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WO2016119220A1 - 杜仲叶提取物及其制备方法和用途 - Google Patents

杜仲叶提取物及其制备方法和用途 Download PDF

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Publication number
WO2016119220A1
WO2016119220A1 PCT/CN2015/071956 CN2015071956W WO2016119220A1 WO 2016119220 A1 WO2016119220 A1 WO 2016119220A1 CN 2015071956 W CN2015071956 W CN 2015071956W WO 2016119220 A1 WO2016119220 A1 WO 2016119220A1
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Prior art keywords
extract
ethanol
eucommia ulmoides
leaves
eluate
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English (en)
French (fr)
Inventor
张洁
黄望
张亮
黄骏
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Sichuan Jiuzhang Biotechnology Co Ltd
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Sichuan Jiuzhang Biotechnology Co Ltd
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Priority to EP15879428.9A priority Critical patent/EP3251683A4/en
Priority to PCT/CN2015/071956 priority patent/WO2016119220A1/zh
Priority to US15/547,319 priority patent/US20180021395A1/en
Publication of WO2016119220A1 publication Critical patent/WO2016119220A1/zh
Anticipated expiration legal-status Critical
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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)
    • A61K36/46—Eucommiaceae (Eucommia family), e.g. hardy rubber tree
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/075—Ethers or acetals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying

Definitions

  • the invention relates to an extract of Eucommia ulmoides leaves and a preparation method and use thereof.
  • Psoriasis commonly known as “psoriasis”
  • psoriasis is a common and easily recurrent chronic inflammatory skin disease characterized by mica-like scaly, red membranous disease, and punctiform hemorrhage.
  • the incidence rate in China is about 0.123%.
  • the incidence of psoriasis is on the rise. It has obvious seasonality at the beginning, and it is winter and heavy in summer. It can be caused by both men and women. The current cause is unknown, and there is still no effective treatment.
  • Psoriasis is currently classified into type 4, psoriasis vulgaris, arthritic psoriasis, erythrodermic psoriasis, and pustular psoriasis, with more than 99% of psoriasis vulgaris.
  • Psoriasis vulgaris is common psoriasis, while the other three types are called special psoriasis.
  • psoriasis vulgaris is generally divided into 4 types: blood heat type (blood heat and wind dry type), blood deficiency type (blood deficiency and wind dry type), blood dry type, blood stasis type (stagnation) Skin type);
  • blood heat type blood heat and wind dry type
  • blood deficiency type blood deficiency and wind dry type
  • blood dry type blood stasis type (stagnation) Skin type
  • the pustular type is generally classified as sepsis type (damp heat type)
  • the erythroder type is venom type (blood heat poison type)
  • the joint type is cold type or Rheumatism and resistance.
  • the main clinical manifestations of psoriasis vulgaris are: white stalks, shiny film and spotted hemorrhage.
  • the main pathological manifestations are: parakeratosis.
  • a small abscess composed of neutrophils can be seen, and the granular layer becomes thin or disappears.
  • the spinous layer is thickened and the epidermis is prolonged.
  • the dermal papillary vessels are twisted and expanded, and the wall is slightly thickened. Mild to moderate inflammatory cell infiltration in the upper part of the dermis.
  • Psoriasis vulgaris is more common, accounting for more than 90%.
  • Pustular psoriasis is a type of condition that is more severe in psoriasis, with generalized psoriasis and localized psoriasis. Clinically, it is characterized by aseptic pustules of miliary size based on generalized erythema around the body, often accompanied by hyperthermia and elevated white blood cells and hypoproteinemia, and may even be life-threatening. This type is rare in clinical practice, accounting for 0.77% of patients with psoriasis.
  • the pathology of psoriasis is that the epidermal keratinocytes are not fully mature, the cells are filled with air, and the refracted light becomes the silvery white mica-like scales seen in the clinic; the dermal papillary swelling in the dermis protrudes into the epidermis and approaches the stratum corneum.
  • Scaling the scales can damage the nipple blood vessels and cause spotted bleeding.
  • Western medicine believes that the occurrence of psoriasis is mainly related to autoimmune, metabolic and endocrine dysfunction, and climate change, mood swings, infection, trauma and so on are predisposing factors.
  • pathogenesis of psoriasis is complex and has not yet been fully elucidated. Its pathogenesis can be broadly summarized as follows: On the basis of a certain genetic background, various pathogenic factors stimulate the immune system of the body to cause immune system dysfunction mainly composed of T cells. Inflammatory cells migrate to the epidermis and locally infiltrate, causing local inflammation and abnormal proliferation of keratinocytes, ultimately leading to pathological changes in psoriasis.
  • Psoriasis is a common chronic inflammatory skin disease with keratinocyte hyperplasia, inflammatory cell infiltration and neovascularization as the main pathological changes. It is a common and frequently-occurring disease in dermatology. The etiology of this disease is unknown and difficult to treat. It is still not completely eradicated. At present, Western medicine uses vitamin A acid and immunosuppressive agents to treat the disease. It has advantages in controlling psoriasis activities, but there are many adverse reactions and recurrence rate after drug withdrawal. Higher problems.
  • Eucommia ulmoides leaves are dried leaves of Eucommia ulmoides Oliv. Go to the liver and kidney. Liver and kidney, strong bones and bones. For liver and kidney deficiency, dizziness, waist and knee pain, weak bones. There is no report on the treatment of psoriasis with Eucommia ulmoides leaf extract.
  • the technical scheme of the present invention provides a novel Eucommia ulmoides leaf extract, a preparation method thereof and use thereof.
  • An extract of Eucommia ulmoides leaves which is a water or ethanol extract of Eucommia ulmoides leaves, wherein the eucommia leaf extract has a chlorogenic acid content of not less than 9%, and geniposide is not less than 1%, and peach leaf coral The glycosides are not less than 1%.
  • the eucommia leaf extract has a chlorogenic acid content of 9.38% to 39.11%, a geniposide content of 1.03% to 10.33%, and a peach saponin content of 1.17% to 10.68%.
  • the eucommia leaf extract has a chlorogenic acid content of 10.21% to 32.37%, a geniposide content of 1.03% to 9.38%, and a peach leaf glycoside of 1.17% to 9.56%.
  • the eucommia leaf extract has a chlorogenic acid content of 10.21% or 32.37%, a geniposide content of 1.03% or 2.24%, and amygdalin of 1.24% or 2.07%.
  • Eucommia ulmoides leaf extract is prepared by the following method:
  • the Eucommia ulmoides leaf extract is prepared by the following method:
  • the leaves of Eucommia ulmoides Oliv. were leached twice with 60% ethanol at 70 ° C for 15 hours each time, and the extracts were combined and concentrated to recover ethanol.
  • the concentrate was saturated with D-101 macroporous adsorption resin. Then, elute with 10%, 30%, 70% and 90% ethanol gradient ethanol, collect the eluate, combine 10% and 30% eluent, or combine 70% and 90% eluent, concentrate and dry , that is,
  • the leaves of Eucommia ulmoides Oliv. are leached twice with 30% ethanol at 60 ° C for 2 hours, each time with 12 volumes, the extracts are combined and concentrated to recover ethanol.
  • the concentrate is HPD-100 macroporous resin resin. After saturated adsorption, elute with 10%, 30%, 70% and 90% ethanol gradient ethanol, collect the eluate, combine 70% and 90% eluent, or take 30% eluent, concentrate and dry. That is;
  • the leaves of Eucommia ulmoides Oliv. are extracted with 20 times water at 80 ° C for 1.5 hours each time, and the extract is saturated with XAD-16 macroporous adsorption resin resin, followed by a gradient of 20% and 60% ethanol. The alcohol was eluted, and 20% of the eluate and 60% of the eluate were separately collected and concentrated to dryness;
  • the leaves of Eucommia ulmoides Oliv. are extracted twice with 12 times water at 60 ° C for 2 hours, and the extract is saturated with AB-8 macroporous adsorption resin resin, and then washed with 10%, 90% ethanol gradient ethanol. The extract was collected, and the eluate was collected, and 10% of the eluate and 90% of the eluate were separately collected and concentrated to dryness.
  • the method for preparing the above extract of Eucommia ulmoides leaves comprises the following steps: taking Eucommia ulmoides leaves, extracting with water or 30-80% ethanol for 1 or 2 times, and adsorbing the extract or its concentrated solution through styrene macroporous adsorption resin The gradient was eluted with a gradient of 10-90% ethanol, and the eluate was collected, combined, and concentrated to dryness.
  • the preparation method of the Eucommia ulmoides leaf extract is as follows:
  • the leaves of Eucommia ulmoides Oliv. were leached twice with 60% ethanol at 70 ° C for 15 hours each time, and the extracts were combined and concentrated to recover ethanol.
  • the concentrate was saturated with D-101 macroporous adsorption resin. Then, elute with 10%, 30%, 70% and 90% ethanol gradient ethanol, collect the eluate, combine 10% and 30% eluent, or combine 70% and 90% eluent, concentrate and dry , you can;
  • the leaves of Eucommia ulmoides Oliv. are leached twice with 30% ethanol at 60 ° C for 2 hours, each time with 12 volumes, the extracts are combined and concentrated to recover ethanol.
  • the concentrate is HPD-100 macroporous resin resin. After saturated adsorption, elute with 10%, 30%, 70% and 90% ethanol gradient ethanol, collect the eluate, combine 70% and 90% eluent, or take 30% eluent, concentrate and dry. Yes;
  • the leaves of Eucommia ulmoides Oliv. are extracted with 20 times water at 80 ° C for 1.5 hours each time, and the extract is saturated with XAD-16 macroporous adsorption resin resin, and then eluted with 20% and 60% ethanol gradient ethanol. , separately collect 20% of the eluate and 60% of the eluent, and concentrate and dry;
  • the leaves of Eucommia ulmoides Oliv. are extracted twice with 12 times water at 60 ° C for 2 hours, and the extract is saturated with AB-8 macroporous adsorption resin resin, and then washed with 10%, 90% ethanol gradient ethanol. The extract was collected, and the 10% eluate and 90% eluate were separately collected and concentrated to dryness.
  • the present invention also provides the use of the aforementioned Eucommia ulmoides leaf extract for the preparation of a medicament for treating psoriasis.
  • the drug is a drug for treating psoriasis vulgaris or pustular psoriasis.
  • the present invention also provides a medicament for treating psoriasis, which comprises the preparation of the aforementioned Eucommia ulmoides leaf extract as an active ingredient, together with a pharmaceutically acceptable adjuvant or auxiliary ingredient.
  • the preparation is an oral preparation or an external preparation.
  • the oral preparation is a tablet, a granule, a powder, a pill, an oral solution, and a capsule.
  • Psoriasis is an immunologically abnormal chronic inflammatory proliferative skin disease induced by multiple genetic factors and stimulated by multiple environmental factors.
  • the prevalence rate of natural population in the world is 0.1%-3%, and the prevalence rate in China The rate was 0.123% and the annual incidence was 0.1%.
  • the disease does not endanger human life, its course is long and stubborn and difficult to treat. In severe cases, it can cause disabling psoriatic arthritis, erythrodermic shoulder disease, and even affect the eyes, liver, kidney, and cardiovascular. Organs such as lungs, which seriously affect the quality of life of patients. Some patients may have psychological disorders such as shame, nervousness, anxiety, depression, etc.
  • Eucommia ulmoides leaf extract is a traditional Chinese medicine extract with small side effects, long-term use, no toxic side effects on the body, safe and effective, improve the quality of life of patients, and has significant curative effect on psoriasis. Therefore, Eucommia ulmoides leaf extract treats psoriasis Has broad application prospects.
  • Eucommia ulmoides leaves were leached twice with 60% ethanol at 70 ° C for 15 hours each time for 1 hour.
  • the extracts were combined and concentrated to recover ethanol.
  • Eucommia ulmoides leaves were leached twice with 30% ethanol at 60 ° C for 12 hours each time for 2 hours.
  • the extracts were combined and concentrated to recover ethanol.
  • the Eucommia ulmoides leaves were leached with 20 times water at 80 ° C for 1.5 hours each time.
  • the Eucommia ulmoides leaves were leached twice with 12 times of water at 60 ° C for 2 hours.
  • the extract sample 2 prepared in Example 1 was weighed according to the prescription amount, and the starch and hydroxypropyl cellulose were added and sieved, and the povidone ethanol solution was wet-granulated, dried, granulated, talcum powder was added, and tableting was carried out.
  • the extract sample 1 prepared in Example 1 was weighed according to the prescription, starch and hydroxypropyl cellulose were added and sieved, and the ethanol was wet-granulated, dried, granulated, magnesium stearate was added, and bagged.
  • the extract sample 1 prepared in Example 2 was weighed according to the prescription, and mannitol was added and sieved to mix and bag.
  • the extract sample 2 prepared in Example 2 was weighed according to the prescription, and starch and hydroxypropyl cellulose were added and sieved, and the carboxymethyl starch sodium water solution was added to prepare a pellet, dried, granulated, magnesium stearate was added, and bagging was carried out.
  • the extract sample 1 prepared in Example 3 was weighed according to the prescription, and the volume was adjusted with water for injection and filled.
  • the extract sample 2 prepared in Example 3 was weighed according to the prescription, granulated with a starch slurry, dried, sized, and filled into capsules.
  • Test Example 1 Efficacy test of the extract of Eucommia ulmoides Oliv. of the present invention for treating psoriasis (psoriasis vulgaris model)
  • mice 50 SCID mice of 6-8 weeks old, with a body mass of 18-22 g, were provided by the Experimental Animal Center of Sichuan University. Animal level: Level 1, license number: No. 10.
  • Drug test sample 2 (Example 1 extract sample 2);
  • Drug test sample 3 (Example 2 extract sample 1);
  • Healthy guinea pigs were randomly divided into 5 groups by random number table method: (1) model control group; (2) positive control group; (3) drug test group 1; (4) drug test group 2; ) Drug test samples 3 groups.
  • SCID mice were divided into male and female, and the mice were able to adapt to the growing environment for 1 week. Patients with plaque or plaque psoriasis were selected (informed consent was signed). Each patient selected a skin-relaxed site containing a 2cm ⁇ 2cm psoriatic lesion for a fusiform incision, cut the entire layer of skin, trimmed into a suitable thickness of 0.6cm ⁇ 0.6cm skin, wrapped in sterile saline gauze for transplantation .
  • mice According to the weight of SCID mice, 3% chloral hydrate anesthesia (0.1ml/10g) was given, the skin was depilated, the epidermis was cut off to the fascia layer, and the trimmed skin was sutured with 3-0 silk thread and impregnated with physiological The cotton balls of saline were pressure-wrapped, and the mice were transplanted in cages after transplantation, and the whole process was completed within 2 hours.
  • PBMC of the ConA-treated graft provider was injected around the lesion 10 days later, and the number of cells was 1 ⁇ 10 6 /piece. After 2 weeks, the external dressing was removed, and the survival condition of the skin piece was observed, and the next experiment was performed after the success was determined.
  • the positive drug control group was given 2.25 mg/kg of Avi A capsule suspension, the amount of gastric perfusion was 1 mL/100 g twice, 2 times/d; the drug test group 1 group, the test group 2 group and the test sample 3
  • the rats were administered by intragastric administration of 1 mL/100 g at a dose of 100 mg/kg once daily.
  • the model control group was intragastrically administered with the same amount of normal saline per day. Continued administration for 10 days.
  • mice were sacrificed by cervical dislocation, and the back graft lesions were taken and fixed with 10% formaldehyde (formalin solution) for 24 hours. Skin lesions were trimmed to appropriate size, dehydrated, embedded, sectioned, and HE stained. The changes of epidermal and dermal inflammatory cells in each group were observed under light microscope. The number of epidermal cells and epidermal thickness were counted in a 200-fold field of view. The number of perivascular lymphocytes and monocytes in the superficial dermis were counted. Backer was calculated. Score, compare the differences between the data of each group.
  • the above guinea pigs were bled before they were sacrificed, centrifuged at 2000 r/rain, serum was taken, and the optical density was measured by an enzyme-linked immunosorbent kit according to the requirements of the kit. The cAMP/cGMP ratio was calculated according to the results.
  • Experimental animal modified Ba ke r scoring method is as follows: stratum corneum keratinization 0.5-1.5 points (light-heavy), hyperkeratosis 1.5 points, epidermal extension is rod-shaped 0.5-1.5 points (light-heavy), acanthosis hypertrophy 1.0 points, the granular layer became thinner or disappeared by 1.0, the inflammatory layer of the dermis was infiltrated by 0.5-1.5 points (light-heavy), the nipple was deformed by 1.0 point, and the telangiectasia was 1.0.
  • the light microscope of each group was changed as follows: positive control group, moderate keratosis, acanthosis about 24-32 layers, epidermis with moderate rod-like hyperplasia, local visible punctate granular layer and hyperkeratosis, small amount of perivascular blood vessels Mononuclear cell infiltration.
  • Drug test group 1 mild keratosis, the acanthosis is about 21-29 layers thick, the epidermis is slightly rod-like hyperplasia, basically no granular layer, a small amount of mononuclear cells infiltrating around the dermis.
  • Drug test group 2 mild keratosis, the acanthosis is about 20-28 layers thick, the epidermis is slightly rod-like hyperplasia, basically no granular layer, a small amount of mononuclear cells infiltrating around the dermis.
  • Drug test group 3 mild keratosis, the acanthosis is about 20-29 layers thick, the epidermis is slightly rod-like hyperplasia, basically no granular layer, a small amount of mononuclear cells infiltrate around the dermis.
  • Negative control group severe parakeratosis, the acanthosis is about 26-38 layers thick, the epidermis is severely bark-like hyperplasia, basically no granular layer, moderate mononuclear cell infiltration around the vascular endovascular.
  • the extract of Eucommia ulmoides leaves can improve the Baker score and reduce the single-nuclear cell count of intradermal infiltration in the animal model of psoriasis, indicating that the extract of Eucommia ulmoides leaves has a good therapeutic effect on psoriasis vulgaris. The best for the test 2 is.
  • the serum cAMP content of psoriasis guinea pig model was significantly decreased, and the cGMP content was significantly increased (P ⁇ 0.01).
  • the cAMP content of the drug test group and the positive control group were significantly increased (P ⁇ 0.01), the cGMP content was decreased (P ⁇ 0.01), and the cAMP/cGMP ratio was increased (P ⁇ 0.01).
  • Serum VEGF in the psoriasis guinea pig model was significantly higher than that in the normal group, and the model was successful.
  • the serum VEGF of each test group and the positive control group were lower than the model group (P ⁇ 0.01), and there were significant differences; the drug test group was lower than the positive control group (P ⁇ 0.01), there was significant difference.
  • the test sample 2 (Extracted sample 2 of Example 1) was optimized.
  • the extract of Eucommia ulmoides leaves of the invention can effectively improve the Baker score of the psoriasis model, increase the content of serum cAMP and cGMP, and decrease the serum VEGF level, indicating that the extract of Eucommia ulmoides leaves can effectively treat psoriasis vulgaris (Liu Yuanlin et al. "Changes in serum E-cad and VEGF levels before and after treatment in patients with psoriasis vulgaris", Chinese Journal of Laboratory Diagnosis, Vol. 12, No.
  • Li Fei "two Therapeutic effect of calcium butyrate cyclic adenosine combined with acitretin on psoriasis vulgaris and its effects on blood cAMP and cGMP", Journal of Dermatology and Venereology, October 2010, Vol. 17, No. 5) 2 has the best effect.
  • Kunming mice were randomly divided into 3 groups.
  • the rats in the drug test group were intragastrically administered with 1 mL/100 g, and the dose was 100 mg/kg; the model control group was given the same amount of normal saline. Observation time points: when not administered (day 0), days 1, 2, 4, and 6 after administration.
  • a 1 cm 2 sterile gelatin sponge was soaked in 0.75% zymosan A for 30 min, surgically placed in the peritoneal cavity of the mice, and normal feeding was given. At 24 hours after operation, 5 mice were sacrificed at the same time point, and the gelatin sponge was taken out and placed in 0.5 ml of cell eluate for 30 min at 37 ° C. The gelatin sponge was rinsed with 0.01 ml/L PBS 0.5 ml respectively.
  • the eluate was collected, centrifuged to a volume of 0.5 ml after passing through a 40 ⁇ m nylon mesh, and the above eluate was subjected to cell sorting and counting by an automatic blood cell meter to read the number of polymorphonuclear leukocytes (PMN).
  • PMN polymorphonuclear leukocytes
  • the extract of Eucommia ulmoides leaves of the present invention can effectively treat psoriasis, especially for psoriasis vulgaris or pustular psoriasis, and provides a new drug for clinical treatment of psoriasis.

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Abstract

公开了一种杜仲叶提取物及其制备方法和用途。该提取物是杜仲叶的水或乙醇提取物，其中绿原酸含量不低于9％，京尼平苷酸不低于1％，桃叶珊瑚苷不低于1％。该提取物对银屑病有疗效。

Description

杜仲叶提取物及其制备方法和用途 技术领域
本发明涉及杜仲叶提取物及其制备方法和用途。
背景技术
银屑病俗称“牛皮癣”，是一种常见的并易复发的慢性炎症性皮肤病，以云母状鳞屑、红膜症、点状出血为临床特征，在我国的发病率约为0.123％。近年来,银屑病发病呈上升趋势，其初期有明显的季节性，呈冬重夏轻，无论男女老幼皆可发病，目前病因未明，尚缺乏有效的治疗方法。
银屑病目前分为4型，寻常型银屑病、关节病型银屑病、红皮病型银屑病和脓疱型银屑病，其中寻常型银屑病占99％以上。寻常型银屑病为普通型银屑病，而其他三型称为特殊型银屑病。根据历代中医名家经验总结,寻常型银屑病一般分为4型：即血热型(血热风燥型)，血虚型(血虚风燥型)，血燥型，血瘀型(瘀滞肌肤型)；特殊型银屑病中，脓疱型一般辨证为脓毒型(湿热蕴毒型)，红皮病型为毒热型(血热毒盛型)，关节型为寒湿型或风湿痹阻型。
寻常型银屑病的主要临床表现为：白色麟屑、发亮薄膜和点状出血。主要病理表现为：角化不全，在角层内或角层下，可见由中性粒细胞构成的小脓肿(Munro小脓肿)，颗粒层变薄或消失。棘层增厚，表皮突延长。真皮乳头部血管扭曲扩张，管壁轻度增厚。真皮上部轻度到中度炎症细胞浸润。寻常型银屑病较为常见，大约占90％以上。
脓疱型银屑病是银屑病中病情较为严重的一种类型,分泛发性银屑病及局限性银屑病。临床上以周身泛发红斑基础上粟粒大小的无菌性脓疱为特征，常伴有高热及白细胞升高和低蛋白血症，甚至可以危及生命。本型在临床上较少见，约占银屑病人的0.77％。
银屑病组织病理为表皮角质细胞不能完全成熟，细胞束之间充满空气，并折射光线成为临床上所见的银白色云母状鳞屑；真皮内的乳头水肿胀大突入表皮接近角质层，临床上搔刮鳞屑可损伤乳头血管而出现点状出血。西医认为，银屑病的发生主要与自身免疫、代谢及内分泌功能紊乱有关，而气候变化、情绪波动、感染、外伤等为诱发因素。
银屑病发病机制复杂，目前尚未完全得以阐明，其发病机制大致可以概括为：在一定遗传背景的基础上，多种致病因子刺激机体免疫系统引起以T细胞为主的免疫系统功能紊乱，炎症细胞移行到表皮并在局部浸润，造成局部炎症和角质形成细胞异常增生，最终导致银屑病病理改变的发生。
银屑病是一种常见的以角质形成细胞过度增生、炎症细胞浸润和新生血管形成为主要病理改变的慢性炎症性皮肤病，为皮肤科常见病、多发病。本病病因不明，治疗困难，尚无法完全根除，目前西药多使用维A酸及免疫抑制剂治疗该病，其在控制银屑病活动方面具有优势，但存在不良反应多、停药后复发率高等问题。
杜仲叶为杜仲科植物杜仲Eucommia ulmoides Oliv.的干燥叶，微辛，温。归肝、肾经。补肝肾，强筋骨。用于肝肾不足，头晕目眩，腰膝酸痛，筋骨痿软。未见杜仲叶提取物治疗银屑病的报道。
发明内容
本发明的技术方案是提供了一种新的杜仲叶提取物及其制备方法和用途。
一种杜仲叶提取物，它是杜仲叶的水或乙醇提取物，所述杜仲叶提取物中，绿原酸含量不低于9％，京尼平苷酸不低于1％，桃叶珊瑚苷不低于1％。
优选的，所述杜仲叶提取物中，绿原酸含量为9.38％～39.11％，京尼平苷酸含量为1.03％～10.33％，桃叶珊瑚苷为1.17％～10.68％。
进一步优选的，所述杜仲叶提取物中，绿原酸含量为10.21％～32.37％，京尼平苷酸含量为1.03％～9.38％，桃叶珊瑚苷为1.17％～9.56％。
再进一步优选的，所述杜仲叶提取物中，绿原酸含量为10.21％或32.37％，京尼平苷酸含量为1.03％或2.24％，桃叶珊瑚苷为1.24％或2.07％。
其中，所述杜仲叶提取物由如下方法制备：
取杜仲叶，用水或30-80％乙醇提取1～2次，每次1～2小时，将提取液或其浓缩液经苯乙烯类大孔吸附树脂吸附后，用10-90％乙醇梯度洗脱，收集洗脱液，合并，浓缩干燥，即得。
优选地，所述杜仲叶提取物由如下方法制备：
取杜仲叶，用60％乙醇于70℃浸提两次，每次用15倍量体积，每次1小时，合并提取液并浓缩回收乙醇，浓缩液用D-101大孔吸附树脂树脂饱和吸附后，依次用10％、30％、70％及90％乙醇梯度浓度乙醇洗脱，收集洗脱液，合并10％及30％洗脱液，或者合并70％及90％洗脱液，浓缩干燥，即得；
或者，取杜仲叶，用30％乙醇于60℃浸提两次，每次2小时，每次用12倍量体积，合并提取液并浓缩回收乙醇，浓缩液用HPD-100大孔吸附树脂树脂饱和吸附后，依次用10％、30％、70％及90％乙醇梯度浓度乙醇洗脱，收集洗脱液，合并70％及90％洗脱液，或者取30％洗脱液，浓缩干燥，即得；
或者，取杜仲叶，用20倍水于80℃浸提一次，每次1.5小时，提取液用XAD-16大孔吸附树脂树脂饱和吸附后，依次用20％及60％乙醇梯度浓度乙 醇洗脱，分别收集20％的洗脱液和60％洗脱液，浓缩干燥，即得；
或者，取杜仲叶，用12倍水于60℃浸提两次，每次2小时，提取液用AB-8大孔吸附树脂树脂饱和吸附后，依次用10％、90％乙醇梯度浓度乙醇洗脱，收集洗脱液，分别收集10％洗脱液和90％洗脱液，浓缩干燥，即得。
本发明制备前述杜仲叶提取物的方法，它包括如下步骤：取杜仲叶，用水或30-80％乙醇提取1～2次，将提取液或其浓缩液经苯乙烯类大孔吸附树脂吸附后，用10-90％乙醇梯度洗脱，收集洗脱液，合并，浓缩干燥，即得。
优选地，所述杜仲叶提取物制备方法如下：
取杜仲叶，用60％乙醇于70℃浸提两次，每次用15倍量体积，每次1小时，合并提取液并浓缩回收乙醇，浓缩液用D-101大孔吸附树脂树脂饱和吸附后，依次用10％、30％、70％及90％乙醇梯度浓度乙醇洗脱，收集洗脱液，合并10％及30％洗脱液，或者合并70％及90％洗脱液，浓缩干燥，即可；
或者，取杜仲叶，用30％乙醇于60℃浸提两次，每次2小时，每次用12倍量体积，合并提取液并浓缩回收乙醇，浓缩液用HPD-100大孔吸附树脂树脂饱和吸附后，依次用10％、30％、70％及90％乙醇梯度浓度乙醇洗脱，收集洗脱液，合并70％及90％洗脱液，或者取30％洗脱液，浓缩干燥，即可；
或者，取杜仲叶，用20倍水于80℃浸提一次，每次1.5小时，提取液用XAD-16大孔吸附树脂树脂饱和吸附后，依次用20％及60％乙醇梯度浓度乙醇洗脱，分别收集20％的洗脱液和60％洗脱液，浓缩干燥，即可；
或者，取杜仲叶，用12倍水于60℃浸提两次，每次2小时，提取液用AB-8大孔吸附树脂树脂饱和吸附后，依次用10％、90％乙醇梯度浓度乙醇洗脱，收集洗脱液，分别收集10％洗脱液和90％洗脱液，浓缩干燥，即可。
本发明还提供了前述的杜仲叶提取物在制备治疗银屑病的药物中的用途。
其中，所述的药物是治疗寻常型银屑病或脓疱型银屑病的药物。
本发明还提供了一种治疗银屑病的药物，它是以前述的杜仲叶提取物为活性成分，加上药学上可接受的辅料或者辅助性成分制备而成的制剂。
其中，所述制剂是口服制剂、外用制剂。
其中，所述口服制剂是片剂、颗粒剂、散剂、丸剂、口服液、胶囊剂。
银屑病是一种由多基因遗传决定的、多环境因素刺激诱导的免疫异常性慢性炎症性增生性皮肤病，在世界范围内自然人群患病率为0.1％-3％，我国患病率为0.123％，年发病率为0.1％。尽管该病不危及人的生命，但其病程长且顽固难治，严重者可发生致残性银屑病性关节炎、红皮病型银肩病，甚至累及眼、肝脏、肾脏、心血管、肺等器官，从而严重影响患者的生活质量。 有些患者可出现心理障碍如羞愧、紧张、焦虑、忧郁等，不愿参加社交活动，影响日常生活，给患者身体和心理带来极大的痛苦。由于该病反复发作，常需反复治疗，因而给患者及其家庭带来沉重的经济负担。目前银屑病无法治愈，传统的治疗除中药外多为免疫抑制剂及维A酸类药物，长期应用这些药物易产生依赖性及肝肾损害等严重的副作用。
杜仲叶提取物为中药提取物，副作用小，可长期使用，对机体不产生毒副作用，安全有效，提高患者的生活质量，对银屑病有显著的疗效，故杜仲叶提取物治疗银屑病有广阔的应用前景。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
具体实施方式
实施例1本发明杜仲叶提取物的制备
杜仲叶用60％乙醇于70℃浸提两次，每次用15倍量体积，每次1小时，合并提取液并浓缩回收乙醇，浓缩液用D-101大孔吸附树脂树脂饱和吸附后，依次用10％、30％、70％及90％乙醇梯度浓度乙醇洗脱，收集洗脱液，合并10％及30％洗脱液，浓缩干燥得提取物样品1(含量：绿原酸＝11.27％，京尼平苷酸＝1.87％，桃叶珊瑚苷＝1.17％)；合并70％及90％洗脱液，浓缩干燥得提取物样品2(含量：绿原酸＝32.37％，京尼平苷酸＝2.24％，桃叶珊瑚苷＝2.07％)
实施例2本发明杜仲叶提取物的制备
杜仲叶用30％乙醇于60℃浸提两次，每次用12倍量体积，每次2小时，合并提取液并浓缩回收乙醇，浓缩液用HPD-100大孔吸附树脂树脂饱和吸附后，依次用10％、30％、70％及90％乙醇梯度浓度乙醇洗脱，收集洗脱液，合并70％及90％洗脱液，浓缩干燥得提取物样品1(含量：绿原酸＝11.24％，京尼平苷酸＝9.38％，桃叶珊瑚苷＝9.56％)；30％洗脱液浓缩干燥得提取物样品2(含量：绿原酸＝38.55％，京尼平苷酸＝1.18％，桃叶珊瑚苷＝1.36％)
实施例3本发明杜仲叶提取物的制备
将杜仲叶用20倍水于80℃浸提一次，每次1.5小时，提取液用XAD-16大孔吸附树脂树脂饱和吸附后，依次用20％及60％乙醇梯度浓度乙醇洗脱，收集洗脱液，20％洗脱液浓缩干燥得提取物样品1(含量：绿原酸＝23.04％，京尼平苷酸＝1.29％，桃叶珊瑚苷＝3.54％)；60％洗脱液浓缩干燥得提取物样 品2(含量：绿原酸＝10.21％，京尼平苷酸＝1.03％，桃叶珊瑚苷＝1.24％)
实施例4本发明杜仲叶提取物的制备
将杜仲叶用12倍水于60℃浸提两次，每次2小时，提取液用AB-8大孔吸附树脂树脂饱和吸附后，依次用10％、90％乙醇梯度浓度乙醇洗脱，收集洗脱液，10％洗脱液浓缩干燥得提取物样品1(含量：绿原酸＝39.11％，京尼平苷酸＝10.33％，桃叶珊瑚苷＝10.68％)；90％洗脱液浓缩干燥得提取物样品2(含量：绿原酸＝9.38％，京尼平苷酸＝1.11％，桃叶珊瑚苷＝2.31％)
实施例5本发明药物的制备(片剂)
处方
Figure PCTCN2015071956-appb-000001
按处方量称取实施例1制备的提取物样品2，加入淀粉、羟丙纤维素过筛混合，加聚维酮乙醇液湿法制粒，干燥，整粒，加滑石粉，压片。
实施例6本发明药物的制备(颗粒剂)
处方
Figure PCTCN2015071956-appb-000002
按处方称取实施例1制备的提取物样品1，加入淀粉、羟丙纤维素过筛混合，乙醇湿法制粒，干燥，整粒，加硬脂酸镁，装袋。
实施例7本发明药物的制备(散剂)
处方
Figure PCTCN2015071956-appb-000003
按处方称取实施例2制备的提取物样品1，加入甘露醇过筛混合，装袋。
实施例8本发明药物的制备(丸剂)
处方
Figure PCTCN2015071956-appb-000004
按处方称取实施例2制备的提取物样品2，加入淀粉、羟丙纤维素过筛混合，加羧甲淀粉钠水液制丸，干燥，整粒，加硬脂酸镁，装袋。
实施例9本发明药物的制备(口服剂)
处方
Figure PCTCN2015071956-appb-000005
按处方称取实施例3制备的提取物样品1，用注射用水调体积，灌装。
实施例10本发明药物的制备(胶囊剂)
处方
Figure PCTCN2015071956-appb-000006
按处方称取实施例3制备的提取物样品2，加淀粉浆制粒，干燥，整粒，填装胶囊。
以下用药效实验进一步说明本发明的有益效果：
试验例1本发明杜仲叶提取物治疗银屑病的药效试验(寻常型银屑病模型)
1.材料
1)动物
6-8周龄的SCID小鼠50只，体质量18-22g，四川大学试验动物中心提供。动物级别：一级，许可证号：第10号。
2)药物
药物供试品1(实施例1提取物样品1)；
药物供试品2(实施例1提取物样品2)；
药物供试品3(实施例2提取物样品1)；
阿维A胶囊，购自重庆华邦制药股份有限公司，批号：2013010
2.实验方法
1)实验分组
健康豚鼠，采用随机数字表法随机分为5组：(1)模型对照组；(2)阳性对照组；(3)药物供试品1组；(4)药物供试品2组；(5)药物供试品3组。
2)动物模型制备
将SCID小鼠雌雄分养，饲养1周，小鼠均能适应生长环境。选取寻常型或斑块型银屑病患者(签定知情同意书)。每位患者选取皮肤松弛部位包含2cm×2cm银屑病皮损做梭形切口，切取皮肤全层，修剪成适宜厚度的0.6cm×0.6cm皮片，用生理盐水纱布包裹无菌包装以备移植。根据SCID小鼠体重给予3％水合氯醛麻醉(0.1ml/10g)，备皮去毛，剪开表皮分离至筋膜层，将修剪好的皮片用3-0丝线缝合，并用浸有生理盐水的棉球加压包扎，移植皮片后小鼠分笼饲养，整个过程在2小时内完成。10天后在皮损周围注射经ConA处理过的移植物提供者的PBMC，细胞数为1×106/只。2周后拆去外部包扎，观察皮片成活情况，确定造成功后进行下一步实验。
3)给药
阳性药对照组给予阿维A胶囊混悬剂2.25mg/kg，灌胃量均为每次1mL/100g，2次/d；药物供试品1组、供试品2组及供试品3组均按1mL/100g灌胃给药，给药剂量100mg/kg，1次/d；模型对照组每天等量生理盐水灌胃。持续给药10天。
4)检测
用药结束后，颈椎脱臼法处死小鼠，取其背部移植皮损，10％甲醛(福尔马林溶液)固定24h。将皮损修剪为适宜大小，脱水，包埋，切片，HE染色。光学显微镜下观察各组移植皮损表皮及真皮内炎症细胞等变化，并在200倍视野内计数表皮细胞层数及表皮厚度，计数真皮浅层血管周围淋巴细胞及单核细胞个数，计算Backer评分，比较各组数据间差异性。
上述豚鼠未处死前取血，离心2000r/rain，取血清，采用酶联免疫试剂盒，按试剂盒说明书要求进行检测光密度，根据标准曲线计算结果，根据结果计算cAMP/cGMP比值。
5)统计学方法
所有数据均用SPSS 13.0统计软件处理，实验结果计量资料用
Figure PCTCN2015071956-appb-000007
表示，以P<0.05为差异具有统计学意义。多样本均数比较采用方差分析。
6)Backer评分
实验动物改良Ba ke r评分法方法如下：角质层角化不全0.5-1.5分(轻-重)，角化过度1.5分，表皮突延伸呈棒状0.5-1.5分(轻-重)，棘层肥厚1.0分，颗粒层变薄或消失1.0分，真皮层炎性细胞浸润0.5-1.5分(轻-重)，乳头上伸杵状变1.0分，毛细血管扩张1.0分
3.结果
(1)各组Baker评分、单一核细胞计数、棘细胞数
各组光镜改变如下：阳性对照组，中度角化不全，棘层约厚24-32层，表皮呈中度棒槌样增生，局部可见点状颗粒层及角化过度，真皮内血管周围少量单一核细胞浸润。药物供试品1组：轻度角化不全，棘层约厚21-29层，表皮呈轻微棒槌样增生，基本无颗粒层，真皮内血管周围少量单一核细胞浸润。药物供试品2组：轻度角化不全，棘层约厚20-28层，表皮呈轻微棒槌样增生，基本无颗粒层，真皮内血管周围少量单一核细胞浸润。药物供试品3组：轻度角化不全，棘层约厚20-29层，表皮呈轻微棒槌样增生，基本无颗粒层，真皮内血管周围少量单一核细胞浸润。阴性对照组：重度角化不全，棘层约厚26-38层，表皮呈重度棒槌样增生，基本无颗粒层，真皮内血管周围中等量单一核细胞浸润。
表1各组Baker评分、单一核细胞计数、棘细胞数
Figure PCTCN2015071956-appb-000008
Figure PCTCN2015071956-appb-000009
注：与模型对照组比较，aP<0.01；与阳性对照组比较bP<0.05；
结果显示，药物供试品1、供试品2及供试品3组与模型对照组有显著性差异(P<0.01)；与阳性对照组相比，药物供试品2及供试品3组有显著差异(P<0.05)。
可以看出，杜仲叶提取物能够改善Baker评分，减轻银屑病动物模型中真皮内浸润的单一核细胞计数，说明本发明杜仲叶提取物对寻常型银屑病具有良好的治疗作用，其中，以供试品2最佳。
(2)对血清cAMP、cGMP的含量及比值的影响
表2对血清cAMP、cGMP的含量及比值的影响(x±s，n＝10)
Figure PCTCN2015071956-appb-000010
注：与空白组比较，aP<0.01；与模型组比较bP<0.01；与阳性组比较cP<0.05
银屑病豚鼠模型血清cAMP含量均显著降低，cGMP含量均显著升高(P<0.01)。与模型组比较，药物供试品各组和阳性对照组cAMP含量均显著升高(P<0.01)，cGMP含量均降低(P<0.01)，cAMP/cGMP比值均升高(P<0.01)；药物供试品1、供试品2及供试品3各组与阳性对照组cGMP含量及cAMP含量比较均有统计学意义(P<0.05)，其中，以供试品2最佳。
(3)对血清VEGF水平的影响
表3对血清VEGF水平的影响(x±s，n＝10)
Figure PCTCN2015071956-appb-000011
注：与空白组比较，aP<0.01；与模型组比较bP<0.01；与阳性组比较cP<0.01；
银屑病豚鼠模型血清VEGF显著高于正常组，造模成功。经治疗，药物各个供试品组及阳性对照组血清VEGF均低于模型组(P<0.01)，有显著差异；药物各个供试品组低于阳性对照组(P<0.01)，有显著差异，其中，以供试品2(实施例1提取物样品2)最佳。
本发明杜仲叶提取物均可以有效改善银屑病模型的Baker评分，提高血清cAMP、cGMP的含量、降低血清VEGF水平，说明本发明杜仲叶提取物可以有效治疗寻常型银屑病(刘元林等，“寻常型银屑病患者治疗前后血清E—cad及VEGF水平的变化”，中国实验诊断学2008年9月第12卷第9期第1162页左栏倒数1～3行；李菲，“二丁酰环磷腺苷钙联合阿维A治疗寻常型银屑病的疗效及对血cAMP和cGMP的影响”，皮肤性病诊疗学杂志2010年10月第17卷第5期)，且以药物供试品2的疗效最优。
试验例2本发明杜仲叶提取物治疗银屑病的药效试验(脓疱型银屑病)
实验研究：
1.材料
1)动物
昆明种小鼠，体质量18-22g，四川大学试验动物中心提供。动物级别：一级，许可证号：第10号。
2)药物
药物供试品1(实施例3提取物样品1)；
药物供试品2(实施例3提取物样品2)；
2.实验方法
1)动物分组
昆明种小鼠随机均分为3组。药物供试品组各鼠按1mL/100g灌胃给药，给药剂量100mg/kg；模型对照组给予等量生理盐水灌胃。观察时间点：未给药时(0天)，给药后第1、2、4、6天。
2)实验方法
将一块1cm2的无菌明胶海绵在0.75％酵母聚糖A中浸泡30min后，手术置入小鼠的腹膜腔中，并给予正常喂饲。术后24小时，断颈处死同一时相点的5只小鼠，取出明胶海绵，分别置于0.5ml的细胞洗脱液中37℃孵育30min，以0.01mol/LPBS0.5ml分别漂洗明胶海绵3次，收集洗脱液，过40μm尼龙筛网后离心定容至0.5ml，以全自动血细胞仪对上述洗脱液进行细胞分类计数，读取多形核白细胞(PMN)的数量。
3.结果
表4药物供试品组在不同时相点的PMN迁移数量(×109/L)
Figure PCTCN2015071956-appb-000012
注：与空白组比较，*P<0.05；△P<0.01；
结果显示，在用药后，药物供试品1组及供试品2组自第二天起PMN明显降低，与空白组相比有显著性差异，说明杜仲叶提取物具有治疗脓疱型银屑病的作用(陈胜平，芳维A酸乙酯治疗脓疱型银屑病的临床观察及实验研究，第三军医大学硕士学位论文2002年)，其中，药物供试品2组(实施例3提取物样品2)的效果相对较优。
综上，本发明杜仲叶提取物可以有效治疗银屑病，特别是对寻常型银屑病或脓疱型银屑病的疗效确切，为银屑病的临床治疗提供了一种新的药物。

Claims (13)

  1. 一种杜仲叶提取物，其特征在于：它是杜仲叶的水或乙醇提取物，所述杜仲叶提取物中，绿原酸含量不低于9％，京尼平苷酸不低于1％，桃叶珊瑚苷不低于1％。
  2. 根据权利要求1所述的杜仲叶提取物，其特征在于：所述杜仲叶提取物中，绿原酸含量为9.38％～39.11％，京尼平苷酸含量为1.03％～10.33％，桃叶珊瑚苷为1.17％～10.68％。
  3. 根据权利要求2所述的杜仲叶提取物，其特征在于：所述杜仲叶提取物中，绿原酸含量为10.21％～32.37％，京尼平苷酸含量为1.03％～9.38％，桃叶珊瑚苷为1.17％～9.56％。
  4. 根据权利要求3所述的杜仲叶提取物，其特征在于：所述杜仲叶提取物中，绿原酸含量为10.21％或32.37％，京尼平苷酸含量为1.03％或2.24％，桃叶珊瑚苷为1.24％或2.07％。
  5. 根据权利要求1～4任意一项所述的杜仲叶提取物，其特征在于：所述杜仲叶提取物由如下方法制备：
    取杜仲叶，用水或30-80％乙醇提取1～2次，每次1～2小时，将提取液或其浓缩液经苯乙烯类大孔吸附树脂吸附后，用10-90％乙醇梯度洗脱，收集洗脱液，合并，浓缩干燥，即得。
  6. 根据权利要求5所述的杜仲提取物，其特征在于：所述杜仲叶提取物由如下方法制备：
    取杜仲叶，用60％乙醇于70℃浸提两次，每次用15倍量体积，每次1小时，合并提取液并浓缩回收乙醇，浓缩液用D-101大孔吸附树脂树脂饱和吸附后，依次用10％、30％、70％及90％乙醇梯度浓度乙醇洗脱，收集洗脱液，合并10％及30％洗脱液，或者合并70％及90％洗脱液，浓缩干燥，即得；
    或者，取杜仲叶，用30％乙醇于60℃浸提两次，每次2小时，每次用12倍量体积，合并提取液并浓缩回收乙醇，浓缩液用HPD-100大孔吸附树脂树脂饱和吸附后，依次用10％、30％、70％及90％乙醇梯度浓度乙醇洗脱，收集洗脱液，合并70％及90％洗脱液，或者取30％洗脱液，浓缩干燥，即得；
    或者，取杜仲叶，用20倍水于80℃浸提一次，每次1.5小时，提取液用XAD-16大孔吸附树脂树脂饱和吸附后，依次用20％及60％乙醇梯度浓度乙醇洗脱，分别收集20％的洗脱液和60％洗脱液，浓缩干燥，即得；
    或者，取杜仲叶，用12倍水于60℃浸提两次，每次2小时，提取液用AB-8大孔吸附树脂树脂饱和吸附后，依次用10％、90％乙醇梯度浓度乙醇洗脱，收集洗脱液，分别收集10％洗脱液和90％洗脱液，浓缩干燥，即得。
  7. 一种制备权利要求1～6任意一项所述杜仲叶提取物的方法，其特征在于：它包括如下步骤：取杜仲叶，用水或30-80％乙醇提取1～2次，每次1～2小时，将提取液或其浓缩液经苯乙烯类大孔吸附树脂吸附后，用10-90％乙醇梯度洗脱，收集洗脱液，合并，浓缩干燥，即得。
  8. 根据权利要求7所述的方法，其特征在于：它包括如下步骤：
    取杜仲叶，用60％乙醇于70℃浸提两次，每次用15倍量体积，每次1小时，合并提取液并浓缩回收乙醇，浓缩液用D-101大孔吸附树脂树脂饱和吸附后，依次用10％、30％、70％及90％乙醇梯度浓度乙醇洗脱，收集洗脱液，合并10％及30％洗脱液，或者合并70％及90％洗脱液，浓缩干燥，即可；
    或者，取杜仲叶，用30％乙醇于60℃浸提两次，每次2小时，每次用12倍量体积，合并提取液并浓缩回收乙醇，浓缩液用HPD-100大孔吸附树脂树脂饱和吸附后，依次用10％、30％、70％及90％乙醇梯度浓度乙醇洗脱，收集洗脱液，合并70％及90％洗脱液，或者取30％洗脱液，浓缩干燥，即可；
    或者，取杜仲叶，用20倍水于80℃浸提一次，每次1.5小时，提取液用XAD-16大孔吸附树脂树脂饱和吸附后，依次用20％及60％乙醇梯度浓度乙醇洗脱，分别收集20％的洗脱液和60％洗脱液，浓缩干燥，即可；
    或者，取杜仲叶，用12倍水于60℃浸提两次，每次2小时，提取液用AB-8大孔吸附树脂树脂饱和吸附后，依次用10％、90％乙醇梯度浓度乙醇洗脱，收集洗脱液，分别收集10％洗脱液和90％洗脱液，浓缩干燥，即可。
  9. 权利要求1～6任意一项所述的杜仲叶提取物在制备治疗银屑病的药物中的用途。
  10. 根据权利要求9所述的用途，其特征在于：所述的药物是治疗寻常型银屑病或脓疱型银屑病的药物。
  11. 一种治疗银屑病的药物，其特征在于：它是以权利要求1～6任意一项所述的杜仲叶提取物为活性成分，加上药学上可接受的辅料或者辅助性成分制备而成的制剂。
  12. 根据权利要求11所述的药物，其特征在于：所述制剂是口服制剂、外用制剂。
  13. 根据权利要求12所述的药物，其特征在于：所述口服制剂是片剂、颗粒剂、散剂、丸剂、口服液、胶囊剂。
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CN115636750A (zh) * 2022-12-03 2023-01-24 湖南一科生物科技有限公司 一种从杜仲中提取绿原酸的方法

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