CN110023342B - Method for preparing rubber tree latex extract and composition thereof - Google Patents
Method for preparing rubber tree latex extract and composition thereof Download PDFInfo
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- CN110023342B CN110023342B CN201680090627.8A CN201680090627A CN110023342B CN 110023342 B CN110023342 B CN 110023342B CN 201680090627 A CN201680090627 A CN 201680090627A CN 110023342 B CN110023342 B CN 110023342B
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/47—Euphorbiaceae (Spurge family), e.g. Ricinus (castorbean)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
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- 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
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- 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 present disclosure provides a process for preparing an extract rich in low molecular weight carbohydrates and having anti-cancer activity from a slurry of calendered acidified hevea latex. The method comprises the following steps: obtaining a slurry released when the acidified hevea brasiliensis latex is calendered; removing debris and microorganisms from the slurry; removing the protein; removing quebrachol and small polar molecules; optionally dissolving the remaining solid powder in water; further removing the protein by treatment with acid; optionally neutralizing excess acid; precipitating with a solvent; collecting the precipitate; and optionally freeze drying the precipitate. The disclosure also provides methods of treating cancer growth and/or metastasis and methods of inhibiting tumor cell proliferation in a patient. In addition, pharmaceutical compositions and functional food compositions comprising the disclosed hevea brasiliensis latex extract are provided.
Description
Technical Field
The present invention relates generally to the field of cancer, including oncology therapy. More particularly, the present invention relates to the prevention and treatment of cancer and cancer metastasis by using extracts of the latex of the Hevea rubber tree (Hevea) rich in low molecular weight carbohydrates. The invention also relates to a pharmaceutical composition for treating cancer and an anticancer functional food composition.
Background
Throughout history, humans have been afflicted with cancer due to the body's inability to control the growth and spread of certain malignant cells. According to the World Health Organization (World Health Organization), cancer as a group is the major non-infectious disease of the first four diseases (cardiovascular disease, cancer, diabetes and chronic lung disease). The american cancer society reports that in 2012, the number of new cases of cancer diagnosed worldwide is 1410 ten thousands, with 820 thousands dying. It was predicted that in 2030, the number of new Cancer cases could reach 2170 million, with 1300 million people dying (iarc.2012., "Global Cancer Facts and charts & regulations"), 3 rd edition, produced by the american Cancer society in collaboration with the international Cancer research institute).
For cancer chemotherapy, different strategies exist. Traditional brute force cytotoxic cancer chemotherapeutic agents often involve considerable side effects on normal cells. Even with monoclonal antibody targeting, some degree of undesirable side effects are expected.
Recently, cancer has been found to be closely related to biological signaling pathways that control somatic cell proliferation and destruction. Chemical agents that manipulate such pathways are useful as anti-cancer agents. Small molecule targeted therapeutic drugs are often inhibitors of catalytic or binding sites on mutated, overexpressed, or other key proteins within cancer cells, but are only applicable to certain types of cancer. Other modern anticancer agents may act by acting as signals that stimulate the patient's immune system.
Many anticancer treatments and drugs based on natural products are known in the prior art. These active therapeutic agents range from small biomolecules to large macromolecules. An example of a small anti-cancer biomolecule found in plant extracts, as disclosed in us patent No. 6,432,452 (anti-cancer compound), is angeloyl-substituted ingenane (ingenane), a macrocyclic diterpene which competitively blocks the carcinogenic effect of phorbol esters, which are themselves another macrocyclic diterpene. On the other hand, peptide allergens in the hevea brasiliensis have been used in pharmaceutical preparations to induce immune responses in the treatment of diseases including cancer. Examples are disclosed in PCT application No. PCT/EP2003/011190 (modular antigen transport molecules for modulating immune responses, related constructs, methods and uses) and No. PCT/EP2013/000291 (pharmaceutical compositions comprising a polymeric carrier cargo complex and at least one protein or peptide antigen), as well as in european patent application No. EP2012/000418 (negatively charged nucleic acids comprising a complex for immune stimulation) and No. EP2012/000420 (pharmaceutical compositions comprising a polymeric carrier cargo complex and at least one protein or peptide antigen).
Between small molecules with a molecular weight of less than one kilodalton (hereinafter referred to as "kD") and large molecules with a molecular weight of more than 10kD are biochemical anti-cancer agents with a molecular weight of a few kD, as shown in european patent publication No. 0589074 (carbohydrate complex for destruction of resistant cancer cells). Some researchers believe that these low molecular weight carbohydrates prevent the growth and metastasis of cancer cells by interfering with cell-cell interactions mediated by cell surface carbohydrate binding molecules, as disclosed in U.S. patent 5,834,442 (a method of inhibiting cancer metastasis by oral administration of soluble modified citrus pectin) and U.S. patent 5,895,784 (a method of treating cancer by oral administration of modified pectin).
It is known in the prior art that certain low molecular weight carbohydrates of animal origin have an anticancer effect. Examples include hyaluronic acid (Ween, M.P., K.hummitzsch, R.J.Rodges, M.K.Oehler and C.Ricciardelli.2011. proteoglycans induce metastatic ovarian Cancer cell behavior that can be inhibited by small hyaluronic acid oligosaccharides (Versican indexes a pro-metastatic ovarian Cancer cell after being inhibited) (Clinical and Experimental Metastasis) 28: 113-125, maltose sulfate (partition, C.R., C.Emamectin, K.J.Brown, D.J.Francis and W.B.Cowuerette. 1999. Using a Novel Angiogenesis and acetyl enzyme Activity test and Growth inhibition of tumors in Vitro (polysaccharide sulfate of polysaccharide sulfate) 33-polysaccharide sulfate and Growth inhibition of heparin sulfate in Vitro (polysaccharide sulfate of polysaccharide sulfate 19) (assay of polysaccharide sulfate 33-polysaccharide sulfate and Growth inhibition of polysaccharide sulfate), m. O.Iqbal, D.A.Hoppenstead, P.Piccolo, S.Ahmad, C.L.Schultz, R.J.Linhardt and J.Fareid.2001. Anticoagulant and Antiprotease Profiles of Novel native heparin-mimetic Mannopentaose Phosphate Sulfate (PI-88) (Anticoagulant and Antiprotease Profiles of a Novel Natural heparin synergistic Phosphate Sulfate (PI-88)). Clinical and Applied Thrombosis/Hemostasis (Clinical and Applied Thrombosis/Hemostasis) 7: 131-. Such anti-cancer effects may be due to a combination of direct cytotoxicity, enhancement of the efficacy of chemotherapeutic drugs, and blocking of carcinogenesis. Anti-proliferative effects have been shown against different types of human cancers, including ovarian cancer, colon cancer, prostate cancer (Khasraw, m., n.pavlakis, s.mccowatt, c.underhill, s.begbie, p.de Souza, a.bouye, f.parans, v.lim, r.harvie and g.marx.2010 PI-88, a heparanase inhibitor in combination with docetaxel in patients with metastatic castration-resistant prostate cancer multi-center phase I/II studies (multicenter phase I/II decision of PI-88, a heparin inhibitor combination with docetaxel with a tumor-resistant cancer cell-cancer cell), annual tumor cell of the neoplasm (astrocyte of 21. astrocyte, c.7. astrocyte.g. c.r.t. astrocyte.g. c.g. astrocyte, c.g. astrocyte.g. c.g. cell binding in subcategorical clinical assays, astrocytomas, anaplastic assays, and glioblastomas, J.Neuropathology and Experimental Neurology (Journal of Neuropathology and Experimental Neurology) 60: 75-84). However, low molecular weight carbohydrates of animal origin are relatively expensive to produce on a large scale and have not been used commercially in anti-cancer formulations.
As disclosed in us patent 8,722,107 (modified pectin, compositions and methods related thereto), it has been shown that some low molecular weight carbohydrates of plant origin, especially modified pectins, also have an anti-cancer effect. Examples include low molecular weight carbohydrates from the fruit of the apple tree. (Li, Q., Z.Zhou, J.lacing, T.Yang, S.Duan, Z.Wang, Q.Mei and L.Liu.2013. apple-derived oligosaccharides induce apoptosis and cell cycle arrest in HT29 human colon cancer cells (Oligosaccharide from apple analogues and cell cycle arrest HT29 human color cells.) Int J.Biol.Macromol.) -57: 245-254 and modified pectins from citrus fruits (US Patents 5,834,442 and 5,895,784 cited above). Disadvantages of using plant-derived low molecular weight carbohydrates, especially modified pectins, in anti-cancer formulations include the cost of raw materials (e.g., apples or lemons) as commercial products and the multi-step extraction and modification of pectin into low molecular weight carbohydrates. Unavoidable trace organic solvent contamination in the final low molecular weight carbohydrate product may also have long-term health concerns.
Instead of raw materials based on edible plants or fruits like citrus or apples, the inventors have surprisingly found that an economically more advantageous raw material for the anti-cancer extract is the slurry obtained when the acidified rubber tree latex is calendered. It has also been surprisingly found that this starting material, in combination with the extraction process disclosed herein, results in a more uniform and reproducible product compared to other processes. Although it is highly unlikely that the chemical composition of the rubber tree extract according to the invention is identical to the chemical composition of the so-called "low molecular weight" carbohydrates cited in the prior art, due to differences in starting materials and extraction processes and conditions, it has been found that the rubber tree extract according to the invention has anticancer activity. In addition, relatively safe alcohols, such as ethanol, are used in the final precipitation step according to the invention to eliminate any long term health problems.
Although the fraction of natural rubber Latex obtained from the soft bark of Hevea brasiliensis (Hevea brasiliensis) has been demonstrated to have some Anti-cancer properties (Latex, k.l. yang, e.sunderasan and m.t.on.2012. Latex C-serous from Hevea brasiliensis induce non-apoptotic Cell death in hepatocellular carcinoma Cell line (HepG2) (Latex C-serum from Hevea brasiliensis is not-apoptotic Cell death in hepatocellular carcinoma Cell line (HepG 2).: Cell proliferation (Cell Prolif.) (45) (6): 577-), (Lee, y.k., l.k.lay, m.s.mauu, t.s.guaiai, s.m.o. and m.o.n.p. alumina, and (Cell proliferation) serous 577-, (Latex b.n.k., t.n.n.p. epithelial Cell proliferation) of Hevea brasiliensis (hep. 75. n. 75. r. C.) (Cell proliferation of Hevea brasiliensis, n. 75. epithelial Cell proliferation, n. kappa. 645), n.abd.rahman, k.l.lam, k.l.yang and m.t.ong.2013. Cell viability assay guide fractionation of natural rubber latex slurries [ journal of rubber research (j.rubber.res.) ] 16(3):195- > 202 ], the use of natural rubber latex itself as starting material is uneconomical due to inefficient utilization of the solid rubber fraction of the latex.
It is well known that when fresh hevea brasiliensis latex is coagulated with an acid such as formic acid and then calendered (extrusion grinding), the liquid released from the grinding process (hereinafter referred to as "slurry") contains excess unreacted formic acid as well as many biomolecules originally present in the rubber latex. Such slurries can be used as inexpensive sources of starting materials for high value-added biomolecules, especially those with therapeutic properties. The present invention utilizes this slurry as a starting material for preparing a low molecular weight carbohydrate mixture.
Unfortunately, in addition to containing carbohydrates, the latex of rubber gum contains a variety of other biomolecules, including allergen proteins such as Hev b1, Hev b2, Hev b4, Hev b5 and Hev b 6.02. (Chen, Z., R.Cremer, A.Posch, M.R.aulf-Heimsoth, H-P.Rihs and X.Baur.1997 "about the allergenicity of Hev b1 in health care workers and spina bifida patients allergic to natural rubber latex" (On the allergenicity of Hev b1 amplitude health workers and patients with spinal bifida alloy to natural rubber latex) ". Journal of Allergy and Clinical Immunology 100 (Journal of Allergy and Clinical Immunology) (5): 684-93; barre, a., r.currier, c.granier, l.selman, w.j.peumans, e.j.m.van Damme, f.bienvenu, j.bienvenu and p.rouge. 2009. "Mapping of IgE-binding epitopes on major latex allergen Hev 2 and cross-reacting 1,3 β -glucanase fruit allergens as the molecular basis for the latex-gum fruit syndrome (Mapping of IgE-binding epitopes on the major latex allergen Hev 2 and cross-reacting 1,3 β -glucanase fruit allergens)". Molecular Immunology 46(8-9): 1595-604; "Structural analysis of glycoprotein allergen Hev b4 from the glycoprotein allergen Hev b4 from natural rubber latex by Mass Spectrometry" by Kolarich, D.D., F.Altmann, and E.Sundersan.2006. 1760(4) from the Biochemical and Biophysics Bulletin (BBA) -General Subjects (Biochimica et Biophysica Acta (BBA) -General Subjects) and 715-20; beezhold, d.h., v.l.hickey, j.e.slater and g.l.sussman.1999, "Human IgE-binding epitopes of latex allergen Hev b5 (Human IgE-binding epitopes of the latex allergen Hev b 5)". 1166-72 in journal of allergy and clinical immunology 103 (6); Reyes-L Lopez, C.A., A.Hern a ndez-Santoyo, M.Pedraza-Escalona, G.Mendoza, A.Hern a ndez-Arana and A.Rodrai guez-Romero.2004. "insight into the conformational epitope of Hev b6.02 (heimin)". Biochemical and Biophysical Research Communications 314 (Biochemical and Biophysical Research Communications) 123-30; Pedraza-Escalona, m., b.becorril-Luj a n, c.agndis, l.dom i anguez-rami i rez, a.pereyra, L.
-Umbarila and A.RodrI guez-romero.2009. "Analysis to reveal B cell epitopes from the allergen Hev 6.02 by using blocking antibodies (Analysis of B-cell epitopes from the allergen Hev 6.02 obtained by using blocking antibodies)". 668-76 in Molecular Immunology 46 (Molecular Immunology). ) Thus, the present invention needs to provide a step of removing proteinaceous material, including enzymes with known allergen epitopes.
Accordingly, it is an object of the present invention to provide a method for preparing a latex extract of hevea brasiliensis having anticancer activity.
It is another object of the present invention to provide a method for preparing a latex extract of hevea brasiliensis having anti-metastatic properties to proliferating cancer cells.
It is another object of the present invention to provide a process for the preparation of low molecular weight carbohydrates from relatively inexpensive biological starting materials.
It is another object of the present invention to provide a process for preparing a rubber tree latex extract rich in low molecular weight carbohydrates.
It is another object of the present invention to provide a process for preparing a hevea brasiliensis latex extract such that the low molecular weight carbohydrates in the final product have a relatively uniform and reproducible molecular composition.
It is another object of the present invention to provide a process for the preparation of rubber tree latex extract such that the final product is free of proteinaceous matter, in particular allergen proteins.
It is another object of the present invention to provide a process for preparing a rubber tree latex extract that is safe for use in pharmaceutical and food compositions. In this case, safety includes the absence of acute, subacute and chronic toxicity as determined by the relevant protocol.
It is another object of the present invention to provide a simple, efficient and relatively inexpensive process for preparing rubber tree latex extracts having anti-cancer and anti-metastatic activity.
It is another object of the present invention to provide a process for preparing a hevea brasiliensis latex extract, which makes the process easily scalable for commercialization.
It is another object of the present invention to take advantage of the anticancer activity of latex extract of hevea brasiliensis and produce products that can be used therapeutically as anticancer drugs and prophylactically as anticancer food supplements.
Disclosure of Invention
The invention described herein relates to providing extracts of hevea brasiliensis latex that are rich in low molecular weight carbohydrates and have anticancer activity.
One aspect of the disclosed invention relates to a method for preparing an extract of hevea brasiliensis latex comprising the steps of: obtaining a slurry released when the acidified hevea brasiliensis latex is calendered; optionally removing debris and microorganisms from the slurry; removing the protein; removing quebrachol and small polar molecules; optionally dissolving the remaining solid powder in water; further removing the protein by treatment with acid; optionally neutralizing excess acid; precipitating with a solvent; collecting the precipitate; and optionally freeze drying the precipitate. Debris and microorganisms are removed by techniques including filtration. The proteins are removed by biochemical techniques including ultrafiltration and/or spray drying. Quebrachitol and small polar molecules are removed by extraction with a solvent such as methanol and collecting the remaining undissolved solid powder. Any remaining protein is further removed by treatment with an acid such as trichloroacetic acid. The remaining mixture is optionally adjusted to neutral pH and the final product is isolated by precipitation with a cold organic solvent such as ethanol. The precipitate was collected by centrifugation and optionally freeze dried.
Another aspect of the invention relates to a method for treating or preventing cancer growth and/or metastasis in a subject, comprising administering to the subject an effective amount of hevea brasiliensis latex extract prepared by the method according to the invention to induce cancer cell death.
In another aspect of the invention, a method for inhibiting tumor cell proliferation in a patient is described. The method comprises administering to a patient an effective amount of a hevea brasiliensis latex extract according to the present invention to achieve a beneficial change in one or more of the following markers: a decrease in CD31 endothelial cell marker; a VEGF angiogenesis marker; a decrease in COX-2 inflammatory markers; decreased EGFR proliferation marker; increased apoptosis of cancer cells; and a reduction in galectin-3 transfer markers.
Another aspect of the invention is a composition comprising a hevea brasiliensis latex extract according to the invention and one or more pharmaceutically acceptable substances. Another aspect of the invention is a composition comprising a hevea brasiliensis latex extract according to the invention and one or more edible ingredients. The use of the word "a/an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one" but it is also consistent with the meaning of "one or more", "at least one" and "one or more than one".
These and other embodiments of the present invention will be better understood and appreciated when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following description, while indicating various embodiments of the present invention and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions and/or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and/or rearrangements.
Drawings
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Figure 1 is a flow diagram showing the source of the raw material rubber gum whey.
Fig. 2 is a flow chart showing the steps of preparing an extract from a latex slurry of a hevea brasiliensis tree according to the present invention.
Fig. 3 is a flowchart showing a best mode embodiment of the present invention.
FIG. 4 is a Fourier transform infrared (FT-IR) spectrum illustrating the possible composition of the extract from hevea brasiliensis latex according to the best mode embodiment of the present invention.
FIG. 5 is a liquid chromatography-mass spectrometry (LC-MS) molecular weight profile of latex extract of Hevea brasiliensis according to a best mode embodiment of the present invention.
Fig. 6 shows the size of the tumor in nude mice measured using a vernier caliper.
FIG. 7 shows the time course of relative tumor volumes in nude mice receiving latex extract of Hevea brasiliensis to illustrate anticancer properties, according to the best mode embodiment of the present invention. (dotted line) experimental group orally gavage 60mg rubber tree latex extract per kg body weight per day for 28 days. (solid line) control group, which received water instead of rubber tree latex extract.
Figure 8 shows how the dorsal skin fold chamber was surgically attached to nude mice in order to visualize angiogenesis. The diameter of the dorsal skin fold chamber is about 7 mm.
Figure 9 shows inhibition of angiogenesis in nude mice with cancer implants in the dorsal skin fold chamber. The mice were first anesthetized. Fluorescein isothiocyanate-dextran (FITC-dextran) solution was then infused into the jugular vein to label the vessels. The dorsal skin fold chamber, as well as the skin with the cancer implant, were surgically removed and viewed using a technique known as "in vivo confocal fluorescence video microscopy". (left) no cancer control group. Among others, cancer implants. (right) extracts of hevea brasiliensis latex with cancer implants and best mode according to the present invention.
Fig. 10 shows the inhibition of angiogenesis in nude mice with cancer implants by oral gavage of 60 milligrams (hereinafter "mg") of rubber tree latex extract per kilogram (hereinafter "kg") of body weight per day, as compared to a control group receiving water. Immunohistochemical visualization was performed using CD31 endothelial cell marker. (left) no cancer control group. Among others, cancer implants. (right) extracts of hevea brasiliensis latex with cancer implants and best mode according to the present invention. Bar 100 microns.
Figure 11 shows the inhibition of angiogenesis in nude mice with cancer implants by oral gavage of 60mg of hevea brasiliensis latex extract per kg body weight per day compared to a control group receiving water. Immunohistochemical visualization was performed using VEGF angiogenic markers. (left) no cancer control group. Among others, cancer implants. (right) extracts of hevea brasiliensis latex with cancer implants and best mode according to the present invention. Bar 100 microns.
Figure 12 shows the reduction of inflammation in nude mice with cancer implants by oral gavage of 60mg of hevea brasiliensis latex extract per kg body weight per day compared to control group receiving water. Immunohistochemical visualization was performed using COX-2 inflammatory markers. (left) no cancer control group. Among others, cancer implants. (right) extracts of hevea brasiliensis latex with cancer implants and best mode according to the present invention. Bar 100 microns.
Figure 13 shows the reduction of cell division in nude mice with cancer implants by oral gavage of 60mg of hevea brasiliensis latex extract per kg body weight per day compared to control group receiving water. Immunohistochemical visualization was performed using EGFR proliferation markers. (left) no cancer control group. Among others, cancer implants. (right) extracts of hevea brasiliensis latex with cancer implants and best mode according to the present invention. Bar 100 microns.
Figure 14 shows the increase in apoptosis in nude mice with cancer implants by oral gavage of 60mg of hevea brasiliensis latex extract per kg body weight per day compared to control group receiving water. Visualization was performed using terminal deoxynucleotidyl transferase (TdT) -mediated deoxyuridine triphosphate nucleotide (dUTP) nick end labeling (TUNEL) method. (left) no cancer control group. Among others, cancer implants. (right) extracts of hevea brasiliensis latex with cancer implants and best mode according to the present invention. Bar 100 microns.
Fig. 15 shows the results of an experimental tail vein transfer assay. Mice were injected with 1X 10 via tail vein5And melanoma B16-F1 cells. The lower panel shows dissected lungs of these mice fed 60mg/kg of rubber tree latex extract daily for 2 weeks. The upper panel shows the lungs from water-fed control mice. The black dots show colonies of metastatic melanoma cells.
FIG. 16 shows galectin-3 (transfer marker)) Results of immunohistochemical staining assay of (a). (left) no cancer control group. (in) mice were injected with 1X 10 through the tail vein5Melanoma B16-F1 cells were treated and fed water instead of rubber tree latex extract. (Right) mice were injected with 1X 10 through the tail vein5Individual melanoma B16-F1 cells, and then 60mg/kg of rubber tree latex extract was fed daily for two weeks. Bar 100 microns.
The figures and written description are not intended to limit the scope of the disclosed methods for preparing the hevea brasiliensis latex extract in any way. Rather, the figures and written description are provided to illustrate to those of ordinary skill in the art the disclosed methods for preparing a hevea brasiliensis latex extract by reference to specific embodiments of the present invention. The invention will now be described with such specificity as to enable any person skilled in the relevant art to practice the invention without undue experimentation.
Detailed Description
Referring to fig. 1, a well-known process for preparing a rubber tree slurry begins with fresh latex 101 collected from a rubber tree. An acid, typically formic acid, is added to coagulate the latex. Coagulation with acid 102 converts the rubbery isoprene polymer particles in the latex into coagulum, which begins to separate from the liquid. This separation is accelerated by extrusion milling or calendering 103, which produces solid rubber, sheet rubber 104 as an output. The liquid released from this extrusion grinding step is referred to as a hevea latex slurry 105. The process according to the invention uses this slurry 105 as starting material.
Referring to fig. 2, the present invention provides a method for preparing a rubber tree latex extract. The method according to the invention comprises the following steps: obtaining a slurry 205 that is released when the acidified hevea brasiliensis latex is calendered (the slurry is the same as hevea brasiliensis latex slurry 105 in fig. 1); optionally removing debris and microorganisms 206; removing the protein 207; removing quebrachol and small polar molecules 208; optionally dissolving the remaining solid powder in water 209; further removal of protein 210 by treatment with acid; optionally neutralizing excess acid 211; precipitating 212 with a solvent; collecting the precipitate 213; and optionally freeze-drying the precipitate 214 to produce a trefoil latex extract 215.
Step 206-214 (including endpoints) comprises conventional biochemical and biophysical techniques known separately to those skilled in the art.
Logically, steps 207, 208, and 210 need not be practiced in this particular order. Those skilled in the art should be able to rearrange, replace, and change the details of these three steps. However, the best mode for carrying out the invention will illustrate that arranging steps 207, 208 and 210 in this particular order has practical and orchestrated advantages when practicing the method.
A detailed description of the sequential steps in fig. 2 is given below:
step 206 is optionally required if the latex slurry is contaminated with debris and microorganisms, such as bacteria, depending on how the treated hevea brasiliensis latex is washed. One example of a removal technique is filtration through a filter having a pore size small enough to exclude debris and microorganisms, typically less than 0.5 microns, and preferably 0.2 microns. Although smaller pore sizes may exclude finer debris and particles, the filtration process slows as the pore size becomes smaller. A filter pore size of 0.2 micron seems to be the best trade-off, as the inventors did not find any negative impact of possible ultrafine debris and nanoparticle contamination.
Step 207 comprises removing proteins, in particular protein allergens naturally present in latex of rubber. This step involves a combination of biochemical techniques such as enzymatic digestion at high temperature, ultrafiltration and spray drying. One example of step 207 is the destruction of proteins by any commercial proteolytic enzyme that is inexpensive and can retain its biological activity in the environment of the hevea latex slurry. Another example of step 207 is the filtration of the liquid through an ultrafiltration membrane with a molecular weight cut-off of at least 3kD and preferably 10 kD. Another example of step 207 is to spray dry the liquid at a temperature above 60 degrees celsius, preferably above 100 degrees celsius and most preferably between 100 and 120 degrees celsius. In the case of serial use of ultrafiltration and spray drying, any protein passing through the ultrafiltration membrane should be denatured and lose its biological activity by high temperature spray drying.
Step 208 comprises removing quebrachol and small polar molecules from the slurry. The importance of this step will be apparent when the latex extract of hevea brasiliensis according to the present invention is combined with one or more pharmaceutically acceptable substances to form a composition for use as an anti-cancer drug. The importance will also be apparent when rubber tree latex extract is combined with one or more edible ingredients to form a composition useful as a functional food against cancer. For a drug or functional food, for carrying out a method for treating cancer growth and/or metastasis to effect death of cancer cells, or for inhibiting proliferation of tumor cells in a patient to effect a beneficial change in one or more suitable markers or indicators of angiogenesis (VEGF reduction), inflammation (COX-2 reduction), proliferation (EGFR reduction), apoptosis, and metastasis (galectin-3 reduction), a cancer patient must be administered an effective amount of the drug or functional food. Patient compliance is dependent, inter alia, on the absence of undesirable side effects, such as diarrhea, which is known to be caused by quebrachol. Thus, the primary purpose of step 208 is to remove such undesirable compounds. Many other small polar molecules, including nucleotides, if present, will also be removed in this step. One example of step 208 is extraction by a suitable mixture of polar solvents (such as methanol) that tends to dissolve polar molecules, including quebrachol, leaving other undissolved less polar molecules. The methanol concentration should be at least 70%, preferably 100%. The extraction may be repeated, preferably twice more, to reduce the concentration of quebrachitol sufficiently for further use.
Step 210 involves further removal of proteins by precipitation with acid to ensure more complete protein removal, especially small proteins and peptides that may remain in step 207. An example of step 210 is precipitation with 1% to 50%, preferably 2% to 10%, and most preferably between 2% and 3% trichloroacetic acid at a temperature above 4 degrees celsius, preferably between 16 and 30 degrees celsius, and most preferably between 20 and 25 degrees celsius. The time required for the protein to precipitate out depends inter alia on the concentration of the acid used and the temperature at which the precipitation takes place. In fact, precipitation was visible to the naked eye within about 0.1 minutes (almost immediately) after the addition of the acid. The optimal incubation time for the precipitation reaction can be determined by one skilled in the art of biochemistry. However, it is advisable to allow sufficient time, preferably 24 hours, for the precipitate to fully form.
Step 209 serves as a transition from step 208 to step 210. In this step, the undissolved solid powder of step 208 is optionally dissolved in a small amount of water to aid the precipitation reaction in step 210.
Step 211 is used to neutralize any excess acid from step 210. Neutralization is usually carried out with addition of sodium hydroxide. This step is considered optional, as the amount of base added depends on the amount of remaining acid.
The above description shows that the method according to the invention automatically enriches latex extracts with low molecular weight carbohydrates without any explicit digestion step for two reasons. First, the use of the slurry 105 or equivalently 205 as starting material has the inherent advantage of at least partially digesting the carbohydrates originally present in the fresh hevea latex 101 into low molecular weight carbohydrates in the acid coagulation step 102. In other words, the feedstock used according to the invention is already partially enriched in low molecular weight carbohydrates compared to fresh hevea latex. Second, if spray drying at elevated temperatures is used in the protein removal step 207, the elevated temperatures may help break down some of the carbohydrates. Finally, further removal of protein by the acid step 210 also helps to further hydrolyze any remaining carbohydrates to low molecular weight carbohydrates. In practice, the time, temperature and reactant concentrations in each step must be controlled to ensure that the resulting extract 215 has a uniform and reproducible low molecular weight carbohydrate composition. The inventors have surprisingly found that the method according to the best mode of the invention gives the most uniform and optimal reproducibility in terms of the composition of low molecular weight carbohydrates compared to other methods and examples. It should also be emphasized that the exact chemical properties of the low molecular weight carbohydrates according to the invention are expected to differ from the so-called "low molecular weight" carbohydrates according to the prior art due to differences in starting materials and extraction methods.
Another advantage of the present invention is that if spray drying at elevated temperature is used in the remove protein step 207, any protein that is not physically removed in other steps will be denatured by heat and will lose the three-dimensional configuration of any possible epitopes thereof, such that such protein will not cause allergy.
The following examples are included to illustrate preferred embodiments or best mode of the invention. It should be appreciated by those of ordinary skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes and best modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. In other words, the best mode and other exemplary embodiments of the present invention should not be construed as limiting the present invention in any way.
Best mode for carrying out the invention
The best mode presently known for carrying out the invention is to use a method according to the following example.
Fig. 3 is provided to illustrate the best mode for carrying out the invention. Best mode begins with obtaining a slurry 305, which slurry 305 is released when the acidified hevea latex is calendered. The starting material 305 refers to the same latex slurry 105 in fig. 1 and 205 in fig. 2. The final product 315 corresponds to the same product 215 in fig. 2.
In this best mode, the filtration step 306 for removing debris and microorganisms comprises filtration through a membrane having a pore size of 0.2 microns.
Also in this best mode, the protein removal step 307 consists of ultrafiltration 316 through an ultrafiltration membrane with a molecular weight cut off of 10kD, followed by spray drying 317 at a temperature of 110 degrees celsius. Since ultrafiltration and spray drying are used in tandem, any protein that passes through the ultrafiltration membrane will denature and lose its biological activity by high temperature spray drying. This allows the use of ultrafiltration membranes with a 10kD cut-off instead of 3kD membranes, which are more expensive and produce slower ultrafiltration rates at the same pressure. Any allergen protein with a molecular weight between 3 and 10kD, such as Hev b6.02, which is a 4.72kD polypeptide, will pass through a 10kD ultrafiltration membrane and will be inactivated by the high temperatures of the spray drying process. The inventors have found that spray drying should be carried out at a temperature above 60 degrees celsius, preferably above 100 degrees celsius and most preferably between 100 and 120 degrees celsius. Although higher spray drying temperatures may allow faster drying, better protein denaturation, and possibly more short chain carbohydrates, they present a risk of altering the sugar structure and product color.
In accordance with the present invention, to remove small polar molecules, including nucleotides and especially quebrachitol, the solid powder from the spray drying step 317 is extracted 308 with a mixture of at least 70% aqueous methanol, which is a relatively polar organic solvent and also relatively inexpensive. In the best mode, extraction is performed with anhydrous methanol at a 1:6 weight to volume ratio (i.e., 1 weight of solid powder to 6 volumes of solvent). The extraction was repeated twice a total of three times with methanol at a weight/volume ratio of 1: 4.
The remaining solid powder from the methanol extraction step 308 is dissolved in distilled water 309 at a 1:8 weight/volume ratio. Trichloroacetic acid is added in step 310 to reach a final concentration of 2.5%. The mixture was stirred at 25 ℃ for 24 hours. The precipitate was removed by centrifugation at 3,000 Xg for 5 minutes and discarded. Although the function of this precipitation step is to remove proteins, macromolecules including nucleic acid polymers, if present, will also be removed.
In the best mode, the supernatant from step 310 is then neutralized with sodium hydroxide 311 and then precipitated 312 with absolute ethanol. The temperature was maintained at-20 degrees celsius. After 24 hours, the precipitate was collected in step 313 by centrifugation at 3,000 Xg for 5 minutes. The precipitate is freeze dried in step 314 to produce a rubber tree latex extract 315, which can be held in a deep freezer for extended periods of time, typically several months.
In the best mode embodiment disclosed above, the stream of protein and small polar molecule removal begins with ultrafiltration 316, followed by spray drying 317, followed by methanol extraction 308, and finally treatment with trichloroacetic acid 310. This sequence ensures that the bulk of the feedstock 305, cleaned of debris and microorganisms by filtration 306, is reduced to a solid powder by spray drying 317, thereby facilitating the steps of methanol extraction 308 and treatment 310 with trichloroacetic acid. The sequence of steps disclosed in this best mode example is the most practical and efficient way known to the inventors to prepare the rubber tree latex extract according to the present invention at the time of filing the PCT application.
Best mode the yield of the example is about 0.17% (weight of latex extract to volume of slurry). In a practical representative preparation using the best mode of the invention, 9,650 liters of fresh hevea brasiliensis latex 101 released 4,147 liters of slurry 105 after calendering 103, which was used as the feedstock 305 for the best mode embodiment of the invention. After the ultrafiltration step 316, the volume was reduced to 3,869 liters. The spray drying step 317 yields 102kg of a solid powder. The ethanol precipitation step 312 and the freeze-drying step 314 yield 7kg of the final product 315.
Described below are representative test results obtained from the products of the best mode embodiments of the present invention. These results are given to show preliminary characterization and preliminary safety test results of the rubber tree latex extract according to the best mode of the invention.
(1) Preliminary characterization of the extracts
Figure 4 shows a fourier transform infrared (FT-IR) spectrum illustrating the possible composition of extracts from hevea brasiliensis latex. The horizontal axis represents the distance from 4000 to well below 1000cm-1The wave number of (c). The vertical axis represents percent transmission. The major absorption peaks included 3392.75, 1619.95, 1418.21, 847.55 and 526.14cm-1. Although 1081.41cm-1Cannot be resolved further, but it is theorized to be 1078.21cm due to the stretching of the beta 1,3 glycosidic bond of the beta-1, 3-glycan-1And 1079.77cm-1The absorption peaks are fairly close.
FIG. 5 shows a liquid chromatography-mass spectrometry (LC-MS) molecular weight profile of latex extract of Hevea brasiliensis. The horizontal axis of this particular histogram has been prepared to represent the mass of molecular fragments below 2000 to 6000 daltons. The vertical axis represents the percentage of a particular fragment in the sample. The figure illustrates the size distribution of the molecular weights of the components in the extract, centered around 3712.1 daltons. Representative molecular mass markers around the central peak are above (3713.6, 3744.8, 3759.1, 3800.1, and 3824.6 daltons) and below (3705.6, 3651.0, 3635.8, and 3554.5 daltons) the peak. If the molecule is actually a carbohydrate, the central molecular weight corresponds to a polymer of about 20 sugar residues.
From fig. 4 and 5 and the fact that the method according to the invention is designed to remove proteins, nucleic acids and other polar molecules, hevea brasiliensis extract 315 may contain low molecular weight carbohydrates of about 20 sugar residues.
(2) Preliminary safety test results
The latex extracts of hevea brasiliensis according to the present invention have been tested for acute toxicity, subacute toxicity for 6 weeks, and chronic toxicity for 9 months.
Rats fed 1 gram of latex extract of hevea brasiliensis per kg body weight did not cause any change in body weight, alertness, respiration, muscle strength, urine color and stool appearance compared to the negative control group according to the acute toxicity test performed under OECD guidelines No. 420.
When 6 male rats were fed 1 gram of latex extract of hevea brasiliensis per kg body weight per day for 6 weeks and compared to a control group of 6 male rats, no subacute toxicity was found with respect to appetite, body weight, liver function, kidney function, white blood cell count, platelet count, hemoglobin and hematocrit, blood glucose level, blood lipid level, fat deposition under the skin and in internal organs such as the liver, and the size of the internal organs (liver, kidney, heart, lung, pancreas, adrenal gland, testis, prostate and seminal vesicles). Furthermore, latex extract of hevea brasiliensis appears to induce nitric oxide release from the vascular lining, resulting in better vascular compliance and recovery of endothelial dysfunction.
Chronic toxicity studies (limit tests) were performed for 9 months according to OECD-OCDC-452 guidelines, revised from the 1981 release and adopted on 9, 7, 2009. 98 male and female weaning rats after 4 weeks were divided into 4 groups. Rats in the experimental group were fed 1 gram of latex extract of hevea brasiliensis per kg body weight per day for 9 months. The rats in the control group were fed the same volume of distilled water as the latex extract of the rubber tree in the experimental group. In addition, two satellite groups (one experimental group and one control group) were provided to monitor the reversibility of any toxicological changes that might be observed in the experimental group. Thus, rats in the experimental satellite group were fed 1 gram of latex extract of hevea brasiliensis per kg body weight per day for 9 months and then distilled water for an additional 28 days, while rats in the control satellite group were fed distilled water for the entire 9 months plus 28 days. No appearance or behavioral abnormalities, such as appetite, were observed. Necropsy did not detect abnormalities in body weight, weight of internal organs (liver, kidney, adrenal gland, left atrium, right atrium, ventricle, spleen, lung, testis, epididymis, prostate, and seminal vesicle), subcutaneous fat deposition, and fat deposition in internal organs (epididymis, prostate, mesentery, and retroperitoneal organs). Histological comparison of the internal organs (liver, kidney, stomach, intestine and adrenal) showed no significant difference between the experimental and control groups. Hematological tests showed no significant differences between the experimental and control groups in white blood cell count (WBC), percent Hematocrit (HCT), hemoglobin concentration (HGB), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), percent Lymphocyte (LYMPH), and platelet concentration (Plt). No adverse reactions were detected with respect to serum levels of glucose, lipids [ 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, bilirubin, aspartate Aminotransferase (AST), alanine Aminotransferase (ALT) and alkaline phosphatase (ALP). In short, no toxicity was found in this 9-month chronic toxicity study.
Industrial applicability of the invention
In the following experiments, it was shown that the rubber tree extract according to the present invention has (1) anticancer activity and (2) anti-metastatic activity. Because of these activities, the present invention is applicable to industries including (1) the pharmaceutical industry and (2) the food industry.
(1) Preliminary results of anticancer Activity
Three sets of experiments have been performed to evaluate the anticancer activity of latex extract of hevea brasiliensis according to the present invention: (a) a change in the volume of a tumor implanted in a nude mouse, (b) an effect on tumor angiogenesis in a nude mouse, and (c) an immunohistochemical study of markers or indicators associated with angiogenesis, inflammation, proliferation and apoptosis.
(a) Volume change of tumor implanted in nude mice
Male BALB/C nude mice (athymic mice) 6 to 8 weeks old weighing between 20 and 25 grams were injected with 10 injections6Individual CaSki cells (cervical cancer cells from human papillomavirus type 16) and allowed their normal life for about one month until the tumor volume is about 100 to 120 cubic millimeters as calculated using the linear measurement of vernier calipers shown in fig. 6. Mice in the experimental group received daily oral gavage of 60mg of latex extract 315/kg body weight of rubber tree for 28 days, while mice in the control group received water. Figure 7 shows the time course of relative tumor volumes in two groups of mice. Although the tumor volume in the control group increased about 7-fold within 4 weeks, the tumor volume in the group receiving rubber tree latex extract 315 remained essentially unchanged.
(b) Effect of angiogenesis on tumors implanted in nude mice
As shown in fig. 8, the 6 to 8 weeks old, weighing between 20 and 25 gramsMale BALB/C nude mice were equipped with 7 mm dorsal skin fold chamber and used 2X 106A CaSki cell was implanted into the chamber. Mice in the experimental group received daily oral gavage of 60mg of rubber tree latex extract per kg of body weight for 14 days, while mice in the control group received water. On day 14, the animals were anesthetized with 50mg/kg sodium pentobarbital. Plasma was labeled with fluorescein isothiocyanate-dextran (FITC-dextran). The dorsal skin fold chamber was carefully removed along with the skin. Angiogenesis was then observed and recorded by in vivo confocal fluorescence video microscopy. The results in fig. 9 qualitatively show that angiogenesis was less in the experimental group. Quantitative Image analysis of the percent capillary density using "Image Pro Plus" computer software (Media Cybernetics, inc., Rockville, MD, USA) demonstrated a statistically significant reduction in angiogenesis around cancer cells in the group receiving the rubber tree latex extract according to the present invention.
(c) Immunohistochemical investigation of markers or indicators associated with angiogenesis, inflammation, proliferation and apoptosis
When tumors from the above experiments were studied by immunohistochemistry, reduced angiogenesis around the tumors was found in the group that received 60mg of rubber tree latex extract per kg body weight per day for 28 days, as shown by the expression of the CD31 endothelial cell marker in fig. 10, and by the presence of the VEGF angiogenesis marker in fig. 11. In addition, the latex extract of Hevea brasiliensis also reduced the levels of the COX-2 inflammatory marker in FIG. 12 and the EGFR proliferation marker in FIG. 13. Finally, as shown in fig. 14, latex extract of hevea brasiliensis enhanced apoptosis of cancer cells.
(2) Preliminary results of anti-metastatic Activity
Metastasis of melanoma cells injected into the tail vein of nude mice was assessed two weeks after injection. At the total tissue level, metastatic melanoma colonies were visible to the naked eye as black spots on a red lung background. At the microscopic level, metastatic melanoma cells can be localized by immunohistochemical staining assay of galectin-3, a metastasis marker. Both of these experiments confirm the presence of anti-metastatic activity in the latex extract of hevea brasiliensis according to the present invention. The details of these experiments are as follows:
the anti-metastatic activity of the latex extract of Hevea brasiliensis according to the invention is shown in figure 105Individual melanoma B16-F1 cells were injected into nude mice in their tail vein. Fig. 15 shows the results of this experiment. In the experimental group that received 60mg/kg of rubber tree latex extract daily for two weeks after cancer cell injection (bottom row), the number of metastatic melanoma black spots was significantly less than in the group that received water instead of rubber tree latex extract (top row).
At the microscopic level, will already use 105Lung sections of nude mice injected with individual melanoma B16-F1 cells into their tail veins were stained for galectin-3 and observed under an optical microscope, as shown in fig. 16. (the staining of galectin-3 in this figure shows brown particles and tones superimposed on light blue cell outlines and dark blue nuclei, somewhat indistinguishable as shown in the black and white photomicrographs.) in the experimental group (right), which received 60mg/kg of rubber tree latex extract per day for two weeks after cancer cell injection, galectin-3 expression accumulated in a brown tone of approximately 50% of that expressed in the untreated group (center) which received water instead of rubber tree latex extract. In the negative control group (left), in which the nude mice were not injected with any melanoma cells, the specimens showed a slightly brown-colored staining of galectin-3, which corresponds to about 17% of the expression of galectin-3 in the untreated group (center). This preliminary result positively confirms the anti-metastatic activity of the latex extract of hevea brasiliensis according to the present invention.
In short, the preliminary results shown above lead to the conclusion that the rubber tree extract according to the present invention has anticancer and anti-metastatic activity. The reduction of angiogenesis and the enhancement of cancer cell apoptosis also indicate that the latex extract of hevea brasiliensis according to the present invention has therapeutic and prophylactic value. Thus, the present invention is applicable to industries including (1) the pharmaceutical industry and (2) the food industry.
(1) Is suitable for pharmaceutical industry
The present invention is applicable to the pharmaceutical industry because the rubber tree extract according to the present invention can be used as an ingredient of a pharmaceutical composition for treating cancer. As mentioned in the above section, acute, subacute and chronic toxicological studies in rats indicate that the rubber tree latex extract according to the present invention is non-toxic. A pharmaceutical composition for treating cancer may be prepared comprising the latex extract of hevea brasiliensis according to the present invention and one or more pharmaceutically acceptable substances selected from the group consisting of additives, binders, carriers, diluents, excipients, fillers, lubricants, solvents, and stabilizers.
Due to the anticancer and anti-metastatic activity of latex extract of hevea brasiliensis in nude mice, we would expect that the extract according to the present invention acts as an anticancer and anti-metastatic agent in a wide range of animals, including humans, as well as in a wide range of cancers, such as aids-related cancers, acoustic neuroma, adenoid cystic carcinoma, adrenocortical carcinoma, idiopathic myelination, alopecia, alveolar soft tissue sarcoma, angiosarcoma, aplastic anemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (bcc), brain stem glioma, carcinoid, childhood cancer, child soft tissue sarcoma, chondrosarcoma, choriocarcinoma, colorectal cancer, skin T-cell lymphoma, skin fibrosarcoma-protuberant small round cell carcinoma, ductal carcinoma, endometrial carcinoma, ependymoma, esophageal carcinoma, Ewing's sarcoma (Ewing's sarcoma), Extrahepatic bile duct cancer, ocular melanoma, retinoblastoma, fallopian tube cancer, Vanconi anemia, fibrosarcoma, gastric cancer, gastrointestinal tract cancer, gastrointestinal carcinoid, genitourinary system cancer, germ cell cancer, gestational trophoblastic disease, glioblastoma, glioma, gynecological cancer, hematological malignancy, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papilloma virus, hydatid fetus, hypopharyngeal carcinoma, intraocular melanoma, insular T cell carcinoma, Kaposi's sarcoma, Langerhan's cell histiocytosis (Langerhan's cell histiocytosis), leiomyosarcoma, leukemia, Li-Freumeti syndrome (Li-Fraumendrome), liposarcoma, lymphedema, lymphoma, Hodgkin's lymphoma (Hodgkin's lymphomas), non-Hodgkin's lymphoma (non-Hodgkin's lymphoma), Breast cancer in males, malignant rhabdoid cancer of the kidney, medulloblastoma, melanoma, merkel cell carcinoma (merkel cell cancer), mesothelioma, metastatic cancer, multiple endocrine tumors, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disease, nasopharyngeal cancer, wilms' tumor, neuroblastoma, neurofibromatosis, nemulin fragmentation syndrome, non-melanoma skin cancer, non-small lung cancer (nsclc), ocular cancer, esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, sinus nasalis, parathyroid cancer, parotid gland cancer, penile cancer, peripheral neuroectodermal cancer, polycythemia vera, rare cancers and related conditions, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rossman Mengdingson syndrome (rothomson syndrome), sarcoma, neuroblastoma, melanoma, neuroblastoma, melanoma, neuroblastoma, melanoma, neuroblastoma, melanoma, Schwannoma, sezary syndrome, small cell lung Cancer (sclc), small bowel Cancer, soft tissue sarcoma, squamous cell carcinoma (sec), synovial sarcoma, transitional cell carcinoma (bladder), transitional cell carcinoma (kidney-pelvis-/-ureter), trophoblastic carcinoma, urinary plaque protein, uterine sarcoma, Waldenstrom's macroglobulinemia, and Wilms' Cancer.
Alternatively, the anticancer and anti-metastatic activity of the latex extract of hevea brasiliensis according to the present invention should be applicable to cancers of various organs and organ systems, such as anus, bladder, bone, intestine, brain, breast, central nervous system, cervix, colon, endocrine glands, ear, endothelial cells, esophagus, eye, gall bladder, head, intestine, kidney, larynx, white blood cells, lip, liver, lung, mouth, nasal cavity, neck, nose, oral cavity, ovary, pancreas, pharynx, pituitary, prostate, rectum, salivary gland, skin, spinal cord, stomach, testis, thymus, thyroid, urethra, urinary system, uterus, vagina and vulva.
In the case where latex extract of hevea brasiliensis is used as an anticancer/anti-metastatic agent, it may be prepared in the form of aqueous solution, tablet, troche, powder, aqueous or oily suspension, emulsion, syrup, elixir. The route of administration can be enteral, intramuscular, intravenous, intranasal, oral, parenteral, rectal, subcutaneous, sublingual, sub-labial, transdermal, and transmucosal.
In addition, the latex extract of hevea brasiliensis may be combined with a therapeutically effective amount or one or more chemotherapeutic agents, such as alkylating agents, antimetabolites, antitumor antibiotics, kinase inhibitors, aromatase inhibitors, mitotic inhibitors, steroid hormones, and topoisomerase inhibitors.
(2) Application to food industry
The present invention is also applicable to the functional food industry, since the rubber tree extract according to the present invention can also be used as a food and beverage supplement having anticancer and cancer-preventing activities. Indeed, the rubber tree latex extract according to the present invention may be mixed with one or more edible ingredients selected from the group consisting of acids, acidity regulators, additives, anti-caking agents, antifoaming agents, antioxidants, baking agents, binders, carriers, color retention agents, diluents, emulsifiers, excipients, fillers, flavors, flavor enhancers, flour treatment agents, food coloring agents, glazing agents, humectants, lubricants, preservatives, solvents, stabilizers, sweeteners and thickeners.
The foregoing description of preferred and other embodiments is not intended to limit or restrict the scope or applicability of the inventive concepts conceived of by the inventors. In exchange for disclosing the inventive concepts contained herein, the inventors intend for all patent rights to be afforded by the appended claims. Accordingly, the inventive concepts contained herein are intended to embrace all such modifications and variations as fall within the scope of the appended claims or the equivalents thereof.
Claims (16)
1. A process for the preparation of an extract from the acidified latex of hevea brasiliensis, comprising the steps of:
obtaining a slurry released when the acidified hevea brasiliensis latex is calendered;
removing debris and microorganisms from the slurry;
removing protein from the slurry;
removing quebrachol and small polar molecules from the slurry;
optionally dissolving the solid powder remaining from the above step in water to form a slurry and water mixture;
further removing protein from the slurry, or the mixture of slurry and water, by treatment with an acid;
neutralizing excess acid;
precipitating low molecular weight carbohydrates with a solvent;
collecting the precipitate to obtain the extract; and
optionally freeze-drying the precipitate to obtain a precipitate,
wherein the removing of proteins comprises ultrafiltration by passage through an ultrafiltration membrane having a molecular weight cut-off of at least 3kD,
wherein the extract has anticancer activity against cervical cancer and melanoma.
2. The method of claim 1, wherein the technique for removing debris and microorganisms comprises filtration through a filter having a pore size of less than 0.5 microns.
3. The method of claim 1, wherein the removing quebrachol and small polar molecules comprises extracting and collecting the remaining solid powder with a mixture of one or more organic solvents in water.
4. The method of claim 1, wherein the further removing proteins by acid treatment comprises precipitating proteins with trichloroacetic acid.
5. The method of claim 1, wherein the alkalinity of the supernatant after the acid treatment step is neutralized by adding an effective amount of a water soluble base.
6. The method of claim 1, wherein the precipitating low molecular weight carbohydrates with a solvent comprises precipitating with a mixture of one or more organic solvents in water.
7. The method of claim 1, wherein the spray drying is performed at a temperature above 60 degrees celsius.
8. The method of claim 3, wherein the mixture of one or more organic solvents in water comprises at least 70% methanol.
9. The method of claim 3, wherein the extracting is optionally repeated.
10. The method of claim 4, wherein the concentration of trichloroacetic acid is from 1% to 50%.
11. The method of claim 4, wherein the acid treatment is performed at a temperature greater than 4 degrees Celsius for at least 0.1 minute.
12. The method of claim 6, wherein the mixture of one or more organic solvents in water comprises at least 70% ethanol.
13. The method of claim 6, wherein the precipitating step is performed at a temperature of less than 4 degrees Celsius for at least 5 minutes.
14. A composition comprising an acidified latex extract from hevea brasiliensis prepared according to any one of claims 1 to 13, said composition comprising one or more pharmaceutically acceptable substances selected from additives and carriers.
15. The composition of claim 14, wherein the additive is selected from at least one of a binder, a diluent, an excipient, a filler, a lubricant, a solvent, and a stabilizer.
16. A composition comprising the acidified latex extract from hevea brasiliensis prepared according to any one of claims 1 to 13 and one or more edible ingredients selected from the group consisting of acids, anti-caking agents, antifoaming agents, antioxidants, baking agents, binders, carriers, color retention agents, diluents, emulsifiers, excipients, fillers, flavors, odorants, flour conditioning agents, food coloring agents, polish, humectants, lubricants, preservatives, solvents, stabilizers and thickeners.
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Also Published As
| Publication number | Publication date |
|---|---|
| EP3510052A4 (en) | 2020-06-17 |
| EP3510052A1 (en) | 2019-07-17 |
| CN110023342A (en) | 2019-07-16 |
| WO2018044242A1 (en) | 2018-03-08 |
| MY197240A (en) | 2023-06-07 |
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