LV13250B

LV13250B - A composition for treating neurocerebrovascular disorders

Info

Publication number: LV13250B
Application number: LVP-04-78A
Authority: LV
Inventors: Madhur Ray; Raghwendra Pal; Satyawan Singh; Nandoo Mal Khanna
Original assignee: Council Scient Ind Res
Priority date: 2001-12-14
Filing date: 2004-07-13
Publication date: 2006-02-20
Also published as: JP2005516930A; DE60233069D1; AU2009212969B2; NO20043000L; LT2004060A; KR20040073478A; CN100528149C; EA007067B1; US20060051438A1; WO2003051380A3; NO332066B1; US20100093870A1; EA200400807A1; CA2473874A1; EE05374B1; WO2003051380A2; LT5284B; BR0214962A; MXPA04005680A; KR101001815B1

Description

i

A COMPOSITION FOR TREATING NEUROCEREBROVASCULAR DISORDERS

Field of the present invention

The present invention relates to the method of producing lipid soluble extract called Curcuma oil in high yield, from rhizomes and leaves of species of zingiberaceae family, particularly Curcuma species and also use of the said oil, its constituents, and novel derivatives of said constituents, for the treatment of Neurocerebrovascular disorders

Background and prior art references

Neurocerebrovascular diseases like cerebrovascular infarction, stroke, ischemic attacks etc. are caused by an interruption of the blood supply resulting from disease of the arteries carrying blood to the brain.

Of the three general types of stroke, cerebral hemorrhage is caused by rupture of a blood vessel vvith bleeding into the brain (intra cerebral hemorrhage) or under its covering membrane, vvhile cerebral thrombosis stems from obstruction of a cerebral blood vessel vvhen a blood clot forms vvithin the vvalls.

The clot may be caused by abnormal thickening of the blood, damage to the vessel vvall from arteriosclerosis, atherosclerosis, inflammation of the arteries or inflammation of the veins.

If the blood supply is stopped completely or is reduced to less than one-fourth its normai Ievel, softening of the brain (cerebral infarction) results, causing permanent brain damage.

Cerebral embolism is obstruction of a cerebral artery by a blood clot or a foreign body migrating from another part of the body’s circulation like vvhen a clot that has formed on the inside vvall of one of the arteries in the neck travels up to the brain and blocks a major artery branch.

Trasient ischemic attacks (TIAs) are brief episodes of symptoms caused by temporary interruptions of the blood supply. Reversible ischemic neurological deficits( RJNDs) are small cerebral infarction. Multiple cerebral infarction can lead to permanent confusion and memory loss. Ischemic stroke is a medical emergency. After TIAs or stroke occur, treatment may be surgical or medical.Surgery may be needed in some cases to remove any blockage of blood vessels going to the brain.

Medication can prevent the formation of blood clots on the atheroscl-erotic plaques within the vessei wall. Brain swelling commonly accompanies brain infarction or hemorrhage.No satisfactory treatment is available.

Currently used drugs in peripheral vascular and cerebral disorders include ergot alkaloids, aspirin, anti-coagulants etc. The latter are used following strokes to prevent further cerebrovascular incidents but their use is contraindicated if the stroke was the result of hemorrhage.

The use of TICLOPIDINE, a highly effective anti-platelet aģent to treat stroke cases is restricted in its long term use due to its adverse side-effects. Tissue plasminogen activator (t-PA) used to treat clots in the coronary arteries (acute heart attack), is a natūrai clot dissolving substance produced by the body which can blow open a blood clot in the brain that causes the acute ischemic brain damage characteristic of a stroke.While t-PA can dissolve the blood clot that causes a blood vessei blockage, there are other complications vvhich occur during ischemic stroke vvhich must be addressed if permanent brain damage is to be prevented. It is critically important to have nitric oxide (NO) and superoxide scavengers in the blood stream when t-PA is administered to reduce the free radical damage that vvill occur vvhen the blood flovv is restricted and even more when the flovv is resumed.

Nitric oxide(NO) and superoxides inflict damage on important biomolecules and their increased production has been implicated in human diseases Iike cerebro-,cardiovascular, inflammatory, neurological dysfunctions and cancer etc.[ Onoda M., Inano H.,Nitrc oxide:Biology and Chemistry, 4,(5),505-515 (2000)].

Most strokes culminate in a core area of celi death (infarction) and the blood flovv is so drastically reduced that the celis usually can not recover.Brain celis die as a result of the actions: calcium activated proteases (enzymes vvhich digest celi proteīns),lipases(enzymes vvhich digest celi membranes) and free radicals formed as a result of the ischemic cascade. Without neuroprotective aģents, nerve celis may be irreversibly damaged vvithin several minūtes. Any disruption of blood flow to the brain causes massive free radical damage that inducēs much of the re-perfusion injury to brain celis, typical of strokes. When blood flow is interrupted and subsequently restored (reperfused), tissues release iron that acts as a catalyst for the formation of free radicals that often permanently damage brain celis. Protecting brain celis from injury caused by blood flow disruption, therefore, is of prime importance. If an ischemic stroke is happening., the use. of large ,quantities of..antboxidants like melatonin, vitamīns and herbs like Ginkgo biloba have been suggested to provide some benefit. Magnesium in an oral dose of 1500 mg. is a safe nutrient to relieve an arterial spasm, a common problem in thrombotic strokes.

The ancient Indian system of medicine- Ayurveda-is concemed with the prevention, diagnosis and cure of disease.The word “dis-ease”- a right translation of illness is viewed as a dysfunction of-the whole body and is attributed to the circulation and transformation of ubiquitous humoral fluids.

Most of the Ayurvedic drugs are products of high repute which act on a number of dysfunctions of the body involving various orgāns and aim at preventing problems or restoring a normai situation, and try to recover the patient completely. Evolved over a long period of time and experimentation, they are the results of a particular combination of certain fundamental elements which determine their properties which in tum are responsible for the Chemical, biological or therapeutic effects of those substances. There is no substance when correctly prepared which can not be used as remedy.

Ayurveda describes a number of beneficial effects of rhizomes and leaves of various species belonging to zingiberaceae family, especially those of Curcuma longa L.syn.Curcurna domestica Valeton, rhizomes and leaves popularly knovvn as Turmeric or Haldi. Prominent among these are the anti-bacterial, antifungal woiind healing and the anti-inflammatory actions vvhich enabled turmeric paste to be used as a house hold remedy to treat wounds and inflammation.

In recent years, its constituents-Curcumin and other curcuminoids have been found to exhibit besides these activities, choleretic, cholagogic, anti-oxidant, anti-cancer, inhibition of leukotriene biosynthesis, 5-lipoxygenase, cyclo-oxygenase, lipid peroxidation, superoxide and nitric oxide (NO) scavenging effects.

Turmeric -a highly reputed herb in Indian system of medicine-Ayurveda-is the rhizome of Curcuma longa L.Syn. Curcuma domestica Valeton (Fani. Zingiberaceae) ,vvhich grovvs abundantly in India. It has long been used as a spice and a colouring aģent in food as well as a naturally occuiring medicme. Its povvder or extracts are recommended to treat vvounds and inflammation.

A major constituent Curcumin vvas developed as an anti-inflammatory aģent [Srimal R.C.,Khanna N.M.,Dhawan B.N., Ind. J. Pharmacol.,3,10 (1971)]. Other therapeutic properties of Curcumin, various curcuminoids and some other constituents of Curcuma species include anti-bacterial, anti-fungal [Sclīraufstatter F., BrentH., Nature, 264,456(1949), Arch.Dermatol..u.Syphilis,735,250 (1949);Lutomski.J.,KedziaB.,Debska W,Planta Med., 26, 9 (1974 ) ; Rao B.G.N., Joseph P., Reichst,Aromen Koerperflegem, 27,405-406(1971); Swada

T., Yamahara J., Shimazu S., Olita T., Shoyakugaku Zasshi, 2 ,11-16(191), Prasad C. R., Sirsi

M. , J.Sci. Ind. Res.,C. 75, 239-41( 1957); Schraufstatter E., Deutsh. S. Z. Naturforsch. 4, 276 ( 1949 ) ; Chopra R. N., Gupta J. C., Chopra G. S.,Ind. J. Med. Res., 29, 769-72 (1941 )], antioxidant [ Ramaswamy T. S., Baneijee Β. N., Ann. Biochem. Exp. Med., 3,55 ( 1948 ) ; Chipault ,J. R., Mizuno G. R., Lundberg W. O., Food Res., 10,209, (1956 )]; inhibition of lipid per-Oxidation [Sharma S.C., Mukhtar H.., Sharma S. K., Krishnamurty C. R., Biochem. Biopharmacol., 21 , 1210-14 ( 1972 ); Zu S., Tang. X. Lin Y., Zhougcuoyev., 22, 2645(1991); Sharma Ο. P., Biochem. Biopharmacol. 25 , 1811(1976 )]; active oxygen species scavenging and prevention of increased free radical formation by Curcumin in the body [Tennesen H.H.,Inter.J. Pharmacol.,50, 67-69(1989), Kunchandy E.., Rao M.N. A., Inter. J. Pharmacol., 58, 237 (1990)]; inhibitory activity for iNOS induction by lipopolysaccharide in the mammary gland and scavenging activity for NO radicals by Curcumin, [ Onoda M., Inano H., Nitric Oxide : Biology and Chemistry, 4 ,505-515 ( 2000)], anti-inflammatory [ Arora R.B., Basu N., Kapoor V., Jain A., Proc. Second Indo Soviet Symposium on. Natūrai Products, Nevv Delhi,1970, p. 170., Ind. J.Med. Res., 59 ,10 ( 1971); Mukhopadhya A., Basu

N. , Ghatak N., Singh Κ. P., Gujral P.D., Proc. Int. Union of Physiol. Sci., 77,241( 1974); GhatakN.N., BasuN., Ind. J. Exp. Biol., 70,235 (1972), ChandraD., Gupta S.S., Ind. J. Med. Res., 60, 138-142 (1972)]; anti-cancer [Soudamini K.K., Kuttan R., J. Ethnopharmacol.

27,221 (1989);Kuttan R. Bhanumatty P., Nirmala K., George M.C., Cancer Lett., 29, 197 (1985)]; antioxidant and antitumor promotor vvhich inducēs apoptosis in human leukemia cellsļRao M.L., Huang T.S, Lin J.K, Biochem. Biophysic. Acta,1817, 98-100(1996)], inhibition of celi grovvth in chinese hamster ovary celi culture and cytotoxicity to lymphocytes and Dalton’s lymphomaCells., [ Cancer Lett. (Ireland), 29,197 (1985) via Chem. Abstr. 104, 61654 ^d (1986)], tumor protecting activity in mouse skin carcinogenesis induced by 7,12dimethyl benz (a) anthracene[Kyoto-Furiton Doigaku Zasshi, 96, 725 (1987)-via Chem. Abstr., 107, 211555³ (1987)], inhibition of HIV protease [ Suz Luz, Craik C.S.,Oritz T., Montanello P.R., Proc. 205 , ACS National Meeting, Denver, colarado, 28 March-2 april, Amer. Chem. Soc. Med. Chem. Div.(1993), Take Y, Inoyya H., Nakamura S., Alauddin H.S., Kuba A, J.Antibiot.,42,107-118 (1989)], inhibition of lipoxygenase, cyclooxygenase [ Tennesen. H.H, Int. J Pharmacol.,50,67 (1989), inhibition of ADP-epinephrine and collagen induced platelet aggregation, [ Srivastava R., Pūri V., Srimal R.C., Dhawn B.N.; Arznei Forsch.,36, 715-717(1986)]; protection against thrombotic challenge [ Srivastava R, Dixit M, Srimal R.C.,Dhawan B.N.,Thromb. Res.,40, 413-17(1985)]; reduction in ratio of total cholesterol / phospholipids in hyperlipidemic rats and elivated HDL-cholesterol and total cholesterol ratio[Ind. J.Physiol.Pharmacol,32,299 (1988)]; anticoagulent activity [Chem. Phaim. Buli.,33, 1499 (1985)]; inhibition of platelet aggregation, metabolic disorders and hyperlipidemia [Lin Y.,U.S. Patent, 4842849;.' Chem. Abstr.,777,160200 (1984); Khanna N.M, Sarin J.P.S., Singh S., Pal R., Seth R.K., Nitya Nand S., Indian Patent 162441(1984)]; vvhich makes it useful to prevent cardiovascular disorders like ischemic heart attacks, myocardial infarction etc. In Īndo-China region, Curcuma extracts are given at parturition on account of their anticoagulent action. Ethyl p-methoxy cinnamate isolated from Curcuma rhizomes essential oil exhibit antifungal activity[ Herba Hung., 28, 95(1989), via Chem. Abstr. 777,191496j (1989)], vvhile furanogermenone and (4S,5S) (+) germacrone 4,5-epoxide also isolated from Curcuma rhizomes essential oil exhibits anti-inflammatory and preventive effect against stress ulceration [Yalcugaku Zasshi, 106, 1137 (1986),Chem. Abstr..706, 95935c (1987); Zhongyao Tungbto, 10, 134 (1983),Chem. Abstr. 103, 115886d(1985)]. The other reputed herb from Zingiberaceae family, Zingiber officinale Rosch, exhibits preventive effects in heart attack or stroke[ Srivastava K.C., Prostaglandins Leukotrienes and Medicines,13,227-235(1964)].

Ohjects of the present Invention:

The main object of the present invention is to use the lipid soluble extract from rhizomes and leaves of Curcuma species, which belong to zingiberaceae family for the treatment of Neurocerebrovascular disorder.

Another object of the present invention is to develop a method to producē lipid soluble extract in high yield from rhizomes and leaves of Curcuma species, vvhich belong to zingiberaceae family.

Yet another object of the present invention is to separate individual components from the Curcuma oil.

Stili another object of the present invention is to develop analogs of the said constituents of the Curcuma oil.

Stili another object of the present invention is to detect the Neurocerebrovascular disorders of the said analogs.

Stili another object of the present invention is to identify active components in Curcuma species for the treatment of neurocerebrovascular disorders.

Stili another object of the present invention is to use active components of Curcuma species for the treatment of ischemia.

Stili another object of the present invention is to use active components of Curcuma species for the treatment of stroke.

Stili another object of the present invention is to use active components of Curcuma species for the treatment of hemorrhage.

Stili another object of the present invention is to use active components of Curcuma species for the treatment of thrombosis.

Summary of the present invention

The present invention relates to the method of producing lipid soluble extract called Curcuma oil in high yield. The source of said oil is rhizomes and leaves of species of zingiberaceae 'family. The particularly species of the said family used to producē said oil is Curcuma species. The said oil is used for the treatment of Neurocerebrovascular disorders. The novel analogs of the constituents of said oil are developed and are also found to have use in the treatment of Neurocerebrovascular disorder.

Detailed deseription of the present invention

Accordingly the present invention relates to an improved method of obtaining high yields of the lipid soluble extract called Curcuma oil and its constituents from rhizomes and leaves of species of Zingiberaceae family particularly Curcuma species.

A composition obtained from the lipid soluble extract of rhizomes and leaves of Curcuma species of Zingiberaceae family, useful for the treatment of neurocerebrovascular disorders, said composition comprising fraction A consisting of ar-turmerone of formula 1, and tunnerone of formula 2, and/or along with fraction B consisting of curcumene and zingiberine, and/or fraction C consisting of germaerone, curcumerone, zedoarone, sedoarondiol, isozdedoaronidiol, curcumenone, and curlone, and/or pharmaceutically acceptable additives.

In stili another embodiment of the present invention, wherein curcuma species is Curcuma domestica Valeton.

In stili another embodiment of the present invention, wherein ratio of fraction A, fraction B, and fraction C is ranging betvveen 1 to 3:1 to3: lto 3.

In stili another embodiment of the present invention, wherein additives are selected from a group comprising melatonin, antioxidants, calcium channel antagonists, tissue plasminogen activator (t-PA), and celi membrane stabilizing aģents.

In stili another embodiment of the present invention, wherein said composition inhibits nitric oxide synthase (NOS) overproduction, prevention calcium overload in neurons, and scavenging free radicals.

In stili another embodiment of the present invention, wherein said composition cerebrovascular disorders are selected from a group comprising ischaemia, strolce, poststroke injury, hemorrhage, reperfusion injury, thrombosis, vasoconstriction, nitric oxideinduced free radical oxidative damage, infraction, inflammation, and Alzheimer’s disease.

In stili another embodiment of the present invention, vvherein fraction A of the composition is most effective.

In stili another embodiment of the present invention, vvherein said disorders are treated using the said composition is the form of various delivery systems selected from a group comprising tablets, capsules, suppository, beads, and aerosols.

In stili another embodiment of the present invention, vvherein an improved method of obtaining high yield lipid soluble extract and its subsequent fractions comprising fraction A consisting of ar-turmerone of formula 1, and turmerone of formula 2, fraction B consisting of curcumene and zingiberine, and fraction C consisting of germacrone, curcumerone, zedoarone, sedoarondiol, isozdedoaronidiol, curcumenone, and curlone, from rhizomes and leaves of Curcuma species Zingiberaceae family, said method comprising steps of:

• povvdering the rhizomes and leaves of the curcuma species in fine pārticies form, • extracting vvith the said povvder vvith polar organic solvent under continuous stirring or sonication for about 24 hours at room temperature, • repeating step (b) tvvo to five times, • removing solvent by distillation under reduced pressure and belovv about 45°C to obtain residual concentrate, • triturating the residual concentrate vvith non-polar solvents, • removing solvent by distillation under reduced pressure and belovv 45°C, • obtaining the said lipid soluble extract, • fractionating the said extract by column chromatography, l

• obtaining fraction A, fraction B, and fraction C, and • fractionating_each of fractions A, B, and C further using HPLC or GLC to obtain the said constituents.

In stili another embodiment of the present invention, vvherein fractioning the extract on silica gel column.

In stili another embodiment of the present invention, vvherein polar solvent is selected from a group comprising alcohol and acetone.

In stili another embodiment of the present invention, vvherein non-polar solvent is selected from a group comprising light petroleum and toluene.

In stili another embodiment of the present invention, vvherein vvherein fractionating the extract using n-hexane, n-hexane: ethyl acetate mixture of ratio 95:5, and ethyl acetate successively.

In stili another embodiment of the present invention, vvherein fraction A constitutes about 75% of the said extract.

In stili another embodiment of the present invention, vvherein ar-turmerone constitutes 95% of the fraction A.

In stili another embodiment of the present invention, vvherein pressure is ranging betvveen 7 and 11 mmHg.

In stili another embodiment of the present invention, vvherein concentration of the extract is about 6%.

In stili another embodiment of the present invention, vvherein a method of treating neurocerebrovascular disorders in animals including humāns using composition of claim 1, by administering therapeutically effective amount of lipid soluble extract.

In stili another embodiment of the present invention, vvherein said method involves inhibiting nitric oxide synthase (NOS) overproduction, prevention calcium overload in neurons, and scavenging free radicals.

In stili another embodiment of the present invention, vvherein vvherein 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.

In stili another embodiment of the present invention, vvherein said diseases are treated using the said composition is the form of various delivery systems selected from a group comprising tablets, capsules, suppository, beads, and aerosols.

In stili another embodiment of the present invention, tvvo novel compounds of formulae 3 and 4.

In stili another embodiment of the present invention, a method of treating Ischaemia in animals including humāns using composition of claim 1, said method comprises step of administering therapeutically effective amount to the subject.

In stili another embodiment of the present invention, vvherein said method helps treat severe brain ischaemia.

In stili another embodiment of the present invention, vvherein the effective amount is ranging betvveen 10-1000 mg/day in divide dosage schedule.

In stili another embodiment of the present invention, vvherein the said composition is administered through various routes comprising i.p., and p.o.

In stili another embodiment of the present invention, vvherein said method prevents overload of calcium ions in the mitochondria.

In stili another embodiment of the present invention, wherein the fraction A is most effective.

In stili another embodiment of the present invention, a method of treating stroke in animals including humāns using composition of claim 1, said method comprises step of administering therapeutically effective amount to the subject.

In stili another embodiment of the present invention, wherein treating strokes selected from a group comprising thrombotic, embolic, and focal.

In stili another embodiment of the present invention, vvherein the effective amount is ranging betvveen 10- In stili another embodiment of the present invention, 1000 mg/day in divide dosage schedule.

In stili another embodiment of the present invention, vvherein the fraction A is most effective.

In stili another embodiment of the present invention, a method of treating hemorrhage in animals including humāns using composition of claim 1, said method comprises step of administering therapeutically effective amount to the subject.

In stili another embodiment of the present invention, vvherein the effective amount is ranging betvveen 10-500 mg/day in divide dosage schedule.

hr stili another embodiment of the present invention, vvherein the said composition is administered through various routes comprising i.p., and p.o.

In stili another embodiment of the present invention, a method of treating thrombosis in animals including humāns using composition of claim 1, said method comprises step of administering therapeutically effective amount to the subject.

In stili another embodiment of the present invention, vvherein thrombosis is selected from a group comprising cerebral, coronary, and deep vein.

In stili another embodiment of the present invention, vvherein the said method brings dovvn the thrombus to one-fourth.

In stili another embodiment of the present invention, A method of treating hypertension in animals including humāns using composition of claim 1, said method comprises step of administering therapeutically effective amount to the subject.

In stili another embodiment of the present invention, vvherein the said method brings dovvn the blood pressure by about 40%.

In stili another embodiment of the present invention, wherein the said method maintains„ the blood pressure of normotensives.

In stili another embodiment of the present'invention, amethod' of treating vasoconstričtion ' in animals including humāns using composition of claim 1, said method comprises step of administering therapeutically effective amount to the subject.

In stili another embodiment of the present invention, and nitric oxide-induced free radical oxidative damage in animals including humāns using composition of claim 1, said method comprises step of administering therapeutically effective amount to the subject.

In stili another embodiment of the present invention, vvherein said method augments the Ievel of oxygen scavenging enzym.es comprising superoxide dismutase (SOD), and catalase.

In stili another embodiment of the present invention, vvherein said method decreases the Ievel of thiobarbituric acid reactive substances (TBARS).

/

In stili another embodiment of the present invention, animals including humāns using composition of claim 1, said method comprises step of administering therapeutically effective amount to the subject.

In stili another embodiment of the present invention, said method involves treating various kinds of edema selected from a group comprising brain and pulmonary edema.

In an embodiment of the present invention povvdering dry rhizomes and leaves into fine pārticies.

In another embodiment of the present invention percolating said povvder vvith organic solvent at room temperature.

In yet another embodiment of the present invention stirring the contents continuously during percolation.

In stili another embodiment of the present invention removing the said organic solvent by distillation under reduced pressure belovv 45°C.

In stili another embodiment of the present invention repeating the above mentioned percolation steps atleast 4-8 times.

In stili another embodiment of the present invention collecting Curcuma oil as orange yellow odoriferous liquid at 5 - 8% yield, and

In stili another embodiment of the present invention separating said oil into its constituents by using techniques like Chromatography and distillation under high vacuum.

In stili another embodiment'of the present invention' Curcuma species is selected from agroup comprising Curcuma longa L.Syn. Curcuma domestica Valeton, and Curcuma aromatica Salisb.

In stili another embodiment of the present invention the organic solvent is non- polar organic solvent selected from a group comprising light petroleum, and toluene.

In stili another embodiment of the present invention the organic solvent is polar organic solvent selected from a group comprising ethanol, and propanol.

In stili another embodiment of the present invention non-polar organic solvents give higher yield as compared to polar organic. solvents.

In stili another embodiment of the present invention the residual concentrate from polar organic solvent extract is extracted with non-polar organic solvent.

In stili another embodiment of the present invention Curcuma oil is separated into its individual constituents comprising ar-d-turmerone (formula 1), turmerones of α and β (formula 2), zingiberine, curcumene, germacrone, curcumenone, and curlone.

In stili another embodiment of the present invention kind of the Chromatography is selected from a group comprising Column Chromatography preferably High Performance Liquid Chromatography, and Gas- Liquid Chromatography.

In stili another embodiment of the present invention the adsorbent for the

Chromatography is selected from a group comprising alumina, and silica gel.

In stili another embodiment of the present invention the elution of the said constituents is vvith organic solvent selected from a group comprising ņ-hexane, ethyl acetate, and nhexane and ethyl acetate mixture in varying proportions.

In stili another embodiment of the present invention molecular weight of the individual constituents of Curcuma oil separated by chromatography is tunnerones (α-,β-) - mol.vvt. 218, ar-d-turmerone - mol.wt.216, zingiberine -mol.wt.204, and Curcumene -mol.wt.202.

In stili another embodiment of the present invention retention time of the individual constituents of Curcuma oil separated by chromatography is turmerones (α-,β-) - retention time 9' -04”, ar-d-turmerone - retention time 8'-08, zingiberine - retention time 5-04, and Curcumene - retention time 4-24.

Novel compound of the formula 3, an analog of compounds comprising ar-d-turmerone, turmerone, and germacrone vvherein, R represents an alkyl, alkenyl, cycloalkane, phenyl, cycloalkene, or cycloalkadiene group, vvith substituents like alkyl, or alkoxy halo, in the phenyl, cycloalkene, cycloalkadiene rings, or hetroaryl like pyridyl nitrogen heterocyclic amine and substituted amines, and Rl represents alkyl or arylalkyl group.

R·

Novel Compound of the formula 4, au analog of compounds comprising Procurcumenol, zedoarondiol, and curcumenone.

CHRļ-CHRi - Phenyl, Substituted phenyl or = a b Δ, H,

R2 = H, or cd Δ,

R3 = Lovver alkyl C 1-3

Pharmaceutical composition useful for treatment of Neurocerebrovascular disorders, said composition comprising effective amount of the lipid soluble extract called Curcuma oil, from rhizomes and leaves of species of plant Zingiberaceae family particularly Curcuma species, either as such or its individual constituents singly or in combination vvith each other or related compounds comprising melatonin, and tissue plasminogen activatior (t-PA), optionally associated vvith pharmaceutically acceptable additives.

In stili another embodiment of the present invention is used to treat, reduce, control and prevent diseases conditions relating to increased production of nitric oxide (NO), injury due to inflammation, increased calcium entry and free radical oxidative damage to important biomolecules.

In stili another embodiment of the present invention vvherein, the additive is selected from a group of nutrients comprising proteīns, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste,’ and/or pharmaceutically acceptable carrier, excipient, diluent, or solvent.

In stili another embodiment of the present invention is administered orally, inhaled, or implanted.

In stili another embodiment of the present invention vvherein, physical state of the said composition for the oral route is in the form of capsule, tablet, syrup, concentrate, powder, granule, aerosol, or beads.

In stili another embodiment of the present invention is administered at dosage Ievel ranging betvveen 5 to 5000 mg/day.

In stili another embodiment of the present invention is used for treating animals or human beings.

In stili another embodiment of the present invention is used for treating hypertension.

In stili another embodiment of the present invention is used for treating cerebral and pulmonary edema vvhich accompanies cerebral and myocardial infarction.

In stili another embodiment of the present invention is used for treating reperfusion injury.

In stili another embodiment of the present invention is used for treating cerebrovascular diseases comprising strokes, and transient ischaemic attacks.

In stili another embodiment of the present invention is used for treating ali kind of strokes comprising thromotic, embolic, focal and recurrent.

In stili another embodiment of the present invention is used for treating subarachnoid and cerebral hemorrhage.

In stili another embodiment of the present invention is used for treating neurological dysfunction.

In stili another embodiment of the present invention is used for treating thrombosis infraction comprising cerebral, coronary, and deep vein.

In stili another embodiment of the present invention is used for treating cancer.

In stili another embodiment of the present invention is used for treating Alzheimer’s disease vvounds.

In stili another embodiment of the present invention is used .for treating Acquired Immunodeficiency Syndrome.

In stili another embodiment of the present invention is used for treating migraine.

In stili another embodiment of the present invention is administered again iņ case of reļapse conditions.

A method of treating a subject for Neurocerebrovascular disorder conditions, said method comprising administering to the subject effective amount of the lipid soluble extract called Curcuma oil, from rhizomes and leaves of species of plant Zingiberaceae family particularly Curcuma species, either as such or its individual constituents singly or in combination vvith each other or related compounds comprising melatonin, and tissue plasminogen activatior (tPA), optionally associated vvith pharmaceutically acceptable additives.

In stili another embodiment of the present invention, is used for treating animals or human beings.

In stili another embodiment of the present invention the additive is selected from a group of nutrients comprising proteīns, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste, and/or pharmaceutically acceptable carrier, excipient, diluent or solvent.

In stili another embodiment of the present invention the composition is administered orally, inhaled, or implanted.

In stili another embodiment of the present invention the physical State of said composition for the oral route is in the form of capsule, tablet, syrup, concentrate, povvder, granule, aerosol, or beads.

In stili another embodiment of the present invention the composition is administered at a dosage Ievel ranging betvveen 5 to 5000 mg/day.

In stili another embodiment of the present invention the composition is used for treating hypertension.

In stili another embodiment of the present invention the composition is used for treating cerebral, and pulmonary edema vvhich accompanies cerebral, and myocardial infarction.

In stili another embodiment of the present invention the composition is used for treating poststroke injury.

In stili another embodiment of the present invention the composition is used for treating reperfusion injury.

In stili another embodiment of the present invention the composition is used for treating cerebrovascular diseases comprising strokes, and transient ischaemic attacks.

In stili another embodiment of the present invention the composition is used for treating ali kind of strokes comprising thromotic, embolic, focal, and recurrent.

In stili another embodiment of the present invention the composition is used for treating subarachnoid, and cerebral hemoirhage.

In stili another embodiment of the present invention the composition is used for treating neurological dysfunction.

In stili another embodiment of the present invention the composition is used for treating thrombosis infraction comprising cerebral, coronary, and deep vein.

In stili another embodiment of the present invention the composition is used for treating cancer.

In stili another embodiment of the present invention the composition is used for treating Alzheimer’s disease vvounds

In stili another embodiment of the present invention the composition is used for treating Acquired Immunodeficiency Syndrome.

In stili another embodiment of the present invention the composition is used for treating migraine.

In stili another embodiment of the present invention the composition is administered again in case of relapse conditions.

In an embodiment of the present ‘invention, · obtain/prepare*-therapeutically effective · medicaments from extracts of Curcuma species rhizomes and leaves which belong to Zingiberaceae family.

In another embodiment of the present invention, more particularly, the lipid soluble extract/fraction of Curcuma longa L. syn. Curcuma domestica Valeton, commonly known as turmeric or Haldi.

In another embodiment of the present invention, in pharmaceutically acceptable formulations / delivery systems such as tablets, capsules, suppository, beads, aerosols, etc. for the treatment and prevention of human diseases in which increased production of Nitric Oxide (NO) and free radical oxidative damage are implicated.

In another embodiment of the present invention, such diseases are neurocerebrovascular disorders like transient ischaemic attacks (ischaemic, hemorrhagic, focal recurrent etc.) thrombosis (cerebral, coronary ,deep vein), infarction, stroke (thrombotic, embolic, focal etc.), Alzheimer’s disease, mflammat-ory, neurological dysfunctions, wounds , carcinogenesis, tumor progression etc.

In another embodiment of the present invention, the superoxide and nitric oxide (NO) scavenging property of the lipid soluble extract / fraction of Curcuma species rhizomes (Family: Zingieraceae) especially Curcuma longa L. Syn. Curcuma domestica Valeton, hereinafter referred to as Curcuma oil either as such or its various constituents singly or in combination with each other which makes them therapeutically effective to control various degenerative diseases, more particularly a drug which is nitric Oxide (NO) and superoxide scavenger with anti-inflammatory activity to combat brain and pulmonary edema / swelling which accompanies brain and myocardial infarction.

In another embodiment of the present invention, Keeping these biological profilēs in vievv and as a follow-up of the holistic vievv of Ayurveda of human diseases, the lipid soluble extract / material of Curcuma species rhizomes and leaves (Zingiberaceae family) hereinafter referred to as Curcuma oil and obtained from Curcuma longa L.syn. Curcuma domestica Valeton, rhizomes and leaves, either as such or its major active constituents, ar-d-turmerone (formula 1), turmerones .(α,-,β-,formula. 2) either .singly or in combiņatioņ vvith each other vvith and vvith the other minor constituents are found to be sīgnificantly beneficial and possess povverful Nitric oxide(NO) and free radical / superoxide scavenging activity.

In another embodiment of the present invention, said lipid soluble extract exhibit/possess potent free radical scavenging/ antioxidant activity vvhich enables them to protect mitochondrial impairment protecting dovvnstream target and they inhibit overproduction of nitric oxide synthase (NOS), avoid injury due to inflammation and reduce calcium entry so that the resultant calcium overload in the neurons does not occur.

In another embodiment of the present invention, another important advantage is that if there is any blockage, the above three parameters vvhich are the major cause of reperfusion injury are. taken care of by these medicaments and the collaterals from the “Circle of Willis” are able to help in the blood flovv and thereby enable the drug to reach the site of injury.

In another embodiment of the present invention, cases vvhere severe brain ischaemia has occurred, administration of Curcuma oil either as such or its individual constituents such as ar-d-turmerone, turmerones etc. singly or in combiņatioņ vvith each other vvith and vvithout other related compounds of the type of formula 3 or 4 and /or other therapeutically beneficial aģents such as melatonin, other antioxidants, calcium channel antagonists, tissue plasminogen activator (t-PA) and celi membrane stabilizing aģents can provide effective protection against cerebral and even coronary damage.

In another embodiment of the present invention, since stroke is one of the main causes of the mortality among hypertensive patients, our finding also underline the importance of the Curcuma oil either as such or its individual constituents either alone or in combiņatioņ vvith each other as an effective anti-hypertensive drug vvith antioxidant and neuro protective activities.

In another embodiment of the present invention, the lipid soluble extract of rhizomes and leaves of Curcuma species of the family zingiberaceae especially Curcuma longa L..syn. Curcuma domestica Valeton hereinbefore referred to as Curcuma oil which is a pale yellow to orange yellow odoriferous oily liquid whose major constituents are: ar-d-turmerone(formula 1), turmerones (α-,β-, formula 2)[Tap Chi Hoa Hoc:25,18(1987) ; Chem.Abstr.,/0£,137682^s(1988)] besides other minor constituents such as zingiberine, curcumene, curlone, curculone, curzenone, α-,β- curcumenolides ,curcumenone, curdione,germacrone, linalool ,camphor, bomeol, zingiberol etc. [Essenze Deriv.Agrum, 54, 117(1984); Chem Abstr.,703, 128791^w(1985)] inhibits increased production of nitric oxide (NO) and is a free radical scavenger/ antioxidant whicb can penetrate the blood brain barrier and provide effective therapeutic protection by combating nitric oxide(NO)and superoxide/free radical induced neuronal injury / damage in human diseases such as neurocerebrovascular dysfunctions, ali types of strokes, thrombosis ( cerebral, coronary, deep vein), infarction, inflammatory and neurological disorders ,certain types of cancer, wounds, Alzheimers disease and other nitric oxide neurotoxicity, byperbaric oxygen exposure etc.

In another embodiment of the present invention, higb yields.of the lipid soluble material is obtained from Curcuma species rhizomes and leaves (Family: Zingiberaceae), particularly Curcuma longa L. syn. Curcuma domestica Valeton, hereinafter referred to as Curcuma oil and isolation of its various constituents.

In another embodiment of the present invention, more particularly this invention relates to nitric oxide (NO) and superoxide scavenging activity and prevention of any changes in cerebral blood flow dynamics by Curcuma oil itself or by its constituents singly or in combination with one another which enables their use as medicaments for the treatment and prevention of neurocerebrovascular disorders and related and unrelated dysfunctions such as ischemic attacks, ali types of stroke, thrombosis, infarction, migraine, Alzheimer’s disease, inflammatory and neurological dysfunctions, carcinogensis, tumor progression wounds and even HIV/AEDS.

Novel compound of the formula 3, an analog of compounds comprising ar-d-turmerone, turmerone, and germacrone wherein, R represents an alkyl, alkenyl, cycloalkane, phenyl, cycloalkene, or cycloalkadiene group, vvith substituents like alkyl, or alkoxy halo, in the phenyl, cycloalkene, cycloalkadiene rings, or hetroaryl like pyridyl nitrogen heterocyclic

R.

R

Ri amine and substituted amines, and R1 represents alkyl or arylalkyl group.

Novel Compound of the formula 4, an analog of compounds comprising Procurcumenol, zedoarondiol, and curcumenone.

CHRi-CHRi = Phenyl, Substituted phenyl or = abA,H,

R2 = H, or cd Δ,

R3 = Lovver alkyl C 1.3

Brief description of the accompany dravvings

Fig.l shovvs prevention of infarction from focal ischaemic rat after using curcuma oil;

Fig.2 shovvs past occlusion complete prevention of infarction in forebrain after using fraction A;

Fig.3 shovvs past occlusion complete prevention of infarction in forebrain after using fraction B;

Fig.4 shovvs Calcium transients in mitochondria (340/380 ratio);

Fig.5 shovvs change in SOD Ievels in mitochondria after using fraction A and fraction B; Fig.6 shovvs Catalase Ievels in mitochondria after using fraction A and fraction B;

Fig.7 shovvs Malondialdehyde Ievels in mitochondria after using fraction A;

Fig.8 shovvs change in percent relaxation to NE induced contraction; and

Fig.9 shovvs change in SNP Ievels in mitochondria after using fraction A.

In another embodiment of the present invention, provides a method of obtaining the lipid soluble extract / fraction of rhizomes and leaves of various species belonging to zingiberaceae family, especially Curcuma. oil from Curcuma - species· in good yield by extraction of powdered rhizomes / leaves of Curcuma longa L. Syn. Curcuma domestica Valeton or Curcuma aromatica Salisb or Curcuma zedoaria Roxb. etc. vvith an organic solvent like alcohol, acetone, ethyl acetate etc. but preferably a non-polar organic solvent like lightpetroleum or toluene under constant stirring and removing the solvent from such extracts by distillation under reduced pressure belovv 45° C.

In another embodiment of the present invention, in the case of extraction by a polar organic solvent such as ethanol, the residual alcoholic concentrate after removal of the solvent is exhaustively extracted vvith a non-polar organic solvent like light-petroleum, toluene etc.

In another embodiment of the present invention, distillation of solvent from such extracts under reduced pressure belovv 45° C yields a pale yellow to orange yellow odoriferous liquid in 5 to 6 per cent yields.

In another embodiment of the present invention, fractionation of this oil by column chromatography over a suitable adsorbent and elution by an appropriate organic solvent or by HPLC or GLC or distillation in vacuum yields the individual constituents such as ar-dturmerone (formula 1), turmerones (ct-,β-, formula 2) as major constituents (about 70 percent as determined by GC-MS) besides other minor constituents like zingiberine, curcumene, zedeorone, germaerone, curlone, curdione etc. ali identified by GC-MS etc.

In another embodiment of the present invention, an improved method of obtaining the lipid soluble extract / material of rhizomes and leaves of various species of Zingiberaceae family especially Curcuma species such as Curcuma longa L.Syn. Curcuma domestica Valeton or Curcuma aromatica Salisb. etc. in high yields by exhaustive extraction of fmely povvdered rhizomes or leaves vvith an appropriate organic solvent under continous gradual stirring or by sonication at ordinary room temperature followed by removal of the solvent from the extract by distillation under reduced pressure belovv 45 ⁰ C. In another embodiment of the present invention, the organic solvent is a non- polar organic solvent such as light petroleum, toluene etc.

In another embodiment of the present invention,'the organic-solvent used is a polar organic solvent such as ethanol, propanol etc.

In another embodiment of the present invention, the residual concentrate after removal of the solvent from the polar organic solvent extract is exhaustively extracted vvith a non-polar organic solvent such as light- petroleum, toluene etc.

In another embodiment of the present invention, the organic solvent is removed from the extracts by distillation under reduced pressure belovv 45 ⁰ C.

In another embodiment of the present invention, the continuos’ stirring is done either manually or by a mechanical stirrer or by an electric motor.

In another embodiment of the present invention, the lipid soluble extract /material of rhizomes or leaves of said species, vvhich, is a pale yellow to orange-yellow odoriferous oily liquid, is separated into its individual constituents such as ar-d-turmerone (formula 1), turmerones (α,β-formula 2), zingiberine, curcumene, germacrone, curcumenone, curlone etc. by chromatography (column, HPLC, GLC etc.) or distillation under high vacuum.

In another embodiment of the present invention, the individual constituents of the Curcuma oil are obtained by column chromatography over a suitable adsorbent such as alumina, silica gel etc·. and elution by appropriate organic solvents such as ņ-hexane, ņ-hexane : ethyl acetate mixture (in varying proportions), ethyl acetate etc..

In another embodiment of the present invention, the individual constituents of Curcuma oil are obtained by HPLC or GLC, e.g. turmerones ( α-,β-), mol.vvt. 218, retention time 9' -04”, ar-d-turmerone (mol.wt.216), retention time 8'-08, zingiberine (mol.wt.204) retention time 5-04, Curcumene (mol.wt.202),. retention time 4-24.

In another embodiment of the present invention, the individual constituents of Curcuma oil, such as ar-d-turmerone(fonnula 1), turmerones(fonnula 2), zinziberine, curcumene, curcumenone, germacrone etc.are obtained by distillation of Curcuma oil in vacuum, e.g. ard-turmerone, b.p. 155-160 °C / 9mm Hg through ar-d-turmerone rich fraction ,b.p. 140-160 ⁰C· /10mm Hg vvhich is about 70 % ofthe whole Curcuma oil

In another embodiment of the present invention, Nitric oxide (NO) and superoxide scavenging (anti-oxidant) property of the said lipid soluble extracts individual constituents such as ar-dturmerone (fig.1), termerones (fig.2), germacrone, curcumenone, zingiberine, curcu-meneetc either as such or it individual constituents.

In another embodiment of the present invention, compounds of the formula 3 as analogs of ard-turmerone or turmerone, germacrone etc. vvhen R represents an alkyl, alkenyl, cycloalkane, phenyl, cycloalk-ene or čycloalkadiene vvith substituents like alkyl, alkoxy halo-etc.in the phenyl or cycloall<ene or cycloalkadiene rings, hetroaryl like pyridyl nitrogen heterocyclic, amine or substituted amine etc. and R1 = alkyl, arylalkyl etc. as nitric oxide (NO), superoxide/free radicals scavengers to combat / prevent nitric oxide (NO), superoxide / free radical oxidative damage to important bio molecules.

Λ

Rf^Rl

In another embodiment of the present invention, compounds of the type- formula 4 as analogs of Procurcumenol, zedoarondiol, curcum-enone etc.-the other minor constituents of the lipid soluble extract of the Curcuma species vvhich incorporate in their molecular architecture the salient features of ar-d-turmerone and turmerone(a-,b-) molecules in a rigid frame vvork as therapeutically beneficial medicaments for the treatment and prevention of ali types of stroke, thrombosis, infarction, neurological dysfunctions etc.

CHR1-CHR1 = Phenyl, Substituted phenyl or = ab^H

R₂ = H, orcdA,

R3 = Lovver alkyl C 1.3

In another embodiment of the present invention, therapeutically beneficial effects of Curcuma oil as such or its individual constituents singly or in combination vvith each other or of related compounds, reduce, control or prevent human diseases in vvhich increased production of nitric oxide (NO) and free radical oxidative damage to important biomolecules is implicated such as ali types of stroke (thrombotic, embolic, focal, ischaemic), thrombosis (cerebral, coronary, deep vein) infarction, neurological dysfunctions etc.

In another embodiment of the present invention, method of treating post-stroke injury in mammals vvhich comprises administering to a subject in need thereof an effective amount of Curcuma oil either as such or its individual constituents singly or in combination vvith each other or related compounds.

In another embodiment of the present invention, method of treating patients of Subarachnoid and cerebral hemorrhagic stroke after 5 to 7 hours of the stroke by administering to a subject in need thereof a therapeutically effective amount of Curcuma oil either as such (vvhole) or its individual constituents singly or in combination vvith each other or related compounds.

In another embodiment of the present invention, method of treating reperfusion injury in mammals that comprises administering to a subject in need thereof an effective amount of Curcuma oil (whole-as such) or its individual constituents singly or in combination vvith each other or related compounds.

In another embodiment of the present invention, method of treating cerebrovascular diseases like ali types of stroke (thrombotic, embolic, focal, recurrent), transient ischaemic attacks etc. by administering to a subject in need thereof an effective amount of Curcuma oil (whole-as such) or its individual constituents singly or in combination with each other or reiated compounds.

In another embodiment of the present invention, method of treating ischaemic diseases and prevent dangerous blood clot formation by administering to a subject in need thereof an effective amount of Curcuma oil (whole) or its individual constituents singly or in combination vvith each other or reiated compounds.

In another embodiment of the present invention, method for treating hypertension in mammals that comprises administering to a subject/ patient in need thereof an effective amount of Curcuma oil (whole) or its individual constituents singly or in combination vvith each other or reiated compounds.

In another embodiment of the present invention, method to combat cerebral and pulmonary edema which accompanies cerebral and myocardial infarction by administering to a subject in need thereof medicaments like Curcuma oil (vvhole) or its individual constituents singly or in combination vvith each other or reiated compounds, vvhich are nitric oxide (NO) and superoxide/free radicals scavengers vvith anti-inflammatory activity.

In another embodiment of the present invention, therapeutically beneficial effects of the SAID lipid soluble extracts, either as such or its individual constituents like ar-d-turmerone, turmerones, germacrone, zinziberine, curcumene, curlone etc. singly or in combination vvith each other vvith and vvithout other therapeutic-ally useful aģents such as melatonin, tissue plasminogen activator (t-PA) administered orally, parentally (individual pure constituents) or in any other appropriate pharmaceutically acceptable delivery system such as tablets, capsules, beads, suppositories aerosols, implants etc in an effective amount (for Stroke, 10 500 mg / daily in divided doses and for other Ailments, 10 -1000 mg/ daily in divided doses),to provide a highly effective cure/treatment for human diseases vvherein nitric oxide (NO) and free radical oxidative damage are implicated such as ali type of stroke, thrombosis, infarction and neurological dysfunctions and vvhich may also be of therapeutic use in certain type of cancer such as leukemia, Alzheimer’s disease vvounds and even HIV./ AIDS.

Examples

The foliowing examples broadly illustrate the invention vvithout in anyway limiting the nature and scope of the invention:

Example 1

This example describes the method of obtaining Curcuma oil and its constituents in high yields and preparation of its dosage formulations. Improved extraction procedure of Curcuma oil and its constituents from Curcuma Icmgs L. syn. Curcuma domestica Valeton or other Curcuma species rhizomes.

The usual extractive procedure employs three or four percolations of dry povvdered Curcuma rhizomes vvith an organic solvent like light petroleum, toluene, alcohol etc. and distillation of the solvent from the percolates. In case of alcoholic extracts, after solvent removal the residual concentrate is triturated vvith a non-polar organic solvent like light petroleum follovved by removal of the solvent by distillation to yield Curcuma oil in 1 to 1.5 percent yields.

Hot extraction (Soxhlet) leads to loss of essential volatile constituents. When these procedures vvere changed to extraction of the dry povvdered Curcuma rhizomes vvith appropriate organic solvents such as light petroleum, acetone, alcohol etc. vvith continous stirring by mechanical stirrers driven by electric motors or manually or agitation vvith sonicator follovved by removal of the solvent from the extracts by distillation under reduced pressure belovv 45° C the yield and quality of the Curcuma oil increased appreciably.

In a typical procedure, dry fmely povvdered Curcuma longa L. rhizomes (1 kg) vvere successively percolated vvith n-hexane (3 liters) in a stainless steel or glass percolator / vessel fittetkv/ith a tap near the“bottoiTrtcrfrrafn-cfukthe-percolate, and the contents vvere stirred under slovv motion continuously for 24 hours each time by a mechanical stirrer driven by an electric motor. The orange-yellow percolate vvas drained out and the procedure repeated four to five times. Solvent vvas distilled off from the percolates under reduced pressure belovv 45 ⁰ C to yield an orange yellow odoriferous liquid (51 gms = 5.1% yield).

Likevvise, initial extraction of finely powdered Curcuma longa L rhizomes (1 kg) vvith acetone or alcohol (5x 3 liters) under continuous stirring for 24 hours each time follovved by removal of the solvent from the percolates by distillation under reduced pressure belovv 45°C and exhaustive trituration of the residual concentrate vvith n-hexane or toluene (6x500 ml) follovved by removal of the solvent by distillation under reduced pressure belovv 45°C yielded an orange yellow odoriferous liquid (60 gm.=6% yield).

Column chromatography of this orange yellow odoriferous liquid (20.0 gm.) over a silica gel column, using n-hexane, n-hexane: ethyl acetate (in varying proportions) mixture and ethyl acetate successively gavē ar-d-turmerone (formula 1, 55%) and turmerones (α-,β- formula 2, 20%) as major constituents (fraction- A.) follovved by curcumene(10%) & zingiberine (fraction- B) and other minor constituents -germacrone, curcumerone, zedoarone, zedoarondiol, isozdedoarondiol, curcumenone, curlone etc. (fraction C) vvhose activity vvas lovv..

Distillation of Curcuma oil (20.0 gms.) under reduced pressure (140-160°C / 9mm Hg) yielded ar-d-turmerone rich major fraction I (formula 1, 15.0 gms) alongvvith other turmerones (α-,β-, formula 2) and other minor constituents (4.2 gms, fraction H) Fraction I

2

Ar-d-turmerone α-turmerone, ab Δ and R= Me β-Tunnerone, R= =0¾ had refractive index (no³⁰) 1.4990, specific optical rotation (L)²⁵+19.6. Curcuma oil itself or its individual constituents obtained by chromatography or distillation under high vacuum are used singly or in combination vvith each other vvith and vvithout other therapeutically beneficial compounds to prepare appropriate clinically effective formulations.

The solid dosage form may be obtained by maceration of Curcuma oil as such or its individual constituents singly or in combination vvith each other particularly ar-d-turmerone, α-,β- turmerones vvith starch and microcrystalline cellulose in suitable proportions in a mixer till the mixture becomes a free flovvable povvder vvhich may be filled in capsules or converted into tablets as per therapeutically desired specifications.

In a typical example, Curcuma oil (10.0 gm.) vvas dissolved in ethanol (lOOml). Starch (5.0gm) and microcrystalline cellulose (85.0 gm) vvere added to this solution. The contents vvere mixed thoroughly and solvent vvas removed by drying belovv 45°C. The resulting product vvas passed through 40-mesh size sieve to obtain free flovving granules. These granules vvere then compressed into tablets of appropriate dosage requirements, e.g. each tablet vveighing 500mg.contain 50mg of Curcuma oil.

EXAMPLE2

Focal cerebral ischaemia;

Male Sprague Dawley rats of 270-375 gm vveight from CDRI Animal House vvere used for this study. Rats vvere housed in a 12-hr. light / dark cycle and vvater vvas given ad libitum. Animals vvere fasted ovemight and anaesthetized vvith pentobarbitone sodium, 30mg/kg. Rectal temperature vvas monitored. Transient ischaemia / reperfusion vvas performed using an intravascular filament to occlude the middle cerebral artery unilaterally [Longa Z.E.,Weinstein P.R., Carlson S., Cummins R.; Reversible middle cerebral artery occlusion vvithout craniectomy in rats : Stroke ,20, 84-91(1989) ] for 2 hours follovved by reperfusion for the remainder of 36 hours. Animals vvere assigned randomly to the follovving groups of n=5 rats (1) Control: Sham operated. (2.) Ischaemic / reflovv- no treatment. (3.) Ischaemic /reflovv-treated group:( i) Curcuma oil (weight/ml., 0. 86gm.), 683.65 mg./kg., given i.p. and P.O. ( ii) . Fraction- A(weight /ml.,0. 88gm.), 569. 56 mg/kg., given , i.p. and P.O. (iii). Fraction -B. (vveight /ml.,0. 91gm.) 938.86mg / kg., given, i.p. and P.O. The animals vvere sacrificed & brains vvere removed and quickly frozen. Eight coronal section of of 2mm thiekness from each brain vvere cut and stained vvith 2,3,5-triphenyltetrazolium chloride at 37° C for 30min. and post frxed by formalin. Each brain slice vvas photographed. The area of infarct in each slice vvas evaluated in a double blind manner. From groups (1,2,&3) rats n=3 , brain vvas removed and processed for mitochondrial Ca ²⁺ estimation .

Experimental Protocol:

Isolation of forebrain mitochondria:

Mitochondria vvere isolated from the rat forebrain according to the method of Lai and Clark [Lai. J.C.K., Clark J.P., Preparation of synaptic and non-synaptic mitochondria from mammalian brain: Method Enzymol.,’ 55, 51-60(1979)] vvith slight modificationš. Rat forebrain vvas immediately removed after decapitation and immersed in ice- cold isolation medium or Phosphate Buffered Saline. Brains vvere minced and rinsed to remove ali the traces of blood. The tissue vvas homogenized (10% w/v) in an appropriate medium using a motorized Teflon homogenizer and immediately centrifuged at 1800g for 10 min. The supematant vvas decanted and the peliet rehomogenized and centrifuged at 1800 g for 10 min. Supematants from the first and the second spins vvere added together and centrifuged at 17,000 g for 20 minūtes. The resultant peliet vvas resuspended in specific mediums and centrifuged at 17000 g. for 5 minūtes.

Determination of mitochondrial content:

Calcium content of mitochondria isolated from forebrain vvas estimated according to the method of Zaidan E. and Sims N.R. [The calcium content of mitochondria from brain sub reģions follovving short term fore brain ischaemia and recirculation in the rat; J. Neurochem., 63,1812-1819(1994)], vvith slight modifications . In brief, mitochondria (0. 3 mg. protein) in succinate mediun vvere loaded vvith Fura-2AM (0-5μΜ) and incubated for 30 min. at 37°C vvith constant shaking . The mitochondria vvere then vvashed tvvice in succinate medium and re-suspended in the same medium.

The ratio of Fura-2 fluorescence at exciting vvavelength of 340 and 380 nm vvith emission at 510 nm vvas determined using a Shimadzu RF '5000 Spectrofluorometer. Mitochondrial Calcium([Ca ²⁺]_m), is presented as tracings of the 340/380 fluorescence ratio[Macleod K.T and Harding S.E.; Effect of phorbolester in contraction, intracellular pH and intracellular Ca in isolated mammalian ventricular myocytes. J. Physiol. (London) ,444,481-498 (1991)].

Result: Infarct from focal ischaemic rat in pretreated group vvas completely prevented as seen in Fig. 1 & 2. In the group vvhere tēst compound / aģent vvas given post occlusion of middle cerebral artery, six out of seven brain sections shows complete prevention (Fig.3), whereas in one about 20% of the area showed up as infarcted. Mitochondria isolated from forebrain from animals made sham., ischaemc and treated with the tēst compound group (i) shovved the intracellular calcium Ievels close to normai (Fig.4).

Example 3

Collagenase-induced intra-cerebral hemorrhage:

Adult male rats (250-350 gm.) from the CDRI-Animal House were used in the follovving experiments. The rats vvere anaesthetized vvith pentobarbitone sodium (30 mg/kg, i.p.) and placed in a stereotaxic frame (for rats, Narashige, Japan). Rosenberg et.al’s method [Rosenberg G.A. Mun-Bryce S., Mary B.S. and Komfeld M., Collagenase-induced intracerebral hemorrhage in rats: Stroke, 21, 801-807 (1990)] vvas follovved. An incision vvas made in the scalp and a 23- guage needle vvas implanted into the caudate nueleus and the putamen (at the coordinates of A5.8, L3.0, Hl). ( A stereotaxic atlas of the rat brain, eds.R. M. Elliot, Gardener Lindzey and Kenneth , MacCorquodale. Meredith Publishing Company, 1967). Rats(n=10) vvere injected vvith collagenase (0.01 IU in 2μ1 of saline) and for sham vvith 2 μΐ of normai saline.After infusion, the needle vvas removed and the vvound vvas sutured. The animals vvere allovved to recover from anesthesia, ķept in a vvann place and allovved access to food and vvater. Eighteen hours later, the animals vvere evaluated for neurological deficit by measuring scoring of abnormal possture and hemiplegia according to Yamamoto et.al. [ Yamamoto A.Tamura, Kirino T., Shimizu M and Sano K. Behavioral changes after focal cerebral ischaemia by left middle cerebral artery occlusion in rats. Brain Research ,452, 323328.(1988)]. Later rats vvere reanaesthetized vvith pentobarbitone sodium, 30-mg/kg i.p. and brain vvas removed. Rats vvere assigned randomly into three groups. Group 1, received.saline. Collagenase (0.01 IU in 2μ1 saline) treated Groups 2 & 3 received fraction A (683. 65mg/kg) after 5 and 7 hours of collagenase treatment by the oral route .

Antioxidant estimations

Mitochondria vvere isolated as described in example 2.For antioxidant estimations, the mitochondria vvere rinsed and suspended in phosphate buffer. For the other estimations mitochondria vvere resuspended in a medium containing sucrose 250 mM, KH2PO4 6 mM and succīnate 6mM, pH 7.2. The isolation procedure vvas carried out at 4°C.

Antioridants:

The oxygen scavenging enzym.es, superoxide dismutase (SOD), catalase (CAT) and thiobarbituric acid reactive substances vvere estimated in mitochondria isolated from forebrain of experimental animals.

SOD: SOD activity vvas measured by the inhibition of NADH, PMS, NBT and absorbance monitored at 560 nm. Enzyme activity is expressed in U/min/mg protein. [Nishikini M., Rao N.A., Yagi K., The occurrence of superoxide anion in the reaction of reduced PMS and molecular oxygen. Biochem Biophysi. Res. Commun., 46, 849- 854 (1972)].

CAT: CAT activity vvas assayed by measuring the UV absorbance change of H2O2 at 240 nm according to Aebi [Aebi H., In Methods of Enzymatic Analysis (Third edition) ed. H.U. Bergmeyer Academic Press, Nevv York and London, Vol.2 pp 673-684. (1974)]

Thiobarbituric acid reactive substance (TBARS):

Mitochondrial TBARS Ievels vvere measured as an index of malondialdehyde and hence lipid peroxidation by the method of Okhavva et. el.[Okhawa H., Ohishi N., Yagi K., Assay of lipid peroxides in animal tissues by thiobarbituric acid reaction: Anal. Biochem., 95,351 (1979)] at 532 nm. Functional deficit vvas estimated according to Bederson [Bederson J.B., Pitts, L.H. Tsiji M., Nishimura M.C. Dāvis R.L., Barkovvisk H., Rat MCAO: Evaluation of modei and development of a neurologic examination, Stroke, 17, 472 (1986)] and vvater contents vvere estimated. Both the parameters vvere found to be significantly reduced as compared to untreated group.

Protein Assay

Mitochondrial protein vvas detennined by the method of Lovvry et.al. [Lovvry O.H., Rosbrough N.J., Farr. A.L., Randall K.J.; Protein measurement vvith folin phenol reaģent: J. Biol. Chem., 193, 265 (1951)] using bovine serum albumin(BSA) as Standard.

Result: The tēst compound (fraction A) given 5 hours after collagenase treatment significantly reduced the edema. NeuroLogical deficit at 5. & 7 hours of treatment vvere scored as grade 4 in untreated group and grade 0-2 in treated group. Mortality in untreated group was 3 out of 5 and in treated group 1 out of 5.

SOD: SOD value in 5 hours vvas almost normai vvhile in case of tēst compound (fraction A) given after 7 hours post collagenase treatment the SOD Ievels vvere augmented (Fig. 5).

Catalase: This enžyme is reported to be presentin minūte amount in brain (Fig. 6)

TBARS: At 5 hours post collagenase treatment, the values vvere close to that of collagenase treated animals, vvhile at 7 hours the values vvere decreased significantly as compared to the normai group indicating the anti-oxidant property of the tēst compound-fraction A (Fig.7). Mitochondria vvere isolated as described in Example 2.

Example 4

Adult male rats (250-350gm.) from the C.D.R.I. Animal house vvere anaesthetized vvith 30mg./ kg. Pentobarbitone sodium. Jugular veins ofthe rats vvere exposed. Five drops of 10 % formalin in 65 % methanol vvas dropped on the vein. Six hours vvere allovved for thrombus formation vvhich vvas then graded according to its presence or absence.[Blake O. R., Ashvvin J.G.,Jaques L.B.,; An assay for the antithrombotic activityofanticoagulants:J.Clin.Pathol.,72,118 (1959)]. Fraction A (ar-d-turmerone and turmerones) vvas given 200pl.i.p./300gm.rat in the treated group,vvhile the untreated (control) group received equivalent amount of saline (i.p).

Result: The thrombus in the untreated group vvas 2.8mg. and in the treated group it vvas

0.75mg. shovving an increase of 373.33 % in untreated versus treated group.

Example 5

Rats vvere made hypertensive according to Goldblatt et. al.[ Goldblatt H., Lynch J., Hanezal R.F., Serville W.W.: Studies on experimental hypertension,: The production of persistent elevation of systolic blood pressure by means of rēnai ischaemia J Exp Med ;59:347-379 (1934)]. Eight vveeks later the hypertensive rats had an average initial blood pressure of 200 mm/Hg. After Curcuma oil , 683.65mg./kg. vvas administered intraperitoneally the blood pressure fell to 115 mm /Hg in 15 min. and stayed at that Ievel for more than 60 min.

Table 1

Rats	Dose (i.p.) (683.65mg/kg).	Blood pressure Fall(%)	Duration (min.)	No.ofExpt.
Hypertensive		38.76+7.19	>60 min.	n = 3
Nonnotensive		No fall		n = 2 '

Resuit:The compound lowers the blood pressure significantly in hypertensive rats and not in the normotensive rats. It reduces blood pressure without bradycardia due to β- adrenergic receptor antagonism or reflex tachycardia common to vasodilator [Nichols A.J,Gallai M.Ruffolo R.P Jr. Studies on the mechanism of arterial vasodilation produced by the noval antihypertensive aģent. Carvediolol. Fundam. Clin. Pharmacol., 5:25-38 (1991) ].

Example6

Abdominal aorta was mounted according to Wolfgang et. al. [Wolfgang Auch-Schvvelk, Zvonimir S.Katusic and Paul M.Vanhoutte : contractions to oxygen derived free radicals are augmented in aorta of the spontaneously hypertensive rats., Hypertension ,13, 859864(1989)]. Aortic rings vvere contracted with norepinephrine 10'⁸ to 10⁵ M.The contracted vessels vvere relaxed by acetylcholine or Curcuma oil, added in a stepvvise manner. Acetylcholine vvas added in a concentration of 10‘⁷ to 10'⁵ M. For Curcuma oil, the final contraction achieved vvas 0.861mg in a 8ml bath (Fig. 8). Protein Kinase C activator, Phorbol 12-Myristate 13- Acetate (PMA) (ΙΟ⁷ M ) induced contraction in the intact and denuded aortic strip preparation. Pretreatment vvith Curcuma. oil ,0.881 mg. completely inhibited PMA induced contraction. It inhibits protein kinase C [Kaczmarck L. K.; The role of Protein Kinase C in regulation of ion channels and neurotransmitter release: Trends in Neurosciences, 10, 3034 (1987); Jin-Moo Lee, Grabb M.C., Zipfel G.J., Choi D. W., J. Clin. Invest., 106, (6), 723731(2000).

Resuit: Curcuma oil and acetylcholine caused complete relaxation in norepinephrine induced contraction shovving a significant vasorelaxant effect.

Example 7

Nitric oxide vvas first characterized in the Central nervous system at the intracellular messenger mediating the increase in cGMP Ievels that follovvs activation of .glutamate receptors (Garthvvaite J, Charles S.L. and Chess-Williams R. Endothelium derived relaxing factor release on activation of NMDA receptors suggest a role as intracellular messenger in the brain. Nature 336, 385-388 (1988). Simultaneous generation of NO and superoxide radicals favours the production of a toxic reaction product peroxynitrite anion (Beckman JS, Beckman TW, Chen J, Marshal PA and Freeman BA). Apparent hydroxyl radical production by peroxynitriye: implication for endothelial injury for nitric oxide and superoxide. Proc. Nati. Acad. Sci. USA 87, 1620 (1990). Once near or inside a celi peroxynitrite can damage or deplete a number of vītai components, DNA by strand scission, lipid by lipid peroxidation aconitase and antioxidant availability (Cuzzocrea S, Riley. DP, Caputi AP and Salvemini D. Antioxidant therapy: a nevv pharmacological approach ion shock, inflammation and ischemia/reperfusion injury. Pharmacol. Rev. 53: 135-159, (2001). To tēst the nitric oxide scavenging property of the tēst aģent invitro testing vvas carried out using an NO donor sodium nitroprusside.

Nitric oxide (NO) scavenging by tēst compounds/agents:

Sodium nitroprusside (SNP) generates Nitric oxide (NO) [Sreejayan and Rao M.N.A: Nitric oxide scavenging by curcuminoids, J.Phann. Pharmacol.,49,105-107(1997)]. fraction -A, 86.14mg vvas mixed in phosphate-buffer saline at different concentration of SNP ( 5-40 mM) Griess reaģent in 1:1 ratio vvas mixed vvith the tēst compound (fraction -A). The absorbance of the above chromophore buffer formed vvith SNP, tēst compound (fraction -A) and Griess reaģent vvas read at 546 nm and refer to the absorbance of Standard solution of potassium nitrite treated in the same way vvith Griess reaģent ( Green L.C, Wagner D.A.,Glogowski J, Skipper P.L., Wishnok J,S.,Tannenbaum S.R., Analysis of nitrate, nitrite and ^b N in biological fluids; Anal. Biochem. 126 , 131(1982). Marcocci L., Maguire J.J, Droy-Lefaix M.T., Packer L.: The nitric oxide scavenging property of Ginkgo bilobci extract EGb 761, Biochem. Biophys. Res. Commun. 207,748 (1994).

Results :SNP generates nitric oxide and tēst compound (fraction A) scavenges the nitric oxide thus generated, The result indicated the tēst compound (fraction A) in focal ischaemia to be a scavenger of nitric oxide (Fig. 9).

Claims

1. A composition obtained from the lipid soluble extract of rhizomes and leaves of Curcuma species of Zingiberaceae family, useful for the treatment of neurocerebrovascular disorders, said composition comprising fraction A consisting of ar-d-turmerone of formula 1,

And α and β turmerone of formula 2, vvherein in α turmerone R=CH3, and in β turmerone R=CH₂ optionally aiong vvith fraction B consisting of curcumene and zingiberine, and fraction C consisting of germacrone, curcumerone, zedoarone, sedoarondiol, isozdedoaronidiol, curcumenone, and curlone, and/ or pharmaceutically acceptable additives.

2. A composition as claimed in claim 1, characterized in that curcuma species iz Curcuma domestica Valeton.

3. A composition as claimed in claim 1, characterized in that ratio of fraction A, fraction B, and fraction C is ranging betvveen 1 to 3:1 to 3:1 to 3.

4. A composition as claimed in claim 1, characterized in that additives are selected from a group comprising melatonin, antioxidants, calcium channel antagonists, tissue plasminogen activator (t-P A), and celi membrane stabilizing aģents.

5. A composition as claimed in claim 1, characterized in that said composition inhibits nitric oxide synthase (NOS) overproduction, prevention calcium overload in neurons and scavenging free radicals.

6. A composition as claimed in claim 1, characterized in that said cerebrovascular disorders are selected from a group comprising ischaemia, stroke, post-stroke injury, hemorrhage, reperfusion injury, thrombosis, vasoconstriction, nitric oxideinduced free radicals oxidative damage, infraction, inflammation and Alzheimer’s disease.

7. A composition as claimed in claim 1, characterized in that fraction A of the composition is most effective.

8. A composition as claimed in claim 1, characterized in that said disorders are treated using the said composition is the form of various delivery systems selected from a group comprising tablets, capsules, suppository, beads, and aerosols.