CN103463185A - Matched artemisia apiacea and agastache rugosaus combination medicine, preparation method and application thereof - Google Patents

Abstract

A combination drug of cyanosides, a preparation method and applications thereof. The invention provides a method for preparing indirubin by using fresh and alive Folium Isatis as raw material, which is characterized in that: adding indolequinone to the crushed fresh and alive Folium Isatis indica raw material; the present invention utilizes indoxyl free radicals The generation of the indirubin can be supplied by the dissolved oxygen in the original soaking liquid. Therefore, the reaction develops towards indirubin through the control of system pH and oxygen control during enzymatic hydrolysis; the method is simple and controllable; the present invention directly utilizes Folium folium The raw material is used as the reaction raw material, which effectively reduces the production cost; the present invention controls the generation of indoxyl free radicals during one-step oxidation, avoids the generation of indoloquinone, but directly adds indole quinone to the reaction raw material, is The response is toward positive equilibrium.

Description

Combination medicine of qinghuo medicine, preparation method and application thereof

technical field

The invention relates to a combination of traditional Chinese medicinal materials and medicines, more specifically, a combination medicine of qinghuo medicines, a preparation method and an application thereof.

Background technique

Artemisia annua is an annual herb of the genus Artemisia in the family Asteraceae. It has the effects of clearing away heat and relieving heat, and eliminating steam and intercepting malaria. The volatile oil is one of its main effective parts. Huoxiang is an annual or perennial herb of Lamiaceae. Its main component contains volatile oil of Huoxiang, which has antibacterial and antiseptic effects, and also has a therapeutic effect on superficial skin fungal infections. According to reports, the clinical application of Artemisia annua and Huoxiang can enhance the efficacy of clearing heat, relieving summer heat and antimalarial.

The medicinal ingredients in Artemisia annua can be divided into two parts: volatile and non-volatile. The non-volatile component is mainly artemisinin, which is a sesquiterpene lactone containing a peroxy group, which is compatible with known antibacterial agents. The chemical structure of malaria drugs is completely different; its various derivatives such as dihydroartemisinin, artesunate, artemether, and artether are all effective monomers for the treatment of malaria. With the in-depth study on the pharmacological effects of artemisinin drugs, it has been confirmed that they have anti-malarial, anti-pregnancy, anti-fibrosis, anti-schistosomiasis, anti-toxoplasma gondii, anti-arrhythmia and tumor cytotoxic effects; although these drugs It has a wide range of functions, but its mechanism of action, characteristics and application are still in its infancy. The volatile components are mainly volatile oils, including ketone, isone, olein, L-camphor, caryophyllene, brigene, borneol, caryophyllene oxide, sesqui-alcohol and other components; among them, camphor, borneol, clove The content of alkenes, ketones, isone ketones, etc. is high; the volatile oil has antipyretic and analgesic, antibacterial and anti-inflammatory effects, cough and asthma and other effects. The volatile components of Huoxiang are mainly methyl piperonol, accounting for more than 80%. It also contains anisole, anisaldehyde, d-limonene, p-methoxycinnamaldehyde, α-pinene, β-pinene, 3-octanone, 3-octanol, p-cymene, etc. Ageratum also includes sophorin, lindenin, mongoside, ageoin, isoagein, and ageoin. In addition, it still contains a small amount of tannin, bitter quality and other ingredients.

For the study of the composition of Artemisia annua and Artemisia annua, Xiong Yunhai et al. used experimental analysis and gas chromatography-mass spectrometry (GC-MS) to measure the diameter of the inhibition zone and the volatile oil components of the volatile oils of Artemisia annua and Artemisia annua and their compounds, respectively. The overlapping chromatographic peaks were analyzed by chemometric analysis method, and the pure chromatographic curves and mass spectra of each component were obtained, and then the analyzed pure components were qualitatively analyzed through the mass spectrum library, and quantified by the total volume integral method. The results showed that the comprehensive antibacterial ability of Huoxiang and Artemisia annua volatile oil was enhanced, and new active compounds were produced. The quality and quantity of the volatile oil compound of Huoxiang and Artemisia annua have significant changes compared with the single herb. The main chemical components of the complex are basically the sum of two single drugs, and the relative content of the single drug components has changed, among which the relative content of Huoxiang component in the compound is relatively high.

Contents of the invention

In order to solve the above-mentioned problems, the present invention provides a medicinal product of Artemisia annua and Artemisia annua on the basis of investigating different extraction parts and different extraction methods of the improved medicinal materials of Artemisia annua and Artemisia annua, and on the basis of the pharmacological activity of the extract obtained by the above method. Combination of extracts.

A method for preparing a combination medicine of Qinghuo medicine. The method is to mix and extract two kinds of medicinal materials, Artemisia annua and Huoxiang, to obtain the components of the volatile oil part and the components of the ethanol extraction part, and mix the components of the volatile oil part and the components of the ethanol extraction part to obtain Qinghuo Drugs versus Combination Drugs.

The method comprises the steps of:

1) Mix the two medicinal materials of Artemisia annua and Huoxiang in equal proportions, and extract the volatile oil parts according to the method of "Pharmacopia" (2010);

2) Extract the residue in step 1) with 70% ethanol under reflux, and combine and recover the ethanol extract;

3) Recover the ethanol in the ethanol extract, collect the paste to a density of 1.3 at 25°C, and obtain the ethanol extraction site;

4) Combining the volatile oil part and the ethanol extraction part to obtain the combination drug of qinghuo medicine.

3. The preparation method of Qinghuo medicine combination medicine according to claim 2, characterized in that: said Artemisia annua and Huoxiang medicinal materials are fresh products within 24 hours after harvesting, and the storage temperature before extraction is lower than 25°C .

A combination medicine of Cincinnia cholera, which is prepared by adopting the method described in claims 1-3.

The application of the combination of the above-mentioned green cholera medicines in the preparation of medicines for treating eczema.

Application of the combination of the above-mentioned green cholera medicines in the preparation of medicines for relieving cough and reducing phlegm.

The beneficial technical effects of the present invention are: the combination drug of cyanosides provided by the invention utilizes the chemical reaction that occurs in the extraction process of the mixed medicinal materials, and plays the beneficial technical effect of the medicinal pair. body, and the combination of the two kinds of drug extraction; Qinghuo medicine has obvious advantages to the combined drug as an external drug for treating eczema and as an internal drug for cough and phlegm; specifically:

1) Compared with the combination of extracts extracted separately from Artemisia annua and Huoxiang separately, it has a significant improvement in the treatment of eczema;

2) Compared with the volatile oil of Artemisia annua and Huoxiang and their mixtures, it has significant progress in relieving cough and reducing phlegm;

3) The combination of volatile oil and alcohol extracts extracted from fresh products of Huoxiang and Artemisia annua has better activity in treating eczema and relieving cough and reducing phlegm.

Detailed ways

The preparation of embodiment 1 Qinghuo extract

Respectively pulverize the dry product raw materials of Huoxiang and Artemisia annua, respectively weigh 200g after crossing a 40-mesh sieve, and extract Huoxiang volatile oil and Artemisia annua volatile oil respectively by the volatile oil extraction method of "The Pharmacopoeia of the People's Republic of China" (2010 edition); The residues of the two medicinal materials were evaporated to dryness, refluxed and extracted with 70% ethanol 5 times the weight of the medicinal materials for 3 times, the extracts were combined respectively, dried, and the extract was prepared to obtain the alcohol extract of Huoxiang and artemisinin. Weigh a certain amount of Huoxiang volatile alcohol extract, Artemisia annua alcohol extract, Huoxiang volatile oil and Artemisia annua volatile oil according to the ratio of 1:1 of Huoxiang and Artemisia annua to obtain the mixed group I of Qinghuo extract; The volatile oil of fenugreek and the volatile oil of Artemisia annua were mixed according to the ratio of 1:1 of the original medicinal materials to obtain the mixed group A of volatile oil of Qinghuo.

Respectively pulverize the dry product raw materials of Huoxiang and Artemisia annua, respectively weigh 200g after crossing a 40-mesh sieve, and extract Huoxiang volatile oil and Artemisia annua volatile oil respectively by the volatile oil extraction method of "The Pharmacopoeia of the People's Republic of China" (2010 edition); The residues of the two medicinal materials were evaporated to dryness, refluxed and extracted with 70% ethanol 5 times the weight of the medicinal materials for 3 times, the extracts were combined respectively, dried, and the extract was prepared to obtain the alcohol extract of Huoxiang and artemisinin. Weigh a certain amount of Huoxiang volatile alcohol extract, Artemisia annua alcohol extract, Huoxiang volatile oil and Artemisia annua volatile oil according to the ratio of 1:1 of Huoxiang and Artemisia annua to obtain the mixed group II of Qinghuo extract; The volatile oil of fenugreek and the volatile oil of Artemisia annua were mixed according to the ratio of 1:1 of the original medicinal materials to obtain the mixed group B of volatile oil of Qinghuo.

Grind the dry raw materials of Huoxiang and Artemisia annua respectively, weigh 200g after passing through a 40-mesh sieve, mix, and extract the mixture of Huoxiang and Artemisia annua volatile oil respectively according to the volatile oil extraction method of "Pharmacopia of the People's Republic of China" (2010 edition); The mixed group C of the volatile oil of Qinghuo Herba is obtained; the residue of the extracted medicinal materials is evaporated to dryness, and the weight of the medicinal materials is 5 times the weight of 70% ethanol for reflux extraction for 3 times, the extracts are combined, dried, and the extract is prepared to obtain the extract of Qinghuo Herba. Mixture Group III.

Grind the raw materials of Huoxiang and Artemisia annua fresh products (within 24 hours of harvesting), weigh 200g each, mix, and extract the mixture of Huoxiang and Artemisia annua volatile oils respectively according to the volatile oil extraction method of "Pharmacopia of the People's Republic of China" (2010 edition) ; Obtain the mixed group D of the volatile oil of Herba Pleurotus chinensis; evaporate the water of the extracted medicinal material residue, and extract 5 times of 70% ethanol reflux with the weight of the material for 3 times, combine the extracts, dry up, prepare the extract, and obtain the medicinal material Extract Mix Group IV.

Grind the raw materials of Huoxiang and Artemisia annua flowers (within 24 hours of harvesting) respectively, weigh 200g each, mix, and extract 3 times with 70% ethanol refluxing with 5 times the weight of the materials, combine the extracts, collect and dry, and prepare the extract. Obtain the mixed group V of the herb extract of Herba Uregonii.

Grind the dry raw materials of Huoxiang and Artemisia annua respectively, weigh 200g each, mix them, and extract 3 times with 70% ethanol refluxing which is 5 times the weight of the materials, combine the extracts, dry them up, prepare the extract, and obtain the Herba Herba Herbaii extract Mixed group VI.

The comparison of embodiment 2 medicines to the volatile oil composition of Psyllium

Gas chromatography/mass spectrometry (GC/MS) method is used to separate and detect the mixed group A of Qinghuo volatile oil, the mixed group B of Qinghuo volatile oil, the mixed group C of the volatile oil of Qinghuo and the mixed group D of the volatile oil of Qinghuo in the embodiment 1. The components of the volatile oil were analyzed, and the two-dimensional GC-MC data were processed by the chemometric resolution method (crave). The chromatographic conditions are: chromatographic column: DB-1 (30m×0.2mm); heating program: the initial temperature is 60° C., and it is maintained for 4 minutes. Rise to 220°C at 4°C/min and maintain for 2min; carrier gas (He) flow rate 1.0mL/min; inlet temperature 280°C; split ratio 100:1; injection volume 10μL. The mass spectrometry conditions are: electron bombardment (EI) ion source; electron energy 70eV, ion source temperature 200°C, interface temperature 250°C. Doubling voltage: 0.80kV, scan range m/z35-500; scan rate: 5.0 times/s, solvent delay 2.5min. The percentage change of each component in the volatile oil is shown in Table 1.

Table 1 The main components of the volatile oil of Qinghuo

The experimental results show that after statistical analysis of the 12 main components of the volatile oil combination of the single herbs of Artemisia annua and Huoxiang and the difference of the 12 main components of the volatile oil of the herb pair Artemisia annua-Huoxiang There were significant differences in the quality and quantity of the volatile oil of Artemisia annua. The volatile oil components of Pinghuo medicine paired mainly came from the single herb Artemisia annua, the types of chemical components were basically different from the sum of the two single herbal medicines, and the content of the volatile oil components of the single herb medicines changed in the medicine pair. Among them, the missing components in the volatile oil of Artemisia annua and Huoxiang medicine are (+)-long camphenone and α-parsnol, and the newly added components are epicyanool, artemisinic acid and allyl anisole. It proves that the composition and ratio relationship of the combination of Artemisia annua and Huoxiang have changed during the extraction process. In addition, the study found that fresh products using Artemisia annua Huoxiang

The application of embodiment 2 Qinghuo medicine to combination medicine in the treatment of eczema

Investigation on Anti-inflammatory Activity of Qinghuoyao on Combination Drugs

表示，用SPSS15.0统计软件分析，2组间差异用t或t’检验，多组间比较采用F检验，P<O.05为差异具有统计学意义；实验结果见表2所示。For the study of the effect on mouse auricle swelling, 80 mice were taken, with a body weight of 18-22g. Male and female were divided into half and half. They were randomly divided into control group, aspirin group, and Qinghuo extract mixed group I and II groups. There are 8 experimental groups, including the extract mixing group III and IV, and the medicine pair Qinghuo extract mixing group V and VI, with 10 rats in each group. The administration group evenly smears each group of Herba Herba Utarii extract obtained in Example 1 on both sides of the left auricle of the mouse. Clean the ointment, apply xylene at the same place on both sides of the right ear with 0.1mL/cause, the left ear is used as a control, and be killed after 30 minutes. Ear slices from the same parts of the left and right ears were taken with a 6mm punch, weighed, and the degree of swelling (mass of the left ear - mass of the right ear) was calculated. for data

Said, analyzed with SPSS15.0 statistical software, the difference between two groups was tested by t or t' test, and the comparison between multiple groups was tested by F test. P<0.05 means that the difference is statistically significant; the experimental results are shown in Table 2.

)The influence of table 2 Qinghuo medicine on the mouse auricle swelling caused by combination drug p-xylene (

)

Note: Compared with the control group, ^* P<O.O1.

Investigation on the antipruritic activity of Qinghuoyao on combined drugs

Get 80 guinea pigs of 300-350g, half male and half male, and randomly divided into blank control group, positive control group, mixed group I and II of Qinghuo extract, mixed group III and IV of medicine-paired Qinghuo extract, and group III and IV of medicine-paired Qinghuo extract. There were 8 experimental groups including Huohu extract mixed group V and VI, with 10 rats in each group. In each administration group, 0.5g·kg ^-1 of the combined drug of Cincinnamon was evenly applied to the right auricle of the mice, and in the positive control group, 2.0g·kg ^-1 of trimidesin cream was evenly applied to the right auricle of the mice. ,, the blank control group evenly smeared 2.0g·kg ^-1 of the blank matrix on the right auricle of the mice, twice a day, 1 day before the experiment, shaved the dorsum of the right hind foot of the guinea pigs in each group, and applied the medicine once. When the experiment was Et, use No. 0 sandpaper to scrape the shaved part of the right hind foot of the animal, with a small amount of blood oozing, and the area is 1cm ² , and then apply the medicine once again. 10 minutes after the last dose. Start to drop 0.01% histamine phosphate 0.05mL/on the wound, with an interval of 3 minutes each time, until the guinea pig turns back to lick its right hind foot, so that the phosphate histamine given when the guinea pig turns back to lick its right hind foot begins to appear The total amount is the most itchy threshold. Record and compare the itching of each group, and perform the t test between groups. The results are shown in Table 7. As can be seen from Table 3, compared with the blank control group, there is a statistically significant difference (P<0.05 or P<0.01). Illustrate that the combination of cyanosides obtained in Example 1 can significantly improve the itching threshold of histamine phosphate on guinea pigs, and the effect is more obvious as the dose increases.

n=10)Table 3 The influence of Chinese cholera on combined drugs on foot itching caused by histamine phosphate (

n=10)

Note: Compared with the blank group, ^* P<O.05, ^# P<O.O1.

Take 60 guinea pigs of 300-350 g, depilate the back of the guinea pigs with 8% sodium sulfide solution, with an area of 4cm×6cm, apply 0.02mL of 1% DNCB acetone solution to the depilated area of each mouse for one time, and then sensitize for the second time after 3 days. On the fourteenth day, it was challenged once again with 0.02 mL of 1% DNCB acetone solution, and allergic contact dermatitis symptoms appeared on the back of guinea pigs where the drug was applied. The above-mentioned guinea pigs were randomly divided into 5 groups, 10 in each group, half male and half male, respectively, the mixed group I and II of Qinghuo extract, the mixed group IIIIIIV of the extract of Qinghuo, and the mixed group of extract of Qinghuo. V, VI groups, positive control group and blank control group. The administration example 2 of Qinghuo medicine to the combination drug group obtains the commercially available product 1.0g kg ⁻¹ ; the mixed group I and II of Qinghuo extract, the mixed group III and IV of the medicine paired with Qinghuo extract, and the mixed group III and IV of the medicine paired with Qinghuo. The Huohu extract mixed group V and VI were evenly applied to the lesion site with 0.5g·kg ^-1 of the combined drug obtained in Example 1, and the positive control group was evenly applied to the lesion site with 4.0g·kg ^-1 Trimetin cream In the blank control group, 4.0 g·kg ^-1 of blank matrix was evenly applied to the lesion, twice a day for 7 consecutive days. After the 8th day, the guinea pigs were executed, and the lesioned skin of the same part was removed with a punch (15 mm in diameter), weighed with an electronic balance, and the skin thickness was measured with a vernier caliper. The results are shown in Table 4. Compared with the blank control group, the skin weight and skin thickness of Qinghuo extract mixed group I and II, medicine pair Qinghuo extract mixed group III and IV, and medicine pair Qinghuo extract group V and VI were statistically The difference (P<0.01) shows that Qinghuo medicine has a certain therapeutic and inhibitory effect on DNCB-induced guinea pig allergic contact dermatitis to combination medicine and each group of positive control group.

n=10)Table 4 Qinghuo medicine on the impact of combination drugs on atopic dermatitis (

n=10)

Note: Compared with blank group, ^* P<O.01.

Eczema is a common skin disease caused by various internal and external factors. It is mainly papules in the acute stage, and a pruritic skin disease that is dominated by epidermal hypertrophy and lichenification in the chronic stage. Western medicine believes that these diseases are mostly related to allergic reactions, and are often accompanied by bacterial infections. Systemic treatment often uses anti-allergic therapy, while local treatment often uses hormone plus antibiotics for treatment. However, due to the toxic and side effects of hormones, the disease has become an intractable disease, which has brought great harm to the patient's physical and mental health. In the present embodiment, by comparing the activity of each group of extracts obtained in Example 1; it is found that in terms of detumescence, antipruritic and atopic dermatitis, the activity of the medicine to the mixed group III and IV of Qinghuo extract is better than that of group III and IV. Herbal extract mixed group V and VI and extract mixed group I and II of Qinghuo.

Example 3 Application of the mixed group of Qinghuo extract in relieving cough and reducing phlegm

According to prior art studies, it has been proved that the volatile oils of Artemisia annua and Huoxiang all have the activity of relieving cough and relieving asthma. The Artemisia annua volatile oil and Huoxiang volatile oil prepared in embodiment 1 are respectively used as Artemisia annua volatile oil group and Huoxiang volatile oil group;

Take 80 female mice weighing 18-20 g, and randomly divide them into 8 groups, 10 in each group, which are blank control group (equal volume of distilled water), Artemisia annua volatile oil group, Huoxiang volatile oil group, and Qinghuo extract mixed group II , medicine pair Qinghuo extract mixed group IV, Qinghu extract mixed group VI and positive control group (dextromethorphan hydrobromide tablets). Animals were administered by intragastric administration according to body weight (5g raw medicinal material/kg), and the administration frequency was 1 time/d, and the administration was continued for 14 days. Before the experiment, the animals in each group were fasted for 10 hours, and the last administration was given. The first 4 groups were gavaged by the above-mentioned method, and the positive control group was given the corresponding medicinal solution by gavage once at a dose of 0.05 g/kg, and 30 minutes after the administration. The mice in each group were put into a self-made cough-inducing instrument (the concentration of ammonia gas was 15%), and were taken out after 20 seconds. The cough latency and the number of coughs within 3 minutes of the mice were observed and recorded. The t-test was used to compare the significant differences in cough latency and cough times between each administration group and the blank control group. The specific results are shown in Table 5.

n=10)The medicine of table 5 is to the antitussive effect of the mixed group of Qinghuo extract on mice (

n=10)

Note: Compared with the blank group, *P﹥0.05, ^** P﹥0.01.

Take 80 male mice of 18-20 g, and randomly divide them into 8 groups, 10 in each group, which are blank control group (equal volume of distilled water), Artemisia annua volatile oil group, Huoxiang volatile oil group, and Qinghuo extract mixed group II , Medicinal herb extract mixed group IV, group VI mixed with extract of Qinghuo and positive control group (ammonium chloride group). Animals were administered by intragastric administration according to body weight (5g raw medicinal material/kg), and the administration frequency was 1 time/d, and the administration was continued for 14 days. Before the experiment, the animals in each group were fasted for 10 hours, and before the last administration, the 4 groups were intragastrically administered according to the above method, and the positive control group was intragastrically administered with the corresponding medicinal solution at a dose of 1 g/kg. Inject 5% phenol red solution into the intraperitoneal cavity according to body weight (0.1mL/10g) 30 minutes after injecting phenol red, kill the animals with ether, carefully peel off the tissues around the trachea, cut off the white thyroid cartilage down to a section of the trachea at the bifurcation of the trachea, and immediately use The prepared 5% sodium bicarbonate solution was rinsed with 0.5mL for 3 consecutive times, the eluates were combined, placed in a 722-type spectrophotometer, and the OD value was measured at a wavelength of 546nm. The t test was used to compare the significance of the difference in absorbance between each treatment group and the blank control group. The specific results are shown in Table 6. The study proved that the mixed group VI of herb extracts of Qinghuo has no activity in relieving cough and reducing phlegm, and the group II mixed with the volatile oil group of Artemisia annua, Huoxiang volatile oil and Qinghuo extract has activity in relieving cough, but not The activity of reducing phlegm, not only has the activity of antitussive, but also has the good activity of reducing phlegm.

n=10)The expectorant effect of table 6 medicines to the mixed group of Qinghuo extract (

n=10)

Note: Compared with the blank group, ^* P﹥0.05, ^** P﹥0.01.

Claims (6)

1. the green grass or young crops preparation method of medicine to composition of medicine suddenly, it is characterized in that: described method is by Herba Artemisiae Annuae and two kinds of medical material mixed extraction of Herba Pogostemonis, obtain volatile oil part composition and ethanol extraction position composition, mixed volatilization oil position composition and ethanol extraction position composition obtain green grass or young crops suddenly medicine to composition of medicine.

2. the green grass or young crops according to claim 1 preparation method of medicine to composition of medicine suddenly, it is characterized in that: described method comprises the steps:

1) Herba Artemisiae Annuae, two kinds of medical material equal proportions of Herba Pogostemonis are mixed, extract volatile oil part;

2), by the residue in step 1), the alcohol reflux with 70%, merge and reclaim ethanol extract;

3) reclaim the ethanol in ethanol extract, receive cream to 25 ℃ density 1.3, obtain the ethanol extraction position;

4) merge volatile oil part and ethanol extraction position, obtain green grass or young crops suddenly medicine to composition of medicine.

3. the green grass or young crops according to claim 2 preparation method of medicine to composition of medicine suddenly is characterized in that: described Herba Artemisiae Annuae and Herba Pogostemonis medical material are the fresh goods in 24 after gathering, and the preservation temperature before extracting is lower than 25 ℃.

A green grass or young crops suddenly medicine to composition of medicine, it is characterized in that: described green grass or young crops suddenly medicine to composition of medicine for adopt the described method of claim 1-3 prepare green grass or young crops suddenly medicine to composition of medicine.

5. green grass or young crops according to claim 4 medicine application in preparation treatment eczema medicine to composition of medicine suddenly.

6. green grass or young crops according to claim 4 medicine application in preparing the relieving cough and resolving phlegm medicine to composition of medicine suddenly.