WO2023024632A1 - 一种治疗过敏性鼻炎的药物组合物及其制备方法 - Google Patents

一种治疗过敏性鼻炎的药物组合物及其制备方法 Download PDF

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WO2023024632A1
WO2023024632A1 PCT/CN2022/096580 CN2022096580W WO2023024632A1 WO 2023024632 A1 WO2023024632 A1 WO 2023024632A1 CN 2022096580 W CN2022096580 W CN 2022096580W WO 2023024632 A1 WO2023024632 A1 WO 2023024632A1
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parts
magnolia
extract
under reduced
reduced pressure
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贾振华
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河北以岭医药研究院有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • A61K35/64—Insects, e.g. bees, wasps or fleas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/232—Angelica
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/237—Notopterygium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/238—Saposhnikovia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/284—Atractylodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/481—Astragalus (milkvetch)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/484—Glycyrrhiza (licorice)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/57—Magnoliaceae (Magnolia family)
    • A61K36/575—Magnolia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A61K36/736—Prunus, e.g. plum, cherry, peach, apricot or almond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9062—Alpinia, e.g. red ginger or galangal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • the invention relates to a traditional Chinese medicine composition for treating allergic rhinitis and a preparation method thereof, belonging to the field of Chinese herbal medicine application.
  • Allergic rhinitis also known as allergic rhinitis (AR), referred to as allergic rhinitis
  • AR allergic rhinitis
  • IgE allergic rhinitis
  • It is a common clinical disease, which used to be divided into seasonal and perennial. In recent years, it is divided into intermittent and persistent according to the characteristics of symptoms. The main clinical features are sudden and recurrent sneezing, runny nose, nasal congestion, and nasal itching. In severe cases, the sense of smell decreases or disappears. Most of the disease occurs in children and young adults, and there is no significant difference in the incidence ratio between males and females.
  • allergic rhinitis is a non-fatal disease, it has a high recurrence rate and is prone to complications such as allergic sinusitis, bronchial asthma, secretory otitis media, and cough variant asthma, which can seriously affect the lives of patients Quality, causing severe insomnia, insomnia at night or obstructive sleep-disordered breathing syndrome, leading to decreased work efficiency, memory loss, etc., bringing functional, emotional and psychosocial negative impacts on daily life and work.
  • a survey on the prognosis of children with allergic rhinitis shows that 19% of children develop asthma, and 40% to 50% of allergic rhinitis patients are combined with asthma. 10% to 39% of patients will develop bronchial asthma or even pulmonary heart disease.
  • allergens including inhalation allergens, food allergens and occupational allergens, etc., which are the direct causes of this disease; should quality.
  • basic links such as immune cells and inflammatory mediators involved in the basic immune response have been elucidated one by one, the pathogenesis of allergic rhinitis has gradually become clear. IgE antibody binding, mast cell degranulation, and release of inflammatory response mediators lead to allergic symptoms in patients; at the same time, there are various cytokine networks regulating the basic immune response.
  • Western medicine mainly includes glucocorticoids, antihistamines, anti-leukotriene drugs, mast cell membrane stabilizers, decongestants, and anticholinergic drugs.
  • the treatment of allergic rhinitis with western medicine mainly adopts anti-allergic and local symptomatic treatment.
  • antihistamine drugs and hormone drugs can temporarily control the symptoms, side effects such as drowsiness, drowsiness, and dry mouth often occur after taking them. Side effects caused by long-term use of hormones It often exceeds the harm of the disease itself to the human body, and the recurrence rate of western medicine is high after taking it, so it is not suitable for long-term application.
  • the composition of the present invention is aimed at the main pathogenesis of allergic rhinitis (nasal rhinitis), although the lesion is in the nose, the lung and spleen are responsible, the lung and spleen are deficient, the defense is not solid, and the wind and cold attack the outside, and it is believed that the lung qi deficiency cannot be declared Wei Qi leads to disharmony between Ying and Wei, loose interstitial muscles, weak guard surface, exogenous evils take advantage of the deficiency and invade the nasal orifice, wind-cold pathogens suppress the lungs, and the lung orifices are blocked, and the lung qi loses its proper descending and clearing function.
  • the prescription is added with pungent and warm products to strengthen the surface, dispel wind and cold, and at the same time add magnolia, angelica dahurica to dispel wind and cold, clear the nose orifices, and galangal to nourish the lung qi , benefit the spleen and stomach, notopterygium dispels wind and cold, cicada slough and paeonol are sweet and cool agents to control the disadvantages of consuming qi and injuring body fluid, ebony acid, astringent and astringent yin to prevent pungent and warm excessive dryness and dark consumption of body fluid.
  • the composition of the present invention is innovative in terms of cubic principle and prescription composition.
  • the preparation technology is advanced, the clinical curative effect is definite, the application is safe, and it has good development and application prospects.
  • the pharmaceutical composition of the present invention is a prescription for treating rhinitis based on the addition and subtraction of the ancient famous prescription Yupingfeng Powder based on many years of experience from the deficiency of the lung and spleen and the invasion of exogenous pathogens, and the clinically proven curative effect.
  • the inventor has been clinically treating rhinorrhea for many years, and has applied several hands-on experience prescriptions, among which the most used ones are the addition and subtraction of Yupingfeng powder combined with Shenling Baizhu powder (heterophylla heterophylla, Poria cocos, Atractylodes macrocephala, Radix Astragali, Fangfeng, galangal, fried white lentils, magnolia, angelica dahurica, moutan cortex, cicada slough), the prescription overlaps with the prescription of the pharmaceutical composition of the present invention with 8 medicines, and the pharmaceutical composition of the present invention removes Radix Pseudostellariae, Poria cocos, white hyacinth bean and adds notopterygium, ebony, At the same time, the amount and proportion of astragalus, atractylodes, and windproofing are adjusted.
  • composition of the present invention even highlighted the jade screen and the lung qi to solidify the lung qi.
  • the prescription of the pharmaceutical composition of the present invention is the representative clinical prescription of the inventor for the main syndrome of deficiency of both the lung and the spleen, and the preliminary clinical research has shown good clinical curative effect and application value.
  • the nose is the portal of breathing, which helps the lungs to breathe. If the nasal orifice is unobstructed, the breathing air will flow in and out smoothly and the lung qi will be unobstructed. Therefore, Yan Yong and "Yan's Jisheng Fang Bimen" in the Song Dynasty said: "The nose of the husband , the master of the lungs, the role of clearing and detoxification, if adjusted properly, the lungs should be smooth, clearing and transporting for their own benefit.” At the same time, the lung governs the breath and communicates with the nose.
  • the lung governs the nose, and the nose is the external orifice of the lung. Its function is just like the “Lingshu ⁇ Pulse Degree Chapter” said: “Lung qi flows through the nose, and when the lungs are in harmony, you can know the smell.” It can be seen that the coordination of lung and nose functions can exert normal physiological functions.
  • the disease is based on the deficiency of the lung and spleen, and the infirmity of the outside, and the invasion of external evils while taking advantage of the deficiency. Therefore, it is effective to treat both the symptoms and the root causes by invigorating qi, consolidating the surface and invigorating the spleen. .
  • the pharmaceutical composition for treating allergic rhinitis comprises the following components in parts by weight: 10-20 parts of Radix Astragali, 5-15 parts of Atractylodes Rhinitis, 5-15 parts of Fangfeng, 5-15 parts of Magnolia Magnolia, and 5-15 parts of Angelica dahurica galangal 5-15 parts, notopterygium 5-15 parts, moutan bark 5-15 parts, cicada slough 5-15 parts, dark plum 5-15 parts, licorice 4-8 parts.
  • composition of the present invention preferably includes the following components by weight: 10 parts of Radix Astragali, 15 parts of Atractylodes Rhizome, 5 parts of Fangfeng, 15 parts of Magnolia Magnolia, 5 parts of Angelica dahurica, 15 parts of Galangal, 5 parts of Notopterygium, 15 parts of Cortex Moutan, and 5 parts of Cicada slough 15 parts of ebony, 4 parts of licorice.
  • composition of the present invention may also preferably include the following components in parts by weight: 20 parts of Radix Astragali, 5 parts of Atractylodes Rhizome, 15 parts of Fangfeng, 5 parts of Magnolia Magnolia, 15 parts of Angelica dahurica, 5 parts of Galangal, 15 parts of Notopterygium, 5 parts of Cortex Moutan, 15 parts of cicada slough, 5 parts of dark plum, 8 parts of licorice.
  • composition of the present invention can also preferably include the following components in parts by weight: 15 parts of Radix Astragali, 10 parts of Atractylodes Rhizome, 10 parts of Fangfeng, 10 parts of Magnolia Magnolia, 10 parts of Angelica dahurica, 10 parts of Galangal, 10 parts of Notopterygium, 10 parts of Cortex Moutan, 10 parts of cicada slough, 10 parts of black plum, 6 parts of licorice.
  • composition of the present invention is more preferably composed of the following components in parts by weight: 16 parts of Radix Astragali, 8 parts of Atractylodes Rhizome, 8 parts of Fangfeng, 8 parts of Magnolia Magnolia, 8 parts of Angelica dahurica, 8 parts of Galangal, 8 parts of Notopterygium, 8 parts of Cortex Moutan, 8 parts of cicada slough, 8 parts of black plum, 5 parts of licorice.
  • compositions of the present invention are capsules, tablets, pills, oral liquids, granules, injections or powders.
  • the active ingredient of the composition of the present invention is made by following steps:
  • the present invention also provides that the preparation process of the Chinese medicine composition into tablets is as follows:
  • E. Use peony cortex fine powder as the base material, granulate according to the conventional process, granulate, and tablet.
  • the preparation technology of this tablet is preferably:
  • E. Use peony cortex fine powder as the base material, granulate according to the conventional process, granulate, and tablet.
  • the invention also provides the application of the pharmaceutical composition in the preparation of anti-inflammation or anti-inflammatory chronic proliferation medicine.
  • the invention also provides the application of the pharmaceutical composition in preparing anti-type I allergy medicine.
  • the invention also provides the application of the pharmaceutical composition in the preparation of medicines for reducing the levels of histamine, total IgE and OVA-specific IgE, IL-10 and IL-12, reducing the expression of IL-4 in nasal mucosa and increasing the expression of IL-2.
  • the invention also provides the application of the pharmaceutical composition in medicines for increasing the level of IFN- ⁇ .
  • the invention also provides the application of the pharmaceutical composition in alleviating allergic symptoms.
  • the present invention adopts the method of "replenishing qi and strengthening the exterior, invigorating the spleen and clearing the orifices".
  • Replenishing qi, consolidating the exterior and invigorating the spleen can cure the root cause, dispelling evils and opening the orifices can treat the symptoms, strengthening the body and expelling evils, and treating both symptoms and root causes can be effective.
  • Astragalus is used as the monarch drug. It is sweet in nature, slightly warm, and its function is to invigorate qi. It is a holy medicine for invigorating qi. Where the evil gathers together, its qi must be deficient. If the lung qi is deficient, the body will not be solid, if the evil and the righteous fight, the sneezing will come out, if the qi is not absorbed, the nose will flow, and the body fluid will stagnate, and the nose will be blocked. Drive it away, but there is no way to prevent it, and you are not afraid of the wind and cold, but you are afraid of its return. Astragalus can nourish the triple burner and protect it. It is the key to resisting the wind and cold in the Xuanfu. It is most suitable to replenish qi and solidify the surface to prevent wind and cold evil from attacking.
  • Fangfeng minister medicine is pungent and sweet in taste, slightly warm in nature, and should be expelled from the outside when wind and cold hit the surface. However, people with loose interstitial joints are more likely to hurt the surface by sweating.
  • Expelling wind evil whichever is the "moisturizing agent in wind medicine"
  • Astragalus and Atractylodes macrocephala to replenish qi and eliminate evil
  • Astragalus can prevent wind and solidify the surface without retaining evil
  • Injury to Zheng has the meaning of nourishing in sparseness and scattered in nourishing, and its merits are obvious. In order to prevent the wind and drive the wind, if you get Astragalus to strengthen the exterior, you will have protection from the outside;
  • Magnolia magnolia and Baizhu Fangfeng are both ministerial medicines, with pungent, warm, light aroma, thin smell, ascending head, with the effect of diverging wind and cold, and clearing the nasal orifices.
  • Tao Hongjing's "Famous Doctors” said: “Warming the middle to relieve the muscles , sharpen the nine orifices, clear nasal congestion and runny nose”, the incense is specially used to enter the lungs, ventilate the lung qi, and is good at clearing the nasal orifices, especially the key medicine for nasal congestion, clear nasal discharge and other symptoms, as stated in "Compendium of Materia Medica” : “Magnolia's pungent temperature moves the qi and enters the lungs, which can help the clear yang in the stomach go up and pass through the sky, so it can warm the middle and treat the diseases of the head, face, nose and nose.”
  • Adjuvant medicine Angelica dahurica, pungent and warm in taste, Xin Neng Xing San, temperature can dispel cold, fragrance travels, assists magnolia to relieve exterior cold, dispels wind and clears nasal orifices, "Compendium of Materia Medica” says: “Angelica dahurica... the aroma is up, Start with Taiyin Lung Meridian”, “Materia Medica Huiyan” says: “Angelica dahurica ascends to the head, descends to the intestines and stomach, reaches the limbs in the middle, penetrates the skin and even the hair orifices, and promotes the release of evil spirits.” Galangal is pungent in taste and hot in nature.
  • “Materia Medica Huiyan” says: “Galangal is a medicine for dispelling cold and dampness and warming the spleen and stomach.” Moderate temperature dispels cold evil, assists Radix Astragali and Atractylodes Rhizoma Rhizoma Atractylodes Rhizoma Rhizoma Rhizoma Rhizoma Atractylodes Rhizoma Rhizome.
  • Notopterygium bitter in taste, warm, has the functions of relieving exterior cold, expelling wind and relieving pain
  • "Lei Gong Pao Medicine Properties Solution” says: “Qi is clear and belongs to yang, good for qi, comfortable but not restrained, ascending and sinking, male and good Scattering, can release evil... use the principle to travel the wind", Qianghuo Xin is used to disperse the wind, clear and upward, directly reaching the head and face, and can dispel wind evil.
  • Cortex Moutan, bitter, pungent, slightly cold has the functions of clearing away heat and cooling blood, promoting blood circulation and dispelling stasis.
  • Cicada sloughs sweet in taste, cold in taste, can search wind and dredge collaterals, ventilate lungs and dissipate heat.
  • Black plum, sour, astringent, flat can "benefit essence and appetizers and transform body fluid" ("Ben Jing Feng Yuan”), and it can be used to prevent excessive body fluid from being overly dry and pungent.
  • Herbs licorice, Ganping, harmonizes urgency, nourishes the lungs, and is good at harmonizing various medicines, so it is used.
  • All medicines are used in combination to invigorate the lung and strengthen the spleen, replenish qi and solidify the surface to cure the root cause, dispel wind and disperse pathogenic factors, activate blood and unblock the orifices to treat the symptoms, treat both the symptoms and root causes, and prevent all symptoms from recurring at the same time.
  • the tablet is a dry solid with stable and controllable quality.
  • Some drugs that are prone to oxidative deterioration and deliquescence can be protected by coating. Light, air, moisture, etc. have little effect on them, and can also cover up the bad smell of traditional Chinese medicine.
  • tablet production is mechanized and has a high degree of automation, which is suitable for industrialization promotion.
  • Astragalus 206g Atractylodes macrocephala 137g, Fangfeng 137g, magnolia 137g, Angelica dahurica 137g, galangal 137g, notopterygium 137g, moutan cortex 137g, cicada slough 137g, ebony plum 137g, licorice 82g.
  • Angelica dahurica and ebony plums weighed according to the prescription amount, add 61 times the amount of 601% ethanol, extract twice, each time for 1.5 hours, filter, extract the ethanol under reduced pressure, concentrate under reduced pressure to a relative density of 1.15 to 1.20, and set aside;
  • E. Use peony cortex fine powder as the base material, granulate according to the conventional process, granulate, and press into 1000 pieces.
  • Astragalus 240g Atractylodes macrocephala 120g, Fangfeng 120g, magnolia 120g, Angelica dahurica 120g, galangal 120g, notopterygium 120g, peony bark 120g, cicada slough 120g, ebony plum 120g, licorice 75g.
  • E uses the fine powder of peony cortex as the base material, granulates according to the conventional process, granulates, and packs into 1000 capsules.
  • Astragalus 137g Atractylodes macrocephala 205g, Fangfeng 68g, magnolia 205g, Baizhi 68g, galangal 205g, notopterygium 68g, moutan cortex 205g, cicada slough 68g, black plum 205g, licorice 55g.
  • Astragalus 300g Atractylodes macrocephala 75g, Fangfeng 225g, magnolia 75g, Angelica dahurica 225g, galangal 75g, notopterygium 225g, moutan bark 75g, cicada slough 225g, black plum 75g, licorice 120g.
  • Astragalus 180g Atractylodes macrocephala 90g, Fangfeng 90g, magnolia 90g, Angelica dahurica 90g, galangal 90g, notopterygium 90g, moutan bark 90g, cicada slough 90g, ebony plum 90g, licorice 60g.
  • E uses peony cortex fine powder as the base material, and makes a spray according to the conventional process.
  • Pseudostellaria 200g Poria 120g, Atractylodes macrocephala 80g, Astragalus 80g, Fangfeng 80g, galangal 100g, fried white lentils 100g, magnolia 100g, angelica dahurica 100g, moutan bark 100g, cicada slough 80g.
  • B Pseudostellariae, Poria cocos, fried white lentils, astragalus membranaceus, and cicada slough, weighed according to the prescription, add 11 times the amount of water and decoct three times, 1 hour each time, filter, extract under reduced pressure and concentrate, combine with volatile oil concentrate under reduced pressure Concentrate to a relative density of 1.15-1.20, set aside;
  • E. Use peony cortex fine powder as the base material, granulate according to the conventional process, granulate, and press into 1000 pieces.
  • Test example 1 the influence of Chinese medicine composition of the present invention on experimental allergic rhinitis in rats
  • the Chinese medicine composition embodiment 1 and embodiment 2 of the present invention can effectively reduce the behavioral score (P ⁇ 0.05); Compared with the model group, there is a significant statistical difference; the histamine, IgE, IL-10 of the Chinese medicine composition embodiment 2 of the present invention has a significant statistical difference (P ⁇ 0.05 or P ⁇ 0.01) compared with the model group. Compared with the model group, IL-12 in each medication group was increased, and there was a significant statistical difference (P ⁇ 0.05) between the Chinese medicine composition embodiment 2 group of the present invention and the model group.
  • the pharmaceutical composition of the present invention can reduce the expression of IL-4 in the nasal mucosa to varying degrees and increase the expression of IL-2.
  • the traditional Chinese medicine composition of the invention can improve the histomorphological lesions of the nasal mucosa to varying degrees.
  • the traditional Chinese medicine composition of the present invention has a certain therapeutic effect on allergic rhinitis induced by ovalbumin in rats.
  • Ovalbumin (OVA) is used to create a rat model of allergic rhinitis, the influence of the traditional Chinese medicine composition of the present invention on the rat model is observed, and its drug efficacy is evaluated.
  • Ketotifen fumarate tablets specification 1mg*60 tablets, produced by Shanghai Fudan Fuhua Pharmaceutical Co., Ltd., batch number: 090701.
  • Comparative example drug prepare according to the comparative example method.
  • OVA-specific IgE test kit BlueGene, Lot: 20100112.
  • Rats were kept in cages, 5 rats/cage, in the New Drug Evaluation Center of Hebei Academy of Integrated Traditional Chinese and Western Medicine, with 12 hours of light per day, temperature of 20-25°C, and relative humidity of 40-70%.
  • the quarantine period for newly arrived animals is 4 days. During the quarantine period, observe the animals' drinking water, food intake and health status, as well as whether there are signs of disease and death.
  • Drinking water fill with ordinary water, and animals can drink freely. Rinse the water bottle and change the water daily.
  • the rats were randomly divided into 6 groups according to body weight, 15 in each group. See Schedule 1.
  • Administration by intragastric administration is basically consistent with the clinical route of administration.
  • GMBY1, GMBY2, and the comparative example were prepared with 0.5% CMC-Na so that the final concentration was 0.244g crude drug/ml (0.0776g medicinal powder/ml). Store at 4°C for later use.
  • ketotifen fumarate is ready-to-use and ready-to-use, take 3 tablets, grind it finely, dilute to 56ml with 0.5% CMC-Na, and mix well.
  • the medicinal solution was extracted with a disposable syringe for intragastric administration, and the administration volume was 1ml/100g body weight.
  • the rats were randomly divided into 6 groups, normal control group, model group, ketotifen group, GMBY1, GMBY2, and control group. Modeling was made according to the following steps: (1) 30 mg of OVA was used as antigen, 3 g of aluminum hydroxide powder was used as adjuvant, and 100 ml of normal saline was added to make a suspension, 1 ml of each mouse was injected intraperitoneally, once every other day, a total of 7 times, as the basis of induction sensitive (the normal control group was replaced by 3% Al(OH) 3 ). (2) The bilateral nasal cavities were challenged with 2% OVA normal saline, 50 ⁇ l on each side, once a day, 7 times in total (the normal control group was replaced by normal saline).
  • Nasal itching Mild: Gently wipe the nose 1-3 times, score 1 point; Severe: Scratch the nose, face more than once, rub everywhere (more than 4), score 2 points; Sneezing: 1-3 points are 1 point, 4- 10 points are 2 points, more than 11 points are 3 points; runny nose: 1 point for flow to the anterior nares, 2 points for more than the anterior nares, 3 points for runny nose.
  • the symptom scores are superimposed. The symptom observation time is within 30 minutes after the last dose of sensitization.
  • Detection of serum histamine, IgE, OVA-specific IgE, IL-10, and IL-12 10% chloral hydrate anesthetized, blood was collected from the abdominal aorta, serum was separated, and tested according to the requirements of the kit.
  • Morphological indicators decapitate, quickly open the back of the nose, take bilateral turbinate and nasal septal mucosa, separate the nasal cavity and nasal septal nasal mucosa in ice bath, put the nasal mucosa tissue into 10% formaldehyde solution to fix, paraffin section, HE staining to observe eosinophilic Morphological changes of granulocytes and nasal mucosa.
  • DP-III slice roasting machine Xiaogan Delisen Electronics Co., Ltd.
  • the experimental data is analyzed and processed by SPSS11.5 statistical software, and the statistical results are expressed as mean ⁇ standard deviation Indicates that the LSD T test was used for homogeneous variance, and the improved Dunnett test was used for heterogeneous variance.
  • histamine, IgE, OVA-specific IgE, and IL-10 in the model group were significantly increased (P ⁇ 0.05 or P ⁇ 0.01), and the above indexes in each medication group were decreased.
  • IgE had significant statistical difference
  • GMBY2 group had significant statistical difference in histamine, IgE, IL-10 compared with the model group (P ⁇ 0.05 or P ⁇ 0.01).
  • IL-12 in the model group was significantly lower (P ⁇ 0.01), and IL-12 in each medication group was higher than that in the model group, and there was a significant statistical difference between the GMBY2 group and the model group (P ⁇ 0.01). ⁇ 0.05).
  • the expression of IL-4 in the nasal mucosa was detected by immunohistochemical method: the positive substance was brownish yellow, granular or filamentous. Positive cells were distributed in the pseudostratified columnar ciliated epithelial cells, Sertoli cells, and basal cells of the mucosal epithelium, as well as endothelial cells, glandular, and ductal cells of the vascular wall in the lamina intestinal and submucosa. Compared with the normal group, the brown-yellow coloration of the model group was darker, and the expression was strongly positive. The brown-yellow granules or filaments in the ketotifen group and the traditional Chinese medicine composition 1 and 2 groups of the present invention were significantly reduced, and the expression was moderately positive.
  • the expression of IL-2 in the nasal mucosa was detected by immunohistochemical method: the positive substance was brownish yellow, granular or filamentous. Positive cells were distributed in the pseudostratified columnar ciliated epithelial cells, Sertoli cells, and basal cells of the mucosal epithelium, as well as endothelial cells, glandular, and ductal cells of the vascular wall in the lamina intestinal and submucosa. Compared with the normal group, the model group had weaker brownish-yellow coloration, and the expression was weakly positive. The brownish-yellow granules or filaments in the ketotifen group and the traditional Chinese medicine composition 1 and 2 groups of the present invention increased significantly, showing moderate positive expression. The results are shown in Table 3.
  • Table 3 The influence of the traditional Chinese medicine composition of the present invention on the serum indexes of rats with experimental allergic rhinitis
  • Fibroblasts and a large number of collagen fibers were seen in the lamina intestinal and submucosa, without inflammatory cell infiltration, and small blood vessels and red blood cells were scattered among them; in the model group, the nasal mucosa was congested and edematous, the epithelial layer was discontinuous, the tissue structure was not clear, and the basement membrane was thickened , edema, destruction, forming inflammatory zone of collagen tissue, which separates the epithelial layer and the subepithelial layer; compared with the model group, the traditional Chinese medicine composition of the present invention can be improved in different degrees from the morphological point of view.
  • the dry ointment powder of the traditional Chinese medicine composition of the invention has a certain therapeutic effect on rat allergic rhinitis induced by ovalbumin.
  • Test example 2 the influence of traditional Chinese medicine composition of the present invention on guinea pig experimental allergic rhinitis
  • guinea pigs were randomly divided into 6 groups, and sensitized with 10% 2,4-diisocyanate (TDI) olive oil solution 10 ⁇ l nasal drops (5 ⁇ l drops in each nasal cavity) for 7 consecutive days After 7 days, drop once every other day to maintain the sensitized state until the end of administration. After the basic sensitization, each group was treated with corresponding drugs for 14 consecutive days. Observe the influence of the dry ointment powder of the Chinese medicine composition of the present invention on behavioral indicators, serological indicators and pathological morphology.
  • TDI 2,4-diisocyanate
  • Example 1 and Example 2 of the Chinese medicine composition of the present invention were significantly lower (P ⁇ 0.05 or P ⁇ 0.01); the Chinese medicine composition of the present invention can reduce histamine, IgE, IL in serum -4 content, embodiment 2 is the most obvious, and wherein IgE, histamine have statistical difference (P ⁇ 0.05) compared with model group, can raise serum IFN- ⁇ level, embodiment 1, embodiment 2 and model group There were significant statistical differences (P ⁇ 0.05); each medication group could improve histomorphological lesions to varying degrees.
  • the traditional Chinese medicine composition of the present invention has a certain therapeutic effect on the model of allergic rhinitis in guinea pigs induced by TDI.
  • TDI 2,4-cresyl diisocyanate
  • Cetirizine hydrochloride capsules specification 10mg, Zhubai Ansheng Phoenix Pharmaceutical Co., Ltd., batch number: 09050206.
  • Comparative example drug prepare according to the comparative example method.
  • Guinea pigs were kept in cages, 3 per cage, in the New Drug Evaluation Center of Hebei Academy of Integrated Traditional Chinese and Western Medicine, with 12 hours of light per day, a temperature of 20-26°C, and a relative humidity of 40-70%.
  • the quarantine period for newly arrived animals is 5 days. During the quarantine period, observe the animals' drinking water, food intake and health status, as well as whether there are signs of disease and death.
  • Drinking water fill with ordinary water, and animals can drink freely. Rinse the water bottle and change the water daily.
  • the guinea pigs were randomly divided into 6 groups according to body weight, 12 in each group. See Schedule 4.
  • Administration by intragastric administration is basically consistent with the clinical route of administration.
  • GMBY1, GMBY2, and the comparison group were formulated with 0.5% CMC-Na to make a final concentration of 0.183g crude drug/ml (0.0582g medicinal powder/ml). Store at 4°C for later use.
  • the positive drug cetirizine hydrochloride capsules are ready-to-use and ready-to-use, take 1 capsule, grind it finely, dilute to 50ml with 0.5% CMC-Na, and mix well.
  • the medicinal solution was extracted with a disposable syringe for intragastric administration, and the administration volume was 1ml/100g body weight.
  • the guinea pigs were randomly divided into 6 groups, normal control group, model group, cetirizine group, GMBY1 and 2 groups, and control group. Modeling was made according to the following steps: 10 ⁇ l nasal drops of 10% TDI olive oil solution (5 ⁇ l drops in each nasal cavity) were used for sensitization for 7 consecutive days, and the sensitized state was maintained by dripping every other day after 7 days until the end of administration. After the basic sensitization, each group was treated with corresponding drugs for 14 consecutive days.
  • Nasal itching Mild: Gently wipe the nose 1-3 times, score 1 point; Severe: Scratch the nose, face more than once, rub everywhere (more than 4), score 2 points; Sneezing: 1-3 points are 1 point, 4- 10 points are 2 points, more than 11 points are 3 points; runny nose: 1 point for flow to the anterior nares, 2 points for more than the anterior nares, 3 points for runny nose.
  • the symptom scores are superimposed. The symptom observation time is within 30 minutes after the last dose of sensitization.
  • Nasal mucosa histomorphology decapitate, quickly open the back of the nose, take bilateral turbinates and nasal septum mucosa, separate the nasal cavity and nasal septum nasal mucosa in ice bath, put the nasal mucosa tissue in 10% formaldehyde solution for fixation, paraffin section, HE staining observation Morphological changes of eosinophils and nasal mucosa.
  • DP-III slice roasting machine Xiaogan Delisen Electronics Co., Ltd.
  • the experimental data is analyzed and processed by SPSS11.5 statistical software, and the statistical results are expressed as mean ⁇ standard deviation Indicates that the LSD T test was used for homogeneous variance, and the improved Dunnett test was used for heterogeneous variance.
  • Table 5 The influence of the traditional Chinese medicine composition of the present invention on the behavior of guinea pig experimental allergic rhinitis
  • Table 6 The influence of the traditional Chinese medicine composition of the present invention on the serum indexes of guinea pigs with experimental allergic rhinitis
  • Fibroblasts and a large number of collagen fibers were seen in the lamina intestinal and submucosa, without inflammatory cell infiltration, and small blood vessels and red blood cells were scattered among them; in the model group, there were large necrosis and defects in the epithelial layer of the nasal mucosa, and the structure was almost unrecognizable.
  • each group of the traditional Chinese medicine composition of the present invention has different degrees of improvement from the morphological point of view.
  • the traditional Chinese medicine composition of the invention has a certain therapeutic effect on the guinea pig allergic rhinitis model induced by TDI.
  • Test example 3 the influence of traditional Chinese medicine composition of the present invention on rat passive skin allergy
  • TCS antiserum inject antiserum (1:10; 1:20 dilution) intradermally on both sides of the dorsal midline of rats 12 days after administration, and inject 0.5% Evans blue prepared by tail vein after 48 hours.
  • Trichosanthin protein solution was used for antigen challenge, animals were sacrificed after 20 min, the back skin was cut off, and the diameter of the coeruleus at each injection point was measured; the coeruleus was cut off, soaked in acetone saline for 48 hours, centrifuged, and the OD value was measured at 620 nm.
  • the traditional Chinese medicine composition of the present invention can alleviate passive skin allergy in rats, and has a certain anti-type I allergy effect.
  • Chlorpheniramine maleate tablets (chlorpheniramine), 4mg/tablet, Hebei Kangtai Pharmaceutical Co., Ltd., batch number 100302, valid until March 1, 2013.
  • Comparative example drug prepare according to the comparative example method.
  • Rats were raised in cages, 6 or 5 rats/cage, raised in the New Drug Evaluation Center of Hebei Academy of Integrated Traditional Chinese and Western Medicine, with 12 hours of light per day, temperature 20-25°C, relative humidity 40-70% .
  • the quarantine period for newly arrived animals is 3 days or 5 days. During the quarantine period, observe the animals' drinking water, food intake and health status, as well as whether there are signs of disease and death.
  • Drinking water fill with ordinary water, and animals can drink freely. Rinse the water bottle and change the water daily.
  • Table 7 Chinese medicine composition of the present invention is to the influence grouping and dosage of rat passive skin allergy
  • Administration by intragastric administration is basically consistent with the clinical route of administration.
  • GMBY1, 2 and the comparative drug were formulated with 0.5% CMC-Na to a final concentration of 0.244 g crude drug/ml (0.0776 g drug powder/ml). Store at 4°C for later use.
  • the positive drug chlorpheniramine is now ready-to-use, take 3 tablets of chlorpheniramine, grind it finely, dilute to 37ml with 0.5% CMC-Na, and mix well.
  • the medicinal solution was extracted with a disposable syringe for intragastric administration, and the administration volume was 1ml/100g body weight.
  • the experimental data is analyzed and processed by SPSS11.5 statistical software, and the statistical results are expressed as mean ⁇ standard deviation Indicates that the difference between groups was compared by One-Way ANOVA LSD T test.
  • Table 8 The influence of traditional Chinese medicine composition of the present invention on the diameter of rat passive skin allergy coeruleus
  • Table 9 The influence of the traditional Chinese medicine composition of the present invention on the absorbance of passive skin allergy coeruleus coeruleus in rats
  • GMBY can alleviate the passive skin allergic reaction in rats, and has a certain inhibitory effect on type I allergic reaction.
  • Test example 4 the influence of Chinese medicine composition of the present invention on mouse ear xenogeneic skin allergy
  • Antiserum of trichosanthin was prepared, mice in each group were sensitized by injection of antiserum in the auricle 12 days after administration, 48 hours later the tail vein was injected with trichosanthin solution prepared by 0.5% Evans blue for challenge, and the mice were killed 30 minutes later , cut off the blue-stained auricle, and put it in KOH solution at 37°C for overnight digestion. The next day, add the acetone phosphate mixture, shake and mix, centrifuge, and measure the OD value of the supernatant at 640nm.
  • the traditional Chinese medicine composition of the present invention has a certain anti-type I allergy effect.
  • Chlorpheniramine maleate tablets (chlorpheniramine), 4mg/tablet, Hebei Kangtai Pharmaceutical Co., Ltd., batch number 100302, valid until March 2, 2013.
  • Comparative example drug prepare according to the comparative example method.
  • the quarantine period for newly arrived animals is 3 days or 5 days. During the quarantine period, observe the animals' drinking water, food intake and health status, as well as whether there are signs of disease and death.
  • Drinking water fill with ordinary water, and animals can drink freely. Rinse the water bottle and change the water daily.
  • mice were randomly divided into 5 groups according to body weight, 14 in each group. See Schedule 10.
  • Administration by intragastric administration is basically consistent with the clinical route of administration.
  • GMBY1, GMBY2, and the comparative drug were prepared with 0.5% CMC-Na to a final concentration of 0.3050 g crude drug/ml (0.0969 g drug powder/ml). Store at 4°C for later use.
  • the positive drug chlorpheniramine is now prepared, take 1 tablet of chlorpheniramine, grind it finely, dilute to 10ml with 0.5% CMC-Na, and mix well.
  • the medicinal solution was extracted with a disposable syringe for intragastric administration, and the administration volume was 0.1ml/10g body weight.
  • the experimental data is analyzed and processed by SPSS11.5 statistical software, and the statistical results are expressed as mean ⁇ standard deviation Indicates that the differences between groups were compared using the One-Way ANOVA Dunnett T3 test.
  • GMBY can reduce the mouse ear xenogeneic skin allergic reaction, and has a certain inhibitory effect on type I allergic reaction.
  • Test example 5 the influence of Chinese medicine composition of the present invention on rat agar granuloma
  • ⁇ Method ⁇ 59 Wistar rats were randomly divided into 5 groups, subcutaneously injected with agar solution, after 14 days of continuous administration, the rats were euthanized by air embolism, the subcutaneous agar granuloma was peeled off and weighed, and the granuloma coefficient was calculated.
  • ⁇ Result ⁇ Aspirin can significantly reduce the weight of granuloma and the coefficient of granuloma, and there is a significant statistical difference compared with the model group (P ⁇ 0.05 or P ⁇ 0.01); Compared with the model group, the granuloma weight of GMBY1 and 2 groups had a significant statistical difference (P ⁇ 0.01); the granuloma coefficient of GMBY2 group had a significant statistical difference compared with the model group (P ⁇ 0.05).
  • the traditional Chinese medicine composition of the present invention has inhibitory effect on the chronic proliferation phase of inflammation.
  • Comparative example drug prepare according to the comparative example method.
  • Rats were raised in cages, 6 or 5 rats/cage, raised in the New Drug Evaluation Center of Hebei Academy of Integrated Traditional Chinese and Western Medicine, with 12 hours of light per day, temperature 20-25°C, relative humidity 40-70% .
  • the quarantine period for newly arrived animals is 4 days. During the quarantine period, observe the animals' drinking water, food intake and health status, as well as whether there are signs of disease and death.
  • Drinking water fill with ordinary water, and animals can drink freely. Rinse the water bottle and change the water daily.
  • the rats were randomly divided into 5 groups according to body weight, with 12 rats in each group (11 rats in the model group). See Schedule 12.
  • Table 12 Chinese medicine composition of the present invention on rat agar granuloma impact grouping and dosage
  • Administration by intragastric administration is basically consistent with the clinical route of administration.
  • GMBY1, GMBY2, and the control group were formulated with 0.5% CMC-Na to make the final concentration 0.244g crude drug/ml (0.0776g medicinal powder/ml). Store at 4°C for later use.
  • the positive drug aspirin is ready-to-use. Take 40 pieces of aspirin, grind it finely, dilute it to 41.7ml with 0.5% CMC-Na, and mix well.
  • the medicinal solution was extracted with a disposable syringe for intragastric administration, and the administration volume was 1ml/100g body weight.
  • 59 Wistar rats were randomly divided into 5 groups, model group, aspirin group, GMBY1, 2, control group. Oral administration, once a day, for 14 consecutive days, the model group was given the same volume of 0.5% CMC-Na. After the first administration, the animals in each group were disinfected on the back skin, and 2 ml of 2% agar was injected subcutaneously at the rear 1/3 of the dorsal midline. 0.5 hours after the last administration, the rats were anesthetized and sacrificed, the back granuloma was stripped and weighed, and the granuloma coefficient was calculated.
  • the experimental data is analyzed and processed by SPSS11.5 statistical software, and the statistical results are expressed as mean ⁇ standard deviation Indicates that the difference between groups was compared by One-Way ANOVA LSD T test.
  • Aspirin can significantly reduce the weight of granuloma and the coefficient of granuloma, and there is a significant statistical difference compared with the model group (P ⁇ 0.05 or P ⁇ 0.01); the weight and coefficient of granuloma in GMBY1, 2 groups and the control group are all reduced, And the weight of granuloma in GMBY1 and 2 groups was significantly different from the model group (P ⁇ 0.01); the granuloma coefficient in GMBY2 group was significantly different from that in the model group (P ⁇ 0.05). See Schedule 13.
  • the traditional Chinese medicine composition of the invention can reduce the weight and coefficient of the agar granuloma, suggesting that it has a certain inhibitory effect on the chronic proliferation phase of inflammation.
  • Test example 6 the effect of the traditional Chinese medicine composition of the present invention on the permeability increase of mouse abdominal cavity capillaries induced by acetic acid
  • Comparative example drug prepared according to the preparation method of the comparative example in the specification.
  • Acetic acid (glacial acetic acid), analytically pure, Beijing Chemical Plant, batch number 20100129.
  • mice were kept in cages, 7 mice/cage, in the New Drug Evaluation Center of Hebei Academy of Integrated Traditional Chinese and Western Medicine, with 12 hours of light per day, a temperature of 20-25°C, and a relative humidity of 40-70%.
  • the quarantine period for newly arrived animals is 3 days. During the quarantine period, observe the animals' drinking water, food intake and health status, as well as whether there are signs of disease and death.
  • Drinking water fill with ordinary water, and animals can drink freely. Rinse the water bottle and change the water daily.
  • mice were randomly divided into 6 groups according to body weight, 14 in each group. See Schedule 14.
  • Table 14 Effects of Chinese medicine composition dry paste powder of the present invention on acetic acid-induced increase in mouse abdominal capillary permeability grouping and dosage
  • Administration by intragastric administration is basically consistent with the clinical route of administration.
  • GMBY1, GMBY2, and the comparative example were prepared with 0.5% CMC-Na so that the final concentration was 0.3050 g crude drug/ml (0.0969 g drug powder/ml). Store at 4°C for later use.
  • the positive drug aspirin is now ready-to-use, take 12 aspirin tablets, grind it finely, dilute to 10ml with 0.5% CMC-Na, and mix well.
  • the medicinal solution was extracted with a disposable syringe for intragastric administration, and the administration volume was 0.1ml/10g body weight.
  • mice were randomly divided into 6 groups, normal control group, model group, aspirin group, GMBY1, GMBY2, and control group. Oral administration, once a day, for 7 consecutive days, the normal control group and the model group were given the same volume of 0.5% CMC-Na. 30 minutes after the last administration, inject 0.5% Evans blue normal saline solution into the tail vein at 0.1ml/10g body weight, and immediately inject 0.6% glacial acetic acid intraperitoneally at 0.1ml/10g body weight (the normal control group is injected with the same amount of normal saline) .
  • mice were sacrificed by breaking the cervical spine, the abdominal cavity was cut open, and the abdominal cavity was washed several times with 6ml of normal saline, the washing liquid was collected and combined to a volume of 10ml, centrifuged at 3000rpm for 10min, and 200 ⁇ l of the supernatant was placed on a 96-well microtiter plate and incubated at 590nm Measure the absorbance (OD value).
  • the experimental data is analyzed and processed by SPSS11.5 statistical software, and the statistical results are expressed as mean ⁇ standard deviation Indicates that the difference between groups was compared by One-Way ANOVA LSD T test.
  • Table 15 The effect of dry ointment powder of traditional Chinese medicine composition of the present invention on the increase in permeability of mouse abdominal cavity capillaries induced by acetic acid
  • the traditional Chinese medicine composition of the invention can significantly reduce the increase of peritoneal capillary permeability caused by acetic acid, suggesting that it has a certain anti-inflammatory effect.
  • Test example 7 the effect of the traditional Chinese medicine composition of the present invention on mouse ear swelling caused by xylene
  • the Chinese medicine composition dry paste powder group of the present invention can reduce swelling, and the middle and high dose groups of the Chinese medicine composition dry paste powder group of the present invention have significant statistical differences compared with the model group (P ⁇ 0.05 or P ⁇ 0.01 ).
  • the dry ointment powder of the traditional Chinese medicine composition of the present invention has a certain anti-inflammatory effect.
  • mice were kept in cages, 6 mice/cage, in the New Drug Evaluation Center of Hebei Academy of Integrated Traditional Chinese and Western Medicine, with 12 hours of light per day, temperature 20-25°C, and relative humidity 40-70%.
  • the quarantine period for newly arrived animals is 4 days. During the quarantine period, observe the animals' drinking water, food intake and health status, as well as whether there are signs of disease and death.
  • Drinking water fill with ordinary water, and animals can drink freely. Rinse the water bottle and change the water daily.
  • Proposed clinical dose 18.3g crude drug/day, orally, based on a human body weight of 60kg, equivalent to 0.305g crude drug/kg.
  • mice were randomly divided into 6 groups according to body weight, 12 in each group. See Schedule 16.
  • Administration by intragastric administration is basically consistent with the clinical route of administration.
  • GMBY1, GMBY2, and the control group were formulated with 0.5% CMC-Na to make the final concentration 0.3050g crude drug/ml (0.0969g medicinal powder/ml). Store at 4°C for later use.
  • the positive drug aspirin is now ready-to-use, take 12 aspirin tablets, grind it finely, dilute to 10ml with 0.5% CMC-Na, and mix well.
  • the medicinal solution was extracted with a disposable syringe for intragastric administration, and the administration volume was 0.1ml/10g body weight.
  • mice 72 KM mice were randomly divided into 6 groups, normal control group, model group, aspirin group, GMBY low, medium and high dose groups. Oral administration, once a day, for 7 consecutive days, the normal control group and the model group were given the same volume of 0.5% CMC-Na. 30 minutes after the last administration, 0.02 ml of 100% xylene was applied to the front and rear sides of the right ear of the mouse (the normal control group was coated with the same amount of normal saline), and the left ear was used as a control. The holers punched round ears on the same site, weighed with an electronic balance, and calculated the degree of swelling and inhibition rate.
  • Swelling degree weight of the right ear of each mouse - weight of the left ear
  • Inhibition rate (average swelling degree of model group-average swelling degree of administration group)/average swelling degree of model group ⁇ 100%
  • the experimental data is analyzed and processed by SPSS11.5 statistical software, and the statistical results are expressed as mean ⁇ standard deviation Indicates that the difference between groups was compared by One-Way ANOVA LSD T test.
  • the swelling degree of the model group was significantly increased (P ⁇ 0.01); compared with the model group, aspirin could significantly reduce the ear swelling degree of xylene-induced inflammation mice (P ⁇ 0.01), and the inhibition rate was 52.60 %; GMBY1, GMBY2, and the control group ear swelling also decreased, and GMBY1, 2 groups had significant statistical differences compared with the model group (P ⁇ 0.05 or P ⁇ 0.01), and the inhibition rates were 19.43%, 37.17%, 34.44%. See Schedule 17.
  • Table 17 The influence of Chinese medicine composition dry ointment powder of the present invention on swelling degree and inhibition rate of mouse ear swelling caused by xylene
  • the experimental results of the ovalbumin-induced allergic rhinitis model in rats show that the traditional Chinese medicine composition of the present invention can effectively reduce the allergic behavior score, reduce serum histamine, total IgE and OVA-specific IgE, IL-10, IL-12 levels, and reduce nasal Mucosal IL-4 expression and increased IL-2 expression can improve nasal mucosa histomorphological lesions to varying degrees; adopt 2,4-cresyl diisocyanate (TDI) to cause guinea pig allergic rhinitis model experimental results show that the traditional Chinese medicine of the present invention
  • the composition can reduce the allergic behavior score, reduce the levels of histamine, IgE, and IL-4 in serum, increase the level of IFN- ⁇ , and improve the morphology of nasal mucosa.
  • the rat passive skin allergy test shows that the Chinese medicine composition of the present invention can alleviate the passive skin allergy of rats
  • the mouse ear xenogeneic skin allergy test shows that the Chinese medicine composition of the present invention can alleviate the mouse ear xenogeneic skin allergy
  • the traditional Chinese medicine composition has a certain anti-type I allergy effect.
  • Rat agar granuloma test shows that the Chinese medicine composition of the present invention has inhibitory effect on the chronic proliferation of inflammation, and the acetic acid-induced mouse mesenteric capillary permeability test shows that the Chinese medicine composition of the present invention can antagonize the inflammation caused by acetic acid;
  • the swelling test shows that the Chinese medicine composition of the present invention can reduce ear swelling, and the above three experiments suggest that the Chinese medicine composition of the present invention has an anti-inflammatory effect.
  • the traditional Chinese medicine composition of the present invention can effectively inhibit the antigen-antibody reaction and the release of inflammatory mediators by regulating the differentiation of Th cells, thereby alleviating allergic symptoms, and at the same time, it has anti-inflammatory and anti-type I allergic reactions.

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Abstract

一种治疗过敏性鼻炎的药物组合物、制备方法及其用途，制成药物组合物的中药原料由黄芪、白术、防风、辛夷、白芷、高良姜、羌活、牡丹皮、蝉蜕、乌梅和甘草组成，用于过敏性鼻炎肺脾两虚、外邪侵袭证。

Description

一种治疗过敏性鼻炎的药物组合物及其制备方法 技术领域
本发明涉及一种治疗过敏性鼻炎的中药组合物及其制备方法，属于中草药应用领域。
背景技术
变态反应性鼻炎又称过敏性鼻炎(allergic rhinitis，AR)，简称变应性鼻炎，是是机体暴露于变应原后主要由IgE介导的鼻黏膜非感染性慢性炎性疾病，为耳鼻喉科临床常见病，既往分为季节性和常年性，近年根据症状出现特征分为间歇性和持续性。临床以突然、反复发作性喷嚏、鼻流清涕、鼻塞、鼻痒为主要特征，严重者嗅觉减退或消失，本病以儿童、青壮年居多，男女性别发病比无明显差异。随着工业化进程、现代生活方式、饮食结构的转变以及生态环境的急剧变化，本病的患病率和发病率显著上升，近十余年来大约影响到全球10％～30％的人群，保守估计全球该病患者超过5亿人可见变应性鼻炎已成为全球性健康问题。该病在大陆地区人口中的患病率为4％-38％，从卫生经济学的角度来看已成为沉重的社会负担。
变应性鼻炎虽然是非致命性疾病，但因具有高复发率的特点，且易出现变应性鼻窦炎、支气管哮喘、分泌性中耳炎、咳嗽变异性哮喘等并发症，并可严重影响患者的生活质量，引起失眠严重者夜不能寐或者发生阻塞性睡眠呼吸紊乱综合征，导致工作效率下降、记忆力减退等，给日常生活工作带来功能上、情感上和社会心理上的负面影响。对患有变应性鼻炎的儿童预后情况调查显示，19％的患儿发展为哮喘，有40％～50％的变应性鼻炎患者合并哮喘，如果得不到正确的治疗或治疗不当，约10％～39％的患者会发展为支气管哮喘，甚至肺心病等。
现代医学认为本病的主要原因一是变应原，包括吸入性变应原、食物变应原和职业性变应原等，是诱发本病的直接原因；二为易感个体，或称特应质。随着参与基本免疫反应的免疫细胞和炎性介质等基本环节被逐个阐明，变应性鼻炎的发病机制逐渐清晰，主要有基本免疫反应机制：即变应原经抗原呈递细胞和肥大细胞表面的IgE抗体结合，肥大细胞脱颗粒，释放炎性反应介质而导致患者出现变态反应症状；同时存在多种细胞因子网络对基本免疫反应的调节机制。
目前西药主要包括糖皮质激素、抗组胺药、抗白三烯药物、肥大细胞膜稳定剂、减充血剂、抗胆碱药物等。西药治疗变应性鼻炎主要采用抗过敏以及局部对症治疗，抗组 胺药物以及激素类药物虽能暂时控制症状，但服用后常常会出现嗜睡、困倦、口干等副作用，长期服用激素产生的副作用往往超过了疾病本身对人体的伤害，且西药服用后复发率高，不宜长期应用。其他如脱敏疗法疗效较好，但是疗程长很多患者难以坚持治疗；对鼻甲粘膜激光照射、射频以及化学烧灼等可降低鼻粘膜敏感性，对增生肥大的下鼻甲做部分切除可改善通气，但均需严格选择适应证。
现代中医对变应性鼻炎的病因病机、治则治法和辨证论治研究颇多，积累了丰富的经验，对减轻临床症状、缓解病情、提高机体免疫功能均显示出独特疗效，弥补了化药的不足，日益受到海内外医学界的高度重视。但是目前已上市变应性鼻炎中成药产品几乎全部定位于风热蕴肺、表虚不固证，绝多数采用祛风清热、解毒通窍之品，且部分药品采用中西药并用(如鼻炎康片、鼻舒适片)。
发明内容
本发明组合物针对变应性鼻炎(鼻鼽)病变部位虽在鼻，但责之肺脾，针对肺脾两虚，卫外不固，风寒外袭的主要病机，认为肺气虚不能宣发卫气，则营卫不和，腠理疏松，卫表不固，外邪乘虚而入侵犯鼻窍，风寒之邪遏肺，肺窍闭塞，肺气失于宜降清肃功能，脾气虚后天失养，致津水外溢，清涕自流，津液停聚则清窍不利而出现鼻塞，喷嚏等，符合临床大多数患者的证候表现。故充分汲取古今医家对肺脾两虚、外邪侵袭致鼻窍不通、鼻流清涕之症的论述，方以益气固表以治本，健脾通窍以治标，扶正驱邪，标本兼治。方中在经典名方玉屏风散基础上，加用辛温之品以益气固表、驱风散寒，同时配以辛夷、白芷祛风散寒、宣通鼻窍，高良姜补肺气、益脾胃，羌活驱风散寒，蝉蜕、丹皮甘凉之剂佐制耗气伤津之弊，乌梅酸、涩敛阴防辛温过燥暗耗津液。适用于肺脾两虚，外邪侵袭证。其作为纯中药制剂，避免了由于用药时间过长由西药所产生的副反应，保证了用药的安全性；与上述同类产品相比，本发明组合物在立方原则、方药组成上具有创新性。制剂工艺先进，临床疗效确切，应用安全，具有较好的开发应用前景。
本发明药物组合物是结合多年经验从肺脾两虚、外邪侵袭论治本病，在古代名方玉屏风散基础上加减并经临床验证疗效确切治疗鼻鼽的处方。
发明人在临床治疗鼻鼽多年，应用了数手经验方，其中应用较多的为采用玉屏风散合参苓白术散加减(太子参、茯苓、白术、黄芪、防风、高良姜、炒白扁豆、辛夷、白芷、牡丹皮、蝉蜕)，该处方与本发明药物组合物处方有8味药物重叠，本发明药物组合物在该方基础上去太子参、茯苓、白扁豆加用羌活、乌梅，同时调整黄芪、白术、防 风用量及比例，本发明组合物更加凸显玉屏风散益肺气以固表之功，肺脾兼治。本发明药物组合物处方作为发明人针对该病肺脾两虚这一主要证型的代表性临床用药处方，前期临床研究显示出良好的临床疗效和应用价值。
发明人认为本病病位在鼻，病变主脏在肺并与脾密切相关，主要病机为肺脾两虚，外邪侵袭。生理上鼻为呼吸之门户，助肺而行呼吸，鼻窍通畅则呼吸之气出入通畅而肺气通利，故宋代严用和《严氏济生方·鼻门》称：“夫鼻者，肺之所主，职司清化，调适得宜，则肺脏宜畅，清运自利”。同时肺主气司呼吸，与鼻相通，通过宣降功能吸清排浊，完成体内外气体交换，肺主鼻，鼻为肺之外窍，鼻主通气和主嗅觉的功能均有赖于肺气的作用，正如《灵枢·脉度篇》言：“肺气通于鼻，肺和则能知臭香矣”，可见肺鼻功能协调方能发挥正常的生理功能。《杂病源流犀烛·卷二十三》载：“鼻为肺窍，外象又属土”，鼻准居面之中央，中央属土，故鼻准属土，脾为后天之本，气血生化之源，肺所主之气，皆属脾运化的水谷精微所化生，故鼻的健旺亦有赖脾气的滋养。可见脾为生气之源，肺为主气之枢，鼻为多气多血之窍，需依肺吸入的清气之养，赖脾运化的气血之濡，若肺脾两虚加之外邪侵袭则易损伤鼻而致鼻鼽。
本病以肺脾气虚卫外不固为本，外邪趁虚侵入为标，故当施以益气固表健脾以治本，散邪通窍以治标之法，扶正驱邪，标本兼治方可奏效。
本发明所述的治疗过敏性鼻炎的药物组合物，包括如下重量份的组分：黄芪10-20份，白术5-15份，防风5-15份，辛夷5-15份，白芷5-15份，高良姜5-15份，羌活5-15份，牡丹皮5-15份，蝉蜕5-15份，乌梅5-15份，甘草4-8份。
本发明组合物优选的包括如下重量份的组分：黄芪10份，白术15份，防风5份，辛夷15份，白芷5份，高良姜15份，羌活5份，牡丹皮15份，蝉蜕5份，乌梅15份，甘草4份。
本发明组合物也可优选为包括以下重量份的组分：黄芪20份，白术5份，防风15份，辛夷5份，白芷15份，高良姜5份，羌活15份，牡丹皮5份，蝉蜕15份，乌梅5份，甘草8份。
本发明组合物还可优选为包括以下重量份的组分：黄芪15份，白术10份，防风10份，辛夷10份，白芷10份，高良姜10份，羌活10份，牡丹皮10份，蝉蜕10份，乌梅10份，甘草6份。
本发明组合物更可优选为包括以下重量份的组分：黄芪16份，白术8份，防风8份，辛夷8份，白芷8份，高良姜8份，羌活8份，牡丹皮8份，蝉蜕8份，乌梅8份，甘 草5份。
本发明组合物可制备成的制剂剂型为胶囊剂、片剂、丸剂、口服液、颗粒剂、注射剂或散剂。
本发明组合物的活性组分是由以下步骤制成：
A将方中白术、羌活粗碎，辛夷粗碎；按处方量称取白术、防风、高良姜、羌活、辛夷，加6-12倍量水，水蒸气蒸馏法提取挥发油，提取6-10小时，收集挥发油，备用；蒸馏后的水溶液滤过，减压浓缩，备用；
B按处方量称取的黄芪、蝉蜕、甘草，加9-13倍量水煎煮三次，每次1小时，滤过，提取液减压浓缩，与挥发油浓缩液合并减压浓缩至相对密度1.15～1.20，备用；
C按处方量称取的白芷、乌梅，加5-8倍量50-80％乙醇，提取二次，第一次2小时，第二次1.5小时，滤过，提取液减压回收乙醇，减压浓缩至相对密度1.15～1.20，备用；
D将牡丹皮粉碎成细粉，灭菌，备用；
步骤A得到的挥发油，步骤B得到的水提液，步骤C得到的醇提液和步骤D的细粉共同构成了本发明组合物的活性组分。
本发明还提供了该中药组合物制备成片剂的制备工艺为：
A将方中白术、羌活粗碎，辛夷粗碎破壳；按处方量称取白术、防风、高良姜、羌活、辛夷，加6-12倍量水，水蒸气蒸馏法提取挥发油，提取6-10小时，收集挥发油，备用；蒸馏后的水溶液滤过，减压浓缩，备用；
B按处方量称取的黄芪、蝉蜕、甘草，加9-13倍量水煎煮三次，每次1-3小时，滤过，提取液减压浓缩，与挥发油浓缩液合并减压浓缩至相对密度1.15～1.20，备用；
C按处方量称取的白芷、乌梅，加5-8倍量50-80％乙醇，提取二次，第一次1-3小时，第二次1-3小时，滤过，提取液减压回收乙醇，减压浓缩至相对密度1.15～1.20，备用；
D将牡丹皮粉碎成细粉，灭菌，备用；
E以牡丹皮细粉为底料，按常规工艺制粒，整粒，压片即得。
该片剂的制备工艺优选为：
A将方中白术、羌活粗碎，辛夷粗碎破壳；按处方量称取白术、防风、高良姜、羌活、辛夷，加8-10倍量水，水蒸气蒸馏法提取挥发油，提取8小时，收集挥发油，备用；蒸馏后的水溶液滤过，减压浓缩，备用；
B按处方量称取的黄芪、蝉蜕、甘草，加10-12倍量水煎煮三次，每次1小时，滤 过，提取液减压浓缩，与挥发油浓缩液合并减压浓缩至相对密度1.15～1.20，备用；
C按处方量称取的白芷、乌梅，加6-7倍量60-70％乙醇，提取二次，第一次2小时，第二次1.5小时，滤过，提取液减压回收乙醇，减压浓缩至相对密度1.15～1.20，备用；
D将牡丹皮粉碎成细粉，灭菌，备用；
E以牡丹皮细粉为底料，按常规工艺制粒，整粒，压片即得。
本发明还提供了该药物组合物在制备抗炎或抑制炎症慢性增殖药物中的应用。
本发明还提供了该药物组合物在制备抗I型变态反应药物中的应用。
本发明还提供了该药物组合物在制备降低组胺、总IgE及OVA特异性IgE、IL-10、IL-12水平降低鼻粘膜IL-4表达及升高IL-2表达药物中的应用。
本发明还提供了该药物组合物在升高IFN-γ水平药物中的应用。
本发明还提供了该药物组合物在缓解过敏症状药物中的应用。
本发明在辨证施治的基础上，以“益气固表，健脾通窍”治法，认为本病以肺脾气虚卫外不固为本，外邪趁虚侵入为标，故当施以益气固表健脾以治本，散邪通窍以治标之法，扶正驱邪，标本兼治方可奏效。
本方中，以黄芪为君药，性甘，微温，功专补气，乃补气之圣药，于内可大补肺脾之气，于外可固表实卫。邪之所凑，其气必虚，肺气亏虚则卫表不固、邪正交争则喷嚏出，气不摄津则流清涕、津液停滞则鼻塞，且治风寒者不患无以驱之，而患无以御之，不畏风寒之不去，而畏之复来，黄芪可补三焦而实卫，为玄府御风寒之关键，以之为君内补肺脾之气，益气固表以防风寒邪外袭最为适宜。
臣药：白术，味苦、甘，温，具有健脾益气，温养分肉之功，肺气的充实，有赖于脾化生水谷之气以供养，如果脾气虚，运化功能失常，不能将精微物质运送到五脏六腑、四肢百骸以及皮毛、筋肉等各脏器，则肺气必弱，鼻易受病。肺卫不固，风寒侵袭反复日久则脾气亦损，方中以之为臣取培土生金之义，母强子亦强，脾气得充则肺气自固。
防风臣药，味辛、甘，性微温，风寒袭表，理当祛之于外，然腠理疏松之人，发汗更易伤表，故方中选用甘温不燥，药性和缓的防风走表而祛风邪，取其乃“风药中之润剂”，防风走表散风寒，合黄芪、白术以益气祛邪，且黄芪得防风固表而不致留邪；防风得黄芪祛邪而不伤正，有补中寓疏，散中寓补之意，其功益彰。以防风之善驱风得黄芪以固表，则外有所卫；得白术以固里，则内有所据，风邪去而不复来。
辛夷与白术防风同为臣药，味辛，温，芳香质轻，气味具薄，上行头目，具发散风寒，宣通鼻窍之效，陶弘景《名医别录》言其：“温中解肌，利九窍，通鼻塞、涕出”， 香窜走上功专入肺，宣通肺气，善通鼻窍，尤为鼻塞、清涕常流等症之要药，正如《本草纲目》所载：“辛夷之辛温走气而入肺，能助胃中清阳上行通于天，所以能温中治头面目鼻之病”。
佐药：白芷，味辛温，辛能行散，温能祛寒，芳香走窜，佐助辛夷解表散寒，祛风通鼻窍，《本草纲目》谓：“白芷……芳香上达，入手太阴肺经”，《本草汇言》谓：“白芷，上行头目，下抵肠胃，中达肢体，遍通肌肤以至毛窍，而利泄邪气”。高良姜，味辛性热，《本草汇言》曰：“高良姜，祛寒湿、温脾胃之药也”，《食物本草》言其具有“补肺气，益脾胃”之功，在方中温散寒邪，佐助黄芪、白术补益肺脾。羌活，味辛苦，温，具有解表散寒，祛风止痛之功，《雷公炮制药性解》云：“气清属阳，善行气分，舒而不敛，升而能沉，雄而善散，可发表邪……以理游风”，羌活辛以散风，轻清上扬，直达头面，可祛风邪。牡丹皮，味苦、辛，微寒，具有清热凉血、活血散瘀之功，《本草经疏》谓：“牡丹皮，其味苦而微辛，其气寒而无毒，辛以散结聚，苦寒除血热，入血分，凉血热之要药也”。临床变应性鼻炎患者多为病程较长，症状反复发作者，根据中医“久病必虚，久病必瘀”之论，加用丹皮活血祛瘀，此外，蝉蜕与丹皮两味药性甘凉可佐制方中辛温发散药物耗气伤津之弊。蝉蜕，味甘，寒，功可搜风通络，宣肺散热。乌梅，酸、涩，平，可“益精开胃化津”(《本经逢原》)，用之以防辛温过燥暗耗津液。
使药：甘草，甘平，和中缓急，润肺，善能调和诸药故以为使。
诸药合用，以补肺健脾，益气固表以治本，驱风散邪，活血通窍以治标，标本兼治，诸症自除同时防止复发。
传统的汤剂煎制麻烦，口感差，质量不稳定，不便于患者携带和服用，使用受到极大限制。因此会选择制成胶囊剂、片剂、颗粒剂、口服液或其他剂型。其中片剂与其它剂型相比辅料用量少，较易成型，体积小，剂量准确，携带、运输和服用均较方便。
另外，片剂为干燥固体，质量稳定可控，某些易氧化变质及潮解的药物可借包衣加以保护，光线、空气、水分等对其影响较小，还可掩盖中药的不良气味。
再者片剂生产机械化、自动化程度高，宜于产业化推广，为了更好的发挥该药的社会价值和经济价值，我们结合处方中药味性质，优选将片剂作为本处方的剂型。
具体实施方式
以下结合实施例详细说明本发明的技术方案，但本发明的保护范围包括但是不限 于此。
实施例1：
黄芪206g，白术137g，防风137g，辛夷137g，白芷137g，高良姜137g，羌活137g，牡丹皮137g，蝉蜕137g，乌梅137g，甘草82g。
A将方中白术、羌活粗碎，辛夷粗碎破壳；按处方量称取白术、防风、高良姜、羌活、辛夷，加8倍量水，水蒸气蒸馏法提取挥发油，提取8小时，收集挥发油，备用；蒸馏后的水溶液滤过，减压浓缩，备用；
B按处方量称取的黄芪、蝉蜕、甘草，加11倍量水煎煮三次，每次1小时，滤过，提取液减压浓缩，与挥发油浓缩液合并减压浓缩至相对密度1.15～1.20，备用；
C按处方量称取的白芷、乌梅，加61倍量601％乙醇，提取二次，每次1.5小时，滤过，提取液减压回收乙醇，减压浓缩至相对密度1.15～1.20，备用；
D将牡丹皮粉碎成细粉，灭菌，备用；
E以牡丹皮细粉为底料，按常规工艺制粒，整粒，压成1000片即得。
实施例2：
黄芪240g，白术120g，防风120g，辛夷120g，白芷120g，高良姜120g，羌活120g，牡丹皮120g，蝉蜕120g，乌梅120g，甘草75g。
A将方中白术、羌活粗碎，辛夷粗碎破壳；按处方量称取白术、防风、高良姜、羌活、辛夷，加10倍量水，水蒸气蒸馏法提取挥发油，提取8小时，收集挥发油，备用；蒸馏后的水溶液滤过，减压浓缩，备用；
B按处方量称取的黄芪、蝉蜕、甘草，加12倍量水煎煮三次，每次1小时，滤过，提取液减压浓缩，与挥发油浓缩液合并减压浓缩至相对密度1.15～1.20，备用；
C按处方量称取的白芷、乌梅，加7倍量70％乙醇，提取二次，第一次2小时，第二次1.5小时，滤过，提取液减压回收乙醇，减压浓缩至相对密度1.15～1.20，备用；
D将牡丹皮粉碎成细粉，灭菌，备用；
E以牡丹皮细粉为底料，按常规工艺制粒，整粒，装入1000粒胶囊即得。
实施例3：
黄芪137g，白术205g，防风68g，辛夷205g，白芷68g，高良姜205g，羌活68g，牡丹皮205g，蝉蜕68g，乌梅205g，甘草55g。
A将方中白术、羌活粗碎，辛夷粗碎破壳；按处方量称取白术、防风、高良姜、羌 活、辛夷，加9倍量水，水蒸气蒸馏法提取挥发油，提取8小时，收集挥发油，备用；蒸馏后的水溶液滤过，减压浓缩，备用；
B按处方量称取的黄芪、蝉蜕、甘草，加11倍量水煎煮三次，每次1小时，滤过，提取液减压浓缩，与挥发油浓缩液合并减压浓缩至相对密度1.15～1.20，备用；
C按处方量称取的白芷、乌梅，加6倍量70％乙醇，提取二次，第一次2小时，第二次1.5小时，滤过，提取液减压回收乙醇，减压浓缩至相对密度1.15～1.20，备用；
D将牡丹皮粉碎成细粉，灭菌，备用；
E以牡丹皮细粉为底料，按常规工艺制粒，整粒，制成1000袋颗粒剂。
实施例4：
黄芪300g，白术75g，防风225g，辛夷75g，白芷225g，高良姜75g，羌活225g，牡丹皮75g，蝉蜕225g，乌梅75g，甘草120g。
A将方中白术、羌活粗碎，辛夷粗碎破壳；按处方量称取白术、防风、高良姜、羌活、辛夷，加10倍量水，水蒸气蒸馏法提取挥发油，提取8小时，收集挥发油，备用；蒸馏后的水溶液滤过，减压浓缩，备用；
B按处方量称取的黄芪、蝉蜕、甘草，加10倍量水煎煮三次，每次1小时，滤过，提取液减压浓缩，与挥发油浓缩液合并减压浓缩至相对密度1.15～1.20，备用；
C按处方量称取的白芷、乌梅，加6倍量60％乙醇，提取二次，第一次2小时，第二次1.5小时，滤过，提取液减压回收乙醇，减压浓缩至相对密度1.15～1.20，备用；
D将牡丹皮粉碎成细粉，灭菌，备用；
E以牡丹皮细粉为底料，按常规工艺制成1000粒丸剂。
实施例5：
黄芪180g，白术90g，防风90g，辛夷90g，白芷90g，高良姜90g，羌活90g，牡丹皮90g，蝉蜕90g，乌梅90g，甘草60g。
A将方中白术、羌活粗碎，辛夷粗碎破壳；按处方量称取白术、防风、高良姜、羌活、辛夷，加10倍量水，水蒸气蒸馏法提取挥发油，提取8小时，收集挥发油，备用；蒸馏后的水溶液滤过，减压浓缩，备用；
B按处方量称取的黄芪、蝉蜕、甘草，加10倍量水煎煮三次，每次1小时，滤过，提取液减压浓缩，与挥发油浓缩液合并减压浓缩至相对密度1.15～1.20，备用；
C按处方量称取的白芷、乌梅，加7倍量70％乙醇，提取二次，第一次2小时，第二次1.5小时，滤过，提取液减压回收乙醇，减压浓缩至相对密度1.15～1.20，备用；
D将牡丹皮粉碎成细粉，灭菌，备用；
E以牡丹皮细粉为底料，按常规工艺制成喷雾剂。
对比例：
太子参200g、茯苓120g、白术80g、黄芪80g、防风80g、高良姜100g、炒白扁豆100g、辛夷100g、白芷100g、牡丹皮100g、蝉蜕80g。
A将方中白术，辛夷粗碎破壳；按处方量称取白术、防风、高良姜、辛夷，加8倍量水，水蒸气蒸馏法提取挥发油，提取8小时，收集挥发油，备用；蒸馏后的水溶液滤过，减压浓缩，备用；
B按处方量称取的太子参、茯苓、炒白扁豆、黄芪、蝉蜕，加11倍量水煎煮三次，每次1小时，滤过，提取液减压浓缩，与挥发油浓缩液合并减压浓缩至相对密度1.15～1.20，备用；
C按处方量称取的白芷，加6倍量60％乙醇，提取二次，每次1.5小时，滤过，提取液减压回收乙醇，减压浓缩至相对密度1.15～1.20，备用；
D将牡丹皮粉碎成细粉，灭菌，备用；
E以牡丹皮细粉为底料，按常规工艺制粒，整粒，压成1000片即得。
试验例：
为了说明本发明药物组合物的功效，用实施例制备的实施例1、实施例2的样品进行了功能性试验。
试验例1：本发明中药组合物对大鼠实验性变应性鼻炎的影响
摘要
【目的】评价本发明中药组合物对卵蛋白致大鼠变应性鼻炎的影响。【方法】将90只SD大鼠随机分为6组，用卵蛋白造成变应性鼻炎模型，给药20天后，进行行为学评分，ELISA法检测血清组胺、IgE、OVA特异性IgE、IL-10、IL-12，HE染色观察组织病理学指标，免疫组化法检测鼻粘膜组织IL-4、IL-2的表达。【结果】与模型组相比，本发明中药组合物实施例1，实施例2可以有效降低行为学评分(P<0.05)；本发明中药组合物实施例1组组胺、IgE与模型组相比有明显统计学差异；本发明中药组合物实施例2组胺、IgE、IL-10与模型组相比有明显统计学差异(P<0.05或P<0.01)。各用药组IL-12与模型组相比均有所升高，本发明中药组合物实施例2组与模型组相比有明显统计学差异(P<0.05)。本发明药物组合物可以不同程度降低鼻粘膜IL-4表达，升高IL-2表达。本发明中药组合物可以不同程度改善鼻粘膜组织形态学病变。【结论】本发明中药组合 物对卵蛋白致大鼠变应性鼻炎有一定的治疗作用。
【关键词】本发明中药组合物，卵蛋白，IgE
实验目的：
采用卵清蛋白(OVA)造成大鼠变应性鼻炎模型，观察本发明中药组合物对此模型大鼠的影响，评价其药效。
1实验材料
1.1供试品
1.1.1名称：本发明中药组合物，实施例1缩写：GMBY1，实施例2缩写GMBY2。
1.1.2理化性质：棕褐色颗粒，气微香、味微酸。
1.1.3拟临床适应症：益气固表，健脾通窍。用于持续性变态反应性鼻炎(本发明中药组合物)。
1.1.4含量及规格：3.146g生药/g干膏粉。
1.1.5来源和批号：石家庄以岭药业股份有限公司提供，090301。
1.1.6供试品保管：密封、干燥。
1.2阳性药、工具药及主要试剂
1.2.1阳性药
富马酸酮替芬片，规格1mg*60片，上海复旦复华药业有限公司生产，批号：090701。
对比例药物：按照对比例方法进行制备。
1.2.2主要试剂
卵蛋白，Sigma，Cat.No.A8040。
氢氧化铝，天津市博迪化工有限公司，批号：20090612。
IgE测试盒，BlueGene，Lot：091209。
OVA特异性IgE测试盒，BlueGene，Lot：20100112。
组胺测试盒，BlueGene，Lot：091209。
IL-10测试盒，BlueGene，Lot：20100809。
IL-12测试盒，BlueGene，Lot：20100809。
IL-2一抗，奥博森，Lot：019082。
IL-4一抗，奥博森。
兔SP检测试剂盒，中杉金桥，Lot：740415A。
DAB，中杉金桥，Lot：614846A。
1.3实验系统
1.3.1动物种系：SD大鼠。
1.3.2动物级别：SPF级。
1.3.3动物性别和数量：90只，雄性。
1.3.4动物体重：140～160g。
1.3.5动物来源：北京维通利华实验动物技术有限公司。
1.3.6动物合格证号及发证单位、接收日期：合格证编号0155254，许可证号SCXK(京)2006-0009，发证单位北京维通利华实验动物技术有限公司，接受日期2009年10月14日。
1.3.7饲养条件：大鼠笼养，5只/笼，饲养于河北省中西医结合医药研究院新药评价中心，光照12小时/天，温度20～25℃，相对湿度40～70％。
1.3.8检疫过程：新到的动物检疫期4天，在检疫期间观察动物饮水、摄食和健康状况，以及是否存在疾病和死亡征兆。
1.3.9饲料：实验动物鼠饲料，中国人民解放军军事医学科学院实验动物中心提供。
1.3.10饮水：灌装普通用水，动物自由饮用。每日冲洗水瓶，并换水一次。
1.3.11垫料：购自河北省实验动物中心，每周更换2次。
1.3.12动物标识：动物识别采用5％苦味酸标记。
2实验方法
2.1实验设计依据
2.1.1采用标准：国家食品药品监督管理局颁布的《药品注册管理办法》附件一、《中药、天然药物注册分类及申报资料要求》和《药物非临床研究质量管理规范》。
2.1.2委托单位提供资料：临床拟用剂量：18.3g生药/天，口服，人体重以60kg计，折合0.305g生药/kg。
2.2剂量与分组
将大鼠按体重随机分为6组，每组15只。见附表1。
表1 本发明中药组合物对大鼠实验性变应性鼻炎的影响分组和给药剂量
Figure PCTCN2022096580-appb-000001
Figure PCTCN2022096580-appb-000002
2.3给药方法
灌胃给药，与临床用药途径基本一致。
2.4供试品配制和保存
将GMBY1、GMBY2、对比例用0.5％CMC-Na配制成终浓度为0.244g生药/ml(0.0776g药粉/ml)。4℃保存备用。
阳性药富马酸酮替芬现用现配，取3片，研细后用0.5％CMC-Na定容至56ml，混匀即可。
2.5供试品的给予
用一次性注射器抽取药液进行灌胃给药，给药体积均为1ml/100g体重。
2.6观测的指标、时间和内容
将大鼠随机分为6组，正常对照组、模型组、酮替芬组、GMBY1、GMBY2、对比例组。按以下步骤造模：(1)用30mg OVA作抗原，氢氧化铝粉末3g作佐剂，加生理盐水100ml制成混悬液，每鼠1ml腹腔注射，隔日一次，共7次，为基础致敏(正常对照组以3％Al(OH) 3代替)。(2)用2％OVA生理盐水攻击双侧鼻腔，每侧50μl，每日一次，共7次(正常对照组以生理盐水代替)。(3)攻击后以2％OVA 100μl滴入左右鼻孔各50μl，隔日1次，维持致敏状态，直至给药结束(正常对照组以生理盐水代替)。各组于基础致敏后给予相应药物进行治疗，连续给药20天。
行为学指标：依据鼻痒、喷嚏、流涕等出现的时间长短、轻重程度、次数为评分标准并评分记录。鼻痒：轻度：轻擦鼻1～3次，记1分；重度：抓挠鼻、面不止，到处磨擦(4个以上)，记2分；喷嚏：1-3个为1分，4-10个为2分，11个以上为3分；流涕：流至鼻前孔为1分，超过鼻前孔为2分，流涕满面为3分。各症状记分叠加。其症状观察时间为末次给药致敏后30min内。
血清组胺、IgE、OVA特异性IgE、IL-10、IL-12检测：10％水合氯醛麻醉，腹主动脉取血，分离血清，按试剂盒要求检测。
鼻粘膜中IL4、IL2检测：免疫组化法检测。
形态学指标：断头，迅速打开鼻背，取双侧鼻甲及鼻中隔黏膜，冰浴分离鼻腔鼻中隔鼻黏膜，将鼻黏膜组织放入10％甲醛溶液中固定，石腊切片，HE染色观察嗜酸性粒细 胞及鼻黏膜形态学变化。
2.7相关工作人员通知
购买动物时通知动物室，在动物出现异常情况时通知病理室进行处理。
2.8仪器系统
DT2000电子天平，常熟市双杰测试仪器厂。
METTLER TOLEDO精密分析天平，梅特勒—托利多仪器(上海)有限公司。
Biofuge primoR高速冷冻离心机，北京华威科仪科技有限公司。
XD711酶标分析仪，上海迅达医疗仪器有限公司。
BX41显微镜：奥林巴斯。
RM2126RT切片机：上海莱卡仪器有限公司。
TSJ-Ⅲ型脱水机：常州中威电子仪器有限公司。
BMJ-Ⅲ型包埋机：常州中威电子仪器有限公司。
DP-Ⅲ摊片烤片机：孝感德立森电子有限公司。
2.9统计方法
实验数据采用SPSS11.5统计软件进行分析处理，统计结果用均数±标准差
Figure PCTCN2022096580-appb-000003
表示，方差齐用LSD T检验，方差不齐用改进的Dunnett检验。
3结果
模型组鼻痒、喷嚏、流涕等症状严重，行为学评分与正常对照组相比明显升高(P<0.01)。各用药组行为学评分与模型组相比均有所降低，其中GMBY1、2组与模型组相比有明显统计学差异(P<0.05)。详见表2。
表2 本发明中药组合物对大鼠实验性变应性鼻炎行为学的影响
Figure PCTCN2022096580-appb-000004
注：与正常对照组相比，**P<0.01；与模型组相比， △P<0.05， △△P<0.01。
模型组组胺、IgE、OVA特异性IgE、IL-10与正常对照组相比均明显升高(P<0.05或P<0.01)，各用药组上述指标均有所降低，GMBY1组组胺、IgE与模型组相比有明显统计学差异；GMBY2组组胺、IgE、IL-10与模型组相比有明显统计学差异(P<0.05或 P<0.01)。模型组IL-12与正常对照组相比明显降低(P<0.01)，各用药组IL-12与模型组相比均有所升高，GMBY2组与模型组相比有明显统计学差异(P<0.05)。
免疫组化法检测鼻粘膜中IL-4的表达：阳性物质为棕黄色，颗粒或丝状。阳性细胞分布在黏膜上皮的假复层柱状纤毛上皮细胞、支持细胞和基底细胞，以及固有层和黏膜下层的血管壁内皮细胞、腺体和导管细胞。模型组与正常组相比棕黄色着色深，表达呈强阳性，酮替芬组与本发明中药组合物1、2组棕黄色颗粒或丝状物明显减少，呈中等阳性表达。
免疫组化法检测鼻粘膜中IL-2的表达：阳性物质为棕黄色，颗粒或丝状。阳性细胞分布在黏膜上皮的假复层柱状纤毛上皮细胞、支持细胞和基底细胞，以及固有层和黏膜下层的血管壁内皮细胞、腺体和导管细胞。模型组与正常组相比棕黄色着色弱，表达呈弱阳性，酮替芬组与本发明中药组合物1、2组棕黄色颗粒或丝状物明显增多，呈中等阳性表达。结果见表3。
表3 本发明中药组合物对实验性变应性鼻炎大鼠血清指标的影响
Figure PCTCN2022096580-appb-000005
注：与正常对照组相比，*P<0.05，**P<0.01；与模型组相比， △P<0.05， △△P<0.01。
镜下观查鼻粘膜组织发现，正常对照组鼻黏膜上皮层连续、完整，组织结构清晰，无充血、水肿、上皮缺损、炎细胞浸润。假复层纤毛柱状上皮，可见纤毛柱状细胞、柱状细胞、杯状细胞、基底细胞，上皮细胞核位于细胞的基底部，卵圆形。固有层和黏膜下层见成纤维细胞及大量的胶原纤维，无炎性细胞浸润，其间散在小血管和红细胞；模型组鼻黏膜充血，水肿，上皮层不连续，组织结构不清晰，基底膜增厚、水肿、破坏，形成胶原组织炎性带，分隔上皮层和上皮下层；本发明中药组合物与模型组相比较，从形态学角度可见不同程度改善。
4结论
本发明中药组合物干膏粉对卵蛋白致大鼠变应性鼻炎有一定的治疗作用。
试验例2：本发明中药组合物对豚鼠实验性变应性鼻炎的影响
摘要
【目的】评价本发明中药组合物对豚鼠变应性鼻炎的影响
【方法】将72只豚鼠随机分为6组，用10％的2,4-二异氰酸甲苯酯(TDI)橄榄油溶液10μl滴鼻(每侧鼻腔各滴5μl)致敏，连续7天，7天后隔天滴一次维持致敏状态，直至给药结束。各组于基础致敏后给予相应药物进行治疗，连续给药14天。观察本发明中药组合物干膏粉对行为学指标，血清学指标及病理形态学的影响。
【结果】本发明中药组合物实施例1，实施例2与模型组相比行为学评分明显降低(P<0.05或P<0.01)；本发明中药组合物可降低血清中组胺、IgE、IL-4含量，实施例2最为明显，其中IgE、组胺与模型组相比有统计学差异(P<0.05)，可升高血清IFN-γ水平，实施例1，实施例2与模型组相比有明显统计学差异(P<0.05)；各用药组可不同程度地改善组织形态学病变。
【结论】本发明中药组合物对TDI所致豚鼠变应性鼻炎模型有一定的治疗作用。
实验目的：
采用2，4—二异氰酸甲苯酯(TDI)造成豚鼠变应性鼻炎模型，观察本发明中药组合物对此模型豚鼠的影响，评价其药效。
1实验材料
1.1供试品
1.1.1名称：本发明中药组合物，缩写：GMBY1，GMBY2。
1.1.2理化性质：棕褐色颗粒，气微香、味微酸。
1.1.3拟临床适应症：益气固表，健脾通窍。用于持续性变态反应性鼻炎(本发明中药组合物)。
1.1.4含量及规格：3.146g生药/g干膏粉。
1.1.5来源和批号：石家庄以岭药业股份有限公司提供，090301。
1.1.6供试品保管：密封、干燥。
1.2阳性药、工具药及主要试剂
1.2.1阳性药
盐酸西替利嗪胶囊，规格10mg，珠海安生凤凰制药有限公司，批号：09050206。
对比例药物：按照对比例方法进行制备。
1.2.2主要试剂
2,4-二异氰酸甲苯酯，INTERNATIONAL LABORATORY，Lot.No.461366。
橄榄油，西班牙亿芭利橄榄油有限公司，批号：20090625。
IgE测试盒，BlueGene，Lot：091205。
组胺测试盒，BlueGene，Lot：090804。
IL-4测试盒，BlueGene，Lot：E100108。
IFN-γ测试盒，BlueGene，Lot：091008A。
1.3实验系统
1.3.1动物种系：DH豚鼠。
1.3.2动物级别：普通级。
1.3.3动物性别和数量：72只，雌雄各半。
1.3.4动物体重：250～300g。
1.3.5动物来源：北京芳元缘养殖场。
1.3.6动物合格证号及发证单位、接收日期：合格证编号0159054，许可证号SCXK(京)2009-0014，发证单位北京芳元缘养殖场，接收日期2010年5月6日。
1.3.7饲养条件：豚鼠笼养，3只/笼，饲养于河北省中西医结合医药研究院新药评价中心，光照12小时/天，温度20～26℃，相对湿度40～70％。
1.3.8检疫过程：新到的动物检疫期5天，在检疫期间观察动物饮水、摄食和健康状况，以及是否存在疾病和死亡征兆。
1.3.9饲料：实验动物豚鼠饲料，中国人民解放军军事医学科学院实验动物中心提供。
1.3.10饮水：灌装普通用水，动物自由饮用。每日冲洗水瓶，并换水一次。
1.3.11垫料：购自河北省实验动物中心，每周更换2次。
1.3.12动物标识：动物识别采用5％苦味酸标记。
2实验方法
2.1实验设计依据
2.1.1采用标准：国家食品药品监督管理局颁布的《药品注册管理办法》附件一、《中药、天然药物注册分类及申报资料要求》和《药物非临床研究质量管理规范》。
2.1.2委托单位提供资料：临床拟用剂量：18.3g生药/天，口服，人体重以60kg计，折合0.305g生药/kg。
2.2剂量与分组
将豚鼠按体重随机分为6组，每组12只。见附表4。
表4 本发明中药组合物对豚鼠实验性变应性鼻炎的影响分组和给药剂量
Figure PCTCN2022096580-appb-000006
Figure PCTCN2022096580-appb-000007
2.3给药方法
灌胃给药，与临床用药途径基本一致。
2.4供试品配制和保存
将GMBY1、GMBY2，对比例组用0.5％CMC-Na配制成终浓度为0.183g生药/ml(0.0582g药粉/ml)。4℃保存备用。
阳性药盐酸西替利嗪胶囊现用现配，取1粒，研细后用0.5％CMC-Na定容至50ml，混匀即可。
2.5供试品的给予
用一次性注射器抽取药液进行灌胃给药，给药体积均为1ml/100g体重。
2.6观测的指标、时间和内容
将豚鼠随机分为6组，正常对照组、模型组、西替利嗪组、GMBY1、2组、对比例组。按以下步骤造模：用10％的TDI橄榄油溶液10μl滴鼻(每侧鼻腔各滴5μl)致敏，连续7天，7天后隔天滴一次维持致敏状态，直至给药结束。各组于基础致敏后给予相应药物进行治疗，连续给药14天。
依据鼻痒、喷嚏、流涕等出现的时间长短、轻重程度、次数为评分标准并评分记录。鼻痒：轻度:轻擦鼻1～3次，记1分；重度：抓挠鼻、面不止，到处磨擦(4个以上)，记2分；喷嚏：1-3个为1分，4-10个为2分，11个以上为3分；流涕：流至鼻前孔为1分，超过鼻前孔为2分，流涕满面为3分。各症状记分叠加。其症状观察时间为末次给药致敏后30min内。
血清组胺、IgE、IL4、IFN-γ检测：10％水合氯醛麻醉，腹主动脉取血，分离血清，按试剂盒要求检测。
鼻粘膜组织形态学，断头，迅速打开鼻背，取双侧鼻甲及鼻中隔黏膜，冰浴分离鼻腔鼻中隔鼻黏膜，将鼻黏膜组织放入10％甲醛溶液中固定，石腊切片，HE染色观察嗜酸性粒细胞及鼻黏膜形态学变化。
2.7相关工作人员通知
购买动物时通知动物室，在动物出现异常情况时通知病理室进行处理。
2.8仪器系统
DT2000电子天平，常熟市双杰测试仪器厂。
METTLER TOLEDO精密分析天平，梅特勒—托利多仪器(上海)有限公司。
Biofuge primoR高速冷冻离心机，北京华威科仪科技有限公司。
XD711酶标分析仪，上海迅达医疗仪器有限公司。
BX41显微镜：奥林巴斯。
RM2126RT切片机：上海莱卡仪器有限公司。
TSJ-Ⅲ型脱水机：常州中威电子仪器有限公司。
BMJ-Ⅲ型包埋机：常州中威电子仪器有限公司。
DP-Ⅲ摊片烤片机：孝感德立森电子有限公司。
2.9统计方法
实验数据采用SPSS11.5统计软件进行分析处理，统计结果用均数±标准差
Figure PCTCN2022096580-appb-000008
表示，方差齐用LSD T检验，方差不齐用改进的Dunnett检验。
3结果
模型组鼻痒、喷嚏、流涕等症状严重，行为学评分与正常对照组相比明显升高(P<0.01)。各用药组行为学评分与模型组相比均有所降低，其中GMBY1组、GMBY2组与模型组相比有明显统计学差异(P<0.05或P<0.01)。结果见表5。
表5 本发明中药组合物对豚鼠实验性变应性鼻炎行为学的影响
Figure PCTCN2022096580-appb-000009
注：与正常对照组相比，**P<0.01；与模型组相比， △P<0.05， △△P<0.01。
模型组组胺、IgE、IL-4与正常对照组相比均明显升高(P<0.05或P<0.01)，各用药组上述指标均有所降低，GMBY2组最为明显，与模型组相比有明显统计学差异(P<0.05，IL-4除外)。模型组IFN-γ与正常对照组相比明显降低(P<0.01)，各用药组与模型组相比均有所升高，GMBY1、2组与模型组相比有明显统计学差异(P<0.05)。 见附表6。
表6 本发明中药组合物对实验性变应性鼻炎豚鼠血清指标的影响
Figure PCTCN2022096580-appb-000010
注：与正常对照组相比，*P<0.05，**P<0.01；与模型组相比， △P<0.05。
镜下观查鼻粘膜组织发现，正常对照组鼻黏膜上皮层连续、完整，组织结构清晰，无充血、水肿、上皮缺损、炎细胞浸润。假复层纤毛柱状上皮，可见纤毛柱状细胞、柱状细胞、杯状细胞、基底细胞，上皮细胞核位于细胞的基底部，卵圆形。固有层和黏膜下层见成纤维细胞及大量的胶原纤维，无炎性细胞浸润，其间散在小血管和红细胞；模型组鼻黏膜上皮层有大片坏死、缺损，结构几乎不可辨识，充血、水肿、上皮间及上皮下大量炎细胞浸润，固有层充血、水肿，胶原纤维排列紊乱，黏膜下层呈病理性腺体增生。本发明中药组合物各组与模型组相比较，从形态学角度可见不同程度改善。
4结论
本发明中药组合物对TDI所致豚鼠变应性鼻炎模型有一定的治疗作用。
试验例3：本发明中药组合物对大鼠被动皮肤过敏的影响
摘要
【目的】评价本发明中药组合物的抗I型变态反应作用。【方法】制备天花粉抗血清，于给药12天后在大鼠背正中线两侧皮内注射抗血清(1:10；1:20稀释)，48小时后尾静脉注射0.5％伊文思蓝配制的天花粉蛋白溶液进行抗原攻击，20min后处死动物，剪下背部皮肤，测定注射各点蓝斑直径；剪下蓝斑，以丙酮生理盐水浸泡48小时，离心，620nm处测定OD值。【结果】蓝斑直径：1:10稀释度扑尔敏组、GMBY2组与模型组相比降低且有明显统计学差异(P<0.05或P<0.01)；1:20稀释度扑尔敏组、GMBY1组、GMBY2组与模型组相比降低且有明显统计学差异(P<0.05或P<0.01)。OD值：两个稀释度下扑尔敏组、GMBY2组、GMBY1组与模型组相比降低且有明显统计学差异(P<0.05或P<0.01)。【结论】本发明中药组合物能减轻大鼠被动皮肤过敏，有一定的抗I型变态反应的作用。
实验目的：
观察本发明中药组合物对大鼠被动皮肤过敏的影响，以评价其抗I型变态反应的作用。
1实验材料
1.1供试品
1.1.1名称：本发明中药组合物，缩写：GMBY1，GMBY2。
1.1.2理化性质：棕褐色颗粒，气微香、味微酸。
1.1.3拟临床适应症：益气固表，健脾通窍。用于持续性变态反应性鼻炎(本发明中药组合物)。
1.1.4含量及规格：3.146g生药/g干膏粉。
1.1.5来源和批号：石家庄以岭药业股份有限公司提供，090301。
1.1.6供试品保管：密封、干燥。
1.2阳性药、工具药及主要试剂
1.2.1阳性药
马来酸氯苯那敏片(扑尔敏)，4mg/片，河北康泰药业有限公司，批号100302，有效期至2013年3月1日。
对比例药物：按照对比例方法进行制备。
1.2.2主要试剂
天花粉蛋白，上海金山制药有限公司，批号080101。
氢氧化铝，Alfa Aesar，Lot：F30R043。
伊文思蓝，Solarbio，Lot.E8010
生理盐水，石家庄四药有限公司，批号100331165。
羧甲基纤维素钠(CMC-Na)，分析纯，天津市永大化学试剂开发中心，批号20080403。
1.3实验系统
1.3.1动物种系：SD大鼠。
1.3.2动物级别：SPF级。
1.3.3动物性别和数量：70只，雌性35只，雄性35只。
1.3.4动物体重：100～180g。
1.3.5动物来源：北京维通利华实验动物技术有限公司。
1.3.6动物合格证号及发证单位、接收日期：合格证编号0174590；0174589，许可证号SCXK(京)2006-0009，发证单位北京维通利华实验动物技术有限公司，接受日期 2010年6月10日。
1.3.7饲养条件：大鼠笼养，6或5只/笼，饲养于河北省中西医结合医药研究院新药评价中心，光照12小时/天，温度20～25℃，相对湿度40～70％。
1.3.8检疫过程：新到的动物检疫期3天或5天，在检疫期间观察动物饮水、摄食和健康状况，以及是否存在疾病和死亡征兆。
1.3.9饲料：实验动物鼠饲料，中国人民解放军军事医学科学院实验动物中心提供，合格证号0025186。
1.3.10饮水：灌装普通用水，动物自由饮用。每日冲洗水瓶，并换水一次。
1.3.11垫料：购自河北省实验动物中心，每周更换2次。
1.3.12动物标识：动物识别采用5％苦味酸标记。
2实验方法
2.1实验设计依据
2.1.1采用标准：国家食品药品监督管理局颁布的《药品注册管理办法》附件一、《中药、天然药物注册分类及申报资料要求》和《药物非临床研究质量管理规范》。
2.1.2委托单位提供资料：临床拟用剂量：18.3g生药/天，口服，人体重以60kg计，折合0.305g生药/kg。
2.2剂量与分组
SD大鼠雌雄各5只，体重100～120g，用于制备抗血清；SD大鼠60只，体重150～180g，雌雄各30只，按体重随机分为5组，每组12只。见附表7。
表7 本发明中药组合物对大鼠被动皮肤过敏的影响分组和给药剂量
Figure PCTCN2022096580-appb-000011
2.3给药方法
灌胃给药，与临床用药途径基本一致。
2.4供试品配制和保存
将GMBY1、2及对比例药物用0.5％CMC-Na配制成终浓度为0.244g生药/ml(0.0776g药粉/ml)。4℃保存备用。
阳性药扑尔敏现用现配，取扑尔敏3片，研细后用0.5％CMC-Na定容至37ml，混匀即可。
2.5供试品的给予
用一次性注射器抽取药液进行灌胃给药，给药体积均为1ml/100g体重。
2.6观测的指标、时间和内容
以5mg天花粉蛋白溶于1ml4％氢氧化铝中。取健康SD大鼠雌雄各5只(体重100～120g)，将天花粉溶液脚掌注射，每个脚掌注射0.1ml。12天后，麻醉动物采血，分离血清，-20℃保存待用。
健康大鼠60只，雌雄各半(150～180g)，按体重随机分为5组，连续给药12天后，在大鼠背正中线两侧，距脊柱1.5cm处剪毛，每侧2点，每点间隔2cm。取抗血清，经生理盐水稀释不同倍数(1:10；1:20)，皮内注射于剪毛各点，每点0.1ml。继续常规给药。48小时后进行抗原攻击，尾静脉注射0.5％伊文思蓝溶液，每毫升含天花粉蛋白1mg，每100g体重注射1ml，20min后处死动物，剪下背部皮肤，测定注射各点的蓝斑直径。剪下蓝斑皮肤，各点分别以5ml丙酮生理盐水(按体积比7：3配制)浸泡48h，3000rpm离心10min，取上清置96孔酶标板，620nm处测定OD值。
2.7相关工作人员通知
购买动物时通知动物室，在动物出现异常情况时通知病理室进行处理。
2.8仪器系统
Biofuge primoR高速冷冻离心机，北京华威科仪科技有限公司。
XD711酶标分析仪，上海迅达医疗仪器有限公司。
DT2000电子天平，常熟市双杰测试仪器厂。
METTLER TOLEDO精密分析天平，梅特勒—托利多仪器(上海)有限公司。
2.9统计方法
实验数据采用SPSS11.5统计软件进行分析处理，统计结果用均数±标准差
Figure PCTCN2022096580-appb-000012
表示，组间差异比较进行One-Way ANOVA LSD T检验。
3结果
与模型组相比各用药组的蓝斑直径均呈降低趋势，1:10稀释度扑尔敏组、GMBY2组与模型组相比有明显统计学差异(P<0.05或P<0.01)；1:20稀释度扑尔敏组、GMBY1组、GMBY2组与模型组相比有明显统计学差异(P<0.05或P<0.01)。见附表8。
表8 本发明中药组合物对大鼠被动皮肤过敏蓝斑直径的影响
Figure PCTCN2022096580-appb-000013
注：与模型组相比， △P<0.05， △△P<0.01。
与模型组相比各用药组蓝斑皮肤浸泡液吸光度值均呈降低趋势，扑尔敏组、GMBY1组、GMBY2组与模型组相比有明显统计学差异(P<0.05或P<0.01)。见附表9。
表9 本发明中药组合物对大鼠被动皮肤过敏蓝斑吸光度的影响
Figure PCTCN2022096580-appb-000014
注：与模型组相比， △P<0.05， △△P<0.01
4结论
GMBY能够减轻大鼠被动皮肤过敏反应，对I型变态反应有一定的抑制作用。
试验例4：本发明中药组合物对小鼠耳异种皮肤过敏反应的影响
摘要
【目的】评价本发明中药组合物的抗I型变态反应作用。
【方法】制备天花粉蛋白抗血清，各组小鼠给药12天后耳廓注射抗血清致敏，48小时后尾静脉注射0.5％伊文思蓝配制的天花粉蛋白溶液进行攻击，30分钟后处死小鼠，剪下蓝染耳廓，置KOH溶液中37℃过夜消化。次日加入磷酸丙酮混合液，震荡混匀，离心，上清液于640nm处测定OD值。
【结果】各用药组OD值与模型组相比均有所降低，其中扑尔敏组、GMBY2组与模型组相比有明显统计学差异(P<0.05或P<0.01)，各用药组抑制百分率均降低。
【结论】本发明中药组合物有一定的抗I型变态反应作用。
实验目的：
观察本发明中药组合物对小鼠耳异种被动皮肤过敏反应的影响，以评价其抗I型变态反应的作用。
1实验材料
1.1供试品
1.1.1名称：本发明中药组合物，缩写：GMBY1、GMBY2。
1.1.2理化性质：棕褐色颗粒，气微香、味微酸。
1.1.3拟临床适应症：益气固表，健脾通窍。用于持续性变态反应性鼻炎(本发明中药组合物)。
1.1.4含量及规格：3.146g生药/g干膏粉。
1.1.5来源和批号：石家庄以岭药业股份有限公司提供，090301。
1.1.6供试品保管：密封、干燥。
1.2阳性药、工具药及主要试剂
1.2.1阳性药
马来酸氯苯那敏片(扑尔敏)，4mg/片，河北康泰药业有限公司，批号100302，有效期至2013年3月2日。
对比例药物：按照对比例方法进行制备。
1.2.2主要试剂
天花粉蛋白，上海金山制药有限公司，批号080101。
氢氧化铝，Alfa Aesar，Lot：F30R043。
伊文思蓝，Solarbio，Lot.E8010
生理盐水，石家庄四药有限公司，批号100331165。
羧甲基纤维素钠(CMC-Na)，分析纯，天津市永大化学试剂开发中心，批号20080403。
1.3实验系统
1.3.1动物种系：SD大鼠；KM小鼠。
1.3.2动物级别：SPF级。
1.3.3动物性别和数量：SD大鼠10只，雌雄各半；KM小鼠70只，雌雄各半。
1.3.4动物体重：100～120g；18～22g。
1.3.5动物来源：北京维通利华实验动物技术有限公司；河北省实验动物中心。
1.3.6动物合格证号及发证单位、接收日期：SD大鼠合格证编号0174589，许可证号SCXK(京)2006-0009，发证单位北京维通利华实验动物技术有限公司，接受日期2010年6月10日。KM小鼠合格证编号1006075许可证号SCXK(冀)2008-1-003，发证单位河北省实验动物中心，接受日期2010年6月9日。
1.3.7饲养条件：大鼠笼养，5只/笼；小鼠笼养，7只/笼，饲养于河北省中西医结合医药研究院新药评价中心，光照12小时/天，温度20～25℃，相对湿度40～70％。
1.3.8检疫过程：新到的动物检疫期3天或5天，在检疫期间观察动物饮水、摄食和健康状况，以及是否存在疾病和死亡征兆。
1.3.9饲料：实验动物鼠饲料，中国人民解放军军事医学科学院实验动物中心提供，合格证号0025186。
1.3.10饮水：灌装普通用水，动物自由饮用。每日冲洗水瓶，并换水一次。
1.3.11垫料：购自河北省实验动物中心，每周更换2次。
1.3.12动物标识：动物识别采用5％苦味酸标记。
2实验方法
2.1实验设计依据
2.1.1采用标准：国家食品药品监督管理局颁布的《药品注册管理办法》附件一、《中药、天然药物注册分类及申报资料要求》和《药物非临床研究质量管理规范》。
2.1.2委托单位提供资料：临床拟用剂量：18.3g生药/天，口服，人体重以60kg计，折合0.305g生药/kg。
2.2剂量与分组
将小鼠按体重随机分为5组，每组14只。见附表10。
表10 本发明中药组合物对小鼠耳异种皮肤过敏反应的影响分组和给药剂量
Figure PCTCN2022096580-appb-000015
2.3给药方法
灌胃给药，与临床用药途径基本一致。
2.4供试品配制和保存
将GMBY1、GMBY2、对比例药物用0.5％CMC-Na配制成终浓度为0.3050g生药/ml(0.0969g药粉/ml)。4℃保存备用。
阳性药扑尔敏现配，取扑尔敏1片，研细后用0.5％CMC-Na定容至10ml，混匀即可。
2.5供试品的给予
用一次性注射器抽取药液进行灌胃给药，给药体积均为0.1ml/10g体重。
2.6观测的指标、时间和内容
以5mg天花粉蛋白溶于1ml4％氢氧化铝中。取健康SD大鼠雌雄各5只(体重100～120g)，将天花粉溶液脚掌注射，每个脚掌注射0.1ml。12天后，麻醉动物采血，分离血清，-20℃保存待用。
连续给药12天后致敏，两耳廓各注射大鼠抗血清20μl。48h后进行抗原攻击，尾静脉注射0.5％伊文思蓝溶液，每毫升含天花粉蛋白1mg，每10g体重注射0.1ml，30min后处死小鼠，剪下蓝染耳廓。两只耳廓置于试管内，加入1mol/L的KOH0.75ml，37℃过夜消化。次日加入3.5ml 0.2mol H 3PO 4与丙酮混合液(体积比5：13)，经漩涡器震摇，再以2500rpm离心15min。上清液置酶标板，640nm处测OD值，并计算。
Figure PCTCN2022096580-appb-000016
2.7相关工作人员通知
购买动物时通知动物室，在动物出现异常情况时通知病理室进行处理。
2.8仪器系统
Biofuge primoR高速冷冻离心机，北京华威科仪科技有限公司。
XD711酶标分析仪，上海迅达医疗仪器有限公司。
DT2000电子天平，常熟市双杰测试仪器厂。
METTLER TOLEDO精密分析天平，梅特勒—托利多仪器(上海)有限公司。
2.9统计方法
实验数据采用SPSS11.5统计软件进行分析处理，统计结果用均数±标准差
Figure PCTCN2022096580-appb-000017
表示，组间差异比较进行One-Way ANOVA Dunnett T3检验。
3结果
各用药组OD值与模型组相比均有所降低，其中扑尔敏组、GMBY2组与模型组相比有明显统计学差异(P<0.05或P<0.01)，各用药组抑制百分率均降低。见附表11。
表11 本发明中药组合物对小鼠耳异种皮肤过敏反应的影响
Figure PCTCN2022096580-appb-000018
Figure PCTCN2022096580-appb-000019
注：与模型组相比， △P<0.05。
4结论
GMBY能减轻小鼠耳异种皮肤过敏反应，对I型变态反应有一定的抑制作用。
试验例5：本发明中药组合物对大鼠琼脂肉芽肿的影响
摘要
【目的】评价本发明中药组合物的抗炎作用。【方法】将59只Wistar大鼠随机分为5组，皮下注射琼脂溶液，连续给药14天后，空气栓塞法对大鼠实施安乐死，剥离皮下琼脂肉芽肿称重，并计算肉芽肿系数。【结果】阿司匹林能够显著降低肉芽肿重量及肉芽肿系数，与模型组相比有显著统计学差异(P<0.05或P<0.01)；GMBY1、2、对比例组肉芽肿重量及系数均有所降低，且GMBY1、2组肉芽肿重量与模型组相比有显著统计学差异(P<0.01)；GMBY2组肉芽肿系数与模型组相比有显著统计学差异(P<0.05)。【结论】本发明中药组合物对炎症慢性增殖相有抑制作用。
实验目的：
观察本发明中药组合物对大鼠琼脂肉芽肿的影响，以评价其抗炎作用。
1实验材料
1.1供试品
1.1.1名称：本发明中药组合物，缩写：GMBY1、GMBY2。
1.1.2理化性质：棕褐色颗粒，气微香、味微酸。
1.1.3拟临床适应症：益气固表，健脾通窍。用于持续性变态反应性鼻炎(本发明中药组合物)。
1.1.4含量及规格：3.146g生药/g干膏粉。
1.1.5来源和批号：石家庄以岭药业股份有限公司提供，090301。
1.1.6供试品保管：密封、干燥。
1.2阳性药、工具药及主要试剂
1.2.1阳性药
阿司匹林肠溶片，25mg/片，石药集团欧意药业有限公司，批号5018090440，有效期至2011年04月28日。
对比例药物：按照对比例方法进行制备。
1.2.2主要试剂
琼脂粉，Solarbio，Cat.No.A8190。
生理盐水，石家庄四药有限公司，批号090530107。
羧甲基纤维素钠(CMC-Na)，分析纯，天津市永大化学试剂开发中心，批号20080403。
1.3实验系统
1.3.1动物种系：wistar大鼠。
1.3.2动物级别：清洁级。
1.3.3动物性别和数量：59只，雌性29只，雄性30只。
1.3.4动物体重：150～180g。
1.3.5动物来源：河北省实验动物中心。
1.3.6动物合格证号及发证单位、接收日期：合格证编号1004070，许可证号SCXK(冀)2008-1-003，发证单位河北省实验动物中心，接受日期2010年4月9日。
1.3.7饲养条件：大鼠笼养，6或5只/笼，饲养于河北省中西医结合医药研究院新药评价中心，光照12小时/天，温度20～25℃，相对湿度40～70％。
1.3.8检疫过程：新到的动物检疫期4天，在检疫期间观察动物饮水、摄食和健康状况，以及是否存在疾病和死亡征兆。
1.3.9饲料：实验动物鼠饲料，中国人民解放军军事医学科学院实验动物中心提供，合格证号0025136。
1.3.10饮水：灌装普通用水，动物自由饮用。每日冲洗水瓶，并换水一次。
1.3.11垫料：购自河北省实验动物中心，每周更换2次。
1.3.12动物标识：动物识别采用5％苦味酸标记。
2实验方法
2.1实验设计依据
2.1.1采用标准：国家食品药品监督管理局颁布的《药品注册管理办法》附件一、《中药、天然药物注册分类及申报资料要求》和《药物非临床研究质量管理规范》。
2.1.2委托单位提供资料：临床拟用剂量：18.3g生药/天，口服，人体重以60kg计，折合0.305g生药/kg。
2.2剂量与分组
将大鼠按体重随机分为5组，每组12只(模型组11只)。见附表12。
表12 本发明中药组合物对大鼠琼脂肉芽肿的影响分组和给药剂量
Figure PCTCN2022096580-appb-000020
Figure PCTCN2022096580-appb-000021
2.3给药方法
灌胃给药，与临床用药途径基本一致。
2.4供试品配制和保存
将GMBY1、GMBY2、对比例组用0.5％CMC-Na配制成终浓度为0.244g生药/ml(0.0776g药粉/ml)。4℃保存备用。
阳性药阿司匹林现用现配，取阿司匹林40片，研细后用0.5％CMC-Na定容至41.7ml，混匀即可。
2.5供试品的给予
用一次性注射器抽取药液进行灌胃给药，给药体积均为1ml/100g体重。
2.6观测的指标、时间和内容
Wistar大鼠59只，随机分为5组，模型组、阿司匹林组、GMBY1、2、对比例组。灌胃给药，每日一次，连续14日，模型组给予相同体积0.5％CMC-Na。各组动物于第1次给药后消毒背部皮肤，将2％的琼脂2ml注射于背正中线后1/3处皮下。末次给药后0.5小时后麻醉处死大鼠，剥取背部肉芽肿称重，并计算肉芽肿系数。
2.7相关工作人员通知
购买动物时通知动物室，在动物出现异常情况时通知病理室进行处理。
2.8仪器系统
DT2000电子天平，常熟市双杰测试仪器厂。
METTLER TOLEDO精密分析天平，梅特勒—托利多仪器(上海)有限公司。
2.9统计方法
实验数据采用SPSS11.5统计软件进行分析处理，统计结果用均数±标准差
Figure PCTCN2022096580-appb-000022
表示，组间差异比较进行One-Way ANOVA LSD T检验。
3结果
阿司匹林能够显著降低肉芽肿重量及肉芽肿系数，与模型组相比有显著统计学差异(P<0.05或P<0.01)；GMBY1、2组及对比例组肉芽肿重量及系数均有所降低，且GMBY1、2组肉芽肿重量与模型组相比有显著统计学差异(P<0.01)；GMBY2组肉芽 肿系数与模型组相比有显著统计学差异(P<0.05)。见附表13。
表13 本发明中药组合物对大鼠琼脂肉芽肿的影响
Figure PCTCN2022096580-appb-000023
注：与模型组相比， △P<0.05， △△P<0.01。
4结论
本发明中药组合物能够降低琼脂肉芽肿的重量及系数，提示其对炎症慢性增殖相有一定的抑制作用。
试验例6：本发明中药组合物对醋酸致小鼠腹腔毛细血管通透性增加的影响
摘要
【目的】评价本发明中药组合物的抗炎作用。【方法】将84只KM小鼠随机分为6组，连续给药7天后，将0.5％伊文思蓝生理盐水溶液按0.1ml/10g体重尾静脉注射，立即按0.1ml/10g体重腹腔注射0.6％冰醋酸。20min后，断颈椎处死小鼠，用生理盐水6ml冲洗腹腔数次，收集洗涤液，合并后定容至10ml，3000rpm离心10min，取上清，于590nm处测OD值。【结果】与正常对照组相比，模型组OD值明显升高(P<0.01)；与模型组相比，阿司匹林能够显著降低OD值(P<0.05)；GMBY1、2组及对比例组OD值亦降低，且GMBY1、2组与模型组相比有显著统计学差异(P<0.05或P<0.01)。【结论】本发明中药组合物有一定的抗炎作用。
【关键词】本发明中药组合物，肿胀度
实验目的：
观察本发明中药组合物对醋酸致小鼠腹腔毛细血管通透性增加的影响，以评价其抗炎作用。
1实验材料
1.1供试品
1.1.1名称：本发明中药组合物，缩写：GMBY1，GMBY2。
1.1.2理化性质：棕褐色颗粒，气微香、味微酸。
1.1.3拟临床适应症：益气固表，健脾通窍。用于持续性变态反应性鼻炎(本发明中 药组合物)。
1.1.4含量及规格：3.146g生药/g干膏粉。
1.1.5来源和批号：石家庄以岭药业股份有限公司提供，090301。
1.1.6供试品保管：密封、干燥。
1.2阳性药、工具药及主要试剂
1.2.1阳性药
阿司匹林肠溶片，25mg/片，石药集团欧意药业有限公司，批号5018090440，有效期至2011年04月28日。
对比例药物：按照说明书中对比例制备方法制得。
1.2.2主要试剂
乙酸(冰醋酸)，分析纯，北京化工厂，批号20100129。
伊文思蓝，Solarbio公司提供，分装Sigma E2129。
生理盐水，石家庄四药有限公司，批号090530107。
羧甲基纤维素钠(CMC-Na)，分析纯，天津市永大化学试剂开发中心，批号20080403。
1.3实验系统
1.3.1动物种系：KM小鼠。
1.3.2动物级别：清洁级。
1.3.3动物性别和数量：84只，雌雄各半。
1.3.4动物体重：18～20g。
1.3.5动物来源：河北省实验动物中心。
1.3.6动物合格证号及发证单位、接收日期：合格证编号1003201，许可证号SCXK(冀)2008-1-003，发证单位河北省实验动物中心，接受日期2010年3月31日。
1.3.7饲养条件：小鼠笼养，7只/笼，饲养于河北省中西医结合医药研究院新药评价中心，光照12小时/天，温度20～25℃，相对湿度40～70％。
1.3.8检疫过程：新到的动物检疫期3天，在检疫期间观察动物饮水、摄食和健康状况，以及是否存在疾病和死亡征兆。
1.3.9饲料：实验动物鼠饲料，中国人民解放军军事医学科学院实验动物中心提供，合格证号0025136。
1.3.10饮水：灌装普通用水，动物自由饮用。每日冲洗水瓶，并换水一次。
1.3.11垫料：购自河北省实验动物中心，每周更换2次。
1.3.12动物标识：动物识别采用5％苦味酸标记。
2实验方法
2.1实验设计依据
2.1.1采用标准：国家食品药品监督管理局颁布的《药品注册管理办法》附件一、《中药、天然药物注册分类及申报资料要求》和《药物非临床研究质量管理规范》。
2.1.2委托单位提供资料：临床拟用剂量：18.3g生药/天，口服，人体重以60kg计，折合0.305g生药/kg。
2.2剂量与分组
将小鼠按体重随机分为6组，每组14只。见附表14。
表14 本发明中药组合物干膏粉对醋酸致小鼠腹腔毛细血管通透性增加的影响分组和给药剂量
Figure PCTCN2022096580-appb-000024
2.3给药方法
灌胃给药，与临床用药途径基本一致。
2.4供试品配制和保存
将GMBY1、GMBY2、对比例用0.5％CMC-Na配制成终浓度为0.3050g生药/ml(0.0969g药粉/ml)。4℃保存备用。
阳性药阿司匹林现用现配，取阿司匹林12片，研细后用0.5％CMC-Na定容至10ml，混匀即可。
2.5供试品的给予
用一次性注射器抽取药液进行灌胃给药，给药体积均为0.1ml/10g体重。
2.6观测的指标、时间和内容
KM小鼠84只，随机分为6组，正常对照组、模型组、阿司匹林组、GMBY1、GMBY2、对比例组。灌胃给药，每日一次，连续7日，正常对照组及模型组给予相同体积0.5％CMC-Na。于末次给药后30min，将0.5％伊文思蓝生理盐水溶液按0.1ml/10g体重尾 静脉注射后，立即按0.1ml/10g体重腹腔注射0.6％冰醋酸(正常对照组注射等量生理盐水)。20min后，断颈椎处死小鼠，剪开腹腔，用生理盐水6ml冲洗腹腔数次，收集洗涤液，合并后定容至10ml，3000rpm离心10min，取上清200μl置96孔酶标板，于590nm处测吸光度(OD值)。
2.7相关工作人员通知
购买动物时通知动物室，在动物出现异常情况时通知病理室进行处理。
2.8仪器系统
DT2000电子天平，常熟市双杰测试仪器厂。
METTLER TOLEDO精密分析天平，梅特勒—托利多仪器(上海)有限公司。
Biofuge primoR高速冷冻离心机，北京华威科仪科技有限公司。
XD711酶标分析仪，上海迅达医疗仪器有限公司。
2.9统计方法
实验数据采用SPSS11.5统计软件进行分析处理，统计结果用均数±标准差
Figure PCTCN2022096580-appb-000025
表示，组间差异比较进行One-Way ANOVA LSD T检验。
3结果
与正常对照组相比，模型组OD值明显升高(P<0.01)；与模型组相比，阿司匹林能够显著降低OD值(P<0.05)；GMBY1、GMBY2、对比例组OD值亦降低，且GMBY1、2组与模型组相比有显著统计学差异(P<0.05或P<0.01)。见附表15。
表15 本发明中药组合物干膏粉对醋酸致小鼠腹腔毛细血管通透性增加的影响
Figure PCTCN2022096580-appb-000026
注：与正常对照组相比，**P<0.01；与模型组相比， △P<0.05， △△P<0.01。
4结论
本发明中药组合物能够显著降低醋酸引起的腹腔毛细血管通透性增加，提示其具有一定的抗炎作用。
试验例7：本发明中药组合物对二甲苯致小鼠耳肿胀的影响
摘要
【目的】评价本发明中药组合物的抗炎作用。【方法】将72只KM小鼠随机分为6组，每组12只，连续给药7天，末次给药后30分钟，用20μl二甲苯涂抹小鼠右耳，左耳作对照，15min后用打孔器取下两耳相同位置的耳片，称重，计算右耳肿胀度和各组的肿胀抑制率。【结果】本发明中药组合物干膏粉组均能降低肿胀度，其中本发明中药组合物干膏粉中、高剂量组与模型组相比有明显统计学差异(P＜0.05或P＜0.01)。【结论】本发明中药组合物干膏粉有一定的抗炎作用。
【关键词】本发明中药组合物，肿胀度
实验目的：
观察本发明中药组合物对二甲苯致小鼠耳肿胀的影响，以评价其抗炎作用。
1实验材料
1.1供试品
1.1.1名称：本发明中药组合物，缩写：GMBY1、GMBY2。
1.1.2理化性质：棕褐色颗粒，气微香、味微酸。
1.1.3拟临床适应症：益气固表，健脾通窍。用于持续性变态反应性鼻炎(本发明中药组合物)。
1.1.4含量及规格：3.146g生药/g干膏粉。
1.1.5来源和批号：石家庄以岭药业股份有限公司提供，090301。
1.1.6供试品保管：密封、干燥。
1.2阳性药、工具药及主要试剂
1.2.1阳性药
阿司匹林肠溶片，25mg/片，石药集团欧意药业有限公司，批号5018090440，有效期至2011年04月28日。
1.2.2主要试剂
二甲苯，分析纯，天津市福晨化学试剂厂，批号20080310。
羧甲基纤维素钠(CMC-Na)，分析纯，天津市永大化学试剂开发中心，批号20080403。
1.3实验系统
1.3.1动物种系：KM小鼠。
1.3.2动物级别：清洁级。
1.3.3动物性别和数量：72只，雄性。
1.3.4动物体重：16～20g。
1.3.5动物来源：河北省实验动物中心。
1.3.6动物合格证号及发证单位、接收日期：合格证编号1002096，许可证号SCXK(冀)2008-1-003，发证单位河北省实验动物中心，接受日期2010年2月26日。
1.3.7饲养条件：小鼠笼养，6只/笼，饲养于河北省中西医结合医药研究院新药评价中心，光照12小时/天，温度20～25℃，相对湿度40～70％。
1.3.8检疫过程：新到的动物检疫期4天，在检疫期间观察动物饮水、摄食和健康状况，以及是否存在疾病和死亡征兆。
1.3.9饲料：实验动物鼠饲料，中国人民解放军军事医学科学院实验动物中心提供，合格证号0025136。
1.3.10饮水：灌装普通用水，动物自由饮用。每日冲洗水瓶，并换水一次。
1.3.11垫料：购自河北省实验动物中心，每周更换2次。
1.3.12动物标识：动物识别采用5％苦味酸标记。
2实验方法
2.1实验设计依据
2.1.1采用标准：国家食品药品监督管理局颁布的《药品注册管理办法》附件一、《中药、天然药物注册分类及申报资料要求》和《药物非临床研究质量管理规范》。
2.1.2实验系统选择说明：药理剂量的雌激素有抗炎与免疫抑制作用，所以本实验全部选用雄性动物。
2.1.3委托单位提供资料：临床拟用剂量：18.3g生药/天，口服，人体重以60kg计，折合0.305g生药/kg。
2.2剂量与分组
将小鼠按体重随机分为6组，每组12只。见附表16。
表16 本发明中药组合物对二甲苯致小鼠耳肿胀的影响分组和给药剂量
Figure PCTCN2022096580-appb-000027
2.3给药方法
灌胃给药，与临床用药途径基本一致。
2.4供试品配制和保存
将GMBY1、GMBY2、对比例组用0.5％CMC-Na配制成终浓度为0.3050g生药/ml(0.0969g药粉/ml)。4℃保存备用。
阳性药阿司匹林现用现配，取阿司匹林12片，研细后用0.5％CMC-Na定容至10ml，混匀即可。
2.5供试品的给予
用一次性注射器抽取药液进行灌胃给药，给药体积均为0.1ml/10g体重。
2.6观测的指标、时间和内容
KM小鼠72只，随机分为6组，正常对照组、模型组、阿司匹林组、GMBY低、中、高剂量组。灌胃给药，每日一次，连续7日，正常对照组及模型组给予相同体积0.5％CMC-Na。于末次给药后30min将100％二甲苯0.02ml涂在小鼠右耳前后两面(正常对照组涂等量生理盐水)，左耳作对照，15min后将小鼠断颈椎处死，用6mm直径打孔器分别在同一部位打下圆耳片，用电子天平称重，计算肿胀度和抑制率。
肿胀度＝每鼠右耳重-左耳重
抑制率＝(模型组平均肿胀度-给药组平均肿胀度)/模型组平均肿胀度×100％
2.7相关工作人员通知
购买动物时通知动物室，在动物出现异常情况时通知病理室进行处理。
2.8仪器系统
DT2000电子天平，常熟市双杰测试仪器厂。
METTLER TOLEDO精密分析天平，梅特勒—托利多仪器(上海)有限公司。
2.9统计方法
实验数据采用SPSS11.5统计软件进行分析处理，统计结果用均数±标准差
Figure PCTCN2022096580-appb-000028
表示，组间差异比较进行One-Way ANOVA LSD T检验。
3结果
与正常对照组相比，模型组肿胀度明显升高(P<0.01)；与模型组相比，阿司匹林能够显著降低二甲苯致炎小鼠的耳肿胀度(P<0.01)，抑制率为52.60％；GMBY1、GMBY2、对比例组耳肿胀度亦降低，且GMBY1、2组与模型组相比有显著统计学差异(P<0.05或P<0.01)，抑制率分别为19.43％、37.17％、34.44％。见附表17。
表17 本发明中药组合物干膏粉对二甲苯致小鼠耳肿胀的肿胀度与抑制率的影响
Figure PCTCN2022096580-appb-000029
注：与正常对照组相比，**P<0.01；与模型组相比， △P<0.05， △△P<0.01。
4结论
二甲苯致小鼠耳肿胀的急性炎症模型建立成功，本发明中药组合物能有效抑制二甲苯导致的小鼠耳肿胀，提示其具有一定的抗炎作用。
结论
卵蛋白致大鼠变应性鼻炎模型实验结果显示本发明中药组合物能有效降低过敏行为学评分，降低血清组胺、总IgE及OVA特异性IgE、IL-10、IL-12水平，降低鼻粘膜IL-4表达及升高IL-2表达，不同程度改善鼻粘膜组织形态学病变；采用2，4—二异氰酸甲苯酯(TDI)造成豚鼠变应性鼻炎模型实验结果显示本发明中药组合物能降低过敏行为学评分，降低血清中组胺、IgE、IL-4水平、升高IFN-γ水平，改善鼻粘膜组织形态，以上二个实验提示本发明中药组合物可通过调节Th细胞分化，有效抑制抗原抗体反应、抑制炎症介质的释放、从而缓解过敏症状。大鼠被动皮肤过敏实验显示本发明中药组合物能减轻大鼠被动皮肤过敏，小鼠耳异种皮肤过敏反应实验显示本发明中药组合物能减轻小鼠耳异种皮肤过敏，以上二个实验提示本发明中药组合物有一定的抗I型变态反应的作用。大鼠琼脂肉芽肿实验显示本发明中药组合物对炎症慢性增殖有抑制作用，醋酸致小鼠肠系膜毛细血管通透性实验显示本发明中药组合物能拮抗醋酸引起的炎症；二甲苯致小鼠耳肿胀实验显示本发明中药组合物能降低耳肿胀度，以上三个实验提示本发明中药组合物有抗炎的作用。
综上所述，本发明中药组合物可通过调节Th细胞分化，有效抑制抗原抗体反应、抑制炎症介质的释放、从而缓解过敏症状，同时具有抗炎、抗I型变态反应。

Claims (14)

  1. 一种治疗过敏性鼻炎的药物组合物，其特征在于该组合物包括如下重量份的组分：黄芪10-20份，白术5-15份，防风5-15份，辛夷5-15份，白芷5-15份，高良姜5-15份，羌活5-15份，牡丹皮5-15份，蝉蜕5-15份，乌梅5-15份，甘草4-8份。
  2. 根据权利要求1所述的组合物，其特征在于该组合物包括如下重量份的组分：黄芪10份，白术15份，防风5份，辛夷15份，白芷5份，高良姜15份，羌活5份，牡丹皮15份，蝉蜕5份，乌梅15份，甘草4份。
  3. 根据权利要求1所述的组合物，其特征在于该组合物包括如下重量份的组分：黄芪20份，白术5份，防风15份，辛夷5份，白芷15份，高良姜5份，羌活15份，牡丹皮5份，蝉蜕15份，乌梅5份，甘草8份。
  4. 根据权利要求1所述的组合物，其特征在于该组合物包括如下重量份的组分：黄芪15份，白术10份，防风10份，辛夷10份，白芷10份，高良姜10份，羌活10份，牡丹皮10份，蝉蜕10份，乌梅10份，甘草6份。
  5. 根据权利要求1所述的组合物，其特征在于该组合物包括如下重量份的组分：黄芪16份，白术8份，防风8份，辛夷8份，白芷8份，高良姜8份，羌活8份，牡丹皮8份，蝉蜕8份，乌梅8份，甘草5份。
  6. 根据权利要求1-5任一所述的组合物，其特征在于该组合物的制剂剂型为胶囊剂、片剂、丸剂、口服液、颗粒剂、注射剂或散剂。
  7. 根据权利要求1-5任一所述的组合物，其特征在于该组合物的活性组分是由以下步骤制成：
    A将方中白术、羌活粗碎，辛夷粗碎；按处方量称取白术、防风、高良姜、羌活、辛夷，加6-12倍量水，水蒸气蒸馏法提取挥发油，提取6-10小时，收集挥发油，备用；蒸馏后的水溶液滤过，减压浓缩，备用；
    B按处方量称取的黄芪、蝉蜕、甘草，加9-13倍量水煎煮三次，每次1小时，滤过，提取液减压浓缩，与挥发油浓缩液合并减压浓缩至相对密度1.15～1.20，备用；
    C按处方量称取的白芷、乌梅，加5-8倍量50-80％乙醇，提取二次，第一次2小时，第二次1.5小时，滤过，提取液减压回收乙醇，减压浓缩至相对密度1.15～1.20，备用；
    D将牡丹皮粉碎成细粉，灭菌，备用；
    步骤A得到的挥发油，步骤B得到的水提液，步骤C得到的醇提液和步骤D的细粉共同构成了本发明组合物的活性组分。
  8. 根据权利要求7所述的组合物，其特征在于片剂的制备工艺为：
    A将方中白术、羌活粗碎，辛夷粗碎破壳；按处方量称取白术、防风、高良姜、羌活、辛夷，加6-12倍量水，水蒸气蒸馏法提取挥发油，提取6-10小时，收集挥发油，备用；蒸馏后的水溶液滤过，减压浓缩，备用；
    B按处方量称取的黄芪、蝉蜕、甘草，加9-13倍量水煎煮三次，每次1-3小时，滤过，提取液减压浓缩，与挥发油浓缩液合并减压浓缩至相对密度1.15～1.20，备用；
    C按处方量称取的白芷、乌梅，加5-8倍量50-80％乙醇，提取二次，第一次1-3小时，第二次1-3小时，滤过，提取液减压回收乙醇，减压浓缩至相对密度1.15～1.20，备用；
    D将牡丹皮粉碎成细粉，灭菌，备用；
    E以牡丹皮细粉为底料，按常规工艺制粒，整粒，压片即得。
  9. 根据权利要求8所述的组合物，其特征在于片剂的制备工艺优选为：
    A将方中白术、羌活粗碎，辛夷粗碎破壳；按处方量称取白术、防风、高良姜、羌活、辛夷，加8-10倍量水，水蒸气蒸馏法提取挥发油，提取8小时，收集挥发油，备用；蒸馏后的水溶液滤过，减压浓缩，备用；
    B按处方量称取的黄芪、蝉蜕、甘草，加10-12倍量水煎煮三次，每次1小时，滤过，提取液减压浓缩，与挥发油浓缩液合并减压浓缩至相对密度1.15～1.20，备用；
    C按处方量称取的白芷、乌梅，加6-7倍量60-70％乙醇，提取二次，第一次2小时，第二次1.5小时，滤过，提取液减压回收乙醇，减压浓缩至相对密度1.15～1.20，备用；
    D将牡丹皮粉碎成细粉，灭菌，备用；
    E以牡丹皮细粉为底料，按常规工艺制粒，整粒，压片即得。
  10. 权利要求1-5任一所述的组合物在制备抗炎或抑制炎症慢性增殖药物中的应用。
  11. 权利要求1-5任一所述的组合物在制备抗I型变态反应药物中的应用。
  12. 权利要求1-5任一所述的组合物在制备降低组胺、总IgE、OVA特异性IgE、IL-10、IL-12水平降低鼻粘膜IL-4表达及升高IL-2表达药物中的应用。
  13. 权利要求1-5任一所述的组合物在制备升高IFN-γ水平药物中的应用。
  14. 权利要求1-5任一所述的组合物在制备缓解过敏症状药物中的应用。
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