Plant extracts for improving cognitive health and function

Abstract

To provide a composition for enhancing, improving, or maintaining cognitive health and / or function.SOLUTION: The present invention provides an oral administration composition for enhancing, improving, or maintaining cognitive health and / or function in a mammal undergoing normal aging cognitive changes, rather than dementia, wherein the composition comprises a water extract of spearmint plant at a dose of 1.6 to 32 mg / kg / day, wherein the water extract comprises about 5 to 15 wt.% of rosmarinic acid, and wherein the extract is administered for a period of at least 90 days.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates generally to plant extracts that enhance, improve or maintain cognitive health and function, and more specifically to the administration of carnosic acid and / or rosmarinic acid to improve learning, memory, attention, vigilance, executive function, verbal fluency, processing speed, and / or cognitive flexibility and related behaviors. [Background technology]

[0002] There is a strong demand for products that can improve cognitive health or function, and the market for these products has continued to grow in recent years despite adverse economic pressures. Part of this growth can be attributed to the growing aging population, especially in Asia and the United States. Worldwide sales of cognitive health ingredients are approximately $455 million. Frost and Sullivan predict an annual growth rate of 12% in this area from 2016 to 2019.

[0003] Leading cognitive health ingredients currently include phosphatidylserine (PS), CoQ10, Omega-3 (marine / algae oils), citicoline, ginkgo, and ginseng. Of the top cognitive health ingredients, phosphatidylserine is the only cognitive health ingredient with a qualified claim approved by the FDA. With growing scientific evidence supporting the claim, the ingredient has enjoyed double-digit growth in sales. In 2010, health claims for DHA and EPA on brain function, heart function, and vision received recommendations from EFSA in Europe. Citicoline is recommended as an ingredient to prevent neuronal degeneration and improve memory.

[0004] Carnosic acid (CA) is one of the most abundant polyphenolic compounds present in Rosmarinus officinalis (rosemary) and Salvia officinalis (sage). Currently, CA is already available on the market as an ingredient or as a finished dietary supplement. The purity of these products varies from 25% to 60%, with some HPLC-grade products listed as 98%. In addition, there are rosemary extracts standardized to CA available in powder form (standardized to 5-30% CA) or as a dietary supplement (300 mg capsules standardized to 6% CA with a recommended dose of 3 capsules per day). The percentage of CA in these rosemary extracts varies from 5 to 60%. Rosemary has a combination of phenolic diterpenes (carnosic acid and carnosol), phenolic acids (caffeic acid and rosemarinic acid), and flavonoids. Diterpenes are combinations of four isoprene units and are known to have antimicrobial and anti-inflammatory properties (Bisio et al. Antimicrobial activity of the ornamental species Salvia corrugata, a potential new crop for extractive purposes. J Agric Food Chem 56: 10468-10472, 2008; Sato et al. Antibacterial novel phenolic diterpenes from Podocarpus macrophyllus D. Don. Chem Pharm Bull (Tokyo) 56: 1691-1697, 2008; Su et al. Anti-inflammatory activities of furanoditerpenoids and other constituents from Fibraurea tinctoria. Bioorg Med Chem 16: 9603-9609, 2008; Wang (E. et al. Triptolide inhibits the differentiation of Th17 cells and suppresses collagen-induced arthritis. Scand J Immunol 68: 383-390, 2008). These isoprene units can be linked together as linear chains or arranged to form rings. CA is thought to be the main active ingredient in Rosmarinus officinalis along with its derivative carnosol.

[0005] [ka]

[0006] The majority of available in vitro studies have evaluated CA for antioxidant protection. Indeed, the antioxidant activity of rosemary has been mainly attributed to CA (Aruoma et al. An evaluation of the antioxidant and antiviral action of extracts of rosemary and provencal herbs. Food and Chemical Toxicology 34: 449-456, 1996; Perez-Fons et al. Rosemary (Rosmarinus officinalis) diterpenes affect lipid polymorphism and fluidity in phospholipid membranes. Archives of Biochemistry and Biophysics 453: 224-236, 2006; Wang et al. Augmentation by Carnosic Acid of Apoptosis in Human Leukaemia Cells Induced by Arsenic Trioxide via Upregulation of the Tumour Suppressor PTEN. The Journal of International Medical Research 36: 682-690, 2008). Neuroprotection of CA The effects were evaluated using a glutamate model of oxidative stress. The study demonstrated that CA protects neurons against severe glutamate-induced damage both in vivo and in vitro (Sato et al. Carnosic acid, an electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1 / Nrf2 pathway via alkylation of targeted cysteines on Keap1. Journal of Neurochemistry 104: 1116-1131, 2008). More recent literature on CA has discussed its possible anti-inflammatory properties.In addition to studies showing that CA has anti-inflammatory activity through downregulation of NF-κB transcription factor, other studies have evaluated additional mechanisms for the anti-inflammatory properties of CA (Laughton et al. Inhibition of mammalian 5-lipoxygenase and cyclo-oxygenase by flavonoids and phenolic dietary additives: Relationship to antioxidant activity and to iron ion-reducing ability. Biochemical Pharmacology 42: 1673-1681, 1991; Poeckel et al. Carnosic acid and carnosol potently inhibit human 5-lipoxygenase and suppress pro-inflammatory responses of stimulated human polymorphonuclear leukocytes. Biochem Pharmacol 76: 91-97, 2008; Rau et al. Carnosic Acid and Carnosol, Phenolic Diterpene Compounds of the Labiate Herbs Rosemary and Sage, are Activators of the Human Peroxisome Proliferator-Activated Receptor Gamma. Planta Med 72: 881-887, 2006).

[0007] The above in vitro data are supported by in vivo data on the antioxidant capacity of CA. Recent in vivo literature has demonstrated the anti-obesity and anti-glycative effects of CA (Alarcon-Aguilar et al. Investigation on the hypoglycaemic effects of extracts of four Mexican medicinal plants in normal and alloxan-diabetic mice. Phytother Res 16: 383-386, 2002; Hsieh et al. Low-density lipoprotein, collagen, and thrombin models reveal that Rosemarinus officinalis L. exhibits potent antiglycative effects. J Agric Food Chem 55: 2884-2891, 2007; Ninomiya et al. Carnosic acid, a new class of lipid absorption inhibitor from sage. Bioorganic & Medicinal Chemistry Letters 14: 1943-1946, 2004; Takahashi et al. Carnosic acid and carnosol inhibit adipocyte differentiation in mouse 3T3-L1 cells through induction of phase 2 enzymes and activation of glutathione metabolism. Biochem Biophys Res Commun 382: 549-554, 2009)。

[0008] Rosmarinic acid (RA) is one of the major components found in spearmint and is an important contributor to its antioxidant capacity (Fletcher et al. Heat stress reduces the accumulation of rosmarinic acid and the total antioxidant capacity in spearmint (Mentha spicata L). Journal of the Science of Food and Agriculture 85: 2429-2436, 2005). RA, a naturally occurring phenolic compound, is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. Its structure consists of a carbonyl group, an unsaturated double bond, and a carboxylic acid between two phenolic rings. RA has exhibited several biological activities such as anti-inflammatory, antimutagenic, antibacterial, antidepressant, HIV-1 inhibitory, antioxidant, and antiviral properties. These properties have made RA an attractive ingredient for the pharmaceutical and cosmetic industries. RA has been used topically in Europe as a nonsteroidal anti-inflammatory drug (Ritschel et al. Percutaneous absorption of rosmarinic acid in the rat. Methods and Findings in Experimental and Clinical Pharmacology 11: 345-352, 1989). Due to its widespread use as a flavoring and preservative in the food industry, RA is considered a safe ingredient for daily consumption (Alkam et al. A natural scavenger of peroxynitrites, rosmarinic acid, protects against impairment of memory induced by A β25-35. Behavioral Brain Research 180: 139-145, 2007).

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[0010] Evidence for the non-specific protective properties of RA has been found in the brain. Enhanced antioxidant activity in the brain was demonstrated after administration of RA to aged mice, which resulted in enhanced superoxide dismutase (SOD) and catalase (CAT) activity in the brain while reducing malondialdehyde (MDA) (Shou et al. Rosmarinic acid attenuates D-galactose induced behavior impairment in mice and its mechanism. 2010, p. 1723-1726). These data demonstrate the nonspecific protective properties of RA as an antioxidant; however, previous data have not demonstrated the ability of RA to affect the brain in specific regions or with specific clinical outcomes.

[0011] In vivo, three studies have evaluated the administration of RA. These studies administered RA either orally or IP in intracranial injury or stress models used to represent specific cognitive disease states (Alkam et al. A natural scavenger of peroxynitrites, rosmarinic acid, protects against impairment of memory induced by Aβ25-35. Behavioural Brain Research 180: 139-145, 2007; Park et al. Subchronic administration of rosmarinic acid, a natural prolyl oligopeptidase inhibitor, enhances cognitive performances. Fitoterapia 81:644-648, 2010; Zhou et al. Rosmarinic acid attenuates D-galactose induced behavior impairment in mice and its mechanism. Intl Conf BMEI 4:1723-1726, 2010). RA has been shown to be beneficial in these models. Although they have been shown to be effective in treating RA-related cognitive disorders, they are not validated models for the assessment of normal age-related cognitive changes (as in SAMP8). In addition, it is unknown whether their mechanism of action is specific or non-specific due to antioxidant effects. Currently, there are no published human studies that have evaluated RA supplementation alone or through the use of spearmint extract.

[0012] Learning and memory can be divided into two main categories: declarative and procedural. Declarative has episodic, spatial and associative memory components. It is related to learning and memory, which has a conscious component that requires attention and vigilance. In humans, it concerns the acquisition, recognition and memory of distinct events, places, people and facts. Declarative learning and memory is currently measured in described animal studies through T-maze acquisition, T-maze retention, and object recognition. Procedural learning and memory may form when a declarative memory task becomes routine or habitual, and was measured in current animal studies through lever pressing. It concerns learning and memory that does not have a conscious component, which in humans is a habit or skill, such as riding a bicycle. Declarative tasks are thought to be initiated in the hippocampus, while procedural tasks are primarily linked to the caudate region of the brain.

[0013] With regard to the assessment of cognitive effects resulting from aging, the senescence accelerated mouse-prone 8 (SAMP8) is a proven model of accelerated aging that develops deficits in learning and memory by 8 months of age (Yagi H, Katoh S, Akiguchi I, Takeda T (1988) Age-related deterioration of ability of acquisition in memory and learning in senescence accelerated mouse: SAM-P / 8 as an animal model of disturbances in recent memory. Neurosci Biobehav Rev. 474, 86-93; Flood JF, Morley JE (1998) Learning and memory in the SAMP8 mouse. Neurosci Biobehav Rev. 22, 1-20). SAMP8 mice have a spontaneous mutation that leads to an age-related increase in amyloid precursor protein (AβPP) and amyloid beta (Aβ) in the brain and increased production of free radicals in the central nervous system, which results in learning and memory deficits (Butterfield DA, Howard BJ, Yatin S, Akkeb KL, Carney JM (1997) Free radical oxidation of brain proteins in accelerated senescence and its modulation by N-tert-butyl- α-phenylnitrone. Prac Natl Acad Sci USA. 94, 674-678; Sato E, Kurokawa T, Oda N, Ishibashi S (1996) Early appearance of abnormality of microperoxisomal enzymes in the cerebral cortex of senescence-accelerated mice. Mech Ageing Dev. 92, 175-184). The SAMP8 model has been reported in the literature to be a model that can be used with a variety of nutritional ingredients, supplements, and drugs. Some examples are alpha-lipoic acid, n-acetylcysteine ​​(Farr SA, Poon HF, Dogrukol-Ak D, Drake J, Banks WA, Eyerman E, Butterfield DA, Morley JE (2003) The antioxidants α-lipoic acid and N-acetylcysteine ​​reverse memory impairment and brain Oxidative stress in aged SAMP8 mice. J Neurochemistry. 84, 1173-1183), Saturated fatty acids (Petursdottir AL, Farr SA, Morley JE, Banks WA, Skuladottir GV (2008) Effect of dietary n-3 polyunsaturated fatty acids on brain lipid fatty acid composition, learning ability, and memory of senescence-accelerated mouse. J Gerontol A Biol Sci Med Sci 63, 1153-1160), and memantine (Zhou, M., Wang, M. -., & Wang, X. -. (2010). Effects of memantine combined with enriched environment therapy on learning and memory abilities and the mental behavior in senescence accelerated mouse. Journal of Clinical Neurology, 23(6), 438-441), all of which showed positive cognitive benefits in follow-up human clinical trials (Volchegorskii, IA, Rassokhina, LM, Kolyadich, MI, & Alekseev, MN (2011). Comparative study of alpha-lipoic acid and mexidol effects on affective status, cognitive functions and quality of life in diabetes mellitus patients. Experimental'Naya i Klinicheskaya Farmakologiya, 74(11), 17-23; Chan, A., Remington, R., Kotyla, E., Lepore, A., Zemianek, J., & Shea, TB (2010). A Vitamin / nutrient formulation impr oves memory and cognitive performance in community-dwelling adults without dementia. Journal of Nutrition, Health and Aging, 14(3), 224-230; Small, GW, Silverman, DHS, Siddarth, P., Ercoli, LM, Miller, KJ, Lavretsky, H., Phelps, ME (2006). Effects of a 14-day healthy lifestyle program on cognition and brain function. American Journal of Geriatric Psychiatry, 14(6), 538-545; Litvinenko, IV, Odinak, MM, Mogil'Naya, VI, & Perstnev, SV (2010). Use of memantine (akatinol) for the correction of cognitive impairments in Parkinson's disease complicated by dementia. Neuroscience and Behavioral Physiology, 40(2), 149-155). Neuroscientists evaluating cognitive components and / or drugs recognize that SAMP8 is a valid animal model that has been successfully utilized to predict their efficacy in follow-up human clinical trials. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Bisio et al. Antimicrobial activity of the ornamental species Salvia corrugata, a potential new crop for extractive purposes. J Agric Food Chem 56:10468–10472

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[0015] The invention consists of the administration of carnosic acid and / or rosmarinic acid extracted from plants to improve learning, memory, executive functions (i.e. working memory, task switching, planning, flexibility, visual attention, mathematical skills, and a person's ability to recognize and adapt to new and changing conditions, environments and situations) and cognitive-related behaviors (i.e. concentration, attention, vigilance, exploration, motivation, etc.). Rosemary extracts containing carnosic acid show improved declarative memory as well as procedural learning and memory. In mammals, this can be observed as enhanced, improved, or maintained memory of distinct events, places, people, and facts. In addition, it can be observed that the ability to learn and maintain habitual information or skills is improved, possibly as a result of an improved ability to make connections. Spearmint extracts containing rosmarinic acid show improvements in declarative learning and memory. In mammals, this can be observed as enhanced, or improved learning of distinct events, places, people, and facts, along with enhanced and maintained memory of these things.

[0016] Oxidative damage is considered one of the hallmarks of the aging process. Neuronal dysfunction present in age-related cognitive impairment is believed to be due in large part to oxidative stress. Both structural and functional damage to mitochondria is present in cognitive impairment, such as Alzheimer's disease, suggesting that antioxidants that penetrate both cells and mitochondria would provide the greatest protection from oxidative stress. This study was designed to test whether two novel proprietary antioxidant-based ingredients, rosemary extract and spearmint extract standardized to carnosic acid and rosmarinic acid, respectively, could improve learning and memory in the SAMP8 mouse model of accelerated aging. We have previously shown the ability of antioxidants to improve learning and memory deficits and reverse signs of oxidative damage in SAMP8 mice; therefore, the SAMP mouse is an excellent model for testing these antioxidant compounds. SAMP mice were placed in one of three extracts. Two rosemary extracts contained carnosic acid (60% carnosic acid and 10% carnosic acid) and one spearmint extract contained 5% rosmarinic acid. Three doses of each extract were tested: 32 mg / kg, 16 mg / kg, and 1.6 mg / kg of the active substance (rosemary). and spearmint extracts (CA or RA, respectively) and vehicle control. A 50% SAMP8 backcross line served as a control, which also received vehicle. After 90 days of treatment, mice were tested in three behavioral tests: T-maze footshock avoidance, object recognition, and lever pressing. Rosemary extract containing 60% carnosic acid was able to improve acquisition at 32 mg / kg CA and retention at all three doses of active substance in T-maze footshock avoidance, and to improve object recognition and lever pressing at 16 and 32 mg / kg CA. Rosemary extract with 10% carnosic acid improved retention in T-maze footshock avoidance (16 mg / kg CA) and lever pressing at 16 and 32 mg / kg CA. Spearmint with 5% rosmarinic acid improved acquisition (16 and 32 mg / kg RA) and retention (at all doses of RA) in the T-maze. In addition, spearmint with 5% rosmarinic acid improved object recognition at 16 and 32 mg / kg RA. 4-Hydroxynonenal (HNE) was reduced in the cerebral cortex after treatment with all three extracts compared to vehicle-treated SAMP8. Protein carbonyls were reduced in the hippocampus after administration of rosemary with 10% carnosic acid and spearmint with 5% rosmarinic acid. Current results show that extracts from spearmint (rosmarinic acid) and rosemary (carnosic acid) have beneficial effects on learning and memory in SAMP8 mice, as well as markers of brain tissue oxidation that occur with aging. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows a chart of the effects of 60% carnosic acid on T-maze acquisition, T-maze retention, object recognition and lever pressing. [Diagram 2]FIG. 2 shows a chart of oxidative stress markers in the brain cortex following administration of rosemary extract containing 60% carnosic acid; results are expressed as percent change from SAMP8 vehicle control; * indicates P<0.05, ** indicates P<0.01. [Diagram 3] FIG. 3 shows a chart of oxidative stress markers in the striatum of the brain following administration of rosemary extract containing 60% carnosic acid; results are expressed as percent change from SAMP8 vehicle control; * indicates P<0.05, ** indicates P<0.01. [Figure 4] FIG. 4 shows a chart of oxidative stress markers in the hippocampus of the brain following administration of rosemary extract containing 60% carnosic acid; results are expressed as percent change from SAMP8 vehicle control; * indicates P<0.05, ** indicates P<0.01. [Diagram 5] FIG. 5 shows a chart of the effects of 10% carnosic acid on T-maze acquisition, T-maze retention, object recognition and lever pressing. [Figure 6] FIG. 6 shows a chart of oxidative stress markers in the brain cortex following administration of rosemary extract containing 10% carnosic acid; results are expressed as percent change from SAMP8 vehicle control; * indicates P<0.05, ** indicates P<0.01. [Figure 7] FIG. 7 shows a chart of oxidative stress markers in the striatum of the brain following administration of rosemary extract containing 10% carnosic acid; results are expressed as percent change from SAMP8 vehicle control; * indicates P<0.05, ** indicates P<0.01. [Figure 8] FIG. 8 shows a chart of oxidative stress markers in the hippocampus of the brain following administration of rosemary extract containing 10% carnosic acid; results are expressed as percent change from SAMP8 vehicle control; * indicates P<0.05, ** indicates P<0.01. [Figure 9] FIG. 9 shows a chart of the effects of 5% rosmarinic acid on T-maze acquisition, T-maze retention, object recognition and lever pressing. [Figure 10] FIG. 10 shows a chart of oxidative stress markers in the brain cortex following administration of rosemary extract containing 5% rosmarinic acid; results are expressed as percent change from SAMP8 vehicle control; * indicates P<0.05, ** indicates P<0.01. [Figure 11] FIG. 11 shows a chart of oxidative stress markers in the striatum of the brain following administration of rosemary extract containing 5% rosmarinic acid; results are expressed as percent change from SAMP8 vehicle control; * indicates P<0.05, ** indicates P<0.01. [Figure 12] FIG. 12 shows a chart of oxidative stress markers in the hippocampus of the brain following administration of rosemary extract containing 5% rosmarinic acid; results are expressed as percent change from SAMP8 vehicle control; * indicates P<0.05, ** indicates P<0.01. DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0018] This study was designed to test whether rosemary extracts (containing CA) and spearmint extracts (containing RA) could improve all aspects of cognitive health and / or function, including but not limited to: attention, vigilance, recognition, recall, reaction time, focus, motivation, behavior, executive function, learning and memory. CA and RA molecules were specifically selected for their unique properties: 1) their ability to cross the blood-brain barrier; 2) different solubility (oil vs. water); and 3) enhanced antioxidant capacity. The two extracts were tested in the SAMP8 mouse strain (a senescence-accelerated mouse model), which shows early onset of learning and memory deficits due to beta-amyloid plaque development and dementia resulting from oxidative stress. 50% SAM mice served as controls.

[0019] definition As used in this application, the following terms have the meanings set forth below.

[0020] Vigilance: a state of close, continuous attention, alertness and readiness to encounter danger or emergency, or of rapid awareness and action.

[0021] Cognitive health: Cognitive health refers to the overall brain, tissue and blood supply and its ability to function properly under various conditions. Good cognitive health is essential for the brain to carry out all mental processes at their best; it is collectively known as cognition, including but not limited to: learning, intuition, judgment, language, attention, vigilance, concentration and memory (both long-term and short-term). Poor cognitive health due to aging, disease and / or other cognitive disorders reduces the brain's ability to function properly, which results in a significant decline in cognitive function and performance.

[0022] Cognitive function: Any mental or intellectual process that involves neurological or symbolic operations, including but not limited to the ability to communicate, perceive, understand, reason, remember, think, be aware, concentrate, pay attention, vigilance, motivate, draw conclusions, executive function, create mental images and make judgments. In animal model systems, cognitive function can be measured by a variety of commonly used methods known in the art, including using the Morris Water Maze (MWM), Barnes Circular Maze, Elevated Radial Arm Maze, T-Maze or any other maze that animals use spatial information. Other tests known in the art can be used to assess cognitive function, such as novel object recognition and odor recognition tasks.

[0023] Executive function: Cognitive processes that control, regulate, and manage other cognitive processes, such as planning, working memory, attention, problem solving, verbal reasoning, mathematical ability, inhibition, mental flexibility, task switching, initiation, flexibility, visual attention, mathematical skills, adaptability to new and changing environments, and monitoring of behavior.

[0024] Learning: the act, process, or experience of acquiring knowledge or skills; especially psychological or behavioral modification by experience or conditioning.

[0025] Memory: a collection of information from past learning or experience that is stored in one's mind. Pieces of information stored in memory, e.g., mental images of experiences. The ability to recall past experiences or learned information includes higher-level mental processes, e.g., learning, retention, recall, and recognition, and results from chemical changes among nerve cells in several different areas of the brain, including the hippocampus. These include: (1) declarative learning or memory, which refers to things that can be consciously recalled, e.g., facts and knowledge; (2) working memory, which refers to the active holding of large numbers of temporary pieces of information in the mind, where they can be manipulated; (3) reference memory, which refers to information obtained from previous experiences, either recent or remote; (4) recognition memory, which is the ability to recognize events, objects, or people previously encountered, and associative memory, which is the ability to learn and recall relationships between unrelated items. Each of these has immediate, short-term, and long-term components. Immediate memory lasts only a few seconds. Short-term memory stores minimally processed information and is available for only a few minutes, such as in remembering a phone number long enough to use it. Short-term memory transfers to long-term memory only if repeated use of the information promotes neurochemical changes that allow it to be retained, which can last for several years.

[0026] Therapeutically effective amount: The amount of the compound of the present invention or its composition or derivative is that amount that will have the intended therapeutic effect when administered to a subject. The full therapeutic effect does not necessarily occur by administration of one dose, but may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The exact effective amount required for a subject will depend, for example, on the subject's size, health and age, the nature and extent of its cognitive impairment, and the combination of therapies selected for administration, and the mode of administration. Those skilled in the art can easily determine the effective amount for a given situation by routine experimentation. In one embodiment, at least one extract of a plant of the Lamiaceae family as described herein, such as CA, RA, or various combinations thereof, is for administration as frequently as once a day, or more than once a day, for example, 2, 3 or 4 times a day.

[0027] Treatment or Treating: Clinical intervention in an attempt to change the natural history of the individual, animal or cell being treated, which can be performed either for prophylaxis or during the course of clinical pathology. Desired effects include prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. A condition or subject refers to taking measures to obtain a beneficial or desired result, including clinical results. Beneficial or desired clinical results include, but are not limited to, enhancing, improving, or maintaining cognitive health and / or function, reducing or improving one or more symptoms associated with mild cognitive impairment or age-related cognitive impairment, delaying or slowing the impairment, improving, alleviating or stabilizing the impairment, and other beneficial results, such as improving cognitive function or reducing the rate of cognitive decline in subjects with or at risk of age-related cognitive impairment. In a preferred embodiment, these terms include a cognitive disorder, such as dyslexia, aspraxia, attention deficit hyperactivity disorder, attention deficit disorder, autism, Alzheimer's disease, Parkinson's disease or stroke, or and other executive function deficits. EXAMPLES

[0028] Oxidative damage is thought to be one of the hallmarks of the aging process [Harman D (2002) Alzheimer's disease: role of aging in pathogenesis. Ann NY Acad Sci. 959, 384-395]. Neuronal dysfunction is present in aging-related diseases, such as Alzheimer's disease. It is believed that most of the damage caused by this condition is due to oxidative stress [Markesbery WR (1997) Oxidative stress hypothesis in Alzheimer's disease. Free Radic Biol Med. 23, 134-147; Polidori MC, Griffiths HR, Mariani E, Mecocci P (2007) Hallmarks of protein Oxidative Damage in Neurodegenerative Diseases: Focus on Alzheimer's Disease. Amino Acids. 32, 553-559]. Structural and functional damage to mitochondria is thought to be a contributing factor in the pathogenesis of Alzheimer's disease. In patients with Heimer's disease, this suggests that antioxidants that readily penetrate both cells and mitochondria would provide the greatest protection from oxidative stress [Skulachev VP, Anisimov VN, Antonenko YN, Bakeeva LE, Chernyak BV, Erichev VP, Filendo OF, Kalinia NI, Kapelko VI, Kolosova NG, Kopin BP, Korshunova GA, Lichinitser MR, Obukhova LA, Pasyukova EG, Pisarenko OI, Roginsky VA, Ruuge EK, Senin II, Severina II, Skulachev MV, Spivak IM, Tashlitsky VN, Tkachuk VA, Vyssokikh MY, Yaguzhinsky LS, Zorov DB (2009) An attempt to prevent senescence: a mitochondrial approach. Biochim Biophys Acta. 1787, 437-461; Suh JH, Shigeno ET, Morrow JD, Cox B, Rocha AE, Frei B, Hagen TM (2001) Oxidative stress in the aging rat heart is reversed by dietary supplementation with (R)-(alpha)-lipoic acid. FASEB J. 15, 700-706]. Senescence-accelerated mouse-prone 8 (SAMP8) showed a reduced ability to learn and remember by 8 months of age. SAMP8 mice are a model of accelerated aging that exhibits defects in memory [Yagi, et al., 1988; Flood, et al., 1998]. ) and have a spontaneous mutation that leads to age-related increases in amyloid beta (Aβ), learning and memory deficits, and oxidative stress. Furthermore, antisense to AβPP enhances memory and learning in these mice [Morley JE, Farr SA, Flood JF (2002) Antibody to amyloid beta protein alleviates impaired acquisition, retention, and memory processing in SAMP8 mice. Neurobiol Learn Mem. 78, 125-38; Kumar VB, Farr SA, Flood JF, Kamelesh V, Franko M, Banks WA, Morley JE (2000) Site-directed antisense oligonucleotide decreases the expression of amyloid precursor protein and reverses deficits in learning and memory in aged SAMP8 mice. Peptides. 21, 1769-1775]. In addition, SAMP8 mice have increased free radical regulating activity in the central nervous system. It has radical generation [Butterfield, et al., 1997; Sato, et al., 1996], which is [Fujibayashi Y, Yamamoto S, Waki ​​A, Jibusgu Hm ​​Yonekura Y (1998) Increased mitochondrial DNA deletion in the brain of SAMP8, a mouse model for spontaneous oxidative stress brain. Neurosci Lett. 254, 109-112] .

[0029] Antioxidants have been shown to reverse learning and memory deficits in SAMP8 mice. Alpha-lipoic acid and n-acetylcysteine ​​enhance memory and reverse markers of oxidative stress in these mice [Farr, et al., 2003]. Alpha-lipoic acid significantly reduced lactate dehydrogenase B, dihydro pyrimindase-like protein, and alpha-enolase carbonyl levels [Poon HF, Farr SA, Thongboonkerd V, Lynn BC, Banks WA, Morley JE, Klein JB, Butterfield DA (2005) Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders. Neurochem Int. 45, 159-68]. Polyphenol antioxidants have also been shown to reverse learning and memory deficits in SAMP8 mice. AMP8 has been shown to improve learning and memory and reverse markers of oxidative stress in mice [Farr SA, Price TO, Domnguez LJ, Motisi A, Saiano F, Niehoff ML, Morley JE, Banks WA, Ercal N, Barbagallo M (2012) Extra virgin olive oil improves learning and memory in SAMP8 mice. J Alzheimer's Dis 28, 81-92].

[0030] Carnosic acid (CA) and rosmarinic acid (RA) have been shown to be neuroprotective and preventive against oxidative stress [Fadel O, El Kirat K, Morandat S (2011) The natural antioxidant rosmarinic acid spontaneously penetrates membranes to inhibit lipid peroxidantion in situ. Biochim Biophys Acta 1808, 2973-2980; Fallarini S, Miglio G, Paoletti T, Minassi A, Amoruso A, Bardelli C, Brunelleschi S, Lombardi G (2009) Clovamide and rosmarinic acid induce neuroprotective effects in invitro models of neuronal death. Br J Pharmacol 157, 1072-1084; Hou CW, Lin YT, Chen YL, Wang YH, Chou JL, Ping LY, Jeng KC (2012) Neuroprotective effects of carnosic acid on neuronal cells Carnosic acid also has an anti-inflammatory effect after administration of LPS.

[19] . and protection against inflammation have been associated with improved memory in diseases of aging [Farr, et al., 2012]. Rosmarinic acid improved memory in a spatial task in the Morris water maze [Park DH, Park SJ, Kim JM, Jung WY, Ryu JH (2010) Subchronic administration of rosmarinic acid, a natural prolyl oligopeptidase inhibitor, enhances cognitive performance. Fitoterapia 81, 644-648].

[0031] Memories are divided into two main categories: declarative (or explicit memory) and procedural (or implicit memory). Declarative memory is further subdivided into semantic memory (facts or meanings) and episodic memory (specific experiences). Semantic memory is generally derived from episodic memory. Declarative memory is thought to be encoded by the hippocampus, while procedural memory is thought to be encoded by the caudate nucleus, a structure within the striatum. Procedural or implicit memories come from learning relationships between responses and rewards. Procedural memories often begin as declarative memories until they become ingrained or habitual.

[0032] The T-maze foot-shock avoidance task represents a declarative episodic memory. It has temporal, spatial and associative memory components. The mouse must learn to associate the opening of a buzzer and a door with the onset of a shock [Farr SA, Banks WA, La Scola ME, Flood JF, Morley JE (2000) Permanent and temporary inactivation of the hippocampus impairs T-maze footshock avoidance acquisition and retention. Brain Res. 872, 242-249]. They must also learn whether they should turn left or right to escape or avoid the shock. Finally, it has a time component, in that they learn that upon the opening of the door and simultaneous onset of a buzzer, they have 5 seconds to run to the goal box (located to the left or right at the end of the corridor) to avoid the shock.

[0033] Object recognition memory is a form of declarative or explicit memory. It is the ability to recognize events, objects, or people previously encountered [Medina JH, Bekinschtein P, Commarota M, Izquierdo I (2008) Do memories consolidate to persist or do they persist to consolidate? Behav Br Res 192:61-69. Mice that have been exposed to the new or novel object will spend more time exploring the new or novel object in the retention test, which is also a test of the hippocampus. Studies have shown that lesions within the hippocampus result in deficits in memory 24 hours after the first exposure [Hammond RS, Tull LE, Stachman RW (2004) On the delay-dependent involvement of the hippocampus in object recognition. Neurobiol Learn Mem 82, 26-34. The experiment involves presenting subjects with a list of words or objects, then during a retention test, some of the words or objects are changed and subjects are asked to identify the words or objects they have previously encountered.

[0034] Lever pressing is a manipulative task. Mice learn to associate pressing a lever with a food reward. Initially, the test uses declarative memory to form the association, but once the association is formed, it becomes a procedural task that primarily involves the caudate region of the brain [Beninger RJ, Ranaldi R (1993) Microinjectiosn of flupenthixol into the caudate-putamen but not the nucleus accumbens, amygdala or frontal cortex of rats produce intra-session declines in food-rewarded operant responding Behav Brain Res 55, 203-212]. Lever pressing is sometimes called the Skinner box, after its inventor.

[0035] This study was designed to test whether these two novel antioxidants found in rosemary and spearmint extracts (carnosic acid and rosmarinic acid, respectively) could improve learning and memory in the SAMP8 mouse model of accelerated aging. We have previously shown the ability of antioxidants to improve learning and memory deficits and reverse signs of oxidative damage in SAMP8 mice [Farr, et al., 2003; Morley JE, Armbrecht HJ, Farr SA, Kumar VB (2012) The senescence accelerated mouse (SAMP8) as a model for oxidative stress and Alzheimer's disease. Biochim Biophy Acta 1822]; therefore, the SAMP mice may be able to tolerate these antioxidant compounds. It is an excellent model for testing [Farr SA, Yamada KA, Butterfield DA, Abdul HM, Xu L, Miller NE, Banks WA, Morley JE (2008) Obesity and hypertriglyceridemia produce cognitive impairment. Endocrinology 149, 2628-2636].

[0036] material and method mouse At the start of treatment, experimental subjects were 9-month-old SAMP8 mice. The control group was 9-month-old 50% SAM mice, a cross between male SAMP8 and female CD-1 mice that do not show age-related impairments in learning and memory. These studies were performed with the approval of the Institutional Animal Care and Use Committee of the VA Medical Center (St. Louis, MO).

[0037] treatment Nine-month-old SAMP8 mice were given rosemary extract containing either 60% or 10% CA (in sunflower oil) or spearmint extract containing 5% RA (in water) by oral gavage once daily for 12 weeks.

[0038] The mice were set up in seven groups: 1. SAMP8 administered rosemary extract containing 60% CA (CA60); 2. SAMP8 administered rosemary extract containing 10% CA (CA10); 3. SAMP8 administered spearmint extract containing 5% RA (RA5); 4. SAMP8 administered sunflower oil as a negative control for groups 1 and 2; 5. SAMP8 administered water as a negative control for group 3; 6. SAMP 50% backcross administered sunflower oil as a positive control for groups 1 and 2; and 7. SAMP 50% backcross treated with water as positive control for group 3.

[0039] Dose-response curves were performed for each extract tested. The doses evaluated were 1.6mg / kg, 16mg / kg, 32mg / kg of active substance (either CA or RA, from rosemary and spearmint, respectively) and a vehicle group for each of the three extracts listed above. Body weights were recorded weekly throughout the study, and behavioral testing began at the 12th week of feeding.

[0040] Behavioral testing Behavioral testing began during week 12 of treatment. Mice were first trained in T-maze foot shock avoidance during week 12 (T-maze acquisition) and T-maze retention of this task was tested during week 13, followed by object recognition at week 13. Lever pressing was performed during weeks 14 and 15.

[0041] T-maze training and testing procedures The T-maze is both a working memory and a learning task based on declarative reference memory tasks. It consisted of a black plastic alley with a start box at one end of a T-shaped arm and two goal boxes at the other end. The start box was separated from the array by a plastic guillotine door that prevented movement down the array until it was raised at the beginning of training. An electrified floor of stainless steel rods ran throughout the maze to deliver mild scrambled foot shocks.

[0042] Mice were not allowed to explore the maze prior to training. A training trial began when the mouse was placed in the start box, the guillotine door was raised, and a signal buzzer sounded; a foot shock was administered 5 s later. The arm of the maze in which the mouse entered on the first trial was designated the "incorrect" box, and a mild foot shock was administered until the mouse entered the other goal box. This goal box was designated as "correct" for all subsequent trials for that particular mouse. At the end of each trial, the mouse was returned to its home cage until the next trial.

[0043] Mice were trained until they performed one avoidance pass (T-maze acquisition). Training was performed with a 35-second interval between trials, the buzzer was a doorbell type sounding at 55 dB, and the shock was set at 0.35 mA (Coulbourn Instruments scrambled grid floor shocker model E13-08). T-maze retention was tested after 1 week by continuing training. Results were reported as the number of trials to criterion for the retention test. Criterion was defined as 5 avoidances of the mild foot shock in 6 consecutive attempts.

[0044] Object-location recognition Object-location recognition, as implemented herein, is a declarative memory task that engages the hippocampus where the retention exposure interval is 24 hours after exposure to the first object [Farr, et al., 2012]. Mice were habituated to an empty apparatus for 5 minutes per day for 3 days before the objects were placed inside. During the training period, mice were exposed to two similar objects (plastic frogs) and allowed to explore them for 5 minutes. The apparatus and objects were cleaned between each mouse. After 24 hours, the mice were exposed to either the original object or a novel object in a new location. The percentage of time spent exploring the object was recorded. The novel object was made of the same material and size as the original object, but had a different shape. This ruled out the possibility that odors associated with certain objects were a confounding factor. The concept underlying the task is based on the tendency of mice to spend more time exploring new and novel objects rather than familiar objects. Thus, the greater the retention / memory at 24 hours, the more time spent on the novel object.

[0045] Lever Press for Milk Reinforcement Lever pressing is a procedural (manipulative) associative learning and memory task. Mice are placed into a fully automated lever-pressing chamber. Pressing a lever on one wall of the compartment causes a light and a liquid dipper with 100 μl of milk to appear on the opposite wall. On day 1, the mouse has 11 s to obtain the reward; On all subsequent days, mice had 6 seconds to obtain their reward. Mice were given 40-minute training periods in M, W, and F for 2 weeks. Mice were food-deprived for 16 hours before the start of the test to provide motivation to perform the task. Food was returned immediately upon completion of the session. Acquisition was measured as the number of rewarded lever presses [Farr SA, Yamada KA, Butterfield DA, Abdul HM, Xu L, Miller NE, Banks WA, Morley JE (2008) Obesity and hypertriglyceridemia produce cognitive impairment. Endocrinology 149, 2628-2636].

[0046] Brain oxidative stress Sample preparation Brain samples were homogenized in 320 mM sucrose, 1% The homogenized samples were briefly homogenized in ice-cold lysis buffer (pH 7.4) containing 1 mM Tris-HCl (pH 8.8), 0.098 mM MgCl2, 0.076 mM EDTA, and the proteinase inhibitors leupeptin (0.5 mg / mL), pepstatin (0.7 μg / ml), aprotinin (0.5 mg / ml), and PMSF (40 μg / ml), as well as a phosphatase inhibitor cocktail. The homogenized samples were then diluted 2-fold in lysis buffer. After homogenization, small aliquots of the homogenized samples were sonicated for 10 seconds at 20% power using a Fisher 550 Sonic Dismembrator (Pittsburgh, PA, USA) and frozen. The remaining homogenate was centrifuged at 3000 g for 5 minutes and the supernatant cytoplasmic and membrane fractions were removed and transferred into another set of tubes. After addition of 400 μl of lysis buffer, the remaining pellet (nuclear fraction) was centrifuged at 3,000 g for 5 min and the supernatant was removed. The pellet was suspended in 20 μl of lysis buffer and inhibitors. The supernatant cytoplasmic and membrane fractions were centrifuged at 10,000 g for 10 min and the resulting supernatant cytoplasmic fraction was transferred into another set of tubes leaving the pellet (membrane fraction). All sonicated samples and fractions were stored at -70°C until used for further experiments. Protein concentration was measured by Pierce bicinchoninic acid (BCA).

[0047] Slot blot analysis Protein carbonyls For detection of protein carbonyls, slot blot analysis of 2,4-dinitrophenylhydrazone (DNP) Schiff base adducts of carbonyls was utilized. Five-μl aliquots of samples were incubated with 5 μl of 12% sodium dodecyl sulfate and 10 μl of 2,4-dinitrophenylhydrazine (from OxyBlottm Protein Oxidation Kit, Chemicon-millipore, Billerica, MA, USA) at room temperature for 20 min, followed by the addition of 7.5 μl of neutralization solution containing Tris (2 M) in 30% glycerol to each sample. After derivatization, samples were diluted to 1 μg / mL with 1× phosphate buffer solution (PBS) containing sodium chloride, mono- and dibasic sodium phosphate. The corresponding sample solutions (250 μl) were rapidly loaded as duplicates onto nitrocellulose membranes by water vacuum pressure. The resulting protein-bound nitrocellulose membrane was then blocked for 90 min with fresh blocking solution containing 750 mg bovine serum albumin (BSA) in 25 ml wash blot (containing 35.2 g sodium chloride, 1.77 g sodium phosphate monobasic, 9.61 g sodium phosphate dibasic, and 1.6 mL TWEEN, diluted to 4 L with deionized water). The membrane was then incubated with polyclonal RbxDNP (OxyBlott™ Protein Oxidation Kit, Chemicon-millipore, Billerica, MA, USA, diluted 1:100) in the wash blot for 2 h. After three 5 min washes with a fresh wash blot, the membrane was then incubated with polyclonal anti-rabbit IgG alkaline phosphatase (Chemicon, Temecula, CA, USA, diluted 1:8000) for 1 h and washed with a fresh wash blot. Blots were washed for 5, 10 and 10 min increments in three increments with 5-bromo-4-chloro-3-indolyl-phosphate / nitroblue tetrazolium reagent solution for alkaline phosphatase secondary antibody. After development, blots were dried and scanned on a CanoScan8800F (Canon) scanner using Adobe Photoshop and analyzed using Scion Image software (Scion Corporation).

[0048] 4-Hydroxy-2-trans-noneal (HNE) The level of protein-bound HNE was used as a marker of lipid peroxidation and was determined as previously described

[29] . For slot blot analysis of protein-bound HNE detection, 5 μl aliquots of samples were incubated with 5 μl of 12% sodium dodecyl sulfate and 10 μl of Laemmli buffer for 20 min at room temperature and subsequently diluted to 1 μg / mL with 1× phosphate buffer solution (PBS) containing sodium chloride, mono- and dibasic sodium phosphate. The corresponding sample solutions (250 μl) were rapidly loaded in duplicate onto nitrocellulose membranes by water vacuum pressure. The resulting protein-bound nitrocellulose membranes were then blocked for 90 min with fresh blocking solution. The membranes were then incubated with polyclonal anti-HNE (Alpha diagnostic, San Antonio, TX, USA, diluted 1:5000) in wash blots for 2 h. After three 5-min washes with a new wash blot, the membrane was then incubated with polyclonal anti-rabbit IgG alkaline phosphatase (Chemicon, Temecula, CA, USA, 1:8000 dilution) for 1 h and washed with a new wash blot for three increments of 5, 10, and 10 min. After washing, the membrane was colorimetrically developed with 5-bromo-4-chloro-3-indolyl-phosphate / nitroblue tetrazolium reagent solution for alkaline phosphatase secondary antibody. After development, the blot was dried and scanned on a CanoScan8800F (Canon) scanner using Adobe Photoshop and analyzed with Scion Image software (Scion Corporation).

[0049] 3-Nitrotyrosine (3-NT) The levels of 3-NT were used as an additional marker of protein oxidative damage [Suh, et al., 2001; Butterfield DA, Stadtman ER (1997) Protein oxidation processes in aging brain. Adv Cell Aging Gerontol 2, 161-191]. Samples (5 μL) were incubated in 5 μL of 12% SDS and 10 μL of Laemmli buffer (0.125 M Trizma base, 4% SDS, 20% glycerol) at room temperature for 20 min. Samples (250 ng protein) per slot were blotted onto nitrocellulose membranes. A primary rabbit antibody (Sigma-Aldrich) specific for 3-NT (1:1000) was used. The same secondary goat anti-rabbit (Sigma-Aldrich) antibody was then used for detection of each primary antibody. Blots were developed and quantified as described above for protein carbonyls. Development and detection was carried out as described above for protein carbonyls.

[0050] Triglycerides Serum triglycerides were quantified using an enzymatic assay system from Pointe Scientific, Inc. (Canton, MI) incorporating a linear end-point color reaction. Triglycerides in the samples were hydrolyzed to glycerol by lipase. The glycerol was then phosphorylated by glycerol kinase and ATP to glycerol-3-phosphate (G 3 P) and ADP. 3 P is converted to dihydroxyacetone phosphate (DAP) and hydrogen peroxide, which is converted to 4-aminoantipyrine (4-AAP) and 3-hydroxy-2,4,6-tribromobenzoic acid (3-hydroxy-2,4, It reacts with 6-tribomobenzoic acid (TBHB) to produce a red quinoneimine dye, and the intensity of the resulting color was measured at 540 nm using a Bio-Rad microplate reader (Hercules, CA).

[0051] statistics Results were analyzed using analysis of variance (ANOVA) to examine differences between groups. Acquisition and retention measures in the T-maze were the number of trials to reach criterion. Results for object recognition are presented as the percentage of total exploration time spent investigating the novel object. Lever pressing was analyzed by two-way repeated measures ANOVA. Results are expressed as the mean plus / minus standard error. Means between groups were compared using Tukey or Bonferroni post hoc analysis. The Dunnett procedure was used for comparisons against the SAMP8 vehicle control group. Parameters of oxidative stress in brain tissue were analyzed using the Mann-Whitney U test.

[0052] result Rosemary extract containing 60% CA (CA60): The results discussed below are shown in FIG.

[0053] Behavioral testing T maze : A one-way ANOVA on trials to criterion in the T-maze acquisition test showed a significant effect of treatment, F(4,48) 8.98, p<0.001. Bonferroni post hoc tests showed that SAMP8 mice given 32 mg / kg CA reached criterion in significantly fewer trials than mice given vehicle. In addition, mice given 32 mg / kg CA were not significantly different from 50% SAM control mice. A one-way ANOVA on trials to criterion in the T-maze retention test showed a significant effect of treatment, F(4,47) = 7.25, p<0.001. Bonferroni post hoc tests showed that SAMP8 mice given 32, 16, and 1.6 mg / kg CA reached criterion in significantly fewer trials than SAMP8 mice given vehicle. Mice given 32, 16, and 1.6 mg / kg CA were not significantly different from 50% SAM mice.

[0054] object recognitionA one-way ANOVA on time spent investigating the novel object in the 24-h retention test yielded a significant treatment effect F(4,46)=4.88, p<0.003. Bonferroni post-hoc tests indicated that SAMP8 mice given 32 and 16 mg / kg CA spent significantly more time investigating the novel object than SAMP8 mice given vehicle. SAMP8 mice given 32 and 16 mg / kg CA were not significantly different from 50% SAM controls.

[0055] Lever press A two-way repeated measures ANOVA on the treatment and day, number of rewarded lever presses ratings yielded significant effects for treatment, F(4,257)=17.27, p<0.001, and day, F(5,257)=15.31, p<0.001. The treatment × day interaction was not significant: F(20,257)=3.78, NS. Tukey's post hoc analysis showed that on days 3, 4, 5, and 6, SAMP8 mice given 32 mg / kg CA were rewarded significantly more than SAMP8 mice given vehicle control. SAMP8 mice given 16 mg / kg CA were rewarded significantly more on days 4, 5, and 6 compared to SAMP8 mice given vehicle control.

[0056] Triglyceride levels One-way ANOVA for triglyceride levels showed that mice receiving CA60 had There was no significant difference in the mean age of the participants: F(4,50) = 2.42.

[0057] Brain oxidative stress Mann-Whitney U tests showed that CA60 significantly reduced 4-hydroxynonenal (HNE) in the cortex at 1.6 and 32 mg / kg CA compared to SAMP8 receiving sunflower oil as a vehicle control. There was no significant effect of CA60 on 3-nitrotyrosine (3-NT) or protein carbonyls in the cortex (see Figure 2). CA60 significantly increased protein carbonyls at 16 mg / kg CA in the striatum but had no effect on HNE or 3-NT (see Figure 3). CA60 significantly increased protein carbonyls at 16 mg / kg CA in the hippocampus. CA60 had no effect on 3-NT in the hippocampus (see Figure 4).

[0058] Rosemary extract containing 10% CA (CA10) The results, discussed below, are shown in FIG.

[0059] Behavioral testing T maze A one-way ANOVA on trials to criterion during T-maze acquisition yielded a significant effect of group: F(4,44) F=5.914, p<0.001. Bonferroni post-hoc tests indicated that 50% SAM mice given vehicle reached criterion in significantly fewer trials than SAMP8 mice given 32, 1.6 mg / kg CA or vehicle. Mice given 16 mg / kg CA were not significantly different from 50% SAM given vehicle or SAMP8 mice given vehicle. An ANOVA on trials to criterion in the T-maze retention test yielded a significant effect of treatment F(4,44)=4.04, p<0.007. Dunnett's post-hoc tests indicated that mice given 16 mg / kg CA reached criterion in significantly fewer trials than SAMP8 mice given vehicle.

[0060] object recognition A one-way ANOVA on the time spent investigating the novel object was not significant: F(4,44)=2.249; p<0.08.

[0061] Lever press A two-way repeated measures ANOVA on the assessment of the number of rewarded lever presses for treatment and day yielded significant effects for treatment, F(4,253)=6.74, p<0.001, and day, F(5,253)=7.53, p<0.001. The treatment × day interaction was not significant: F(20,253)=1.83. Tukey's post hoc analysis showed that on days 3, 4, 5, and 6, mice receiving 32 mg / kg and 16 mg / kg CA received significantly more rewards than mice receiving vehicle.

[0062] Triglyceride levels One-way ANOVA on triglyceride levels showed a significant effect after administration of CA10: F(4,47)=3.11, p<0.02. Tukey's post-hoc test showed that the mean ± SD was 50%. SAM mice were shown to have significantly higher triglyceride levels than SAMP8 mice fed 1.6 mg / kg CA. There were no other significant differences between any of the other groups.

[0063] Brain oxidative stress Mann-Whitney U tests showed that CA10 significantly decreased HNE in the cortex at 32 mg / kg CA, 3-NT in the cortex at 16 mg / kg CA, and significantly increased protein carbonyls at 16 and 32 mg / kg CA compared to vehicle-treated controls of SAMP8 (see Figure 6). had no effect on HNE, 3-NT, or protein carbonyls in the striatum (see FIG. 7). Finally, CA10 significantly reduced 3-NT and protein carbonyls in the hippocampus at 16 mg / kg CA compared to vehicle-treated SAMP8 controls (see FIG. 8).

[0064] Spearmint Extract containing 5% RA (RA5): The results discussed below are shown in FIG.

[0065] Behavioral testing T mazeA one-way ANOVA on trials to criterion in the T-maze acquisition test showed a significant effect of treatment, F(4,52) 6.38, p<0.001. Bonferroni post hoc tests showed that SAMP8 mice given 32 and 16 mg / kg RA reached criterion in significantly fewer trials than mice given vehicle. A one-way ANOVA on trials to criterion in the T-maze retention test showed a significant effect of treatment, F(4,50)=12.77, p<0.001. Bonferroni post hoc analyses showed that SAMP8 mice given 32, 16, and 1.6 mg / kg RA reached criterion in significantly fewer trials than SAMP8 mice given vehicle. Mice given 32, 16, and 1.6 mg / kg RA were not significantly different from 50% SAM control mice.

[0066] object recognition A one-way ANOVA on time spent investigating the novel object yielded a significant effect of treatment, F(4,47)=2.79; p<0.03. Dunnett's post-hoc test showed that mice receiving 32 and 16 mg / kg RA spent significantly more time investigating the novel object than SAMP8 mice receiving vehicle.

[0067] Lever press A two-way repeated measures ANOVA on the assessment of the number of rewarded lever presses for treatment and day yielded significant effects for treatment F(4,257) = 6.18, p < 0.001 and day F(5,257) = 40.98, p < 0.001. The treatment × day interaction was not significant: F(20,257) = 2.44. Tukey's post hoc analysis showed that there were no significant differences between SAMP8 mice given RA5 and SAMP8 mice given vehicle.

[0068] Triglyceride levels One-way ANOVA on triglyceride levels after treatment yielded a significant effect: F(4,44)=4.06, p<0.006. Tukey's post-hoc analysis showed that 50% SAM control mice had significantly higher triglyceride levels compared to SAMP8 mice given vehicle and SAMP8 mice given 1.6 mg / kg RA. There were no differences between the other groups.

[0069] Brain oxidative stress Mann-Whitney U tests showed that RA5 significantly reduced HNE in the cortex of mice given 16 and 32 mg / kg RA, and reduced 3-NT at 32 mg / kg RA. RA5 had no effect on protein carbonyls in the cortex (see Figure 10). In addition, RA5 had no effect on HNE or 3-NT in the striatum. Mice given RA5 had significantly higher protein carbonyl levels in the striatum compared to vehicle-treated control SAMP8 mice at 1.6 and 16 mg / kg RA (see Figure 11). RA5 significantly reduced 3-NT and protein carbonyl levels at 16 mg / kg RA in the hippocampus (see Figure 12).

[0070] Essay : In this study, extracts containing CA or RA improved learning and memory. Overall, CA had effects on both declarative and procedural memory. The T-maze and object recognition both tested declarative memory, while manipulative lever pressing tested procedural learning and memory. We found that rosemary extracts containing 60% CA improved both declarative and procedural learning and memory. Rosemary extracts with 10% CA improved both declarative memory and procedural learning and memory as assessed by the lever pressing task. Spearmint extracts with 5% RA improved both declarative learning and memory. None of the compounds had effects on body weight.

[0071] Both CA and RA acted in a dose-response manner. This is not surprising since most memory-enhancing compounds exhibit subthreshold enhancing effects, a phenomenon in which there is an optimal dose for memory enhancement below which the compound is ineffective and above which the compound produces impairment, thus resulting in a so-called "inverted U" shaped dose-response curve. This has been previously reported in the water maze test with rosmarinic acid and in its anxiolytic potential when tested in the elevated plus maze [Park, et al., 2010; Butterfield, et al., 1997]. Our previous study found cognitive enhancing potential using "stress" conditions in a normal healthy non-aged mouse model. Rosmarinic acid protected against Aβ toxicity in Aβ-injected mice [Pereira P, Tysca D, Oliveria P, da Silva Brum LF, Picada JN, Ardenghi P (2005) Neurobehavioral and genotoxic aspects of rosmarinic acid. Pharmacol Res 52, 199-203]. In this study, we found that rosmarinic acid prevented memory loss in a mouse model that naturally overproduces Aβ.

[0072] The findings in this study are similar to previous findings following antioxidant supplementation in SAMP8 mice. Alpha-lipoic acid improved learning and memory in 12-month-old SAMP8 mice after only one week of treatment, and in 18-month-old SAMP8 mice after just two weeks [Farr, et al., 2003; Pereira, et al., 2005]. The antioxidant docosahexaenoic acid also improved learning and memory in SAMP8 mice [Alkam, et al., 2007]. Supplementation with mulberry extract rich in alpha-lipoic acid resulted in improved avoidance learning and memory, reduced cholesterol and reduced indices of oxidative stress in SAMP8 mice [Farr SA, Price TO, Banks WA, Ercal N, Morley JE (2012) Effect of Alpha-Lipoic Acid on Memory, Oxidation and Lifespan in SAMP8 Mice. J Alzheimer's Dis (In Press)]. In addition, these findings suggest that some of the above mentioned molecules / extracts may have cognitive benefits. Follow-up human clinical trials on , which show positive effects, are encouraging as they demonstrate the predictive and translatable nature of this model from rodents to humans [Petursdottir, et al., 2008; Shih PH, Chan YC, Liao JW, Wang MF, Yen GC (2010) Antioxidant and cognitive effects promotion of anthocyanin-rich mulberry (Morus atropurpurea L.) on senescence-accelerated mice and prevention of Alzheimer's disease. J Nutr Biochem 21, 598-605].

[0073] Protein and lipid oxidation occurs with age in the brains of SAMP8 mice [Butterfield, et al., 1997; Poon, et al., 2005; Pereira, et al., 2005]. Proteins and lipids play important roles in the normal structure and function of cells [Butterfield DA, Reed T, Sultana R (2011) Roles of 3-nitrotyrosine- and 4-hydroxynonenal-modified brain proteins in the progression and pathogensis of Alzheimer's disease. Free Radic Res 45, 59-72]. Abnormal cellular function and ultimately cell death can occur through oxidative modification of proteins in cells. [Lee HC, Wei YH (2012) Mitochondria and aging. Adv Exp Med Biol 942, 311-327]. In this study, we used a highly sensitive immunochemical method to detect antioxidants We determined whether rosemary extract containing 10% CA or spearmint extract containing 5% RA had any effect on protein carbonyl levels. Our results showed a reduction in protein oxidation in the hippocampus (after administration of rosemary extract containing 10% CA or spearmint extract containing 5% RA) and lipid oxidation in the cortex (after administration of all extracts). The hippocampus and cortex have been shown to be important regions for learning and memory in the T-maze [Farr SA, Banks WA, La Scola ME, Flood JF, Morley JE (2000) Permanent and temporary inactivation of the hippocampus impairs T-maze footshock avoidance acquisition and retention . Brain Res 872, 242-249; Farr SA, Uezu K, Creonte TA, Flood JF, Morley JE (2000) Modulation of memory processing in the cingulate cortex of mice. Pharmacol Biochem Behav 65, 363-368]. In addition Studies have shown that the hippocampus is important for memory in object recognition using a 24-hour retention delay [Hammond, et al., 2004]. Our data suggest that the use of carnosic acid We show that both rosemary extract and spearmint extract containing rosmarinic acid can help reverse the oxidative changes that cause aging and cognitive decline in SAMP8 mice.

[0074] In this study, we orally supplemented rosemary extract containing the antioxidants carnosic acid and spearmint extract containing rosmarinic acid to determine whether they have beneficial effects on learning and memory in the SAMP8 mouse model of cognitive impairment. The extracts resulted in an inverted U-shaped dose-response curve as exhibited by most memory-enhancing compounds, and effective doses of both extracts were identified. The extracts tested showed beneficial effects on both declarative and procedural memory, both of which have been reported to be affected by aging and disease. To our knowledge, this is the first study to show prevention of cognitive decline following administration of either rosemary extract containing carnosic acid or spearmint extract containing rosmarinic acid in a mouse model of accelerated age-related cognitive decline. These findings suggest that rosemary extract containing carnosic acid and spearmint extract containing rosmarinic acid are potential treatments for age-related cognitive decline.

[0075] All three extracts improved learning and memory in at least two of the three tasks. Rosemary extract (containing CA) had effects on both declarative and procedural memory. T-maze and object recognition both tested declarative memory. Manipulative lever pressing tests procedural learning and memory. Spearmint extract (containing RA) improved all declarative memory tasks tested (T-maze acquisition and learning and object recognition) but had no effect on procedural manipulation learning and memory. None of the compounds had an effect on body weight. The use of CA and / or RA is now directly linked to cognitive outcome measures. Rosemary supplements that discuss energy and cognition exist, but none are supported by scientific studies and none directly link causation to carnosic acid. This is shown in our study, as increasing the carnosic acid content increased the effectiveness of the extract.

[0076] No cognitive behavioral outcome measures related to learning or memory were linked to CA, and no learning outcome measures were linked to RA.

[0077] These results are surprising because of the nature of how two completely different molecules with different chemical structures and polarities can cross the blood-brain barrier and penetrate different regions of the brain. Once these compounds penetrate various regions of the brain, they result in significant performance changes in cognitive performance. Prior to this study, these molecules were only thought to have a general non-specific antioxidant effect in the brain. Previously, these molecules had not been associated with specific cognitive effects resulting from different regions of the brain. For the first time, this study demonstrates that they can affect behavioral cognitive performance outcomes. In addition, the two compounds are able to link specific brain regions that are responsible for those cognitive outcomes. These results demonstrate the specificity of the ability of these molecules to act independently of the general non-specific antioxidant effects attributed to other molecules.

[0078] Example 2 90-day toxicity study of carnosic acid The objective of this study was to evaluate the toxicity of the test article: a suspension (in sunflower oil) of rosemary extract containing approximately 10% carnosic acid (active ingredient) when administered by oral gavage once or twice daily for at least 90 days to rats.

[0079] Male and female Hsd:Sprague Dawley SD rats were assigned to groups and doses were administered as shown in the table below. Animals were dosed by oral gavage.

[0080] [Table 1]

[0081] Toxicity assessment was based on mortality, clinical signs, body weight, body weight change, food consumption, ophthalmologic examination, functional observation battery (FOB), and clinical and anatomic pathology.

[0082] Oral administration of rosemary extract containing approximately 10% carnosic acid in sunflower oil to Sprague Dawley rats was well tolerated at dose levels below 180 mg / kg / day. The frequency of administration (SID or BID) in animals given 130 mg / kg / day did not appear to have any substantial difference to the nature or magnitude of the effects. Doses greater than 65 mg / kg / day resulted in non-adverse test-article related effects on hematology and clinical chemistry parameters; organ weight changes; and microscopic findings in the liver, kidney, non-glandular stomach, colon, and cecum. Based on this pattern of findings, 180 mg carnosic acid / kg / day is considered to be the maximum no observed adverse effect level (NOAEL) when given orally for 90 consecutive days. Using the NOAEL of 180 mg / kg / day of carnosic acid and a CA concentration in the extract of exactly 11.55%, the NOAEL would translate to 1,558 mg / kg / day of rosemary extract. Using a 100-fold safety factor, this gives a human equivalent dose of 15.58 mg / kg / day, or 1.0 mg / kg per day for a 70 kg human. This would be converted to 1091 mg.

[0083] Example 3 A 90-day toxicity study of rosmarinic acid The objective of this study was to evaluate the toxicity of the test article: a dry extract of rosemary containing approximately 15% (w / w) rosmarinic acid (active ingredient) dissolved in distilled water, when administered once daily by oral gavage to rats for at least 90 days.

[0084] Male and female Hsd:Sprague Dawley SD rats were assigned to groups and doses were administered as shown in the table below. Animals were dosed by oral gavage.

[0085] [Table 2]

[0086] Toxicity assessment was based on mortality, clinical signs, body weight, body weight change, food consumption, ophthalmologic examination, functional observation battery (FOB), and clinical and anatomic pathology.

[0087] Daily oral administration of dry spearmint extract (containing approximately 15% RA as the active ingredient) to male and female Sprague Dawley rats at doses up to 300 mg RA / kg bw / day for 90 days was well tolerated. No treatment-related clinical signs or adverse effects were observed in any of the parameters studied, including body weight, food consumption, neurological parameters, hematology, clinical chemistry, gross and histopathology. Weight increases were observed in the pituitary and thyroid lands, but they were within historical norms for age and strain, and had no corresponding histopathological changes. Thus, the "No Observed Adverse Effect Level (NOAEL)" for the test item under the test conditions and doses used was found to be 300 mg RA / kg bw / day (corresponding to 1948.2 mg / kg bw / day of dry spearmint extract). Using a 100-fold safety factor, this corresponds to a NOAEL of dry spearmint extract for the human equivalent dose of 19.48 mg / kg / day, which would be equivalent to 1363.74 mg / day for a 70 kg human.

[0088] The foregoing description and drawings include illustrative embodiments of the present invention. The foregoing embodiments and methods described herein may be varied based on the ability, experience, and preferences of those of ordinary skill in the art. The mere recitation of the steps of the method in a particular order does not constitute any limitation to the order of the steps of the method. The foregoing description and drawings merely explain and illustrate the present invention, and the present invention is not limited thereto, except as the claims are so limited. Those skilled in the art having the foregoing disclosure will be able to make modifications and variations therein without departing from the scope of the present invention. [1] A study to improve cognitive health and function in mammals involving the oral administration of an effective amount of an extract from a plant of the Lamiaceae family. and / or methods for enhancing, improving, or maintaining function. [2] The method according to [1], wherein the mammal is selected from the list consisting of humans and companion animals. [3] The method according to [2], wherein the effective amount of the extract is 0.01 to 50 mg / kg / day. [4] The method according to [1], wherein the extract contains carnosic acid. [5] The method of [4], wherein cognitive health and / or cognitive function refers to procedural learning (due to improved concentration, attention, and vigilance) and memory, and wherein memory is selected from the group consisting of reference memory, recognition memory, short-term memory, long-term memory and associative memory, which have both declarative and procedural components. [6] The method described in [4], wherein cognitive health and / or cognitive function refer to executive function. [7] The method according to [2], wherein the extract contains rosmarinic acid. [8] The method according to [7], wherein the effective amount of the extract is 0.01 to 50 mg / kg / day. [9] The method according to [7], wherein rosmarinic acid is associated with cognitive health and / or cognitive function, such as improved focus, attention, and vigilance, leading to improved declarative learning.

[10] The method according to [7], in which rosmarinic acid is involved in cognitive health and / or cognitive function, referring to both declarative memory, short-term and long-term memory.

[11] The method described in [7], wherein cognitive health and / or cognitive function refer to executive function.

[12] The method of [1], wherein the extract comprises carnosic acid and rosmarinic acid.

[13] The method of

[12] , wherein enhancing, enhancing, and maintaining cognitive health and / or function comprises enhancing, enhancing, and maintaining learning, wherein learning is selected from the group consisting of declarative learning and procedural learning.

[14] The method of

[12] , wherein enhancing, improving, and maintaining cognitive health and / or function includes enhancing, improving, and maintaining memory, wherein memory is selected from the group consisting of reference memory, which has both declarative and procedural components, recognition memory, short-term memory, long-term memory, and associative memory.

[15] The method according to [1], wherein the plant is selected from the group consisting of basil, mint, sage, savory, marjoram, oregano, thyme, lavender, spearmint, and rosemary.

[16] The method according to [4] for the prevention or treatment of cognitive disorders, such as dyslexia, aspraxia, attention deficit hyperactivity disorder, attention deficit disorder, autism, Alzheimer's disease, Parkinson's disease or stroke, or other disorders of executive function.

[17] The method according to [7] for the prevention or treatment of cognitive disorders, such as dyslexia, aspraxia, attention deficit hyperactivity disorder, attention deficit disorder, autism, Alzheimer's disease, Parkinson's disease or stroke, or other disorders of executive function.

[18] The method according to [4] for the prevention or treatment of cognitive disorders or deficits associated with the normal aging process.

[19] The method according to [7] for the prevention or treatment of cognitive disorders or deficits associated with the normal aging process.

[20] The method according to [4] for maintaining normal cognitive function under stressful conditions such as disruptions to circadian rhythms such as those occurring in conditions selected from the list consisting of jet lag, time zone changes, pregnancy, drug therapy, changes in routine and shift work.

[21] The method according to [7] for maintaining normal cognitive function under stressful conditions such as disruptions to circadian rhythms, such as those occurring in conditions selected from the list consisting of time zone changes, pregnancy, drug therapy, changes in routine work and shift work.

[22] A method for treating or preventing a decline in cognitive health and / or function in a mammal comprising orally administering an effective amount of an extract of a plant of the Lamiaceae family.

Claims

1. 1. An orally administered composition comprising 1.6 to 32 mg / kg / day of an aqueous extract of spearmint plant, said aqueous extract comprising about 5 to 15% by weight of rosmarinic acid, said extract being administered for at least 90 days to enhance, improve or maintain cognitive health and / or function in a mammal not suffering from dementia and experiencing normal age-related cognitive changes.

2. The composition of claim 1, wherein said mammal is selected from the list consisting of humans and companion animals.

3. The composition of claim 1, wherein cognitive health and / or cognitive function refers to procedural learning through improved focus, attention, and vigilance.

4. 2. The composition of claim 1, wherein cognitive health and / or cognitive function refers to both declarative memory, short-term and long-term memory.

5. The composition of claim 1 , wherein cognitive health and / or cognitive function refers to executive function.

6. 2. The composition of claim 1, wherein enhancing, improving, and maintaining cognitive health and / or function comprises enhancing, improving, and maintaining learning, wherein learning is selected from the group consisting of declarative learning and procedural learning.

7. 2. The composition of claim 1, wherein enhancing, improving, and maintaining cognitive health and / or function includes enhancing, improving, and maintaining memory, wherein memory is selected from the group consisting of reference memory, which has both declarative and procedural components, recognition memory, short-term memory, long-term memory, and associative memory.

8. The composition of claim 1 for alleviating symptoms of cognitive impairment or deficits associated with the normal aging process.

9. The composition of claim 1 for alleviating symptoms of cognitive impairment or deficits associated with the normal aging process.

10. 2. The composition of claim 1 for maintaining normal cognitive function under stressful conditions such as disruptions to circadian rhythms such as those occurring in conditions selected from the list consisting of jet lag, time zone changes, pregnancy, medication, changes in routine work and shift work.

11. 2. The composition of claim 1 for maintaining normal cognitive function under stressful conditions such as disruptions to circadian rhythms such as those occurring in conditions selected from the list consisting of time zone changes, pregnancy, drug therapy, changes in routine work and shift work.

12. 1. An orally administered composition comprising 1.6-32 mg / kg / day of an extract of spearmint plant, said extract comprising about 5-15% by weight rosmarinic acid, said extract being administered for at least 90 days for treating or preventing decline in cognitive health and / or function in a mammal not suffering from dementia and experiencing normal age-related cognitive changes.

13. The composition of claim 12, wherein said mammal is selected from the list consisting of humans and companion animals.

14. The composition of claim 12, wherein the extract contains at least 5% by weight of rosmarinic acid.

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