CN103585193B - The application of Aleuritopteris argentea (Gmel.) Fee extract in preparation treatment leukemia medicament - Google Patents
The application of Aleuritopteris argentea (Gmel.) Fee extract in preparation treatment leukemia medicament Download PDFInfo
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Abstract
The present invention relates to the application of a kind of Aleuritopteris argentea (Gmel.) Fee extract in preparation treatment leukemia medicament, belong to field of medicaments.The application of Aleuritopteris argentea (Gmel.) Fee extract in preparation treatment leukemia medicament; Aleuritopteris argentea (Gmel.) Fee extract is preparing the application by improving in NCK, IL-2, IL-6, IFN-gamma activity treatment leukemia medicament.Described Aleuritopteris argentea (Gmel.) Fee extract is prepared as follows: take Aleuritopteris argentea (Gmel.) Fee as raw material, uses 30%-80% ethanol extraction, is obtained by high-speed countercurrent chromatography, macroporous adsorbent resin isochromatic spectrum purification.
Description
Technical field
The invention belongs to field of medicaments, relate to the application of a kind of Chinese medicinal material extract in preparation treatment leukemia medicament, specifically relate to the application of Aleuritopteris argentea (Gmel.) Fee extract in preparation treatment leukemia medicament.
Background technology
Leukemia is origin hemopoietic system, to produce and to gather the malignant disease that a large amount of inmature and abnormal white cell is feature in bone marrow or other hemopoietic tissue.Viewed from form, leukaemia's great majority are immaturity and paramorph leukocyte; Functionally, leukaemia has the feature identical with tumor cell: leukaemia is tumprigenicity increment, uncontrollable, often cause suppression and the exhaustion of hemopoietic function of bone marrow, there is anemia, neutrophilic granulocyte and thrombocytopenia in pathogenic people, leukaemia can infiltrate the various tissue of whole body and internal organs, makes the function of each internal organs impaired, as spleen, liver, lymph node, meninges, skin etc., produce clinical symptoms and the signs such as liver, spleen, lymphadenectasis, headache, neck rigidity; Also can show as the tumor of local, such as, when infiltrating subperiosteum, light green lump can be gathered into, be commonly called as chloroma, as mastoid process chloroma, cerebral dura mater chloroma, vertebra chloroma, pelvis chloroma etc., and can be clinical early than coming across before leukemia.Because leukaemia comes from blood system, there is again the feature of tumor cell, so people claim him to be leukemia traditionally.Leukemic distribution is global, accounts for about 5% of cancer total incidence, fall ill with child and youth in the majority.China leukaemic is about 3-4 people/100,000 populations, and male is more than women.In the mortality rate of each age group malignant tumor of China, leukemia accounts for the 6th (male) and the 8th (women), in the crowd of child and less than 35 years old, then account for the 1st.Leukemia is pressed leukocyte fully-developed degree and is distinguished, and can be divided into acute, chronic two kinds; Dissimilarly to distinguish by leukocytic, lymphocytic type, granulocyte type, monocyte type, plasma cell type, megakaryocytic leukemia can be divided into, sometimes also can be formed by two kinds of mixing with cells, as grain one single cell leukemia.
Current leukemic treatment mainly relies on chemotherapy, treat leukemic medicine also many, Chinese medicine Western medicine all has, common Western medicine has methotrexate, cyclophosphamide, vincristine, harringtonine, interferon etc., Chinese patent medicine has precious balosam capsule, FUFANG BANMAO JIAONANG etc., these medicines are treated leukemia by different mechanism, wherein have cytotoxic drug, differentiation-inducing medicine etc.Although cytotoxic drug can kill and wound a large amount of leukaemias, reduce the leukemia load in body, side effect is more, as killed and wounded, suppressing normal hematopoetic cells, causes anemia, hemorrhage; Suppress normal immunological function, immunity of organisms declines, easy concurrent infection; Damage gastrointestinal tract mucous, cause anorexia Nausea and vomiting; Also can affect darling renal function, but also the second Tumor incidence can be made to increase.In addition, these drug doses are large again, also can not kill the leukaemia in body completely, and residual leukaemia is the root of palindromia.Induction-differential therapy refers to that application can promote that leukaemia's differentiation and maturation maybe can regulate leukaemia's phenotype to strengthen its differentiation-inducing agents to drug susceptibility.Current application the most effectively Treated with All-trans Retinoic Acid acute promyelocytic leukemia, all-trans-retinoic acid has become the choice drug of acute promyelocytic leukemia inducer remission.
Interferon is the cytokine that a kind of mononuclear cell and lymphocyte produce.Be divided into α, β, γ 3 kinds.Since the eighties, many research displays, interferon (especially alpha-interferon and gamma interferon), except having antiviral, immunoregulatory effect, also has obvious anti-cell increment effect.Current interferon has been used to treat multiple leukemia.The chronic leukemias such as the treatment of interferon mainly shows slow grain (chronic phase), hairy cell leukemia, slow pouring are helpful.Because interferon therapy somewhat expensive, heavy dose are side effect many (as ability to act reduces, administers and go down), current domestic application is still not general.
What said medicine had has stronger toxicity, leukemicly has lethal effect to normal Cell sap, some DeGrains, although some effects are good expensive in treatment simultaneously.Chinese medicine has shown certain effect in aortic arch syndrome treatment, and due to Chinese materia medica tocixity low, few side effects and more and more receiving publicity.
Aleuritopteris argentea (Gmel.) Fee system Sinopteridaceae plant Aleuritopteris argentea (Gmel.) Fee (Aleuritopterisargentea(Gmel.) Fee) dry herb.Autumn gathers, and removing impurity dries.Record in 1998 " Drug Standard of Ministry of Public Health of the Peoples Republic of China mongolian medicine fascicle ".Its property is put down, and mildly bitter flavor, has the effects such as improving eyesight, wound healing, hemostasis, promoting blood flow to regulate menstruation, cough-relieving, dampness removing, removing toxic substances and promoting subsidence of swelling, can be used for conjunctival congestion, visual deterioration, thoracic injury, bone injury, pulmonary tuberculosis cough, haematemesis.The common drug of the minority areas such as illiteracy is hidden for China.But about the basic research of Aleuritopteris argentea (Gmel.) Fee is still very limited, the follow-up promotion and application of this medical material are restricted.Modern study shows, Aleuritopteris argentea (Gmel.) Fee contains alepterolic acid (alepterolicacid), sucrose (sucrose) and flavone compound, and other chemical compositions are not quite clear; Pharmacological research shows, Aleuritopteris argentea (Gmel.) Fee flavone is that a kind of natural effective free radical scavenger Aleuritopteris argentea (Gmel.) Fee flavone has very strong scavenging action to hydroxyl radical free radical, ultra-oxygen anion free radical, hydrogen peroxide, its EC
50be respectively 0.0099,0.0060 and 0.0190mg/mL, and in concentration-dependent relation, other pharmacological actions have no report.
Number of patent application be CN96102241.8 patent discloses a kind of specific medicament prescription for the treatment of liver and bile stone and anti-recurrence, wherein containing Aleuritopteris argentea (Gmel.) Fee.Number of patent application is that application discloses of CN200310110176.4 discloses a kind of medicament composing prescription for the treatment of sinusitis, wherein containing Aleuritopteris argentea (Gmel.) Fee.Number of patent application be CN200310110531.8 patent discloses a kind of herbal mediciment being used for the treatment of aplastic anemia, it is formulated that it adds Chinese liquor by nearly 20 kinds of Chinese herbal medicine such as HUISHENGCAO, Radix Rhodiolae, Aleuritopteris argentea (Gmel.) Fee through water infusion.Number of patent application be CN97107327.9 patent discloses a kind of Chinese herbal medicine for the treatment of dysmenorrhes, the composition of formula comprises: Aleuritopteris argentea (Gmel.) Fee, Rhizoma Atractylodis, Endothelium Corneum Gigeriae Galli etc.Number of patent application be CN201210440734.2 patent discloses a kind of medicine for the treatment of the optimum myositis of children acute, its composition of raw materials and weight proportion are: Aleuritopteris argentea (Gmel.) Fee 6 ~ 10 parts, Retinervus Luffae Fructus 8 ~ 15 parts, the Herba Blumeae Laciniatae 8 ~ 15 parts etc.Number of patent application be CN201210462718.3 patent discloses a kind of medicine for the treatment of cystic hyperplasia of breast, it is characterized in that component and weight proportion are: Aleuritopteris argentea (Gmel.) Fee 1: Fructus Seu Herba Pubescentis 2: the Rhizoma Atractylodis Macrocephalae 1.5 etc.
Above-mentioned document and patent etc., there is not yet Aleuritopteris argentea (Gmel.) Fee or the Aleuritopteris argentea (Gmel.) Fee extract report for the preparation for the treatment of leukemia medicament.
Summary of the invention
The object of the present invention is to provide the application of Aleuritopteris argentea (Gmel.) Fee extract in preparation treatment leukemia medicament.
The present invention is achieved through the following technical solutions:
The present invention's Aleuritopteris argentea (Gmel.) Fee used is Sinopteridaceae plant Aleuritopteris argentea (Gmel.) Fee (Aleuritopterisargentea(Gmel.) Fee) dry herb.
The application of Aleuritopteris argentea (Gmel.) Fee extract of the present invention in preparation treatment leukemia medicament, the preparation method of described Aleuritopteris argentea (Gmel.) Fee extract is:
(1) Aleuritopteris argentea (Gmel.) Fee, with concentration 30% ~ 80% ethanol as solvent, extract at 30 DEG C ~ 70 DEG C warm macerating, extraction time is 2 ~ 5 times, and each extraction time is 2 ~ 6 hours, and each solvent load is 8 ~ 15 times of Aleuritopteris argentea (Gmel.) Fee weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Aleuritopteris argentea (Gmel.) Fee extract A;
(2) get the Aleuritopteris argentea (Gmel.) Fee extract A that step (1) obtains and add 6 ~ 18 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A; By precipitate A, adopt high-speed countercurrent chromatography (HSCCC), with cyclohexane-ethyl acetate-n-butanol-water (3:2:3:1) for solvent system, carry out separation and purification, recycling design, concentrate drying, obtain Aleuritopteris argentea (Gmel.) Fee extract B;
(3) by the medicinal liquid A that step (2) obtains, by macroporous adsorptive resins, wash with water, then carry out eluting with the alcoholic solution of concentration 50% ~ 85%, collect ethanol elution, concentrate drying, obtains Aleuritopteris argentea (Gmel.) Fee extract C;
(4) above-mentioned Aleuritopteris argentea (Gmel.) Fee extract B, Aleuritopteris argentea (Gmel.) Fee extract C, wherein one or both mix by a certain percentage, obtain Aleuritopteris argentea (Gmel.) Fee extract of the present invention.
The preparation method of Aleuritopteris argentea (Gmel.) Fee extract is:
(1) Aleuritopteris argentea (Gmel.) Fee, with concentration 30% ~ 80% ethanol as solvent, extract at 30 DEG C ~ 70 DEG C warm macerating, extraction time is 2 ~ 5 times, and each extraction time is 2 ~ 6 hours, and each solvent load is 8 ~ 15 times of Aleuritopteris argentea (Gmel.) Fee weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Aleuritopteris argentea (Gmel.) Fee extract A;
(2) the Aleuritopteris argentea (Gmel.) Fee extract A that step (1) obtains is added 6 ~ 18 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A; By precipitate A, adopt high-speed countercurrent chromatography (HSCCC), with cyclohexane-ethyl acetate-n-butanol-water (3:2:3:1) for solvent system, carry out separation and purification, recycling design, concentrate drying, obtain Aleuritopteris argentea (Gmel.) Fee extract B;
(3) by the medicinal liquid A that step (2) obtains, by macroporous adsorptive resins, wash with water, then carry out eluting with the alcoholic solution of concentration 50% ~ 85%, collect ethanol elution, concentrate drying, obtains Aleuritopteris argentea (Gmel.) Fee extract C;
(4) above-mentioned Aleuritopteris argentea (Gmel.) Fee extract B and Aleuritopteris argentea (Gmel.) Fee extract C mix, and obtain Aleuritopteris argentea (Gmel.) Fee extract of the present invention.
The preparation method of preferred Aleuritopteris argentea (Gmel.) Fee extract is:
(1) Aleuritopteris argentea (Gmel.) Fee, with concentration 60% ethanol as solvent, extract at 50 DEG C of warm macerating, extract 4 times, each extraction time is 4 hours, and each solvent load is 12 times of Aleuritopteris argentea (Gmel.) Fee weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Aleuritopteris argentea (Gmel.) Fee extract A;
(2) the Aleuritopteris argentea (Gmel.) Fee extract A that step (1) obtains is added 12 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A.By precipitate A, adopt high-speed countercurrent chromatography (HSCCC), with cyclohexane-ethyl acetate-n-butanol-water (3:2:3:1) for solvent system, carry out separation and purification, recycling design, concentrate drying, obtain Aleuritopteris argentea (Gmel.) Fee extract B;
(3) by the medicinal liquid A that step (2) obtains, by X-5 macroporous adsorptive resins, wash with water, then carry out eluting with the alcoholic solution of concentration 70%, collect ethanol elution, concentrate drying, obtains Aleuritopteris argentea (Gmel.) Fee extract C;
(4) above-mentioned Aleuritopteris argentea (Gmel.) Fee extract B and Aleuritopteris argentea (Gmel.) Fee extract C mix, and obtain Aleuritopteris argentea (Gmel.) Fee extract of the present invention.
In Aleuritopteris argentea (Gmel.) Fee extract of the present invention, Aleuritopteris argentea (Gmel.) Fee extract B mainly contains: alepterolic acid (alepterolicacid); 8 (17), 13 (E)-labdadien-15,19-dioicacid; Methyl8 (17), 13 (E)-labdadien-19-oicacid-15-oate; 15-acetoxy-8 (17), 13 (E)-labdadi-en-19-oicacid; 15-hydroxy-8 (17), 13 (E)-labdadien-19-oicacid; 8 (17), 13 (E)-labdadien-15,19-diol; Totally 6 labdane diterpenes compositions.
In Aleuritopteris argentea (Gmel.) Fee extract of the present invention, Aleuritopteris argentea (Gmel.) Fee extract C mainly contains: onychin (Onychin); Onychin-2'-O-β-D-Glucose glycosides; Onychin-2'-O-β-D-Azloglycoside; High mountain onychium japonicum Kunze A prime; High mountain onychium japonicum Kunze B prime; Isomatteucinol (5,7-dihydroxy-6,8-dimethyl-4-methoxyl group-flavanone); De-methoxy isomatteucinol (5,7-dihydroxy-6,8-dimethyl-flavanone); Totally 7 flavanones and flavanone methods of glycosides.
Aleuritopteris argentea (Gmel.) Fee extract of the present invention, its preparation method is characterised in that: the macroporous adsorbent resin adopted is X-5 macroporous adsorbent resin, NKA macroporous adsorbent resin, ADS-8 macroporous adsorbent resin.
Aleuritopteris argentea (Gmel.) Fee extract of the present invention, by adding the various adjuvants that pharmaceutics allows, makes the peroral dosage forms such as the tablet on pharmaceutics, granule, capsule.
The present invention is first with Sinopteridaceae plant Aleuritopteris argentea (Gmel.) Fee (Aleuritopterisargentea(Gmel.) Fee) dry herb be that raw material is prepared extract and is used for the treatment of leukemia.The present invention's exploratory development first and be separated obtain Aleuritopteris argentea (Gmel.) Fee have treatment leukemic extract.Gained Aleuritopteris argentea (Gmel.) Fee extract is by regulatory T-cell subset proportions and promote that the secretion of these cytokines plays its immunoregulation effect, recover self suppressed immunologic function and kill and wound residual leukaemia, thus confirm that Aleuritopteris argentea (Gmel.) Fee extract has and induce IL-2, the effect of IL-6 and IFN-γ, has certain effect to leukemic therapy apparatus immunity function restructuring.Our experimental result also shows, Aleuritopteris argentea (Gmel.) Fee extract has enhancing IL-2 and NCK active function, illustrates that Aleuritopteris argentea (Gmel.) Fee extract has certain protective effect to the immunosuppressant of P388 leukemia mouse after chemotherapy.Infer its mechanism of action may with enhancing Exploration of Red Cell Immuno-adhesive Function, impel erythrocyte to discharge NCK activity factor, regulate NCK-IFN-IL-2 immunological network relevant.
Aleuritopteris argentea (Gmel.) Fee extract of the present invention is preparing the application by improving in NCK, IL-2, IL-6, IFN-gamma activity treatment leukemia medicament.Aleuritopteris argentea (Gmel.) Fee extract of the present invention can be used for preparation treatment leukemia medicament, especially improves NCK, IL-2, IL-6, IFN-gamma activity, treats leukemic medicine.
The application in preparation treatment leukemia medicament of Aleuritopteris argentea (Gmel.) Fee extract A of the present invention, Aleuritopteris argentea (Gmel.) Fee extract B, Aleuritopteris argentea (Gmel.) Fee extract C.
The treatment leukemia medicament that Aleuritopteris argentea (Gmel.) Fee extract of the present invention and chemical drugs or Chinese medicine or natural drug form.
Detailed description of the invention
Below by specific experiment example and embodiment, the application of Aleuritopteris argentea (Gmel.) Fee extract in preparation treatment leukemia medicament is described further, but is not limited to the present invention.
Embodiment 1: Aleuritopteris argentea (Gmel.) Fee extract and monomeric compound preparation
(1) Aleuritopteris argentea (Gmel.) Fee 20kg, with concentration 60% ethanol as solvent, extract at 50 DEG C of warm macerating, extract 4 times, each extraction time is 4 hours, and each solvent load is 12 times of Aleuritopteris argentea (Gmel.) Fee weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Aleuritopteris argentea (Gmel.) Fee extract A;
(2) the Aleuritopteris argentea (Gmel.) Fee extract A that step (1) obtains is added 12 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A.By precipitate A, adopt high-speed countercurrent chromatography (HSCCC), with cyclohexane-ethyl acetate-n-butanol-water (3:2:3:1) for solvent system, carry out separation and purification, recycling design, concentrate drying, obtain Aleuritopteris argentea (Gmel.) Fee extract B;
(3) by the medicinal liquid A that step (2) obtains, by X-5 macroporous adsorptive resins, wash with water, then carry out eluting with the alcoholic solution of concentration 70%, collect ethanol elution, concentrate drying, obtains Aleuritopteris argentea (Gmel.) Fee extract C;
(4) above-mentioned Aleuritopteris argentea (Gmel.) Fee extract B and Aleuritopteris argentea (Gmel.) Fee extract C mix, and obtain Aleuritopteris argentea (Gmel.) Fee extract of the present invention.
Method described above prepares Aleuritopteris argentea (Gmel.) Fee extract B, again through 200-300 order silica gel column chromatography, with petroleum ether-ethyl acetate gradient elution (10:1-3:1), thin layer tracing detection, merge same stream part, leave standstill crystallize, sucking filtration, petroleum ether-acetone (1:1) recrystallization, obtains alepterolic acid (alepterolicacid) respectively; 8 (17), 13 (E)-labdadien-15,19-dioicacid; Methyl8 (17), 13 (E)-labdadien-19-oicacid-15-oate; 15-acetoxy-8 (17), 13 (E)-labdadi-en-19-oicacid; 15-hydroxy-8 (17), 13 (E)-labdadien-19-oicacid; The compositions such as 8 (17), 13 (E)-labdadien-15,19-diol, the chemical constitution of above each compound is all through the wave spectrum such as mass spectrum and nuclear magnetic resonance, NMR means confirmation, and purity detects through high performance liquid chromatography and is all greater than 98%.It is mass fraction 35% that content detects the content of labdane diterpenes composition in Aleuritopteris argentea (Gmel.) Fee extract B such as alepterolic acid through high performance liquid chromatography.
Method described above prepares Aleuritopteris argentea (Gmel.) Fee extract C, then through 200-300 order silica gel column chromatography, with chloroform-methanol elution gradient (100:1-3:1), thin layer tracing detection, merges same stream part, leaves standstill crystallize, sucking filtration, recrystallizing methanol, obtains onychin (Onychin) respectively; Onychin-2'-O-β-D-Glucose glycosides; Onychin-2'-O-β-D-Azloglycoside; High mountain onychium japonicum Kunze A prime; High mountain onychium japonicum Kunze B prime; Isomatteucinol (5,7-dihydroxy-6,8-dimethyl-4-methoxyl group-flavanone); The compositions such as de-methoxy isomatteucinol (5,7-dihydroxy-6,8-dimethyl-flavanone), the chemical constitution of above each compound is all through the wave spectrum such as mass spectrum and nuclear magnetic resonance, NMR means confirmation, and purity detects through high performance liquid chromatography and is all greater than 98%.Content detects the flavanones such as onychin through high performance liquid chromatography and the content of flavanone methods of glycosides in Aleuritopteris argentea (Gmel.) Fee extract C is mass fraction 80%.
Embodiment 2: Aleuritopteris argentea (Gmel.) Fee extract and monomeric compound preparation
(1) Aleuritopteris argentea (Gmel.) Fee 10kg, with concentration 30% ethanol as solvent, extract at 70 DEG C of warm macerating, extraction time is 2 times, and each extraction time is 2 hours, and each solvent load is 8 times of Aleuritopteris argentea (Gmel.) Fee weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Aleuritopteris argentea (Gmel.) Fee extract A;
(2) the Aleuritopteris argentea (Gmel.) Fee extract A that step (1) obtains is added 6 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A.By precipitate A, adopt high-speed countercurrent chromatography (HSCCC), with cyclohexane-ethyl acetate-n-butanol-water (3:2:3:1) for solvent system, carry out separation and purification, recycling design, concentrate drying, obtain Aleuritopteris argentea (Gmel.) Fee extract B;
(3) by the medicinal liquid A that step (2) obtains, by NKA macroporous adsorptive resins, wash with water, then carry out eluting with the alcoholic solution of concentration 50%, collect ethanol elution, concentrate drying, obtains Aleuritopteris argentea (Gmel.) Fee extract C;
(4) above-mentioned Aleuritopteris argentea (Gmel.) Fee extract B and Aleuritopteris argentea (Gmel.) Fee extract C mix by a certain percentage, obtain Aleuritopteris argentea (Gmel.) Fee extract of the present invention.Wherein Aleuritopteris argentea (Gmel.) Fee extract B accounts for 25%, and Aleuritopteris argentea (Gmel.) Fee extract C accounts for 75%.
Method described above prepares Aleuritopteris argentea (Gmel.) Fee extract B, again through 200-300 order silica gel column chromatography, with petroleum ether-ethyl acetate gradient elution (10:1-3:1), thin layer tracing detection, merge same stream part, leave standstill crystallize, sucking filtration, petroleum ether-acetone (1:2) recrystallization, obtains alepterolic acid (alepterolicacid) respectively; 8 (17), 13 (E)-labdadien-15,19-dioicacid; Methyl8 (17), 13 (E)-labdadien-19-oicacid-15-oate; 15-acetoxy-8 (17), 13 (E)-labdadi-en-19-oicacid; 15-hydroxy-8 (17), 13 (E)-labdadien-19-oicacid; The compositions such as 8 (17), 13 (E)-labdadien-15,19-diol, the chemical constitution of above each compound is all through the wave spectrum such as mass spectrum and nuclear magnetic resonance, NMR means confirmation, and purity detects through high performance liquid chromatography and is all greater than 98%.It is mass fraction 56% that content detects the content of labdane diterpenes composition in Aleuritopteris argentea (Gmel.) Fee extract B such as alepterolic acid through high performance liquid chromatography.
Method described above prepares Aleuritopteris argentea (Gmel.) Fee extract C, then through 200-300 order silica gel column chromatography, with chloroform-methanol elution gradient (100:1-3:1), thin layer tracing detection, merges same stream part, leaves standstill crystallize, sucking filtration, recrystallizing methanol, obtains onychin (Onychin) respectively; Onychin-2'-O-β-D-Glucose glycosides; Onychin-2'-O-β-D-Azloglycoside; High mountain onychium japonicum Kunze A prime; High mountain onychium japonicum Kunze B prime; Isomatteucinol (5,7-dihydroxy-6,8-dimethyl-4-methoxyl group-flavanone); The compositions such as de-methoxy isomatteucinol (5,7-dihydroxy-6,8-dimethyl-flavanone), the chemical constitution of above each compound is all through the wave spectrum such as mass spectrum and nuclear magnetic resonance, NMR means confirmation, and purity detects through high performance liquid chromatography and is all greater than 98%.Content detects the flavanones such as onychin through high performance liquid chromatography and the content of flavanone methods of glycosides in Aleuritopteris argentea (Gmel.) Fee extract C is mass fraction 70%.
Embodiment 3: Aleuritopteris argentea (Gmel.) Fee extract and monomeric compound preparation
(1) Aleuritopteris argentea (Gmel.) Fee 5kg, with concentration 80% ethanol as solvent, extract at 30 DEG C of warm macerating, extraction time is 5 times, and each extraction time is 6 hours, and each solvent load is 15 times of Aleuritopteris argentea (Gmel.) Fee weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Aleuritopteris argentea (Gmel.) Fee extract A;
(2) the Aleuritopteris argentea (Gmel.) Fee extract A that step (1) obtains is added 18 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A.By precipitate A, adopt high-speed countercurrent chromatography (HSCCC), with cyclohexane-ethyl acetate-n-butanol-water (3:2:3:1) for solvent system, carry out separation and purification, recycling design, concentrate drying, obtain Aleuritopteris argentea (Gmel.) Fee extract B;
(3) by the medicinal liquid A that step (2) obtains, by ADS-8 macroporous adsorptive resins, wash with water, then carry out eluting with the alcoholic solution of concentration 85%, collect ethanol elution, concentrate drying, obtains Aleuritopteris argentea (Gmel.) Fee extract C;
(4) above-mentioned Aleuritopteris argentea (Gmel.) Fee extract B, the mixing of Aleuritopteris argentea (Gmel.) Fee extract C, obtain Aleuritopteris argentea (Gmel.) Fee extract of the present invention.
Method described above prepares Aleuritopteris argentea (Gmel.) Fee extract B, again through 200-300 order silica gel column chromatography, with petroleum ether-ethyl acetate gradient elution (10:1-3:1), thin layer tracing detection, merge same stream part, leave standstill crystallize, sucking filtration, petroleum ether-acetone (1:2) recrystallization, obtains alepterolic acid (alepterolicacid) respectively; 8 (17), 13 (E)-labdadien-15,19-dioicacid; Methyl8 (17), 13 (E)-labdadien-19-oicacid-15-oate; 15-acetoxy-8 (17), 13 (E)-labdadi-en-19-oicacid; 15-hydroxy-8 (17), 13 (E)-labdadien-19-oicacid; The compositions such as 8 (17), 13 (E)-labdadien-15,19-diol, the chemical constitution of above each compound is all through the wave spectrum such as mass spectrum and nuclear magnetic resonance, NMR means confirmation, and purity detects through high performance liquid chromatography and is all greater than 98%.It is mass fraction 65% that content detects the content of labdane diterpenes composition in Aleuritopteris argentea (Gmel.) Fee extract B such as alepterolic acid through high performance liquid chromatography.
Method described above prepares Aleuritopteris argentea (Gmel.) Fee extract C, then through 200-300 order silica gel column chromatography, with chloroform-methanol elution gradient (100:1-3:1), thin layer tracing detection, merges same stream part, leaves standstill crystallize, sucking filtration, recrystallizing methanol, obtains onychin (Onychin) respectively; Onychin-2'-O-β-D-Glucose glycosides; Onychin-2'-O-β-D-Azloglycoside; High mountain onychium japonicum Kunze A prime; High mountain onychium japonicum Kunze B prime; Isomatteucinol (5,7-dihydroxy-6,8-dimethyl-4-methoxyl group-flavanone); The compositions such as de-methoxy isomatteucinol (5,7-dihydroxy-6,8-dimethyl-flavanone), the chemical constitution of above each compound is all through the wave spectrum such as mass spectrum and nuclear magnetic resonance, NMR means confirmation, and purity detects through high performance liquid chromatography and is all greater than 98%.Content detects the flavanones such as onychin through high performance liquid chromatography and the content of flavanone methods of glycosides in Aleuritopteris argentea (Gmel.) Fee extract C is mass fraction 50%.
Embodiment 4: the preparation of Aleuritopteris argentea (Gmel.) Fee extract tablet
Example 1 Aleuritopteris argentea (Gmel.) Fee extract 315g, adds starch 60g, mixing, granulates, dry, sieves, adds 15g microcrystalline Cellulose, 0.5g magnesium stearate, and mixing, is pressed into 1000, obtains Aleuritopteris argentea (Gmel.) Fee extract tablet.
Embodiment 5: the preparation of Aleuritopteris argentea (Gmel.) Fee extract particles
Example 2 Aleuritopteris argentea (Gmel.) Fee extract 315g, adds dextrin 150g, mixing, granulates, dry, granulate, i.e. Aleuritopteris argentea (Gmel.) Fee extract particles.
Embodiment 6: the preparation of Aleuritopteris argentea (Gmel.) Fee extract capsule
Example 3 Aleuritopteris argentea (Gmel.) Fee extract 305g, adds starch 50g, mixing, granulates, dry, sieves, adds Magnesium Stearate proper quantity, and mixing is encapsulated, obtains Aleuritopteris argentea (Gmel.) Fee extract capsule.
Experimental example 1: Aleuritopteris argentea (Gmel.) Fee extracts L
615the test of pesticide effectiveness of leukemia mouse
1. modeling method: aseptically get L
615leukemia mouse spleen makes 25% normal saline cell suspension, gives the L that grows up
615mouse hypodermic inoculation, all there is leukemia in result, transplants continuously, set up stable L with morbidity mice spleen oncocyte at Syngenic mice
615leukemia Model.
L
615the foundation of leukemia cell line, removes L
615the spleen of leukemia mouse, shreds spleen in Hanks liquid, makes leukaemia free out, by collected by centrifugation free cell, then uses RPMI1640 culture fluid (containing 10% calf serum) to be diluted to 8*10
6the Cell sap of/ml, cultivates at 37 DEG C.Cultivate 20d and find there is adherent fusiform cells in culture, around it, have the round cell island of propagation.When separately being cultivated by cells different for two kinds of forms, spindle cell can normal adherent growth, and suspension cell growth is slow; Again by two kinds of cells Mixed culture again, two kinds of Growth of Cells are vigorous, after 45d, by the round cell of suspension growth single culture again, pass to for 12 generations always, and Growth of Cells is good, thus sets up this cell strain.
2. the Aleuritopteris argentea (Gmel.) Fee extract for treating leukemia test of pesticide effectiveness
Material: Kunming mouse (Shandong Province's Experimental Animal Center).(embodiment 1 method prepares for Aleuritopteris argentea (Gmel.) Fee extract, Aleuritopteris argentea (Gmel.) Fee extract A, Aleuritopteris argentea (Gmel.) Fee extract B, Aleuritopteris argentea (Gmel.) Fee extract C, lot number is respectively: 20120102,20120103,20120104,20120105), cyclophosphamide (Hengrui Medicine Co., Ltd., Jiangsu Prov.), standard RhIL-2 (Beijing Shuanglu Pharmaceutical Co., Ltd.), school of life and health sciences cellular resources center, Yackl and IL-2 dependent cells strain CTLL(Chinese Academy of Sciences Shanghai), RPMII640 cell culture fluid and hyclone (Huamei Bio-Engrg Co.); Tetramethyl azo azoles salt (MTT), dodecyl sodium sulfate and canavaline, dimethyl formamide is (Sigma Products); Sample injector, CO2 incubator, ELX-800 microplate reader (Bio-Tek company of the U.S.); The 96 flat Tissue Culture Plates in hole, Epics flow cytometer (Beckman company of the U.S.).
Method: modeling as stated above.Experiment grouping first group is Normal group, and 20 615 is mice, after conventional raising 1w, and intraperitoneal injection of saline 0.2ml/d, normal saline enema 0.3ml, each 1 time of upper and lower noon.2nd group is model group, after conventional raising 1w, and inoculation L
615leukaemia, the same Normal group of inoculation post processing.3rd group is Western medicine group, and 20 conventional raisings, after one week, inoculate L
615leukaemia, after inoculation the 3rd day starts ip cyclophosphamide, 200mg/kg, 1 time/d.4th group of-7 groups is respectively Aleuritopteris argentea (Gmel.) Fee extract group, Aleuritopteris argentea (Gmel.) Fee extract A group, Aleuritopteris argentea (Gmel.) Fee extract B group, Aleuritopteris argentea (Gmel.) Fee extract C group, get respectively 20 conventional raise 1d after, inoculation L
615leukaemia, gives Aleuritopteris argentea (Gmel.) Fee extract on the 3rd day, Aleuritopteris argentea (Gmel.) Fee extract A, Aleuritopteris argentea (Gmel.) Fee extract B, Aleuritopteris argentea (Gmel.) Fee extract C gavage, 0.3ml, each 1 time of upper and lower noon after inoculation respectively.Above each treated animal often group get 10 in the 5th day put to death, get spleen and carry out related immune Indexs measure, remaining each animal carries out observation life cycle.
Observation index: 1. observe life cycle.2. T cell Subsets.3. IL-2, IL-6, IFN-gamma activity measures.
Statistical method: data represent with x ± s, adopts Mi-crosoftExcel to carry out t inspection.
Result:
Life cycle: Aleuritopteris argentea (Gmel.) Fee each extract group life cycle, comparatively model group and Western medicine group obviously extended, statistics there were significant differences P<0.01, in table 1.
Table 1 each experimental group compares life cycle
Note: compared with model group
*p<0.01, compared with Western medicine group
△p<0.01.
T cell subgroup: each extract group CD4 of Aleuritopteris argentea (Gmel.) Fee
+cell comparatively model group and Western medicine group high.CD8
+leukopenia, CD4/CD8 ratio recovers normal.In table 2.
Table 2 respectively group T cell subgroup change
Note: compared with Normal group
*p<0.01, compared with model group
△p<0.01, compared with Western medicine group
▲p<0.01.
IL-2, IL-6, IFN-gamma activity: after Western medicine group leukemia mouse chemotherapy, it secretes IL-2, IL-6, the level of IFN-γ significantly declines, and the secretion level of each extract group IL-2 of Aleuritopteris argentea (Gmel.) Fee, IL-6, IFN-γ obviously increases, both have significant difference (P<0.01), in table 3 at contrast.
Table 3 respectively organizes IL-2, and IL-6, IFN-gamma activity changes
Note: compared with Normal group
*p<0.01, compared with model group
△p<0.01, compared with Western medicine group
▲p<0.01.
The whether normal of immunologic function be one of key factor of tumor development, and immunologic function is that the ratio of T cell subgroup also plays an important role in stable regulation immunologic function by performed by the cytokine secreted by immunocyte.T cell subset proportions imbalance after leukemia mouse chemotherapy, the levels of cytokine secretion such as IL-2, IL-6, IFN-γ decline, and point out the immunologic function of now body to be in inhibitory state.And by after use Aleuritopteris argentea (Gmel.) Fee extract for treating, T cell subset proportions recovers normal, IL-2, IL-6, the levels of cytokine secretion such as IFN-γ go up, illustrate that Aleuritopteris argentea (Gmel.) Fee extract is by regulatory T-cell subset proportions and promote that the secretion of these cytokines plays its immunoregulation effect, recover self suppressed immunologic function and kill and wound residual leukaemia, thus confirm that Aleuritopteris argentea (Gmel.) Fee extract has and induce IL-2, the effect of IL-6 and IFN-γ, has certain effect to leukemic therapy apparatus immunity function restructuring.
Experimental example 2: Aleuritopteris argentea (Gmel.) Fee extracts the pharmacodynamics test to P388 leukemia mouse
1. modeling method: get ascitic type P
388leukemia mouse, de-cervical vertebra is put to death, and abdominal part is sterilized, suction ascites, and add normal saline mixing, it is (1-1.5) * 10 that microscope lowers intact cells concentration
7individual/ml, the inoculum density of every Mus 0.2ml(Kunming mouse and dosage) carry out lumbar injection.
2. Aleuritopteris argentea (Gmel.) Fee extract is to P
388the impact of natural killer cell (NCK), interleukin-2 (IL-2) activity after leukemia mouse chemotherapy.
Material: Kunming kind one-level mice (Shandong Province's Experimental Animal Center), 50, male and female half and half, body weight 18-22g.Aleuritopteris argentea (Gmel.) Fee extract prepared by embodiment 1, cyclophosphamide (Hengrui Medicine Co., Ltd., Jiangsu Prov.), standard RhIL-2 (Beijing Shuanglu Pharmaceutical Co., Ltd.), school of life and health sciences cellular resources center, Yackl and IL-2 dependent cells strain CTLL(Chinese Academy of Sciences Shanghai), RPMII640 cell culture fluid and hyclone (Huamei Bio-Engrg Co.); Tetramethyl azo azoles salt (MTT), dodecyl sodium sulfate and canavaline, dimethyl formamide is (Sigma Products); Sample injector, CO2 incubator, ELX-800 microplate reader (Bio-Tek company of the U.S.); The 96 flat Tissue Culture Plates in hole, Epics flow cytometer (Beckman company of the U.S.).
Modeling and group technology: get 40 mices, inoculation P
388the strain of Lymphocytic leukemia tumor (inoculation method: get ascitic type P388 leukemia mouse, de-cervical vertebra is put to death, and abdominal part is sterilized, suction ascites, adds normal saline mixing, and it is 1.5*107/ml that microscope lowers intact cells concentration, every mice right oxter injection 0.2ml), be divided into 4 groups at random.Another 10 is normal blank group.
Processing method: 1. normal blank group: with 0.4ml/20g normal saline ig; 2. P
388leukemia Model group: with 0.4ml/20g normal saline ig; 3. chemotherapy group: with 0.4ml/20g normal saline ig; 4. chemotherapy adds Aleuritopteris argentea (Gmel.) Fee extract low dose group: respectively with 50mg extract in 0.4ml normal saline ig; 5. chemotherapy adds Aleuritopteris argentea (Gmel.) Fee extract high dose group: be dissolved in 0.4ml normal saline ig with 100mg extract.3. the continuous ig14d of each group above, give simultaneously, 4., 5. organize ip cyclophosphamide 35mg/kg/d, be used in conjunction 6d.In the 15th day, de-cervical vertebra was put to death, and aseptically got spleen NCK, IL-2 active.
Detection method: NCK Activity determination is with reference to Jiang Zhonghua method, and IL-2 activity test method is with reference to Qian Yukun method.
Statistical method: adopt many group means to compare, namely first do variance analysis, remake q inspection.
Result: result shows, it is active that Aleuritopteris argentea (Gmel.) Fee extract can improve NCK and IL-2.In table 4
Table 4 is group NCK and IL-2 expression activitiy respectively
Note: compare with normal group, * P<0.05, * * P<0.01; Compare with model group,
△p<0.05,
△ △p<0.01;
Compare with chemotherapy group,
#p<0.05,
##p<0.01.
NCK is one of three major types lymphocyte, be the important composition composition of the early stage antineoplastic immune supervisory role of body, be a group broad-spectrum anti-tumor cell, its killing activity does not need the prior sensitization of antigen, do not rely on antibody and thymus, not by the restriction of major histocompatibility antigen (MHC) yet.NCK does not need specific antigen to stimulate for virus infected cell and tumor cell in vivo, immune complex and target cell surface structure all directly can bring out the immunoreation of NCK, the a large amount of cytokine of rapid secretion, as interferon-γ (IFN-γ), tumor necrosis factor-alpha (TNF-α), GM-CSF (GMCSF), IL-2 etc., regulates phagocyte and T, B cell function.IL-2, IFN can increase again the activity of NCK simultaneously, therefore body exists NCK-IFN-IL-2 immunological network to have scholar to think.The glycoprotein of the 15KD that IL-2 is mainly produced by T cell, and act on T cell, impel T cell to breed.IL-2 is main, the most strong SCIF in body.IL-2 can induce or promote the activity of various kinds of cell toxic cell, as NCK, cytotoxic T lymphocyte (CTL), Lymphatic circulation (LAK) and tumor infiltrating lymphocyte (TIL) etc., these cells are most important in the immune surveillance and antineoplastic immune of body.Our experimental result shows, P388 leukemia mouse self and immunity function after chemotherapy are suppressed, NCK and IL-2 activity all has reduction in various degree, and after chemotherapy, P388 leukemia mouse is starkly lower than normal group and model group.This is because chemotherapeutic is on immune impact, also kills and wounds or inhibit caused by LAK cell while killing tumor cell.Recent study finds, the NCK of tumor mice activity reduces, may with body lotus tumor time, the cytokine network antitumor action of its immunocyte is not normal relevant, and as immunosuppressive factor in serum exists and increased activity, immunological enhancement factor active declines.Can by suppressing the generation of IFN thus making NCK activity be suppressed further after giving chemotherapeutic on this basis.In normal body, IL-2 has self forward immunoregulation effect, but at most of tumor mice, IL-2 system exists obviously suppression or disorderly.Most researcher is thought, the ability and all having IL-2 reactivity that tumor patient produces IL-2 reduces in various degree, may SMXO and Ts function strengthen relevant in or host hyperfunction with IL-2 catabolism.After giving chemotherapeutic, inducing of IL-2 is suppressed further, thus increases the weight of the decline of tumor mice immunity.After giving Aleuritopteris argentea (Gmel.) Fee extract for treating, high dose group NCK and IL-2 is all increased significantly.Our experimental result shows, Aleuritopteris argentea (Gmel.) Fee extract has enhancing IL-2 and NCK active function, illustrates that Aleuritopteris argentea (Gmel.) Fee extract has certain protective effect to the immunosuppressant of P388 leukemia mouse after chemotherapy.Infer its mechanism of action may with enhancing Exploration of Red Cell Immuno-adhesive Function, impel erythrocyte to discharge NCK activity factor, regulate NCK-IFN-IL-2 immunological network relevant.The above-mentioned effect of Aleuritopteris argentea (Gmel.) Fee extract, for the immunosuppressant caused by control chemotherapy of tumors, effectively ensures completing of chemotherapy, has positive meaning.
Claims (8)
1. the application of Aleuritopteris argentea (Gmel.) Fee extract in preparation treatment leukemia medicament, the preparation method of described Aleuritopteris argentea (Gmel.) Fee extract is:
(1) Aleuritopteris argentea (Gmel.) Fee, with concentration 30% ~ 80% ethanol as solvent, extract at 30 DEG C ~ 70 DEG C warm macerating, extraction time is 2 ~ 5 times, and each extraction time is 2 ~ 6 hours, and each solvent load is 8 ~ 15 times of Aleuritopteris argentea (Gmel.) Fee weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Aleuritopteris argentea (Gmel.) Fee extract A;
(2) get the Aleuritopteris argentea (Gmel.) Fee extract A that step (1) obtains and add 6 ~ 18 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A; By precipitate A, adopt high-speed countercurrent chromatography (HSCCC), with the cyclohexane-ethyl acetate-n-butanol-water of portion rate 3:2:3:1 for solvent system, carry out separation and purification, recycling design, concentrate drying, obtain Aleuritopteris argentea (Gmel.) Fee extract B;
(3) by the medicinal liquid A that step (2) obtains, by macroporous adsorptive resins, wash with water, then carry out eluting with the alcoholic solution of concentration 50% ~ 85%, collect ethanol elution, concentrate drying, obtains Aleuritopteris argentea (Gmel.) Fee extract C;
(4) above-mentioned Aleuritopteris argentea (Gmel.) Fee extract B, Aleuritopteris argentea (Gmel.) Fee extract C, wherein one or both mix by a certain percentage, obtain Aleuritopteris argentea (Gmel.) Fee extract.
2. application according to claim 1, is characterized in that the preparation method of described Aleuritopteris argentea (Gmel.) Fee extract is:
(1) Aleuritopteris argentea (Gmel.) Fee, with concentration 30% ~ 80% ethanol as solvent, extract at 30 DEG C ~ 70 DEG C warm macerating, extraction time is 2 ~ 5 times, and each extraction time is 2 ~ 6 hours, and each solvent load is 8 ~ 15 times of Aleuritopteris argentea (Gmel.) Fee weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Aleuritopteris argentea (Gmel.) Fee extract A;
(2) the Aleuritopteris argentea (Gmel.) Fee extract A that step (1) obtains is added 6 ~ 18 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A; By precipitate A, adopt high-speed countercurrent chromatography (HSCCC), with the cyclohexane-ethyl acetate-n-butanol-water of portion rate 3:2:3:1 for solvent system, carry out separation and purification, recycling design, concentrate drying, obtain Aleuritopteris argentea (Gmel.) Fee extract B;
(3) by the medicinal liquid A that step (2) obtains, by macroporous adsorptive resins, wash with water, then carry out eluting with the alcoholic solution of concentration 50% ~ 85%, collect ethanol elution, concentrate drying, obtains Aleuritopteris argentea (Gmel.) Fee extract C;
(4) above-mentioned Aleuritopteris argentea (Gmel.) Fee extract B and Aleuritopteris argentea (Gmel.) Fee extract C mix, and obtain Aleuritopteris argentea (Gmel.) Fee extract.
3. application according to claim 1, is characterized in that the preparation method of described Aleuritopteris argentea (Gmel.) Fee extract is:
(1) Aleuritopteris argentea (Gmel.) Fee, with concentration 60% ethanol as solvent, extract at 50 DEG C of warm macerating, extract 4 times, each extraction time is 4 hours, and each solvent load is 12 times of Aleuritopteris argentea (Gmel.) Fee weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Aleuritopteris argentea (Gmel.) Fee extract A;
(2) the Aleuritopteris argentea (Gmel.) Fee extract A that step (1) obtains is added 12 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A; By precipitate A, adopt high-speed countercurrent chromatography (HSCCC), with the cyclohexane-ethyl acetate-n-butanol-water of portion rate 3:2:3:1 for solvent system, carry out separation and purification, recycling design, concentrate drying, obtain Aleuritopteris argentea (Gmel.) Fee extract B;
(3) by the medicinal liquid A that step (2) obtains, by X-5 macroporous adsorptive resins, wash with water, then carry out eluting with the alcoholic solution of concentration 70%, collect ethanol elution, concentrate drying, obtains Aleuritopteris argentea (Gmel.) Fee extract C;
(4) above-mentioned Aleuritopteris argentea (Gmel.) Fee extract B and Aleuritopteris argentea (Gmel.) Fee extract C mix, and obtain Aleuritopteris argentea (Gmel.) Fee extract.
4. application according to claim 1, is characterized in that described Aleuritopteris argentea (Gmel.) Fee extract in preparation by improving the application in NCK, IL-2, IL-6, IFN-gamma activity treatment leukemia medicament.
5., according to application according to claim 1 or claim 2, it is characterized in that the macroporous adsorbent resin that the preparation method of described Aleuritopteris argentea (Gmel.) Fee extract adopts is X-5 macroporous adsorbent resin, NKA macroporous adsorbent resin, ADS-8 macroporous adsorbent resin.
6. application according to claim 1, is characterized in that the treatment leukemia medicament that described Aleuritopteris argentea (Gmel.) Fee extract and chemical drugs or Chinese medicine form.
7. application according to claim 1, is characterized in that the application in preparation treatment leukemia medicament of described Aleuritopteris argentea (Gmel.) Fee extract B, Aleuritopteris argentea (Gmel.) Fee extract C.
8. application according to claim 1, is characterized in that described Aleuritopteris argentea (Gmel.) Fee extract makes tablet oral on pharmaceutics, granule, capsule by the various adjuvants adding pharmaceutics permission.
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