method 2204A of the General Rules of the 2020 Edition of the Chinese Pharmacopoeia) to extract the volatile oil. Collect the volatile oil, dissolve it with petroleum ether and make up to the volume in a 20ml volumetric flask, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Separately, take appropriate amounts of Nardostachys grandiflora H and Aristolochicone reference substances, accurately weigh them, and add petroleum ether to make a solution containing 50μg per 1ml, which is used as the reference substance mixed solution.

[0085] Take appropriate amounts of galbanum ketone H, aristolochicone, and galbanum ketone 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 serrata slices, accurately weigh them, add 10 times the weight of water to soak for 30 minutes, then boil for 30 minutes and filter while hot; add 8 times the amount of water to the filtered residue and boil for 25 minutes, filter while hot, and combine the filtered decoction to obtain the standard decoction of Nardostachys serrata slices. The standard decoction of Nardostachys serrata slices is kept 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, 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] Determination method: Accurately aspirate 2μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0088] The test results are shown in Table 3. The chromatogram corresponding to the test solution showed 10 characteristic peaks, of which Peak 5 corresponded to the retention time of the galbanum H reference peak, Peak 7 corresponded to the retention time of the aristolochicone reference peak, and Peak 8 corresponded to the retention time of the galbanum H reference peak. The peak corresponding to the galbanum H reference peak was the S peak. The relative retention times of Peaks 1 to 4 and Peaks 6 to 10 were calculated and were within ±10% of the specified values. The specified values ​​were: 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 suitability parameters indicate that the method is feasible, the test sample chromatographic peaks can correspond to the reference, and the resolution of each chromatographic peak is good.

[0089] Table 3 System adaptability parameters

[0090]

[0091]

[0092] Example 2

[0093] 18 batches of Nardostachys serrata 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 serrata slices for quality control of Nardostachys serrata slices. The specific method of using the control characteristic spectrum for quality control is as follows:

[0098] pass Figure 2-3 、 Figure 14 It can be seen that there are 10 characteristic peaks in the characteristic spectrum of the Nardostachys serrata slice, which correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material. Among them, Peak 5 is the reference peak of the reference substance ...

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

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

[0101]

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

[0103]

[0104] Table 7 Similarity results of characteristic spectra of 18 batches of Gansong decoction pieces

[0105]

[0106]

[0107] Example 3

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

[0109] The results are shown in Table 8 below. The results show that the content of naringenone H in the volatile oil of multiple batches of naringenan formula granules ranges from 0.7% to 1.7%, and the content of aristolochone ranges from 0.7% to 1.6%. This multi-batch testing data allows us to determine the proportions of these two components in naringenan formula granules, providing a basis for the formulation of quality standards for naringenan 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 and inject it into the liquid chromatograph. Select the chromatogram under different absorption wavelengths commonly used in the study of nard. The other chromatographic conditions are as shown in Example 1. The results are shown in Figure 9 As shown in the table below, the optimal absorption wavelength was selected based on the amount of detected chromatographic peak information and system suitability parameters. It was determined that the difference in detection results was minimal between 230nm and 270nm. Peaks were well resolved between 230nm and 270nm, so detection at 230nm to 270nm was the preferred wavelength.

[0116] Table 9 Investigation results of different detection wavelengths

[0117]

[0118]

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

[0120] Take 5 g of Gansong formula granules, add 200 ml 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 with petroleum ether and make up to the volume in a 20 ml volumetric flask, shake well, filter, and take the filtrate to prepare the test solution.

[0121] The liquid 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 Gansong formula granules. The other chromatographic conditions are shown in Example 1. The results are shown in Figure 10As shown in the table below, it was determined that the differences in detection were small in phosphoric acid, formic acid, and acetic acid. Therefore, this method can be used for detection in 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, 2.1×100 mm, 1.9 μm; chromatographic column 2: Waters HSS T3 (1.8 μm, 2.1×100 mm); chromatographic column 3: Shim-pack GIST-HPC18-AQ (1.9 μm, 2.1×100 mm). The other chromatographic conditions were the same as in Example 1. The results are shown in FIG. Figure 11 As shown in the table below, the results show that different chromatographic columns have significant differences in detection. The Agilent InfinityLab Poroshell120SB-C18 column has the best detection effect and good separation of each chromatographic peak, so this column is the preferred choice 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: 100 mm, inner diameter: 2.1 mm, particle size: 1.9 μm); acetonitrile was used as mobile phase A, and 0.2% aqueous phosphoric acid was used as mobile phase B. Gradient elution was performed according to the requirements 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 essential oil of Nardostachys quinoa granules were investigated. The test solution was prepared using the method described in Experimental Example 1, Item 2, and then subjected to HPLC. The only difference was the extraction solvent. By comparing the chromatograms of the test sample obtained with different extraction solvents and the system adaptability parameters, the appropriate extraction solvent was selected. The results are shown in Figure 12 As shown in the table below, all three extraction solvents can produce characteristic spectra with minimal differences. However, petroleum ether provides the best extraction results for the test sample, with relatively superior system adaptability parameters. Therefore, petroleum ether (60-90°C) was selected as the extraction solvent.

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

[0139]

[0140] 2. Injection volume investigation

[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 Table 1. Figure 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 25g of Nardostachys odoratus 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 decoction, and keep the decoction slightly boiling for 5 hours according to the volatile oil determination method (Method 2204A of the General Rules of the 2020 Edition of the Chinese Pharmacopoeia) to extract the volatile oil. Collect the volatile oil, dissolve it with petroleum ether and make up to a 20ml volumetric flask, shake well, filter, and take the filtrate.

[0147] Experimental Example 3 Methodology 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 according to the chromatographic conditions in Example 1 for detection. 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 (naringone 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 galanginone H and aristolochicone is less than 2.0%, indicating that the instrument precision 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 glutinosa 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. 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 reproducibility.

[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 glutinosa slices to prepare test solutions according to the preparation method of the test solution in Example 1. The prepared test solutions were measured with the same equipment for the relative retention time and peak area of ​​each common peak, and the relative retention time and relative peak area were calculated.

[0165] The results are shown in Table 19. As can be seen from the results, 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 are shown in Figure 4-6 As can be seen from the figure, there is no interference in the negative test, and the method has good specificity.

[0171] 5. Stability inspection

[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 detected 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 values ​​of the relative retention times of each characteristic peak and the reference S peak are less than 2.0%, and the RSD values ​​of the peak areas are 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, except 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. As can be seen from the results, at different flow rates, 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 robust 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 injected and tested basically according to the chromatographic conditions in Example 1, except that the measurement was carried out at column temperatures of 20°C, 25°C, and 30°C to examine the separation effect of the characteristic peaks of this product when the column temperature changed.

[0189] The results are shown in Tables 24-25 below. As can be seen from the results, 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 robust at different column temperatures.

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

[0191]

[0192] Table 25 Results of different column temperatures

[0193]

[0194] 7. Linear relationship investigation

[0195] Study on the linear relationship of naringenone H reference concentration Take an appropriate amount of naringenone H reference substance, accurately weigh it, and 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 measure the peak area. The standard curve is drawn 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 showed that the linear relationship was good in the injection concentration range of 3.14μg / ml to 313.7μg / ml for naringenone H.

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

[0197]

[0198] Study on the linear relationship of aristolochicone reference concentration Take an appropriate amount of aristolochicone reference substance, accurately weigh it, and add petroleum ether to make solutions containing 142.4μg, 56.9μg, 28.5μg, 14.2μg, 5.7μg, 2.85μg, and 1.42μg per 1mL respectively. Accurately draw 2μL of the above aristolochicone reference substance solutions of different concentrations and inject them into the liquid chromatograph to measure the peak area. The standard curve is drawn 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 showed that the linear relationship was good in the range of aristolochicone injection concentration from 1.42 μg / ml to 142.4 μg / ml.

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

[0200]

[0201] 8. Accuracy inspection

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

[0203] The results are shown in Tables 28-29 below. It can be seen from the results in the table that the average recovery rate of galantone 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, the method has good accuracy.

[0204] Table 28 Nardone 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 glutinosa slices established in Example 1, the peak height of each characteristic peak is affected to a certain extent under different high-performance liquid chromatography 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 galanginone H in standard decoctions of galangin slices.

[0210] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic spectrum of volatile oil in a water extract of spikenard and its preparation, characterized in that: The following steps are involved: (1) Preparation of test solution; 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 a test solution; the construction method further comprises the step of preparing a reference solution using at least one reference substance selected from the group consisting of galbanum H, aristolochicone, and galbanum; (2) The test solution and the reference solution were tested by high performance liquid chromatography, using octadecylsilane bonded silica gel as the filler and acetonitrile and acid-containing aqueous solution as the mobile phase; Gradient elution: the gradient elution program includes: 0 → 30 min → 35 min, the volume percentage of acetonitrile in the mobile phase is 40% → 50% → 55%; Step (1) further satisfies the following conditions: in step 1), the volatile oil is extracted by a hot reflux extraction method; in step 2), the organic solvent is selected from at least one of petroleum ether, cyclohexane, and dichloromethane.

2. The construction method according to claim 1, characterized in that Step (1) also satisfies any one or more of the following AB: A. In step 1), the test sample is selected from an aqueous extract of nardus truncatus or a preparation made from an aqueous extract of nardus truncatus; B. Preparation: The volume ratio of the raw material nardus 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, wherein The volume percentage of phosphoric acid in the acid-containing aqueous solution is 0.18-0.25%; and / or the chromatographic column has a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8-1.9 μm.

4. The construction method according to claim 1, 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.

5. The construction method according to claim 4, characterized in that The detection wavelength is 230-270nm.

6. The construction method according to claim 1, characterized in that The concentration of the reference solution is 10-100 μg / mL.

7. The construction method according to any one of claims 1 to 6, characterized in that: The characteristic spectrum of the volatile oil in the nardostachys water extract and its preparation has 10 characteristic peaks, the peak corresponding to the peak of the nardostachys H reference substance is peak No. 5, and peak No. 5 is used as the reference peak. The relative retention times of the characteristic peaks 1-4 and peaks 6-10 and the reference peak are within the range of ±10% of the specified values; the specified values ​​of peaks 1-4 and peaks 6-10 are: 0.32, 0.70, 0.84, 0.88, 1.17, 1.31, 1.45, 1.66, and 1.90, respectively.

8. A quality control method for a spikenard aqueous extract and a preparation thereof, characterized in that: The method comprises the steps of taking a nard product to be tested and constructing a characteristic spectrum according to the construction method described in any one of claims 1 to 7.

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