COMPOSITION COMPRISING THE EXTRACT OF HERBS FOR PREVENTING OR TREATING NEURODEGENERATIVE DISORDERS

Abstract

This invention relates to a pharmaceutical composition and a health functional food for preventing or improving neurodegenerative disorders comprising mixed herb extracts of Dioscorea Rhizoma and Dioscorea nipponica in a weight ratio of 3.5:1 (w/w). The herb extracts mixed Dioscorea Rhizoma and Dioscorea nipponica in a weight ratio of 3.5:1 have the synergetic effects on increasing the amount of nerve growth factor in vivo, increasing the neural cell proliferation, promoting the formation of neuritis and enhancing cognitive abilities. Thus, the herb extracts of the present invention may be used for a pharmaceutical composition and a health functional food for preventing or treating neurodegenerative disorders.

Claims

1. A method for preventing or treating a neurodegenerative disorder in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising: an aqueous alcohol solvent extract of a mixture of Dioscorea Rhizoma and the rhizome of Dioscorea nipponica in a weight ratio of 3.5:1 (w/w) as an active ingredient.

2. The method of claim 1, wherein the neurodegenerative disorder is selected from among Alzheimer's disease, Creutzfeldt-Jakob disease, Huntington's disease, multiple sclerosis, Guillain-Barre syndrome, Parkinson's disease, Lou Gehrig's disease, paralytic dementia caused by gradual nerve cell death, and diseases caused by progressive incontinentia.

3. The method of claim 1, wherein the aqueous alcohol solvent extract is obtained by extracting the mixture of 3.5:1 Dioscorea Rhizoma: the rhizome of Dioscorea nipponica (w/w) with 50% ethanol.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0041] FIG. 1 is a photograph of a hippocampus region of the brain tissue illustrating the result from the evaluation of the secretion effect of nerve growth factor using NGF immunohistochemistry according to EXAMPLE 1.

MODE FOR THE INVENTION

[0042] The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the example.

EXAMPLE 1

[0043] Preparation of the Mixed Herb Extracts of Dioscorea Rhizoma and Dioscorea nipponica.

[0044] Dioscorea Rhizoma and Dioscorea nipponica, both in a dry condition, were purchased from a herb medicine shop in Kyoungdong market, Korea. After impurities were removed therefrom, the herbs were chopped with a cutter and mixed at a weight ratio of 3.5:1 Dioscorea Rhizoma: Dioscorea nipponica. To 2 kg of the mixture was added 10 L of a 50% ethanol solution, followed by incubation at room temperature for 48 hours with stirring. The herb mixture was removed by filtration, and the filtrate was concentrated in a vacuum and freeze dried to afford a mixed herb extract (crude extract) (see Table 1).

TABLE-US-00001 TABLE 1 Yield of the mixed herb extracts Amount Amount D. D. of Extraction Extraction Result Yield Rhizoma nipponica Solvent solvent wash temperature time (g) (%) Example 1.55 kg 0.45 kg 50% 10 L 1 L Room 2 days 212.85 10.64 1 EtOH Temperature

COMPARATIVE EXAMPLES 1 and 2

[0045] Preparation of Crude Herb Extracts

[0046] 1. Preparation of Crude Extract of Dioscorea Rhizoma

[0047] 2 kg of the same Dioscorea Rhizoma used in EXAMPLE 1 and stirred 2 kg of the same Dioscorea Rhizoma used in Example 1 and stirred 10 L of 50% ethanol solution was added to for 48 hours at room temperature. The extract of Dioscorea Rhizoma crude extract was finally obtained by lyophilization after extracting, filtering and concentrating under reduced pressure (See Table 2).

TABLE-US-00002 TABLE 2 Yield of the extract of Dioscorea Rhizoma Amount Amount Extraction Extraction Result Yield of herb Solvent of solvent Clean Temperature Time (g) (%) Comparative 2 kg 50% 10 L 1 L Room 2 days 253.8 12.69 Example 1 EtOH temperature

[0048] 2. Preparation of Crude Extract of Dioscorea nipponica

[0049] 10 L of 50% ethanol solution was added to 2 kg of the same Dioscorea nipponica used in Example 1 and stirred for 48 hour at room temperature. The extract of Dioscorea nipponica (crude extract) was finally obtained by lyophilization after extracting, filtering and concentrating under reduced pressure (See Table 3).

TABLE-US-00003 TABLE 3 Yield of the extract of Dioscorea nipponica Amount Amount Extraction Extraction Result Yield of herb Solvent of solvent Wash Temperature Time (g) (%) Comparative 2 kg 50% 10 L 1 L Room 2 days 160 8.00 Example 2 EtOH temperature

COMPARATIVE EXAMPLES 3 to 10

[0050] Preparation of the Mixed Herb Extract of Dioscorea Rhizoma and Dioscorea nipponica

[0051] The same herbs Dioscorea Rhizoma and Dioscorea nipponica as used in Example 1 were used. Dioscorea Rhizoma and Dioscorea nipponica were chopped with a cutter and mixed at the weight ratios listed in Table 4. To 2 kg of each of the mixtures was added 10 L of a 50% ethanol solution, followed by incubation at room temperature for 48 hours with stirring. The herb mixtures were removed by filtration, and the filtrate was concentrated in a vacuum and freeze dried to afford mixed herb extracts (crude extracts). (See Table 4).

TABLE-US-00004 TABLE 4 Yield of the mixed herb extracts. Amount of herb (kg) D. D. Amount Extraction Extraction Result Yield Rhizoma nipponica Solvent of solvent Wash Temperature Time (g) (%) Comparative 1 1 50% 10 L 1 L Room 2 days 179.05 8.95 Example 3 EtOH temperature Comparative 1.33 0.67 50% 10 L 1 L Room 2 days 135.96 6.80 Example 4 EtOH temperature Comparative 1.67 0.33 50% 10 L 1 L Room 2 days 160.07 8.00 Example 5 EtOH temperature Comparative 1 .82 0.18 50% 10 L 1 L Room 2 days 138.75 6.93 Example 6 EtOH temperature Comparative 0.67 1.33 50% 10 L 1 L Room 2 days 153.56 7.68 Example 7 EtOH temperature Comparative 0.45 1.55 50% 10 L 1 L Room 2 days 139.15 6.96 Example 8 EtOH temperature Comparative 0.33 1.67 50% 10 L 1 L Room 2 days 181.06 9.05 Example 9 EtOH temperature Comparative 0.18 1.82 50% 10 L 1 L Room 2 days 146.43 7.32 Example 10 EtOH temperature

EXPERIMENTAL EXAMPLE 1

[0052] Effect of the Herb Extracts on Increasing the Secretion of Nerve Growth Factor

[0053] The effects on increasing and enhancing the secretion of NGF by Example 1 was identified by using C6 glioma, a cell strain of neuroglioma of rats producing NGF. Comparative examples 1 to 10 were the control groups of this experiment.

[0054] C6 cell was cultured in 24-well cell culture plate with medium, DMEM (adding 5% FBS, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 50 μg streptomycin, 100 U/ml penicillin) in 37° C. and 5% of CO.sub.2. After adjusting the cultured cell in the 2×10.sup.5 cell/well concentrations, Example 1, Comparative examples 1 to 10 were treated by 100, 250, and 500 μg/ml. In the same culture condition, the cell was cultured for 2 days and then the concentration of NGF in the medium was measured by using ELISA. (See Table 5).

TABLE-US-00005 TABLE 5 Effects on increasing the secretion of NGF D. Rhizoma:D. nipponica Concentration compared to the total Concentration of NGF weight (μg/ml) (% of control) Example 1 3.5:1   100 122 250 149 500 156 Comparative D. Rhizoma crude 100 112 Example 1 extract 250 126 500 135 Comparative D. nipponica crude 100 102 Example 2 extract 250 121 500 130 Comparative 1:1 100 108 Example 3 250 124 500 141 Comparative 2:1 100 117 Example 4 250 131 500 136 Comparative 5:1 100 115 Example 5 250 129 500 134 Comparative 10:1  100 112 Example 6 250 129 500 134 Comparative 1:2 100 107 Example 7 250 116 500 129 Comparative   1:3.5 100 106 Example 8 250 118 500 130 Comparative 1:5 100 107 Example 9 250 116 500 126 Comparative  1:10 100 105 Example 10 250 116 500 125

[0055] As shown in Table 5, the concentration of NGF was increased according to the concentration of Dioscorea Rhizoma and Dioscorea nipponica. Also, it was shown that the concentration of NGF in Dioscorea Rhizoma (Comparative example 1) was more increased than in Dioscorea nipponica (Comparative example 2) so the former showed more significant effect.

[0056] Although the concentration of Dioscorea Rhizoma 25% less than comparative example 1 (D.Rhizoma 200) was injected to Example 1 (D.Rhizoma:D.nipponica=155:45), 8.9 to 15.5% of NGF content was increased in the same concentration.

[0057] Furthermore, the concentration of NGF in Example 1 was more increased approximately 4.2 to 16.4% than Comparative example 5 (D.Rhizoma:D.nipponica=167:33, 5:1) which Dioscorea Rhizoma was injected 7.7% more and it was more increased 8.9 to 16.4% than Comparative example 6 (D.Rhizoma:D.nipponica=182:18, 10:1) which Dioscorea Rhizoma was injected 17.4% more). The amount of NGF in Example 1 was more increased approximately 4.4 to 12.9% than Comparative example 4 (D.Rhizoma:D.nipponica=133:67, 2:1) which Dioscorea Rhizoma was injected 14.2% less. It showed the significantly increasing content of NGF than other Comparative examples.

[0058] Therefore it is found that a pharmaceutical composition of the present invention disorders comprising a mixed extract of Dioscorea Rhizoma and Dioscorea nipponica in a weight ratio of 3.5:1 can increase the concentration of NGF efficiently compared to other mixed ratio. Thus, it could be used in treating or preventing neurodegenerative disorders.

[0059] Hereinafter, the enhancing effects on cell proliferation, formation of neurite and cognitive abilities according to the contents of NGF in the herb extracts of Dioscorea Rhizoma and Dioscorea nipponica are comparatively experimented. So, the availability for preventing or treating neurodegenerative disorders will be discussed.

EXPERIMENTAL EXAMPLE 2

[0060] Effect of the Herb Extracts on Improving Cognitive Abilities

[0061] ICR mice (male, 6 age of the weeks, 25-28 g) provided from Daehan Biolink (Chungbuk, Korea) were using after 7 days of being raised and adapted in a clean cage of college of pharmacy in Kyunghee university. They were allowed to have feeds (Daehan Biolink, Chungbuk, Korea) and water freely. The temperature (22±2° C.), humidity (53±3%) and cycle of light and dark (12 hours) were controlled automatically.

[0062] 10 mice were assigned to each group. Saline was orally administered to mice in the control group in 5 mL per kg of weight. 10, 100 mg/kg of EXAMPLE 1, Comparative examples 1 to 10 respectively melted in saline solution were orally administered to mice in the treatment group. Oral administration was done once a day and 1 hour after the last oral administration, passive avoidance test were conducted as follows.

[0063] Passive avoidance test was conducted in the A modified shuttle with two communicating (7×7 cm sliding door built into the separating wall) compartments of equal size and a stainless steel bar floor was used. The right-hand compartment (shock compartment) was painted black to obtain a dark chamber. The left-hand compartment was illuminated by a bulb (24 V; 5 W) installed on the top Plexiglass cover. First day, a light turns on in a bright room and turns off in a dark room. And the inventors opened guillotine door after letting mice stay in a bright room for 10 seconds then measured the time until mice entered a dark room. When mice moved into a dark room, guillotine door closed and 0.3 mA of electrical stimulation was given to them for 3 seconds. After 24 hours, passive avoidance test was demonstrated with the same method and latency time of staying in a bright room was measured. The results are shown in Table 6.

TABLE-US-00006 TABLE 6 Effect on improving cognitive abilities in passive avoidance test D. Rhizoma:D. nipponica compared to the total Amount Latency weight (mg/kg) Time (sec) Control Group — 166.18 ± 13.83 Example 1 3.5:1   10 220.17 ± 25.63 100 275.87 ± 25.40 Comparative D. Rhizoma 10 202.84 ± 19.65 Example 1 crude extract 100 225.28 ± 20.21 Comparative D. nipponica 10 199.62 ± 17.01 Example 2 crude extract 100 218.24 ± 18.47 Comparative 1:1 10 194.86 ± 9.43  Example 3 100 204.19 ± 10.72 Comparative 2:1 10 202.69 ± 11.24 Example 4 100 231.42 ± 16.55 Comparative 5:1 10 211.43 ± 18.21 Example 5 100 235.71 ± 21.60 Comparative 10:1  10 205.33 ± 15.08 Example 6 100 219.93 ± 16.14 Comparative 1:2 10 186.72 ± 1061  Example 7 100 204.68 ± 14.32 Comparative   1:3.5 10 196.93 ± 16.58 Example 8 100 209.61 ± 14.41 Comparative 1:5 10 205.89 ± 12.60 Example 9 100 215.99 ± 18.96 Comparative  1:10 10 193.53 ± 16.08 Example 10 100 211.74 ± 20.57

[0064] As shown in Table 6, the retention latency in the Dioscorea Rhizoma administrating group was increased than that of Dioscorea nipponica administrating group, Example 1 (D.Rhizoma:D.nipponica=155:45) increased retention latency 8.9 to 22.2% than Comparative example 1 (D.Rhizoma 200), although Example 1 was administered 25% less Dioscorea Rhizoma than Comparative example 1 (D.Rhizoma 200).

[0065] Also, the retention latency according to Example 1 was increased 4.2 to 19% than Comparative example 5 (D.Rhizoma:D.nipponica=167:33, 5:1) which Dioscorea Rhizoma was injected 7.7% more and it was more increased 7.3 to 25.6% than Comparative example 6 (D.Rhizoma:D.nipponica=182:18, 10:1) which Dioscorea Rhizoma was injected 17.4% more). The retention latency according to Example 1 was increased 8.9 to 19% than Comparative example 4 (D.Rhizoma:D.nipponica=133:67, 2:1) which Dioscorea Rhizoma was injected 14.2% less. It showed the memory was increased significantly than other Comparative examples.

[0066] Therefore it is found that a pharmaceutical composition of the present invention disorders comprising a mixed extract of Dioscorea Rhizoma and Dioscorea nipponica in a weight ratio of 3.5:1 has the effects on improving the cognitive abilities compared to other mixed ratio.

EXPERIMENTAL EXAMPLE 3

[0067] Effect the Herb Extracts on Enhancing Generation of Neurons

[0068] Hippocampi were separated from mice of each group after completing the test of Experimental example 2. 5-bromo-2-deoxyuridine (BrdU, santa cruz, rat origin 1:500) as a first antibody was reacted for one night after dehydrating separated hippocampus with hydrogen peroxide. And then, biotinylated anti-rat (vector, goat origin) was used as a second antibody, and color was developed by using Diaminobenzidine after ABC reaction (ABC kit, vector). The effect on enhancing neuronal cell generation was identified by counting BrdU-positive cells in the area of dentate gyrus in hippocampus. The results are shown in Table 7.

TABLE-US-00007 TABLE 7 Effect on enhancing cell generation D. Rhizoma:D. nipponica BrdU-positive compared to the total Amount cells weight (mg/kg) (% of control) Example 1 3.5:1   10 153.79 ± 15.24 100 158.59 ± 9.45  Comparative D. Rhizoma crude 10 123.81 ± 10.14 Example 1 extract 100 129.42 ± 11.36 Comparative D. nipporica crude 10 118.60 ± 8.37  Example 2 extract 100 121.59 ± 8.13  Comparative 1:1 10 124.25 ± 10.93 Example 3 100 125.86 ± 9.06  Comparative 2:1 10 128.44 ± 11.65 Example 4 100 136.70 ± 13.28 Comparative 5:1 10 129.54 ± 12.91 Example 5 100 134.08 ± 14.61 Comparative 10:1  10 121.08 ± 9.54  Example 6 100 128.57 ± 11.77 Comparative 1:2 10 120.68 ± 9.27  Example 7 100 124.16 ± 11.36 Comparative   1:3.5 10 126.39 ± 11.17 Example 8 100 130.28 ± 13.49 Comparative 1:5 10 124.70 ± 10.25 Example 9 100 128.06 ± 11.59 Comparative  1:10 10 121.84 ± 13.18 Example 10 100 124.12 ± 12.85

[0069] As shown in Table 7, Dioscorea Rhizoma (Example 1) showed increased the number of BrdU-positive cells than Dioscorea nipponica (Comparative example 2) when counting BrdU-positive cells in the area of dentate gyrus in hippocampus. Although the amount of Dioscorea Rhizoma was injected to Example 1 25% less than Comparative example 1, the number of BrdU-positive cells were increased approximately 25% more.

[0070] Furthermore, the number of BrdU-positive cells were increased approximately 25% more in Example 1 than Comparative example 5 which Dioscorea Rhizoma was injected 7.7% more than Example 1 and Comparative example 6 which Dioscorea Rhizoma was injected 17.4% more than Example 1. The number of BrdU-positive cells were increased approximately 20% more in Example 1 than Comparative example 4 which Dioscorea Rhizoma was injected 14.2% less than Example 1. It showed the significantly increasing the number of BrdU-positive cells than other Comparative examples.

[0071] Therefore it is found that a pharmaceutical composition of the present invention disorders comprising a mixed extract of Dioscorea Rhizoma and Dioscorea nipponica in a weight ratio of 3.5:1 has the effects on enhancing the neuronal cell generation in the area of dentate gyrus compared to other mixed ratio.

EXPERIMENTAL EXAMPLE 4

[0072] Effect of the Herb Extracts on Neuronal Cell Differentiation and Enhancing Neurite Growth

[0073] Hippocampi were separated from mice of each group after completing the test of Experimental example 2. Doublecortin (DCX, santa cruz, goat origin 1:500) as a first antibody was reacted for one night after dehydrating separated hippocampus with hydrogen peroxide. And then, biotinylated anti-goat (vector, horse origin) was used as a second antibody, and color was foiiiiulated by using Diaminobenzidine after ABC reaction (ABC kit, vector). The effect on cell differentiation and enhancing neurite growth was identified by counting DCX-positive cells in the area of dentate gyrus in hippocampus.

[0074] The results are shown in Table 8.

TABLE-US-00008 TABLE 8 Effect on cell differentiation and enhancing neurite growth D. Rhizoma:D. nipponica DCX-positive Length of compared to the Amount cell neurite total weight (mg/kg) (% of control) (% of control) Example 1 3.5:1   10 156.24 ± 9.40  125.38 ± 2.82 100 183.04 ± 12.67 142.59 ± 1.66 Comparative D. Rhizoma 10 126.84 ± 7.05  115.42 ± 3.60 Example 1 crude extract 100 132.81 ± 9.69  121.34 ± 2.56 Comparative D. nipponica 10 118.60 ± 8.37  120.48 ± 3.74 Example 2 crude extract 100 121.59 ± 8.13  120.57 ± 2.36 Comparative 1:1 10 127.19 ± 7.23  120.48 ± 3.74 Example 3 100 132.16 ± 9.61  129.63 ± 2.16 Comparative 2:1 10 130.80 ± 8.67  124.08 ± 2.48 Example 4 100 141.64 ± 7.85  131.75 ± 1.81 Comparative 5:1 10 132.54 ± 10.34 121.64 ± 1.34 Example 5 100 144.10 ± 9.32  130.12 ± 2.73 Comparative 10:1  10 127.06 ± 11.46 123.39 ± 1.89 Example 6 100 138.42 ± 9.29  129.61 ± 2.48 Comparative 1:2 10 125.47 ± 14.38 119.42 ± 1.99 Example 7 100 131.37 ± 11.94 126.74 ± 2.65 Comparative   1:3.5 10 126.88 ± 10.43 120.74 ± 1.84 Example 8 100 133.05 ± 9.59  122.38 ± 1.69 Comparative 1:5 10 130.15 ± 9.29  125.42 ± 2.88 Example 9 100 135.53 ± 10.74 131.41 ± 2.15 Comparative  1:10 10 124.06 ± 9.86  121.72 ± 1.61 Example 10 100 126.91 ± 10.34 124.63 ± 2.83

[0075] As shown in Table 8, in the area of dentate gyrus in hippocampus, Dioscorea Rhizoma (Example 1) showed increased the number of DCX-positive cells and the length of DCX-positive neurites than Dioscorea nipponica (Comparative example 2). Although the amount of Dioscorea Rhizoma was injected to Example 1 25% less than Comparative example 1, the number of DCX-positive cells and the length of DCX-positive neurites were increased approximately 25 to 40% and 1.6 to 17.3% more, respectively.

[0076] Furthermore, the number of DCX-positive cells and the length of DCX-positive neurites were increased approximately 18 to 27% and 3.3 to 9.2% more, respectively in Example 1 than Comparative example 5 (Dioscorea Rhizoma was injected 7.7% more than Example 1). The number of DCX-positive cells and the length of DCX-positive neurites were increased approximately 22.8 to 32.6% and 3.3 to 10% more, respectively in Example 1 than Comparative example 6 (Dioscorea Rhizoma was injected 17.4% more than Example 1). The number of DCX-positive cells and the length of DCX-positive neurites were increased approximately 20 to 30% and 1 to 8% more, respectively in Example 1 than Comparative example 4 (Dioscorea Rhizoma was injected 14.2% less than Example 1). It showed the significantly increasing the number of DCX-positive cells and the length of DCX-positive neurites than other Comparative examples.

[0077] Therefore it is found that a pharmaceutical composition of the present invention disorders comprising a mixed extract of Dioscorea Rhizoma and Dioscorea nipponica in a weight ratio of 3.5:1 has the effects on neuronal cell differentiation and enhancing neurite growth in the area of dentate gyrus compared to other mixed ratio.

EXPERIMENTAL EXAMPLE 5

[0078] Effect of the Herb Extracts on Enhancing the Secretion of NGF In Vivo

[0079] Hippocampi were separated from mice of the control group and the treatment group which was injected the mixed extract (D.Rhizoma:D.nipponica=3.5:1 extract) after completing the test of Experimental example 2. Anti-Nerve growth factor (NGF, abcam, rabbit origin 1:500) as a first antibody was reacted for one night and then biotinylated anti-rat (vector, goat origin) was used as a second antibody. After that, fluorescene-color was formulated by using streptavidin after ABC reaction (ABC kit, vector). The effect on enhancing the secretion of NGF was identified by counting NGF-positive cells in the area of hilus in hippocampus. The results are shown in FIG. 1.

[0080] As shown in FIG. 1, the herb extracts of Example 1 has the significant effect on enhancing the secretion of NGF in hippocampus.

[0081] Therefore it is found that a pharmaceutical composition of the present invention comprising a mixed extract of Dioscorea Rhizoma and Dioscorea nipponica in a weight ratio of 3.5:1 has the synergism compared to other mixed ratio. Thus, a phaimaceutical composition of the present invention may be used for preventing or treating neurodegenerative disorders including Alzheimer's disease, Creutzfeldt-Jakob disease, Huntington's disease, multiple sclerosis, Guillain-Barre syndrome, Parkinson's disease, Lou Gehrig's disease, paralytic dementia caused by gradual nerve cell death, diseases caused by progressive incontinentia, abnormalities in posture and exercise, progressive ataxia, muscular atrophy and weakness, and sensation and movement disorders.

[0082] Now, the composition comprising the herb extracts disclosed in EXAMPLE 1 will described in further detail by Formulation Examples. There have been no intentions to limit the claims but explain specifically.

FORMULATION EXAMPLE 1

Preparation of Injection

[0083]

TABLE-US-00009 Extract of Example 1 100 mg  Sodium metabisulfite 3.0 mg Methylparaben 0.8 mg Propylparaben 0.1 mg Sterile water for injection q.s.

[0084] To a mixture of the ingredients was added sterile water to form a total volume of 2 mL, and the solution was loaded to a 2 mL ampule and sterilized to give an injection.

FORMULATION EXAMPLE 2

Preparation of Tablet

[0085]

TABLE-US-00010 Extract of Example 1 200 mg Lactose 100 mg Starch 100 mg Mg stearate q.s.

[0086] The ingredients were mixed and compressed into a tablet using a tableting method.

FORMULATION EXAMPLE 3

Preparation of Capsule

[0087]

TABLE-US-00011 Extract of Example 1 100 mg Lactose  50 mg Starch  50 mg Talc  2 mg Mg Stearate q.s.

[0088] The ingredients were mixed and loaded to a gelatin capsule according to a typical method to afford a capsule.

FORMULATION EXAMPLE 4

Preparation of Liquid

[0089]

TABLE-US-00012 Extract of Example 1 1000 mg Sugar 20 g Isomerase 20 g Lemon Flavor q.s. Purified water added to form a total volume of 100 mL

[0090] The above ingredients were mixed, loaded into a 100 mL brown vial and sterilized to afford a liquid formulation.