COMPOSITION

Abstract

A composition comprising or consisting of L-theanine and one or more fructooligosaccharides, wherein the ratio of L-theanine to froctooligosaccharide(s) is of about 2:1 to 99:1. A composition in the form of a food or nutraceutical, and also in the form of a powder, granules, suspension, tablet, capsule, lozenge, bakery item, sweets, drink, beverage or oral preparation.

Claims

1. A composition comprising or consisting of L-theanine and one or more fructooligosaccharides, wherein the ratio of L-theanine to froctooligosaccharide(s) is of about 2:1 to 99:1.

2. A composition according to claim 1 wherein the ratio of L-theanine to froctooligosaccharide(s) is of about 2:1 to 50:1, such as 2:1 to 20:1, 2:1 to 10:1, 2:1 to 5:1, 2:1 to 4:1.

3. A composition according to claim 1 or 2 wherein the fructooligosaccharide(s) comprise between 3 and 9 fructose residues.

4. A composition according to any preceding claim for use as a medicament.

5. A composition as claimed in any preceding claim for use in enhancing memory or improving cognitive performance.

6. A composition as claimed in any preceding claim for use in reducing anxiety or ameliorating mood.

7. A composition as claimed in any of claims 4 to 6, wherein said composition is administered to humans in a dose of up to 900 mg/person/day, preferably in a dose of about 420 to 860 mg/person/day.

8. A composition as claimed in any of the preceding claims, wherein said composition further comprises: amino acids (essential and/or non-essential), taurine, vitamins (such as vitamin A, B, C, D, E), metal supplements (such as sodium, potassium, calcium and/or magnesium), flavouring agents (sweeteners and/or flavours), fillers, lubricants and/or binders.

9. A composition as claimed in any preceding claim wherein the fructooligosaccharide is extracted from Morinda officinalis.

10. A pharmaceutical composition comprising a composition as claimed in any preceding claim and pharmaceutically acceptable excipients.

11. A composition as claimed in any of claims 1 to 8 in the form of a food or nutraceutical.

12. A composition as claimed in any preceding claim in the form of a powder, granules, suspension, tablet, capsule, lozenge, bakery item, sweets, drink, beverage or oral preparation.

Description

[0040] There are further presented a series of examples of the invention, in connection also with the FIGURES, which represent:

[0041] FIG. 1a. Average escape time of mice during testing phase.

[0042] FIG. 1b. average error count for mice during testing phase in water maze test

[0043] FIG. 1c. Average latency of mice in passive avoidance test during retention phase

[0044] FIG. 1d. Average error count of mice in passive avoidance test during testing phase

[0045] The following examples are used to demonstrate possible formulations to utilise the present invention, and the application of the present invention is not restricted by the given examples. It should be noted that modifications or improvements can be made based on following examples; however, such modifications or improvements while not deviating from the application or effectiveness of the present invention are still covered by this patent.

Embodiment 1. A Composition Used to Improve Memory

[0046] Fructooligosaccharide: 10-500 mg

[0047] L-Theanine: 10-500 mg

[0048] The fructooligosaccharide(s) may include one or more trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptaasaccharide, octasaccharide and nonasaccharide sugar molecules, and may consist of FructoseN and/or Glucose-FructoseN where N is between 3 and 9.

[0049] The above composition can be used to prepare orally administrable products including powders, granules, dry suspension, tablet, effervescent tablet, capsules, lozenges, sustained-release preparations, drinks, beverages and oral solutions using conventional techniques.

Embodiment 2. A Composition Used to Improve Memory

[0050] Fructooligosaccharide 10-500 mg

[0051] L-Theanine 10-500 mg

[0052] Vitamin B3: 1-10 mg

[0053] Vitamin B6:0.2-3 mg

[0054] The fructooligosaccharide(s) may include one or more trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptaasaccharide, octasaccharide and nonasaccharide sugar molecules, and may consist of FructoseN and/or Glucose-FructoseN where N is between 3 and 9.

[0055] The above composition can be used to prepare orally administered products such as powders, granules, dry suspensions, tablets, effervescent tablets, capsules, lozenges, sustained-release preparations, drinks, beverages and oral solutions using conventional techniques.

Embodiment 3. A Powder or Dry Suspension Preparation or Granules Including the Present Invention

[0056] Fructooligosaccharide 10-500 mg

[0057] L-Theanine 10-500 mg

[0058] L-Tryptophan 70 mg

[0059] Taurine 100 mg

[0060] Magnesium Citramate 1.5 g

[0061] Vitamin B3 5 mg

[0062] Vitamin B6 0.6 mg

[0063] Vitamin B12 4.0 μg

[0064] Lactose 2.8 g

[0065] Flavouring and sweetener

[0066] The fructooligosaccharide(s) may include one or more trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptaasaccharide, octasaccharide and nonasaccharide sugar molecules, and may consist of FructoseN and/or Glucose-FructoseN where N is between 3 and 9.

Embodiment 4. A Suspension Preparation Containing Present Invention

[0067] Fructooligosaccharide 10-500 mg

[0068] L-Theanine 10-500 mg

[0069] Taurine 100 mg

[0070] Lactose 2.8 g

[0071] Flavouring and sweetener

[0072] The fructooligosaccharide(s) may include one or more trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptaasaccharide, octasaccharide and nonasaccharide sugar molecules, and may consist of FructoseN and/or Glucose-FructoseN where N is between 3 and 9.

Effectiveness of the Invention in Standard Animal Tests

[0073] The effectiveness of the invention has been demonstrated by the following rigorous animal testing regimen as described below.

[0074] The Passive Avoidance Test (Solomon and Wynne, 1954; Solomon and Turner, 1960) is a fear-aggravated task employed to evaluate learning and memory in rodent models. In this test, subjects learn to avoid an environment in which an aversive stimulus (such as a foot-shock) was previously delivered. The test chamber is divided into a light compartment and a dark compartment, with a gate between the two. Animals are allowed to explore both compartments on the first day. On the following day, they are given a mild foot shock in one of the compartments. Animals will learn to associate certain properties of the chamber with the foot shock. In order to test their learning and memory, the mice are then placed back in the compartment where no shock was delivered. Mice with normal learning and memory will avoid entering the chamber where they had previously been exposed to the shock. This is measured by recording the latency to cross through the gate between the compartments. “Learning” is the period where mice were trained in this test (Table 1 and 2). Mice were tested at 24 or 48 hours after learning and the results were recorded (“Test column” in Table 1 and 2). “Fade” indicates re-testing of mice 5 days after learning (“Fade column” Table 1 and 2). Error percentage indicates the percentage of mice that have been shocked during the test.

[0075] Our research was undertaken with KumMing (KM) mice (Dai et al. 2012; Ma et al. 2013). In each test, 48 KM mice were separated into 4 groups, 12 mice per group. A negative-control group was also included in the test and mock treated. Our data indicate that neither fructooligoscharide nor L-theanine alone have a positive impact on rodent memory (Table 1). However, treatment of mice with a dose of embodiment 1, consisting of a combination of both fructooligoscharide and L-theanine, enhances memory in a statistically significant fashion in the mouse passive avoidance test (Table 1).

[0076] We subsequently, tested different doses of embodiment 1, 0.1, 0.2 or 0.3 g/kg of mouse body weight (bw) of embodiment 1, consisting of a combination of fructooligoscharide and L-theanine, in the passive avoidance test. Mice administered 0.2 g/kg.bw or 0.3 g/kg.bw of embodiment 1 (containing 0.134 g/kg.bw of L-theanine and 0.066 g/kg.bw of fructooligosaccahride or 0.201 g/kg.bw of L-theanine and 0.099 g/kg.bw of fructooligosaccahride, respectively) showed a statistically significant reduction in error count (p<0.01) (Table 2). Thus, our data indicates that doses of 0.2 g/kg.bw and 0.3 g/kg.bw of embodiment 1, compromised of a combination of both fructooligosaccahride and L-theanine, enhances memory in a statistically significant fashion in the mouse passive avoidance test.

[0077] To confirm and extend these findings, we also determined if different doses of embodiment 1, compromised of a combination of both fructooligosaccahride and L-theanine, could positively impact memory in an alternative assay, the Morris water maze test (D'Hooge, R. and De Deyn, P. P. 2001; Vorhees, C. and Williams, M. 2006). This test is designed to test both spatial memory and long-term memory by observing and recording escape latency, thigmotaxis duration, distance moved and velocity during the time spent in the test tank. Tempera paint is added into the water until it becomes opaque. A hidden platform, 1/10 the length of the diameter of the water body, is placed about 1 cm below the water surface. Three fourths of the water tank is surrounded by privacy blinds with 3 visual cues. The subjects are monitored by a video tracking system directly above the water tank as they swim and parameters are measured using Ethovision software. “Learning” is the period where mice are trained in this test (Table 3). Mice were tested 24 hours after learning and the results recorded (“Test column” in Table 3). The “Fade” column indicates re-testing of mice 5 days after learning (Table 3). The percentage of mice that finished the maze was also recorded (Table 3).

[0078] Our research was undertaken with K M mice (Dai et al. 2012; Ma et al. 2013). In each test, 48 KM mice were separated into 4 groups, 12 mice per group. A negative-control group was also included in the test and mock treated. Our data indicate that mice in all groups (0.1, 0.2 or 0.3 g/kg.bw of embodiment 1, containing 0.067, 0.134 or 0.201 g/kg.bw, respectively, of L-theanine and 0.033, 0.066 and 0.099 g/kg.bw, respectively, of fructooligosaccharide) show a statistically significantly reduction in the time to finish this test. Thus, indicating embodiment 1, containing a combination of fructooligosaccharide and L-theanine, enhances memory, as determined by the Morris water maze test.

Effectiveness of the Invention in a Human Clinical Trial

[0079] To confirm and extend our findings that an embodiment consisting of both oligofructosaccharide and L-theanine can enhance memory in two distinct classical mouse models designed to test this capacity, we undertook an independent, double-blind, 30-day clinical trial. This was employed to determine if this embodiment can also enhance human memory and or associated brain function. Thus, 120 healthy adults, 35 males and 81 females, were recruited voluntarily to participate. Of these recruits, 116 participants completed the trial. The Clinical Memory Scale was used to assess participants' memory throughout the trial, following Technical Standards for Testing and Assessment of Health Food (2003) (Table 4-8).

[0080] Clinical memory, associative learning, graphic memory, the recognition of meaningless images and portrait retrieval are different individual memory tests that were conducted during the trial (He et al., 2008).

[0081] In the clinical memory test, the examinee reads 24 words, in which 12 of them belongs to the same category while the other 12 words are not associated with the given category. The examinee is then asked to recall the 12 words that belong to the same category. In the associative learning test, the examinee reads 12 word pairs, in which 6 of them are semantically related, with random order, 3 times. The examinee is subsequently given the first word of the pair and asked to recall the second. In the graphic memory test, the examinee is given 2 sets of pictures, with each set containing 15 pictures. They are then asked to recall the pictures within 2 minutes. In the recognition of meaningless images test, the examinee is shown 20 pictures with 5 different patterns of drawing. Afterwards, the examinee is shown a further 40 pictures to recall if they have seen the same pattern in the first 20 pictures. In the portrait retrieval test, the examinee is shown 6 human portraits and the associated name of the individual, their occupation and characteristics. The examinee is subsequently asked to recall the name, occupation and habit of the person while shown the relevant portrait, although the order of portraits is changed (He et al. 2008). Total memory measurement is a combined score of individual scores derived from the above individual tests. The memory quotient is calculated using total memory measurement according to the equivalent memory quotient conversion table. Memory quotient is the benchmark for human memory.

[0082] Firstly, memory was compared between participants separated into either the placebo group or the treatment group before commencement of the clinical trial. No statistically significant difference could be detected in clinical memory, associative learning or graphic memory in either the placebo or the treatment group before the onset of the clinical trial (Table 4). Thus, establishing an indistinguishable baseline memory between the subsequent placebo group and treatment group to be utilised in the clinical trial.

[0083] Importantly, the placebo group showed no increase in memory during the duration of the clinical trial (Table 5). In contrast, participants who were administered the present invention showed statistically significant improvement in memory test scores, indicating the present invention can enhance human memory. This was evidenced by a series of well-established memory indicators. Participants who were administered the present invention showed statistically significant improvement in clinical memory, associative learning, graphic memory, the recognition of meaningless images and portrait retrieval (Table 6 and 7). Thus, administration of the current invention to participants enhanced the performance of human memory, as scored by multiple indicators.

[0084] To confirm and extend these findings we next scored the participants in this clinical trial for possible enhancement of memory quotient and total memory measurement following administration of the current invention. Both memory quotient and total memory measurement exhibited striking statistically significant enhancement following administration of the current invention (Table 8).

TABLE-US-00001 TABLE 1 A combination of both fructooligosaccharide and L-theanine but not each molecule alone enhances memory in the passive avoidance test Number Learning Test Fade Dose of Incubation Error Incubation Error Incubation Error (g/kg .Math. bw) Mice period (Sec) Count period (Sec) Count period (Sec) Count Control 12 52.60 ± 16.52 3.00 ± 1.83 214.56 ± 59.15 2.20 ± 1.32 227.60 ± 43.56 1.84 ± 1.14 0.01 Embodiment 1 12 39.20 ± 14.99 2.70 ± 1.88 248.82 ± 75.27  0.80 ± 0.79* 318.00 ± 53.06  0.35 ± 0.33* 0.0067 L-Theanine 12 48.30 ± 10.71 3.90 ± 1.52 232.13 ± 70.71 1.22 ± 1.14 267.80 ± 51.03 1.40 ± 1.07 0.0033 12 49.62 ± 12.86 4.02 ± 1.93 230.30 ± 72.63 1.76 ± 1.01 241.30 ± 43.26 1.50 ± 0.99 Fructooligosaccharide Passive avoidance test to demonstrate activity of present invention. Mice administered with 0.01 g/kg .Math. bw embodiment 1 (containing 0.0067 g/kg .Math. bw L-theanine and 0.0033 g/kg .Math. bw true tooligosaccahride) showed statistically significant less (indicated as *p < 0.05) errors in passive avoidance test comp are to fructooligosaccharide, L-theanine and the control group, Neither L-theanine or fruictooligosaccahride alone showed any sstatistically significant impact on memory enhancement.

TABLE-US-00002 TABLE 2 Different doses of both fructooligosaccharide and L-theanine enhance memory in the mouse passive avoidance test Learning Test Fade Number Incubation Incubation Incubation Dose of period Error period Error period Error Error Percentage (%) (g/kg .Math. bw) Mice (Sec) Count (Sec) Count (Sec) Count Learning Test Fade Control 12 64.58 ± 2.58 ± 165.42 ± 0.83 ± 242.17 ± 1.00 ± 100.00 50.00 41.67 49.48 1.31 141.02 0.94 58.39 0.95 0.1 12 67.33 ± 3.58 ± 169.08 ± 1.00 ± 288.83 ± 0.08 ± 100.00 58.33 8.33 36.42 1.88 122.12 1.13 38.68 0.29 0.2 12 84.67 ± 3.00 ± 178.08 ± 1.58 ± 280.75 ± 0.08 ±  91.67 66.67 8.33 76.86 1.65 111.09 1.44 66.68 0.29 ** 0.3 12 67.42 ± 3.42 ± 171.33 ± 1.33 ± 271.92 ± 0.17 ± 100.00 58.33 16.67 59.32 1.62 124.31 1.30 78.35 0.39 ** Passive avoidance test to demonstrate activity of present invention. As expected, in learning and testing stage, mice administered with different doses of the present invention showed no difference compared to the control group (p > 0.05). In memory reproduction stage, however, mice administered of 0.2 g/kg .Math. bw and 0.3 g/kg .Math. bw embodiment 1 (containing 0.134 g/kg .Math. bw L-theanine and 0.066 g/kg .Math. bw fructocligosaccahride, 0.201 g/kg .Math. bw L-theanine and 0.099 g/kg .Math. bw fructooligosaccahride respectively) showed less error count (indicated as **p < 0.01). Thus, indicating embodiment 1 improves memory in a rodent model system.

TABLE-US-00003 TABLE 3 A combination of both fructooligosaccharide and L-theanine enhance memory in the Morris water maze test Learning (5 times) Test Reproduction Total Time Time Percentage of mouse time to to to that finish maze Number finish finish finish in 2 min (%) Dose of maze Error maze Error maze Error Learning (g/kg .Math. bw) Mouse (Sec) Count (Sec) Count (Sec) Count (5 times) Test Fade Control 12 281.67 ± 12.17 ± 91.08 ± 4.67 ± 78.83 ± 2.83 ± 83.33 50.00 75.00 88.84 4.26 36.18 2.61 35.22 1.34 0.1 12 253.83 ± 12.92 ± 67.67 ± 3.08 ± 39.00 ± 1.33 ± 86.67 83.33 100.00 85.18 4.58 38.13 1.98 15.27 * 0.89 0.2 12 211.17 ± 10.33 ± 58.83 ± 2.42 ± 47.17 ± 2.33 ± 95.00 91.67 100.00 88.91 4.27 32.99 1.93 20.83 * 1.56 0.3 12 229.00 ±  9.75 ± 82.83 ± 3.17 ± 63.25 ± 2.67 ± 91.67 58.33 75.00 52.61 3.96 37.30 1.64 37.81 * 2.15 Morris water maze test to demonstrate activity of present invention. As expected, at the learning and testing stage, mice administered with different doses of the present invention show no difference compared to the control group (p > 0.05). However, in the memory reproduction stage, mice in all groups (0.1/0.2/0.3 g/kg .Math. bw of embodiment 1, containing 0.067/0.134/0.201 g/kg .Math. bw of L-theanine and 0.033/0.066/0.099 g/kg .Math. bw of fructooligosaccharide) showed statistically significant less time to finish the maze (indicated as * p < 0.05). Thus, indicating embodiment 1 improves memory in a rodent model system.

TABLE-US-00004 TABLE 4 No significant difference in memory scores of participants separated into either a placebo group or a treatment group before commencement of an independent, double-blind, clinical trial The recognition Number of Clinical associative Graphic of meaning- Portrait Group participants Memory learning memory less images retrieval Placebo 58 13.17 ± 3.38 13.97 ± 2.88 13.83 ± 3.65 14.66 ± 3.21 14.50 ± 2.36 Treatment 58 13.71 ± 3.23 13.41 ± 3.33 13.72 ± 3.83 14.41 ± 3.08 14.16 ± 2.67 Memory scores of participants separated into a placebo group or a treatment group before commencement of a clinical trial. There was no difference in clinical memory, associative learning or graphic memory between these two groups in the absence of any treatment or placebo before initiation of the clinical trial.

TABLE-US-00005 TABLE 5 No memory enhancement in the placebo group during a clinical trial The recognition Number of Clinical associative Graphic of meaning- Portrait Group participants Memory learning memory less image retrieval Before 58 13.17 ± 3.38 13.97 ± 2.88 13.83 ± 3.65 14.66 ± 3.21 14.50 ± 2.36 After 58 13.12 ± 2.93 13.81 ± 2.29 14.17 ± 2.71 14.50 ± 2.96 14.00 ± 1.84 Memory score of participants in placebo group before and al ter trial. Participants in the placebo group showed no changes in memory score before and after trial, indicating the placebo has no effect on memory improvement.

TABLE-US-00006 TABLE 6 Treatment with embodiment 1 enhances human memory in a clinical trial The recognition Number of Clinical associative Graphic of meaning- Portrait Group participants Memory learning memory less image retrieval Before 58 13.71 ± 3.23   13.41 ± 3.33 13.72 ± 3.83   14.41 ± 3.08   14.16 ± 2.67   After 58 14.60 ± 2.67 ** 14.34 ± 2.82 17.33 ± 3.10 ** 15.03 ± 2.77 ** 15.12 ± 2.30 ** Memory score of participants in treatment group before and after clinical trial. Participants who were administered the present invention (0.52 g/person/day of embodiment 1, containing 0.36 g L-theanine and 0.18 g fructooligosaccharide) showed statistically significant improvement in memory test scores (indicated as ** p < 0.01 compared to before trial), indicating the present invention can improve human memory, as evidenced by a number of well-established indicators.

TABLE-US-00007 TABLE 7 Administration of embodiment 1 enhances human memory relative to administration of a placebo The recognition Number of Clinical associative Graphic of meaning- Portrait Group participants Memory learning memory less image retrieval Placebo 58 13.12 ± 2.93   13.81 ± 2.29 14.17 ± 2.71   14.50 ± 2.96 14.00 ± 1.84   Treatment 58 14.60 ± 2.67 ** 14.34 ± 2.82 17.33 ± 3.10 ** 15.03 ± 2.77 15.12 ± 2.30 ** Memory scores of participants in both placebo group and treatment group after trial. Participants who were administered the present invention (0.52 g/person/day of embodiment 1, containing 0.36 g L-theanine and 0.18 g fructooligosaccharide) showed improved performance (indicated as ** p < 0.01 compared placebo group after trial), indicating the present invention can improve memory, as evidenced by a number of well-established indicators.

TABLE-US-00008 TABLE 8 Comparison of total memory measurement and memory quotient of participants before and after administration of the current invention Embodiment 1 (n = 58) Placebo (n = 58) Group Before trial After trial Before trial After trial Total measurement 69.41 ± 6.63 76.43 ± 5.68 **{circumflex over ( )}{circumflex over ( )} 70.12 ± 6.79 69.60 ± 6.37 Memory quotient 80.12 ± 7.24 85.98 ± 6.59 **{circumflex over ( )}{circumflex over ( )} 82.67 ± 7.17 82.31 ± 6.85 Comparison of total memory measurement and memory quotient. Participants who were administered the present invention (0.52 g/person/day of embodiment 1, containing 0.36 g L-theanine and 0.18 g fructooligosaccharide) showed a statistically significant enhancement in both memory score and memory quotient, compared to participants in a placebo group after the clinical trial (indicated as **, p < 0.01). Further, there is a statistically significant enhancement in both memory score and memory quotient of participants following administration of the invention (indicated as {circumflex over ( )}{circumflex over ( )}, p < 0.01). Thus, the present invention can improve both total memory measurement and memory quotient.

[0085] A series of safety tests have also been conducted to demonstrate the safety of the invention. These include the acute oral toxicity test (Walum, 1998; OECD 2001), repeated dose 28-day oral toxicity test (OECD, 2008) and the genetic toxicity test (Proudlock, 2016). The resulting data suggest that our invention composition is non-toxic and safe to consume. The acute oral toxicity test indicates that the LD.sub.50 (median lethal dose) is >10 g/kg.bw. From a repeated dose 28-day oral toxicity test, the harmful dose of the present invention was established at larger than 2.47 g/kg.bw. Three independent tests were conducted for genetic toxicity, including the Bacterial Reverse Mutation Assay (Ames test), In Vivo Rodent Micronucleus Assay and The In Vitro Chromosome Aberration Test (Proudlock., 2016). The resulting findings from all three test indicate no genetic toxicity was associated with the present invention.

[0086] In conclusion, collectively, our findings show that neither fructooligosaccharides nor L-theanine improved mammalian memory in a series of well-established memory tests in a rodent model. However, a composition containing both fructooligosaccharides and L-theanine enhanced mammalian memory in a statistically significantly fashion in a series of rodent memory tests. To confirm extend these findings, we undertook an independent, double-blind, clinical trial to determine if this composition of fructooligosaccharides and L-theanine could positively impact human memory. Participants who were administered the present invention showed a statistically significant improvement in clinical memory, associative learning, graphic memory, the recognition of meaningless images and portrait retrieval. Furthermore, both memory quotient and total memory measurement also exhibited a statistically significant enhancement following administration of the current invention. Therefore, the current invention enhances human memory, as scored by multiple indicators.

[0087] Furthermore, to test the hypothesis that administration of M. officinalis FOS and L-theanine in a ratio of about 2:1 L-theanine:FOS synergistically enhance mice memory, we have performed a step-down passive avoidance test and a water maze test. This was employed to assess the effect of M. officinalis FOS and L-theanine individually on mice memory compared to their collective effect when administrated in combination in a ratio of about 2:1 L-theanine:FOS (MT-01) (FIG. 1).

[0088] In the step-down passive avoidance test, [0089] neither the FOS-only nor L-theanine-only group show any significant difference in latency during the retention phase compared to the control group at a low dose. [0090] in contrast, mice treated with the mentioned MT-01 combination showed a significantly longer (p<0.05, FIG. 1c) latency in the retention phase compared to individual treatments and control data. [0091] In addition, mice treated with the MT-01 combination showed significantly less error during the passive avoidance test compared to individual treatments and control data (p<0.01, FIG. 1d).

[0092] *In addition, mice treated with FOS alone showed a statistically significant higher latency and less error compared to the control group, but only at a high dose (p<0.05, FIGS. 1c and d).

[0093] *Also, the administration of L-theanine alone showed less error and higher latency in a passive avoidance test at high dose (FIGS. 1c and 1d).

[0094] *Administration of MT-01 at a low and medium dose showed significantly longer latency compared to all other test groups, but at a high dose, the performance of mice administrated with MT-01 showed no statistical difference compared to other treatment groups, although significantly better than the control group, during the retention phase.

[0095] With regard to error count (FIG. 1c), administration of MT-01 in a medium dose showed significantly less error during the testing phase compared to all other groups. Administration of MT-01 in a low and high dose resulted in significantly less error compare to the FOS treatment group, but is not significantly different to the L-theanine group (FIG. 1d).

[0096] Maze test was performed to further confirm the hypothesis.

[0097] Compared to control group, all mice with MT-01, L-theanine or FOS treatment at all doses exhibited significantly (p<0.05) less time to escape during the test (FIG. 1a). In addition, mice administrated with MT-01 show a significantly reduced escape time compared to other groups in the retention phase (p<0.05, FIG. 1a). —At a medium and high dose, no significant differences were observed in escape time between mice treated with MT-01, L-theanine or FOS in escape time during test. [0098] Mice treated with a low dose of MT-01 showed a significantly reduced error count in a maze test during the testing phase, compared to all other groups (FIG. 1b). However, all other groups showed no significant difference compared to the control group or between each other (FIG. 1b).

[0099] We analysed the synergy between FOS and L-theanine in the formulation by calculating the coefficient of drug interaction (CDI), as shown in Table 1.

TABLE-US-00009 TABLE 1 Coefficient of drug interaction between FOS and L-theanine in passive avoidance test and water maze test. Passive avoidance test Water maze test Dose(mg/kg .Math. bw) Error count during testing phase Error count during testing phase Group L-theanine FOS L-theanine FOS MT-01 CDI L-theanine FOS MT-01 CDI 0 0 2.33 2.33 2.33 4.11 4.11 4.11 Low 66 33 0.83 1.25 1.75 0.89 2.45 3.83 3.77 0.70 Medium 134 66 1.00 1.92 1.83 0.33 2.95 2.98 2.84 1.44 High 200 100 0.50 1.08 1.00 1.08 2.41 2.98 2.25 1.47

[0100] Table 1. Coefficient of drug interaction between FOS and L-theanine in passive avoidance test and water maze test.

[0101] Table 1 depicts the Coefficient of drug interaction (CDI) between FOS and L-theanine on error counts in water maze test during retention phase. CDI suggests L-theanine and FOS act antagonistically (CDI>1) at high doses in the passive avoidance test (CDI=1.08) and a medium dose (CDI=1.44) and a high dose (CDI=1.47) in the water maze test. L-theanine and FOS act synergistically (CDI<1) at a low dose in both tests, where CDI is 0.89 and 0.70, respectively.

[0102] Thus, our data suggests that, during the passive avoidance test, at low (66 mg/kg L-theanine and 34 mg/kg FOS in combination) and medium (134 mg/kg L-theanine and 66 mg/kg FOS) doses, L-theanine and FOS act synergistically (CDI<1) in enhancing mouse memory, resulting in a reduced error count during the testing phase.

[0103] However, L-theanine and FOS may act antagonistically (CDI>1) at high (200 mg/kg L-theanine and 100 mg/kg FOS) doses. Similar results were obtained in the maze test, L-theanine and FOS act synergistically at a low dose, resulting in lower errors during the testing phase and act antagonistically at medium and high doses.

[0104] By translating the above dosages from Animal experiment to Human, based on “Nair A B, Jacob S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm. 2016; 7(2):27-31. doi:10.4103/0976-0105.177703”, the effective low dose (66 mg/kg L-theanine and 34 mg/kg FOS, 100 mg/kg total) and medium dose (200 mg/kg total) observed to show synergy in the animal test (Mice), convert, by calculation, into a human effective dose (HED) of from 427 mg/day to 854 mg/day of composition with 1:2 ratio between FOS and L-theanine. These doses are very close to the range we have found to show surprising synergic effects as compared to employing L-theanine or OS separately. Our example is also 520 mg/person/day, which is in that range.

[0105] To summarize, we have demonstrated that FOS isolated from M. officinalis and L-theanine act synergistically at a low dose to enhance mice memory in the maze test and also act synergistically at low and medium doses to enhance mice memory in the passive avoidance test. In both tests, FOS and L-theanine appear to act antagonistically at a high dose.

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