WO2005017134A2 - Isozyme of autoclavable superoxide dismutase (sod) derived from curcuma longa l - Google Patents

Isozyme of autoclavable superoxide dismutase (sod) derived from curcuma longa l Download PDF

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Publication number
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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French (fr)
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WO2005017134A3 (en
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 WO2005017134A3 publication Critical patent/WO2005017134A3/en

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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 tibetana, 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.

Abstract

The invention relates to the isolation and characterization of a novel heat stable, autoclavable, microwaveable purified superoxide dismutase (SOD) isoenzyme, extracted from the leaves and rhizomes of Curcuma longa L. It has free radicals scavenging property and the scavenging activity remains intact before and after autoclaving (6-20 bars) upto 30min; heating (0-60 min at 30-1000°C) and microwaving (1-5 min). The form of SOD is stable with 33% of 02 scavenging activity remaining up to 6 days at room temperature (25-30 °C). The form is stable at least for 18 months at 4°C having 62% of the activity and 78% activity at -10 to -20°C containing 30% glycerol in a freezer without any infection or contamination. The enzyme has been purified by affinity chromatography. This isoenzyme of SOD has a molecular mass of 32 kDa under non-denaturing conditions with similar mass under denaturing conditions, thus, showing its monomeric nature.The inhibitor studies have shown that this isoform of the enzyme requires Cu/Zn as a co-factor and has antifungal, anti inflammatory and antibacterial properties. The method for the preparation of the purified isoenzyme of autoclavable superoxide dismutase and formulations containing the said autoclavable superoxide dismutase are given.

Description

Novel and stable enzymatic antioxidant defense system in Curcuma longa L.
Description:
FIELD OF INVENTION
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. The invention also relates to a process for the extraction of the said SOD and its use in preparing many cosmetic, pharmaceutical and food compositions.
BACKGROUND AND PRIOR ART REFERENCES TO THE INVENTION
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 02 to oxygen and reduction of another 0 to hydrogen peroxide as per the chemical reaction given below.
2 0.sub.2.sup.-. +2 H.sup.+ =H.sub.2 O.sub.2 +O.sub.2
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).
In pharmaceutical applications, 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). Hence, SOD in animal system has following implications:
(1) anti-inflammatory agent in wounds, burns etc. (Nimrod, A. Ezov, N., Parizada, L., Weiss, L., Tochner, Z., Slavin, S., Panet, A. and Gorecki, M. In Frontiers of Reactive Oxygen Species in Biology and Medicine (Eds. Asada, K. and Yoshikawa, T.) Excerpta Medica, Amsterdam, 1994, pp. 383-387); Halliwell B, Hoult JRS, Blake Dr 1989 FASEB J 2: 2867-2873.
(2) suppressors of asthamatic response (Ihaku, D., Tanimukai, T., Kitada, O., Taniguchi, N., and Sugita. M. In Frontiers of Reactive Oxygen Species in Biology and Medicine (Eds. Asada, K. and Yoshikawa, T.) Excerpta Medica, Amsterdam, 1994, pp. 407-408); Bolli R 1988 J Am Coli Cardiol 12: 239-249. McCard JM 1985 N Eng J Med 312 159-163.
(3) suppressors of side-effects of anti-cancerous agents and in enhancing the life of tumor- bearing animals (Fugimoto, J. In Frontiers of Reactive Oxygen Species in Biology and Medicine (Eds. Asada, K. and Yoshikawa, T.) Excerpta Medica, Amsterdam, 1994, pp. 411- 412); Breimer LH 1988 Br J Cancer 57, 1-18.
(4) in relieving rheumatoid arthritis, SOD as a drug is administrated intra-articularly (Goebel, K. M. and Storck, U. 1983. Am. J. Med. 74: 124-128); Halliwell B, Hoult JRS, Blake Dr 1989 FASEB J 2: 2867-2873.
(5) in reducing the harmful effects of treatment with ionizing radiations (Edsmyr, F., Huber, W. and Menander, K. B. 1976. Curr. Ther. Res. Clin. Exp. 19: 198-211); Breimer LH 1988 Br J Cancer 57, 1-18
(6) in conferring cardiac protection during heart surgery, heart transplantation, kidney transplantation, and during transplantation of other organs such as skin, lung, liver, and pancreas (mentioned in Marklund; Stefan; Edlund; Thomas, 1998. U.S. Pat. No. 5,788,961);
(7) in prolonging the survival of the perfused isolated rabbit cornea (Neuwirth, L. O., and Dikstein, S. 1985. Curr. Eye. Res. 4: 153-154); Kretzer FL, Metha RS, Johnson AT, Hunter DG, Broen ES, Hittner HM 1984 Nature (Lond) 309: 793-795.;
(8) in protecting the isolated lens against photo-peroxidation (Varma, S. D. 1982. Ophthalmic Res. 14: 167-175) Kretzer FL, Metha RS, Johnson AT, Hunter DG, Broen ES, Hittner HM 1984 Nature (Lond) 309: 793-795;
(9) injecting of SOD is helpful in reducing the frequency of intraventricular brain hemorrhage following hypotension (Ment, L. R., Stewart, W. B., and Duncan, C. C. 1984. J. Neurosurg. 62: 563-569) Halliwell B 1991 American Journal of Medicine 1991; 3-14;
(10) in ameliorating hepatitis in rats induced by injection in general as induced by bacteria such as Corynebacterium parvum (Arthur, M. J., Bentley, I. S., Tanner, A. R., Saunders, P. K., Milluard-Sadlor, G. H., and Wright, R. 1985. Gastroenterology, 89: 1114-1122); Halliwell B 1991 American Journal of Medicine 1991; 3-14;
(11) in protecting kidneys against acute pyelonephritis (Robert, J. A., Roth, J. K. Jr., Domingue, G., Lewis, R. W., Kaack, B., Baskin, G. 1982. J. Urology, 128: 1394-1400) and nephrotoxic nephritis in rats (Rehan., A., Johnson, K. J., Wiggin, R. C, Kunkel, R. G. and Ward, P. A.1984. Lab. Invest. 51: 396-403); Halliwell B 1991 American Journal of Medicine 1991; 3-14;
(12) in ameliorating the functional and morphological abnormalities caused because of high blood pressure (Kontos, H. A. 1985. Circ. Res. 57: 508-516); Halliwell B 1991 American Journal of Medicine 1991; 3-14;
(13) in protection against diabetes mellitus and diabetogenic activity of alloxan (Grankvist, K., Marklund, S., Sehlin, J. and Taljedal, I. B. 1979. Biochem J, 782: 17-24.); Wolff SP, Dean RT 1987 Biochemical J 245: 243-250.
(14) in protection of tracheal cells against respiratory disorders caused by asbestos and other pollutants (Mossman, B. T., and Landesman, J. M. 1983. Chest, 835: 50s-51s); Halliwell B
1991 American Journal of Medicine 1991; 3-14;
(15) in relieving the depressor effect of spinal cord injury (Taoka, Y., Urakado, M., Koyanagi, E., Naruo, M., Inoue, M. In Frontiers of Reactive Oxygen Species in Biology and Medicine (Eds. Asada, K. and Yoshikawa, T.) Excerpta Medica, Amsterdam, pp. 241-242); Halliwell B 1991 American Journal of Medicine 1991; 3-14;
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. For storage of organs at low temperature, 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. Apart from the use of autoclaved SOD in pharmaceuticals and medical fields, sterile SOD will also be a choice in the cosmetic and food industry (for preventing oxygen disorders) as well.
(a) 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 δO.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. and Yoshikawa, T.), Excerpta Medica, Amsterdam, pp. 129-130)], stability without adding an external stabilizer [the addition of hydrogen peroxide trapping agent, polyols, and sugars etc. are required to stabilise the enzyme from other sources such as germinated plant seeds (Bresson-Rival; Delphine; Boivin; Patrick; Linden; Guy; Perrier; Erric; Humbert; Gerard; 1999; U.S. Pat. No. 5,904,921)] to function to above 50.degree. C. temperature [temperature range for SOD activity has been reported between 5 to 45. degree. C. most of the workers (Burke, J. J. and Oliver, M. J. (Plant Physiol. 1992. 100: 1595-1598); Hakam, N. and Simon, J. P. (Physiol. Plant. 1996. 97: 209-216)] a wide range of stability of the enzyme is extremely useful and safe to humans.
The formulations/compositions mentioned below, but not limited to those mentioned below, have included SOD as one of the active ingredients:
(a)Hersh, T. in U.S. Pat. No. 5,922 dated Jul. 13, 1999 disclosed a composition for ameliorating free radical damage induced by tobacco products and environmental pollutants. The composition 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.
(b)U.S. Pat. No. 5,904,921, describes a composition for cosmetic use as antiaging antiwrinkle and antistress cream for skin . The composition was inclusive of SOD along with peroxidase with a peroxidase specific reducing substrate. The SOD was obtained from germinated seeds of barley, soya, wheat and peas, whereas peroxidase was obtained from black radish (or horseradish peroxidase) that was combined with an enzymatic substrate constituted by uric acid. Apart from horseradish peroxidases, 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.
(c) Another U.S. Pat. No. 6,011,067 issued on Jan. 4, 2000 by Hersh, T. discribes an antioxidant composition 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%).
(d) 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).
Further, 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.
(e) 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. Inventors described other formulations and enormous sources of SOD, and various types of SOD. 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. They form especially 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. The 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. Besides the conventional active ingredients 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. Inventors mentioned that the synergistic combination of SOD and melanin could be incorporated as a main or secondary ingredient, in various 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). The cosmetic composition of the invention may also be for oral and dental use, for example a toothpaste. In this case 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.
(f) 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.
(g) 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. Inventors mentioned that 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.
(h) 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.
(i) 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. 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. These included but are not limited to the anti-oxidants, tocopherols (vitamin E), green tea and pycnogenols and also steroids, non-steroidal anti- inflammatories, capsaicin extract, tissue respiratory factor and the local anesthetics of the caine family.
(j) 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. Since 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.
In addition to the use of SOD in various pharmaceutical, cosmetic and food industry, the enzyme plays crucial roles in plant industry as well. Thus, for example, but not limited to, a SOD with long storage life will aid in protecting the plant against oxidative stress during winter months. And, a high thermal stability of the enzyme would be a desirable feature for the plant experiencing intense photoinhibition during hot summer and drought stress.
Reference may be made to 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.
Given below is the state of art in relation to stability of enzyme at high rnge of temperatures for its function:
Reference may be made to a document by Burke, J. J. and Oliver, M. J. (Plant Physiol. 1992.
100: 1595-1598) wherein SOD is described to possess properties from pea (Pisum sativum L. var Progress No. 9) assayed at temperature varying between lO.degree. C. to 45.degree. C.
Chloroplast localised Cu/Zn-SOD was found to have highest activity at 10C, whereas Mn-
SOD and cytosolic Cu/Zn-SOD showed no change in activity between lO.degree. C. - 30.degree. C. The enzyme activity was lowest at 45.degree. C.
Reference may be made to another document by Hakam, N. and Simon, J. P. (Physiol. Plant. 1996. 97: 209—216) wherein is described SOD properties assayed at two temperatures of 5 and 25. degree. C. from a C.sub.4 grass Echinochloa crus-galli (L.) Beauv. No change in the enzyme activity was observed at these two temperatures.
Reference is made to US patent No 6485950 reveals a purified isozyme of SOD from the leaves of Potentilla atrosanguinea a high altitude plant which is autoclavable and functions at sub zero to 80 C temperature which is endegered and it is difficult to acclimatization to the hot Indian plan conditions therefore the heat stable autloclavable, microwaveable isoenzyme described in our patent application from curcuma leaves and rhizomes is better since it functions at a wide range of temperature at 100 C.
Reference may be made to yet another document by Bonaccorsi di Patti, M. C, Giartosio, A., Musci, G., Carlini, P. and Calabrese, L. (In Frontiers of Reactive Oxygen Species in Biology and Medicine. 1994. (Eds. Asada, K. and Yoshikawa, T.), Excerpta Medica, Amsterdam, pp. 129-130) wherein thermostability of Cu/Zn-SOD has been analysed from ox, sheep, shark, yeast, and Xenopus laevis and showed conformational melting temperatures to be 88.05, 87.1, 84.1, 73.1 and 71.15.degree. C, respectively. However, there was no mention of the enzyme activity at various temperatures. Also, the enzymes were reported to be denatured when heated beyond transition peak.
Another reference from Bueno P., Verla, J., Gallego, G. G., and Rio del A. L. (Plant Physiol. 1995. 108: 1151-1160) wherein the thermostability of Cu/Zn SOD isolated from the cotyledon of water melon has been shown, SOD activity reduced by 40% after 4 hour of incubation at 50.degree. C; by 50% after 15 minute of incubation at 70.degree. C; by 80% after 60 minute of incubation at SO.degree. C; and by 100% after 15 minute of incubation at lOO.degree. C.
Reference may be made to Document by Miyata, K., Maejima, K., and Tomoda, K. (U.S. Pat. No. 4,563,349; Jan. 7,1986) wherein SOD has been reported from a microorganism belonging to genus Serratia having the thermostability characters as follows:
(a) Stable at 37.degree. C. for 60 minutes; Inactivated by 50% when incubated at 50- 60.degree. C. for 60 minutes; and Inactivated by 100%) when incubated at 80.degree. for 5 minutes.
Reference may be made to Document by Gudin; Claude; Trezzy; Claudine (U.S. Pat. No. 5,536,654; Jul. 16, 1996) which describes the production and extraction of SOD from a photosynthetic microorganism culture, which is thermostable upto 80.degree. C.
The drawbacks of the SOD as reported in the prior art are: There is no reported SOD which is heat stable (50-100 degree C ) could be autoclaved, and microwaved to ensure a germ free sterile preparation and, at the same time, can catalyze dismutation of 0.sub.2.sup.-. at lower temperature. The maximum thermostability of SOD reported so far is at 80.degree. C.
(Gudin; Claude; Trezzy; Claudine 1996. U.S. Pat. No. 5,536,654; U.S. Pat. No. 6485950)
(Hakam, N. and Simon, J. P. 1996. Physiol. Plant. 97: 209-216). (b) There is no reported SOD which can catalyze dismutation of 0.sub.2.sup.-. when microwaved.
(c) Prior art procedures for carrying out enzyme assay do not describe any method to study activity in leaves after microwaving /autoclaving /or storage for more than 8 months (U.S. Pat. No. 64859504).
(d) Prior art procedures do not describe the method to identify the isoenzymes encompassing unique properties of sub-zero and higher temperature functionality before the purification of the enzyme could be taken up.
(e) Reported SODs do not retain their activity at ambient temperature unless stabilized by the addition of polyols, sugars or any other stabilizing agent (Bresson-Rival;
Delphine; Boivin; Patrick; Linden; Guy; Perrier; Erric; Humbert; Gerard; 1999; U.S. Pat. No. 5,904,921).
(f) Prior art procedures for SOD purification do not ensure complete elimination of the associated proteins.
(g) Although there are several procedures for purification of SOD (Beaman, B. L.; Scates, S. M.; Moring, S. E.; Deem, R.; And Misra, H. P.; Journal ot'Biological Chemistry, 258: 91-96, 1983; Steinman, H. M.; Journal of Biological Chemistry, 257, 10283-10293).
h) We have not encountered any reference wherein a particular isoenzyme of SOD has been shown to possess anti-inflammatory, antifungal, antibiotic properties besides heat stability (30-100 C) autoclavibility, micrwavibility in the same isoenzyme.
Other References
Reference is also made to four US patents (5401504), which are filed for anti- inflammatory, wound healing, food additive and cosmetic property of Curcuma. US patents were withdrawn in 1998.
Reference is made to US patent No. 6485950. Nov. 2002. Kumar et al. Beuno et al. Peroxisomal copper, zinc super oxide dismutase, characterization of the isoenzyme from watermelon cotyledons (1995) Plant Physiol., vol. 108, pp. 1151-1160.* Gupta et al., Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase (1993) PNAS, USA, vol. 90, pp. 1629-1633.
Therefore, we are claiming in this application for the first time in a simple, reliable and reproducible manner of the isolation and characterization of an antioxidant isoenzyme of SOD from leaves and rhizomes of curcuma longa combining the properties as mentioned above in h.
OBJECTS OF THE INVENTION
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 another object of the present invention is to provide a SOD which is stable at ambient temperature (25. degree. C.) at least for 7 days without adding any stabilizing agent. Yet another object of the present invention is to provide a process for more complete purification of SOD to eliminate the proteins carrying same charge but different molecular weight.
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
SUMMARY OF THE INVENTION
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,
(b) is stable to boiling in distilled water for 30 minutes , and
(c) has free radical scavenging capability ranging between zero (O.degree. C.) to +1 OO.degree. C. Particularly, 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.-..
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, 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:
(a) 0.sub.2.sup.-. scavenging activity remains high before and after autoclaving;
(b) scavenges 0,sub.2.sup.-. from temperature 25.degree. C. to high temperature of +1 OO.degree. C; (c) 0.sub.2.sup.-. . scavenging activity at 25.degree. C. for 7 days without adding any stabilizing agent such as polyols or sugars;
(d) stable from -20 degree C to 4.degree. C.to for at least 18 months;
(e) contamination free and infection free from any living micro- and/ or macro-organism after autoclaving;
(f) possesses a molecular weight of 32 kDa under non-denaturating and denaturing conditions; and
(g) requires Cu/Zn as a co-factor;
(h) possesses ani- fungal properties;
(i) possesses anti-bacterial properties;
(j) possesses anti-inflammatory properties.
Further, the invention provides a method for identification of the target isoenzyme of the superoxide dismutase said method comprising the steps of:
(a) localizing various isoenzymes of SOD in the crude extract of the leaf on 7-12% native polyacrylamide gel;
(b) after elecfrophoresis, rinsing the gel with distilled water followed by incubation for 30 minutes in 2.5 mM NBT;
(c) immersing the gel in 1.17.times.10.sup.-6 M riboflavin for 20 minute while shaking and removed later on to a petri plate to expose to a light intensity of 25-1000 .mu. Einstein/m.sup.2 /second using a fluorescent light source to develop purple color throughout the gel except for the locations where SOD was localized;
(d) incubating with nitroblue tetrazolium and riboflavin, and exposing to light at room temperature
(e) when stored at -20.degree. C for longer periods, adding glycerol (30% final concentration) in the solution to avoid freezing and loss of activity;
(f) identifying the most prominent isoenzyme at all the temperatures and selecting for the purpose of purification.
In an embodiment, the invention provides a method for the preparation of purified novel isoenzyme of SOD wherein the said method comprises the steps of:
a) homogenizing leaf tissue in a homogenizing buffer at pH 7.0-7.8 and at a temperature range between 4-8.degree. C;
b) filtering the homogenate through 8 layers of cheese cloth and centrifuging the filtrate at 8,000-13,000 rpm for 10-30 minutes at 4-8.degree. C;
c) collecting the supernatant by decantation for purification of SOD;
d) precipitating SOD with 30-60%) ammonium sulfate;
e) dissolving the precipitate in a 10 to 100 mM buffer at pH 7 to 7.8 and dialyzing for 10-36 hours with 6-12 changes of the buffer;
f) loading the dialyzed protein onto a SephadexG-100 column and assaying fractions for SOD activity;
g) loading the dialyzed protein or the pooled fraction from Sephadex G-100 column to an ion exchange / affinity (Blue Sepharose) column and eluted with 5-20 μM NBT (Blue Sepharose Column) or 100-500 mM KC1 (ion exchange column) prepared in a buffer (all autoclaved or non-autoclaved);
h) assaying and pooling SOD containing fractions; i) fractionating SOD containing fractions on a fast protein liquid chromatography (FPLC)1 using mono Q column and eluting with 100-200 mM KC1 prepared in 10-50 mM phosphate buffer (all autoclaved or non-autoclaved) with a flow rate of 1-10 ml per minute;
j) assaying each peak for SOD activity. SOD peak, obtained after FPLC and concentrating using a protein concentrator column / glycerol;
k) assaying concentrated protein for SOD activity at room temperature after heating at different temperatures ranging between 25 to +100 degree. C;
1) localizing the purified SOD on 7 to 12% polyacrylamide gel by known methods;
In yet another embodiment, 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
In still an other embodiment, the invention provides that the purified isoenzyme in young leaves possess anti-inflammatory and anti-bacterial property.
In yet an other embodiment, the invention provides that the purified isoenzyme in mature leaves possess anti-inflammatory and anti-bacterial property.
In an other embodiment, the invention provides that the purified isoenzyme in dried leaves /rhizomes possess anti-inflammatory and anti-bacterial property.
In an other embodiment, the invention provides that the novel SOD is present in young, mature fresh or dried , heated, autoclaved, microwaved leaves/rhizomes
In an other embodiment, 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. In still another embodiment, 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.
In another embodiment, 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 tibetana, Arnaebia euchroma, Amaranthus, Chenopodium,
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.
In still another embodiment, 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.
In yet another embodiment, 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
In yet another embodiment, the invention also provides a 88% higher amount of peroxidase in the leaves of Curcuma longa as compared to 12% in rhizome.
In still another embodiment, 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.
In yet another embodiment, 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. In another embodiment, 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.
Use of SOD for preparation of formulations involving SOD such as 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.
Use of SOD for preparation of formulations involving SOD such as 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.
Use of the isoenzyme of SOD for preparation of gels, lozenges, tablets and gums wherein the isoenzyme of SOD is mixed with gums, tablets to ensure a germ free sterile preparation.
Accordingly ,the present invention provides 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.
In one of the embodiment of the invention 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. BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
FIG. 1 represents effect of heat on isoenzymes of SOD (Superoxide dismutase)in young and old leaves and rhizome of Curcuma longa L. 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. In India, 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.
In an advantageous embodiment, 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. In a preferred embodiment, 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.
In still an other embodiment, 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. Yet in another embodiment, 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 H202
It will be possible to use the product of the invention in the formulations/compositions mentioned below, but not be limited to those mentioned below, which have included SOD as one of the active ingredients:
The present invention will be illustrated in greater details by the following examples. These examples are presented for illustrative purposes only and should not be construed as limiting the invention, which is properly delineated in the claims.
EXAMPLE 1
Preparation of the Crude Extract and Identification of Optimal pH for SOD Activity
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 β-mercaptoethanol(βme), 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. 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).
A control reaction was always performed wherein all the steps and components were exactly the same as described above except that crude enzyme was replaced with equal volume of homogenizing buffer. SOD competes with NBT for 0.sub.2.suρ.-., hence presence of SOD inhibits the color development. Activity of SOD is expressed as per cent inhibition in colour development as compared to the control reaction (higher the inhibition, higher the SOD activity), the enzyme showed pH optima of 7.8. Therefore, all the further experiments were performed at pH of 7.8. EXAMPLE 2
Superoxide dismutase (SOD), peroxidase (POD) and Catalase (CAT) activity in leaves and rhizomes of Curcuma longa L.
The crude enzyme extracts from leaves and rhizomes of Curcuma longa L. were assayed for SOD (method described in example 1) , peroxidase (POD) and Catalase (CAT) activities by the known methods (Kochhar, S., Kochhar, V.K. and Khanduja, S.D.(1979). Ameri. J, Enol. Vitcul. 30:275-277); Catalase (CAT) by the method of Aebi, H. E. (1983). Methods Enzymol.105: 121-126.
As can be seen from table- 1, the percent activity of SOD, POD and CAT is much higher in leaves (L) as compared to rhizome (R) of Curcuma longa L.
Table - 1 : Superoxide dismutase (SOD), peroxidase (POD) and Catalase (CAT) activity in leaves and rhizomes of Curcuma.
Figure imgf000025_0001
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.
EXAMPLE 3
Effect of Temperature on Crude SOD activity in Curcuma longa L. The crude enzyme was assayed at room temperatures after preheating the enzyme at temperatures ranging between 40 to 1 OO.degree. C. by the method as described in Example 1. As can be seen from Table 2, another novel finding is that the enzyme remained stable even at boiling temperatures (100 degree. C). As compared to 100% activity at room temperature leaf enzyme showed 98% activity at 40C , 80% at 60C, 70% at 80C and 57% at 100C respectively. As compared to 100% activity at room temperature the enzyme from rhizome showed 88% activity at 40C, 70% at 60C, 52% at 80C and 20% at lOOC. The applicants feel that the heat stable leaf SOD isoenzyme will be functional even at temperatures below -lO.degree and higher than 100 degree C. Control reactions, as mentioned in Example 1, were always performed separately with all the enzyme assays.
Table 2: Effect of temperature on the antioxidant enzymes (superoxide dismutase- SOD) in leaves and rhizomes of Curcuma longa L.
Figure imgf000026_0001
The data represents the mean values of three different independent preparations.
EXAMPLE 4
Effect of different temperatures on POD activity from leaves and rhizomes of Curcuma longa L.
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.
In contrast to SOD activity , 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/
Table 3. Effect of different temperatures on POD activity from leaves and rhizomes of Curcuma.
Figure imgf000027_0001
The data represents the mean values of three different independent preparations.
EXAMPLE 5
Effect of autoclaving on SOD activity of leaves and rhizome of Curcuma longa L
A rigorous test for 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.
Table 4. Effect of autoclaving on Crude SOD Activity
Figure imgf000027_0002
Figure imgf000028_0001
The data represents the mean values of three different independent preparations.
The above table 4 explains that the percentage superoxide dismutase (SOD) activity of leaf enzyme was 57%) after 30min autoclaving as compared to 31% in rhizome when autoclaved for 30min. Therefore, the antioxidant enzyme activity in the leaves was much more stable to autoclaving. EXAMPLE 6
Effect of microwaving on SOD activity in leaves and rhizome of Curcuma longa L.
A rigorous test for 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.
Table 5 Effect of microwaving on crude SOD activity
Figure imgf000028_0002
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.
To achieve this:
(a) various isozymes of SOD in the crude extract of the leaf and rhizome were localized on 10% native polyacrylamide gel after heating as described by Beauchamp and Fridovich (Anal. Biochem. 1971.44,276-287).
(b) After electrophoresis, gel was rinsed with distilled water followed by 30 minute incubation in 2.5 mM NBT. Gel was then immersed in 1.17.times.l0.sup.-6 M riboflavin for 20 minute and removed later onto a petri plate to expose to a light intensity of 1000 .mu. Einstein/m.sup.2 /second using a fluorescent light source. Light exposure led to photogeneration of O.sub.2. sup.-., which converts NBT into insoluble purple colored formazan. As a result, purple color is developed throughout the gel except for the locations where SOD was localized.
(c) Incubation with NBT and riboflavin, and light exposure was carried out after heating the leaf (both young and old) and rhizome enzyme extract at 80 degree C for 0,10 and 20minutes respectively.
(d) As can be seen from FIG. 1 that 4 isoenzymes with very high SOD activity were detected in both young and old leaves at room temperature whereas two sharp and two faded bands were detected in rhizome. The SOD isoenzyme having molecular mass of 32 kDa was found to be most heat stable both in the leaves as well as in rhizomes. While this heat stable isoenzyme band was much sharper and denser in young leaves, its intensity was lower in old leaves and still lower in rhizomes.
e) Staining intensity of rest of the isoenzymes was negligible. Therefore, the isoenzyme with molecular mass of 32kDa was identified as a most stable isoenzyme and considered to be novel. This isoenzyme was targeted for the purpose of purification. EXAMPLE 8
Effect of heat on isoenzymes of peroxidase in Curcuma leaves and rhizome
Various isoenzymes of POD in the crude extract of the leaf and rhizome were localized on 10%> native polyacrylamide gel as described by activities by the known methods (Kochhar, S., Kochhar, V.K. and Khanduja, S.D.(1979). Ameri. J, Enol. Vitcul. 30:275-277). The enzyme extract from leaves (young and old) and rhizome was loaded on 10% Native polyacrylamide gel after heating at 80C for 2 -5 min as POD was much unstable than SOD. Two isoenzymes of POD with molecular masses of 25 and 17 kDa were found to be heat stable upto 5 minutes (Fig. 2). These were not detectable in the rhizome enzyme extract. The inventors feel that leaves of Curcuma leaves have much higher rate of detoxification of toxic compounds like H sub.2 O sub.2.
EXAMPLE 9
Process for Purification of SOD
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.
(a) homogenizing leaf tissue (500 g) in 1 L of homogenizing buffer (0.05 M potassium phosphate buffer, pH 7.8 (autoclaved); 2%PVPP; 1 mM B-mercaptothanol ) at 4.degree. C;
(b) filtering the homogenate through 8 layers of muslin cloth and centrifuging the filtrate at 10, 000 rpm for 20 minutes at 4,degree. C;
(c) Collecting by decantation of the supernatant for purification of SOD; (d) precipitating SOD with 30-60% ammonium sulfate (30-60%) fraction);
(e) dissolving precipitate in phosphate buffer (10 - 100 mM potassium phosphate buffer, pH 7.8; autoclaved) and dialyzing for. 18-36 hours with 6-12 changes of phosphate buffer; (f) loading the above said fraction at (e) on to a Sepahdex G-100 column (50cm, 2.5 cm dia) and eluting the enzyme with the same buffer as in (e);
(g) loading the dialyzed protein onto a DEAE-Cellulose column and eluting with 50 mM of 250 ml of potassium chloride (KCl) solution followed by 250 ml of 200 mM KCl solution and finally eluting with 250 ml of 500 mM of KCl Solution (solution of KCl was prepared in 10 mM phosphate buffer; all autoclaved).
(h) assaying fractions containing protein for SOD, pooling the fractions showing SOD activity, (i) loading the above said fraction at (h) on to a Sepahdex G-100 column (50cm, 2.5 cm dia) and eluting the enzyme with the same buffer as in (e); (j) assaying fractions containing protein for SOD, pooling the fractions showing SOD activity, (k) Fractionating SOD containing fractions on an affinity column (Blue Sepharose, 15 cm, 2.5 cm dia),washing the column with buffer as described at (e); (1) Eluting the column with 0.05M phosphate buffer (pH 7.8) containing 5-20 uM NBT. (m) Assaying each fraction for SOD activity, concentrating in glycerol; (n) The purified fraction at (m) was further purified using FPLC through mono Q column. Each fraction was assayed for SOD activity, heat stability and auto clavability.
Table 6. Purification of heat stable, autoclavable and microwavable SOD from Curcuma longa L leaves.
Figure imgf000031_0001
Figure imgf000032_0001
EXAMPLE 10 Confirmation of the Purified Isozyme of the SOD as a Single Protein
To confirm the purified isozyme of the SOD as a single protein, it was localized on a 10% SDS polyacrylamide gel as described by Laemmeli, U. K. (1970; Nature, 227: 680-685). Delta-one SDS PAGE molecular weight markers purchased from Amersham Pharmacia Biotech, USA, suitable for SDS polyacrylamide gels were also loaded in an adjacent well. Thus after completing the electrophoresis, 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).
EXAMPLE 11 Absorption Spectrum of SOD
Absorption spectrum was recorded using Bausch and Lomb Spectronic 2000UV/Visible spectrophotometer. Purified SOD exhibited strong absorption in UV range (190-340 nm). A UV absorption spectrum of the purified SOD exhibited peak at 268 nm which shows the presence of phenyl alanine amino acid (hydrophobic in nature) in the protein.
EXAMPLE 12
Effect of Temperature on Single band SOD
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).
Table 6 Activity of purified heat stable and autoclavable band excised from native gel.
Figure imgf000033_0001
The data represents the mean values of three different independent preparations.
EXAMPLE 13
Effect of Inhibitors on SOD Activity
Purified SOD was completely inhibited either by potassium cyanide (3mM) or hydrogen peroxide (5mM). It is known that depending upon the co-factor requirements, the SOD can be Mn-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) and Fe-SOD (SOD requiring iron as a co-factor; sensitive to hydrogen peroxide but insensitive to potassium cyanide) (Bowler, C, Montagu, M. V. and Inze, D. 1992. Annual Review of Plant Physiol. and Mol. Biol. 43: 83-116). The SOD reported in the present invention is inhibited by both KCN and H. sub.2 O.sub.2, and hence represents Cu/Zn SOD.
Table-7 Effect of KCN and H202 (SOD inhibitors) on the activity of purified SOD assayed at room temperature.
Figure imgf000034_0001
The data represents the mean values of three different independent preparations.
EXAMPLE 14
Effect of Storage Conditions on SOD Activity
Purified SOD was tested for its longevity at 4 and 25.degree. C. 62 %> SOD activity was stable even after 18 months of storage at 4.degree. C. At room temperature (25.degree. C), the enzyme activity was reduced by 50%> in 5 days. The activity at -10 to -20 C was found to 78 % after 18 months of the original activity (See Table 4).
Table-8 Effect of storage temperature on the activity of purified SOD assayed at room temperature.
Figure imgf000035_0001
The data represents the mean values of three different independent preparations.
EXAMPLE 15
Anti-inflammatory activity of purified SOD isoenzyme
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
Part used Paw volume (ml) at
60 min 120 min 180 min 240 min 2005/017134
Figure imgf000036_0001
The data represents the mean values of three different independent preparations.
Values are mean + SEM; P: a< 0.05, b< 0.01 and c< 0.001 compared to respective control groupThe standard drug used is nimusilide. It is a synthetic compound having significant anti-inflammatory activity but prolonged use of the drug causes mucosal damage and inactivates or saturates the enzymes responsible for inflammation in the body. EXAMPLE 16 Antifungal activity of purified SOD from leaves and rhizomes of Curcuma. Antifungal activity: The potato dextrose agar plates (for anti-fungal activity) were prepared by dissolving agar powder (4.1 gm/100 ml) in distilled water. It was then sterilized by autoclaving for 15 minutes. The sterile agar was then allowed to cool down till its temperature came down to 50° C before pouring. Aspergillus niger- a fungal strain was spread with the help of loop wire over plate. The disks of uniform 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 ui of extract was poured onto disks and were placed in the petri dishes with the fungal 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 (Table 10). The antifungal 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 (fig 4)..
Table 10: The anti-fungal effect of the antioxidant enzymes of Curcuma leaves and rhizomes
Part of plant Used Antifungal activity (%>) 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.
.EXAMPLE 17
Antibacterial property of antioxidant enzymes of Curcuma leaves and rhizomes Antibiotic property of curcuma leaves and rhizomes, 3.5 gm of L.B. Agar (Luria Broth Agar) per 100 ml of distilled water was used without the addition of antibiotic for the preparation of plates. Rest of the procedure was the same as described above. DH5α strain of E-coli was used as bacterial strain (Table 11). The disks of unifoπn 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
Part of curcuma used Antimicrobial (%>) Leaf(L) 63 Rhizome (R) 52 Ampicillin (Antibiotic 100 Control)
The data represents the mean values of three different independent preparations. Explanation of the Table 11 : The circles around the disks were observed to find out the antibiotic 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. 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).
EXAMPLE 18
A comparative account of SOD activity from crude extracts of leaves and rhizome of Curcuma, leaves of Spinach, Phaseolus and rhizomes of Ginger.
SOD activities from leaves and rhizomes of Curcuma have been compared with the SOD activity of Spinach leaf, phaseolus lleaf and ginger.
Table 12 Comparative account of SOD activity from crude extracts of leaves and rhizome of Curcuma, leaves of Spinach, Phaseolus and rhizomes of Ginger
Figure imgf000038_0001
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.
EXAMPLE 19 Effect of heating on SOD activity of Phaseolus and Spinach Leaf Tissue Crude enzyme from the leaf tissue of phaseolus and spinach was extracted and assayed essentially as described in Examples 1, 2 and 3 at different temperatures for 20 minutes,
Table 13 A comparison of SOD activity from the leaf tissues of Phaseolus and spinach
Plant Activity (Percent)
Material
Initial 40C 60C 80C 100C
Phaseolus 100 70 0 0 0
Spinach 100 60 35 20 0
The data rer )resents the ir lean values o 'three different independe nt preparatic
As can be seen from Table 13 that the enzyme did not show any activity in phaseolus at 60- 100 degree C and Spinach also lost 80% activity at 80c and 100% at 100 degree C. The investors feel that Curcuma leaves exhibit ideal enzymatic antioxidant defense system.
EXAMPLE 20
Effect of autoclaving on spinach and phaseolus SOD
Table 14- Effect of autoclaving on Phaseolus and Spinach SOD from leaf tissue
Plant Leaf Effect of Autoclaving for the time Initial lmin 2min 5 min lOmin 20 min
Phaseolus 100 0 0 0 0 0
Spinach 100 0 0 0 0 0
The data ref )resents the n lean values o ■ three differe nt independe nt preparatioi is.
As can be seen from Table 14 that the enzyme did not show any activity both in phaseolus and spinach after autoclaving for 1 minute. The investors feel that Curcuma leaves exhibit ideal enzymatic antioxidant defense system. EXAMPLE 21
SOD Activity in ginger another Plant Species belonging to family zingiberaceae
As can be seen in Table 14, the activity of the SOD enzyme from ginger rhizome, extract prepared as mentioned in Example 1, and assayed as mentioned in example 2 and 3, showed that: (a) That 62 % activity remains after heating the extract at 40 degree C for 20 min; 38% after heating the extract at 60 degree C for 20 min; 20% after heating the extract at 80 degree C for 20 min; 0% after heating the extract at 100 degree C for 20 min;
Table 16 Effect of heating on SOD activity from Ginger rhizome
Figure imgf000040_0001
The data represents the mean values of three different independent preparations.
(b) Effect of autoclaving on SOD activity from Ginger rhizome
As can be seen in Table 16, the activity of the SOD enzyme from ginger rhizome, extract prepared as mentioned in Example 1, and assayed as mentioned in example 2 and 3, showed that:
Table 17 Effect of autoclaving on SOD activity from ginger rhizome
Figure imgf000040_0002
Figure imgf000041_0001
The data represents the mean values of three different independent preparations.
The main advantages of the present invention are:
1. For the first time, 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.
2. The identified SOD isoenzyme functions more efficiently from young leaves than old leaves or rhizome.
3. The feature of heat stability, autoclavability and microwavablity is shown by the same SOD.
4. It has been shown for the first time that SOD from leaves and rhizome of Curcuma can function even at boiling temperatures. 5. The autoclaved preparation of the SOD is free from microbial contamination, hence, will be of immense use in medical, cosmetic and food industry.
6. 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.
7. The SOD having anti-inflammatory activity and is of great use in medicines.
8. The SOD having anti-bacterial activity and is extremely useful in medicines. 9. The SOD having anti-fungal activity and is of great advantage in medicines.
10. A one step quick method to identify the isoenzyme showing the desired properties before the purification is taken up.
I A process for more complete purification of SOD to eliminate the proteins carrying same charge but different molecular weight.
12. This isoenzyme can be easily obtained from the Curcuma plant which grows easily under varying environmental gradients.
13. 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.
14. 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.
15. 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. COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
SHEET NO 1/5
Fig. 1. SOD activity in native gel in Curcuma longa Mr
Figure imgf000043_0001
M C H H H H C H H 10 m 20 m 10 m 20 m 10 m 20 m 80°C 80°C 80°C ■4 ► ■4 ► ► Young Leaves Old leaves Rhizome
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.
COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH SHEET NO 2/5
Fig. 2. Peroxidase activity in native gel in curcuma
Figure imgf000044_0001
M C H H C H H C H H 2M 5M 2M 5M 2M 5M -4 ► -4 ► -4 ► Young Leaves Old Leaves Rhizome
Fig 2 shows the POD fingeφrints of crude enzyme from leaves (young and old) and rhizome when heated for 10 and 20 minutes at 80 degree C.
COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
SHEET NO 3/5
Fig 3. Activity of Purified Heat stable Isoenzyme from Curcuma Leaves and rhizome
Mr x 10 -3
Figure imgf000045_0001
M YL OL
M= Mol. Mass Marker; YL=Young leaves; OL=01d leaves; R= Rhizome
Fig 3 Shows one band purification of heat stable, autoclavable and microwavable SOD isoenzyme from young , old leaves and rhizome of Curcuma longa L. COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
SHEET NO 4/5
Figure imgf000046_0001
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.
COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
SHEET NO 5/5
Figure imgf000047_0001
Antibacterial activity of curcuma leaf extract against DH 5 a.
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.

Claims

What is claimed is:
1. An autoclavable heat stable, and microwavable isoenzyme of superoxide dismutase (SOD) present in crude extract as well as in purified form from leaves and rhizomes of Curcuma longa L
2. An autoclavable heat stable, and microwavable isoenzyme of superoxide dismutase (SOD) present in crude extract as well as in purified form from leaves and rhizomes of Curcuma longa L, having the following characteristics:
(a) high O { scavenging activity between 30-40 units per mg (100%) protein before and 15- 30 units per mg protein (50-70%) after autoclaving at 5-20 bars, 30min in leaves;
(b) 02 " scavenging activity in rhizome being 15-28 units per mg (100%) protein before and 5-10 units per mg protein (30-40%>) after autoclaving at 5-20 bars, 30min;
(c ) scavenging O 2 " even when exposed to high temperature of 40-100 °C;
(d) contamination free and infection free from any living micro- and/or macro-organism after autoclaving;
(e) high O 2 scavenging activity remains high 30-40 units per mg (100%) protein before and 12-20 units per mg protein (40-50%) after exposing the leaf enzyme to microwave for 1-5 minutes.
(f ) high O 2 scavenging activity betweenl5-28 units per mg (100%) protein before and 3-9 units per mg protein (20-30%>) after exposing the rhizome enzyme to microwave for 1-5 minutes.
(g) The isoenzyme being detected in intact leaves heated at 50-80 °C, autoclaved at 5-20 bars, 30min; or microwaved for 1-5 minutes.
(h) The isoenzyme being 20-30 %» more active in young 10-30 days old leaves than mature 31-150 days old leaves. (i) The isoenzyme being present in rhizomes (both young and mature) but the activity being 40-50% lower as compared to the activity in leaves.
(j) The crude extract of leaves exhibiting higher detoxification of H sub.20 sub.2 because of 88 % higher peroxidase activity than the rhizome POD.
(k) The isoenzyme from leaves being a non staining protein
3. The purified isoenzyme of SOD as claimed in claim 1, being stable at 4°C for at least 18 months.
4. The purified isoenzyme of SOD as claimed in claim 1, is extracted from the leaves of Curcuma longa L.
5. The purified isoenzyme of SOD as claimed in claim 1, is extracted from the rhizomes of Curcuma longa L.
6. The activity of purified isoenzyme as claimed in claim 1, being between 60-90%> in leaves and 40-60% In rhizomes with increasing temperature of 40-100 °C and in the range of 0-
60minutes.
7. The activity of purified isoenzyme as claimed in claim 1, being between 50-70% in leaves and 30-40% in rhizomes after autoclaving upto 20 minutes at 5-20 bar pressure and 120 C temperature.
8. The purified isoenzyme as claimed in claim 1, in young leaves / rhizome possessing anti- inflammatory and anti-bacterial property.
9.The purified isoenzyme as claimed in claim 1, in mature leaves / rhizome possessing anti- inflammatory and anti-bacterial property.
10. The isoenzyme as claimed in claim 1, in dried leaves / rhizome possessing anti- inflammatory and anti-bacterial property.
11. A formulation as comprising the isoenzyme of SOD as an active ingredient.
12. The formulation as claimed in claim 11, comprising the isoenzyme of SOD further comprising at least one of the members of the group comprising of reduced glutathione, selenium, a source of selenium, a carrier, a flavoring agent and antioxidants.
13. A formulation as claimed in claim 12, wherein the selenium source chosen from the group consisting of elemental selenium, selenomethionine and selenocysteine.
14. A formulation as claimed in claim 11, wherein the antioxidant is selected from the group consisting of vitamin C, vitamin E, alpha tocopherol, vitamin A, and beta carotene.
15. A formulation as claimed in claim 11, wherein the formulation is selected from the group consisting of solutions, talcum powders, lotions, creams, oils, gels, sticks, toothpick, sprays, ointments, balms, shampoo, serums, mousses, emollient, aerosol, roll-on, patches, lozenges, tablets, gums, dye compositions, pastes, and combinations thereof.
16. A formulation as claimed in claim 15, wherein the loranzes consists of a carrier that enables the loranzes to slowly dissolve in a user's mouth releasing said active ingredients in concentrations effective for reducing free radical damage.
17. A formulation as claimed in claim 11, further comprising at least one amino acid selected from the group comprising of cysteine, methionine, taurine, arginine, and zinc gluconate.
18. A formulation as claimed in claim 11, further comprising approximately 30 to 70% by weight of a gum composition.
19. A formulation as claimed in claim 18, wherein the gum composition is an elastomer, a polyvinyl acetate polymer, an acetylated monoglyceride, a wax with melting point below approximately OO.degree. C, an elastomer solvent, a plasticizer or a filler.
20. A formulation as claimed in claim 11, further comprising a sweetener selected from the group consisting of xylitol, lactitol, sucrose, lactose and a saccharide.
21. A formulation as claimed in claim 11, further comprising at least one of 1-15%> peroxidase, enzymatic substrate or peroxidase specific substrates.
22. A formulation as claimed in claim 21, wherein the peroxidase is selected from the group consisting of black radish peroxidase, horseradish peroxidase, Curcuma leaves Peroxidase, lactoperoxidase, glutathione peroxidase and spinal cord peroxidase.
23. A formulation as claimed in claim 21, wherein the enzymatic substrate is selected from the group consisting of uric acid, glutathione, phenol, guaiacol, mesitol, 3,5-dichloro-2- hydroxybenzenesulfonic acid, aniline, dihydroxymaleic acid, cytochrome C, phenolphthalein, vitamin C, an iodide, a chloride, a bromide, 2-2'-azido-di-3-ethylbenzo-thiazoline-6-sulfonic acid, and SCN. sup.-.
24. A formulation as claimed in claim 11, further comprising a lipophilic antioxidant in an effective antioxidizing amount, selected from the group consisting of tocopherol, tocopherol acetate, tocopherol linoleate, and tocopherol phosphate.
25. A formulation as claimed in claim 11, further comprising antioxidants, L-glutathione at 0.001% to 15% by weight, selenomethionine or selenium in a suitable carrier, wherein said formulation is suitable for topical application for the treatment of psoriasis, eczema, seborrhoeic dermatitis and related skin and scalp conditions.
26. A formulation as claimed in claim 25 further comprising at least zinc pyrithione, N- acetyl-L-cysteine, zinc oxide, vitamin E, or vitamin C.
27. A formulation as claimed in claim 25 capable of being encapsulated in protective membranes selected from the group consisting of liposomes, nanospheres and glycospheres.
28. A formulation as claimed in claim 25 dispersed or dissolved in at least one solvent selected from the group comprising of water, 1,2,4-butanetriol, propylene glycol, sorbitol esters, butylene glycol, polyethylene glycol, polypropylene glycol, glycerol, 1,2,6- hexanetriol, ethanol, isopropanol, butanediolmethanol, propanol, butanol, and ethylene glycol.
29. A formulation as claimed in claim 11, including or excluding melanin pigments, wherein the formulation is a cosmetic or pharmaceutical composition.
30. The formulation as claimed in claim 11, further comprising of medium fatty acid glyceride, higher fatty acid glyceride, or both.
31. The formulation as claimed in claim 11, further comprising at least one member selected from the group consisting of mono-, di- and triglycerides of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, linoleic acid, and linolenic acid or tar products.
32. The formulation as claimed in claim 11, further comprising liposomes, optionally mixed hyaluronic acid, at least one physiologically acceptable carrier, or mixed with both hyaluronic acid and at least one physiologically acceptable carrier, and wherein said formulation is a pharmaceutical composition.
33. A formulation as claimed in claim 13, further comprising at least one of steroids, non- steroidal anti-inflammatories, capsaicin extract, tar products, tissue respiratory factor or the local anesthetics of the caine family.
34. A formulation as claimed in claim 11, further comprising vitamin E, pyruvate, .beta.- carotene, selenium, N-acetylcysteine, vitamin C, catalase, glutathione peroxidase, glutathione reductase, or alpha tocopherol alone or in combinations thereof.
35. A formulation comprising the isoenzyme of SOD as claimed in claim 1 together with a cosmetically acceptable peroxidase, cosmetically acceptable peroxidase substrate, hydrogen peroxide, ascorbate solvents, carriers and additives.
36. A formulation as claimed in claim 35 made as a cosmetic emulsion further comprising of one member selected from the group consisting of steareth-2, steareth-21, propylene glycol- 15 stearyl ether, cetearyl alcohol, butylene alcohol, water, a preservative, parabens, phenoxyethanol and tocopherol.
37. A formulation comprising the isoenzyme of SOD as claimed in claim 1 further comprising at least one member selected from the group consisting of surfactants, colorants, perfumes, preserving agents, emulsifiers, synthetic oil, mineral oil, vegetable oil, fatty acids, fatty alcohols, a liquid carrier, water, fatty substances forming the fatty phase of an emulsion, milks creams, and resins, wherein the at least one substance is suitable for dermopharmaceutical purposes.
38. A formulation as claimed in claim 37 wherein the fatty phase of the emulsion is formed by employing compounds selected from the group consisting of mineral oils, vegetable oils, synthetic oils, waxes, fatty alcohols and fatty acids.
39. A formulation as claimed in claim 37 wherein the mineral oil is liquid paraffin.
40. A formulation as claimed in claim 37 wherein the synthetic oil is at least one of the groups consisting of ethyl palmitates, isopropyl palmitates, alkyl myristates, isopropyl myristate, butyl myristate, cetyl myristate, hexyl stearate, triglycerides of octanoic acids, triglycerides of decanoic acids, cetyl ricinoleate, stearyl octanoate, purcellin oil, and hydroxylated polyisobutene octanoate.
41. A formulation as claimed in claim 37 wherein the vegetable oil or wax is selected from the group consisting of sweet almond oil, avocado oil, coconut oil, wheat-germ oil, corn oil, castor oil, olive oil, palm oil, sesame oil, soya oil, argan oil, evening primrose oil, borage oil, jajoba oil, essential oils, vegetable waxes, beeswax, synthetic waxes, and silicone waxes.
42. A formulation as claimed in claim 37 wherein the fatty alcohol is selected from the group consisting of cetyl alcohol, stearyl alcohol, myristyl alcohol, hydroxystearyl alcohol, oleyl alcohol, isostearyl alcohol, lauryl alcohol, hexadecyl alcohol, ricinoleyl alcohol, behenyl alcohol, erucyl alcohol and 2-octyl-dodecanol.
43. A formulation as claimed in claim 37 wherein the fatty acids are selected from the group consisting of stearic acid, myristic acid, palmitic acid, oleic acid, linoleic acid, lauric acid, isostearic acid, hydroxy-stearic acid, ricinoleic acid, arachidic acid, behenic acid, erucic acid and lanolinic acids.
44. A formulation as claimed in claim 37 wherein the vegetable oil is selected from the group consisting of olive oil, maize oil, sesame oil, camellia oil, coconut oil and palm oil.
45. A formulation as claimed in claim 37 further comprising at least one amphiphilic agent, natural amphoteric surfactant, polyglycerin-fatty acid ester, polyoxyethylenesorbitan fatty acid ester, sorbitan fatty acid ester or polyethylene glycol.
46. A formulation as claimed in claim 37 further comprising of at least one amphoteric surfactant, soybean phospholipid, yolk lecithin, phosphatidyicholine, yolk lecithin, soybean lecithin, or phosphatidylethanolamine.
47. A formulation for topical application comprising an isozyme of a SOD as claimed in claim 1, wherein the formulation is in the form of a lotion, a serum, a liquid, semiliquid or milk emulsion wherein the emulsion is obtained by dispersing a fatty phase in an aqueous phase of oil-in-water or water-in-oil or suspensions, cream emulsions, gel emulsions, microgranulates, or vesicular dispersions that are ionic or nonionic.
48. A formulation as claimed in claim 47 further comprising of at least one member chosen from the group consisting of an antioxidant, vitamin E, alpha-d-tocopherol, alpha-dl- tocopherol, alpha-d-tocopherol acetate, alpha-dl-tocopherol acetate or alpha-d-tocopherol acid succinate, ascorbic acid, beta-carotene, polymer, mixture of polymers and selenium.
49. A formulation as claimed in claim 47 further consisting of at least one member selected from the group consisting of an antioxidant, vitamin C, acetyl-L-carnitine and glutathione reductase.
50. A formulation as claimed in claim 47 further comprising of free radical scavengers, antioxidants, anti-inflammatory agents, or a local anesthetic.
51. A formulation as claimed in claim 47 further consisting of an agent selected from the group consisting of 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, zmc tetratluoroborate, zinc gluconate, and zinc glycinate.
52. A formulation as claimed in claim 47 further comprising of an antioxidant together with the enzymes of the pentose monophosphate shunt pathway that regenerates or produces reduced nicotinamide adenosine diphosphate (NADPH).
53. A formulation as claimed in claim 47, wherein said formulation is capable of being administered in at least one route selected from the group consisting of: topical administration; oral administration; and parenteral administration.
54. A drug delivery system comprising of the isoenzyme of SOD as claimed in claim 1 and a polymer, and optionally comprising an antioxidant within the matrix of the polymer, wherein the matrix does not interact with the antioxidant.
55. A drug delivery system as claimed in claim 54 wherein the drug delivery system comprises of a natural, modified natural, synthetic, hydrophilic or hydrophobic polymer.
56. A drug delivery system as claimed in claim 54 wherein the polymer is selected from the group consisting of gelatin, hydrophilic polymers, hydrophobic polymers, ovalbumin, soybean proteins, gum arabic, modified starch, methylcellulose, hydroxypropyl cellulose, and hydroxypropylmethyl.
57. A drug delivery system as claimed in claim 54 wherein the polymer is selected from the group consisting of polyamide, polyacrylate, polyurethane, waxes, polypropylene, and ethyl cellulose.
58. A drug delivery system as claimed in claim 54 adapted to a dosage form (s) for local administration, ophthalmic administration, and transdermal administration.
59. A drug delivery system as claimed in claim 54 adapted to dosage form (s) for implants which would release the antioxidant in a controlled manner.
60. A drug delivery system as claimed in claim 54 made in the form of a film.
61. A drug delivery system as claimed in claim 60 wherein the film is prepared from polymer solutions in ethanol or chloroform.
62. The formulation as claimed in 11,35,25,29,37,47 or 54 at is in the from of a day cream, night cream, makeup removal cream, foundation cream, sun cream, fluid foundation, makeup removal milk, body protection or care milk, sun milk, lotion, gel, cleansing lotion, sun lotion, artificial tanning lotion, composition for the bath or a deodorizing composition wherein the formulation may further comprise a bactericidal agent.
63. The formulation as claimed in claims 11,35,25,29,37,47 or 54 that is in the form of a shampoo that may also further comprise of at least one member selected from the group consisting of melanin pigments, a cationic, anionic or nonionic detergent, a dye, a composition for the reduction step of a deformation of hair, mercaptans, sulfites, and a composition for slowing down the loss of hair and for promoting fresh growth of hair, minoxidil (2,4-diamino-6-piperidino-3-pyrimidine oxide) and its derivatives, diazoxide (7- chloro-3 -methyl- 1,2,4-benzothiadiazine, 1,1-dioxide) and phenytoin (5,5-diphenyl imidazolidine-2,4-dione) .
64. The formulation as claimed in claims 11,35,25,29,37,47 or 54 that is in the form of an oral composition, a dental composition, or both an oral and dental composition, that further comprising of at least one member selected from the group consisting of toothpaste, an adjuvant or additive for compositions for oral use, a surface-active agents, a thickening agent, a moisturizer, a polishing agent, silica, a fluoride, sodium fluoride, a sweetening agents and sodium saccharinate.
65. The formulation as claimed in claims 11,35,25,29,37,47 or 54 further comprising of an cosmetic compositions capable of maintaining the keratinous structure of the skin or of the hair.
66. An isoenzyme of SOD as claimed in claim 1, in a composition comprising of an endogenous antioxidant, glutathione, an amino acid with selenium, selenemethionine or selenocysteine.
67. An isoenzyme of SOD as claimed in claim 1, that is in the form of a cream, lotion, solution, spray, aerosol, ointment, gel, balm, patch, or emulsion.
68. An isoenzyme of SOD as claimed in claim 1, further comprising of an antioxidant, vitamin E, green tea or pycnogenols.
69. A toothpick having the novel isoenzyme of SOD as an active ingredient as claimed in claim 1.
70. 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.
71. An isoenzyme of SOD as claimed in claim 1, wherein the isoenzyme is capable of being immobilized onto a suitable medium selected from the group consisting of: (a) a polymer matrix, a polymer film, wherein the polymer matrix or film are capable of being be used in biomedical/cosmetic/food science/field/industry as a scavenger of O.sub.2 ;
(b) a water-soluble polysaccharide, dextran, dextrin;
(c) a protein, serum albumin, casein, amylases, trypsins; and
(d) a synthetic polymer, polyethylene glycol, polyvinyl alcohol.
72. A purified isoenzyme of SOD as claimed in claim 1, wherein said enzyme has the following characteristics:
(d) 02 " scavenging activity at 25. degree. C. for 5 days without adding any stabilizing agent polyols or sugars; and (e) stable at 4.degree. C. for at least 18 months.
73. A method for identification of the target isoenzyme of claim 1 comprising
(a) localizing various isoenzymes of SOD in the crude extract of the leaf or rhizome of Curcuma on 7-12% native polyacrylamide gel;
(b) after electrophoresis, rinsing the gel with distilled water followed by incubation for 30 minutes in 2.5 mM NBT;
(c) immersing the" gel in 1.17.times.l0.sup.-6 M riboflavin for 20 minutes and removed later onto a petri plate to expose to a light intensity of 25-1000.mu. Einstein/m.sup.2 /second using a fluorescent light source to develop purple color throughout the gel except for the locations where SOD was localized;
(d) incubating with nitroblue tetrazolium and riboflavin, and exposing to light at room temperature.
(e) Incubating the gel with continuous shaking on a gel rocker .
(f) identifying the most prominent isoezymes for the purpose of purification.
74. A method for the preparation of the isoezymes of claim 1, comprising
(a) homogenizing leaf tissue in a homogenizing buffer at pH 7.0-7.8 and at a temperature range between 4-8. degree. C;
(b) filtering the homogenate and centrifuging the filtrate at 8,000-13,000 rpm for 10-30 minutes at 4-8.degree. C;
(c) decanting the supernatant for purification of SOD;
(d) precipitating SOD with 30-60% ammonium sulfate;
(e) dissolving the precipitate in a 10 to 100 mM phosphate buffer at pH 7 to 7.8 and dialyzing for 18-36 hours with 6-12 changes of the buffer; . (f) loading the dialyzed protein onto a column with Sephadex G 100
(g) loading the protein from Sephadex G-100 column onto an affinity (Blue Sepharose) /ion exchange column and eluted with 5-20 μM NBT (Blue Sepharose Column) or 100-500 mM
KCl prepared in a buffer (all autoclaved or non-autoclaved);
(g) assaying fractions containing protein for SOD activity; (h) fractionating SOD containing fractions on a fast protein liquid chromatography (FPLC) using mono Q column and eluted with 50-200 mM KCl prepared in 10-50 mM phosphate buffer (all autoclaved or non-autoclaved) with a flow rate of 5.0-10.0 ml per minute;
(i) assaying each peak was assayed for SOD activity SOD peak, obtained after FPLC and concentrating using a protein concentrator column/ glycerol; (j) assaying concentrated protein for SOD activity at room temperatures before and after heating at varying temperatures ranging between 40 to 100 degree. C;
(k) localizing the purified SOD on 7 to 12% polyacrylamide gel by known methods; (1) identifying the target isoenzyme by the method of claim72 and;
(m) recovering the most prominent isoenzyme n) the isoenzyme was characterized further by treating the gel with 2-10mM KCN /2-10 mM
H sub.2 Osub. 2.
PCT/IN2004/000248 2003-08-19 2004-08-19 Isozyme of autoclavable superoxide dismutase (sod) derived from curcuma longa l WO2005017134A2 (en)

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* Cited by examiner, † Cited by third party
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6485950B1 (en) * 2000-07-14 2002-11-26 Council Of Scientific And Industrial Research Isozyme of autoclavable superoxide dismutase (SOD), a process for the identification and extraction of the SOD in cosmetic, food and pharmaceutical compositions
WO2003080091A1 (en) * 2002-03-27 2003-10-02 All India Institute Of Medical Sciences Herbal ophthalmic formulation for preventing cataract
US20040033277A1 (en) * 2001-12-13 2004-02-19 Council Of Scientific & Industrial Research Herbal medicaments for the treatment of neurocerebrovascular disorders

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6485950B1 (en) * 2000-07-14 2002-11-26 Council Of Scientific And Industrial Research Isozyme of autoclavable superoxide dismutase (SOD), a process for the identification and extraction of the SOD in cosmetic, food and pharmaceutical compositions
US20040033277A1 (en) * 2001-12-13 2004-02-19 Council Of Scientific & Industrial Research Herbal medicaments for the treatment of neurocerebrovascular disorders
WO2003080091A1 (en) * 2002-03-27 2003-10-02 All India Institute Of Medical Sciences Herbal ophthalmic formulation for preventing cataract

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GHONEIM A I ET AL: "Protective effects of curcumin against ischaemia/reperfusion insult in rat forebrain" PHARMACOLOGICAL RESEARCH, ACADEMIC PRESS, LONDON, GB, vol. 46, no. 3, September 2002 (2002-09), pages 273-279, XP002284470 ISSN: 1043-6618 *
SREEKANTH KAVITHA SIVARAMAN ET AL: "Antioxidant activity of Smoke Shield in-vitro and in-vivo." JOURNAL OF PHARMACY AND PHARMACOLOGY, vol. 55, no. 6, June 2003 (2003-06), pages 847-853, XP009042801 ISSN: 0022-3573 *
SUBRAMANIAN LALITHA ET AL: "Prevention of CCl4-induced hepatotoxicity by aqueous extract of turmeric" NUTRITION RESEARCH, vol. 19, no. 3, March 1999 (1999-03), pages 429-441, XP002314161 ISSN: 0271-5317 *

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