Synergistic herbal composition with intelligence-promoting activity for improving cognitive function and gaming performance

Abstract

Disclosed is a synergistic herbal composition comprising: a first ingredient selected from an extract of centella asiatica, said extract containing at least one ingredient selected from the group consisting of a magnesium salt or complex of asiatic acid, a magnesium salt or complex of madecassic acid, or a mixture thereof, while also containing asiaticoside and madecassoside; a second component selected from the group consisting of an extract, fraction, phytochemicals of cocoa or mixtures thereof; the invention also discloses a preparation method and a treatment method of the composition, and application of the intelligence-improving composition to improvement of cognitive function or mental ability and game performance.

Description

Technical Field

[0001] The present invention relates to a synergistic herbal composition, which comprises: a first component selected from Centella asiatica extract, the extract containing at least one component selected from the group consisting of magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and also containing asiaticoside and madecassoside; and a second component selected from extracts, fractions, phytochemicals derived from cocoa or mixtures thereof; the composition is used for enhancing nootropic activities crucial for improving cognitive function and enhancing gaming performance. Background Art

[0002] Cognition is defined as the ability of the brain to acquire, process, store, and retrieve information. It involves multiple key elements such as attention, perception, memory, motor skills, executive function, and language skills. Age-related cognitive decline has been well documented in the scientific literature. Oxidative stress and inflammation are associated with aging and age-related cognitive decline in neurodegenerative diseases, including Alzheimer's disease (AD). Acetylcholine (ACh) is the first neurotransmitter discovered and is used by all cholinergic neurons, playing a crucial role in the peripheral nervous system and the central nervous system (CNS). The cholinergic system plays a key role in higher cognitive functions, especially in the processes of learning and decision-making. ACh is involved in neuromodulation through differential activation of nicotinic and muscarinic receptors. Multiple studies have shown that ACh is very important for the regulation of memory as well as acquisition, encoding, concentration, consolidation, and reconsolidation. ACh is rapidly inactivated by acetylcholinesterase (AChE).

[0003] In recent years, esports or video gaming has become a very popular entertainment and professional activity globally, especially among young people. Esports requires task switching, rapid responses to multiple audiovisual cues, decision-making, executive functions, memory, spatial awareness, fine motor skills, and mood improvement. Esports is emerging as a research area focusing on the cognitive processes contributing to executive functions and their impact on competitive gaming performance. Since the participants in esports competitions usually play for several hours per match and the competitions last for several days, it is crucial to maintain cognitive and executive functions, attention, and fine motor skills. For this reason, many video game players rely on chemicals such as caffeine or caffeinated beverages to improve cognitive functions, especially executive functions, memory, creativity, motivation, alertness, and delay fatigue. However, excessive intake of caffeine may cause nervousness and / or interfere with fine motor skills, or have no effect on esports performance. It may also cause side effects such as caffeine crashes and caffeine-induced irritability. Therefore, there is an increasing interest in developing non-caffeinated herbal extracts or plant compositions that can be used to improve gaming performance and mood while having no adverse effects common to caffeine or caffeinated products.

[0004] Indian Patent Publication No. IN304584 discloses a synergistic pharmaceutical composition for inhibiting serotonin reuptake, wherein the composition comprises pentacyclic triterpenoid saponins, preferably asiaticoside and madecassoside, and an excipient.

[0005] Another Indian Patent Publication No. 1633 / MUM / 2011 discloses a method for treating a disorder selected from Cushing's syndrome, headache disorders, neuropathic pain, or any combination thereof, the method comprising the act of administering to a subject in need a pharmaceutically effective amount of a composition comprising asiaticoside and madecassoside, and optionally further comprising at least one excipient.

[0006] Another Indian Patent Publication No. 202011020939 discloses a herbal composition for relieving obsessive-compulsive disorder and general mental disorders, the herbal composition comprising a plurality of herbs including whole Centella asiatica, whole Convolvulus arvensis, Acorus calamus root, Glycyrrhiza glabra root, Tinospora cordifolia stem, tubers and roots of Asparagus cochinchinensis, roots of Nardostachys jatamansi, roots of Aucklandia lappa, Betula platyphylla bark, Celastrus orbiculatus seeds, roots of Withania somnifera, and whole Ocimum tenuiflorum; wherein the content of each of the herbs in the herbal composition is 4-20% (weight / weight), and the herbal composition is administered to the subject in the form of herbal smoke generated upon burning.

[0007] Another Indian Patent Publication No. 1167 / CHE / 2009 discloses a medicinal or herbal composition comprising about 30% Acorus calamus, about 30% Clitoria ternatea, about 10% Glycyrrhiza glabra, about 10% Terminalia catappa, about 10% Bacopa monnieri and about 10% Centella asiatica by weight of the composition.

[0008] Another Indian Patent Publication No. IN236752 discloses a herbal formulation comprising extracts in pharmacologically active form obtained from Tinospora cordifolia, Centella asiatica, Withania somnifera, Erythrina variegata and Curcuma longa, present in pharmaceutically acceptable doses and optionally further containing an additive useful as a brain tonic for the treatment of senile dementia and Alzheimer's disease.

[0009] Other Indian Patent Publication No. IN238309 discloses a synergistic herbal composition as a brain tonic, cognitive enhancer, helpful for recalling thoughts and as an antioxidant, capable of treating or preventing amnesia and having the property of improving memory; the composition comprises a pharmaceutically acceptable amount of plant extracts, wherein the content range of Centella asiatica extract is from 1% to 15%, the content range of Sesame extract is from 2% to 20%, and optionally contains pharmaceutically acceptable salts, carriers or diluents as described herein.

[0010] Chinese Patent Publication No. CN102512431 discloses the application of Centella asiatica triterpenoids in the preparation of products for treating craniocerebral injury, namely the new uses of asiaticoside, asiatic acid aglycone, madecassoside and madecassic acid aglycone in the preparation of drugs for treating craniocerebral trauma and its sequelae.

[0011] Another Chinese Patent Publication No. CN102389437 discloses the application of Centella asiatica triterpenoids in the preparation of products for improving sequelae of stroke, especially the new uses of Centella asiatica α - amyrin triterpenoids (including asiaticoside, asiatic acid, madecassoside and madecassic acid) in the preparation of products for improving sequelae of stroke.

[0012] Korean Patent Publication No. KR960022436 discloses a madecassic acid used as a medicament for treating wounds, which is made from madecassic acid derived from Centella asiatica.

[0013] However, the prior art fails to provide a precise and effective composition for enhancing nootropic activity which is crucial for improving cognitive function and enhancing gaming performance. Summary of the Invention

[0014] Accordingly, a main object of the present invention is to provide a synergistic and safe herbal composition, the composition comprising: a first component selected from extracts of Centella asiatica, the extract containing at least one component selected from the group consisting of magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and further containing asiaticoside and madecassoside; a second component selected from extracts of Theobroma cacao, fractions, phytochemicals or mixtures thereof. In addition, the present invention also relates to a method for preparing the composition and a therapeutic method for enhancing nootropic activity, the enhancement of nootropic activity being crucial for improving cognitive function and enhancing gaming performance.

[0015] Accordingly, in one aspect, the present invention provides a synergistic herbal composition, the composition comprising: a first component selected from extracts of Centella asiatica, the extract containing at least one component selected from the group consisting of magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and further containing asiaticoside and madecassoside; a second component selected from extracts of Theobroma cacao, fractions thereof, phytochemicals or mixtures thereof; the composition for enhancing nootropic activity which is crucial for improving cognitive function and enhancing gaming performance.

[0016] In another aspect, the present disclosure provides a synergistic herbal composition, the composition comprising: a first component selected from extracts of Centella asiatica, the extract containing at least one component selected from the group consisting of magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and further containing asiaticoside and madecassoside; a second component selected from extracts of Theobroma cacao, fractions thereof, phytochemicals or mixtures thereof; and optionally further comprising at least one component selected from pharmaceutically, nutraceutically or dietetically acceptable excipients, carriers and diluents.

[0017] In another aspect, the present disclosure provides a method for preparing a composition, the composition comprising: a first component selected from extracts of Centella asiatica, the extract containing at least one component selected from the group consisting of magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and further containing asiaticoside and madecassoside; a second component selected from extracts of Theobroma cacao, fractions thereof, phytochemicals or mixtures thereof; and optionally further comprising at least one component selected from pharmaceutically, nutraceutically or dietetically acceptable excipients, carriers and diluents.

[0018] In a further aspect, the present disclosure provides a method of improving at least one benefit related to cognitive function, mental ability, and / or nootropic activity, said benefit being selected from the group consisting of alertness, vigilance, attention, focus / concentration, neuromotor function (reaction time), memory (verbal, working, visual, logical), intelligence, learning, acquisition, retention, recall of information / communication, perception, reasoning, problem-solving, decision-making; reducing mental fatigue and improving game performance; wherein the method comprises administering to a person in need an effective dose of a composition comprising: a first component selected from Centella asiatica extract, said extract containing at least one component selected from the group consisting of magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and further containing asiaticoside and madecassoside; a second component selected from cocoa extract, its fractions, phytochemicals, or mixtures thereof; and optionally further comprising at least one component selected from pharmaceutically, nutraceutically, or dietetically acceptable excipients, carriers, and diluents.

[0019] In an even further aspect, the present disclosure provides the use of the herbaceous composition of the present invention, said composition comprising: a first component selected from Centella asiatica extract, said extract containing at least one component selected from the group consisting of magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and further containing asiaticoside and madecassoside; a second component selected from cocoa extract, its fractions, phytochemicals, or mixtures thereof; and optionally further comprising at least one component selected from pharmaceutically, nutraceutically, or dietetically acceptable excipients, carriers, and diluents; said composition for improving at least one benefit related to cognitive function, mental ability, and / or nootropic activity, said benefit being selected from the group consisting of alertness, vigilance, attention, focus / concentration, neuromotor function (reaction time), memory (verbal, working, visual, logical), intelligence, learning, acquisition, retention, recall of information / communication, perception, reasoning, problem-solving, decision-making; reducing mental fatigue and improving game performance. Detailed Description

[0020] The present invention will now be described in detail in connection with certain preferred and optional embodiments in order to more fully understand and appreciate its various aspects.

[0021] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For the purpose of describing the present invention, certain terms are specifically defined herein as follows.

[0022] The terms "metal salt", "metal complex", and "metal chelate" have the same meaning and are used interchangeably in this specification. Therefore, a centella asiatica extract containing madecassic acid magnesium salt and asiatic acid magnesium salt; a centella asiatica extract containing madecassic acid magnesium complex and asiatic acid magnesium complex; a centella asiatica extract containing madecassic acid magnesium chelate and asiatic acid magnesium chelate have the same meaning and are used interchangeably in this specification. The term "asiaticoside" or "total asiaticoside" includes asiaticoside, madecassoside, madecassic acid, and asiatic acid; they have the same meaning and are used interchangeably in this specification.

[0023] Unless otherwise stated, words such as "comprising", "including", "including... within", and "containing... within" mean "including but not limited to" and should not be construed as limiting any general statement that follows. The terms "improve" and "better" have the same meaning and are used interchangeably herein. The term "method for obtaining a health benefit" refers to inhibiting, preventing, or arresting the development of a pathology (disease, disorder, or condition), and / or causing a reduction, alleviation, or regression of the pathology. Those skilled in the art will understand that various methods and assays can be used to evaluate the development of a pathology, and similarly, various methods and assays can be used to evaluate the reduction, alleviation, or regression of a pathology.

[0024] The herbal sources used in the present invention are as follows:

[0025] 1. Centella asiatica was collected from Aswaraopet Village, Aswaraopet Mandal, Bhadradri-Kothagudem District, Telangana State, India.

[0026] 2. Theobroma cacao was collected from Kondalaraopalem Village, Pedavegi Mandal, Eluru District, Andhra Pradesh State, India.

[0027] Centella asiatica: It is a perennial aromatic plant belonging to the Apiaceae family and is commonly known as gotu kola or Indian pennywort. It has been used in the ancient Indian medical system as a medicine for enhancing memory, treating skin diseases, and neurological diseases. In Chinese literature, it has been reported as one of the "miracle drugs of life" known more than 2,000 years ago. In addition to its common use as a medicinal plant, it can be eaten fresh as a salad, cooked as a vegetable, or mixed into a drink. Centella asiatica is used to treat various diseases such as skin diseases, rheumatism, inflammation, syphilis, mental diseases, epilepsy, hysteria, asthma, dehydration, and diarrhea. The medicinal value of this plant is mainly attributed to the triterpenoids it contains, such as madecassic acid, asiatic acid, asiaticoside, and madecassoside (Figure 1). These four components are collectively referred to as asiaticoside. Other components also exist in centella asiatica, including flavonoids and some phytosterols, flavonoids such as quercetin, kaempferol, and phytosterols such as campesterol, sitosterol, and stigmasterol.

[0028]

[0029] Figure 1: Chemical structures of asiatic acid, madecassic acid, asiaticoside and madecassoside

[0030] Many drug molecules containing organic acids exist in the form of metal salts or metal complexes. This is the simplest way to alter the undesirable properties of organic acids. By pairing metal ions with the salt form or complex of the parent compound, the typically undesirable physicochemical or biopharmaceutical properties of organic acids can be improved.

[0031] Magnesium is one of the essential minerals that is abundant in the human body. Magnesium is a cofactor for more than 300 enzyme systems, including protein synthesis, muscle and nerve function, blood sugar control, and blood pressure regulation. Magnesium is necessary for the synthesis of DNA, RNA, and the antioxidant glutathione. It is crucial in the process by which cells generate sufficient energy. It also plays a role in the active transport of calcium and potassium ions across cell membranes, which is very important for nerve impulse conduction, neuromuscular coordination, muscle contraction, and normal heart function. In addition, magnesium also plays an important role in energy balance, metabolism, and brain function. Adenosine triphosphate (ATP) is the energy currency of the body; however, in order to exert its activity or generate energy, ATP must bind to a magnesium ion, usually to one of its phosphate groups.

[0032] The inventors speculated that metal salts, complexes or chelates containing asiatic acid and madecassic acid, as well as asiaticoside and madecassoside in centella asiatica extract can improve nootropic activity.

[0033] Therefore, in view of the importance of magnesium, the inventors of the present invention prepared a novel centella asiatica extract that naturally contains magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, as well as asiaticoside and madecassoside.

[0034] Magnesium salts or complexes containing asiatic acid and madecassic acid, and centella asiatica extract containing asiaticoside and madecassoside: Formation of salts or complexes:The aerial parts of Centella asiatica were extracted with 70% aqueous ethanol, and the extract was treated with magnesium oxide. The used raw materials were extracted with water, and the extract was also treated with magnesium oxide. The mixture of the two extracts formed an extract (CAMg-1) containing the magnesium salt or complex of asiatic acid, the magnesium salt or complex of madecassic acid, asiaticoside, and madecassoside. The extract thus obtained contained 13.03% asiaticoside and 1.09% magnesium (Example-1). For comparison, a mixture of a 70% aqueous ethanol extract of Centella asiatica without magnesium and an extract of Centella asiatica after extraction with water (Comparative Example-1A) was also prepared. Similarly, a 70% aqueous ethanol extract of Centella asiatica (CAMg-2) containing the magnesium salt or complex of asiatic acid and madecassic acid, asiaticoside, and madecassoside was prepared. This extract contained 13.69% asiaticoside and 0.61% magnesium (Example-2). For comparison, a 70% aqueous ethanol extract of Centella asiatica without magnesium salt or complex (Comparative Example-2A) was also prepared. In addition, a water extract of Centella asiatica containing magnesium (CAMg-3), a 70% aqueous methanol extract containing magnesium (CAMg-4), and a 70% aqueous acetone extract containing magnesium (CAMg-5) were prepared (Examples 3-5).

[0035] Cocoa seed extract: The chemical structures of asiatic acid and madecassic acid contain a carboxylic acid group (see Figure 1) and can form metal salts or complexes with bases. When the Centella asiatica extract is treated with metal oxides, metal hydroxides, metal carbonates, etc., metal salts or complexes of asiatic acid and madecassic acid are formed. For example, when the Centella asiatica extract is treated with magnesium oxide and then the solution is filtered to remove the unreacted magnesium oxide, a Centella asiatica extract containing the magnesium salt or complex of asiatic acid, the magnesium salt or complex of madecassic acid, asiaticoside, and madecassoside is obtained. The presence of magnesium (1.09%) was detected by inductively coupled plasma mass spectrometry (ICP-MS), confirming the formation of the magnesium salt. Since magnesium oxide is actually insoluble in 70% aqueous ethanol or water, the only possible way for magnesium to be present in the product is to form salts with asiatic acid and madecassic acid.

[0036] In addition, the pH value of the Centella asiatica extract containing the magnesium salt or complex of asiatic acid and madecassic acid, asiaticoside, and madecassoside is about 7.2, while the pH value of the Centella asiatica extract containing asiatic acid, madecassic acid, asiaticoside, and madecassoside is about 4.5. The difference in pH values clearly shows that asiatic acid and madecassic acid form salts with magnesium.

[0037] The inventors surprisingly found that, compared with the Centella asiatica extract without magnesium (CA-1), the Centella asiatica extract (CAMg-1) containing magnesium salts of asiatic acid and madecassic acid, as well as asiaticoside and madecassoside, exhibited better nootropic activities, including activities related to cognitive functions.

[0038] Evaluate the nootropic activities (enhancing cognitive function, shortening reaction time, and improving alertness) of Centella asiatica extracts with and without magnesium in a Sprague-Dawley rat in vivo model

[0039] In the T-maze reward alternation test, compared with the Centella asiatica extract without magnesium (CA-1), rats supplemented with a mixture (CAMg-1) of a 70% aqueous ethanol solution and a water extract successively obtained from the aerial parts of Centella asiatica, which contained magnesium salts of asiatic acid and madecassic acid, as well as asiaticoside and madecassoside, showed a significant improvement in working memory. As shown in Table 1, the working memory index of rats supplemented with CAMg-1 (Group G3) increased by 36.1% compared to that of the control animals. In contrast, the working memory index of rats supplemented with CA-1 (Group G2) only increased by 21.3%. This observation indicates that the magnesium-containing extract mixture is significantly more effective than the magnesium-free similar extract mixture (Example 6). Similarly, the 70% aqueous ethanol solution extract (CAMg-2) of the aerial parts of Centella asiatica containing magnesium salts of asiatic acid and madecassic acid, as well as asiaticoside and madecassoside, also showed better efficacy than the magnesium-free extract (CA-2), as shown in Table 1 below.

[0040] The working memory index is calculated using the following formula:

[0041] Working memory index = Number of correct choices / Total number of trials

[0042] Table 1: Effects of herbal extract supplements on the working memory index of Sprague-Dawley rats in the T-maze reward alternation test

[0043]

[0044] Note: The higher the working memory index, the better the efficacy.

[0045] The T-maze Oriented Response Test (ORT) is a psychometric test used to detect attention-related behaviors in experimental animals (including rats). A shorter reaction time indicates improved attention and concentration in the experimental animals. In this experiment, compared with rats supplemented with a mixture of Centella asiatica extract without magnesium (CA-1), rats supplemented with an extract mixture of the aerial parts of Centella asiatica (CAMg-1) obtained by sequential extraction with 70% aqueous ethanol solution and water, containing asiatic acid and magnesium salt of hydroxyasiatic acid, as well as asiaticoside and hydroxyasiaticoside, showed a significantly shorter reaction time. As shown in Table 2, the reaction time of rats supplemented with CAMg-1 (Group G3) was shortened by 33% compared with that of the control animals (Group G1); in contrast, the reaction time of rats supplemented with CA-1 (Group G2) was only shortened by 23% compared with that of Group G1 rats. The observation that rats supplemented with the magnesium-containing extract showed higher efficiency was surprising. Similarly, the 70% aqueous ethanol solution extract of the aerial parts of Centella asiatica (CAMg-2) containing asiatic acid and magnesium salt of hydroxyasiatic acid, as well as asiaticoside and hydroxyasiaticoside, also significantly shortened the reaction time compared with the 70% aqueous ethanol solution extract without magnesium (CA-2). Overall, in experimental rats, supplementation with magnesium-containing extracts (CAMg-1 and CAMg-2) showed higher efficiency than the corresponding magnesium-free extracts (CA-1 and CA-2) in enhancing cognitive function, improving attention and concentration.

[0046] Table 2: Effects of herbal extract supplements on reaction time of Sprague Dawley rats in the Oriented Response Test (ORT)

[0047]

[0048] Note: The shorter the reaction time, the better the efficacy.

[0049] Therefore, the Centella asiatica extract of the present invention containing asiatic acid and magnesium salt of hydroxyasiatic acid, as well as asiaticoside and hydroxyasiaticoside, with the total asiaticoside (asiaticosides) content in the range of 2-40% and the magnesium content in the range of 0.2-5.0%, unexpectedly showed better efficacy in nootropic activity compared with the ordinary extract without magnesium salt.

[0050] Composition

[0051] Inspired by the enhancing effect of the Centella asiatica extract containing asiatic acid and magnesium salt of hydroxyasiatic acid, as well as asiaticoside and hydroxyasiaticoside, in nootropic activity, the inventors prepared several compositions of Centella asiatica extract containing magnesium, which were combined with at least one ingredient selected from cocoa extract, fractions, phytochemicals or mixtures thereof.

[0052] Therefore, various solvent extracts of cocoa seeds were prepared using different solvents.

[0053] Composition 33 (the ratio of CAMg-1 to Th-1 is 2:1): The cocoa seeds were pulverized, and the powder was extracted with various solvents such as 50% aqueous ethanol solution, 90% aqueous ethanol solution, ethanol, water, methanol, and acetone, respectively, to obtain the 50% aqueous ethanol extract (Th-1), 90% aqueous ethanol extract (Th-2), ethanol extract (Th-3), water extract (Th-4), methanol extract (Th-5), and acetone extract (Th-6) of cocoa seeds, respectively. These extracts were standardized to theobromine by the analytical HPLC method, and the specific details are summarized in Examples 7-10.

[0054] Then, as described in Examples 11-20, a variety of compositions (C1-C32) were prepared, which contained centella asiatica extract containing asiatic acid and magnesium salt of hydroxyasiatic acid, as well as asiaticoside and hydroxyasiaticoside, in combination with at least one component selected from cocoa extract, fractions, phytochemicals, or mixtures thereof.

[0055] Acetylcholinesterase (AChE): Acetylcholinesterase (AChE) is a cholinergic enzyme that is mainly present at the neuromuscular junction, especially in muscles and nerves. AChE hydrolyzes acetylcholine (Ach) into acetic acid and choline. The main role of AChE is to terminate synaptic transmission and signal conduction between neurons. The activation of AChE reduces the level of Ach in the brain, which is related to the pathophysiological process of cognitive dysfunction and can lead to neurodegenerative diseases such as Alzheimer's disease. Therefore, inhibiting AChE can reduce the decomposition of Ach and increase its level in the brain. This increased level of Ach activates nicotinic and muscarinic receptors, which helps to relieve cognitive dysfunction and improve memory, learning, information recall / communication, attention, concentration, reasoning, and problem-solving abilities. Several AChE inhibitors, such as tacrine, donepezil, rivastigmine, and galantamine, have been clinically proven to be effective in reducing the symptoms of cognitive dysfunction, thus improving cognitive ability and executive function.

[0056] The acetylcholinesterase (AChE) inhibitory activity of the above compositions (Compositions 1 to 32) was evaluated compared with the corresponding individual components (Example 21). The data of in vitro AChE assays showed that these compositions exhibited unexpectedly better efficacy in enzyme inhibition compared with the corresponding individual components, indicating that the compositions containing centella asiatica extract containing asiatic acid and magnesium salt of hydroxyasiatic acid, as well as asiaticoside and hydroxyasiaticoside, in combination with cocoa extract tended to show a synergistic effect.

[0057] For example, Centella asiatica extract (CAMg-1) with magnesium in a 70% ethanol aqueous solution and water extract at a concentration of 12.5 μg / mL, and Theobroma cacao seed 50% ethanol aqueous solution extract (Th-1) at a concentration of 12.5 μg / mL had AChE inhibition rates of 13.75% and 6.70%, respectively. Surprisingly, Composition 3 containing these two extracts in a ratio of 1:1 had an AChE inhibition rate of 41.20% at a concentration of 25 μg / mL, significantly superior to the additive effect of these two components (13.75% + 6.70% = 20.45%), indicating that Centella asiatica extract (CAMg-1) with magnesium in a 70% ethanol aqueous solution and water extract and Theobroma cacao extract (Th-1) had a synergistic effect in inhibiting AChE. In contrast, a similar composition (Composition 3A) containing Centella asiatica extract (CA-1) without magnesium in a 70% ethanol aqueous solution and water extract and Theobroma cacao extract (Th-1) in the same ratio of 1:1 had an AChE inhibition rate increase of 29.45% at 25 μg / mL, much lower than the similar composition containing magnesium complex (Composition 3). This indicates that magnesium had a synergistic effect on this composition. Similarly, Compositions 1, 2, 4, and 5, which were obtained by mixing Centella asiatica extract (CAMg-1) with magnesium in a 70% ethanol aqueous solution and water extract and Theobroma cacao seed 50% ethanol aqueous solution extract (Th-1) in ratios of 3:1, 2:1, 1:2, and 1:3, respectively, also showed a synergistic effect in AChE inhibition compared to the inhibition rates shown by their corresponding individual components, as summarized in Table 4. The enhancement of the inhibitory effect of the composition of the present invention on acetylcholinesterase (AChE) is surprising and unexpected.

[0058] Similarly, Compositions 6 to 8 containing Centella asiatica extract (CAMg-1) with magnesium in a 70% ethanol aqueous solution and water extract and Theobroma cacao seed 90% ethanol aqueous solution extract (Th-2); Compositions 9 to 11 containing Centella asiatica extract (CAMg-1) with magnesium in a 70% ethanol aqueous solution and water extract and Theobroma cacao seed ethanol extract (Th-3); Compositions 12 to 14 containing Centella asiatica extract (CAMg-1) with magnesium in a 70% ethanol aqueous solution and water extract and Theobroma cacao seed water extract (Th-4); also showed a synergistic effect in acetylcholinesterase inhibition compared to the inhibition rates shown by their corresponding individual components, as summarized in Table 5.

[0059] Similarly, compositions 16 and 17 containing a 70% aqueous ethanol solution containing magnesium and a water-extracted Centella asiatica extract (CAMg-1) and a methanol extract of Theobroma cacao seeds (Th-5); compositions 18 to 20 containing a 70% aqueous ethanol solution containing magnesium and a water-extracted Centella asiatica extract (CAMg-1) and an acetone extract of Theobroma cacao seeds (Th-6); compositions 21 to 23 containing a 70% aqueous ethanol extract of Centella asiatica containing magnesium (CAMg-2) and a 50% aqueous ethanol extract of Theobroma cacao seeds (Th-1); compositions 24 to 26 containing a water extract of Centella asiatica containing magnesium (CAMg-3) and an ethanol extract of Theobroma cacao seeds (Th-3); compositions 27 to 29 containing a 70% aqueous methanol extract of Centella asiatica containing magnesium (CAMg-4) and an acetone extract of Theobroma cacao seeds (Th-6); compositions 30 to 32 containing a 70% aqueous acetone extract of Centella asiatica containing magnesium (CAMg-5) and a methanol extract of Theobroma cacao seeds (Th-5); also showed a synergistic effect in terms of AChE inhibition compared to the inhibition rates shown by their corresponding individual components, as summarized in Tables 6-8.

[0060] Formulation: The synergistic herbal composition comprises: a first component selected from Centella asiatica extracts, which extracts contain at least one component selected from magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and also contain asiaticoside and madecassoside; a second component selected from Theobroma cacao extracts, fractions thereof, phytochemicals or mixtures thereof; the composition may also be formulated with at least one component selected from pharmaceutically, nutraceutically or dietetically acceptable excipients, carriers and diluents.

[0061] The co - composition can be formulated using pharmaceutically, nutraceutically or dietetically acceptable excipients, carriers and diluents; these substances are selected from monosaccharides such as glucose, dextrose, fructose, galactose, etc.; disaccharides such as but not limited to sucrose, maltose, lactose, lactulose, trehalose, cellobiose, chitobiose, etc.; polysaccharides such as starch and modified starches such as sodium carboxymethyl starch, pre - gelatinized starch, soluble starch, super - dispersant A and other modified starches; dextrins produced by hydrolysis of starch or glycogen such as yellow dextrin, white dextrin, maltodextrin, glucose dextrin 12D, rice maltodextrin, tapioca maltodextrin, etc.; polyols or sugar alcohols such as but not limited to sorbitol, mannitol, inositol, xylitol, isomaltulose, etc.; cellulose - based derivatives such as but not limited to microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, etc.; silicates such as but not limited to New Zealand gum, magnesium aluminum silicate, talc, colloidal silica, silica sol, etc.; metal stearates such as but not limited to calcium stearate, magnesium stearate, zinc stearate, etc.; organic acids such as citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L - ascorbic acid, etc.; esters of fatty acid esters and polysorbates, natural gums such as but not limited to gum arabic, carrageenan, guar gum, xanthan gum, etc.; B - group vitamins, niacinamide, calcium pantothenate, amino acids, proteins such as but not limited to casein, gelatin, pectin, agar; organometallic salts such as but not limited to sodium chloride, calcium chloride, calcium hydrogen phosphate, zinc sulfate, zinc chloride, etc.; natural pigments, flavors, class I and class II preservatives, and aqueous, alcoholic, hydroalcoholic, organic solutions of the above components alone or in combination.

[0062] For example, the preparation method of composition 33 - 35 is as follows (Example 22): The 70% aqueous ethanol extract of the aerial parts of Centella asiatica and the extract of the residue after water extraction (CAMg - 1), which successively contains asiatic acid and magnesium salt of hydroxyasiatic acid as well as asiaticoside and hydroxyasiaticoside, is mixed with the 50% aqueous ethanol extract of cocoa seeds (Th - 1) in three ratios (2:1, 1:1 and 1:2), and excipients such as rice maltodextrin and colloidal silica are added in a suitable solvent, and then the solvent is evaporated.

[0063] Preparation method: A method for preparing a composition comprising the following combinations of components: A first component selected from Centella asiatica extracts, which extract contains at least one component selected from the magnesium salts or complexes of asiatic acid, the magnesium salts or complexes of hydroxyasiatic acid, or mixtures thereof, and also contains asiaticoside and hydroxyasiaticoside; a second component selected from cocoa extracts, their fractions, phytochemicals or mixtures thereof; the composition may optionally further comprise at least one component selected from pharmaceutically, nutraceutically or dietetically acceptable excipients, carriers and diluents; the preparation method comprises the following steps:

[0064] (a) Extracting the dried Centella asiatica powder with a suitable solvent;

[0065] (b) Treat the solution obtained in step (a) with a magnesium source;

[0066] (c) Filter the solution in step (b) to obtain a first extract and the used Centella asiatica powder;

[0067] (d) Extract the used Centella asiatica powder with a second solvent;

[0068] (e) Treat the solution obtained in step (d) with a magnesium source;

[0069] (f) Filter the solution in step (e) to obtain a second extract;

[0070] (g) Combine the two extracts obtained in steps (c) and (f);

[0071] (h) Evaporate the solvent in step (g) and dry the residue to obtain a Centella asiatica extract mixture containing asiatic acid and magnesium salts or complexes of hydroxyasiatic acid, as well as madecassoside and magnesium salts or complexes of hydroxymadecassoside;

[0072] (i) Optionally, omit steps (d) to (h), evaporate the filtrate in step (c) and dry the residue to obtain a Centella asiatica extract containing asiatic acid and magnesium salts or complexes of hydroxyasiatic acid, as well as madecassoside and magnesium salts or complexes of hydroxymadecassoside;

[0073] (j) Mix the extract in step (h) or step (i) with a cocoa extract in the presence of a suitable solvent; and optionally add a suitable excipient;

[0074] (k) Dry the product to obtain the desired composition.

[0075] According to the present invention, suitable solvents for preparing a Centella asiatica extract containing asiatic acid and magnesium salts or complexes of hydroxyasiatic acid, as well as madecassoside and magnesium salts or complexes of hydroxymadecassoside, are selected from, but not limited to, C1-C5 alcohols such as ethanol, methanol, and n-butanol; water and its mixtures; ketones selected from acetone and methyl isobutyl ketone; chlorinated solvents selected from dichloromethane and chloroform; water and its mixtures; C1-C7 hydrocarbons such as, but not limited to, hexane and heptane; esters such as, but not limited to, ethyl acetate and its mixtures. The second solvent is selected from water. The magnesium source used in this method is selected from its oxides, hydroxides, or carbonates; examples include magnesium oxide, magnesium hydroxide, and magnesium carbonate.

[0076] Similarly, according to the present invention, suitable solvents for preparing cocoa extracts are selected from, but not limited to, C1-C5 alcohols such as ethanol, methanol, and n-butanol; water and its mixtures; ketones selected from acetone and methyl isobutyl ketone; chlorinated solvents selected from dichloromethane and chloroform; water and its mixtures; C1-C7 hydrocarbons such as, but not limited to, hexane and heptane; esters such as, but not limited to, ethyl acetate and its mixtures.

[0077] After determining the enhanced efficacy of the magnesium-containing Centella asiatica extract, a composition (comp33-35) containing different proportions of the magnesium-containing Centella asiatica extract, cocoa seed extract, and excipient, as well as its individual components, were evaluated for cognition-related parameters in an animal model, as described below.

[0078] The nootropic activities (enhancing cognitive function, shortening reaction time, and improving alertness) of the magnesium-containing Centella asiatica extract, cocoa seed extract, and their composition were evaluated in Sprague-Dawley rats: The data from the in vivo study were as expected. Compared with the corresponding individual components, the composition showed better efficacy in enhancing nootropic activity, further confirming the tendency of synergistic effects when the magnesium-containing Centella asiatica extract and cocoa seed extract were combined (Example 23).

[0079] In the T-maze reward alternation test, compared with rats supplemented with individual components, the working memory of rats supplemented with a composition containing the following components was significantly improved: 1) a 70% aqueous ethanol extract of Centella asiatica containing asiatic acid and magnesium salt of hydroxyasiatic acid, as well as asiaticoside and hydroxyasiaticoside, and an extract of the residue after water extraction (CAMg-1); 2) a 50% aqueous ethanol extract of cocoa seeds (Th-1).

[0080] For example, when the dose of both the magnesium-containing Centella asiatica extract (CAMg-1) and the 50% aqueous ethanol extract of cocoa seeds (Th-1) was 50 mg, compared with the control group animals, the working memory indices increased by 29.8% and 24.5% respectively. Composition 33, containing these two extracts in a ratio of 2:1, had a working memory index increase of 45.8%, which was significantly higher compared to the increases shown by the individual components, thus indicating a synergistic effect between the two extracts. Similarly, Compositions 34 and 35, obtained by mixing these two components in ratios of 1:1 and 2:1 respectively, also showed synergistic effects compared to their corresponding single components, as specifically summarized in Table 9.

[0081] In addition, the compositions (Compositions 33-35) containing the magnesium-containing Centella asiatica extract (CAMg-1) and the 50% aqueous ethanol extract of cocoa seeds (Th-1) also showed synergistic efficacy in shortening reaction time in the T-maze orientation response test (ORT), as shown in Table 10.

[0082] These data clearly show, in aggregate, that Composition 33-35 synergistically enhanced cognitive function, shortened reaction time, and increased alertness in rats.

[0083] These compositions also showed synergistic efficacy in increasing three key neurohormones in the brain tissue and serum of experimental animals compared to animals supplemented with a single extract, as described below.

[0084] Norepinephrine (NE), also known as noradrenaline, is an excitatory neurotransmitter produced and released into the bloodstream by the brainstem, hypothalamus, and adrenal glands. Norepinephrine is biosynthesized from dopamine by the action of dopamine β-monooxygenase, mainly within neurotransmitter vesicles. Norepinephrine can enhance motor neuron function. An increase in norepinephrine levels in the brain raises alertness and wakefulness.

[0085] Dopamine (DA) is a neurotransmitter secreted by neurons located in the substantia nigra of the midbrain. Since it has both excitatory and inhibitory effects depending on the type of receptor to which it binds, it is regarded as a special type of neurotransmitter. Dopamine affects many systems in the central nervous system, from motor control to cognitive executive functions and emotional limbic activities. Overall, dopamine and norepinephrine are involved in major functions in the brain, such as sensory processing, motor planning, plasticity, and memory encoding. They are also crucial in mood maintenance, motivation, and attention or concentration. An increase in the levels of these key neurotransmitters (neurohormones) is essential for improving motor performance and cognitive function.

[0086] Serotonin (5-hydroxytryptamine, 5-HT) is an inhibitory neurotransmitter involved in mood regulation. It is secreted by neurons in the brainstem and neurons that innervate the gastrointestinal tract (enteric nervous system). It regulates body temperature, pain perception, mood, and sleep cycles. Insufficient serotonin secretion can lead to a range of mood disorders, such as depression, anger control problems, and obsessive-compulsive disorder; these effects can have a negative impact on attitude and motivation and downregulate motor performance.

[0087] Interestingly, the compositions of the present invention showed a synergistic improvement in the levels of the above neurotransmitters in supplemented animals. For example, in rats supplemented with Centella asiatica magnesium-containing extract (CAMg-1) and 50% aqueous ethanol extract of Theobroma cacao seeds (Th-1), the norepinephrine (NE) levels increased by 13.1% and 18.4% respectively compared to control animals. In contrast, the norepinephrine (NE) level in rats supplemented with Composition 33 increased by 27.7%, which is a synergistic improvement compared to the efficacy shown by single extracts such as CAMg-1 and Th-1 (Table 11). Similarly, Compositions 34 and 35 also showed a synergistic increase in NE concentration compared to their respective individual components.

[0088] In addition, the compositions (Compositions 33 - 35) containing magnesium - containing Centella asiatica extract (CAMg - 1) and Theobroma cacao seed extract (Th - 1) also showed a synergistic effect in increasing serotonin and dopamine levels in supplemented rats, as shown in Table 11.

[0089] In this study, compared with rats supplemented with the corresponding individual extracts, Compositions 33 - 35 synergistically increased the levels of dopamine and norepinephrine in the brain tissues of supplemented rats and the level of serotonin in the serum. Given the physiological activity concepts of the above - mentioned neurotransmitters, the observations of this animal study suggest that supplementing with Compositions 33 - 35 provides a significant synergistic improvement in cognitive function, mood, motivation, alertness, and reaction time. Notably, the observations of the biomarkers provide a physiological basis for the nootropic activity of the above - mentioned compositions.

[0090] Therefore, the herbal composition of the present invention comprises: a first component selected from Centella asiatica extracts, which contain at least one component selected from the magnesium salts or complexes of asiatic acid, the magnesium salts or complexes of madecassic acid, or mixtures thereof, and also contain asiaticoside and madecassoside; a second component selected from Theobroma cacao extracts, their fractions, phytochemicals, or mixtures thereof; the composition may also optionally comprise at least one component selected from pharmaceutically, nutraceutically, or dietetically acceptable excipients, carriers, and diluents; the composition shows enhanced nootropic activity crucial for improving cognitive function and enhancing gaming performance.

[0091] Therefore, in an important embodiment, the present invention provides a synergistic herbal composition, which comprises: a first component selected from Centella asiatica extracts, which contain at least one component selected from the magnesium salts or complexes of asiatic acid, the magnesium salts or complexes of madecassic acid, or mixtures thereof, and also contain asiaticoside and madecassoside; a second component selected from Theobroma cacao extracts, their fractions, phytochemicals, or mixtures thereof; the composition is used to enhance nootropic activity crucial for improving cognitive function and enhancing gaming performance.

[0092] In a preferred embodiment, the synergistic herbal composition provided by the present invention comprises: a first component selected from Centella asiatica extracts, which contain at least one component selected from the magnesium salts or complexes of asiatic acid, the magnesium salts or complexes of madecassic acid, or mixtures thereof, and also contain asiaticoside and madecassoside; a second component selected from Theobroma cacao extracts, their fractions, phytochemicals, or mixtures thereof; and may also optionally comprise at least one additional component selected from biologic agents, nootropic agents, pharmaceutically acceptable active ingredients, vitamins, minerals, pharmaceutically, nutraceutically, or dietetically acceptable excipients, carriers, or diluents.

[0093] In other preferred embodiments, the synergistic herbal composition provided by the present invention comprises: a first component selected from centella asiatica extract, which extract contains at least one component selected from the magnesium salt or complex of asiatic acid, the magnesium salt or complex of madecassic acid, or a mixture thereof, and also contains asiaticoside and madecassoside; a second component selected from cocoa extract, its fractions, phytochemicals or a mixture thereof; wherein the concentration of centella asiatica extract in the composition is in the range of 10%-90% by weight, and the concentration of cocoa extract is in the range of 90%-10% by weight.

[0094] In another embodiment, the present invention discloses the above-mentioned synergistic herbal composition, wherein the composition is standardized to contain at least one centella asiatica phytochemical marker selected from asiatic acid, madecassic acid, asiaticoside, madecassoside or total asiaticosides, and contains at least one cocoa phytochemical reference marker compound or pharmacological activity marker, but not limited to theobromine.

[0095] In another embodiment, the synergistic herbal composition provided by the present invention comprises: a first component selected from centella asiatica extract, which extract contains at least one component selected from the magnesium salt or complex of asiatic acid, the magnesium salt or complex of madecassic acid, or a mixture thereof, and also contains asiaticoside and madecassoside; a second component selected from cocoa extract, its fractions, phytochemicals or a mixture thereof; wherein the content of total asiaticosides (asiaticoside group) is in the range of 2.0%-40%, and the magnesium content is in the range of 0.2%-5.0%.

[0096] In another embodiment, the present invention discloses the above-mentioned synergistic herbal composition, wherein the composition contains asiatic acid in the range of 0.1%-8%, madecassic acid in the range of 0.1%-8%, asiaticoside in the range of 0.1%-11%, madecassoside in the range of 0.1%-13% and magnesium in the range of 0.2%-5.0%.

[0097] In another embodiment, the present invention discloses the above-mentioned synergistic herbal composition, wherein the composition is standardized to contain at least one centella asiatica phytochemical component marker selected from asiatic acid, madecassic acid, asiaticoside, madecassoside or total asiaticosides; and contains at least one cocoa tree phytochemical reference marker compound or pharmacological activity marker, and is not limited to theobromine; wherein the concentration range of the phytochemical marker compound or a group of phytochemical compounds in the extract is 0.01% to 50% by weight. For example, cocoa tree extract, its fractions or a mixture thereof are standardized in terms of theobromine, and the theobromine content is in the range of 0.5% to 50%.

[0098] In another embodiment, in the above-disclosed compositions, the phytochemical reference marker compounds, the pharmacological activity markers or a set of markers and their magnesium salts can be determined by suitable analytical techniques known in the art, such as but not limited to high performance liquid chromatography (HPLC), gravimetry, ultraviolet spectrophotometry (UV), gas chromatography (GC) and inductively coupled plasma mass spectrometry (ICP-mass).

[0099] In yet another embodiment, the present invention provides the above-mentioned synergistic herbal composition; wherein the extracts or fractions of Centella asiatica and Theobroma cacao are obtained from at least one plant part selected from the group consisting of leaves, stems, shoots, twigs, aerial parts, whole fruits, pericarp, husk, immature fruits, seeds, flower heads, roots, bark, hardwood, rhizomes or whole plants or mixtures thereof.

[0100] In another embodiment, the present invention provides the above-disclosed synergistic herbal composition, wherein the extract, fraction or mixture thereof is prepared using at least one solvent selected from the following: C1-C5 alcohols such as ethanol, methanol, n-propanol, isopropanol; ketones such as acetone, methyl isobutyl ketone; chlorinated solvents such as dichloromethane and chloroform; water and mixtures thereof; C1-C7 hydrocarbons such as but not limited to hexane, heptane; esters such as but not limited to ethyl acetate and mixtures thereof.

[0101] In other embodiments, the extraction process of Centella asiatica and Theobroma cacao employs techniques known in the art, such as maceration, digestion, decoction, infusion, percolation, Soxhlet extraction, reflux extraction, continuous extraction, ultrasound-assisted extraction, microwave-assisted extraction, pressurized liquid / fluid extraction, supercritical carbon dioxide extraction. In addition, the extract or fraction can be further purified by column chromatography or resin chromatography using microporous resins.

[0102] In another embodiment, the present invention discloses a method for preparing a composition, which composition comprises: a first component selected from Centella asiatica extract, which extract contains at least one component selected from the group consisting of magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and also contains asiaticoside and madecassoside; a second component selected from Theobroma cacao extract, its fractions, phytochemicals or mixtures thereof; and the composition may further optionally comprise at least one component selected from pharmaceutically, nutraceutically or dietetically acceptable excipients, carriers and diluents.

[0103] In yet another embodiment, the present invention provides a method for obtaining at least one benefit related to cognitive function, mental ability, and / or nootropic activity, said benefits being selected from alertness, vigilance, attention, concentration / focus, neuromotor function (reaction time), memory (verbal, working, visual, logical), intelligence, learning, acquisition, retention, recall of information / communication, perception, reasoning, problem-solving, decision-making; reducing mental fatigue and improving gaming performance; wherein the method comprises administering to a person in need an effective dose of a composition comprising: a first component selected from extracts of Centella asiatica, said extract containing at least one component selected from magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and further containing asiaticoside and madecassoside; a second component selected from cocoa extracts, fractions thereof, phytochemicals, or mixtures thereof; the composition optionally further comprising at least one component selected from pharmaceutically, nutraceutically, or dietetically acceptable excipients, carriers, and diluents.

[0104] In yet another embodiment, the present invention provides the use of a synergistic herbal composition comprising: a first component selected from extracts of Centella asiatica, said extract containing at least one component selected from magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and further containing asiaticoside and madecassoside; a second component selected from cocoa extracts, fractions thereof, phytochemicals, or mixtures thereof; the composition optionally further comprising at least one component selected from pharmaceutically, nutraceutically, or dietetically acceptable excipients, carriers, and diluents; for obtaining at least one benefit related to cognitive function, mental ability, and / or nootropic activity, said benefits being selected from alertness, vigilance, attention, concentration / focus, neuromotor function (reaction time), memory (verbal, working, visual, logical), intelligence, learning, acquisition, retention, recall of information / communication, perception, reasoning, problem-solving, decision-making; reducing mental fatigue and improving gaming performance.

[0105] In another embodiment of the present invention, the composition disclosed above is formulated into a dosage form selected from dry powder form, liquid form, beverage, food, dietary supplement, or any suitable form, such as tablets, capsules, soft chewables, gummies, or gummy sticks.

[0106] In another embodiment of the present invention, the composition disclosed above is formulated into a nutritional / dietary supplement and can be designed / formulated into the dosage form of a health food or a food for specific health uses, such as solid foods like chocolate or nutrition bars, semi-solid foods like cream, jam, or gel, or beverages such as refreshing drinks, lactic acid bacteria drinks, drops, candies, chewing gums, gummies, yogurt, ice cream, pudding, red bean soft cakes, jelly, cookies, tea, soft drinks, fruit juices, milk, coffee, cereal foods, snack bars, etc.

[0107] In another embodiment of the present invention, the above-disclosed composition is formulated into a controlled-release tablet by using controlled-release polymer-based coating technologies (including nanotechnology, microencapsulation, hot melt extrusion, colloidal carrier systems, and other drug delivery systems) to obtain the desired therapeutic effect.

[0108] Those of ordinary skill in the art will understand that the above embodiments can be changed without departing from the broad concept of the present invention. Therefore, it can be understood that the present invention is not limited to the specific embodiments or examples disclosed herein, but is intended to cover modifications within the scope and objectives of the present invention as defined in this specification. These examples are provided for illustrative purposes only and should not be construed as a limitation of the present disclosure, as many variations are possible without departing from the spirit of the present disclosure.

[0109] Examples

[0110] Example 1: Preparation of the aerial part extract of Centella asiatica (CAMg-1), which is a mixture of the 70% ethanol aqueous solution extract and the extract of the residue after water extraction, containing magnesium salts of asiatic acid and madecassic acid, as well as asiaticoside and madecassoside.

[0111] The dried raw material of the aerial part of Centella asiatica was pulverized, 50 grams of the powder was taken, and extracted with 400 ml of 70% ethanol aqueous solution under heating conditions for 2 hours. The mixture was filtered, and the extraction process was repeated with 70% ethanol aqueous solution (2 × 300 ml). 280 mg of magnesium oxide was added to the combined 70% ethanol aqueous solution extract, and the mixture was stirred at room temperature for 2 hours. The solution was filtered, and the filtrate was evaporated under reduced pressure to the minimum volume. The extracted raw material was extracted with 400 ml of water at room temperature for 3 hours. The mixture was filtered, and the extraction process was repeated with water (2 × 300 ml). 90 mg of magnesium oxide was added to the combined water extract, and the mixture was stirred at room temperature for 2 hours. The mixture was filtered, and the filtrate was combined with the concentrated 70% ethanol aqueous solution extract. The combined extract was evaporated under reduced pressure to obtain the magnesium-containing Centella asiatica extract (CAMg-1), which is a brown solid (15.0 grams).

[0112] The asiatic acid, madecassic acid, asiaticoside, and madecassoside (i.e., the total amount of asiaticosides) in the product were analyzed by analytical high performance liquid chromatography (HPLC), and the magnesium content was determined by inductively coupled plasma mass spectrometry (ICP-MS). It was found that the product contains 1.36% of asiatic acid, 2.20% of madecassic acid, 3.59% of asiaticoside, and 5.88% of madecassoside (i.e., the total amount of asiaticosides is 13.03%) and 1.09% of magnesium.

[0113] Comparative Example 1A (CA-1): The Centella asiatica extract (CA-1) for comparison is a mixture of 70% aqueous ethanol extract and water extract sequentially extracted, containing asiatic acid, madecassic acid, asiaticoside, and madecassoside. Its preparation method is the same as that of Example 1, but the magnesium oxide treatment step is not carried out.

[0114] Analytical HPLC was used to analyze asiatic acid, madecassic acid, asiaticoside, and madecassoside (i.e., total asiaticosides) in the product (CA-1), and ICP-MS was used to determine the magnesium content. It was found that the product contained 1.23% asiatic acid, 1.96% madecassic acid, 2.82% asiaticoside, and 4.50% madecassoside (i.e., the total amount of asiaticosides was 10.51%).

[0115] Example 2: Prepare a 70% aqueous ethanol extract of the aerial parts of Centella asiatica, which contains magnesium salts of asiatic acid, magnesium salts of madecassic acid, asiaticoside, and madecassoside (CAMg-2).

[0116] The dried raw material of the aerial parts of Centella asiatica was pulverized, 50 g of the powder was taken, and extracted with 400 ml of 70% aqueous ethanol under heating conditions for 2 hours. The mixture was filtered, and the extraction process was repeated with 70% aqueous ethanol (2 × 300 ml). 280 mg of magnesium oxide was added to the combined 70% aqueous ethanol extract, and stirred at room temperature for 2 hours. The mixture was filtered, and the filtrate was evaporated under reduced pressure to obtain a brown solid product (CAMg-2) (11.3 g).

[0117] Analytical HPLC was used to analyze asiatic acid, madecassic acid, asiaticoside, and madecassoside (i.e., total asiaticosides) in the product, and ICP-MS was used to determine the magnesium content. It was found that the product contained 1.85% asiatic acid, 2.13% madecassic acid, 3.76% asiaticoside, and 5.95% madecassoside (i.e., the total amount of asiaticosides was 13.69%) and 0.61% magnesium.

[0118] Comparative Example 2A (CA-2): The 70% aqueous ethanol extract of the aerial parts of Centella asiatica (CA-2) for comparison was prepared in the same manner as in Example 2, but the magnesium oxide treatment step was not carried out.

[0119] Analytical HPLC was used to analyze asiatic acid, madecassic acid, asiaticoside, and madecassoside (i.e., total asiaticosides) in the product, and ICP-MS was used to determine the magnesium content. It was found that the product contained 1.46% asiatic acid, 2.34% madecassic acid, 3.56% asiaticoside, and 5.53% madecassoside (i.e., the total amount of asiaticosides was 12.88%).

[0120] Example 3: Preparation of the aqueous extract from the aerial parts of Centella asiatica (L.) Urb. (CAMg-3), which contains asiaticoside magnesium salt, madecassoside magnesium salt, asiaticoside and madecassoside.

[0121] The dried raw material of the aerial parts of Centella asiatica (L.) Urb. was pulverized, and 50 g of the powder was taken and extracted with 400 mL of water for 2 hours under heating conditions. The mixture was filtered, and the extraction process was repeated with 2 × 300 mL of water. 230 mg of magnesium oxide was added to the combined aqueous extract, and the mixture was stirred at room temperature for 2 hours. The mixture was filtered, and the filtrate was evaporated under reduced pressure to obtain a brown solid product (CAMg-3) (13.0 g).

[0122] The asiaticoside content in the product was determined by HPLC, and the magnesium content was determined by ICP-MS. It was found that the extract contained 7.95% asiaticosides and 1.45% magnesium.

[0123] Example 4: Preparation of the 70% aqueous methanol extract from the aerial parts of Centella asiatica (L.) Urb. (CAMg-4), which contains asiaticoside magnesium salt, madecassoside magnesium salt, asiaticoside and madecassoside.

[0124] The dried raw material of the aerial parts of Centella asiatica (L.) Urb. was pulverized, and 50 g of the powder was taken and extracted with 400 mL of 70% aqueous methanol under heating conditions for 2 hours. The mixture was filtered, and the extraction process was repeated with 70% aqueous methanol (2 × 300 mL). 280 mg of magnesium oxide was added to the combined 70% aqueous methanol extract, and the mixture was stirred at room temperature for 2 hours. The mixture was filtered, and the filtrate was evaporated under reduced pressure to obtain a brown solid product (CAMg-4) (14.2 g).

[0125] Analytical high performance liquid chromatography (HPLC) was used to analyze asiatic acid, madecassic acid, asiaticoside and madecassoside (i.e., asiaticosides) in the product, and inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the magnesium content. It was found that the product contained 2.86% asiatic acid, 2.01% madecassic acid, 0.99% asiaticoside and 0.58% madecassoside (i.e., the total asiaticoside content was 6.44%) and 1.02% magnesium.

[0126] Example 5: Preparation of the 70% aqueous acetone extract from the aerial parts of Centella asiatica (L.) Urb. (CAMg-5), which contains asiaticoside magnesium salt, madecassoside magnesium salt, asiaticoside and madecassoside.

[0127] The aerial parts of dry Centella asiatica grass as raw materials were pulverized, and powder (50 g) was taken and extracted with 70% aqueous acetone solution (400 mL) under heating conditions for 2 hours. The mixture was filtered, and then the extraction process was repeated with 70% aqueous acetone solution (twice, 300 mL each time). Magnesium oxide (280 mg) was added to the combined 70% aqueous acetone solution extract, and it was stirred at room temperature for 2 hours. The mixture was filtered, and the filtrate was evaporated under reduced pressure to obtain a brown solid product (CAMg-5) (5.8 g).

[0128] The asiatic acid, madecassic acid, asiaticoside, and madecassoside (total asiaticosides) in this product were analyzed by high performance liquid chromatography (HPLC), and at the same time, magnesium element was analyzed by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that this product contained 3.23% asiatic acid, 3.13% madecassic acid, 4.33% asiaticoside, and 2.90% madecassoside (i.e., the total content of total asiaticosides was 13.58%), and contained 1.21% magnesium element.

[0129] Example 6: Evaluation of the nootropic activity of herbal extracts in Sprague-Dawley rats

[0130] Treatment method: Thirty healthy male Sprague-Dawley rats (age: 6 - 8 weeks) were randomly divided into five groups (n = 6 in each group). For 30 days, animals were respectively supplemented with 50 mg / kg of CA-1 (Group G2), CAMg-1 (Group G3), CA-2 (Group G4), or CAMg-2 (Group G5) contained in 10 mL of 0.5% sodium carboxymethyl cellulose (CMC) aqueous solution by gavage every day. During the study period, rats in the control group (Group G1) only received the excipient (10 mL of 0.5% CMC aqueous solution). On the 31st day of the study, all animals were subjected to the T-maze test.

[0131] Test parameters: According to the procedures disclosed in the literature, the T-maze reward alternation test (working memory) and the orienting response test (ORT, attention and reaction time) were evaluated, and the results are shown in Table 1 and Table 2. The working memory index was calculated using the following formula.

[0132] Working memory index = Number of correct choices / Total number of trials or attempts

[0133] Table 1: Effects of herbal extract supplements on the working memory index of Sprague-Dawley rats in the T-maze reward alternation test

[0134]

[0135]

[0136] Note: The higher the working memory index value, the better the efficacy.

[0137] Table 2: Effects of Herbal Extract Supplements on Response Time of Sprague-Dawley Rats in the Orienting Response Test (ORT)

[0138]

[0139] Note: The shorter the response time, the better the efficacy.

[0140] Example 7: Preparation of 50% Aqueous Ethanol Extract (Th-1) of Cocoa Seeds

[0141] The cocoa seeds (100 g) were pulverized, and the resulting powder was extracted with 700 ml of 50% aqueous ethanol under heating conditions for 1 hour. The extract was filtered, and the extracted raw material was extracted twice again with 50% aqueous ethanol (2 × 500 ml) under the same conditions. The extracts were combined and filtered, and then concentrated under vacuum to obtain the residue of the 50% aqueous ethanol extract (Th-1; 11.2 g).

[0142] Example 8: Preparation of 90% Aqueous Ethanol, Ethanol, and Water Extracts of Cocoa Seeds

[0143] Using an extraction method similar to that described in Example 7, 90% aqueous ethanol, ethanol, and water were used as extraction solvents, respectively, to obtain 90% aqueous ethanol extract (Th-2; 4.5 g), ethanol extract (Th-3; 4.1 g), and water extract (Th-4; 12.2 g) of cocoa seeds.

[0144] Example 9: Preparation of Methanol and Acetone Extracts of Cocoa Seeds

[0145] Using an extraction method similar to that described in Example 7, methanol and acetone were used as extraction solvents, respectively, to obtain methanol extract (Th-5; 5.8 g) and acetone extract (Th-6; 27 g) of cocoa seeds.

[0146] Example 10: Standardization of Cocoa Seed Extracts

[0147] Using analytical high performance liquid chromatography (HPLC), various cocoa seed extracts were standardized with theobromine as the standard, and the results are summarized in Table 3.

[0148] Table 3: HPLC Analysis Results of Cocoa Seed Extracts

[0149]

[0150] Example 11: Prepare various compositions, which contain a mixture (CAMg-1) of a 70% aqueous ethanol solution and a water extract of the aerial parts of Centella asiatica containing madecassoside magnesium salt, hydroxymadecassoside magnesium salt, asiaticoside and hydroxyasiaticoside obtained successively, and a 50% aqueous ethanol extract of Theobroma cacao seeds (Th-1).

[0151] Composition 1 (Comp-1): C-1 is prepared by mixing CAMg-1 and Th-1 in a ratio of 3:1.

[0152] Composition 2 (Comp-2): C-2 is prepared by mixing CAMg-1 and Th-1 in a ratio of 2:1.

[0153] Composition 3 (Comp-3): C-3 is prepared by mixing CAMg-1 and Th-1 in a ratio of 1:1.

[0154] Composition 4 (Comp-4): C-4 is prepared by mixing CAMg-1 and Th-1 in a ratio of 1:2.

[0155] Composition 5 (Comp-5): C-5 is prepared by mixing CAMg-1 and Th-1 in a ratio of 1:3.

[0156] Control Composition (comp-3A): Comp-3A is prepared by mixing a mixture (CA-1) of a 70% aqueous ethanol solution and a water extract of the aerial parts of Centella asiatica without magnesium obtained successively with a 50% aqueous ethanol extract of Theobroma cacao seeds (Th-1) in a ratio of 1:1.

[0157] Example 12: Prepare various compositions, which contain a mixture (CAMg-1) of a 70% aqueous ethanol solution and a water extract of the aerial parts of Centella asiatica containing madecassoside magnesium salt, hydroxymadecassoside magnesium salt, asiaticoside and hydroxyasiaticoside obtained successively, and a 90% aqueous ethanol extract of Theobroma cacao seeds (Th-2).

[0158] Composition 6 (Comp-6): C-6 is prepared by mixing CAMg-1 and Th-2 in a ratio of 2:1.

[0159] Composition 7 (Comp-7): C-7 is prepared by mixing CAMg-1 and Th-2 in a ratio of 1:1.

[0160] Composition 8 (Comp-8): C-8 is prepared by mixing CAMg-1 and Th-2 in a ratio of 1:2.

[0161] Example 13: Prepare various compositions containing a mixture (CAMg-1) of a 70% ethanol aqueous solution and a water extract of the aerial parts of Centella asiatica containing madecassoside magnesium salt, asiaticoside magnesium salt, asiaticoside, and madecassoside obtained successively, and an ethanol extract of Theobroma cacao seeds (Th-3).

[0162] Composition 9 (Comp-9): C-9 is prepared by mixing CAMg-1 and Th-3 in a ratio of 2:1.

[0163] Composition 10 (Comp-10): C-10 is prepared by mixing CAMg-1 and Th-3 in a ratio of 1:1.

[0164] Composition 11 (Comp-11): C-11 is prepared by mixing CAMg-1 and Th-3 in a ratio of 1:2.

[0165] Example 14: Prepare various compositions containing a mixture (CAMg-1) of a 70% ethanol aqueous solution and a water extract of the aerial parts of Centella asiatica containing madecassoside magnesium salt, asiaticoside magnesium salt, asiaticoside, and madecassoside obtained successively, and a water extract of Theobroma cacao seeds (Th-4).

[0166] Composition 12 (Comp-12): C-12 is prepared by mixing CAMg-1 and Th-4 in a ratio of 2:1.

[0167] Composition 13 (Comp-13): C-13 is prepared by mixing CAMg-1 and Th-4 in a ratio of 1:1.

[0168] Composition 14 (Comp-14): C-14 is prepared by mixing CAMg-1 and Th-4 in a ratio of 1:2.

[0169] Example 15: Prepare various compositions containing a mixture (CAMg-1) of a 70% ethanol aqueous solution and a water extract of the aerial parts of Centella asiatica containing madecassoside magnesium salt, asiaticoside magnesium salt, asiaticoside, and madecassoside obtained successively, and a methanol extract of Theobroma cacao seeds (Th-5).

[0170] Composition 15 (Comp-15): C-15 is prepared by mixing CAMg-1 and Th-5 in a ratio of 2:1.

[0171] Composition 16 (Comp-16): C-16 is prepared by mixing CAMg-1 and Th-5 in a ratio of 1:1.

[0172] Composition 17 (Comp-17): C-17 is prepared by mixing CAMg-1 and Th-5 in a ratio of 1:2.

[0173] Example 16: Prepare various compositions containing a mixture (CAMg-1) of a 70% ethanol aqueous solution and a water extract of the aerial parts of Centella asiatica containing madecassoside magnesium salt, asiaticoside magnesium salt, asiaticoside, and madecassoside obtained successively, and an acetone extract of Theobroma cacao seeds (Th-6).

[0174] Composition 18 (Comp-18): C-18 is prepared by mixing CAMg-1 and Th-6 in a ratio of 2:1.

[0175] Composition 19 (Comp-19): C-19 is prepared by mixing CAMg-1 and Th-6 in a ratio of 1:1.

[0176] Composition 20 (Comp-20): C-20 is prepared by mixing CAMg-1 and Th-6 in a ratio of 1:2.

[0177] Example 17: Prepare various compositions containing a 70% ethanol aqueous extract (CAMg-2) of the aerial parts of Centella asiatica containing madecassoside magnesium salt, asiaticoside magnesium salt, asiaticoside, and madecassoside, and a 50% ethanol aqueous extract of Theobroma cacao seeds (Th-1).

[0178] Composition 21 (Comp-21): C-21 is prepared by mixing CAMg-2 and Th-1 in a ratio of 2:1.

[0179] Composition 22 (Comp-22): C-22 is prepared by mixing CAMg-2 and Th-1 in a ratio of 1:1.

[0180] Composition 23 (Comp-23): C-23 is prepared by mixing CAMg-2 and Th-1 in a ratio of 1:2.

[0181] Example 18: Prepare various compositions containing a water extract (CAMg-3) of the aerial parts of Centella asiatica containing madecassoside magnesium salt, asiaticoside magnesium salt, asiaticoside, and madecassoside, and an ethanol extract of Theobroma cacao seeds (Th-3).

[0182] Composition 24 (Comp-24): C-24 is prepared by mixing CAMg-3 and Th-3 in a ratio of 2:1.

[0183] Composition 25 (Comp-25): C-25 is prepared by mixing CAMg-3 and Th-3 in a ratio of 1:1.

[0184] Composition 26 (Comp-26): C-26 is prepared by mixing CAMg-3 and Th-3 in a ratio of 1:2.

[0185] Example 19: Preparation of various compositions containing a 70% methanol aqueous extract of the aerial parts of Centella asiatica containing asiaticoside magnesium salt, madecassoside magnesium salt, asiaticoside and madecassoside (CAMg-4), and an acetone extract of Theobroma cacao seeds (Th-6).

[0186] Composition 27 (Comp-27): C-27 is prepared by mixing CAMg-4 and Th-6 in a ratio of 2:1.

[0187] Composition 28 (Comp-28): C-28 is prepared by mixing CAMg-4 and Th-6 in a ratio of 1:1.

[0188] Composition 29 (Comp-29): C-29 is prepared by mixing CAMg-4 and Th-6 in a ratio of 1:2.

[0189] Example 20: Preparation of various compositions containing a 70% acetone aqueous extract of the aerial parts of Centella asiatica containing asiaticoside magnesium salt, madecassoside magnesium salt, asiaticoside and madecassoside (CAMg-5), and a methanol extract of Theobroma cacao seeds (Th-5).

[0190] Composition 30 (Comp-30): C-30 is prepared by mixing CAMg-5 and Th-5 in a ratio of 2:1.

[0191] Composition 31 (Comp-31): C-31 is prepared by mixing CAMg-5 and Th-5 in a ratio of 1:1.

[0192] Composition 32 (Comp-32): C-32 is prepared by mixing CAMg-5 and Th-5 in a ratio of 1:2.

[0193] Example 21: General method for the determination of acetylcholinesterase inhibitory activity

[0194] The activity of acetylcholinesterase was determined using the substrate acetylthiocholine iodide, which is converted to thiocholine. Thiocholine reacts with the chromogenic substrate 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to produce the yellow anion of 2-thio-2-nitrobenzoic acid, which has a strong absorbance at 412 nm. The determination of acetylcholinesterase (AChE) was carried out according to the method of Ellman et al. with some minor modifications, using acetylthiocholine iodide as the substrate (Lee J. H. et al., Arch. Pharm. Res. 2004, 27(1):53 - 56). The Ellman reaction mixture contained 1.0 mmol / L acetylthiocholine iodide and 0.5 mmol / L 5,5'-dithiobis(2-nitrobenzoic acid) in 50 mmol / L sodium phosphate buffer (pH 8.0). The assay mixture contained 50 μL of 50 mmol / L phosphate buffer at pH 8.0, 30 μL of the test substance at different concentrations, and 20 μL of the enzyme (100 mU / mL). For the blank group, the enzyme was replaced with phosphate buffer. After thoroughly mixing the reaction mixture, 100 μL of the Ellman reagent was added and incubated at room temperature for 10 minutes. The absorbance was measured at 412 nm using a microplate reader. The percentage of enzyme activity inhibition was calculated by comparing the optical density (OD) of the test wells with that of the control wells using the following formula: Inhibition percentage = [(control - sample) / control]×100. The results of the acetylcholinesterase inhibitory activities of the single extracts and their combinations are summarized in Tables 4 - 8.

[0195] Table 4: Acetylcholinesterase inhibitory activities of compositions containing mixtures of the aerial parts extracts of Centella asiatica with magnesium (CAMg-1) or without magnesium (CA-1) sequentially extracted with 70% aqueous ethanol and water, and 50% aqueous ethanol extract of Theobroma cacao seeds (Th-1)

[0196]

[0197] Table 5: Acetylcholinesterase inhibitory activities of compositions containing mixtures of the aerial parts extracts of Centella asiatica with magnesium (CAMg-1) sequentially extracted with 70% aqueous ethanol and water, and 90% aqueous ethanol extract (Th-2), ethanol extract (Th-3) or water extract (Th-4) of Theobroma cacao seeds

[0198]

[0199]

[0200] Table 6: Acetylcholinesterase inhibitory activity of the composition containing a mixture of the aerial part extract of Centella asiatica containing magnesium (CAMg-1) successively extracted with 70% aqueous ethanol solution and water, and the methanol extract (Th-5) or acetone extract (Th-6) of Theobroma cacao seeds

[0201]

[0202] Table 7: Acetylcholinesterase inhibitory activity of the composition containing the 70% aqueous ethanol extract of Centella asiatica containing magnesium (CAMg-2) and the 50% aqueous ethanol extract of Theobroma cacao seeds (Th-1), and the composition containing the aqueous extract of Centella asiatica containing magnesium (CAMg-3) and the ethanol extract of Theobroma cacao seeds (Th-3)

[0203]

[0204] Table 8: Acetylcholinesterase inhibitory activity of the composition containing the 70% aqueous methanol extract of Centella asiatica containing magnesium (CAMg-4) and the acetone extract of Theobroma cacao seeds (Th-6), and the composition containing the 70% aqueous acetone extract of Centella asiatica containing magnesium (CAMg-5) and the methanol extract of Theobroma cacao seeds (Th-5)

[0205]

[0206] Example 22: Formulation of the composition

[0207] Composition 34 (the ratio of CAMg-1 to Th-1 is 1:1): The mixture of the aerial part extract of Centella asiatica (CAMg-1, 6.0 kg) was stirred with water (14 L) at room temperature for 10 to 15 minutes. The 50% aqueous ethanol extract of Theobroma cacao seeds (Th-1, 3.0 kg) was slowly added to the mixture, and it was stirred for another 10 to 15 minutes. After thoroughly mixing the contents, rice maltodextrin (0.8 kg) was added, and it was further stirred for 20 to 30 minutes. The contents were dried under reduced pressure at room temperature to obtain a flaky product. These flakes were uniformly mixed with colloidal silica (0.2 kg) in a polyethylene container or in a suitable stirrer. The material was further pulverized and sieved through a 40-mesh sieve to obtain a fine powder composition 33.

[0208] Composition 35 (the ratio of CAMg-1 to Th-1 is 1:2):Stir a mixture of CAMg-1 (4.5 kg) and water (10 L) at room temperature for 10 to 15 minutes. Slowly add Th-1 (4.5 kg) to this mixture and stir for another 10 to 15 minutes. After thoroughly mixing the contents, add rice maltodextrin (0.8 kg) and continue stirring for 20 to 30 minutes. Dry the contents under reduced pressure at room temperature to obtain a flaky product. Uniformly mix these flakes with colloidal silica (0.2 kg) in a polyethylene container or in a suitable stirrer. Further pulverize the material and screen it through a 40-mesh sieve to obtain a fine powder composition 34.

[0209] ​ Stir a mixture of CAMg-1 (3.0 kg) and water (10 L) at room temperature for 10 to 15 minutes. Slowly add Th-1 (6.0 kg) to this mixture and stir for another 10 to 15 minutes. After thoroughly mixing the contents, add rice maltodextrin (0.8 kg) and continue stirring for 20 to 30 minutes. Dry the contents under reduced pressure at room temperature to obtain a flaky product. Uniformly mix these flakes with colloidal silica (0.2 kg) in a polyethylene container or in a suitable stirrer. Further pulverize the material and screen it through a 40-mesh sieve to obtain a fine powder composition 35.

[0210] Example 23: Evaluation of the nootropic activity of herbal extracts and their compositions in Sprague-Dawley rats

[0211] Treatment method: Randomly divide thirty-six healthy male Sprague-Dawley rats (age: 6 to 8 weeks) into six groups (n = 6 per group). Over a period of 30 days, supplement these rats by gavage with 50 mg / kg of CAMg-1 (Group G2), Th-1 (Group G3), Comp-33 (Group G4), Comp-34 (Group G5), or Comp-35 (Group G6) contained in 10 mL of an aqueous solution of 0.5% carboxymethylcellulose sodium (CMC). During the study period, the rats in the control group (Group G1) received only the excipient (10 mL of 0.5% CMC aqueous solution). On the 31st day of the study, perform a T-maze test on all rats.

[0212] Test parameters: Evaluate the T-maze reward alternation test (working memory) and the orienting response test (ORT, attention and reaction time) according to the procedures disclosed in the literature. The results are shown in Tables 9 and 10. The working memory index is calculated using the following formula:

[0213] Working memory index = Number of correct choices / Total number of trials or attempts

[0214] Table 9: Effects of supplementation with herbal extracts and their combinations on the working memory index of Sprague-Dawley rats in the T-maze reward alternation test

[0215]

[0216] Note: The higher the value, the better the efficacy.

[0217] Table 10: Effects of supplementation with herbal extracts and their combinations on the reaction time of Sprague-Dawley rats in the orienting response test (ORT)

[0218]

[0219] Note: The lower the value, the better the efficacy.

[0220] Measurement of norepinephrine: Measure the norepinephrine level in the brain tissue of the cerebral cortex region. Weigh approximately 30 mg of cerebral cortex tissue and collect it in 1-fold phosphate buffer (1XPBS, 270 μL, pH 7.4). Homogenize the tissue using a Dounce homogenizer under ice-cold conditions. After homogenization, sonicate the tissue in a probe-type sonicator for 30 seconds. Centrifuge the tissue homogenate at 21952 x g for 15 minutes at 4°C. Collect the supernatant and measure the protein content using the Pierce TM BCA Protein Assay Kit (Catalog No. 23225, Thermo Fisher Scientific, Waltham, Massachusetts, USA). Use the NA / NE (norepinephrine / noradrenaline) enzyme-linked immunosorbent assay (ELISA) kit (Catalog No. E-EL-0047) from Wuhan Elabscience Biotechnology Co., Ltd. to detect norepinephrine according to the manufacturer's protocol. The brief steps are as follows: In a 96-well microplate strip, add 50 μL of the standard, blank control, and tissue lysate samples to the corresponding wells respectively. Immediately add 50 μL of the biotinylated detection antibody working solution to all wells, seal the microplate, and incubate at 37°C for 45 minutes. Wash the microplate 3 times with the wash buffer; add 100 μL of the horseradish peroxidase (HRP) conjugate solution, seal the microplate, and incubate at 37°C for 30 minutes. After washing (3 times) with the wash buffer, add 90 μL of the substrate reagent, seal the microplate, and incubate at 37°C for 15 minutes. Finally, add 50 μL of the stop solution to each well, mix well, and measure the absorbance at 450 nm in a microplate reader (SpectraMax5e, Molecular Devices, San Jose, California, USA) with a correction wavelength of 650 nm. Determine the norepinephrine concentration in the tissue lysate according to the standard curve plotted using the norepinephrine standard and normalize it according to the protein content in the tissue lysate.

[0221] Measurement of Serotonin: Measure the serotonin level in the brain tissue of the hippocampal region. Weigh approximately 30 mg of brain hippocampal tissue and collect it in 1-fold phosphate buffered saline (1XPBS, 270 μL, pH 7.4). Homogenize the tissue using a Dounce homogenizer under cold conditions. After homogenization, sonicate the tissue in a probe sonicator for 30 seconds. Centrifuge the tissue homogenate at 21952 x g for 15 minutes at 4°C. Collect the supernatant and measure the protein content using the Pierce TM BCA Protein Assay Kit (Cat. No. 23225, Thermo Fisher Scientific, Waltham, Massachusetts, USA). Use the ST / 5-HT (Serotonin / 5-Hydroxytryptamine) Enzyme-Linked Immunosorbent Assay (ELISA) Kit (Cat. No. E-EL-0033) from Wuhan Elabscience Biotechnology Co., Ltd. to detect serotonin according to the manufacturer's protocol. The brief steps are as follows: In a 96-well microplate strip, add 50 μL of the standard, blank control, and tissue lysate samples to the corresponding wells respectively. Immediately add 50 μL of the biotinylated detection antibody working solution to all wells, seal the microplate, and incubate at 37°C for 45 minutes. Wash the microplate 3 times with the wash buffer; add 100 μL of the horseradish peroxidase (HRP) conjugate solution, seal the microplate, and incubate at 37°C for 30 minutes. After washing (3 times) with the wash buffer, add 90 μL of the substrate reagent, seal the microplate, and incubate at 37°C for 15 minutes. Finally, add 50 μL of the stop solution to each well, mix well, and measure the absorbance at 450 nm in a microplate reader (SpectraMax 5e, Molecular Devices, San Jose, California, USA) with a correction wavelength of 650 nm. Determine the serotonin concentration in the tissue lysate according to the standard curve plotted using the serotonin standard and normalize it according to the protein content in the tissue lysate.

[0222] Measurement of dopamine: The dopamine level in serum samples was measured using the LDN Dopamine Quick ELISA Kit (product number BAE - 6300) from LDN in Nordhorn, Germany, according to the manufacturer's protocol. The brief steps are as follows: Dopamine in serum samples or standards was extracted and acylated using the extraction and acylation buffers provided in the kit. In a 96 - well dopamine microplate strip, 25 μL of enzyme solution, the extracted standards, controls, and extracted serum samples were added respectively, and incubated on a shaker at room temperature (RT) for 30 minutes. After incubation, 25 μL of dopamine antiserum was added, the microplate was covered with sticky foil, and incubated on a shaker at room temperature for 2 hours. The microplate was washed 3 times with wash buffer; 100 μL of enzyme conjugate was added and incubated on a shaker at room temperature for 30 minutes. After washing (3 times) with wash buffer, 100 μL of substrate was added to the microplate and incubated on a shaker at ambient temperature (20 - 25 °C) for 30 minutes. Finally, 100 μL of stop solution was added to each well, mixed well, and the absorbance was measured at 450 nm in a microplate reader (SpectraMax5e, Molecular Devices, San Jose, California, USA), with a correction wavelength of 650 nm. The dopamine concentration in serum samples was calculated according to the standard curve plotted using dopamine standards. The results are shown in Table 11.

[0223] Table 11: Biomarker data

[0224]

[0225]

[0226] Note: The higher the average concentration value, the better the efficacy.

[0227] Although the present invention has been described in detail above for purposes of illustration, it should be understood that these details are solely for that purpose, and those skilled in the art can make various changes therein without departing from the spirit and scope of the present invention, unless such changes may be limited by the claims.

Claims

1. A synergistic herbal composition, characterized in that, The composition comprises: a first component selected from Centella asiatica extract, said extract containing at least one component selected from the following: magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and also containing asiaticoside and madecassoside; a second component selected from extracts, fractions, phytochemicals of Theobroma cacao or mixtures thereof; the composition is for enhancing nootropic activities crucial for improving cognitive function and enhancing gaming performance.

2. The synergistic herbal composition according to claim 1, characterized in that, The concentration of the Centella asiatica extract is in the range of 10% - 90% by weight, and the concentration of the Theobroma cacao extract is in the range of 90% - 10% by weight.

3. The synergistic herbal composition according to claim 1, characterized in that, The total asiaticoside content is in the range of 1.0% - 40%.

4. The synergistic herbal composition according to claim 3, characterized in that, The total asiaticoside content includes: asiatic acid content in the range of 0.1% - 8.0%, madecassic acid content in the range of 0.2 - 8.0%, asiaticoside content in the range of 0.1 - 11.0%, madecassoside content in the range of 0.1 - 13.0%.

5. The synergistic herbal composition according to claim 1, characterized in that, The magnesium content in the salts or complexes of asiatic acid and the salts or complexes of madecassic acid is in the range of 0.1% to 10%.

6. The synergistic composition according to claim 1, characterized in that, The extract, fraction, active compound or phytochemical corresponding to each component in the composition is obtained from at least one plant part including leaves, stems, shoots, twigs, aerial parts, whole fruits, pericarp, seeds, roots, bark, bulbs, hardwoods, gum resins or whole plants or mixtures thereof.

7. The synergistic herbal composition according to claim 6, characterized in that, The composition is standardized to contain at least one Centella asiatica marker selected from asiatic acid, madecassic acid, asiaticoside and madecassoside or total asiaticosides; and contain at least one Theobroma cacao phytochemical reference marker compound or pharmacological activity marker, but not limited to theobromine.

8. The synergistic composition according to claim 1, characterized in that, The solvents for the extracts, fractions or mixtures thereof are selected from C1 - C5 alcohols such as ethanol, methanol, n - propanol, isopropanol; ketones such as acetone, methyl isobutyl ketone; chlorinated solvents such as dichloromethane and chloroform; C1 - C7 hydrocarbons such as but not limited to hexane, heptane; esters such as but not limited to ethyl acetate and mixtures thereof; water and mixtures thereof.

9. The synergistic composition according to claim 1, characterized in that, Optionally, the composition comprises at least one additional component selected from the following: biologics, nootropics; pharmaceutically acceptable active ingredients, vitamins, minerals; Pharmaceutically, nutraceutically or dietetically acceptable excipients, carriers or diluents.

10. The synergistic composition according to claim 9, characterized in that, Pharmaceutically, nutraceutically or dietetically acceptable excipients, carriers and diluents are selected from: monosaccharides such as glucose, dextrose, fructose, galactose, etc.; disaccharides such as but not limited to sucrose, maltose, lactose, lactulose, trehalose, cellobiose, chitobiose, etc.; polysaccharides such as starch and modified starches such as sodium carboxymethyl starch, pregelatinized starch, soluble starch, superdispersant A and other modified starches; dextrins produced by hydrolysis of starch or glycogen such as yellow dextrin, white dextrin, maltodextrin, glucose dextrin 12D, rice maltodextrin, tapioca maltodextrin, etc.; polyols or sugar alcohols such as but not limited to sorbitol, mannitol, inositol, xylitol, isomalt, etc.; cellulose-based derivatives such as but not limited to microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, etc.; silicates such as but not limited to neusilin, veegum bentonite, talc, colloidal silica, sylodent, etc.; metal stearates such as but not limited to calcium stearate, magnesium stearate, zinc stearate, etc.; organic acids such as citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, etc.; esters of fatty acid esters and polysorbates; natural gums such as but not limited to gum arabic, carrageenan, guar gum, xanthan gum, etc.; B vitamins, niacinamide, calcium pantothenate; amino acids, proteins such as but not limited to casein, gelatin, pectin, agar; organometallic salts such as but not limited to sodium chloride, calcium chloride, calcium hydrogen phosphate, zinc sulfate, zinc chloride, etc.; natural pigments, flavoring agents, first and second class preservatives, and aqueous, alcoholic, hydroalcoholic, organic solutions of the ingredients listed above alone or in combination.

11. The synergistic herbal composition according to claim 1, characterized in that, The composition is formulated into a dosage form selected from dry powder form, liquid form, beverage, food, dietary supplement, or any suitable form such as tablets, capsules, soft chewables, gummies or gummy sticks.

12. The synergistic herbal composition according to any one of the preceding claims, characterized in that, The composition is formulated into a nutritional / dietary supplement and can be designed / formulated into the dosage form of a health food or a food for specific health use, such as solid foods like chocolate or nutrition bars, semi-solid foods like cream, jam or gel, or beverages like energy drinks, lactic acid bacteria drinks, drops, candies, chewing gums, gummy candies, yogurts, ice creams, puddings, soft red bean jelly, jellies, cookies, teas, soft drinks, fruit juices, milk, coffee, cereal foods, snack bars, etc.

13. The synergistic herbal composition according to claim 1, characterized in that, The composition is made into a controlled-release tablet with a coating based on a controlled-release polymer, using techniques including nanotechnology, microencapsulation, hot melt extrusion, colloidal carrier systems and other drug delivery systems, etc., to obtain the desired therapeutic effect.

14. A method for preparing a synergistic herbal composition, characterized in that, The composition comprises: a first component selected from extracts of Centella asiatica, the extract containing at least one component selected from the following: magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and also containing asiaticoside and madecassoside; a second component selected from extracts, fractions, phytochemicals of Theobroma cacao or mixtures thereof; and optionally further comprises at least one component selected from pharmaceutically, nutritionally or dietetically acceptable excipients, carriers and diluents; the method comprising the steps of: (a) Extract the dried Centella asiatica powder with a suitable solvent; (b) Treat the solution from step (a) with a magnesium source; (c) Filter the solution from step (b) to obtain a first extract and the used Centella asiatica powder; (d) Extract the used Centella asiatica powder with a second solvent; (e) Treat the solution from step (d) with a magnesium source; (f) Filter the solution from step (e) to obtain a second extract; (g) Combine the two extracts obtained in steps (c) and (f); (h) Evaporate the solvent from step (g) and dry the residue to obtain a Centella asiatica extract mixture containing magnesium salts or complexes of asiatic acid and madecassic acid, as well as asiaticoside and madecassoside; (i) Optionally, omit steps (d) to (h), evaporate the filtrate from step (c), and dry the residue to obtain a Centella asiatica extract containing magnesium salts or complexes of asiatic acid and madecassic acid; also containing asiaticoside and madecassoside; (j) Mix the extract from step (h) or step (i) with a cocoa extract in the presence of a suitable solvent, and optionally mix with a suitable excipient; (k) Dry the product to obtain the desired composition.

15. The method for preparing a synergistic composition according to claim 14, wherein Wherein the suitable solvent is selected from but not limited to C1-C5 alcohols such as ethanol, methanol, n-butanol, water and their mixtures; ketones selected from acetone and methyl isobutyl ketone; chlorinated solvents selected from dichloromethane and chloroform; water and their mixtures; C1-C7 hydrocarbons such as but not limited to hexane, heptane; esters such as but not limited to ethyl acetate and their mixtures.

16. The method for preparing a synergistic composition according to claim 15, wherein Wherein the magnesium source is selected from oxides, hydroxides or carbonates; examples include magnesium oxide, magnesium hydroxide and magnesium carbonate.

17. A method for obtaining at least one benefit related to cognitive function, mental ability, and / or nootropic activity, wherein The benefits are selected from alertness, vigilance, attention, concentration / focus, neuromotor function (reaction time), memory (verbal, working, visual, logical), intelligence, learning ability, knowledge acquisition, knowledge retention, information recall / communication, perception ability, reasoning ability, problem-solving ability, decision-making ability; and alleviating mental fatigue and improving game performance; wherein the method comprises supplementing an effective dose of a composition to a person in need thereof, the composition comprising a combination of the following components: a first component selected from Centella asiatica extracts, the extract containing at least one component selected from magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, also containing asiaticoside and madecassoside; a second component selected from extracts, fractions, phytochemicals of cocoa or mixtures thereof; and the composition may optionally further comprise at least one component selected from pharmaceutically, nutraceutically or dietetically acceptable excipients, carriers and diluents.

18. The use of a synergistic herbal composition, wherein The composition comprises: a first component selected from extracts of Centella asiatica, said extracts containing at least one component selected from the group consisting of magnesium salts or complexes of asiatic acid, magnesium salts or complexes of madecassic acid, or mixtures thereof, and also containing asiaticoside and madecassoside; a second component selected from extracts, fractions, phytochemicals of Theobroma cacao or mixtures thereof; and optionally further comprises at least one component selected from pharmaceutically, nutraceutically or dietetically acceptable excipients, carriers and diluents; the composition is for obtaining at least one benefit related to cognitive function, mental ability and / or nootropic activity, said benefits being selected from alertness, vigilance, attention, concentration / focus, neuro-motor function (reaction time), memory (verbal, working, visual, logical memory), intelligence, learning, acquisition, retention, recall of information / communication, perception, reasoning, problem-solving ability, decision-making ability, and alleviating mental fatigue and improving game performance.

Images