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WO2020263075A1 - Formulation qui réduit le stress oxydatif - Google Patents

Formulation qui réduit le stress oxydatif Download PDF

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Publication number
WO2020263075A1
WO2020263075A1 PCT/MY2019/000036 MY2019000036W WO2020263075A1 WO 2020263075 A1 WO2020263075 A1 WO 2020263075A1 MY 2019000036 W MY2019000036 W MY 2019000036W WO 2020263075 A1 WO2020263075 A1 WO 2020263075A1
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WIPO (PCT)
Prior art keywords
formulation
content
juice
acid
juices
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PCT/MY2019/000036
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English (en)
Inventor
Suzana SHAHAR
Wan Aida WAN MUSTAPHA
Nor Fadilah RAJAB
Hasnah HARON
Hanisah ROSLI
Munirah AHMAD MUNAWAR
Siti Nur 'Aqilah ABDUL MALEK
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Universiti Kebangsaan Malaysia (Ukm)
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Publication of WO2020263075A1 publication Critical patent/WO2020263075A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/385—Concentrates of non-alcoholic beverages
    • A23L2/39—Dry compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/02—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof containing fruit or vegetable juices
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/61—Myrtaceae (Myrtle family), e.g. teatree or eucalyptus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants

Definitions

  • This invention relates to a formulation that reduces oxidative stress in a mammal patient suffering cognitive status.
  • 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.
  • oxidative stress such as cancers, cardiovascular diseases and impaired cognitive.
  • 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.
  • compositions for supporting healthy memory and optimizing mental energy and methods for improving, preventing, and treating mental disorders or deterioration 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 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.
  • 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%.
  • DPPH 2,2-Diphenyl-1-Picrylhydrazyl
  • 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.
  • FIG. 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).
  • PCA principal component analysis
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • AD Alzheimer's disease
  • Ab amyloid-beta peptide
  • Redox active metal ions as for example copper
  • ROS Reactive Oxygen Species
  • 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.
  • 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.
  • subjects were supplemented with 532 ml of a commercialized grape juice (203.8 mg of anthocyanins daily) for 12 weeks.
  • anthocyanins are the main component of flavonoids that is responsible for the improvement of cognitive status related to aging.
  • the formulation of the invention is selected from tropical fruits namely pomegranate, white guava and roselle.
  • 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.
  • no placebo-controlled intervention studies that examined the effects of polyphenols and cognitive status have been conducted involving tropical fruits among Asian populations.
  • the formulation consists of 1/3 pomegranate, 1/3 white guava, and 1/3 roselle.
  • the formulation consists of 2/3 pomegranate juice, 1/6 white guava juice, and 1 /6 roselle juice.
  • the formulation consists of 1/6 pomegranate, 2/3 white guava, and 1/6 roselle.
  • 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
  • pomegranate Pierica granatuni
  • 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.
  • LDL low-density lipoprotein
  • Hibiscus sabdariffa Roselle
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 (SecomamTM). 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.
  • TTA Titratable Acidity
  • 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:
  • Analyses method for determination of moisture, ash, protein, fat and carbohydrate content are based on AOAC (2000).
  • Protein (%) [(Volumeb lank x Weight samPle x 14.007) /Volume HCI x 10] x 6.25
  • 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.
  • 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
  • A absorbance difference
  • e cyanidine-3-glucoside molar absorbance
  • L cell path length
  • MW is anthocyanin molecular weight
  • DF is dilution factor
  • 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).
  • ABTS 2,2-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid
  • solution A 7 mM ABTS radical cation stock solution
  • K8S208 potassium persulfate
  • Another antioxidant capacity value was estimated by 2,2-diphenyl- 1-picrylhydrazylassay (DPPH) assay. About 200 m ⁇ 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).
  • DPPH 2,2-diphenyl- 1-picrylhydrazylassay
  • 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.
  • RTD ready-to-drink
  • the addition of organic acid into commercial juice was intended to lower the original pH of the juices.
  • 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.
  • the lower the pH of fruit juices the greater the heat effect given to the microorganisms, especially in terms of pressure and radiation levels.
  • TTA titratable acidity
  • TTA can describe the effect of acid on taste of the food better than pH does.
  • TTA value can also be used to determine the rate of ripening of fruit.
  • 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
  • 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.
  • 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.
  • TPC total phenolic content
  • TMA total monomeric anthocyanin
  • 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.
  • TPC value 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.
  • TPC test 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.
  • 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.
  • 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.
  • EA ellagic acid
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • Beta-amyloid is defined as a small piece of a larger protein called "amyloid precursor protein” (APP).
  • APP amyloid precursor protein
  • APP extends from the inside of brain cells to the outside by passing through the fatty membrane around the cell.
  • APP is "activated” to do its normal job, it is cut by other proteins into separate, smaller sections that stay inside and outside cells.
  • APP can be cut; under some circumstances, one of the pieces produced is beta-amyloid in which is triggered by the oxidative stress.
  • 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).
  • FRAP ferric reducing antioxidant power
  • DPPH 2-diphenyl-l- picrylhydrazyl
  • TPC total phenolic content
  • 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.
  • 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 454TM).
  • Loctite 454TM cyanoacrylate Loctite glue
  • AZETTM mini osmotic pump
  • OFT Open field test
  • NOR novel object recognition task
  • 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
  • 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 CorpTM) respectively. Absorbance was measured at wavelength of 450 nm by 96 well microplate reader (Bio-RadTM).
  • 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.
  • BSA bovine serum albumin
  • 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- RadTM). Histological Analysis of Hippocampus
  • the hippocampus of the brains was first sectioned and isolated by using brain matrices (TedpellaTM]. 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.
  • 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.
  • F9 has the lowest (p ⁇ 0.05) DPPH radical removal activity when compared to L-ascorbic acid (positive control) as shown in Figure lb.
  • 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).
  • CA1 Comus Ammonis
  • 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).
  • 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.
  • IBFAb Ibuprofen
  • 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.
  • 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.
  • RAVLT Digit Span and the Comprehensive Trail Making Test
  • CMT Comprehensive Trail Making Test
  • 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.
  • 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.
  • Trail 4 there were certain circles with numbers in digits and rectangles with numbers spelt in words, starting from 1 to 20.
  • 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).
  • MCI mild cognitive impairment
  • the sensitivity and specificity were 0.68 and 0.67 (for composite index), respectively. Cronbach's alpha was 0.88.
  • lipid peroxidation 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.
  • BHT butylated hydroxytoluene
  • TAA thiobarbituric acid
  • MDA malondialdehyde
  • 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 m ⁇ 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 m ⁇ of the urine sample was then mixed with 200 m ⁇ 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 m ⁇ of the samples was transferred into a 5 mm NMR tube.
  • phosphate buffer pH 7.4
  • 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).
  • the water signal was suppressed by running ID NOESY-presat experiments. The number of scans was set at 64 for the urine samples.
  • PCA principal component analysis
  • mice 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the intervention group showed higher difference in the concentration of urinary thyroxine as compared to baseline than the placebo group.
  • urinary thyroxine showed good correlation with free thyroxine in the serum and provides clear indication of the thyroid functions.
  • TTR human transthyretin
  • 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.
  • various small molecules were developed by the pharmacological industry in order to bind to thyroxine binding site and developed the stability of TTR.
  • 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.

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Abstract

La présente invention concerne une formulation qui réduit le stress oxydatif chez un patient mammifère souffrant d'un état cognitif, la formulation comprenant : une teneur en eau entre 89.38 % et 92,93 %, une teneur en cendres de 0,12 % à 0,15 %, une teneur en protéines de 0,080 % à 0,16 %, une teneur en matières grasses de 0,00 % à 0,01 %, une teneur en glucides de 6,86 % à 10,31 %, une teneur en sucre de 109,27 mg/ml, la formulation étant riche en différents types de polyphénols, d'antioxydants et d'anthocyanine. Le jus de fruit est formulé à partir de fruits tropicaux à savoir la grenade, la goyave et la roselle.
PCT/MY2019/000036 2018-09-03 2019-08-30 Formulation qui réduit le stress oxydatif WO2020263075A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
KR20100061245A (ko) * 2008-11-28 2010-06-07 고려대학교 산학협력단 석류 추출물을 포함하는 신경계 질환 예방 또는 치료용 조성물
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KR20100061245A (ko) * 2008-11-28 2010-06-07 고려대학교 산학협력단 석류 추출물을 포함하는 신경계 질환 예방 또는 치료용 조성물
US20120164243A1 (en) * 2010-12-23 2012-06-28 Amazentis Sa Compositions and Methods for Improving Mitochondrial Function and Treating Neurodegenerative Diseases and Cognitive Disorders
US20140348925A1 (en) * 2011-12-20 2014-11-27 Consejo Superior De Investigaciones Científicas (Csic) Antioxidant Ingredient With Low Calorie Content, Method for Obtaining Same and Use Thereof
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SITI NUR AQILAH ABDUL MALEK, HASNAH HARON, WAN AIDA WAN MUSTAFA, SUZANA SHAHAR: "Physicochemical Properties, Total Phenolic and Antioxidant Activity of Mixed Tropical Fruit Juice, TP 3 in 1TM", JOURNAL OF AGRICULTURAL SCIENCE, vol. 9, no. 13, pages 50, XP055770771, ISSN: 1916-9752, DOI: 10.5539/jas.v9n13p50 *

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