阅读说明：本技术 益智仁配方颗粒的特征图谱构建方法及其质量检测方法 (Characteristic spectrum construction method and quality detection method of fructus alpiniae oxyphyllae formula granules ) 是由 刘燎原 邓颂 李燕珍 崔婷 干丽 魏梅 程学仁 孙冬梅 于 2020-04-24 设计创作，主要内容包括：本发明涉及一种益智仁配方颗粒的特征图谱构建方法及其质量检测方法。所述构建方法包括如下步骤：对照品溶液的制备：取圆柚酮、山柰素对照品,加溶剂溶解,得对照品溶液；供试品溶液的制备：取益智仁配方颗粒,加入提取溶剂进行提取,提取液过滤,取续滤液,得供试品溶液；将所述对照品溶液和供试品溶液注入超高效液相色谱仪中进行检测,所述超高效液相色谱仪采用的流动相A为乙腈,流动相B为体积分数为0.05～0.15％的甲酸水溶液,洗脱方式为梯度洗脱。该构建方法构建获得的特征图谱能够较全面的反映益智仁配方颗粒的特征,特别是实现了益智仁配方颗粒中挥发油的检测,能够很好地对益智仁配方颗粒的质量进行控制。(The invention relates to a method for constructing a characteristic spectrum of fructus alpiniae oxyphyllae formula granules and a quality detection method thereof. The construction method comprises the following steps: preparation of control solutions: dissolving pomelo ketone and kaempferide reference substances in solvent to obtain reference substance solution; preparation of a test solution: adding an extraction solvent into the fructus alpiniae oxyphyllae formula granules for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test solution; and injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, wherein a mobile phase A adopted by the ultra-high performance liquid chromatograph is acetonitrile, a mobile phase B is a formic acid aqueous solution with the volume fraction of 0.05-0.15%, and the elution mode is gradient elution. The characteristic spectrum constructed by the construction method can comprehensively reflect the characteristics of the fructus alpiniae oxyphyllae formula particles, particularly realizes the detection of volatile oil in the fructus alpiniae oxyphyllae formula particles, and can well control the quality of the fructus alpiniae oxyphyllae formula particles.)

1. A method for constructing a characteristic spectrum of fructus alpiniae oxyphyllae formula granules is characterized by comprising the following steps:

preparation of control solutions: dissolving pomelo ketone and kaempferide reference substances in solvent to obtain reference substance solution;

preparation of a test solution: adding an extraction solvent into the fructus alpiniae oxyphyllae formula granules for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test solution;

detecting the reference solution and the test solution by using an ultra-high performance liquid chromatography, wherein the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, the mobile phase B is a formic acid aqueous solution with the volume fraction of 0.05-0.15%, and the elution mode is gradient elution.

2. The feature map construction method according to claim 1, wherein the gradient elution includes the following procedure:

the volume percentage of the mobile phase A is increased from 8% to 16% in 0-10 min;

the volume percentage of the mobile phase A is increased from 16% to 23% in 10-25 min;

the volume percentage of the mobile phase A is increased from 23% to 73% within 25-35 min;

and (3) reducing the volume percentage of the mobile phase A from 73% to 8% in 35-38 min.

3. The feature map construction method according to claim 1, wherein the conditions of the ultra high performance liquid chromatography include:

the chromatographic column takes octadecylsilane chemically bonded silica as a filler, the detection wavelength is 250-260 nm, the flow rate is 0.25-0.35 m L/min, and the column temperature is 25-35 ℃.

4. The feature map construction method according to claim 3, wherein the conditions of the ultra high performance liquid chromatography include:

the chromatographic column uses octadecylsilane chemically bonded silica as filler, the detection wavelength is 254nm, the flow rate is 0.3m L/min, and the column temperature is 30 ℃.

5. The method for constructing a feature map according to any one of claims 1 to 4, wherein the extraction solvent is a methanol aqueous solution having a volume fraction of 45% to 55% in the preparation of the sample solution.

6. The feature map construction method according to claim 5, wherein the extraction method is ultrasonic extraction: the ultrasonic power is 200-300W, and the frequency is 20-30 kHZ; the extraction time is 25-35 min.

7. A quality detection method of fructus alpiniae oxyphyllae formula granules is characterized by comprising the following steps:

preparation of index component reference solution: dissolving a nootkatone reference substance in a solvent to prepare index reference substance solutions with different concentrations;

preparing a sample solution to be tested: adding an extraction solvent into the fructus alpiniae oxyphyllae formula granules to be detected for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test sample solution;

establishing a standard curve: detecting the index component reference substance solutions with different concentrations by using ultra-high performance liquid chromatography, and constructing a concentration-peak area standard curve of the index component;

and (3) detection: detecting the sample solution to be detected by using ultra-high performance liquid chromatography, and comparing a detection map with a concentration-peak area standard curve of the index component;

wherein, the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05 percent to 0.15 percent, and the elution mode is gradient elution.

8. The quality detection method according to claim 7, wherein the gradient elution comprises the following procedure:

the volume percentage of the mobile phase A is increased from 8% to 16% in 0-10 min;

the volume percentage of the mobile phase A is increased from 16% to 23% in 10-25 min;

the volume percentage of the mobile phase A is increased from 23% to 73% within 25-35 min;

and (3) reducing the volume percentage of the mobile phase A from 73% to 8% in 35-38 min.

9. The quality detection method according to claim 7, wherein the conditions of the ultra high performance liquid chromatography include:

the chromatographic column takes octadecylsilane chemically bonded silica as a filler, the detection wavelength is 250-260 nm, the flow rate is 0.25-0.35 m L/min, and the column temperature is 25-35 ℃.

10. The quality detection method according to claim 9, wherein the conditions of the ultra high performance liquid chromatography include:

the chromatographic column uses octadecylsilane chemically bonded silica as filler, the detection wavelength is 254nm, the flow rate is 0.3m L/min, and the column temperature is 30 ℃.

11. The quality detection method according to any one of claims 7 to 10, wherein in the preparation of the sample solution to be detected, the extraction solvent is a methanol aqueous solution with a volume fraction of 45% to 55%.

12. The quality detection method according to claim 11, wherein the extraction method is ultrasonic extraction, the ultrasonic power is 200-300W, and the frequency is 20-30 kHZ; the extraction time is 25-35 min.

Technical Field

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a characteristic spectrum construction method and a quality detection method of fructus alpiniae oxyphyllae formula granules.

Background

Fructus Alpinae Oxyphyllae (Alpinia Oxyphylla Miq) is dry mature fruit of perennial herb of Zingiberaceae, has warm nature and pungent taste, and has effects of invigorating spleen and kidney channels, warming spleen, relieving diarrhea, arresting saliva, warming kidney, stopping nocturnal emission, and reducing urination, and can be used for treating abdominal pain, vomiting, diarrhea, anorexia, excessive saliva, etc. caused by kidney qi deficiency and failure to arrest. Mainly produced in Hainan, Guangdong, Guangxi, etc., is one of the genuine medicinal materials in Hainan and also one of the four great south medicinal materials. Modern pharmacology indicates that fructus alpiniae oxyphyllae has various pharmacological effects, and meanwhile, more and more researches indicate that the main components of the fructus alpiniae oxyphyllae playing the drug effect are volatile oil: as people think of Luoqin and the like, the volatile oil of fructus alpiniae oxyphyllae has obvious bacteriostatic action and has larger potential in the aspect of antibiosis; research of Liuchanghong iris and the like shows that the chemical component of the nootropic volatile oil, namely nootkatone, has a protective effect on chronic kidney injury and explains the kidney tonifying and urination reducing mechanism of the nootkatone; in addition, the active ingredients of the sharpleaf galangal fruit which have the effects of warming spleen and relieving diarrhea are volatile oil. In conclusion, the volatile oil component of fructus alpiniae oxyphyllae is an important component of fructus alpiniae oxyphyllae for exerting the drug effect.

The traditional Chinese medicine decoction is a prescription which is prepared for patients on the basis of dialectical treatment by a clinical traditional Chinese medicine doctor under the guidance of the traditional Chinese medicine theory, is continuously used up to now since commercial Yi creation decoction, and makes a great contribution to the medical health of Chinese nationality. But with the development of modern society and the improvement of technological level. The traditional Chinese medicine decoction cannot meet the fast-paced life style of modern people, and the traditional Chinese medicine formula granules are produced at the same time. The traditional Chinese medicine formula particle is a new clinical use form of pure traditional Chinese medicine prepared by taking traditional Chinese medicine decoction pieces which are processed by a modern processing technology and accord with the traditional processing specification as raw materials, and applying modern scientific technology to extraction, solid-liquid separation, concentration, drying, granulation and packaging; the functions, indications, properties and taste of the traditional Chinese medicine decoction pieces are consistent with those of the traditional Chinese medicine decoction pieces, the efficacy and the property of the traditional Chinese medicine decoction pieces are kept, and the traditional Chinese medicine decoction pieces have the characteristics of convenience for taking and dispensing of patients.

According to the regulation of the 2015 edition of Chinese pharmacopoeia, the content of the volatile oil in the sharpleaf galangal fruit decoction pieces is not less than 1.0%. However, the fructus alpiniae oxyphyllae volatile oil is unstable and volatile, so that most of the volatile oil is lost in the production and storage processes of the preparation, and the drug effect is influenced, therefore, the quality of the existing fructus alpiniae oxyphyllae formula particles is difficult to ensure to be equal to that of the traditional Chinese medicine decoction, particularly the content of the volatile oil in the fructus alpiniae oxyphyllae formula particles is difficult to achieve the drug effect equal to that of the traditional Chinese medicine decoction if the content of the volatile oil in the fructus.

At present, the quality research on the fructus alpiniae oxyphyllae formula particles is less, and particularly, the volatile oil detection is carried out, so that a better quality standard is lacked to ensure the stable quality of the fructus alpiniae oxyphyllae formula particles. The method for acquiring comprehensive Chinese medicine chemical component characteristic information by utilizing the characteristic map is widely applied to the quality control of the Chinese medicine, and the Chinese medicine characteristic map expresses and reflects the integral chemical characteristics of the Chinese medicine, so that the quality of the Chinese medicine preparation can be effectively monitored, and the curative effect is improved and stabilized. Therefore, it is necessary to establish a characteristic map of the fructus alpiniae oxyphyllae formula granules.

Disclosure of Invention

Based on this, there is a need for a method for constructing a characteristic map of fructus alpiniae oxyphyllae formula granules. The characteristic spectrum constructed by the construction method can comprehensively reflect the characteristics of the fructus alpiniae oxyphyllae formula particles, particularly realizes the detection of volatile oil in the fructus alpiniae oxyphyllae formula particles, has comprehensive and reliable quality detection and short time, and can well control the quality of the fructus alpiniae oxyphyllae formula particles.

A method for constructing a characteristic spectrum of fructus alpiniae oxyphyllae formula granules comprises the following steps:

preparation of control solutions: dissolving pomelo ketone and kaempferide reference substances in solvent to obtain reference substance solution;

preparation of a test solution: adding an extraction solvent into the fructus alpiniae oxyphyllae formula granules for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test solution;

detecting the reference solution and the test solution by using an ultra-high performance liquid chromatography, wherein the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, the mobile phase B is a formic acid aqueous solution with the volume fraction of 0.05-0.15%, and the elution mode is gradient elution.

In one embodiment, the gradient elution comprises the following procedure:

the volume percentage of the mobile phase A is increased from 8% to 16% in 0-10 min;

the volume percentage of the mobile phase A is increased from 16% to 23% in 10-25 min;

the volume percentage of the mobile phase A is increased from 23% to 73% within 25-35 min;

and (3) reducing the volume percentage of the mobile phase A from 73% to 8% in 35-38 min.

In one embodiment, the conditions of the ultra high performance liquid chromatography comprise:

the chromatographic column takes octadecylsilane chemically bonded silica as a filler, the detection wavelength is 250-260 nm, the flow rate is 0.25-0.35 m L/min, and the column temperature is 25-35 ℃.

In one embodiment, the conditions of the ultra high performance liquid chromatography comprise:

the chromatographic column uses octadecylsilane chemically bonded silica as filler, the detection wavelength is 254nm, the flow rate is 0.3m L/min, and the column temperature is 30 ℃.

In one embodiment, the sample solution is prepared by using a methanol aqueous solution with a volume fraction of 45-55%.

In one embodiment, the method of extraction is ultrasonic extraction: the ultrasonic power is 200-300W, and the frequency is 20-30 kHZ; the extraction time is 25-35 min.

The invention also provides a quality detection method of the fructus alpiniae oxyphyllae formula granules, which comprises the following steps:

preparation of index component reference solution: dissolving a nootkatone reference substance in a solvent to prepare index reference substance solutions with different concentrations;

preparing a sample solution to be tested: adding an extraction solvent into the fructus alpiniae oxyphyllae formula granules to be detected for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test sample solution;

establishing a standard curve: detecting the index component reference substance solutions with different concentrations by using ultra-high performance liquid chromatography, and constructing a concentration-peak area standard curve of the index component;

and (3) detection: detecting the sample solution to be detected by using ultra-high performance liquid chromatography, and comparing a detection map with a concentration-peak area standard curve of the index component;

wherein, the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05 percent to 0.15 percent, and the elution mode is gradient elution.

In one embodiment, the gradient elution comprises the following procedure:

the volume percentage of the mobile phase A is increased from 8% to 16% in 0-10 min;

the volume percentage of the mobile phase A is increased from 16% to 23% in 10-25 min;

the volume percentage of the mobile phase A is increased from 23% to 73% within 25-35 min;

and (3) reducing the volume percentage of the mobile phase A from 73% to 8% in 35-38 min.

In one embodiment, the conditions of the ultra high performance liquid chromatography comprise:

the chromatographic column takes octadecylsilane chemically bonded silica as a filler, the detection wavelength is 250-260 nm, the flow rate is 0.25-0.35 m L/min, and the column temperature is 25-35 ℃.

In one embodiment, the conditions of the ultra high performance liquid chromatography comprise:

the chromatographic column uses octadecylsilane chemically bonded silica as filler, the detection wavelength is 254nm, the flow rate is 0.3m L/min, and the column temperature is 30 ℃.

In one embodiment, in the preparation of the sample solution to be tested, the extraction solvent is a methanol aqueous solution with a volume fraction of 45% to 55%.

In one embodiment, the extraction method is ultrasonic extraction, the ultrasonic power is 200-300W, and the frequency is 20-30 kHZ; the extraction time is 25-35 min.

Compared with the prior art, the invention has the following beneficial effects:

(1) the characteristic spectrum construction method of the fructus alpiniae oxyphyllae formula particles takes ultra-high performance liquid chromatography (UP L C) as a technical means, adopts a proper mobile phase for gradient elution, can obtain the characteristic spectrum of the fructus alpiniae oxyphyllae formula particles, particularly detects volatile oil components in the fructus alpiniae oxyphyllae formula particles, can comprehensively reflect main chemical component information of the fructus alpiniae oxyphyllae formula particles, has comprehensive and reliable quality detection, can accurately detect and evaluate the quality of the fructus alpiniae oxyphyllae formula particles, and has important significance for ensuring the clinical curative effect.

(2) The quality detection method provided by the invention can comprehensively reflect the main chemical component information of the fructus alpiniae oxyphyllae formula particles, can accurately measure the content of nootkatone in the fructus alpiniae oxyphyllae formula particles, is convenient for comprehensively monitoring the quality of the fructus alpiniae oxyphyllae formula particles, can synchronously perform qualitative and quantitative analysis, can accurately and effectively distinguish whether the volatile oil equivalent to the volatile oil in the fructus alpiniae oxyphyllae standard decoction is added in the fructus alpiniae oxyphyllae formula particles, and can ensure the safety, effectiveness and controllable quality of the medicine as a whole.

Drawings

FIG. 1 is a chromatogram of a control kaempferide;

FIG. 2 is a chromatogram of control nootkatone;

fig. 3 is a characteristic map of the constructed alpinia oxyphylla formula granules;

FIG. 4 is a chromatogram of standard decoction of fructus Alpinae Oxyphyllae;

fig. 5 is a chromatogram of fructus alpiniae oxyphyllae formula granules prepared by a conventional process;

FIG. 6 is a chromatogram of β -cyclodextrin negative control solution in methodological validation;

FIG. 7 is a chromatogram of a silica negative control solution in a methodological assay;

FIG. 8 is a chromatogram of a 50% methanol negative control solution in a methodological assay.

Detailed Description

The method for constructing the characteristic spectrum of the fructus alpiniae oxyphyllae formula particle and the quality detection method thereof are further described in detail with reference to specific examples.

The embodiment of the invention provides a method for constructing a characteristic spectrum of fructus alpiniae oxyphyllae formula granules, which comprises the following steps:

preparation of control solutions: dissolving pomelo ketone and kaempferide reference substances in solvent to obtain reference substance solution;

preparation of a test solution: adding an extraction solvent into the fructus alpiniae oxyphyllae formula granules for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test solution;

and injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, wherein a mobile phase A adopted by the ultra-high performance liquid chromatograph is acetonitrile, a mobile phase B is a formic acid aqueous solution with the volume fraction of 0.05-0.15%, and the elution mode is gradient elution.

In one specific embodiment, the gradient elution comprises the following procedure:

the volume percentage of the mobile phase A is increased from 8% to 16% in 0-10 min;

the volume percentage of the mobile phase A is increased from 16% to 23% in 10-25 min;

the volume percentage of the mobile phase A is increased from 23% to 73% within 25-35 min;

and (3) reducing the volume percentage of the mobile phase A from 73% to 8% in 35-38 min.

In one specific embodiment, the conditions of the ultra high performance liquid chromatograph include:

the chromatographic column takes octadecylsilane chemically bonded silica as a filler, the detection wavelength is 250-260 nm, the flow rate is 0.25-0.35 m L/min, and the column temperature is 25-35 ℃.

More specifically, the conditions of the ultra high performance liquid chromatograph include:

the chromatographic column uses octadecylsilane chemically bonded silica as filler, the detection wavelength is 254nm, the flow rate is 0.3m L/min, and the column temperature is 30 ℃.

In one specific embodiment, the conditions of the ultra high performance liquid chromatograph further comprise: the theoretical plate number is not less than 100000 calculated according to the nootkatone peak.

In one specific embodiment, in the preparation of the test solution, the extraction solvent is a methanol aqueous solution with a volume fraction of 45% to 55%. Correspondingly, the extraction method can be ultrasonic extraction, and more specifically, the process conditions of the ultrasonic extraction include: the ultrasonic power is 200-300W, the frequency is 20-30 kHZ, and the extraction time is 25-35 min.

In one specific embodiment, the solvent used in the preparation of the control solution can be an alcohol solvent, preferably methanol, and more specifically, the concentrations of the nootkatone and the kaempferide in the control solution are 105-115 μ g/m L and 80-90 μ g/m L.

In one specific embodiment, in the feature map constructed by the construction method, the integration parameter is set to 6000 as the minimum peak area.

The embodiment of the invention also provides a quality detection method of the fructus alpiniae oxyphyllae formula granules, which comprises the following steps:

preparation of index component reference solution: dissolving a nootkatone reference substance in a solvent to prepare index reference substance solutions with different concentrations;

preparing a sample solution to be tested: adding an extraction solvent into the fructus alpiniae oxyphyllae formula granules to be detected for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test sample solution;

establishing a standard curve: respectively injecting the index component reference substance solutions with different concentrations into an ultra-high performance liquid chromatograph for detection, and constructing a concentration-peak area standard curve of the index component;

and (3) detection: injecting the sample solution to be detected into an ultra-high performance liquid chromatograph for detection, and comparing a detection map with a concentration-peak area standard curve of an index component;

wherein, the mobile phase A adopted by the ultra-high performance liquid chromatograph is acetonitrile, the mobile phase B is formic acid water solution with the volume fraction of 0.05 percent to 0.15 percent, and the elution mode is gradient elution.

Specifically, the method for preparing the sample solution to be tested is the same as the method for preparing the sample solution to be tested in the construction of the characteristic map, and the conditions related to the ultra performance liquid chromatography in the standard curve establishment and detection steps are the same as the construction of the characteristic map, which is not repeated herein.

In one specific embodiment, the solvent used in the preparation of the index component control solution may be an alcohol solvent, preferably methanol, and more specifically, the concentrations of the index component control solutions with different concentrations may be set to 80-90 μ g/m L and 75-85 μ g/m L.

In one specific embodiment, the quality detection method further comprises the steps of constructing a characteristic map of the fructus alpiniae oxyphyllae formula granules according to the construction method; and comparing the obtained detection map of the sample to be detected with the characteristic map. Namely, the quality detection method can realize the detection of the characteristic spectrum and the nootkatone content of the fructus alpiniae oxyphyllae formula particles, and simply, conveniently and comprehensively monitor the quality of the fructus alpiniae oxyphyllae formula particles.

The following specific examples are provided, and the raw materials used in the examples are all commercially available unless otherwise specified.

The experimental materials and instruments used in the specific examples were as follows:

experimental materials:

a round teak reference substance (6-JTN-173-1, Shenzhen Lijing);

kaempferide reference (110861-201209, China institute for testing food and drug); acetonitrile, 0.1% formic acid.

Fructus alpiniae oxyphyllae formula granules are provided by Guangdong party pharmaceutical company Limited, 10 batches in total, and the preparation method is as follows:

selecting fructus Alpinae Oxyphyllae, removing impurities to obtain fructus Alpinae Oxyphyllae decoction pieces, decocting, collecting volatile oil, clathrating the volatile oil with β -cyclodextrin to obtain β -cyclodextrin clathrate, collecting residues, decocting twice, vacuum concentrating the filtrate under reduced pressure to obtain fluid extract, spray drying the fluid extract, mixing the spray dried powder with β -cyclodextrin clathrate, and dry granulating.

The batch numbers are shown in Table 1 below:

TABLE 1

The instrument comprises an ultra-high performance liquid chromatograph Waters ACQUITY UP L C, a chromatographic column of Agilent ZORBAX SB-C18 chromatographic column with the thickness of 2.1mm, the thickness of × 100mm and the thickness of 1.8um, an XP26 millionth and plano (METT L ER TO L EOD) and an ultrasonic instrument of KQ-500DE type ultrasonic cleaner (300W and 40 Hz).