Combination with orientin-2''-O-beta-L-galactoside as main ingredient and use of combination

Technical Field

The present invention relates to heat-clearing, antibacterial anti-inflammatory effect, raids the lungwind hot evil poisonous used for the treatment, of the extracted upper respiratory tract infections, pharyngitis, tonsillitis Globeflower diseases such as pharmaceutical composition. Also relates to pharmaceutical formulations of the drug, the invention belongs to the medicine Technical Field.

Background Art

Currently marketed Trollflower preparation, are using Globeflower crude drugs with water extraction, concentration, then adding different accessories of various preparations, tablets and capsules and the main, the contents are simple crude extract, the active ingredient is not clear, there is no clear effective substance, the dose is large, which is not beneficial to improving modern traditional Chinese medicine research, to improve overall level of health of the community.

Trollflower the Ranunculaceae plant Trollflower (  Bge. Trollius   chinensis) dry flower. Globeflower beginning is contained in the book Zhao Xuemin the Qing Dynasty "bencao gangmu theft by finding". "Bencao gangmu theft by finding" in that their "bitter, cold, non-toxic", can be "ruling Aphtha, the throat swells , floats the hot tooth to proclaim , ear pain, item of pain ", "improving eyesight, xie Lanzhang " has the effect of. Trollflower the resources are abundant, with antibacterial, anti-virus, for treating respiratory tract and widely used for such as an intestinal infection (China's modern traditional Chinese medicine, 2007 years 9 roll 3 a view to 29-32 page). In the past, have been reported in the study of the method for preparing Globeflower component, Zhao Yimin the reports extracting flavonoid compound with an organic reagent used for rheumatic arthritis or diseases such as rheumatoid arthritis (CN1872084). High spring equal reports, Trollflower Decoct with water alcohol precipitation, macroporous resin preparation Trollflower injection method for separating (CN1876031), is a kind of the purity is low, the effective components are not explicitly extract, made into injection of officinal property is poor. Short petal fan Guorong globeflower those reports for the ethanol extraction, concentration, concentrate through macroporous resin adsorption, eluting with ethanol, high-speed countercurrent chromatography (HSCCC) and preparation liquid chromatographic purification, to obtain high-purity [...] , vitexin (CN1660864). For the the reports yang Yaxin globeflower 70% ethanol extraction, with n-butanol extraction, then a SephadexLH 20 column separation to obtain the identified -2 [...] -O-β-L-galactopyranoside, [...] (Heilongjiang medicine, 2006 years, 19 (2), 93-94), however, there is no clear pharmacodynamic study. Flooding of Chinese globeflower warm cloud sea the such studies with anti-virus function extract (Chinese mikrobiologiya and amynologic magazine, 1999, 19 (1): 21); the such studies su Lianjiethe Chinese globeflower is mellow extract has anti-virus effect in vivo, has jiere role, relieving cough (Chinese herbal medicine, 2007, 38 (7): 1062-1063, China's technology, 2009, 16 (1): 65, Chinese medical technology, 2010, 17 (6): 476); su Lianjie , the such studies zhang Hongbo Globeflower flavonid and [...] having anti-virus activity (2010 year, Heilongjiang master's thesis University of traditional Chinese medicine), of the total flavonoids in this study is in order to through the polyamide separation, identified -2 the removed [...] -O-β-L-galactopyranoside; for those reports cai Jinna the injection process for the preparation of Chinese globeflower total flavonoids (CN101234147), is water or ethanol extraction, concentration, then through macroporous resin, polyamide separation to obtain the total flavonoids, wherein, the total flavonoids do not contain the same identified-"-O-β-L-galactoside.

We have found that unexpected of experimental research using conventional polyamide column separation, purification Globeflower flavonid is not -2 to the identified [...] -O-β-L-galactopyranoside and -2 the identified [...] -O-β-arabinoside the. Our experimental studies have shown, -2 the identified [...] -O-β-L-galactopyranoside and -2 the identified [...] -O-β-arabinoside is almost not be polyamide the adsorption, the loss of two xylosides almost all. Therefore, in our polyamide column of water in the eluent to obtain a large number of identified -2 the- [...] O-β-L-galactopyranoside and part 2 the-identified [...] -O-β-arabinoside. Therefore, total flavone Trollflower of that reported in the past do not contain the above two xylosides.

In order to identified -2 the- [...] O-β-L-galactoside as main component with the composition by wind hot evil poisonousraids the lung , caused by the extracted upper respiratory tract infections, pharyngitis, tonsillitis the role of the diseases, and in the past, the extracted Trollflower injection flavonid and Globeflower crude extract preparation made with great technical progress and nature of the difference. So far there is no literature reports of the in -2 identified [...] -O-β-L-galactopyranoside 30%-65%, [...] 5%-30%, vitexin 2%-10% and -2 the identified [...] -O-β-arabinoside 1%-6% of the composition by wind hot evil poisonousraids the lung , caused by the extracted upper respiratory tract infections, pharyngitis, tonsillitis study and application of the disease. We in the literature and patents retrieved on the basis of the, -2 to the identified [...] -O-β-L-galactopyranoside as the main ingredient of the composition of the antibiotic, anti-viral, anti-inflammatory experiment, shows that the preparation than Globeflower crude extract, the crude extract and literature reports has better pharmacological action.

Not found before the completion of the present invention relating to the identified -2 [...] -O-β-L-galactopyranoside as the main component a composition as a and is raids the lungwind hot evil poisonous , caused by the extracted upper respiratory tract infections, pharyngitis, tonsillitis reports of diseases, containing mainly been found also identified -2 the- [...] O-β-L-galactopyranoside composition raids the lungwind hot evil poisonous used for the prevention and the treatment, the extracted upper respiratory tract infections, pharyngitis, tonsillitis disease clinical aspects of the application.

Content of the invention

The purpose of this invention is to supplement existing Globeflower crude extract the deficiency of the preparation, to provide

A high bioavailability, curative effect is precise, small dosage, high drug content, clearly effective component of the pharmaceutical composition, a significant technical improvement, and Globeflower crude extract preparation has the distinction of the nature.

The beneficial effect of the invention is to offer a kind of Globeflower drug flavone, composite more traditional Trollflower oral preparation and Globeflower injection is greatly improved, the active ingredient is clear, controllable quality, small dosage, officinal property is strong, with a traditional Chinese medicine it can be used for clinical innovative drug, has very strong application value.

Another beneficial effects Globeflower flavone composition of literature reports runic object is provided with a clear components, high content of flavone components, identified -2 the enriched [...] -O-β-L-galactopyranoside and -2 the identified [...] -O-β-arabinoside ingredient, with a chemical composition.

Another object of the present invention and is characterized in that: provides a plant rich in resources, can be industrialized production Globeflower flavone composition, this composition is characterized in that: by weight -2 the count identified [...] -O-β-L-galactopyranoside 30%-65%, [...] 5%-30%, vitexin 2%-10% and -2 the identified [...] -O-β-arabinoside 1%-6%, with obvious that by the test of the antibacterial, anti-inflammatory actions, and no obvious toxic side effects; the performance is stable, can be made into various different dosage forms; composition principal ingredients are carbon glucoside flavone, with that reported in the past has obvious flavonid Trollflower of the different, our test shows that the, -2 the identified [...] -O-β-L-galactoside is not is polyamide the adsorption, the literature reports of the use of the Chinese globeflower total flavonoids polyamide separation, therefore, does not contain the identified -2 [...] -O-β-L-galactopyranoside, we of the isolation and purification of the largest contribution to the composition is enriched -2 the identified [...] -O-β-L-galactopyranoside and -2 the identified [...] -O-β-arabinoside, and in the composition up to 50% more, two xylosides is Trollflower flavone the major active substance in the composition, this is also a difference in the past Globeflower flavone flavonid outstanding characteristics. This composition has very strong clearing away heat and toxic material, antibacterial anti-inflammatory effect, raids the lungwind hot evil poisonous used for the treatment, of the extracted upper respiratory tract infections, pharyngitis, tonsillitis of the present invention is also a major innovative point.

Another purpose of this invention is to provide the -2 containing the identified [...] -O-β-L-galactopyranoside composition of the pharmaceutical preparation. Major drug form such as a tablet, capsule, pill, granule, suspension, pill, any of the injection.

The invention is characterized in that the identified -2 mainly containing [...] -O-β-L-galactopyranoside composition is from the Ranunculaceae Globeflower belongs to plant Trollflower Trollius   chinensisBunge, short flap Trollflower (  Reichb.   ledebouri Trollius) and long petal Trollflower (  Fr.Schmidt   macropetalus Trollius) extract of dried flowers, its production process in the previous Patent to the inventor (Patent number CN101559066) to the in, it needs to be pointed out that is, in the meaning of this Patent -2 the identified [...] -O-β-L-galactopyranoside as the main component of the composition may be a product obtained by the process of the Patent, can also be further on the basis of the use of adsorption, desorption, column chromatography, recrystallization and other technology and its assembling method refining the obtained products. Specific experimental steps are as follows:

Globeflower crude drugs with 30-95% ethanol reflux extraction 2-3 time, each 1-3 hours. Directly by D101, AB -8 the macroporous resin column or polyamide column adsorption to purify, effluent and eluent recovering ethanol to mellow content of less than 10%, and then through D101 or AB-8 of macroporous resin column adsorption purification. Washing with water first, then using 20-80% ethanol elution, collecting 20-80% ethanol eluent, and recovering the solvent to dry, using 60-95% ethanol heating dissolved, add 0.3-3.0% (W/V) pharmaceutically active carbon, filtration, filtrate directly purified through silica gel, and using 60-95% ethanol elution, and eluent of collecting the effluent liquid, and recovering the solvent to dry, the -2 containing identified [...] -O-β-L-galactopyranoside pharmaceutical composition.

-2 the identified [...] -O-β-L-galactopyranoside structural formula

To achieve the above object, the present invention provides the following technical solutions.

The content of the composition is measured through the following method:

1, -2 the identified [...] -O-β-L-galactopyranoside, -2 the identified [...] -O-β-arabinoses, [...] , vitexin utilizes high-performance liquid chromatography to determine the: to octadecyl silane bonding silica gel as a filler; to acetonitrile -0.35% aqueous solution of phosphoric acid (the 13 [...] 87) as the mobile phase; detecting a wavelength of 345 nanometer. Theoretical plate number to the identified -2 [...] -O-β-L-galactopyranoside peak calculation of not less than 3000.

Preparation of control solution respectively [...] -2 the precision that [...] -O-β-arabinoses, [...] , vitexin reference substance, the capacity in the bottle is, with 60% methanol dissolved into 1 milliliter -2 containing the identified [...] -O-β-arabinoses 0.005 milligram, -2 the identified [...] -O-β-L-galactopyranoside 0.04 milligram, [...] 0.02 milligram, vitexin 0.01 milligram reference substance solution.

the system is on a waiting list composition of the sample solution 5 milligram, precision stability, article 50 milliliter amount in the bottle, with 60% methanol 40 milliliter, ultrasonic 30 minute, is placed to the room temperature, 60% methanol constant volume to scale, shaking, as for sample solution.

Precision draw method for measuring and reference solution, respectively for the test solution the 10  l, into the liquid chromatograph, measuring, get.

2, composition by ultraviolet colorimetric determination of the total flavonoids:

Preparation of control solution [...] -2 the precision that [...] -O-β-L-galactopyranoside reference substance, is made of ethanol is dissolved for 0.005 milligram/ml reference substance solution.

the system is on a waiting list composition of the sample solution 5 milligram, precision stability, article 100 milliliter amount in the bottle, adding ethanol 80 milliliter, ultrasonic 30 minute, is placed to room temperature, ethanol constant volume to scale, shaking, precision rectifier 1 milliliter, article 10 milliliter in measuring flask, add alcohol to scale, as the test sample solution.

Determination of the reference solution, respectively, for the sample solution, a blank control. As spectrophotometry ("Chinese Pharmacopoeia" 2010 year version   B a appendix V), at a wavelength of 345 nanometer are respectively measuring absorbance, computing.

Globeflower flavone composition containing flavonid to in -2 the identified [...] -O-β-L-galactopyranoside meter is not lower than 80%.

In order to identified -2 the- [...] O-β-L-galactopyranoside as the main component of the composition (identified -2 the [...] -O-β-L-galactoside content is 54.3%, [...] content of 21.7%, male jing Gan content of 4.6%, identified -2 the [...] -O-β-arabinoside 2.5%) raids the lungwind hot evil poisonous with and is, the extracted upper respiratory tract infections, pharyngitis, tonsillitis the role of diseases, these pharmacological action, through the following pharmocodynamics test example to be confirmed.

1, Globeflower flavone the anti-inflammatory effects of the composition

1.1 Globeflower flavone composition of the mouse to the xylene swelling caused by the impact of the auricle inflammation

takes the mouse 50 only, male, body weight (20 ± 2) g, random divided into 5 groups of, in accordance with table 1 intragastrically to the medicine, every day 1 time, continuous 7d, the last administration 1h the rear, the xylene 0.3 milliliter melted and right ear of the mouse, is not coated with the do with the left ear. 2h neck dislocations executed after the mouse, with a diameter of 9 mm the puncher on the left respectively, lay ear provided with a circular lug, weighing, calculating the left, the weight difference as right ear pieceswelling , compare differences between groups. Results are shown in table 1.

Table 1 para-xylene Globeflower flavone composition due to the impact of the mouse's auricle inflammation turgesce

**P <0.01, *P <0.05. Compare

From table 1 it can be seen, Globeflower flavone composition high, middle, low three dose to the xylene swelling MICE auricle inflammation due to the role of prominent inhibition, indicating that they have significant anti-inflammatory effect. Similarly, the positive control drug indol the United States after xin Guanwei , it has prominent anti-inflammatory effect.

1.2 Globeflower flavone composition forming a cotton ball granuloma to the impact of the RAT

takes the big mouse 50 only, according to the gender and weight random is divided into 5 groups. According to cotton ball into the method of the operation steps for implantation of a subcutaneous the groin (30 ± 1) milligram of the sterilized cotton ball. Postoperative day administration, dosage and table 2, continuous 7d, in section 8d kill animals, stripping and taking out the cotton ball granulation tissue, article 60 the drying oven [...] 12h after weighing, swelling and inhibition is calculated.

Table 2, Globeflower flavone composition forming a cotton ball granuloma to the impact of the RAT

**P <0.01, *P <0.05. Compare

From table 2 it can be seen, Globeflower flavone composition high, middle, low-dose, the RAT having a cotton ball granuloma role of prominent inhibition, with significant anti-inflammatory effect. The control group compared with the physiological saline, display the granulation proliferative inflammation has significant anti-inflammatory effect. The indomethacin granulation proliferative inflammation also has significant anti-inflammatory effect.

2, Globeflower flavone composition the antibacterial action of the

Experimental materials : (1) strain: Staphylococcus aureus, pneumococcus, Staphylococcus epidermidis, pathogenic Escherichia coli, bacillus subtilis, fujinaka huang Wei club by jlu provided for ; (2) culture medium: nutrient agar, nutrient broth, calf serum, fresh sheep blood by jlu provide.

Experimental methods and results : (1) preparation of medicines: will Globeflower flavone composition plus sterile physiological saline soaking sleepovers after dissolving the spare. (2) preparation of the bacterium: respectively in the above-mentioned strain inoculation serum bouillon tube and broth tube 37 the culture [...] 18 hr, McFar tube compared to muddy   Land's turbidity standards to 3 billion/ml of spare bacterium fluid. (3) the dilution of the test tube, add sterile culture fluid, and test bacterial drug stock solution according to the respective operating the dilution rate, the 37 [...] 18 hours after the cultivation, the tube liquid transfer blood flat in 18 hours later, observed result, the respectively to the MIC of various fungi : Staphylococcus aureus 40 subsidence g/ml, Pneumococcal 55 subsidence g/ml, epidermial staphylococcus 20 subsidence g/ml, pathogenic Escherichia coli 180 subsidence g/ml, bacillus subtilis 80 subsidence g/ml, fujinaka huang Wei coccobacilli 60 subsidence g/ml, have a different degree of the bacteriostatic effect.

3, Globeflower flavone composition role of anti-virus

3.1 Globeflower flavone composition of the mouse to the influenza virus infection protection function

Selecting health mouse, the male half, random packet, each group of 25 only, to ig the 3d (blank control group and virus control ig equivalent distilled water), each day 1 time, at the time of infection, the diluted virus liquid ethyl ether under mild anesthesia Collunarium infection mouse, each mouse 0.05 milliliter, to continue administration after infection, every day 1 time, continuous 7d, and record each day and deaths animal disease conditions, observation period to 14d. The death rate is calculated. Results are shown in table 3.

= death rate (control group mortality-the group mortality) × 100%

Table 3, Globeflower flavone composition to the death of mouse influenza virus infection protection function

**P <0.01. The control group compared with the virus

Table 3 results show, Globeflower flavone composition caused by the influenza virus infection the death of MICE with the remarkable role of protection, high, middle, low each dose makes the mouse mortality is obviously reduced, ribavirin death rate of the mouse can be obviously reduced.

3.2 Globeflower flavone composition on the influenza virus infection the quality of the mouse lung

Selecting health mouse, the male half, random packet, each group of 20 only, to ig the 3d (blank control group and virus control ig equivalent distilled water), each day 1 time, at the time of infection, the diluted virus liquid ethyl ether under mild anesthesia Collunarium infection mouse, each mouse 0.05 milliliter, to continue administration after infection, every day 1 time, section after the infection 4 days (pre-prohibiting food water 8h), after weighing body weight of the mouse quickly beheaded bloodlettings executed, taking out the whole lung, is placed into water washing 2 times, ablation trachea, lung tissues such as lymph nodes, dry with filter paper of the surface moisture, said quality. Lung index and inhibition is calculated. Results are shown in table 4.

Lung index = lung mass (g)/ weight (g) × 100%

Lung index inhibition rate = (control group mean lung index-the group average lung index)/ the control group × 100% the average lung index

Table 4, Globeflower flavone composition influenza virus infection to the mouse lung index inhibiting effect

**P <0.01, compared with the virus control; P △△ <0.01, compared with the blank control group

Table 4 the result shows that, Globeflower flavone composition can significantly reduce the dose of influenza virus infection of the mouse lung quality, reduced lung index, ribavirin is high, the invention can significantly reduce the influenza virus infection of the mouse lung index.

From the above that the pharmacodynamics experiment can be known, in order to identified -2 the- [...] O-β-L-galactoside as main component with the composition by wind hot evil poisonousraids the lung , caused by the extracted upper respiratory tract infections, pharyngitis, tonsillitis the role of such diseases.

The present invention is the adoption of the following experimental construct (confirmed) can be realized

Embodiment 1, the identified -2 containing [...] -O-β-L-galactopyranoside composition preparation of:

Globeflower crude drugs with 50% ethanol reflux extraction 3 times, every 2 hours. Filtering, AB-8 directly from the filtrate of macroporous resin column adsorption purification, effluent and 50% ethanol eluent recovering ethanol to mellow content of less than 10%, the concentrate AB-8 of macroporous resin column adsorption purification. First washing with water, then with 50% ethanol elution, collecting 50% ethanol eluent, and recovering the solvent to dry, with 85% ethanol heating dissolved, plus 1% pharmaceutically active carbon, mixing, filtration, filtrate directly purified through silica gel, and the 85% ethanol elution, and eluent of collecting the effluent liquid, and recovering the solvent to dry, the -2 containing identified [...] -O-β-L-galactopyranoside composition. In this composition the identified -2 [...] -O-β-L-galactoside content is 54.3%, [...] content of 21.7%, male jing Gan content of 4.6%, -2 the identified [...] -O-β-arabinoside 2.5%.

Embodiment 2, (capsule)

The raw materials of composition 100g, medicinal starch proper amount, mixing, granulation, drying, the whole grain, into 1, capsule, into 1000 granulata, get. Each 2 granulata, daily 2 times.

Embodiment 3, (tablet)

The raw materials of composition 100g, medicinal proper starch, dextrin proper amount, mixing, granulation, drying, the whole grain, drying, adding lubricant proper amount, tableting, made 1000 sheet, to obtain. Each 2 sheet, daily 2 times.

Embodiment 4, (pill)

Weighing 30g polyethylene glycol 4000, melts the in the water bath, compound raw material adds the group 20g, stir, in put thermal insulation pipe, regulating thermostatic device, the liquid medicine in the 80-90 under [...] drop in cooling liquid paraffin, after the dipping, blotting at the filter paper into the pill paraffin oil, into 1000 granulata, get. Each 10 granulata, daily 2 times.

Embodiment 5, (granule)

The raw materials of composition 100g, powdered sugar 400g, mix, with appropriate amount of ethanol granulation, dry, granulate, subpackages, made 500 bag, is obtained. Each 1 bag, daily 2 times.