Composition of Withania somnifera to improve memory and cognition.

Abstract

The present invention relates to a medicinal composition derived from withania somnifera extract comprising withanolides and sominone wherein said sominone in the extract is standardized in the range of 0.1-30%. Withanolides in the said composition are in the range of 10-60%. It is further comprising withanolide glycosides and withaferin A. Medicinal composition along with pharmaceutically acceptable excipients are formulated into a dosage form. It also discloses a method of preparation of said medicinal composition. Said composition is used to improve memory and cognition when administered to subjects in the appropriate dosage. Said composition is capable of enhancing the bioavailability of its constituents.

Description

1 / 60 Composition of Withania somnifera to improve memory and cognition. Field of Invention

[001] The present invention relates to a medicinal composition derived from Withania somnifera extract comprising withanolides and sominone. The composition further comprises withanolide glycosides and withapherin A. Said composition is used to improve memory and cognition when administered to individuals at the appropriate dosage. Fundamentals

[002] Memory is a fundamental mental process, and without memory, we are incapable of anything beyond simple reflexes and stereotyped behavior. Stress, anxiety, or depression can cause forgetfulness, confusion, difficulty concentrating, and other problems that hinder daily activities. Long-term potentiation (LTP), a groundbreaking discovery made in 1966, suggested that memories may be encoded in the potency of synaptic signals between neurons. As a result, memory began to be understood as a neurochemical phenomenon. Recent advances in science and technology indicate that the notion of memory should be expanded and unified. Thus, many people now believe that cognition is also extended, meaning that memory can be stored elsewhere. Petition 870250098597, dated 10 / 28 / 2025, page 9 / 74 2 / 60 that is not the brain.

[003] Modern psychology defines memory as the ability to encode, store, and retrieve information. Three main types of memory have been identified by psychologists: sensory, short-term, and long-term. Each of these types of memory has unique properties. The term sensory memory describes the ability to recall details of the senses. Information processed quickly is called short-term memory. This processing is carried out in working memory (WM). The central executive (attention control), the visuospatial sketchpad (creates and maintains a visuospatial representation), phonological temporary storage (stores and consolidates new words), and episodic temporary storage are the four components of working memory that process, store, and integrate information from different sources. It is possible to retain information in long-term memory for a long time.A conscious (explicit memory) or unconscious retrieval of this knowledge is possible (implicit memory). Both episodic memory (time-related experiences) and semantic memory (concepts and meanings) are components of explicit memory. Associative memory (classical and operant conditioning), non-associative memory (sensitization and habituation), procedural memory. Petition 870250098597, dated 10 / 28 / 2025, page 10 / 74 3 / 60 (motor and executive skills) and priming (a primary stimulus influencing a secondary one) are all components of implicit memory.

[004] With advancing age, cognition and memory generally deteriorate. Although a wide variation in cognitive aging can be observed even in healthy individuals, it may be linked to chronic somatic and neurodegenerative disorders. Mild cognitive impairment (MCI) is a neurocognitive disorder involving cognitive impairments beyond those expected based on an individual's age and education, but which are not significant enough to interfere with instrumental activities of daily living. According to a recent 10-year study, cognitive impairment often begins between the ages of 45 and 55. Mild cognitive impairment is classified into two subtypes: amnestic and non-amnestic. Amnestic mild cognitive impairment is a clinically significant memory impairment that does not meet the criteria for dementia.Non-amnestic mild cognitive impairment is characterized by a subtle decline in functions unrelated to memory, affecting attention, language use, or visuospatial skills.

[005] Mild cognitive impairment (MCI) can be caused by changes in the brain triggered during Petition 870250098597, dated 10 / 28 / 2025, page 11 / 74 4 / 60 the early stages of Alzheimer's disease or other forms of dementia.

[006] For thousands of years, traditional alternative medicine systems such as Ayurveda and Traditional Chinese Medicine (TCM) have prescribed herbal treatments to improve memory and cognition.

[007] Ashwagandha, also known as Withania somnifera (WS) (L.) Dunal, is a popular medicine categorized as an adaptogen or a Rasayana in the Indian Ayurvedic pharmaceutical system. In Ayurveda, the roots of W. somnifera are used to prepare many herbal medicines. The biologically active chemical constituents of Withania somnifera are alkaloids (isopelletiurine, anapherine), steroidal lactones (withanolides, withapherines), saponins containing an additional acyl group (sitoindoside VII and VIII), and withanolides with glucose at carbon 27 (sitoindoside XI and X). Other plant constituents of the genus Withania, namely fatty acids and volatile compounds, have also been reported.

[008] Several constituents of Withania somnifera and their activities are known. But a medicinal composition derived from Withania somnifera extract comprising withanolides and sominone is not disclosed in any of the data of the art. Furthermore, this composition also contains withanolide glycosides and withapherin A. It has been found that Petition 870250098597, dated 10 / 28 / 2025, page 12 / 74 5 / 60 This compound improves memory and cognition when administered to individuals at the appropriate dosage. Summary

[009] Memory is the cornerstone of cognitive function, without which our abilities are reduced to mere reflexes and stereotyped behaviors. Conditions such as stress, anxiety, or depression can lead to forgetfulness, confusion, and difficulty concentrating, which disrupts daily activities. A medicinal composition derived from Withania somnifera extract, comprising withanolides and sominone, has been formulated with the aim of improving memory function.

[0010] This composition is used to improve memory and cognition when administered to individuals at the appropriate dosage. The composition additionally comprises withanolide glycosides and withapherin A.

[0011] Thus, a medicinal composition derived from Withania somnifera extract is disclosed, comprising withanolides and sominone, wherein said sominone in the extract is standardized in the range of 0.1-30%. In said medicinal composition, the withanolides are in the range of 10-60%. Said composition further comprises withanolide glycosides and withapherin A, preferably in the range of 10-25% and 2-10%, respectively. According to one embodiment of the invention, said medicinal composition comprises not less than 2% sominone, not less than 20% Petition 870250098597, dated 10 / 28 / 2025, page 13 / 74 6 / 60 of withanolide glycosides, not less than 6% withapherin A and not less than 30% withanolides. In another embodiment, said medicinal composition comprises 2-3% sominone, 20-25% withanolide glycosides, 68% withapherin A and 30-40% withanolides. In another embodiment of the invention, said medicinal composition comprises 2.3% sominone, 23.5% withanolide glycosides and 6.5% withapherin A, and 34.9% withanolides.

[0012] It has been found that the medicinal composition, when administered to individuals, improves the bioavailability of its constituents.

[0013] The invention further relates to a dosage form comprising a medicinal composition derived from Withania somnifera extract with withanolides and sominone, wherein said sominone in the extract is standardized in the range of 0.130%. Said dosage form has withanolides in the range of 10-60%. It further comprises withanolide glycosides and withapherin A, preferably in the range of 10-25% and 2-10%, respectively. Said medicinal composition comprises 2-3% sominone, 20-25% withanolide glycosides, 6-8% withapherin A and 30-40% withanolides. Said medicinal composition comprises not less than 2% sominone, not less than 20% withanolide glycosides, not less than 6% of Petition 870250098597, dated 10 / 28 / 2025, page 14 / 74 7 / 60 withaferin A and not less than 30% withanolides. In one embodiment of the invention, said dosage form comprises 2.3% sominone, 23.5% withanolide glycosides and 6.5% withaferin A, and 34.9% withanolides. The dosage form comprising said medicinal composition is used to improve the bioavailability of its constituents.

[0014] The invention also relates to a method of preparing a medicinal composition derived from Withania somnifera comprising: a) Clean the parts of the Ashwagandha plant (roots and leaves); (b) dry the cleaned parts of the plant and spray them; (c) extract the powdered parts using a polar solvent; (d) collect the supernatant after extraction and then concentrate it; (e) subject the said concentrate to liquid-liquid extraction using a non-polar solvent; (f) separate the aqueous and solvent portions after liquid-liquid extraction; (g) subject the aqueous part of step (f) to column purification using water and methanol; (h) collect the water and methanol eluate and set aside; i) subject the solvent portion collected in step f) to purification by column elution with hexane-methanol in Petition 870250098597, dated 10 / 28 / 2025, page 15 / 74 8 / 60 different ratios and collect the different fractions; and (j) mix the methanol eluate from step (h) and one of the fractions containing a higher composition of Ashwagandha, resulting in the said composition.

[0015] The mixing ratio of the methanol eluate from step (h) and one of the fractions containing a higher Ashwagandha composition from step (i) varies from 20:1 to 2:1 and preferably 9:1.

[0016] The aforementioned dosage form comprising the above-mentioned medicinal composition is used for: 1. To improve learning and behavioral activity; 2. Improve immediate memory, general memory, working memory, and visuospatial memory; 3. Improve verbal episodic memory; 4. Improve verbal learning and memory skills; 5. Improve visual memory and recognition skills; 6. Improve working memory and cognitive processing skills; 7. Improve visuospatial working memory and attention skills by improving spatial amplitude scores; 8. Improve visual memory and skills Petition 870250098597, dated 10 / 28 / 2025, page 16 / 74 9 / 60 visuospatial; 9. improve the individual's spatial visualization skills as assessed by an increase in the score on the Shepard Mental Rotation Task; and 10. Improve cognitive impairment by improving scores on the Montreal Cognitive Assessment (MoCA) and the Mini-Mental State Examination (MMSE).

[0017] The medicinal composition of the invention is made in dosage forms selected from capsules, tablets, mini-tablets, granules, sachets, powder, paste, infusion, injection, ampoule, solution, suspension, emulsion, pills or cream. The dosage for administration to a mammal, especially humans, ranges from about 30 mg to about 2000 mg per day. The dosage form further comprises pharmaceutically acceptable excipients selected from the group comprising diluents, binders, wetting agents, disintegrants and lubricants and mixtures thereof. Brief Description of the Drawings

[0018] These and other attributes, aspects and advantages of the present invention will be better understood when the detailed description is read together with the following attached drawing.

[0019] Figure 1 represents the Montreal Cognitive Assessment (MoCA) score after administration of Ashwagandha extract and placebo. Petition 870250098597, dated 10 / 28 / 2025, page 17 / 74 10 / 60

[0020] Figure 2 represents the Mini-Mental State Examination (MMSE) score after administration of Ashwagandha extract and placebo.

[0021] Figure 3 represents the score of the Shepard Mental Rotation Task after administration of Ashwagandha extract and Placebo.

[0022] Figure 4A represents the Wechsler Memory Scale-III (WMS-III) - Immediate Memory after administration of Placebo.

[0023] Figure 4B represents the Wechsler Memory Scale-III (WMS-III) - Immediate Memory after administration of Ashwagandha extract.

[0024] Figure 5A represents the Wechsler Memory Scale-III (WMS-III) - General Memory after administration of Placebo.

[0025] Figure 5B represents the Wechsler Memory Scale-III (WMS-III) - General Memory after administration of Ashwagandha extract.

[0026] Figure 6A represents the Wechsler Memory Scale-III (WMS-III) - Working Memory after administration of Placebo.

[0027] Figure 6B represents the Wechsler Memory Scale-III (WMS-III) - Working Memory after administration of Ashwagandha extract.

[0028] Figure 7A represents the Memory Scale of Petition 870250098597, dated 10 / 28 / 2025, page 18 / 74 11 / 60 Wechsler MS-III (WMS-III) - Visuospatial processing and response after administration of placebo.

[0029] Figure 7B represents the Wechsler Memory Scale-III (WMS-III) - Visuospatial processing and response after administration of Ashwagandha extract. Detailed Description

[0030] The invention is directed to a medicinal composition obtained from Withania somnifera (Ashwagandha) and its use to improve memory and cognition. The composition of the Withania somnifera extract comprises withanolides and sominone. The withanolides in the composition of the Withania somnifera extract are the sum of withanolide glycosides and withanolide aglycones. The composition of the Withania somnifera extract comprises 10-60% withanolides, and the sominone in the extract is standardized in the range of 0.1-30%. The composition of the Withania somnifera extract comprises withanolides and sominone, and said sominone in the extract is standardized to at least 2%. The composition further comprises withanolide glycosides and withaferin A. The invention provides a process for preparing the Ashwagandha extract.

[0031] The words “Withania somnifera” and “Ashwagandha” mean the same thing and may be used interchangeably throughout the descriptive report. The term Petition 870250098597, dated 10 / 28 / 2025, page 19 / 74 12 / 60 "Withania" refers to plants in the group, but is not limited to, Acnistus, Datura, Deprea, Dunalis, Iochroma, Jaborosa, Lycium, Nicandra, Physalis, Salpichroa, Tubocapsicum, Discopodium, Trechonaetes, Withania, Witheringia, and any plant of the genus Withania or a plant containing withanolides.

[0032] As used in this document, the term individual refers to the target of the administration, whether it is a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the individual for the methods disclosed in this document may be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not indicate a specific age range or sex. Thus, adult and newborn individuals, whether male or female, should be covered unless otherwise specified.

[0033] Ashwagandha plant material contains a negligible level of sominone. Sominone, an aglycone of withanoside IV, has been identified as an active metabolite following oral administration of withanoside IV. Sominone can also be synthetically prepared from dehydroepiandrosterone based on an RCM strategy. Sominone is an important metabolite of withanosides, but there is no report on the standardization of Ashwagandha memory-enhancing formulations with sominone as a marker. From Petition 870250098597, dated 10 / 28 / 2025, page 20 / 74 13 / 60 According to the present invention, a medicinal composition derived from Ashwagandha extract comprises withanolides and sominone. The composition further comprises withanolide glycosides and withapherin A. The composition, when administered to individuals, improves memory and cognitive ability.

[0034] The medicinal composition derived from Withania somnifera extract comprises 1% to 20% sominone, 10% to 25% withanolide glycoside, 2% to 10% withapherin A and 10-40% withanolides.

[0035] The aforementioned medicinal composition derived from Withania somnifera extract preferably comprises 10-60% withanolides, 0.1% to 30% sominone, 10% to 25% withanolide glycoside and 2% to 10% withapherin A.

[0036] In another embodiment, the said medicinal composition comprises not less than 2% sominone, not less than 20% withanolide glycosides, not less than 6% withapherin A and not less than 30% withanolides. In another embodiment of the invention, the medicinal composition derived from Withania somnifera extract comprises 2-3% sominone, 20-25% withanolide glycosides, 6-8% withapherin A and 30-40% withanolides.

[0037] In another embodiment of the invention, the medicinal composition derived from Withania somnifera extract Petition 870250098597, dated 10 / 28 / 2025, page 21 / 74 14 / 60 comprises 2.3% sominone, 13.5% withanolide glycosides and 5.4% withapherin A, withanolides 20.5%.

[0038] In yet another embodiment, the medicinal composition derived from Withania somnifera extract comprises 2.3% sominone, 23.5% withanolide glycosides, 6.5% withapherin A and 34.9% withanolides

[0039] The present invention also provides an extraction process for Ashwagandha comprising withanolides and sominone wherein the sominone in the extract is standardized to contain at least 2% sominone.

[0040] The present invention also provides a process for extracting the Ashwagandha composition with withanolides and at least 2% sominone, comprising the following steps: (a) Clean the parts of the Ashwagandha plant (roots and leaves). (b) Dry the cleaned parts of the plant and spray them. (c) Extract the powdered parts using a polar solvent. (d) Collect the supernatant after extraction and then concentrate. (e) Subject the said concentrate to liquid-liquid extraction using a non-polar solvent. (f) Separate the aqueous and solvent portions after liquid-liquid extraction. g) Subject the aqueous part of step (f) to purification. Petition 870250098597, dated 10 / 28 / 2025, page 22 / 74 15 / 60 in a column using water and methanol. (h) Collect the water and methanol eluate and set aside. (i) Subject the portion of solvent collected in step (f) to column purification by elution with hexane-methanol in different ratios and collect the different fractions. (j) Mix the methanol eluate from step (h) and one of the fractions containing the highest percentage of active ingredients obtained in step (i) to form the Ashwagandha composition.

[0041] The mixing ratio of the methanol eluate and the fraction containing the higher percentage of active ingredients varies from 20:1 to 2:1. The preferred mixing ratio is 9:1.

[0042] The adsorption column can be silica, SP700, HP20, HP2MGL, SA10A, WA10, CRB03, CRB05, CR20, XAD 7HP, FP66, SK1B or SP825L.

[0043] The polar solvents used to extract Ashwagandha are selected from water, lower alcohols such as methanol, ethanol, acetone, isopropyl alcohol, etc., ketones such as acetone, methyl ethyl ketone, diethyl ketone, ethyl butyl ketone, etc., ethers such as dimethyl ether, diethyl ether, tetrahydrofuran (THF) and their combinations.

[0044] The nonpolar solvents used to extract Ashwagandha are selected from a group of chloroform, hexane, toluene, ethyl acetate, carbon tetrachloride and their combinations. Petition 870250098597, dated 10 / 28 / 2025, page 23 / 74 16 / 60

[0045] The polar solvent used in the above-mentioned process is preferably methanol. The non-polar solvent used in the above-mentioned process is preferably ethyl acetate.

[0046] In another embodiment, Withania somnifera extract, together with pharmaceutically acceptable excipients, is formulated into an oral dosage form.

[0047] Consequently, pharmaceutically acceptable excipients are selected from the group comprising diluents, binders, wetting agents, disintegrants and lubricants and mixtures thereof as delivery systems.

[0048] The medicinal composition derived from Withania somnifera extract is in the selected dosage form from capsule, tablet, mini-tablet, granule, sachet, powder, paste, infusion, injection, ampoule, solution, suspension, emulsion, pills or cream.

[0049] In addition, a dosage form of the medicinal composition derived from Withania somnifera extract is disclosed for administration in a dosage ranging from about 30 mg to about 2000 mg per day to an individual.

[0050] In one embodiment, the said dosage of the medicinal composition derived from Withania somnifera extract comprises 10-60% withanolides and 0.1-30% sominone. According to another embodiment, the said dosage Petition 870250098597, dated 10 / 28 / 2025, page 24 / 74 17 / 60 of the medicinal composition derived from Withania somnifera extract comprises 10-60% withanolides and at least 2% sominone.

[0051] The medicinal composition, when administered to individuals, is capable of improving the bioavailability of its constituents. The aforementioned medicinal composition for improving the bioavailability of constituents comprises Ashwagandha extract with 10-60% withanolides and 0.1-30% sominone. A method for improving the bioavailability of constituents by administering Ashwagandha extract with 10-60% withanolides and 0.1-30% sominone is also disclosed.

[0052] The efficacy of Ashwagandha composition on learning and memory activity (behavioral) is studied in rats. Behavioral testing is performed at the end of treatment using the Elevated Plus Maze Apparatus, Y-Maze Model, Cooks' Pole Climbing Method, and Morris Water Maze Method. Rats treated with Ashwagandha extract showed transfer latency almost similar to the Piracetam control group.

[0053] In a pole climbing test, early escape to the shock-free zone (avoiding the shock) indicates retention of the learned task. Rats treated with Ashwagandha extract showed an impressive retention effect. Petition 870250098597, dated 10 / 28 / 2025, page 25 / 74 It has an 18 / 60 memory effect and is almost identical to the standard medication Piracetam.

[0054] The retention of the learned task, as well as the number of entries into the arm, are observed using the Y-maze in rats. The retention latency is significant in the group treated with Ashwagandha extract. Data on the number of entries into the arm also showed a significant difference for rats treated with Ashwagandha extract.

[0055] The effect on scopolamine-induced amnesia in the Morris Water Maze (MWM) experiment showed that pretreatment with Ashwagandha extract in rats exhibited improvement in scopolamine-induced amnesia. The improving effect of Ashwagandha extract is evident in the significant decrease in escape latency compared to the scopolamine-treated group on day 10. Piracetam also significantly reversed scopolamine-induced amnesia, as evidenced by the decrease in escape latency compared to the scopolamine-treated group.

[0056] The transfer latency of rats in the scopolamine-treated group improved significantly after scopolamine injection. All extracts, as well as the standard drug Piracetam, are able to significantly reverse the amnesic effect of scopolamine. Rats treated with Ashwagandha extract showed a Petition 870250098597, dated 10 / 28 / 2025, page 26 / 74 19 / 60 transfer latency, almost similar to the standard Piracetam group.

[0057] In another modality, the effect of Ashwagandha composition on memory improvement in adults with mild cognitive impairment is studied. The primary outcome measures are the Wechsler Memory Scale (WMS), visuospatial processing and response, and the Shepard Mental Rotation Task. The secondary outcome measures are the Wisconsin Card Sorting Test (WCST), Eriksen Flanker Task, Trail Making Test (Part A), Mackworth Clock Drawing Test, Montreal Cognitive Assessment (MOCA), and Mini-Mental State Examination (MMSE). Wechsler Memory Scale (WMS)

[0058] The WMS-III is an individually administered battery of 11 subtests of learning, memory, and working memory. Six of the subtests are included in the core battery, and five subtests are considered optional supplementary subtests. Only six of the WMS-III subtests are considered primary (Logical Memory I and II, Verbal Paired Associations I and II, Letter-Number Sequencing are presented auditorily, and Faces I and II, Family Figures I and II, and Spatial Amplitude are presented visually), and all must be administered to calculate the WMS-III index scores. The primary subtests may Petition 870250098597, dated 10 / 28 / 2025, page 27 / 74 20 / 60 to be administered over 30-35 minutes. The Information and Orientation, Word Lists I and II, and Mental Control, Digit Span subtests are optional in the auditory presentations, and the Visual Reproduction I and II subtests are considered optional in the visual presentation. None of these optional indices contribute to the WMS-III index scores, but they are used to obtain supplementary information. The Family Pictures subtest is new to the WMS-III and is considered the visual analogue of the Logical Memory subtest. The WMS-III Faces subtest uses a recognition paradigm to assess immediate and delayed visual memory. The Letter and Number Sequencing subtest is new to the WMS-III and is a measure of auditory working memory. The WMS-III provides the calculation of eight primary index scores and four Auditory Process Composite scores. Visuospatial Processing and Response

[0059] Visual-spatial processing is the ability to tell where objects are in space. This includes your own body parts. It also involves being able to tell how far away objects are from you and from each other.

[0060] These skills are usually assessed by copying two-dimensional or three-dimensional figures with paper and pencil. Shepard's Mental Rotation Task Petition 870250098597, dated 10 / 28 / 2025, page 28 / 74 21 / 60

[0061] The experiment specifically tested mental rotation of three-dimensional objects. Each individual was presented with several pairs of three-dimensional, asymmetrical objects with lines or cubes. The experiment was designed to measure how long it would take each individual to determine whether the pair of objects was in fact the same object or two different objects. The increase in the score on Shepard's Mental Rotation Task shows the individual's spatial visualization skills. Wisconsin Card Sorting Test (WCST)

[0062] The Wisconsin Card Sorting Test (WCST) is a neuropsychological test frequently used to measure higher-level cognitive processes such as attention, perseverance, WM, abstract thinking, CF, and set shifting. It is particularly used in clinical settings to measure perseverative behaviors, which refer to an individual's persistence in wrong behavior. Furthermore, to be able to shift categories, one needs to have high intellectual flexibility and concept-forming ability.

[0063] The WCST consists of two packs of cards with four stimulus cards and 64 response cards in each. Each card measures 7*7 cm, and there are various geometric shapes in different columns and numbers. Participants are expected to accurately classify each response card with one of the Petition 870250098597, dated 10 / 28 / 2025, page 29 / 74 22 / 60 four stimulus cards were given feedback (right or wrong) based on a rule. Among the various versions, the 128-card WCST version, developed by Heaton, was used in this study. The test was administered individually, and 12 scores were obtained (Heaton, Chelune, Talley, Kay, and Curtiss, 1993). A reliability study could not be performed due to the nature of the test. Validity studies, on the other hand, were conducted on sick and healthy groups, and the test was shown to be valid for a Turkish sample (Kafadar, 2004). Eriksen's Flanker Task

[0064] Executive functions are a key dimension of cognition and comprise components such as selective attention, response inhibition, and working memory. Different tests have been well established to examine individual executive functions. The flanker task requires selective (spatial) attention and executive control. In this task, irrelevant stimuli must be inhibited to respond to a relevant target stimulus. Eriksen's flanker task is a set of tests to assess selective attention and inhibitory function. In this task, the target is positioned in the center and is flanked by non-target stimuli. The individual is asked to press the left or right arrow key according to the direction of the target. Petition 870250098597, dated 10 / 28 / 2025, page 30 / 74 23 / 60 Trail Formation Test (Part A)

[0065] The Trail Making Test is a neuropsychological test of visual attention and task switching. Assessing an individual's visual attention and task switching skills can provide information about visual search speed, processing speed, and mental flexibility, as well as executive functioning. Part A is a 20.32 x 27.94 cm (8 x 11) page on which the numbers 1 to 25 are scattered in circles. The patient is instructed to draw lines connecting the numbers in order as quickly as possible. TMT A results are reported as the number of seconds required to complete the task; therefore, higher scores reveal greater impairment. Mackworth's Clock Test

[0066] Mackworth's Clock is an experimental device used in the field of experimental psychology to study the effects of long-term surveillance on signal detection. The device has a large black pointer on a large circular background like a clock. The pointer moves in short jumps like the second hand of an analog clock, approximately every second. At infrequent and irregular intervals, the pointer makes a double jump, for example, 12 times every 30 seconds. The task is to detect when the double jumps occur by pressing a button. Petition 870250098597, dated 10 / 28 / 2025, page 31 / 74 24 / 60 Typically, participants in the Mackworth test would perform this task for two hours. The Mackworth Clock was used to establish one of the fundamental findings in the literature on vigilance and sustained attention: the vigilance decay (accuracy of signal detection) decreases markedly after 30 minutes on the task. The test continues to be used today in vigilance research in various forms, including computer-displayed versions. Montreal Cognitive Assessment (MoCA)

[0067] The Montreal Cognitive Assessment (MoCA) is a widely used screening assessment for detecting cognitive impairment. It can help identify those at risk of developing Alzheimer's disease and other forms of dementia. The MoCA contains 30 questions and takes about 10 to 12 minutes to complete. The MoCA test was validated as a highly sensitive tool for early detection of mild cognitive impairment (MCI) in 2000. The MoCA has subsequently been adopted in clinical settings worldwide and is widely used as a scale in academic and non-academic research. The fundamentals of this test include short-term memory, executable performance, attention, focus, and more. MoCA scores range from 0 to 30. A score of 26 or higher is normal. Mini Mental State Examination (MMSE)

[0068] The Mini-Mental State Examination (MMSE) or test of Petition 870250098597, dated 10 / 28 / 2025, page 32 / 74 25 / 60 The Folstein Test is a 30-point questionnaire widely used in clinical and research settings to measure cognitive impairment. It is commonly used in medicine and allied health to screen for dementia. It is also used to estimate the severity and progression of cognitive impairment and to track the course of cognitive changes in an individual over time; thus, making it an effective way to document an individual's response to treatment. Administering the test takes between 5 and 10 minutes and examines functions that include simple questions and problems in several areas: time and place of testing, repetition of word lists, arithmetic such as serial sevens, language use and comprehension, and basic motor skills. Any score of 24 or more (out of 30) indicates normal cognition. Below that, scores may indicate severe (9 points), moderate (10-18 points), or mild (19-23 points) cognitive impairment.

[0069] The improvements observed after administration of the Ashwagandha composition are improved memory in adults with mild cognitive impairment (MCI) and without any impairment of Activities of Daily Living, improved subjective complaint of a memory disorder, reduced memory deficit, improved cognitive functions, improved activities of daily living, and reduced dementia. Other improvements include improved immediate auditory index with Petition 870250098597, dated 10 / 28 / 2025, page 33 / 74 26 / 60 long-term verbal memory and verbal learning, improve immediate visual index with long-term nonverbal memory and nonverbal learning, improve immediate memory index, improve delayed auditory index with delayed verbal memory and verbal learning, improve delayed visual index with delayed nonverbal memory and nonverbal learning, improve auditory recognition delay index with verbal information recognition, improve general memory, improve working memory with auditory processing, executive function of attention / concentration and visual-spatial processing.

[0070] No significant variability in MOCA was observed with placebo treatment during days 30 and 60 compared to baseline. Significant improvement was observed with Ashwagandha treatment during days 30 (percent change = 7.83%) and 60 (percent change = 14.77%) compared to baseline. Day 60 showed significantly higher MOCA (percent change = 6.43%) compared to day 30 with Ashwagandha treatment. The result showed an improvement in cognitive impairment after administration with Ashwagandha extract.

[0071] No significant variability in the MMSE was observed with placebo treatment during days 30 and 60 compared to baseline. Day 60 showed no change. Petition 870250098597, dated 10 / 28 / 2025, page 34 / 74 Significant variability was observed in the 27 / 60 MMSE compared to day 30 with placebo treatment. Significant improvement was observed in the MMSE with Ashwagandha treatment during days 30 (percent change = 9.26%) and 60 (percent change = 19.21%) compared to baseline. The result showed an improvement in cognitive impairment after administration with Ashwagandha extract.

[0072] The placebo group showed no significant change in Shepard Mental Rotation Task scores on days 30 and 60 compared to baseline. Significant improvement was observed with Ashwagandha treatment on day 30 (percent change = 12.22%) compared to baseline. Day 60 showed a significantly higher Shepard Mental Rotation Task score (percent change = 31.67%) compared to baseline with Ashwagandha treatment. Day 60 showed a significantly higher Shepard Mental Rotation Task score (percent change = 17.37%) compared to day 30 with Ashwagandha treatment. The results showed an improvement in the individual's spatial visualization skills after administration with Ashwagandha extract.

[0073] Significant improvement is observed in logical memory I (percentage change - 2.63%) and II (percentage change = 4.96%) with treatment with Ashwagandha during Petition 870250098597, dated 10 / 28 / 2025, page 35 / 74 Day 60 showed significantly higher Logical Memory I (percent change = 6.5%) and II (percent change = 12.8%) compared to baseline with Ashwagandha treatment. Day 60 showed significantly higher Logical Memory I (percent change = 3.7%) and II (percent change = 7.5%) compared to day 30 with Ashwagandha treatment. No significant variability was observed with placebo treatment during days 30 and 60. The results showed an improvement in verbal episodic memory after administration of Ashwagandha extract.

[0074] Significant improvement in Paired Verbal Association I (percent change = 2.64%) and II (percent change = 11.02%) is observed with Ashwagandha treatment during days 30 and 60 compared to baseline. Day 60 showed significantly higher Paired Verbal Association I (percent change = 6.93%) and II (percent change = 19.21%) compared to day 30 with Ashwagandha treatment. No significant variability is observed with placebo treatment during days 30 and 60. The results showed an improvement in verbal learning and memory skills after administration of Ashwagandha extract.

[0075] No significant variability is observed Petition 870250098597, dated 10 / 28 / 2025, page 36 / 74 29 / 60 in FACES I & II with placebo treatment on days 30 and 60 compared to baseline. Significant variability was observed in FACES I (percent change = 0.73%) and II (percent change = 3.7%) with Ashwagandha treatment on day 30 compared to baseline. Day 60 showed significantly higher FACES I (percent change = 7.06%) and II (percent change = 14.36%) compared to baseline with Ashwagandha treatment. Day 60 showed significantly higher FACES I (percent change = 6.28%) and II (percent change = 10.33%) compared to day 30 with Ashwagandha treatment. The results showed an improvement in visual memory and recognition skills after administration of Ashwagandha extract.

[0076] No significant variability in FAMILY PICTURES I and II is observed with placebo treatment during days 30 and 60 compared to baseline. Significant variability in FAMILY PICTURES I and II is observed with Ashwagandha treatment during days 30 (percent change = 0.5%) and 60 (percent change = 0.25%) compared to baseline. Day 60 showed significantly higher (percent change = 9.25%) FAMILY PICTURES I and FAMILY PICTURES II (percent change = 12.3%) compared to baseline. Petition 870250098597, dated 10 / 28 / 2025, page 37 / 74 Day 30 / 60 was the baseline with Ashwagandha treatment. Day 60 showed significantly higher (percentage change = 8.706%) FAMILY PICTURES I and II (percentage change = 12%) compared to day 30 with Ashwagandha treatment. The results showed an improvement in visual memory and recognition skills after administration of the Ashwagandha extract.

[0077] No significant variability in number and letter sequencing was observed with placebo treatment during day 30 (percent change = 0.27%) and day 60 (percent change = 0.53%) compared to baseline. Significant improvement was observed with Ashwagandha treatment during day 30 (percent change = 7.24%) and day 60 (percent change = 16.35%) compared to baseline. Day 60 showed significantly higher NUMBER AND LETTER SEQUENCING compared to day 30 with Ashwagandha treatment (percent change = 8.5%). The results showed an improvement in working memory and cognitive processing skills after administration of Ashwagandha extract.

[0078] No significant variability is observed in SPATIAL AMPLITUDE with placebo treatment during day 30 (percent change = 0.86%) and day 60 (percent change = 1.14%) compared to baseline. Petition 870250098597, dated 10 / 28 / 2025, page 38 / 74 31 / 60 Significant improvement was observed with Ashwagandha treatment on days 30 (percent change = 12.9%) and 60 (percent change = 20.8%) compared to baseline. Day 60 showed significantly greater SPATIAL AMPLITUDE compared to day 30 with Ashwagandha treatment (percent change = 6.9%). The results showed an improvement in visuospatial working memory and attention skills after administration of Ashwagandha extract.

[0079] No significant improvement is observed in VISUAL REPRODUCTION I and II with placebo treatment during day 30 and day 60 compared to baseline. Significant improvement is observed in VISUAL REPRODUCTION I (percent change = 7.24%) and II (percent change = 6.1%) with Ashwagandha treatment during day 30 compared to baseline. Day 60 showed significantly higher VISUAL REPRODUCTION I and II (percent change = 13.9%) (percent change = 14.9%) compared to baseline with Ashwagandha treatment. Day 60 showed significantly higher VISUAL REPRODUCTION I (percent change = 6.25%) and II (percent change = 8.25%) compared to day 30 with Ashwagandha treatment. The results showed an improvement in visual memory and visuospatial skills after administration of the extract of Petition 870250098597, dated 10 / 28 / 2025, page 39 / 74 32 / 60 Ashwagandha. Example 1

[0080] 100 kg of Ashwagandha roots and leaves were collected and cleaned. Cleaned roots and leaves were refluxed with 80% methanol at the boiling point (60-70 °C) of methanol for one hour to obtain the first residue and the first supernatant. The first residue was then extracted twice more with four times the amount of methanol each time. The residue and supernatants were separated. All supernatants were pooled and concentrated in a stirred thin-film evaporator (ATFE) to form a concentrated methanolic extract. (Sample 1) (yield 15%).

[0081] 15 kg of sample 1 were partitioned with ethyl acetate in a liquid-liquid extractor. Aqueous and ethyl acetate layers were formed, and each layer was collected separately. The aqueous portion was passed through the resin column and eluted first with water, followed by methanol. The methanol eluate was concentrated and dried to form Ashwagandha methanol eluate powder. (Sample 2, Yield 40%)

[0082] The ethyl acetate layer was fractionated by passing it through a silica column. The column was eluted with different hexane:methanol ratios. The first fraction (F1) was a 95:5 hexane:methanol eluate (yield of 3.8%). Petition 870250098597, dated 10 / 28 / 2025, page 40 / 74 33 / 60 the second fraction (F2) was a 90:10:methanol hexane eluate (12.6% yield), the third fraction (F3) was an 85:15:methanol hexane eluate (15.4% yield) and the final fraction (F4) was an 80:20:methanol hexane eluate (67.5% yield), fraction F3 was concentrated and dried to form Ashwagandha hexane-methanol eluate powder (sample 3).

[0083] Sample 2 and sample 3 were mixed in a 9:1 ratio to form the Ashwagandha composition (sample 4).

[0084] Sample 4 has 2.3% sominone, 23.5% withanolide glycosides, 6.5% withapherin A and 34.9% withanolides. Example 2

[0085] Efficacy of Ashwagandha extract on learning and memory activity (behavioral) in experimental rats.

[0086] Twenty-four male SD rats were divided into four groups comprising 6 rats in each group. The animals were treated according to the following details: Group 1: Normal control (0.5% Polysorbate 80; 10 ml / kg) Group 2: Untreated control (scopolamine; 0.6 mg / kg i.p.) Group 3: Piracetam - 200 mg / kg + Scopolamine 0.6 mg / kg i.p. Petition 870250098597, dated 10 / 28 / 2025, page 41 / 74 34 / 60 Group 4: Ashwagandha Extract (Sample 4) - 100 mg / kg + Scopolamine 0.6 mg / kg i.p.

[0087] The vehicle / extract / standard was administered daily for 10 consecutive days. Swimming habituation was performed on day 6 of the experiment. Scopolamine was injected 30 minutes after the vehicle / extract / standard in the designated groups. The following behavioral test was performed at the end of treatment using the elevated plus maze apparatus, Y-maze model, and Cooks' pole climbing method. Assessment of memory activity

[0088] The elevated plus maze consists of two open arms and two closed arms (50 x 10 x 40 cm each) raised to a height of 50 cm. Rats were placed individually at the end of an open arm facing away from the central platform, and the time they took to move from there to any of the closed arm's transfer latencies was recorded. If the animal did not enter the closed arms within 90 seconds, it was gently nudged into one of the two closed arms, and the transferred latency was assigned as 90 seconds. Rats were allowed to explore the maze for a further 10 seconds and then returned to their home cage. The test substances and the standard drug were administered daily for 10 consecutive days. On day 10, the standard and standard substances were administered. Petition 870250098597, dated 10 / 28 / 2025, page 42 / 74 35 / 60 tests were administered orally 30 minutes before the latency of transfer of the scopolamine injection was recorded.

[0089] Pole Climbing Test: Rats were trained to climb a pole (shock-free zone) within 30 seconds to avoid a shock. The shock was preceded by a bell ringing for 15 seconds. The animals were trained to climb the pole upon hearing the bell (conditioned avoidance response). At specific intervals, 20 trials were given to each animal, and the average shock avoidance and errors were recorded. The trained animals were tested for conditioned avoidance responses.

[0090] Y-Maze Model - Treatment was initiated (according to the groups mentioned) for the animals from day 1 and continued until day 8. On day 8, after 30 minutes of amnestic agent induction, Y-maze trails were performed and retention was observed on day 8. Briefly, each time the animals were placed directly inside arm B facing away from the center and allowed to move through the apparatus for 8 minutes while being monitored by the tracking system. The test was terminated at the specified time duration. Each entry into the arm (defined as all four paws entering the arm) was scored and recorded. The animals were returned to Petition 870250098597, dated 10 / 28 / 2025, page 43 / 74 36 / 60 cage origin and the number of fecal pellets was counted in the Y-maze and the data were recorded. The Y-maze was cleaned with alcohol between each animal's attempts. Morris Water Maze (MWM) Procedure

[0091] The MWM consists of a circular pool (180 cm in diameter and 60 cm high) with an insignificant internal surface. The circular pool was filled with water (23 ± 1°C) to a height of 40 cm. The pool was divided into four quadrants of equal area. The escape platform was centered in one of the four quadrants. On the day before the training experiments (day 6), the animals were habituated to swimming training for 60 s in the absence of the platform. The animals were treated with vehicle / extract / standard drug for 6 days. On day 7, 30 min after administration of the vehicle / extract / standard drug, scopolamine (0.6 mg / kg, ip) was injected. The training session was initiated 30 min after scopolamine administration. The rats received four test sessions per day for four consecutive days (days 7-10), with a 15 min interval between trials, and escape latencies were recorded.Once the rat located the platform, it was allowed to remain on it for 15 seconds to observe vital cues. If the rat did not locate the platform within 120 seconds, it was guided to the platform. Petition 870250098597, dated 10 / 28 / 2025, p. 44 / 74 37 / 60 and then left on it for 15 s, after which it was removed from the pool. On day 11, the probe test was performed by removing the platform from the pool and a time limit of 60 s was maintained for exploration. The time spent by the animal in the target quadrant was recorded. The longer the time spent in the quadrant, the better the retention capacity as the animal remembers the position of the platform. Results Elevated Cross Labyrinth:

[0092] The transfer latency of the rats in Group 2 improved significantly after scopolamine injection compared to normal control rats. All extracts, as well as the standard drug Piracetam, were able to significantly reverse the amnesic effect of scopolamine. Rats treated with Ashwagandha extract (sample 4) showed transfer latency almost similar to the standard Piracetam group. Table 1: Effect of Test Products on Transfer Latency in Aged Rats Using Elevated Plus Maze Treatment Groups Transfer Latency (sec) Group 1 Normal control 6.17±1.47*** Group 2 Untreated control 35.50±8.29 Group 3 Reference Standard (Piracetam) 7.00±0.89*** Group 4 Ashwagandha Extract 7.50±1.38*** Petition 870250098597, dated 10 / 28 / 2025, page 45 / 74 38 / 60

[0093] Data represented as Mean±SEMR (n = 6). Statistical significance was compared between the untreated control (Group 2) versus other groups (*** P-value < 0.0001). Pole Climbing Test:

[0094] In a pole climbing test, early escape to the shock-free zone (avoiding the shock) indicates retention of the learned task. As detailed in Table 2, the escape latency of rats in Group 2 increased significantly (13.83 s) after scopolamine injection compared to normal control rats (5.17 s). Prior administration of the test product, as well as the standard drug Piracetam, was able to significantly reverse the amnesic effect of scopolamine. Rats treated with Ashwagandha extract (sample 4) showed an impressive memory retention effect and it was almost similar to the standard drug Piracetam. Table 2: Effect of Test Products on Avoidance / Escape Latency of Aged Rats Using the Pole Climbing Test Treatment Groups Avoidance / Escape Latency (sec) Group 1 Normal control 5.17±0.75*** Group 2 Untreated control 13.83±0.75 Group 3 Reference Standard (Piracetam) 6.00±0.89*** Petition 870250098597, dated 10 / 28 / 2025, page 46 / 74 39 / 60 Group 4 Ashwagandha Extract 6.50±1.05***

[0095] Data represented as Mean±SEMR (n = 6). Statistical significances were compared between the untreated control (Group 2) versus other groups (*** P-value < 0.0001). Y-Maze Test

[0096] The retention of the learned task, as well as the number of entries into the arm, were observed using the Y-maze in rats. The untreated control rats (Group 2) showed the longest retention latency and the lowest number of entries into the arm among all rats (Table 3). The retention latency was significant in the group treated with Ashwagandha extract (2.53 min). The data on the number of entries into the arm also showed a significant difference for the rats treated with Ashwagandha extract (4.50). Table 3: Effect of Test Products on Transfer Latency in Aged Rats Using a Y-Maze Treatment Groups Retention Latency (minutes) Number of Arm Entries Group 1 Normal Control 2.94±0.49*** 5.17±0.75*** Group 2 Untreated Control 7.09±0.34 1.83±0.75 Group 3 Reference Standard (Piracetam) 2.07±0.46*** 5.00±0.89*** Group 4 Ashwagandha Extract 2.53±0.32*** 4.50±0.55***

[0097] Data represented as Mean±SEMR (n = 6). As Petition 870250098597, dated 10 / 28 / 2025, page 47 / 74 40 / 60 statistical significances were compared between the untreated control (Group 2) versus other groups (*** P-value < 0.0001). Effect on scopolamine-induced amnesia in MWM:

[0098] It is evident from the results (Table 4) that the animals in the normal control group showed a significant learning and retention response, as indicated by a decrease in escape latency (5.67 s) compared to the animals treated with scopolamine (57.92 s) on day 10. Pretreatment with Ashwagandha extract at 100 mg / kg in rats showed improvement in scopolamine-induced amnesia. The improving effect of the Ashwagandha extract was evident in the significant decrease (p<0.05) in escape latency (7.17 s) compared to the scopolamine-treated group (57.92 s) on day 10. Piracetam also significantly reversed (p<0.05) scopolamine-induced amnesia, as evidenced by the decrease in escape latency (5.25 s) compared to the scopolamine-treated group. Table 4: Time to reach the escape platform on training days (average value in seconds) Treatment Groups Day 7 Day 8 Day 9 Day 10 Group 1 Normal Control 44.50 17.17 7.58 5.67 Group 2 Untreated Control 120.00 110.50 93.17 57.92 Group 3 Reference Standard 74.25 47.33 9.83 5.25 Petition 870250098597, dated 10 / 28 / 2025, page 48 / 74 41 / 60 (Piracetam) Group 4 Ashwagandha Extract 76.08 24.67 11.42 7.17

[0099] Based on the results above, it is evident that the group treated with Ashwagandha exhibited better activities in improving learning and memory (behavioral) than other groups when compared to the untreated control group. Example 3

[00100] 40 healthy individuals with mild cognitive impairment were randomized in a 1:1 ratio (n=20 each) to receive treatment with Ashwagandha extract with 2% sominone (250 mg once daily) or placebo for 60 consecutive days.

[00101] Group 1: Ashwagandha Extract (sample 4) (Dosage 250 mg once daily for 60 days)

[00102] Group 2: Placebo (dosage of 250 mg once daily for 60 days)

[00103] The primary outcomes were the change (baseline to 30 days and 60 days) in cognitive function assessed using outcome measures, Wechsler Memory Scale III (WMS-III) and Shepard Mental Rotation Task.

[00104] Secondary outcomes were the change (baseline to 30 days and 60 days) in MoCA and MMSE scores. Petition 870250098597, dated 10 / 28 / 2025, page 49 / 74 42 / 60

[00105] Within-group analysis (placebo group and Ashwagandha group) - Comparison between day 30 and baseline, day 60 with baseline, and day 60 with day 30. Montreal Cognitive Assessment (MOCA)

[00106] No significant variability was observed with placebo treatment on day 30 compared to baseline (percent change = -0.45%). Day 60 showed no significant variability in MoCA compared to baseline with placebo treatment (percent change = 0.90%). Day 60 showed no significant variability in the MOCA task compared to day 30 with placebo treatment (percent change = 1.36%). Significant improvement was observed with ashwagandha treatment on day 30 compared to baseline (percent change = 7.83%). Day 60 showed significantly higher MOCA compared to baseline with ashwagandha treatment (percent change = 14.77%). Day 60 showed significantly higher MOCA compared to day 30 with ashwagandha treatment (percent change = 6.43%).The results showed an improvement in cognitive impairment after administration of Ashwagandha extract. The results above are represented in Figure 1. Mini Mental State Examination (MMSE)

[00107] No significant variability was Petition 870250098597, dated 10 / 28 / 2025, page 50 / 74 43 / 60 observed with placebo treatment on days 30 and 60 compared to baseline (percentage change = 2.08% and 2.77%, respectively). Day 60 showed no significant variability in MMSE compared to day 30 with placebo treatment (percentage change = 0.68%). A significant improvement was observed with Ashwagandha treatment on day 30 compared to baseline (percentage change = 9.26%). Day 60 showed significantly higher MMSE compared to baseline with Ashwagandha treatment (percentage change = 19.21%). Day 60 showed significantly higher MMSE compared to day 30 with Ashwagandha treatment (percentage change = 9.11%). The result showed an improvement in cognitive impairment after administration with Ashwagandha extract. The above results are represented as Figure 2. Shepard's Mental Rotation Task

[00108] No significant variability was observed with placebo treatment on day 30 compared to baseline (percent change = -0.46%). Day 60 showed a significantly lower Shepard mental rotation task compared to baseline with placebo treatment (percent change = -9.216%). Day 60 showed a significantly lower Shepard mental rotation task compared to day 30. Petition 870250098597, dated 10 / 28 / 2025, page 51 / 74 44 / 60 with placebo treatment (percentage change = -8.796%). Significant improvement was observed with ashwagandha treatment on day 30 compared to baseline (percentage change = 12.22%). Day 60 showed a significantly higher Shepard mental rotation task compared to baseline with ashwagandha treatment (percentage change = 31.67%). Day 60 showed a significantly higher Shepard mental rotation task compared to day 30 with ashwagandha treatment (percentage change = 17.34%). The results showed an improvement in the individual's spatial visualization skills after administration with ashwagandha extract. The above results are represented as Figure 3. Wechsler Memory Scale III (WMS III) - Immediate Memory Logical Memory I

[00109] Significant improvement was observed with Ashwagandha treatment on day 30 compared to baseline. There was a 2.63% improvement in logical memory on day 30 compared to baseline. Day 60 showed significantly higher (6.5%) logical memory I compared to baseline with Ashwagandha treatment. Day 60 showed significantly higher (3.7%) logical memory I compared to day 30. Petition 870250098597, dated 10 / 28 / 2025, page 52 / 74 45 / 60 with Ashwagandha treatment. No significant variability was observed with placebo treatment during day 30 (-0.235%) and day 60 (-2.117%) compared to baseline. Day 60 showed a significant reduction in LOGICAL MEMORY I compared to day 30 with placebo treatment (percent change = -1.886%). The results showed an improvement in verbal episodic memory after administration of Ashwagandha extract. The above results are represented as Figures 4A and 4B. b. Paired Verbal Association I

[00110] A significant improvement (2.64%) was observed with Ashwagandha treatment on day 30 compared to baseline. Day 60 showed significantly higher PAIRED VERBAL ASSOCIATION I compared to baseline with Ashwagandha treatment (6.93 percent change). Day 60 showed significantly higher PAIRED VERBAL ASSOCIATION I compared to day 30 with Ashwagandha treatment (4.1 percent change).

[00111] No significant variability was observed with placebo treatment during day 30 (0% change) compared to baseline. Day 60 showed no significant variability in the PAIRED VERBAL ASSOCIATION I compared to baseline with placebo treatment (percent change = -0.982%). Day 60 Petition 870250098597, dated 10 / 28 / 2025, pp. 53 / 74 46 / 60 showed a significant reduction in the PAIRED VERBAL ASSOCIATION I compared to day 30 with placebo treatment (percentage change = -0.982%). The results showed an improvement in verbal learning and memory skills after administration of the Ashwagandha extract. The above results are represented as Figures 4A and 4B. c. Faces I

[00112] No significant variability was observed with placebo treatment during day 30 (0.242%) and day 60 (-0.242%) compared to baseline. Day 60 showed no significant variability in FACES I compared to day 30 with placebo treatment (percent change = 0%). No significant variability was observed with Ashwagandha treatment during day 30 compared to baseline (percent change = 0.729%). Day 60 showed significantly higher FACES I compared to baseline with Ashwagandha treatment (percent change = 7.06%). Day 60 showed significantly higher FACES I compared to day 30 with Ashwagandha treatment (percent change = 6.280%). The results showed an improvement in visual memory and recognition skills after administration of Ashwagandha extract. The results above are represented as Figures 4A and 4B. Petition 870250098597, dated 10 / 28 / 2025, pp. 54 / 74 47 / 60 d. Family Engravings I

[00113] No significant variability in FAMILY PICTURES I was observed with placebo treatment during day 30 (percent change = 0.25%) and day 60 (percent change = 0.5%) compared to baseline. Day 60 showed no significant variability in FAMILY PICTURES I compared to day 30 with placebo treatment (percent change = 0.249%). Significant variability was observed with Ashwagandha treatment during day 30 compared to baseline (percent change = 0.5%). Day 60 showed significantly higher (percent change = 9.25%). FAMILY IMAGINATION I compared to baseline with Ashwagandha treatment. Day 60 showed significantly higher (percent change = 8.706%) FAMILY IMAGINATION I compared to day 30 with Ashwagandha treatment. The results showed an improvement in visual memory and recognition skills after administration of the Ashwagandha extract. The above results are represented as Figures 4A and 4B. Wechsler Memory Scale III (WMS III) - General Memory Logical Memory II

[00114] A significant improvement (percentage change = 4.96%) was observed in LOGICAL MEMORY II with the Petition 870250098597, dated 10 / 28 / 2025, page 55 / 74 48 / 60 treatment with Ashwagandha during day 30 compared to baseline. Day 60 showed significantly higher Logical Memory II (12.8%) compared to baseline with Ashwagandha treatment. Day 60 showed significantly higher Logical Memory II (7.5%) compared to day 30 with Ashwagandha treatment. No significant variability was observed in Logical Memory II with placebo treatment during day 30 (0.263%) and day 60 (1.319%) compared to baseline. Day 60 showed a significant reduction in Logical Memory II compared to day 30 with placebo treatment (percent change = 1.052%). The results showed an improvement in verbal episodic memory after administration of Ashwagandha extract. The above results are represented as Figures 5A and 5B. b. Paired Verbal Association II

[00115] A significant improvement (11.02%) was observed in Paired Verbal Association II with Ashwagandha treatment on day 30 compared to baseline. Day 60 showed significantly higher Paired Verbal Association II compared to baseline with Ashwagandha treatment (percent change = 19.21%). Day 60 showed significantly higher Paired Verbal Association II compared to day 30 with Ashwagandha treatment (percent change = 7.38%). Petition 870250098597, dated 10 / 28 / 2025, pp. 56 / 74 49 / 60

[00116] No significant variability was observed with placebo treatment on day 30 (1.40% change) compared to baseline. Day 60 showed no significant variability in PAIRED VERBAL ASSOCIATION II compared to baseline with placebo treatment (percent change = 2.53%). Day 60 showed a significant reduction in PAIRED VERBAL ASSOCIATION II compared to day 30 with placebo treatment (percent change = 1.11%). The results showed an improvement in verbal learning and memory skills after administration of Ashwagandha extract. The above results are represented as Figures 5A and 5B. c. Faces II

[00117] No significant variability was observed with placebo treatment on day 30 (1.32%) compared to baseline. Day 60 showed no significant improvement in FACES II compared to baseline with placebo treatment. Day 60 showed no significant variability in FACES II compared to day 30 with placebo treatment (percent change = 1.8%). Significant improvement was observed with Ashwagandha treatment on day 30 compared to baseline (percent change = 3.7%). Day 60 showed significantly higher FACES II compared to baseline. Petition 870250098597, dated 10 / 28 / 2025, pp. 57 / 74 Day 50 / 60 baseline with Ashwagandha treatment (percentage change = 14.36%). Day 60 showed significantly higher FACES II scores compared to day 30 with Ashwagandha treatment (percentage change = 10.33%). The results showed an improvement in visual memory and recognition skills after administration of the Ashwagandha extract. The above results are represented as Figures 5A and 5B. d. Family engravings II

[00118] No significant variability in FAMILY PICTURES II was observed with placebo treatment during day 30 (percent change = 0%) and day 60 (percent change = 1.3%) compared to baseline. Day 60 showed no significant variability in FAMILY PICTURES II compared to day 30 with placebo treatment (percent change = 1.25%). Significant variability was observed with Ashwagandha treatment during day 30 compared to baseline (percent change = 0.25%). Day 60 showed significantly higher (percent change = 12.3%) FAMILY PICTURES II compared to baseline with Ashwagandha treatment. Day 60 showed significantly higher (percent change = 12%) FAMILY PICTURES II compared to day 30 with Ashwagandha treatment. The results showed a Petition 870250098597, dated 10 / 28 / 2025, pp. 58 / 74 51 / 60 improvement in visual memory and recognition skills after administration of Ashwagandha extract. The above results are represented as Figures 5A and 5B. Wechsler Memory Scale III (WMS III) - Working Memory Sequencing Letters and Numbers

[00119] No significant variability in letter and number sequencing was observed with placebo treatment during day 30 (percent change = 0.27%) and day 60 (percent change = 0.53%) compared to baseline. Day 60 showed no significant variability in NUMBER AND LETTER SEQUENCING compared to day 30 with placebo treatment (percent change = 0.27%). Significant improvement was observed with Ashwagandha treatment on day 30 (percent change = 7.24%) and day 60 (percent change = 16.35%) compared to baseline. Day 60 showed significantly higher LETTER NUMBER SEQUENCING compared to day 30 with Ashwagandha treatment (percent change = 8.5%). The results showed an improvement in working memory and cognitive processing skills after administration of Ashwagandha extract.The results above are represented as Figures 6A and 6B. b. Spatial amplitude Petition 870250098597, dated 10 / 28 / 2025, page 59 / 74 52 / 60

[00120] No significant variability was observed in SPATIAL RANGE with placebo treatment during day 30 (percent change = 0.86%) and day 60 (percent change = 1.14%) compared to baseline. Day 60 showed no significant variability in SPATIAL RANGE compared to day 30 with placebo treatment (percent change = 0.28%). Significant improvement was observed with ashwagandha treatment during day 30 compared to baseline (percent change = 12.9%). Day 60 showed significantly greater SPATIAL RANGE compared to baseline with ashwagandha treatment (percent change = 20.6%). Day 60 showed significantly greater SPATIAL RANGE compared to day 30 with ashwagandha treatment (percent change = 6.84%).The results showed an improvement in visuospatial working memory and attention skills after administration of the Ashwagandha extract. The results above are represented as Figures 6A and 6B. Wechsler Memory Scale - III (WMS III) Visuospatial Processing and Response Visual Reproduction I

[00121] No significant improvement was observed in VISUAL REPRODUCTION with placebo treatment during day 30 (percent change = 1.626%) and day 60 (change Petition 870250098597, dated 10 / 28 / 2025, pp. 60 / 74 53 / 60 percent = 1.084%) compared to baseline. Day 60 showed no significant variability in VISUAL REPRODUCTION compared to day 30 with placebo treatment (percent change = -0.533%). A significant improvement was observed with Ashwagandha treatment during day 30 compared to baseline (percent change = 7.24%). Day 60 showed significantly greater VISUAL REPRODUCTION (percent change = 13.941%) compared to baseline with Ashwagandha treatment. Day 60 showed significantly greater VISUAL REPRODUCTION compared to day 30 with Ashwagandha treatment (percent change = 6.25%). The above results are represented as Figures 7A and 7B. b. Visual Reproduction II

[00122] No significant variability was observed in VISUAL REPRODUCTION II with placebo treatment during day 30 (percent change = 1.061%) and day 60 (percent change = 0%) compared to baseline. Day 60 showed no significant variability in VISUAL REPRODUCTION II compared to day 30 with placebo treatment (percent change = -104%). Significant improvement was observed with Ashwagandha treatment during day 30 compared to baseline (percent change = 6.1%). Day 60 showed significantly higher VISUAL REPRODUCTION II compared Petition 870250098597, dated 10 / 28 / 2025, pp. 61 / 74 54 / 60 with the baseline with Ashwagandha treatment (percentage change = 14.854%). Day 60 showed significantly higher VISUAL REPRODUCTION II compared to day 30 with Ashwagandha treatment (percentage change = 8.25%). The above results are represented as Figures 7A and 7B.

[00123] The results showed an improvement in visual memory and visuospatial skills after administration of Ashwagandha extract. Between-Group Analysis for Ashwagandha and Placebo Groups. Wechsler Memory Scale - III (WMS III) Logical Memory I

[00124] Treatment with Ashwagandha showed significantly higher logical memory I than placebo treatment during observations on day 30 (mean difference (Ashwagandha-Placebo) = 3). During day 60, treatment with Ashwagandha showed significantly higher logical memory I than placebo treatment (mean difference (Ashwagandha-Placebo) = 9). b. Paired verbal association I

[00125] Treatment with Ashwagandha showed significantly higher paired verbal association I than placebo treatment during observations on day 30 (mean difference (Ashwagandha-Placebo) = 2.75). During day 60, treatment with Ashwagandha showed association Petition 870250098597, dated 10 / 28 / 2025, pp. 62 / 74 55 / 60 verbal paired I significantly higher than placebo treatment (mean difference (Ashwagandha-Placebo) = 8). c. Faces I

[00126] Treatment with Ashwagandha showed significantly higher Faces I than placebo treatment during observations on day 30 (mean difference (Ashwagandha-Placebo) = 1). During day 60, treatment with Ashwagandha showed significantly higher Faces I than placebo treatment (mean difference (Ashwagandha-Placebo) = 7.5). d. Family Engravings I

[00127] Treatment with Ashwagandha showed no significant variability in familiar images I compared with placebo treatment during observations on day 30 (mean difference (Ashwagandha-Placebo) = 0.25). During day 60, treatment with Ashwagandha showed significantly more familiar images I than placebo treatment (mean difference (Ashwagandha-Placebo) = 8.75). e. Logical Memory II

[00128] Treatment with Ashwagandha showed significantly greater logical memory II than treatment with placebo during observations on day 30 (mean difference (Ashwagandha-Placebo) = 4.5). During day 60, treatment with Ashwagandha showed logical memory Petition 870250098597, dated 10 / 28 / 2025, pp. 63 / 74 56 / 60 II significantly higher than placebo treatment (mean difference (Ashwagandha-Placebo) = 11). f. Paired Verbal Association II

[00129] Treatment with Ashwagandha showed significantly higher Paired Verbal Association II than placebo treatment during observations on day 30 (mean difference (Ashwagandha-Placebo) = 6.083). During day 60, treatment with Ashwagandha showed significantly higher Paired Verbal Association II than placebo treatment (mean difference (Ashwagandha-Placebo) = 14.75). g. Faces II

[00130] Treatment with Ashwagandha showed no significant variability in Faces II compared with placebo treatment during observations on day 30 (mean difference (Ashwagandha-Placebo) = 2.25). During day 60, treatment with Ashwagandha showed significantly higher Faces II than treatment with placebo (mean difference (Ashwagandha-Placebo) = 10.75). h. Family Engravings II

[00131] Treatment with Ashwagandha showed no significant variability in familiar images II compared with placebo treatment during observations on day 30 (mean difference (Ashwagandha-Placebo) = 0.25). During day 60, treatment with Ashwagandha Petition 870250098597, dated 10 / 28 / 2025, pp. 64 / 74 57 / 60 showed significantly higher Family Tracing II scores than the placebo treatment (mean difference (Ashwagandha-Placebo) = 11). Sequencing Letters and Numbers

[00132] Treatment with Ashwagandha showed significantly greater number and letter sequencing than placebo treatment during observations on day 30 (mean difference (Ashwagandha - Placebo) = 6.5). During day 60, treatment with Ashwagandha showed significantly greater number and letter sequencing than placebo treatment (mean difference (Ashwagandha - Placebo) = 14.75). Spatial Amplitude

[00133] Treatment with Ashwagandha showed a significantly greater spatial amplitude than placebo treatment during observations on day 30 (mean difference (Ashwagandha-Placebo) = 6.5). During day 60, treatment with Ashwagandha showed a significantly greater spatial amplitude than placebo treatment (mean difference (Ashwagandha-Placebo) = 14.75). Visual Reproduction I

[00134] Treatment with Ashwagandha showed significantly greater visual reproduction I than treatment with placebo during observations on day 30 (mean difference (Ashwagandha-Placebo) = 5.25). During the Petition 870250098597, dated 10 / 28 / 2025, pp. 65 / 74 58 / 60 day 60, treatment with Ashwagandha showed significantly greater visual reproduction I than treatment with placebo (mean difference (Ashwagandha-Placebo) = 12). Visual Reproduction II

[00135] Treatment with Ashwagandha showed significantly higher visual reproduction II than placebo treatment during observations on day 30 (mean difference (Ashwagandha-Placebo) = 4.75). During day 60, treatment with Ashwagandha showed significantly higher visual reproduction II than placebo treatment (mean difference (Ashwagandha-Placebo) = 14). Shepard's Mental Rotation

[00136] Treatment with Ashwagandha (Ashwagandha) showed significantly greater Shepard mental rotation than placebo treatment during observations on day 30 (mean difference (Ashwagandha-Placebo) = 1.4). During day 60, treatment with Ashwagandha showed significantly greater Shepard mental rotation than placebo treatment (mean difference (Ashwagandha-Placebo) = 4.5). Girl

[00137] Treatment with Ashwagandha showed a significantly higher MoCA than placebo treatment during observations on day 30 (mean difference (Ashwagandha-Placebo) = 1.8). During day 60, the treatment Petition 870250098597, dated 10 / 28 / 2025, pp. 66 / 74 59 / 60 with Ashwagandha showed a significantly higher MoCA than the placebo treatment (mean difference (Ashwagandha / Placebo) = 3.1). MEEM

[00138] Treatment with Ashwagandha showed significantly higher MMSE than placebo treatment during observations on day 30 (mean difference (Ashwagandha-Placebo) = 1.8). During day 60, treatment with Ashwagandha showed significantly higher MMSE than treatment with placebo (mean difference (Ashwagandha-Placebo) = 3.1).

[00139] Based on the above results, the increase in WMS-III subtest scores indicates a significant improvement in immediate, long-term, and working memory with Ashwagandha treatment compared to placebo. Visuospatial performance was also increased after treatment with Ashwagandha extract. Ashwagandha treatment showed an increase in MMSE scores, supporting the improvement in cognitive functions. In the above study, the MoCA score was significantly improved at 30 and 60 days with Ashwagandha treatment compared to placebo.

[00140] Reduced visuospatial working memory is an early sign of MCI, which may be due to shrinkage / atrophy of the hippocampus, leading to an effect Petition 870250098597, dated 10 / 28 / 2025, pp. 67 / 74 60 / 60 deleterious in visual-grapheme-phoneme conversion. The significant improvement in visuospatial working memory in the study above indicates a positive effect of Ashwagandha on visual-grapheme-phoneme conversion in the fusiform gyrus of the hippocampus. Studies have reported that improved working memory results in the effective conversion of short-term memory into long-term memory (Alescio-Lautier, 2007; Missonnier, 2007; Yetkin, 2006).

[00141] The above results suggest that Ashwagandha therapy standardized with sominone led to significant improvements in enhancing both immediate and general memory in people with Mild Cognitive Impairment (MCI), as well as in improving cognitive function, such as attention and information processing speed. Petition 870250098597, dated 10 / 28 / 2025, pp. 68 / 74

Claims

1 / 7 CLAIMS 1. Medicinal composition derived from Withania somnifera extract, characterized in that it comprises withanolides and sominone, wherein said sominone in the extract is standardized in the range of 0.1-30%.

2. Medicinal composition, according to claim 1, characterized in that the withanolides are in the range of 10-60%.

3. Medicinal composition according to claim 1, characterized in that said composition further comprises withanolide glycosides and withapherin A.

4. Medicinal composition, according to claim 3, characterized in that withanolide glycosides are in the range of 10-25% and withapherin A is in the range of 2-10%.

5. Medicinal composition, according to claim 3, characterized in that said composition comprises not less than 2% sominone, not less than 20% withanolide glycosides, not less than 6% withapherin A and not less than 30% withanolides.

6. Medicinal composition, according to claims 1 to 5, characterized in that it is subjected to the addition of pharmaceutically acceptable excipients selected from the group comprising diluents, binders, wetting agents, disintegrants and lubricants and mixing thereof with dispensing systems.

7. Method for preparing the medicinal composition derived from Withania somnifera extract, characterized in that it comprises the steps of (a) cleaning the parts of the Ashwagandha plant (roots and leaves); (b) drying the cleaned parts of the plant and turning them into powder; (c) extracting the powdered parts using a polar solvent; (d) collecting the supernatant after extraction, followed by concentrating it; (e) subjecting said concentrate to liquid-liquid extraction using a non-polar solvent; (f) separating the aqueous and solvent fractions after liquid-liquid extraction; (g) subjecting the aqueous fraction in step (f) to column purification using water and methanol; (h) collecting the water and methanol elute and keeping it aside; (i) subjecting the solvent fraction collected in step (f) to column purification by eluting with hexane-methanol in different ratios and collecting different fractions;and (j) mix the methanol eluted material from step (h) and one of the fractions containing a higher concentration of Ashwagandha composition, resulting in said composition.

8. Method for preparing the medicinal composition derived from Withania somnifera extract, according to claim 7, characterized in that the mixing ratio of the methanol eluate from step (h) and one of the fractions containing a higher Ashwagandha composition from step (i) varies from 20:1 to 2:1 and preferably 9:

1.

9. Dosage form, characterized by the fact that it comprises a medicinal composition derived from Withania somnifera extract, containing withanolides and sominone, wherein the sominone in the extract is standardized in the range of 0.1% to 30%.

10. Dosage form according to claim 9, characterized in that said composition further comprises withanolide glycosides and withapherin A.

11. Dosage form according to claim 10, characterized in that the withanolide glycosides are in the range of 10-25% and withapherin A is in the range of 2-10%.

12. Dosage form according to claim 10, characterized in that it comprises 2.3% sominone, 23.5% withanolide glycosides, 6.5% withapherin A, and 34.9% withanolides.

13. Dosage form, according to claims 9 to 12, characterized in that it is subjected to the addition of pharmaceutically acceptable excipients selected from the group comprising diluents, binders, wetting agents, disintegrants and lubricants and a mixture thereof as dispensing systems.

14. Dosage form, according to claims 9 to 13, characterized in that the dose varies from about 30 mg to about 2000 mg per day for administration to an individual.

15. Dosage form according to claim 14, characterized in that it is made in a selected form of capsule, tablet, mini-tablet, granule, sachet, powder, paste, infusion, injection, ampoule, solution, suspension, emulsion, pills or cream.

16. Dosage form, according to claim 15, characterized in that it increases the bioavailability of its constituents when administered to individuals.

17. A method for improving learning and behavioral activity, characterized by the fact that it is achieved through the administration of a medicinal composition derived from Withania somnifera extract, made in a dosage form as defined in claims 9 to 15.

18. Method for improving immediate memory, general memory, working memory and visuospatial memory, characterized by the fact that it is through the administration of the medicinal composition, which is derived from the extract of Withania somnifera made in a dosage form, as defined in claims 9 to 15.

19. Method for improving verbal episodic memory, characterized by the fact that it is achieved through the administration of a medicinal composition derived from Withania somnifera extract, made in a dosage form as defined in claims 9 to 15.

20. Method for improving verbal learning and memory skills, characterized by the fact that it is achieved through the administration of a medicinal composition derived from Withania somnifera extract, made in a dosage form as defined in claims 9 to 15.

21. Method for improving visual memory and recognition skills, characterized by the fact that it is achieved through the administration of a medicinal composition derived from Withania somnifera extract made in a dosage form as defined in claims 9 to 15.

22. Method for improving working memory and cognitive processing skills, characterized by the fact that it is achieved through the administration of a medicinal composition derived from Withania somnifera extract made in a dosage form as defined in claims 9 to 15.

23. Method for improving visuospatial working memory and attention skills, characterized by the fact that it is achieved through improvement in spatial amplitude scores through the administration of a medicinal composition derived from Withania somnifera extract made in a dosage form as defined in claims 9 to 15.

24. Method for improving visual memory and visuospatial skills, characterized by the fact that it is achieved through the administration of a medicinal composition derived from Withania somnifera extract, made in a dosage form as defined in claims 9 to 15.

25. A method for improving an individual's spatial visualization skills, characterized by being assessed by an increase in the score on Shepard's Mental Rotation Task through the administration of a medicinal composition derived from Withania somnifera extract made in a dosage form as defined in claims 9 to 15.

26. Method for improving cognitive impairment by improving the Montreal Cognitive Assessment (MoCA) score, characterized by the fact that it is achieved through the administration of a medicinal composition derived from Withania somnifera extract made in a dosage form as defined in claims 9 to 15.

27. Method for improving cognitive impairment by improving scores on the Mini-Mental State Examination (MMSE), characterized by the fact that it is achieved through the administration of a medicinal composition derived from Withania somnifera extract made in a dosage form as defined in claims 9 to 15.

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