CN110575479A

CN110575479A - Application of herba patriniae and traditional Chinese medicine composition for treating porcine reproductive and respiratory syndrome

Info

Publication number: CN110575479A
Application number: CN201910999174.6A
Authority: CN
Inventors: 朱善元; 秦枫; 刘云; 吴双; 郭长明; 吴植; 王安平; 王永娟; 封琦; 洪伟鸣
Original assignee: Jiangsu Agri Animal Husbandry Vocational College
Current assignee: Jiangsu Agri Animal Husbandry Vocational College
Priority date: 2019-10-21
Filing date: 2019-10-21
Publication date: 2019-12-17
Anticipated expiration: 2039-10-21
Also published as: CN110575479B

Abstract

the invention relates to the technical field of traditional Chinese medicines, and discloses application of herba patriniae and a traditional Chinese medicine composition for treating porcine reproductive and respiratory syndrome. The invention utilizes Western-Blot, qRT-PCR, apoptosis and other technologies to prove through experiments of various layers that the patrinia extract can obviously inhibit PRRSV infection and replication, has obvious in-vivo and in-vitro PRRSV virus resistance, can become a novel medicament for preventing and treating the porcine reproductive and respiratory syndrome, and has good application prospect in the prevention and treatment of the porcine reproductive and respiratory syndrome.

Description

Application of herba patriniae and traditional Chinese medicine composition for treating porcine reproductive and respiratory syndrome

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to application of herba patriniae and a traditional Chinese medicine composition for treating porcine reproductive and respiratory syndrome.

Background

Porcine Reproductive and Respiratory Syndrome (PRRSV), commonly known as Porcine Reproductive and Respiratory Syndrome virus, is an acute, contact, highly contagious disease caused by the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). The primary infected subjects of PRRSV are pregnant sows, which present a reproductive barrier, and piglets, which, in addition to severe respiratory symptoms and persistent infections, also cause growth retardation and neurological symptoms. The disease was first reported in 1987 in the united states, followed by successive reports in germany, the netherlands, canada, etc., and then rapidly spread to asia and other regions, with enormous losses to the swine industry worldwide.

the main currently effective means of managing and preventing this syndrome is vaccination. Currently, the PRRSV vaccines on the market mainly comprise inactivated vaccines and attenuated vaccines, but both vaccines have serious efficacy and safety problems. The first vaccine used for clinical treatment of PRRSV was the inactivated vaccine. The inactivated vaccine has safety, does not cause virus transmission and epidemic diseases, and has the advantages of convenient storage and transportation. However, the protective effect of inactivated vaccines is not ideal. After the inactivated vaccine is used, only humoral immunity can be generated, so that secondary immunity is needed to play a certain protection role, the time from immunization to immune protection of the inactivated vaccine is long, the generation amount of neutralizing antibodies in the period is possibly little, or the neutralizing antibodies are not generated, so that the condition of ineffective immunity occurs, and the inactivated vaccine cannot effectively control the attack and the spread of PRRSV. In contrast, the attenuated vaccine can be induced to generate neutralizing antibody rapidly, and has the advantages of long immunization time, strong resistance and the like. However, attenuated vaccines present the risk of virus spread and may be virulent back during the course of immunization.

The traditional Chinese medicines are various in types and rich in resources, and due to the characteristics of safety, low toxicity, difficulty in generating drug resistance and the like, antiviral research is increasingly emphasized, and more traditional Chinese medicines have an immunoregulation effect and can play an antiviral role by improving the resistance of an organism. The anti-PRRSV effect of the traditional Chinese medicine comprises the ways of blocking, inhibiting, killing and the like, and the antiviral mechanism of the traditional Chinese medicine mainly comprises two effects of direct antiviral and indirect antiviral. The direct antiviral effect is the effect of resisting viruses by directly blocking the virus proliferation process, and the indirect antiviral effect is the effect of resisting viruses by enhancing the immunity of the organism. The antiviral action mechanism mainly comprises the following mechanisms: (1) inhibiting the adsorption, penetration and shelling of viruses; (2) inhibiting transcription and replication of viral nucleic acids; (3) inhibiting synthesis of viral proteins; (4) blocking a cellular receptor; (5) inhibiting viral assembly; (6) promoting the body resistance; (7) enhancing the antiviral ability of the host, and the like. The traditional Chinese medicine is used for treating virus infectious diseases, not only aiming at direct antiviral action, but also paying attention to the relationship of virus-organism-traditional Chinese medicine. Not only can directly kill virus and prevent the virus from being absorbed, penetrated, duplicated and transfected, but also can enhance the immunity of the organism and stimulate all immune defense systems of the organism to play an antiviral role. Multiple ways and multiple links, and can improve the adverse reaction and symptoms of organisms caused by viruses, which is the unique advantage of the traditional Chinese medicine for resisting viruses.

disclosure of Invention

In view of the above, the present invention aims to provide the application of herba patriniae and its extract in the preparation of drugs for treating and/or preventing porcine reproductive and respiratory syndrome;

The invention also aims to provide the application of the patrinia and the extract thereof in preparing the medicament for resisting the porcine reproductive and respiratory syndrome virus;

Another object of the present invention is to provide a Chinese medicinal composition for treating porcine reproductive and respiratory syndrome virus, comprising herba patriniae.

herba Patriniae is dry whole plant of Patrinia villosa juss of Patrinia. Cool in nature, pungent and bitter in flavor. Has the effects of clearing away heat and toxic materials, eliminating phlegm and expelling pus, and is commonly used for treating intestinal abscess, pulmonary abscess, dysentery, postpartum abdominal pain due to blood stasis, carbuncle swelling and furuncle; in the prior art, although traditional Chinese medicine preparations for treating porcine reproductive and respiratory syndrome containing herba patriniae exist, the traditional Chinese medicine preparations have more components, the antiviral action mechanism of the traditional Chinese medicine preparations is not fundamentally clarified, and whether the herba patriniae plays a key role in treatment or not and can directly resist the porcine reproductive and respiratory syndrome virus or not is not clearly clarified.

the invention finds that the patrinia extract has obvious antiviral effect on porcine reproductive and respiratory syndrome virus, specifically analyzes the antiviral effect of the patrinia extract on the PRRSV by different action modes and different action times of the patrinia extract on the PRRSV, performs in-vitro in-vivo tests by taking ribavirin as a positive control medicament, and proves that the patrinia extract can obviously inhibit the infection and the replication of the PRRSV by the Western-Blot, qRT-PCR, apoptosis and other technologies through experiments of various layers, and has obvious in-vivo and in-vitro anti-PRRSV effect.

Therefore, the invention provides the application of the patrinia or the extract thereof in preparing the medicines for treating and/or preventing the porcine reproductive and respiratory syndrome virus and the medicines for resisting the porcine reproductive and respiratory syndrome virus;

More specifically, the application provided by the invention is the application of the patrinia or the extract thereof in the preparation of the medicine for treating and/or preventing the porcine reproductive and respiratory syndrome virus; or the application of the combination of the dahurian patrinia herb or the extractive thereof and other traditional Chinese medicine components excluding the following traditional Chinese medicine components in preparing the medicine for treating and/or preventing the porcine reproductive and respiratory syndrome virus, wherein the excluded traditional Chinese medicine components comprise the following components:

american ginseng, codonopsis pilosula, astragalus, bighead atractylodes rhizome, panax notoginseng, medlar, semen cuscutae, lucid ganoderma, tripterygium wilfordii, rhubarb, ligusticum wallichii, liquorice, eclipta alba, sea buckthorn, curcuma zedoary, oldenlandia diffusa, houttuynia cordata, honeysuckle, red paeony root, mint, reed rhizome, gardenia, eupatorium, fructus forsythiae, salvia miltiorrhiza, radix scrophulariae, subprostrate sophora, rhizoma atractylodis, isatis root, folium isatidis, schizonepeta, radix puerariae, radix paeoniae alba, lophatherum gracile, cortex dictamni, cynanchum atratum, Chinese pulsatilla root, schizonepeta, geranium wilfordii, scutellaria baicalensis, reed rhizome, angelica;

Preferably, the other Chinese medicinal components are selected from one or more of radix rehmanniae Preparata, radix Pseudostellariae, radix Berberidis, pericarpium Citri Tangerinae, radix Curcumae, herba Euphorbiae Humifusae, Ficus Simplicissima lour, herba Gynostemmatis, radix seu herba Spiranthis Lanceae, radix Acanthopanacis Senticosi, rhizoma Polygonati Odorati, herba Lobeliae chinensis, calyx seu fructus Physalis, and rhizoma paridis; more preferably, three or more selected from radix rehmanniae Preparata, radix Pseudostellariae, radix Berberidis kunmingensis, pericarpium Citri Tangerinae, radix Curcumae, herba Euphorbiae Humifusae, Ficus Simplicissima lour, herba Gynostemmatis, radix seu herba Spiranthis Lanceae, radix Acanthopanacis Senticosi, rhizoma Polygonati Odorati, herba Lobeliae chinensis, calyx Seu fructus Physalis, and rhizoma paridis; in a specific embodiment of the invention, the plant is selected from three or four of prepared rhizome of rehmannia, radix pseudostellariae, radix berberidis, dried orange peel, radix curcumae, humifuse euphorbia herb, hispid fig root, gynostemma pentaphylla, discorea radix, acanthopanax, rhizoma polygonati, polygonatum odoratum, Chinese lobelia, calyx seu fructus physalis and rhizoma paridis.

preferably, the patrinia extract is an aqueous extract of patrinia, and is prepared by a water decoction method, and the following can be specifically referred to:

Pulverizing herba Patriniae, weighing 10g, adding 10 times of water, decocting for three times, extracting, filtering for 30min each time, mixing filtrates, concentrating to 1g/mL, freeze drying, and vacuum drying to obtain the extract.

Preferably, the porcine reproductive and respiratory syndrome virus is directly resistant to the porcine reproductive and respiratory syndrome virus.

Meanwhile, the invention also provides a traditional Chinese medicine composition for treating porcine reproductive and respiratory syndrome, which comprises extracts of dahurian patrinia herb and one or more than two traditional Chinese medicine components selected from prepared rhizome of rehmannia, radix pseudostellariae, radix berberidis, dried orange peel, radix curcumae, humifuse euphorbia herb, radix fici simplicissimae, gynostemma pentaphylla, discorea, acanthopanax, rhizoma polygonati, polygonatum odoratum, Chinese lobelia, calyx seu fructus physalis and rhizoma paridis.

Preferably, the traditional Chinese medicine composition comprises patrinia and extracts of three or more than four traditional Chinese medicine components selected from radix rehmanniae preparata, radix pseudostellariae, radix berberidis, dried orange peel, radix curcumae, humifuse euphorbia herb, hispid fig root, gynostemma pentaphylla, discordant ginseng, acanthopanax root, rhizoma polygonati, polygonatum odoratum, Chinese lobelia, calyx seu fructus physalis and rhizoma paridis; in a specific embodiment of the invention, the traditional Chinese medicine composition comprises extracts of herba patriniae and three or four traditional Chinese medicine components selected from radix rehmanniae preparata, radix pseudostellariae, radix berberidis, pericarpium citri reticulatae, radix curcumae, humifuse euphorbia herb, hispid fig, gynostemma pentaphylla, radix salviae miltiorrhizae, acanthopanax, rhizoma polygonati, polygonatum odoratum, Chinese lobelia, calyx seu fructus physalis and rhizoma paridis.

Preferably, the dosage of each traditional Chinese medicine component is as follows:

10-20 parts of dahurian patrinia herb, 10-20 parts of prepared rehmannia root, 10-20 parts of radix pseudostellariae, 10-15 parts of radix berberidis, 6-10 parts of dried orange peel, 6-10 parts of radix curcumae, 10-15 parts of humifuse euphorbia herb, 10-20 parts of hispid fig root, 10-20 parts of gynostemma pentaphylla, 10-20 parts of Chinese ladybell root, 10-20 parts of acanthopanax root, 10-20 parts of rhizoma polygonati, 10-20 parts of fragrant solomonseal rhizome, 10-15 parts of Chinese lobelia herb, 10-15 parts of calyx seu fructus physalis and 10-15 parts of paris polyphylla rhizome.

Preferably, the extract is an aqueous extract and is prepared by a water decoction method.

In a specific embodiment of the invention, the traditional Chinese medicine components in the traditional Chinese medicine composition are selected from one of the following combination forms:

(1) Radix fici simplicissimae, herba Patriniae, herba Lobeliae chinensis, and pericarpium Citri Reticulatae; (2) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (3) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (4) radix fici simplicissimae, herba Patriniae, herba Lobeliae chinensis, and radix Curcumae; (5) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, radix Curcumae; (6) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and radix Curcumae; (7) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (8) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (9) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and pericarpium Citri Reticulatae; (10) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (11) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (12) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and radix Curcumae; (13) gynostemma pentaphyllum, herba patriniae, Chinese lobelia herb and dried orange peel; (14) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb and dried orange peel; (15) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb, Chinese lobelia herb and dried orange peel; (16) gynostemma pentaphyllum, herba patriniae, Chinese lobelia herb and radix curcumae; (17) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb and radix curcumae; (18) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb, Chinese lobelia herb and radix curcumae; (19) gynostemma pentaphyllum, herba patriniae, radix berberidis, and pericarpium citri reticulatae; (20) gynostemma pentaphyllum, herba patriniae, radix berberidis, and radix curcumae; (21) gynostemma pentaphyllum, herba patriniae, radix berberidis, Chinese lobelia herb and dried orange peel; (22) gynostemma pentaphyllum, herba patriniae, radix berberidis, humifuse euphorbia herb and dried orange peel; (23) gynostemma pentaphyllum, herba patriniae, radix berberidis, humifuse euphorbia herb and radix curcumae; (24) gynostemma pentaphyllum, herba patriniae, radix berberidis, Chinese lobelia herb and radix curcumae; (25) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (26) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (27) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (28) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Lobeliae chinensis, and radix Curcumae; (29) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, radix Curcumae; (30) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and radix Curcumae; (31) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (32) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (33) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (34) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (35) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (36) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and radix Curcumae; (37) acanthopanax root, herba patriniae, Chinese lobelia herb and dried orange peel; (38) acanthopanax, herba patriniae, humifuse euphorbia herb and dried orange peel; (39) acanthopanax, herba patriniae, humifuse euphorbia herb, Chinese lobelia herb and dried orange peel; (40) acanthopanax, herba patriniae, Chinese lobelia and radix curcumae; (41) acanthopanax, herba patriniae, humifuse euphorbia herb and radix curcumae; (42) acanthopanax, herba patriniae, humifuse euphorbia herb, Chinese lobelia herb and radix curcumae; (43) acanthopanax, herba patriniae, radix berberidis and dried orange peel; (44) acanthopanax, herba patriniae, radix berberidis and radix curcumae; (45) acanthopanax, herba patriniae, radix berberidis, Chinese lobelia herb and dried orange peel; (46) acanthopanax, herba patriniae, radix berberidis, humifuse euphorbia herb and dried orange peel; (47) acanthopanax, herba patriniae, radix berberidis, humifuse euphorbia herb and radix curcumae; (48) acanthopanax, herba patriniae, radix berberidis, Chinese lobelia herb and radix curcumae; (49) rhizoma Polygonati, herba Patriniae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (50) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (51) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (52) rhizoma Polygonati, herba Patriniae, herba Lobeliae chinensis, and radix Curcumae; (53) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (54) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and radix Curcumae; (55) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (56) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (57) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (58) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (59) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (60) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and radix Curcumae; (61) rhizoma Polygonati Odorati, herba Patriniae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (62) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (63) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (64) rhizoma Polygonati Odorati, herba Patriniae, herba Lobeliae chinensis, and radix Curcumae; (65) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (66) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and radix Curcumae; (67) rhizoma Polygonati Odorati, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (68) rhizoma Polygonati Odorati, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (69) radix polygonati officinalis, herba patriniae, radix berberidis, Chinese lobelia and dried orange peel; (70) radix polygonati officinalis, herba patriniae, radix berberidis, humifuse euphorbia herb and dried orange peel; (71) radix polygonati officinalis, herba patriniae, radix berberidis, humifuse euphorbia herb and radix curcumae; (72) rhizoma Polygonati Odorati, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and radix Curcumae; (73) radix rehmanniae Preparata, herba Patriniae, herba Lobeliae chinensis, and pericarpium Citri Reticulatae; (74) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (75) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (76) radix rehmanniae Preparata, herba Patriniae, herba Lobeliae chinensis, and radix Curcumae; (77) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (78) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and radix Curcumae; (79) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (80) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (81) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (82) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (83) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (84) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and radix Curcumae; (85) radix Pseudostellariae, herba Patriniae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (86) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (87) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (88) radix Pseudostellariae, herba Patriniae, herba Lobeliae chinensis, and radix Curcumae; (89) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (90) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and radix Curcumae; (91) radix Pseudostellariae, herba Patriniae, radix Berberidis, and pericarpium Citri Tangerinae; (92) radix Pseudostellariae, herba Patriniae, radix Berberidis, and radix Curcumae; (93) radix Pseudostellariae, herba Patriniae, radix Berberidis, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (94) radix Pseudostellariae, herba Patriniae, radix Berberidis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (95) radix Pseudostellariae, herba Patriniae, radix Berberidis, herba Euphorbiae Humifusae, and radix Curcumae; (96) radix Pseudostellariae, herba Patriniae, radix Berberidis, herba Lobeliae chinensis, and radix Curcumae; (97) radix fici simplicissimae, herba Patriniae, herba Lobeliae chinensis, and pericarpium Citri Reticulatae; (98) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (99) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (100) radix fici simplicissimae, herba Patriniae, herba Lobeliae chinensis, and radix Curcumae; (101) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, radix Curcumae; (102) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and radix Curcumae; (103) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (104) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (105) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and pericarpium Citri Reticulatae; (106) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (107) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (108) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and radix Curcumae; (109) gynostemma pentaphyllum, herba patriniae, Chinese lobelia herb and dried orange peel; (110) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb and dried orange peel; (111) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb, Chinese lobelia herb and dried orange peel; (112) gynostemma pentaphyllum, herba patriniae, Chinese lobelia herb and radix curcumae; (113) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb and radix curcumae; (114) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb, Chinese lobelia herb and radix curcumae; (115) gynostemma pentaphyllum, herba patriniae, radix berberidis, and pericarpium citri reticulatae; (116) gynostemma pentaphyllum, herba patriniae, radix berberidis, and radix curcumae; (117) gynostemma pentaphyllum, herba patriniae, radix berberidis, Chinese lobelia herb and dried orange peel; (118) gynostemma pentaphyllum, herba patriniae, radix berberidis, humifuse euphorbia herb and dried orange peel; (119) gynostemma pentaphyllum, herba patriniae, radix berberidis, humifuse euphorbia herb and radix curcumae; (120) gynostemma pentaphyllum, herba patriniae, radix berberidis, Chinese lobelia herb and radix curcumae; (121) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (122) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (123) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (124) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Lobeliae chinensis, and radix Curcumae; (125) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, radix Curcumae; (126) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and radix Curcumae; (127) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (128) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (129) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (130) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (131) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (132) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and radix Curcumae; (133) acanthopanax root, herba patriniae, Chinese lobelia herb and dried orange peel; (134) acanthopanax, herba patriniae, humifuse euphorbia herb and dried orange peel; (135) acanthopanax, herba patriniae, humifuse euphorbia herb, Chinese lobelia herb and dried orange peel; (136) acanthopanax, herba patriniae, Chinese lobelia and radix curcumae; (137) acanthopanax, herba patriniae, humifuse euphorbia herb and radix curcumae; (138) acanthopanax, herba patriniae, humifuse euphorbia herb, Chinese lobelia herb and radix curcumae; (139) acanthopanax, herba patriniae, radix berberidis and dried orange peel; (140) acanthopanax, herba patriniae, radix berberidis and radix curcumae; (141) acanthopanax, herba patriniae, radix berberidis, Chinese lobelia herb and dried orange peel; (142) acanthopanax, herba patriniae, radix berberidis, humifuse euphorbia herb and dried orange peel; (143) acanthopanax, herba patriniae, radix berberidis, humifuse euphorbia herb and radix curcumae; (144) acanthopanax, herba patriniae, radix berberidis, Chinese lobelia herb and radix curcumae; (145) rhizoma Polygonati, herba Patriniae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (146) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (147) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (148) rhizoma Polygonati, herba Patriniae, herba Lobeliae chinensis, and radix Curcumae; (149) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (150) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and radix Curcumae; (151) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (152) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (153) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (154) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (155) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (156) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and radix Curcumae; (157) rhizoma Polygonati Odorati, herba Patriniae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (158) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (159) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (160) rhizoma Polygonati Odorati, herba Patriniae, herba Lobeliae chinensis, and radix Curcumae; (161) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (162) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and radix Curcumae; (163) rhizoma Polygonati Odorati, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (164) rhizoma Polygonati Odorati, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (165) radix polygonati officinalis, herba patriniae, radix berberidis, Chinese lobelia and dried orange peel; (166) radix polygonati officinalis, herba patriniae, radix berberidis, humifuse euphorbia herb and dried orange peel; (167) radix polygonati officinalis, herba patriniae, radix berberidis, humifuse euphorbia herb and radix curcumae; (168) rhizoma Polygonati Odorati, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and radix Curcumae; (169) radix rehmanniae Preparata, herba Patriniae, herba Lobeliae chinensis, and pericarpium Citri Reticulatae; (170) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (171) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (172) radix rehmanniae Preparata, herba Patriniae, herba Lobeliae chinensis, and radix Curcumae; (173) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (174) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and radix Curcumae; (175) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (176) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (177) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (178) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (179) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (180) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, herba Lobeliae chinensis, and radix Curcumae; (181) radix Pseudostellariae, herba Patriniae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (182) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (183) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (184) radix Pseudostellariae, herba Patriniae, herba Lobeliae chinensis, and radix Curcumae; (185) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (186) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, herba Lobeliae chinensis, and radix Curcumae; (187) radix Pseudostellariae, herba Patriniae, radix Berberidis, and pericarpium Citri Tangerinae; (188) radix Pseudostellariae, herba Patriniae, radix Berberidis, and radix Curcumae; (189) radix Pseudostellariae, herba Patriniae, radix Berberidis, herba Lobeliae chinensis, and pericarpium Citri Tangerinae; (190) radix Pseudostellariae, herba Patriniae, radix Berberidis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (191) radix Pseudostellariae, herba Patriniae, radix Berberidis, herba Euphorbiae Humifusae, and radix Curcumae; (192) radix Pseudostellariae, herba Patriniae, radix Berberidis, herba Lobeliae chinensis, and radix Curcumae; (193) radix fici simplicissimae, herba Patriniae, calyx seu fructus physalis, and pericarpium Citri Reticulatae; (194) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (195) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, calyx seu fructus physalis, and pericarpium Citri Tangerinae; (196) radix fici simplicissimae, herba Patriniae, calyx seu fructus physalis, and radix Curcumae; (197) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, radix Curcumae; (198) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, calyx seu fructus physalis, and radix Curcumae; (199) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (200) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (201) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, calyx seu fructus physalis, and pericarpium Citri Tangerinae; (202) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (203) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (204) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, calyx seu fructus physalis, and radix Curcumae; (205) gynostemma pentaphyllum, herba patriniae, calyx seu fructus physalis, and pericarpium Citri Reticulatae; (206) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb and dried orange peel; (207) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb, calyx seu fructus physalis, and pericarpium citri reticulatae; (208) gynostemma pentaphyllum, herba patriniae, calyx seu fructus physalis, and radix Curcumae; (209) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb and radix curcumae; (210) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb, calyx seu fructus physalis, and radix curcumae; (211) gynostemma pentaphyllum, herba patriniae, radix berberidis, and pericarpium citri reticulatae; (212) gynostemma pentaphyllum, herba patriniae, radix berberidis, and radix curcumae; (213) gynostemma pentaphyllum, herba patriniae, radix berberidis, calyx seu fructus physalis, and pericarpium citri reticulatae; (214) gynostemma pentaphyllum, herba patriniae, radix berberidis, humifuse euphorbia herb and dried orange peel; (215) gynostemma pentaphyllum, herba patriniae, radix berberidis, humifuse euphorbia herb and radix curcumae; (216) gynostemma pentaphyllum, herba patriniae, radix berberidis, calyx seu fructus physalis, and radix curcumae; (217) radix seu herba Spiranthis Lanceae, herba Patriniae, calyx seu fructus physalis, and pericarpium Citri Tangerinae; (218) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (219) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, calyx Seu fructus Physalis, and pericarpium Citri Tangerinae; (220) radix seu herba Spiranthis Lanceae, herba Patriniae, calyx seu fructus physalis, and radix Curcumae; (221) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, radix Curcumae; (222) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, calyx Seu fructus Physalis, and radix Curcumae; (223) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (224) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (225) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, calyx seu fructus physalis, and pericarpium Citri Tangerinae; (226) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (227) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (228) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, calyx seu fructus physalis, and radix Curcumae; (229) acanthopanax, herba patriniae, calyx seu fructus physalis and pericarpium citri reticulatae; (230) acanthopanax, herba patriniae, humifuse euphorbia herb and dried orange peel; (231) acanthopanax, herba patriniae, humifuse euphorbia herb, calyx seu fructus physalis and dried orange peel; (232) acanthopanax, herba patriniae, calyx seu fructus physalis, radix curcumae; (233) acanthopanax, herba patriniae, humifuse euphorbia herb and radix curcumae; (234) acanthopanax, herba patriniae, humifuse euphorbia herb, calyx seu fructus physalis and radix curcumae; (235) acanthopanax, herba patriniae, radix berberidis and dried orange peel; (236) acanthopanax, herba patriniae, radix berberidis and radix curcumae; (237) acanthopanax, herba patriniae, radix berberidis, calyx seu fructus physalis and dried orange peel; (238) acanthopanax, herba patriniae, radix berberidis, humifuse euphorbia herb and dried orange peel; (239) acanthopanax, herba patriniae, radix berberidis, humifuse euphorbia herb and radix curcumae; (240) acanthopanax, herba patriniae, radix berberidis, calyx seu fructus physalis and radix curcumae; (241) rhizoma Polygonati, herba Patriniae, calyx Seu fructus Physalis, and pericarpium Citri Tangerinae; (242) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (243) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, calyx Seu fructus Physalis, and pericarpium Citri Tangerinae; (244) rhizoma Polygonati, herba Patriniae, calyx Seu fructus Physalis, and radix Curcumae; (245) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (246) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, calyx Seu fructus Physalis, and radix Curcumae; (247) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (248) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (249) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, calyx seu fructus physalis, and pericarpium Citri Tangerinae; (250) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (251) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (252) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, calyx seu fructus physalis, and radix Curcumae; (253) rhizoma Polygonati Odorati, herba Patriniae, calyx Seu fructus Physalis, and pericarpium Citri Tangerinae; (254) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (255) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, calyx Seu fructus Physalis, and pericarpium Citri Tangerinae; (256) rhizoma Polygonati Odorati, herba Patriniae, calyx Seu fructus Physalis, and radix Curcumae; (257) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (258) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, calyx Seu fructus Physalis, and radix Curcumae; (259) rhizoma Polygonati Odorati, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (260) rhizoma Polygonati Odorati, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (261) radix polygonati officinalis, herba patriniae, radix berberidis, calyx seu fructus physalis and pericarpium citri reticulatae; (262) radix polygonati officinalis, herba patriniae, radix berberidis, humifuse euphorbia herb and dried orange peel; (263) radix polygonati officinalis, herba patriniae, radix berberidis, humifuse euphorbia herb and radix curcumae; (264) radix polygonati officinalis, herba patriniae, radix berberidis, calyx seu fructus physalis and radix curcumae; (265) radix rehmanniae Preparata, herba Patriniae, calyx Seu fructus Physalis, and pericarpium Citri Tangerinae; (266) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (267) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, calyx Seu fructus Physalis, and pericarpium Citri Tangerinae; (268) radix rehmanniae Preparata, herba Patriniae, calyx Seu fructus Physalis, and radix Curcumae; (269) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (270) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, calyx Seu fructus Physalis, and radix Curcumae; (271) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (272) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (273) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, calyx seu fructus physalis, and pericarpium Citri Tangerinae; (274) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (275) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (276) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, calyx seu fructus physalis, and radix Curcumae; (277) radix Pseudostellariae, herba Patriniae, calyx Seu fructus Physalis, and pericarpium Citri Tangerinae; (278) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (279) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, calyx Seu fructus Physalis, and pericarpium Citri Tangerinae; (280) radix Pseudostellariae, herba Patriniae, calyx Seu fructus Physalis, and radix Curcumae; (281) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (282) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, calyx Seu fructus Physalis, and radix Curcumae; (283) radix Pseudostellariae, herba Patriniae, radix Berberidis, and pericarpium Citri Tangerinae; (284) radix Pseudostellariae, herba Patriniae, radix Berberidis, and radix Curcumae; (285) radix Pseudostellariae, herba Patriniae, radix Berberidis, calyx seu fructus Physalis, and pericarpium Citri Tangerinae; (286) radix Pseudostellariae, herba Patriniae, radix Berberidis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (287) radix Pseudostellariae, herba Patriniae, radix Berberidis, herba Euphorbiae Humifusae, and radix Curcumae; (288) radix Pseudostellariae, herba Patriniae, radix Berberidis, calyx seu fructus physalis, and radix Curcumae; (289) radix fici simplicissimae, herba Patriniae, rhizoma paridis, and pericarpium Citri Reticulatae; (290) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (291) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, rhizoma paridis, and pericarpium Citri Reticulatae; (292) radix fici simplicissimae, herba Patriniae, rhizoma paridis, and radix Curcumae; (293) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, radix Curcumae; (294) radix fici simplicissimae, herba Patriniae, herba Euphorbiae Humifusae, rhizoma paridis, and radix Curcumae; (295) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (296) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (297) radix fici simplicissimae, herba Patriniae, radix Berberidis, rhizoma paridis, and pericarpium Citri Reticulatae; (298) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (299) radix fici simplicissimae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (300) radix fici simplicissimae, herba Patriniae, radix Berberidis, rhizoma paridis, and radix Curcumae; (301) gynostemma pentaphyllum, herba patriniae, rhizoma paridis, and pericarpium citri reticulatae; (302) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb and dried orange peel; (303) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb, rhizoma paridis and pericarpium citri reticulatae; (304) gynostemma pentaphyllum, herba patriniae, rhizoma paridis, and radix curcumae; (305) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb and radix curcumae; (306) gynostemma pentaphyllum, herba patriniae, humifuse euphorbia herb, rhizoma paridis and radix curcumae; (307) gynostemma pentaphyllum, herba patriniae, radix berberidis, and pericarpium citri reticulatae; (308) gynostemma pentaphyllum, herba patriniae, radix berberidis, and radix curcumae; (309) gynostemma pentaphyllum, herba patriniae, radix berberidis, rhizoma paridis, and pericarpium citri reticulatae; (310) gynostemma pentaphyllum, herba patriniae, radix berberidis, humifuse euphorbia herb and dried orange peel; (311) gynostemma pentaphyllum, herba patriniae, radix berberidis, humifuse euphorbia herb and radix curcumae; (312) gynostemma pentaphyllum, herba patriniae, radix berberidis, rhizoma paridis, and radix curcumae; (313) radix seu herba Spiranthis Lanceae, herba Patriniae, rhizoma paridis, and pericarpium Citri Reticulatae; (314) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (315) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, rhizoma paridis, and pericarpium Citri Tangerinae; (316) radix seu herba Spiranthis Lanceae, herba Patriniae, rhizoma paridis, and radix Curcumae; (317) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, radix Curcumae; (318) radix seu herba Spiranthis Lanceae, herba Patriniae, herba Euphorbiae Humifusae, rhizoma paridis, and radix Curcumae; (319) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (320) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (321) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, rhizoma paridis, and pericarpium Citri Tangerinae; (322) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (323) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (324) radix seu herba Spiranthis Lanceae, herba Patriniae, radix Berberidis kunmingensis, rhizoma paridis, and radix Curcumae; (325) radix Acanthopanacis Senticosi, herba Patriniae, rhizoma paridis, and pericarpium Citri Tangerinae; (326) acanthopanax, herba patriniae, humifuse euphorbia herb and dried orange peel; (327) acanthopanax, herba patriniae, humifuse euphorbia herb, rhizoma paridis and dried orange peel; (328) acanthopanax root, herba patriniae, rhizoma paridis and radix curcumae; (329) acanthopanax, herba patriniae, humifuse euphorbia herb and radix curcumae; (330) acanthopanax, herba patriniae, humifuse euphorbia herb, rhizoma paridis and radix curcumae; (331) acanthopanax, herba patriniae, radix berberidis and dried orange peel; (332) acanthopanax, herba patriniae, radix berberidis and radix curcumae; (333) radix Acanthopanacis Senticosi, herba Patriniae, radix Berberidis kunmingensis, rhizoma paridis, and pericarpium Citri Tangerinae; (334) acanthopanax, herba patriniae, radix berberidis, humifuse euphorbia herb and dried orange peel; (335) acanthopanax, herba patriniae, radix berberidis, humifuse euphorbia herb and radix curcumae; (336) radix Acanthopanacis Senticosi, herba Patriniae, radix Berberidis kunmingensis, rhizoma paridis, and radix Curcumae; (337) rhizoma Polygonati, herba Patriniae, rhizoma paridis, and pericarpium Citri Reticulatae; (338) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (339) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, rhizoma paridis, and pericarpium Citri Tangerinae; (340) rhizoma Polygonati, herba Patriniae, rhizoma paridis, and radix Curcumae; (341) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (342) rhizoma Polygonati, herba Patriniae, herba Euphorbiae Humifusae, rhizoma paridis, and radix Curcumae; (343) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (344) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (345) rhizoma Polygonati, herba Patriniae, radix Berberidis, rhizoma paridis, and pericarpium Citri Tangerinae; (346) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (347) rhizoma Polygonati, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (348) rhizoma Polygonati, herba Patriniae, radix Berberidis, rhizoma paridis, and radix Curcumae; (349) rhizoma Polygonati Odorati, herba Patriniae, rhizoma paridis, and pericarpium Citri Reticulatae; (350) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (351) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, rhizoma paridis, and pericarpium Citri Tangerinae; (352) rhizoma Polygonati Odorati, herba Patriniae, rhizoma paridis, and radix Curcumae; (353) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (354) rhizoma Polygonati Odorati, herba Patriniae, herba Euphorbiae Humifusae, rhizoma paridis, and radix Curcumae; (355) rhizoma Polygonati Odorati, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (356) rhizoma Polygonati Odorati, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (357) rhizoma Polygonati Odorati, herba Patriniae, radix Berberidis, rhizoma paridis, and pericarpium Citri Tangerinae; (358) radix polygonati officinalis, herba patriniae, radix berberidis, humifuse euphorbia herb and dried orange peel; (359) radix polygonati officinalis, herba patriniae, radix berberidis, humifuse euphorbia herb and radix curcumae; (360) rhizoma Polygonati Odorati, herba Patriniae, radix Berberidis, rhizoma paridis, and radix Curcumae; (361) radix rehmanniae Preparata, herba Patriniae, rhizoma paridis, and pericarpium Citri Reticulatae; (362) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (363) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, rhizoma paridis, and pericarpium Citri Tangerinae; (364) radix rehmanniae Preparata, herba Patriniae, rhizoma paridis, and radix Curcumae; (365) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (366) radix rehmanniae Preparata, herba Patriniae, herba Euphorbiae Humifusae, rhizoma paridis, and radix Curcumae; (367) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, and pericarpium Citri Tangerinae; (368) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, and radix Curcumae; (369) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, rhizoma paridis, and pericarpium Citri Tangerinae; (370) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (371) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, herba Euphorbiae Humifusae, and radix Curcumae; (372) radix rehmanniae Preparata, herba Patriniae, radix Berberidis kunmingensis, rhizoma paridis, and radix Curcumae; (373) radix Pseudostellariae, herba Patriniae, rhizoma paridis, and pericarpium Citri Reticulatae; (374) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (375) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, rhizoma paridis, and pericarpium Citri Tangerinae; (376) radix Pseudostellariae, herba Patriniae, rhizoma paridis, and radix Curcumae; (377) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, and radix Curcumae; (378) radix Pseudostellariae, herba Patriniae, herba Euphorbiae Humifusae, rhizoma paridis, and radix Curcumae; (379) radix Pseudostellariae, herba Patriniae, radix Berberidis, and pericarpium Citri Tangerinae; (380) radix Pseudostellariae, herba Patriniae, radix Berberidis, and radix Curcumae; (381) radix Pseudostellariae, herba Patriniae, radix Berberidis, rhizoma paridis, and pericarpium Citri Tangerinae; (382) radix Pseudostellariae, herba Patriniae, radix Berberidis, herba Euphorbiae Humifusae, and pericarpium Citri Tangerinae; (383) radix Pseudostellariae, herba Patriniae, radix Berberidis, herba Euphorbiae Humifusae, and radix Curcumae; (384) radix pseudostellariae, herba patriniae, radix berberidis, rhizoma paridis and radix curcumae.

According to the technical scheme, Western-Blot, qRT-PCR, apoptosis and other technologies are utilized to prove through experiments of various layers that the patrinia extract can obviously inhibit PRRSV infection and replication, has obvious in-vivo and in-vitro PRRSV virus resistance, can be used as a novel medicament for preventing and treating porcine reproductive and respiratory syndrome, and has a good application prospect in the prevention and treatment of the porcine reproductive and respiratory syndrome.

Drawings

FIG. 1 is a gel electrophoresis of the recombinant plasmid containing the N gene;

FIG. 2 shows that 400. mu.g/mL of herba Patriniae extract blocks PRRSV, and after 24h, 48h and 72h, the expression level of N gene is detected by qRT-PCR, P is <0.05 and P is < 0.01;

FIG. 3 shows that 400. mu.g/mL of herba Patriniae extract has an inhibitory effect on PRRSV, and after 24h, 48h and 72h, the expression level of N gene is detected by qRT-PCR, P is <0.05 and P is < 0.01;

FIG. 4 shows that 400. mu.g/mL of herba Patriniae extract directly inactivates PRRSV, and after 24h, 48h and 72h, the expression levels of N gene are detected by qRT-PCR, P is 0.05 and P is 0.01;

FIG. 5 shows that 400. mu.g/mL of herba Patriniae extract blocks PRRSV, and the expression level of N protein is detected by Western Blot after 24h, 48h and 72 h; wherein, 1: s extract group; 2: a positive control ribavirin group; 3: PRRSV control group; 4: a cell control group;

FIG. 6 shows that 400. mu.g/mL of herba Patriniae extract has an inhibitory effect on PRRSV, and the expression level of N protein is detected by Western Blot after 24 hours, 48 hours and 72 hours of the effect; wherein, 1: s extract group; 2: a positive control ribavirin group; 3: PRRSV control group; 4: a cell control group;

FIG. 7 shows that 400. mu.g/mL of herba Patriniae extract directly inactivates PRRSV, and the expression level of N protein is detected by Western Blot after 24h, 48h and 72 h; wherein, 1: s extract group; 2: a positive control ribavirin group; 3: PRRSV control group; 4: a cell control group;

FIG. 8 shows the apoptosis detected by flow cytometry after 24h, 48h and 72h incubation of uninfected PRRSV and infected PRRSV, respectively; wherein the lower left quadrant in the figure represents live cells, the lower right quadrant represents early apoptotic cells, the upper right quadrant represents late apoptotic or dead cells, and the upper left quadrant represents dead cells; LL is the living cell rate, LR is the early apoptosis rate, UR is the late apoptosis or death rate, and UL is the cell death rate; n1, N2 and N3 respectively represent cell control groups of 24h, 48h and 72h, and D1, D2 and D3 respectively represent virus control groups of 24h, 48h and 72 h;

FIG. 9 shows that 400. mu.g/mL of Patrinia extract blocked PRRSV, and after 24h, 48h, and 72h, the effect on apoptosis was detected by flow cytometry; wherein the lower left quadrant in the figure represents live cells, the lower right quadrant represents early apoptotic cells, the upper right quadrant represents late apoptotic or dead cells, and the upper left quadrant represents dead cells; LL is the living cell rate, LR is the early apoptosis rate, UR is the late apoptosis or death rate, and UL is the cell death rate; a1, A2 and A3 respectively represent S extract groups of 24h, 48h and 72h, B1, B2 and B3 respectively represent positive control drug ribavirin groups of 24h, 48h and 72 h;

FIG. 10 shows that 400. mu.g/mL of Patrinia extract inhibited PRRSV, and after 24h, 48h, and 72h, the effect on apoptosis was detected by flow cytometry; wherein the lower left quadrant in the figure represents live cells, the lower right quadrant represents early apoptotic cells, the upper right quadrant represents late apoptotic or dead cells, and the upper left quadrant represents dead cells; LL is the living cell rate, LR is the early apoptosis rate, UR is the late apoptosis or death rate, and UL is the cell death rate; a1, A2 and A3 respectively represent S extract groups of 24h, 48h and 72h, B1, B2 and B3 respectively represent positive control drug ribavirin groups of 24h, 48h and 72 h;

FIG. 11 shows that 400. mu.g/mL of Patrinia extract directly inactivates PRRSV, and after 24h, 48h, and 72h, the effect on apoptosis was detected by flow cytometry; wherein the lower left quadrant in the figure represents live cells, the lower right quadrant represents early apoptotic cells, the upper right quadrant represents late apoptotic or dead cells, and the upper left quadrant represents dead cells; LL is the living cell rate, LR is the early apoptosis rate, UR is the late apoptosis or death rate, and UL is the cell death rate; a1, A2 and A3 respectively represent S extract groups of 24h, 48h and 72h, B1, B2 and B3 respectively represent positive control drug ribavirin groups of 24h, 48h and 72 h;

FIG. 12 shows monitoring of changes in body temperature of groups of piglets after infection with virus;

FIG. 13 shows the clinical symptom scores of groups of piglets after infection with virus;

FIG. 14 shows lung lesion observations of experimental groups of piglets;

FIG. 15 shows lung pathology slice observations of experimental groups of piglets;

FIG. 16 shows the dynamic monitoring of serum viremia for experimental piglets;

figure 17 shows the tissue organ viral load RT-PCR assay of experimental piglets, # P <0.05, # P < 0.01;

Figure 18 shows PRRSV antibody detection of test piglets.

Detailed Description

the invention discloses application of herba patriniae and a traditional Chinese medicine composition for treating porcine reproductive and respiratory syndrome, and can be realized by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the invention can be practiced and applied by modifying or appropriately combining the applications and Chinese medicinal compositions described herein without departing from the spirit and scope of the invention.

In a specific embodiment of the present invention, an antiviral test is performed with an aqueous extract of herba Patriniae, as follows:

the herba patriniae traditional Chinese medicinal material is purchased from Shang nationality drug source commercial and trade company Limited, is identified by animal drug academy of sciences of Jiangsu farming science and technology and profession in the professor of Shenglan, and the specimen is stored in the high-tech research focus laboratory of veterinary biological products in Jiangsu province of the Jiangsu farming science and technology and profession.

Pulverizing herba Patriniae, weighing 1000g, adding 10 times of water, decocting for three times, extracting, filtering, each time for 30min, mixing filtrates, concentrating to 1g/mL, and vacuum drying in a drying oven for use.

The mass of the dried herba Patriniae extract is 290g, and the extraction rate is 29.0%.

statistical analysis of the following examples of the invention: all experiments were repeated at least 3 times independently, and single-factor analysis of variance was performed using software SPSS17.0, all data expressed as Mean ± SD, Student's test statistical analysis. And analyzing the obtained grey value of the protein band by using Image J, and dividing the grey value of the target band by the corresponding grey value of the beta-actin band to obtain a ratio which is a relative expression value. All histograms were plotted using graph Prism 5.

The invention is further illustrated by the following examples.

example 1: cytotoxicity of Patrinia extract and ribavirin

1. Material

(1) Ribavirin is purchased from south-bound aerospace biotechnology limited.

(2) tetramethyltetrazolium blue (MTT) was purchased from Solebao technologies, Inc., Beijing. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystalline Formazan (Formazan) and deposit the Formazan in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and an enzyme linked immunosorbent assay detector is used for measuring the light absorption value of formazan at 490nm wavelength, so that the quantity of living cells can be indirectly reflected.

2. Test method

cells grown to a monolayer in cell flasks were digested with DMEM containing 10% FBS and diluted to approximately 2X 10⁵Perml, add to 96-well plate, 100. mu.L cell suspension per well. And (3) after the cells grow to a single layer, removing the old culture solution, sequentially adding the diluted liquid medicine from high concentration to low concentration in the 1 st to 10 th rows, repeating 4 holes in each concentration, adding 100 mu L of the cell culture solution in each hole, and adding the cell culture solution in the 11 th to 12 th rows to serve as cell control. Culturing for 72h, discarding the medicinal liquid, adding MTT solution, culturing for 4h, discarding the MTT solution, adding DMSO to dissolve the crystal completely, and measuring OD_490nmThe value is obtained. The calculation formula is as follows:

Growth inhibition (%) (cell control OD)_490nmvalue-drug group OD_490nmValue)/cell control OD_490nmThe value is multiplied by 100%

When the inhibition rate is 0, the drug has no toxic effect on cells at that concentration, and the maximum safe concentration of the drug is determined, and the results are shown in table 1.

TABLE 1 inhibition ratio (%) -145 cells by different drug concentrations

Note: the initial concentration of the herba Patriniae extract is 25mg/mL, and the initial concentration of ribavirin is 1 mg/mL.

3. Results

As is clear from Table 1, when the concentration of the Patrinia scabiosaefolia extract is 400. mu.g/mL or less, and the concentration of ribavirin is 15.63. mu.g/mL or less, it is not toxic to Marc-145 cells. Therefore, the initial concentration of Patrinia scabiosaefolia extract used in the subsequent experiments was 400. mu.g/mL and the initial concentration of ribavirin was 15.63. mu.g/mL.

Example 2: viral Titer (TCID)₅₀) Measurement of

sequentially diluting virus solution to 10 times by using DMEM culture solution without FBS according to 10 times ratio^-1～10^-88 concentration gradients, seeded on 96 well cell culture plates at 100. mu.L/well. 8 wells were inoculated at each dilution, and a virus control group and a cell control group were simultaneously established and placed in 5% CO₂culturing in an incubator at 37 ℃ for 2h, discarding virus solution, adding cell maintenance solution, observing for 5-7 days, recording cytopathic condition, and calculating TCID by Reed-Muench method₅₀The calculation formula is as follows:

LgTCID₅₀Log dilution of virus above 50% lesion rate + (above 50% lesion rate-50%)/(above 50% lesion rate-below 50% lesion rate) x log of dilution multiple

The results of the virus titer determination are shown in Table 2, and the TCID of PRRSV on Marc-145 cells is calculated according to the formula₅₀Value of 10^-4.60.1mL, i.e., 0.1mL of virus was inoculated at a concentration of 10^-4.6the virus liquid can cause 50% of cells to be diseased.

TABLE 2PRRSV TCID₅₀measurement results

Example 3: MTT method for detecting in-vitro anti-PRRSV effect of patrinia extract

the in vitro anti-PRRSV effect of the patrinia extract is detected by 3 action modes of blocking, inhibiting and directly inactivating. The 1 st mode is that the medicine is added firstly and then the virus is added (blocked), so as to observe whether the medicine can be combined with the cell membrane surface receptor to prevent the adsorption and entry of the virus; the 2 nd mode of action is that the virus is added firstly and then the medicine is added (inhibited), aiming at observing whether the medicine can enter the cell to act on the virus in the cell and enter the cell to inhibit the biosynthesis and the replication of the virus; the 3 rd mode of action is that the medicine and the virus are added after a period of time of action (direct inactivation), so as to observe whether the medicine has direct inactivation effect on the virus or not, and directly inhibit and kill the virus outside host cells before infection. The 3 action modes are respectively matched with the design of clinical prevention, treatment and disinfection tests, and clinically, the 1 st action mode is the prevention action of the medicament on the viral diseases, and the last two action modes are the treatment action of the medicament on the viral diseases.

(1) Blocking effect of herba patriniae extract on PRRSV

After Marc-145 cells grow to a single layer in a 96-well plate, discarding old culture solution, adding Chinese medicinal extract solution with maximum safe mass concentration as initial concentration, diluting with 2 times of the solution at 100 μ L per well for 4 concentration gradients, culturing for 4h, discarding medicinal solution, adding 100TCID₅₀And culturing PRRSV solution in each well for 2h in 100 mu L, discarding virus solution, and adding DMEM growth maintenance solution containing 2% FBS. And calculating the inhibition rate of the medicament on the virus through a formula.

The results are shown in table 3, the maximum inhibition rates of the patrinia extract and ribavirin on PRRSV at the maximum safe concentrations are 117.94% and 63.31%, respectively, and the inhibition rate of the patrinia extract on ribavirin is significantly higher than that of ribavirin (P <0.01), which indicates that the patrinia extract has a stronger blocking effect on PRRSV.

TABLE 3 inhibition of PRRSV by blocking action of drugs (%)

Note: c1, C2, C3, C4 represent 4 different concentrations diluted 2-fold from the maximum safe concentration of the drug, all data are expressed as Mean ± standard deviation (Mean ± SD). P compared to ribavirin control group<0.05，**P<0.01 (enhanced action);^aP<0.05，^aaP<0.01 (reduced action). The same applies below.

(2) inhibition of PRSV by Patrinia scabiosaefolia extract

After Marc-145 cells grew to a monolayer in a 96-well plate, the old culture solution was discarded, and 100TCID was added₅₀100 mu L of PRRSV solution per hole, culturing for 2h, discarding virus solution, adding a traditional Chinese medicine extract solution with the maximum safe mass concentration as the initial concentration, diluting by 2 times with 100 mu L per hole for 4 concentration gradients, culturing for 4h, discarding liquid medicine, and adding DMEM growth maintenance solution containing 2% FBS. And calculating the inhibition rate of the medicament on the virus through a formula.

The results are shown in table 4, the maximum inhibition rates of the patrinia extract and the ribavirin on the PRRSV are 109.14% and 68.85%, respectively, and the inhibition rate of the patrinia extract on the ribavirin is significantly higher than that of the ribavirin (P <0.01), which indicates that the patrinia extract has a stronger inhibition effect on the PRRSV.

TABLE 4 inhibition of drug on PRRSV (%)

(3) Direct inactivation of PRRSV by Patrinia extract

After Marc-145 cells grew to a monolayer in a 96-well plate, the old culture solution was discarded, and 50. mu.L of the Chinese medicinal extract solution and 50. mu.L of the virus solution (the maximum concentration of the liquid drug was the maximum safe mass concentration and the final concentration of the virus solution was 100 TCID) were added simultaneously₅₀) After culturing for 4h, the liquid medicine is discarded, and a DMEM growth maintenance liquid containing 2% FBS is added. And calculating the inhibition rate of the medicament on the virus through a formula.

The results are shown in table 5, the maximum inhibition rates of the patrinia extract and the ribavirin on the PRRSV are 113.25% and 65.46%, respectively, and the inhibition rate of the patrinia extract on the ribavirin is significantly higher than that of the ribavirin (P <0.01), which indicates that the patrinia extract has stronger direct inactivation effect on the PRRSV.

TABLE 5 inhibition of PRRSV by direct inactivation of the drug (%)

Example 4: influence of herba Patriniae extract on N gene, N protein expression and apoptosis under PRRSV action

1. Influence of herba Patriniae extract on N gene expression under PRRSV action

1.1 primer design

PRRSV N gene primers were designed using the software Primer Premier 5.0, see table 6, with reference to the NCBI PubMed gene bank.

TABLE 6 primer sequences and PCR product sizes

1.2 preparation of standards

extracting RNA from the virus liquid, carrying out reverse transcription on the extracted RNA, carrying out PCR amplification by using a specific primer aiming at the N gene code, connecting a pMD-18T vector, transforming DH5 alpha competent cells, extracting a plasmid by using the kit, and measuring the concentration. And carrying out PCR amplification on the plasmid, detecting the size of the amplified product fragment by agarose gel electrophoresis, determining the concentration of the N gene recombinant plasmid, and calculating the template amount of the standard substance.

Sequencing results show that the homology of the recombinant plasmid gene sequence and the corresponding sequence of the NCBI database reaches 99.6 percent. The recombinant plasmid was amplified by ordinary PCR, and the amplified product was electrophoresed to see a clean band, which was identical to the expected band size, as shown in FIG. 1. The recombinant plasmid OD260/OD280 value was 1.89, and the copy number of the recombinant plasmid was calculated to be 2.94X 1 based on the conversion formula of the concentration and the copy number0⁹copies/μL。

1.3 recombinant plasmid concentration determination and Standard Curve establishment

a standard curve was prepared. A standard substance (10-1 to 10-6) diluted by 10 times of EASY Dilution is used as a template, an ABI 7300 fluorescence quantitative PCR instrument is used for amplification, and the reaction system is shown in the following table 7:

TABLE 7 fluorescent quantitative PCR reaction System

Performing fluorescent quantitative PCR reaction by a two-step method: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing and extension at 60 ℃ for 31s, 40 cycles, ending extension, and collecting fluorescence signals; and finally, preparing a melting curve in the process of 60-90 ℃.

1.4 Effect of Patrinia extract on N Gene expression under PRRSV action

Discarding old culture solution from a six-hole plate of the Marc-145 cells growing to a single layer, and adopting a mode of adding the liquid medicine first and then adding the virus solution, adding the virus solution first and then adding the liquid medicine and simultaneously adding the virus solution and the liquid medicine to ensure that the concentration of the liquid medicine is the maximum safe concentration and the final concentration of the virus solution is 100TCID₅₀，5％CO₂Incubations were continued for 24h, 48h, 72h at 37 ℃ respectively, with 3 replicates per time point. Meanwhile, a positive drug ribavirin control group (ribavirin and virus solution), a cell control group (only adding cell maintenance solution) and a virus control group (only adding virus solution) are arranged.

Extracting total RNA from cells according to the TaKaRa MiniBEST Universal RNAextraction Kit, reverse-transcribing the cDNA of each treatment group, and using RNase-free H₂And O, diluting the samples of each group to the loading concentration, and performing reaction according to the following reaction system: SYBR Green Premix ExTaq II (2X) 10. mu.L, PCRFR Primer (10. mu.M) 0.8. mu.L, PCR Reverse Primer (10. mu.M) 0.8. mu.L, ROX Reference Dye (50X) 0.4. mu.L, cDNA 2. mu.L, sterile water 6. mu.L, and 20. mu.L total reaction timeAnd calculating the copy number of the PRRSV N gene in each group of samples by using the standard curve made in the same batch and referring to the Ct value of each group. The results are shown in figure 2, figure 3 and figure 4, the patrinia extract and the ribavirin can both inhibit the synthesis of the N gene, and the patrinia extract has stronger effect than the ribavirin, which indicates that the patrinia extract has the effect of resisting PRRSV.

2. influence of herba Patriniae extract on N protein expression under PRRSV action

Culturing cells according to the method in the step 1.4, removing the culture solution, completely sucking, adding 4 ℃ precooled PBS (PBS), slightly washing, removing, placing the plate on ice, adding 100 mu L of lysis solution containing PMSF (1mL of lysis solution containing 100 mu L of PMSF) into each cell, lysing for 30min on ice, and frequently shaking to fully lyse the cells; taking beta-actin as an internal reference to carry out western blot detection.

The results are shown in figure 5, figure 6 and figure 7, the herba patriniae extract and ribavirin can both inhibit the expression of the N protein, and the herba patriniae extract has stronger effect than ribavirin, and the results are consistent with the gene level detection results.

3. Effect of Patrinia extract on apoptosis under PRRSV action

culturing cells according to the method in the 1.4, collecting cell samples according to the instructions of the Annexin V-EGFP/PI double-staining apoptosis detection kit, and detecting the influence of the patrinia extract on apoptosis after the cells are infected with PRRSV by using a flow cytometer.

As shown in fig. 8, fig. 9, fig. 10, fig. 11, table 8, table 9, table 10, and table 11, the cells showed apoptosis 24h after PRRSV infection, but not very significant, apoptosis 48h and 72h after virus infection, and significant late apoptosis 72h in the virus control group. Within 24-72 h, the results of the research on the PRRSV resistance by the three action modes of the patrinia extract show that the late apoptotic cell rate is not significantly different from that of a cell control group, and the total apoptotic rate is significantly lower than that of a virus control group (P < 0.05). This suggests that the herba Patriniae extract may achieve the effect of resisting PRRSV by inhibiting late apoptosis.

TABLE 8 apoptosis assay results for Virus control and cell control groups

Note: compared with the cell control,^#P<0.05，^##P<0.01；

TABLE 9 apoptosis assay results in the blocking Effect

Note: compared with the PRRSV control group,^*P<0.05，^**P<0.01; compared with the cell control group,^#P<0.05，^##P<0.01; compared with the ribavirin control group,^aP<0.05，^aaP<0.01；

TABLE 10 apoptosis test results in inhibiting effect

Note: compared with the PRRSV control group,^*P<0.05，^**P<0.01; compared with the cell control group,^#P<0.05，^##P<0.01; compared with the ribavirin control group,^aP<0.05，^aaP<0.01

TABLE 11 apoptosis assay results with direct inactivation

example 5: in vivo assay

1. test pig pathogen detection

Purchasing 12 large-growing ternary hybrid weaned pigs of 28-day old from a PRRS non-immune pig farm, feeding for 3-5 days by using a negative pressure isolator, and observing whether the pigs are abnormal or not; before experiment, the vena cava is used for blood sampling and serum separation, DNA/RNA is extracted by using a kit, a PRRSV specific primer (F: GGGGAATGGCCAGYCAGTCAA, R: GCCAGRGGAAAATGKGGCTTCTC) is synthesized, a virus genome stored in a laboratory is used as a positive control, the pathogen infection condition of a test pig is detected by using PCR/RT-PCR, and a PRRSV specific antibody is detected by using a Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) enzyme-linked immunosorbent assay kit.

2. Experimental protocol

Dividing 9 32-day-old weaned piglets into 4 groups, feeding 3 piglets in each group in 4 negative pressure isolators respectively, injecting herba Patriniae extract in group 1, injecting ribavirin medicinal liquid as positive control medicament in group 2, injecting no toxicity counteracting control group in group 3, and injecting no toxicity counteracting control group in group 4. After the 4 th day of administration, the piglets of the 1 st, 2 nd and 3 rd groups injected with intramuscular injection and inoculated with nasal drip SH1705 strain PRRSV (10)⁵TCID₅₀)。

3. Body temperature monitoring

After the experimental piglets are infected with the PRRSV, the rectal temperature is measured once every day at regular time, data are recorded, and a body temperature change trend graph is drawn. The results are shown in fig. 12, the body temperature of piglets in the challenge control group and the drug group was significantly increased at the start of day 4 of the virus infection, and the body temperature was decreased and gradually returned to normal at day 11. The body temperature of each group of piglets is observed, and the effect of the patrinia extract on resisting PRRSV in vivo cannot be seen.

4. Clinical symptom scoring

Clinical symptoms were observed 2 times daily and recorded, and scored according to the clinical symptom scoring criteria of table 12.

TABLE 12 clinical symptom Scoring criteria

The results are shown in fig. 13, the piglets in the challenge control group began to have clinical symptoms such as diarrhea, emaciation and the like (1-4 points) on day 3, then gradually became serious (4-10 points), had clinical symptoms such as rash, skin ulceration, shortness of breath, hair loss and the like, and had symptom relief on day 17 and continued to day 22; the positive control drug ribavirin group piglets begin to have clinical symptoms such as diarrhea, rash and the like on the 8 th day and continue to the 22 th day (2-3 min); the piglets of the patrinia extract group show slight symptoms and gradually return to normal (1-3 points) on the 3 rd day, and the clinical symptom score is obviously lower than that of the challenge control group (P <0.05) on the 4 th-16 th day. The placebo pigs did not develop significant symptoms during the trial.

5. Sample collection and detection

Collecting 5mL of blood separated serum from the anterior vena cava of each piglet on days 1, 3, 5, 8, 11, 15, 18 and 22 after PRRSV infection, and collecting 5mL of anticoagulation blood for flow detection on days 8, 15 and 22, and meanwhile, re-suspending 1g of feces collected from each group by PBS; performing cesarean examination on the killed or experimental animal, collecting samples of heart, liver, spleen, lung, kidney, lymph node, etc., and freezing at-80 deg.C for later use. Collecting lung tissue blocks, and cutting into pieces of 1mm³the small blocks are fixed by 4% buffer solution at 4 ℃, fixed tissue samples are embedded by paraffin, and pathological histological changes of the small blocks are observed after conventional HE staining.

5.1 Lung dissection and pathological section Observation

The lung changes of the piglets in each group are shown in fig. 14, and the pathological section observation results are shown in fig. 15. As can be seen from FIG. 14, the lungs of the piglets in the toxicity counteracting control group had significant bleeding and swelling, and a large number of necrotic foci were visible on the surface, while the lungs of the piglets in the herba Patriniae extract group and the positive control drug ribavirin group had no significant macroscopic change, and only a small number of bleeding spots and necrotic foci were visible. FIG. 15 shows: interstitial pneumonia of lung of the virus attacking control piglet is obvious, alveolar walls of the lung are thickened, lymphocytes are infiltrated, a large amount of exudates appear in bronchus, pathological damage of a positive control group is reduced compared with that of the virus attacking control group, pathological damage of a herba patriniae group is lighter than that of the positive control group, lung of the piglet of a blank control group is normal, and no pathological damage is found, so that the herba patriniae extract and the positive control medicament ribavirin can reduce tissue damage and tissue lesion caused by PRRSV infection in vivo.

5.2 viral load detection

Viral RNA was extracted from each sample using TaKaRa MiniBEST viral RNA/DNA Extraction Kit, wherein 200. mu.L of serum was taken, 100mg of tissue and organ were taken, and the number of copies of viral genome in each sample was determined by the above-established fluorescent quantitative RT-PCR.

The viremia test result is shown in fig. 16, the serum of the piglet of the challenge control group is positive in the PRRSV test at the 3 rd day, the copy number of the virus RNA is increased to the maximum (4.6-5.1log10/mL) at the 8 th day, no piglet dies in the period, and the serum virus content of the piglet is reduced to 1.1-1.6log10/mL at the 22 th day; the positive control medicament ribavirin group and the patrinia extract group also detect positive PRRSV at 3 days after infection, the copy number of virus RNA is increased to the maximum at 8 days, the serum virus content is respectively 4.1-5.1log10/mL and 4.0-4.8log10/mL, and is respectively reduced to 0.4-0.9log10/mL and 0.1-0.9log10/mL at 22 days, and the serum virus content of the patrinia extract group is obviously lower than that of the positive control medicament ribavirin group and the infection group (P <0.05) at 12 days-20 days.

The organ viral load detection results are shown in fig. 17, and the viral loads of heart, liver, spleen, lung, kidney and lymph node of the challenge control group are respectively 1.9-4.2, 1.2-1.9, 4.1-4.2, 4.1-4.7, 1.2-2.1 and 4.4-4.6log 10/g; the positive control drug ribavirin groups are respectively 0.9-2.0, 0.5-1.6, 4.1-4.7, 1.4-2.3, 1.4-1.7 and 3.5-4.9log10/g, wherein the viral load of the liver and the lung is obviously lower than that of the challenge control group (P <0.05 and P < 0.01); the herba patriniae extract groups are respectively 0.6-2.5, 1.1-1.3, 3.2-3.6, 1.8-4.0, 0.9-1.6 and 2.7-3.8log10/g, wherein the viral load of organs such as spleen, lung and lymph node is obviously lower than that of the challenge control group (P <0.05), the viral load of lymph node is obviously lower than that of the positive control medicament ribavirin group (P <0.05), and the viral load of lung is obviously higher than that of the positive control medicament ribavirin group (P < 0.05). The results show that the positive control drugs of ribavirin and patrinia extract have the function of inhibiting PRRSV in vivo, and the PRRSV resistant effect of the patrinia extract is slightly stronger than that of ribavirin.

5.3PRRSV antibody detection

After infecting PRRSV SH1705, the serum of each group of piglets is periodically collected, and the PRRSV antibody level is detected according to the specification of a Jianglai Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) enzyme-linked immunosorbent assay kit.

The results are shown in fig. 18, at day 1 after challenge, antibody detection of the challenge control group, the positive control medicament ribavirin group and the patrinia extract group are positive, then antibody levels of the challenge control group and the patrinia extract group are rapidly increased, the antibody levels reach the highest values at day 8, and at days 3-8, the antibody level of the patrinia extract group is significantly higher than that of the challenge control group (P <0.05), which indicates that the patrinia extract can stimulate body immunity and promote antibody production, so that the antibody level is rapidly increased. The antibody levels in the field of the Patrinia extract decreased gradually at days 9-15, and tended to level off at days 15-22, indicating that the Patrinia extract might exert a direct virucidal effect during this period. Similarly, the positive control drug ribavirin antibody level was significantly lower than the challenge control at days 1-15 (P <0.05), indicating that ribavirin does not have an immune modulating effect, but rather exerts its direct virucidal effect. The results of the PRRSV antibody detection of the piglets of the blank control group in the test period are all negative (not shown).

The results show that the patrinia extract has the function of resisting PRRSV virus in vivo and in vitro. In vivo, it is achieved by blocking viral adsorption, inhibiting viral replication, killing viruses directly, and inhibiting PRRSV-induced apoptosis. In addition, the patrinia extract can also play a role in resisting PRRSV virus by inhibiting N gene synthesis and N protein expression. In order to further verify the effect of the patrinia extract on resisting PRRSV, a virus challenge protection test of a PRRSV SH1705 strain is carried out, and results of lung lesion observation, viremia, organ virus load and the like show that the patrinia extract can effectively inhibit the proliferation of PRRSV in vivo and has the effect of regulating the immunity of an organism.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. Application of herba Patriniae or its extract in preparing medicine for treating and/or preventing porcine reproductive and respiratory syndrome virus and medicine for resisting porcine reproductive and respiratory syndrome virus is provided.

2. The use of claim 1, wherein the herba Patriniae extract is an aqueous herba Patriniae extract.

3. The use according to claim 1, wherein the porcine reproductive and respiratory syndrome virus is a direct porcine reproductive and respiratory syndrome virus.

4. The use of claim 1, wherein the herba Patriniae extract is an aqueous herba Patriniae extract.

5. A Chinese medicinal composition for treating porcine reproductive and respiratory syndrome is characterized by comprising herba Patriniae and extracts of one or more Chinese medicinal components selected from radix rehmanniae Preparata, radix Pseudostellariae, radix Berberidis kunmingensis, pericarpium Citri Tangerinae, radix Curcumae, herba Euphorbiae Humifusae, radix fici simplicissimae, herba Gynostemmatis, radix seu herba Spiranthis Lanceae, radix Acanthopanacis Senticosi, rhizoma Polygonati Odorati, herba Lobeliae chinensis, calyx seu fructus physalis and rhizoma paridis.

6. The Chinese medicinal composition according to claim 5, wherein the Chinese medicinal composition comprises herba Patriniae and extracts of three or more Chinese medicinal components selected from radix rehmanniae Preparata, radix Pseudostellariae, radix Berberidis kunmingensis, pericarpium Citri Tangerinae, radix Curcumae, herba Euphorbiae Humifusae, Ficus Simplicissimae, herba Gynostemmatis, radix seu herba Spiranthi Lanceae, radix Acanthopanacis Senticosi, rhizoma Polygonati Odorati, herba Lobeliae chinensis, calyx Seu fructus Physalis, and rhizoma paridis.

7. The traditional Chinese medicine composition according to claim 5 or 6, wherein the traditional Chinese medicine components comprise the following components in parts by weight:

8-25 parts of dahurian patrinia herb, 8-25 parts of prepared rehmannia root, 8-25 parts of heterophylly falsestarwort root, 6-20 parts of radix berberidis, 5-15 parts of dried orange peel, 5-15 parts of turmeric root-tuber, 6-20 parts of humifuse euphorbia herb, 8-25 parts of hispid fig root-bark, 8-25 parts of gynostemma pentaphylla, 8-25 parts of Chinese ladybell root, 8-25 parts of acanthopanax root, 8-25 parts of rhizoma polygonati, 8-25 parts of fragrant solomonseal rhizome, 6-20 parts of Chinese lobelia herb, 6-20 parts of calyx seu fructus physalis and 6-20 parts of paris polyph.

8. The Chinese medicinal composition of claim 5 or 6, wherein the extract is an aqueous extract.