AMELIORATIVE EFFECTS OF A WHOLE COFFEE FRUIT EXTRACT ON AGE-RELATED NEURODEGENERATIVE DISEASE

Abstract

The invention provides a method of extracting whole coffee fruit, an extract obtained from the method, and method of ameliorating age-related neurodegenerative diseases using said extract. The whole coffee fruit extract may be extracted by water, methanol, ethanol, or acetone and may comprise chlorogenic acid (CA) and procyanidine. The expression levels of p-CREB, BDNF, p-eIF2, BACE-1, A, NLRP3, caspase-1, IL-1 and COX-2 which may relate to age-related neurodegenerative diseases can be modulated.

Claims

1. A method of producing a whole coffee fruit extract, comprising the steps of: (a) providing freshly collected whole coffee fruit, optionally drying the whole coffee fruit so that the water content is 20% or less; (b) subjecting the dried whole coffee fruit of step (a) to extraction by a solvent selected from water, methanol, ethanol, and acetone; and (c) recovering the extract of step (b).

2. The method of claim 1, wherein the extraction in step (b) is conducted under the temperature of about 50 to about 100 C.

3. The method of claim 1, wherein the extraction in step (b) is conducted for about 0.5 to about 5 hours.

4. The method of claim 1, wherein the solid to liquid ratio in the extraction in step (b) is about 1:20 to about 1:5.

5. The method of claim 1, wherein the extraction in step (b) is performed by about 25 to about 30% ethanol at a solid to liquid ratio of about 1:10 to about 1:5 at about 70 to about 80 C. for about 3.5 to about 4 hours.

6. A whole coffee fruit extract obtained from the method of claim 1.

7. The whole coffee fruit extract of claim 6, comprising chlorogenic acid (CA) and procyanidine.

8. A method of ameliorating age-related neurodegenerative diseases, comprising administering to a subject in need thereof a therapeutically effective amount of a whole coffee fruit extract.

9. The method of claim 8, wherein the expression levels of p-CREB and BDNF are increased and the expression levels of p-eIF2, BACE-1, A, NLRP3, caspase-1, IL-1 and COX-2 are decreased.

10. The method of claim 9, wherein A deposition in Alzheimer's disease is reduced.

11. The method of claim 8, wherein the age-related neurodegenerative diseases are selected from Alzheimer's disease, Parkinson's disease, ischemic dementia, and Huntington's disease.

12. The method of claim 11, wherein the age-related neurodegenerative disease is Alzheimer's disease.

13. The method of claim 8, wherein the therapeutically effective amount of the whole coffee fruit extract is about 50 to about 400 mg/kg.

14. The method of claim 13, wherein the therapeutically effective amount of the whole coffee fruit extract is about 200 m/kg.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGS. 1A and 1B demonstrate the ratio of expression level of p-eIF2 to -actin in the hippocampus and cortex, respectively, of 3-month old male SAMP8 mice after feeding with different chews for 12 weeks (a: Control, b: 200 mg/kg whole coffee fruit extract).

[0018] FIGS. 2A and 2B demonstrate the ratio of expression level of BACE-1 to -actin in the hippocampus and cortex, respectively, of 3-month old male SAMP8 mice after feeding with different chews for 12 weeks (a: Control, b: 200 mg/kg whole coffee fruit extract).

[0019] FIGS. 3A and 3B demonstrate the percentage of area deposition of A in the hippocampus and whole brain, respectively, of 3-month old male SAMP8 mice after feeding with different chews for 12 weeks (a: Control, b: 200 mg/kg whole coffee fruit extract).

[0020] FIGS. 4A and 4B demonstrate the ratio of expression level of p-CREB to CREB in the hippocampus and cortex, respectively, of 3-month old male SAMP8 mice after feeding with different chews for 12 weeks (a: Control, b: 200 mg/kg whole coffee fruit extract).

[0021] FIGS. 5A and 5B demonstrate the ratio of expression level of BDNF to -actin in the hippocampus and cortex, respectively, of 3-month old male SAMP8 mice after feeding with different chews for 12 weeks (a: Control, b: 200 mg/kg whole coffee fruit extract).

[0022] FIGS. 6A and 6B demonstrate the ratio of expression level of NLRP3 to -actin in the hippocampus and cortex, respectively, of 3-month old male SAMP8 mice after feeding with different chews for 12 weeks (a: Control, b: 200 mg/kg whole coffee fruit extract).

[0023] FIGS. 7A and 7B demonstrate the ratio of expression level of Caspase-1 p20 to -actin in the hippocampus and cortex, respectively, of 3-month old male SAMP8 mice after feeding with different chews for 12 weeks (a: Control, b: 200 mg/kg whole coffee fruit extract).

[0024] FIGS. 8A and 8B demonstrate the ratio of expression level of IL-1 to -actin in the hippocampus and cortex, respectively, of 3-month old male SAMP8 mice after feeding with different chews for 12 weeks (a: Control, b: 200 mg/kg whole coffee fruit extract).

[0025] FIGS. 9A and 9B demonstrate the ratio of expression level of COX-2 to -actin in the hippocampus and cortex, respectively, of 3-month old male SAMP8 mice after feeding with different chews for 12 weeks (a: Control, b: 200 mg/kg whole coffee fruit extract).

[0026] FIG. 10 shows the results of immunohistochemical (IHC) staining for the levels of A deposition in the brain of 3-month old male SAMP8 mice after feeding with normal chew (A) and the whole coffee fruit extract (200 mg/kg) of the present invention (B) for 12 weeks.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedence over any dictionary or extrinsic definition.

[0028] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

[0029] The term whole coffee fruit as used herein refers to the entire fruit of the coffee tree (Coffea spec.) in which the exocarp and the outer mesocarp (i.e., the pulp) surround the inner mesocarp (i.e. the mucilage) and endocarp (i.e., the hull), which in turn surround the seeds (i.e., the beans). Thus, the term whole coffee fruit specifically refers to a whole coffee fruit, which may or may not include the seed of the fruit.

[0030] The term whole coffee fruit extract as used herein refers to the product of any whole coffee fruit extraction process known in the art. Any whole coffee fruit extract is deemed suitable for use herein. For example, a whole coffee fruit extract may be prepared with an aqueous and/or alcoholic solvent to obtain a solution enriched in one or more desirable components (and/or to obtain a material depleted of one or more undesired components). So prepared extracts can further be refined and/or enriched in a specific component using chromatographic methods (e.g., ion exchange, size exclusion, or filtration), or addition of a component or flavoring agent of the whole coffee fruit. Depending on the desired component(s), it should further be recognized that the extracts may also be prepared from selected portions of a coffee cherry (e.g., at least one or more of the bean of the coffee cherry, the pulp, the mucilage, and/or the hull of the whole coffee fruit). In one preferred embodiment, a whole coffee fruit extract is prepared by the method disclosed herein. In another preferred embodiment, the whole coffee fruit extract which is prepared by the method disclosed herein is that under the product name, Anthochlorogin.

[0031] The term ameliorating as used herein refers to delaying the onset of the symptoms of a susceptible subject or reducing the occurrence of a disease, reducing and/or improving the symptoms of a susceptible subject or increasing the survival rate of the subject with certain lethal disorders or conditions.

[0032] The term age-related neurodegenerative diseases as used herein refers to the neurodegenerative diseases that progress as age increases, for example, brain function and cognition decline with aging. For example, such diseases include Alzheimer's disease, Parkinson's disease, ischemic dementia, and Huntington's disease.

[0033] The term subject as used herein denotes animals, especially mammals. In one preferred embodiment, the term subject denotes humans.

[0034] The term therapeutically effective amount as used herein refers to the amount of an active ingredient used alone or in combination with other treatments/medicaments for treating age-related neurodegenerative diseases that shows therapeutic efficacy.

[0035] Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0036] The inventors of the invention found that a whole coffee fruit extract can enhance the expression of p-CREB and BDNF and decrease the expression of p-eIF2, BACE-1, A, NLRP3, caspase-1, IL-1 and COX-2. As these parameters may be associated with age-related neurodegenerative disease, the present invention may thus provide a novel therapy for age-related neurodegenerative disease. In a preferred embodiment, the age-related neurodegenerative disease is Alzheimer's disease.

[0037] In addition to the data on the enzymes, in situ data showing the reduced deposition of A in the brain of the mice further evidence the efficacy of the whole coffee fruit extract in diseases associated with A deposition. In an embodiment, the disease associated with A deposition is Alzheimer's disease.

[0038] Having now generally described the invention, the same may be more readily understood through reference to the following examples, which provide exemplary protocols for the production of the whole coffee fruit extract of the invention and its use in the amelioration of age-related neurodegenerative disease. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

EXAMPLES

Example 1

Preparation of Whole Coffee Fruit Extract

[0039] Coffee (Coffee Arabica) berry raw materials were obtained from the coffee garden of farmers or agricultural production and marketing groups in Tainan, Pingtung, Hualien and Taitung. The coffee fruits (fresh fruit) were sterilized after receipt so as to reduce surface microbes. Then, the coffee fruits were delivered to the lab by a preservation technology. Right after arrival, the coffee fruits were subjected to cold wind or freeze drying so that the fresh fruits were dried to reach a water content of 20% or less. After that, the coffee fruits were stored or grounded into powder for extraction.

[0040] During extraction, the whole coffee fruit powders were dissolved in ethanol for extraction. The extract was subjected to high performance liquid chromatography (HPLC) for analysis of possible active ingredients. The results reveal that the extract comprises 5-CQA, CA and procyanidine. The extract was freeze-dried and a brown to deep brown powder was obtained, which is named as Anthochlorogin. Before evaluation on the biological activities of the extract, the dried product was dissolved in corn oil and adjusted to desired concentrations in the chew.

Example 2

Experimental Design and Composition of Feedstuff

[0041] Senescence accelerated mice P8 (SAMP8) was used as the animal model for evaluating amelioration of Alzheimer's disease. This mice model is characterized in having age-related memory defects. Whether whole coffee fruit extract has efficacy in improving learning and memory capacities and the effect on related molecular mechanism were explored. 3-month old SAMP8 were raised in 30 (W)20 (D)10 (H) cm transparent plastic cages. The temperature was kept at 222 C., relative humidity kept at 655% and the room had automatically controlled light periods, where 7:00-19:00 was the dark period and 19:00-7:00 was the light period. Before testing, animals were accommodated for 3-5 days. At the beginning of the experiment, mice were separated into a control group taking standard diet (20% Casein, 5% corn oil, 1% vitamin mixture (AIN93-G), 5% mineral mixture (AIN93-G), 2% cellulose powder, and 2.5% choline) and an experimental group taking an additional 200 mg/kg whole coffee fruit extract. Said 200 mg/kg is a human daily dose and was converted to mice dose for administration. Feedstuff and water were taken freely. During the experiment, the amounts of food intake and body weight were recorded. After feeding for 12 weeks, the mice were sacrificed and brain samples were subjected to analysis for amyloid (A), phospho-eukaryotic initiation factor 2 (p-eIF2), beta-site amyloid precursor protein-cleaving enzyme-1 (BACE-1), brain-derived neurotrophic factor (BDNF) and phospho-cAMP response element-binding protein (p-CREB). Levels of the inflammation markers NLRP3, caspase-1, IL-1, and COX2 were also measured.

Example 3

Western Blot Analysis

[0042] Brain tissue samples were homogenized. According to the molecular weight of the target protein to be observed, different concentrations of SDS-PAGE gels were prepared for electrophoresis. The samples were heated at 100 C. and sequentially loaded into the wells of the SDS-PAGE gels. Separation was conducted at 65V 100 mins and 100V 80 mins for the target protein to be able to run to the desired position. After electrophoresis, SDS-PAGE gels and PVDF membranes were placed in a cassette for transferring the target protein. The transfer was conducted in a 4 C. cold room.

[0043] After completion of transfer, the PVDF membrane was cut according to the molecular weight of the target protein and placed in small box for blocking for 1 hour. The membrane was then washed by wash buffer on a shaker for several times. After that, primary antibody solution was added to soak the membrane on a shaker in a 4 C. cold room and recovered after 8-16 hours of incubation. Secondary antibody solution was then added to soak the membrane on a shaker at room temperature for 1 hour. The PVDF membrane was then incubated with an enhanced chemiluminescence (ECL) substrate and placed in a chemiluminescence imaging system for taking photos of the images.

[0044] The data of each experimental group in this example were analyzed by SPSS software. The results are represented by meanS.E.M. Differences of protein expression among each group were analyzed using one-way analysis of variance (one-way ANOVA). P<0.05 represents significant difference.

Example 3.1

Analysis of the Expression Level of the A Related Factor p-eIF2

[0045] As shown in FIGS. 1A and 1B, in 3-month old male SAMP8 mice fed with different chews (a: Control, b: 200 mg/kg whole coffee fruit extract) for 12 weeks, the ratio of expression levels of p-eIF2 to -actin in the hippocampus (FIG. 1A) and cortex (FIG. 1B) of the mice brain are decreased. The decreases reach statistical significance.

Example 3.2

Analysis of the Expression Level of A Regulatory Factor BACE-1

[0046] As shown in FIGS. 2A and 2B, in 3-month old male SAMP8 mice fed with different chews (a: Control, b: 200 mg/kg whole coffee fruit extract) for 12 weeks, the ratio of expression levels of BACE-1 to -actin in the hippocampus (FIG. 2A) and cortex (FIG. 2B) of the mice brain are decreased. The decreases reach statistical significance.

Example 3.3

Analysis of the Deposition of A in the Brain

[0047] As shown in FIGS. 3A and 3B, in 3-month old male SAMP8 mice fed with different chews (a: Control, b: 200 mg/kg whole coffee fruit extract) for 12 weeks, the percentage of area deposition of A in the hippocampus (FIG. 3A) and whole brain (FIG. 3B) of the mice brain are decreased. The decreases reach statistical significance.

Example 3.4

Analysis of the Expression Level of Neurotrophy Factor p-CREB

[0048] As shown in FIGS. 4A and 4B, in 3-month old male SAMP8 mice fed with different chews (a: Control, b: 200 m/kg whole coffee fruit extract) for 12 weeks, the ratio of expression levels of p-CREB to CREB in the hippocampus (FIG. 4A) and cortex (FIG. 4B) of the mice brain are increased. The increases reach statistical significance.

Example 3.5

Analysis of the Expression Level of Neurotrophy Factor BDNF

[0049] As shown in FIGS. 5A and 5B, in 3-month old male SAMP8 mice fed with different chews (a: Control, b: 200 m/kg whole coffee fruit extract) for 12 weeks, the ratio of expression levels of BDNF to -actin in the hippocampus (FIG. 5A) and cortex (FIG. 5B) of the mice brain are increased. The increases reach statistical significance.

Example 3.6

Analysis of the Expression Level of Inflammation Related Factor NLRP3

[0050] As shown in FIGS. 6A and 6B, in 3-month old male SAMP8 mice fed with different chews (a: Control, b: 200 m/kg whole coffee fruit extract) for 12 weeks, the ratio of expression levels of NLRP3 to -actin in the hippocampus (FIG. 6A) and cortex (FIG. 6B) of the mice brain are decreased. The decreases reach statistical significance.

Example 3.7

Analysis of the Expression Level of Inflammation Related Factor Caspase-1

[0051] As shown in FIGS. 7A and 7B, in 3-month old male SAMP8 mice fed with different chews (a: Control, b: 200 m/kg whole coffee fruit extract) for 12 weeks, the ratio of expression levels of caspase-1 p 20 to -actin in the hippocampus (FIG. 7A) and cortex (FIG. 7B) of the mice brain are decreased. The decreases reach statistical significance.

Example 3.8

Analysis of the Expression Level of Inflammation Related Factor IL-1

[0052] As shown in FIGS. 8A and 8B, in 3-month old male SAMP8 mice fed with different chews (a: Control, b: 200 m/kg whole coffee fruit extract) for 12 weeks, the ratio of expression levels of IL-1 to -actin in the hippocampus (FIG. 8A) and cortex (FIG. 8B) of the mice brain are decreased. The decreases reach statistical significance.

Example 3.9

Analysis of the Expression Level of Inflammation Related Factor COX-2

[0053] As shown in FIGS. 9A and 9B, in 3-month old male SAMP8 mice fed with different chews (a: Control, b: 200 m/kg whole coffee fruit extract) for 12 weeks, the ratio of expression levels of COX-2 to -actin in the hippocampus (FIG. 9A) and cortex (FIG. 9B) of the mice brain are decreased. The decreases reach statistical significance.

Example 4

Immunohistochemical (IHC) Staining for A

[0054] After mice were sacrificed, brain tissues were obtained and immediately soaked in 10% formalin for paraffin embedding and tissue slicing (3-5 m/slice). Before performing IHC staining, the slices were placed in xylene for dewaxing and treated with ethanol for removing xylene. The slices were soaked in Trilogy and heated at 121 C. for 15 minutes to restore antigenicity followed by IHC staining. At first, goat serum-PBS solution was added for blocking. After that, diluted monoclonal anti-A antibody was added and incubated at 4 C. overnight. The next day, superenhancer solution was added for reaction at room temperature in a dark place followed by reaction with poly-HRP reagent at room temperature in the dark place. DAB was used for 1.5 minutes for colorification and then hematoxylin was used for counter stain. After completion, the slices were placed on an optical microscope to observe the density of a brownish substance which is equivalent to the amount of A deposition.

[0055] The results are shown in FIG. 10 which represents the level of deposition of A in the brain of 3-month old male SAMP8 mice fed with different diets for 12 weeks. FIG. 10A is the result of the control group in which mice were fed with normal chew. FIG. 10B is the result of the experimental group in which mice were fed with chew additionally added with 200 mg/kg whole coffee fruit extract of the present invention. The results in FIG. 10 clearly demonstrate that the level of A deposition in FIG. 10A is obviously more intense than that in FIG. 10B. This result indicates that mice fed with 200 m/kg whole coffee fruit extract of the present invention have significantly reduced amount of A deposition in the brain of SAMP8 mice. Thus, diseases or symptoms associated with A deposition may be ameliorated in SAMP8 mice fed with the whole coffee fruit extract of the present invention.

[0056] Age-related neurodegenerative diseases may be caused due to oxidative stress, neuron cell damage, or decreased levels of neurotrophy factors. From the examples demonstrated above, it was surprisingly found that by ingesting the whole coffee fruit extract of the present invention, the levels of neurotrophy factors p-CREB and BDNF were increased, and p-eIF2 and BACE-1 that regulate A were decreased. Eventually, decreased deposition of A was observed. Further, the expression levels of inflammation factors NLRP3, caspase-1, IL-1 and COX-2 were decreased. Thus, the whole coffee fruit extract of the present invention, which may comprise chlorogenic acid (CA) and procyanidine, may be useful for ameliorating age-related neurodegenerative diseases.

[0057] Numerous modifications and variations of the invention as set forth in the above illustrative examples are expected to occur to those skilled in the art. Consequently, only such limitations as appear in the appended claims should be placed on the invention.