CN110023342A - The method and combinations thereof for being used to prepare latex of panama rubber tree extract - Google Patents

The method and combinations thereof for being used to prepare latex of panama rubber tree extract Download PDF

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Publication number
CN110023342A
CN110023342A CN201680090627.8A CN201680090627A CN110023342A CN 110023342 A CN110023342 A CN 110023342A CN 201680090627 A CN201680090627 A CN 201680090627A CN 110023342 A CN110023342 A CN 110023342A
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cancer
latex
cell
carcinoma
rubber tree
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CN110023342B (en
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拉皮潘·维蒂苏瓦纳库尔
博隆普罗·尤伊松尼
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PRINCE OF SONGKLA UNIVERSITY
Thai Centre For Excellent Scientific Life
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PRINCE OF SONGKLA UNIVERSITY
Thai Centre For Excellent Scientific Life
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08C—TREATMENT OR CHEMICAL MODIFICATION OF RUBBERS
    • C08C1/00—Treatment of rubber latex
    • C08C1/02—Chemical or physical treatment of rubber latex before or during concentration
    • C08C1/04—Purifying; Deproteinising
    • 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)
    • A61K36/47—Euphorbiaceae (Spurge family), e.g. Ricinus (castorbean)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • 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

Abstract

The disclosure provides the method for the extract by the slurries preparation of the acidification latex of panama rubber tree rolled rich in low molecular weight carbohydrate and with anticancer activity.Method comprises the steps of: the slurries for obtaining and discharging when the latex of panama rubber tree of acidification is calendered；Clast and microorganism are removed from slurries；Remove protein；Remove quebrachitol and small polar molecule；Optionally remaining solid powder is dissolved in water；Protein is further removed by being handled with acid；Optionally neutralize excessive acid；Use solvent deposition；Collect sediment；Optionally it is freeze-dried sediment.The disclosure also provides the method for the treatment of cancer growth and/or transfer and inhibits the method for the tumor cell proliferation in patient.Further it is provided that pharmaceutical composition and functional food composition comprising disclosed latex of panama rubber tree extract.

Description

The method and combinations thereof for being used to prepare latex of panama rubber tree extract
Technical field
Present invention relates generally to the fields for the cancer for including tumor therapy.It is more particularly related to by using The extract of rubber tree (Hevea) latex rich in low molecular weight carbohydrate prevents and treats cancer and and cancer metastasis. The invention further relates to the pharmaceutical compositions and anti-cancer function food compositions for treating cancer.
Background technique
Throughout history, the mankind are constantly subjected to the puzzlement of cancer, this is because body is unable to control the life of certain malignant cells Caused by long and diffusion.According to the World Health Organization (World Health Organization), the cancer as a group is Main non-communicable diseases in the first four place disease (cardiovascular disease, cancer, diabetes and chronic lung disease).American Cancer Society Report claims, and 2012, it was 14,100,000 that the whole world, which is diagnosed to be new cancer number of cases, wherein there is 8,200,000 people dead.Predict 2030 Year, new cancer number of cases is likely to be breached 21,700,000, wherein 13,000,000 people dead (IARC.2012. " the global cancer fact and chart (Global Cancer Facts&Figures) ", the 3rd edition, by American Cancer Society and international cancer research institution's cooperation Make).
For cancer chemotherapy, there are different strategies.Traditional brute-force cytotoxic cancer chemotherapeutant is usual It is related to sizable side effect to normal cell.Even if using monoclonal antibody target, it is also contemplated that some degree are not intended to Side effect.
Recently, it has been found that cancer and the biological signal conduction approach of control somatic cell proliferation and destruction are closely related.Manipulation The chemical reagent of this class.path can be used as anticancer agent.Small molecule target therapeutic agent is mutated in cancer cell, is overexpressed Or catalysis or binding site in other key protein matter inhibitor, but be only applicable to certain form of cancer.Other are existing It can be worked and serving as the signal of stimulation patients immune system for anticancer agent.
Anticancer therapy and drug of many based on natural products are known in the prior art.These active therapeutic agents Range is from small biomolecule to big macromolecular.Such as disclosed in U.S. Patent No. 6,432,452 (anticancer compound), The example of the small Anti-cancer biologic molecule found in plant extracts be angeloyl groups replace ingane (ingenane), one Kind macrocyclic diterpene, competitively blocks the carcinogenesis of phorbol ester, phorbol ester itself is another macrocyclic diterpene.It is another Aspect, the peptide anaphylactogen in rubber tree have been used in pharmaceutical preparation, are exempted from inducing in the treatment of the disease including cancer Epidemic disease response.Example is disclosed in PCT application the PCT/EP2003/011190th, and (modular antigen for adjusting immune response turns Transferrin molecule, related constructs, method and purposes) and No. PCT/EP2013/000291 (comprising polymeric carrier cargo complex and The pharmaceutical composition of at least one protein or peptide antigen) in, and in No. EP2012/000418 (packet of European Patent Application No. Negatively charged nucleic acid containing the compound for immunostimulation) and No. EP2012/000420 it is (multiple comprising polymeric carrier cargo Close the pharmaceutical composition of object and at least one protein or peptide antigen) in.
It less than the small molecule and molecular weight of a kilodalton (hereinafter referred to as " kD ") is more than the big of 10kD in molecular weight Between molecule is the biochemical anticancer agent that molecular weight is several kD, such as at European Patent Publication case the 0th 589 074 (for destroying The carbohydrate compound of resistant cancer cells) in show.Some researchers think that these low molecular weight carbohydrates are logical Cross interference mediated by Cell surface carbohydrate binding molecule cell-ECM interaction come prevent cancer cell growth and Transfer, such as United States Patent (USP) 5,834,442 (passing through the method that soluble modified citrus pectin is administered orally and inhibits cancer metastasis) and Disclosed in United States Patent (USP) 5,895,784 (method by the way that modified pectin treating cancer is administered orally).
The low molecular weight carbohydrate of certain animal origins well known in the art has antitumaous effect.Example includes Hyaluronic acid (Ween, M.P., K.Hummitzsch, R.J.Rodgers, M.K.Oehler and C.Ricciardelli.2011. Proteoglycans induction can be by rush Metastatic carcinoma in the ovary cell behavior (Versican induces a that small hyaluronic acid oligosaccharide inhibits pro-metastatic ovarian cancer cell behavior which can be inhibited by small Hyaluronan oligosaccharides) " clinical and experimental metastasis (Clinical and Experimental Metastasis) " 28:113-125), sulfuric acid maltose (Parish, C.R., C.Freeman, K.J.Brown, D.J.Francis and W.B.Cowden.1999. is identified using novel angiogenesis and the active in vitro test of heparitinase Inhibitor (the Identification of Sulfated based on sulfated oligosaccharide of growth and metastasis of tumours Oligosaccharide-based Inhibitors of Tumor Growth and Metastasis Using Novel In Vitro Assays for Angiogenesis and Heparanase Activity) " cancer research (Cancer Research) " 59:3433-3441) and heparin simulation phosphomannose pentasaccharides sulfate (PI-88) (Demir, M., O.Iqbal, D.A.Hoppensteadt, P.Piccolo, S.Ahmad, C.L.Schultz, R.J.Linhardt and J.Fareed.2001. anticoagulation and the antiprotease spectrum of new type natural heparin simulation mannopentaose phosphosulphate (PI-88) (Anticoagulant and Antiprotease Profiles of a Novel Natural Heparinomimetic Mannopentaose Phosphate Sulfate (PI-88)) " clinical and application thrombosis/hemostasis (Clinical and Applied Thrombosis/Hemostasis)"7:131–140).Such antitumaous effect may be due to direct cytotoxicity, The combination of the blocking of the enhancing and carcinogenesis of chemotherapeutics effect.It has shown that for the anti-of different types of human cancer Proliferation function, including oophoroma, colon cancer, prostate cancer (Khasraw, M., N.Pavlakis, S.McCowatt, C.Underhill, S.Begbie, P.de Souza, A.Boyce, F.Parnis, V.Lim, R.Harvie and G.Marx.2010.PI-88, a kind of heparanase inhibitors joint Docetaxel is before intractable with metastatic castration The multicenter I/II phase in column adenocarcinoma patients studies (Multicentre phase I/II study of PI-88, a heparanase inhibitor in combination with docetaxel in patients with Metastatic castrate-resistant prostate cancer) " oncology yearbook (Annals of Oncology) " 21:1302-1307 and neuroastrocytoma (Camby, I., C.Decaestecker, L.Gordower, R.DeDecker,Y.Kacem,A.Lemmers,H.C.Siebert,N.V.Bovin,P.Wesseling,A.Danguy, I.Salmon, H.J.Gabius, R.Kiss.2001. pilocytic astrocytoma, astrocytoma, anaplastic astrocytoma on curtain To significant difference (the Distinct differences in of the binding ability of sugared epitope in cytoma and glioblastoma binding capacity to saccharide epitopes in supratentorial pilocytic Astrocytomas, astrocytomas, anaplastic astrocytomas, and glioblastomas) " neuropathy Of science and experimental neurology magazine (Journal of Neuropathology and Experimental Neurology) " 60:75–84).However, the low molecular weight carbohydrate large-scale production of animal origin is relatively expensive, and not yet in business It is upper to be used in anticancer agent.
As disclosed in United States Patent (USP) 8,722,107 (modified pectin, relative composition and method), show Show the low molecular weight carbohydrate of some plant origins, especially modified pectin, it may have antitumaous effect.Example includes The low molecular weight carbohydrate of fruit from apple tree.(Li,Q.,Z.Zhou,J.Jing,T.Yang,S.Duan, The oligosaccharides of Z.Wang, Q.Mei and L.Liu.2013. from apple induces cell apoptosis and carefully in HT29 human colon cancer cells Born of the same parents' cycle arrest (Oligosaccharide from apple induces apoptosis and cell cycle arrest In HT29 human colon cancer cells) " international bio macromolecular magazine (Int.J.Biol.Macromol.) " 57:245-254) and the modified pectin of the fruit from mandarin tree (United States Patent (USP) 5,834,442 and 5,895 as cited above, 784).The shortcomings that low molecular weight carbohydrate, especially modified pectin of plant origin is used in anticancer agent is comprising making It is extracted for the cost and pectin of the raw material (such as apple or lemon) of commodity as the multistep of the carbohydrate of low molecular weight And modification.Inevitable trace organic solvents pollution may also have long-term in final low molecular weight carbohydrate product Health problem.
Instead of based on edible plants or fruit as citrus or the raw material of apple, it is surprisingly found by the inventors that, be used for anticancer The economically more favorable raw material of extract is the slurries obtained when the latex of panama rubber tree of acidification is calendered.Also send out in surprise It is existing, be compared with other methods, this starting material with extracting method disclosed herein in conjunction with, generation more evenly with reproducible production Product.Although due to starting material and the difference of extraction process and condition, the chemical group of rubber tree extract according to the present invention At it is identical as the chemical composition for so-called " low molecular weight " carbohydrate quoted in the prior art be impossible, but sent out Existing rubber tree extract according to the present invention has anticancer activity.In addition, being used in final settling step according to the present invention Comparatively safe alcohol, such as ethyl alcohol, to eliminate any chronic health problems.
Although the portion of the natural rubber latex obtained from the soft bark of Para rubber tree (Hevea brasiliensis) Point have been demonstrated there are some anticancer properties that (Lam, K.L., K.L.Yang, E.Sunderasan and M.T.Ong.2012. come from Non- apoptotic cell death (Latex C- in the latex C- slurries inducing hepatocyte cancerous cell line (HepG2) of Para rubber tree serum from Hevea brasiliensis induces non-apoptotic cell death in Hepatocellular carcinoma cell line (HepG2)) 45 (6) " cell Proliferation (Cell Prolif.) ": 577- 585；Lee, Y.K., L.K.Lay, M.S.Mahsufi, T.S.Guan, S.Elumalai and O.M.Thong.2012. paragutta Inhibited proliferation (Anti-proliferation effect of of the gummy cream B- slurries to human mammary epithelial cell Hevea brasiliensis latex B-serum on human breast epithelial cells) " Pakistan Pharmaceutical science magazine (Pak.J.Pharm.Sci.) " 25 (3): 645-650；Sunderasan,E.,N.Abd.Rahman, The classification separation of K.L.Lam, K.L.Yang and M.T.Ong.2013. cell viability measurement guidance natural rubber latex whey liquid (Cell viability assay guided fractionation of natural rubber latex sera) " rubber Glue studies magazine (J.Rubb.Res.) " 16 (3): 195-202), due to the inefficient utilization of the solid rubber portion of latex, use It is uneconomic that natural rubber latex itself, which is used as starting material,.
It is well known that when sour (such as formic acid) cohesion of fresh latex of panama rubber tree, when then rolling (crush and grind), from The liquid (hereinafter referred to as " slurries ") discharged in process of lapping is containing excessive unreacted formic acid and is initially present in Many biomolecule in rubber latex.This slurries can be used as the cheap next of the starting material of high additive value biomolecule Source especially has those of therapeutic properties biomolecule.The present invention is using this slurries as preparation low molecular weight carbon aquation The starting material of polymer mixtures.
Regrettably, other than containing carbohydrate, elastomer gum whey liquid includes various other biomolecule, packet Include allergen protein such as Hev b1, Hev b2, Hev b4, Hev b5 and Hev b6.02.(Chen,Z.,R.Cremer, A.Posch, M.Raulf-Heimsoth, H-P.Rihs and X.Baur.1997. are " about Hev b 1 to natural rubber latex mistake Sensitization (On the allergenicity of Hev b 1among in quick health care worker and spina bifida patient health care workers and patients with spina bifida allergic to natural rubber latex)"." allergy and clinical immunology magazine (Journal of Allergy and Clinical Immunology) " 100 (5):684–93；Barre,A.,R.Culerrier,C.Granier,L.Selman,W.J.Peumans,E.J.M.Van " IgE- combination epitope is in main latex allergy original Hev b by Damme, F.Bienvenu, J.Bienvenu and P.Roug é .2009. 2 and 1,3 1,4 beta-glucanase fruit anaphylactogen of cross reaction on mapping as cream-glue fruit syndrome molecular basis (Mapping of IgE-binding epitopes on the major latex allergen Hev b 2 and thecross-reacting 1,3β-glucanase fruit allergens as a molecular basis for the latex-fruit syndrome)"." molecular immunology (Molecular Immunology) " 46 (8-9): 1595-604； Kolarich, D., F.Altmann, and E.Sunderasan.2006. are " with mass spectrography to the sugar in natural rubber latex Protein allergies original Hev b 4 carries out structural analysis (Structural analysis of the glycoprotein allergen Hev b 4 from natural rubber latex by mass spectrometry)"." biochemistry with Biophysics journal (BBA)-subject included in general education (Biochimica et Biophysica Acta (BBA)-General Subjects)"1760(4):715–20；Beezhold, D.H., V.L.Hickey, J.E.Slater and G.L.Sussman.1999. " human IgE-of latex allergy original Hev b 5 combines epitope (Human IgE-binding epitopes of the latex allergen Hev b 5)""." allergy and clinical immunology magazine " 103 (6): 1166- 72；Reyes-López,C.A.,A.Hernández-Santoyo,M.Pedraza-Escalona,G.Mendoza,A.Herná Ndez-Arana and A.Rodr í guez-Romero.2004. is " to the opinion of Hev b 6.02 (hevein) comformational epitope (Insights into a conformational epitope of Hev b 6.02(hevein))"." biochemistry and life Object physical study communicates (Biochemical and Biophysical Research Communications) " 314 (1): 123–30；Pedraza-Escalona,M.,B.Becerril-Luján,C.Agundis,L.Domínguez-Ramírez, A.Pereyra,L.- Umbarila and A.Rodr í guez-Romero.2009. " are come by using blocking antibody announcement From analysis (the Analysis of B-cell epitopes from the of the B cell epitope of anaphylactogen Hev b 6.02 allergen Hev b 6.02revealed by using blocking antibodies)"." molecular immunology (Molecular Immunology)"46(4):668–76.) therefore, the present invention needs to provide the step of removing protein material, Including the enzyme with known allergen epitope.
Therefore, it is an object of the present invention to provide the sides for being used to prepare the latex of panama rubber tree extract with anticancer activity Method.
It is a further object to provide be used to prepare the latex of panama rubber tree for having transfer resistance to proliferating cancer cells The method of extract.
It is a further object to provide prepare low molecular weight carbon hydrate by relatively cheap biological starting material The method of object.
It is a further object to provide the latex of panama rubber tree extracts that preparation is rich in low molecular weight carbohydrate Method.
It is a further object to provide the methods for being used to prepare latex of panama rubber tree extract, so that in final products Low molecular weight carbohydrate have relatively uniform and reproducible molecular composition.
It is a further object to provide the methods for being used to prepare latex of panama rubber tree extract, so that final products are not Containing protein material, especially allergenic protein.
It is a further object to provide be used to prepare the elastomer gum being used safely in drug and food compositions The method of newborn extract.In this case, safety includes acute, subacute and chronic there is no being determined by related protocol Toxicity.
It is a further object to provide be used to prepare to extract with the latex of panama rubber tree of anticancer and antimetastatic activity Simple, effective and relatively cheap method of object.
It is a further object to provide the methods for being used to prepare latex of panama rubber tree extract, so that this method is easy to Extension is for being commercialized.
Another object of the present invention is can be in the treatment using the anticancer activity and production of latex of panama rubber tree extract As anticancer drug and prophylactically as the product of anticancer food replenishers.
Summary of the invention
Invention as described herein is related to providing rich in low molecular weight carbohydrate and with the rubber tree of anticancer activity The extract of latex.
The one aspect of present invention disclosed is related to the method for being used to prepare the extract of latex of panama rubber tree, comprising following Step: the slurries discharged when the latex of panama rubber tree of acidification is calendered are obtained；Clast and microorganism are removed optionally from slurries； Remove protein；Remove quebrachitol and small polar molecule；Optionally remaining solid powder is dissolved in water；Pass through use Acid processing further removes protein；Optionally neutralize excessive acid；Use solvent deposition；Collect sediment；Optionally freeze Dry sediment.Clast and microorganism are removed by the inclusion of the technology of filtering.By the inclusion of the biology of ultrafiltration and/or spray drying Chemical technology removes protein.By with the solvent extraction of such as methanol and collect remaining undissolved solid powder remove it is white Sparrow another name for and small polar molecule.By further removing any remaining protein with acid such as trichloroacetic acid processing.Optionally Remaining mixture is adjusted to neutrality pH by ground, and by separating final product with cold organic solvent such as ethanol precipitation. By the way that sediment is collected by centrifugation and is optionally freeze-dried.
Another aspect of the present invention relates to the method for treating or preventing the growth of cancers in subject and/or transfer, Comprising to subject application it is a effective amount of by latex of panama rubber tree extract prepared according to the methods of the invention to induce cancer thin Born of the same parents are dead.
In another aspect of the invention, the method for inhibiting the tumor cell proliferation in patient is described.Method packet Containing a effective amount of latex of panama rubber tree extract according to the present invention is applied to patient to realize one of following marker or more The advantageous variation of kind: CD31 endothelial cell marker object is reduced；VEGF angiogenesis marker；COX-2 Markers of inflammation is reduced； EGFR proliferation marker is reduced；The Apoptosis of cancer cell increases；It is reduced with galectin-3 metastatic marker object.
Another aspect of the present invention be comprising latex of panama rubber tree extract according to the present invention and it is one or more pharmaceutically The composition of acceptable substance.Another aspect of the present invention is comprising latex of panama rubber tree extract according to the present invention and one kind Or the composition of a variety of comestible compositions.When being used in combination in claims and/or specification with term "comprising", word The use of language "/kind (a/an) " can indicate "/kind ", but it also with "/kind or multiple/kind ", " at least one A/kind " consistent with the meaning of "/kind or more than one/kind ".
When combining the following description and drawings to consider, these and other implementations of the invention will be better appreciated and understood Example.Although indicating various embodiments of the present invention and its many details it should be appreciated, however, that being described below, lead to Way of illustration and not limitation is crossed to provide.Without departing from the spirit of the invention, can within the scope of the invention into Row it is many replace, modifies, adds and/or rearranges, and the present invention include it is all it is such replacement, modification, add and/or again New arrangement.
Detailed description of the invention
The following drawings forms part of this specification, and is included to further illustrate certain sides of the invention Face.By reference to the detailed description of one or more of these attached drawings and combination specific embodiment given herein, Ke Yigeng Understand the present invention well.
Fig. 1 is the flow chart for showing the source of raw material elastomer gum whey liquid.
Fig. 2 is the flow chart for showing the step of preparing extract from elastomer gum whey liquid according to the present invention.
Fig. 3 is the flow chart for showing optimal mode embodiment of the invention.
Fig. 4 is Fourier transform infrared (FT-IR) spectrum, and which illustrate coming for optimal mode embodiment according to the present invention From the possibility composition of the extract of latex of panama rubber tree.
Fig. 5 is the liquid chromatography-mass spectrography (LC- of the latex of panama rubber tree extract of optimal mode embodiment according to the present invention MS) molecular weight characteristic figure.
Fig. 6 shows the size of tumour in the nude mice using vernier caliper measurement.
Fig. 7 shows phase in the nude mice for receiving latex of panama rubber tree extract of optimal mode embodiment according to the present invention To the time course of gross tumor volume, to illustrate anticancer property.(dotted line) experimental group, daily every kg bodyweight p tube feed 60mg rubber Gum latex extract continues 28 days.(solid line) control group receives water rather than latex of panama rubber tree extract.
Fig. 8, which shows skin of back folding room and how to pass through surgical operation, is attached to nude mice to visualize angiogenesis. The diameter of skin of back folding room is about 7 millimeters.
Fig. 9 shows the inhibition that the nude mice medium vessels in skin of back folding room with cancer implantation material generate.First Mouse is anaesthetized.Then fluorescein isothiocyanate-dextran (FITC- glucan) solution is infused into jugular vein to mark Remember blood vessel.Skin of back folding room and skin with cancer implantation material are removed by surgical operation and use referred to as " living body The technology of interior confocal fluorescent videomicroscopy " is observed.(left side) does not have cancer control group.(in) there is cancer implantation material.(right side) tool There is the extract of the latex of panama rubber tree of cancer implantation material and optimal mode according to the present invention.
Figure 10 is shown compared with the control group for receiving water, passes through daily every kilogram of (hereinafter referred to as " kg ") weight mouth It is raw in the nude mice medium vessels with cancer implantation material to take 60 milligrams of tube feed (hereinafter referred to as " mg ") latex of panama rubber tree extract At inhibition.Immunohistochemistry visualization is carried out using CD31 endothelial cell marker object.(left side) does not have cancer control group.(in) There is cancer implantation material.(right side) has the extract of the latex of panama rubber tree of cancer implantation material and optimal mode according to the present invention.Item =100 microns.
Figure 11 is shown compared with the control group for receiving water, passes through daily every kg bodyweight p tube feed 60mg latex of panama rubber tree The inhibition that extract is generated in the nude mice medium vessels with cancer implantation material.Immune group is carried out using VEGF angiogenesis marker Weave chemistry visualization.(left side) does not have cancer control group.(in) there is cancer implantation material.(right side) has cancer implantation material and according to this hair The extract of the latex of panama rubber tree of bright optimal mode.Item=100 micron.
Figure 12 is shown compared with the control group for receiving water, passes through daily every kg bodyweight p tube feed 60mg latex of panama rubber tree The reduction of extract inflammation in the nude mice with cancer implantation material.Immunohistochemistry is carried out using COX-2 Markers of inflammation Visualization.(left side) does not have cancer control group.(in) there is cancer implantation material.(right side) have cancer implantation material and it is according to the present invention most The extract of the latex of panama rubber tree of good mode.Item=100 micron.
Figure 13 is shown compared with the control group for receiving water, passes through daily every kg bodyweight p tube feed 60mg latex of panama rubber tree Extract fissional reduction in the nude mice with cancer implantation material.Immuning tissue is carried out using EGFR proliferation marker Learn visualization.(left side) does not have cancer control group.(in) there is cancer implantation material.(right side) has cancer implantation material and according to the present invention The extract of the latex of panama rubber tree of optimal mode.Item=100 micron.
Figure 14 is shown compared with the control group for receiving water, passes through daily every kg bodyweight p tube feed 60mg latex of panama rubber tree The increase of extract Apoptosis in the nude mice with cancer implantation material.It is situated between using terminal deoxynucleotidyl transferase (TdT) Deoxyuridine triphosphate nucleotide (dUTP) Nick End label (TUNEL) method led is visualized.(left side) does not have cancer Control group.(in) there is cancer implantation material.(right side) has the latex of panama rubber tree of cancer implantation material and optimal mode according to the present invention Extract.Item=100 micron.
Figure 15 shows the result of experimental tail vein transfer assay.By tail vein to mouse injection 1 × 105A melanin Tumor B16-F1 cell.Lower row illustrates the dissection lung of these mouse, and the daily feeding 60mg/kg latex of panama rubber tree of these mouse mentions Take object for 2 weeks.Upper row illustrates the lung of the control mice from fed with water.Stain shows the collection of the melanoma cells of transfer It falls.
Figure 16 shows the result of the immunohistochemical staining measurement of galectin-3 (metastatic marker object).(left side) There is no cancer control group.(in) pass through tail vein to mouse injection 1 × 105A melanoma B16-F1 cell, and fed with water generation For latex of panama rubber tree extract.(right side) is by tail vein to mouse injection 1 × 105A melanoma B16-F1 cell, and so Daily feeding 60mg/kg latex of panama rubber tree extract continues two weeks afterwards.Item=100 micron.
Attached drawing and written description, which are not intended to limit in any way, disclosed is used to prepare latex of panama rubber tree extract Method range.On the contrary, provide attached drawing and written description be in order to by reference to the particular embodiment of the present invention to this field Those of ordinary skill illustrates the disclosed method for being used to prepare latex of panama rubber tree extract.It will be described now with such particularity The present invention, so that any technical staff of related fields can practice the present invention without many experiments.
Specific embodiment
With reference to Fig. 1, the well known method for being used to prepare rubber tree slurries is opened with the fresh latex 101 collected from rubber tree Begin.Addition acid, usual formic acid is so that emulsion condensation.The rubber isoprene copolymer particle in latex is turned with 102 cohesion of acid It is melted into coagulation, starts to separate with liquid.By crush and grind or roll 103 this separation of acceleration, generation solid rubber, Sheet rubber 104 is used as output.The liquid discharged from this crush and grind step is known as elastomer gum whey liquid 105.According to Method of the invention uses the slurries 105 as starting material.
Referring to fig. 2, the present invention provides the methods for being used to prepare latex of panama rubber tree extract.It wraps according to the method for the present invention Containing following steps: obtaining (the rubber tree in the slurries and Fig. 1 of slurries 205 discharged when the latex of panama rubber tree of acidification is calendered Latex slurries 105 are identical)；Optionally remove clast and microorganism 206；Remove protein 207；Remove quebrachitol and small pole Property molecule 208；Remaining solid powder is optionally dissolved in water 209；Protein is further removed by being handled with acid 210；Optionally neutralize excessive sour 211；With solvent deposition 212；Collect sediment 213；Optionally it is freeze-dried sediment 214, to generate para-caoutchouc cream extract 215.
Step 206-214 (including endpoint) includes that those skilled in the art separate known conventional biochemical and biological object Reason technology.
In logic, step 207,208 and 210 are not needed with particular order practice.Those skilled in the art should be able to Rearrange, replace and change the details of these three steps.However, optimal mode for carrying out the present invention is by explanation when practice When this method, there is practical and arrangement coordination advantage with the alignment step 207,208 and 210 of the particular order.
The detailed description of consecutive steps in Fig. 2 is shown below:
If latex slurries optionally need step 206 by clast and microorganism such as germ contamination, this is depended on such as What cleans processed latex of panama rubber tree.Removing technology another example is be small enough to exclude clast and micro- life by aperture The filter of object filters, and usually less than 0.5 micron, and preferably 0.2 micron.Although lesser aperture can exclude more tiny Clast and particle, but as aperture becomes smaller, filter process is slack-off.The ultra-fine clast and nanometer not had found that it is likely that due to inventor Any negative effect of particle pollution, therefore the tradeoff that 0.2 micron of filter pore size is seemingly best.
Step 207 includes to remove protein, the protein allergens being especially naturally present in latex of panama rubber tree.The step Rapid includes the combination of Measurement for Biochemistry, such as enzymic digestion, ultrafiltration and spray drying at high temperature.One example of step 207 It is that protein is destroyed by any business proteolytic enzyme, the proteolytic enzyme is cheap and can be in elastomer gum whey liquid Its bioactivity is kept in environment.It is at least 3kD and preferred 10kD that another example of step 207, which is by molecular cut off, Ultrafiltration membrance filter liquid.Another example of step 207 is higher than 60 degrees Celsius, preferably higher than 100 degrees Celsius and most It is preferred that the spray-drying liq at a temperature of between 100 and 120 degrees Celsius.The case where being used in series ultrafiltration and spray drying Under, it should be denaturalized by any protein of ultrafiltration membrane and its bioactivity is lost by high temperature spray-drying.
Step 208 includes that quebrachitol and small polar molecule are removed from slurries.When elastomer gum according to the present invention When newborn extract is combined with one or more pharmaceutically acceptable substances to form the composition for being used as anticancer drug, the step Importance will be apparent.When latex of panama rubber tree extract is combined with one or more comestible compositions to be formed and be used as When the composition of anti-cancer function food, importance also be will be apparent.For drug or functional food, in order to implement to be used for The method for the treatment of cancer growth and/or transfer is to realize the death of cancer cell, or for inhibiting tumor cell proliferation in patient Method to realize angiogenesis (VEGF reduction), inflammation (COX-2 reduction), proliferation (EGFR reduction), Apoptosis and transfer The advantageous variation of one or more suitable markers or indicant of (galectin-3 reduction), it is necessary to allow cancer patient Eat a effective amount of drug or functional food.The compliance of patient particularly depends on no undesirable side effect, such as known The diarrhea as caused by quebrachitol.Therefore, the main purpose of step 208 is to remove this undesirable compound.It is many other Small polar molecule, including nucleotide, if it exists, will also be removed in this step.Step 208 another example is It being extracted by suitable polar solvent (such as methanol) mixture, the polar solvent mixture tends to dissolve polar molecule, Including quebrachitol, other undissolved lesser molecules of polarity are left.Methanol concentration should be at least 70%, preferably 100%.It mentions Taking can repeat, and preferably carry out again twice, for further use with the concentration for sufficiently reducing quebrachitol.
Step 210 includes further to remove protein to ensure that more complete protein removes, especially by being precipitated with acid It is the little albumen matter that possible retain in step 207 and peptide.The example of step 210 is to be higher than 4 degrees Celsius, and preferably 16 and 30 take the photograph Between family name's degree, and with 1% to 50%, preferably 2% to 10% most preferably at a temperature of between 20 and 25 degrees Celsius, and most It is preferred that the trichloroacetic acid precipitation between 2% and 3%.Time needed for being settled out protein particularly depend on acid used concentration and The temperature precipitated.In fact, about 0.1 minute (almost immediately) interior some visual precipitation after addition of the acid.Precipitation reaction Best incubative time can be determined by the technical staff of biochemical field.However, wise is to allow the sufficient time, preferably 24 Hour, so that sediment is formed completely.
Step 209 is used as the transition from step 208 to step 210.It in this step, will be undissolved solid in step 208 Body powder is optionally dissolved in a small amount of water, to facilitate the precipitation reaction in step 210.
Step 211 is for neutralizing any excessive acid from step 210.Usually in the case where adding sodium hydroxide into Row neutralizes.The step is considered optional, because the amount of the alkali of addition depends on the amount of residual acid.
Step 212 and 213 is related to separating final product, without protein but is rich in low molecular weight carbohydrate, example Such as, by low temperature with suitable solvent mixture such as ethyl alcohol and water sedimentation.Concentration of alcohol should be at least 70%, preferably 100%.It is deposited in lower than 4 degrees Celsius, preferably shorter than -10 degrees Celsius, and is most preferably carried out under -20 degrees Celsius.Although It can be seen that precipitating in about 5 minutes, but suggest the sufficient time of permission 24 hours, to obtain maximum precipitate.The receipts of final product Collection can be for example, by least 3,000 × g is centrifuged at least 5 minutes, realize within preferably 10 minutes.Optionally will in step 214 Sediment freeze-drying.Final product is anticancer latex of panama rubber tree extract 215.It, can if to use extract immediately To omit step 214.
Above description shows due to two, according to the method for the present invention in no any specific digestion step In the case of automatically make latex extract be enriched with low molecular weight carbohydrate.Firstly, using slurries 105 or equally 205 making Have the advantages that for starting material intrinsic, i.e., makes to be initially present in fresh latex of panama rubber tree 101 in sour agglomeration step 102 Carbohydrate be at least partly digested to low molecular weight carbohydrate.In other words, with fresh latex of panama rubber tree phase Than raw material used according to the invention is partially rich in low molecular weight carbohydrate.Secondly, if at elevated temperatures Spray drying is for removing in protein step 207, then high temperature potentially contributes to decompose some carbohydrate.Finally, passing through Sour step 210 further removes protein and also contributes to further by any remaining carbohydrate inversion at low molecular weight carbon Hydrate.In practice, it is necessary to time, temperature and the reactant concentration in each step are controlled, it is extract obtained to ensure 215 have uniform and reproducible low molecular weight carbohydrate composition.Inventor is it was unexpectedly observed that with other methods and implementation Example compare, the method for optimal mode according to the present invention provided in terms of the composition of low molecular weight carbohydrate most it is uniform most Good reproducibility.It is same it is emphasized that due to starting material and extracting method difference, it is contemplated that low molecule according to the present invention The definite chemical property for measuring carbohydrate is different from " low molecular weight " carbohydrate so-called according to prior art.
It is a further advantage of the invention that if be spray-dried at high temperature for removing in protein step 207, Any protein that non-physics removes in other steps will pass through heat denatured and will lose its any possible epitope Three-dimensional configuration, so that this proteinoid is not caused allergy.
Including following example to illustrate the preferred embodiment of the present invention or optimal mode.Those of ordinary skill in the art answer It solving, technology disclosed in following example represents the technology of inventor's discovery worked well in the practice of the invention, and It and it can be considered that is the preference pattern and optimal mode for constituting practice.However, those skilled in the art answer according to the disclosure Understand, without departing from the spirit and scope of the present invention, many changes can be carried out to disclosed specific embodiment And still obtain the same or similar result.In other words, optimal mode of the invention and other exemplary embodiments are not answered It is interpreted any limitation of the invention.
The best mode embodiment of the present invention
The best mode embodiment of the present invention being currently known is using the method according to following example.
Fig. 3 is for illustrating the best mode embodiment of the present invention.Since optimal mode obtaining slurries 305, and slurries 305 are in acid The release when latex of panama rubber tree of change is calendered.Starting material 305 refers to the identical latex slurries 105 in Fig. 1 and 205 in Fig. 2. Final products 315 correspond to the like products 215 in Fig. 2.
In the optimal mode, it is 0.2 micron that the filtration step 306 for removing clast and microorganism, which includes by aperture, Film filtering.
Equally in the optimal mode, protein remove step 307 by by molecular cut off be 10kD ultrafiltration membrane into Row ultrafiltration 316, then in 110 degrees Celsius of at a temperature of 317 composition of spray drying.Since ultrafiltration and spray drying are used in series, Therefore it can be all denaturalized by any protein of ultrafiltration membrane and its bioactivity is lost by high temperature spray-drying.This allows to make With the ultrafiltration membrane with 10kD molecular cut off rather than 3kD film, 3kD film is more expensive and generates at the same pressure slower Ultrafiltration rate.Any allergen protein of the molecular weight between 3 and 10kD, such as Hev b 6.02 are 4.72kD polypeptides, It will will be inactivated by the high temperature in spray-drying process by 10kD ultrafiltration membrane.Inventor has found spray drying Ying Gao In 60 degrees Celsius, preferably higher than 100 degrees Celsius and most preferably carried out at a temperature of between 100 and 120 degrees Celsius.Although compared with High spray drying temperature can permit faster dry, preferable protein denaturation, and there may be more short chain carbon Hydrate, but there is the risk for changing sugared structure and product colour in them.
According to the present invention, in order to remove small polar molecule, including nucleotide and especially quebrachitol, at least 70% The mixture of methanol aqueous solution extracts 308 solid powders from spray drying step 317, and the methanol is a kind of relative polarity Organic solvent it is also relatively cheap.In optimal mode, with 1:6 w/v, (i.e. the solid powder of 1 weight is than 6 volumes Solvent) anhydrous methanol extract.It repeats to extract twice, in total three times with the weight/volume of 1:4 with methanol again.
Remaining solid powder from methanol extraction procedure 308 is dissolved in 309 in distilled water with 1:8 weight/volume. Add trichloroacetic acid in the step 310 to reach 2.5% ultimate density.Mixture is stirred 24 hours under 25 degrees Celsius. It is centrifuged by 3,000 × g and removes within 5 minutes and abandon sediment.Although the function of the settling step is to remove protein, such as If fruit exists, the macromolecular including nucleic acid polymers will be also removed.
In optimal mode, the supernatant from step 310 then is neutralized with sodium hydroxide 311, then uses dehydrated alcohol Precipitating 312.Temperature is maintained at -20 degrees Celsius.After 24 hours, with 3,000 × g is centrifuged 5 minutes and collects precipitating in step 313 Object.Sediment is freeze-dried to generate latex of panama rubber tree extract 315 in a step 314, it can be in deep freezer Kept for the extended time, usually some months.
In optimal mode embodiment disclosed above, the logistics that protein and small polar molecule remove starts from ultrafiltration 316,317 are then spray-dried, then methanol extracts 308, and finally with trichloroacetic acid processing 310.This sequentially ensures to pass through The big content of starting materials 305 for removing clast and microorganism by filter 23 06 is reduced to solid powder by spray drying 317, to promote The step of extracting 308 into methanol and handling 310 with trichloroacetic acid.The sequence of steps disclosed in the optimal mode example is to mention The most practical and effective mode known for inventor for preparing latex of panama rubber tree extract according to the present invention when friendship PCT application.
The yield of optimal mode embodiment is about 0.17% (the weight ratio slurry volume of latex extract).Using this hair In the practical representative preparation of bright optimal mode, 9,650 liters of fresh latex of panama rubber tree 101 discharge 4,147 after calendering 103 Slurries 105 are risen, the raw material 305 of optimal mode embodiment of the invention is used as.After ultrafiltration step 316, volume reduce to 3,869 liters.Spray drying step 317 obtains the solid powder of 102kg.Methanol precipitation step 312 and freeze-drying step 314 obtain To 7kg final product 315.
Described below is the representative test result obtained from the product of optimal mode embodiment of the invention.Provide this A little results are tested with the preliminary characterization and preliminary safety that show the latex of panama rubber tree extract of optimal mode according to the present invention As a result.
(1) preliminary characterization of extract
Fig. 4 shows fourier-transform infrared (FT-IR) spectrum, which illustrate the extract from latex of panama rubber tree can It can composition.Horizontal axis is indicated from 4000 to far below 1000cm-1Wave number.The longitudinal axis indicates percent transmittance.Major absorbance peak packet Containing 3392.75,1619.95,1418.21,847.55 and 526.14cm-1.Although 1081.41cm-1Absorption peak cannot be further It differentiates, but it and β -1, the caused theoretical 1078.21cm that stretches of 1,3 glycosidic bond of β of 3- glycan-1And 1079.77cm-1It inhales It is fairly close to receive peak.
Fig. 5 shows liquid chromatography-mass spectrography (LC-MS) molecular weight characteristic figure of latex of panama rubber tree extract.It has been prepared for The horizontal axis of the specific histogram is to indicate the quality of the molecule fragment lower than 2000 to 6000 dalton.The longitudinal axis indicates in sample The percentage of specific fragment.The figure illustrates the size distribution of the molecular weight of component in extract, and center is in 3712.1 dalton Left and right.Representative molecular mass around central peak marks at the upper surface of described peak (3713.6,3744.8,3759.1,3800.1 With 3824.6 dalton) and it is following (3705.6,3651.0,3635.8 and 3554.5 dalton).If the molecule is actually Carbohydrate, then central element amount corresponds to the polymer of about 20 saccharide residues.
It is designed to from Figure 4 and 5 and according to the method for the present invention remove protein, nucleic acid and other polar molecules The fact, rubber tree extract 315 may contain the low molecular weight carbohydrate of about 20 saccharide residues.
(2) preliminary safety test result
The acute toxicity of latex of panama rubber tree extract according to the present invention, 6 weeks subacute toxicities and 9 months are tested Chronic toxicity.
Compared with negative control group, the acute toxic test carried out according to OECD guide the 420th, 1 gram of rubber of feeding rats Gum latex extract/kg weight not will lead to weight, alertness, breathing, muscle strength, urine color and excrement appearance and occur Any variation.
When 1 gram of latex of panama rubber tree extract of the daily feeding of 6 male rats/kg continued weight 6 weeks and with 6 male rats Control group is compared, thin relative to appetite, weight, liver function, renal function, white blood cell count(WBC), platelet count, hemoglobin and blood Born of the same parents' specific volume, blood glucose level, blood lipid level, the subcutaneous and fat deposition in the internal organs of such as liver and internal organs (liver, Kidney, heart, lung, pancreas, adrenal gland, testis, prostate and seminal vesicle) size, do not find subacute toxicity.In addition, rubber tree Latex extract seems that induced nitrous oxide is discharged from blood vessel inner layer, leads to better blood vessel compliance and endothelial dysfunction Restore.
It is scheduled to last according to OECD-OCDC-452 guide (being used from version revisions in 1981 and on September 7th, 2009) Chronic toxicity research (limit test) in 9 months.By 98 males and after female 4 weeks, weaning rat is divided into 4 groups.It is big in experimental group 1 gram of latex of panama rubber tree extract of the daily feeding of mouse/kg continued weight 9 months.Rubber in control group in feeding rats and experimental group The distilled water of gum latex extract same volume.In addition, additionally providing two satellite groups (experimental group and a control Group), with monitor in experimental group can it is observed that the variation of any toxicology invertibity.Therefore, rat is every in experimental satellite group 1 gram of latex of panama rubber tree extract of its feeding/kg continued weight 9 months, then other 28 days of feeding distilled water, and compare satellite group Middle feeding rats distilled water continues entire 9 months plus 28 days duration.Appearance or abnormal behavior is not observed, such as eats It is intended to.Ptomatopsia be not detected weight, internal organs (liver, kidney, adrenal gland, atrium sinistrum, atrium dextrum, ventricle, spleen, lung, testis, Epididymis, prostate and seminal vesicle) weight, subcutaneous fat deposits and internal organs (device after epididymis, prostate, mesenterium and peritonaeum Official) in fat deposition in terms of exception.The histology of internal organs (liver, kidney, stomach, intestines and adrenal gland) compares display experimental group It is not significantly different between control group.In white blood cell count(WBC) (WBC), blood between hematology test display experimental group and control group Specific volume of cell percentage (HCT), hemoglobin concentration (HGB), mean corpuscular volume (MCV) (MCV), mean corpuscular hemoglobin (MCH), in terms of mean corpuscular hemoglobin concentration (MCHC) (MCHC), cent lymphocytes (LYMPH) and platelet concentration (Plt) Without significant difference.Do not detect glucose, lipid [triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), Low density lipoprotein cholesterol (LDL-C) and LDL/HDL ratio], urea nitrogen (BUN), creatinine, total protein, albumin, gallbladder it is red Element, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) serum levels side The adverse reaction in face.In brief, without discovery toxicity in this 9 months chronic toxicity research.
Industry practicability of the invention
In following experiment, show that there is (1) anticancer activity and (2) anti-rotation to move and live for rubber tree extract according to the present invention Property.Due to these activity, the present invention is suitable for inclusion in the industry of (1) pharmacy industry He (2) grocery trade.
(1) PRELIMINARY RESULTS of anticancer activity
Three groups of experiments have been carried out to assess the anticancer activity of latex of panama rubber tree extract according to the present invention: (a) planting In the volume change of the intracorporal tumour of nude mice, (b) influence to the intracorporal Tumor Angiongesis of nude mice is implanted in, and (c) and blood vessel Generation, inflammation, the immunohistochemistry research of proliferation and apoptosis-related marker or indicant.
(a) it is implanted in the volume change of the intracorporal tumour of nude mice
By 6 to 8 week old male BALB/C nude mices (athymic mouse) injection 10 of the weight between 20 and 25 grams6It is a CaSki cell (cervical cancer cell from 16 type human papilloma virus) simultaneously allows its normal life about one month until tumour body About 100 to 120 cubic millimeters of product, as calculated using the linear measurement of vernier caliper shown in fig. 6.Mouse connects in experimental group By 60 milligrams of daily oral tube feed latex of panama rubber tree extract 315/kg continued weight 28 days, and mouse receives water in control group. Fig. 7 shows the time course of relative tumour volume in two groups of mouse.Although the gross tumor volume in control group increases about within 4 weeks 7 times, but the gross tumor volume received in the group of latex of panama rubber tree extract 315 is held essentially constant.
(b) influence to the angiogenesis for being implanted in the intracorporal tumour of nude mice
As shown in figure 8,6 to 8 week old male BALB/C nude mices of the weight between 20 and 25 grams are equipped with 7 millimeters of back skins Skin folding room, and with 2 × 106A CaSki cell is implanted into the room.Mouse receives daily 60 milligrams of oral tube feed in experimental group Latex of panama rubber tree extract/kg continued weight 14 days, and mouse receives water in control group.At the 14th day, compared with penta bar of 50mg/kg Appropriate sodium anesthetized animal.Blood plasma is marked with fluorescein isothiocyanate-dextran (FITC- glucan).Skin of back folding room and skin Skin carefully removes together.Then angiogenesis is observed and recorded by vivo confocal fluorescent videomicroscopy.In Fig. 9 As a result the generation of experimental group medium vessels is qualitatively shown less.Use " Image Pro Plus " computer software (Maryland, US The media cybernetics company (Media Cybernetics, Inc., Rockville, MD, USA) of state Rockwell) to blood capillary Pipe density percent is than carrying out quantitative image analysis, it was demonstrated that cancer is thin in the group for receiving latex of panama rubber tree extract according to the present invention The statistically significant reduction that born of the same parents' peripheral vessels generate.
(c) immunohistochemistry with angiogenesis, inflammation, proliferation and apoptosis-related marker or indicant is ground Study carefully
When by immunohistochemistry research from the tumour of above-mentioned experiment, discovery is receiving 60mg elastomer gum daily Angiogenesis in newborn extract/28 days groups of kg continued weight around tumour is reduced, such as CD31 endothelial cell marker in Figure 10 Shown in the expression of object, and as shown in the presence of VEGF angiogenesis marker in Figure 11.In addition, latex of panama rubber tree extract is also Reduce the level of the COX-2 Markers of inflammation in Figure 12 and the EGFR proliferation marker in Figure 13.Finally, as shown in figure 14, Latex of panama rubber tree extract enhances the Apoptosis of cancer cell.
(2) PRELIMINARY RESULTS of antimetastatic activity
The transfer for the melanoma cells being injected into nude mice tail vein is assessed within two weeks after injection.In total tissue level, meat Eye is it can be seen that stain of the metastatic melanoma colony as red lung background.In microscopic scale, the melanoma cells of transfer It can be measured by a kind of immunohistochemical staining of galectin-3 (metastatic marker object) to position.The two experiments All confirm that there are antimetastatic activities in latex of panama rubber tree extract according to the present invention.The details of these experiments is as follows:
The antimetastatic activity of latex of panama rubber tree extract according to the present invention is shown in 105A melanoma B16-F1 is thin Born of the same parents are injected into the nude mice in its tail vein.Figure 15 shows the result of the experiment.Receive 60mg/ daily after injecting cancer cell In the experimental group (bottom row) that kg latex of panama rubber tree extract continues two weeks, the quantity of the melanoma stain of transfer, which is considerably less than, to be received Water replaces the group (top row) of latex of panama rubber tree extract.
It, will be with 10 in microscopic scale5Lung of a melanoma B16-F1 cell infusion to the nude mice in its tail vein Slice dyes galectin-3, and observes under an optical microscope, as shown in figure 16.(the galactolipin agglutination in the figure - 3 dyeing of element is shown as brown particle and tone, is superimposed upon on light blue cell outline and navy blue core, as shown, black Some in white microphoto are difficult to differentiate between.) in experimental group (right side), receive 60mg/kg rubber daily after cancer cell injection Gummy cream extract continues two weeks, and the accumulation of galectin-3 expression about receives water in brown tone instead of elastomer gum 50% expressed in the untreated fish group (center) of newborn extract.In negative control group (left side), wherein nude mice is not injected any black Pigment oncocyte, sample show that the slight galectin-3 in brown tone dyes, correspond to untreated fish group (in The heart) in galectin-3 expression about 17%.The PRELIMINARY RESULTS confirms elastomer gum according to the present invention for certain The antimetastatic activity of newborn extract.
In brief, PRELIMINARY RESULTS shown above is drawn a conclusion, and rubber tree extract according to the present invention has anticancer And antimetastatic activity.The reduction of angiogenesis and the enhancing of cancer cell-apoptosis also indicate that latex of panama rubber tree according to the present invention extracts Object, which has, treats and prevents value.Therefore, the present invention is suitable for inclusion in the industry of (1) pharmacy industry He (2) grocery trade.
(1) pharmacy industry is applicable in
The present invention is suitable for pharmacy industry, because rubber tree extract according to the present invention can be used as the medicine group for the treatment of cancer Close the ingredient of object.Described in part as above, acute, the subacute and chronotoxicology of rat is studies have shown that rubber according to the present invention Gum latex extract is nontoxic.The medical composition for treating cancer can be prepared, it includes rubbers according to the present invention Gum latex extract and one or more pharmaceutically acceptable substances, the pharmaceutically acceptable substance are selected from addition The group that agent, binder, carrier, diluent, excipient, filler, lubricant, solvent and stabilizer form.
Due to anticancer and antimetastatic activity of the latex of panama rubber tree extract in nude mice, we should it is expected according to this hair Bright extract plays the role of anticancer and anti-transfer agent in extensive animal (including the mankind) and in extensive cancer, The cancer such as AIDS-related cancers, adenoid cystic carcinoma, adrenocortical carcinoma, agnogenic myeloid metaplasia, take off acoustic neurinoma Hair, alveolar soft tissue sarcoma, angiosarcoma, alpastic anemia, astrocytoma, asynergy-capillary dilation, Basal-cell carcinoma (bcc), brain stem glioma, class cancer, children with cancer, children soft tissue sarcoma, chondrosarcoma, choriocarcinoma, colon The carcinoma of the rectum, skin T cell lymphoma, dermatofibrosarcoma-protrusion, Hypertrophic small circle cell cancer, duct carcinoma, carcinoma of endometrium, Ependymoma, the cancer of the esophagus, ewing's sarcoma (Ewing's sarcoma), cholangiocarcinoma, ophthalmo melanoma, retina are female thin Born of the same parents' tumor, carcinoma of fallopian tube, Fanconi anemia, fibrosarcoma, gastric cancer, gastrointestinal cancer, gastrointestinal associated cancers, genitourinary cancers, Germinocarcinoma, gestational trophoblastic disease, glioblastoma, glioma, gynecological cancer, Malignancy, liver Cell cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease (Hodgkin's disease), human papilloma virus, Vesicular mole, hypopharyngeal cancer, intraocular melanoma, island T cell cancer, Kaposi sarcoma (Kaposi's sarcoma), Lang Gehan Schwann Cells histocytosis (Langerhan's cell histiocytosis), leiomyosarcoma, leukaemia, Li-Buddhist Beautiful Buddhist nun's syndrome (Li-Fraumeni syndrome), embryonal-cell lipoma, lymphedema, lymthoma, Hodgkin lymphoma (Hodgkin's lymphoma), non-Hodgkin lymphoma (non-Hodgkin's lymphoma), male breast carcinoma, kidney are disliked Property rhabdoid tumor cancer, medulloblastoma, melanoma, merkel's cells cancer (merkel cell cancer), celiothelioma, Metastatic carcinoma, MEN,muitiple endocrine neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disease, Nasopharyngeal carcinoma, the nephroblastoma, neuroblastoma, neurofibromatosis, how Makin breakage syndrome, non-black melanoma skin Cancer, non-small thin lung cancer (nsclc), eye cancers, the cancer of the esophagus, carcinoma of mouth, oropharyngeal cancer, osteosarcoma, neostomy oophoroma, pancreas Cancer, nasal sinus cancer, parathyroid carcinoma, carcinoma of parotid gland, carcinoma of penis, peripheral nerve ectoderm cancer, polycythemia vera, rare cancer With associated disease, clear-cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rosemond thomson syndrome (rothmund Thomson syndrome), sarcoma, neurinoma, Sezary syndrome (sezary syndrome), Small Cell Lung Cancer (sclc), carcinoma of small intestine, soft tissue sarcoma, squamous cell carcinoma (sec), synovial sarcoma, transitional cell carcinoma (bladder), transitional cell carcinoma (kidney-pelvis -/- ureter), choriocarcinoma, urine plaque albumen, sarcoma of uterus, Walden Si Telunshi macroglobulinemia (Waldenstrom's macroglobulinemia) and Weir nurse Si Shi cancer (Wilms'Cancer).
Alternatively, the anticancer of latex of panama rubber tree extract according to the present invention and antimetastatic activity should apply to Different Organs With the cancer of tract, such as anus, bladder, bone, intestines, brain, breast, central nervous system, cervix, colon, endocrine Gland, ear, endothelial cell, esophagus, eye, gall-bladder, head, intestines, kidney, larynx, leucocyte, lip, liver, lung, oral area, nasal cavity, neck, nose, Oral cavity, ovary, pancreas, pharynx, hypophysis, prostate, rectum, salivary gland, skin, spinal cord, stomach, testis, thymus gland, thyroid gland, urethra, Urinary system, uterus, vagina and vulva.
Latex of panama rubber tree extract be used as anticancer/anti-transfer agent in the case where, can be prepared into aqueous solution, tablet, Pastille, powder, aqueous or oily suspensions, lotion, the form of syrup, elixir.Administration method can be enteral, intramuscular, quiet Arteries and veins is interior, under intranasal, oral, parenteral, rectum, subcutaneous, sublingual, labia, it is transdermal and transmucosal.
In addition, latex of panama rubber tree extract can with therapeutically effective amount or one or more chemotherapeutic combinations, such as Alkylating agent, antimetabolite, antitumor antibiotics, kinase inhibitor, aromatase inhibitor, mitotic inhibitor, steroid hormone And topoisomerase enzyme inhibitor.
(2) grocery trade is applicable in
It is suitable for functional food industry, because rubber tree extract according to the present invention also is used as with anticancer With the food and beverage replenishers of cancer preventive activity.In fact, latex of panama rubber tree extract according to the present invention can be with one Kind or the mixing of a variety of comestible compositions, the comestible composition are selected from acid, acidity regulator, additive, anti-caking agent, defoaming Agent, bakes agent, binder, carrier, color preserving agent, diluent, emulsifier, excipient, filler, fragrance, flavour enhancing at antioxidant Agent, Flour ingredient, food color, glazing agent, moisturizer, lubricant, preservative, solvent, stabilizer, sweetener and thickening The group of agent composition.
The foregoing description of preferred embodiment and other embodiments be not intended to limit or constrain it is contemplated that the present invention The range or applicability of design.As the exchange of the open inventive concept being contained herein, inventor wishes to be wanted by appended right Book is asked to provide institute patented.Therefore, the inventive concept being contained herein be intended to include fall into the appended claims or its etc. All modifications and change in the range of jljl.

Claims (29)

1. a kind of method for the extract for being used to prepare latex of panama rubber tree, the method comprise the steps of:
Obtain the slurries that are discharged when the latex of panama rubber tree of acidification is calendered is removed optionally from the slurries clast with Microorganism；
Remove protein；
Remove quebrachitol and small polar molecule；
Optionally remaining solid powder is dissolved in water；
Protein is further removed by being handled with acid；
Optionally neutralize excessive acid；
Use solvent deposition；
Collect sediment；And
Optionally it is freeze-dried the sediment.
2. according to the method described in claim 1, wherein the technology for removing clast and microorganism includes to be less than by aperture 0.5 micron, preferably 0.2 micron of filter filtering.
3. according to the method described in claim 1, wherein the technology for removing protein includes in ultrafiltration and spray drying It is at least one.
4. according to the method described in claim 1, wherein the removing quebrachitol and small polar molecule include with a kind of or The mixture of a variety of organic dissolutions in water extracts and collects the remaining solid powder.
5. according to the method described in claim 1, by acid processing, further removing protein includes with three chloroethenes described in wherein Sour precipitating proteins.
6. according to the method described in claim 1, wherein the basicity of the supernatant after the acid treatment step is by being added with The water-soluble alkali of effect amount, preferably sodium hydroxide neutralize.
7. according to the method described in claim 1, wherein the separation of latex of panama rubber tree extract includes with one or more organic The mixture precipitation of solvent in water.
8. according to the method described in claim 3, wherein by molecular cut off be at least 3kD, preferably the ultrafiltration membrane of 10kD into Row ultrafiltration.
9. according to the method described in claim 3, being wherein higher than 60 degrees Celsius, preferably higher than 100 degrees Celsius, and most preferably The spray drying is carried out at a temperature of between 100 degrees Celsius and 120 degrees Celsius.
10. according to the method described in claim 4, wherein the mixture of one or more organic solvents in water includes extremely Few 70% methanol, preferably 100% methanol.
11. according to the method described in claim 4, wherein the extraction optionally repeats, preferably in total three times.
12. according to the method described in claim 5, wherein the concentration of trichloroacetic acid be 1% to 50%, preferably 2% to 10%, and And most preferably between 2% and 3%.
13. according to the method described in claim 5, wherein be higher than 4 degrees Celsius, preferably between 16 and 30 degrees Celsius, and The acid processing is most preferably carried out at a temperature of between 20 and 25 degrees Celsius continues at least 0.1 minute, preferably 24 hours.
14. pass through at least 3 with the sediment that the sour processing is formed according to the method described in claim 5, wherein passing through, 000 × g centrifugation continues at least 5 minutes, removes within preferably 10 minutes.
15. according to the method described in claim 7, wherein the mixture of one or more organic solvents in water includes extremely Few 70% ethyl alcohol, preferably 100% ethyl alcohol.
16. according to the method described in claim 7, being wherein lower than 4 degrees Celsius, preferably shorter than -10 degrees Celsius, and most preferably The settling step is carried out under -20 degrees Celsius continues at least 5 minutes, preferably 24 hours.
17. according to the method described in claim 7, wherein the precipitating in the organic solvent is by least 3,000 × g Centrifugation continues at least 5 minutes to collect.
18. a kind of method for treating or preventing the growth of cancers in subject and/or transfer, the method includes to described Subject's application is a effective amount of by according to claim 1 to the rubber of the method preparation described in any one of 17 (including endpoints) The extract of gummy cream is with inducing cancer cell death.
19. a kind of for inhibiting the method for the tumor cell proliferation in patient, the method includes effective to patient application Amount according to claim 1 to the latex of panama rubber tree extract of the method preparation described in any one of 17 (including endpoints), it is described The advantageous variation instruction that effective quantity passes through one or more suitable markers or indicant.
20. according to the method for claim 19, wherein the advantageous variation of the suitable marker is in CD31 Epithelial cell marker object is reduced, VEGF angiogenesis marker is reduced, COX-2 Markers of inflammation is reduced, EGFR proliferation marker subtracts Less, Apoptosis indicant increases and galectin-3 metastatic marker object reduces the group of composition.
21. according to the method for claim 18, wherein the cancer is selected from group consisting of: AIDS-related cancers, Acoustic neurinoma, adenoid cystic carcinoma, adrenocortical carcinoma, agnogenic myeloid metaplasia, alopecia, alveolar soft tissue sarcoma, angiosarcoma, Alpastic anemia, astrocytoma, asynergy-capillary dilation, basal-cell carcinoma (bcc), brain stem glioma, Class cancer, children with cancer, children soft tissue sarcoma, chondrosarcoma, choriocarcinoma, colorectal cancer, skin T cell lymphoma, skin Fibrosarcoma-protrusion, Hypertrophic small circle cell cancer, duct carcinoma, carcinoma of endometrium, ependymoma, the cancer of the esophagus, ewing's sarcoma (Ewing's sarcoma), cholangiocarcinoma, ophthalmo melanoma, retinoblastoma, carcinoma of fallopian tube, Fanconi anemia, fibre Tie up sarcoma, gastric cancer, gastrointestinal cancer, gastrointestinal associated cancers, genitourinary system carcinoma, germinocarcinoma, gestational trophoblastic disease, glue Matter blastoma, glioma, gynecological cancer, Malignancy, hepatocellular carcinoma, hereditary breast cancer, tissue are thin Born of the same parents' increase disease, Hodgkin's disease (Hodgkin's disease), human papilloma virus, vesicular mole, hypopharyngeal cancer, intraocular melanoma, Island T cell cancer, Kaposi sarcoma (Kaposi's sarcoma), Langerhan's cell histiocytosis (Langerhan's cell histiocytosis), leiomyosarcoma, leukaemia, Li-Fo Meini syndrome (Li- Fraumeni syndrome), embryonal-cell lipoma, lymphedema, lymthoma, Hodgkin lymphoma (Hodgkin's lymphoma), Non-Hodgkin lymphoma (non-Hodgkin's lymphoma), male breast carcinoma, the pernicious rhabdoid tumor cancer of kidney, at nerve channel Cytoma, melanoma, merkel's cells cancer (merkel cell cancer), celiothelioma, metastatic carcinoma, Multiple Endocrine Tumour, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disease, nasopharyngeal carcinoma, the nephroblastoma, mind Through blastoma, neurofibromatosis, how Makin breakage syndrome, nonmelanoma skin cancer, non-small cell lung cancer (nsclc), eye cancer, the cancer of the esophagus, carcinoma of mouth, oropharyngeal cancer, osteosarcoma, neostomy oophoroma, cancer of pancreas, nasal sinus cancer, by first shape Gland cancer, carcinoma of parotid gland, carcinoma of penis, peripheral nerve ectoderm cancer, polycythemia vera, rare cancer and associated disease, kidney are thin Born of the same parents' cancer, retinoblastoma, rhabdomyosarcoma, Rosemond thomson syndrome (rothmund Thomson Syndrome), sarcoma, neurinoma, Sezary syndrome (sezary syndrome), Small Cell Lung Cancer (sclc), small intestine Cancer, soft tissue sarcoma, squamous cell carcinoma (sec), synovial sarcoma, transitional cell carcinoma (bladder), transitional cell carcinoma (kidney-pelvis -/- Ureter), choriocarcinoma, urine plaque albumen, sarcoma of uterus, Walden Si Telunshi macroglobulinemia (Waldenstrom's macroglobulinemia) and Weir nurse Si Shi cancer.
22. according to the method for claim 18, wherein the group of cancer cancer selected from the following composition: anus, bladder, Bone, intestines, brain, breast, central nervous system, cervix, colon, endocrine gland, ear, endothelial cell, esophagus, eye, gall-bladder, head, It is intestines, kidney, larynx, leucocyte, lip, liver, lung, oral area, nasal cavity, neck, nose, oral cavity, ovary, pancreas, pharynx, hypophysis, prostate, straight Intestines, salivary gland, skin, spinal cord, stomach, testis, thymus gland, thyroid gland, urethra, urinary system, uterus, vagina and vulva.
23. according to the method for claim 18, wherein administration method be selected from enteral, intramuscular, it is intravenous, intranasal, oral, Parenteral, rectum, it is subcutaneous, sublingual, under labia, the group of transdermal and transmucosal composition.
24. according to the method for claim 18, wherein the form of the latex of panama rubber tree extract is selected from aqueous solution, piece Agent, pastille, powder, aqueous or oily suspensions, lotion, the group of syrup and elixir composition.
25. according to the method for claim 18, wherein the subject is selected from the mankind, mammal and animal composition Group.
26. according to the method for claim 18, wherein by one kind of the latex of panama rubber tree extract and therapeutically effective amount Or a variety of chemotherapeutic combinations are applied to the subject.
27. according to the method for claim 26, wherein the chemotherapeutant is selected from alkylating agent, antimetabolite, antitumor Antibiotic, kinase inhibitor, aromatase inhibitor, mitotic inhibitor, steroid hormone and topoisomerase enzyme inhibitor composition Group.
28. a kind of composition, the composition includes according to claim 1 to the rubber of any one of 17 (including endpoints) preparation Gummy cream extract and one or more pharmaceutically acceptable substances, the pharmaceutically acceptable substance be selected from additive, The group of binder, carrier, diluent, excipient, filler, lubricant, solvent and stabilizer composition.
29. a kind of composition, the composition includes according to claim 1 to the rubber of any one of 17 (including endpoints) preparation Gummy cream extract and one or more comestible compositions, the comestible composition are selected from acid, acidity regulator, additive, resist Agglomeration agent, defoaming agent, antioxidant, bake agent, binder, carrier, color preserving agent, diluent, emulsifier, excipient, filler, Fragrance, flavoring agent, Flour ingredient, food color, glazing agent, moisturizer, lubricant, preservative, solvent, stabilizer, sweet tea The group of taste agent and thickener composition.
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WO2018044242A1 (en) 2018-03-08

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