CN117462571A

CN117462571A - Pharmaceutical composition and application thereof

Info

Publication number: CN117462571A
Application number: CN202310773604.9A
Authority: CN
Inventors: 王永香; 李萍; 杨华; 孙永城; 张艳军; 赵恒利
Original assignee: Nanjing Hailing Chinese Medicine Pharmaceutical Technology Research Co ltd; Yangzijiang Pharmaceutical Group Jiangsu Longfengtang Traditional Chinese Medicine Co ltd
Current assignee: Nanjing Hailing Chinese Medicine Pharmaceutical Technology Research Co ltd; Yangzijiang Pharmaceutical Group Jiangsu Longfengtang Traditional Chinese Medicine Co ltd
Priority date: 2023-06-28
Filing date: 2023-06-28
Publication date: 2024-01-30

Abstract

The present application provides a pharmaceutical composition and uses thereof, the pharmaceutical composition comprising: 12-50 parts of flavonoids, 0.7-7 parts of alkaloids, 0.5-3.5 parts of limonoids and 38-100 parts of iridoid glycosides. The pharmaceutical composition is prepared by combining effective parts of traditional Chinese medicines according to a certain proportion, and by adopting limonoids, saccharides and/or organic acids to act together in coordination with flavonoids, alkaloids and iridoid glycosides, has good antiviral and anti-inflammatory activity, has the effect of treating upper respiratory tract infection, has clear pharmacodynamic substance basis, accords with clinical practice of traditional Chinese medicine and can meet clinical requirements; in addition, the pharmaceutical composition can be used as a standard extract or a control extract for quality standard and quality control of Chinese patent medicines.

Description

Pharmaceutical composition and application thereof

Technical Field

The application relates to the technical field of medicinal chemistry, in particular to a pharmaceutical composition and application thereof.

Background

The traditional Chinese medicine clinic treatment for viral infectious fever mainly adopts methods of pungent and cool exterior syndrome relieving, pungent and warm exterior syndrome relieving, summer heat clearing, dampness removing, exterior syndrome relieving, lung heat clearing, fever relieving, heat and toxin clearing, sore throat relieving, detumescence, qi clearing, blood cooling and the like. The common medicinal materials for treating heat and toxic materials such as radix Isatidis, herba Andrographitis, coptidis rhizoma, scutellariae radix, herba Taraxaci, herba Violae, fructus Gardeniae, cortex Phellodendri, folium Isatidis, fructus Arctii, rhizoma anemarrhenae, semen Scaphii Lychnophori, fructus Aurantii Immaturus, notopterygii rhizoma, herba Menthae, rhizoma Phragmitis, radix et rhizoma Rhei, and flos Lonicerae have effects of clearing heat and toxic materials, cooling blood and relieving swelling.

The clinical application of the traditional Chinese medicine is to pay attention to overall regulation and diagnosis and treatment, and has the function characteristics of multi-component and multi-target integration. However, the drug effect substance basis of the traditional Chinese medicine is difficult to clear, and the drug effect substance of the traditional Chinese medicine needs to be researched by utilizing modern science, so as to establish an active component discovery theory and technical system which accords with the integral action characteristics of the traditional Chinese medicine. In a complex chemical system of traditional Chinese medicine, which components can represent the efficacy of the traditional Chinese medicine, which equivalent component groups are equivalent to the efficacy of the original traditional Chinese medicine aiming at a certain disease, and which are core effective parts of the traditional Chinese medicine are all fundamental problems which need to be solved urgently in basic research of the traditional Chinese medicine. The modern technology and means are adopted to extract each active site for drug effect activity screening and safety evaluation, and scientific formula is carried out according to the synergistic effect of different active sites on the curative effect, so that the modern Chinese herbal compound preparation which has the characteristic of distinct curative effect and takes the group of the effective sites of the Chinese herbal as the substance basis is developed, and the modern Chinese herbal compound preparation has become one of the directions of research and development of the modern Chinese herbal compound preparation. At present, an effective part group which can meet the characteristics of traditional Chinese medicines and has obvious curative effect and can treat upper respiratory tract infection is required to be searched to meet clinical requirements.

Disclosure of Invention

The application aims at providing a pharmaceutical composition which accords with clinical practice of traditional Chinese medicine and has obvious curative effect and is formed by combining effective parts of traditional Chinese medicine according to a certain proportion. The specific technical scheme is as follows:

a first aspect of the present application provides a pharmaceutical composition comprising: 12-50 parts of flavonoids, 0.7-7 parts of alkaloids, 0.5-3.5 parts of limonoids and 38-100 parts of iridoid glycosides.

In some embodiments of the present application, the pharmaceutical composition comprises: 15-35 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-2.0 parts of limonoids and 38-90 parts of iridoid glycosides;

preferably, the pharmaceutical composition comprises: 20-33 parts of flavonoids, 1-5 parts of alkaloids, 0.5-1.5 parts of limonoids and 45-75 parts of iridoid glycosides.

In some embodiments of the present application, the pharmaceutical composition further comprises: 0.4-3 parts by weight of organic acids and/or 5-50 parts by weight of saccharides;

preferably, the pharmaceutical composition further comprises: 0.4-1.5 parts by weight of organic acids and/or 5-25 parts by weight of saccharides;

more preferably, the pharmaceutical composition further comprises: 0.4-1.3 parts by weight of organic acids and/or 5-18 parts by weight of saccharides.

In some embodiments of the present application, the pharmaceutical composition comprises: 15-35 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-2.0 parts of limonoids, 38-90 parts of iridoid glycosides and 0.4-1.5 parts of organic acids;

preferably, the pharmaceutical composition comprises: 20-33 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-1.5 parts of limonoids, 45-75 parts of iridoid glycosides and 0.4-1.3 parts of organic acids;

more preferably, the pharmaceutical composition comprises: 19.74-32.40 parts of flavonoids, 1.35-4.49 parts of alkaloids, 0.50-1.14 parts of limonoids, 49.07-74.99 parts of iridoid glycosides and 0.49-1.30 parts of organic acids.

In some embodiments of the present application, the pharmaceutical composition comprises: 15-35 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-2.0 parts of limonoids, 38-90 parts of iridoid glycosides and 5-25 parts of saccharides;

preferably, the pharmaceutical composition comprises: 20-33 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-1.5 parts of limonoids, 45-75 parts of iridoid glycosides and 5-18 parts of saccharides.

In some embodiments of the present application, the pharmaceutical composition comprises: 15-35 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-2.0 parts of limonoids, 38-90 parts of iridoid glycosides, 0.4-1.5 parts of organic acids and 5-25 parts of saccharides;

preferably, the pharmaceutical composition comprises: 20-33 parts of flavonoids, 1-5 parts of alkaloids, 0.5-1.5 parts of limonoids, 45-75 parts of iridoid glycosides, 0.4-1.3 parts of organic acids and 5-18 parts of saccharides;

more preferably, the pharmaceutical composition comprises: 19.74-32.40 parts of flavonoids, 1.35-4.49 parts of alkaloids, 0.5-1.14 parts of limonoids, 49.07-74.99 parts of iridoid glycosides, 0.49-1.30 parts of organic acids and 5.49-17 parts of saccharides.

In some embodiments of the present application, the flavonoid comprises at least one of baicalein, baicalin, wogonin, rutin, and oroxylin a;

preferably, the flavonoids comprise (0.025-0.1) by weight: (0.08-1.4): (0.03-0.06): (0.01-0.03): (0.005-0.02) baicalein, baicalin, wogonin, rutin, and oroxylin A;

More preferably, the flavonoids comprise (0.025-0.1) by weight: 1: (0.03-0.06): (0.01-0.03): (0.005-0.01) baicalein, baicalin, wogonin, rutin and oroxylin A.

In some embodiments of the present application, the flavonoid further comprises at least one of chrysin-7-O- β -D-glucuronide, isoquercitrin, scutellarin, baicalein II, and calycosin;

preferably, the weight ratio of baicalin to chrysin-7-O-beta-D-glucuronide is (0.08-1.4): (0.01-0.03), preferably 1: (0.01-0.03); and/or

The weight ratio of baicalin to isoquercitrin is (0.08-1.4): (0.002-0.007), preferably 1: (0.002-0.007); and/or

The weight ratio of baicalin to scutellarin is (0.08-1.4): (0.004-0.02), preferably 1: (0.004-0.02); and/or

The weight ratio of baicalin to baicalein II is (0.08-1.4): (0.008-0.02); preferably 1: (0.008-0.02); and/or

The weight ratio of baicalin to calycosin is (0.08-1.4): (0.004-0.01), preferably 1: (0.004-0.01).

In some embodiments of the present application, the flavonoids comprise (0.025-0.1) by weight: (0.08-1.4): (0.03-0.06): (0.01-0.03): (0.005-0.02): (0.01-0.03): (0.008-0.02): (0.004-0.01) baicalein, baicalin, wogonin, rutin, oroxylin A, chrysin-7-O-beta-D-glucuronide, baicalein II and calycosin;

Preferably, the flavonoids comprise (0.025-0.1) by weight: (0.08-1.4): (0.03-0.06): (0.01-0.03): (0.005-0.02): (0.01-0.03): (0.002-0.007): (0.004-0.02): (0.008-0.02): (0.004-0.01) baicalein, baicalin, wogonin, rutin, oroxylin A, chrysin-7-O-beta-D-glucuronide, isoquercitrin, scutellarin, baicalein II and calycosin.

In some embodiments of the present application, the alkaloids comprise at least one of berberine hydrochloride and adenosine;

preferably, the weight ratio of berberine hydrochloride to adenosine is (0.5-1.5): (0.7-5.0), preferably 1: (0.7-5.0).

In some embodiments of the present application, the alkaloids further comprise indirubin, berberine hydrochloride, and indirubin in a weight ratio of (0.5-1.5): (0.005-0.12);

preferably, the weight ratio of berberine hydrochloride, adenosine and indirubin is (0.5-1.5): (0.7-5.0): (0.005-0.12), preferably 1: (0.735-5.0): (0.005-0.12).

In some embodiments of the present application, the alkaloids further comprise at least one of phellodendrine hydrochloride, magnolol, palmatine hydrochloride, jatrorrhizine hydrochloride, uridine, and guanosine;

Preferably, the weight ratio of berberine hydrochloride to phellodendrine hydrochloride is (0.5-1.5): (0.5-5), preferably 1: (0.5-4.5); and/or

The weight ratio of the berberine hydrochloride to the magnolol is (0.5-1.5): (0.4-3.5), preferably 1: (0.4-3.5); and/or

The weight ratio of the berberine hydrochloride to the palmatine hydrochloride is (0.5-1.5): (0.02-0.2), preferably 1: (0.024-0.2); and/or

The weight ratio of the berberine hydrochloride to the jateorhizine hydrochloride is (0.5-1.5): (0.01-0.2), preferably 1: (0.015-0.15); and/or

The weight ratio of the berberine hydrochloride to the uridine is (0.5-1.5): (0.3-2.5), preferably 1: (0.35-2.5); and/or

The weight ratio of the berberine hydrochloride to the guanosine is (0.5-1.5): (0.3-2.0), preferably 1: (0.33-2.0).

In some embodiments of the present application, the alkaloids comprise (0.5-1.5) by weight: (0.7-5.0): (0.005-0.12): (0.5-5): (0.4-3.5): (0.02-0.2): (0.01-0.2): (0.3-2.5): (0.3-2.0) berberine hydrochloride, adenosine, indirubin, phellodendrine hydrochloride, magnaline, palmatine hydrochloride, jateorhizine hydrochloride, uridine and guanosine;

preferably, the alkaloids comprise 1 in weight ratio: (0.735-5.0): (0.005-0.12): (0.5-4.5): (0.4-3.5): (0.024-0.2): (0.015-0.15): (0.35-2.5): (0.33-2.0) berberine hydrochloride, adenosine, indirubin, phellodendrine hydrochloride, magnaline, palmatine hydrochloride, jateorhizine hydrochloride, uridine and guanosine;

More preferably, the alkaloids comprise 1 in weight ratio: (0.735-4.3): (0.005-0.12): (0.669-4.37): (0.446-3.13): (0.024-0.16): (0.019-0.135): (0.38-2.447): (0.33-1.898) berberine hydrochloride, adenosine, indirubin, phellodendrine hydrochloride, magnaline, palmatine hydrochloride, jateorhizine hydrochloride, uridine and guanosine.

In some embodiments of the present application, the limonoids comprise at least one of limonin and phellodendron ketone;

preferably, the weight ratio of limonin to phellodendron ketone is (10-30): (0-1.1), preferably (12-28.5): 1.

in some embodiments of the present application, the iridoid glycoside comprises geniposide.

In some embodiments of the present application, the organic acid comprises at least one of chlorogenic acid, ferulic acid, and benzoic acid;

preferably, chlorogenic acid accounts for less than or equal to 100 weight percent of the organic acids, preferably less than or equal to 92 weight percent;

more preferably, the organic acid comprises (4-12) by weight: (0.8-1.2) chlorogenic acid and ferulic acid;

still more preferably, the organic acid comprises (4-12) by weight: chlorogenic acid and ferulic acid of 1.

In some embodiments of the present application, the saccharide comprises an oligosaccharide; preferably, the oligosaccharide comprises stachyose.

In some embodiments of the present application, the pharmaceutical composition comprises: 19.74-32.40 parts by weight of flavonoids, 1.35-4.49 parts by weight of alkaloids, 0.5-1.14 parts by weight of limonoids and 49.07-74.99 parts by weight of iridoid glycosides; wherein the flavonoids comprise (0.025-0.1) by weight: 1: (0.03-0.06): (0.01-0.03): (0.005-0.02) baicalein, baicalin, wogonin, rutin, and oroxylin A; the alkaloid comprises the following components in percentage by weight: (1.0-5.0) berberine hydrochloride and adenosine; the limonin comprises limonin; the iridoid glycoside comprises geniposide;

preferably, the pharmaceutical composition comprises: 19.74-32.40 parts by weight of flavonoids, 1.35-4.49 parts by weight of alkaloids, 0.5-1.14 parts by weight of limonoids, 49.07-74.99 parts by weight of iridoid glycosides, 0.49-1.30 parts by weight of organic acids and 5.49-17 parts by weight of saccharides; wherein the flavonoids comprise (0.025-0.1) by weight: 1: (0.03-0.06): (0.01-0.03): (0.005-0.02) baicalein, baicalin, wogonin, rutin, and oroxylin A; the alkaloid comprises the following components in percentage by weight: (1.0-5.0): (0.01-0.1) berberine hydrochloride, adenosine and indirubin; the limonin comprises the following components in percentage by weight (12-27): 1 limonin and phellodendron ketone; the iridoid glycoside comprises geniposide; the organic acid comprises chlorogenic acid; the saccharide comprises stachyose;

Preferably, the pharmaceutical composition comprises: 19.74-32.40 parts by weight of flavonoids, 1.35-4.49 parts by weight of alkaloids, 0.5-1.14 parts by weight of limonoids, 49.07-74.99 parts by weight of iridoid glycosides, 0.49-1.30 parts by weight of organic acids and 5.49-17 parts by weight of saccharides; wherein the flavonoids comprise (0.025-0.1) by weight: 1: (0.03-0.06): (0.01-0.03): (0.005-0.02): (0.01-0.03): (0.002-0.007): (0.004-0.02): (0.008-0.02): (0.004-0.01) baicalein, baicalin, wogonin, rutin, oroxylin A, chrysin-7-O-beta-D-glucuronide, isoquercitrin, scutellarin, baicalein II and calycosin; the alkaloid comprises the following components in percentage by weight: (0.735-5.0): (0.005-0.12): (0.5-4.5): (0.4-3.5): (0.024-0.2): (0.015-0.15): (0.35-2.5): (0.33-2.0) berberine hydrochloride, adenosine, indirubin, phellodendrine hydrochloride, magnaline, palmatine hydrochloride, jateorhizine hydrochloride, uridine and guanosine; the limonin comprises the following components in percentage by weight (12-28.5): 1 limonin and phellodendron ketone; the iridoid glycoside comprises geniposide; the organic acid comprises the following components in percentage by weight (4-12): chlorogenic acid and ferulic acid of 1; the saccharide comprises stachyose;

Preferably, the pharmaceutical composition comprises: 19.74-32.40 parts by weight of flavonoids, 1.35-4.49 parts by weight of alkaloids, 0.5-1.14 parts by weight of limonoids, 49.07-74.99 parts by weight of iridoid glycosides, 0.49-1.30 parts by weight of organic acids and 5.49-17 parts by weight of saccharides; wherein the flavonoid component comprises the following components in percentage by weight (0.025-0.1): 1: (0.03-0.06): (0.01-0.03): (0.005-0.02): (0.01-0.03): (0.008-0.02): (0.004-0.01) baicalein, baicalin, wogonin, rutin, oroxylin A, chrysin-7-O-beta-D-glucuronide, baicalein II and calycosin; the alkaloid comprises the following components in percentage by weight: (0.7-5.0): (0.005-0.12): (0.5-5): (0.4-3.5): (0.02-0.20): (0.01-0.2): (0.3-2.5): (0.3-2.0) berberine hydrochloride, adenosine, indirubin, phellodendrine hydrochloride, magnaline, palmatine hydrochloride, jateorhizine hydrochloride, uridine and guanosine; the limonin comprises the following components in percentage by weight (10-30): limonin and phellodendron ketone of (0-1.1); the iridoid glycoside comprises geniposide; the organic acid comprises the following components in percentage by weight (4-12): (0.8-1.2) chlorogenic acid and ferulic acid; the saccharide comprises stachyose.

In some embodiments of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient;

preferably, the pharmaceutical composition can be prepared into a pharmaceutically acceptable preparation, and the dosage form of the preparation comprises capsules, oral liquid, tablets, granules or dripping pills.

A second aspect of the present application provides the use of a pharmaceutical composition according to the first aspect of the present application for the manufacture of a medicament for the prevention and/or treatment of upper respiratory tract infections;

preferably, the upper respiratory tract infection comprises a cold, pharyngitis, laryngitis, angina, tonsillitis; wherein the pharyngitis comprises acute pharyngitis, chronic pharyngitis, viral pharyngitis and bacterial pharyngitis; the laryngitis comprises acute laryngitis, chronic laryngitis, viral laryngitis and bacterial laryngitis; the angina comprises herpetic angina; the tonsillitis comprises acute tonsillitis, chronic tonsillitis, viral tonsillitis and bacterial tonsillitis.

A third aspect of the present application provides the use of a pharmaceutical composition according to the first aspect of the present application for controlling the quality of a chinese patent medicine; preferably, the pharmaceutical composition is used as a standard extract or a control extract.

The pharmaceutical composition provided by the application is prepared by combining the effective part groups of the traditional Chinese medicines according to a certain proportion, and by adopting limonoids, saccharides and/or organic acids to act together in cooperation with flavonoids, alkaloids and iridoid glycosides, the pharmaceutical composition has good antiviral and anti-inflammatory activity effects, has the effect of treating upper respiratory tract infection, has clear pharmacodynamic substance basis, accords with clinical practice of traditional Chinese medicine, and can meet clinical requirements; in addition, the pharmaceutical composition can be used as a standard extract or a control extract for quality standard and quality control of Chinese patent medicines.

Of course, not all of the above-described advantages need be achieved simultaneously in practicing any one of the products or methods of the present application.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following description will briefly introduce the drawings that are required to be used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other embodiments may also be obtained according to these drawings to those skilled in the art.

FIG. 1 is a graph showing changes in the morphology of RAW264.7 cells (×200 times) following Lipopolysaccharide (LPS) stimulation and administration of the pharmaceutical compositions of examples 7-8 in anti-inflammatory cell experiments.

FIG. 2 is a graph showing the change in NO content of RAW264.7 cells after Lipopolysaccharide (LPS) stimulation and administration of the pharmaceutical compositions of examples 7-8 in anti-inflammatory cell experiments. * Represents (p < 0.01) compared to the blank (NC); # represents (p < 0.01) compared to the model group (LPS).

FIG. 3 is a graph showing the variation of the IL-6 content of RAW264.7 cells after administration of the pharmaceutical compositions of examples 7-8, as measured by ELISA for Lipopolysaccharide (LPS) stimulation in anti-inflammatory cell assay one.

FIG. 4 is a graph showing the variation of the IL-1β content of RAW264.7 cells after administration of the pharmaceutical compositions of examples 7-8, as measured by ELISA for Lipopolysaccharide (LPS) stimulation in anti-inflammatory cell assay one.

FIG. 5 is a graph showing the change in the amount of TNF- α in RAW264.7 cells after administration of the pharmaceutical compositions of examples 7-8, as measured by ELISA in anti-inflammatory cell assay I.

In fig. 3 to 5, p < 0.01 compared to the blank (NC); # represents (p < 0.01) compared to the model group (LPS).

FIG. 6 is a graph showing the phosphorylation levels of β -actin, TLR4, NF- κ B p65 and p-NF- κ B p65 proteins in RAW264.7 cells after Lipopolysaccharide (LPS) stimulation by Western blot analysis in anti-inflammatory cell experiment one.

FIG. 7 is a graph showing the quantitative analysis of β -actin, TLR4, NF- κ B p65 and p-NF- κ B p65 proteins in RAW264.7 cells after Lipopolysaccharide (LPS) stimulation by Western blot in anti-inflammatory cell experiment one. * Represents (p < 0.01) compared to the blank (NC); # denotes (p < 0.01) compared to model group (LPS), and # denotes (p < 0.05) compared to model group (LPS).

FIG. 8 is a graph showing changes in RAW264.7 cell morphology (×200 times) following Lipopolysaccharide (LPS) stimulation in anti-inflammatory cell experiments II and administration of the pharmaceutical compositions of examples 1-6.

FIG. 9 is a graph showing the change in NO content of RAW264.7 cells after Lipopolysaccharide (LPS) stimulation and administration of the pharmaceutical compositions of examples 1-6 in inflammatory cell experiments II. * Represents (p < 0.01) compared to the blank (NC); # represents (p < 0.01) compared to the model group (LPS).

FIG. 10 is a graph showing the variation of the IL-6 content of RAW264.7 cells after administration of the pharmaceutical compositions of examples 1-6, as measured by ELISA in anti-inflammatory cell experiment II.

FIG. 11 is a graph showing the variation of the IL-1β content of RAW264.7 cells after administration of the pharmaceutical compositions of examples 1-6, as measured by ELISA in anti-inflammatory cell experiment II.

FIG. 12 is a graph showing the change in the amount of TNF- α in RAW264.7 cells after ELISA detection of Lipopolysaccharide (LPS) stimulation in anti-inflammatory cell experiment II and administration of the pharmaceutical compositions of examples 1-6.

In fig. 10 to 12, p < 0.01 compared to the blank (NC); # represents (p < 0.01) compared to model group (LPS); # represents (p < 0.05) compared to the model group (LPS).

Fig. 13 is a graph showing the effect of the drug group on the pharyngeal mucosa of rats with acute pharyngitis in an animal experiment with pharyngitis.

Fig. 14 is a statistical plot of apparent scores of the pharyngeal portion of rats in an animal experiment with pharyngitis. * Represents (p < 0.01) compared to the blank (NC); # represents (p < 0.01) compared to the model group (AP) group; ns indicates no significant difference.

FIG. 15 is a graph showing the effect of the drug group on acute pharyngitis rat serum inflammatory factor IL-6 in an animal experiment with pharyngitis.

FIG. 16 is a graph showing the effect of the drug group on acute pharyngitis rat serum inflammatory factor IL-1. Beta. In an animal experiment with pharyngitis.

FIG. 17 is a graph showing the effect of a drug group on acute pharyngitis rat serum inflammatory factor TNF- α in an animal experiment with pharyngitis.

In fig. 15 to 17, p < 0.01 compared to the blank (NC); # represents (p < 0.01) compared to model group (AP); # represents (p < 0.05) compared to model group (AP); ns indicates no significant difference.

Fig. 18 is a view of pathological section of pharyngeal tissue of rats in pharyngitis animal experiments.

FIG. 19 is a graph showing the phosphorylation levels of beta-actin, TLR4, NF-kappa B p and p-NF-kappa B p proteins in pharyngeal tissues of rats with acute pharyngitis by Western blot analysis in animal experiments with pharyngitis.

FIG. 20 is a quantitative graph of β -actin, TLR4, NF- κ B p65 and p-NF- κ B p65 proteins in pharyngeal tissue of rats with acute pharyngitis by Western blot analysis in animal experiments with pharyngitis. * Represents (p < 0.01) compared to the blank (NC) and represents (p < 0.05) compared to the blank (NC); # denotes (p < 0.01) compared to model group (AP), and # denotes (p < 0.05) compared to model group (AP); ns indicates no significant difference.

Detailed Description

The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all, of the embodiments of the present application. All other embodiments obtained based on the present application by a person skilled in the art are within the scope of the protection of the present application.

In some embodiments of the present application, the pharmaceutical composition comprises: 15-35 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-2.0 parts of limonoids and 38-90 parts of iridoid glycosides.

In some embodiments of the present application, the pharmaceutical composition comprises: 20-33 parts of flavonoids, 1-5 parts of alkaloids, 0.5-1.5 parts of limonoids and 45-75 parts of iridoid glycosides.

In some embodiments of the present application, the pharmaceutical composition further comprises: 0.4-3 parts by weight of organic acids and/or 5-50 parts by weight of saccharides.

In some embodiments of the present application, the pharmaceutical composition further comprises: 0.4-1.5 parts by weight of organic acids and/or 5-25 parts by weight of saccharides.

In some embodiments of the present application, the pharmaceutical composition further comprises: 0.4-1.3 parts by weight of organic acids and/or 5-18 parts by weight of saccharides.

In some embodiments of the present application, the pharmaceutical composition comprises: 15-35 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-2.0 parts of limonoids, 38-90 parts of iridoid glycosides and 0.4-1.5 parts of organic acids.

In some embodiments of the present application, the pharmaceutical composition comprises: 20-33 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-1.5 parts of limonoids, 45-75 parts of iridoid glycosides and 0.4-1.3 parts of organic acids.

In some embodiments of the present application, the pharmaceutical composition comprises: 19.74-32.40 parts of flavonoids, 1.35-4.49 parts of alkaloids, 0.50-1.14 parts of limonoids, 49.07-74.99 parts of iridoid glycosides and 0.49-1.30 parts of organic acids.

In some embodiments of the present application, the pharmaceutical composition comprises: 15-35 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-2.0 parts of limonoids, 38-90 parts of iridoid glycosides and 5-25 parts of saccharides.

In some embodiments of the present application, the pharmaceutical composition comprises: 20-33 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-1.5 parts of limonoids, 45-75 parts of iridoid glycosides and 5-18 parts of saccharides.

In some embodiments of the present application, the pharmaceutical composition comprises: 15-35 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-2.0 parts of limonoids, 38-90 parts of iridoid glycosides, 0.4-1.5 parts of organic acids and 5-25 parts of saccharides.

In some embodiments of the present application, the pharmaceutical composition comprises: 20-33 parts of flavonoids, 1-5 parts of alkaloids, 0.5-1.5 parts of limonoids, 45-75 parts of iridoid glycosides, 0.4-1.3 parts of organic acids and 5-18 parts of saccharides.

In some embodiments of the present application, the pharmaceutical composition comprises: 19.74-32.40 parts of flavonoids, 1.35-4.49 parts of alkaloids, 0.5-1.14 parts of limonoids, 49.07-74.99 parts of iridoid glycosides, 0.49-1.30 parts of organic acids and 5.49-17 parts of saccharides.

In some embodiments of the present application, the flavonoid comprises at least one of baicalein, baicalin, wogonin, rutin, and oroxylin a.

In some embodiments of the present application, the flavonoids comprise (0.025-0.1) by weight: (0.08-1.4): (0.03-0.06): (0.01-0.03): (0.005-0.02) baicalein, baicalin, wogonin, rutin and oroxylin A.

In some embodiments of the present application, the flavonoids comprise (0.025-0.1) by weight: 1: (0.03-0.06): (0.01-0.03): (0.005-0.01) baicalein, baicalin, wogonin, rutin and oroxylin A.

In some embodiments of the present application, the flavonoid further comprises at least one of chrysin-7-O- β -D-glucuronide, isoquercitrin, scutellarin, baicalein II, and calycosin.

In some embodiments of the present application, the weight ratio of baicalin to chrysin-7-O- β -D-glucuronide is (0.08-1.4): (0.01-0.03), preferably 1: (0.01-0.03); and/or

In some embodiments of the present application, the flavonoids comprise (0.025-0.1) by weight: (0.08-1.4): (0.03-0.06): (0.01-0.03): (0.005-0.02): (0.01-0.03): (0.008-0.02): (0.004-0.01) baicalein, baicalin, wogonin, rutin, oroxylin A, chrysin-7-O-beta-D-glucuronide, baicalein II and calycosin.

In some embodiments of the present application, the flavonoids comprise (0.025-0.1) by weight: (0.08-1.4): (0.03-0.06): (0.01-0.03): (0.005-0.02): (0.01-0.03): (0.002-0.007): (0.004-0.02): (0.008-0.02): (0.004-0.01) baicalein, baicalin, wogonin, rutin, oroxylin A, chrysin-7-O-beta-D-glucuronide, isoquercitrin, scutellarin, baicalein II and calycosin.

In some embodiments of the present application, the alkaloids comprise at least one of berberine hydrochloride and adenosine.

In some embodiments of the present application, the weight ratio of berberine hydrochloride to adenosine is (0.5-1.5): (0.7-5.0), preferably 1: (0.7-5.0).

In some embodiments of the present application, the alkaloids further comprise indirubin, berberine hydrochloride, and indirubin in a weight ratio of (0.5-1.5): (0.005-0.12).

In some embodiments of the present application, the weight ratio of berberine hydrochloride, adenosine, indirubin is (0.5-1.5): (0.7-5.0): (0.005-0.12), preferably 1: (0.735-5.0): (0.005-0.12).

In some embodiments of the present application, the alkaloids further comprise at least one of phellodendrine hydrochloride, magnolone, palmatine hydrochloride, jateorhizine hydrochloride, uridine, and guanosine.

In some embodiments of the present application, the weight ratio of berberine hydrochloride to phellodendrine hydrochloride is (0.5-1.5): (0.5-5), preferably 1: (0.5-4.5); and/or

In some embodiments of the present application, the alkaloids comprise (0.5-1.5) by weight: (0.7-5.0): (0.005-0.12): (0.5-5): (0.4-3.5): (0.02-0.2): (0.01-0.2): (0.3-2.5): (0.3-2.0) berberine hydrochloride, adenosine, indirubin, phellodendrine hydrochloride, magnaline, palmatine hydrochloride, jateorhizine hydrochloride, uridine and guanosine.

In some embodiments of the present application, the alkaloids comprise a weight ratio of 1: (0.735-5.0): (0.005-0.12): (0.5-4.5): (0.4-3.5): (0.024-0.2): (0.015-0.15): (0.35-2.5): (0.33-2.0) berberine hydrochloride, adenosine, indirubin, phellodendrine hydrochloride, magnaline, palmatine hydrochloride, jateorhizine hydrochloride, uridine and guanosine.

In some embodiments of the present application, the alkaloids comprise a weight ratio of 1: (0.735-4.3): (0.005-0.12): (0.669-4.37): (0.446-3.13): (0.024-0.16): (0.019-0.135): (0.38-2.447): (0.33-1.898) berberine hydrochloride, adenosine, indirubin, phellodendrine hydrochloride, magnaline, palmatine hydrochloride, jateorhizine hydrochloride, uridine and guanosine.

In some embodiments of the present application, the limonoids comprise at least one of limonin and phellodendron.

In some embodiments of the present application, the weight ratio of limonin to phellodendron ketone is (10-30): (0-1.1), preferably (12-28.5): 1.

In some embodiments of the present application, the organic acid comprises at least one of chlorogenic acid, ferulic acid, and benzoic acid.

In some embodiments of the present application, chlorogenic acid comprises less than or equal to 100%, preferably less than or equal to 92% by weight of the organic acids.

In some embodiments of the present application, the organic acids comprise (4-12) by weight: (0.8-1.2) chlorogenic acid and ferulic acid.

In some embodiments of the present application, the organic acids comprise (4-12) by weight: chlorogenic acid and ferulic acid of 1.

In some embodiments of the present application, the saccharide comprises an oligosaccharide.

In some embodiments of the present application, the oligosaccharide comprises stachyose.

In some embodiments of the present application, the pharmaceutical composition comprises: 19.74-32.40 parts by weight of flavonoids, 1.35-4.49 parts by weight of alkaloids, 0.5-1.14 parts by weight of limonoids and 49.07-74.99 parts by weight of iridoid glycosides; wherein the flavonoids comprise (0.025-0.1) by weight: 1: (0.03-0.06): (0.01-0.03): (0.005-0.02) baicalein, baicalin, wogonin, rutin, and oroxylin A; the alkaloid comprises the following components in percentage by weight: (1.0-5.0) berberine hydrochloride and adenosine; limonin comprises limonin; the iridoid glycoside contains geniposide.

In some embodiments of the present application, the pharmaceutical composition comprises: 19.74-32.40 parts by weight of flavonoids, 1.35-4.49 parts by weight of alkaloids, 0.5-1.14 parts by weight of limonoids, 49.07-74.99 parts by weight of iridoid glycosides, 0.49-1.30 parts by weight of organic acids and 5.49-17 parts by weight of saccharides; wherein the flavonoids comprise (0.025-0.1) by weight: 1: (0.03-0.06): (0.01-0.03): (0.005-0.02) baicalein, baicalin, wogonin, rutin, and oroxylin A; the alkaloid comprises the following components in percentage by weight: (1.0-5.0): (0.01-0.1) berberine hydrochloride, adenosine and indirubin; the limonin comprises the following components in percentage by weight (12-27): 1 limonin and phellodendron ketone; the iridoid glycoside comprises geniposide; the organic acids comprise chlorogenic acid; the saccharide comprises stachyose.

In some embodiments of the present application, the pharmaceutical composition comprises: 19.74-32.40 parts by weight of flavonoids, 1.35-4.49 parts by weight of alkaloids, 0.5-1.14 parts by weight of limonoids, 49.07-74.99 parts by weight of iridoid glycosides, 0.49-1.30 parts by weight of organic acids and 5.49-17 parts by weight of saccharides; wherein the flavonoids comprise (0.025-0.1) by weight: 1: (0.03-0.06): (0.01-0.03): (0.005-0.02): (0.01-0.03): (0.002-0.007): (0.004-0.02): (0.008-0.02): (0.004-0.01) baicalein, baicalin, wogonin, rutin, oroxylin A, chrysin-7-O-beta-D-glucuronide, isoquercitrin, scutellarin, baicalein II and calycosin; the alkaloid comprises the following components in percentage by weight: (0.735-5.0): (0.005-0.12): (0.5-4.5): (0.4-3.5): (0.024-0.2): (0.015-0.15): (0.35-2.5): (0.33-2.0) berberine hydrochloride, adenosine, indirubin, phellodendrine hydrochloride, magnaline, palmatine hydrochloride, jateorhizine hydrochloride, uridine and guanosine; limonin comprises the following components in percentage by weight (12-28.5): 1 limonin and phellodendron ketone; the iridoid glycoside comprises geniposide; the organic acid comprises the following components in percentage by weight (4-12): chlorogenic acid and ferulic acid of 1; the saccharide comprises stachyose.

In some embodiments of the present application, the pharmaceutical composition comprises: 19.74-32.40 parts by weight of flavonoids, 1.35-4.49 parts by weight of alkaloids, 0.5-1.14 parts by weight of limonoids, 49.07-74.99 parts by weight of iridoid glycosides, 0.49-1.30 parts by weight of organic acids and 5.49-17 parts by weight of saccharides; wherein the flavonoid component comprises the following components in percentage by weight (0.025-0.1): 1: (0.03-0.06): (0.01-0.03): (0.005-0.02): (0.01-0.03): (0.008-0.02): (0.004-0.01) baicalein, baicalin, wogonin, rutin, oroxylin A, chrysin-7-O-beta-D-glucuronide, baicalein II and calycosin; the alkaloid comprises the following components in percentage by weight: (0.7-5.0): (0.005-0.12): (0.5-5): (0.4-3.5): (0.02-0.20): (0.01-0.2): (0.3-2.5): (0.3-2.0) berberine hydrochloride, adenosine, indirubin, phellodendrine hydrochloride, magnaline, palmatine hydrochloride, jateorhizine hydrochloride, uridine and guanosine; the limonin comprises the following components in percentage by weight (10-30): limonin and phellodendron ketone of (0-1.1); the iridoid glycoside comprises geniposide; the organic acid comprises the following components in percentage by weight (4-12): (0.8-1.2) chlorogenic acid and ferulic acid; the saccharide comprises stachyose.

The application combines the analysis of the composition components, the identification of in vivo metabolites and pharmacokinetics, and selects 45 micromolecular compounds (including 14 flavone and flavonoid glycoside, 6 iridoid glycoside, 7 benzyl isoquinoline alkaloid, 4 sulfur-containing alkaloid, 2 indole alkaloid, 4 nucleoside, 2 phenylpropanoids, 2 limonene triterpenoids, 2 crocin diterpenes, 1 monoterpenoid and 1 oligosaccharide compound) with pharmacodynamic activity for compatibility; by adopting specific components and specific proportions, the components have synergistic effect to obtain the pharmaceutical composition with good antiviral and anti-inflammatory activity and obvious curative effect on upper respiratory tract infection. The medicinal composition has clear medicinal effect substance foundation and can meet clinical requirements.

The application has no limitation on the sources of the components adopted, namely, geniposide, stachyose, baicalein, baicalin, wogonin, rutin, oroxylin A, chrysin-7-O-beta-D-glucuronide, baicalein II, calycosin, berberine hydrochloride, adenosine, indirubin, phellodendrine hydrochloride, magnaline, palmatine hydrochloride, jatrorrhizine hydrochloride, uridine, guanosine, limonin, phellodendron, chlorogenic acid, ferulic acid, isoquercitrin and scutellarin, and can be obtained by a chemical synthesis process or extraction and purification of medicinal materials. The medicinal materials include, but are not limited to, at least one of radix Isatidis, scutellariae radix, fructus Gardeniae, cortex Phellodendri, semen Scaphii Lychnophori, herba Taraxaci, herba Violae, herba Andrographitis, coptidis rhizoma, fructus Aurantii Immaturus, notopterygii rhizoma, herba Menthae, rhizoma Phragmitis, cortex Phellodendri, radix et rhizoma Rhei, scutellariae radix, flos Lonicerae, folium Isatidis, fructus Arctii, rhizoma anemarrhenae, fructus forsythiae or folium Perillae.

The preparation method of the pharmaceutical composition disclosed by the application can be as follows: and uniformly mixing the raw material components in the first aspect of the application to obtain the pharmaceutical composition.

The pharmaceutical composition described herein can also be prepared by the following method: decocting at least one of the above materials with water and/or alcohol, and heating under reflux or percolating to obtain extractive solution; the extract is concentrated, purified and dried to obtain the extract containing the pharmaceutical composition.

The pharmaceutical composition described herein can also be prepared by the following method: extracting the medicinal materials with water, precipitating with ethanol, collecting supernatant, concentrating, and drying to obtain the extract containing the pharmaceutical composition.

The pharmaceutical composition provided by the application has good antiviral and anti-inflammatory activity by adopting limonoids and/or saccharides to act together in coordination with flavonoids, alkaloids and iridoid glycosides.

In some embodiments of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient, such as a solvent, diluent, disintegrant, precipitation inhibitor, surfactant, glidant, binder, lubricant, dispersant, suspending agent, isotonic agent, thickener, emulsifier, preservative, stabilizer, hydrating agent, emulsification accelerator, buffer, absorbent, colorant, flavoring agent, sweetener, ion exchanger, mold release agent, coating agent, flavoring agent, or antioxidant, etc., and specifically, dextrin, sucralose, magnesium stearate, microcrystalline cellulose, sodium carboxymethyl starch, sodium benzoate, tween 80, polyethylene glycol 6000, polyethylene glycol 4000, or dimethylsilicon, etc.

In some embodiments of the present application, the pharmaceutical composition may be formulated into pharmaceutically acceptable formulations, and the dosage forms of the pharmaceutical composition include, but are not limited to, capsules, oral liquids, tablets, granules or drop pills.

For example, adding a proper amount of dextrin, sucralose and magnesium stearate into the pharmaceutical composition of the first aspect of the application, granulating by a dry method, and sieving and granulating to obtain granules; adding a proper amount of microcrystalline cellulose, sodium carboxymethyl starch and magnesium stearate into the pharmaceutical composition of the first aspect of the application, uniformly mixing, and tabletting to obtain tablets; dissolving the pharmaceutical composition in water, standing overnight, adding appropriate amount of sodium benzoate and tween 80, mixing, adding water to 1000mL, stirring, boiling, refrigerating, filtering, adjusting pH to 6-7, packaging, and sterilizing to obtain oral liquid; adding appropriate amount of polyethylene glycol 6000 and polyethylene glycol 4000 into the pharmaceutical composition of the first aspect of the application, heating and melting, stirring uniformly, dripping into simethicone coolant, washing, drying, and selecting pills to obtain dripping pills.

A second aspect of the present application provides the use of a pharmaceutical composition according to the first aspect of the present application for the manufacture of a medicament for the prophylaxis and/or treatment of upper respiratory tract infections.

A third aspect of the present application provides a medicament for the prevention and/or treatment of upper respiratory tract infections; wherein the medicament comprises a pharmaceutical composition according to the first aspect of the present application.

In a fourth aspect the present application provides a method for the prophylaxis and/or treatment of upper respiratory tract infections comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to the first aspect of the present application.

In some embodiments of the present application, the upper respiratory tract infection comprises a cold, pharyngitis, laryngitis, angina, tonsillitis; wherein the pharyngitis comprises acute pharyngitis, chronic pharyngitis, viral pharyngitis and bacterial pharyngitis; laryngitis includes acute laryngitis, chronic laryngitis, viral laryngitis and bacterial laryngitis; angina includes herpetic angina; tonsillitis includes acute tonsillitis, chronic tonsillitis, viral tonsillitis, and bacterial tonsillitis.

A fifth aspect of the present application provides the use of a pharmaceutical composition according to the first aspect of the present application for controlling the quality of a chinese patent medicine.

In some embodiments of the present application, the pharmaceutical composition is used as a standard extract or a control extract.

Examples

Hereinafter, embodiments of the present application will be described more specifically by way of examples. The various tests and evaluations were carried out according to the following methods. Unless otherwise specified, "parts" and "%" are mass references.

Adenosine (Adenosine, 98%), baicalein (Baicalein, 98%), wogonin (Wogonin, 98%), rutin (Rutin, 98%), berberine hydrochloride (Berberine, 98%), baicalin (Baicalin, 98%), limonin (Limonin, 98%), oroxylin a (orexin a, 98%), geniposide (Geniposide, 98%), phellodendrone (Obacunone, 98%), stachyose (Stachyose, 98%), chlorogenic Acid (Chlorogenic Acid, 98%), indirubin (indiubin, 97%), isoquercitrin (isoquercitin, 98%), palmatine hydrochloride (Palmatine Chloride, 98%), phellodine hydrochloride (Phellodendrine Chloride, 98%), jatrorrhizine hydrochloride (Jatrorrhizine Hydrochloride, 98%), scutellarin (Scutellarin, 95%), ferulic Acid (feruacid, 98%) are commercially available from all technologies, limited; chrysin-7-O-beta-D-glucuronide (Chrysin 7-O-beta-D-glucopyranoside, 98%), magnoflorine (98%), calycosin (98%) were purchased from adzuki biosciences, inc; baicalein II (Skullcapflavone II, 98%) was purchased from ChemFaces; uridine (98%) and Guanosine (98%) were purchased from Shanghai source leaf biotechnology limited.

Example 1

Taking 1479mg of flavonoids, 76.8mg of alkaloids, 40.4mg of limonoids, 3854.7mg of iridoid glycosides, 32.6mg of organic acids and 439.4mg of saccharides, wherein: the flavonoids comprise baicalein 41.3mg, baicalin 1358.6mg, wogonin 46mg, oroxylin A9.3mg and rutin 23.8mg; the alkaloids comprise berberine hydrochloride 14.9mg, adenosine 61.5mg, indirubin 0.4mg; limonin comprises limonin 38.6mg and phellodendron ketone 1.8mg; the iridoid glycoside comprises geniposide 3854.7mg; the organic acids include chlorogenic acid 32.6mg; the saccharide includes stachyose 439.4mg. Mixing the above materials uniformly to obtain the pharmaceutical composition.

Example 2-example 34

The procedure of example 1 was repeated except that the types of the raw material components and the contents of the raw material components were adjusted as shown in tables 1, 2 and 3, respectively.

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Example 35-example 68

65% dextrin, 0.5% sucralose and 1% magnesium stearate (calculated by taking the mass of the pharmaceutical composition as 100%) are respectively added into the pharmaceutical compositions of the examples 1-34, and the mixture is mixed, granulated by a dry method, sieved and granulated to obtain granules.

Example 69-example 102

70% microcrystalline cellulose, 4% sodium carboxymethyl starch and 0.5% magnesium stearate (calculated by taking the mass of the pharmaceutical composition as 100%) are respectively added into the pharmaceutical compositions of the examples 1-34, and the mixture is uniformly mixed and tabletted to obtain tablets.

Examples 103 to 136

Dissolving the pharmaceutical compositions of examples 1-34 in water, standing overnight, adding 0.5% sodium benzoate and 1% Tween 80 (based on 100% of the mass of the pharmaceutical composition), mixing, adding water to 1000mL, stirring, boiling, refrigerating, filtering, adjusting pH to 6-7, packaging, and sterilizing to obtain oral liquid.

Examples 137 to 170

Respectively weighing polyethylene glycol (polyethylene glycol comprises polyethylene glycol 6000 and polyethylene glycol 4000 with mass ratio of 3:1) twice of the pharmaceutical composition, heating and melting, adding into the pharmaceutical compositions of examples 1-34, stirring, dripping into simethicone coolant, washing, drying, and selecting pill to obtain dripping pill.

Anti-inflammatory cell experiment one

1. Experimental materials

1. Experimental cell

RAW264.7 mouse macrophages (catalog number: TCM 13) were purchased from the China academy of sciences typical culture Collection Committee cell bank.

2. Experimental reagent

Dexamethasone acetate (DXM) was purchased from zhejiang sony pharmaceutical company, inc.

Fetal bovine serum (Fetal bovine serum, FBS) was purchased from Gibco corporation (Grand Island, USA).

DMEM medium containing dual-antibody high sugar was purchased from Jiangsu Kaiki biotechnology Co., ltd.

Dimethyl sulfoxide (DMSO) and Lipopolysaccharide (LPS) were purchased from Sigma.

Mouse tumor necrosis factor-alpha (TNF-alpha) ELISA kit (E-EL-M3063), mouse interleukin-6 (IL-6) ELISA kit (E-EL-M0044 c), and mouse interleukin-1β (IL-1β) ELISA kit (E-EL-M0037) were purchased from Wohan Irettan Biotech Co.

CCK-8 detection kit was purchased from APExIBO corporation.

BCA protein quantification kit, nitric oxide detection kit, western and IP cell lysate were purchased from shanghai bi yun biotechnology limited.

Protease inhibitors and phosphatase inhibitors were purchased from Shanghai Luo's pharmaceutical Co.

Skimmed milk powder was purchased from inner mongolian illi real group limited.

Nitrocellulose membranes NC were purchased from Millipore corporation.

Protein markers were purchased from Nanjinopran biotechnology Co.

Sodium Dodecyl Sulfate (SDS), tris, 4 XTris-HCl (pH 8.8), 4 XTris-HCl (pH 6.8), ready-to-use TBS powder, glycine, ammonium persulfate, tetramethyl ethylenediamine (TEMED) and 30% acrylamide solution were purchased from Shanghai Ala Biochemical technologies Co., ltd.

Tween 20 was purchased from south tokyo chemical agents inc.

Primary antibodies to TLR4 (mouse, sc-293072) and NF- κ B p65 (mouse, sc-8008) are available from Santa Cruz Biotechnology.

Red loading buffer (3X, 7723) and an anti-p-NF- κ B p65 (Rabbit, 3033) are available from Cell signaling technology.

Internal control beta-actin (Rabbit, 20536-1-AP), HRP-goat anti-Rabbit IgG (SA 00001-2) and HRP-goat anti-mouse IgG (SA 00001-1) were purchased from Proteintech.

ECL chemiluminescent substrates were purchased from Shanghai, energy technology limited.

3. Experimental instrument

BT25S one ten thousandth balance (Sartorius, gottingen, germany); a multi-functional microplate reader (BERTHOLD TECHNOLOGIES); full-automatic chemiluminescence/fluorescence image analysis systems (Shanghai Techno Co., ltd.); HH-4 digital display constant temperature water bath (national electric appliance Co., ltd.); inverted fluorescence microscope (Nanjing Gauss instruments Co., ltd.); centrifuge (zemoeimer technologies (china) limited); carbon dioxide cell incubator (Hereaus, germany); biosafety cabinet (Thermo Fisher Scientific, USA).

2. Experimental method and experimental result

Experimental grouping: (1) blank (NC); (2) a model group (LPS); (3) pharmaceutical compositions of the present application ECS-1 group: the pharmaceutical composition of example 7; (4) pharmaceutical compositions of the present application ECS-2 group: the pharmaceutical composition of example 8; (5) positive Drug (DXM) group: dexamethasone acetate.

Preparation of sample solution: (1) culture solution: DMEM high-sugar culture solution containing 5vol% of Fetal Bovine Serum (FBS) and antibiotics (100U/mL penicillin, 100. Mu.g/mL streptomycin).

Culture broth containing the pharmaceutical composition of example 7: the pharmaceutical composition of example 7 was added to 1000mL of the above culture solution, wherein 0.1327mg/mL of baicalin, 2.150mg/mL of baicalin, 0.1182mg/mL of wogonin, 0.0194mg/mL of oroxylin, 0.03661mg/mL of rutin, 0.02574mg/mL of berberine hydrochloride, 0.088mg/mL of adenosine, 0.001540mg/mL of indirubin, 0.09117mg/mL of limonin, 0.005180mg/mL of phellodendron, 5.640mg/mL of gardenoside, 0.06917mg/mL of chlorogenic acid, and 1.062mg/mL of stachyose.

Culture broth containing the pharmaceutical composition of example 8: the pharmaceutical composition of example 8 was added to 1000mL of the above culture medium, wherein 0.1327mg/mL of baicalin, 2.150mg/mL of baicalin, 0.1182mg/mL of wogonin, 0.0194mg/mL of oroxylin A, 0.03661mg/mL of rutin, 0.02574mg/mL of berberine hydrochloride, 0.088mg/mL of adenosine, 0.09117mg/mL of limonin and 5.640mg/mL of gardenoside.

Culture broth of DXM-containing pharmaceutical composition: DXM was added to 1000mL of the above culture broth, wherein the final concentration of DXM was 4.4. Mu.g/mL.

(2) Phosphate buffered saline (1×pbs): taking the premixed powder, dissolving and uniformly mixing the premixed powder with deionized water to 1L, and sterilizing.

Cell culture and passage: RAW264.7 cells were cultured in DMEM high-sugar culture medium containing 10vol% of Fetal Bovine Serum (FBS) and antibiotics (100U/mL penicillin, 100. Mu.g/mL streptomycin), and the incubator culture conditions were setCO at a volume fraction of 5% ₂ The growth of cells was observed daily at 37 ℃, and the cells were changed and passaged. When RAW264.7 cells grow to 80-90% fusion degree, discarding old cell culture solution, sucking new cell culture solution by a suction pipe, adding the new cell culture solution, repeatedly gently blowing the attached cells to fall off and suspend, transferring the attached cells into a 15mL centrifuge tube, centrifuging (1000 rpm,3 min), discarding supernatant, adding the new culture solution, and repeatedly gently blowing to suspend. The cells were then conditioned as per 1: 2-1: 3 inoculating in a new culture dish, and placing in CO with volume fraction of 5% ₂ Culturing in an incubator at 37 ℃. Various cell experiments were performed when the cell growth was in logarithmic growth phase, with LPS (100 ng/mL) used as a model group of inflammation.

And (3) data processing: statistical analysis was performed on the data using GraphPad Prism 9.0, and experimental results were expressed as mean ± standard error (mean ± SEM). Comparison between two sets of data used the t-test (Student's t-test) and comparison between multiple sets of data used the One-way ANOVA. The difference of p < 0.05 has statistical significance ^* p＜0.05， ^** p＜0.01； ^# p＜0.05， ^## p is less than 0.01; ns = no significance).

1. Changes in RAW264.7 cell morphology following LPS stimulation

A blank (NC), model (LPS), pharmaceutical composition of the application ECS-1, pharmaceutical composition of the application ECS-2 and dexanabinol acetate (DXM) were set up, 2 duplicate wells per group.

RAW264.7 cells were seeded in 6-well plates (1X 10) ⁶ Cells/well), 2mL per well, 5% co by volume at 37 °c ₂ After 24h incubation, the cell supernatant was discarded, and 1800. Mu.L of fresh culture medium was added to the NC group and LPS group, and the same volumes of culture medium containing the corresponding drugs were added to each of the ECS-1 group, ECS-2 group and DXM group. After 2h, 200. Mu.L LPS (final LPS concentration 100 ng/mL) was added to the NC group, and 200. Mu.L LPS was added to each of the LPS group, ECS-1 group, ECS-2 group and DXM group. After 16h of co-culture, the cells were observed for morphological changes on an inverted fluorescence microscope and photographed.

The experimental results are shown in FIG. 1, which is a graph showing changes (x 200-fold) in the morphology of RAW264.7 cells after Lipopolysaccharide (LPS) stimulation and administration of the pharmaceutical compositions of examples 7-8, as can be seen from FIG. 1: compared with NC group, RAW264.7 cells in LPS group are stimulated by LPS induction, cell morphology is changed, and cells stretch out of antenna to generate polarization state; the cell morphology of the dosed groups (ECS-1, ECS-2 and DXM) all showed different degrees of improvement compared to the LPS group; the above results indicate that different administration groups can effectively improve the cell morphology of the LPS-induced inflammation model.

2. NO content change in RAW264.7 cells after LPS stimulation tested by nitric oxide detection kit

Blank (NC), model (LPS), pharmaceutical composition ECS-1, pharmaceutical composition ECS-2 and dexamethasone acetate positive Drug (DXM) were set up, 8 wells per group.

RAW264.7 cells were seeded in 48-well plates (3X 10) ⁵ Cells/well), 200 μl per well, 5% co by volume at 37 °c ₂ After 24h incubation, the cell supernatant was discarded, 180. Mu.L of fresh medium was added to the NC group and LPS group, and the same volumes of medium containing the corresponding drugs were added to each of ECS-1 group, ECS-2 group and DXM group. After 2h, 20. Mu.L LPS (final LPS concentration 100 ng/mL) was added to the NC group, and 20. Mu.L LPS was added to each of the LPS group, ECS-1 group, ECS-2 group and DXM group. After 16h co-cultivation, the cell supernatants were collected, centrifuged (1000 Xg, 20 min), the supernatants were taken and then tested for NO content according to the nitric oxide detection kit instructions.

The results of the experiment are shown in FIG. 2, and FIG. 2 is a graph showing the change in NO content of RAW264.7 cells after administration of Lipopolysaccharide (LPS) stimulation and the pharmaceutical compositions of examples 7-8, as can be seen from FIG. 2: compared with NC group, the release amount of NO in LPS group is obviously increased, which indicates that the inflammation model is successfully constructed; the expression level of NO in ECS-1 group, ECS-2 group and DXM group was reduced to a different extent than in LPS group; the above results indicate that different administration groups can effectively inhibit the release of NO from an inflammation model induced by LPS, thereby producing good anti-inflammatory effects.

3. ELISA (enzyme Linked immunosorbent assay) for detecting change of inflammatory factor content in RAW264.7 cells after LPS stimulation

Blank (NC), model (LPS), pharmaceutical composition ECS-1, pharmaceutical composition ECS-2 and dexamethasone acetate positive Drug (DXM) were set up, 8 duplicate wells per group.

RAW264.7 cells were seeded in 48-well plates (3X 10) ⁵ Cells/well), 200 μl per well, 5% co by volume at 37 °c ₂ After 24h incubation, the cell supernatant was discarded, 180. Mu.L of fresh medium was added to the NC group and LPS group, and the same volumes of medium containing the corresponding drugs were added to each of ECS-1 group, ECS-2 group and DXM group. After 2h, 20. Mu.L of PBS was added to the NC group, and 20. Mu.L of LPS (final LPS concentration of 100 ng/mL) was added to each of the LPS group, ECS-1 group, ECS-2 group and DXM group. After 16h of co-culture, the cell supernatants were collected, centrifuged (1000 Xg, 20 min) and the supernatants were taken and tested for the content of each inflammatory factor according to the instructions of the mouse IL-6, TNF- α, IL-1β detection kit.

The expression level of inflammatory factors IL-6, IL-1 beta and TNF-alpha are important indexes of acute pharyngitis. The experimental results are shown in fig. 3-5, and fig. 3 is a graph showing the change of the content of inflammatory factor IL-6 in RAW264.7 cells after the administration of the pharmaceutical compositions of examples 7-8 in accordance with the detection of Lipopolysaccharide (LPS) stimulation by ELISA; FIG. 4 is a graph showing the variation of the IL-1β content of RAW264.7 cells after ELISA for Lipopolysaccharide (LPS) stimulation and administration of the pharmaceutical compositions of examples 7-8; FIG. 5 is a graph showing the change in the amount of TNF- α in RAW264.7 cells after administration of the pharmaceutical compositions of examples 7-8, as can be seen in FIGS. 3-5, in ELISA for Lipopolysaccharide (LPS) stimulation: compared with NC group, the content of the inflammatory factors IL-6, IL-1 beta and TNF-alpha in LPS group is extremely higher than that in blank group (p is less than 0.01), which shows that the inflammatory model is successfully constructed; compared with LPS group, the levels of cell inflammatory factors IL-6, IL-1 beta and TNF-alpha in ECS-1 group, ECS-2 group and DXM group are obviously reduced (p is less than 0.01), and the result shows that the pharmaceutical composition provided by the application can exert good anti-inflammatory effect.

4. Western blot method for detecting expression of related proteins in TLR4/NF- κB signal path

A blank (NC), model (LPS), pharmaceutical composition of the application ECS-1, pharmaceutical composition of the application ECS-2 and dexanabinol acetate (DXM) were set up, 3 duplicate wells per group.

RAW264.7 cells were seeded in 6-well plates (1×)10 ⁶ Cells/well), 2mL per well, 5% co by volume at 37 °c ₂ After 24h incubation, the cell supernatant was discarded, and 1800. Mu.L of fresh culture solution was added to the NC group and LPS group, and the same volumes of culture solution containing the corresponding drugs were added to each of the ECS-1 group, ECS-2 group and DXM group. After 2h, 200. Mu.L of PBS was added to the NC group, and 200. Mu.L of LPS (final LPS concentration of 100 ng/mL) was added to each of the LPS group, ECS-1 group, ECS-2 group and DXM group. After 16h of co-culture, the cell culture supernatant was discarded and the cells were collected. Adding 120 μl of cell lysate (Western and IP cell lysate: 100×protease inhibitor: 100×phosphatase inhibitor at volume ratio of 100:1:1), performing lysis on ice, centrifuging the lysate in a centrifuge precooled at 4deg.C at 12000r/min for 10min, and collecting supernatant. Protein concentration is measured by the BCA protein quantitative kit, and a quantitative protein sample is added with 3 Xprotein loading buffer solution in a certain proportion to dilute the buffer solution into 1X, and the buffer solution is denatured at a high temperature of 100 ℃ for 15min and preserved at-80 ℃. And detecting the expression of beta-actin, TLR4, NF-kappa B p65 and p-NF-kappa B p proteins in the cells by adopting a Western blot method. According to the quantitative protein concentration of a sample, 10 mug of the protein per hole is sampled, after 80V electrophoresis is carried out for 30min, 120V electrophoresis is carried out for 1h,350mA membrane transfer is carried out for 1h,5% (w/V) skimmed milk powder is sealed at 4 ℃, primary antibody is diluted in proportion and incubated for overnight at 4 ℃, TBST buffer solution is used for washing the membrane for 3X 10min the next day, secondary antibody is diluted in proportion and incubated at 4 ℃, ECL hypersensitive luminescent solution is added after TBST buffer solution is used for washing the membrane, and beta-actin, TLR4, NF-kappa B p65 and p-NF-kappa B p protein signals are acquired by exposure in a darkroom.

The experimental results are shown in FIG. 6 and FIG. 7, wherein FIG. 6 is a graph of the phosphorylation levels of beta-actin, TLR4, NF-kappa B p65 and p-NF-kappa B p protein in RAW264.7 cells after Lipopolysaccharide (LPS) stimulation by Western blot, FIG. 7 is a graph of the quantitative analysis of beta-actin, TLR4, NF-kappa B p65 and p-NF-kappa B p protein in RAW264.7 cells after Lipopolysaccharide (LPS) stimulation by Western blot, and in FIG. 7, p-NF-kappa B p 65/NF-kappa B p refers to the ratio of p-NF-kappa B p65 to NF-kappa B p protein, beta-actin is an internal reference, and the ratio is used for judging the protein amount. As can be seen from fig. 6 and 7: lipopolysaccharide (LPS) can stimulate TLR4 and p-NF-kappa B p protein in RAW264.7 cells to be obviously up-regulated (p is less than 0.01), and ECS-1, ECS-2 and DXM can inhibit TLR4 and p-NF-kappa B p protein expression (p is less than 0.05) in RAW264.7 cells after LPS induction. The above results indicate that the pharmaceutical compositions provided herein exert substantially the same anti-inflammatory effect. Without being limited to any theory, the inventors believe that: the anti-inflammatory effects exerted by the various component groups in the pharmaceutical composition provided by the application may be related to inhibiting the TLR4/NF- κB signaling pathway in RAW264.7 cells.

Anti-inflammatory cell experiment two

1. Experimental materials

The same experimental materials as "anti-inflammatory cell experiment one".

2. Experimental method and experimental result

Experimental grouping: (1) blank (NC); (2) a model group (LPS); (3) pharmaceutical compositions 9+4-L of the present application: the pharmaceutical composition of example 1; (4) pharmaceutical compositions 9+4-M group of the present application: the pharmaceutical composition of example 2; (5) pharmaceutical compositions 9+4-H of the present application: the pharmaceutical composition of example 3; (6) pharmaceutical composition 9-L of the present application: the pharmaceutical composition of example 4; (7) pharmaceutical compositions 9-M of the present application: the pharmaceutical composition of example 5; (8) pharmaceutical composition 9-H of the present application: the pharmaceutical composition of example 6; (9) positive Drug (DXM) group: dexamethasone acetate.

Culture broth containing the pharmaceutical composition of example 1: the pharmaceutical composition of example 1 was added to 1000mL of the above culture medium, wherein 0.0413mg/mL of baicalin, 1.3586mg/mL of baicalin, 0.046mg/mL of wogonin, 0.0093mg/mL of oroxylin A, 0.0238mg/mL of rutin, 0.0149mg/mL of berberine hydrochloride, 0.0615mg/mL of adenosine, 0.0004mg/mL of indirubin, 0.0386mg/mL of limonin, 0.0018mg/mL of phellodendron, 3.8547mg/mL of gardenoside, 0.0326mg/mL of chlorogenic acid, and saccharides including stachyose 0.4394mg/mL.

Culture broth containing the pharmaceutical composition of example 2: the pharmaceutical composition of example 2 was added to 1000mL of the above culture solution, wherein 0.1765mg/mL of baicalin, 2.3211mg/mL of baicalin, 0.0899mg/mL of wogonin, 0.0196mg/mL of oroxylin A, 0.0366mg/mL of rutin, 0.0257mg/mL of berberine hydrochloride, 0.0880mg/mL of adenosine, 0.0015mg/mL of indirubin, 0.0912mg/mL of limonin, 0.0052mg/mL of phellodendron, 5.6389mg/mL of gardenoside, 0.0692mg/mL of chlorogenic acid, and 1.0617mg/mL of stachyose.

Culture broth containing the pharmaceutical composition of example 3: the pharmaceutical composition of example 3 was added to 1000mL of the above culture solution, wherein 0.2568mg/mL of baicalin, 3.2261mg/mL of baicalin, 0.1419mg/mL of wogonin, 0.0280mg/mL of oroxylin A, 0.0653mg/mL of rutin, 0.1447mg/mL of berberine hydrochloride, 0.1159mg/mL of adenosine, 0.0030mg/mL of indirubin, 0.1263mg/mL of limonin, 0.0103mg/mL of phellodendron, 8.9997mg/mL of gardenoside, 0.1432mg/mL of chlorogenic acid, and 2.0403mg/mL of stachyose.

Culture broth containing the pharmaceutical composition of example 4: the pharmaceutical composition of example 4 was added to 1000mL of the above culture medium, wherein 0.0413mg/mL of baicalin, 1.3586mg/mL of baicalin, 0.046mg/mL of wogonin, 0.0093mg/mL of oroxylin A, 0.0238mg/mL of rutin, 0.0149mg/mL of berberine hydrochloride, 0.0615mg/mL of adenosine, 0.0386mg/mL of limonin and 3.8547mg/mL of gardenoside.

Culture broth containing the pharmaceutical composition of example 5: the pharmaceutical composition of example 5 was added to 1000mL of the above culture medium, wherein 0.1765mg/mL of baicalin, 2.3211mg/mL of baicalin, 0.0899mg/mL of wogonin, 0.0196mg/mL of oroxylin A, 0.0366mg/mL of rutin, 0.0257mg/mL of berberine hydrochloride, 0.0880mg/mL of adenosine, 0.0912mg/mL of limonin and 5.6389mg/mL of gardenoside.

Culture broth containing the pharmaceutical composition of example 6: the pharmaceutical composition of example 6 was added to 1000mL of the above culture solution, wherein 0.2568mg/mL of baicalin, 3.2261mg/mL of baicalin, 0.1419mg/mL of wogonin, 0.0280 mg/mL of oroxylin A, 0.0653mg/mL of rutin, 0.1447mg/mL of berberine hydrochloride, 0.1159mg/mL of adenosine, 0.1263mg/mL of limonin, and 8.9997mg/mL of gardenoside.

Cell culture and passage: the same as the cell culture and passage of "anti-inflammatory cell experiment one".

And (3) data processing: the same data processing as for "anti-inflammatory cell experiment one".

1. Changes in RAW264.7 cell morphology following LPS stimulation

Blank (NC), model (LPS), different drug combination groups (9+4-H, 9+4-M, 9+4-L, 9-H, 9-M, 9-L) and dexamethasone acetate positive drug group (DXM) were set, 2 multiple wells per group.

RAW264.7 cells were seeded in 12-well plates (5X 10) ⁵ Cells/well), 2mL per well, 5% co by volume at 37 °c ₂ After 12H incubation, the cell supernatant was discarded, 1800. Mu.L of fresh culture solution was added to the NC group and LPS group, and the same volumes of culture solution containing the corresponding drugs were added to the different drug composition groups (9+4-H, 9+4-M, 9+4-L, 9-H, 9-M, 9-L) and DXM group, respectively. After 2H, 200. Mu.L of PBS was added to the NC group, and 200. Mu.L of LPS (final concentration 100 ng/mL) was added to the NC group, the LPS group, the different pharmaceutical composition groups (9+4-H, 9+4-M, 9+4-L, 9-H, 9-M, 9-L) and DXM group, respectively. After 16h of co-culture, the cells were observed for morphological changes on an inverted fluorescence microscope and photographed.

The experimental results are shown in fig. 8, which is a graph showing changes (x 200-fold) in the morphology of RAW264.7 cells after Lipopolysaccharide (LPS) stimulation and administration of the pharmaceutical compositions of examples 1 to 6, as can be seen from fig. 8: compared with NC group, RAW264.7 cells in LPS group are stimulated by LPS induction, cell morphology is changed, and cells stretch out of antenna to generate polarization state; the cell morphology of the dosed group was improved to a different extent compared to the LPS group. The pharmaceutical composition provided by the application can effectively improve the cell morphology of an LPS-induced inflammation model.

Blank (NC), model (LPS), different drug composition (9+4-H, 9+4-M, 9+4-L, 9-H, 9-M, 9-L) and Dexamethasone (DXM) groups were set, 4 duplicate wells each.

RAW264.7 cells were seeded in 48-well plates (3X 10) ⁵ Individual cells/wells),200 mu L per well, volume fraction 5% CO at 37 DEG C ₂ After 12H incubation, the cell supernatant was discarded, 180. Mu.L of fresh culture solution was added to the NC group and LPS group, and the same volumes of culture solution containing the corresponding drugs were added to the different drug composition groups (9+4-H, 9+4-M, 9+4-L, 9-H, 9-M, 9-L) and DXM group, respectively. After 2H, 20. Mu.L of PBS was added to the NC group, and 20. Mu.L of LPS (final concentration 100 ng/mL) was added to the NC group, the LPS group, the different pharmaceutical composition groups (9+4-H, 9+4-M, 9+4-L, 9-H, 9-M, 9-L) and DXM group, respectively. After 16h co-cultivation, the cell supernatants were collected, centrifuged (1000 Xg, 20 min), the supernatants were taken and then tested for NO content according to the nitric oxide detection kit instructions.

The results of the experiment are shown in FIG. 9, and FIG. 9 is a graph showing the change in NO content of RAW264.7 cells after Lipopolysaccharide (LPS) stimulation and administration of the pharmaceutical compositions of examples 1-6, as can be seen from FIG. 9: compared with NC group, the release amount of NO in LPS group is obviously increased, which indicates that the inflammation model is successfully constructed; the expression level of NO in the administration group was reduced to a different extent compared to the LPS group, indicating that the release of NO in the inflammatory model induced by LPS can be effectively inhibited by the different administration groups, thereby producing a good anti-inflammatory effect.

RAW264.7 cells were seeded in 48-well plates (3X 10) ⁵ Cells/well), 200 μl per well, 5% co by volume at 37 °c ₂ After 12H incubation, the cell supernatant was discarded, 180. Mu.L of fresh culture solution was added to the NC group and LPS group, and the same volumes of culture solution containing the corresponding drugs were added to the different drug composition groups (9+4-H, 9+4-M, 9+4-L, 9-H, 9-M, 9-L) and DXM group, respectively. After 2H, 200. Mu.L of PBS was added to the NC group, and 20. Mu.L of LPS (final LPS concentration of 100 ng/mL) was added to each of the LPS group, the different drug composition groups (9+4-H, 9+4-M, 9+4-L, 9-H, 9-M, 9-L) and DXM group. After 16h of co-culture, the cell supernatants were collected, centrifuged (1000 Xg, 20 min) and the supernatants were taken and then assayed according to the instructions of the mouse IL-6, TNF-alpha, IL-1 beta assay kitThe content of inflammatory factors was tested.

The experimental results are shown in fig. 10-12, and fig. 10 is a graph showing the change of the content of inflammatory factor IL-6 in RAW264.7 cells after the administration of the pharmaceutical compositions of examples 1-6 in accordance with the detection of Lipopolysaccharide (LPS) stimulation by ELISA; FIG. 11 is a graph showing the variation of the IL-1β content of RAW264.7 cells after ELISA for Lipopolysaccharide (LPS) stimulation and administration of the pharmaceutical compositions of examples 1-6;

FIG. 12 is a graph showing the change in the amount of TNF-. Alpha.in RAW264.7 cells after administration of the pharmaceutical compositions of examples 1-6, as seen in FIGS. 10-12, which shows the stimulation by ELISA to detect Lipopolysaccharide (LPS). Compared with NC group, the content of the inflammatory factors IL-6, IL-1 beta and TNF-alpha in LPS group is extremely higher than that in blank group (p is less than 0.01), which shows that the inflammatory model is successfully constructed; compared with LPS group, the levels of the cell inflammatory factors IL-6, IL-1 beta and TNF-alpha in different drug combination groups (9+4-H, 9+4-M, 9+4-L, 9-H, 9-M and 9-L) and DXM group are obviously reduced (p < 0.01). The results show that the pharmaceutical composition provided by the application can exert good anti-inflammatory effect.

Animal experiment for pharyngitis

1. Experimental materials

1. Experimental animal

Sprague-Dawley (SD) male rats (200+ -20 g) purchased from Thejiang Venetuno laboratory animal technologies Co., ltd., animal license number: SCXK (Zhe) 2019-0001. Adaptive feeding was given for one week prior to the formal experiment, during which time diet and water were free. The raising environment temperature is 24+/-2 ℃, and the daily illumination is 12 hours. All animal experiments were conducted under the guidance of "Experimental animal management regulations" in Jiangsu province. The experimental ethics is 2022-09-007.

2. Experimental reagent

Dexamethasone acetate was purchased from Zhejiang Xianju pharmaceutical Co., ltd.

Isoflurane was purchased from ravode life technologies limited in Shenzhen.

Tween 20 and ammonia were purchased from Nanjing Chemicals Inc.

4% paraformaldehyde universal tissue fixative was purchased from biosharp corporation.

Rat tumor necrosis factor-alpha (TNF-alpha) ELISA kit (ml 002859), rat interleukin-6 (IL-6) ELISA kit (ml 102828), rat interleukin-1 beta (IL-1 beta) ELISA kit (ml 003057) were purchased from Shanghai enzyme-linked biotechnology Co.

BCA protein quantification kit, western and IP cell lysate and RIPA lysate were purchased from Shanghai Biyun biotechnology Co.

Skimmed milk powder was purchased from inner mongolian illi real group limited.

Nitrocellulose membranes NC were purchased from Millipore corporation.

Protein markers were purchased from Nanjinopran biotechnology Co.

Sodium carboxymethyl cellulose, SDS, tris, 1.5M Tris-HCl (pH 8.8), 0.5M Tris-HCl (pH 6.8), ready-to-use TBS powder, glycine, ammonium persulfate, tetramethyl ethylenediamine (TEMED) and 30% acrylamide solution were purchased from Shanghai Ala Latin Biochemical technologies Co., ltd.

3. Experimental instrument

Ear-nose-throat atomizing spray gun (Xinnuoer medical instruments Co., ltd., buddha); small animal endoscope (Shanghai Jade research science instruments Co., ltd.); small animal anesthesia machine (Shenzhen Ruiwod life technologies Co., ltd.); a multi-functional microplate reader (BERTHOLD TECHNOLOGIES); inverted fluorescence microscope (Nanjing Gauss instruments Co., ltd.); BT25S one ten thousandth balance (Sartorius, gottingen, germany); MJXFSPRP-24 tissue grinder (Shanghai Jijing Xingzhi Co., ltd.); centrifuge (zemoeimer technologies (china) limited); 5810R high-speed cryocentrifuge (eppendorf); full-automatic chemiluminescence/fluorescence image analysis system (Shanghai Techno Co., ltd.).

2. Experimental method and experimental result

Experimental grouping: SD rats were divided into blank (NC), model (AP), pharmaceutical composition of the present application example 7 (ECS-1), example 8 (ECS-2), example 34 (ECS-3) and dexamethasone acetate positive (DXM).

Molding and drug administration: the method for stimulating the pharynx of the rat by using ammonia water is adopted to prepare an acute pharyngitis model of the rat, ammonia water with the mass fraction of 20% is sprayed to the pharynx of the rat by using an ear-nose-throat atomization spray gun, each spray is carried out 1 time in the morning and afternoon (timing 10:00 and 16:00) each time, and the spray is continuously carried out for 3 days under each spray for 2-3 days, so that the mucosa of the pharynx of the rat is acutely red and swollen due to the stimulation of the ammonia water to form the acute pharyngitis model, and physiological saline is sprayed in a blank group in the same way. After the molding was completed, the rats were randomly grouped into 8 rats each. Starting on day 4, ECS-1, ECS-3 and ECS-2 groups were given by gavage at clinical dose (5.40 mL/kg), respectively, DXM group was given by gavage at clinical dose (1.5 mg/kg), 1 time per day for 5 days, and NC group and AP group were replaced with physiological saline.

Drawing materials: after 2 hours of last administration, the pharyngeal condition of the rats was observed and photographed by a small animal endoscope, and after 12 hours of overnight fast, the rats were anesthetized with isoflurane, and abdominal aortic whole blood and pharyngeal tissues were collected. Centrifuging (3500 rpm,10 min) the blood sample, collecting supernatant, fixing part of pharyngeal tissue with 4% paraformaldehyde fixing solution, immediately placing the rest tissue into liquid nitrogen, and preserving at-80deg.C.

1. Acute pharyngitis rat pharyngeal appearance evaluation

The photographed pharyngeal condition of the rats is shown in fig. 13, and fig. 13 is a graph showing the effect of the drug group on the pharyngeal mucosa of the rats with acute pharyngitis, as can be seen from fig. 13: NC group mucous membrane is smooth, light red in color and blood vessel light red; compared with NC group, AP group has serious blood vessel congestion, rough mucous membrane and increased saliva; compared with the AP group, the DXM group has reduced mucus, improved mucous membrane state and light red blood vessel recovery; compared with the AP group, the mucous membranes of the ECS-3 group and the ECS-1 group are smooth, and the mucous membranes are obviously improved; the ECS-2 group showed improved mucosal status, slightly less gloss and slightly more mucus retention than the AP group.

The pharyngeal appearance scoring criteria were as follows: 0 minutes, the pharyngeal tissue is light red, the surface is moist and glossy, secretion and congestion are avoided, and other pathological phenomena are avoided; 1 minute, poor gloss of pharyngeal tissue and small amount of secretion; 2, darkening tissue color, poor color and luster, and pathological phenomena such as secretion, acute congestion and the like; 3, the pharyngeal tissues are dark red, secretion is more, acute congestion is serious, and pathological phenomena such as mucosal ulcer and the like appear.

The pharyngeal appearance was scored, and the results are shown in fig. 14, and fig. 14 is a statistical graph of pharyngeal appearance scores of rats. As can be seen from fig. 14: according to the pharyngeal appearance scoring criteria, the AP group score is significantly higher compared to the NC group; the scores of the different dosing groups were significantly lower than those of the AP group, indicating that the pharmaceutical compositions in the dosing groups all had an effect of improving the pharyngeal condition of rats.

2. ELISA test for expression of serum inflammatory factor of acute pharyngitis rat

The collected serum was subjected to detection of inflammatory factors strictly according to the protocol of each kit.

The experimental results are shown in fig. 15-17, and fig. 15 is a graph showing the influence of a drug group on acute pharyngitis rat serum inflammatory factor IL-6; FIG. 16 is a graph showing the effect of the drug group on the serum inflammatory factor IL-1β of acute pharyngitis rats; FIG. 17 is a graph showing the effect of the drug group on the inflammatory factor TNF- α in the serum of rats with acute pharyngitis, as can be seen from FIGS. 15 to 17: compared with NC group, the expression of IL-6, TNF-alpha and IL-1 beta inflammatory factors of AP group is extremely obviously increased (p is less than 0.01); ECS-3, ECS-1, ECS-2 and DXM IL-6 and TNF-alpha inflammatory factor expression were significantly down-regulated (p < 0.01); compared with the AP group, the expression of the IL-1 beta inflammatory factors of the ECS-3 group and the DXM group is extremely obviously down-regulated (p is less than 0.01), and the expression of the IL-1 beta inflammatory factors of the ECS-1 group and the ECS-2 group is obviously down-regulated (p is less than 0.05); there was no significant difference (p > 0.05) between ECS-1, ECS-2 and ECS-3 groups. Without being limited to any theory, the inventors believe that: the pharmaceutical composition provided by the application can play a good role in resisting acute pharyngitis by regulating the expression level of inflammatory factors such as IL-6, TNF-alpha, IL-1 beta and the like.

3. Acute pharyngitis rat pharyngeal histopathology

(1) Manufacturing paraffin sections of pharyngeal tissues: and (3) taking out the tissue with the concentration of 4% paraformaldehyde fixed for more than 48 hours, leveling the target part and putting the target part into a dehydration box. The tissue-filled dehydration box was placed in a dehydrator, sequentially immersed in 75vol% ethanol for 4h,85vol% ethanol for 2h,90vol% ethanol for 2h,95vol% ethanol for 1h, absolute ethanol for 2 times, 30min each time, alcohol benzene for 10min, xylene for 2 times, 10min each time, and wax for 3h. Embedding the waxed tissue, cooling and solidifying the wax block at-20deg.C, trimming the wax block, cooling at-20deg.C, cutting into 4 μm slices, spreading the tissue in 40 deg.C warm water, taking out the tissue with glass slide, baking at 60deg.C, and storing at normal temperature.

(2) H & E staining: the paraffin sections were sequentially put into xylene for 2 times, 20min each time, 2 times in absolute ethanol, 5min each time, 75vol% ethanol for 5min, and were washed with tap water. Then placing the slices into hematoxylin dye solution for dyeing, washing with tap water after 5min, adding differentiation solution for differentiation, washing with tap water, adding blue returning solution for returning blue, and washing with running water. Then, the slices are sequentially soaked in 85vol% ethanol and 95vol% ethanol for 5min for gradient dehydration, and then are put into eosin dye solution for dyeing for 5min. Sequentially soaking in absolute ethanol for 3 times (5 min each time) and in xylene for 2 times (5 min each time) until the slice is transparent, sealing with neutral resin, and preserving at normal temperature.

The experimental results are shown in fig. 18, fig. 18 is a pathological section diagram of pharyngeal tissues of rats with acute pharyngitis, and fig. 18 shows that: NC group rat pharyngeal tissue mucous membrane layer and submucosal gland are all normal; compared with NC group, the mucosa layer of pharyngeal tissue of the rat in AP group is thickened, a great amount of inflammatory cells in submucosa are gathered, glands are enlarged, and fibers of the muscle layer are destroyed; the pharyngeal tissue mucous membrane layer of the DXM group rat is thinned, a small amount of inflammatory cells infiltrate into the submucosa, the gland is basically normal, and the myometrium fiber is improved; the pharyngeal tissue mucous membrane layer of the ECS-3 group rats is basically normal, inflammatory cells of submucosa are obviously reduced, and glands are reduced; the pharyngeal tissue mucous membrane layer of the ECS-1 group rats is improved, the glands of submucosa are contracted, inflammatory cells are reduced, and the arrangement of fibers of the myometrium is basically recovered; the pharyngeal tissue mucous membrane epithelium of ECS-2 group rats is basically flat, inflammatory cells of submucosa are reduced, and the arrangement of muscle layer fibers is basically restored. The pharmaceutical compositions in the administration groups can improve the pathological state of the pharyngeal tissues of rats with acute pharyngitis to different degrees.

4. Western blot analysis of expression of related proteins in pharyngeal tissues of rats with acute pharyngitis

The pharyngeal tissue is weighed 20mg, placed in a 1.5mL centrifuge tube, added with magnetic beads, added with 200 mu L of lysate (RIPA lysate: 100 Xproteinase inhibitor: 100 Xphosphatase inhibitor volume ratio: 100:1:1), ground in a tissue grinder (frequency 55Hz, grinding 55s, interrupt 10s, grinding 5 times), placed at 4 ℃ for 30min, and the lysate is centrifuged at 12000r/min for 10min in a centrifuge precooled at 4 ℃ to obtain the supernatant. Protein concentration was measured using BCA protein quantification kit, and the quantified protein samples were diluted 1× with a proportion of 3× protein loading buffer, denatured at 100 ℃ for 15min at-80 ℃ and stored. Detecting the expression of beta-actin, TLR4, NF-kappa B p65 and p-NF-kappa B p proteins in pharyngeal tissues by adopting a Western blot method, loading 15 mug of sample per hole protein according to the quantitative protein concentration of a sample, transferring to 120V electrophoresis for 1h after 80V electrophoresis for 30min, transferring to a film for 350mA, sealing at 5% (w/V) of skimmed milk powder at 4 ℃, incubating overnight at 4 ℃ after the primary antibody is diluted in proportion, washing the film for 3X 10min on the next day, incubating at 4 ℃ after the secondary antibody is diluted in proportion, adding ECL hypersensitive luminescent liquid after the film is washed by TBST buffer, and acquiring the beta-actin, TLR4, NF-kappa B p65 and p-NF-kappa B p protein signals by exposure in a darkroom.

The experimental results are shown in FIG. 19, FIG. 19 is a graph of the protein expression levels of beta-actin, TLR4, NF-kappa B p65 and p-NF-kappa B p in pharyngeal tissues of rats suffering from acute pharyngitis by Western blot analysis, and FIG. 20 is a graph of the protein expression quantification of beta-actin, TLR4, NF-kappa B p65 and p-NF-kappa B p in pharyngeal tissues of rats suffering from acute pharyngitis by Western blot analysis, wherein p-NF-kappa B p/NF-kappa B p shown in FIG. 20 is the ratio of p-NF-kappa B p to NF-kappa B p, beta-actin is an internal reference, and the ratio is used for judging the protein quantity. As can be seen from fig. 19 and 20: compared with the NC group, the expression of TLR4 and p-NF-kappa B p65 protein in the AP group is obviously up-regulated (p is less than 0.05); compared with the AP group, the expression of TLR4 and p-NF-kappa B p65 proteins in the ECS-3 group, the ECS-1 group, the ECS-2 group and the DXM group is inhibited (p is less than 0.05), and the inhibition effect of the ECS-3 group, the ECS-1 group and the ECS-2 group is basically the same. Without being limited to any theory, the inventors believe that: the effect of each component group in the pharmaceutical composition provided by the application on resisting acute pharyngitis is possibly related to inhibiting TLR4/NF- κB signaling in RAW264.7 cells.

The test results show that the pharmaceutical composition has the effect of improving acute pharyngitis and can inhibit the expression of inflammatory factors TNF-alpha, IL-6 and IL-1 beta in the serum of rats with acute pharyngitis; in addition, the pharmaceutical composition of the present application can inhibit the expression of TLR4, p-NF- κ B p65 key proteins in pharyngeal tissues, and the present inventors believe, without being limited to any theory: the anti-acute pharyngitis effect of the pharmaceutical composition provided by the application can be realized by inhibiting the activation of TLR4/NF- κB signaling pathway.

The foregoing description of the preferred embodiments of the present invention is not intended to limit the invention to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, alternatives, and alternatives falling within the spirit and scope of the invention.

Claims

1. A pharmaceutical composition comprising: 12-50 parts of flavonoids, 0.7-7 parts of alkaloids, 0.5-3.5 parts of limonoids and 38-100 parts of iridoid glycosides;

preferably, the pharmaceutical composition comprises: 15-35 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-2.0 parts of limonoids and 38-90 parts of iridoid glycosides;

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises: 0.4-3 parts by weight of organic acids and/or 5-50 parts by weight of saccharides;

preferably, the pharmaceutical composition further comprises: 0.4-1.3 parts by weight of organic acids and/or 5-18 parts by weight of saccharides;

Preferably, the pharmaceutical composition comprises: 15-35 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-2.0 parts of limonoids, 38-90 parts of iridoid glycosides and 0.4-1.5 parts of organic acids;

preferably, the pharmaceutical composition comprises: 19.74-32.40 parts by weight of flavonoids, 1.35-4.49 parts by weight of alkaloids, 0.50-1.14 parts by weight of limonoids, 49.07-74.99 parts by weight of iridoid glycosides and 0.49-1.30 parts by weight of organic acids;

preferably, the pharmaceutical composition comprises: 15-35 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-2.0 parts of limonoids, 38-90 parts of iridoid glycosides and 5-25 parts of saccharides;

preferably, the pharmaceutical composition comprises: 20-33 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-1.5 parts of limonoids, 45-75 parts of iridoid glycosides and 5-18 parts of saccharides;

preferably, the pharmaceutical composition comprises: 15-35 parts of flavonoids, 0.7-5 parts of alkaloids, 0.5-2.0 parts of limonoids, 38-90 parts of iridoid glycosides, 0.4-1.5 parts of organic acids and 5-25 parts of saccharides;

preferably, the pharmaceutical composition comprises: 19.74-32.40 parts of flavonoids, 1.35-4.49 parts of alkaloids, 0.5-1.14 parts of limonoids, 49.07-74.99 parts of iridoid glycosides, 0.49-1.30 parts of organic acids and 5.49-17 parts of saccharides.

3. The pharmaceutical composition according to claim 1 or 2, wherein the flavonoids comprise at least one of baicalein, baicalin, wogonin, rutin and oroxylin a;

preferably, the flavonoids comprise (0.025-0.1) by weight: 1: (0.03-0.06): (0.01-0.03): (0.005-0.01) baicalein, baicalin, wogonin, rutin and oroxylin A;

preferably, the flavonoid further comprises at least one of chrysin-7-O-beta-D-glucuronide, isoquercitrin, scutellarin, baicalein II and calycosin;

The weight ratio of baicalin to calycosin is (0.08-1.4): (0.004-0.01), preferably 1: (0.004-0.01);

preferably, the flavonoids comprise (0.025-0.1) by weight: (0.08-1.4): (0.03-0.06): (0.01-0.03): (0.005-0.02): (0.01-0.03): (0.008-0.02): (0.004-0.01) baicalein, baicalin, wogonin, rutin, oroxylin A, chrysin-7-O-beta-D-glucuronide, baicalein II and calycosin;

4. The pharmaceutical composition according to claim 1 or 2, wherein the alkaloids comprise at least one of berberine hydrochloride and adenosine;

preferably, the weight ratio of berberine hydrochloride to adenosine is (0.5-1.5): (0.7-5.0), preferably 1: (0.7-5.0);

preferably, the alkaloid further comprises indirubin, and the weight ratio of berberine hydrochloride to indirubin is (0.5-1.5): (0.005-0.12);

preferably, the weight ratio of berberine hydrochloride, adenosine and indirubin is (0.5-1.5): (0.7-5.0): (0.005-0.12), preferably 1: (0.735-5.0): (0.005-0.12);

preferably, the alkaloids further comprise at least one of phellodendrine hydrochloride, magnolol, palmatine hydrochloride, jateorhizine hydrochloride, uridine and guanosine;

The weight ratio of the berberine hydrochloride to the guanosine is (0.5-1.5): (0.3-2.0), preferably 1: (0.33-2.0);

preferably, the alkaloids comprise (0.5-1.5) by weight: (0.7-5.0): (0.005-0.12): (0.5-5): (0.4-3.5): (0.02-0.2): (0.01-0.2): (0.3-2.5): (0.3-2.0) berberine hydrochloride, adenosine, indirubin, phellodendrine hydrochloride, magnaline, palmatine hydrochloride, jateorhizine hydrochloride, uridine and guanosine;

preferably, the alkaloids comprise 1 in weight ratio: (0.735-4.3): (0.005-0.12): (0.669-4.37): (0.446-3.13): (0.024-0.16): (0.019-0.135): (0.38-2.447): (0.33-1.898) berberine hydrochloride, adenosine, indirubin, phellodendrine hydrochloride, magnaline, palmatine hydrochloride, jateorhizine hydrochloride, uridine and guanosine.

5. The pharmaceutical composition according to claim 1 or 2, wherein the limonoids comprise at least one of limonin and phellodendron ketone;

preferably, the weight ratio of limonin to phellodendron ketone is (10-30): (0-1.1), preferably (12-28.5): 1, a step of;

preferably, the iridoid glycoside comprises geniposide.

6. The pharmaceutical composition of claim 2, wherein the organic acid comprises at least one of chlorogenic acid, ferulic acid, and benzoic acid;

preferably, the organic acid comprises (4-12) by weight: (0.8-1.2) chlorogenic acid and ferulic acid;

preferably, the organic acid comprises (4-12) by weight: chlorogenic acid and ferulic acid of 1;

preferably, the saccharide comprises an oligosaccharide; preferably, the oligosaccharide comprises stachyose.

7. The pharmaceutical composition of claim 1, comprising: 19.74-32.40 parts by weight of flavonoids, 1.35-4.49 parts by weight of alkaloids, 0.5-1.14 parts by weight of limonoids and 49.07-74.99 parts by weight of iridoid glycosides; wherein the flavonoids comprise (0.025-0.1) by weight: 1: (0.03-0.06): (0.01-0.03): (0.005-0.02) baicalein, baicalin, wogonin, rutin, and oroxylin A; the alkaloid comprises the following components in percentage by weight: (1.0-5.0) berberine hydrochloride and adenosine; the limonin comprises limonin; the iridoid glycoside comprises geniposide;

8. The pharmaceutical composition of any one of claims 1-7, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient;

9. Use of a pharmaceutical composition according to any one of claims 1-8 for the preparation of a medicament for the prevention and/or treatment of upper respiratory tract infections;

10. Use of the pharmaceutical composition of any one of claims 1-8 for controlling the quality of a chinese patent medicine; preferably, the pharmaceutical composition is used as a standard extract or a control extract.