FIELD OF THE INVENTION

This invention relates to a formulation that reduces oxidative stress in a mammal patient suffering cognitive status.

BACKGROUND OF THE INVENTION

Over the course of human history, life expectancy at birth has increased substantially. The continuing increase in life expectancy gives rise to modifications on the population structure and steady increase in the elderly population. Of all the changes which occur during the aging process, deterioration in cognitive status is perhaps the single most disabling condition.

In recent years, researches have strongly supported the role for polyphenols in the prevention of degenerative diseases, particularly neurodegenerative diseases including cognitive status. A commercialized grape juice was given to elderly subjects with poor cognitive status. The study was among the first intervention study which examined the effects of polyphenols-rich juice towards cognitive status. In the study, subjects were supplemented with 532 ml of a commercialized grape juice (203.8 mg of anthocyanins daily] for 12 weeks. There was significant improvement in the measure of verbal learning and non-significant enhancement of verbal and spatial recall. Previous researches have shown that anthocyanins are the main component of flavonoids that is responsible for the improvement of cognitive status related to aging.

As antioxidants, polyphenols may protect cell constituents against oxidative damage and, therefore, limit the risk of various degenerative diseases associated to oxidative stress, such as cancers, cardiovascular diseases and impaired cognitive. Due to the increased oxidative damage in aged brain and the detrimental effects of oxygen radicals on crucial neuronal components, nutritional approach to address age-related neuronal dysfunctions have started to focus on dietary interventions of antioxidant-rich food. Initial studies in nutritional neuroscience demonstrated that antioxidant-rich diets could delay and even reverse age-related cognitive decline in laboratory animals. Subsequent work has established that supplementation with berries such as blueberries and strawberries can induce dramatic changes in the brains of aged animals above and beyond strictly antioxidant actions. U.S Application No. US 12/027, 652 to Smith discloses compositions for supporting healthy memory and optimizing mental energy and methods for improving, preventing, and treating mental disorders or deterioration. The compositions of the invention can be formulated from various fruits as nutritional or dietary supplements. This patent teaches how to produce a supplement using a group of fruits. This patent, however does not suggest the use of tropical fruits juices as the source polyphenols, antioxidants and anthocyanin to be incorporated in the supplement.

U.S. Pat No. US 6,642,277 to Howard et al. discloses a plant-derived flavonol-containing dry composition suitable for human consumption, wherein at least 25% of the plant-derived material in the composition comprises polyphenols, together with uses thereof. This patent however, utilizes wine and grape juices in order to obtain the polyphenols and it does not suggest the use of tropical fruits juices as the nutrient sources to be consumed as the way the invention is consumed.

U.S. Pat. No. US 6,106,874 to Liebrecht et al. disclose a low pH nutritional beverage that utilizes pectin-free fruit juice as a major component and a source of calcium selected from natural milk mineral, calcium lactate gluconate and mixtures thereof. The beverage also contains water soluble vitamins, flavors and carbohydrates. The use of a pectin-free and clarified pear juice in a preferred embodiment of the beverage provides a fat-free beverage with a substantially clear appearance and a light, refreshing mouthfeel. The beverage is preferably produced using a "cold water process that results in excel lent physical stability of the beverage over shelf life and the reduction of browning. This reference fails to suggest or disclose the utilization of tropical fruits juice as the main ingredients in producing this formulation.

U.S. Pat. No. 6,086,910 to Howard et al. discloses a flavonol-containing dry composition suitable for human consumption, together with uses thereof. This patent however fails to suggest the utilization of tropical fruits juices as the main ingredients for the sources of nutrients to improve cognitive status of a person affected by diseases caused by oxidative stress in the brain.

While the prior art disclosed the supplementation of juice-based drinks with nutrients sources that will assist in reducing the effect of oxidative stress in the brain such as polyphenols, anthocyanin and antioxidants and the like, they have failed to appreciate that juices from the tropical fruits may also contain similar nutrients, if not higher nutrients content to cater the needs for supplements to Asians. Whilst the sources of fruits in the prior art are mostly available in Western countries, it is important to note that for Asian countries, these fruits are not readily available and expensive. This is contrary to the present invention wherein the formulation can be produced from the tropical fruits at lower costs, as well as obtaining similar nutrient contents for the application in reducing the effects of oxidative stress. In view of the present disclosures or through the practice of the present invention, other advantages or solutions to other problems will become apparent.

SUMMARY OF THE INVENTION

This invention relates to a formulation that reduces oxidative stress in a mammal patient suffering cognitive status, the formulation including: a water content between 89.38% to 92.93%, ash content of 0.12% to 0.15%, protein content of 0.08% to 0.16%, fat content of 0.00% to 0.01%, carbohydrates content of 6.86% to 10.31%, a phenolic acid content between 609 to 690.27 mg GAE/L, an anthocyanin content between 12.94 to 18.79 mg/L, wherein an antioxidant activity of the formulation measured by 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) assay is 88.9%. The advantages and elements of the present invention will be described explicitly from the description of the preferred embodiments which illustrate the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure la shows the Ferric reducing antioxidant power (FRAP) antioxidant capacity assay among different mixture juices formulation of three fruits.

Figure lb shows 2, 2-diphenyl- 1-picrylhydrazyl (DPPH) antioxidant capacity assay among different mixture juices formulation of three fruits.

Figure lc shows a total phenolic content (TPC) among different mixture juices formulation of three fruits.

Figure 2 shows the b-amyloidl-42 (Ab1-42) concentration in brain homogenate among different rats group.

Figure 3 shows the CRH concentration in brain homogenate among different rats group.

Figure 4 shows the intensity relative of inducible nitric oxide synthase (iNOS) expression in brain homogenate among different rats group.

Figure 5 shows the histology in cornus ammonis (CA1) region of hippocampus brain tissue under Nissl staining (Cresyl violet) among rats group.

Figure 6 shows a neuronal count among different rats’ group.

Figure 7 depicts the changes in malondialdehyde (MDA) concentration from baseline for both intervention and placebo groups.

Figure 8a shows a score plot of principal component analysis (PCA) at baseline for juice supplementation group (green) and placebo group (blue).

Figure 8b shows a loading plot of PCA at baseline.

Figure 9a shows a score plot of PCA at week 5 for juice supplementation group (green) and placebo group (blue).

Figure 9b shows a loading plot of PCA at week 5 of supplementation. Figure 10a shows a score plot of PCA at week 10 for juice supplementation group (green) and placebo group (blue). Figure 10b shows a loading plot of PCA at week 10 of supplementation.

Figure 11 depicts the changes in the concentration of hippuric add from baseline in percentage for both intervention and placebo groups. Figure 12 depicts the changes in the concentration of benzoic acid from baseline in percentage for both intervention and placebo groups.

Figure 13 depicts the changes in the concentration of glycolic acid from baseline in percentage for both intervention and placebo groups.

Figure 14 depicts the changes in the concentration of 3-methyladenine from baseline in percentage for both intervention and placebo groups.

Figure 15 depicts the changes in the concentration of thyroxine from baseline in percentage for both intervention and placebo groups.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a formulation that reduces oxidative stress in a mammal patient suffering cognitive status wherein the formulation mainly contains water, ash, protein, carbohydrates, natural sugar and low fat and further formulated to be rich in phenolic acids, antioxidants and anthocyanin. It is known that, one of the dietary aspects which has received attention in the field of cognitive research is dietary polyphenols. In recent years, many studies have strongly supported the role for polyphenols in the prevention of degenerative diseases, particularly neurodegenerative diseases including cognitive status.

Before the present invention is described in greater detail, it should be appreciated that formulation and other formulations in accordance with this disclosure may have any of numerous different specific formulations or constitutions. The formulation in accordance with this disclosure can vary to a certain extent, depending upon such factors as the formulation’s intended market segment, its desired nutritional characteristics, flavor profile and the like. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Unless defined otherwise, 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing of the present invention, the preferred methods and materials are described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. It is noted that, as used herein and in the appended claims, the singular forms "a”, "an", and "the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely,” "only” and the like in connection with the recitation of claim elements, or use of a "negative” limitation.

As it will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible

In further describing the subject invention, the formulation according to the embodiments of the invention are described first in greater detail. Next systems that find use in practicing various embodiments of the formulation of the invention are reviewed.

It is known by those in the medical field that oxidative stress plays an important role in the pathogenesis of a number of diseases. Particularly, it is linked to the Alzheimer's disease (AD) is an age-related neurodegenerative disease and the main common cause of dementia in the elderly. Histopathological hallmarks of AD are intracellular neurofibrillary tangles and extracellular formation of senile plaques composed of the amyloid-beta peptide (Ab) in aggregated form along with metal-ions such as copper, iron or zinc. Redox active metal ions, as for example copper, can catalyze the production of Reactive Oxygen Species (ROS) when bound to the amyloid-b (Ab). The ROS thus produced, in particular the hydroxyl radical which is the most reactive one, may contribute to oxidative damage on both the Ab peptide itself and on surrounding molecule (proteins, lipids, ...).

As reviewed above, the invention provides a formulation for human beings wherein this formulation has a potential to improve the cognitive status among middle-aged women, namely the learning, memory, processing speed, sequencing, mental flexibility and visual-motor skills. The metabolites identified were related to polyphenols consumption and cognitive functions. In a prior invention, a commercialized grape juice was given to elderly subjects with poor cognitive status. This was among the first intervention study which examined the effects of polyphenols- rich juice towards cognitive status. In the study, subjects were supplemented with 532 ml of a commercialized grape juice (203.8 mg of anthocyanins daily) for 12 weeks. There was a significant improvement in the measure of verbal learning and non-significant enhancement of verbal and spatial recall. It has been confirmed that the anthocyanins are the main component of flavonoids that is responsible for the improvement of cognitive status related to aging.

In a preferred embodiment, the formulation of the invention is selected from tropical fruits namely pomegranate, white guava and roselle. The utilization of tropical fruits for the role of polyphenols and cognitive status has been fairly limited as polyphenols-rich fruit that are usually employed are only easily available for the Western population, such as berries. Tropical fruits, such as pomegranate ( Punicagranatum ) and dragon fruit ( Hylocereusundatus ) which are more accessible by the Asians have similar, if not higher content of polyphenols. These fruits have some potential to improve cognitive status during aging. However, to the best of the inventors’ knowledge, no placebo-controlled intervention studies that examined the effects of polyphenols and cognitive status have been conducted involving tropical fruits among Asian populations.

In one of the embodiments, the formulation consists of 1/3 pomegranate, 1/3 white guava, and 1/3 roselle.

In other embodiment of the formulation, it consists of 2/3 pomegranate juice, 1/6 white guava juice, and 1 /6 roselle juice.

In another embodiment, the formulation consists of 1/6 pomegranate, 2/3 white guava, and 1/6 roselle.

In an alternative embodiment, the formulation consists 1/6 pomegranate, 1/6 white guava, and 2/3 roselle.

Tropical fruits are valuable sources of dietary fiber, vitamins and natural phenolic antioxidant One example of tropical fruits is pomegranate (Punica granatuni) under Punicaceae family, a fruit native to the Middle East Pomegranate is a phytochemical dense fruit containing anthocyanins and hydrolysable tannins. Different parts of this plant are used in indigenous Indian medicine to cure various diseases, particularly diabetes.

Guava ( Psidium guajava) fruit is considered as highly nutritious since it contains high levels of ascorbic acid that is three to six times higher than orange. Guava could be used as a lipid level controller because of its ability to reduce both total cholesterol and low-density lipoprotein (LDL) levels in the subjects tested by 18.8% and 19.4%, respectively, compared to their baseline levels upon consumption for 4 weeks. In additional of having high amount of vitamin C, Hibiscus sabdariffa (Roselle) is one such plant which flowers are used to prepare juices. The roselle extract has a unique red colour, good flavour, low sugar and high acidic content. The acidity makes the juice sour hence the need for addition of sweetening products. Calyx extract of roselle has also been used as an effective treatment for patients with kidney stones due to its uricosuric effect

The fact that pomegranate is one of the fruits which always mentioned in many health related study while guava and roselle seems to be popular tropical juice enjoyed nowadays. The synergistic effect from mixing two or more of such fruit juices cannot be over emphasized. The increase or decrease in the content of bioactive compounds or antioxidant activity can be related to chemical reactions that can occur among the fruits, which should be better studied. Thus, the mixture is expected to have acceptable physicochemical quality and have higher phenolic content compared to its single juice. It is important to note that the present invention is not only limited to concentrated fresh fruits juices wherein concentrated fruits extracts will produce similar results as well.

In one of the embodiments, the phenolic acids identified to be present in the formulation include gallic acid, ellagic acid, catechin, epicatechin, P-coumaric acid, chlorogenic acid, procyanidin B2 and kuromanin chloride. For example, the inventors had specifically identified eight (8) types of polyphenols present in the invention and this information is tabulated in Table 4.

The formulation of this present invention will soon be available either in the form of a liquid concentrate, a tablet, granules, a powder, or a solution, depending on the requirement provided by the desired manufacturers.

In order to understand the physico-chemical properties of the formulation, it was further tested in a separate experiment and the efficacy of the formulation is further illustrated in the following working examples in animal and human testing.

EXAMPLE 1

Experiments to confirm the formulation's physico-chemical properties

Materials and Method

A. Preparation of Tropical Fruits Juices Extraction

Pomegranate and guava fruits were cleaned using tap water followed by extraction of juice using heavy duty electric juicer {Breville, Australia ] while calyx of the seedless roselle was extracted in hot water for 15 minutes. All fruit extracts were mixed and filtered with muslin cloth before it went through low temperature long time (LTLT) pasteurization process. Finished product were kept in high-density barrier type of bottle and stored in 4-8 °C prior to analyses. The results for nutrient contents were expressed as the average ± standard deviation of the two replicates from six batches of finished product.

B. Physicochemical Analysis of Juice

In order to determine the pH value for the mixture of the tropical juices, a digital pH meter was utilized. About 15 ml sample was used for the test for each of these samples. The pH meter (Mettler Toledo, Switzerland] was calibrated before use.

1. Total Soluble Solid (TSS)

Brix value (°Brix] or TSS for all samples has been determined using table-top refractometer (Abbe, Germany ) and the lens is adjusted to be midway between the bright and dark side lines.

2. Colour intensity

Approximately 30 ml of homogenized samples was poured into a test tube for measurement using hand-held chromameter {Minolta, Japan). The chromameter was calibrated prior to samples measurement. The test tube was placed into a special hollow holder in front of the chromameter and the reading was taken for three times to get average value.

3. Vitamin C Content

Determination of vitamin C using titration method from the Association of Analytical Communities (AOAC, 2000] was done in this study. Approximately 0.1% ascorbic acid solution was titrated with 1 ml of 0.01% DCPIP solution until the original blue color of the DCPIP solution faded. The steps were repeated three times for each sample to get average reading. 4. Total Sugar

Total sugar analysis was done according to International Commission for Uniform Methods of Sugar Analysis method. About 1 ml of 5% phenol reagent solution was added to 1 ml juice sample which had been diluted for 1000 times. About 5 ml of concentrated H ₂ SO ₄ was added slowly into the mixture and was incubated for 30 minutes at room temperature. The mixture absorption was measured at 490 nm using UV-visible spectrophotometer (Secomam™). Absorbance of samples was compared with glucose serial dilution (0.02-0.10 mg/ml) by using standard curve. The amount of total sugar content was reported in g/100 ml.

5. Titratable Acidity (TTA)

Titratable acidity of the juice was determined with some modification. About 10 ml of the sample was transferred into volumetric flask (100 ml) which was then topped up with distilled water until it reached the volume. About 10 ml solution from the flask was placed into a conical flask and three drops of 2% phenolphthalein were added into the sample. The sample was then titrated with 0.1N NaOH until the pink color appeared for 30 seconds. The volume of NaOH used was recorded and this step was repeated for three times for each sample. The content of the TTA in the sample was determined based on the following equation:

6. Proximate Analysis of luice

Analyses method for determination of moisture, ash, protein, fat and carbohydrate content are based on AOAC (2000).

7. Moisture Content

About 1.5 g sample was weighed using analytical balance (Mettler Toledo, Switzerland) prior to drying the samples in the oven at 105 °C for overnight Samples were then left to cool in the desiccator and the samples were reweighed until it reached a consistent weight Moisture content was calculated in percentage based on wet weight basis.

8. Ash Content

About 5 g sample was placed in crucibles and the sample was turned to ash in a furnace (Carbolite, UK) at 550 °C for overnight Samples were then left to cool in the desiccator and the samples were reweighed until reached a consistent weight. Total ash content was calculated in percentage based on initial sample weight 9. Protein Content

About 4 g sample was weighed and placed in the digestive tube. About 7 g K2SO4, 0.8 g CuS0 ₄ and 12 ml H2SO4 (catalyst) were added into the sample as well. The sample was then heated at 420 °C for 60 minutes in the digestive unit (Foss, Sweden). Sample mixture was left to cool and 75 ml of distilled water were added into the digestive tube prior to the distillation process. A total of 25 ml of boric acid (4%) was added into the conical flask. The distillation process started with the addition of 50 ml NaOH into the distillation unit (Foss, Sweden). The boric acid was then titrated with 0.2 N HC1 until the original color of boric acid was obtained. The percentage of protein content was determined based on following equation:

Protein (%) = [(Volumeb _lank x Weight _samPle x 14.007) /Volume _HCI x 10] x 6.25

10. Fat Content

About 5 g of sample was placed into the ceramic cup and weighed using analytical balance.

About 2-3 spatulas of Celite 545® were added into samples and were dried over hot water bath for two hours. Samples were then mixed with 2-3 spatulas of Na ₂ SO ₄ before been transferred into the thimbles. The thimbles were placed in the fat extractor system (Foss, Sweden) and 70 ml petroleum ether (40%-60%) was added into the extraction cups before they were placed in the fat extractor system. When the extraction process finished, extraction cups were dried in the oven at 100 °C for 15 minutes. The extraction cups were left to cool in the desiccator and they were reweighed until consistent weight has been reached. The fat content was calculated in percentage based on initial sample weight 11. Carbohydrate and Calorie Content

Differential method has been used to determine the carbohydrate content in the sample while calorie content was determined according to Recommended Dietary Allowance (RDA) calculation: Total carbohydrate = 100% - ( Moisture % + Ash % + Protein % + fat %)

Calorie (kcal/100 g) = ( Protein % x 4) + ( Fat % x 9) + ( Carbohydrate % x 4]

C. Determination of Juice Antioxidant Content

1. Total Polyphenol Content (TPC) Analysis TPC value of the juice was determined using spectrophotometer ( Secomam , France ) based on method by Singleton, Orthofer and Lamuela-Raventos (1999] with a slight modification. About 0.5 ml of diluted sample was mixed with 2.5 ml diluted Folin-Ciocalteu reagent (1:10). In interval time of 4-8 minutes, 2 ml saturated NaCO ² solution (75 g/L) was added into the mixture prior to 2 hours of incubation in the dark at room temperature. The absorbance of the sample was measured at 760 nm and was compared to gallic acid serial dilution (0.2-1 mM) by using standard curve. The value was calculated in mg of GAE/100 ml.

2. Total Monomeric Antocvanin (TMA) Analysis

The method was based on Lee, Durst, and Wrolstad (2005). About 1 ml of liquid sample (1:10) was poured into a flask containing 25 ml pH 4.5 buffer solution (this mixture was stable for 4 hours at room temperature). Same thing was done on a flask containing pH 1.0 buffer solution and absorbance for both pH solutions was taken at 510 nm and 700 nm. The value of TMA was determined by using below calculation:

TMA (mg C3G/L) = [DA/eL) x MW x 10 ³ x DF

Where, A is absorbance difference, e is cyanidine-3-glucoside molar absorbance, L is cell path length MW is anthocyanin molecular weight, and DF is dilution factor.

3. Antioxidant Capacity hy Trolox Equivalents fTEAC) Analysis

The TEAC value was estimated using 2,2-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) reagent assay, based on method by Thaipong et al. (2006). 7 mM ABTS radical cation stock solution (solution A) and 2.45 mM potassium persulfate (K8S208) stock solution (solution B) were prepared and then were mixed based on 1:1 ratio to be used as working solution. The working solution was allowed to stand in the dark at room temperature for 12-16 hours before it was diluted to an absorbance of 1.1 [A] at 734 nm. About 0.2 ml sample aliquot was diluted with 3.8 ml ABTS radical cation working solution prior to 2 hours incubation in the dark. Absorbance of the sample mixture was measured and was compared with Trolox serial dilution (0-300 mM) standard curve. Result was reported in mM Trolox Equivalent (TE)/g.

4. Antioxidant Capacity hv 2.2-Diphenyl-l-Picrylhvdrazyl Assay (DPPH) Analysis

Another antioxidant capacity value was estimated by 2,2-diphenyl- 1-picrylhydrazylassay (DPPH) assay. About 200 mΐ sample was mixed with 0.1 mM DPPH stock solution which was previously prepared by dissolving the DPPH powder in methanol to an absorbance 0.70 ± 0.01 at 516 nm. The absorption of the mixture was measured after 30 minutes against a blank. Percentage of antiradical action toward DPPH was estimated by the difference in absorbance with or without the sample (control).

5. Polyphenols Compounds Determination bv Hiah-Performance Liquid Chromatography (HPLC) Reverse phase HPLC with photodiode array detection (DAD) was used to characterize the polyphenols compounds based on their Ultra-violet (UV) spectra. This method was based on Abad-Garc’ia et al. (2007) with some modification. Eight polyphenols compounds analyzed were consisted of hydroxybenzoic acid (gallic acid and ellagic acid), hydroxycinnamic acid (chlorogenic acid and p-coumaric acid), flavan-3-ol (catechin, epicatechin, and procyanidin B2) and anthocyanidin (kuromanin chloride). All polyphenol standard solutions (ranging from 25 to 250 mg/ ml) were prepared in methanol and all were stored at 4°C in darkness. Chromatographic analysis was performed by HPLC equipped with a DAD detector, and controlled by Empower software. An Atlantis C18 (150 x 4.6 mm, i.d., 5 mm) column with mobile phase A (acetic acid- water, 0.5:99.5, v/v) and B (methanol) were used. The applied elution conditions were in gradient mode, with flow rate of 1.0 ml/min and injection volume of 10 pi. Hydroxybenzoic acids were monitored and quantified at 254 nm while flavan-3-ols and hydroxycinnamic acids were monitored at 280 nm and 320 nm, respectively.

Results and Discussions

1. Physicochemical Characteristic of the Beverage

The pH value of the juice is important to be measured. It represents the degree of acidity and alkalinity of a substance. The beverage has low pH value of 3.69 (Table 1). This indicated that the juice was in acidic condition and suitable to be served as ready-to-drink (RTD) beverages. In beverage industry, the addition of organic acid into commercial juice was intended to lower the original pH of the juices. However, in preparation of fruits juices there was no addition of organic acid into it Despite of that, pathogens such as Escherichia coli (0157: H7), Salmonella sp., and parasitic protozoa such as Cryptosporidium parvum can still reproduce in juices having pH less than 4.6. They are not only shortening product shelf life but also can cause food-borne illnesses and death. Therefore, pasteurization treatment process is a must to be carried out properly in all beverages produced. In a typical embodiment, the lower the pH of fruit juices, the greater the heat effect given to the microorganisms, especially in terms of pressure and radiation levels.

Table 1. Physicochemical characteristics of the beverage

Measurement of food colour is usually performed for quality control purposes. The value L of the beverage was 33.25 while a and b values were 3.16 and -4.63, respectively. Colour of the juice was highly depending on the types of the fruit that contained in the mixture. It has been reported the value of polyphenol content was in line with the colour brightness and polyphenol content will increase when the fruit is matured or cooked. Storage condition and time can also cause colour changes on food product At the beginning of the storage period, the brightness colour of the juice will increase but then decreases over the period of storage. This condition was normally caused by non-enzymatic browning reaction. The main soluble solid in fruit juices normally consisted of sugar. Other than that, organic compounds, amino acids and pectin that are present in fruit juice also can be considered as soluble solids. In this study, corresponding value for total soluble solids (TSS) of the beverage was 8.1 °Brix. According to Malaysian Food Act 1983 and Food Regulations 1985, the number of soluble solids for fruit juices shall not be less than 8 g/100 ml at 20 °C. At 20 °C, 1 °Brix usually coincides with 1 g of sucrose in every 100 ml of solution. This means that the beverage can still be consider as fruit juice because it contained 8.1 g/100 ml of TSS at 20°C.

Each 100 ml of the juice also contained 10.92% total sugar, 4.0 mg vitamin C and 0.44% titratable citric acid. This value complies with the Malaysian Food Act 1983 and Food Regulations 1985, which outlines that the acidity of fruit juices should be lower than 3.5% citric acid. The value of titratable acidity (TTA) was measured to determine the degree of acidity of fruit juices caused by acid production by polysaccharides, pectic materials, and uric acid. According to Hussain, Zeb, Shakir, and Sattar Shah (2008), oxidation of reducing sugar during maturity process can contribute to the increase of fruit acidity. In food analysis, the value of TTA has an indirect relationship with the pH value. Each of these values was determined separately in different ways and each has its own effect on the quality of the food where TTA can describe the effect of acid on taste of the food better than pH does. However, correlation analysis of pH and TTA values obtained in this study showed no significant correlation (r = -0.210) (p > 0.01) between them. TTA value can also be used to determine the rate of ripening of fruit.

Previously, it was found vitamin C or ascorbic acid content decreased when there was a change in temperature during process of pasteurisation and the amount of sugar increased during the storage period of the juice. It cannot be denied that there was a change in temperature throughout the processing and storing of the juice even though the precaution measures have been taken. The results for total sugar and soluble solids content of TP 3 in 1TM juice showed a strong and significant positive correlation (p < 0.01). This clearly indicated that the number of soluble solids in the juice was contributed by the sugar content. The correlation value between total sugar and soluble solids was r = 0.892.

2. Proximate Composition of the Beverage

The moisture content in food or drink is the same as the water content, where water is one of the important macronutrients in the daily diet. According to FDA (2016), high water content food or drink normally contains 85% or more moisture value. Table 2 showed beverage contained 89.38% water. This moisture value is normal and within the same range of most freshly made mixed juice product The total ash content of this juice was 0.15%. Ash content in food can be referring to the residue of inorganic substances such as minerals in a food. The higher ash content indicates higher mineral content. Table 2. Proximate proportion of the beverage

Note. Results were reported in average ± standard deviation of 6 replicates.

Result showed that fat content was absence in this juice. This may be due to the type of fruits used in the juice formulation, when there was absolutely no fat or the content was not significant to be detected by the Soxhlet method. The Soxhlet method has a lower limit detection of 0.1%, in which food samples that have lower than 0.1% fat cannot be determined by this tool. This also indicated that the combination of these fruit juices was very healthy because it has very low-fat content The beverage has 0.16% protein and 10.31% of total carbohydrates. The juice was also low in calories, which contained only 42 kcal per 100 ml and suitable to be included in a daily diet Calories in the beverage was mainly contributed by carbohydrate and protein contents. 0. K. Ahmed and S. E. D. Ahmed (2014) reported that matured fruits were usually sweeter. Therefore, the level of fruit sweetness can be measured based on the content of sugar or the amount of carbohydrate. The value of carbohydrates in a diet usually not only consisted of sugar content but also dietary fiber. Natural sugar in fresh fruits is commonly known as fructose, a type of simple sugar monosaccharide while dietary fibers can also be categorized as complex polysaccharides. Generally, some nutrients such as protein, fat and fiber will be slightly lower in fruit juice compared to its fresh fruit because these nutrients were reduced during the processing of the fruit juice. Fortification and enrichment of any nutrient can be carried out if needed. 3. Total Phenolic Content and Antioxidant Capacity of the Beverage

Generally, it was known that total phenolic content is highly correlated with antioxidant activity and bioavailability of polyphenols. In this study, two phenolic tests were carried out on the beverage which were total phenolic content (TPC) and total monomeric anthocyanin (TMA). In general, TPC quantifies total amount phenol group including mono-phenol, phenol, tri-phenol and polyphenol compound while anthocyanin only quantities polyphenols under the subgroups of flavonoids. Therefore, both tests were done to get extra information about the possible difference between phenolic and anthocyanin value. Table 3 shows TMA value for the beverage and it was found to be 12.94 mg cyanidin-3-glucoside (C3G)/100 ml. The intensity of the red color (a] in the beverage indicated of higher anthocyanin content in it. TPC values in fruit juices can be divided into several groups according to certain values. TPC values less than 500 mg GAE/100 ml can be categorized as low while moderate TPC values range were ranging from 500 to 2000 mg GAE/100 ml. The TPC value is considered high if the content exceeds 2000 mg GAE/100 ml. Table 3 shows TPC value of beverage, which was 609 mg gallic acid equivalent (GAE)/100 ml.

Malaysian guava fruit has much higher TPC value, which is 1394.94 mg GAE/100 g. There were many factors that were likely influenced the phenolic content Some of the factors were the time of fruit was harvested, the fruit ripening state, the environmental factor and the physical factors during the processing and storage operation. Environmental factors such as soil and weather conditions have the most significant effect on polyphenol content. Exposure to sunlight affects almost all types of flavonoids may also effect on polyphenol content There is a possibility that the differences in flavonol concentration may exist among several fruit seeds on the same tree. This was due to exposure to unbalanced sunlight on all part of the tree which showed a variance in their polyphenol content The total antioxidant (free radical scavenging) activity of fruits was mainly attributed to the additive and synergistic effects of inherent phytochemicals notably, the phenolic compounds.

Table 3. Antioxidant activity of the beverage

Note. Results were reported in average + standard deviation of 6 replicates.

It is recommended TPC test and at least two different antioxidant tests to be carried out in order to confirm the final results of antioxidant activity in the food samples. Antioxidant value obtained through DPPH and ABTS tests in the formulation were 88.90% and 472.44 mM Trolox equivalent (TE] per ml, respectively. Fruit juices normally have medium antioxidant capacity but have higher value for phenolic compounds, carotenoids and vitamin C. There has been a positive correlation between TPC and antioxidant assays (r = 0.808 and r = 0.826, respectively]. The DPPH test was easy to use and widely used to determine the activity of phenolic and antioxidant of pigmented compounds while ABTS tests were found to be suitable for samples in acidic condition and contained hydrophilic components. However, these antioxidant tests are very sensitive and need to be carried out with care. Precautions should be taken such as not exposing reagents and samples to direct lights as well as using fresh solutions each time during the analysis. It is important to run multiple antioxidant test in order to get better estimation of antioxidant capacity and to substantiate in vitro results with clinical studies. A slight difference in the method of antioxidant used may have caused variability in the results. Factor such as pasteurisation condition used in preparing juice may also affect the changes of the antioxidant capacity of juice. Other than that, temperature variation during sample incubation and different specification of spectrophotometer used can also leads to significant internal variability.

4. Polyphenol Compounds in the Beverage

Eight types of polyphenols were selected to be determined in the beverage. Presently, ellagic acid (EA] was the highest component in the juice with a value of 633.73 mg/100 ml (Table4], The results also indicated that individual polyphenol such as procyanidin B2 (375.99 mg], chlorogenic acid (327.59 mg], epicatechin (291.10 mg], gallic acid (195.39 mg] and catechin (117.22 mg] were abundantly found in each 100 ml juice. In addition, p-coumaric acid (0.13 mg] and kuromanin chloride (15.19 mg] were also present in every 100 ml of the beverages. EA and chlorogenic acid were found to be the most predominant and common polyphenol in this formulation. EA is a main component of plant cell wall. It is a dimeric derivative of gallic acid, occurs in fruits (fresh and processed] and nuts in either its free form, as EA-glycosides, or bound as ellagitannins. Medicinally, EA was used to prevent cancer, treat viral and bacterial infections. Previous study, had used pomegranate juice as the main supplement for EA in his human study. Chlorogenic acid was found abundantly in fruit cultivar of western countries but not in tropical fruit cultivar. It has been reported to exist in varieties of apples and berries. The wide range of potential health benefits of chlorogenic acid has been reported including its anti-diabetic, anti- carcinogenic, anti-inflammatory and anti-obesity impact Procyanidin B2 was normally found in red colour juices such as pomegranate and roselle juices. As such procyanidin B2 is normally a reference compound for anthocyanidin. Table 4. Targeted polyohenol compounds in the beverage

Note. Results were reported in average ± standard deviation of 6 replicates. CONCLUSION

In conclusion, this present invention reported that the formulation has acceptable physical properties and contained significant amount of phenolic and antioxidant content Pomegranate, guava and roselle juice individually contained various nutrient and a combination of these three fruit juices may provide beneficial activity towards health.

EXAMPLE 2

The testing of the formulation towards animals

Materials and Methods

Animals

In this example, the mammals chosen for the testing were 40 male Wistar rats weighing 200 to 250 g, following an ethical approval from Universiti Kebangsaan Malaysia Animal Ethics

Commitee (FSK/BIOMED/2013/NOR/20-MARCH/50-MARCH-2013-MARCH-2015]. Rats were kept at constant room temperature 24°C under a 12-hour light/dark cycle (lights on from 7:00 AM to 7:00 PM). The animals were acclimatized for seven days prior to testing and further divided into five groups of eight rats per group. The groups were 1] sham-operated control group (dPBS) treated with distilled water (dH ₂ 0) (5 ml/kg oral), 2) b-amyloid control group (dAb) (0.1 mg/ml intracerebroventricular or i.c.v.) treated with dH ₂ O, 3) sham-operated control treated with the formulation (5 ml/kg oral) and PBS (JPBS), 4) b-amyloid control groups which were given the formulation (JAb), and 5) b-amyloid control groups which were given Ibuprofen (IBFAb). The administration of Ibuprofen was given daily via oral gavage at a modified dosage of 5 ml/kg body weight and 1 mg/kg/10 ml respectively for four weeks, followed by Ab injection for two weeks in the rats.

Chemicals

The rats were injected with synthetic b -amyloid b-amyloid or Beta-amyloid is defined as a small piece of a larger protein called "amyloid precursor protein” (APP). In its complete form, APP extends from the inside of brain cells to the outside by passing through the fatty membrane around the cell. When APP is "activated" to do its normal job, it is cut by other proteins into separate, smaller sections that stay inside and outside cells. There are several different ways APP can be cut; under some circumstances, one of the pieces produced is beta-amyloid in which is triggered by the oxidative stress.

Preparation of the formulation

In the preparation of the formulation of this invention, tropical fruits i.e. pomegranate, white guava and roselle juice were utilized. All the single fruit juices were mixed according to 10 points centroid-simplex design modified method with different ratios for each formulation. By "10 points centroid-simplex design” means that this design method is commonly applied in industrial product formulations such as food processing, chemical formulations, textile fibers and pharmaceutical drugs. This method also can be used to evaluate the effects of chemicals in a formulation. By practice, this method is applied preparing the following formulations in which the outcomes would be compared. In this case, the formulations were prepared using 4 different formulations at different ratios of the tropical fruits. The ratios were as follows:

a) 1/3 pomegranate juice, 1/3 white guava juice, and 1/3 roselle juice (F7)

b) 2/3 pomegranate juice, 1/6 white guava juice, and 1/6 roselle juice (F8)

c) 1/6 pomegranate juice, 2/3 white guava juice, and 1/6 roselle juice (F9)

d) 1/6 pomegranate juice, 1/6 white guava juice, and 2/3 roselle juice (F10).

The above formulations were stored at -40°C prior to further analysis for the antioxidant capacity assay namely ferric reducing antioxidant power (FRAP) and 2, 2-diphenyl-l- picrylhydrazyl (DPPH) and total phenolic content (TPC).

Analysis of Tropical Fruit Juice Mixture

Upon preparing the above formulations, each of the formulation was analyzed using various analysis methods that consisted of FRAP, DPPH and TPC. These analysis methods were used to determine the best formulation in terms of antioxidant capacity and TPC content in the formulation. The best formulation was selected for further used in the in vivo test Supplementation of Tropical Fruit Juice Mixture and Ibuprofen

Tropical fruit juice mixture and Ibuprofen were given daily via oral gavage at a modified dosage of 5 ml/kg body weight and 1 mg/kg/10 ml respectively for four weeks, followed by Ab injection for two weeks.

Intracerebroventricular Administration o/b-Amyloid

Synthetic Ab _1-42 (0.1 mg/ml) was dissolved in phosphate buffered saline (PBS) and incubated at 37 ^º C for three days to allow the formation of Abi aggregation. The rats were anaesthetized using a mixture of ketamine, tiletamine and xylazine (KTX) (0.1ml/ 200g) via intraperitoneal (i.p.). Ab was injected intracerebroventricularly using a bone microdrill (21, 30). A small incision was made on the head of the anaesthetized rats to expose the skull. The exposed skull was then drilled with one hole (anteroposterior +1.2 mm from Bregma, mediolateral +2.0 mm, dorsoventral +4.0 mm) with the position of the hole determined stereotaxically. The cannula was affixed to the skull by using cyanoacrylate Loctite glue (Loctite 454™). Following this procedure, a subcutaneous pocket was prepared in the midscapular region of the back of the rat to receive the mini osmotic pump (ALZET™). The pump was then implanted in the subcutaneous pocket and was attached to the brain cannula via polyvinylchloride tubing. Ab solution and vehicle (PBS) was spontaneously infused into the left lateral cerebral ventricle supplied by the mini osmotic pump for two weeks at a constant rate (0.5 ml/hour). The wound was then stapled closed with wound clips (Reflex 7™). When the pump was properly placed, the tubing should have a generous amount of slack to permit free motion of the animal’s head and neck. The animal required no restraint or handling during the delivery period.

Open Field Test

All the animals were trained two days prior to drug and juice administration. Open field test (OFT) was performed before novel object recognition task (NOR). OFT was done to measure locomotor activity in rats and exploration time in the central square of arena. Experiment was conducted in a sound proofed room illuminated with red light (20 watt) for easier recording of rats’ activity. Each rat was placed in an open field arena (72 cm x 72 cm x 38 cm) and its exploration activity was recorded for the duration of five minutes. Recorded videos of the rats were analyzed and their behaviors were scored based on the frequency of line crossing, rearing, freezing and time spent in the central square zone. Locomotor activity was measured by total frequency of line crossing and rearing. Novel Object Recognition

The test was carried out on day 7th and 14th following the Ab injection. Each rat was acclimatized for 45 minutes in a room inside the cage and one minute in an empty arena before the experiment NOR was divided to three phases; habituation, familiarization and novel with duration of seven days (modification of 33). The first phase was habituation for five minutes (day 1st and 2nd), followed by familiarization phase for 15 minutes from the 3rd day until 6th day and lastly novel phase for 10 minutes at day 7th. Rats were given two objects with similar shape and size during the familiarization phase but one of the familiar objects was replaced with a novel object during the novel phase. The object exploration was scored from video recording as time spent exploring each object. Exploration was scored when the distance between the rat’s nose was one cm from the placed object and there was movement of vibrissae. Rats’ preference for novel object was expressed as percentage of NOR exploration time compared to familiar object. b-amyloid and Corticotropin Releasing Hormone (CRH) ELISA Assay Kit Determination

Brain Ab and plasma CRH concentration were determined by using 96 well specialized microplate assay kit pre-coated with antibody according to the manufacturer’s instructions (SensoLyte® & Cloud-Clone Corp™) respectively. Absorbance was measured at wavelength of 450 nm by 96 well microplate reader (Bio-Rad™).

Western Blot

Rats from each group were decapitated, and their brains were rapidly removed by using the aid of dried ice to maintain the necessary temperature. Following dissection, each hippocampus area was weighed and homogenized with lysis buffer. Brain lysate containing protein was used for preparation of loading samples. Samples were separated on 10% SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) and transferred to PVDF (polyvinylidene difluoride) membranes. Using a conventional method, the membrane was blocked first with 4% bovine serum albumin (BSA) in tris-buffered saline with tween (TBST) and proceeded with membrane washing using TBST for five times with five mins per washing. The membrane was incubated with primary antibodies from Abeam: rabbit polyclonal anti-iNOS (1:1000 dilutions overnight (16 hours) at 4°C before being washed with TBST for five times with five mins/washing. Subsequently, the membrane was incubated with Abeam HRP-conjugated anti-rabbit for secondary antibody (1:1000) for 1 hour and 50 mins at room temperature and the washing process was repeated. Finally, the membrane was exposed, and the resultant bands were visualized using the chromogenic substrate (Bio- Rad™). Histological Analysis of Hippocampus

The hippocampus of the brains was first sectioned and isolated by using brain matrices (Tedpella™]. After fixing with 10% formaldehyde, the hippocampus regions were dehydrated, embedded in paraffin and sliced into 5 mm thick sections before staining with Nissl stain. Prepared slides were examined under a light microscope and neuronal count was done within the selected per 100 pm ² area of brain tissue.

Statistical Analysis

Statistical analysis was conducted by using SPSS version 22 (IBM™). The results were expressed as mean ± standard error means (S.E.M) with the p value less than 0.05 was considered as significant. For the behavioural test, the ANOVA repeated measures were carried out to determine the significant differences between different rat’s groups and day of b-amyloid infusion. The results from the biochemical tests and the neuronal count was obtained from one- way ANOVA and followed by Bonferonni and Tukey HSD post-hoc analysis respectively for parametric data. For non-parametric data, Kruskal-Wallis was done and followed by Mann- Whitney U test to define the significant differences between rat’s groups.

Results

Analyses of the Formulations

Based on the FRAP analysis in Figure la, F9 (4725.25 ± 158.70 mg/ml LAA) has significantly higher FRAP value (p<0.05) than F7 (4075.98 ± 159.18 mg/ml LAA) and F10 (3808.09 ± 150.67 mg/ml LAA), respectively for the combination of the three fruits. This formulation contained higher percentage of white guava, while F10 was dominated by bigger fraction of roselle. On the other hand, F9 has the lowest (p<0.05) DPPH radical removal activity when compared to L-ascorbic acid (positive control) as shown in Figure lb. For TPC (Figure 1c), the highest phenolic content was reported in the fraction of F9 (690.27 ± 21.18 mg GAE/ml). Both F8 and F9 were reported to have higher TPC and FRAP value compared to other mixture formulation, however, the percentage removal of free radicals DPPH of F8 was higher than F9. Hence, the best formulation chosen for this study was F8 due to its higher phenolic content, FRAP value and removal activity of DPPH free radicals.

Locomotor Activity in Open Field Test

Only effect of time differences (main effect of day) were observed following Ab infusion for 7 days as compared to 14 days, where decreased locomotor activity was observed in all rats group (dPBS, dAb, JPBS, JAb, IBFAb) following Ab infusion; with F (1, 7) = 6.940, p<0.05 and F (1, 7) = 7.152, p<0.05 as shown in Table 5 below. However, after 14 days of Ab infusion, the JAp group showed higher locomotor activity as compared to Ab control group ( dAb).

Table 5 Locomotor activity in open field test (OFT) among different rats' groups after 7 and 14 days of Ab infusion.

Note: Sham-operated control (dPBS), b-amyloid control (dAb), juice mixture + PBS (JPBS), juice mixture + b-amyloid + (JAb), Ibuprofen + b-amyloid (IBFAb), and * = significant differences (p<0.05) as compared to after 7 days of Ab infusion using ANOVA repeated measures. Data are presented as mean ± S.E.M (n=8).

Percentage of Novel Object Recognition

Table 6 shows NOR percentage between rat groups after 7 and 14 days of Ab infusion. Only time differences (main effect of day) showed significant decreases in the percentage of NOR after 14 days of Ab infusion as compared to 7 days of Ab infusion; where F (1, 7) = 7.152, p<0.05.

Note: Sham-operated control (dPBS), b-amyloid control (dAb), juice mixture + PBS QPBS), juice mixture + b-amyloid + (JAb), Ibuprofen + b-amyloid (IBFAb), and *p<0.05 as compared to after 7 days of Ab infusion using ANOVA repeated measures. Data are presented as mean ± S.E.M with n=8 per treatment group.

Concentration of b-amyloid in Brain Homogenate

Concentration of Ab (Figure 2) showed no significant difference (p>0.05) in the brain homogenates between the animal groups. Concentration of CRH in Blood Plasma

The concentration of plasma CRH of different rats’ groups is as shown in as shown in Figure 3. CRH concentration was lowered significantly (p<0.05) in JAp (244.88 ± 89.16 pg/ml] and IBFAb (336.08 ± 87.86 pg/ml) as compared to dPBS (859.11 ± 188.55 pg/ml). JAb and IBFAb group also have lower (p<0.01) CRH concentration compared to dAb (792.41 ± 97.33 pg/ml).

Intensity Relative of iNOS Expression in Brain Homogenate

Based on Figure 4, infusion of Ab (dAb) increased the expression of iNOS level. However, supplementation of fruits juice mixture in the JAb group was able to decreased the expression of iNOS (p<0.05) as compared to dAb control group

Histological Analysis of Comus Ammonis (CA1) Region in Hippocampus (Nissl)

Cross sectional histology of hippocampus tissue in cornus ammonis (CA1) region at higher magnification of 400X is as shown in Figure 5. In Figure 5B, a prominent tissue damage and shrinkage of neuronal cells were observed following infusion of Ab via i.c.v. In addition, neuron cells were not orderly arranged with presence of gaps or spaces in between the cells. However, CA1 region of hippocampus for Ab rats group treated with fruits juice showed the presence of normal shaped neuron cells even after Ab infusion (Figure 5D). This indicates that tropical fruit juice mixture able to prevent neuronal shrinkage and damage. Similar results were also seen in JPBS group (Figure 5C) as compared to dPBS group (Figure 5A). On the other hand, Ibuprofen treatment showed an order and compact arrangemet of neuron cells (Figure 5E) as compared to fruits juice- treated (Figure 5D) of Ab-induced condition.

Neuronal Count in Comus Ammonis (CA1) Region of Hippocampus (Nissl)

Figure 6 shows the results of the neuronal count of cornus ammonis 1 (CA1) region of the hippocampus. Infusion of Ab by i.c.v. into the brain hippocampus caused significant reduction (p<0.05) in neuronal count per 100 pm ² as observed in dAb group (38.00 ± 2.00) as compared to the sham-controlled group dPBS (77.00 ± 1.00). However, treatment with tropical fruit juice mixture enabled an increase in the neuronal cells number either in normal (p>0.05) or Ab neurotoxicity-induced condition with a mean value of 93.50 ± 6.50 and 71.50 ± 6.50 respectively. On the other hand, supplementation of Ibuprofen (IBFAb) also caused a significant increment (p<0.05) in the neuronal count (93.00 ± 4.00) as compared to b-amyloid control (<1Ab) and juice- treated (ίAb) group. Conclusion

In conclusion, supplementation of the formulation in doses of 5 ml/kg body weight/day has the ability to improve locomotor activity and object recognition (spatial memory) in both open field test and novel object recognition. The depression level was also reduced accompanied by the reduction of CRH and iNOS pro-inflammatory protein expressions. This was further supported by the increased in neuronal count and less neuronal damage in histological analysis of the brain tissues (hippocampus). Thus, the embodiment in F8 has the potential to be developed as a neuroprotective agent in improving neuroinflammation and other neurodegenerative related conditions.

EXAMPLE 3

Testing of the formulation in human Methodology

A randomised control trial among middle-aged women (aged 45-59 years) with signs of poor cognitive status as assessed using Rey’s Auditory Verbal Learning Test (RAVLT) was conducted to determine efficacy of the formulation supplementation on cognitive status, lipid peroxidation and metabolomics profile. The exclusion criteria were history of mental illnesses, physical disability, pregnant and lactating women and those with chronic diseases including diabetes, cancers, hypertension and hyperlipidemia.

Subjects were randomised to be in either placebo or intervention group. Subjects in both groups were provided with 1500 ml of the formulation (500 ml before breakfast, lunch and dinner) daily, three days in a week, for a period of ten weeks. For the placebo beverage, it was formulated to contain no juice or natural polyphenol but look and taste like the beverage with the same energy content (Table 7). The beverage was provided to the subjects in a weekly basis.

Table 7 Comparison of the daily composition of polyphenols and other nutrients from the formulation and placebo with the composition of a commercialized grape juice (532 ml)

Outcomes of interest were cognitive status as assessed by several cognitive batteries including RAVLT, Digit Span and the Comprehensive Trail Making Test (CTMT). RAVLT was proven useful in evaluating verbal learning and memory, including proactive inhibition, retroactive inhibition, retention, encoding versus retrieval, and subjective organization. The RAVLT consists of two different lists (A and B) of 15 concrete nouns. Subjects were presented with list A for five times (Recall 1 to Recall 5) at a rate of one item per second. Free verbal recall was tested immediately after each presentation. Then list B was presented followed by a free recall of list B. Thereafter, recall of list A (A6) was examined without prior presentation of list A. The Malay version of RAVLT showed good validity (factor analysis 0.66 to 0.98) and test-retest reliability (Pearson’s correlation ranged from 0.24 to 0.84). Additionally, in a prior study, the Cronbach’s alpha values for Malay version RAVLT was 0.74 and 0.84 for learning and memory sections, respectively.

Digit Span is composed of two tasks administered independently of each other: Digits Forward and Digits Backward. On both tasks, the researcher read a series of number sequences to the subjects. The subjects were required to repeat the number sequence in the same order as read by the researcher for Digits Forward. Whilst for Digits Backward, the subjects were required to repeat the number sequence in the reverse order. The Digits Forwards and Digits Backwards test were discontinued after a score of zero on both trials of any item. The sensitivity and specificity of Digit Span were 51% and 91%, respectively, when validated among healthy adults. Additionally, the value of Cronbach’s alpha was 0.74 when validated in Malaysian elderly population. The Digit Span task evaluated verbal working memory and short-term memory. Working memory refers to the dynamic relationship between passive storage and active manipulation or transformation of information held in memory.

The CTMT consists of five trails. For Trail 1, 2 and 3, the subjects were required to draw lines sequentially connecting circles starting from circle 1 until circle 25, distributed on a sheet of paper. As the subjects proceed to the following trails, they would face more distractors. For Trail 4, there were certain circles with numbers in digits and rectangles with numbers spelt in words, starting from 1 to 20. For Trail 5, the subj ects were required to draw lines connecting alternately between numbers and letters. The score on each part represents the amount of time required to complete the task. The five visual search and sequencing tasks showed the subjects’ attention, concentration, resistance to distraction, and cognitive flexibility (or set-shifting). The sensitivity and specificity of CTMT ranged from 45.1% and 91.1%, respectively, when validated among elderly subjects with mild cognitive impairment (MCI). In another study involving healthy adults, the sensitivity and specificity were 0.68 and 0.67 (for composite index), respectively. Cronbach's alpha was 0.88.

For the analysis of lipid peroxidation, a total of 5 ml of venous blood drawn into EDTA tubes was centrifuged at 3,000 rpm for 10 minutes at 4°C for plasma separation. The plasma was transferred into multiple aliquots of 1.5 ml microcentrifuge tubes. It was then stored at -30°C and thawed only prior to the analysis. Analysis of lipid peroxidation was conducted using a kit by ABCAM (USA). The kit was kept at -20°C immediately after it was received from the supplier. The kit consisted of phosphotungtic acid solution, butylated hydroxytoluene (BHT) (100X), thiobarbituric acid (TBA) solution and malondialdehyde (MDA) standard (4.17 M). Microplate spectrophotometer was used for the measurement of absorption. All components of the kit were thawed to room temperature prior to analysis.

Urine samples were collected in urine collection bottles for metabolomics analysis. As the first void urine was rather more variable than the subsequent voids, the second void urine was used in this study. Urine samples were collected in sterile urine collection bottles (60 ml) and later transferred into multiple aliquots of 4 ml in 5 ml sterile screw cap centrifuge tubes. A total of 10 mΐ of sodium azide (0.1% wt/vol) was added to the aliquots of urine samples. The centrifuge tubes were sealed tightly and stored at -80 °C until analysis.

Urine samples were thawed and equilibrated to room temperature prior to the analysis. Once thawed, the samples were centrifuged for 3000 rpm for 15 minutes. The supernatant was collected and used in subsequent analysis. A total of 400 mΐ of the urine sample was then mixed with 200 mΐ of phosphate buffer (pH 7.4) in a microcentrifuge tube, and later centrifuged at 12,000g for 5 minutes at 4°C. A total of 550 mΐ of the samples was transferred into a 5 mm NMR tube.

The protocol of NMR analysis for human urine samples using 600 MHz Ultrashield Plus NMR spectrometer (Bruker Biospin™) was used in this metabolomics profiling experiment.

The NMR experiments were acquired at constant temperature of 32.2°C with acquisition time of 7 minutes 35 seconds. The samples were loaded into the autosampler and the temperature was calibrated and kept constant before each experiment. Subsequently, the reference signal of the deuterated solvent was locked and the magnetic field was homogenized (shimming). In order to observe the dynamic range of metabolites concentrations efficiently, the water signal was suppressed by running ID NOESY-presat experiments. The number of scans was set at 64 for the urine samples.

All data were analysed using Statistical Package for Social Sciences (SPSS) version 21. In order to analyse the difference between treatment and placebo groups, independent student t-test and chi square test were conducted with the result presented as means and standard deviation with 95% confidence interval. In addition, repeated measures analysis of variance (ANOVA) was carried out to determine the effects of the beverage consumption at baseline, week 5 and week 10 (time effect), group effect and interaction effect.

All the FID files obtained from NMR were imported to ACD/NMR Processor Academic Version and stacked together before Fourier transformation was carried out in order to convert the spectrum from time domain to frequency domain. The spectrum was then auto-phase and auto-baseline corrected. It was then integrated into equal 1000 bins.

The data which had been pre-processed were subjected to principal component analysis (PCA) through SIMCA software. PCA is an unsupervised mathematical algorithm and showed the summary of the difference of the metabolites profiles between subjects receiving the beverage supplementation and placebo. Metabolites responsible for the difference between the intervention and placebo groups were identified and quantified by referring to The Human Metabolome Database (HMDB) (www.hmdb.ca). PCA, through the score and loading plots, established the parts of spectra that were similar or dissimilar between the groups. Analysis of concentration of metabolites identified in HMDB was carried out by referring the resonance of metabolites to the TSP and its concentration using two-way repeated measure ANOVA. Pathway analysis was conducted by referring to the Metabolomics Workbench (www.metabolomicsworkbench.org) and other literature search.

Results and Discussions

A total of 16 and 15 middle-aged women (aged 50.8 ± 3.7 years) were randomly assigned to intervention and placebo groups, respectively. There were no dropouts in both groups throughout the study period. Generally, all subjects showed good compliance towards the study protocols. Most of the subjects were married, had education until primary and secondary levels, not working and living with family members (Table 8). Additionally, most subjects can be categorized as physically inactive and did not engage in regular social activities.

As shown in Table 9, there was a significant improvement for interaction effects in the RAVLT immediate recall score for the intervention group (ղp ² =0.294; power=0.80; p<0.05). This was similar to the result in previous studies using Concord grape juice and wild blueberry juice. RAVLT is a cognitive measurement tool to measure verbal learning and memory, which include proactive inhibition, retroactive inhibition, retention, encoding versus retrieval, and subjective organization. Additionally, Table 6 also shows that there was a significant reduction in the time required for the intervention group to complete the CTMT Trail 4 (ղp ² =0.319; power=0.81; p<0.05). To the best of our knowledge, no previous studies have used CTMT to assess cognitive status in study involving supplementation of polyphenols. CTMT are used to evaluate various cognitive aspects, such as processing speed, sequencing, mental flexibility and visual-motor skills.

Previously, adult male mice were introduced intraperitoneally with polyphenols-rich extract of wild blueberries at 30 and 60 mg/kg body weight for 7 days to study the effects on cognitive performance and reduced glutathione levels in whole brain homogenates and acetylcholinesterase activity. Mice treated with 60 mg/kg body weight of wild blueberries extract showed significant improvement in learning and memory. Both groups of mice also showed significant decrease in salt and detergent soluble acetylcholinesterase activity. Acetylcholinesterase is responsible for the breakdown of neurotransmitter acetylcholine and some other choline esters. Loss of cholinergic neurons associated learning and memory deficits was observed in the forebrain of patients with Alzheimer’s disease. The usage of agonists of cholinergic receptors and inhibitors of acetylcholinesterase are among the methods to overcome cognitive impairments which occur during aging.

Table 10 shows the concentration of malondialdehyde (MDA) which represented lipid peroxidation process. There was a significant time effect (ղp ² =0.17; power=0.63, p<0.05) with the intervention group showed a decreasing trend of MDA concentration.

Table 8. Demographic, socioecomomic and social profiles of subjects

In addition, Figure 7 shows the changes in MDA concentration from baseline for both intervention and placebo groups. There was a sharp reduction in the MDA concentration until week 5 for the intervention group, however, the concentration increased by week 10 of supplementation.

Figure 8a and Figure 8b are the score plot and loading plot, respectively, of the subjects during baseline. There was no clear separation of the score plot during baseline. The loading plot shows the variables which contribute to the formation of scores. As can be seen in Figure 8b, there were some points which show separation in both PCI and PC2. However, since the pattern separation was not seen in the score plot, the resonance was not identified further as the pattern may be contributed by other factors, such as the presence of outliers.

Subsequently, the PCA analysis was continued at week 5 of supplementation. Figure 3a and Figure 3b are the score plot and loading plot, respectively, of the subjects during week 5 of supplementation. There was an assembly of observation along PC2 without clear separation. There was still an outlier located outside the eclipse. There were some variables which show separation at both PCI and PC2 at the loading plot

Figure 9a and Figure 9b indicates the score and loading plots, respectively, by the end of the supplementation period. The score plot indicates there is some separation, in both PCI and PC2, between the two groups. The pattern is clearly separated with no outliers observed outside of the eclipse. As can be seen in the loading plot, the variables which cause separation in PCI are 3.03-3.06 ppm and 4.04-4.07 ppm, whilst resonance 3.96-3.99, 7.55-7.58 and 7.82-7.85 ppm are responsible for the variation in PC2.

Following the analysis of PCA, analysis of NMR peaks which were found to be responsible for the separation and clustering appearing in the score and loading plots of the urine samples were conducted. The peaks were compared with the data in the Human Metabolome Database (HMDB) (Wishart et al. 2007], Subsequently, analysis of concentration of the metabolites was carried out as shown in Table 11.

As there were no significant interaction effects in the concentration of metabolites, further analysis on the percentage of the difference in the concentration of metabolites from baseline concentration was conducted. Figures 11, 12, 13, 14 andl5 show that the changes in the concentration of all metabolites identified over the intervention period were higher and more prominent in the intervention group as compared to the placebo group. The insignificant interaction effects in this study could be due to bioavailability and metabolism of polyphenols itself. The absorption of aglycones of polyphenols starts in the small intestine. However, in food, polyphenols are available in the form of esters, glycosides or polymers that cannot be absorbed. Intestinal enzymes and colonic microflora hydrolyse the natural form of polyphenols to be absorbed. However, the absorption efficacy of polyphenols is reduced as the flora will also degrade the aglycones produced to multiple aromatic acids. Additionally, some polyphenols may be conjugated by hepatic or intestinal phase II enzymes. As a consequence of these modifications, many kinds of polyphenolic structures may yield a limited pattern of circulating metabolites.

Hippuric acid, one of the metabolites identified in the current study, is a product of colonic microbial catabolism of polyphenols. The catabolites are absorbed and conjugated by phase II enzymes, producing hippuric acid. Hippuric acid has been recognized as a biomarker of polyphenols consumption. In a study by Vetrani et al. (2014), a total of 78 adults were supplemented with isocaloric meals with different polyphenolics content for 8 weeks. Low and high polyphenols meals provided 363 and 2868 mg/ day of total polyphenols. In group receiving the high polyphenols diet, there was a significant urinary hippuric acid concentration after 8 weeks of supplementation as compared to the baseline concentration (p<0.001). Additionally, hippuric acid has been used to determine the intake of fruit and vegetables among patients with kidney stones and healthy children and adolescents.

Additionally, the current study found that subjects from the intervention group had higher difference in the concentration of urinary benzoic acid as compared to the placebo group after 10 weeks of supplementation. Benzoic acid is the glycine conjugate of hippuric acid. In the study by Vetrani et al. (2014) as discussed above, there was a significant urinary benzoic acid concentration after 8 weeks of supplementation as compared to the baseline concentration in group receiving the high polyphenols diet (p<0.05).

Glycolic acid, a metabolite identified in this study, is in the group of a-hydroxy acid. It is an organic acid, naturally found in various types of fruit. The elevated level of glycolic acid among the supplementation group could be due to its presence in the fruit available in the juice. Being easily soluble in water, it is excreted from the body through urine, thus, giving rise to its concentration as urine metabolites. In addition, the intervention group showed higher difference in the concentration of urinary thyroxine as compared to baseline than the placebo group. In a study by Orden et al. (1987], urinary thyroxine showed good correlation with free thyroxine in the serum and provides clear indication of the thyroid functions. The increased urinary concentration of thyroxine in subjects receiving polyphenols- rich juice could be explained by the action of human transthyretin (TTR], Human TTR, a tetramer, carries thyroxine in the body. It is an amyloidgenic protein which denaturation, misfolding and dissociation of the tetramer into monomer causes amyloid fibril formation, which is responsible with many diseases with aging, including Alzheimer’s disease. TTR consisted of two thyroxine binding sites, in which the binding of thyroxine with TTR contributed to the stability of TTR. As the result, various small molecules were developed by the pharmacological industry in order to bind to thyroxine binding site and developed the stability of TTR. Interestingly, recent studies have shown that natural polyphenols may reduce the fibrillogenesis of TTR by specifically interacting with thyroxine binding sites. In a study by Florio et al. (2015), comparative analysis was conducted to determine the interaction and ability to maintain the chemical stability of TTR using resveratrol and thyroxine. The result showed the preferential binding of resveratrol and thyroxine towards TTR, in a negative binding cooperativity. Additionally, the binding site of resveratrol showed an interesting feature as compared to the thyroxine binding site where when one of the two ligands attaches to TTR, the other ligand is notable to displace it, even if added at high concentration. In relation to the current study, with higher polyphenols consumed by the intervention group, there would be more free thyroxine unbounded to TTR, thus, giving rise to its concentration in the body, and subsequently, in the urinary excretion as seen in the intervention group.

Additionally, the current study found that the intervention group had a higher percentage difference in the urinary excretion of 3-methyladenine at week 10 as compared to baseline than the placebo group. Several studies have shown that 3-methyladenine may affect cognitive functions associated with aging through autophagy. Autophagy is a catabolic pathway to destroy cellular components through the functions of lysosome. It is an intracellular recycling pathway and its inactivation is associated with reducing the risk of Alzheimer’s disease.

As shown in Figure 10, the consumption of the beverage for three times a week for 10 weeks using similar dosage of anthocyanins as previous earlier study, but higher total polyphenols content was able to act as neuroprotective agent through reducing oxidative stress and increase excretion of metabolites associated with Alzheimer’s Disease, i.e. thyroxine and 3- methyladenine. Both of these metabolites provide stability to human TTR and activates the autophagy process. Conclusion

Polyphenols-rich tropical fruit juice in this formulation has the potential to improve the cognitive status among middle-aged women, namely the learning, memory, processing speed, sequencing, mental flexibility and visual-motor skills. The metabolites identified were related to polyphenols consumption and cognitive functions.