WO2013179309A1 - Novel compounds as memory enhancers - Google Patents

Abstract

The present invention relates to a new series of chemical compounds with a novel norneolignan skeleton (chemical formula I) possessing a biological activity on the central nervous system (CNS), especially memory-enhancing and acetylcholinesterase inhibitory activity, individually, or in combination thereof. The novel compounds of a new series have been named as Clitorienolactones. (The chemical formula should be inserted here.) wherein R₁, R₂ is H, OH or OCH₃; Ring A and B may be mono- to polysubstituted

Description

NOVEL COMPOUNDS AS MEMORY ENHANCERS

FIELD OF THE INVENTION

The field of invention pertains to a new series of chemical compounds (chemical formula I) with biological role of improving central nervous system (CNS) functions, especially enhancing learning and memory in normal and memory deficit animals. The compounds of this new series with a new chemical skeleton have been named as clitorienolactones. These have been isolated from the roots of the plant, Clitoria ternatea L. (Family - Fabaceae) but can also be synthesized chemically.

wherein R_/, ¾ is , OH or OCH₃; Ring A and B may be mono- to polysubstituted

(Chemical formula I)

BACKGROUND OF THE INVENTION

Mental disorders account for approximately 12 % of all diseases, worldwide, and include depression, mental retardation, epilepsy, dementia, Alzheimer's disease (AD) and schizophrenia in decreasing order of prevalence [WHO, Geneva, Switzerland, 2001 ; Ernst et al. Am J Public Health 84, 1261-1264, 1994]. Dementia accounts for most common mental disorder characterized by progressive loss of intellectual skills and other cognitive skills [Bird et al, Harrison's principles of internal medicine, The McGraw-Hill Companies, Inc., New York, pp. 2393-2406, 2006]. Learning and memory are the most common and most important cognitive abilities that are lost in dementia [Grady et al, Curr Opin Neurobiol 10, 224-231 , 2000 & Zitnik et al, J Neurosci Res 70, 258-263, 2002]. Alzheimer's disease is the most common cause of dementia accounting for more than 50 % of all cases followed by vascular causes, Parkinson's disease, brain injury and several more. Pharmacotherapy is focused on symptomatic relief, leading to improved memory, and slowing disease progression. It does not provide cure from cognitive disorders.

The functioning of human brain is extremely complex involving billions of neurons and trillions of synapses. Comparatively very little is known with certainty about the intricate functioning of nervous system. As a result very few knowledge-based drugs have been discovered to treat mental disorders. Among various functions of brain, learning and memory are two vital functions in the overall well-being of a person. A normal or a diseased state of nervous system can have memory deficit and drugs capable of enhancing memory form a valued category, which is of immense use in all age groups for correcting the memory deficit or improving the memory.

The use of acetylcholinesterase inhibitors such as donepezil, [Matsui et al., Drug Metab Dispos 27, 1406-1414, 1999 & osasa et al., Eur J Pharmacol 386, 7- 13, 1999], tacrine [McGleenon et al, Br J Clin Pharmacol 48, 471 -480, 1999], rivastigmine [Suh et al, Pharmacol Rev 54, 469-525, 2002] and galantamine [Auchus et al, Neurol 69, 448-458, 2007] are common therapies used for assisting or improving failing memory.

The other options available to treat these ailments are use of nootropics [Schindler et al. Drug Dev Res 4,567-576, 1984], antioxidants [Polidori et al, Amino acids 32, 55-3-55¾-20.07-]-and-herbaLdrugs Singh_et^^

1997; Bhakuni et al, Phytother Res 10, 170-171 , 1996; Sierpina et al. Am Fam Physician 68, 923-926, 2003 & Kennedy et al, Pharmacol Biochem Behav 75, 687- 700, 2003]. Besides these options, as discussed above, the upcoming memory enhancers, under trial, for treatment of dementia are estrogen replacement therapy [Birge et al. The Am J Med 103, 36S-45S, 1997], non-steroidal anti-inflammatory agents [Broe et al. Arch Neurol 57, 1586-1591 , 2000 & Etminan et al, Br Med J 327, 128-132, 2003] and anti-amyloid agents [Cherny et al., Neuron 30, 665-676, 2001]. All these options have limitations because of one or other reason.

The use of plants for the prevention and treatment of human ailments has been in practice from times immemorial. In an analysis published in 2007, it was shown that 275 of 1010 drugs (27 %) approved during the period 1981-2006 (25 years) were natural products or their derivatives [ ewmann et al., J Nat Prod 70, 461 -477, 2007]. There is vast experience-based knowledge in traditional medicines including Ayurveda. The holistic approach of Ayurveda is more rational in management of CNS disorders [Mukherjee et al., Expert Opin Drug Discov 2633-2657, 2007], which warrants exploration of these drugs using modern scientific tools. Ayurveda has described a separate category of drugs to improve intellect, known as 'medhya rasayanas ' [Sharma P.V., Dravyaguna Vijnana, Chaukhambha Bharati Academy, Varanasi, 2, pp 3- 17, 2001]. A number of plants have been mentioned under the category of medhya rasayanas. Some of the more important plants like Centella asiatica, Bacopa monniera and Acorus calamus of the category have been extensively investigated with valuable findings, while other plants like the plant of this invention, did not produce any significant results though its use in treatment of mental disorders is known [Pandey G., Dravyaguna Vijnana 2, pp.161 -164, 1998].

A number of investigations on Clitoria ternatea have been carried out in the past but none could yield useful results to exploit its medicinal potential. A number of pharmacological activities have been reported for the different extracts of the different parts of the plant. However, the specific compounds involved for biological effects remained unknown and have not been disclosed in the prior art. It was, therefore logical to investigate this plant and invent phytoconstituents with potential to develop drug molecules. The chemical and biological work reported on this plant is summarized below.

The aqueous extract of the root of white-flowered variety of this plant at 100 mg / kg dose has been shown to possess learning and memory enhancing activity [Rai et al., Indian J Physiol Pharmacol 45, 305-313, 2001 ; Rai et al., Fitoterapia 73, 685-689, 2002; & Rai et al., Phytother Res 19, 592-598, 2005]. Further, above articles discloses phytochemical screening of aqueous extract and reported results show the presence of amino acids, proteins and carbohydrates without identifying any specific constituents and absence of alkaloids, saponins, flavonoids, coumarins and lignans in the aqueous extract.

In other reports, the alcoholic extracts of root and aerial parts at different doses have been shown to attenuate the electroshock-induced amnesia producing significant memory retention. The effect has been shown to be more pronounced with root extract than aerial parts, but the report does not describe any chemical constituents responsible for the activity [Taranalli et al., Pharm. Biol. 38, 51 -56, 2000; Taranalli et al, J Cell Tissue Res. 7, 949-952, 2007].

The methanolic extract of the root of the plant C. ternatea has been shown to possess neuroleptic activity. However, no information has been provided on the nature of compounds present in the extract or in the root or responsible for the activity [Boominathan et al, Nat Prod Sci 9, 260-263, 2003]. The nootropic, anxiolytic and CNS-depressant activity of the roots of the plant is also reported [Malik et al, Pharm Biol 49, 1234-1242, 201 1 ]. The methanol extract of roots of blue variety of the plant has been shown to possess antipyretic [Parimaladevi et al, Fitoterapia 74, 345-349, 2003; Parimaladevi et al, Phyotmedicine 1 1 , 323-326, 2004]; anti-inflammatory and analgesic activity [Parimaladevi et al, Fitoterapia 74, 345-349, 2003]; these investigations reported the presence of following types of phytoconstituents during phytochemical screening of the methanolic extract: flavonoids, tannins, steroids and saponins. In one of the earlier reports, roots have been shown to possess diuretic activity [Piala et al, Experientia 18, 89, 1962]. The roots are also shown to possess antiasthmatic activity and preliminary phytochemical study of ethanol extract of Clitoria ternatea observed the presence of steroids, saponin, flavonoids and glycosides [Taur et al, J Ethnopharmacol 136, 374-376, 201 1].

The aerial parts are reported to have local anesthetic, analgesic, decreased locomotor and exploratory and antipyretic activity in animal model [ ulkarni et al, Indian J Exp Biol 26, 957-960, 1988]. The aerial parts have also been shown to possess nootropic, anxiolytic, antidepressant, anticonvulsant and antistress activity [Jain et al., Pharmacol Biochem Behav 75, 529-536, 2003].

The seeds have been shown to possess antimicrobial, insecticidal and larvicidal activity. The flowers have been shown to inhibit platelet aggregation and relaxes vascular smooth muscles [Honda et al., Japananese okai Tokkyo oho JP 03014596, 1991 ] and are found to be antidiabetic [Sharma et al., Calcutta Med J 87, 168-171 , 1990]. The anti-inflammatory and analgesic activity of flower extract [Shyamkumar et al., Int Res J Pharm 3, 208-210, 2012] of Clitoria ternatea has been reported recently.

It has been observed that only two pentacyclic triterpenoids, taraxerol [Banerjee et al., Bull Calcutta Sch Trop Med 1 1, 106-107, 1963] and taraxerone [Banerjee et al., Bull Calcutta Sch Trop Med 12, 23, 1964] have been reported from the roots of Clitoria ternatea. A number of workers have correlated the various biological activities of the plant to the presence of earlier known triterpenoid, taraxerol [Kumar et al., Planta Med 73, P479, 2007; Shyamkumar et al., Int Res J Pharm 3, 208-210, 2012].

The triterpenoid, taraxerol is found commonly in the plant kingdom. Much of the work on this plant relates to isolation of several anthocyanins namely ternatins and flavonol glycosides from the aerial parts or flowers. A phytochemical analysis of the methanol extract of root has shown the presence of resins, saponins, flavonoids, alkaloids, steroids, phenol, protein, carbohydrates, glucosides and absence of tannins [Manalisha et al., Int Res J Pharm 2, 139-140, 201 1]. The root and seed extracts (50 % v/v alcohol) have shown the presence of glycosides, tannins, saponins, phenolics, flavonoids, proteins and carbohydrates [Solanki et al., Global J Sci Front Res 10, 2-8, 2010]. Another study on the methanol extract of root has shown the presence of alkaloids, carbohydrates, phenols, terpenoids and tannins and absence of saponins, flavonoids, phlobatannins and proteins [Mathew et al., Parasitol Res 104, 1017-1025, 2009].

In view of the above, it is evident that the specific chemical compounds responsible for the CNS activity of the plant have not been identified except for taraxerol, which itself cannot be considered a specific marker of CNS activity of this plant as this chemical compound commonly occurs in number of other plants. Therefore, there is enough reason to investigate Clitoria ternatea to identify and isolate the compounds specifically responsible for the CNS activity for using them in the treatment of the ailments related to brain.

The present invention relates to isolation and characterization of a new series of chemical compounds with novel skeleton, from the plant Clitoria ternatea, which enhance the learning and memory in normal or memory deficit mammals. These compounds can also be obtained from other sources including chemical synthesis. These compounds belong to a category of lignans and in particular to norneolignans.

OBJECTS OF THE INVENTION

It is an object of the invention to disclose a new series of compounds with new chemical skeleton having central nervous system (CNS) activity.

It is an object of the invention to disclose a new series of compounds with new chemical skeleton having central nervous system (CNS) activity especially learning and memory enhancement.

One more object is to disclose the isolation of these compounds from the plant Clitoria ternatea.

One more object is to disclose the dosage of the compounds for possible therapeutic use in mammals.

One more object is to enrich a fraction of the plant extract with bioactive compounds.

SUMMARY OF THE INVENTION

The present invention relates to isolation and characterization of a new series of chemical compounds, which enhance learning and memory in normal and memory deficit mammals. The compounds of this new series with a new chemical skeleton of norneolignans, have been isolated from the plant Clitoria ternatea L. The compounds have been named as clitorienolactones according to the guidelines on the nomenclature of natural products. The structure of these compounds is given below in chemical formula I.

In another embodiment, present invention provides a method of obtaining a compound of formula I from a plant Clitoria ternatea, family Fabaceae, comprises, extracting the pulverized plant part with one or more organic solvents to form one or more plant extracts,

partitioning the extracts between aqueous and one or more organic solvents, concentrating the organic phase, and

subjecting the residue to column chromatography to obtain pure compound. DETAILED DESCRIPTION OF THE INVENTION

As used herein the term "Clitoria ternatea " refers to plant Clitoria ternatea L. and its blue variety.

The present invention relates to isolation and characterization of a new series of CNS active chemical compounds, which enhance learning and memory in normal and memory deficit mammals. These compounds of a new series of norneolignans have been isolated from the plant Clitoria ternatea L. The compounds have been named as clitorienolactones according to the guidelines on the nomenclature of natural products. Novelty aspect of the new compounds:

The present invention provides the isolation and characterization of new compounds of a novel series of norneolignans with chemical structure represented above in chemical formula I. The compounds have not been disclosed in the prior art and are not known in nature or otherwise. In present invention, they have been isolated from the roots of Clitoria ternatea but they can be found in other sources and their chemical synthesis can also be carried out. The new compounds have the potential to be developed as drugs for treating disorders of central nervous system and to enhance learning and memory in normal and memory-deficit mammals.

Structure and nomenclature:

The new compounds belong to a group of norneolignans. The lignans are compounds derived from two phenylpropane moieties (C6-C3) linked through β- carbons of their side chains. In neolignans the two phenylpropane moieties are linked by a bond other than the β,β'-bond. The suffix nor in norneolignans refers to the loss of a carbon atom.

The compounds of new series are named clitorienolactones (individual compounds named clitorienolactone A, clitorienolactone B and so on) according to guidelines on nomenclature of natural products and lignans [Giles, Pure Appl. Chem. 71 , 587-643, 1999; Moss, Pure Appl. Chem. 72, 1493-1523, 2000]. These compounds include their racemates or isomers. Particularly compound of formula I can be characterized as 8R or 85 or racemic mixture at a chiral carbon C-8.

EXAMPLES

Method of extraction

The process involves the following steps:

Mechanical disruption: Taking the dried roots of Clitoria ternatea and grinding them to a coarse powder. The mechanical disruption of the plant comprises drying, crushing and grinding. Extraction: The roots of Clitoria ternatea, after disrupting are extracted by maceration with an alcoholic solvent, preferably methanol. The alcoholic extract is concentrated in rotary vacuum evaporator.

Partitioning: The concentrated alcoholic extract is then partitioned between water and a non-polar organic solvent. Non-polar organic solvent can be selected from hydrocarbon, chlorinated hydrocarbons, esters and the like. The partitioning is continued with a series of solvents in increasing polarity. The preferred series is hexane, chloroform which is followed optionally by ethyl acetate. Thereafter, the aqueous portion is extracted with n-butanol. The partitioning is carried out to enrich the bioactive constituents in one fraction. The n-butanol and other fractions of the extract are then subjected to suitable chromatographic separations.

Chromatography: The resulting residue is then chromatographed to isolate pure compounds, which can optionally be further purified by recrystallization from suitable solvent, wherever required. The suitable chromatographic separations include column chromatography and / or medium pressure liquid chromatography and / or high performance liquid chromatography and / or preparative thin layer chromatography and the like. Preferably the separation of norneolignans is performed by column chromatography and medium pressure liquid chromatography. Column chromatography and medium pressure liquid chromatography can be carried out using silica gel as a stationary phase and a mixture of chloroform and methanol or a combination of similar polarity as an eluting solvent. Silica gel can be of size ranging from 60-400 mesh, preferably 60-120 or 100-200 in case of column chromatography and mesh size 230-400 in case of medium pressure liquid chromatography. Elution of the compounds from the column depends upon both the polarity of the solvent as well as polarity of the compound to be eluted. The polarity of eluting solvent is increased by increasing the amount of methanol in the mixture.

Isolation of bioactive compounds: The fractions eluted in chloroform containing 3 % methanol or more from the column, contain new compounds of a novel skeleton belonging to norneolignans, represented by chemical structure shown above in chemical formula I. The isolated compounds are further purified by repeating column chromatography or by re-crystallization from a suitable solvent, wherever required. The preferred solvent for re-crystallization is a mixture of chloroform and methanol in suitable proportion, preferred ratio being 5 to 15 % of methanol in chloroform.

Characterization and identification: The structural confirmation of the new compounds is carried out by using several spectroscopic technique such as ultra violet (UV), infra red (IR), nuclear magnetic resonance (NMR) (I D and 2D experiments), electrospray-ionization mass spectrometry (ESIMS) and high- resolution mass spectrometry (HRMS). The skeleton is further confirmed through single-crystal X-ray crystallography of one of the compounds of the series.

The optical rotation was determined on a Rudolph Research Autopol IV Polarimeter at room temperature. The UV spectra are recorded on Perkin Elmer Lambda 650. The IR spectroscopy is carried out using KBr disc in Perkin Elmer RX1 FTIR Spectrophotometer. The proton nuclear magnetic resonance (Ή NMR) and carbon nuclear magnetic resonance (^l3C NMR) spectra are recorded in CD₃OD or DMSO using Bruker spectrometer (400 MH_Z for Ή NMR and 100 MH_Z ¹³C NMR). Mass spectra are obtained on an Applied Biosystems API 2000™ Mass spectrophotometer. The HRMS are recorded on an Agilent 6520-QTOF LCMS (ESI, Positive mode).

Structural details of some of the novel compounds isolated as above, are given below:

Compound of chemical formula II (Clitorienolactone A, CigHjsOe).

The compound of chemical formula II, eluted in chloroform containing 3 % methanol, is characterized by the following data: UV λ _max (MeOH), nm : 323.7, 287.7

IR max (KBr) cm^"1 : 3368, 3260, 2944, 2841, 1717, 1607 fal * : +42.4 (c 0.375, MeOH) Ή NMR (400 MHz, CD₃OD) (δ): 2.74 (Ha-7) [dd, J = 14.5 & 5.0 Hz], 3.10 (Hb-7) [dd, J = 14.5 & 3.9 Hz], 3.58 (OCH₃) [s], 3.75 (OCH₃) [s], 5.81 (H-8) [m], 5.98 (H- 8') [d, J = \ . \ Hz], 6.26 (H-6) [dd, J= 8.0 & 1.9 Hz], 6.32 (H-2) [dJ= 1 .9 Hz], 6.43 (Η-5') [dd, J = 8.5 & 2.2 Hz], 6.46 (Η-3') [d, J = 2.2 Hz], 6.50 (H-5) [d, J = 8.0 Hz],

l ³C NMR (100 MHz, CD₃OD) (δ): 39.52 (C₇), 56.15 (OCH₃), 56.05 (OCH₃), 85.35 (C₈), 100.43 (C₃-), 109.43 (C₅ ), 1 12.19 (C_r), 1 13.75 (C₈ ), 1 14.36 (C₂), 1 15.73 (C₅), 123.48 (C₆), 127.58 (C), 132.51 (C₆), 146.3 1 (C₄), 148.34 (C₃), 161.39 (C₄ ), 163.70 (Cy), 167.38 (C₇) and 177.10 (C₉ )

ESI-MS: m/z [M+Hf 343.1, [M-H]^" 341.1

HRESI-MS m/z [M+H]⁺ 343.1 181 (calculated for Ci₉Hi₈0₆ , 343.1 181)

(Chemical formula II) Systematic name:

5-(4-Hydroxy-3-methoxybenzyl)-4-(4-hydroxy-2-methoxyphenyl)furan-2(5H)-one

Semi-systematic name:

9-Nor-4,4'-dihydroxy-3,2'-dimethoxy-8,7'-neolign-7'-eno-8,9'-lactone

Compound of chemical formula III (Clitorienolactone B, CtsHieOs).

The compound, of chemical formula III, eluted in chloroform containing 5 % methanol is characterized by the following data: UV λ max (MeOH), nm : 323.3, 287

IR v_max (KBr) cm^"1 : 3287, 2943, 1718, 1616, 1568

Ή NMR (400 MHz, CD₃OD) (5): 2.68 (Ha-7) [dd, J = 14.5 & 5.6 Hz], 3.08 (Hb-7) [dd, J= 14.6 & 3.8 Hz], 3.77 (OCH₃) [s], 5.79 (H-8) [m], 6.00 (Η-8') \d J = 1 1 Hz], 6.43 (Η-5') [dd, J = 8.5 & 2.2 Hz], 6.47 (Η-3') [d, J = 2.2], 6.51 (H-3 & H-5) [2H, AA'BB' system], 6.68 (H-2 & H-6) [2H, AA'BB' system], 7.23(H-6') [d, J= 8.4 Hz]

¹³C NMR (100 MHz, CD₃OD) (δ): 39.42 (C₇), 56.07 (OCH₃), 85.58 (C₈), 100.44 (C_y), 109.45 (C₅-), 1 12.08 (C_r), 1 13.60 (C₈-), 1 15.81 (C₃ & C₅), 127.26 (Q), 131 .86 (C₂ & C₆), 132.46 (Cff), 157.21 (C₄), 161 .37 (C₄ ), 163.70 (C₂), 167.59 (C_T) and

ESI-MS: m/z [M+H]⁺ 313.1 , [M-H]^" 31 1.1

HRESI-MS m/z [M+H]⁺ 313.1065 (calculated for Ci₈H|₆0₅, 3 13.1076)

(Chemical formula III)

Systematic name:

5-(4-Hydroxybenzyl)-4-(4-hydroxy-2-methoxyphenyl)furan-2(5H)-one

Semi-systematic name:

9-Nor-4,4'-dihydroxy-2'-methoxy-8,7'-neolign-7'-eno-8,9'-lactone Compound of chemical formula IV (Clitorienolactone C, CisHi 0₆).

The fraction eluted in chloroform containing 10 % methanol and its sub-fraction eluted in chloroform containing 3 % methanol in medium pressure liquid chromatography contains another compound of chemical formula IV, characterized by the following data:

UV λ _max (MeOH), nm : 326.9, 287.4

IR v_max (KBr) cm^"1 : 3483, 3255, 2927, 1694, 1604

Ml⁵ : +1 12.3 (c 0.25, MeOH)

Ή NMR (400 MHz, CD₃OD) (δ): 2.79 (Ha-7) [dd, J = 14.5 & 5.2 Hz], 3.16 (Hb-7) [dd, J = 14.5 & 3.7 Hz], 3.60 (OCH₃) [s], 5.86 (H-8) [m], 6.00 (Η-8') [d, J= 1.2 Hz], 6.33 (Η-3') [m], 6.33 (Η-5') [m], 6.33 (H-6) [m], 6.38 (H-2) [d, J= 1.9], 6.50 (H-5) [d, J = 8.0 Hz], 7.19 (Η-6') [d, J= 6.6 Hz]

¹³C NMR (100 MHz, CD₃OD) (δ): 39.71 (C₇), 56.1 1 (OCH₃), 85.52 (C₈), 103.91 (C₃.), 109.16 (Cs , 1 10.87 (C_r), 1 12.64 (C₈>), 1 14.33 (C₂), 1 15.69 (C₅), 123.53 (C₆), 127.90 (C , 132.26 (C_ff), 146.30 (C₄), 148.34 (C₃), 159.83 (C₄>), 163.31 (C₂), 168.21 (C_T) and 177.29 (C₉)

ESI-MS: m/z [M+H]⁺ 329.1 , [M-H]^' 327

HRESI-MS m/z [M+H]⁺ 329.101 1 (calculated for C,₈H₁₆0₆, 329.1025)

(Chemical formula IV) Systematic name:

5-(4-Hydroxy-3-methoxybenzyl)-4-(2,4-dihydroxyphenyl)furan-2(5H)-one

Semi-systematic name:

9-Nor-4,2',4'-trihydroxy-2-methoxy-8,7'-neolign-7'-eno-8,9'-lactone

Compound of chemical formula V (Clitorienolactone D, C17H1 O5).

The fraction eluted with 10 % methanol in chloroform and its sub-fraction eluted in chloroform containing 4 % methanol in medium pressure liquid chromatography contains another compound of chemical formula V, which is characterized by the following data:

UV λ _max (MeOH), nm: 326.0, 285.2

IR v_max (KBr) cm^"1 : 2922, 2854, 1708, 1464

: 0 (c 0.15, MeOH)

Ή NMR (400 MHz, CD₃OD) (δ): 2.70 (Ha-7) [dd, J = 14.5 & 6.0 Hz], 3.1 5 (Hb-7) [dd, J = 14.5 & 3.6 Hz], 5.82 (H-8) [m], 6.02 (Η-8') [d, J = 1.2], 6.34 (Η-3') [m], 6.34 (Η-5') [m], 6.52 (H-3 & H-5) [2H, AA'BB' system], 6.75 (H-2 & H-6) [2H, AA'BB' system], 7.20 (Η-6') [d, J= 6.9 Hz]

^, C NMR (100 MHz, CD₃OD) (δ): 38.16 (C₇), 84.30 (C₈), 102.43 (C₃), 107.70 (C₅ ), 109.24 (C_r), 1 10.91 (C&-), 1 14.30 (C₃ & C₅), 126.17 (Q), 130.34 (C₂ & C₆), 130.73 (Ce , 155.71 (C₄), 158.28 (C₄'), 161.77 (C₂), 166.98 (C_T) and 175.68 (C9-)

ESl-MS: m/z [M+H]⁺ 299.1 , [M-H]- 297.1

HRESI-MS m/z [M+H]⁺ 299.0904 (calculated for C 17H14O5, 299.0919)

(Chemical formula V)

Systematic name:

5-(4-Hydroxybenzyl)-4-(2,4-dihydroxyphenyl)furan-2(5H)-one

Semi-systematic name:

9-Nor-4,2',4'-trihydroxy-8,7'-neolign-7'-eno-8,9'-lactone

DETERMINATION OF BIOACTIVITY

Animals for pharmacological studies

LACA mice (20-30 g) of either sex were used in the study after due approval of the Institutional Animals Ethics Committee. After procurement, the animals were divided into different groups and left for one week for acclimatization to experimentation room and were maintained on standard conditions of housing and diet. The animals were divided into groups of five each for experimentation. Preparation of dosages of test and standard drugs for administration

All the test substances for the administration were suspended in the normal saline solution containing 2 % Tween 80 to deliver desired quantity of the test substance in the required volume. Donepezil at a dose of 1 mg / kg was used as a standard drug. Scopolamine at a dose of 0.6 mg / kg was used to produce amnesia. Administration of the test drugs, standard drugs and vehicle

The freshly prepared doses of the test drugs (extracts, fractionated extracts, chemical isolates), standard drug (donepezil) and the vehicle were administered orally for 14 days to the animals using a tuberculin syringe fitted with oral canula. Scopolamine was administered intra peritoneally, only to scopolamine group, 30 minutes prior to experimentation, on day 14 and day 15.

Evaluation of memory enhancing activity

Memory enhancing activity was determined using Morris water maze model in the mice. A 15-day protocol was used in the study. The animals were divided into different groups of control, standard, scopolamine and test. The activity was evaluated by recording escape latency on day 14, and the time spent in the target quadrant on day 15. The results were compared with standard drug, donepezil and are given below in Table 1.

Table 1 : Evaluation of memory enhancing activity

Clitorieno- 10.0 ± 40.6 ±

6 20 * 64.5 * 48.7 lactone B 4.8 5.9

Clitorieno- 19.2 ± 39.8 ±

7 5 50.3 75.3^# lactone A + ^eS 6.6" 5.3"

Clitorieno- 14.8 ± 39.5 ±

8 20 _ * * 61.7 ** 74.0

lactone B + ^eS 7.0 2.9 ^a« = 5; P < 0.05 vs control P < 0.05 vs scopolamine ; ^ff percent decrease from scopolamine group; ^bEL = escape latency in sec; °per cent change in EL or TSTQ = (I -te t/ control) x 100; ^dTSTQ = time spent in target quadrant in sec;

eS = scopolamine.

The learning and memory improving effect of activity-enriched n-butanol fraction, clitorienolactone A and clitorienolactone B was apparent from a significant decrease in escape latency in treated animals (Group no 4 to 6) as compared to control (Group no 1) and amnesic (Group no 3) animals. Clitorienolactone A and clitorienolactone B significantly decreased the escape latency and increased the time spent in target quadrant in comparison to the control group signifying increase in learning and memory; and increased retention and retrieval of memory. The novel compounds also completely reversed the scopolamine induced amnesia as evident from the comparison of escape latency of control (Group no 1), amnesic (Group no 3) and treated groups (Group no 7 & 8).

Estimation of acetylcholinesterase inhibitory activity

The memory is related to the levels of acetylcholine (ACh) in the brain. The acetylcholinesterase (AChE) degrades the ACh through hydrolysis. Cholinergic dysfunction can be assessed by measuring the activity of AChE. The quantitative measurement of acetylcholinesterase levels in mice brain were performed according to the method of Ellman et al. (1961). The assay mixture contained 0.05 ml of supernatant obtained from homogenized brain tissue, 3 ml of 0.01M sodium phosphate buffer (pH 8), 0.10 ml of acetylthiocholine iodide and 0.10 ml of 5,5'- dithiobis(2-nitrobenzoic acid) (Ellman's reagent). The change in absorbance was measured at 412 nm for 2 min. Results were calculated using molar extinction coefficient of chromophore (1.36 ^χ 10⁴ M ¹ cm ') and expressed as percentage of control.

Acetylcholinesterase inhibitory activity. Based on the knowledge that cholinergic system plays a crucial role in spatial memory, the effect of clitorienolactone A and clitorienolactone B, on the activity of AChE, an indicator of cholinergic function was evaluated. It was found that clitorienolactone A and clitorienolactone B significantly decreased the AChE activity in the brain homogenate (P < 0.05 compared to control).

For therapeutic use, the compounds can be made into formulations, pharmaceutical preparations and dietary supplements which may be provided in the form of powder, granules, tablets, capsules, liquid preparations or the likes and can be administered per orally, parenterally, by inhalation, locally or the like.

The examples above are set forth to aid in understanding the invention but are not intended to, and should not be construed to limit its scope in any way. The examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinal skill in the art and are described in numerous publications.

Claims

CLAIM:

1. A compound of chemical formula I:

[Chemical formula I] wherein R_t, R₂ is H, OH or OC¾; Ring A and B may be mono- to

polysubstituted

2. The compound as claimed in claim 1 , is a norneolignan.

3. The compound of formula 1 as claimed in claim 1 is a compound of chemical formula II:

The compound of formula I as claimed in claim 1 is a compound of chemical formula III.

5. The compound of formula I as claimed in claim 1 is a compound of chemical formula IV.

6. The compound of formula I as claimed in claim 1 is a compound of chemical formula V:

7. A pharmaceutical composition comprising a compound as claimed in claims 1 -6, individually or in a mixture thereof, with one or more pharmaceutically acceptable excipients.

8. The compound as claimed in claims 1-6, for use in the manufacture of medicament for use in the treatment of memory disorders in normal and memory deficit mammals.

9. The compound as claimed in claims 1-6, for use in the manufacture of memory enhancing formulations.

10. A method of obtaining a compound of formula I from a plant Clitoria ternatea, family Fabaceae, comprises,

extracting the pulverized plant part with one or more organic solvents to form one or more plant extracts,

partitioning the extracts between aqueous and one or more organic solvents, concentrating the organic phase, and subjecting the residue to column chromatography to obtain pure compound.