CN113341048A - Quality evaluation method of nigella sativa prescription - Google Patents

Abstract

The invention relates to the technical field of quality evaluation of traditional Chinese medicine component formulas, in particular to a quality evaluation method of a nigella sativa L prescription, which takes nigella sativa L seeds or nigella sativa L seed extracts as main material components and comprises the following steps: extracting effective components in nigella sativa formula to obtain formula extract; detecting the actual content of thymoquinone in the nigella sativa formula extract, and calculating the actual content W of thymoquinone in the nigella sativa formula; detecting the theoretical content W of thymoquinone in nigella sativa seeds or nigella sativa seeds extract in nigella sativa formula₀(ii) a Mixing W with W₀The closer the two are compared, the better the quality of the prescription is. According to the invention, effective components in the nigella sativa L prescription are extracted, and the content of thymoquinone in the nigella sativa L prescription is detected and compared with the theoretical content, so that whether the prescription with nigella sativa L seeds or nigella sativa L seed extract as a main substance component is effective quality evaluation is realized.

Description

Quality evaluation method of nigella sativa prescription

Technical Field

The invention relates to the technical field of quality evaluation of traditional Chinese medicine component formulas, in particular to a method for evaluating the quality of a prescription by taking nigella sativa seeds or nigella sativa seed extracts as main material components.

Background

The black grass seeds are dry mature seeds of black grass (Nigella sativa) of Ranunculaceae, are mainly distributed in Xinjiang and Yunnan of China, are used for thousands of years as medicinal materials commonly used by Uyghur and Dai nationality, are collected in the pharmacopoeia of the people's republic of China, and have the effects of tonifying kidney, strengthening brain, stimulating the menstrual flow, inducing diuresis and the like. Modern pharmacological research shows that nigella sativa L.is often used as a medicine with seeds rich in various components such as oil, volatile oil, saponin, flavone, alkaloid and the like, has various pharmacological effects such as anti-inflammatory, antibacterial, antioxidant, antitumor, immunoregulation and the like, and has a good protection effect on organs such as kidney, liver and the like. The composition is widely applied to the industries of food, medicines, spices and the like at home and abroad, and is also one of common compatible medicinal materials of Dai, Mongolian, Tibetan and Uygur medicines. However, when the nigella sativa seeds are used as a main component and are combined with other medicinal components to prepare a medicinal formula, the medicinal effect of the nigella sativa seeds in the formula cannot be fully exerted due to the influence of active ingredients in different medicinal components on the medicinal effect of the nigella sativa seeds, so that the treatment effect of the formula containing the nigella sativa seeds on diseases is greatly reduced, namely, the theoretical treatment effect and the actual treatment effect are seriously deviated.

For the above reasons, in the preparation of a pharmaceutical formulation using nigella sativa seeds, it is necessary to evaluate the quality of the formulation consisting of nigella sativa seeds and other compatible drugs for further study. In the prior art, the evaluation method for the effect of the nigella sativa seed formula mainly comprises clinical tests or animal in-vivo tests, the verification process is complex and the cost is high, and the wide application of the nigella sativa seeds in the field of treatment of traditional Chinese medicine diseases is greatly influenced.

Based on the method, if a simple and effective quality evaluation method of the black grass seed prescription can be established, the medicine effect of the prescription can be preliminarily estimated on the premise of not carrying out clinical tests or animal living tests, so that the preliminary screening of the medicine formula of the black grass seed prescription is realized, and the method has great significance for the application of the black grass seeds in the traditional Chinese medicine prescription.

Disclosure of Invention

The technical purpose of the invention is to provide a method for evaluating the quality of a prescription by taking nigella sativa seeds or nigella sativa seed extracts as main material components. After the effective components in the nigella sativa L.prescription are extracted, the content of thymoquinone in the nigella sativa L.prescription is detected and compared with the theoretical content, so that the effective quality evaluation of the prescription with nigella sativa L.seed or nigella sativa L.seed extract as the main material component is realized.

The technical scheme of the invention is as follows: a quality evaluation method of a nigella sativa prescription, which takes nigella sativa seeds or extracts of the nigella sativa seeds as main material components, comprises the following steps:

extracting effective components in the nigella sativa prescription to obtain a nigella sativa prescription extract;

detecting the actual content of thymoquinone in the nigella sativa formula extract, and calculating the actual content W of thymoquinone in the nigella sativa formula;

detecting the theoretical content W of thymoquinone in nigella sativa seeds or nigella sativa seeds extract in nigella sativa formula₀；

Mixing W with W₀The closer the two are compared, the better the quality of the nigella sativa formula is.

Further, the extraction of the effective components in the nigella sativa formula specifically comprises the following steps: grinding the nigella sativa L prescription into powder, uniformly mixing and soaking in hot water, centrifuging to obtain supernatant, drying with nitrogen, redissolving with ethanol, and filtering to obtain prescription extract.

Further, the feed-liquid ratio of the nigella sativa formula to hot water is 3 g: 10 mL; the temperature of the hot water is 90 ℃, and the soaking time is 10 min; centrifugation conditions: 3000rpm, 10 min; filtration through a 0.22 μm filter.

Further, the method for detecting the actual content of thymoquinone in the nigella sativa formula extract specifically comprises the following steps:

and (3) detecting the actual content of thymoquinone in the nigella sativa formula extract by using an external standard method and a gas chromatography-mass spectrometer.

Further, the conditions for detecting thymoquinone in the nigella sativa prescription extract by the gas chromatography-mass spectrometry combination instrument are as follows:

gas chromatography conditions: SH Rxi-5il MS chromatographic column (30m is multiplied by 0.25mm is multiplied by 0.25 mu m), the carrier gas is high-purity helium, the flow rate is 1.2mL/min, and the injection port temperature is 230 ℃; temperature programming: maintaining the initial temperature at 50 deg.C for 1 min; heating to 150 deg.C at 25 deg.C/min, heating to 300 deg.C at 10 deg.C/min, and maintaining for 15 min; the sample introduction amount is 1 mu L, and the sample introduction is not carried out by shunting;

mass spectrum conditions: the ion source is an electron bombardment source; transmission line temperature: 250 ℃; ion source temperature: 230 ℃; scanning mode: fully sweeping; scanning the mass range of 50-500 au; the solvent is delayed for 5 min.

Further, when X > 70%, the formula is effective, wherein X ═ W/W₀。

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

the nigella sativa seeds are rich in various bioactive components such as saponin, flavone and alkaloid and have rich pharmacological effects, effective component analysis after compatibility of the nigella sativa seeds and other medicinal components shows that different medicinal formulas can cause changes of the effective components in the formula, so that the treatment effects of the nigella sativa seed formula are different, and in the formulas, the content of thymoquinone is greatly influenced by the formula composition, so that the actual content and the theoretical content of the thymoquinone in the formula are inconsistent, and further research shows that the changes of the content of the thymoquinone are main factors influencing the effectiveness of the formula. Based on the above, the invention provides a quality evaluation method of a formula with nigella sativa seeds or nigella sativa seeds extract as a main material component, which determines the effectiveness of the formula by comparing the actual content and the theoretical content of thymoquinone in the formula, so that the drug effect of the nigella sativa seeds or nigella sativa seeds extract formula can be accurately evaluated on the premise of no clinical test or animal in vivo test.

Drawings

FIG. 1 is a standard graph of thymoquinone in example 1 of the present invention.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

(1) Determining the black grass seed formula: 80g of black grass seeds, 26.6g of liquorice, 26.6g of fennel seeds and 26.6g of black tea;

the theoretical content of thymoquinone in black grass seeds of China is calculated by referring to the contents of chemical component analysis and thymoquinone quantification of volatile oil of black grass seeds of Gunn-Dong, Zhaofeng, Lanjia, Fuguo, J, Chinese traditional medicine journal, 2009,34(22): 2887-2890':

(2) extracting effective components of the formula: grinding 3.00g of black sedge seeds, 1.00g of liquorice, 1.00g of fennel seeds and 1.00g of black tea (the dosage proportion is the same as the step (1)) into powder respectively, putting the powder into a 50mL centrifuge tube, adding 20mL of boiled water (90 ℃), covering the centrifuge tube, vortex and uniformly mixing the powder for 30s, soaking the powder for 10min, centrifuging the powder for 10min at 3000rpm, and taking supernatant. Taking 2mL of the supernatant fluid in a 10mL nitrogen blowing pipe, drying the supernatant fluid by using nitrogen, redissolving the supernatant fluid by using 2mL of ethanol, uniformly mixing the redissolved supernatant fluid and the redissolved supernatant fluid for 10 seconds by vortex, and filtering the redissolved supernatant fluid by using a 0.22 mu m filter membrane for standby use.

(3) Detecting the content of thymoquinone by GC-MS:

weighing a proper amount of thymoquinone standard substance, dissolving with ethanol, and preparing into standard substance stock solution of 10 μ g/mL for use. The stock solutions were diluted with ethanol to 0.1. mu.g/mL, 0.2. mu.g/mL, 0.5. mu.g/mL, 1.0. mu.g/mL, 2.0. mu.g/mL, respectively, and subjected to GC-MS on a 0.22 μm filter to prepare a thymoquinone standard curve (FIG. 1);

gas chromatography conditions: SH Rxi-5il MS chromatographic column (30m × 0.25mm × 0.25 μm), high purity helium as carrier gas, flow rate of 1.2mL/min, and injection port temperature of 230 deg.C. Temperature programming: maintaining the initial temperature at 50 deg.C for 1 min; raising the temperature to 150 ℃ at a speed of 25 ℃/min, raising the temperature to 300 ℃ at a speed of 10 ℃/min, and keeping the temperature for 15 min. The sample introduction amount is 1 mu L, and the sample introduction is not carried out by shunting;

mass spectrum conditions: the ion source is an electron impact source (EI); transmission line temperature: 250 ℃; ion source temperature: 230 ℃; scanning mode: fully sweeping; scanning the mass range of 50-500 au; the solvent is delayed for 5 min.

The curve equation y is obtained as 30384.42x +0.0 (R)²＝0.9998)；

Wherein x is the content of the component to be detected, mu g/mL; y is the peak area of the measured component.

Preparing samples in the two steps (2) in parallel by adopting the same method, detecting the content of thymoquinone in the two samples under the same conditions in the step (I) (performing two parallel tests), measuring the peak area of the component to be detected, and then calculating the content of the component to be detected;

the thymoquinone content of the formulations was determined as shown in table 1;

TABLE 1

Sample (I)	Area A of measured peak	Measured concentration C (μ g/mL)
			1	11494	0.37829
2	9527	0.31355

The actual content of thymoquinone extracted from nigella sativa in the formula is as follows:

in the above data, 2mL is the volume determined by the volume on the machine, 10 is the dilution factor (2 mL on the machine in 20 mL), and 3g is the mass of nigella sativa in the formula.

Comparing results: in the formula, X is 2.30613/3.08179 is 74.8%. Therefore, the prescription is effective.

Example 2

Crushing 80g of black grass seeds, adding 2.5 times of distilled water, soaking in a water bath at 50-60 ℃ for 12h, extracting under reflux at 85 ℃ for 2 times, each time for 1 hour, centrifuging the extracting solution at 3000rpm for 10min, and taking the supernatant; concentrating the supernatant by rotary evaporation to a relative density of 1.14 (50-60 ℃) to obtain a liquid I;

pulverizing and uniformly mixing 26.6g of liquorice, 26.6g of fennel seeds and 26.6g of black tea, adding 2.5 times of distilled water, soaking in a water bath at 50-60 ℃ for 12 hours, carrying out reflux extraction at 85 ℃ for 2 times, carrying out reflux extraction for 1 hour each time, and centrifuging the extracting solution at 3000rpm for 10min to obtain a supernatant; vacuum freeze-drying and concentrating the supernatant to a relative density of 1.2 (50-60 ℃) to obtain a liquid II;

mixing the solution I and the solution II, adding 2 times of 95% ethanol, stirring, and refrigerating at 2-6 deg.C for 24 hr; collecting supernatant, recovering ethanol under reduced pressure, diluting with water to 400mL, standing, collecting supernatant, filtering, canning, each 10mL, and sterilizing to obtain Chinese medicinal composition oral liquid.

Comparative example 1

The difference from example 2 is that only 133.2g of black grass seeds and 26.6g of black tea are contained. The results of the tests conducted in example 1 show the values of X and the values of the observed values in the formula shown in Table 2.

Comparative example 2

The difference from example 2 is that licorice root was replaced with moxibustion licorice root and fennel seed was replaced with clove. The calculated values of the measured values and X values in the obtained formulations are shown in Table 2.

Comparative example 3

The same as example 2, except that 80g of black grass seeds, 20g of liquorice, 20g of fennel seeds and 20g of black tea. The calculated values of the measured values and X values in the obtained formulations are shown in Table 2.

TABLE 2 statistical table of thymoquinone content in formulas of comparative examples 2-4

Effect test example 1

Tests are carried out on the anti-allergic rhinitis effect and the immunity improving effect of the traditional Chinese medicine composition oral liquid in the embodiment 2 and the comparative examples 1-3, and the specific steps are as follows:

(1) dose design: taking the traditional Chinese medicine composition oral liquid, wherein the concentration of the traditional Chinese medicine composition oral liquid is 0.2g/mL according to the concentration of the black grass seeds; diluting the oral liquid to 0.1g/mL and concentrating to 0.4g/mL respectively to obtain three measured test solutions, namely low (0.1g/mL), medium (0.2g/mL) and high (0.4 g/mL).

(2) Experimental animals and feeding conditions: BALB/C mice, male, 18-22 grams, clean grade (SPF grade) animals provided by shanghai jihui laboratory animals ltd [ license number: SCXK (Shanghai) 2017-0012, certificate number: 20170012007896]. The temperature of the experimental animal feeding room is 23 +/-2 ℃, the relative humidity is 40-70%, the animal feed is provided by Hangzhou Hebei science and technology limited, and the mice need to be adaptively fed in the animal room environment for one week before the experiment.

(3) The administration route is as follows: performing intragastric administration, wherein the intragastric administration volume is 0.2mL/20g of body weight;

(4) the experimental method comprises the following steps: 108 BALB/C mice were weighed one by one and randomly divided into 9 groups of 12 mice each: blank control group, model group, traditional Chinese medicine low dose group, traditional Chinese medicine medium dose group, traditional Chinese medicine high dose group, dexamethasone group, comparative example 1 group, comparative example 2 group and comparative example 3 group. The blank control group was replaced by PBS for sensitization and nasal drip, and eight other groups were constructed as mouse models of allergic rhinitis by OVA stimulation. The molding method comprises the following steps: mice were sensitized by intraperitoneal injection of Ovalbumin (OVA) 100. mu.g + aluminum hydroxide 1mg gel at time points D0, D4, D7, D10, D14, D18, and D21, each 0.2mL, seven times, D22 was administered once a day, D28 was administered by nasal drip with 2% OVA, 10. mu.l of each 20. mu.l of each nostril, nasal drip was continued to 42 days, and the duration of challenge was 2 weeks, once a day, before drawing material.

(5) The experimental results are as follows:

a. weight: starting on day 4W (D22), mice in the administration group were gavaged once a day for 3 weeks (21 days) and the weight of the mice was weighed weekly; the effect of each sample on the body weight of the experimental animals is shown in table 3;

TABLE 3 Effect of samples on body weight of test animals

Note: p < 0.05 compared to placebo; compared with the model group, # P < 0.05, # P < 0.01.

As can be seen from Table 3, the body weight growth rate of the model mice was reduced after the model formation, and the body weight was significantly smaller than that of the blank control group at 7W (P < 0.05) as compared with that of the blank control group. Compared with the model group mice, the mice in each administration group have almost no weight increase or even weight reduction after the 4W administration, and are probably related to the drug, the weight reduction of the mice in the high-dose group is significant at the time points of 6W and 7W (P < 0.05), and the weight reduction of the mice in the dexamethasone group is very significant at the time points of 5W, 6W and 7W (P < 0.01). The weight changes before and after administration of the drugs in the comparative example 1, comparative example 2 and comparative example 3 were not significant, and there was no statistically significant difference compared with the model group.

b. Sneezing and nasal number of flexions: after 3W administration, 2W after continuous nasal drip, sneezing and nasal scratching were observed and recorded within 10min after the last nasal drip with 2% ovalbumin solution, and the results are shown in Table 4.

TABLE 4 Effect of samples on rhinitis symptoms in Experimental animals

Group of	Number of sneezes	Number of times of scratching the nose
			Blank control group	1.25±0.25	1.17±0.27
Model set	19.33±1.51**	15.73±1.42**
			Low dose group	12.83±1.60##	11.25±0.77#
Middle dose group	9.00±1.04##	7.55±0.62##
			High dose group	9.45±1.43##	6.73±1.14##
Dexamethasone group	3.00±0.44##	3.17±0.51##
			Comparative example 1 group	18.84±1.22	14.16±1.18
Comparative example 2 group	17.87±1.31	13.83±1.24
			Comparative example 3 group	15.69±1.27	11.69±1.58

Note: p < 0.05 compared to placebo; compared with the model group, # P < 0.05, # P < 0.01

As can be seen from Table 4, the number of sneezes and nasal flexions in the mice in the model group was significantly increased compared to the blank control group, indicating that the allergic rhinitis model was very successful. Compared with the model group mice, the numbers of sneezing and nasal scratching of the mice in each administration group are obviously reduced (P is less than 0.05 or less than 0.01), and the sample of the example 2 can obviously relieve the allergic rhinitis symptoms of the experimental animals. The sneezing and nasal scratching times of the mice in the comparative example 1 group, the comparative example 2 group and the comparative example 3 group are similar to those of the model group, and have no statistical difference.

c. Thickness of nasal mucosa: after recording rhinitis symptoms, the experimental mice draw blood and lavage fluid, then 5 mice in each group and nasal bones fix the whole nasal mucosa, pathological sections are made, 5 visual fields are randomly selected in each group to measure the thickness of the nasal mucosa, and the results are shown in table 5;

TABLE 5 Effect of samples on nasal mucosa thickness of Experimental animals

Group of	Thickness of nasal mucosa (mum)
		Blank control group	34.40±9.22
Model set	66.80±12.46**
		Low dose group	42.00±7.64##
Middle dose group	39.00±6.87##
		High dose group	36.60±3.98##
Dexamethasone group	37.80±2.79##
		Comparative example 1 group	61.58±5.68
Comparative example 2 group	57.69±6.11
		Comparative example 3 group	55.85±5.17

Note: p < 0.05 compared to placebo; compared with the model group, # P < 0.05, # P < 0.01

As can be seen from table 5, the nasal mucosa thickness was significantly increased in the model group mice compared to the blank control group, indicating that the allergic rhinitis model was very successful. Compared with the model group mice, the thickness of the nasal mucosa of each administration group mouse is obviously reduced (P is less than 0.01), which indicates that the sample of the example 2 can obviously relieve the pathological symptoms of allergic rhinitis nasal mucosa thickening of the experimental animal. The thicknesses of the nasal mucosa of the mice in the comparative example 1 group, the comparative example 2 group and the comparative example 3 group are similar to those of the model group, and no statistical difference exists, so that the traditional Chinese medicine composition in the comparative examples 1-3 has little relieving effect on the thickening of the nasal mucosa of the allergic rhinitis mice.

d. Lavage fluid inflammatory cells: the results are shown in tables 6 and 7;

TABLE 6 Effect of samples on inflammatory cells of nasal lavage fluid in Experimental animals

Note: p < 0.05 compared to placebo; compared with the model group, # P < 0.05, # P < 0.01

As can be seen from table 6, the nasal inflammatory cells were significantly increased in the mice of the model group compared to the blank control group, indicating that the model of allergic rhinitis was very successful. Compared with the mice in the model group, the total nasal cavity inflammatory cells of the mice in each administration group are remarkably reduced (P is less than 0.01), which shows that the sample in the example 2 can remarkably relieve the symptoms of the increase of the nasal cavity inflammatory cells of the allergic rhinitis of the experimental animals, especially the high-dose group. Compared with the model group, the number and the total number of various inflammatory cells in the nasal cavity of mice in the comparative example 1 group, the comparative example 2 group and the comparative example 3 group are reduced, but no statistical difference exists, which shows that the traditional Chinese medicine composition in the comparative examples 1-3 has no obvious relieving effect on the symptoms of the increase of the number of inflammatory cells in the nasal cavity.

Table 7 effect of samples on alveolar lavage fluid inflammatory cells in experimental animals.

Control group	Eosinophils	Neutrophils	Lymphocytes	Macrophage cell	Total number of cells
						Blank control group	0.47±0.08	0.20±0.05	2.93±0.15	0.47±0.15	2.96±0.15
Model set	1.60±0.25##	1.33±0.33##	13.05±0.14#	1.60±0.21	18.07±0.14##
						Low dose group	0.80±0.21	0.53±0.14	8.13±0.37*	0.47±0.06*	8.83±0.37*
Middle dose group	0.80±0.16	0.27±0.08*	6.00±0.23**	0.40±0.08*	6.55±0.23**
						High dose group	0.53±0.13*	0.15±0.02**	5.27±0.18**	0.27±0.04*	5.97±0.18**
Dexamethasone group	0.27±0.07**	0.07±0.03**	2.93±0.17**	0.23±O.08*	3.10±0.17**
						Comparative example 1 group	1.35±0.15	1.12±0.07	12.15±0.14	1.51±0.07	17.12±0.10
Comparative example 2 group	1.24±0.11	1.08±0.06	11.23±0.17	1.43±0.05	15.86±0.11
						Comparative example 3 group	1.16±0.14	0.87±0.07	10.64±0.15	1.22±0.06	14.38±0.14

Note: p < 0.05 compared to placebo; compared with the model group, # P < 0.05, # P < 0.01

As can be seen from table 7, the alveolar inflammatory cells of the mice in the model group were significantly increased compared to the blank control group, indicating that the allergic rhinitis model was very successful. Compared with the mice in the model group, the total number of the alveolar inflammatory cells of the mice in each administration group is extremely reduced (P < 0.01), which shows that the sample in the example 2 can obviously relieve the symptoms of the increase of the number of the alveolar inflammatory cells of the allergic rhinitis of the experimental animals, and particularly, the number of the lymphocytes in the alveoli of the mice is extremely reduced (P < 0.01) by the medium and high dose groups. The mouse pulmonary alveoli of the comparative example 1 group, the comparative example 2 group and the comparative example 3 group have reduced numbers and total numbers of various inflammatory cells compared with the model group, but have no statistical difference, which shows that the traditional Chinese medicine composition of the comparative examples 1-3 has no obvious relieving effect on the symptoms of the increase of the pulmonary alveoli inflammatory cells

e. The immunity improving effect is as follows: the levels of immunoglobulins IgE, IgG1 and IgG2a in the sera of experimental animals are shown in Table 8.

TABLE 8 Effect of samples on the serum levels of immunoglobulins IgE, IgG1 and IgG2a in experimental animals

Note: p < 0.05 compared to placebo; compared with the model group, # P < 0.05, # P < 0.01

As shown in Table 8, compared with the blank control group, the serum levels of immunoglobulin IgE and IgG1 in the mice of the model group are obviously increased (P < 0.01), and the level of IgG2a is obviously reduced (P < 0.01), which indicates that the immunity of the mice with allergic rhinitis is obviously reduced. Compared with the model group mice, the serum levels of immunoglobulin IgE and IgG1 in the mice of each administration group are reduced, wherein the IgE level of the medium-high dose group is obviously reduced (P < 0.05), and the IgG1 level of the high dose group is obviously reduced (P < 0.05). In addition, the level of IgG2a in each administration group is obviously increased, wherein the level of IgG2a in the medium and high dose groups is obviously increased (P is less than 0.01), which shows that the traditional Chinese medicine composition can obviously improve the level of immunoglobulin in the serum of experimental animals and enhance the immunity, and particularly the level of the IgG2 in the medium and high dose groups is obviously improved. Compared with the model group, the levels of immunoglobulin IgE and IgG1 in the serum of mice in the comparative example 1 group, the comparative example 2 group and the comparative example 3 group are reduced, the level of IgG2a is increased, but no statistical difference exists, and the traditional Chinese medicine composition of the comparative examples 1-3 has no obvious effect of enhancing the immunity of the mice.

f. The immunity improving effect is as follows: the levels of interleukins IL-4, IL-5 and IL-13 in the tissues of the experimental animals are shown in Table 9.

TABLE 9 Effect of samples on IL-4, IL-5 and IL-13 levels in tissues of Experimental animals

Note: p < 0.05 compared to placebo; compared with the model group, # P < 0.05, # P < 0.01

As can be seen from Table 9, the levels of IL-4, IL-5 and IL-13 in the nasal mucosa and lung tissues of the model mice were significantly increased (P < 0.01) compared to the blank control group, indicating that the immunity of the allergic rhinitis mice was significantly reduced. Compared with the model group mice, the water levels of the interleukins IL-4, IL-5 and IL-13 in the nasal mucosa and the lung tissue of each administration group mice are reduced, wherein the level of the interleukins IL-4 in the nasal mucosa tissue of the medium and high dose groups is obviously reduced (P is less than 0.05), and the level of the IL-4 in the lung tissue is extremely obviously reduced (P is less than 0.01); the IL-5 level in the lung tissue of the medium and high dose groups is obviously reduced (P is less than 0.05), and the IL-5 level in the nasal mucosa tissue of the high dose group is obviously reduced (P is less than 0.05); IL-13 level in lung tissue of medium and high dose groups is reduced remarkably (P < 0.01), and IL-13 level in nasal mucosa tissue of high dose groups is reduced remarkably (P < 0.05). The results show that the traditional Chinese medicine composition can reduce the levels of interleukins IL-4, IL-5 and IL-13 in nasal mucosa and lung tissues of experimental animals, enhance the immunity and particularly obviously reduce the level of interleukins in a high-dose group. Compared with the model group, the levels of interleukins IL-4, IL-5 and IL-13 in nasal mucosa and lung tissues of the group of the comparative example 1, the group of the comparative example 2 and the group of the comparative example 3 are all reduced, but have no statistical difference, which shows that the traditional Chinese medicine composition of the comparative examples 1 to 3 has no obvious effect of enhancing the immunity of mice and is consistent with the judgment result of thymoquinone.

In conclusion, after the samples of example 2 are orally administered to mice with different dosages for 21 days, the effects of resisting allergic rhinitis and improving immunity are achieved; therefore, the traditional Chinese medicine composition can slow down the weight reduction trend of allergic rhinitis of experimental animals, obviously reduce sneezing and nose scratching times, obviously relieve the symptoms of nasal mucosa thickening of the allergic rhinitis, obviously reduce the number of inflammatory cells in nasal cavities and alveoli, obviously improve the level of immunoglobulin in serum of the experimental animals, and obviously reduce the level of interleukin in nasal mucosa and lung tissues, and the result is effectively consistent with the judgment result of the embodiment 1.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included therein.

Claims (6)

1. A quality evaluation method of a nigella sativa prescription, which takes nigella sativa seeds or extracts of the nigella sativa seeds as main material components, is characterized by comprising the following steps:

extracting effective components in the nigella sativa prescription to obtain a nigella sativa prescription extract;

detecting the actual content of thymoquinone in the nigella sativa formula extract, and calculating the actual content W of thymoquinone in the nigella sativa formula;

detecting the theoretical content W of thymoquinone in nigella sativa seeds or nigella sativa seeds extract in nigella sativa formula₀；

Mixing W with W₀The closer the two are compared, the better the quality of the nigella sativa formula is.

2. The method for evaluating the quality of a nigella sativa formula according to claim 1, wherein the extracting the effective components of the nigella sativa formula specifically comprises: grinding the nigella sativa L.prescription into powder, uniformly mixing and soaking in hot water, centrifuging to obtain supernatant, drying with nitrogen, redissolving with ethanol, and filtering to obtain nigella sativa L.prescription extract.

3. The method for evaluating the quality of a nigella sativa formula according to claim 2, wherein the ratio of the nigella sativa formula to hot water is 3 g: 10 mL; the temperature of the hot water is 90 ℃, and the soaking time is 10 min; centrifugation conditions: 3000rpm, 10 min; filtration through a 0.22 μm filter.

4. The method for evaluating the quality of a nigella sativa formula as claimed in claim 1, wherein the step of detecting the actual content of thymoquinone in the nigella sativa formula extract comprises the following steps:

and (3) detecting the actual content of thymoquinone in the nigella sativa formula extract by using an external standard method and a gas chromatography-mass spectrometer.

5. The quality evaluation method of a nigella sativa prescription according to claim 4, wherein the conditions for detecting thymoquinone in the nigella sativa prescription extract by using a gas chromatography-mass spectrometry are as follows:

gas chromatography conditions: SH Rxi-5il MS chromatographic column (30m is multiplied by 0.25mm is multiplied by 0.25 mu m), the carrier gas is high-purity helium, the flow rate is 1.2mL/min, and the injection port temperature is 230 ℃; temperature programming: maintaining the initial temperature at 50 deg.C for 1 min; heating to 150 deg.C at 25 deg.C/min, heating to 300 deg.C at 10 deg.C/min, and maintaining for 15 min; the sample introduction amount is 1 mu L, and the sample introduction is not carried out by shunting;

mass spectrum conditions: the ion source is an electron bombardment source; transmission line temperature: 250 ℃; ion source temperature: 230 ℃; scanning mode: fully sweeping; scanning the mass range of 50-500 au; the solvent is delayed for 5 min.

6. The method for evaluating the quality of a nigella sativa formula according to claim 1, wherein X > 70% is effective, wherein X ═ W/W₀。

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