阅读说明：本技术 一种组合物的功效成分检测方法 (Method for detecting functional components of composition ) 是由 程刚 于 2020-12-31 设计创作，主要内容包括：本发明涉及一种组合物的功效成分检测方法,公开了一种同时检测酸枣仁皂苷A和灵芝酸A含量的方法,本发明的分析方法用于一种含γ-氨基丁酸、酸枣仁和灵芝提取物的组合物的制备和分析。(The invention relates to a method for detecting functional components of a composition, and discloses a method for simultaneously detecting the contents of spina date seed saponin A and ganoderic acid A.)

1. The method for detecting the functional components of the composition is characterized by comprising the following steps: method for simultaneously detecting content of spina date seed saponin A and content of ganoderic acid A

Preparation of Standard solutions

Stock solution of jujuboside a: accurately weighing 10mg of jujuboside A reference substance in a 50ml volumetric flask, dissolving with methanol, fixing the volume, and shaking up for use;

stock solution of ganoderic acid a: accurately weighing 10mg of ganoderic acid A standard substance in a 50ml volumetric flask, dissolving with methanol, fixing the volume, and shaking up for later use;

preparation of test solution

Accurately weighing 800mg of a test sample in a Soxhlet extractor, adding a proper amount of petroleum ether (60-90 ℃) for heating and refluxing for 2h, discarding petroleum ether liquid, volatilizing the solvent of medicine residues, transferring to a conical flask, adding 20mL of 70% ethanol, heating and refluxing for 2h, filtering, washing filter residues with 70% ethanol, combining washing liquid and filtrate, recovering the solvent until the solvent is dry, dissolving the residues with methanol, transferring to a 5mL volumetric flask, diluting to the constant volume with methanol, and shaking uniformly for later use;

chromatographic conditions

A chromatographic column: acclaim (TM) 120C 18, 5 μm, 4.6 × 250 mm;

flow rate: 1.0 mL/min;

column temperature: 40 ℃;

sample introduction amount: 10 mu L of the solution;

corona Ultra CAD detector atomization chamber temperature: 35 deg.C

Mobile phase gradient elution:

and (4) sample determination.

2. The sleep composition is characterized by comprising the following components in parts by weight: 1-5 parts of gamma-aminobutyric acid; 30-50 parts of spina date seeds; 10-30 parts of lucid ganoderma; a detection assay according to the method of claim 1.

3. The sleep composition is characterized by comprising the following components in parts by weight: 1-5 parts of gamma-aminobutyric acid; 30-50 parts of spina date seeds; 10-30 parts of lucid ganoderma, 1-3 parts of mannitol and 1-2 parts of magnesium stearate; a detection assay according to the method of claim 1.

Technical Field

The invention belongs to the field of component detection of medicines, and relates to a method for detecting functional components of a composition and application of the composition in a preparation.

Background

The Chinese medicine supervision department encourages the development of multi-target medicines, the multi-target medicines are often multi-component medicines, and the multi-component medicines play an integral role and are not simple addition of single components. For the quality control of the composition, there are two paths, one is to control the functional components and the other is to control the content components, and in the practice of drug development, the types and contents of the chemical components are often reflected by the peak positions, areas or proportions of the main characteristic peaks of the map.

According to the survey of the world health organization, about 10-49% of people worldwide suffer from sleep disorders with different degrees, about 27% of patients who see primary medical treatment have sleep problems. 20-30% of people in China suffer from sleep diseases of different degrees, the incidence rate of the sleep diseases of the old people is up to 40%, and the problems of insufficient sleep and sleep quality become one of the important worldwide public health problems. In 2001, the global sleep and health program sponsored by the international mental health organization identified day 21 of 3 months per year as the "world sleep day", and in 2003, chinese sleep research formally introduced the "world sleep day" into china.

Semen Ziziphi Spinosae is dry mature seed of Ziziphus jujuba Mill.var. spinosa (Bunge) Hu ex H.F.Chou belonging to Rhamnaceae. Sweet in nature, sour and neutral. It enters liver, gallbladder and heart meridians. Nourish heart and tonify liver, calm heart and induce tranquilization, arrest sweating, promote fluid production. It is used for restlessness and insomnia due to deficiency, palpitation and dreaminess, asthenia and hyperhidrosis, body fluid deficiency and thirst, and is a key herb for tranquilizing mind by nourishing heart and treating restlessness and insomnia due to deficiency, and praised as "eastern sleep fruit" by doctors and patients. The Chinese date seed recorded in Ben Cao gang mu is used for treating insomnia due to gallbladder deficiency and dysphoria with thirst and sweating due to debility; unprocessed herbs for gallbladder heat and good sleep are also indicated for Jueyin shaoyang herbs. Modern pharmacology proves that the spina date seed total saponin can increase the total sleep time, the total flavone has the sedative-hypnotic effect, and the polysaccharide has the effect of reducing the autonomous activity.

Ganoderma lucidum is dried fruiting body of Ganoderma lucidum (leys. ex Fr.) Karst. or Ganoderma sinense Garoderma sinense Zhao, Xu et Zhang, belonging to Polyporaceae. Sweet in nature and mild in nature. It enters heart, lung, liver and kidney meridians. Tonify qi and induce tranquilization, relieve cough and dyspnea. Can be used for treating restlessness, insomnia, palpitation, cough and asthma due to lung deficiency, asthenia, short breath, and anorexia. From ancient times, there are the beautiful names of "Xiancao", "Ruicao", "Huanyang" and "Lingcao", etc. From Shen nong Ben Cao Jing of Dong Han, Ganoderma is listed as the top grade, and it has the effects of tranquilizing, replenishing vital essence, enhancing intelligence, and treating the middle and middle of the chest, and can be taken for a long time to lighten the body and make it not old. Ganoderma lucidum (Ganoderma lucidum, Ganoderma sinense) fruiting body is collected from pharmacopoeia of people's republic of China as legal traditional Chinese medicinal material. Ganoderma lucidum was recorded in United states herbal pharmacopoeia and treatment summary of 2000 edition. Ganoderma has obvious tranquilizing effect on central nervous system, can reduce spontaneous activity of animals, relax muscle slightly, and enhance central inhibition effect of barbiturate, but has no hypnotic or anesthetic effect.

The electrospray detector (CAD) is a detector which develops rapidly in recent years, and has the advantages of high sensitivity, good repeatability, consistent signal response, wide dynamic detection range, intuitive and simple operation and the like compared with the traditional ultraviolet detector, evaporation light detector and differential refraction detector, so that the electrospray detector is more and more widely applied to the detection of the functional components of the pharmacy and the health-care food.

At present, spina date seeds in health-care food are generally detected by adopting an HPLC method of the first part of the 'Chinese pharmacopoeia' 2015 edition or total saponins detected by the 'health-care food inspection and evaluation technical specification' 2003 edition. Ganoderma lucidum generally adopts polysaccharide detection in the first edition of the Chinese pharmacopoeia 2015 or adopts high performance liquid chromatography for determining the content of ganoderic acid in Ganoderma lucidum product in NY/T2278 and 2012. The detection of the total saponins and the polysaccharides adopts a spectrophotometry, and the accuracy is relatively low without specificity although the method is quick, simple and convenient for analyzing a certain kind of components. The formula of the health food simultaneously contains the spina date seed and the lucid ganoderma, the detection operation is complicated and time-consuming, and if the spina date seed and the lucid ganoderma in the product can be detected simultaneously by the detection method, the analysis time is greatly simplified, and the analysis cost is reduced.

In the prior art, although a method for simultaneously and quantitatively measuring 9 blood-entering components in a spina date seed aqueous extract is reported in a literature, the sources of active components are only limited to spina date seeds, and therefore, the detection method in the literature cannot constitute a technical suggestion for the invention.

In the prior art, although documents report a nerve-soothing tea prepared by matching malt, lily, lucid ganoderma and spina date seed; although documents report that the traditional Chinese medicine is prepared by using astragalus root, lucid ganoderma, spina date seed, tuckahoe, Chinese date, liquorice and pig blood as raw materials, the indications of the traditional Chinese medicine comprise palpitation, insomnia, amnesia, dreaminess, easy awakening and the like caused by heart-spleen deficiency; although the literature reports that the deep sleep promoter is obtained by matching polyrhachis vicina Roger dry powder, red ganoderma lucidum dry powder and fried spina date seed dry powder; although there are reports in the literature that pharmaceutical compositions for improving sleep and treating insomnia are prepared by using ganoderan and spina date seed total saponins; although there are reports on sleep-aiding health foods consisting of ganoderma lucidum, poria cocos and spina date seeds, there are reports on sleep-improving compositions consisting of lignan extracts of schisandra chinensis, aqueous extracts of ganoderma lucidum and alcohol extracts of spina date seeds. However, since the effective components of the extracts obtained by different extraction methods are different from each other, and since these documents do not disclose how to detect the effective components, it is unexpected that effective component analysis methods having high efficiency, high stability and strong specificity are obtained from the prior art and used for developing compositions for improving sleep.

Gamma-aminobutyric acid (GABA) is a naturally occurring non-protein amino acid, is an important inhibitory neurotransmitter in the central nervous system, is widely present in the cortex, hippocampus, thalamus, basal ganglia and cerebellum of the human brain, plays a role in information transmission, and has an extremely important physiological function. It has effects in strengthening brain, improving intelligence, delaying brain aging, relieving epilepsy, and improving sleep. With the increase of age and mental stress, the synthesis of GABA in human brain is reduced, when the human body is lack, symptoms such as anxiety, uneasiness, fatigue, insomnia and the like can be generated, generally, people who are in a high-pressure competitive environment for a long time easily lack GABA, and need to be supplemented in time to reduce the activity of neurons and prevent nerve cells from overheating so as to relieve the pressure. A large number of studies at home and abroad show that the time for falling asleep can be shortened and the time for deep sleep can be prolonged after GABA is supplemented, and the medicine has the effects of antianxiety, sedation and the like. It is also called "natural sedative for the brain" because it can induce the brain to calm down.

The Food and Drug Administration (FDA) has indicated that the addition of GABA to foods is safe according to the results of toxicological experiments, and the range of uses includes beverages, coffee, tea, chewing gum, and the like, but does not allow the addition to infant foods, meat products, or meat-containing products.

The 2009 publication No. 12 of the Ministry of health of China approved that GABA is a new resource food, and the GABA is produced by using L-sodium glutamate as a raw material and through the steps of lactobacillus hilgardii fermentation, heating sterilization, cooling, activated carbon treatment, filtering, adding starch serving as an auxiliary material, spray drying and the like. However, the intake of GABA should not exceed 500mg/d, and the GABA can be used in the range of beverages, cocoa products, chocolate and its beverages, candies, baked goods and puffed foods, but cannot be added to infant foods.

According to the search of health food prescription databases, 229 documents of the prior sleep improvement health food which takes spina date seeds and extracts thereof as main raw materials, 85 documents of the prior sleep improvement health food which takes lucid ganoderma and extracts thereof as main raw materials, only 1 document of the prior sleep improvement health food which takes gamma-aminobutyric acid as a main raw material, and no health food compounded by the three documents are available. Therefore, the invention is valuable through deeply developing health-care food which is prepared by compounding the three components, and the development of the product needs to monitor the content of GABA, spina date seed active ingredients and lucid ganoderma active ingredients, which is the technical problem to be solved by the invention.

Disclosure of Invention

The invention discloses a method for simultaneously detecting the content of spina date seed saponin A and ganoderic acid A, which adopts the following technical scheme:

1. preparation of Standard solutions

Stock solution of jujuboside a: accurately weighing 10mg of jujuboside A reference substance in a 50ml volumetric flask, dissolving with methanol, metering volume, and shaking up for use.

Stock solution of ganoderic acid a: accurately weighing 10mg of ganoderic acid A standard substance in a 50ml volumetric flask, dissolving with methanol, fixing the volume, and shaking up for later use.

2. Preparation of test solution

Accurately weighing 800mg of a sample to be tested in a Soxhlet extractor, adding a proper amount of petroleum ether (60-90 ℃) for heating and refluxing for 2h, discarding petroleum ether liquid, volatilizing the solvent of medicine residues, transferring to a conical flask, adding 20mL of 70% ethanol, heating and refluxing for 2h, filtering, washing filter residues with 70% ethanol, combining washing liquid and filtrate, recovering the solvent until the solvent is dry, dissolving the residues with methanol, transferring to a 5mL volumetric flask, diluting to the constant volume with methanol, and shaking uniformly for later use.

3. Chromatographic conditions

A chromatographic column: acclaim^TM 120 C18，5μm，4.6×250mm；

Flow rate: 1.0 mL/min;

column temperature: 40 ℃;

sample introduction amount: 10 mu L of the solution;

corona Ultra CAD detector atomization chamber temperature: 35 deg.C

Mobile phase gradient elution:

4. sample assay

And (3) carrying out HPLC analysis on the standard solution, carrying out integral calculation to obtain the retention time and chromatographic peak area of the standard solution, and drawing a standard curve by taking the concentration of the standard solution as a horizontal coordinate and the peak area of the standard solution as a vertical coordinate. And (3) carrying out HPLC analysis on the test solution under the same conditions, carrying out qualitative analysis according to the retention time of the standard solution, carrying out integral calculation to obtain the chromatographic peak area of the test solution, substituting the chromatographic peak area into a standard curve equation to obtain the concentration of the test solution, and calculating the content of the substance to be detected.

The invention aims to solve the problems and provides a composition containing gamma-aminobutyric acid, spina date seed and lucid ganoderma extracts to solve the problem of poor sleep improvement effect of a single component.

The sleep improving composition provided by the invention comprises the following components in parts by weight: 1-5 parts of gamma-aminobutyric acid; 30-50 parts of spina date seeds; 10-30 parts of lucid ganoderma.

The sleep improving composition provided by the invention comprises the following components in parts by weight: 1-5 parts of gamma-aminobutyric acid; 30-50 parts of spina date seeds; 10-30 parts of lucid ganoderma, 1-3 parts of mannitol and 1-2 parts of magnesium stearate.

The innovation points of the invention are as follows:

innovation points 1: preferred process for improving the parts by weight of a sleep composition

ICR mice were randomly divided into a blank control group and a test substance group, and 12 mice were each group. After the oral gavage for 30 days, the sleep improvement effect of the test object is evaluated through direct sleep, prolongation of the sleep time of pentobarbital sodium, subliminal dose hypnosis of the barbital sodium and a sleep latency period experiment of the barbital sodium. The research finds that compared with a blank control group, the direct sleep experiment results of all the tested substances are negative, the single component has different sleep improvement effects, and the three components are used together, so that the sleep improvement effect is more remarkable.

Innovation points 2: preferred procedure for mobile phase in chromatographic conditions

At 30-40min, the mobile phase was acetonitrile: 0.1% formic acid solution 30:70(V/V), and ganoderic acid a did not reach baseline separation (fig. 1); when acetonitrile is changed to 0.1% formic acid solution (20: 80 (V/V)), the ganoderic acid A achieves baseline separation and better peak pattern (figure 2).

Drawings

One of the screens for mobile phase in chromatographic conditions of FIG. 1

FIG. 2 second screening of mobile phase in chromatographic conditions

Detailed Description

Example 1

Preparing the following raw materials in parts by weight: 3 parts of gamma-aminobutyric acid; 40 parts of spina date seeds; 20 parts of lucid ganoderma; 2 parts of mannitol; and 1 part of magnesium stearate.

The preparation method comprises the following steps:

1. extraction: the spina date seed and the lucid ganoderma decoction pieces are weighed according to the formula proportion and decocted for 2 times by adding water. Adding 15 times of water for the 1 st time, and extracting under reflux for 2 h; adding 10 times of water for the second time, and extracting for 1h under reflux.

2. And (3) filtering: filtering the two extractive solutions with 200 mesh sieve, and mixing filtrates.

3. Concentration: concentrating the filtrate under reduced pressure at 60-70 deg.C and-0.08-0.1 MPa to density of 1.05-1.10 g/cm³；

4. And (3) drying: and (3) spray-drying the concentrated solution at the inlet temperature of 95-105 ℃ and the outlet temperature of 75-85 ℃.

5. Mixing: and uniformly mixing the gamma-aminobutyric acid, the mannitol and the magnesium stearate with the spray-dried powder according to the formula proportion.

6. Tabletting: tabletting with a tabletting machine to obtain 0.45 g/tablet;

7. inner packaging: 60 pieces/bottle;

8. and (3) outer packaging: packing with cartons conforming to single corrugated cartons and double corrugated cartons for GB/T6543 transport packaging;

11. quality inspection: inspecting the finished product by a quality inspector;

12. warehousing; and (7) warehousing and storing.

Example 2

Preparing the following raw materials in parts by weight: 1 part of gamma-aminobutyric acid; 50 parts of spina date seeds; 30 parts of lucid ganoderma; 1 part of mannitol; and 1 part of magnesium stearate.

The preparation method is the same as example 1.

Example 3

Preparing the following raw materials in parts by weight: 5 parts of gamma-aminobutyric acid; 30 parts of spina date seeds; 10 parts of lucid ganoderma; 3 parts of mannitol; and 2 parts of magnesium stearate.

The preparation method is the same as example 1.

Example 4

Preferred process for improving the parts by weight of a sleep composition

1. Materials and reagents

1.1 animals

The SPF grade ICR female mouse has the weight of 18-22 g and is provided by animal research institute of cooperative medical science institute of China medical science institute.

1.2 materials and instruments

Lucid ganoderma and spina date seed aminobutyric acid tablets are prepared in laboratories;

sodium pentobarbital and sodium barbital are analytically pure and purchased from Shanghai chemical reagents.

XS105 electronic balance, METTLER TOLEDO.

2. Content of the experiment

2.1 animal grouping and handling

The recommended intake for an adult (60kg) is 1.8 g/person d, which corresponds to 30mg/kg d. Mice were randomly grouped into groups of 12 mice each. The test group is administered by intragastric administration with an intragastric administration amount of 20mL/kg, and is continuously administered for 30 days, and the blank control group is administered by intragastric administration with distilled water with the same volume.

2.2 direct sleep test

And (3) giving the test object to each group of animals, observing whether the mice have sleep phenomenon within 1h after the gavage, judging the mice to sleep if the righting reflex disappearance of the animals exceeds 60s, and comparing the influence of the blank control group and each group of the test object on the direct hypnosis of the mice.

2.3 experiment for prolonging sleep time of sodium pentobarbital

Refer to "inspection and evaluation of health food" technical Specifications^[2]After the animals are subjected to the last gastric lavage for 15min, 50mg/kg bw sodium pentobarbital is injected into the abdominal cavity of each group of miceThe injection amount was 0.2mL/20g · bw. And (3) taking the disappearance of the mouse righting reflex for more than 60s as a sleep onset judgment standard, taking the time from the disappearance of the mouse righting reflex to the recovery of the mouse righting reflex as the sleep time of the animal, and comparing whether the test object prolongs the sleep time of the pentobarbital sodium injection mouse.

2.4 Pentobarbital sodium subthreshold dose hypnosis test

Refer to "inspection and evaluation of health food" technical Specifications^[2]After the animals are subjected to the last gastric lavage for 15min, 25 mg/kg-bw sodium pentobarbital is injected into the abdominal cavity of each group of mice, and the injection amount is 0.2mL/20 g-bw. And taking the disappearance of the righting reflex of the mouse as a sleep onset judgment standard for more than 60s, recording the number of animals falling asleep in each group within 30min and calculating the sleep incidence. The incidence of sleep (%) ═ 100% (number of animals falling asleep/number of animals per group). The subjects were compared for increasing the incidence of sleep in mice injected with subthreshold dose of sodium pentobarbital.

2.5 Barbituric sodium sleep latency test

Refer to "inspection and evaluation of health food" technical Specifications^[2]After the animals are subjected to the last gastric lavage for 15min, 250mg/kg · bw of barbital sodium is injected into the abdominal cavity of each group of mice, and the injection amount is 0.2mL/20g · bw. And (3) taking the disappearance of the righting reflex of the mouse as a sleep onset judgment standard for more than 60s, taking the time from the injection of the barbital sodium into the mouse to the disappearance of the righting reflex as a sleep latency period, and comparing whether the test object can shorten the sleep latency period of the barbital sodium injection mouse.

2.6 statistical methods

The experimental results are established by Excel software, SPSS 17.0 statistical software is used for analysis, data are expressed by mean +/-standard deviation (x +/-s), chi-square test is adopted for counting data, one-way ANOWA (one-way ANOWA) or rank sum test is adopted for metering data, and the difference with P less than 0.05 has statistical significance.

3. Results of the experiment

3.1 direct sleep test

After the administration is finished, all the mice in the blank control group are in a clear-headed state; some mice in the experimental group appeared quiet, less active and sleepy, but none of them appeared asleep (i.e. no mice with missing righting reflex). The experimental result shows that the ganoderma lucidum wild jujube seed aminobutyric acid tablets have no direct sleep effect on mice, and the experimental result is negative.

3.2 experiment for prolonging sleep time of sodium pentobarbital

According to the technical specification for health food inspection and evaluation, on the basis of pentobarbital sodium hypnosis, if the sleep time of mice is prolonged after the intervention of the formula, the formula and the pentobarbital sodium have synergistic effect, the result is shown in table 1, the sleep time of mice in a blank control group is (31.78 +/-3.65) min, and the prognosis sleep time of mice in a aminobutyric acid group, a spina date seed extract group, a lucid ganoderma extract group and a lucid ganoderma spina date seed aminobutyric acid group is (37.20 +/-2.80), (36.48 +/-3.50), (41.93 +/-3.47) and (42.16 +/-4.24) min. Compared with the blank control group, the sleeping time of the mice in each group is respectively prolonged by 17.05%, 14.79%, 16.21% and 24.48%, and the mice in each group have statistical differences (P < 0.01). The formula with a certain dosage has a synergistic effect with the pentobarbital sodium, so that the sleep time of the mouse can be remarkably prolonged, and the effect of the three in cooperation is better.

TABLE 1 Effect of prolonging the sleep time of pentobarbital sodium after continuous gavage for 30 days

Experimental group	Total number of mice per group (only)	Sleep time (min)
			Blank space	12	31.78±3.65
Aminobutyric acid group	12	37.20±2.80
			Wild jujube seed extract group	12	36.48±3.50
Ganoderma lucidum extract group	12	36.93±3.47
			Lucid ganoderma and spina date seed aminobutyric acid tablet group	12	39.56±4.24

3.3 Pentobarbital sodium subthreshold dose hypnosis test

The experimental results in table 2 show that the blank control group of mice has a sleep incidence of 16.67%, meets the requirements of the experiment for 10% -20% of animals to fall asleep, and shows that 30mg/kg · bw can be used for the test of the sodium pentobarbital to meet the subthreshold dosage. The sleep incidence rates of the aminobutyric acid group, the spina date seed extract group, the lucid ganoderma extract group and the lucid ganoderma spina date seed aminobutyric acid group are respectively 33.33 percent, 41.67 percent and 66.67 percent, which are higher than the sleep incidence rate of the blank control group. The sleep incidence was increased and significantly different (P <0.05) for each group compared to the placebo group, with a dose-effect relationship.

TABLE 2 Effect on sleep incidence of barbiturate after 30 days of continuous gavage

Experimental group	Total number of mice per group (only)	Sleep-inducing mouse	Incidence of sleep (%)
				Blank space	12	2	16.67
Aminobutyric acid group	12	4	33.33
				Wild jujube seed extract group	12	4	33.33
Ganoderma lucidum extract group	12	5	41.67
				Lucid ganoderma and spina date seed aminobutyric acid tablet group	12	8	66.67

3.4 Barbituric sodium sleep latency test

As can be seen from the experimental results in table 3, in the barbital sodium sleep latency experiment, after the aminobutyric acid group, the spina date seed extract group, the lucid ganoderma extract group and the lucid ganoderma spina date seed aminobutyric acid group are subjected to dry prognosis, the sleep latencies of the mice are respectively (48.12 ± 4.16), (39.27 ± 6.35), (42.38 ± 3.87) and (36.69 ± 5.61) min, compared with the blank control, the sleep latency time of each group is respectively shortened by 5.72%, 23.06%, 16.97% and 28.12%, and the difference has statistical significance (P < 0.05). The experimental result shows that each formula has good effect of promoting the mice to fall asleep, the compatibility effect of the three is better, and the barbital sodium has the effect of shortening the sleep latency period.

TABLE 3 Effect of continuous gavage for 30 days on sleep latency in mice

Experimental group	Total number of mice per group (only)	Sleep latency period
			Blank space	12	51.04±5.02
Aminobutyric acid group	12	48.12±4.16
			Wild jujube seed extract group	12	39.27±6.35
Ganoderma lucidum extract group	12	42.38±3.87
			Lucid ganoderma and spina date seed aminobutyric acid tablet group	12	36.69±5.61

Example 5

The method for simultaneously detecting the jujuboside A and the ganoderic acid in the sleep improving tablet comprises the following steps:

1. materials and reagents

Lucid ganoderma and spina date seed aminobutyric acid tablets are prepared in laboratories;

jujuboside A, ganoderic acid A, China institute for food and drug testing;

methanol, formic acid, ethanol, acetonitrile, MREDA;

petroleum ether, Tianjin, maozen chemical reagent plant;

2. apparatus and device

XS105 electronic balance, METTLER TOLEDO;

KQ-500DE ultrasonic cleaner, ultrasonic instruments Inc. of Kunshan;

HH-8 digital display constant temperature water bath pot, Shanghai Haozhuang Instrument Co., Ltd;

model U3000 HPLC, Corona Ultra CAD detector, Thermo Scientific.

3. Experimental methods

(1) Preparation of Standard solutions

Stock solution of jujuboside a: accurately weighing 10mg of jujuboside A reference substance in a 50ml volumetric flask, dissolving with methanol, metering volume, and shaking to obtain stock solution.

Stock solution of ganoderic acid a: accurately weighing 10mg of ganoderic acid A standard substance in a 50ml volumetric flask, dissolving with methanol, fixing the volume, and shaking up to obtain stock solution.

(2) Preparation of test solution

Accurately weighing 800mg of a sample to be tested in a Soxhlet extractor, adding a proper amount of petroleum ether (60-90 ℃) for heating and refluxing for 2h, discarding petroleum ether liquid, volatilizing the solvent of medicine residues, transferring to a conical flask, adding 20mL of 70% ethanol, heating and refluxing for 2h, filtering, washing filter residues with 70% ethanol, combining washing liquid and filtrate, recovering the solvent until the solvent is dry, dissolving the residues with methanol, transferring to a 5mL volumetric flask, diluting to the constant volume with methanol, and shaking uniformly for later use.

(3) Chromatographic conditions

A chromatographic column: acclaim^TM 120 C18，5μm，4.6×250mm；

Flow rate: 1.0 mL/min;

column temperature: 40 ℃;

sample introduction amount: 10 mu L of the solution;

corona Ultra CAD detector atomization chamber temperature: 35 deg.C

Mobile phase gradient elution:

4. methodology investigation

(ii) precision test

And (3) taking the prepared sample solution, and continuously injecting samples for 6 times according to chromatographic conditions, wherein the RSD of the relative peak area of the result is less than 3%, and the experimental precision is good.

② repeatability test

6 parts of test sample are precisely weighed, test sample solution is prepared according to the method, sample introduction is respectively carried out under chromatographic conditions, RSD of the result relative peak area is less than 3%, and the experimental repeatability is good.

Stability test

The prepared test solution is taken and measured at 8 different time points of 0, 1, 2, 4, 8, 12, 18 and 24h respectively according to chromatographic conditions, the RSD of the relative peak area of the result is less than 3%, and the experimental stability is good.

Investigation of linear relationship

And (3) drawing a standard curve of the spina date seed saponin A: accurately sucking a proper amount of standard solution, diluting with methanol, preparing standard working solution with mass concentration of 10, 25, 50, 100, 200 μ g/mL, determining according to chromatographic conditions, and drawing a standard curve by taking the mass concentration of the jujuboside A as abscissa and the corresponding peak area as ordinate.

The content of the jujuboside A in the sample is calculated according to a formula I.

In the formula:

omega represents the mass fraction of the jujuboside A in the sample, mg/g;

rho is the mass concentration of the jujuboside A in the test solution calculated according to the standard curve, mu g/mL;

v is the volume of the test solution with constant volume, mL;

m-mass of sample, mg.

Drawing a standard curve of ganoderic acid A: accurately sucking a proper amount of standard solution, diluting with methanol, preparing standard working solution with mass concentration of 10, 25, 50, 100, 150 μ g/mL, determining according to chromatographic conditions, and drawing a standard curve by taking the mass concentration of ganoderic acid A as abscissa and the corresponding peak area as ordinate.

The content of ganoderic acid A in the sample is calculated according to formula II.

In the formula:

omega represents the mass fraction of ganoderic acid A in the sample, mg/g;

rho is the mass concentration of ganoderic acid A in the test solution calculated according to the standard curve, mu g/mL;

v is the volume of the test solution with constant volume, mL;

m-mass of sample, mg.

The result shows that the retention time of the jujuboside A is 16.2min, the retention time of the ganoderic acid A is 32.5min, the retention time is stable, and the peak pattern is better.

Example 6

Preferred procedure for mobile phase in chromatographic conditions:

the experimental procedure was the same as 3 in example 5. In 30-40min, when the mobile phase is acetonitrile: 0.1% formic acid solution (30: 70 (V/V)), ganoderic acid a does not reach baseline separation (as shown in fig. 1); when acetonitrile is changed to 0.1% formic acid solution (20: 80 (V/V)), the ganoderic acid A achieves baseline separation and better peak pattern (as shown in figure 2).

Example 7: detection method for improving GABA in sleep tablets

Although the key point of deeply developing health-care food related to the compound of the three is to detect the jujuboside A and the ganoderic acid at the same time, so that the detection efficiency can be improved, the GABA content still needs to be monitored, so that the comprehensiveness of the detected components can be improved.

1. Materials and reagents

Lucid ganoderma and spina date seed aminobutyric acid tablets are prepared in laboratories;

gamma-aminobutyric acid, usp;

ethanol, acetonitrile, HPLC grade, MREDA;

sodium bicarbonate, sodium acetate trihydrate, AR grade, shinyleaf science and technology development ltd;

4-dimethylaminoazobenzene-4-sulfonyl chloride, AR grade, Beijing YinoKai science, Inc.

2. Apparatus and device

XS105 electronic balance, METTLER TOLEDO;

KQ-500DE ultrasonic cleaner, ultrasonic instruments Inc. of Kunshan;

VM-2500 Multi-tube vortex Mixer, American restricted technologies;

HH-8 digital display constant temperature water bath pot, Shanghai Haozhuang Instrument Co., Ltd;

model U3000 HPLC, Thermo Scientific.

3. Experimental methods

(1) Preparation of Standard solutions

Standard stock solution of γ -aminobutyric acid: accurately weighing 10mg of gamma-aminobutyric acid standard substance in a 10ml volumetric flask, dissolving with acetonitrile, fixing the volume, and shaking up to obtain a stock solution.

Gamma-aminobutyric acid standard use solution: diluting the standard stock solution into 2.0, 5.0, 10.0, 50.0, 100.0 mg/L.

(2) Preparation of test solution

Sodium bicarbonate solution: 0.4g of sodium bicarbonate was weighed, dissolved in water and diluted to 10 mL.

DABS-Cl solution: 20mg of 4-dimethylaminoazobenzene-4-sulfonyl chloride was weighed out, dissolved in acetonitrile and diluted to 10 mL.

Sodium acetate trihydrate solution: 3.4g of sodium acetate trihydrate were weighed, dissolved and diluted with water to 500mL and filtered through a 0.45 μm filter.

(3) Preparation of test solution

Weighing 250mg of a test sample into a 100mL volumetric flask, adding a proper amount of extracting solution (ethanol + water is 4+1), carrying out ultrasonic treatment for 30min, cooling to room temperature, fixing the volume, and shaking up for later use.

(4) Derivatization

Accurately sucking 1mL of test solution or standard solution into a test tube with a plug, adding 0.2mL of sodium bicarbonate solution and 0.4mL of DABS-Cl solution, mixing uniformly, performing derivatization reaction in a water bath at 70 ℃ for 20min, and filtering with a 0.45-micron filter membrane.

(5) Chromatographic conditions

A chromatographic column: acclaim^TM 120 C18，5μm，4.6×250mm；

Detection wavelength: 436 nm;

column temperature: 30 ℃;

sample introduction amount: 10 mu L of the solution;

mobile phase: acetonitrile + sodium acetate trihydrate solution (35+ 65);

flow rate: 1.0 mL/min;

the result shows that the retention time of gamma-aminobutyric acid at 9.6min is stable, and the peak shape is better.

Reference to the literature

[1] China pharmacopoeia [ M ] Beijing, China pharmaceutical science and technology Press 2015.

[2] Inspection and evaluation of health food technical Specification [ M ] Beijing, national public health Press, 2003.