CN114097911B

CN114097911B - Tea bag for preventing and treating pharyngitis and detection method thereof

Info

Publication number: CN114097911B
Application number: CN202111617379.7A
Authority: CN
Inventors: 张志锋; 李婧; 张英秀
Original assignee: Southwest Minzu University
Current assignee: Southwest Minzu University
Priority date: 2021-12-28
Filing date: 2021-12-28
Publication date: 2023-10-03
Anticipated expiration: 2041-12-28
Also published as: CN114097911A

Abstract

The invention provides a teabag with the function of preventing and treating pharyngitis, which consists of six raw materials of liquorice, platycodon grandiflorum, chinese olive, dark plum, peppermint and black tea. The product has effects in nourishing yin, moistening lung, benefiting throat, and clearing lung-heat, and can be used for treating chronic pharyngolaryngitis such as dry throat, less phlegm and pharyngitis due to lung yin deficiency, body fluid failure, and laryngopharynx lesions. Meanwhile, the invention also provides a quality control method of the teabag.

Description

Tea bag for preventing and treating pharyngitis and detection method thereof

Technical Field

The invention relates to a natural plant product with a certain effect and an analysis method thereof.

Background

The tea agent is an oral preparation prepared by mixing decoction pieces or extract with tea or other auxiliary materials, and comprises blocky tea agent, bagged tea agent and decocted tea agent. Chinese is the hometown of tea, the tea culture is a great source of history in China, and the medicinal tea is an important component in the traditional Chinese medicine treasury. In recent years, the number of pharyngitis treatment modes is not counted, and health care products with protection function are also available.

Disclosure of Invention

Specifically, the invention provides a teabag with the function of preventing and treating pharyngitis, which consists of six raw materials of liquorice, platycodon grandiflorum, chinese olive, dark plum, peppermint and black tea. The product has effects in nourishing yin, moistening lung, benefiting throat, and clearing lung-heat, and can be used for treating chronic pharyngolaryngitis such as dry throat, less phlegm and pharyngitis due to lung yin deficiency, body fluid failure, and throat experiment.

In the recipe, licorice root, radix Glycyrrhizae Praeparata, spleen invigorating qi, heat-clearing and detoxicating, phlegm-eliminating and cough-relieving, and pain-relieving, and harmonizing the drugs. Dark plum fruit, fructus mume, astringing lung, astringing intestine, promoting fluid production and relieving ascariasis. Is used for treating lung deficiency chronic cough, chronic diarrhea, deficiency heat diabetes, ascariasis vomiting and abdominal pain. Fructus Canarii albi, clearing heat and detoxicating, relieving sore throat, promoting fluid production. Can be used for treating sore throat, cough, phlegm, dysphoria with smothery sensation, thirst, and poisoning of fish and crab. Radix Platycodi, has effects of dispersing lung qi, relieving sore throat, eliminating phlegm, and expelling pus. Can be used for treating cough with excessive phlegm, chest distress, pharyngalgia, hoarseness, pulmonary abscess, and pus discharge.

At present, common tea drinks prepared from natural extracts are prepared by adding auxiliary materials such as filling agents, the concentration of the products is easily reduced, the taste cannot be truly reduced, the filling agents are needed to be used, and the cost is increased.

In order to avoid the problems, in the preparation process of the product, the extracting solution of the raw materials and the coarse powder of the raw materials are mixed and dried. Firstly, the problems caused by adding auxiliary materials are avoided; secondly, the extracting solution contained in the product is concentrated, so that the extracting solution can be dissolved and diluted firstly in the subsequent use, the effect is quickly achieved, and meanwhile, the natural taste of each raw material can be tasted in the initial use period; meanwhile, the extracting solution is matched with coarse powder for use, and the coarse powder can gradually release active ingredients and flavor components after being brewed, so that the times and time of each time the product can be brewed are prolonged.

The main preparation method comprises the following steps: pulverizing Glycyrrhrizae radix, fructus Canarii albi, radix Platycodi, herba Menthae, and black tea, retaining coarse powder of 10-20 mesh, extracting the rest powder with mume fructus with water, concentrating to relative density of 1.1-1.2, mixing the medicinal liquid with the coarse powder, standing, drying, spraying 75% ethanol, and drying.

The product is simply unrecognizable from the appearance after being prepared into tea, and therefore, the invention also provides a detection method which is convenient for effectively controlling the quality of the product and mainly adopts High Performance Liquid Chromatography (HPLC) for detection. However, in previous experiments, it was found that the separation and detection of the components of the product was not effective using a series of more conventional chromatographic conditions, and for this reason, the inventors have made extensive conditionals such as:

mobile phase screening (see fig. 1-3):

(1) Mobile phase 1:

methanol 5% and water 95% in 0-5 min; the method comprises the steps of carrying out a first treatment on the surface of the

Methanol 20% and water 80% for 5-10 min;

methanol 40% and water 60% for 10-15 min;

methanol 60% and water 40% for 15-20 min;

methanol 80% and water 20% for 20-25 min;

methanol 99% and water 1% for 25-30 min;

(2) Mobile phase 2:

Methanol 10% and water 90% for 5-10 min;

methanol 30% and water 70% for 10-15 min;

15-20min of methanol 50% and water 50%;

methanol 60% and water 40% for 20-25 min;

methanol 70% and water 30% for 25-30 min;

30-35min of methanol 80% and water 20%;

methanol 99% and water 1% for 35-40 min;

(3) Mobile phase 3:

methanol 5% and water 95% in 0-10 min; the method comprises the steps of carrying out a first treatment on the surface of the

10-13min of methanol 10% and 90% of water;

13-20min of methanol 20% and water 80%;

methanol 30% and water 70% for 20-40 min;

40-45min of methanol 50% and water 50%;

methanol 60% and water 40% for 45-50 min;

50-55min of methanol 70% and water 30%;

55-60min of methanol 80% and water 20%;

methanol 90% and water 10% for 60-65 min;

methanol 99% and water 1% for 65-75 min;

the separation, identification and detection of chromatographic peaks cannot be satisfied from the chromatogram of the mobile phase gradient of screening alone. Based on the above, various ways of adjusting temperature, flow rate, acid addition and the like are tried to detect, and finally, the concentration of the acid species is found to have a significant effect on the detection result, and after the specific content (0.4%) of glacial acetic acid is added, the chromatographic peak separation effect is obviously improved, and even if compared with the addition of 0.2% of glacial acetic acid, the method has significant advantages (see fig. 4-6).

The wavelengths were screened, such as 325nm, 252nm, 230nm, etc., and finally the detection chromatographic peaks at 252nm were found to be more, see FIGS. 7-9.

After multiple attempts on different conditions, according to the summary and experience adjustment of the inventor, the preferred conditions are finally obtained: chromatograph: shimadzu liquid chromatograph LC-16

A detector: SPD-16 ultraviolet-visible detector

Work station: labsolution 5.88

Chromatographic column: c18, kromasil C18 (4.6X250 mm,5 μm), column temperature 30 ℃,

mobile phase: gradient elution is carried out on methanol-0.4% glacial acetic acid solution, the flow rate is 1ml/min, and the sample injection amount is 10ul

Wavelength of 252nm

Time program: methanol 5% for 0-10min, 0.4% glacial acetic acid solution 95%; the method comprises the steps of carrying out a first treatment on the surface of the

10-13min of methanol 10%,0.4% glacial acetic acid solution 90%;

13-20min methanol 20%,0.4% glacial acetic acid solution 80%;

methanol 30% for 20-40min, and 0.4% glacial acetic acid solution 70%;

methanol 50% for 40-45min, 0.4% glacial acetic acid solution 50%;

methanol 60% for 45-50min, and glacial acetic acid solution 40% of 0.4%;

methanol 70% and 0.4% glacial acetic acid 30% in 50-55 min;

methanol 80% for 55-60min, and glacial acetic acid solution 20% 0.4%;

methanol 90% for 60-65min, and glacial acetic acid solution 10% 0.4%;

methanol 99% and 0.4% glacial acetic acid solution 1% for 65-75 min.

The HPLC method is adopted in the research to establish the fingerprint of the teabag, and the similarity evaluation and the principal component analysis are carried out to screen out the conditions causing the difference of the teabag components in different batches, and the standard fingerprint is formed, so that the quality control of the teabag is realized.

Drawings

FIG. 1 chromatogram of mobile phase 1

FIG. 2 chromatogram of mobile phase 2

FIG. 3 chromatogram of mobile phase 3

FIG. 4 chromatogram without acid addition

FIG. 5 chromatogram of 0.2% glacial acetic acid

FIG. 6 chromatogram of 0.4% glacial acetic acid

FIG. 7 chromatogram at 325nm wavelength

FIG. 8 chromatogram at 252nm wavelength

FIG. 9 chromatogram at 230nm wavelength

FIG. 10 S1-S10 sample chromatography overlay

FIG. 11 HPCL of teabag superimposed a multipoint correction pattern and a control pattern (R)

FIG. 12 HPLC standard fingerprint of teabag, 9. Chlorogenic acid, 13. Glycyrrhizin, 16. Quercetin, 21. Chrysophanol

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

Example 1

Prescription: 200g of liquorice, 200g of platycodon grandiflorum, 200g of Chinese olive

300g of peppermint, 700g of black tea and 100g of dark plum

The preparation process comprises the following steps:

weighing 200g of liquorice, green fruits and platycodon grandiflorum respectively, 100g of dark plum, 300g of peppermint and 700g of black tea. Mixing the weighed Glycyrrhrizae radix, fructus Canarii albi, radix Platycodi, herba Menthae and black tea uniformly, pulverizing together, reserving coarse powder with 10-20 mesh sieve for use, decocting the rest powder and mume fructus together with 6-8 times of water for 3 times and half an hour each time, mixing the decoctions, concentrating to appropriate volume of medicinal liquid with density of 1.15, pouring the medicinal liquid into the coarse powder, stirring uniformly, soaking overnight, drying in oven at 60deg.C, spraying 75% alcohol on tea surface, and drying in oven.

Example 2

Pharmacological tests prove that the product has obvious inhibition effect on acute exudative inflammation and chronic proliferative inflammation of mice and rats under the treatment dosage.

Test materials

GJ-01 teabag (prepared in example 1) contains crude drug 2.3g/g, and soaking in boiled water to obtain 8.33% and 16.67% leaching solution (0.2 g/ml and 0.4 g/ml). Compound Scutellariae radix tablet (Zhaoqing star lake pharmaceutical Co., ltd., batch No. 202008) is formulated into 0.1 tablet/ml suspension. Animals, kunming mice, chengdu Biotechnology Co., ltd, qualification number SCXK 2018-0008.

Method and results:

(one) acute anti-inflammatory test

1. The ear swelling method of mice is that the body weight of the female Kunming mice is 18-22 g, the mice are randomly divided into 4 groups, a normal control group, a GJ-01 teabag high-dose group, a low-dose group and a compound baical skullcap root tablet buccal tablet group. The stomach was filled with 0.2ml/L0g.2 times, 18h after the first administration, 30min after the 2 nd administration, animals were sacrificed 30min after molding with 50. Mu.L of xylene per right ear of the mice, left and right ears were removed with a stainless steel punch with a diameter of 8mm, and the swelling ratio was calculated. The results are shown in Table 1

TABLE 1 influence of GJ-01 teabag on swelling of mouse ear

(II) chronic anti-inflammatory test

1. The effect on chronic granuloma of mice caused by agar is that Kunming sex mice, weight of 18-22 g, are subcutaneously injected with 0.2 ml/piece of 0.2% agar at back, and then are randomly divided into 4 groups, which are the same as above. Granuloma was taken and weighed on day 11 for 10 days with 0.2ml/10g of ig administration, and the results are shown in Table 2.

TABLE 2 influence on the formation of granulomas in mice by agar

The results show that the high dose group can significantly inhibit granuloma formation.

(III) GJ-01 teabag acute toxicity test

The GJ-01 teabag is given to the mice for 38g/kg (calculated by the amount of the contained crude drug) and is 240 times of the dosage for human, and the continuous observation is carried out for 7 days, so that 40 mice die, the stool, the fur and the activity and the passive reflection of the mice are not obviously changed, the weight change is not obviously different after weighing again on the 7 th day of the experiment. Experimental results prove that the product has no obvious toxicity to animals when being administrated at a certain dosage. The dosage of the experiment is 240 times of daily dosage of a person, the person takes the medicine 2 to 3 times per day, 1 bag is used each time, and each bag contains 5g of crude drug. The clinical quasi-dosage is a safe and nontoxic dosage.

(IV) summary and evaluation

The GJ-01 teabag is processed by adopting a modern preparation process, and experimental results prove that the teabag has obvious inhibition effect on acute exudative inflammation and chronic proliferative inflammation of mice at doses of 4g/kg and 8g/kg, and all medicinal materials are medicinal and edible medicinal materials, so that the teabag has no toxic or side effect. The pharmacological experiment results prove that the product has the efficacy on the treatment of pharyngitis.

Example 3

The research adopts an HPLC method to establish the fingerprint of the teabag, and carries out similarity evaluation and principal component analysis to screen out the condition of causing the difference of the teabag components in different batches, thereby providing scientific basis for the quality control of the teabag.

1. Instrument and materials

1.1 instruments

Island body fluid phase chromatograph LC-16: AE240 electronic balance (meltrele-tolido company, switzerland); KQ-250B ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); QE-100 Chinese medicine pulverizer (Zhejiang Yi Gong trade Co., ltd.).

1.2 materials

The reference substances chlorogenic acid, liquiritin, quercetin and chrysophanol were purchased from Sichuan Kangbang biotechnology Co.

The balloonflower root, the liquorice, the Chinese olive, the dark plum and the mint are purchased from the medicinal material market of the adult lotus pool and are identified as genuine products through the teaching of the university of southwest of China Zhang Zhifeng; black tea (producing place: anxi county, long pit village, three villages, production license number: SC 11435052401202)

2. Method and results

2.1 chromatographic conditions

By C ₁₈ Kromasil C ₁₈ (4.6X250 mm,5 μm) column, gradient elution (0-10 min methanol 5%,0.4% glacial acetic acid solution 95%, 10-13min methanol 10%,0.4% glacial acetic acid solution 90%, 13-20min methanol 20%,0.4% glacial acetic acid solution 80%, 20-40min methanol 30%,0.4% glacial acetic acid solution 70%, 0-45min methanol 50%,0.4% glacial acetic acid solution 50%, 45-50min methanol 60%,0.4% glacial acetic acid solution 40%, 50-55min methanol 70%,0.4% glacial acetic acid solution30% of solution; methanol 80% for 55-60min, and glacial acetic acid solution 20% 0.4%; methanol 90% for 60-65min, and glacial acetic acid solution 10% 0.4%; methanol 99% for 65-75min, 0.4% glacial acetic acid solution 1%); flow rate: 1ml/min; wavelength: 252nm; column temperature is 30 ℃; the sample injection amount is 10 mu L.

2.2 preparation of control solution

Chlorogenic acid, liquiritin, quercetin and chrysophanol reference substance are weighed precisely, dissolved by adding methanol with ultrasound, and prepared into reference substance solution of 0.1 μg/ml.

2.3 preparation of sample solutions

1 sample was weighed from each batch prepared "2.1" into a 250ml Erlenmeyer flask, labeled S1, S2, S3, S4, S5, S6, S7, S8, S9, S10. Adding 10ml of 80% methanol solution into each sample, setting the temperature at 30 ℃, carrying out ultrasonic treatment for 30min, filtering by filter paper, taking 1ml of each sample, and filtering by a microporous filter membrane with the thickness of 0.22 mu m.

2.4 fingerprint Studies

2.4.1 establishment of finger print

Taking 0.2g of each of 10 teabags, preparing a sample solution according to the method under the item "2.3", carrying out sample injection measurement according to the chromatographic condition under the item "2.2", and recording each chromatogram. The HPLC profile was recorded using a "traditional chinese medicine chromatographic fingerprint similarity evaluation system (2012.13072 version)", see fig. 10:

2.4.2 similarity analysis

And (3) performing similarity evaluation on HPLC fingerprints of 10 sample teabags by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.130723 edition). And (3) taking the S1 sample spectrum as a reference spectrum, performing peak matching by a median method and multipoint correction for 0.1min to generate a reference fingerprint spectrum (R), wherein the result is shown in FIG. 11, and the similarity calculation is performed, and the result is shown in Table 3. As shown in FIG. 7, the finger print of 10 samples all have a similarity of > 0.966, indicating that the difference between 10 samples is small.

Table 3 table of similarity of HPLC fingerprints of 10 six-ingredient sore throat relieving tea

2.4.3 principal component analysis, namely using the common peak area of the S1-S10 samples as a variable, carrying out principal component analysis on the 10 samples by using SPSS software, and calculating the eigenvalue and variance contribution ratio of the correlation matrix. Taking the eigenvalue >1 as a standard, 5 components are obtained, the cumulative variance contribution rate is 90.974%, most of the information of the sample can be represented, and the results are shown in tables 4 and 5. Chromatographic peaks 1, 6, 8, 10, 11, 12, 13, 15 have higher positive loading on component 1, wherein peak No. 8 is chlorogenic acid and peak No. 13 is glycyrrhizin; chromatographic peaks 2, 4, 7, 16, 17, 18, 19, 20, 21 have a higher positive load on component 2, wherein peak 16 is quercetin and peak 21 is chrysophanol; chromatographic peaks 3, 9 have a higher positive load on component 3; the chromatographic peaks 5, 14 have a relatively high positive load on component 5. The standard fingerprint is shown in fig. 12.

The total variance explained in Table 4

TABLE 5 component matrix

Claims

1. The method for detecting the teabag is characterized in that the teabag comprises the following raw materials: 15-25 parts of liquorice, 15-25 parts of platycodon grandiflorum, 15-25 parts of Chinese olive, 25-35 parts of mint, 60-80 parts of black tea and 80-120 parts of dark plum; the detection was performed by HPLC, including the following chromatographic conditions:

chromatographic column: C18C

Mobile phase: methanol-0.4% glacial acetic acid solution, gradient procedure was as follows:

methanol 5% for 0-10min, 0.4% glacial acetic acid solution 95%;

10-13min of methanol 10%,0.4% glacial acetic acid solution 90%;

13-20min methanol 20%,0.4% glacial acetic acid solution 80%;

methanol 30% for 20-40min, and 0.4% glacial acetic acid solution 70%;

methanol 50% for 40-45min, 0.4% glacial acetic acid solution 50%;

methanol 60% for 45-50min, and glacial acetic acid solution 40% of 0.4%;

methanol 70% and 0.4% glacial acetic acid 30% in 50-55 min;

methanol 80% for 55-60min, and glacial acetic acid solution 20% 0.4%;

methanol 90% for 60-65min, and glacial acetic acid solution 10% 0.4%;

methanol 99% for 65-75min, 0.4% glacial acetic acid solution 1%;

column temperature: 30 ℃ +/-2 ℃;

flow rate: 1+/-0.2 ml/min;

wavelength: 252+ -2 nm;

in HPLC detection, glycyrrhetinic acid, isoleucine, quercetin catechin and gallic acid are used as reference substances.

2. The method of claim 1, wherein: the preparation method of the teabag comprises the following steps: pulverizing Glycyrrhrizae radix, fructus Canarii albi, radix Platycodi, herba Menthae, and black tea, retaining coarse powder of 10-20 mesh, extracting the rest powder with mume fructus with water, concentrating to relative density of 1.1-1.2, mixing the medicinal liquid with the coarse powder, standing, drying, spraying 75% ethanol, and drying.

3. The method of claim 1, wherein: the column size was 4.6X250 mm,5 μm.

4. The method of claim 1, wherein: the preparation method of the test sample comprises the following steps: taking the teabag, and extracting with 75-85% methanol.

5. The method of claim 1, wherein: the method comprises the following steps of detecting by adopting a fingerprint spectrum: comparing the sample to be detected with a standard fingerprint, wherein the similarity is more than 0.9, namely the qualified teabag, and the standard fingerprint comprises the following characteristic peaks: chlorogenic acid, glycyrrhizin, quercetin and chrysophanol.