WO2025133675A1

WO2025133675A1 - Method for producing nasal formulation comprising aescin and rutin

Info

Publication number: WO2025133675A1
Application number: PCT/IB2023/062974
Authority: WO
Inventors: Seyed Mohammad MOUSAVI MIRZAEI; Zahra KIANI; Mojgan TAVAKOLI; Mohammad Shafiei
Original assignee: Melikoglu Ahmet
Current assignee: Melikoglu Ahmet
Priority date: 2023-12-20
Filing date: 2023-12-20
Publication date: 2025-06-26
Anticipated expiration: 2026-06-20
Also published as: EP4611763A1

Abstract

A method for producing a nasal formulation comprising aescin with a final concentration between 10 g/l and 30 g/l and rutin with a final concentration between 4.5 g/l and 5.5 g/l. the method may comprise forming a first mixture comprising propylene glycol and aescin; shaking the first mixture; leaving the shaken first mixture; forming a second mixture comprising propylene glycol and rutin; shaking the second mixture; leaving the shaken second mixture; forming a third mixture comprising the first and the second mixture; adding a preservative to the third mixture; adding doubled-distilled water to the third mixture to form an aqueous solution; shaking the aqueous solution; adjusting the pH level of the shaken aqueous solution between 6-8; and filtering the aqueous solution using a 0.45µm syringe filter.

Description

METHOD FOR PRODUCING NASAL FORMULATION COMPRISING AESCIN

AND RUTIN

TECHNICAL FIELD

[0001] The present disclosure is generally related to an exemplary method for producing nasal formulation comprising aescin and rutin.

BACKGROUND

[0002] The most prevalent sleep-related breathing illness, sleep apnea, has a number of adverse effects, including higher healthcare expenditures, metabolic and cardiovascular problems, accidents in vehicles, and a higher chance of mortality. Obstructive Sleep Apnea (OSA), the most prevalent type of sleep apnea, happens when the muscles in the throat loosen and temporarily block the airway while you are sleeping. According to estimates, 10% of postmenopausal women and 20% of adult males have moderate-to- severe obstructive OSA.

[0003] Different symptoms, polysomnographic patterns, anthropometric inclinations, comorbidities, and long-term results can all be used to diagnose OSA. Snoring, which may be brought on by swelling and inflammation of the upper respiratory tract walls as you sleep, is the most common sign of OSA. The major treatment for OSA is positive airway pressure (PAP), which has been shown to reduce OSA symptoms and potentially improve cardiovascular health. However, PAP has limitations, including inconsistent or long-term adherence, poor responsiveness in some populations, and no proof that it increases survival rates. Therefore, it is necessary to create alternative therapies based on substances from nature that have been shown to be secure, reliable, and efficient in regaining normal health.

SUMMARY [0004] This summary is intended to provide an overview of the subject matter of the present disclosure, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. Its sole purpose is to present some concepts of one or more exemplary aspects in a simplified form as a prelude to the more detailed description that is presented later. The proper scope of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0005] One or more exemplary embodiments describe an exemplary method for producing an exemplary nasal formulation. In an exemplary embodiment, an exemplary nasal formulation may comprise aescin with a final concentration between 10 g/1 and 30 g/1 and rutin with a final concentration between 4.5 g/1 and 5.5 g/1. In an exemplary embodiment, an exemplary method for producing an exemplary nasal formulation may comprise: forming an exemplary first mixture comprising propylene glycol and aescin with a volume/weight ratio of 4:1 (propylene glycol: aescin); shaking an exemplary first mixture at a temperature level between 20 °C and 30 °C, with a speed range between 65 rpm and 75 rpm, and for a time duration between 15 minutes and 25 minutes; leaving an exemplary shaken first mixture at a temperature level between 20 °C and 30 °C, and for a time duration between 55 minutes and 65 minutes; forming an exemplary second mixture comprising propylene glycol and rutin with a volume/weight ratio of 4:1 (propylene glycol: rutin); shaking an exemplary second mixture at a temperature level between 20 °C and 30 °C, with a speed range between 65 rpm and 75 rpm, and for a time duration between 15 minutes and 25 minutes; leaving an exemplary shaken second mixture at a temperature level between 20 °C and 30 °C, and for a time duration between 55 minutes and 65 minutes; forming an exemplary third mixture comprising aescin and rutin; adding an exemplary preservative to an exemplary third mixture while stirring on a stirrer, at a temperature level of 25 °C, and with a speed of about 70 rpm; forming an exemplary aqueous solution comprising aescin and rutin; shaking an exemplary aqueous solution comprising aescin and rutin at a temperature level between 20 °C and 30 °C, with a speed range between 65 rpm and 75 rpm, and for a time duration between 15 minutes and 25 minutes; adjusting the pH level of an exemplary shaken aqueous solution between 6-8; and filtering an exemplary aqueous solution using a 0.45pm syringe filter.

[0006] In an exemplary embodiment, forming an exemplary third mixture may comprise mixing an exemplary shaken first mixture and an exemplary shaken second mixture while stirring on a stirrer, at temperature level of 25 °C, with a speed of about 70 rpm, and for a time duration of 20 minutes. In an exemplary embodiment, forming an exemplary aqueous solution may comprise adding doubled-distilled water to an exemplary third mixture with a volumetric ratio of 1:1 (third mixture: doubled distilled water). In an exemplary embodiment, an exemplary nasal formulation may comprise aescin with a final concentration of 25 g/1 and rutin with a final concentration of 5 g/1. In an exemplary embodiment, shaking an exemplary first mixture may comprise shaking an exemplary first mixture at a temperature level of 25 °C, with a speed of 70 rpm, and for a time duration of 20 minutes. In an exemplary embodiment, leaving an exemplary shaken first mixture may comprise leaving an exemplary shaken first mixture at a temperature level of 25 °C, and for a time duration of 1 hour. In an exemplary embodiment, shaking an exemplary second mixture may comprise shaking an exemplary second mixture at a temperature level of 25 °C, with a speed of about 70 rpm, and for a time duration of 20 minutes. In an exemplary embodiment, leaving an exemplary shaken second mixture may comprise leaving an exemplary shaken second mixture at a temperature level of 25 °C, and for a time duration of 60 minutes. In an exemplary embodiment, shaking an exemplary aqueous solution comprising aescin and rutin may comprise shaking an exemplary aqueous solution comprising aescin and rutin at a temperature level of 25 °C, with a speed of about 70 rpm, and for a time duration of 20 minutes. In an exemplary embodiment, an exemplary preservative may be selected from the group consisting of benzalkonium chloride, chlorobutanol, and parabens. In an exemplary embodiment, an exemplary preservative may comprise an exemplary double distilled water solution of benzalkonium chloride with a final concentration of 0.1 g/1.

[0007] This Summary may introduce a number of concepts in a simplified format; the concepts are further disclosed within the “Detailed Description” section. This Summary is not intended to configure essential/key features of the claimed subject matter, nor is intended to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The novel features which are believed to be characteristic of the present disclosure, as to its structure, organization, use and method of operation, together with further objectives and advantages thereof, will be better understood from the following drawings in which an exemplary embodiment will now be illustrated by way of example. It is expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the present disclosure. Exemplary embodiments will now be described by way of example in association with the accompanying drawings in which: [0009] FIG. 1 illustrates an exemplary flowchart of an exemplary method for producing an exemplary nasal formulation comprising aescin and rutin, consistent with one or more exemplary embodiments of the present disclosure;

[00010] FIG. 2 illustrates an exemplary flowchart of exemplary first method for producing an exemplary dried powder of aescin, consistent with one or more exemplary embodiments of the present disclosure;

[00011] FIG. 3 illustrates an exemplary flowchart of exemplary second method for producing an exemplary dried powder of aescin, consistent with one or more exemplary embodiments of the present disclosure; [00012] FIG. 4 illustrates an exemplary flowchart of exemplary third method for producing an exemplary dried powder of aescin, consistent with one or more exemplary embodiments of the present disclosure; and

[00013] FIG. 5 illustrates an exemplary flowchart of exemplary fourth method for producing an exemplary dried powder of aescin, consistent with one or more exemplary embodiments of the present disclosure.

DETAILED DESCRIPTION

[00014] In the following detailed description, numerous specific details are set forth by way of examples to provide a thorough understanding of the relevant teachings related to the exemplary embodiments. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and/or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[00015] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in one or more exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be plain to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein. [00016] Disclosed herein is an exemplary method for producing nasal formulation comprising aescin and rutin. In an exemplary embodiment, an exemplary nasal formulation may improve the symptoms of one or more exemplary sleep disorders including, but not limited to, sleep apnea and snoring. “Sleep apnea” may refer to a form of sleep disorders where shallow or irregular breathing during sleep or breathing pauses may happen. Obstructive sleep apnea (OSA) and central sleep apnea (CSA) are just a few examples of sleep apnea. “Nasal formulation” may refer to a formulation in exemplary dosage forms including, but not limited to solutions, suspensions, emulsions, sols, solids, gels, colloids, oily liquid, and/or non-aqueous (water-free) formulated for delivery of an exemplary active ingredient to nasal cells such as mucosa. In one or more exemplary embodiments, improving the symptoms of an exemplary sleep disorder may include: the prevention of an exemplary sleep disorder in a patient or subject who may be at risk of getting one; preventing the development of an exemplary sleep disorder; and/or resolving an exemplary sleep problem, i.e., causing the disorder's progression to slow and/or reverse. The terms "subject" and "patient" may refer to both people and other animals, notably mammals. Thus, an exemplary nasal formulation may have an indication for both human and animals. In an exemplary embodiment, an exemplary nasal formulation may comprise aescin with a final concentration between 10 g/1 and 30 g/1 by volume of an exemplary nasal formulation, and rutin with a final concentration between 4.5 g/1 and 5.5 g/1 by volume of an exemplary nasal formulation.

[00017] In an exemplary embodiment, an exemplary nasal formulation may further comprise an exemplary preservative selected from the group consisting of chlorobutanol, benzalkonium chloride, and parabens. In one or more exemplary embodiment, parabens may include, but are not limited to, ethyl paraben, butyl paraben, methyl paraben, propyl paraben, heptyl paraben, their sodium salts, and a combination thereof. In an exemplary embodiment, an exemplary nasal formulation may comprise an exemplary preservative with a final concentration between 0.1 g/1 and 0.15 g/1. In an exemplary embodiment, an exemplary nasal formulation may comprise benzalkonium chloride with a final concentration of about 0.1 g/1.

[00018] In an exemplary embodiment, an exemplary nasal formulation may be formulated as an exemplary nasal spray. In an exemplary embodiment, an exemplary nasal spray may be applied between 1 puff and 5 puffs every 24 hours a day in a patient with obstructive sleep apnea. In an exemplary embodiment, an exemplary nasal spray may be applied between 1 puff and 5 puffs every 12 hours a day in a patient with obstructive sleep apnea. In an exemplary embodiment, an exemplary nasal spray may be applied between 1 puff and 5 puffs every 8 hours a day in a patient with obstructive sleep apnea. In an exemplary embodiment, an exemplary nasal spray may be applied between 1 puff and 5 puffs every 6 hours a day in a patient with obstructive sleep apnea. In an exemplary embodiment, an exemplary nasal formulation may be formulated as an exemplary nasal drop. In an exemplary embodiment, an exemplary nasal drop may be applied between 1 drop and 5 drops every 24 hours a day in a patient with obstructive sleep apnea. In an exemplary embodiment, an exemplary nasal drop may be applied between 1 drop and 5 drops every 12 hours a day in a patient with obstructive sleep apnea. In an exemplary embodiment, an exemplary nasal drop may be applied between 1 drop and 5 drops every 8 hours a day in a patient with obstructive sleep apnea. In an exemplary embodiment, an exemplary nasal drop may be applied between 1 drop and 5 drops every 6 hours a day in a patient with obstructive sleep apnea.

[00019] “Rutin” or “rutoside” or “Quercetin” or “3-rutinoside” with a molecular formula C27H30O16 and a molecular weight of 610.5 g/mol is the glycoside that is combining the flavonol quercetin and the disaccharide rutinose. “Aescin” or “beta-Escin” with a molecular formula C55H86O24 and a molecular weight of 1131.3 g/mol is the main active component of the plant Aesculus hippocastanum (the horse chestnut), that has anti-inflammatory, vasoconstrictor and vasoprotective effects. [00020] Referring to the figures, FIG. 1 illustrates an exemplary flowchart of an exemplary method 100 for producing an exemplary nasal formulation comprising aescin and rutin, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, an exemplary method 100 may include: forming an exemplary first mixture comprising propylene glycol and aescin with a predetermined volume/weight ratio (step 102); shaking an exemplary first mixture at a predetermined temperature level, with a predetermined speed range, and for a predetermined time duration (step 104); leaving an exemplary shaken first mixture at a predetermined temperature level, and for a predetermined time duration (step 106); forming an exemplary second mixture comprising propylene glycol and rutin with a predetermined volume/weight ratio (step 108); shaking an exemplary second mixture at a predetermined temperature level, with a predetermined speed range, and for a predetermined time duration (step 110); leaving an exemplary shaken second mixture at a predetermined temperature level, and for a predetermined time duration (step 112); forming an exemplary third mixture comprising aescin with a predetermined concentration and rutin with a predetermined concentration (step 114); adding an exemplary preservative to an exemplary third mixture (step 116); forming an exemplary aqueous solution comprising aescin and rutin (step 118); shaking an exemplary aqueous solution comprising aescin and rutin at a predetermined temperature level, with a predetermined speed range, and for a predetermined time duration (step 120); adjusting a pH level of an exemplary shaken aqueous solution between 6 and 8 (step 122); and filtering an exemplary shaken aqueous solution (step 124).

[00021] In further detail with respect to step 102, step 102 may include forming an exemplary first mixture comprising propylene glycol and aescin with a predetermined volume/weight ratio. In an exemplary embodiment, forming an exemplary first mixture comprising propylene glycol and aescin with a predetermined volume/weight ratio may include forming an exemplary first mixture comprising propylene glycol and aescin with a predetermined volume/weight ratio of 4:1 (propylene glycol: aescin). In an exemplary embodiment, forming an exemplary first mixture comprising propylene glycol and aescin with a predetermined volume/weight ratio of 4:1 (propylene glycol: aescin) may include adding an exemplary dried powder of aescin (with a purity of 95 % (w/w)) (e.g., using spatula) to an exemplary liquid solvent comprising propylene glycol in an exemplary laboratory container including, but not limited to tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc. For example, forming an exemplary first mixture comprising propylene glycol and aescin with a predetermined volume/weight ratio may include adding a dried powder of aescin (with a purity of 95 % (w/w)) (e.g., using spatula) to an exemplary liquid solvent comprising propylene glycol with a volume/weight ratio of 4:1 (propylene glycol: aescin) in an exemplary laboratory container, such as a beaker. In an exemplary embodiment, the dried powder of aescin may be weighed by an exemplary laboratory scale.

[00022] In further detail with respect to step 104, step 104 may include shaking an exemplary first mixture at a predetermined temperature level, with a predetermined speed range, and for a predetermined time duration. In an exemplary embodiment, shaking an exemplary first mixture at a predetermined temperature level, with a predetermined speed range, and for a predetermined time duration may include shaking/mixing an exemplary first mixture at a predetermined temperature level between 20 °C and 30 °C, with a predetermined speed range between 65 rpm and 75 rpm, and for a predetermined time duration between 15 and 25 minutes. For example, shaking/mixing an exemplary first mixture may include shaking/mixing exemplary first mixture at a temperature level of 25°C, with a speed of 70 rpm, and for a time duration of 20 minutes using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00023] In further detail with respect to step 106, step 106 may include leaving an exemplary shaken first mixture at a predetermined temperature level, and for a predetermined time duration. In an exemplary embodiment, leaving an exemplary shaken first mixture at a predetermined temperature level, and for a predetermined time duration may include leaving an exemplary shaken first mixture at a predetermined temperature level between 20 °C and 25 °C and for a predetermined time duration between 55 minutes and 65 minutes. For example, leaving an exemplary shaken first mixture at a predetermined temperature level and for a predetermined time duration may include leaving an exemplary shaken first mixture at a temperature level of 20 °C, and for a time duration of 1 hour.

[00024] In further detail with respect to step 108, step 108 may include forming an exemplary second mixture comprising propylene glycol and rutin with a predetermined volume/weight ratio. In an exemplary embodiment, forming an exemplary second mixture comprising propylene glycol and rutin with a predetermined volume/weight ratio may include forming an exemplary second mixture comprising propylene glycol and rutin with a predetermined volume/weight ratio of 4:1 (propylene glycol: rutin). In an exemplary embodiment, forming an exemplary second mixture comprising propylene glycol and rutin with a predetermined volume/weight ratio of 4:1 (propylene glycol: rutin) may include adding an exemplary dried powder of rutin (with a purity of 80 % (w/w)) (e.g., using spatula) to an exemplary liquid solvent comprising propylene glycol in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc. For example, forming an exemplary second mixture comprising propylene glycol and rutin with a predetermined volume/weight ratio may include adding a dried powder of rutin (with a purity of 80 % (w/w)) (e.g., using spatula) to a liquid solvent comprising propylene glycol with a volume/weight ratio of 4:1 (propylene glycol: rutin) in an exemplary laboratory container, such as a beaker.

[00025] In further detail with respect to step 110, step 110 may include shaking an exemplary second mixture at a predetermined temperature level, with a predetermined speed range, and for a predetermined time duration. In an exemplary embodiment, shaking an exemplary second mixture at a predetermined temperature level, with a predetermined speed range, and for a predetermined time duration may include shaking an exemplary second mixture at a predetermined temperature level between 20 °C and 30 °C, with a predetermined speed range between 65 rpm and 75 rpm, and for a predetermined time duration between 15 and 25 minutes. For example, shaking an exemplary second mixture may include shaking/mixing an exemplary second mixture at a temperature level of 25°C, with a speed of 70 rpm, and for a time duration of 20 minutes using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00026] In further detail with respect to step 112, step 112 may include leaving an exemplary shaken second mixture at a predetermined temperature level, and for a predetermined time duration. In an exemplary embodiment, leaving an exemplary shaken second mixture at a predetermined temperature level, and for a predetermined time duration may include leaving an exemplary shaken second mixture at a predetermined temperature level between 20 °C and 25 °C and for a predetermined time duration between 55 minutes and 65 minutes. For example, leaving an exemplary shaken second mixture at a predetermined temperature level and for a predetermined time duration may include leaving an exemplary shaken second mixture at a temperature level of 20 °C, and for a time duration of 1 hour.

[00027] In further detail with respect to step 114, step 114 may include forming an exemplary third mixture comprising aescin with a predetermined concentration and rutin with a predetermined concentration. In an exemplary embodiment, forming an exemplary third mixture comprising aescin with a predetermined concentration and rutin with a predetermined concentration may include forming an exemplary third mixture comprising aescin with a predetermined concentration between 1 % (w/w) and 3 % (w/w) and rutin with a predetermined concentration between 0.45% (w/w) and 0.55 % (w/w). In an exemplary embodiment, forming an exemplary third mixture comprising aescin with a predetermined concentration between 1 % (w/w) and 3 % (w/w) and rutin with a predetermined concentration between 0.45 % (w/w) and 0.55 % (w/w) may include mixing an exemplary shaken first mixture and an exemplary shaken second mixture in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc. while stirring on a stirrer a temperature level between 20 °C and 30 °C, with a speed range between 65 rpm and 75 rpm, and for a time duration between 14 minutes and 25 minutes.

[00028] In further detail with respect to step 116, step 116 may include adding an exemplary preservative to an exemplary third mixture. In an exemplary embodiment, adding an exemplary preservative to an exemplary third mixture may include adding an exemplary preservative to an exemplary third mixture in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc. while stirring on a stirrer at temperature level between 20 °C and 30 °C, with a speed range between 60 rpm and 100 rpm. In an exemplary embodiment, an exemplary preservative may be selected from the group consisting of benzalkonium chloride, chlorobutanol, and parabens. In one or more exemplary embodiment, parabens may include, but are not limited to, ethyl paraben, methyl paraben, butyl paraben, propyl paraben, heptyl paraben, their sodium salts, and a combination thereof. In an exemplary embodiment, adding an exemplary preservative to an exemplary third mixture may include adding an exemplary preservative comprising an exemplary double distilled water solution of benzalkonium chloride to an exemplary third mixture with a volumetric ratio of 1:0.1 (an exemplary third mixture: an exemplary double-distilled water solution of benzalkonium chloride) in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc. while stirring on a stirrer a temperature level of 25 °C, with a speed of about 70 rpm. After adding an exemplary preservative to an exemplary third mixture, an exemplary third mixture may have benzalkonium chloride with a final concentration of 0.1 g/1.

[00029] In further detail with respect to step 118, step 118 may include forming an exemplary aqueous solution comprising aescin and rutin. In an exemplary embodiment, forming an exemplary aqueous solution comprising aescin and rutin may include adding doubled-distilled water to an exemplary third mixture with a volumetric ratio of 1 : 1 (an exemplary third mixture: doubled distilled water) in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc.

[00030] In further detail with respect to step 120, step 120 may include shaking an exemplary aqueous solution comprising aescin and rutin at a predetermined temperature level, with a predetermined speed range, and for a predetermined time duration. In an exemplary embodiment, shaking an exemplary aqueous solution comprising aescin and rutin at a predetermined temperature level, with a predetermined speed range, and for a predetermined time duration may include shaking an exemplary aqueous solution comprising aescin and rutin at a predetermined temperature level between 20 °C and 30 °C, with a predetermined speed range . For example, shaking an exemplary aqueous solution comprising aescin and rutin may include shaking an exemplary aqueous solution comprising aescin and rutin at a temperature level of 25°C, with a speed of 70 rpm, and for a time duration of 20 minutes using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00031] In further detail with respect to step 122, step 122 may include adjusting a pH level of an exemplary shaken aqueous solution between 6-8. In an exemplary embodiment, adjusting a pH level of an exemplary shaken aqueous solution between 6-8 may include adding hydrochloric acid or sodium hydroxide dropwise to an exemplary shaken aqueous solution in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc. while stirring on a stirrer a temperature level between 25 °C, and with a speed between 60 rpm and 100 rpm.

[00032] In further detail with respect to step 124, step 124 may include filtering an exemplary shaken aqueous solution. In an exemplary embodiment, filtering an exemplary shaken aqueous solution may include passing an exemplary shaken aqueous solution through an exemplary syringe filter with a pore size of 0.45pm.

[00033] Referring to the figures, FIG. 2 illustrates an exemplary flowchart of exemplary first method 200 for producing an exemplary dried powder of aescin, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, exemplary first method 200 may include: forming an exemplary dried powder of Aesculus hippocastanum seeds (step 202); forming an exemplary ethanolic mixture of Aesculus hippocastanum seeds with a concentration of 1000 g/1 (step 204); shaking an exemplary ethanolic mixture of Aesculus hippocastanum seeds (step 206); filtering an exemplary shaken ethanolic mixture of Aesculus hippocastanum seeds (step 208); forming an exemplary second ethanolic extract solution of Aesculus hippocastanum seeds (step 210); shaking an exemplary second ethanolic extract solution of Aesculus hippocastanum seeds (step 212); filtering an exemplary shaken second ethanolic extract solution of Aesculus hippocastanum seeds (step 214); forming an exemplary fourth ethanolic extract solution of Aesculus hippocastanum seeds (step 216); shaking an exemplary fourth ethanolic extract solution of Aesculus hippocastanum seeds (step 218); filtering an exemplary shaken fourth ethanolic extract solution of Aesculus hippocastanum seeds (step 220); forming an exemplary sixth ethanolic extract solution of Aesculus hippocastanum seeds (step 222); shaking an exemplary sixth ethanolic extract solution of Aesculus hippocastanum seeds (step 224); filtering an exemplary shaken sixth ethanolic extract solution of Aesculus hippocastanum seeds (step 226); forming an exemplary eighth ethanolic extract solution of Aesculus hippocastanum seeds (step 228); forming an exemplary final ethanolic extract solution of Aesculus hippocastanum seeds (step 230); forming an exemplary aescin extract solution (step 232); and concentrating an exemplary aescin extract solution (step 234).

[00034] In further detail with respect to step 202, step 202 may include forming an exemplary dried powder of Aesculus hippocastanum seeds. In an exemplary embodiment, forming an exemplary dried powder of Aesculus hippocastanum seeds may include grinding (e.g., using a pestle, grinding mill, etc.) dried Aesculus hippocastanum seeds in an exemplary laboratory plate (e.g., a pestle).

[00035] In further detail with respect to step 204, step 204 may include forming an exemplary ethanolic mixture of Aesculus hippocastanum seeds with a concentration of 1000 g/1. In an exemplary embodiment, forming an exemplary ethanolic mixture of Aesculus hippocastanum seeds with a concentration of 1000 g/1 may include adding an exemplary dried powder of Aesculus hippocastanum seeds (e.g., using spatula) to an ethanol solution (with a concentration of at least 50% (v/v)) in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc.

[00036] In further detail with respect to step 206, step 206 may include shaking an exemplary ethanolic mixture of Aesculus hippocastanum seeds. In an exemplary embodiment, shaking an exemplary ethanolic mixture of Aesculus hippocastanum seeds may include shaking/mixing an exemplary ethanolic mixture of Aesculus hippocastanum seeds at a temperature level between 20 °C and 30 °C, for a time duration between about 55 minutes and 65 minutes, and with a speed range between 65 rpm and 75 rpm, using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00037] In further detail with respect to step 208, step 208 may include filtering an exemplary shaken ethanolic mixture of Aesculus hippocastanum seeds. In an exemplary embodiment, filtering an exemplary shaken ethanolic mixture of Aesculus hippocastanum seeds may include passing an exemplary shaken ethanolic mixture of Aesculus hippocastanum seeds through an exemplary cellulose filter paper to form an exemplary first ethanolic extract solution of Aesculus hippocastanum seeds and an exemplary first residue of Aesculus hippocastanum seeds, separately.

[00038] In further detail with respect to step 210, step 210 may include forming an exemplary second ethanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, forming an exemplary second ethanolic extract solution of Aesculus hippocastanum seeds may include adding an exemplary first residue of Aesculus hippocastanum seeds to an ethanol solution (with a concentration of at least 50% (v/v)) in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc.

[00039] In further detail with respect to step 212, step 212 may include shaking an exemplary second ethanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, shaking an exemplary second ethanolic extract solution of Aesculus hippocastanum seeds may include shaking/mixing an exemplary second ethanolic extract solution of Aesculus hippocastanum seeds at a temperature level between 22 °C and 30 °C, for a time duration between about 55 minutes and 65 minutes, and with a speed range between 65 rpm and 75 rpm, using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00040] In further detail with respect to step 214, step 214 may include filtering an exemplary shaken second ethanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, filtering an exemplary shaken second ethanolic extract solution of Aesculus hippocastanum seeds may include passing an exemplary shaken second ethanolic extract solution of Aesculus hippocastanum seeds through an exemplary cellulose filter paper to form an exemplary third ethanolic extract solution of Aesculus hippocastanum seeds and an exemplary second residue of Aesculus hippocastanum seeds, separately.

[00041] In further detail with respect to step 216, step 216 may include forming an exemplary fourth ethanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, forming an exemplary fourth ethanolic extract solution of Aesculus hippocastanum seeds may include adding an exemplary second residue of Aesculus hippocastanum seeds to an ethanol solution (with a concentration of at least 50% (v/v)) in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc.

[00042] In further detail with respect to step 218, step 218 may include shaking an exemplary fourth ethanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, shaking an exemplary fourth ethanolic extract solution of Aesculus hippocastanum seeds may include shaking/mixing an exemplary fourth ethanolic extract solution Aesculus hippocastanum seeds at a temperature level between 22 °C and 30 °C, for a time duration between about 55 minutes and 65 minutes, and with a speed range between 65 rpm and 75 rpm, using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00043] In further detail with respect to step 220, step 220 may include filtering an exemplary shaken fourth ethanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, filtering an exemplary shaken fourth ethanolic extract solution of Aesculus hippocastanum seeds may include passing an exemplary shaken fourth ethanolic extract solution of Aesculus hippocastanum seeds through an exemplary cellulose filter paper to form an exemplary fifth ethanolic extract solution of Aesculus hippocastanum seeds and an exemplary third residue of Aesculus hippocastanum seeds, separately.

[00044] In further detail with respect to step 222, step 222 may include forming an exemplary sixth ethanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, forming an exemplary sixth ethanolic extract solution of Aesculus hippocastanum seeds may include adding an exemplary third residue of Aesculus hippocastanum seeds to an ethanol solution (with a concentration of at least 50% (v/v)) in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc.

[00045] In further detail with respect to step 224, step 224 may include shaking an exemplary sixth ethanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, shaking an exemplary sixth ethanolic extract solution of Aesculus hippocastanum seeds may include shaking/mixing an exemplary sixth ethanolic extract solution of Aesculus hippocastanum seeds at a temperature level between 22 °C and 30 °C, for a time duration between about 55 minutes and 65 minutes, and with a speed range between 65 rpm and 75 rpm, using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00046] In further detail with respect to step 226, step 226 may include filtering an exemplary shaken sixth ethanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, filtering an exemplary shaken sixth ethanolic extract solution of Aesculus hippocastanum seeds may include passing an exemplary shaken sixth ethanolic extract solution of Aesculus hippocastanum seeds through an exemplary cellulose filter paper to form an exemplary seventh ethanolic extract solution of Aesculus hippocastanum seeds and an exemplary fourth residue of Aesculus hippocastanum seeds, separately.

[00047] In further detail with respect to step 228, step 228 may include forming an exemplary eighth ethanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, forming an exemplary eighth ethanolic extract solution of Aesculus hippocastanum seeds may include adding an exemplary first, third, fifth, and seventh ethanolic extract solution of Aesculus hippocastanum seeds to an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc. while shaking/mixing at a temperature level between 22 °C and 30 °C, and for a time duration between about 10 minutes and 20 minutes, using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00048] In further detail with respect to step 230, step 230 may include forming an exemplary final ethanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, forming an exemplary final ethanolic extract solution of Aesculus hippocastanum seeds may include increasing the ethanol concentration of an exemplary eighth ethanolic mixture from 50% (v/v) to 65% (v/v) by adding ethanolic solution to an exemplary eighth ethanolic mixture in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc while shaking/mixing at a temperature level between 22 °C and 30 °C, for a time duration between about 10 minutes and 20 minutes, using a stirrer or agitation machine (e.g., a magnetic stirrer). [00049] In further detail with respect to step 232, step 232 may include forming an exemplary aescin extract solution. In an exemplary embodiment, forming an exemplary aescin extract solution may include adding an exemplary final ethanolic extract solution of Aesculus hippocastanum seeds to a cation exchange column with a height of 50 cm comprising cation resin (for about 0.66% volume of column) and ethanol (for about 1.3% volume of column).

[00050] In further detail with respect to step 234, step 234 may include concentrating an exemplary aescin extract solution. In an exemplary embodiment, concentrating an exemplary aescin extract solution may include evaporating ethanol of an exemplary aescin extract solution using vacuum rotary evaporator for a time duration between 2 hours and 6 hours. In an exemplary embodiment, evaporating ethanol of an exemplary aescin extract solution may result in forming an exemplary dried powder of aescin with a purity of 80 % (w/w).

[00051] Referring to the figures, FIG. 3 illustrates an exemplary flowchart of exemplary second method 300 for producing an exemplary dried powder of aescin, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, exemplary second method 300 may include: forming an exemplary dried powder of Aesculus hippocastanum seeds (step 302); forming an exemplary methanolic mixture of Aesculus hippocastanum seeds with a concentration of 125 g/1 (step 304); shaking an exemplary methanolic mixture of Aesculus hippocastanum seeds (step 306); filtering an exemplary shaken methanolic mixture (step 308); forming an exemplary second methanolic extract solution of Aesculus hippocastanum seeds (step 310); shaking an exemplary second methanolic extract solution of Aesculus hippocastanum seeds (step 312); filtering an exemplary shaken second methanolic extract solution of Aesculus hippocastanum seeds (step 314); forming an exemplary fourth methanolic extract solution of Aesculus hippocastanum seeds (step 316); shaking an exemplary fourth methanolic extract solution of Aesculus hippocastanum seeds (step 318); filtering an exemplary shaken fourth methanolic extract solution of Aesculus hippocastanum seeds (step 320); forming an exemplary sixth methanolic extract solution of Aesculus hippocastanum seeds (step 322); adjusting a pH level of an exemplary sixth methanolic extract solution of Aesculus hippocastanum seeds between 2 and 3 (step 324); shaking an exemplary sixth methanolic extract solution of Aesculus hippocastanum seeds (step 326); leaving an exemplary shaken sixth methanolic extract solution of Aesculus hippocastanum seeds at a predetermined temperature level, and for a predetermined time duration to produce an exemplary precipitate (step 328); forming an exemplary final methanolic extract solution of Aesculus hippocastanum seeds (step 330); shaking an exemplary final methanolic extract solution of Aesculus hippocastanum seeds (step 332); concentrating an exemplary shaken final methanolic extract solution of Aesculus hippocastanum seeds (step 334); and filtering an exemplary dried powder of aescin (step 336).

[00052] In further detail with respect to step 302, step 302 may include forming an exemplary dried powder of Aesculus hippocastanum seeds. In an exemplary embodiment, forming an exemplary dried powder of Aesculus hippocastanum seeds may include grinding (e.g., using a grinding mill, pestle, etc.) dried Aesculus hippocastanum seeds in an exemplary laboratory plate (e.g., a pestle).

[00053] In further detail with respect to step 304, step 304 may include forming an exemplary methanolic mixture of Aesculus hippocastanum seeds with a concentration of 125 g/1. In an exemplary embodiment, forming an exemplary methanolic mixture of Aesculus hippocastanum seeds with a concentration of 125 g/1 may include adding an exemplary dried powder of Aesculus hippocastanum seeds (e.g., using spatula) to a methanol solution (with a concentration of at least 10% (v/v)) in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc.

[00054] In further detail with respect to step 306, step 306 may include shaking an exemplary methanolic mixture of Aesculus hippocastanum seeds. In an exemplary embodiment, shaking an exemplary methanolic mixture of Aesculus hippocastanum seeds may include shaking/mixing an exemplary methanolic mixture at a temperature level between 22 °C and 30 °C, for a time duration between about 45 hours and 50 hours, and with a speed range between 65 rpm and 75 rpm, using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00055] In further detail with respect to step 308, step 308 may include filtering an exemplary shaken methanolic mixture. In an exemplary embodiment, filtering an exemplary shaken methanolic mixture may include passing an exemplary shaken methanolic mixture through an exemplary cellulose filter paper to form an exemplary first methanolic extract solution of Aesculus hippocastanum seeds and an exemplary first residue of Aesculus hippocastanum seeds, separately.

[00056] In further detail with respect to step 310, step 310 may include forming an exemplary second methanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, forming an exemplary second methanolic extract solution of Aesculus hippocastanum seeds may include adding an exemplary first residue of Aesculus hippocastanum seeds to a methanol solution (with a concentration of at least 10% (v/v)) in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc.

[00057] In further detail with respect to step 312, step 312 may include shaking an exemplary second methanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, shaking an exemplary second methanolic extract solution of Aesculus hippocastanum seeds may include shaking/mixing an exemplary second methanolic extract solution of Aesculus hippocastanum seeds at a temperature level between 22 °C and 30 °C, for a time duration between about 22 hours and 26 hours, and with a speed range between 65 rpm and 75 rpm, using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00058] In further detail with respect to step 314, step 314 may include filtering an exemplary shaken second methanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, filtering an exemplary shaken second methanolic extract solution of Aesculus hippocastanum seeds may include passing an exemplary shaken second methanolic extract solution of Aesculus hippocastanum seeds through an exemplary cellulose filter paper to form an exemplary third methanolic extract solution of Aesculus hippocastanum seeds and an exemplary second residue of Aesculus hippocastanum seeds, separately.

[00059] In further detail with respect to step 316, step 316 may include forming an exemplary fourth methanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, forming an exemplary fourth methanolic extract solution of Aesculus hippocastanum seeds may include adding an exemplary second residue of Aesculus hippocastanum seeds to a methanol solution (with a concentration of at least 10% (v/v)) in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc.

[00060] In further detail with respect to step 318, step 318 may include shaking an exemplary fourth methanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, shaking an exemplary fourth methanolic extract solution of Aesculus hippocastanum seeds may include shaking/mixing an exemplary fourth methanolic extract solution of Aesculus hippocastanum seeds at a temperature level between 22 °C and 30 °C, for a time duration between about 22 hours and 26 hours, and with a speed range between 65 rpm and 75 rpm, using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00061] In further detail with respect to step 320, step 320 may include filtering an exemplary shaken fourth methanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, filtering an exemplary shaken fourth methanolic extract solution of Aesculus hippocastanum seeds may include passing an exemplary shaken fourth methanolic extract solution of Aesculus hippocastanum seeds through an exemplary cellulose filter paper to form an exemplary fifth methanolic extract solution of Aesculus hippocastanum seeds and an exemplary third residue of Aesculus hippocastanum seeds, separately.

[00062] In further detail with respect to step 322, step 322 may include forming an exemplary sixth methanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, forming an exemplary sixth methanolic extract solution of Aesculus hippocastanum seeds may include adding an exemplary first, third, and fifth methanolic extract solution of Aesculus hippocastanum seeds to an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc. while shaking/mixing at a temperature level between 22°C and 30 °C, for a time duration between about 10 minutes and 20 minutes, using a stirrer or agitation machine (e.g., a magnetic stirrer). [00063] In further detail with respect to step 324, step 324 may include adjusting a pH level of an exemplary sixth methanolic extract solution of Aesculus hippocastanum seeds between 2 and 3. In an exemplary embodiment, adjusting a pH level of an exemplary sixth methanolic extract solution of Aesculus hippocastanum seeds between 2 and 3 may include adding sulfuric acid (with a concentration between 0.5 M and 4 M) dropwise to an exemplary sixth methanolic extract solution of Aesculus hippocastanum seeds in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc.

[00064] In further detail with respect to step 326, step 326 may include shaking an exemplary sixth methanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, shaking an exemplary sixth methanolic extract solution of Aesculus hippocastanum seeds may include shaking/mixing an exemplary sixth methanolic extract solution of Aesculus hippocastanum seeds at a temperature level between 22 °C and 30 °C, for a time duration between 50 minutes and 70 minutes, and with a speed range between 65 rpm and 75 rpm, using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00065] In further detail with respect to step 328, step 328 may include leaving an exemplary shaken sixth methanolic extract solution of Aesculus hippocastanum seeds at a predetermined temperature level and for a predetermined time duration to produce an exemplary precipitate. In an exemplary embodiment, leaving an exemplary shaken sixth methanolic extract solution of Aesculus hippocastanum seeds at a predetermined temperature level and for a predetermined time duration to produce an exemplary precipitate may include leaving an exemplary shaken sixth methanolic extract solution of Aesculus hippocastanum seeds at a predetermined temperature level between 20 °C and 25 °C, and for a predetermined time duration between 10 hours and 14 hours to produce an exemplary precipitate. [00066] In further detail with respect to step 330, step 330 may include forming an exemplary final methanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, forming an exemplary final methanolic extract solution of Aesculus hippocastanum seeds may include adding an exemplary precipitate to a methanol solution (with a concentration of at least 10% (v/v)) in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc.

[00067] In further detail with respect to step 332, step 332 may include shaking an exemplary final methanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, shaking an exemplary final methanolic extract solution of Aesculus hippocastanum seeds may include shaking/mixing an exemplary final methanolic extract solution of Aesculus hippocastanum seeds at a temperature level between 22 °C and 30 °C, for a time duration between about 15 minutes and 25 minutes, and with a speed range between 65 rpm and 75 rpm, using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00068] In further detail with respect to step 334, step 334 may include concentrating an exemplary shaken final methanolic extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, concentrating an exemplary shaken final methanolic extract solution of Aesculus hippocastanum seeds may include evaporating methanol of an exemplary shaken final methanolic extract solution of Aesculus hippocastanum seeds using vacuum rotary evaporator for a time duration between 30 minutes and 4 hours. In an exemplary embodiment, evaporating methanol of an exemplary shaken final methanolic extract solution of Aesculus hippocastanum seeds may result in forming an exemplary dried powder of aescin.

[00069] In further detail with respect to step 336, step 336 may include filtering an exemplary dried powder of aescin. In an exemplary embodiment, filtering an exemplary dried powder of aescin may include passing an exemplary dried powder of aescin through an exemplary solution of active charcoal followed by drying an exemplary powder of aescin in an exemplary oven with a temperature level between 20 °C and 25°C, that may result in producing an exemplary dried powder of aescin with a purity of 80 % (w/w).

[00070] Referring to the figures, FIG. 4 illustrates an exemplary flowchart of an exemplary third method 400 for producing an exemplary dried powder of aescin, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, exemplary method 400 may include: forming an exemplary dried powder of Aesculus hippocastanum seeds (step 402); forming an exemplary ethanolic mixture of Aesculus hippocastanum seeds with a concentration of 334 g/1 (step 404); shaking an exemplary ethanolic mixture of Aesculus hippocastanum seeds (step 406); filtering an exemplary shaken ethanolic mixture of Aesculus hippocastanum seeds (step 408); concentrating an exemplary ethanolic extract solution of Aesculus hippocastanum seeds (step 410); shaking an exemplary extract solution of Aesculus hippocastanum seeds (step 412); leaving an exemplary shaken extract solution of Aesculus hippocastanum seeds at a predetermined temperature level, and for a predetermined time duration (step 414); filtering twice an exemplary shaken extract solution of Aesculus hippocastanum seeds (step 416); concentrating an exemplary solution of aescin (step 418); freezing an exemplary mixture comprising an exemplary sediment (step 420); and freeze drying an exemplary frozen mixture (step 422).

[00071] In further detail with respect to step 402, step 402 may include forming an exemplary dried powder of Aesculus hippocastanum seeds. In an exemplary embodiment, forming an exemplary dried powder of Aesculus hippocastanum seeds may include grinding (e.g., using a grinding mill, pestle, etc.) dried Aesculus hippocastanum seeds in an exemplary laboratory plate (e.g., a pestle).

[00072] In further detail with respect to step 404, step 404 may include forming an exemplary ethanolic mixture of Aesculus hippocastanum seeds with a concentration of 334 g/1. In an exemplary embodiment, forming an exemplary ethanolic mixture of Aesculus hippocastanum seeds with a concentration of 334 g/1 may include adding an exemplary dried powder of Aesculus hippocastanum seeds to an ethanol solution (with a concentration of at least 50% (v/v)) in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc.

[00073] In further detail with respect to step 406, step 406 may include shaking an exemplary ethanolic mixture of Aesculus hippocastanum seeds. In an exemplary embodiment, shaking an exemplary ethanolic mixture of Aesculus hippocastanum seeds may include shaking/mixing an exemplary ethanolic mixture of Aesculus hippocastanum seeds at a temperature level between 22 °C and 30 °C, for a time duration between 46 hours and 50 hours, and with a speed range between 55 rpm and 65 rpm, using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00074] In further detail with respect to step 408, step 408 may include filtering an exemplary shaken ethanolic mixture of Aesculus hippocastanum seeds. In an exemplary embodiment, filtering an exemplary shaken ethanolic mixture of Aesculus hippocastanum seeds may include passing an exemplary shaken ethanolic mixture of Aesculus hippocastanum seeds through an exemplary cellulose filter paper to form an exemplary ethanolic extract solution of Aesculus hippocastanum seeds and an exemplary residue of Aesculus hippocastanum seeds, separately.

[00075] In further detail with respect to step 410, step 410 may include concentrating an exemplary ethanolic extract solution of Aesculus hippocastanum seeds and. In an exemplary embodiment, concentrating an exemplary ethanolic extract solution of Aesculus hippocastanum seeds may include evaporating ethanol of an exemplary ethanolic extract solution of Aesculus hippocastanum seeds using vacuum rotary evaporator for a time duration between 1 hours and 3 hours, that may result in producing an exemplary extract solution of Aesculus hippocastanum seeds.

[00076] In further detail with respect to step 412, step 412 may include shaking an exemplary extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, shaking an exemplary extract solution of Aesculus hippocastanum seeds may include shaking/mixing an exemplary extract solution of Aesculus hippocastanum seeds at a temperature level between 25 °C and 30 °C, for a time duration between about 15 minutes and 25 minutes, and with a speed range between 65 rpm and 75 rpm, using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00077] In further detail with respect to step 414, step 414 may include leaving an exemplary shaken extract solution of Aesculus hippocastanum seeds at a predetermined temperature level, and for a predetermined time duration. In an exemplary embodiment, leaving an exemplary shaken extract solution of Aesculus hippocastanum seeds at a predetermined temperature level, and for a predetermined time duration may include leaving an exemplary shaken extract solution of Aesculus hippocastanum seeds at a predetermined temperature level between 20 °C and 25 °C, and for a predetermined time duration between 50 minutes and 75 minutes.

[00078] In further detail with respect to step 416, step 416 may include filtering twice an exemplary shaken extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, filtering an exemplary shaken extract solution of Aesculus hippocastanum seeds may include passing an exemplary shaken extract solution of Aesculus hippocastanum seeds through an exemplary cellulose filter paper to form an exemplary solution of aescin.

[00079] In further detail with respect to step 418, step 418 may include concentrating an exemplary solution of aescin. In an exemplary embodiment, concentrating an exemplary solution of aescin may include evaporating ethanol of an exemplary solution of aescin using vacuum rotary evaporator for a time duration between 5 hours and 7 hours, at a temperature level between 35 °C and 38 °C, and with a speed range between 30 rpm and 35 rpm. In an exemplary embodiment, evaporating ethanol of an exemplary solution of aescin may result in producing an exemplary mixture comprising an exemplary sediment.

[00080] In further detail with respect to step 420, step 420 may include freezing an exemplary mixture comprising an exemplary sediment. In an exemplary embodiment, freezing an exemplary mixture comprising an exemplary sediment may include placing an exemplary mixture comprising an exemplary sediment inside a freezer chamber and producing an exemplary frozen mixture.

[00081] In further detail with respect to step 422, step 422 may include freeze drying an exemplary frozen mixture. In an exemplary embodiment, freeze drying an exemplary frozen mixture may include placing an exemplary frozen mixture inside a freeze-drying chamber of an exemplary freeze dryer; freezing an exemplary frozen mixture with liquid nitrogen (-196 °C); and equilibrated at a temperature level between about -44 °C and -48 °C for about 48 hours, that may result in producing an exemplary dried powder of aescin with a purity of 80 % (w/w).

[00082] Referring to the figures, FIG. 5 illustrates an exemplary flowchart of exemplary fourth method 500 for producing an exemplary dried powder of aescin, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, exemplary fourth method 500 may include: forming an exemplary dried powder of Aesculus hippocastanum seeds (step 502); forming an exemplary aqueous solution of Aesculus hippocastanum seeds with a concentration of 334 g/1 (step 504); shaking an exemplary aqueous solution of Aesculus hippocastanum seeds (step 506); filtering an exemplary shaken aqueous solution of Aesculus hippocastanum seeds (step 508); shaking an exemplary aqueous extract solution of Aesculus hippocastanum seeds (step 510); leaving an exemplary shaken aqueous extract solution of Aesculus hippocastanum seeds at a predetermined temperature level, and for a predetermined time duration (step 512); filtering twice an exemplary shaken aqueous extract solution of Aesculus hippocastanum seeds (step 514); concentrating an exemplary aqueous solution of aescin (step 516); freezing an exemplary mixture comprising an exemplary sediment (step 518); and freeze drying an exemplary frozen mixture (step 520).

[00083] In further detail with respect to step 502, step 502 may include forming an exemplary dried powder of Aesculus hippocastanum seeds. In an exemplary embodiment, forming an exemplary dried powder of Aesculus hippocastanum seeds may include grinding (e.g., using a grinding mill, pestle, etc.) dried Aesculus hippocastanum seeds in an exemplary laboratory plate (e.g., a pestle).

[00084] In further detail with respect to step 504, step 504 may include forming an exemplary aqueous solution of Aesculus hippocastanum seeds with a concentration of 334 g/1. In an exemplary embodiment, forming an exemplary aqueous solution of Aesculus hippocastanum seeds with a concentration of 334 g/1 may include adding an exemplary dried powder of Aesculus hippocastanum seeds to water in an exemplary laboratory container including, but not limited to, tins, beakers, flasks, buckets, bottles, bowls, basins, tubes vials, canisters, barrels, etc.

[00085] In further detail with respect to step 506, step 506 may include shaking an exemplary aqueous solution of Aesculus hippocastanum seeds. In an exemplary embodiment, shaking an exemplary aqueous solution of Aesculus hippocastanum seeds may include shaking/mixing an exemplary aqueous solution of Aesculus hippocastanum seeds at a temperature level between 22 °C and 30 °C, for a time duration between 46 hours and 50 hours, and with a speed range between 55 rpm and 65 rpm, using a stirrer or agitation machine (e.g., a magnetic stirrer). [00086] In further detail with respect to step 508, step 508 may include filtering an exemplary shaken aqueous solution of Aesculus hippocastanum seeds. In an exemplary embodiment, filtering an exemplary shaken aqueous solution of Aesculus hippocastanum seeds may include passing an exemplary shaken aqueous solution of Aesculus hippocastanum seeds through an exemplary cellulose filter paper to form an exemplary aqueous extract solution of Aesculus hippocastanum seeds and an exemplary residue of Aesculus hippocastanum seeds, separately.

[00087] In further detail with respect to step 510, step 510 may include shaking an exemplary aqueous extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, shaking an exemplary aqueous extract solution of Aesculus hippocastanum seeds may include shaking/mixing an exemplary aqueous extract solution of Aesculus hippocastanum seeds at a temperature level between 25 °C and 30 °C, for a time duration between about 15 minutes and 25 minutes, and with a speed range between 65 rpm and 75 rpm, using a stirrer or agitation machine (e.g., a magnetic stirrer).

[00088] In further detail with respect to step 512, step 512 may include leaving an exemplary shaken aqueous extract solution of Aesculus hippocastanum seeds at a predetermined temperature level, and for a predetermined time duration. In an exemplary embodiment, leaving an exemplary shaken aqueous extract solution of Aesculus hippocastanum seeds at a predetermined temperature level, and for a predetermined time duration may include leaving an exemplary shaken aqueous extract solution of Aesculus hippocastanum seeds at a predetermined temperature level between 20 °C and 25 °C, and for a predetermined time duration between 50 minutes and 75 minutes.

[00089] In further detail with respect to step 514, step 514 may include filtering twice an exemplary shaken aqueous extract solution of Aesculus hippocastanum seeds. In an exemplary embodiment, filtering twice an exemplary shaken aqueous extract solution of Aesculus hippocastanum seeds may include passing an exemplary shaken aqueous extract solution of Aesculus hippocastanum seeds through an exemplary cellulose filter paper to form an exemplary aqueous solution of aescin.

[00090] In further detail with respect to step 516, step 516 may include concentrating an exemplary aqueous solution of aescin. In an exemplary embodiment, concentrating an exemplary aqueous solution of aescin may include evaporating water of an exemplary aqueous solution of aescin using vacuum rotary evaporator for a time duration between 5 hours and 7 hours, at a temperature level between 35 °C and 38 °C, and with a speed range between 30 rpm and 35 rpm. In an exemplary embodiment, evaporating water of an exemplary aqueous solution of aescin may result in producing an exemplary mixture comprising an exemplary sediment.

[00091] In further detail with respect to step 518, step 518 may include freezing an exemplary mixture comprising an exemplary sediment. In an exemplary embodiment, freezing an exemplary mixture comprising an exemplary sediment may include placing an exemplary mixture comprising an exemplary sediment inside a freezer chamber and producing an exemplary frozen mixture.

In further detail with respect to step 520, step 520 may include freeze drying an exemplary frozen mixture. In an exemplary embodiment, freeze drying an exemplary frozen mixture may include placing an exemplary frozen mixture inside a freeze-drying chamber of an exemplary freeze dryer; freezing an exemplary frozen mixture with liquid nitrogen (-196 °C); and equilibrated at a temperature level between about -44 °C and -48 °C for about 48 hours, that may result in producing an exemplary dried powder of aescin with a purity of 80 % (w/w).

EXAMPLES

[00092] Hereinafter, one or more exemplary embodiments will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples may be for illustrative purposes only and are not to be interpreted to limit the scope of the present disclosure.

Example 1: producing an exemplary nasal formulation comprising aescin and rutin

[00093] In order to produce an exemplary nasal formulation comprising aescin and rutin, two mixtures were prepared. At first, a dried powder of aescin was added to propylene glycol with a volume/weight ratio of 4:1 (propylene glycol: aescin) in a beaker, that may result in producing a first mixture. Then, a dried powder of rutin was added to propylene glycol with a volume/weight ratio of 4:1 (propylene glycol: rutin) in a beaker, that may result in producing a second mixture. Each first and second mixture was shaken at a temperature level of 25 °C, with a speed of about 70 rpm, and for a time duration of 20 minutes and was leaved at a temperature level of 25 °C, and for a time duration of 1 hour. After that, a third mixture was formed by mixing the first and the second mixture while stirring on a stirrer, at a temperature level of 25 °C, with a speed of about 70 rpm, and for a time duration of 15 minutes. Then, the preservative comprising a double-distilled water solution of benzalkonium chloride was added to the third mixture with a volumetric ratio of 1:0.1 (the third mixture: the double-distilled water solution of benzalkonium chloride) while stirring on a stirrer, at temperature level of 25 °C, and with a speed of about 70 rpm. After adding an exemplary preservative to an exemplary third mixture, an exemplary third mixture may have benzalkonium chloride with a final concentration of 0.1 g/1. After that, doubled-distilled water was added to the third mixture with a volumetric ratio of 1:1 (third mixture: doubled-distilled water) and was shaken at a temperature level of 25 °C, with a speed of about 70 rpm, and for a time duration of 20 minutes and the pH level was adjusted between 6 and 8. Finally, the shaken aqueous solution was passed through a 0.45pm syringe filter and an exemplary nasal formulation was prepared.

Example 2: Evaluating improvement in sings of obstructive sleep apnea with a clinical trial after applying an exemplary nasal formulation comprising aescin and rutin [00094] For this purpose, forty patients with obstructive sleep apnea were included in the clinical trial. These patients were examined with STOP- Bang with an instrument called Viatom Checkme^TR O2 Pulse Oximeter. Moreover, these patients were evaluated for depression and anxiety with Beck and Hamilton depression scale and sleep quality with Pitsburgh Sleep Quality Index (PSQI). Those patients whom were pregnant or with drug sensitivity, renal failure, and liver failure were excluded from the clinical trial. The nasal spray of the nasal formulation of the present disclosure were applied in the patients in the clinical trial. For this purpose, the nasal spray of the pharmaceutical formulation of the present disclosure was applied two puffs in each nasal cavity every 8 hours a day for 30 days. After 30 days, the assessment described above was performed in the patients. Table 1 below shows the results of the tests in patients at day 0 and 30 after applying the exemplary nasal formulation, consistence with exemplary embodiments of the present disclosure. As shown in Table 1, the mean O2 saturation was increased in patients after 30 days. Moreover, the mean Score of PSQI and Beck and Hamilton depression scale was decreased after 30 days. It is be noted that the mean frequency of stop in breathing during sleep was reduced.

Table 1: The results of the tests in patients at day 0 and 30 after applying the exemplary nasal formulation, consistence with exemplary embodiments of the present disclosure.

[00095] While the foregoing has described what are considered to be the best mode and/or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings.

[00096] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[00097] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[00098] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[00099] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[000100] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[000101] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study, except where specific meanings have otherwise been set forth herein. Relational terms such as “first” and “second” and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[000102] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it may be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter. [000103] While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and/or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.

Claims

What is claimed is:

1. A method for producing a nasal formulation comprising aescin with a final concentration of 25 g/1 and rutin with a final concentration of 5 g/1, the method comprising: forming a first mixture comprising propylene glycol and aescin with a volume/weight ratio of 4:1 (propylene glycol: aescin); shaking the first mixture at a temperature level of 25 °C, with a speed of about 70 rpm, and for a time duration of 20 minutes; leaving the shaken first mixture at a temperature level of 25 °C, and for a time duration of 1 hour; forming a second mixture comprising propylene glycol and rutin with a volume/weight ratio of 4:1 (propylene glycol: rutin); shaking the second mixture at a temperature level of 25 °C, with a speed of about 70 rpm, and for a time duration of 20 minutes; leaving the shaken second mixture at a temperature level of 25 °C, and for a time duration of 60 minutes; forming a third mixture comprising aescin and rutin, wherein forming the third mixture comprises mixing the shaken first mixture and the shaken second mixture while stirring on a stirrer, at temperature level of 25 °C, with a speed of about 70 rpm, and for a time duration of 20 minutes; adding a preservative comprising a double-distilled water solution of benzalkonium chloride to the third mixture with a volumetric ratio of 1:0.1 (the third mixture: the doubledistilled water solution of benzalkonium chloride) while stirring on a stirrer, at temperature level of 25 °C, and with a speed of about 70 rpm, wherein after adding the double distilled water solution of benzalkonium chloride to the third mixture, the third mixture benzalkonium chloride with a final concentration of 0.1 g/1; forming an aqueous solution comprising aescin and rutin, wherein forming the aqueous solution comprises adding doubled-distilled water to the third mixture with a volumetric ratio of 1:1 (third mixture: doubled-distilled water); shaking the aqueous solution comprising aescin and rutin at a temperature level of 25 °C, with a speed of about 70 rpm, and for a time duration of 20 minutes; adjusting the pH level of the shaken aqueous solution between 6-8; and filtering the aqueous solution using a 0.45pm syringe filter.

2. A method for producing a nasal formulation comprising aescin with a final concentration between 10 g/1 and 30 g/1 and rutin with a final concentration between 4.5 g/1 and 5.5 g/1, the method comprising: forming a first mixture comprising propylene glycol and aescin with a volume/weight ratio of 4:1 (propylene glycol: aescin); shaking the first mixture at a temperature level between 20 °C and 30 °C, with a speed range between 65 rpm and 75 rpm, and for a time duration between 15 minutes and 25 minutes; leaving the shaken first mixture at a temperature level between 20 °C and 30 °C, and for a time duration between 55 minutes and 65 minutes; forming a second mixture comprising propylene glycol and rutin with a volume/weight ratio of 4:1 (propylene glycol: rutin); shaking the second mixture at a temperature level between 20 °C and 30 °C, with a speed range between 65 rpm and 75 rpm, and for a time duration between 15 minutes and 25 minutes; leaving the shaken second mixture at a temperature level between 20 °C and 30 °C, and for a time duration between 55 minutes and 65 minutes; forming a third mixture comprising aescin and rutin, wherein forming the third mixture comprises mixing the shaken first mixture and the shaken second mixture while stirring on a stirrer, at temperature level between 20 °C and 35 °C, with a speed range between 65 rpm and 75 rpm, and for a time duration between 15 minutes and 25 minutes; adding a preservative to the third mixture while stirring on a stirrer, at a temperature level of 25 °C, and with a speed of about 70 rpm; forming an aqueous solution comprising aescin and rutin, wherein forming the aqueous solution comprises adding doubled-distilled water to the third mixture with a volumetric ratio of 1:1 (third mixture: doubled distilled water); shaking the aqueous solution comprising aescin and rutin at a temperature level between 20 °C and 30 °C, with a speed range between 65 rpm and 75 rpm, and for a time duration between 15 minutes and 25 minutes; adjusting the pH level of the shaken aqueous solution between 6-8; and filtering the aqueous solution using a 0.45pm syringe filter.

3. The method for producing a nasal formulation of claim 2, wherein the nasal formulation comprises aescin with a final concentration of 25 g/1 and rutin with a final concentration of 5 g/1-

4. The method for producing a nasal formulation of claim 2, wherein shaking the first mixture comprises shaking the first mixture at a temperature level of 25 °C, with a speed of 70 rpm, and for a time duration of 20 minutes.

5. The method for producing a nasal formulation of claim 2, wherein leaving the shaken first mixture comprises leaving the shaken first mixture at a temperature level of 25 °C, and for a time duration of 1 hour.

6. The method for producing a nasal formulation of claim 2, wherein shaking the second mixture comprises shaking the second mixture at a temperature level of 25 °C, with a speed of about 70 rpm, and for a time duration of 20 minutes.

7. The method for producing a nasal formulation of claim 2, wherein leaving the shaken second mixture comprises leaving the shaken second mixture at a temperature level of 25 °C, and for a time duration of 60 minutes.

8. The method for producing a nasal formulation of claim 2, wherein shaking the aqueous solution comprising aescin and rutin comprises shaking the aqueous solution comprising aescin and rutin at a temperature level of 25 °C, with a speed of about 70 rpm, and for a time duration of 20 minutes.

9. The method for producing a nasal formulation of claim 2, wherein the preservative may be selected from the group consisting of benzalkonium chloride, chlorobutanol, and parabens.

10. The method for producing a nasal formulation of claim 2, wherein the preservative comprises benzalkonium chloride with a final concentration of 0.1 g/1.