Sagelic Acid and Compositions Containing Sagelic Acid for Use in the Prevention and / or Treatment of Age-Related Disorders in Humans or Animals

Abstract

The present invention relates to sagelic acid and compositions containing sagelic acid for use in the prevention and / or treatment of age-related disorders in humans and animals.

Description

[Technical Field]

[0001] The present invention relates to sagelic acid and compositions containing sagelic acid for use in the prevention and / or treatment of senescence-related disorders in humans and animals. [Background technology]

[0002] Aging is a multifactorial process characterized by a progressive decline in physiological function that leads to an increased susceptibility to age-related diseases. Several theories have been proposed to explain the nature of aging, and there is currently agreement on how to classify the different mechanisms involved in aging.

[0003] One of the best-known identifies free radicals generated by mitochondrial metabolism as the cause of cell damage 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, non-coding RNA, and histone modifications, play important roles 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. Sirtuins are histone diacylation enzymes that protect genetic material from damage induced by various factors and have been shown to strongly positively mediate the effects of calorie restriction (2).

[0005] Another aspect of aging is protein glycation (3). Generally speaking, all proteins interact with reducing sugars in a passive, spontaneous reaction that does not involve enzymes, resulting in the formation of more or less irreversible condensation compounds. This reaction is known as the Maillard reaction, and its mechanism was first described by J.E. Hodge in 1953 (4). This reaction results in several rearranged compounds (notably Amadori bodies). Upon completion, the products are called "advanced glycation end products," or AGEs.

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

[0007] During glycation, proteins undergo "cross-linking," which alters their functionality and stiffens structural proteins such as collagen and elastin. These proteins are usually degraded by proteases, allowing them to be regenerated. The more glycated, the less sensitive the protein is to the effects of proteases. Slowly regenerated proteins, such as those found in joints or the brain, undergo particularly intense glycation in relation to this slow regeneration rate.

[0008] Aging therefore passively induces increased glycation of under-regenerated proteins. Diabetes produces the same results due to high blood sugar levels. In both cases, organs that depend on connective tissue for functioning, especially the vascular system, are weakened.

[0009] As a result, diabetic disease is often accompanied by various vascular disorders, especially in the eyes and kidneys.

[0010] Glycation-related disorders are highly diverse and vary in severity, whether they are normal, i.e., correlated with physiological aging, or pathological, as in the case of diabetes. (5) One example is the loss of elasticity of blood and lymphatic vessels, which leads to all forms of vascular damage, with diseases such as stroke, atheroma, macular degeneration, renal failure (6), and respiratory failure. Aesthetic damage to the integumentary system, for example, sagging skin, is also a problem.

[0011] Generally speaking, it also seems acceptable that glycation 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 induces aortic remodeling. These age-related changes may be related to increased activity of metalloproteinases, especially collagenase and elastase, in large-diameter arteries (9). Therefore, it is important to fully regulate the activity of these enzymes. This can be achieved by protecting arterial connective tissue proteins, primarily collagen and elastin, from the excessive action of these enzymes.

[0013] As a result of the above, to effectively combat aging in the body, it is desirable to simultaneously address what appear to be four fundamental axes of aging: Action against oxidative stress by using antiradical molecules Action on protein glycation by molecules capable of reversing glycation that has already occurred Activation of sirtuins protects DNA Protection of connective tissue by inhibiting proteases that degrade connective tissue, particularly elastase and collagenase.

[0014] Molecules capable of acting on one or more of these four axes have already been described.

[0015] Antioxidants and free radical scavengers Since the beginning of the 20th century, numerous antioxidants and free radical scavengers have been described, especially in the plant kingdom, the most common of which are polyphenols. There are also molecules such as water-soluble vitamin C and fat-soluble vitamin E that have good antiradical properties.

[0016] A simple and rapid method to assess antiradical efficacy involves the incorporation of the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and colorimetric measurement of the decomposition of DPPH by the molecule being tested.

[0017] Glycation inhibitors and protein deglycants Currently, numerous molecules are known to inhibit protein glycation (10). Because glycation involves an oxidation step, the simplest act as antioxidants. Thus, for example, numerous polyphenols have been recognized as potential inhibitors of protein glycation. Other molecules, such as aminoguanidine, carnosine (a monopeptide found in red meat), and pyridoxamine, act as competitive inhibitors of amino radicals from sugars involved in glycation.

[0018] Protein glycation inhibitors are beneficial to health by limiting glycation reactions and reducing the formation of new cross-links. However, glycation slows down protein regeneration, leading to protein accumulation and the decline of the function of the organs they are associated with. Some proteins, especially collagen and elastin, which are connective tissue proteins that play an important role in the viscoelastic properties of the cardiovascular system, joints, and dermis, regenerate themselves slowly. Therefore, it is very important to discover molecules that can reverse glycation, cut existing cross-links, and allow tissues to regain at least part of their original viscoelastic properties.

[0019] In particular, the loss of elasticity in the cardiovascular system is the most important problem during aging, and the main cause of this loss of elasticity is the formation of cross-links between AGEs and protein molecules. The use of glycation inhibitors cannot restore the elasticity lost over time in the cardiovascular system. In order to restore the elasticity lost over time, an agent that cuts cross-links is required.

[0020] Therefore, there is a clear need for products that are capable of cleaving protein cross-links caused by glycation without any adverse side effects.

[0021] Such products have many applications. Most importantly, they address vascular disorders, particularly those associated with nephropathy, heart disease, and retinopathy; improve vascular and lymphatic function in the elderly; improve skin elasticity, thereby improving appearance; and address the loss of tendon and cartilage elasticity that leads to decreased mobility due to joint inflammation and pain. All of these pathological conditions are age-related in normal subjects and exacerbated by diabetes. Another example is the improvement of venous and lymphatic function following injury resolution due to improved functionality of connective tissue proteins resulting from reduced glycation rates of these proteins.

[0022] Finally, aging is accompanied by the degradation of vascular connective tissue, which leads to the hardening of blood and lymphatic vessels and the creation of atherosclerotic plaques.

[0023] Certain molecules of the thiazolium group have been described as having protein deglycation activity in vitro and in animal pharmacological tests in Patent Document 1 (22, 23). Patent Document 2 has revealed a family of polyphenol molecules with protein deglycation effects (11).

[0024] Sirtuin activators (12) The sirtuin family of NAD+-dependent lysine deacylase proteins regulates various physiological functions, from energy metabolism to stress responses. Human sirtuin isoforms, SIRT1-SIRT7, are specifically activated during calorie restriction and are considered attractive therapeutic targets for age-related diseases such as type 2 diabetes, inflammatory diseases, and neurodegenerative disorders. Pharmacological activators of sirtuin-1 and other isoforms have been described, and initial clinical trials are underway.

[0025] Connective tissue protector (13) Epidemiological studies have suggested an association between the consumption 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 many types of cancer. It now appears to be proven that polyphenols protect proteins of the extracellular matrix from exposure to free radicals and attack by proteases overexpressed during inflammation.

[0026] However, none of these molecules alone can simultaneously affect the four fundamental aspects of aging: Action against oxidative stress by using antiradical molecules Action on protein glycation by molecules capable of reversing glycation that has already occurred Activation of sirtuins protects DNA Protection of connective tissue by inhibiting proteases that degrade connective tissue, particularly elastase and collagenase.

[0027] Sagelic acid was first discovered and described in common sage (Salvia officinalis) by Yinrong Lu and L. Yeap Foo in 1999 (17).

[0028] Since then, sagelic acid has been identified in numerous plant species in the Lamiaceae family, including Melissa officinalis (18), Rosmarinus officinalis (19), Orthosiphon grandiflorus (20), Helicteres hirsuta (21), and Plectranthus amboinicus (15).

[0029] Sage and lemon balm are plants with numerous applications and properties, and these numerous effects are generally due to the presence of various molecules in the extracts.

[0030] The efficacy of sagelic acid as an inhibitor of COVID-19 was predicted by in silico simulations, but no physical evidence of this activity has been obtained (14).

[0031] Furthermore, in patent document 3 (15), a composition containing an extract of Plectranthus amboinicus is claimed as an anti-inflammatory drug, and sagelic acid is cited as one of the possible active ingredients.

[0032] The potential anti-leishmanial and anti-inflammatory activities of sagelic acid have been described ( 16 ). [Prior art documents] [Patent documents]

[0033] [Patent Document 1] U.S. Patent No. 5,853,703 [Patent Document 2] International Publication No. 2011042890 [Patent Document 3] U.S. Patent No. 8,105,636 Summary of the Invention

[0034] The present invention is based on the serendipitous discovery of the effects of sagelic acid on four fundamental aspects of aging. During in vitro testing, it was surprisingly observed that a single molecule of sagelic acid possesses all four of the above properties.

[0035] The present invention is applicable to the prevention and treatment of age-related disorders.

[0036] Aging-related disorders include, but are not limited to: Connective tissue diseases that affect cartilage, such as the non-inflammatory component of osteoarthritis and rheumatoid arthritis (degradation of connective tissue rather than an immune response causing inflammation), Diseases of the vascular connective tissue: stroke, vascular nephritis and retinitis, macular degeneration, atherosclerotic disease, Diseases directly associated with protein glycation, such as cataracts, Alzheimer's disease, skin healing disorders and presbycusis, Aesthetic skin disorders such as lipofuscinosis, pigmented spots, erythema and loss of skin elasticity.

[0037] Sagelic acid can be applied in pure form or as an extract in combination with suitable excipients for oral, parenteral, topical, rectal or inhaled use.

[0038] Therefore, the present invention proposes sagelic acid for use in the prevention and / or treatment of age-related disorders in humans or animals.

[0039] In a first embodiment, these disorders are due to oxidative stress.

[0040] In a second embodiment, the disorders are due to protein glycation.

[0041] In a third embodiment, the disorders are due to alterations in DNA.

[0042] In a fourth embodiment, the disorders result from alterations in proteins of arterial connective tissue.

[0043] The present invention also provides sagelic acid for use as an antioxidant and / or free radical scavenger.

[0044] The present invention also provides sagelic acid for use as a protein deglycating agent.

[0045] The present invention also provides sagelic acid for use as a DNA protecting agent.

[0046] The present invention also proposes sagelic acid for use as a protease inhibitor.

[0047] In a preferred embodiment, the present invention proposes sagelic acid to be used for the simultaneous prevention and / or treatment of disorders caused by oxidative stress, and disorders caused by protein glycation, and disorders caused by DNA alterations, and disorders caused by alterations of proteins in arterial connective tissue in humans or animals.

[0048] Another subject of the present invention is a composition comprising sagelic acid for use in the prevention and / or treatment of age-related disorders in humans or animals.

[0049] In a first embodiment of the composition of the present invention, the damage is caused by oxidative stress.

[0050] In a second embodiment of the composition of the invention, the disorder results from protein glycation.

[0051] In a third embodiment of the composition of the invention, the disorder is due to an alteration in DNA.

[0052] In a fourth embodiment of the composition of the invention, the disorder results from alterations in proteins of arterial connective tissue.

[0053] A preferred subject of the present invention is a composition comprising sagelic acid for use in the simultaneous prevention and / or treatment of disorders caused by oxidative stress, disorders caused by protein glycation, disorders caused by DNA alterations, and disorders caused by alterations of proteins in arterial connective tissue in humans or animals.

[0054] The invention will be better understood and its features and advantages will become apparent on reading the following description and examples of use. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0055] Example 1: Preparation of purified sagelic acid Soak 170 g of dried lemon balm (Melissa officinalis) leaves in 2 L of deionized water at room temperature with constant stirring for 24 hours.

[0056] The extract solution was separated by filtering through filter paper and then freeze-dried. The weight of the extract residue was 32.3 g, giving a yield of 19%.

[0057] 10 g of the freeze-dried extract is dissolved in 50 mL of deionized water and the solution is subjected to chromatography on a 45 mm diameter, 24 cm long column packed with approximately 200 g of Daiaon HP20 resin (Thermo Scientific reference number 046488.A1) using 2 L of deionized water as the eluent.

[0058] 2 L of eluate are removed from the column. Elution is then carried out with 2 L of methanol (Thermo Scientific Reference Number L13255). The entire eluate is collected and evaporated under reduced pressure until the methanol has completely evaporated. The remainder is made up to 100 mL of aqueous phase with deionized water, 10 mL of concentrated hydrochloric acid (Honeywell Fluka Reference Number 320331) is added, and three countercurrent extractions are carried out with 100 ml of ethyl acetate (Roth Reference Number 73361).

[0059] The organic phase is dried over anhydrous sodium sulfate (Sigma Aldrich, ref. 238597), filtered and evaporated to dryness to obtain approximately 700 mg of partially purified sagelic acid.

[0060] 700 mg of crude sagelic acid was then chromatographed in batches of 100 mg dissolved in 1 mL of deionized water by preparative HPLC on a 22 mm diameter, 250 mm long C18 column (Vydac Denali 10 μm) using the following gradient: Flow rate: 20 mL per minute Solvent A: Deionized water containing 0.1% formic acid (Fischer Chemical A117-50) Solvent B: Methanol (Thermo Scientific Reference Number L13255)

[0061] TIFF2025531314000001.tif31170

[0062] 10 ml fractions are collected.

[0063] Fractions containing sagelic acid are identified by their UV spectrum (maximum). Fractions containing sagelic acid are retained and other fractions are removed.

[0064] After 700 mg had passed through the column, the fractions were combined and evaporated to dryness under reduced pressure to give 165 mg of residue.

[0065] The remaining 165 mg was then subjected to chromatography in the same manner as above, resulting in 23 mg of sagelic acid with a purity of 92%.

[0066] Example 2: Preparation of lemon balm extract enriched with sagelic acid Soak 170 g of dried lemon balm leaves in 2 L of deionized water at room temperature with constant stirring for 24 hours.

[0067] The extract solution was separated by filtering through filter paper and then freeze-dried. The weight of the extract residue was 32.3 g, giving a yield of 19%.

[0068] 10 g of the freeze-dried extract was dissolved in 50 mL of deionized water and subjected to chromatography on a 45 mm diameter, 24 cm long column packed with approximately 200 g of Daiaon HP20 resin (Thermo Scientific reference number 046488.A1) using 2 L of deionized water as the eluent.

[0069] 2 L of eluate are removed from the column. Elution is then carried out with 2 L of methanol (Thermo Scientific Reference Number L13255). The entire eluate is collected and evaporated under reduced pressure until the methanol has completely evaporated. The remainder is made up to 100 mL of aqueous phase with deionized water, 10 mL of concentrated hydrochloric acid (Honeywell Fluka Reference Number 320331) is added, and three countercurrent extractions are carried out with 100 ml of ethyl acetate (Roth Reference Number 73361).

[0070] The organic phase is dried over anhydrous sodium sulfate (Sigma Aldrich, ref. 238597), filtered and evaporated to dryness. Approximately 700 mg of lemon balm extract containing 8% sagelic acid is obtained.

[0071] Example 3: Evaluation of the effects of sagelic acid and ascorbic acid on oxidative stress 3.1. Principle The principle of the method for assessing the effect of sagelic acid in comparison with the reference molecule ascorbic acid is based on the scavenging of the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH Sigma Aldrich Ref. D9132).

[0072] 3.2. Equipment and Materials Jasco V-730 Dual Beam UV-Visible Spectrophotometer Stopwatch Standard Laboratory Equipment Precision scale: ML104 (Mettler Toledo) Automatic variable volume pipette.

[0073] Reagents Monopotassium phosphate (Riedel de Haen, ref. 04243) Sodium hydrogen phosphate dihydrate (Sigma Aldrich reference number 30435) purified water DPPH (Sigma Aldrich, reference number D9132) Methanol Optima™ LC / MS grade (Fisher Chemical, ref. A456-212) Toluene, Reference (Carl Roth Reference No. 4445.1) Pharmethyl 96% Ethyl Alcohol (Cristalco Reference Number 11371014)

[0074] 3.4. Preparation of reagent solutions: Phosphate buffer, pH 7 Dissolve 0.177 g of potassium phosphate monobasic and 0.730 g of sodium hydrogen phosphate dihydrate in 50 ml of purified water. Adjust the pH if necessary. Solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) in methanol A solution of exactly 0.0027% (weight / volume) DPPH in methanol is made. Antioxidant Solution A solution of ascorbic acid (Riedel-de-Haen, reference number 33034) is made at exactly 0.1% (weight / volume) in ethyl alcohol. This solution is diluted to 0.0007% (volume / volume) in ethyl alcohol. Make a solution of exactly 0.1% (weight / volume) sagelic acid (prepared according to Example 5.1) in water. Dilute this solution to 0.0007% (volume / volume) in water.

[0075] 3.5.Operation Mode 3.5.1. Control Solution: Place the following into a series of sealed pillboxes:

[0076] TIFF2025531314000002.tif32170

[0077] Testing: A series of sealed pillboxes contain:

[0078] TIFF2025531314000003.tif39170

[0079] Regarding samples and blanks: After addition, stir on a magnetic stirrer for 5 minutes. Add 4 ml of toluene Stir for 30 seconds using a magnetic stirrer. Let stand for 20 seconds to 1 minute (if necessary) Using an automatic pipette, collect the organic phase from the top, being careful not to collect any of the aqueous phase. The OD of the organic phase is read at 519 nm using a spectrophotometer

[0080] 3.6.Results

[0081] TIFF2025531314000004.tif24170

[0082] These results indicate that sagelic acid is almost twice as effective as ascorbic acid in scavenging free radicals and therefore in preventing and / or treating damage caused by oxidative stress.

[0083] Example 4: Evaluation of the effects of sagelic acid and alagebrium as protein deglycating agents 4.1. Principle A 0.25% (wt / vol) solution of the test product was placed in contact with a 0.375% (wt / vol) solution of 1-phenyl-1,2-propanedione (PPD) for 24 hours at 37° C. HPLC dosing at 229 nm after passing through an Ascentis™ Express C18 column was performed at time TO and again after 24 hours to determine the benzoic acid content.

[0084] The activity of the deglycosylating agent is determined by calculating the percentage μmol benzoic acid / μmol PPD in the reaction medium.

[0085] 4.2. Equipment and Materials Ultimate 3000 HPLC system (Thermo Scientific - Dionex) equipped with a diode array detector and column dryer. Ascentis™ Express C18 HPLC column (Supelco reference number 53816-U) Dimensions: 15cm x 3.0mm Particle size: 2.7μm BD 53 / E2 Dryer (Binder) PVDF (polyvinyl fluoride) filter with a pore size of 0.45 μm (Millex™-HV, reference number SLHVX13NL) Standard Laboratory Equipment Precision scale: ML104 (Mettler Toledo) Automatic variable volume pipette

[0086] Reagents Water suitable for UHPLC-UV Optima™ LC / MS (Fisher, ref. W6-212) Acetonitrile Optima™ LC / MS grade (Fisher Chemica, ref. A955-212) Methanol Optima™ LC / MS grade (Fisher Chemical, ref. A456-212) Pharmethyl 96% Ethyl Alcohol (Cristalco, Reference Number 11371014) Sodium acetate trihydrate (Sigma Aldrich reference number S8625) Acetic acid (Carl Roth reference number HN55.1) Benzoic acid, reference 242381 (Sigma Aldrich reference number 242381) Monopotassium phosphate (Riedel de Haen, ref. 04243) Sodium hydrogen phosphate dihydrate (Sigma Aldrich reference number 30435) 1-Phenyl-1,2-propanedione (PPD) (Aldrich Reference No. 223034)

[0087] 4.4. Preparation of reagent solutions Phosphate buffer, pH 7.4 Dissolve 0.178 g of potassium phosphate monobasic and 0.955 g of sodium hydrogen phosphate dihydrate in 100 ml of purified water. Adjust the pH if necessary. Phosphate buffer, pH 7.4-methanol Prepare a mixture of 50 ml of phosphate buffer, pH 7.4, and 50 ml of methanol. 1-phenyl-1,2-propanedione (PPD) solution A solution of exactly 0.375% (weight / volume) PPD is made in a mixture of phosphate buffer, pH 7.4-methanol (50 / 50).

[0088] 4.5. Preparation of solutions of the products to be tested A solution of approximately 0.25% (weight / volume) of the product to be tested is made in a phosphate buffer, pH 7.4-methanol mixture (50 / 50).

[0089] Products tested: Alagebrium chloride (MedChem Express, reference number HY-106024B), sagelic acid (prepared according to Example 5.1).

[0090] 4.6. Benzoic Acid Calibration Range 4.6.1. Standard solutions Make a stock solution of approximately 0.08% (weight / volume) benzoic acid in ethyl alcohol. Dilute this solution 1:10 (volume / volume) in purified water (D).

[0091] (D) is then diluted 1:5, 2:5, 3:5 and 4:5 (volume / volume) in purified water (D1, D2, D3 and D4).

[0092] D1, D2, D3, D4 and D are passed through a PVDF filter with a pore size of 0.45 μm.

[0093] 4.6.2 Mobile phase A: Sodium acetate buffer: Weigh out 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. B: Acetonitrile

[0094] 4.6.3 Analysis conditions Column: Ascentis™ Express C18 15 cm x 3.0 mm 2.7 μm Flow rate: 0.5 ml per minute at 25°C Mode: Gradient (see Table 1) Detection: 229nm ultraviolet (UV) Injection: 10 μl for solutions D1, D2, D3, D4 and D.

[0095] TIFF2025531314000005.tif45170

[0096] A calibration line was established: benzoic acid peak area as a function of concentration, in % (weight / volume), observed at 229 nm with a retention time of 9.17 min.

[0097] 4.7. Action on PPD Operation Mode: A series of sealed pillboxes contain:

[0098] TIFF2025531314000006.tif32170

[0099] Stir after each addition.

[0100] At time T0, 0.05 ml was taken from each pillbox and the benzoic acid content was measured. The pillboxes were resealed and placed in a desiccator at 37°C for a minimum of 21 hours and a maximum of 24 hours.

[0101] Upon removal from the oven, an equal volume is taken for measurement.

[0102] The time at 37°C must be exactly the same for the control and all test articles, and therefore the control and all test articles are prepared as they go along.

[0103] HPLC measurements for benzoic acid are also performed on an ongoing basis.

[0104] 5.4.7.2. HPLC determination of released benzoic acid: Dilute 0.05 ml of sample 1:5 (volume / volume) in purified water, i.e. add 0.2 ml.

[0105] The dilutions are passed through a PVDF filter with a pore size of 0.45 μm before injection.

[0106] 5.4.7.2.1.Analysis conditions: Column: Ascentis™ Express C18 15 cm x 3.0 mm 2.7 μm Flow rate: 0.5 ml per minute at 25°C Mode: Gradient (see Table 2) Detection: 229nm ultraviolet (UV) Injection: 10μl

[0107] TIFF2025531314000007.tif59170

[0108] 4.8 Results Using the calibration line, calculate the benzoic acid content in g per 100 ml of reaction medium for: PPD control at TO and 24 hours: T T0 and T 24h Test items at TO and 24 hours: E T0 and E 24h

[0109] Convert these concentrations to μmol of benzoic acid per ml of reaction medium (PM benzoic acid = 122.12) T T0 ×10000 / 122.12

[0110] The concentration of PPD in μmol per ml of reaction medium is: 0.375×1000000 / 100×2×148.16=12.655(PM PPD=148.16)

[0111] Calculate the % of μmol of benzoic acid / μmol of PPD in the reaction medium: T T0 x10000 x 100 / 122.12 x 12.655

[0112] Values ​​obtained at different times for the control and trial are compared.

[0113] The deglycosylation activity of sagelic acid was compared with that of the reference molecule, Alagebrium .

[0114] TIFF2025531314000008.tif24170

[0115] These results indicate that sagelic acid has excellent activity in scavenging free radicals as well as good protein deglycation activity.

[0116] Example 5: Evaluation of the effects of sagelic acid and resveratrol on sirtuin-1 5.1. Principle The SIRT1 inhibitor / activator screening kit involves deacetylating a substrate with SIRT1, followed by cleavage of the deacetylated substrate to release a fluorescent group, which is detected by fluorimetry at Ex / Em = 400 / 505 nm.

[0117] SIRT1 activators enhance SIRT1 activity, resulting in a higher fluorescent signal compared to the control. This kit provides a rapid, simple, sensitive, and reliable test suitable for high-throughput screening of SIRT1 activators.

[0118] 5.2. Equipment and Materials Gemini EM Spectrofluorometer Stopwatch Standard Laboratory Equipment Precision scale: ML104 (Mettler Toledo) Automatic variable volume pipette Innovens 28EU1 dryer, Jouan / Thermo

[0119] 5.3 Reagents Kit solution: Reference ab283377 (Abcam) buffer solution substrate SIRT1 NAD DTT DMSO Reference 34869 (Honeywell Riedel-de-Haen)

[0120] 5.4. Preparation of reagent solutions SIRT1 enzyme: Store at -80°C Substrate: Store at -20°C. Avoid repeated freeze / thaw cycles. Use a fresh aliquot each time. Mix 1 μl of substrate with 9 μl of DMSO. NAD: Store at -80°C. Avoid repeated freezing / thawing. Mix 2 μl of NAD stock solution with 58 μl of buffer without DTT. 1M DTT: Store at -20°C. Thaw and keep on ice during use. Buffer: Store at 4°C or -20°C. Warm to 37°C. Must be prepared immediately before use. Preparation of DTT buffer solution: Mix 0.002 ml of DTT with 0.998 ml of buffer solution. Revealing: Store at -20°C. Avoid repeated freeze / thaw cycles. Keep on ice during use.

[0121] 5.5. Preparation of the solution of the product to be tested (activator) A solution of exactly 2.28% (weight / volume) of activators (resveratrol (Sigma Aldrich, ref. R5010) and sagelic acid prepared according to Example 5.1.) is made in DMSO. Dilute 1:250 in DTT buffer solution.

[0122] 5.6.Operation Mode Preparation of enzyme solution: To three separate wells make the following additions:

[0123] TIFF2025531314000009.tif17170

[0124] Preparation of screening compounds, inhibitor controls, enzyme controls and blank controls: 1. Add SIRT1 enzyme solution to wells as follows: 25 μl of undiluted activator solution (2.28% solution) is added to one well as an activator control (AC). 25 μl of buffer without DTT is added to one well as an enzyme control (EC). 25 μl of the diluted activator solution is added as assay (S).

[0125] 2. To another well, add 50 μl of buffer without DTT as a blank control (no enzyme).

[0126] 3. Mix the wells and incubate the plaques at 37°C for 5 minutes.

[0127] Substrate preparation: After incubation, add the following to each well:

[0128] TIFF2025531314000010.tif32170

[0129] Mix and incubate at 37°C for 30-60 minutes.

[0130] Developing: Add 10 μl of revealing to each well. Mix wells and incubate for 10 minutes at 37°C protected from light.

[0131] Evaluation criteria: Read the fluorescence (Ex / Em=400 / 505 nm).

[0132] 5.7.Results The activation of sirtuin-1 by sagelic acid was compared to the activation of sirtuin-1 by the reference molecule, resveratrol.

[0133] TIFF2025531314000011.tif24170

[0134] These results indicate that sagelic acid has a good effect on sirtuin activation, i.e., a good effect in preventing and / or treating DNA alterations, as well as good protein deglycation activity and excellent free radical scavenging activity.

[0135] Example 6: Evaluation of the effects of sagelic acid and witch hazel as protectors of connective tissue proteins 6.1. Principle A collagenase activity assay kit (Sigma Aldrich MAK293) was used, which allows screening of collagenase inhibitors by measuring collagenase activity using a synthetic peptide (FALGPA) that mimics the structure of collagen.

[0136] 6.2. Equipment and Materials Jasco V-730 Dual Beam UV-Visible Spectrophotometer Stopwatch Standard Laboratory Equipment Precision scale: ML104 (Mettler Toledo) Automatic variable volume pipette Innovens 28EU1 dryer, Jouan / Thermo

[0137] Reagents Kit solution: Reference MAK293-1KT (Sigma Aldrich) Collagenase buffer (MAK293A-KC) Collagenase solution (MAK293B-KC) Collagenase substrate (MAK293C-KC) Pharmethyl 96% Ethyl Alcohol, Reference 11371014 (Cristalco) purified water

[0138] 6.4. Preparation of solutions of the products (inhibitors) to be tested A solution of exactly 1% (weight / volume) hamamelitannin (Extrasynthesis reference number 0958) in ethyl alcohol is made.

[0139] A solution of exactly 1% (weight / volume) sagelic acid (prepared according to Example 5.1) is made in purified water.

[0140] 6.5.Operation Mode 6.5.1. Control solution A series of sealed pillboxes contain:

[0141] TIFF2025531314000012.tif39170

[0142] Stir manually immediately after addition. Place in a desiccator and leave at 37°C for 30 minutes. Add 1.6 mL of purified water. Mix the wells. Read DO: sample against blank by spectrophotometer at 345 nm.

[0143] 6.5.2.Results Collagenase activity The anti-collagenase activity of sagelic acid was compared with that of the reference molecule hamamelitannin. Absolute value: Y=[(DO ブランク2 -DO 試験物 )-(DO ブランク1 -DO 対照 )] / (DO ブランク1 -DO 対照 )] x 100

[0144] TIFF2025531314000013.tif25170

[0145] These results indicate that sagelic acid has an inhibitory effect on proteases, such as collagenase, that is more than twice as strong as that of the reference molecule hamamelitannin.

[0146] This protease inhibitory effect is in addition to the excellent free radical scavenging activity, the good activity against sirtuin activation, i.e., the good effect in preventing and / or treating DNA alterations, and the good protein deglycosylation activity.

[0147] Therefore, sagelic acid has excellent activity in the prevention and / or treatment of age-related disorders in humans and animals, since it simultaneously exerts effects on four fundamental aspects of ageing: action against oxidative stress due to its excellent antiradical activity, action against protein glycation due to its ability to reverse existing glycation in proteins, DNA protection due to its ability to activate sirtuins, and protection of connective tissue due to its excellent inhibition of proteases that degrade connective tissue, particularly elastase and collagenase.

[0148] The application of sagelic acid to the treatment of the four major axes of aging is based on dosages determined relative to the dosages required when the four reference molecules used are administered together.

[0149] Recommended daily oral doses are between 200 mg and 1 g per day for ascorbic acid (24), between 250 mg and 1 g per day for resveratrol (25), between 700 mg and 2 g per day for hamamelitannin (26 and 27), and between 100 mg and 300 mg per day for alagabrium (28).

[0150] A daily oral dose of sagelic acid between 10 mg and 2 g per day provides the recommended amounts of ascorbic acid, resveratrol, hamamelitannin, and alagebrium.

[0151] One advantage of the present invention is that a single dose of sagelic acid is equivalent to separate doses of ascorbic acid, resveratrol, alagebrium and hamamelitannin.

[0152] Example 7. Preparation of Melissa officinalis extract capsules enriched with sagelic acid 1 kg of Melissa officinalis dried leaf extract obtained according to Example 2 was thoroughly mixed with 10 g of sodium carbonate (E500) in a knife grinder.

[0153] This mixture is then used to fill No. 00 capsules (0.9 mL), each containing 550 mg of extract, which is equivalent to 44 mg of sagelic acid.

[0154] Example 8. Herbal formulation of a gel adapted for use on the face and body to treat aesthetic disorders Purified water: 91.69% Carbomer (Carbopol 980, Lubrizol): 2% Melissa officinalis dried leaf extract as described in Example 1: 5% Benzyl alcohol (Geogard 221, Lonza): 0.87% Dehydroacetic acid (Geogard 221, Lonza): 0.09% Sodium hydroxide (Sigma-Aldrich): 0.35%

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Claims

1. A composition comprising sagelic acid, antioxidants and / or free radical scavengers, protein deglycosylating agents, DNA protecting agents, and protease inhibitors, A composition for preventing and / or treating aesthetic skin disorders, which is used as a

2. A composition comprising sagelic acid for use as an elastase and / or collagenase inhibitor for the prevention and / or treatment of aesthetic skin disorders.

3. 3. The composition according to claim 1, wherein the aesthetic skin disorders and changes are selected from lipofuscinosis, pigmented spots, erythema and loss of skin elasticity.

4. 3. The composition according to claim 1, wherein the composition is applied topically.

5. A medicament comprising sagelic acid for the prevention and / or treatment of age-related diseases caused by alterations in DNA and / or alterations in proteins of connective tissue in humans or animals.

6. The pharmaceutical composition according to claim 5, characterized in that the disease is further caused by oxidative stress and / or protein glycation.

7. The pharmaceutical composition according to claim 5 or 6, for the prevention and / or treatment of aging-related disorders.

8. connective tissue diseases that affect cartilage, diseases of the vascular connective tissue, Diseases directly linked to protein glycation, The pharmaceutical composition according to claim 7, for the prevention and / or treatment of:

9. the connective tissue disease affecting cartilage is selected from osteoarthritis and the non-inflammatory component of rheumatoid arthritis; The disease of vascular connective tissue is selected from vascular disorders, stroke, vascular nephritis and retinitis, macular degeneration, atherosclerotic disease, and The disease directly associated with protein glycation is selected from cataracts, Alzheimer's disease, skin healing disorders, and presbycusis. The pharmaceutical composition according to claim 8.

10. 6. The medicament according to claim 5, characterized in that it is used for administration orally, parenterally, topically, rectally or by inhalation.