Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
  • A61K31/05—Phenols
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/075—Ethers or acetals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/24—Antidepressants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

• This invention relates to a method for treating, alleviating or preventing anxiety by administering a pharmaceutical composition comprising a therapeutically effective amount of pterosti!bene, an analog of resveratrol, found in grapes and some Vaccinium berries.
• Anxiety disorders are common in community settings and in primary and secondary medical care, and frequently turn into chronic clinical conditions (Nutt et a!, 2002. Int. J. Neuropsychopharmacol. 5:315-325). Anxiety disorders are the most common type of psychiatric disorders, with an incidence of 18.1 % and a lifetime prevalence of 28.8% (Kessler et al. 2005. Arch. Gen. Psychiatry 82: 617-627: Ohayon, M. M. 2006. J. Psychiatr. Res. 40: 475-476). Anti-anxiety drugs have been used by human beings for thousands of years.
• Benzodiazepine group of drugs are fast acting, effective and the most commonly prescribed anxiolytics (Bandelow et al. 2008. World J. Biol. Psychiatry 9:248-312; Baldwin et al. 2005. J. Psychopharrnacoi. 19:567-596;
• Stiibenes are a group of phytochemicais having an ⁇ , ⁇ -diphenylethylene core structure. Stiibenes have been reported in a large number of unrelated plant genera including grapes, peanuts, and Vaccinlum berries (Chong et al. 2009. Plant Sci. 177:143-155).
• Resveratroi a widely studied stilbene, has been reported to exert antioxidant, anti-inflammatory, chemopreventive, and anti-aging effects in a number of biological systems (Aggarwal et al. 2004. Anticancer Res. 24:2783-2840; Baur and Sinclair. 2008. Nat Rev. Drug Disco. 5:493-506; Bishayee, A. 2009. Cancer Prev. Res. 2:409-418). Recently, pterostiibene, a natural analog of resveratroi, found in some Vaccinium berries such as blueberries and deerberries (Rimando et al. 2004. J. Agric. Food Chem.
• Pterostiibene has analgesic, antidiabetic, antioxidant, anti-inflammatory, hypolipidemic, and cancer chemopreventive properties (Am a math Sateesh and Pari. 2006. J. Pharm. Pharmacol. 58: 1483-1490; Remsberg et ai. 2008. Phytotherapy Res. 22: 169-179; Rimando et ai. 2002. J. Agric. Food Chem. 50:3453-3457; Rimando et ai. 2005. J. Agric. Food Chem. 53:3403-3407).
• Pterostiibene also has significant effect on colon cancer development (Paul et al. 2009. Cancer Prev. Res. 2:650-657), invasion and metastasis (Pan et ai. 2009.
• kits comprising a pharmaceutical composition containing pterostilbene; and instructions for the use of the kit.
• Figure 1 shows the chemical structure of pterostilbene.
• Figures 2A and 2B show the effect of oral administration of pterostilbene on behavior in the elevated plus-maze (EPM) test.
• EPM elevated plus-maze
• Male Swiss Webster mice were injected with vehicle or pterostilbene (1 -10 mg/kg) by oral gavage 80 min before testing on the EPM for 5 min.
• Each column represents the mean ⁇ SEM.
• n 6-10 per dose.
• Figure 3 depicts the effect of intraperitoneal (i.p) administration of diazepam on behavior in the elevated plus-maze test.
• Male Swiss Webster mice were injected with vehicle or diazepam (0,5-2 mg/kg) by i.p. 30 min before testing on the EPM for 5 min.
• Each column represents the mean ⁇ SEM.
• n 8-10 per dose. ** p ⁇ 0.01 compared to vehicle control (one-way ANOVA followed by Dunnett's test).
• Figure 4 depicts the effect of oral administration of pterostilbene on mouse locomotor activity.
• Male Swiss Webster mice were injected with vehicle or pterostilbene (1 -10 mg/kg) by oral gavage 60 min before testing on the elevated plus-maze for 5 min.
• Figures 5A and 5B depict the effect of oral administration of pterostilbene on phosphorylation of ERK1/2 in the hippocampus.
• Fig. 5A shows representative data on phosphory!ated ERK1/2 levels for mice sacrificed immediately after EPM test.
• Fig. 5B shows the densitometry analysis of the changes in ERK 1/2 phosphorylation for mice sacrificed immediately after training. Values are expressed as the mean ⁇ SEM of at least three animals. * p ⁇ 0.05, ** p ⁇ 0.01 compared to vehicle control (one-way ANOVA followed by Dunnett's test).
• Figure 6 depicts the effect of oral administration of pterostilbene on
• Diazepam one of the most frequently used anxiolytic compounds was used as a positive control in this study.
• the EPM is a widely used model of anxiety employed by a large number of investigators (Peliow et al., supra; Hogg, supra), it has been proposed that normal exploratory behavior of rodents is in favor of the enclosed arms of the maze in the elevated plus-maze model, and that aversion towards the anxiety-provoking open arms is the basis of EPM model (Peliow et al., supra; Ohl, F. 2003. Clin. Neruosci. Res. 3: 233-238).
• a large body of evidence has established that the administration of anxiolytic compounds reduces the natural aversion to the open arms and promotes the exploration thereof (Peliow et al., supra; Hogg, supra).
• Pterostilbene manifested anxiolysis at 1 and 2 mg/kg doses: the per cent Permanence Time in Open Arms (PTOA), and Number of Entries in Open Arms
• NEOA which are critical determinants and considered to be correiated with anxiety
• TDOA percent Traveled Distance in Open Arms
• PTEA percent Permanence Time in the Enclosed Arms
• pterostilbene showed comparable effects to 2 mg/kg of diazepam, at least, in the EPM.
• the locomotor activity of the animals was evaluated to demonstrate that the anxiolytic activity of pterostilbene at lower doses (1 and 2 mg/kg) was not a secondary consequence of depressive action of the compound on the motor activity of the animals.
• the absence of anxiolytic effect at the higher doses (5 and 10 mg/kg) was also not due to motor impairment. Therefore, no stimulant or depressive actions were recorded, suggesting that it is very unlikely that the observed anxiolytic effects are false positives.
• Akt1 , Akt2, and Akt3 three genes in the Akt family (Akt1 , Akt2, and Akt3) code for enzymes that are members of the
• the brain contains a variety of receptors like NMDA, GABA and ion channels that can potentially alter the phosphorylation of MAP kinase.
• NMDA neuropeptide-like kinase
• GABA GABA-like kinase
• ion channels that can potentially alter the phosphorylation of MAP kinase.
• Studies showing the activation of MAP kinase by NMDA receptors have been well documented (Xia et al. 1996. J. Neurosci. 16:5425-5436; Orban et al. 1999. Trends Neurosci. 22:38-44).
• studies implicated the ERK/MAPK pathway as a negative modulator of GABA receptor function (Bell-Horner et al. 2006. J. Neurobiol. 66:1467-1474). Whether the decrease in ERK1/2 phosphorylation is mediated by blockade of NMDA receptors or activation of GABA receptors, is a subject
• the compounds of the present invention are therefore of use to treat, alleviate, or prevent anxiety in a subject in need thereof.
• the compounds of the present invention are therefore of use to treat, alleviate, or prevent anxiety in a subject in need thereof.
• the compounds of the present invention are therefore of use to treat, alleviate, or prevent anxiety in a subject in need thereof.
• the compounds of the present invention are therefore of use to treat, alleviate, or prevent anxiety in a subject in need thereof.
• anxiety disorders such as panic disorder with or without agoraphobia, agoraphobia without history of panic disorder, animal and other phobias including social phobias, obsessive-compulsive disorder, and generalized or substance-induced anxiety disorder: stress disorders including post-traumatic and acute stress disorder: sleep disorders; memory disorder; neuroses; convulsive disorders, for example epilepsy, seizures, convulsions, or febrile convulsions in children; migraine; mood disorders; depressive or bipolar disorders, for example depression, single-episode or recurrent major depressive disorder, dysthymic disorder, bipolar disorder, bipolar I and bipolar I manic disorders, and cyclothymic disorder, psychotic disorders, including schizophrenia; neurodegeneration arising from cerebral ischemia; attention deficit hyperactivity disorder; pain and nociception, e.g.
• emesis including acute, delayed and anticipatory emesis, in particular emesis induced by chemotherapy or radiation: motion sickness, post-operative nausea and vomiting; eating disorders including anorexia nervosa and bulimia nervosa; premenstrual syndrome: neuralgia, e.g.
• trigeminal neuralgia muscle spasm or spasticity, e.g. in paraplegic patients; the effects of substance abuse or dependency, including alcohol withdrawal; cognitive disorders, such as Alzheimer's disease; cerebral ischemia, stroke, head trauma; tinnitus: and disorders of circadian rhythm, e.g. in subjects suffering from the effects of jet lag or shift work.
• a composition in accordance with the present invention containing pterostilbene, or a pharmaceutically acceptable salt of pterostilbene can be prepared by conventional procedures for blending and mixing compounds.
• the composition also includes an excipient, most preferably a pharmacuetical excipient.
• Compositions containing an excipient and incorporating the pterostilbene can be prepared by procedures known in the art.
• pterostilbene can be formulated into tablets, capsules, powders, suspensions, solutions for oral administration and solutions for parenteral administration including intravenous, intradermal, intramuscular, and subcutaneous administration, and into solutions for application onto patches for transdermal application with common and conventional carriers, binders, diluents, and excipients.
• the invention provides pharmaceutical compositions comprising the chemical compound of the invention, or a pharmaceutically acceptable salt or derivative thereof, together with one or more pharmaceutically acceptable carriers, and, optionally, other therapeutic and/or prophylactic ingredients, known and used in the art.
• the carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof.
• compositions of the invention may be those suitable for oral, rectal, bronchial, nasal, pulmonal, topical (including buccal and sub-lingual),
• transdermal, vaginal or parenteral including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebral, intraocular injection or infusion
• sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compound of the invention, which matrices may be in form of shaped articles, e.g. films or microcapsules.
• the chemical compound of the invention together with a conventional adjuvant, carrier, or diluent, may thus be placed into the form of pharmaceutical compositions and unit dosages thereof.
• Such forms include solids, and in particular tablets, filled capsules, powder and pellet forms, and liquids, in particular aqueous or non-aqueous solutions, suspensions, emulsions, elixirs, and capsules filled with the same, ail for oral use, suppositories for rectal administration, and sterile injectable solutions for parenteral use.
• compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
• the chemical compound of the present invention can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a chemical compound of the invention or a pharmaceutically acceptable salt of a chemical compound of the invention.
• pharmaceutically acceptable carriers can be either solid or liquid.
• Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersibie granules.
• a solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubiiizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
• the carrier is a finely divided solid, which is in a mixture with the finely divided active component.
• the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
• the powders and tablets preferably contain from five or ten to about seventy percent of the active compound.
• Suitable carriers are magnesium carbonate,
• magnesium stearate talc
• sugar lactose
• pectin dextrin
• starch gelatin
• tragacanth methylceliuiose
• sodium carboxymethylcellulose a low melting wax
• cocoa butter and the like.
• preparation is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it.
• cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.
• Liquid preparations include solutions, suspensions, and emuisions, for example, water or water-propyiene glycol solutions.
• parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.
• the chemical compound according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative.
• compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilising and/or dispersing agents.
• the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
• Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents, as desired.
• Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylceiluiose, sodium carboxymethylcel!uiose, or other well known suspending agents.
• the chemical compound of the invention may be formulated as ointments, creams or lotions, or as a transdermal patch.
• Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
• Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
• compositions suitable for topical administration in the mouth include lozenges comprising the active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerine or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
• compositions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray.
• the compositions may be provided in single or multi-dose form.
• the compound In compositions intended for administration to the respiratory tract, including intranasal compositions, the compound will generally have a small particle size for example of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization.
• the pharmaceutical preparations are preferably in unit dosage forms, in such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component.
• the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules.
• the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
• compositions are preferred compositions.
• compositions Solutions or suspensions for application to the nasal cavity or to the respiratory tract are preferred compositions.
• Transdermal patches for topical administration to the epidermis are preferred.
• the invention provides a method for the treatment, prevention or alleviation of anxiety in a subject in need thereof, and which method comprises administering to such a subject, including a human, in need thereof an effective amount of the pterostilbene of the invention.
• a therapeutically effective dose refers to that amount of active ingredient, which ameliorates the symptoms or condition.
• Therapeutic efficacy and toxicity e.g. ED 50 and LD50, may be determined by standard pharmacologica! procedures in ceil cultures or experimental animals. The dose ratio between therapeutic and toxic effects is the therapeutic index and may be expressed by the ratio LD 50 / ED 50 . Pharmaceutical compositions exhibiting large therapeutic indexes are preferred.
• the dosage of compounds used in accordance with the invention varies depending on the compound and the condition being treated.
• the age, lean body weight, total weight, body surface area, and clinical condition of the recipient patient; and the experience and judgment of the clinician or practitioner administering the therapy are among the factors affecting the selected dosage. Other factors include the route of administration, the patient's medical history, the severity of the disease process, and the potency of the particular compound.
• the dose should be sufficient to ameliorate symptoms or signs of the disease treated without producing unacceptable toxicity to the patient.
• the dosage may be varied by titration of the dosage to the particular
• HED Animal dose (mg/kg) multiplied by Human K m where the K m factor, body weight (kg) divided by BSA (m 2 ), is used to convert the mg/kg dose used in the animal study to an mg/ m 2 .
• the K m factor for mouse is 3 and the K m factor for human is 37.
• a table (Table 1 , Reagan-Shaw et a/,, supra) lists K m factors calculated for several animal species based on data from FDA Guidelines.
• the pterostiibene is present in the composition in an amount sufficient to treat, alleviate, or prevent anxiety in a subject in need thereof. In a most preferred
• the pterostiibene is present in the composition in an amount sufficient to treat, alleviate, or prevent anxiety by itself.
• the active ingredient may be administered in one or several doses per day.
• a satisfactory result can, in certain instances, be obtained at a dosage as low as the human equivalent dose (HED) of 0.0405 mg/kg p.o. or a dose of about 3 mg/day p.o. for a 80 kg human patient to a dose of about 15 mg/day p.o. for a 60 kg human patient.
• HED human equivalent dose
• an appropriate range can be from about 10 mg/day p.o. to about 100 mg/ day p.o. for said human patient.
• suitable dosage ranges are 10-100 mg daily for human patients, dependent as usual upon the exact mode of administration, form in which administered, the indication toward which the administration is directed, the subject involved and the body weight and body surface area of the subject involved, and further the preference and experience of the physician or veterinarian in charge.
• the dosing regimen may be reduced.
• Pterostiibene was synthesized as previously described (Joseph et a/., supra). Briefly, pterostiibene was synthesized by condensation of 3,5-dimethoxybenzaldehyde and 4-hydroxyphenylacetic acid in acetic anhydride and triethylamine. The reaction mixture was heated (150 °C) under an atmosphere of nitrogen and continuously stirred. After 20 h, the reaction was stopped and cooled to room temperature, and concentrated hydrochloric acid (5 mL) was added. A precipitate formed, and this was dissolved in 50 mL of chloroform and then extracted with 10% aqueous sodium hydroxide.
• the aqueous extract was acidified to pH 1 with concentrated hydrochloric acid and stirred for at least 6 h, resulting in the precipitation of the intermediate product, a-[(3,5- dimethoxyphenyI)methyl ene]-4-hydroxy-(aZ)-benzeneacetic acid.
• This intermediate product was heated with 1 .0 g of copper in 10 mL of quinoline (200 °C, 6 h, under nitrogen). The reaction mixture was cooled to room temperature and filtered. To the filtrate was added 5 N hydrochloric acid (25 mL), which was stirred for 1 h and then extracted with chloroform.
• the chloroform extract containing impure pterostiibene was purified by flash chromatography on a Horizon HPFC system (Biotage, inc.,
• Pterostiibene was dissolved in a mixture of cremophor, ethanoi, and saline (1 :1 : 18) in doses between 1 and 10 mg/kg body weight. A volume of 10 mL/kg was administered orally 1 h before the behavioral tests.
• mice Eight-week old male Swiss Webster mice (Harlan, IN, USA) weighing 24-30 gm at the time of testing were used for ail studies. Animals were housed in groups of five, were given food and water ad libitum, and maintained under a 12:12 h day:light cycle. All mice were randomly selected for each treatment group. Housing, handling and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Mississippi and adhered to the regulations of the National Institutes of Health Guide for Care and Use of Laboratory Animals.
• IACUC Institutional Animal Care and Use Committee
• the EPM apparatus consisted of two open arms (30 x 5 cm) and two enclosed arms (30 x 5 cm, wails 15 cm high) which extended from a common central platform (5 x 5 cm).
• the configuration formed the shape of a plus-sign with comparable arms arranged opposite one another.
• the apparatus was elevated 50 cm above the floor level
• mice were acclimatized for 30 min in the EPM room before pterosti!bene or diazepam treatment While pterostiibene was given orally, diazepam was administered intraperitoneal!y (i.p.).
• mice were allowed to stay in the same room for 80 min and 30 min, respectively, prior to the EPM trial. Thereafter, mice were placed individually in the center of the apparatus facing an open arm, and allowed to freely explore for 5 min.
• the Traveled Distance in Open Arms (TDOA), the Traveled Distance in Enclosed Arms (TDEA); Permanence Time in the Open Arms (PTOA), Permanence Time in the Enclosed Arms (PTEA), Number of Entries in the Open Arms (NEOA) and Number of Entries in the Enclosed Arms (NEEA) were measured; i.e., the percentage of distance traveled, and time spent on both open and enclosed arms, and the number of open- and enclosed-arm entries were quantified using a computer- assisted video tracking system (San Diego Instruments, CA, USA). The maze was wiped clean with glass cleaner and dried after each trial. An arm entry was recorded when all four paws of the mouse were in the arm. The percentage of time spent in the open arms and the number of open arm entries are considered to be critical determinants, and pure measures of anxiety (Hogg, supra).
• pterostiibene (Fig. 1 ) dose-dependently exhibited anxiolytic-like action in EPM.
• the anxiolytic action was apparent at 1 mg/kg dose of the compound, reached the maximum at 2 mg/kg, and then declined at 5, and 10 mg/kg doses.
• mice 8- 10/group were administered with pterostilbene by oral gavage. Each mouse was then placed in the locomotor chamber and acclimated in the Plexiglas enclosure for 30 min. After the 30 min acclimation period, locomotor activity was measured for 30 min using an automated activity monitoring system (San Diego Instruments, CA, USA). Total activity was expressed as the total number of interruptions of 16 ceil photobearns per chamber.
• the supernatant was collected, supplemented with sample buffer, and boiled at 95 °C for 5 min.
• the prepared sample was thus ready for Western blot experiments.
• An equal amount of protein (30 pg) was subjected to SDS-polyacrylamide gel electrophoresis (12% gels), and the blotted membrane was blocked in TBST buffer containing 5% skim milk for 1 h at room temperature.
• the membrane was then incubated with anti ⁇ phospho ⁇ ERK (Thr 202/Tyr 204, Cell Signaling Technologies, USA) antibody.
• the other gel was run with the same sample, and the membrane was incubated with anti-beta-actin antibody (Cell signaling Technologies, USA).
• the expression level of phospho-Akt in hippocampus was checked by using anti-phospho-Akt (Ser 473, Cell signaling Technologies, USA) antibody followed by re-probing with anti-$-actin antibody (Cell signaling Technologies, USA). Bound antibodies were detected with horseradish peroxidase-iinked anti-rabbit antibody (Cell Signaling Technologies, USA) and developed using VersaDoc imaging system (Bio-Rad, USA). The relevant immunoreactive bands were quantified using Quantity One 1 -D Analysis Software (Bio-Rad, USA). To evaluate ERK activation, the phospho-ERK levels were normalized to that of ⁇ -actin.
• Plasma were kept at -80°C, and thawed on ice prior to extraction. Plasma were
• Hippocampus tissues were kept at ⁇ 80°C until time of extraction. Hippocampus (left and right) from 2-3 animals was combined in an Eppendorf tube, and processed as one sample. To the tube was added 200 ⁇ _ phosphate buffer (0.2M NaH 2 P0 4 : 0.2M Na 2 HP0 4 . and the tissues were homogenized manually for 2 minutes. To 100 ⁇ aliquot, 50 ⁇ _ ⁇ -glucuronidase (5000 U/mL potassium phosphate buffer) was added. The mixture was vortex-mixed, and incubated (37 °C, 20 hours) while shaking (750 rpm). The samples were then centrifuged (15 min, 7000g, 4 °C). The supernatant was collected and partitioned with ethyl acetate (200 ⁇ [_, twice). The ethyl acetate layers were combined, dried under a stream of nitrogen, and used for GC-MS analysis.
• 200 ⁇ _ phosphate buffer 0.2M NaH 2 P0 4
• the GC temperature program was: initial 190 °C, increased to 240 °C at 20 °C/min rate, increased to 280 °C at the rate of 2.5 °C/min, then finally increased to 300 °C at the rate of 30 -C/min and held at this temperature for 0.5 min.
• the carrier gas was ultrahigh purity helium, at 1 mL/min flow rate.
• the injection port, GC-MS interface, and ionization chamber were kept at 250, 230, and 230 °C, respectively.
• the volume of injection was 2 juL (splitless injection).
• the mass spectrum was acquired in the positive, selected ion-monitoring mode (mlz 328, 313, and 297); electron impact 70 eV.
• GC-MS analyses were in duplicates.
• the retention time of pterosti!bene was 10.2 min.
• Quantitation of pterostiibene was done using an external standard of a synthetic sample of pterostiibene previously characterized for structure and purity.
• the plasma level of pterostiibene at 1 mg/kg dose was found to be 10.08 ⁇ 8.87 ng/mL, and increased correspondingly with dose reaching 130.26 ⁇ 40.25 ng/mL at 10 mg/kg (Table 1 ).
• the level of pterostiibene in the hippocampus tissues at 1 mg/kg dose were detectable but below the limit of quantitation (0.0840 ng).
• the levels were quantifiable, and ranged from 0.2627 - 1 .1217 ng/hippocampus (Table 2). Only trace levels of pterostiibene (unquantifiable) were found in the prefrontal cortex tissues for ail the doses administered.

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