WO2005017134A2 - Systeme de defense antioxydant enzymatique nouveau et stable dans curcuma longa l - Google Patents

Systeme de defense antioxydant enzymatique nouveau et stable dans curcuma longa l Download PDF

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WO2005017134A2
WO2005017134A2 PCT/IN2004/000248 IN2004000248W WO2005017134A2 WO 2005017134 A2 WO2005017134 A2 WO 2005017134A2 IN 2004000248 W IN2004000248 W IN 2004000248W WO 2005017134 A2 WO2005017134 A2 WO 2005017134A2
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Prior art keywords
sod
formulation
isoenzyme
acid
zinc
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PCT/IN2004/000248
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English (en)
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WO2005017134A3 (fr
Inventor
Deeksha Dixit
Palpu Pushpangadan
Vinod Kumar Kochhar
Sunita Kochhar
Chandana Venketeshwara Rao
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Council Of Scientific And Industrial Research
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Publication of WO2005017134A2 publication Critical patent/WO2005017134A2/fr
Publication of WO2005017134A3 publication Critical patent/WO2005017134A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9706—Algae
    • A61K8/9717—Rhodophycota or Rhodophyta [red algae], e.g. Porphyra
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9794—Liliopsida [monocotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses

Definitions

  • the present invention relates to the isolation and characterization of a novel purified isoezyme of a heat stable, autoclavable, moicrowavable, superoxide dismutase (EC 1.15.1.1; hereinafter, referred to "SOD"), a process for the identification and extraction of the said superoxide dismutase from leaves/ rhizome of Curcuma longa L belonging to family Zingiberaceae.
  • SOD superoxide dismutase
  • Reactive oxygen species are constantly produced in plant, animal and human system.
  • Enzymatic antioxidant such as SOD is present in plant and animal cells to protect the cellular components against the deleterious effects caused by superoxide radical (hereinafter, referred to 0.sub.2.sup.-.).
  • SOD removes superoxide radicals by catalyzing a dismutation reaction, involving oxidation of the 0 2 to oxygen and reduction of another 0 to hydrogen peroxide as per the chemical reaction given below.
  • O.sub.2.sup.-. If O.sub.2.sup.-. is not removed, it reacts with H.sub.2 O.sub.2 and produces a highly reactive hyroxyl free radical, which causes deoxy ribonucleic acid DNA damage, membrane disruption, release of Calcium ions with in the cells leading to activation of proteases and nuc leases causing protein denaturation, lipid peroxidation in activation of membrane receptors and DNA mutation.
  • a living system is under oxidative stress, when such active oxygen mediated reactions are not being taken care of by enzyme systems.
  • SOD is an extremely important enzyme to manage oxidative stress in plants, animal and human systems.
  • the SOD can be Mn-SOD (SOD requiring manganese as a co-factor; localised in mitochondria; insensitive to potassium cyanide and hydrogen peroxide), Cu/Zn- SOD (SOD requiring copper and zinc as co-factors; localised in cytoplasm and chloroplast; sensitive to potassium cyanide and hydrogen peroxide) and Fe-SOD (SOD requiring iron as a co-factor; detected in microbes, blue-green algae cyanobacteria and in some higher plants).
  • SOD is also an important enzyme identified for imparting chilling tolerance/high temperature to the plants and in stresses for example, water stress, low temperature stress, light stress (particularly, high light intensity), salt stress, radiation stress and all other stresses wherein O.sub.2.sup.-. is generated in excess quantity to damage the system (Foyer, C. H., Descourvieres, P. and Kunert, K. J. 1994. Plant Cell Environ. 17: 507-523; Allen, R. D., 1995. Plant Physiol. 107: 1049-1054; Rizhsky L, Liang H and Mittler R. 2002 Plant Physiology 130: 1143-1151).
  • the enzyme has implications in all those diseases wherein 0.sub.2.sup.-. is produced in a quantity causing damage to the system such as radiation damage, mechanism of intestinal fat absorption, adult respiratory syndrome, rhemutoid arthritis (Halliwell B 1991 American Journal of Medicine 1991; 3-14).
  • SOD in animal system has following implications:
  • a sterile composition would be needed when SOD is to be injected in the body, and for that a heat stable, autoclavable, SOD would be an ideal choice.
  • an autoclavable SOD would be required which can function efficiently.
  • SOD with a long storage life functioning act at varying temperature will be an asset.
  • sterile SOD will also be a choice in the cosmetic and food industry (for preventing oxygen disorders) as well.
  • SOD is being used in a number of formulations developed for pharmaceutical and cosmetic applications using SOD as one of the important antioxidant ingredients. Availability of a SOD with heat stability, autoclavability and microvability will ensure a germ free sterile preparation with long shelf life [the maximum thermostability of SOD described so far is at ⁇ O.degree. C. (Gudin; Claude; Trezzy; Claudine 1996. U.S. Pat. No. 5,536,654); Bonaccorsi di Parti, M. C, Giartosio, A., Musci, G., Carlini, P. and CalabreH, L. (In Frontiers of Reactive Oxygen Species in Biology and Medicine. 1994. (Eds. Asada, K.
  • compositions for ameliorating free radical damage induced by tobacco products and environmental pollutants included, as active ingredients, reduced glutathione (0.5 mg) and a source of selenium (5 .mu.g) selected from the group consisting of elemental selenium, selenomethionine and selenocysteine.
  • the active ingredients were combined with suitable carriers and flavorings for their intraoral administration in concentrations for reducing free radical damage induced by tobacco products and other environmental pollutants to the oral cavity, pharynx and upper respiratory tract of a user and secondary smokers.
  • a germ free sterile preparation (because of autoclavability of SOD) will ensure no further infection to the smoker/secondary smoker to the affected portion.
  • compositions for cosmetic use as antiaging antiwrinkle and antistress cream for skin were inclusive of SOD along with peroxidase with a peroxidase specific reducing substrate.
  • SOD was obtained from germinated seeds of barley, soya, wheat and peas
  • peroxidase was obtained from black radish (or horseradish peroxidase) that was combined with an enzymatic substrate constituted by uric acid.
  • the other groups of peroxidases included lactoperoxidase, glutathione peroxidase and spinal cord peroxidase.
  • the composition also included a lipophilic antioxidant such as tocopherol, tocopherol acetate, tocopherol linoleate, tocopherol phosphate in an effective antioxidizing amount.
  • compositions for the treatment of psoriasis, seborrhoeic dermatitis and related skin and scalp conditions.
  • the said composition comprised L-glutathione (0.001% to 15% by weight) and selenomethionine a source of selenium in a suitable carrier for topical application.
  • the composition further included zinc pyrithione, N-acetyl-L-cysteine, SOD, zinc oxide, zinc pyrithione, vitamin E, and vitamin C.
  • the composition is encapsulated in protective membranes consisting of liposomes, nanospheres and glycospheres.
  • the suitable carrier was in the form, but not limited to, of a member selected from the group consisting of a solutions, lotions, creams, oils, gels, sticks, sprays, ointments, balms, shampoo and pastes.
  • the cream consisted of L-glutathione (reduced, 0.20%, L-selenomethionine (0.05%), N- acetyl-L-cysteine (0.25%), A,C,E Liposome (2.50%), Superoxide dismutase (0.25%)and Zinc pyrithione (0.25%).
  • the spray also consisted to these active ingredients.
  • the active ingredients in shampoo included L-glutathione (reduced, 0.20%, L-selenomethionine (0.025%), N-acetyl-L-cysteine (0.25%), A,C,E Liposome (2.00%), SOD (0.10%), Dex panthenol (0.5%) and Zinc pyrithione (1.0%).
  • U.S. Pat. No. 5,296,500 discusses formulations as an aerosol requiring the propellent to be added to a solution to be applied to the skin as a spray and topical pharmaceutical compositions as an pharmaceutically acceptable emollient (materials used for the prevention or relief of dryness, as well as for the protection of the skin).
  • U.S. Pat. No. 5,470,876 discribes the use of SOD as a topical anti-alopecia agent compounded in a topical formulation.
  • the pharmaceutical carriers for dispersion of SOD which were mentioned included water, urea, alcohols and glycols such as methanol, ethanol, propanol, butanol, ethylene glycol, propylene glycol, and the like.
  • Suitable water-in-oil emulsions that are commercially available were also mentioned which included Aquaphor, cold cream, Eucerin, hydrous lanolin, Hydrosorb hydrophilic petrolatum, Nivea, Polysorb, Qualatum and Velvachol.
  • Suitable oil-in-water emulsions that are commercially available were also mentioned and it included acid mantle cream, Almay emulsion cream, Cetaphil, Dermabase, Dermavan, hydrophilic ointment, Keri cream, Lubriderm cream, Multi base cream, Neobase cream, Unibase cream, Vanibase cream and Wibi.
  • the carrier described contained various other emollients, emulsifiers, water, perfumes, colorants, preservatives, and the like.
  • the topical formulation mentioned was in the form of a cream, lotion, shampoo, cream rinse, or the like.
  • Inventor selected SOD active compound from one or more of copper salicylate, copper aspirinate, indomethacin-copper and a complex of an amino acid or peptide and a transition metal.
  • the amino acid was selected from one or more of copper, iron, zinc and manganese.
  • the peptide consisted of glycine, histidine, lysine, arginine, cysteine or methionine.
  • U.S. Pat. No. 5,925,363 used SOD in combination with melanin pigments in a cosmetic, hygienic or pharmaceutical compositions to be employed topically to combat cutaneous aging and to protect the skin against the effects of the free radicals induced, for example, by atmospheric pollutants and/or by ultraviolet radiation, composition was also intended to protect the hair and mucosa against the effects of the free radicals.
  • Der opharmaceutical formulations apart from the SOD and melanin and active ingredient, mentioned in the patent included surfactants, colorants, perfumes, preserving agents, emulsifiers, liquid carriers such as water, fatty substances intended to form the fatty phase of emulsions (such as milks or creams), resins and the like. These compositions were prepared by the usual methods.
  • cleansing creams for protecting or care of the face, the hands or the body (for example day creams, night creams, makeup removal creams, foundation creams, sun creams), fluid foundations, makeup removal milks, body protection or care milks, sun milks, lotions, gels or mousses for skin care, such as cleansing lotions, sun lotions, artificial tanning lotions, compositions for the bath or deodorizing compositions containing a bactericidal agent.
  • the compositions could also consist of solid preparations forming soaps or cleansing cakes.
  • the compositions could also be packaged in the form of an aerosol composition also containing a pressurized propellent agent.
  • compositions for hair could be presented in the form of aqueous, alcoholic or hydroalcoholic solutions or in the form of creams, gels, emulsions, mousses or else in the form of an aerosol composition also containing a pressurized propellent agent.
  • various adjuvants were also mentioned that are usually present in these compositions for hair, for example liquid or gel-form carriers, perfumes, dyes, preserving agents, thickening agents and the like.
  • compositions for hair care forming, for example, creams, lotions, gels, serums or mousses for the care of the scalp, shampoos, hairsetting lotions, treating lotions, styling creams or gels, dye compositions (especially oxidation dyes) optionally in the form of dyeing shampoos, restructuring lotions for hair, permanent wave compositions (especially compositions for the first step of a permanent waving), lotions or gels to combat hair loss, and the like.
  • the compounds of the invention may be especially: shampoos containing, besides a SOD and the melanin pigments a cationic, anionic or nonionic detergent, dyeing compositions including coloring shampoos which contain dyes or usual dye precursors, compositions for the first step (reduction step) of a deformation of hair, containing reducing derivatives such as mercaptans, sulphites and the like, compositions for slowing down the loss of hair and for promoting fresh growth of hair, containing compounds such as minoxidil (2,4-diamino-6-piperidino-3-pyrimidine oxide) and its derivatives, diazoxide (7-chloro-3-methyl-l,2,4-benzothiadiazine, 1,1-dioxide) and phenytoin (5,5-diphenyl imidazolidine-2,4-dione).
  • dyeing compositions including coloring shampoos which contain dyes or usual dye precursors, compositions for the first step (reduction step) of a deformation of hair,
  • the cosmetic composition of the invention may also be for oral and dental use, for example a toothpaste.
  • the composition would contain usual adjuvants and additives for compositions for oral use and especially surface-active agents, thickening agents, moisturizers, polishing agents such as silica, various active ingredients such as fluorides, in particular sodium fluoride, and optionally sweetening agents such as sodium saccharinate.
  • the cosmetic treatment was intended to be used in the form of creams, of gels, of serums, of lotions, of makeup removal milks or antisun compositions to the skin or to the hair, application of a hair lotion to wet hair, shampooing or application of toothpaste to the gums to obtain the desired protection effect.
  • This cosmetic treatment process was intended in particular to maintain the keratinous structure of the skin or of the hair so as to avoid their degradation and the harmfull effects of such a degradation under the influence of the free radicals induced especially by atmospheric pollutants.
  • U.S. Pat. No. 5,137,820 describes the use of medium higher fatty acid glyceride for preparation of the composition for oral administration of SOD.
  • Representative examples of such fatty acid glyceride include the mono-, di- and triglycerides of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, linoleic acid, linolenic acid or the like. These fatty acid glycerides can be used singly or in combination.
  • U.S. Pat. No. 5,897,879 discloses a sustained-release pharmaceutical delivery system for the administration of an antioxidant drug to a patient in need of such drug to reduce increased formation of active oxygen species.
  • the delivery system comprised antioxidant drug in combination with a polymeric matrix which does not interact with the antioxidant drug or a mixture of such polymers.
  • the various formulations may be prepared by mixing the polymer or mixture of polymers with the active antioxidant drug, by methods as described in the following examples as well as by other methods known to the man versed in the art.
  • the delivery system of the present invention may be adapted to dosage forms for local, for example opthalmic, and transdermal administration, as. well as implants which will release the active antioxidant drug in a controlled manner.
  • Particular forms suitable for such administration include, for example, films.
  • the film could be prepared from ethanolic or chloroformic solutions of the polymers.
  • the drug was also . released faster from films comprising polyethylene glycol.
  • the amount of active antioxidant drug could vary as desired for a therapeutically effective amount and might depend on the patient's age, sex, weight, physical condition, disease or condition to be treated, and other medical criteria as well as on the relative efficacy of the drug. This effective amount may be determined by techniques known in the art. For example, in case the antioxidant drug is vitamin E, the amount of the drug is a dosage unit form may be from about 10 IU to about 1000 IU.
  • U.S. Pat. No. 5,942,245 describes the use of SOD in liposomes, optionally mixed with hyaluronic acid and/or at least one physiologically acceptable carrier, and other optional additives, to prepare a pharmaceutical composition useful against increased concentrations of superoxide radicals and/or the damage caused thereby.
  • These compositions can be administered topically, orally and/or parenterally to prevent and/or heal burns, acne, skin lesions due to radiation, inflammations, rheumatic and arthritic diseases, bronchitis, ARDS, emphysema, allergic oedemas and other inflammatory process, possibly trigged by microbial infections.
  • Another U.S. Pat. No. 5,827,886 reveales a composition and method of reducing the inflammation and pain of various clinical entities including, but not limited to, the arthritis of rheumatoid arthritis and the other so-called autoimmune diseases, and osteoarthritis, the common syndrome of low back pain, myalgias, neuropathies, such as that of diabetes, and neuralgias, such as after shingles (herpes) as well as any cutaneous manifestations, if any, of these conditions.
  • various clinical entities including, but not limited to, the arthritis of rheumatoid arthritis and the other so-called autoimmune diseases, and osteoarthritis, the common syndrome of low back pain, myalgias, neuropathies, such as that of diabetes, and neuralgias, such as after shingles (herpes) as well as any cutaneous manifestations, if any, of these conditions.
  • the preparations described may be in the forms of creams, lotions, solutions including sprays and aerosols and in roll-on dispensing bottles, ointments, gels, balms, patches, or emulsions as are known in this industry.
  • Other free radical scavengers, antioxidants, anti-inflammatory agents, and local anesthetics, particularly capsaicin could be included in the composition to deal with the inflammation and chronic pain characteristic of these diseases and clinical syndromes.
  • vitamins E tocopherols
  • green tea and pycnogenols and also steroids, non-steroidal anti- inflammatories, capsaicin extract, tissue respiratory factor and the local anesthetics of the caine family.
  • U.S. Pat. No. 5,875,798 reveales a method of treating oral and systemic diseases which included impregnating or coating a toothpick with active therapeutic agents and rubbing the toothpick against mouth tissue to release the active therapeutic agents onto the tissue for penetration through the tissue.
  • the active therapeutic agent was selected from the group consisting of, but not limited to: zinc sulfate, zinc chloride, zinc acetate, zinc phenol sulfonate, zinc borate, zinc bromide, zinc nitrate, zinc glycerophosphate, zinc benzoate, zinc carbonate, zinc citrate, zinc hexafluorosilicate, zinc diacetate trihydrate, zinc oxide, zinc peroxide, zinc salicylate, zinc silicate, zinc stannate, zinc tannate, zinc titanate, zinc tetrafluoroborate, zinc gluconate, and zinc glycinate.
  • An additional therapeutic agent may also be impregnated or coated on the toothpick, for example, antimicrobials, antibiotics, antioxidants, anti-plaque agents, analgesics, anti-tartar agents, anti-caries agents, hemostatic agents, anti-inflammatory agents, hormones, bleaching agents, vitamins, vaccines, caffeine and monoclonal antibodies.
  • antioxidants enhance the healing of infected and noninfected- wounds by reducing the damage caused by oxygen radicals, these include but are not limited to: vitamin E, pyruvate .beta.-carotene, selenium, N-acetylcysteine, vitamin C, antioxyenzymes such as SOD, catalase, glutathione peroxidase, and glutathione reductase together with the enzymes of the pentose monophosphate shunt pathway that regenerate NADPH.
  • the enzyme plays crucial roles in plant industry as well.
  • a SOD with long storage life will aid in protecting the plant against oxidative stress during winter months.
  • a high thermal stability of the enzyme would be a desirable feature for the plant experiencing intense photoinhibition during hot summer and drought stress.
  • WO 03/051380 wherein a composition obtained from the lipid soluble extract of rhizomes and leaves oi Curcuma species of Zingiberaceae family, useful for the treatment of neurocerebro vascular disorders, said composition comprising fraction A consisting of arturmerone and turmerone, and/or along with fraction B consisting of curcumene and zingiberine, and/or fraction C consisting of germacrone, curcumerone, zedoarone, sedoarondiol, isozdedoaronidiol, curcumenone, and curlone, and/or pharmaceutically acceptable additives and a method of treating neurocerebrovascular disorders in animals including humans using said composition by administering therapeutically effective amount of lipid soluble extract.
  • Chloroplast localised Cu/Zn-SOD was found to have highest activity at 10C, whereas Mn-
  • This invention relates to the isolation, purification and characterization of a novel heat stable, autoclavable and microwavable (enzymatic antioxidant) isoenzyme of superoxide dismutase extracted from the leaves/rhizomes of Curcuma longa L. an easily growing plant in varying climatic conditions.
  • Another objective is a free oxygen radical scavenging enzyme and also can be used in cheap germ-free sterile preparations for pharmaceuticals, cosmetics and food industry.
  • Yet another object of the present invention is to provide a SOD, which can function efficiently at varying temperatures.
  • Still another object of the present invention is to provide SOD in which the feature of autoclavability and functioning at varying temperatures , is possessed by the same SOD.
  • Yet another object of the present invention is to provide a method to identify the isoenzyme which show the activity at temperatures higher than +1 OO.degree. C.
  • Another object of the present invention is to provide a process to purify an autoclavable SOD enzyme which can function between the temperatures ranging between 25 C to +100 degree. C.
  • Yet an other object of the present invention is to provide a simple, single step procedure to identify and characterize the novel heat stable, autoclavable and microwavable SOD
  • the present invention relates to the process for identification and the extraction of SOD from Curcuma leaves/rhizomes which,
  • (a) is autoclavable. under a pressure of 5-20 bars ; 30min to ensure a germ free sterile SOD,
  • (c) has free radical scavenging capability ranging between zero (O.degree. C.) to +1 OO.degree. C.
  • this invention describes the procedure to purify one of the isoenzymes of SOD showing the above mentioned properties from the plant Curcuma and can be used in medical, cosmetic and food industry/research, and also in producing transgenic plant resistance/tolerance to biotic and abiotic stresses in which, the damage is mediated through the production of 0.sub.2.sup.-..
  • the invention provides a novel purified isoenzyme of a heat stable, microwavable and autoclavable superoxide dismutase extracted from the leaves/rhizome of Curcuma longa L having the following characteristics:
  • the invention provides a method for identification of the target isoenzyme of the superoxide dismutase said method comprising the steps of:
  • the invention provides a method for the preparation of purified novel isoenzyme of SOD wherein the said method comprises the steps of:
  • SOD peak obtained after FPLC and concentrating using a protein concentrator column / glycerol
  • the invention provides a method for the preparation of novel heat stable, autoclavable and microwave able isoenzyme of SOD present in crude extract as well as in purified form from leaves and rhizomes of Curcuma longa L
  • the invention provides that the purified isoenzyme in young leaves possess anti-inflammatory and anti-bacterial property.
  • the invention provides that the purified isoenzyme in mature leaves possess anti-inflammatory and anti-bacterial property.
  • the invention provides that the purified isoenzyme in dried leaves /rhizomes possess anti-inflammatory and anti-bacterial property.
  • the invention provides that the novel SOD is present in young, mature fresh or dried , heated, autoclaved, microwaved leaves/rhizomes
  • the invention provides a quick one step method to identify , characterize, and isolate the novel SOD by excising the target band from the gels and eluting the gel in buffer.
  • the invention provides a method for the preparation of novel isoenzyme of SOD wherein the source of novel SOD may be selected from other plants.
  • the invention provides a method where the source of novel SOD may be further selected from Aconitum sp., Artemisia sp., Trigonella emodi, Hippophae rhamnoides, Hippophae ***a, Arnaebia Vietnameseroma, Amaranthus, Chenopodium,
  • Dactylorhiza hatagirea Dactylorhiza hatagirea, Aquilegia sp., Ranunculus sp., Rosa webbiana, Podophyllum sp., Ephedra gerardiana, Caragana jubata, Geum elatum, Picrorhiza kurooa, Ginger, Spinach, Phaseolus and other flora and micro flora, and fauna which would also yield novel SOD.
  • the invention also provides a formulation comprising a plant superoxide dismutase (SOD) in isoenzyme as an active ingredient, together with reduced glutathione, source of selenium, carriers, flavouring agents and oxidants.
  • SOD plant superoxide dismutase
  • the invention also provides a formulation comprising a plant superoxide dismutate (SOD) isoenzyme together with an effective amount of cosmetically acceptable peroxidase, cosmetically acceptable peroxidase cofactor, solvents, carriers and conventional additives
  • SOD plant superoxide dismutate
  • the invention also provides a 88% higher amount of peroxidase in the leaves of Curcuma longa as compared to 12% in rhizome.
  • the invention also provides a formulation comprising isoenzyme of SOD, along with antioxidants such as, but not limited to, L-glutathione (0.001% to 15%) by weight) and selenomethionine a source of selenium in a suitable carrier for topical application for the treatment of psoriasis, seborrhoeic dermatitis and related skin and scalp conditions.
  • antioxidants such as, but not limited to, L-glutathione (0.001% to 15%) by weight) and selenomethionine a source of selenium in a suitable carrier for topical application for the treatment of psoriasis, seborrhoeic dermatitis and related skin and scalp conditions.
  • the invention provides a formulation comprising plant superoxide dismutase (SOD) isoenzyme as claimed in claim 1 and capable of being used for topical application either as, but not limited to, solutions or dispersions of the lotion or serum type, emulsions of liquid or semiliquid consistency of the milk type, which are obtained by dispersing a fatty phase in an aqueous phase of oil-in-water or vice versa i.e. water-in-oil or suspensions-or emulsions of soft consistency of the cream or gel type, or else microgranulates, or vesicular dispersions of ionic and/or nonionic type.
  • the invention provides a drug delivery system comprising purified isoenzyme of SOD together with antioxidant drug in combination with a polymeric matrix, which does not interact with the antioxidant drug or a mixture of such polymers.
  • SOD for preparation of formulations involving SOD
  • water-in-oil emulsions that are commercially available such as, but not limited to, Aquaphor, cold cream, Eucerin, hydrous lanolin, Hydrosorb, hydrophilic petrolatum, Nivea, Polysorb, Qualatum and Velvachol.
  • SOD oil-in-water emulsions selected from acid mantle cream, Almay emulsion cream, Cetaphil, Dermabase, Dermavan, hydrophilic ointment, Keri cream, Lubriderm cream, Multibase cream, Neobase cream, Unibase cream, Vanibase cream and Wibi.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isoenzyme of SOD as claimed in claim 1 , a pharmaceutically acceptable carrier and optionally at least one therapeutic agent selected from the group consisting of antimicrobials, antibiotics, antioxidants, anti-plaque agents, analgesics, anti-tartar agents, anti-caries agents, hemostatic agents, anti-inflammatory agents, anti-HIV agent , anti-cancer agent, a therapeutic agent for the treatment of neurovascular disorders, hormones, bleaching agents, vitamins, vaccines, caffeine and monoclonal antibodies.
  • a therapeutic agent selected from the group consisting of antimicrobials, antibiotics, antioxidants, anti-plaque agents, analgesics, anti-tartar agents, anti-caries agents, hemostatic agents, anti-inflammatory agents, anti-HIV agent , anti-cancer agent, a therapeutic agent for the treatment of neurovascular disorders, hormones, bleaching agents, vitamins, vaccines, caffeine and monoclonal antibodies.
  • the cerebrovascular disorders are selected from a group comprising ischaemia, stroke, post-stroke injury, hemorrhage, reperfusion injury, thrombosis, vasoconstriction, nitric oxide-induced free radical oxidative damage, infraction, inflammation, and Alzheimer's disease.
  • FIG. 1 represents effect of heat on isoenzymes of SOD (Superoxide dismutase)in young and old leaves and rhizome of Curcuma longa L.
  • SOD Superoxide dismutase
  • the enzyme preparation in each case was heated at 80 degree C for 10 and 20 minutes.
  • FIG. 2 represents effect of heat on isoenzymes of POD (Peroxidase) in young and old leaves and rhizome of Curcuma longa L.
  • the enzyme preparation in each case was heated at 80 degree C for 2 and 5 minutes.
  • FIG. 3 represents the SOD activity of purified heat stable, autoclavable and microwavable isoenzyme from Curcuma Longa L leaves and rhizomes.
  • the isoenzyme shows native molecularmass of 32 kDa as deduced from the protein marker.
  • First lane molecular mass marker for native gel
  • lane 2 purified isoenzyme from young leaves
  • lane 3 purified isoenzyme from old leaves
  • lane 4 purified isoenzyme from rhizome.
  • FIG. 4 represents the antifungal activity of purified isoenzyme of SOD from young, mature leaves and rhizome of Curcuma longa L.
  • the potato dextrose agar plate was spread with the fungal strain (Aspergillus niger) and the sterilized whatman 3 filter paper disks were soaked in the concentrated purified SOD isoenzyme preparation and were placed on the fungal spread plate. The clear areas around the disks represent the antifungal activity.
  • FIG. 5 represents the antibacterial activity of purified isoenzyme of SOD from young, mature leaves and rhizome of Curcuma longa L.
  • the LB (Luria broth) agar plate was spread with the bacterial strain (E coli strain DH5 alpha) and the sterilized whatman 3 filter paper disks were soaked in the concentrated purified SOD isoenzyme preparation and were placed on the bacterial spread plate. The clear areas around the disks represent the antimicrobial activity.
  • SOD as disclosed in the present invention is extracted from leaves and rhizome of Curcuma longa L.
  • Turmeric plant (Curcuma longa L.) belonging to family Zingiberaceae is a perennial, herbaceous tropical plant indigenous to Southern-Asia and is cultivated for its underground rhizomes.
  • the turmeric plant is commonly called haldi; it is known in Chinese as jiang huang.
  • the turmeric rhizome is very aromatic, with a musky odour and yellow colour.
  • Turmeric plant (Curcuma longa) is a robust perennial with a short stem and tufted leaves. The pale-yellow flowers are found in dense spikes, topped by a tuft of pinkish bracts.
  • the rhizomes which yield the colorful condiment, are short and thick with blunt tubers.
  • Turmeric plant is extensively cultivated all over India.
  • India is the principal supplier of turmeric to the world markets.
  • the total acreage under turmeric in India has been estimated variously from 60,000 to 100,000 acres, and the production is nearly 100,000 tons of rhizomes per annum.
  • the Curcuma leaves which are considered a post harvest waste have been used as source for novel isoenzyme of SOD, which is heat stable (shows the activity at temperatures higher than +50.degree. C), is autoclavable, microwaveable.
  • the enzyme has been purified and characterized.
  • a more complete purification of SOD is accomplished by size fractionation on a size exclusion column of the extract obtained after affinity chromatography /ion exchange in order to eliminate the proteins carrying same charge but different molecular weight. Size fractionation has been accomplished using a fast protein liquid chromatography system to save on time.
  • a one step purification has been achieved by excising the target gel band with SOD activity and eluting in Tris-Hcl buffer (pH 6.8, 0.125mM ) containing 1 mM EDTA.
  • SOD has been characterized in terms of its molecular weight and, the isoenzyme was characterized further by inhibitor studies by treating the gel with 2- lOmM KCN /2-10 mM H 2 0 2
  • the first step in the extraction of this novel enzymatic antioxidant involves the extraction of these enzymes. Curcuma leaves 500 gm are taken and chopped into small pieces. These are then ground using liquid nitrogen in a grinder or pestle and mortar using phosphate buffer (2 ml for 1.0 g of fresh weight of leaf tissue , 50-100 mM, pH 7.0-7.8, temp 0-4°C containing ImM ⁇ -mercaptoethanol( ⁇ me), 2% polyvinyl-polypyrrolidone (PVP) and ImM EDTA). The filtrate is then filtered through 8 layers of cheesecloth. The homogenate is then centrifuged at 8000-12,000 rpm for 20 at 4°C. The supernatant is then used as crude enzyme extract.
  • phosphate buffer 2 ml for 1.0 g of fresh weight of leaf tissue , 50-100 mM, pH 7.0-7.8, temp 0-4°C containing ImM ⁇ -mercaptoethanol( ⁇ me), 2% polyvinyl-polypyr
  • the enzyme activity is measured by light reaction of NBT using the method Beauchamp, C and Fridovich. Inhibition in photoreduction of nitroblue tetrazolium (NBT) by SOD as described by Beauchamp and Fridovich (Anal. Biochem. 1971; 44: 276-287).
  • Assay mixture contained 0.05 M potassium phosphate buffer (pH ranging between 6.5 to 9.0), 5J.times.10.sup.-5 M nitroblue tetrazolium (NBT), 9.9.times.l0.sup.-3 M methionine, 1.17.times.l0.sup.-6 M riboflavin and 0.025%) Triton X-100 in a total volume of 3.0 ml.
  • Reaction (performed in 10- 20 ml glass vial) was initiated by illuminating the reaction with light intensity of 1000 ,mu. Einstein/m.sup.2 /second using fluorescent tubes. The reaction was terminated after 10 min and the absorbance was read at 560 nm in a spectrophotometer ( Bausch and Lomb Spectronic 2000).
  • SOD superoxide dismutase
  • POD peroxidase
  • CAT Catalase
  • Table - 1 Superoxide dismutase (SOD), peroxidase (POD) and Catalase (CAT) activity in leaves and rhizomes of Curcuma.
  • the data represents the mean values of three different independent preparations.
  • YL Young Leaf
  • OL Mature Leaf
  • R Rhizome
  • SOD Superoxide dismutase
  • POD Peroxidase activity
  • CAT Catalase.
  • Table 2 Effect of temperature on the antioxidant enzymes (superoxide dismutase- SOD) in leaves and rhizomes of Curcuma longa L.
  • the data represents the mean values of three different independent preparations.
  • the crude enzyme was assayed for POD activity at room temperatures after preheating the enzyme at temperatures ranging between 40 to 1 OO.degree. C. by the method as described in Example 2.
  • the peroxidase activity was 74.5% at 40°C, 70% at 60C , 10% at 80C and 2.8% at 100 C.
  • the activity of rhizome was much lower ,2.6% at 40C; 1.1% at 60C; 0.2% at 80C and 0% at 100 C/
  • the data represents the mean values of three different independent preparations.
  • thermo stability was performed by autoclaving the crude enzyme from leaves and rhizome for varying time periods and then performing assay at room temperature.
  • the assay was done by the method described in example 1.
  • the data represents the mean values of three different independent preparations.
  • thermo stability was performed by microwaving the crude enzyme from leaves and rhizome for varying time periods and then performing assay at room temperature.
  • the assay was done by the method described in example 1.
  • the data represents the mean values of three different independent preparations.
  • EXAMPLE 7 Method of Identification of the Target heat stable, autoclavable and microwavable Isoenzyme of the SOD for the Purpose of Purification.
  • the above Examples 3and 5 are suggestive of novel SOD in Curcuma leaves and rhizomes not described hitherto. Hence, it was essential to know if all the isozymes or any one of them depicts the above mentioned properties.
  • a method was, therefore, developed to monitor the activity of various isoezymes at vaiying temperatures (25-100 degree C). The isoezymes showing good activity at these temperatures was/were targeted for the purpose of purification and tested for autoclavability. Since crude extract shows the SOD activity after autoclaving, it was contemplated that any isoenzyme showing prominent activity at this temperature amplitude should show the property of autoclavability as well.
  • the targeted novel isozyme of SOD was purified as follows not described hitherto. Hence, it was essential to purify the enzyme and then study the properties.
  • the gel was soaked in a fixative solution (400 ml of methanol, 70 ml of acetic acid and 530 ml of water; all mixed together) for 2 hours and then soaked in a staining solution (0.25 g Coomassie Brilliant Blue R dissolved in 500 ml of fixative solution) for 18 hours.
  • the gel was destained by dipping in fixative solution for 20 hours. Four to five changes of the fixative solution were required for proper de-staining of the gel.
  • Gel was then transferred into 7% acetic acid solution for storage. A single protein band with molecular mass of 32 kDa was obtained after purification (as shown in Fig.3).
  • the band was cut with a razor blade froml0% Native gel , soaked for 30 minutes with the occasional swirling in 10 ml of (0.125 M Tris-Hcl buffer pH 7.0) buffer. SOD activity was checked after heating at 80 degreeC for 20 min. and autoclaving at 20 bars for 10 min. (method described in example 1).
  • the data represents the mean values of three different independent preparations.
  • SOD SOD requiring manganese as a co-factor, insensitive to potassium cyanide ana hydrogen peroxide
  • Cu/Zn-SOD SOD requiring copper and zinc as co-factors; sensitive to potassium cyanide and hydrogen peroxide
  • Fe-SOD SOD requiring iron as a co-factor; sensitive to hydrogen peroxide but insensitive to potassium cyanide
  • the data represents the mean values of three different independent preparations.
  • Table-8 Effect of storage temperature on the activity of purified SOD assayed at room temperature.
  • the data represents the mean values of three different independent preparations.
  • Rats were injected with 0.1 ml of 1 %> carrageenan into the subplantar region of the left hind paw (Winter et al., 1962).
  • the paw was marked with ink at the level of lateral malleolus and dipped in perspex cell up to this mark.
  • the paw volume was measured with Ugo Basile Plethysmometer (No: 6142, 7140 Comerio-varese, Italy) before and 60,120,180 and 240 minute's after injecting the carrageenan suspension.
  • the anti-inflammatory activity is observed in the leaves and a very little effect is seen in the rhizomes. In terms of statistical significance the results obtained in the leaves (antioxidant enzymes) are statistically significant (Table 9).
  • Table 9 The anti-inflammatory effect of the antioxidant enzymes of Curcuma leaves and rhizomes
  • the data represents the mean values of three different independent preparations.
  • Leaf 55 Rhizome 45 The data represents the mean values of three different independent preparations. Explanation of the Table 10: The circles around the disks were observed to find out the antifungal activity. The clear zones around the disc of the agar medium in the leaf (L) and rhizome (R) enzymes were measured and there %> inhibition is higher in the leaf than that of the rhizome. Aspergillus niger was used as standard fungal strain.
  • Antibacterial property of antioxidant enzymes of Curcuma leaves and rhizomes Antibiotic property of curcuma leaves and rhizomes, 3.5 gm of L.B.
  • Agar Lia Broth Agar
  • DH5 ⁇ strain of E-coli was used as bacterial strain (Table 11).
  • the disks of unifo ⁇ n size were cut from sterilized what man 3 filter paper.
  • SOD extracts from leaf and rhizome tissues (preparation method described earlier) were used for the test. 5-10 ul of extract was poured onto disks and were placed in the petri dishes with the bacterial strain spread over it.
  • the petridishes were then sealed and kept overnight at 37° C in an incubator. Whole procedure was done under sterilized conditions. The antibacterial activity was taken from the diameter of clear area around the filter paper disks soaked in the purified enzyme. The percent activity is given in arbitrary units depending upon the diameter of the clear area.
  • Table 11 The antibacterial activity of the antioxidant enzymes of Curcuma leaves and rhizomes
  • the data represents the mean values of three different independent preparations.
  • Table 11 The circles around the disks were observed to find out the antibiotic activity.
  • Ampicillin was used as standard synthetic antibiotic.
  • the percent activity is given in arbitrary units depending upon the diameter of the clear area (fig 5).
  • Table 12 Comparative account of SOD activity from crude extracts of leaves and rhizome of Curcuma, leaves of Spinach, Phaseolus and rhizomes of Ginger
  • the data represents the mean values of three different independent preparations.
  • Table 12 shows that leaves of Curcuma have the highest amount of SOD activity in terms of Units/mg protein and hence are the best source of SOD.
  • Phaseolus 100 0 0 0 0 0 0 0 0
  • the data ref resents the n lean values o ⁇ three differe nt independe nt preparatioi is.
  • the data represents the mean values of three different independent preparations.
  • the data represents the mean values of three different independent preparations.
  • a SOD enzyme has been identified from leaves and rhizomes of Curcuma longa Lwhich is heat stable, autoclawable and microwavable.
  • the isozyme can be autoclaved to give a germ free sterile preparation. It can function at temperature ranging from zero to, 1 OO.degree. C.
  • the identified SOD isoenzyme functions more efficiently from young leaves than old leaves or rhizome.
  • a SOD which remains stable at room temperature (25.degree. C.) for five days without adding any stabilizing agent. It remains stable without losing appreciable activity between 4to -20-degree C for 18 months.
  • This isoenzyme can be easily obtained from the Curcuma plant which grows easily under varying environmental gradients.
  • the activity of isoenzyme has been compared with SOD from phaseolus and spinach.
  • the SOD from phaseolus and spinach leaves is much less stable, being completely inactivated at boiling temperatures and is not autoclavable and microwable as the activity is completely lost on autoclaving and microwaving.
  • Novel SOD isoenzyme from Curcuma leaves and rhizome has also been compared with SOD from ginger rhizome.
  • the activity from ginger rhizome is not heat stable, autoclavable or microwable.
  • Curcuma longa L grows in a wide range of environmental regimes and can be easily acclamatized to any temperature change.
  • the novel SOD isoenzyme described in this patent is heat stable and retains its activity from -20 degree-C to 100 degree C without any appreciable loss of activity. This isoenzyme can be stored more than 18 months at -10 to 4 degree C.
  • This isoenzyme is also autoclavable ( retains 50-70 %> activity in crude form and 60-90 % in purified form when autoclaved for 1 to 30 min), retains appreciable activity even after exposing to 25-100 degreeC and also retains more than 50%> activity when microwaved for 1-5 minutes.
  • Fig. 1 shows the SOD fingerprints of crude enzyme from leaves (young and old) and rhizome when heated for 10 and 20 minutes at 80 degree C.
  • Fig 2 shows the POD finge ⁇ rints of crude enzyme from leaves (young and old) and rhizome when heated for 10 and 20 minutes at 80 degree C.
  • Fig 3 Shows one band purification of heat stable, autoclavable and microwavable SOD isoenzyme from young , old leaves and rhizome of Curcuma longa L.
  • Figure 4 The anti-fungal effect of the antioxidant enzymes of Curcuma leaves and rhizomes. Nos. 1 No enzyme; 2 -4 SOD from young leaves of Curcuma; 5-6 SOD from young rhizome of Curcuma; 7-8 SOD from old leaves of Curcuma; 9-10 SOD from old rhizome of Curcuma.
  • Figure 5. show the antibacterial activity of the purified isoenzyme.
  • C No enzyme
  • YL SOD from young leaves of curcuma
  • OL SOD from old leaves of Curcuma
  • OR SOD from old rhizome
  • YR Sod from young rhizome.

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Abstract

La présente invention concerne l'isolation et la caractérisation d'une iso-enzyme dismutase superoxyde (SOD) purifiée micro-ondable, résistant à l'autoclave et thermiquement stable, extraite de feuilles et de rhizomes de Curcuma longa L. Elle possède des propriétés d'élimination de radicaux libres et cette activité d'élimination reste intacte avant et après un passage à l'autoclave (6-20 bars) sur une durée allant jusqu'à 30 minutes, un chauffage (0-60 min à 30-1000°C) et un passage au micro-onde (1-5 min). La forme de SOD est stable avec 33 % d'une activité d'élimination de 02 conservée jusqu'à six jours à température ambiante (25-30 °C). Cette forme est stable au moins pendant dix-huit mois à 4 °C, gardant 62 % de l'activité et, 78 % de l'activité à -10 à 20 °C, contenant 30 % de glycérol dans un congélateur sans aucune infection ni aucune contamination. Cette enzyme a été purifiée par chromatographie par infinité. Cette iso-enzyme de SOD possède une masse moléculaire de 32 kDa dans des conditions non dénaturantes avec une masse similaire dans des conditions dénaturantes, montrant ainsi sa nature monomère. Les études d'inhibiteur ont montré que cette isoforme d'enzyme nécessite Cu/Zn comme cofacteur et possèdent des propriétés antifongiques, anti-inflammatoires et antibactériennes. Cette invention concerne aussi le procédé de préparation de cette iso-enzyme purifiée de dismutase super peroxyde résistant à l'autoclave et des préparations contenant cette dismutase superoxyde résistant à l'autoclave.
PCT/IN2004/000248 2003-08-19 2004-08-19 Systeme de defense antioxydant enzymatique nouveau et stable dans curcuma longa l WO2005017134A2 (fr)

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