Salilic acid and composition comprising same for use in prevention and / or treatment of senescence-related disorders in humans or animals

Abstract

The present invention relates to salic acid and a composition comprising salic acid for use in the prevention and / or treatment of aging-related disorders in humans and animals.

Description

[0001] The present invention relates to sagerinic acid and compositions containing sagerinic acid for use in preventing and / or treating disorders associated with aging in humans and animals.

[0002] Aging is a multifactorial process characterized by a progressive loss of physiological functions, leading to susceptibility to age-related diseases. Several theories have been proposed to explain the nature of aging, and there is now a consensus on how to categorize the different mechanisms involved in aging.

[0003] One of the most famous discoveries is that free radicals generated by mitochondrial metabolism are responsible for cellular disorders and damage to genetic material (1).

[0004] Another well-known aspect of aging is the change in genetic material over time. There is considerable evidence that epigenetic modifications, such as DNA methylation, noncoding RNA, and histone modifications, play an important role in the molecular mechanisms of aging. Calorie restriction is an intervention that has beneficial effects on the health of aging organisms and significantly extends their lifespan in all animal species to which it has been applied. Studies have shown that sirtuins, a type of histone diacylase that protects genetic material from damage by various factors, are closely associated with the effects of calorie restriction (2).

[0005] Another aspect of aging is protein glycation (3). In general, all proteins interact with reducing sugars in a passive, spontaneous reaction (which does not involve enzymes), resulting in more or less irreversible condensation compounds. This reaction is called the Maillard reaction, and its reaction mechanism was first proposed by JE Hodge in 1953 (4). This reaction produces multiple rearranged compounds (Amadori bodies, etc.). When this reaction is completed, we call them "advanced glycation end products" or AGEs (Advanced Glycation Endproducts).

[0006] This reaction is universal. It occurs in all cases, but with varying intensity depending on the number of free amino radicals present on the sugar and protein molecules involved. The reaction is amplified by heat and depends on the concentration of sugar in the medium.

[0007] During glycosylation, proteins undergo "cross-linking", which changes their function and stiffens structural proteins such as collagen and elastin. These proteins are normally eliminated by proteases that allow them to turn over. The more heavily glycosylated, the less sensitive the protein is to the action of proteases. Proteins that turn over slowly (such as those found in joints or the brain) undergo particularly intense glycosylation associated with this slow turnover rate.

[0008] Thus, aging passively leads to an increase in the glycosylation of proteins with a low turnover. Diabetes leads to the same result due to high blood sugar levels. In both cases, organs whose function depends on connective tissues, especially the vascular system, become weakened.

[0009] Therefore, diabetes is often associated with various vascular (especially eye and kidney) pathologies.

[0010] Disorders associated with glycation, whether normal (i.e., associated with physiological aging) or pathological (such as diabetes) (5) are very different and have varying degrees of severity. One example is the loss of elasticity of blood and lymphatic vessels, which leads to various forms of vascular disease associated with diseases such as stroke, atherosclerosis, macular degeneration, renal failure (6) and respiratory failure. Aesthetic damage to the integumentary system (such as sagging skin) is also a problem.

[0011] In general, it seems to be accepted that glycosylation of collagen, the most important protein in mammals, is a reliable marker of biological age and can be used to determine life expectancy (7, 8).

[0012] Aging leads to remodeling of large arteries. These age-related changes may be associated with increased activity of metalloproteinases, especially collagenase and elastase in large-diameter arteries (9). Therefore, it is important to perfectly regulate the activity of these enzymes. This regulation can be achieved by protecting the proteins of the arterial connective tissue (mainly collagen and elastin) from excessive action of these enzymes.

[0013] For the reasons stated above, in order to effectively combat the aging of the body, it is advisable to act simultaneously on four fundamental aspects of aging:

[0014] – Oxidative stress through the use of anti-radical molecules

[0015] – By being able to reverse the glycosylation of molecules and proteins that have already been glycosylated

[0016] – Protects DNA by activating sirtuins

[0017] – Protects connective tissue by inhibiting the proteases that break down connective tissue (particularly elastase and collagenase).

[0018] Molecules capable of acting in one or more of these four areas have been described.

[0019] Antioxidants and free radical scavengers

[0020] Since the beginning of the twentieth century, many antioxidants and free radical scavengers have been described, especially in the plant kingdom, polyphenols being the most common representatives. There are also molecules with good anti-free radical properties (such as water-soluble vitamin C and fat-soluble vitamin E).

[0021] A simple and rapid method for evaluating the anti-radical effect is to use the stable free radical 2,2-diphenyl 1-pycrylhydrazyl (DPPH) and measure its degradation by the test molecule using a colorimetric method.

[0022] Glycosylation inhibitors and protein deglycant

[0023] A large number of molecules are known to inhibit protein glycation (10). Most act only as antioxidants, since the glycation reaction includes an oxidation stage. Thus, for example, a large number of polyphenols have been suggested to be inhibitors of protein glycation. Others, such as aminoguanidine, carnosine (a monopeptide found in red meat), and pyridoxamine, act as competitive inhibitors of the amino radicals of the sugars involved in glycation.

[0024] Glycosylation inhibitors of proteins benefit health by limiting this reaction and reducing the generation of new cross-links. However, glycation slows down the turnover of proteins, leading to their accumulation and functional degradation of the organs with which they are associated. Some proteins are slow to renew themselves, especially the connective tissue proteins, collagen and elastin, which play a key role in the viscoelastic properties of the cardiovascular system, joints and dermis. Therefore, it is extremely important to find molecules that can reverse glycation in order to sever pre-existing cross-links and allow tissues to at least partially restore their original viscoelastic properties.

[0025] In particular, the reduction in the elasticity of the cardiovascular system is the most important problem during aging, and the main reason for this reduction is the formation of AGEs and cross-links between protein molecules. The use of glycation inhibitors cannot restore the elasticity of this system that is lost over time. This requires agents that are able to cut cross-links.

[0026] Therefore, there is a clear need for a product that can break down protein cross-links resulting from glycation without producing any deleterious side effects.

[0027] The applications of such products are quite extensive. The most important among them are to combat vascular diseases (especially related nephropathy, heart disease and retinopathy), to improve blood and lymphatic function in the elderly, to improve skin elasticity and thus appearance, and to combat the loss of elasticity of tendons and cartilage, which leads to reduced mobility due to inflammation and pain in the joints. In normal subjects, all of these conditions are age-related and are aggravated by diabetes. Another example is to improve venous and lymphatic function in the resolution of trauma by improving the function of connective tissue proteins due to a reduction in their glycosylation rate.

[0028] Finally, aging is accompanied by degradation of vascular connective tissue, leading to vascular and lymphatic stiffness and the development of atherosclerotic plaques.

[0029] In in vitro and animal pharmacology studies, certain molecules in the thiazolium group have been described in US Pat. No. 5,853,703A (22, 23) with this activity. Patent WO2011042890A2 discloses a family of polyphenolic molecules with protein deglycosylation effects (11).

[0030] Sirtuin Activators (12)

[0031] Lysine deacylase proteins that depend on NAD+ of the sirtuin family regulate a range of physiological functions, from energy metabolism to stress responses. Human isoforms of sirtuins, SIRT1-7, which are particularly activated during calorie restriction, are considered attractive therapeutic targets for age-related diseases such as type 2 diabetes, inflammatory diseases, and neurodegenerative diseases. Pharmacological activators of sirtuin-1 and other isoforms have been described, and preliminary clinical trials have been performed.

[0032] Connective tissue protectants (13)

[0033] Epidemiological studies have shown a link between the intake of foods or beverages containing polyphenols and the prevention of certain human diseases, such as chronic obstructive pulmonary disease and heart disease, chronic inflammation, and a reduced risk of various cancers. It now appears that polyphenols protect the proteins of the extracellular matrix from exposure to free radicals and from attack by proteases that are overexpressed during inflammation.

[0034] However, none of these molecules can simultaneously act on four fundamental aspects of aging:

[0035] – Oxidative stress through the use of free radical molecules

[0036] – By being able to reverse the glycosylation of molecules and proteins that have already been glycosylated

[0037] – Protects DNA by activating sirtuins

[0038] – Protects connective tissue by inhibiting the proteases that break down connective tissue (particularly elastase and collagenase).

[0039] Thaliric acid is a molecule first discovered and described in the sage plant (Salvia officinalis) by Yinrong Lu and L. Yeap Foo in 1999 (17).

[0040] Since then, salicylic acid has been found in a number of plant species in the Lamiaceae family, including Melissa officinalis (18), rosemary (Rosmarinus officinalis) (19), cat’s whisker (Orthosiphon grandiflorus) (20), Helicteres hirsuta (21), and Plectranthus amboinicus (15).

[0041] Sage and lemon balm are plants with multiple uses and properties, and these multiple effects are often due to the different molecules present in the extracts.

[0042] The effectiveness of salicyclic acid as a COVID-19 inhibitor was predicted by computer simulations, but there is no physical evidence that such activity has been produced (14).

[0043] Furthermore, US Pat. No. 8,105,636 B2 (15) claims a composition comprising an extract of Plectranthus amboinicus as an anti-inflammatory agent, in which salicylic acid is listed as one of the possible active ingredients.

[0044] Potential anti-leishmanial and anti-inflammatory activities of salicylic acid have been described (16).

[0045] The present invention is based on the serendipitous discovery of the effects of salicylic acid on these four fundamental aspects of ageing: During in vitro experiments it was observed that, surprisingly, a single molecule of salicylic acid possesses the four properties mentioned above.

[0046] The present invention is useful in preventing and treating disorders associated with aging.

[0047] These include, but are not limited to, the following:

[0048] – connective tissue diseases affecting cartilage, such as osteoarthritis of the joints and the non-inflammatory component of rheumatoid arthritis (degeneration of connective tissue rather than an immune response leading to inflammation),

[0049] – Vascular connective tissue diseases: stroke, vasculitis nephritis and retinitis, macular degeneration, atherosclerotic disease,

[0050] – Diseases directly associated with protein glycosylation, such as cataracts, Alzheimer’s disease, skin healing disorders, and presbycusis,

[0051] – Aesthetic skin disorders such as lipofuscin accumulation, pigmentation spots, erythema, and loss of skin elasticity.

[0052] Saliric acid can be used in pure form or as an extract in combination with excipients suitable for its use: oral, parenteral, topical, rectal or by inhalation.

[0053] Therefore, the present invention provides thalinic acid for use in preventing and / or treating disorders associated with aging in humans or animals.

[0054] In a first embodiment, these disorders are caused by oxidative stress.

[0055] In a second embodiment, the disorders are due to protein glycosylation.

[0056] In a third embodiment, the disorders are caused by changes in the DNA.

[0057] In a fourth embodiment, the disorders are caused by changes in proteins of the arterial connective tissue.

[0058] The present invention also provides salicylic acid used as an antioxidant and / or a free radical scavenger.

[0059] The present invention also provides salicylic acid used as a protein deglycosylation agent.

[0060] The invention also provides salicylic acid used as a DNA protecting agent.

[0061] The invention also provides salicylic acid used as a protease inhibitor.

[0062] In a preferred embodiment, the present invention provides thalinic acid for simultaneously preventing and / or treating disorders caused by oxidative stress, disorders caused by protein glycosylation, disorders caused by changes in DNA, and disorders caused by changes in proteins of arterial connective tissue in humans or animals.

[0063] Another object of the present invention is a composition for preventing and / or treating disorders associated with aging in humans or animals, which composition comprises thalinic acid.

[0064] In a first embodiment of the composition of the invention, these disorders are caused by oxidative stress.

[0065] In a second embodiment of the composition of the invention, the disorders are caused by protein glycosylation.

[0066] In a third embodiment of the compositions of the invention, the disorders are caused by changes in the DNA.

[0067] In a fourth embodiment of the composition of the invention, the disorders are caused by changes in proteins of the arterial connective tissue.

[0068] A preferred object of the present invention is a composition for simultaneously preventing and / or treating disorders caused by oxidative stress, disorders caused by protein glycosylation, disorders caused by changes in DNA and disorders caused by changes in proteins of arterial connective tissue in humans or animals, which composition includes thalinic acid.

[0069] The invention will be better understood and its characteristics and advantages will become more apparent on reading the illustrative description and examples of use which follow. Example

[0070] Example 1: Preparation of purified salicyclic acid

[0071] 170 g of dried lemon balm (Melissa officinalis) leaves were soaked in 2 L of deionized water at room temperature with constant stirring for 24 hours.

[0072] The extract was separated by filtration on paper and then freeze-dried. The mass of the extract residue was 32.3 g, and the yield was 19%.

[0073] 10 g of freeze-dried extract was dissolved in 50 mL of deionized water, and the solution was chromatographed on a 45 mm diameter, 24 cm long column containing about 200 g of HP20 resin (Thermo Scientific, reference number 046488.A1), with 2 L of deionized water as the eluent.

[0074] Remove 2L of the eluted from the column. Then elute with 2L of methanol (Thermo Scientific, reference number L13255). All the eluent is recovered and evaporated under reduced pressure until the methanol is completely evaporated. The remaining 100mL of aqueous phase consists of deionized water and 10mL of concentrated hydrochloric acid (Honeywell Fluka, reference number 320331), and is extracted three times with 100ml of ethyl acetate in countercurrent (Roth, reference number 73361).

[0075] The organic phase was dried over anhydrous sodium sulfate (Sigma-Aldrich, reference number 238597), filtered and evaporated to dryness. About 700 mg of partially purified salicylic acid were obtained.

[0076] Then, 700 mg of crude thaliric acid was batch chromatographed by preparative HPLC on a C18 column (Vydac Denali 10 μm) with a diameter of 22 mm and a length of 250 mm, dissolving each 100 mg of crude thaliric acid in 1 mL of deionized water using the following gradient:

[0077] Flow rate: 20mL / min

[0078] Solvent A: Deionized water containing 0.1% formic acid (Fisher Chemical A117-50)

[0079] Solvent B: Methanol (Thermo Scientific, reference number L13255)

[0080]

[0081] Fractions of 10 ml were collected.

[0082] The fraction containing salicylic acid was identified by UV spectroscopy (max). It was retained and the other fractions were discarded.

[0083] After exceeding 700 mg, the fractions were combined and evaporated to dryness under reduced pressure. 165 mg of a residue were obtained.

[0084] The remaining 165 mg was then chromatographed in the same manner as above. The final product was 23 mg of salicylic acid with a purity of 92%.

[0085] Example 2: Preparation of Lemon Balm Extract Rich in Thaliric Acid

[0086] 170 g of dried lemon balm leaves were soaked in 2 L of deionized water at room temperature under constant stirring for 24 hours. The extract was separated by filtration on paper and then freeze-dried. The mass of the extract residue was 32.3 g, and the yield was 19%.

[0087] 10 g of freeze-dried extract was dissolved in 50 mL of deionized water, and the solution was chromatographed on a 45 mm diameter, 24 cm long column containing about 200 g of D HP20 resin (Thermo Scientific, reference number 046488.A1), with 2 L of deionized water as the eluent.

[0088] Remove 2L of the eluted from the column. Then elute with 2L of methanol (Thermo Scientific, reference number L13255). All the eluent is recovered and evaporated under reduced pressure until the methanol is completely evaporated. The remaining 100mL of aqueous phase consists of deionized water and 10mL of concentrated hydrochloric acid (Honeywell Fluka, reference number 320331), and is extracted three times with 100ml of ethyl acetate in countercurrent (Roth, reference number 73361).

[0089] The organic phase was dried over anhydrous sodium sulfate (Sigma-Aldrich, reference number 238597), filtered and evaporated to dryness. Approximately 700 mg of a lemon balm extract containing 8% salicylic acid was obtained.

[0090] Example 3: Evaluation of the effects of salicylic acid and ascorbic acid on oxidative stress

[0091] 3.1. Principle

[0092] The principle of the method used to evaluate the effect of thalinic acid in comparison with ascorbic acid as a reference molecule is based on the elimination of a stable free radical, 2,2-diphenyl-1-trinitrophenylhydrazine (DPPH, Sigma-Aldrich, reference number D9132).

[0093] 3.2. Instruments and materials

[0094] –JASCO V-730 Double Beam UV Visible Spectrophotometer

[0095] – Stopwatch

[0096] – Standard laboratory equipment.

[0097] – Precision scale: ML104 (Mettler-Toledo).

[0098] – Automatic variable volume pipettes.

[0099] Reagents

[0100] – Potassium dihydrogen phosphate (Riedel de Haen, reference number 04243)

[0101] – Disodium hydrogen phosphate dihydrate (Sigma-Aldrich, reference number 30435)

[0102] – Purified water

[0103] –DPPH (Sigma-Aldrich, reference number D9132)

[0104] – Methanol LC / MS grade (Fisher Chemical, reference number A456-212)

[0105] – Toluene, reference (Carl Roth, reference number 4445.1)

[0106] – PharmEthyl 96% alcohol (Cristalco, reference number 11371014)

[0107] 3.4. Preparation of reagent solution:

[0108] – Phosphate buffer pH 7

[0109] Dissolve 0.177 g potassium dihydrogen phosphate and 0.730 g disodium hydrogen phosphate dihydrate in 50 ml purified water. Adjust the pH if necessary.

[0110] –2,2-Diphenyl-1-trinitrophenylhydrazine (DPPH) in methanol

[0111] An exact 0.0027% (w / v) DPPH solution in methanol was prepared.

[0112] – Antioxidant solution

[0113] A solution of exactly 0.1% (w / v) ascorbic acid was prepared in ethanol (Riedel-de- Reference No. 33034). The solution was diluted to 0.0007% (V / V) in ethanol.

[0114] A solution of exactly 0.1% (w / V) salicylic acid (prepared according to Example 5.1) was prepared in water. This solution was diluted to 0.0007% (V / V) in water.

[0115] 3.5. Operation Mode

[0116] 3.5.1. Control solution:

[0117] A series of sealed pill boxes filled with:

[0118] sample blank Buffer solution (ml) 1.50 1.50 DPPH solution (ml) 3.00 0.00 Methanol(ml) 0.00 3.00

[0119] 3.5.2. Testing:

[0120] A series of sealed pill boxes filled with:

[0121] sample blank Buffer solution (ml) 1.35 1.35 DPPH solution (ml) 3.00 0.00 Methanol(ml) 0.00 3.00 Antioxidant solutions to be tested 0.15 0.15

[0122] For samples and blanks:

[0123] – After addition, stir on a magnetic stirrer for 5 minutes

[0124] – Add 4 ml of toluene

[0125] – Stir with a magnetic stirrer for 30 seconds

[0126] – Wait 20 seconds to 1 minute (if necessary)

[0127] – Use an automated pipette to recover the organic phase from the top, taking care not to recover any of the aqueous phase

[0128] – Read the OD of the organic phase using a spectrophotometer at 519 nm

[0129] Results

[0130] Tested molecules Activity (arbitrary dimensionless unit) Ascorbic acid (reference) 5.26 Thaliric acid 9.70

[0131] These results indicate that salicylic acid is almost twice as effective as ascorbic acid in scavenging free radicals and thus in preventing and / or treating disorders caused by oxidative stress.

[0132] Example 4: Evaluation of the effects of salicylic acid and alagebrium as protein deglycation agents.

[0133] 4.1. Principle

[0134] The test product in a 0.25% (w / V) solution was contacted with a 0.375% (w / V) solution of 1-phenyl-1,2-propanedione (PPD) at 37°C for 24 hours. After the ExpressC18 column, HPLC quantification was performed at 229 nm at T0 and again after 24 hours to determine the content of benzoic acid.

[0135] The activity of the deglycating agent was determined by calculating the percentage of μmol benzoic acid / μmol PPD in the reaction medium.

[0136] 4.2. Apparatus and materials

[0137] – Ultra3000 HPLC system (Thermo Scientific-Dionex) equipped with a diode array detector and a column oven.

[0138] – Express C18 HPLC column (Supelco, reference number 53816-U)

[0139] – Dimensions: 15cm x 3.0mm

[0140] – Particle size: 2.7 μm

[0141] – BD 53 / E2 drying oven (Binder).

[0142] – 0.45 μm pore size PVDF (polyvinyl fluoride) filter ( -HV, reference number SLHVX13NL).

[0143] – Standard laboratory equipment.

[0144] – Precision scale: ML104 (Mettler-Toledo).

[0145] – Automatic variable volume pipette.

[0146] Reagents

[0147] – Water suitable for UHPLC-UV LC / MS (Fisher, reference number W6-212)

[0148] – Acetonitrile LC / MS grade (Fisher Chemical Company, reference number A955-212)

[0149] – Methanol LC / MS grade (Fisher Chemical Company, reference number A456-212)

[0150] – PharmEthyl 96% alcohol (Cristalco, reference number 11371014)

[0151] – Sodium acetate trihydrate (Sigma-Aldrich, reference number S8625)

[0152] – Acetic acid (Carl Roth, reference number HN55.1)

[0153] – Benzoic acid, reference number 242381 (Sigma-Aldrich, reference number 242381)

[0154] – Potassium dihydrogen phosphate (Riedel de Haen, reference number 04243)

[0155] – Disodium hydrogen phosphate dihydrate (Sigma-Aldrich, reference number 30435)

[0156] – 1-Phenyl-1,2-propanedione (PPD) (Aldrich, reference number 223034)

[0157] 4.4. Preparation of reagent solutions

[0158] – Phosphate buffer pH 7.4

[0159] Dissolve 0.178 g potassium dihydrogen phosphate and 0.955 g disodium hydrogen phosphate dihydrate in 100 ml purified water. Adjust the pH if necessary.

[0160] – Phosphate buffer pH 7.4-methanol

[0161] A mixture was prepared with 50 ml of phosphate buffer (pH 7.4) and 50 ml of methanol.

[0162] –1-Phenyl-1,2-propanedione (PPD) solution

[0163] An exact 0.375% (w / V) PPD solution was prepared in a mixed buffer of pH 7.4-methanol (50 / 50).

[0164] 4.5. Preparation of the test product solution

[0165] The product to be tested was prepared at about 0.25% (w / V) in a mixed buffer of pH 7.4-methanol (50 / 50).

[0166] Products to be tested: Alagebrium chloride (MedChem Express, reference number HY-106024B), salicylic acid (prepared according to Example 5.1).

[0167] 4.6. Benzoic acid calibration range

[0168] 4.6.1. Standard solution

[0169] A stock solution of approximately 0.08% (w / v) benzoic acid was prepared in ethanol. This solution was diluted 1:10 (v / v) in purified water (D).

[0170] (D) Then diluted 1:5, 2:5, 3:5 and 4:5 (V / V) in purified water. (D1, D2, D3 and D4).

[0171] D1, D2, D3, D4 and D passed through a PVDF filter with a porosity of 0.45 μm.

[0172] 4.6.2 Mobile phase

[0173] A: Sodium acetate buffer: Weigh 3.18 g of sodium acetate trihydrate, add 10 ml of acetic acid, make up to 500 ml with "Optima" water, and stir until completely dissolved.

[0174] B: Acetonitrile

[0175] 4.6.3 Analysis conditions

[0176] Chromatographic column: Express C18 15cm x 3.0mm 2.7μm

[0177] Flow rate: 0.5 ml / min at 25°C

[0178] Mode: Gradient (see Table 1)

[0179] Detection: Ultraviolet (UV) at 229nm

[0180] Injection: 10 μl of solutions D1, D2, D3, D4 and D.

[0181] Table 1

[0182] Time (minutes) Mobile phase A Mobile phase B 0 90 10 0-15 90→75 10→25 15-17 75→90 25→10 17-30 No gradient at 90 locations 10 places without gradient

[0183] A linear calibration curve was established: the relationship between the peak area of ​​benzoic acid, with a retention time of 9.17 min, observed at 229 nm, and the concentration (w / V).

[0184] 4.7. Activity against PPD

[0185] 4.7.1. Operation mode:

[0186] A series of sealed pill boxes filled with:

[0187] Comparison test Solution of product to be tested (ml) 0 1 DPPH solution (ml) 1 1 Buffered phosphate-methanol (50 / 50) (ml) 1 0

[0188] Stir after each addition.

[0189] 0.05 ml was removed from each pill box and the benzoic acid content was measured at T 0. The pill boxes were resealed and placed in an oven at 37°C for a minimum of 21 hours and a maximum of 24 hours.

[0190] When leaving the oven, take the same volume for dosing.

[0191] The time at 37°C for the control and all tests must be exactly the same so that they are ready during the run.

[0192] HPLC quantitative feeding of benzoic acid was also performed on an ongoing basis.

[0193] 5.4.7.2. HPLC quantitative feed of released benzoic acid:

[0194] 0.05 ml of sample was diluted in purified water to 1:5 (V / V), i.e., 0.2 ml was added.

[0195] They were passed through a 0.45 μm pore size PVDF filter before injection.

[0196] 5.4.7.2.1.Analysis conditions:

[0197] Chromatographic column: Express C18 15cm x 3.0mm 2.7μm

[0198] Flow rate: 0.5 ml / min at 25°C

[0199] Mode: Gradient (see Table 2)

[0200] Detection: Ultraviolet (UV) at 229nm

[0201] Injection: 10 μl

[0202] Table 2

[0203] Time (minutes) Mobile phase A Mobile phase B 0 90 10 0-12 90→78 10→22 12-14 78→0 22→100 14-20 No gradient at 0 100% No Gradient 20-22 0→90 100→10 22-35 90 no gradients 10 places without gradient

[0204] 4.8 Results

[0205] Using the calibration curve, calculate the benzoic acid content in g / 100 ml of reaction medium.

[0206] PPD control at T0 and 24 h: T T0 and T 24h

[0207] Tests were performed at T0 and 24 hours: E T0 and E 24h

[0208] These concentrations were converted to μmol benzoic acid / ml reaction medium

[0209] (PM benzoic acid = 122.12)

[0210] T T0 x 10000 / 122.12

[0211] The concentration of PPD in μmol / ml reaction medium is as follows:

[0212] 0.375x 1000000 / 100x 2x 148.16=12.655(PM PPD=148.16)

[0213] Calculate the percentage of μmol benzoic acid / μmol PPD in the reaction medium:

[0214] T T0 x 10000x 100 / 122.12x 12.655

[0215] The values ​​obtained for the control and the test at different times were compared.

[0216] The deglycosylation activity of salicylic acid was compared with that of alachloramide as a reference molecule.

[0217] Tested molecules Activity (arbitrary dimensionless unit) Alachloramine (reference) 28.79 Thaliric acid 16.78

[0218] These results indicate that salicylic acid has good protein deglycosylation activity in addition to its good free radical scavenging activity.

[0219] Example 5: Evaluation of the effects of salicylic acid and resveratrol on sirtuin-1

[0220] 5.1. Principle

[0221] The SIRT1 Inhibitor / Activator Screening Kit deacetylates a substrate with SIRT1, and then cleaves the deacetylated substrate to release a fluorescent group, which is detected by fluorescence measurement at Ex / Em=400 / 505 nm.

[0222] SIRT1 activators enhance SIRT1 activity compared to controls, resulting in higher fluorescence signals. This kit provides a fast, easy, sensitive, and reliable test suitable for high-throughput screening of SIRT1 activators.

[0223] 5.2. Apparatus and materials

[0224] –Gemini EM Fluorescence Spectrophotometer

[0225] – Stopwatch

[0226] – Standard laboratory equipment.

[0227] – Precision scale: ML104 (Mettler-Toledo).

[0228] –Automatic variable volume pipette

[0229] – Oven Innovens 28EU1 Jouan / Thermo

[0230] 5.3 Reagents

[0231] – Solution for the kit: Reference number ab283377 (Abcam)

[0232] Buffer

[0233] Substrate

[0234] SIRT1

[0235] NAD

[0236] ·DTT

[0237] –DMSO Reference No. 34869 (Honeywell Riedel-de- )

[0238] 5.4. Preparation of reagent solutions

[0239] SIRT1 enzyme: Store at -80°C

[0240] Substrate: Store at -20°C and avoid repeated freezing / thawing. Use a fresh sample each time.

[0241] Mix 1 μl of substrate and 9 μl of DMSO.

[0242] NAD: Store at -80°C and avoid repeated freezing / thawing.

[0243] Mix 2 μl of NAD stock solution with 58 μl of buffer without DTT.

[0244] 1M DTT: Store at -20°C, thaw and keep frozen between uses.

[0245] Buffer: Store at 4°C or -20°C, warm to 37°C, must be prepared before use.

[0246] Preparation of DTT buffer: Mix 0.002 ml of DTT with 0.998 ml of buffer.

[0247] Revealing: Store at -80°C and avoid repeated freezing / thawing. Keep frozen during use.

[0248] 5.5. Preparation of the test product (activator) solution

[0249] A solution of exactly 2.28% (w / V) of activator (resveratrol (Sigma-Aldrich, Ref. R5010)) and salicylic acid prepared according to Example 5.1 was prepared in DMSO. Diluted to 1:250 in DTT buffer solution.

[0250] 5.6. Operation Mode

[0251] Preparation of enzyme solution: Add the following to 3 separate wells:

[0252] SIRT1 Dosing Buffer (with DTT) 23μL SIRT1 2μL

[0253] Screening Compounds, Inhibitor Controls, Enzyme Controls, and Blank Control Formulations:

[0254] 1. Add to the SIRT1 enzyme solution well:

[0255] – 25 μl of undiluted activator solution (2.28% solution) in each well as reagent control (AC)

[0256] – 25 μL of DTT-free buffer in each well as enzyme control (EC)

[0257] – 25 μL of diluted activator solution as test(s)

[0258] 2. In another well, add 50 μl of DTT-free buffer as a blank control (no enzyme).

[0259] 3. Mix well and incubate the plaque at 37°C for 5 minutes

[0260] Preparation of substrate: After incubation, add the following to each well:

[0261] entry volume SIRT1 Dosing Buffer (with DTT) 36μL Diluted substrate 2μL Diluted NAD 2μL

[0262] Mix and incubate at 37 °C for 30-60 minutes.

[0263] development:

[0264] Add 10 μl of exposure reagent to each well, mix well and incubate at 37°C for 10 minutes, protected from light.

[0265] Measurement:

[0266] The fluorescence was read (Ex / Em=400 / 505 nm).

[0267] Results

[0268] The sirtuin-1 activity of salicylic acid was compared with the sirtuin-1 activity of resveratrol as a reference molecule.

[0269] Tested molecules Activity (arbitrary dimensionless unit) Resveratrol (reference) 80.54 Thaliric acid 26.56

[0270] These results indicate that salicylic acid has a good effect on the activation of sirtuins, that is, has a good effect on the prevention and / or treatment of changes in DNA, and has good protein deglycosylation activity and excellent free radical scavenging activity.

[0271] Example 6: Evaluation of the effect of salicylic acid and witch hazel as connective tissue protein protectants price.

[0272] 6.1. Principle

[0273] A collagenase activity assay kit (Sigma-Aldrich MAK293) was used. This kit allows screening of collagenase inhibitors by measuring collagenase activity using a synthetic peptide (FALGPA) that mimics the structure of collagen.

[0274] 6.2. Apparatus and materials

[0275] –JASCO V-730 Double Beam UV Visible Spectrophotometer

[0276] – Stopwatch

[0277] – Standard laboratory equipment.

[0278] – Precision scale: ML104 (Mettler-Toledo).

[0279] –Automatic variable volume pipette

[0280] – Oven Innovens 28EU1 Jouan / Thermo

[0281] Reagents

[0282] – Solution of the kit: Reference MAK293-1kT (Sigma-Aldrich)

[0283] ●Collagenase buffer (MAK293A-KC)

[0284] ●Collagenase solution (MAK293B-KC)

[0285] ●Collagenase substrate (MAK293C-KC)

[0286] – PharmEthyl 96% alcohol, reference number 11371014 (Cristco)

[0287] – Purified water

[0288] 6.4. Preparation of test product (inhibitor) solution

[0289] The solution was made of an exact 1% (w / v) solution of Hamamelis virginiana tannin (Extrasynsis, Ref. 0958) in ethanol.

[0290] A solution of exactly 1% (w / V) salicylic acid (prepared according to Example 5.1) was prepared in purified water.

[0291] 6.5. Operation Mode

[0292] 6.5.1. Control solution

[0293] A series of sealed pill boxes filled with:

[0294] Blank 1 Comparison Blank 2 test Buffer (μl) 320 300 316 296 Collagenase (μl) 0 20 0 20 Substrate (μl) 80 80 80 80 Inhibitor (μl) 0 0 4 4

[0295] – After adding, stir quickly by hand

[0296] – Place in oven at 37°C for 30 minutes

[0297] – Add 1.6 mL of purified water. Mix well.

[0298] – Read DO: Sample against blank, spectrophotometer at 345nm.

[0299] Results

[0300] Collagenase activity

[0301] The anti-collagenase activity of salicylic acid was compared with that of witch hazel tannin as a reference molecule.

[0302] In absolute value: Y = [(DO 空白2 -DO 测试 )-(DO 空白1 -DO 对照 )] / (DO 空白1 -DO 对照 )]x 100

[0303] Tested molecules Activity (arbitrary dimensionless unit) Witch Hazel Tannins (reference) 21.43 Thaliric acid 46.43

[0304] These results indicate that salicylic acid has an inhibitory effect on proteases, represented by collagenase, which is more than two times greater than the inhibitory effect of the reference molecule Hamamelis tannin.

[0305] Furthermore, this has excellent free radical scavenging activity, good activity on sirtuin activation (ie, good effect on preventing and / or treating DNA alterations), and good protein deglycosylation activity.

[0306] Therefore, salicylic acid itself has excellent activity in preventing and / or treating disorders associated with aging in humans and animals because it acts simultaneously on four fundamental aspects of oxidative stress in aging: due to its excellent anti-free radical effect; protein glycation, due to its ability to reverse glycation already present in proteins; protection of DNA, due to its ability to activate sirtuins; and protection of connective tissue, due to its excellent inhibitory effect on proteases that degrade connective tissue (particularly elastase and collagenase).

[0307] Application of salicylic acid to the four major axes of treatment of aging will be based on doses defined relative to the doses necessary to administer the four reference molecules used together.

[0308] The recommended daily oral dose of ascorbic acid is 200 mg to 1 g per day (24), resveratrol is 250 mg to 1 g per day (25), witch hazel tannins are 700 mg to 2 g per day (26-27), and alachloramine is 100 mg to 300 mg per day (28).

[0309] Oral administration of 10 mg to 2 g of thalinic acid per day provides the recommended amounts of ascorbic acid, resveratol, hamamelitannin, and alachloramine.

[0310] An advantage of the present invention is that a single dose of salicylic acid corresponds to separate doses of ascorbic acid, resveratrol, alachloramine and witch hazel tannins.

[0311] Example 7. Preparation of Melissa officinalis extract capsules rich in salicylic acid.

[0312] 1 kg of the dried leaf extract of Melissa officinalis obtained according to Example 2 was intimately mixed with 10 g of sodium carbonate (E500) in a knife grinder.

[0313] This mixture was then used to fill size 00 capsules (0.9 mL). Each capsule contained 550 mg of the extract, equivalent to 44 mg of thalinic acid.

[0314] Example 8. Galenic formula of a gel for the treatment of aesthetic disorders, suitable for application to the face and body.

[0315]

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[0336] 16.Oliver A.Radtke,Lai Yeap Foo,Yinrong Lu,Albrecht F.Kiderlen,andHerbert Kolodziej Evaluation of Sage Phenolics for Their AntileishmanialActivity and Modulatory Effects on Interleukin-6,Interferon and TumourNecrosis Factor-α-Release in RAW 264.7 Cells.Z.Naturforsch.2003,58c,395-400.

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Claims

1. Thaliric acid for use in preventing and / or treating disorders associated with aging in humans or animals.

2. The salinic acid according to claim 1, characterized in that The disorder is caused by oxidative stress.

3. The salicylic acid according to claim 1, characterized in that The disorder is caused by protein glycosylation.

4. The salicylic acid according to claim 1, characterized in that The disorder is caused by changes in the DNA.

5. The salicylic acid according to claim 1, characterized in that The disorder is caused by changes in proteins in the connective tissue of the arteries.

6. Thaliic acid for use as an antioxidant and / or free radical scavenger.

7. Thaliric acid for use as a protein deglycosylation agent.

8. A salicyclic acid used as a DNA protecting agent.

9. Thaliric acid for use as a protease inhibitor.

10. Thaliric acid for use in the simultaneous prevention and / or treatment of disorders caused by oxidative stress, disorders caused by protein glycosylation, disorders caused by changes in DNA and disorders caused by changes in proteins of arterial connective tissue in humans or animals.

11. A composition for preventing and / or treating disorders associated with aging in humans or animals, said composition comprising thalinic acid.

12. The composition according to claim 10, characterized in that The disorder is caused by oxidative stress.

13. The composition according to claim 10, characterized in that The disorder is caused by protein glycosylation.

14. The composition according to claim 10, characterized in that The disorder is caused by changes in the DNA.

15. The composition according to claim 10, characterized in that The disorder is caused by changes in proteins in the connective tissue of the arteries.

16. A composition for simultaneously preventing and / or treating disorders caused by oxidative stress, disorders caused by protein glycation, disorders caused by changes in DNA, and disorders caused by changes in proteins of arterial connective tissue in humans or animals, the composition comprising thalinic acid.

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