WO2017041734A1 - 一种治疗糖尿病视网膜病变的中药组合物 - Google Patents

一种治疗糖尿病视网膜病变的中药组合物 Download PDF

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WO2017041734A1
WO2017041734A1 PCT/CN2016/098510 CN2016098510W WO2017041734A1 WO 2017041734 A1 WO2017041734 A1 WO 2017041734A1 CN 2016098510 W CN2016098510 W CN 2016098510W WO 2017041734 A1 WO2017041734 A1 WO 2017041734A1
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parts
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chinese medicine
extract
traditional chinese
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金明
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金明
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    • 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
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/62—Leeches; Worms, e.g. cestodes, tapeworms, nematodes, roundworms, earth worms, ascarids, filarias, hookworms, trichinella or taenia
    • 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)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/254—Acanthopanax or Eleutherococcus
    • 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)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • 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
    • 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)
    • A61K36/36—Caryophyllaceae (Pink family), e.g. babysbreath or soapwort
    • 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)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/537—Salvia (sage)
    • 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)
    • A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
    • 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)
    • A61K36/64—Orobanchaceae (Broom-rape family)
    • 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)
    • A61K36/80—Scrophulariaceae (Figwort family)
    • A61K36/804—Rehmannia
    • 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)
    • A61K36/894—Dioscoreaceae (Yam family)
    • 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)
    • A61K36/894—Dioscoreaceae (Yam family)
    • A61K36/8945—Dioscorea, e.g. yam, Chinese yam or water yam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the invention relates to a traditional Chinese medicine composition, in particular to a traditional Chinese medicine composition for treating diabetic retinopathy, belonging to the field of traditional Chinese medicine.
  • Diabetic retinopathy is one of the most important diseases in diabetic microvascular complications, and it has accounted for the first place in blindness and visual disability. How to delay the occurrence and development of DR has become the focus of attention and research in the medical community.
  • One of the important pathogenesis of DR is microcirculatory disorder. From the perspective of the whole process of diabetic retinopathy, comprehensive methods such as drugs, lasers, and surgery are its governance projects.
  • the virtual standard refers to the majority of diabetic patients experiencing early yin deficiency and heat, medium-term qi and yin deficiency, and late yin and yang deficiency. Stage; "standard” reflects "qi stagnation and blood stasis” throughout microvascular disease. Diabetic retinopathy usually occurs 10-20 years after the onset of illness. Qi and yin deficiency, qi stagnation and blood stasis are the main pathogenesis.
  • the main target is to cure the microvascular hemangioma, micro-hemorrhagic foci, hard exudation, edema and other signs.
  • the treatment is important in promoting blood circulation and removing blood stasis.
  • the general symptoms of qi and yin deficiency are common to both patients and the disease.
  • Qi Yang Yin blood circulation and phlegm. Under this basic principle, there are many researches on sugar nets. Including the comparison of different clinical syndrome types, the effects of different experimental rules on retinal microvascular disease, etc., due to irregularities or non-systemic drawbacks.
  • Another object of the present invention is to provide the use of the traditional Chinese medicine composition for preparing a medicament for treating diabetic retinopathy
  • Another object of the present invention is to provide a use of the traditional Chinese medicine composition for the preparation of a medicament for treating macular edema.
  • the invention relates to a traditional Chinese medicine composition for treating diabetic retinopathy.
  • the raw material composition of the traditional Chinese medicine composition comprises Astragalus, Radix Pseudostellariae, Guizhi, Angelica, Sanqi, Yam, Salvia, Dilong, Dihuang and Atractylodes.
  • the raw material composition of the traditional Chinese medicine composition is: 10 to 80 parts by weight of Astragalus, 5 to 30 parts by weight of Radix Pseudostellariae, 3 to 25 parts by weight of Guizhi, 8 to 60 parts by weight of Angelica, 2 to 15 parts by weight of Panax notoginseng. 10 to 80 parts by weight of yam, 10 to 80 parts by weight of salvia miltiorrhiza, 2 to 15 parts by weight of earthworm, 8 to 60 parts by weight of rehmannia, and 8 to 60 parts by weight of atractylodes.
  • composition of the raw material of the traditional Chinese medicine composition is: 15 to 60 parts by weight of Astragalus, 6 to 25 parts by weight of Radix Pseudostellariae, 5 to 20 parts by weight of Guizhi, 10 to 50 parts by weight of Angelica, and 3 to 12 weights of Panax notoginseng. Parts, yam 15 to 60 parts by weight, salvia miltiorrhiza 15 to 60 parts by weight, earthworm 3 to 12 parts by weight, rehmannia 10 to 50 parts by weight, and atractylodes 10 to 50 parts by weight.
  • composition of the raw material of the traditional Chinese medicine composition is: 20-50 parts by weight of Astragalus, 8-20 parts by weight of Radix Pseudostellariae, 8-18 parts by weight of Guizhi, 12-40 parts by weight of Angelica, 4-10 weight of Panax notoginseng.
  • composition of the raw material of the traditional Chinese medicine composition is: 25-40 parts by weight of Astragalus, 10-18 parts by weight of Radix Pseudostellariae, 9-15 parts by weight of Guizhi, 15-30 parts by weight of Angelica, and 5-8 weights of Panax notoginseng. Parts, yam 25 to 40 parts by weight, salvia miltiorrhiza 25 to 40 parts by weight, earthworm 5 to 8 parts by weight, rehmannia 15 to 30 parts by weight, and atractylodes 15 to 30 parts by weight.
  • composition of the raw material of the traditional Chinese medicine composition is: 30 parts by weight of Astragalus, Radix Pseudostellariae 12 parts by weight, 10 parts by weight of cassia twig, 20 parts by weight of angelica, 6 parts by weight of ginseng, 30 parts by weight of yam, 30 parts by weight of salvia miltiorrhiza, 6 parts by weight of earthworm, 20 parts by weight of rehmannia, 20 parts by weight of atractylodes;
  • Astragalus 38 parts by weight of Astragalus, 11 parts by weight of Radix Pseudostellariae, 14 parts by weight of cassia twig, 16 parts by weight of Angelica, 7 parts by weight of ginseng, 28 parts by weight of yam, 35 parts by weight of Salvia miltiorrhiza, 5 parts by weight of Dilong, and 28 parts by weight of Rehmannia glutinosa , Atractylodes 16 parts by weight.
  • the ginseng can be replaced by ginseng and American ginseng.
  • the atractylodes is preferably atracting atractylodes; the rehmannia is preferably rehmannia.
  • the traditional Chinese medicine composition of the present invention may be a composition obtained by directly pulverizing the above-mentioned raw material medicine after mixing, or may be an extract obtained by mixing a raw material medicine according to a conventional extraction method, or an effective part obtained by further purifying and purifying the extract. Or a conventional oral dosage form prepared according to a conventional formulation process;
  • the conventional extraction method comprises immersion extraction, decoction extraction, reflux extraction, osmosis extraction, ultrasonic extraction, etc.
  • the extraction solvent comprises water, 20-95% ethanol solution
  • the purification and purification process includes alcohol precipitation, extraction, and silica gel Column separation, macroporous resin column separation, etc.
  • the conventional oral dosage forms include powders, tablets, capsules, granules, oral liquids, pills, and the like.
  • the preparation method of the traditional Chinese medicine composition of the invention is:
  • the raw materials are taken in proportion, extracted by water or organic solvent according to a conventional extraction method, and prepared into a conventional oral dosage form according to a conventional preparation process.
  • the above conventional extraction method includes any one of conventional extraction methods such as boiling extraction, reflux extraction, immersion extraction, ultrasonic extraction or osmotic extraction, or a combination of different extraction methods; the organic solvent is 20 to 95% ethanol. Solution.
  • the conventional oral dosage forms include granules, tablets, powders, capsules, oral liquids, pills, and the like.
  • Filling agents include: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrating agents include: starch, pregelatinized starch, microcrystalline cellulose, carboxymethyl Base starch sodium, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silica, etc.
  • Suspension agents include: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; sweet Flavoring agents include: sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various flavors; preservatives include: parabens, benzoic acid, sodium benzoate, Sorbic acid and its salts, benzalkonium bromide, chlorhexidine acetate, eucalyptus oil, etc.; the matrix includes: PEG6000, PEG4000, insect wax and the like.
  • the traditional Chinese medicine composition of the present invention can also adopt the extract of the above-mentioned original medicinal material (effective part) in addition to the forms of the raw materials of Astragalus, Radix Pseudostellariae, Guizhi, Angelica, Sanqi, Yam, Salvia, Dilong, Dihuang, and Atractylodes.
  • the present invention further discloses a traditional Chinese medicine composition for treating diabetic retinopathy:
  • a traditional Chinese medicine composition for treating diabetic retinopathy comprising the following raw materials: 10 to 80 parts by weight of astragalus extract, 5 to 30 parts by weight of extract of Radix Pseudostellariae, 3 to 25 parts by weight of cassia twig extract, 8 to 60 parts by weight of Angelica sinensis extract, 2 to 15 parts by weight of Panax notoginseng extract, 10 to 80 parts by weight of yam extract, 10 to 80 parts by weight of Salvia miltiorrhiza extract, 2 to 15 parts by weight of Dijon extract, and Rehmannia glutinosa extract 8 to 60 parts by weight, and 8 to 60 parts by weight of atractylodes extract.
  • the composition of the raw material of the traditional Chinese medicine composition is: 15 to 60 parts by weight of the astragalus extract, 6 to 25 parts by weight of the extract of Radix Pseudostellariae, 5 to 20 parts by weight of the osmanthus extract, and 10 to 50 parts by weight of the angelica extract.
  • the extract is 10 to 50 parts by weight.
  • the raw material composition of the traditional Chinese medicine composition is: 25 to 40 parts by weight of the astragalus extract, 10 to 18 parts by weight of the extract of Radix Pseudostellariae, 9 to 15 parts by weight of the osmanthus extract, and 15 to 30 parts by weight of the angelica extract. 5 parts to 8 parts by weight of extract of Panax notoginseng, 25 to 40 parts by weight of yam extract, 25 to 40 parts by weight of Salvia miltiorrhiza extract, 5 to 8 parts by weight of Dilong extract, 15 to 30 parts by weight of Rehmannia glutinosa extract, and Atractylodes macrocephala L.
  • the extract is 15 to 30 parts by weight.
  • composition of the raw material of the traditional Chinese medicine composition is: 30 parts by weight of Astragalus membranaceus extract, 12 parts by weight of Radix Pseudostellariae extract, 10 parts by weight of cassia twig extract, 20 parts by weight of Angelica sinensis extract, and 6 weight of Panax notoginseng extract. And 30 parts by weight of yam extract, 30 parts by weight of Salvia miltiorrhiza extract, 6 parts by weight of Dilong extract, 20 parts by weight of Rehmannia glutinosa extract, and 20 parts by weight of Atractylodes macrocephala extract;
  • the raw material drug extract may be an aqueous extract of each raw material drug, or an organic solvent extract, or a purified product obtained by further purifying and purifying the aqueous extract/organic solvent extract.
  • the organic solvent is a 20 to 95% ethanol solution
  • the extraction method for preparing the above extract includes any one of conventional extraction methods in the field such as decoction extraction, reflux extraction, impregnation extraction, ultrasonic extraction or percolation extraction, or a combination of different extraction methods; the purification purification process Including alcohol precipitation, extraction, silica gel column separation, macroporous resin column separation.
  • the traditional Chinese medicine composition of the invention is mainly composed of medicines such as astragalus, ginseng, cassia twig, panax notoginseng, salvia miltiorrhiza and earthworm.
  • Astragalus, Ginseng and Guizhi have the functions of warming the yang and strengthening the spleen and replenishing yang, which can speed up the blood flow and improve the blood supply quality.
  • Sanqi, Danshen and Dilong both stop bleeding and promote blood circulation, both collaterals and phlegm, and have reduced platelets.
  • A normal group
  • B model group
  • C experimental group
  • D control group
  • A normal group
  • B model group
  • C experimental group
  • D control group
  • A normal group
  • B model group
  • C experimental group
  • D control group
  • A normal group
  • B model group
  • C experimental group
  • D control group
  • the subjects were from 80 patients (128 eyes) diagnosed with diabetic macular edema (DME) from January 2014 to July 2015. Among them, 49 patients (80 eyes) in the treatment group and 31 patients (48 eyes) in the control group.
  • the treatment group consisted of 18 males (29 eyes) and 31 females (51 eyes) with an average age of 59.80 ⁇ 10.21 years and a course of 7.41 ⁇ 4.62 years.
  • the control group consisted of 11 males (17 eyes) and 20 females (31 eyes). The age was 57.63 ⁇ 12.12 years old and the course of disease was 8.17 ⁇ 5.34 years.
  • Diabetic macular edema Refer to the "International Classification of Diabetic Macular Edema": a total of mild, moderate and severe: mild DME: the posterior pole retina has a certain degree of thickening and hard exudation, But far from the center of the macula; moderate DME: the posterior pole retina has a certain degree of thickening and hard exudation, close to the center of the macula but not involving the center; severe DME: retinal thickening and hard exudation, involving the center of the macula.
  • FFA fundus fluorescein angiography
  • ETDRS American DR early treatment research group standard
  • local edema type macular Localized edema, macular telangiectasia and microaneurysm leakage fluorescence
  • diffuse edema type range ⁇ 2PD, and accumulated avascular area in the center of the macula.
  • a large number of capillary leaks can be diffused to the upper and lower vascular arches; cystic edema type: severe edema, although the capillary leakage is not diffused to the upper and lower vascular arch, but the late FFA fluorescein leakage is distributed around the arch ring Petals or ring-shaped high fluorescence.
  • the patients in the treatment group were orally administered with the traditional Chinese medicine composition of the present invention (prepared according to the method of Example 1) twice a day, 100 ml each time, orally for three months.
  • Compound Xueshuantong Capsule is contained in the Chinese Pharmacopoeia (2015 edition). Its raw materials are composed of Sanqi, Astragalus, Salvia, and Scrophulariaceae. It has the effect of promoting blood circulation, replenishing qi and nourishing yin; Retinal vein occlusion of yin deficiency syndrome, seeing vision loss or visual abnormalities, fundus blood stasis and other symptoms.
  • OCT measures the foveal thickness of the macula (TOPCON3DOCT-1000).
  • the visual acuity evaluation criteria were formulated as follows: Using an international standard visual acuity chart, the best corrected visual acuity was stable within one week. There is no light perception to the light sensor 2 lines, and the light sense, manual, index, 0.02, 0.04, 0.06, 0.08, and 0.1 intervals are all counted. After treatment, the visual acuity improvement ⁇ 2 behavior is effective; the visual acuity is improved by 1 line or the visual acuity is unchanged and the fundus condition is improved; the visual acuity is invalid.
  • the distribution of visual acuity of patients before treatment is shown in Table 1.
  • the visual acuity changes after 1 month of treatment were as shown in Table 2.
  • the visual acuity of the treatment group and the control group were improved compared with those before treatment.
  • the difference was statistically significant (P ⁇ 0.05).
  • ⁇ 2 test there was no significant difference in visual acuity between the treatment group and the control group (P>0.05).
  • the visual acuity changes after 3 months of treatment were as shown in Table 3.
  • the visual acuity of the treatment group and the control group were significantly improved compared with those before treatment (P ⁇ 0.05).
  • Using the ⁇ 2 test there was no significant difference in visual acuity between the two groups (P>0.05). It can be seen that both the treatment group and the control group can effectively improve the visual acuity of DME patients, but there is no significant difference in the degree of visual acuity improvement between the two.
  • Table 4 The thickness of the fovea before and after treatment
  • the number of retinal microangiomas was observed by color fundus photography before and after treatment. See Table 5 for details. After non-parametric test, the number of microangiomas in the treatment group was not significantly different from that in the control group after 1 month of treatment (P>0.05). After 3 months of treatment, the number of microangiomas decreased, and the treatment group was significantly better than the control group. Statistically significant (P ⁇ 0.05). It is indicated that the traditional Chinese medicine composition of the present invention is combined with laser treatment of DR, and the change of microangioma can only take advantage in three months.
  • * indicates that P>0.05 compared with the treatment group , indicating P ⁇ 0.05 compared with the treatment group.
  • the changes of retinal leakage area were calculated by fundus fluorescein angiography before and after treatment. See Table 6 for details. After non-parametric test, after treatment for 1 month, the leakage area of the treatment group was significantly better than that of the control group, the difference was statistically significant (P ⁇ 0.05). After 3 months of treatment, the leakage area of the two groups was reduced compared with the treatment group. The degree was significant and the difference was statistically significant (P ⁇ 0.05). It can be seen that the treatment group can better reduce the leakage area and the effect is stable.
  • the traditional Chinese medicine composition has the curative effect on the patients with macular edema, can effectively improve the vision of the patient, reduce the thickness of the fovea, reduce the leakage area, and reduce the number of microhemangioma.
  • VAF male Sprague-Dawley rats weighing 140-160 g were purchased from Vitallihua Experimental Animal Technology Co., Ltd. and were raised in the Experimental Animal Center of the Institute of Clinical Medicine of China-Japan Friendship Hospital. Rats were free to eat and drink during the experiment. Standard rat feed was purchased from Beijing Jiujiang Granule Feed Factory, and squirrel cages and litter were purchased from Beijing Kelin Experimental Animal Service Center. After 1 week of adaptive feeding in rats, there were 70 rats weighing 180-200 g. Except the normal group, the rats before the model were fasted for 12 hours. After intraperitoneal injection of streptozotocin (STZ), STZ was used.
  • STZ streptozotocin
  • a sterile 0.1 mmol/L, pH 4.4 sodium citrate buffer solution was used to prepare a 1% STZ solution, and 65 mg/Kg rat body weight was injected into the lower left abdominal cavity.
  • the normal control group was injected with the same volume of physiological saline. After 72 hours, the tail vein blood was taken, and the blood glucose was measured with a fast blood glucose meter (Roche blood glucose meter, Lecon full blood glucose test strip).
  • a model of diabetic rats is a non-fasting blood glucose ⁇ 16.7mmol/L.
  • the diabetic rats were randomly divided into a model group of 10, an experimental group of 10 (the traditional Chinese medicine composition prepared according to Example 1), and a normal group of 10 rats.
  • the experimental period is 300 days.
  • VEGF vascular endothelial cells
  • IAM-1 intercellular adhesion molecule-1
  • PEDF pigment epithelium-derived factor
  • AGEs advanced glycation end products
  • the fresh eyeball was lysed and the mRNA levels of VEGF mRNA, PEDF mRNA, ICAM-1 mRNA and RAGE were detected by RT-PCR. Immunohistochemical color intensity was measured with an image analyzer for quantitative analysis. The data was analyzed and processed by SPSS 13.0 statistical software.
  • E Group n Endothelial cells
  • P Pericytes
  • E/P normal group 10 796.40 ⁇ 18.48 203.50 ⁇ 18.54 3.95 ⁇ 0.45
  • Model group 10 919.00 ⁇ 10.22* 81.10 ⁇ 10.06* 11.50 ⁇ 1.56 * test group 9 874.78 ⁇ 15.75* ⁇ 125.22 ⁇ 15.75* ⁇ 7.10 ⁇ 0.98 * ⁇
  • Model group, experimental group and normal group * P ⁇ 0.01; treatment group compared with model group: ⁇ P ⁇ 0.01.
  • the traditional Chinese medicine composition can reduce the expression of AGEs and RAGE mRNA in the retina of diabetic rats, reduce the deposition of AGEs in endothelial cells, pericytes and basement membrane, reduce the stimulation of endothelial cells, thereby reducing the production of VEGF and thereby reducing the tube. Wall dysfunction.
  • the adhesion of leukocytes to vascular endothelial cells was alleviated by reducing the expression of ICAM-1 and retinal ICAM-1 mRNA in the retinal capillaries.
  • the permeability of blood vessels is reduced and the expression of ICAM-1 is down-regulated. It can increase the expression of retinal PEDF and retinal PEDF mRNA, thereby inhibiting neovascularization.
  • mice 48 SPF-class six-week-old SD rats, male, weighing (220 ⁇ 20) g, were purchased from Huakang Biotechnology Co., Ltd. After 1 week of adaptive feeding, the rats were randomly divided into 36 models and 12 rats in the normal group. The model of experimental diabetic rats was induced by fasting for 12 hours before modeling and intraperitoneal injection of 1% STZ solution 60 mg/kg body weight. Rats in the normal group were injected with the same volume of normal saline. Blood glucose was monitored 72 hours later, blood was taken from the tail vein, and blood glucose ⁇ 16.7mmol/L for 3 consecutive days was successful. After successful modeling, the rats were randomly divided into 12 model groups and 12 Chinese herbal medicine experimental groups.
  • the rats in the experimental group and the control group were administered the next day after successful modeling.
  • the rats in the experimental group were given 10 ml/(kg ⁇ d); the rats in the control group were administered at 250 mg/(kg ⁇ d).
  • the Dobes powder was dissolved in 2 ml of distilled water.
  • Rats in the normal group and the model group were intragastrically administered with 2 ml of distilled water per day. During the experiment, all rats were fed feed and water regularly every day; the body weight and blood glucose changes were monitored monthly. After 9 months, take the material.
  • the retinal digested patch specimens of 6 rats in each group were randomly prepared.
  • the preparation method was as follows: after the eyeball was fixed in 4% paraformaldehyde solution for 48 hours, the running water was gently washed for 5 min, and the sclera was cut by the serrated ring. The anterior segment of the eye was removed; the posterior cup was cut into 3 pieces with the nipple as the center, and the retina was gently separated; PBS (0.01 mol/L pH 7.4) was rinsed for 10 min; 3% trypsin was incubated at 37 °C. 3h; transferred into distilled water for rinsing and shaking several times, leaving only a layer of transparent retinal vascular network; floated and tiled on a glass slide, PAS staining, observed under an Olympus optical microscope.
  • Paraffin sections were prepared from the bilateral eyeballs of the remaining 6 rats: the eyeball and optic nerve were fixed in 4% paraformaldehyde (4 ° C, 48 h), and the eye wall was cut along the limbus to remove the cornea, crystal, vitreous, gradient. Alcohol dehydration, xylene transparent, paraffin embedded, serial sectioning, slice thickness 10 ⁇ m, HE staining and immunohistochemical staining, observed under Olympus optical microscope.
  • the other eyeballs of 6 rats were removed and fixed in 2.5% glutaraldehyde (4 °C) for 72 hours.
  • the central region of the retina was excised 2 mm ⁇ 2 mm.
  • 1% citric acid fixed, acetone dehydrated, Epon812 ring Oxygen resin was embedded, and a thin slice of 1 ⁇ m was cut for light microscopy, and then ultrathin sections were taken and double stained with uranyl acetate and lead citrate, and observed under Hitachi H-600 transmission electron microscope.
  • ⁇ means P>0.05 compared with normal group
  • * means P ⁇ 0.05 compared with normal group
  • Retinal digestive patching under microscope normal rat retinal capillaries distribution rule
  • the direction is straight, the diameter of the tube is uniform and uniform; the endothelial cells are generally located in the central part of the capillaries, the nucleus is larger, the stain is shallower, and the shape is elliptical or irregular.
  • the long axis is mostly parallel to the capillaries; the pericytes are located in the capillary tube Outside the cavity, the nucleus is small, the staining is deep, and it is mostly spherical or triangular (see Figure 1-A).
  • the retinal capillary network was disordered, the orientation was extremely irregular, the thickness of the lumen was uneven, the multiple capillaries were twisted into sputum, the segmental expansion, the nucleus of the pericytes was pyknosis, and the endothelial cells and extracellular matrix were obviously proliferated.
  • Characteristic changes in diabetic retina such as cell-free capillaries, periculocytes, and endothelial cell apoptosis (see Figure 1-B).
  • retinal microangiopathy was significantly protected or delayed: no typical early DR lesions such as acellular capillaries and pericytes, capillary vessels The distribution is relatively regular, the thickness is relatively uniform, and there is no obvious distortion and expansion.
  • HE staining of retinal paraffin sections was observed under light microscope: the layers of the retinas in the normal group were clear and arranged neatly (see Figure 2-A).
  • the layers of the retina were extremely disordered, the telangiectasia was dilated, the edema of the nerve fiber layer was thickened, the number of retinal ganglion cells was reduced, vacuolar degeneration, nucleus pyknosis, chromatin edge aggregation, and partial retinal ganglion cell necrosis. , nuclear dissolution, internal plexiform edema, core layer vacuolar degeneration (see Figure 2-B).
  • the rat retinal interstitial was mildly edematous and the cells were arranged neatly (see Figure 2-C).
  • the retinal reticular structure was loose, the inner and outer granular layers were disordered, telangiectasia and interstitial edema (see Figure 2-D).
  • vascular endothelial growth factor The immunohistochemically labeled vascular endothelial growth factor (VEFG) was widely expressed in the retina of rats, and the positive expression site was brownish yellow or tan.
  • the gray value of VEGF expression in each group of retinas was significantly higher in the model group (see Figure 3-A) than in the normal group (see Figure 3-B) (P ⁇ 0.01), and the experimental group (see Figure 3-C).
  • the expression of VEGF in the control group and the control group (Fig. 3-D) was significantly lower than that in the model group (P ⁇ 0.01), which was statistically significant, but the difference between the experimental group and the control group was not significant (see Table 9).
  • test group 6 0.42 ⁇ 0.86 *# ⁇ Control group 6 0.43 ⁇ 0.09 *#
  • the retinal capillary endothelial cells of the model group were obviously swollen, the cell body became round, and the cell body became round, protruding into the lumen, mitochondria swelling, vacuolization, thickening of the basement membrane, deformation of the ganglion cell body, obvious condensation of the nucleus, disappearance of the nuclear membrane, cytoplasm
  • vacuoles of varying sizes mitochondria swelling, markedly reduced cell surface protrusions, and a marked decrease in polysomes and rough endoplasmic reticulum (see Figure 4-B).
  • the retinal capillary wall was still smooth, the basement membrane was slightly thickened, a few pericytes were edematous, some endothelial cells were slightly hyperplasia, the ganglion cells were deformed, the mitochondria were slightly swollen, and the structure was slightly ill (see Figure 4-C).
  • the retinal capillary lumen wall was still smooth, the basement membrane was thickened, a few pericytes were edematous, some endothelial cells were swollen, some mitochondria were vacuolated, ganglion cell bodies were deformed, mitochondria were slightly swollen, and chromatin was dissolved. The gap is more expanded or even disappears (see Figure 4-D).
  • the retinal angiogenesis of experimental diabetic rats has obvious pathological damage; the traditional Chinese medicine composition has a certain protective effect on the retinal blood vessels of diabetic rats, can inhibit the expression of VEGF in the retina of diabetic rats, and helps to reduce the retinal blood vessels. damage.
  • the molding method was the same as Experimental Example 2.
  • the diabetic rats were randomly divided into 5 model groups, 5 experimental groups (Chinese medicine composition prepared according to Example 1), and 5 rats in the normal group.
  • the experimental period is 300 days.
  • GFAP retinal fibrillary acidic protein
  • RGCs retinal ganglion cells
  • GFAP glutamate transporter
  • GS glutamine synthetase
  • LSAB assay immunohistochemistry
  • NT-3 neurotrophin-3
  • the determination of positive apoptotic RGCs cells the cells were stained with brownish yellow (brown) color as positive cells.
  • the counting method of positive apoptotic RGCs cells under high magnification (10 ⁇ 40 times) binocular microscope, along the side of the optic disc, the zigzag edge, continuous 5 fields of view, taking the mean value as apoptotic RGCs cells/high power field, reading continuously Three sections, the average number of RGCs cells as the number of apoptosis of RGCs in the sample. The results of this experiment showed that the rat retinal apoptosis-positive cells were only found in the RGCs layer and the inner nuclear layer by TUNEL assay. The effects of each group on the apoptosis of RGCs are shown in Table 10.
  • the above results showed that compared with normal rats, the number of RGCs apoptosis in the model group was significantly increased (P ⁇ 0.01). Compared with the model group, the number of RGCs apoptosis in the experimental group was significantly reduced (P ⁇ 0.05), suggesting the present invention.
  • the composition can inhibit the apoptosis of RGCs in model rats and has neuroprotective effects.
  • the molding was the same as Experimental Example 2.
  • diabetic rats were randomly divided into 20 model groups, 30 experimental groups (Chinese medicine composition prepared according to Example 1), 20 control groups (Dobes group), and 12 normal rats.
  • the experimental period is 300 days.
  • Observation methods and indicators using iris blood flow perfusion scan and iris fluorescein angiography to observe iris blood flow velocity, iris vessel diameter, iris microvessel density, iris microvascular endothelial structure, iris microvascular permeability and iris unit area perfusion the amount.
  • ** indicates P ⁇ 0.01 compared with the normal group
  • ⁇ indicates P ⁇ 0.01 compared with the model group.
  • 2.2 iris vessel diameter the results are shown in Table 12. The results showed that the diameter of the iris vessels in the normal group was uniform, and the diameter of the vessels in the model group was significantly expanded compared with the normal group. The experimental group was significantly lower than the model group (P ⁇ 0.01). There was no significant difference between the Dobes group and the model group.
  • ** indicates P ⁇ 0.01 compared with the normal group
  • ⁇ indicates P ⁇ 0.01 compared with the model group.
  • ** indicates P ⁇ 0.01 compared with the normal group
  • ⁇ indicates P ⁇ 0.05 compared with the model group
  • the experimental composition of the present invention can accelerate the blood flow of the iris, inhibit the abnormal expansion of the blood vessel and improve the abnormal increase of the permeability of the iris microvessel, and can increase the perfusion of the unit surface and alleviate the ischemic state.
  • composition of raw materials Astragalus 30g, Radix Pseudostellariae 12g, Guizhi 10g, Angelica 20g, Sanqi 6g, Yam 30g, Salvia 30g, Dilong 6g, Rehmannia 20g, Fried Atractylodes 20g;
  • the preparation method comprises the following steps: taking the raw materials according to the ratio, adding boiling water twice, each time for 1.5 hours, combining the decoction, filtering, and concentrating to 200 ml;
  • Dosage twice daily, 100ml each time.
  • composition of raw materials Astragalus 28g, Radix Pseudostellariae 16g, Guizhi 9g, Angelica 28g, Sanqi 5g, Yam 35g, Salvia 28g, Dilong 7g, Rehmannia 16g, Fried Atractylodes 28g;
  • the preparation method comprises the following steps: taking the raw material medicine in proportion and ultrasonically extracting twice with 70% ethanol, the first time is 1 hour, the second time is 0.5 hour, the combined extracts are filtered, concentrated under reduced pressure, and the extract is used as an excipient with dextrin. Mix and mix according to the ratio of 3:1, mix well, make granules, dry, and obtain granules.
  • composition of raw materials Astragalus 38g, Radix Pseudostellariae 11g, Guizhi 14g, Angelica 16g, Sanqi 7g, Yam 28g, Salvia 35g, Dilong 5g, Rehmannia 28g, Fried Atractylodes 16g;
  • the preparation method comprises the following steps: taking the raw material medicine in proportion, adding water to cook twice, each time for 1.5 hours, combining the decoction, filtering, and forming an oral liquid according to a conventional process.
  • composition of raw materials Astragalus 22g, Ginseng 18g, Guizhi 9g, Angelica 35g, Sanqi 5g, Yam 45g, Salvia 22g, Dilong 8g, Rehmannia 15g, Atractylodes 35g;
  • Preparation method take the raw material according to the ratio, add water and reflux to extract twice, each time for 1.5 hours, combine the decoction, filter, concentrate, add 60% ethanol solution to alcohol precipitation, let stand, filter, concentrate, dry under reduced pressure, to paste Fine excipients, mixed according to the ratio of 3:1, mixed, made into granules, dried, and obtained granules.
  • composition of raw materials Astragalus 45g, Ginseng 10g, Guizhi 15g, Angelica 13g, Sanqi 8g, Yam 22g, Salvia 48g, Dilong 5g, Rehmannia 38g, Atractylodes 15g;
  • Preparation method taking the raw materials in proportion, mixing, pulverizing through a 60-80 mesh sieve to prepare a powder.
  • composition of raw materials Astragalus 16g, American ginseng 24g, Guizhi 6g, Angelica 45g, Sanqi 4g, Yam 55g, Danshen 16g, Dilong 10g, Rehmannia 12g, Atractylodes 45g;
  • the preparation method comprises the following steps: taking the raw material medicine in proportion, adding water to cook twice, each time for 1.5 hours, combining the decoction, filtering, and forming an oral liquid according to a conventional process.
  • composition of raw materials Astragalus 55g, Radix Pseudostellariae 7g, Guizhi 18g, Angelica 12g, Sanqi 11g, Mountain 18g of medicine, 55g of salvia miltiorrhiza, 4g of earthworm, 45g of rehmannia, 12g of fried atractylodes;
  • the preparation method comprises the following steps: taking the raw material medicine in proportion and ultrasonically extracting twice with 70% ethanol, the first time is 1 hour, the second time is 0.5 hour, the combined extracts are filtered, concentrated under reduced pressure, and the D101 macroporous resin is purified on the concentrated liquid, first Elution with 2BV water, elution with 5BV 50% ethanol solution, collection of 50% ethanol eluate, recovery of ethanol, the extract was prepared into a tablet by a conventional process, adding conventional excipients.
  • composition of raw materials Astragalus 12g, Pseudostellariae 28g, Guizhi 4g, Angelica 55g, Sanqi 3g, Yam 75g, Salvia miltiorrhiza 12g, Dilong 14g, Rehmannia 9g, Fried Atractylodes 55g;
  • Preparation method take the raw materials according to the ratio, add boiling water twice, each time for 1.5 hours, combine the decoction, filter, concentrate, dry under reduced pressure, use dextrin as excipient, mix according to the ratio of 3:1 , mix, make granules, dry, and obtain granules.
  • composition of raw materials Astragalus 75g, Radix Pseudostellariae 6g, Guizhi 24g, Angelica 8g, Sanqi 14g, Yam 12g, Salvia 75g, Dilong 3g, Rehmannia 55g, Fried Atractylodes 10g;
  • the preparation method comprises the following steps: taking the raw material medicine in proportion, adding 6 times of 75% ethanol and refluxing for 2 times, each time for 1.5 hours; combining the extracts, filtering, adding a conventional auxiliary material to prepare an oral liquid.
  • composition of raw materials Astragalus extract 30g, Radix Pseudostellariae extract 12g, Guizhi extract 10g, Angelica extract 20g, Panax notoginseng extract 6g, Yam extract 30g, Salvia miltiorrhiza extract 30g, Dilong extract 6g, Dihuang extract 20g, Atractylodes macrocephala extract 20g;
  • the above-mentioned raw material drug extracts are water extracts obtained by adding boiling water to the raw material medicine, and the aqueous extracts of the raw material medicines are combined, filtered, and an oral liquid is prepared according to a conventional process.
  • composition of raw materials Astragalus extract 28g, ginseng extract 16g, Guizhi extract 9g, Angelica extract 28g, Panax notoginseng extract 5g, Yam extract 35g, Salvia miltiorrhiza extract 28g, Dilong extract 7g, Rehmannia extract 16g, fried atractylodes extract 28g;
  • Each of the above raw material drug extracts is an alcohol extract obtained by refluxing the raw material medicine with a 70% ethanol solution, and the alcohol extract of each raw material medicine is concentrated, dried under reduced pressure, and uniformly mixed, and the dextrin is used as an excipient. 3:1 is mixed, mixed, granulated, dried, and obtained granules.
  • composition of raw materials Astragalus extract 38g, American ginseng extract 11g, Guizhi extract 14g, Angelica extract 16g, Panax notoginseng extract 7g, Yam extract 28g, Salvia miltiorrhiza extract 35g, Dilong extract 5g, Rehmannia extract 28g, fried Atractylodes Rhizome extract 16g;
  • Each of the above-mentioned raw material drug extracts is an aqueous extract obtained by adding water to a raw material medicine.
  • the aqueous extracts of the raw materials are concentrated, dried under reduced pressure, and uniformly mixed.
  • the dextrin is used as an excipient, mixed at a ratio of 3:1, mixed, and granulated, dried, and obtained granules.

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Abstract

一种治疗糖尿病视网膜病变的中药组合物，所述中药组合物由下列重量的原料制备而成：黄芪10～80、太子参5～30、桂枝3～25、当归8～60、三七2～15、山药10～80、丹参10～80、地龙2～15、地黄8～60、白术8～60。该中药组合物可治疗或减轻黄斑水肿，提高患者的视力、视野，缓解眼底病变。

Description

一种治疗糖尿病视网膜病变的中药组合物 技术领域
本发明涉及一种中药组合物，具体涉及一种治疗糖尿病视网膜病变的中药组合物，属于中医药领域。
背景技术
糖尿病是目前世界上对人类健康造成严重威胁的重大疾病之一，患病率高达10％，约50-60％糖尿病患者死于糖尿病的大、小血管并发症。糖尿病视网膜病变(DR)就是糖尿病微血管并发症中最重要的病变之一，目前已占失明和视力致残第一位。如何延缓DR的发生和发展已经成为当今医学界关注和研究的重点。DR的重要发病机制之一为微循环障碍。从糖尿病性视网膜病变全程控制角度看，药物、激光、手术等综合手段是其治理工程。早期糖尿病性视网膜病变是中药干预的关键环节，特别是合并黄斑水肿导致中心视力下降给糖尿病患者带来的痛苦比疾病本身更大。合理应用中医药可以延缓糖尿病视网膜病变发展速度并拦截早期黄斑水肿，对于维护视力具有优势。尽管糖尿病性黄斑水肿一直是眼科治疗难点，但不可否认中药发挥了极大作用。通过临床观察和实验研究，充分利用“转化平台”，深入探讨中药作用机制为临床用药、推广提供客观依据。
综观消渴目病的发病过程，中医大家认可的病因病机为“本虚标实”，“本虚”指的是糖尿病患者大多经历早期阴虚燥热，中期气阴两虚，晚期阴阳俱虚阶段；“标实”体现“气滞血瘀”贯穿微血管病变始终。糖尿病性视网膜病变一般多在患病后10-20年发生，气阴两虚、气滞血瘀是其主要病机。围绕病机以“治标”为主，抓住眼底微血管瘤、微小出血灶、硬性渗出、水肿等征象，施治重在活血化瘀；全身出现气阴不足症候者，标本兼治，重在益气养阴、活血化瘀。在这个基本原则下有关糖网的研究很多，包 括临床不同证型比较、实验不同治则对视网膜微血管病变影响等，因不规范或不系统存在弊端。
发明内容
本发明的目的在于提供一种治疗糖尿病视网膜病变的中药组合物。
本发明的另一个目的在于提供该中药组合物在制备治疗糖尿病视网膜病变药物中的应用；
本发明的另一个目的在于提供该中药组合物在制备治疗黄斑水肿药物中的应用。
本发明的目的是通过如下技术方案实现的：
一种治疗糖尿病视网膜病变的中药组合物，该中药组合物的原料药组成包括黄芪、太子参、桂枝、当归、三七、山药、丹参、地龙、地黄、白术。
进一步，所述中药组合物的原料药组成为：黄芪10～80重量份、太子参5～30重量份、桂枝3～25重量份、当归8～60重量份、三七2～15重量份、山药10～80重量份、丹参10～80重量份、地龙2～15重量份、地黄8～60重量份、白术8～60重量份。
更进一步，所述中药组合物的原料药组成为：黄芪15～60重量份、太子参6～25重量份、桂枝5～20重量份、当归10～50重量份、三七3～12重量份、山药15～60重量份、丹参15～60重量份、地龙3～12重量份、地黄10～50重量份、白术10～50重量份。
更进一步，所述中药组合物的原料药组成为：黄芪20～50重量份、太子参8～20重量份、桂枝8～18重量份、当归12～40重量份、三七4～10重量份、山药20～50重量份、丹参20～50重量份、地龙4～10重量份、地黄12～40重量份、白术12～40重量份。
更进一步，所述中药组合物的原料药组成为：黄芪25～40重量份、太子参10～18重量份、桂枝9～15重量份、当归15～30重量份、三七5～8重量份、山药25～40重量份、丹参25～40重量份、地龙5～8重量份、地黄15～30重量份、白术15～30重量份。
更进一步，所述中药组合物的原料药组成为：黄芪30重量份、太子参 12重量份、桂枝10重量份、当归20重量份、三七6重量份、山药30重量份、丹参30重量份、地龙6重量份、地黄20重量份、白术20重量份；
或，黄芪28重量份、太子参16重量份、桂枝9重量份、当归28重量份、三七5重量份、山药35重量份、丹参28重量份、地龙7重量份、地黄16重量份、白术28重量份；
或，黄芪38重量份、太子参11重量份、桂枝14重量份、当归16重量份、三七7重量份、山药28重量份、丹参35重量份、地龙5重量份、地黄28重量份、白术16重量份。
所述太子参可用人参、西洋参替代。
所述白术优选炒白术；所述地黄优选生地黄。
本发明所述中药组合物可以是上述原料药混合后直接粉碎得到的组合物，也可以是原料药混合后按常规提取方法提取得到的提取物，或提取物进一步经过精制纯化工艺得到的有效部位，或进一步按照常规制剂工艺制备得到的常规口服剂型；
其中，所述常规提取方法包括浸渍提取、煎煮提取、回流提取、渗漉提取、超声提取等；提取溶剂包括水、20-95％乙醇溶液；所述精制纯化工艺包括醇沉、萃取、硅胶色谱柱分离、大孔树脂柱分离等；所述常规口服剂型包括散剂、片剂、胶囊剂、颗粒剂、口服液、丸剂等。
本发明中药组合物的制备方法为：
按比例取各原料药，以水或有机溶剂按常规提取方法提取，并按常规制剂工艺制备成常规口服剂型。
上述常规提取方法包括煎煮提取、回流提取、浸渍提取、超声提取或渗漉提取等常规提取方法中的任意一种方式，或者不同提取方法的组合；所述有机溶剂为20～95％的乙醇溶液。所述常规口服剂型包括颗粒剂、片剂、散剂、胶囊剂、口服液、丸剂等。
为使本发明所述剂型能够实现，需在制备这些剂型时加入药学可接受的辅料，例如：填充剂、崩解剂、润滑剂、助悬剂、粘合剂、甜味剂、矫味剂、防腐剂、基质等。填充剂包括：淀粉、预胶化淀粉、乳糖、甘露醇、甲壳素、微晶纤维素、蔗糖等；崩解剂包括：淀粉、预胶化淀粉、微晶纤维素、羧甲 基淀粉钠、交联聚乙烯吡咯烷酮、低取代羟丙纤维素、交联羧甲基纤维素钠等；润滑剂包括：硬脂酸镁、十二烷基硫酸钠、滑石粉、二氧化硅等；助悬剂包括：聚乙烯吡咯烷酮、微晶纤维素、蔗糖、琼脂、羟丙基甲基纤维素等；粘合剂包括：淀粉浆、聚乙烯吡咯烷酮、羟丙基甲基纤维素等；甜味剂包括：糖精钠、阿斯帕坦、蔗糖、甜蜜素、甘草次酸等；矫味剂包括：甜味剂及各种香精；防腐剂包括：尼泊金类、苯甲酸、苯甲酸钠、山梨酸及其盐类、苯扎溴铵、醋酸氯乙定、桉叶油等；基质包括：PEG6000，PEG4000，虫蜡等。为使上述剂型能够实现中药药剂学，需在制备这些剂型时加入药学可接受的其它辅料(范碧亭《中药药剂学》，上海科学出版社1997年12月第1版中各剂型记载的辅料)。
本发明中药组合物除了以黄芪、太子参、桂枝、当归、三七、山药、丹参、地龙、地黄、白术原药材投料的形式外，还可以采用以上述原药材的提取物(有效部位)投料的形式，因此本发明进一步公开了一种治疗糖尿病视网膜病变的中药组合物：
一种治疗糖尿病视网膜病变的中药组合物，该中药组合物由如下原料药组成：黄芪提取物10～80重量份、太子参提取物5～30重量份、桂枝提取物3～25重量份、当归提取物8～60重量份、三七提取物2～15重量份、山药提取物10～80重量份、丹参提取物10～80重量份、地龙提取物2～15重量份、地黄提取物8～60重量份、白术提取物8～60重量份。
更进一步，所述中药组合物的原料药组成为：黄芪提取物15～60重量份、太子参提取物6～25重量份、桂枝提取物5～20重量份、当归提取物10～50重量份、三七提取物3～12重量份、山药提取物15～60重量份、丹参提取物15～60重量份、地龙提取物3～12重量份、地黄提取物10～50重量份、白术提取物10～50重量份。
更进一步，所述中药组合物的原料药组成为：黄芪提取物25～40重量份、太子参提取物10～18重量份、桂枝提取物9～15重量份、当归提取物15～30重量份、三七提取物5～8重量份、山药提取物25～40重量份、丹参提取物25～40重量份、地龙提取物5～8重量份、地黄提取物15～30重量份、白术提取物15～30重量份。
更进一步，所述中药组合物的原料药组成为：黄芪提取物30重量份、太子参提取物12重量份、桂枝提取物10重量份、当归提取物20重量份、三七提取物6重量份、山药提取物30重量份、丹参提取物30重量份、地龙提取物6重量份、地黄提取物20重量份、白术提取物20重量份；
上述原料药提取物可以是各原料药的水提物，或有机溶剂提取物，或水提物/有机溶剂提取物经过进一步精制纯化工艺得到的精制物。
所述有机溶剂为20～95％的乙醇溶液；
制备上述提取物所用的提取方法包括煎煮提取、回流提取、浸渍提取、超声提取或渗漉提取等本领域常规提取方法中的任意一种方式，或者不同提取方法的组合；所述精制纯化工艺包括醇沉、萃取、硅胶色谱柱分离、大孔树脂柱分离等。
本发明中药组合物主要由黄芪、人参、桂枝、三七、丹参、地龙等药物组成。其中黄芪、人参、桂枝具有温阳健脾益气升阳的作用，可以加快血流速度和改善供血质量，三七、丹参、地龙既止血又活血，既通络又化瘀，同时具有降低血小板聚集，降低血液粘滞度，清除自由基，减轻缺血性损害，改善微循环，提高机体对缺氧的耐受力的作用、具有扩张血管增加血流量,改善缺血状态，抑制血液凝固，促进迁溶，改善血液流变性；降低血浆粘度，减少血小板聚集，抑制血栓形成；改善微循环、促进发生障碍的微循环血流速度加快的作用。整体上符合中医益气温阳活血通络的治疗原则。临床实验表明本发明中药组合物对黄斑水肿患者疗效确切，能有效提高其视力、视野，缓解眼底病变，对减轻黄斑水肿有积极的作用，其效果优于复方血栓通胶囊。
附图说明
图1视网膜消化铺片显微镜观察结果
A、正常组；B、模型组；C、实验组；D、对照组
图2视网膜石蜡切片光镜观察结果
A、正常组；B、模型组；C、实验组；D、对照组
图3视网膜石蜡切片免疫组织化学染色结果
A、正常组；B、模型组；C、实验组；D、对照组
图4视网膜电镜观察结果
A、正常组；B、模型组；C、实验组；D、对照组
实验例1本发明中药组合物治疗糖尿病视网膜病变的临床实验
1资料和方法
1.1一般资料。研究对象均来自2014年1月到2015年7月确诊为糖尿病黄斑水肿(DME)患者80例(128只眼)。其中治疗组49例(80只眼)，对照组31例(48只眼)。治疗组男性18例(29眼)，女性31例(51眼)，平均年龄59.80±10.21岁，病程7.41±4.62年；对照组男性11例(17眼)，女性20例(31眼)，平均年龄57.63±12.12岁，病程8.17±5.34年。
1.2诊断标准：参照《DME国际临床分型》、《美国DR早期治疗研究组ETDRS》、《中药新药临床研究指导原则》“治疗糖尿病性视网膜病变的临床研究指导原则”制定。
临床诊断：
(1)病史：有糖尿病视网膜病变病史。
(2)症状：早期眼部有轻度视物模糊自觉症状，病久可有不同程度视力减退，眼前黑影飞舞，或视物变形，甚至失明。
(3)体征：糖尿病黄斑水肿：参照《糖尿病黄斑水肿国际临床分型标准》：共分为轻度、中度和重度：轻度DME：后极部视网膜有一定程度增厚及硬性渗出,但距黄斑中心较远；中度DME：后极部视网膜有一定程度增厚硬性渗出，接近黄斑中心但未累及中心；重度DME：视网膜增厚及硬性渗出，累及黄斑中心。
(4)眼底荧光血管造影(FFA)：参照《美国DR早期治疗研究组标准ETDRS》，分为局部水肿型、弥散水肿型和囊样水肿型(归属弥散水肿型统计)：局部水肿型：黄斑呈局限性水肿，黄斑毛细血管扩张及微动脉瘤渗漏荧光；弥漫水肿型：范围≥2PD，并累计黄斑中心无血管区。大量毛细血管渗漏，可弥散至上、下血管弓；囊样水肿型：局限性水肿严重者，虽然毛细血管渗漏未弥散至上、下血管弓，但FFA晚期荧光素渗漏分布在拱环外围呈花瓣状或环形高荧光。
1.3纳入标准。
(1)经眼底荧光血管造影(FFA)选择I-III期糖尿病性视网膜病变同时伴黄斑水肿病例；
(2)符合西医黄斑水肿诊断标准，
(3)年龄18-70岁；
(4)2周内未参加其他临床试验者；
(5)2周内未服用本病相关治疗药物者；
(6)签署知情同意书。
1.4治疗方法
治疗组患者口服本发明中药组合物(按实施例1方法制备)，每日两次，每次100ml，口服三个月。
对照组患者口服复方血栓通胶囊，1次3粒，1日3次。复方血栓通胶囊为《中国药典》(2015版)所收载，其原料组成为三七、黄芪、丹参、玄参，其具有活血化瘀、益气养阴之功效；用于血瘀兼气阴两虚证的视网膜静脉阻塞，症见视力下降或视觉异常、眼底瘀血等症。
1.5观察指标
(1)治疗前后最佳矫正视力(国际标准视力表)；
(2)OCT测量黄斑中心凹厚度(TOPCON3DOCT-1000)。
(3)眼底彩色照相计数微血管瘤数(Topcon TRC-50DX)
(4)眼底荧光造影测量渗漏面积(Topcon Corp)
视力疗效评价标准制定如下：采用国际标准视力表，最佳矫正视力在一周内稳定。无光感至光感计2行，光感、手动、指数、0.02、0.04、0.06、0.08、0.1间隔均计1行。治疗后视力提高≥2行为有效；视力提高1行或视力不变而眼底病情有所改善者为稳定；视力减退者为无效。
1.6统计学方法
运用SPSS19.0软件进行统计学处理，治疗前后的正态分布数据采用配对样本t检验，非正态分布数据采用非参数检验，结果以均值±标准差
Figure PCTCN2016098510-appb-000001
表示，以P<0.05为差异有统计学意义。
2结果
2.1视力
治疗前患者视力分布情况如表1。治疗1月后视力变化情况如表2，组内比较，用t检验，与治疗前相比治疗组和对照组视力均有提高，差异有统计学意义(P<0.05)，组间比较，用χ2检验，治疗组和对照组相比视力改变无显著差异(P>0.05)。治疗3月后视力变化情况如表3，组内比较，用t检验，与治疗前相比治疗组和对照组视力均有显著提高，差异有统计学意义(P<0.05)，组间比较，用χ2检验，二组视力改变无显著差异(P>0.05)。可见治疗组和对照组均可有效提高DME患者视力，但二者对视力改善程度无显著差异。
表1治疗前视力分布
Figure PCTCN2016098510-appb-000002
表2治疗1月后视力变化
Figure PCTCN2016098510-appb-000003
注：#表示与治疗前比较P<0.05*表示与治疗组比较P>0.05
表3治疗3月后视力变化
Figure PCTCN2016098510-appb-000004
Figure PCTCN2016098510-appb-000005
注：#表示与治疗前比较P<0.05*表示与治疗组比较P>0.05
2.2黄斑中心凹厚度
对所有患者在治疗前后进行了OCT的检查，黄斑中心凹厚度变化情况如表4，用非参数检验，统计结果显示，治疗1月后，治疗组黄斑中心凹下降幅度大于对照组，差异有统计学意义(P<0.05)。治疗3月与对照组相比，治疗组黄斑中心凹厚度下降更明显，差异有统计学意义(P<0.05)。可见治疗组黄斑水肿消退情况优于对照组，且疗效稳定。
表4治疗前后黄斑中心凹厚度
Figure PCTCN2016098510-appb-000006
组别 治疗前 治疗1月后 治疗3月后
治疗组 437.05±96.48 317.50±99.96 268.20±78.62
对照组 439.63±100.42 355.38±92.06# 312.25±76.34#
注：#表示与治疗组比较P<0.05
2.3微血管瘤数
治疗前后行彩色眼底照相观察视网膜微血管瘤数目，具体情况见表5。用非参数检验，治疗1月后，治疗组微血管瘤数目变化情况和对照组相比无显著差异(P>0.05)，治疗3月后，微血管瘤数目减少趋势治疗组明显优于对照组，差异有统计学意义(P<0.05)。说明本发明中药组合物联合激光治疗DR，对于微血管瘤的改变在3个月才能发挥优势。
表5治疗前后微血管瘤数
Figure PCTCN2016098510-appb-000007
组别 治疗前 治疗1月后 治疗3月后
治疗组 22.83±12.84 20.86±10.33 18.75±9.96
对照组 23.17±12.89 21.96±11.46* 21.58±10.96#
注：*表示与治疗组比较P>0.05#表示与治疗组比较P<0.05
2.4渗漏面积
治疗前后通过眼底荧光素造影计算视网膜渗漏面积变化情况，具体见表6。用非参数检验，治疗1月后，治疗组渗漏面积缩小程度明显优于对照组，差异有统计学意义(P<0.05)，治疗3月后，二组相比治疗组渗漏面积缩小 程度显著，差异有统计学意义(P<0.05)。可见治疗组能更好的缩小渗漏面积，且疗效稳定。
表6治疗前后渗漏面积
Figure PCTCN2016098510-appb-000008
组别 治疗前 治疗1月后 治疗3月后
治疗组 1.71±0.83 1.33±0.82 1.28±0.83
对照组 1.72±0.92 1.50±0.80# 1.49±0.79#
注：#表示与治疗组比较P<0.05
3结论
本发明中药组合物对黄斑水肿患者疗效确切，能有效提高患者视力、降低黄斑中心凹厚度，缩小渗漏面积，减少微血管瘤数目。
实验例2本发明中药组合物治疗糖尿病大鼠视网膜血管病变的实验研究
(一)
1实验方法
1.1造模及分组：选用VAF级雄性SD大鼠，体重140-160克，购自维通利华实验动物技术有限责任公司，饲养于中日友好医院临床医学研究所实验动物中心。实验期间大鼠自由进食和饮水。标准大鼠饲料购自北京九江颗粒饲料厂，鼠笼及垫料购自北京柯林实验动物服务中心。大鼠适应性饲养1周后，体重180-200克共70只，除正常组外，其余造模前大鼠禁食12小时，经腹腔注射链脲佐菌素(STZ)，STZ在临用时用无菌的0.1mmol/L，pH 4.4柠檬酸钠缓冲液配成1％STZ溶液，按65mg/Kg大鼠体重，左下腹腔内注射，正常对照组注射同等体积的生理盐水。72小时后取尾静脉血，用快速血糖仪(Roche血糖仪，乐康全血糖检测试纸)测量血糖。凡非空腹血糖≥16.7mmol/L者即为糖尿病大鼠模型。
造模成功后糖尿病大鼠随机分为模型组10只，实验组10只(按实施例1制备的中药组合物)，正常组大鼠10只。实验周期300天。
1.2观测方法及指标：动物处死后将大鼠的眼球固定在4％多聚甲醛溶液中，制作视网膜消化铺片标本，PAS-H染色，观察血管内皮细胞(E)、周细胞(P)计数及E/P比较。石蜡包埋制作视网膜切片，观察血管内皮生 长因子(VEGF)、细胞间粘附分子-1(ICAM-1)、色素上皮衍生因子(PEDF)和糖基化终产物(AGEs)的表达。新鲜眼球溶浆，RT-PCR检测VEGFmRNA、PEDFmRNA，ICAM-1mRNA和RAGE的mRNA水平。用图象分析仪测量免疫组化显色强度，进行定量分析。采用SPSS 13.0统计软件对数据进行分析处理。
2实验结果
2.1视网膜消化铺片内皮细胞(E)、周细胞(P)计数及E/P比较结果见表7。
表7内皮细胞(E)、周细胞(P)计数及E/P比较
组别 n 内皮细胞(E) 周细胞(P) E/P
正常组 10 796.40±18.48 203.50±18.54 3.95±0.45
模型组 10 919.00±10.22* 81.10±10.06* 11.50±1.56*
实验组 9 874.78±15.75*Δ 125.22±15.75*Δ 7.10±0.98*Δ
注：模型组、实验组与正常组比较：*P＜0.01；治疗组与模型组比较：ΔP＜0.01。
上述结果表明，模型组同正常组比内皮细胞增生明显，周细胞明显减少(P＜0.01)。而实验组、正常组同模型组比较这种改变明显减轻(P＜0.01)，E/P有显著性差异。提示实验药物有抑制内皮细胞增生，减少周细胞选择性丢失的作用，从而延缓糖尿病视网膜病变的进展。
2.2作用机制研究
研究表明，本发明中药组合物可以通过降低糖尿病大鼠视网膜AGEs和RAGE mRNA的表达，减轻AGEs在内皮细胞、周细胞及基底膜沉积，减少对内皮细胞的刺激，从而减少VEGF的产生从而减轻管壁的功能障碍。通过降低视网膜毛细血管ICAM-1和视网膜ICAM-1mRNA的表达，减轻白细胞与血管内皮细胞粘附。通过降低视网膜、毛细血管VEGF和视网膜VEGF mRNA的表达，减轻血管的通透性，下调ICAM-1的表达。能增加视网膜PEDF和视网膜PEDF mRNA的表达，从而抑制新生血管形成。
实施例3本发明中药组合物治疗糖尿病大鼠视网膜血管病变的实验研究
(二)
1材料和方法
1.1实验动物和分组
SPF级六周龄SD大鼠48只，雄性，体重(220±20)g，购自华阜康生物科技股份有限公司。大鼠适应性饲养1周后，随机分为造模组36只，正常组12只。造模前禁食12小时，一次性腹腔注射1％STZ溶液60mg/kg体重，诱发实验性糖尿病大鼠模型。正常组大鼠注射同等体积的生理盐水。72小时后监测血糖，尾尖静脉取血，连续3天血糖≥16.7mmol/L即为造模成功，再把造模成功后大鼠随机分为模型组12只，中药组合物实验组12只(简称实验组)，多贝斯对照组(简称对照组)12只。实验组和对照组大鼠在造模成功后次日开始给药，实验组大鼠给药量为10ml/(kg·d)；对照组大鼠给药量为250mg/(kg·d)，多贝斯粉末用2ml蒸馏水溶解。正常组与模型组大鼠每日蒸馏水灌胃2ml。实验期间，所有大鼠每天定时添加饲料和饮水；每月监测大鼠体重及血糖变化。9个月后取材。
1.2视网膜消化铺片
随机取各组6只大鼠一侧眼球制作成视网膜消化铺片标本，制备方法如下：眼球固定在4％多聚甲醛溶液中48h后，流水轻轻冲洗5min，经锯齿缘环形剪开巩膜，去除眼前节部分；将后眼杯以视乳头为中心桔瓣样切成3块，轻轻分离出视网膜；PBS(0.01mol/L pH7.4)漂洗10min；3％胰蛋白酶37℃孵箱消化3h；移入蒸馏水中漂洗与振荡多次，仅剩下一层透明的视网膜血管网；漂取并平铺于载玻片、行PAS染色，在Olympus光学显微镜下观察。
1.3视网膜石蜡切片光镜标本制备
摘取其余6只大鼠的双侧眼球制备石蜡切片：将眼球和视神经置于4％多聚甲醛(4℃，48h)固定，沿角膜缘剪开眼球壁，去除角膜、晶体、玻璃体，梯度酒精脱水，二甲苯透明，石蜡包埋，连续切片，切片厚度为10μm，行HE染色及免疫组化染色，在Olympus光学显微镜下观察。
1.4视网膜电镜观察
摘取6只大鼠的另一侧眼球，置于2.5％戊二醛(4℃)固定72h后，切取视网膜中央区组织2mm×2mm。1％锇酸固定，丙酮逐级脱水，Epon812环 氧树脂包埋，切1μm半薄切片作光镜定位，再作超薄切片，经醋酸铀和枸橼酸铅双重染色，在日立H-600透射电镜下观察。
1.5统计学分析
实验中收集的所有数据，均采用SPSS19.0统计软件进行分析处理，采用t检验、方差分析或非参数检验，统计结果以均值±标准差表示，以α＝0.05作为检验标准，即以P≤0.05作为差异具有统计学意义。
2结果
2.1体重和血糖
结果见表8。实验期间观察到，与正常组大鼠相比，造模组大鼠体重增长缓慢，毛色枯槁、体型消瘦，尿量增多、大便稀溏。造模前各组大鼠血糖无明显差异(P>0.05)；与正常组对比，成模后大鼠在各时间段的血糖均明显升高，差异显著(P<0.01)。
表8各组大鼠不同时间段的血糖变化(单位：mmol/L)
Figure PCTCN2016098510-appb-000009
时间 正常组 实验组
造模前 6.05±0.49 6.09±0.45△
成模后 6.42±0.15 28.03±2.29*
1月 6.92±0.94 29.30±1.85*
2月 6.63±0.71 30.29±2.43*
3月 6.48±0.58 31.16±1.62*
4月 6.18±0.49 29.04±1.95*
5月 5.53±0.56 29.00±2.74*
6月 5.12±0.67 29.71±3.13*
7月 5.58±0.43 29.99±1.57*
8月 6.00±0.25 30.01±2.95*
9月 5.95±0.46 29.63±3.19*
注：△表示与正常组比较P>0.05，*表示与正常组比较P<0.05
2.2视网膜消化铺片
显微镜下观察视网膜消化铺片：正常大鼠视网膜毛细血管分布规则，走 向较直，管径粗细均匀一致；内皮细胞一般位于毛细血管中央部位，核较大，染色较浅，成椭圆形或不规则形，其长轴多与毛细血管平行；周细胞位于毛细血管管腔外侧，核较小，染色深，多成球形或三角形(见图1-A)。模型组大鼠视网膜毛细血管网排列紊乱，走向极不规则，管腔粗细不均，多根毛细血管扭曲成袢，节段性膨大，周细胞核固缩，内皮细胞及细胞外基质明显增生，可见无细胞毛细血管、影周细胞及内皮细胞凋亡等糖尿病视网膜特征性改变(见图1-B)。实验组(见图1-C)和对照组(见图1-D)干预后，视网膜微血管病变得到明显的保护或延缓：未见无细胞毛细血管、影周细胞等早期DR典型病变，毛细血管分布比较规则，粗细比较均匀，无明显扭曲扩张。
2.3视网膜石蜡切片HE染色
光镜下观察视网膜石蜡切片HE染色：正常组大鼠视网膜各层组织层次清楚，排列整齐(见图2-A)。模型组大鼠视网膜各层组织排列极度紊乱，毛细血管扩张明显，神经纤维层水肿增厚，视网膜神经节细胞数量减少，空泡变性，细胞核固缩，染色质边缘聚集，部分视网膜神经节细胞坏死，核溶解，内丛状层水肿，内核层空泡变性(见图2-B)。实验组大鼠视网膜间质轻度水肿，细胞排列基本整齐(见图2-C)。对照组大鼠视网膜网状结构疏松，内、外颗粒层排列紊乱，毛细血管扩张、间质水肿(见图2-D)。
2.4视网膜石蜡切片免疫组化
经免疫组织化学法标记染色的血管内皮生长因子(VEFG)在大鼠视视网膜内广泛表达，阳性表达部位呈棕黄色或棕褐色。VEGF在各组视网膜上表达的灰度值比较：在模型组(见图3-A)明显高于正常组(见图3-B)(P＜0.01)，实验组(见图3-C)和对照组(见图3-D)VEGF表达明显低于模型组(P＜0.01)，有统计学意义，但实验组和对照组组间差异不显著(见表9)。
表9视网膜全层VEGF组间表达比较(灰度值)
Figure PCTCN2016098510-appb-000010
组别 眼数 平均光密度值
正常组 6 0.28±0.12
模型组 6 0.77±0.10*
实验组 6 0.42±0.86*#△
对照组 6 0.43±0.09*#
注：*表示与正常组比较P<0.01，#表示与模型组比较P<0.01，△表示实验组与对照组比较P>0.05。
2.5视网膜电镜
视网膜超微结构观察：正常大鼠视网膜内皮细胞形态正常，呈梭形，细胞间连接完整，基底膜连续、不厚。神经纤维层微管和线粒体清晰，神经节细胞胞体呈卵圆形，核大而明显，胞浆中有较多的粗面内质网、多聚核糖体、线粒体，细胞表面有较多的突起(见图4-A)。模型组大鼠视网膜毛细血管内皮细胞明显肿胀，胞体变圆，突向管腔，线粒体肿胀，空泡化，基底膜增厚，神经节细胞胞体变形，细胞核明显固缩，核膜消失，胞浆内可见许多大小不等的空泡，线粒体肿胀，细胞表面突起明显减少，多聚核糖体及粗面内质网明显减少(见图4-B)。实验组大鼠视网膜毛细血管管腔壁尚光整，基底膜轻度增厚，少数周细胞水肿,部分内皮细胞轻度增生，神经节细胞胞体变形，线粒体轻度肿胀，结构稍欠清(见图4-C)。对照组大鼠视网膜毛细血管管腔壁尚光整，基底膜增厚，少数周细胞水肿，部分内皮细胞肿胀，部分线粒体可见空泡化，神经节细胞胞体变形，线粒体轻度肿胀,染色质溶解,嵴间隙更扩张甚至消失(见图4-D)。
3结论
实验性糖尿病大鼠的视网膜血管发生明显的病理性损害；本发明中药组合物对糖尿病大鼠的视网膜血管有一定的保护作用，能抑制糖尿病大鼠视网膜VEGF的表达，有助于减轻视网膜血管的损坏。
实验例4本发明中药组合物治疗糖尿病大鼠视神经传导病变的实验研究
1实验方法
1.1造模和分组
造模方法同实验例2。
造模成功后糖尿病大鼠随机分为模型组5只，实验组5只(按实施例1制备的中药组合物)，正常组大鼠5只。实验周期300天。
1.2观测方法及指标：动物处死后制备视网膜组织切片进行形态学检查；在电镜观察视网膜神经细胞超微结构；RT-PCR法检测视网膜胶质纤维酸性蛋白(GFAP)mRNA表达水平；利用TUNEL法检测视网膜神经节细胞(RGCs)及内核层细胞的凋亡并记数细胞凋亡数量；利用免疫组织化学技术LSAB法检测GFAP、谷氨酸转运体(GLAST)、谷氨酰胺合成酶(GS)、神经营养因子-3(NT-3)在视网膜的表达，观察RGCs及内核层细胞功能的改变。数据分析方法同实验例2。
2实验结果
2.1RGCs的细胞凋亡研究：
阳性凋亡RGCs细胞的判定：以细胞核呈棕黄(褐)色染色为阳性细胞。阳性凋亡RGCs细胞的记数方法：高倍(10×40倍)双目显微镜下，沿视盘一侧向锯齿缘，连续5个视野，取其均值作为凋亡RGCs细胞/高倍视野，读取连续3张切片，其RGCs细胞平均数作为该样本的RGCs细胞凋亡数。本实验结果显示：用TUNEL法检测，大鼠视网膜凋亡阳性细胞仅见于RGCs层和内核层。各组对RGCs凋亡的影响见表10。
表10本发明组合物对RGCs凋亡的影响
Figure PCTCN2016098510-appb-000011
组别 例数 RGCs凋亡数
正常组 5 4.92±0.16
模型组 5 11.98±1.56*
实验组 5 8.14±0.88*#
注：*表示与正常组相比P<0.01，#表示与模型组相比P<0.05
上述结果表明，与正常大鼠相比，模型组大鼠RGCs凋亡数量显著增加(P<0.01)；与模型组相比，实验组RGCs凋亡数量明显减少(P<0.05)，提示本发明组合物能抑制模型大鼠RGCs的凋亡，对其具有神经保护作用。
2.2作用机制研究
研究表明，本发明中药组合物抑制Müller细胞GFAP的过度表达，抑制胶质细胞反应性增生，减轻DM对Müller细胞功能的影响，减轻视网膜神经元的损害；能够促进Müller细胞GLAST及GS的表达，及时有效清除细胞外过量谷氨酸，减轻高浓度谷氨酸的兴奋性毒性作用；能促进神经营养因 子NT-3的表达，减少神经细胞的凋亡。
实验例5本发明中药组合物治疗糖尿病大鼠虹膜微循环病变的实验研究
1实验方法
1.1造模及分组：
造模同实验例2。
造模成功后糖尿病大鼠随机分为模型组20只，实验组30只(按实施例1制备的中药组合物)，对照组20只(多贝斯组)，正常组大鼠12只。实验周期300天。
1.2观测方法及指标：利用虹膜血管血流灌注扫描及虹膜荧光血管造影观察虹膜血流速度、虹膜血管管径、虹膜微血管密度、虹膜微血管内皮结构、虹膜微血管通透性和虹膜单位面积血流灌注量。
2实验结果
2.1虹膜血流速度：结果见表11。结果表明，正常组虹膜血流速度稳定，模型组虹膜血流速度明显降低，实验组较模型组明显加快(P<0.01)，多贝斯组与模型组相比无统计学差异。
表11治疗前、后虹膜血流速度比较
Figure PCTCN2016098510-appb-000012
Figure PCTCN2016098510-appb-000013
注：**表示与正常组相比P<0.01；△△表示与模型组相比P<0.01。
2.2虹膜血管管径：结果见表12。结果表明，正常组虹膜血管管径均匀，模型组较正常组血管管径明显扩张，实验组较模型组明显减低(P<0.01)，多贝斯组与模型组无统计学差异。
表12治疗前、后虹膜血管直径比较
Figure PCTCN2016098510-appb-000014
Figure PCTCN2016098510-appb-000015
Figure PCTCN2016098510-appb-000016
注：**表示与正常组相比P<0.01；△△表示与模型组相比P<0.01。
2.3虹膜血流灌注量：结果见表13。结果表明，激光多普勒扫描成像显示正常组虹膜单位面积血流灌注量充足，扫描成像均匀，模型组虹膜血流灌注量较正常组明显降低(P<0.01)，治疗组血流灌注量较模型组增多(P<0.05)，对照组与模型组相比无统计学差异。
表13治疗前、后单位面积血流灌注量
Figure PCTCN2016098510-appb-000017
Figure PCTCN2016098510-appb-000018
注：**表示与正常组相比P<0.01；△表示与模型组相比P<0.05；
上述结果表明本发明实验组合物可以促进虹膜血流加快，抑制血管异常扩张及改善虹膜微血管的通透性异常增高状态，并且可以增加单位面血流灌注量，缓解缺血状态。
具体实施方式
实施例1
原料药组成：黄芪30g、太子参12g、桂枝10g、当归20g、三七6g、山药30g、丹参30g、地龙6g、地黄20g、炒白术20g；
制备方法：按比例取原料药，加水煎煮两次，每次1.5小时，合并煎液，滤过，浓缩为200ml；
用法用量：每日早晚两次，每次100ml。
实施例2
原料药组成：黄芪28g、太子参16g、桂枝9g、当归28g、三七5g、山药35g、丹参28g、地龙7g、地黄16g、炒白术28g；
制备方法：按比例取原料药，70％乙醇超声提取二次，第一次1小时，第二次0.5小时，合并提取液，滤过，减压浓缩，提取物以糊精为赋形剂，按药辅比3:1进行混合，混匀，制成颗粒，干燥，既得颗粒剂。
实施例3
原料药组成：黄芪38g、太子参11g、桂枝14g、当归16g、三七7g、山药28g、丹参35g、地龙5g、地黄28g、炒白术16g；
制备方法：按比例取原料药，加水煎煮二次，每次1.5小时，合并煎液，滤过，按常规工艺制成口服液。
实施例4
原料药组成：黄芪22g、人参18g、桂枝9g、当归35g、三七5g、山药45g、丹参22g、地龙8g、地黄15g、白术35g；
制备方法：按比例取原料药，加水回流提取二次，每次1.5小时，合并煎液，滤过，浓缩，加入60％乙醇溶液醇沉，静置，过滤，浓缩，减压干燥，以糊精为赋形剂，按药辅比3:1进行混合，混匀，制成颗粒，干燥，既得颗粒剂。
实施例5
原料药组成：黄芪45g、人参10g、桂枝15g、当归13g、三七8g、山药22g、丹参48g、地龙5g、地黄38g、白术15g；
制备方法：按比例取原料药，混合，粉碎过60-80目筛，制成散剂。
实施例6
原料药组成：黄芪16g、西洋参24g、桂枝6g、当归45g、三七4g、山药55g、丹参16g、地龙10g、地黄12g、白术45g；
制备方法：按比例取原料药，加水煎煮二次，每次1.5小时，合并煎液，滤过，按常规工艺制成口服液。
实施例7
原料药组成：黄芪55g、太子参7g、桂枝18g、当归12g、三七11g、山 药18g、丹参55g、地龙4g、地黄45g、炒白术12g；
制备方法：按比例取原料药，70％乙醇超声提取二次，第一次1小时，第二次0.5小时，合并提取液，滤过，减压浓缩，浓缩液上D101大孔树脂纯化，先用2BV水洗脱，再用5BV 50％乙醇溶液洗脱，收集50％乙醇洗脱液，回收乙醇，提取物通过常规工艺，加入常规辅料，制成片剂。
实施例8
原料药组成：黄芪12g、太子参28g、桂枝4g、当归55g、三七3g、山药75g、丹参12g、地龙14g、地黄9g、炒白术55g；
制备方法：按比例取原料药，加水煎煮两次，每次1.5小时，合并煎液，滤过，浓缩，减压干燥，以糊精为赋形剂，按药辅比3:1进行混合，混匀，制成颗粒，干燥，既得颗粒剂。
实施例9
原料药组成：黄芪75g、太子参6g、桂枝24g、当归8g、三七14g、山药12g、丹参75g、地龙3g、地黄55g、炒白术10g；
制备方法：按比例取原料药，加6倍量75％乙醇回流提取2次，每次1.5小时；合并提取液，滤过，加入常规辅料制成口服液。
实施例10
原料药组成：黄芪提取物30g、太子参提取物12g、桂枝提取物10g、当归提取物20g、三七提取物6g、山药提取物30g、丹参提取物30g、地龙提取物6g、地黄提取物20g、白术提取物20g；
上述各原料药提取物为原料药加水煎煮得到的水提取物，将各原料药水提液合并，滤过，按常规工艺制成口服液。
实施例11
原料药组成：黄芪提取物28g、人参提取物16g、桂枝提取物9g、当归提取物28g、三七提取物5g、山药提取物35g、丹参提取物28g、地龙提取物7g、地黄提取物16g、炒白术提取物28g；
上述各原料药提取物为原料药加70％乙醇溶液回流提取得到的醇提物，将各原料药醇提物浓缩，减压干燥后混合均匀，以糊精为赋形剂，按药辅比3:1进行混合，混匀，制成颗粒，干燥，既得颗粒剂。
实施例12
原料药组成：黄芪提取物38g、西洋参提取物11g、桂枝提取物14g、当归提取物16g、三七提取物7g、山药提取物28g、丹参提取物35g、地龙提取物5g、地黄提取物28g、炒白术提取物16g；
上述各原料药提取物为原料药加水煎煮得到的水提取物。将各原料药水提物浓缩，减压干燥后混合均匀，以糊精为赋形剂，按药辅比3:1进行混合，混匀，制成颗粒，干燥，既得颗粒剂。

Claims (10)

  1. 一种治疗糖尿病视网膜病变的中药组合物，其特征在于，所述中药组合物的原料药组成包括黄芪、太子参、桂枝、当归、三七、山药、丹参、地龙、地黄、白术。
  2. 如权利要求1所述的中药组合物，其特征在于，所述中药组合物的原料药组成为：黄芪10～80重量份、太子参5～30重量份、桂枝3～25重量份、当归8～60重量份、三七2～15重量份、山药10～80重量份、丹参10～80重量份、地龙2～15重量份、地黄8～60重量份、白术8～60重量份。
  3. 如权利要求2所述的中药组合物，其特征在于，所述中药组合物的原料药组成为：黄芪15～60重量份、太子参6～25重量份、桂枝5～20重量份、当归10～50重量份、三七3～12重量份、山药15～60重量份、丹参15～60重量份、地龙3～12重量份、地黄10～50重量份、白术10～50重量份；
    或，所述中药组合物的原料药组成为：黄芪20～50重量份、太子参8～20重量份、桂枝8～18重量份、当归12～40重量份、三七4～10重量份、山药20～50重量份、丹参20～50重量份、地龙4～10重量份、地黄12～40重量份、白术12～40重量份；
    或，所述中药组合物的原料药组成为：黄芪25～40重量份、太子参10～18重量份、桂枝9～15重量份、当归15～30重量份、三七5～8重量份、山药25～40重量份、丹参25～40重量份、地龙5～8重量份、地黄15～30重量份、白术15～30重量份。
  4. 如权利要求3所述的中药组合物，其特征在于，所述中药组合物的原料药组成为：黄芪30重量份、太子参12重量份、桂枝10重量份、当归20重量份、三七6重量份、山药30重量份、丹参30重量份、地龙6重量份、地黄20重量份、白术20重量份；
    或，黄芪28重量份、太子参16重量份、桂枝9重量份、当归28重量份、三七5重量份、山药35重量份、丹参28重量份、地龙7重量份、地黄16重量份、白术28重量份；
    或，黄芪38重量份、太子参11重量份、桂枝14重量份、当归16重量 份、三七7重量份、山药28重量份、丹参35重量份、地龙5重量份、地黄28重量份、白术16重量份。
  5. 如权利要求2～4任一项所述的中药组合物，其特征在于，所述太子参可用人参或西洋参替代；所述白术为炒白术；所述地黄为生地黄。
  6. 如权利要求1-5任一项所述中药组合物的制备方法，其特征在于，该制备方法为：按比例取各原料药，以水或有机溶剂按常规提取方法提取，并按常规制剂工艺制备成常规口服剂型。
  7. 如权利要求6所述的制备方法，其特征在于，所述常规口服剂型包括颗粒剂、片剂、散剂、胶囊剂、口服液或丸剂。
  8. 一种治疗糖尿病视网膜病变的中药组合物，其特征在于，所述中药组合物由如下原料药组成：黄芪提取物15～60重量份、太子参提取物6～25重量份、桂枝提取物5～20重量份、当归提取物10～50重量份、三七提取物3～12重量份、山药提取物15～60重量份、丹参提取物15～60重量份、地龙提取物3～12重量份、地黄提取物10～50重量份、白术提取物10～50重量份。
  9. 如权利要求1-5或8任一项所述中药组合物在制备治疗糖尿病视网膜病变药物中的应用；
  10. 如权利要求1-5或8任一项所述中药组合物在制备治疗黄斑水肿药物中的应用。
PCT/CN2016/098510 2015-09-10 2016-09-09 一种治疗糖尿病视网膜病变的中药组合物 WO2017041734A1 (zh)

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