Composition for prevention, amelioration, or treatment of vascular diseases, comprising fermented morinda citrifolia, and method for preparing same

Abstract

The present application provides: a composition for prevention, amelioration, or treatment of vascular diseases, comprising fermented Morinda citrifolia; and a preparation method therefor. More specifically, the present application provides: a composition for prevention, amelioration, or treatment of vascular diseases, comprising Morinda citrifolia fermented with lactic acid bacteria; and a preparation method therefor.

Description

Composition for preventing, improving, or treating vascular diseases containing fermented noni and method for preparing the same

[0001] The present invention relates to a composition for preventing, improving, or treating vascular diseases comprising fermented noni, and a method for preparing the same. More specifically, the present invention relates to a composition for preventing, improving, or treating vascular diseases comprising fermented noni fermented with lactic acid bacteria, and a method for preparing the same.

[0002] With the entry into an aging society, the incidence of vascular diseases is rapidly increasing, placing a serious national and social burden. One of the fundamental causes of these vascular diseases is hemodynamic disorder and endothelial dysfunction, which are key elements of Virchow's triad.

[0003] Hemodynamic disorders are characterized by insufficient blood supply to tissues and organs due to vascular occlusion, rupture, or dysfunction, and blood flow stasis or turbulence promotes thrombus formation.

[0004] These hemodynamic disorders are also associated with cardiovascular disease (CVD). Cardiovascular disease is one of the leading causes of death worldwide, and its major causes include endothelial dysfunction, increased blood viscosity, excessive coagulation reactions that lead to thrombosis, and decreased vascular elasticity. Endothelial dysfunction is considered a fundamental cause of impaired blood circulation.

[0005] Endothelial dysfunction is defined by increased oxidative stress, disruption of the balance between vasodilation and vasoconstriction, and reduced anticoagulant and anti-inflammatory functions, and reduced nitric oxide (NO) production acts as a major pathological factor, increasing the risk of thrombosis from the early stages of atherosclerosis.

[0006] Nitric oxide is essential for vasodilation, anti-inflammatory responses, and antithrombotic effects, and a deficiency of nitric oxide can lead to endothelial-dependent vasodilation disorders. Therefore, restoring nitric oxide production is considered an important mechanism for improving vasodilation and preventing endothelial dysfunction.

[0007] To date, drug therapies including angiotensin-converting enzyme (ACE) inhibitors and beta-blockers are commonly used to correct vasodilation imbalances. However, long-term use of such drugs can cause side effects such as chronic dry cough and bronchoconstriction. Therefore, natural bioactive compounds are emerging as a safer alternative.

[0008] As background technology of the present invention, Korean Registered Patent No. 10-0695590 discloses the therapeutic use and delivery mechanism of a drug that stimulates the production of nitric oxide or prostacyclin.

[0009] The purpose of the present invention is to provide a composition for the prevention, improvement, or treatment of vascular diseases that is safe even with long-term use.

[0010] Another objective of the present invention is to provide a composition for the prevention, improvement, or treatment of vascular diseases that efficiently generates nitric oxide in the body.

[0011] Another objective of the present invention is to provide a method for efficiently manufacturing a composition for the prevention, improvement, or treatment of vascular diseases.

[0012] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0013] According to one aspect, a food composition for preventing and / or improving vascular disease is provided, comprising a fermented noni extract fermented with lactic acid bacteria including Lactobacillus plantarum.

[0014] According to one embodiment, in the food composition for preventing and / or improving vascular disease of the present invention, the lactic acid bacteria may further include one or more of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus reuteri, or Lactococcus lactis.

[0015] According to one embodiment, the fermented noni extract can increase the production of nitric oxide in the body.

[0016] According to one embodiment, the fermented noni extract can activate a signaling pathway mechanism to increase nitric oxide production by activating PI3K to phosphorylate AKT and activating eNOS.

[0017] According to one embodiment, the fermented noni extract can relax blood vessels.

[0018] According to one embodiment, the food composition for preventing and / or improving vascular disease may further include one or more of fermented celery extract or fermented beet extract.

[0019] According to another aspect, a pharmaceutical composition for the prevention and / or treatment of vascular disease is provided, comprising a fermented noni extract fermented with lactic acid bacteria including Lactobacillus plantarum.

[0020] According to one embodiment, in the pharmaceutical composition for the prevention and / or treatment of vascular disease of the present invention, the lactic acid bacteria may further include one or more of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus reuteri, or Lactococcus lactis.

[0021]

[0022] According to another aspect, a method for preparing a composition for preventing and / or improving vascular diseases is provided, comprising the steps of: inoculating noni with lactic acid bacteria including Lactobacillus plantarum to ferment and age it; and preparing a fermented noni extract from the fermented noni obtained by fermentation and ageing.

[0023] According to one embodiment, the fermentation and aging step may include inoculating the lactic acid bacteria at a weight of 1 to 5 percent relative to the noni, and fermenting and aging at 35 to 40°C for 30 to 90 days.

[0024] According to one embodiment, the composition for preventing, improving, or treating vascular diseases of the present invention utilizes a natural product, noni extract, so that the possibility of side effects is low even when taken for a long period, allowing for safe prevention or improvement of vascular diseases.

[0025] According to one embodiment, the composition for preventing, improving, or treating vascular diseases of the present invention can prevent, improve, or treat vascular diseases by efficiently generating nitric oxide in the body using a noni extract fermented with lactic acid bacteria.

[0026] According to one embodiment, the method for preparing a composition for preventing, improving, or treating vascular diseases according to the present invention can efficiently prepare a composition for preventing, improving, or treating vascular diseases in which a noni extract fermented with lactic acid bacteria facilitates the smooth generation of nitric oxide in the body.

[0027] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0028] FIG. 1 is a diagram schematically illustrating a signal transduction mechanism in which a fermented noni extract according to one embodiment of the present invention promotes vasodilation in vascular endothelial cells.

[0029] Figure 2 is a chromatogram showing the results of analyzing fermented noni extract and noni extract by HPLC.

[0030] Figure 3 is a graph showing the results of analyzing the antioxidant effects of fermented noni extract and noni extract using (a) ABTS radical scavenging assay and (b) DPPH free radical scavenging assay.

[0031] Figure 4 is a graph showing the cell viability of HUVECs treated with fermented noni extract and noni extract.

[0032] Figure 5 is a graph showing the results of analyzing the effect of fermented noni extract and noni extract on nitric oxide production in HUVEC.

[0033] Figure 6 is a diagram showing the results of analyzing the effects of fermented noni extract and noni extract on PI3K expression.

[0034] Figure 7 is a diagram showing the results of analyzing the effects of fermented noni extract and noni extract on p-AKT / AKT expression.

[0035] Figure 8 is a diagram showing the results of analyzing the effects of fermented noni extract and noni extract on p-eNOS / eNOS expression.

[0036] Figure 9 is a graph showing the results of analyzing the effects of fermented noni extract and noni extract on AKT mRNA expression in HUVEC.

[0037] Figure 10 is a graph showing the results of analyzing the effects of fermented noni extract and noni extract on eNOS mRNA expression in HUVEC.

[0038] Figure 11 is a graph showing the effect of fermented noni extract on nitric oxide production in Raw 264.7 cells.

[0039] Figure 12 is a graph showing the change in absolute body weight of experimental animals when a fermented noni extract (FMCE) according to one embodiment of the present invention is administered orally.

[0040] Figure 13 is a graph showing the change in relative body weight of experimental animals when a fermented noni extract (FMCE) according to one embodiment of the present invention is administered orally.

[0041] Figure 14 is a graph showing the change in systolic blood pressure of experimental animals when a fermented noni extract (FMCE) according to one embodiment of the present invention is administered orally.

[0042] Figure 15 is a graph showing the change in average blood pressure of experimental animals when a fermented noni extract (FMCE) according to one embodiment of the present invention is administered orally.

[0043] Figure 16 is a graph showing the change in diastolic blood pressure of experimental animals when a fermented noni extract (FMCE) according to one embodiment of the present invention is administered orally.

[0044] Figure 17 is a graph showing changes in the pulse of experimental animals when a fermented noni extract (FMCE) according to one embodiment of the present invention is administered orally.

[0045] Figure 18 is a graph showing the change in absolute organ weight (liver, kidney, heart) of experimental animals when a fermented noni extract (FMCE) according to one embodiment of the present invention is administered orally.

[0046] Figure 19 is a graph showing the change in relative organ weight (liver, kidney, heart) of experimental animals when a fermented noni extract (FMCE) according to one embodiment of the present invention is administered orally.

[0047] Figure 20 is a graph showing the change in blood Angiotensin II concentration in experimental animals when a fermented noni extract (FMCE) according to one embodiment of the present invention is orally administered.

[0048] Figure 21 is a graph showing the change in blood Renin concentration of experimental animals when a fermented noni extract (FMCE) according to one embodiment of the present invention is orally administered.

[0049] Figure 22 is a graph showing the nitric oxide production ability of spinach, onion, carrot, garlic, celery, and beet by fermentation strain.

[0050] Figure 23 is a graph showing the cell viability of HUVECs treated with a mixture of fermented noni extract (FMCE) and fermented beet extract according to one embodiment of the present invention.

[0051] Figure 24 is a figure showing the results of analyzing the effect on eNOS protein expression after treating HUVEC with a mixture of fermented noni extract (FMCE) and fermented beet extract according to one embodiment of the present invention.

[0052] FIG. 25 is a graph showing the results of analyzing the nitric oxide production effect in HUVEC treated with a mixture of fermented noni extract (FMCE) and fermented beet extract according to one embodiment of the present invention.

[0053] The objects, advantages, and features of the present invention will become more apparent from the following detailed description and embodiments associated with the attached tables and drawings.

[0054] Prior to this, terms and words used in this specification and claims should not be interpreted in their ordinary and dictionary meanings, but should be interpreted in a meaning and concept consistent with the technical spirit of this disclosure, based on the principle that the inventor can appropriately define the concept of the terms to best describe his invention.

[0055] The terms used herein are merely for describing specific embodiments and are not intended to limit the disclosure. The singular expression includes the plural expression unless the context clearly indicates otherwise.

[0056] In this application, terms such as 'comprising' or 'having' are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0057] In this application, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0058] In this specification, descriptions following "e.g." should not limit the embodiments of the invention according to various embodiments of the invention, such as variations including tolerances, measurement errors, limits of measurement accuracy, and other commonly known factors, as the information presented, such as cited characteristics, variables, or values, may not exactly match.

[0059] The present invention is capable of various modifications and may have various embodiments; specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of related prior art may obscure the essence of the present invention, such detailed description is omitted.

[0060] In this specification, "prevention" refers to anything that suppresses or delays symptoms of vascular disease through the administration of the above composition.

[0061] In this specification, "improvement" means at least reducing parameters related to the condition treated by the administration of the composition, such as the degree of symptoms.

[0062] In this specification, "treatment" means at least reducing parameters related to the condition treated by the administration of the composition, such as the degree of symptoms.

[0063]

[0064] According to one aspect, a food composition for preventing and / or improving vascular diseases of the present invention comprises a fermented noni extract fermented with lactic acid bacteria including Lactobacillus plantarum.

[0065] Noni contains various bioactive compounds such as phenols, flavonoids, and iridoids, making it widely used as a key ingredient in functional health products. Deacetylasperulosidic acid (DAA), the main active ingredient in noni, can promote nitric oxide (NO) synthesis and improve endothelial function. Therefore, consuming noni can be beneficial for vascular health.

[0066] Fermentation refers to the process in which microorganisms break down organic matter using enzymes to produce substances useful to humans, and it improves bioavailability by promoting structural changes in physiologically active compounds.

[0067] Although not limited to this, when noni is fermented and used, diacetylasperulosidic acid, the main active ingredient of noni, can be activated in a larger amount compared to non-fermented noni.

[0068] Although not limited thereto, the fermented noni extract of the present invention utilizes Lactobacillus plantarum, and the Lactobacillus plantarum may be Lactobacillus plantarum NST1805 (accession number: KCCM12833P). Although not limited thereto, the use of the above-mentioned Lactobacillus plantarum NST1805 (accession number: KCCM12833P) may enhance the preventive, improvement, or therapeutic effects of vascular diseases by the fermented noni extract.

[0069] Although not limited to this, since fermented noni extract fermented with Lactobacillus plantarum is a natural product and does not cause adverse effects on the human body even when administered in excess, the quantitative upper limit of fermented noni fermented with Lactobacillus plantarum included in the composition of the present invention may be selected and implemented by a person skilled in the art within an appropriate range.

[0070] To date, research on the vasodilating properties of fermented noni extracts has been limited, necessitating further studies. Accordingly, this institution provides a fermented noni extract fermented with lactic acid bacteria, a method for its preparation, and a molecular mechanism based on the fermented noni extract, which can enhance the vasodilating effect by increasing the bioavailability of major physiologically active components of noni, including DAA, and improving antioxidant properties.

[0071]

[0072] Although not limited to this, the lactic acid bacteria of the present invention may use a single strain of Lactobacillus plantarum or a complex strain.

[0073] Although not limited thereto, the above lactic acid bacteria may further include one or more of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus reuteri, or Lactococcus lactis subsp. lactis.

[0074] Although not limited thereto, the above lactic acid bacteria may comprise 60 to 90 weight% of Lactobacillus plantarum and may further comprise one or more of Lactobacillus paracasei 1 to 10 weight%, Lactobacillus rhamnosus 1 to 10 weight%, Lactobacillus casei 1 to 10 weight%, Lactobacillus fermentum 1 to 10 weight%, Lactobacillus reuteri 1 to 10 weight%, and / or Lactococcus lactis 1 to 10 weight%.

[0075] Although not limited to this, if Lactobacillus plantarum is less than 60% by weight, the active ingredients of noni, which are useful for preventing and improving vascular diseases, may not be sufficiently activated.

[0076] Although not limited thereto, a composition for preventing and / or improving vascular diseases may further include one or more of fermented celery extract or fermented beet extract.

[0077] Although not limited to this, using a red bit may be most suitable for the above bit.

[0078] The above fermented celery extract and / or the above fermented beet extract may be fermented with Bacillus subtilis (B. subtilis MI385) or Bacillus amyloliquefaciens (Bacillus amyloliquefaciens FBB1).

[0079] Although not limited thereto, a composition for preventing or treating vascular disease may further include a fermented extract comprising spinach, onion, carrot, garlic, or one or more of these.

[0080] Fermented celery extract and / or fermented beet extract may have a superior amount of nitric oxide production, though not limited thereto. However, the celery and / or beet may be fermented with one or more of lactic acid bacteria, Bacillus subtilis, or Bacillus amyloliquefaciens. However, the celery and / or beet may have the best amount of nitric oxide production when fermented with Bacillus amyloliquefaciens.

[0081] Although not limited thereto, the fermented noni extract:fermented celery and / or fermented beet extract may be suitablely included in a ratio of 1:1 to 10:1 by weight%, 2:8 to 8:2, 3:7 to 7:3, and most suitablely included in a ratio of 1:2 to 2:1.

[0082] Although not limited to this, fermented noni extract: fermented celery and / or fermented beet extract can generate nitric oxide more efficiently when included in different weights rather than in equal amounts.

[0083] Nitric oxide is produced within cells from the amino acid L-arginine by nitric oxide synthase (NOS), and as a signaling molecule, it is involved in various physiological activities such as immunity and vasodilation. In particular, by inducing the production of cGMP, it influences vasodilation, thereby helping to lower blood pressure and improve blood flow. Therefore, nitric oxide is effective in preventing strokes and heart attacks such as myocardial infarction by blocking blood clots in the cardiovascular system.

[0084] Although not limited thereto, the food composition for the prevention and / or improvement of vascular diseases of the present invention may increase the production of nitric oxide in the body. Although not limited thereto, the composition for the prevention and / or improvement of vascular diseases of the present invention increases the production of nitric oxide in the body, causing the nitric oxide to dilate blood vessels, and thereby may play a role in improving the cardiovascular system, improving inflammation and immunity, transmitting neurotransmitters, and preventing blood coagulation.

[0085] FIG. 1 is a schematic diagram illustrating the signaling mechanism by which a fermented noni extract according to one embodiment of the present invention promotes vasodilation in vascular endothelial cells. Referring to FIG. 1, but not limited thereto, a food composition for the prevention and / or improvement of vascular diseases according to the present invention may activate a signaling pathway mechanism to increase nitric oxide production by activating PI3K to phosphorylate AKT and activating eNOS (endothelial nitric oxide synthase). The PI3K-AKT-eNOS signaling pathway is an important molecular mechanism for nitric oxide synthesis during vasodilation. Endothelial cell damage can disrupt this pathway, leading to vascular dysfunction and serious diseases including hypertension and cardiovascular disease. PI3K activation promotes the migration of AKT to the cell membrane, thereby activating and phosphorylating AKT (p-AKT). p-AKT activates nitric oxide synthesis by phosphorylating the Ser1177 site of eNOS.

[0086] Meanwhile, although not limited thereto, the food composition for the prevention and / or improvement of vascular diseases of the present invention may inhibit the expression of iNOS (Inducible Nitric Oxide Synthase). iNOS may amplify inflammation by producing excessive amounts of nitric oxide.

[0087] Although not limited thereto, the food composition for the prevention and / or improvement of vascular diseases of the present invention may relax blood vessels. Although not limited thereto, the composition for the prevention and / or improvement of vascular diseases of the present invention may induce the production of nitric oxide in the body, and the generated nitric oxide may diffuse into vascular smooth muscle cells to cause vasodilation and increased blood flow.

[0088]

[0089] In this document, the term "food composition" refers to a natural product or processed product containing one or more nutrients, preferably one that has undergone a certain degree of processing to become ready for direct consumption, and includes, in the conventional sense, all of food, food additives, functional foods, and beverages.

[0090] In addition, in this invention, the term "food composition for prevention and / or improvement" refers to a group of foods to which added value has been imparted by using physical, biochemical, or biotechnological methods to make the functions of the food act or manifest for a specific purpose, or to a food processed by designing it to fully express in the body the in vivo regulatory functions regarding the regulation of biological defense rhythms, disease prevention, and recovery possessed by the food composition.

[0091] Food compositions for the prevention and / or improvement of vascular diseases according to the present invention include, for example, various food products, beverages, chewing gum, tea, vitamin complexes, health functional foods, etc. Furthermore, the food composition of the present invention may contain additional ingredients such as various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages, and said additional ingredients may be used independently or in combination. The said additional ingredients may be added in an appropriate amount within a range that does not impair the efficacy of fermented noni for the prevention or improvement of vascular diseases. For example, said additional ingredients may be included in an amount of 0.001 to 1 weight% based on the total weight of said food composition for the prevention or improvement of vascular diseases.

[0092] In addition, the amount of fermented noni extract or powder added to the health functional food of the present invention varies depending on the type of health functional food to be added and cannot be uniformly specified, but it may be added within a range that does not impair the original taste of the food, and may typically be in the range of 0.01 to 100 weight%, 0.1 to 80 weight%, or 0.1 to 50 weight% with respect to the food to be added. In addition, in the case of health functional foods in the form of beverages, pills, granules, tablets, or capsules, it may typically be added in the range of 0.1 to 100 weight%, preferably 0.5 to 80 weight%.

[0093] In addition, the food composition for the prevention and / or improvement of vascular disease of the present invention may include food-grade acceptable food additives and may further include suitable carriers, excipients, and diluents commonly used in the preparation of food compositions for prevention and / or improvement.

[0094] The food composition for the prevention and / or improvement of vascular diseases of the present invention can be formulated in various forms, so the form is not particularly limited. Preferably, the food composition for the prevention and / or improvement of vascular diseases can be formed into any one of the formulations selected from beverages, granules, tablets, powders, pills, and capsules.

[0095] The food compositions of the present invention for the prevention and / or improvement of vascular diseases, having such beverage, granule, tablet, powder, pill, and capsule formulations, are easy to carry and easy to consume at any time and anywhere.

[0096] The composition for the prevention and / or treatment of vascular disease of the present invention may be used alone or in combination with other compositions for the prevention or treatment of vascular disease other than the present invention.

[0097]

[0098] According to another aspect, the pharmaceutical composition for the prevention and / or treatment of vascular disease of the present invention comprises a fermented noni extract fermented with lactic acid bacteria including Lactobacillus plantarum.

[0099] The pharmaceutical composition for the prevention or treatment of vascular disease of the present invention may be used alone or in combination with other compositions for the prevention or treatment of vascular disease other than the present invention.

[0100] Although not limited thereto, the pharmaceutical composition for the prevention or treatment of vascular diseases of the present invention may include fermented noni fermented with Lactobacillus plantarum NST1805 (accession number: KCCM12833P) as an active ingredient.

[0101] Meanwhile, the term "containing as an active ingredient" in this invention means containing an amount sufficient to achieve the efficacy or activity of a fermented noni extract fermented with lactic acid bacteria. In one embodiment of this invention, fermented noni fermented with Lactobacillus plantarum NST1805 (accession number: KCCM12833P) in the composition of the present invention may be included, for example, at an amount of 0.001 mg / kg or more, preferably 0.1 mg / kg or more, more preferably 10 mg / kg or more, even more preferably 100 mg / kg or more, even more preferably 250 mg / kg or more, and most preferably 0.1 g / kg or more. Since fermented noni fermented with Lactobacillus plantarum NST1805 (accession number: KCCM12833P) is a natural product and does not cause adverse effects on the human body even when administered in excess, the quantitative upper limit of fermented noni fermented with Lactobacillus plantarum NST1805 (accession number: KCCM12833P) included in the composition of the present invention may be selected and implemented by a person skilled in the art within an appropriate range.

[0102] The pharmaceutical composition of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, and the adjuvants may include excipients, disintegrants, sweeteners, binders, coatings, leavening agents, lubricants, lubricants, or flavoring agents.

[0103] The above pharmaceutical composition may preferably be formulated into a pharmaceutical composition by including one or more additional pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

[0104] The formulation form of the above pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops, or injectable liquids. For example, for formulation into the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, or water. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants, and colorants may also be included in the mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose, or beta-lactose; corn sweeteners; natural and synthetic gums such as acacia, trackercanth, or sodium oleate; sodium stearate; magnesium stearate; sodium benzoate; sodium acetate; sodium chloride; etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc.

[0105] Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, provided that they are sterile and biocompatible. Additionally, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0106] Furthermore, it can be preferably formulated according to the ingredients using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

[0107] The pharmaceutical composition of the present invention may be administered orally or parenterally. In the case of parenteral administration, it may be administered via intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, transdermal infusion, etc., and preferably orally.

[0108] Suitable dosages of the pharmaceutical composition of the present invention vary depending on factors such as the method of formulation, mode of administration, age, body weight, sex, pathological condition, food, time of administration, route of administration, rate of excretion, and responsiveness, and a physician of ordinary skill can easily determine and prescribe a dosage effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention may be 0.001 to 10 g per 1 kg of the weight of the subject to administration.

[0109] Although not limited to this, the effective dose (per day) of the fermented noni extract of the present invention for humans may be 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 60 mg / kg or more, 70 mg / kg or more, 80 mg / kg or more, 90 mg / kg or more, 100 mg / kg or more, 110 mg / kg or more, 120 mg / kg or more, 130 mg / kg or more, 140 mg / kg or more, 150 mg / kg or more, 160 mg / kg or more, 170 mg / kg or more, 180 mg / kg or more, 190 mg / kg or more, or 200 mg / kg or more.

[0110] There is no specific upper limit on the effective daily dose of the fermented noni extract (40 to 80 Brix) of the present invention for humans. For example, the effective daily dose of the fermented noni extract (40 to 80 Brix) of the present invention for humans may be 30 mg / kg to 120 mg / kg and 40 mg / kg to 100 mg / kg. The effective dose of the fermented noni concentrate (60 Brix) may be 3 g / day to 6 g / day based on a 60 kg adult.

[0111] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-volume container by formulation using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0112] Although not limited thereto, the fermented noni extract of the pharmaceutical composition for the prevention and / or treatment of vascular diseases of the present invention may be fermented with lactic acid bacteria including one or more of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus reuteri, or Lactococcus lactis.

[0113]

[0114] According to another aspect, a method for preparing a composition for preventing and / or improving vascular diseases according to the present invention comprises the steps of: inoculating noni with lactic acid bacteria including Lactobacillus plantarum to ferment and age it; and preparing a fermented noni extract from the fermented noni obtained by fermentation and ageing.

[0115] Although not limited thereto, in the method for preparing a composition for preventing and / or improving vascular diseases according to the present invention, the lactic acid bacteria may further include one or more of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus reuteri, or Lactococcus lactis subsp. lactis, and may include all of these.

[0116] Although not limited thereto, the above lactic acid bacteria may be fermented by further including 60 to 90 weight% of Lactobacillus plantarum, 1 to 10 weight% of Lactobacillus paracasei, 1 to 10 weight% of Lactobacillus rhamnosus, 1 to 10 weight% of Lactobacillus casei, 1 to 10 weight% of Lactobacillus fermentum, 1 to 10 weight% of Lactobacillus reuteri, and / or 1 to 10 weight% of Lactococcus lactis.

[0117] Although not limited thereto, the above Noni (Morinda citrifolia L.) may refer to the fruit, flower, or leaf, or may include all of these. Although not limited thereto, the above Noni may exhibit the most excellent antioxidant effect when the fruit is fermented and aged.

[0118] Although not limited thereto, the fermentation and aging step may include inoculating the lactic acid bacteria at a weight of 1 to 5% relative to the noni and fermenting and aging at 35°C to 40°C for 30 to 90 days. The lactic acid bacteria may be inoculated at a weight of 1 to 3% relative to the noni and fermented and aged for 30 to 70 days. If the lactic acid bacteria are inoculated at less than 1% by weight relative to the noni or fermented and aged for less than 30 days, sufficient fermentation may not occur. Furthermore, if the lactic acid bacteria are inoculated at more than 5% by weight relative to the noni or fermented and aged for more than 90 days, over-fermentation may occur, which may actually reduce the beneficial components and degrade the taste.

[0119] Although not limited to this, if the above fermentation and aging temperature is less than 35℃, fermentation may not proceed normally, and if it exceeds 40℃, the fermentation and aging time may be prolonged, and lactic acid bacteria may die, making it difficult to efficiently produce a composition with excellent efficacy in preventing and / or improving vascular diseases of fermented noni extract in large quantities, and the efficacy and aroma may be reduced.

[0120]

[0121] Although not limited thereto, the method for preparing a composition for the prevention or treatment of vascular diseases according to the present invention may further include a step of preparing a concentrated filtrate, wherein the fermented noni fruit is juiced, filtered to remove particles, and then the filtrate is concentrated to 5 Brix to 20 Brix. If the filtrate is concentrated to less than 5 Brix, it becomes too diluted, which may result in reduced drying efficiency and a decrease in yield and / or recovery rate. If the filtrate is concentrated to more than 20 Brix, the viscosity may increase excessively, which may worsen processability, and during the heat / concentration process, protein denaturation, reduced enzyme activity, and / or reduced microbial viability may occur.

[0122] Although not limited thereto, the method for preparing a composition for the prevention or treatment of vascular diseases according to the present invention may further include the step of freeze-drying the concentrated filtrate to extract the powder.

[0123]

[0124] A method for preparing a composition for preventing and / or improving vascular diseases, which is not limited thereto, comprises the steps of: inoculating celery or beets with lactic acid bacteria or Bacillus bacteria to ferment and mature them; preparing the fermented celery or fermented beet extract obtained by fermentation and maturation; and may further include beets.

[0125] Fermented celery and / or fermented beet extracts may be fermented with lactic acid bacteria together with noni, though this is not limited to this.

[0126] For the preparation of fermented celery extract or fermented beet extract, although not limited thereto, dried and ground raw material (celery or beet) is mixed with purified water to a concentration of 5% (w / v) and sterilized, and then a pre-prepared Bacillus amyloliquefaciens FBB1 culture solution is inoculated to a concentration of 1% to 5% (v / v) or 1% to 3% (v / v), and fermented and aged at 30°C to 50°C for 10 to 30 hours or 15 to 25 hours.

[0127] Although not limited to this, fermented celery or fermented beet extract powder can be obtained by fermenting and aging the above-mentioned celery or beet, juicing and filtering the resulting liquid, concentrating it to 10 to 30 Brix or 15 to 25 Brix, and then freeze-drying it.

[0128] Although not limited thereto, the above-mentioned fermented celery or fermented beet extract may further include a step of mixing with the fermented noni extract of the present invention.

[0129] Although not limited thereto, if the fermented noni extract of the present invention further includes the fermented celery or the fermented beet extract, the nitric oxide production efficiency of the composition for preventing or improving vascular diseases of the present invention may be further increased.

[0130] Although not limited thereto, the ratio of fermented noni extract to fermented beet extract may be 1:10 to 10:1 by weight, 1:5 to 5:1, or 1:2 to 2:1. Although not limited thereto, if the ratio of fermented noni extract is lower than 1:10, the beneficial components of fermented noni may not be sufficient, resulting in less nitric oxide production and other synergistic effects, and if it exceeds 10:1, it may be uneconomical.

[0131]

[0132] The present invention will be described in more detail below through examples. However, these examples are intended solely to illustrate the present invention, and the scope of the present invention should not be interpreted as being limited by these examples.

[0133]

[0134] Example 1 and Comparative Example 1. Preparation of Fermented Noni Extract

[0135] Noni fruits were harvested from the NSTBIO farm in Indonesia and used as raw materials for the preparation of fermented noni extract (FMCE, Example 1) and noni extract (MCE, Comparative Example 1). The fermented noni extract was produced by inoculating noni fruits with a 2% culture of AON1805 probiotics (Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus reuteri, and Lactococcus lactis subsp. lactis) and fermenting at 37°C for 60 days. After fermentation, the noni fruits were juiced and filtered to remove particles, and the filtrate was concentrated to 10 Brix. The concentrated filtrate was freeze-dried to obtain a powder extract. MCE was prepared by immediately extracting juice from fresh noni and freeze-drying it to obtain a powder extract.

[0136]

[0137] Experimental Example 1. HPLC Analysis

[0138] Quantification and verification of asperulosidic acid (AA), diesoacetic acid (DAA), and scopoletin (SCP) were performed in MCE and FMCE.

[0139] The HPLC system (Shimadzu Corporation, Kyoto, Japan) consisted of an LC-20AD pump and an SPD-20A UV / VIS detector. Separation was performed using a Symmetry C18 column (250 x 4.6 mm, 5 μm), and the column temperature was maintained at 25°C. The binary mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile. The flow rate was maintained at 1.0 mL / min for a total of 40 minutes. The gradient elution protocol was as follows: 0–5 min, 0% B; 5–40 min, 35% B. The sample injection volume was 5 μL. Each compound was recognized and quantified by comparing the peak retention period to a reference standard.

[0140]

[0141] Figure 2 is a chromatogram showing the results of HPLC analysis of noni fermented product and noni extract. Referring to Figure 2, the major compounds were identified and quantified by comparing the retention times of the analyte peaks in MCE and FMCE with the retention times of standard compounds.

[0142] As a result of the analysis, compounds were detected in the order of deacetylasperulosidic acid (DAA), asperulosidic acid (AA), and scopoletin (SCP). Quantitative analysis showed that the DAA content in FMCE (15.93 ± 0.11 mg / g) was higher than that in MCE (12.93 ± 0.10 mg / g).

[0143] The AA content of MCE (9.47 ± 0.06 mg / g) was significantly higher than that of FMCE (1.19 ± 0.02 mg / g). There was no significant difference in SCP concentration between the two extracts, and it was measured as 0.45 ± 0.01 mg / g in MCE and 0.43 ± 0.01 mg / g in FMCE.

[0144] These results demonstrate that fermentation alters the composition of bioactive compounds, causing MCE and FMCE to exhibit different functional characteristics. This difference highlights the potential of fermentation processes to enhance the functionality of bioactive compounds, establishes a scientific foundation for developing functional materials through optimized fermentation processes, and provides important insights for future applied research.

[0145]

[0146] Experimental Example 2. Measurement of Antioxidant Effect

[0147] 2-1. Total Polyphenol Content (TPC)

[0148] Total polyphenol content (TPC) was measured using the modified Folin and Denis (1912) method. 0.2 mL of a sample diluted 1:9 with EGM-2 medium (Lonza, Basel, Switzerland) for Morinda citrifolia L. extract was mixed with 1 mL of 2N Folin-Ciocalteu reagent diluted 1:29.

[0149] After adding 0.8 mL of 7.5% sodium carbonate (Na2CO3) solution, the mixture was left at room temperature for 1 hour in the dark.

[0150] Absorbance at 765 nm was measured using an ELISA microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). A standard curve was established using gallic acid, and total phenol content (TPC) was expressed as gallic acid equivalent (mg GAE / g).

[0151] 2-2. Total Flavonoid Content (TFC)

[0152] Total flavonoid content (TFC) was quantified. 0.05 mL of MCE, FMCE, DAA, and 0.03 mL of sodium nitrite were added, and the mixture was left in a dark room at room temperature for 10 minutes. 0.06 mL of 2% aluminum chloride hexahydrate (AlCl3·6H2O) solution and 0.1 mL of 1N sodium hydroxide (NaOH) solution were mixed in a 96-well plate and left at room temperature in a light-blocked environment for 11 minutes. Absorbance at 415 nm was measured using an ELISA microplate reader. Total flavonoid content (TFC) was quantified as quercetin equivalent (mg QE / g) using a standard curve established with quercetin as the reference compound, and quercetin was used as the reference compound.

[0153] 2-3. 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) radical scavenging activity

[0154] ABTS (2,2'-azino-bis(3-ethylbenzthiazolin-6-sulfonic acid)) radical scavenging analysis was performed according to the modified procedure established by Re et al. (1999).

[0155] The ABTS reagent was prepared by mixing 7.4 mM ABTS and 2.6 mM potassium persulfate (K2S2O3), and then incubated in the dark at room temperature for 16 hours. To obtain an absorbance of 0.7 ± 0.02 at 734 nm, the ABTS solution was first diluted with PBS buffer (pH 7.4). Samples were prepared at various concentrations of 25, 50, 100, and 200 μg / mL using DMSO, or at a concentration of 20 μg / mL using DAA. 250 μL of ABTS reagent and 50 μL of sample solution were added to each well, and the samples were incubated in the dark at room temperature for 30 minutes. Absorbance was measured at 734 nm using an ELISA microplate reader. Each experiment was repeated three times, and L-ascorbic acid (1 mM) was used as a positive control. The ABTS radical scavenging activity of the samples was determined as follows.

[0156]

[0157] 2-4,1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity

[0158] The DPPH free radical scavenging activity analysis was performed by slightly modifying the method of Blois (1958).

[0159] MCE and FMCE were diluted to various concentrations (25, 50, 100, 200 μg / mL) or DAA was diluted to 20 μg / mL using EMG-2 medium (Lonza, Basel, Switzerland).

[0160] 100 μl of 0.4 mM DPPH solution (ethanol solution) was mixed with 100 μl of each sample in a 96-well plate and incubated at 37°C for 30 minutes under light blockage. 1 mM L-ascorbic acid was used as a positive control, and absorbance was measured at 517 nm using an ELISA microplate reader.

[0161] Each experiment was repeated three times. The DPPH radical scavenging activity of the samples was determined as follows.

[0162]

[0163] 2-5. Antioxidant Effects of MCE and FMCE

[0164] Figure 3 is a graph showing the results of analyzing the antioxidant effects of fermented noni extract and noni extract using (a) ABTS radical scavenging assay and (b) DPPH free radical scavenging assay. Values ​​are expressed as mean ± standard deviation. **** P < 0.0001 (compared to Ascorbic acid (AA)). Here, AA is ascorbic acid; DAA is deacetylasperulosidic acid; ABTS is 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); DPPH is 1,1-diphenyl-2-picryl hydrazyl; MCE is Morinda citrifolia L. extracts; FMCE is Fermented Morinda citrifolia L. extracts; and DAA, deacetylasperulosidic acid.

[0165] Referring to Figure 3 and Table 3, the total phenolic content (TPC) of MCE, FMCE, and DAA was measured as gallic acid equivalents per gram (mg GAE / g), and the total flavonoid content (TFC) was quantified as quercetin equivalents per gram (mg QE / g). In this study, the TPCs of MCE, FMCE, and DAA were 55.44 ± 0.60, 83.71 ± 0.80, and 2.89 ± 0.01 mg GAE / g, respectively. The TFC values ​​were 2.34 ± 0.05, 3.57 ± 0.13, and 0.01 ± 0.00 mg QE / g, respectively.

[0166] In addition, since flavonoids are a type of phenolic compound, the total flavonoid content (TFC) is naturally lower than the total phenolic content (TPC).

[0167] MCE and FMCE were evaluated at concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, and DAA was evaluated at a concentration of 20 μg / mL. Referring to Figure 3, the ABTS radical scavenging activity of MCE was quantified as 27.28 ± 0.28%, 34.33 ± 0.65%, 45.66 ± 0.60%, and 56.19 ± 0.31% at concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. The ABTS radical scavenging activity of FMCE was quantified as 30.19 ± 0.58%, 39.49 ± 0.62%, 49.24 ± 0.61%, and 61.81 ± 0.99% at each concentration.

[0168] The ABTS radical scavenging activity of DAA was quantified as 24.44 ± 0.27% at a concentration of 20 μg / mL. MCE showed DPPH radical scavenging activity values ​​of 22.80 ± 1.11%, 28.37 ± 0.55%, 40.05 ± 0.20%, and 52.29 ± 0.39% at concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. FMCE showed values ​​of 26.42 ± 0.24%, 34.65 ± 1.11%, 46.29 ± 0.37%, and 57.44 ± 0.22% at the same concentration ranges, respectively. At a concentration of 20 μg / mL, DAA shows 17.33 ± 0.49% DPPH radical scavenging activity.

[0169] In numerous studies, if ABTS and DPPH radical scavenging activity is 50% or higher, it indicates significant antioxidant activity. MCE and FMCE exhibit significant antioxidant activity, such as showing a scavenging rate of 50% or higher at a concentration of 200 μg / mL.

[0170]

[0171] FMCE exhibits ABTS radical scavenging activity approximately 1.08–1.15 times higher and DPPH radical scavenging activity approximately 1.10–1.22 times higher than MCE. The total phenolic content (TPC) of FMCE is approximately 1.51 times higher and the total flavonoid content (TFC) is approximately 1.53 times higher than that of MCE. Fermentation enhances antioxidant activity, which is likely due to the high DAA content found in FMCE.

[0172] FMCE, which exhibits superior antioxidant activity compared to MCE, is expected to more effectively reduce oxidative stress, protect endothelial cells, and increase nitric oxide production, thereby promoting vasodilation.

[0173] These results suggest that the enhanced antioxidant properties of FMCE provide an important scientific basis for the development of functional materials and future research.

[0174] Table 1 below shows the total phenolic and flavonoid content of noni fermented product and noni extract.

[0175]

[0176] The figures are expressed as mean ± standard deviation. 1) mg gallic acid equivalents per gram. 2) mg quercetin equivalents per gram.

[0177]

[0178] Experimental Example 3. Culture of Human Umbilical Vein Endothelial Cells (HUVEC)

[0179] Human umbilical vein endothelial cells (HUVEC or HUVECs) were purchased from Lonza (Basel, Switzerland) and cultured under standard culture conditions of 37°C, 5% CO2, and 20% O2.

[0180] Cells were cultured in 100 mm × 15 mm cell culture dishes (SPL Life Sciences, Pocheon, Korea) in EGM-2 medium (Lonza, Basel, Switzerland) supplemented with 5% heat-treated inactivated fetal bovine serum (FBS).

[0181] The culture medium was changed three times a week, and cells from the 3rd to 9th passages were used in the experiment. HUVECs were purchased from Lonza (Basel, Switzerland) and cultured according to standard protocols at 37°C, 5% CO2, and 20% O2. Cells were cultured in 100mm × 15mm cell culture dishes (SPL Life Sciences, Pocheon, Korea) using EGM-2 medium (Lonza, Basel, Switzerland) supplemented with 5% heat-treated inactivated fetal bovine serum (FBS). Cells from the 3rd to 9th passages were used in the experiment, and the culture medium was changed three times a week.

[0182]

[0183] Experimental Example 4. Cell viability

[0184] The cytotoxic effects of MCE and FMCE at concentrations of 20, 100, and 200 μg / mL and DAA at concentrations of 20 and 100 μg / mL on HUVECs were evaluated.

[0185] MCE and FMCE (20, 100, 200 μg / mL) and DAA (20, 100 μg / mL) were added to each well and cultured for 24 hours. Cells were cultured in EGM-2 (Lonza, Basel, Switzerland) and EZ-Cytox test reagent (Dogen Bio, Seoul, Korea) for 30 minutes, and then washed with HEPES buffer (Lonza, Basel, Switzerland).

[0186] Absorbance at 450 nm was measured using an ELISA microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Each analysis was repeated three times.

[0187] Figure 4 is a graph showing the cell viability of HUVECs treated with fermented noni extract and noni extract. The graph shows HUVECs (1.5 × 10⁻⁶ / ml) treated with 20 μg / mL, 100 μg / mL, and 200 μg / mL of MCE and FMCE, 20 μg / mL and 100 μg / mL of DAA, and a control group (EGM-2) for 24 hours. Data are expressed as mean ± standard deviation (n=3). *** p < 0.001 and **** p < 0.0001 represent results compared to the control group. Here, HUVECs refer to human umbilical vein endothelial cells. Referring to Figure 4, the effects of MCE, FMCE, and DAA on cell viability were evaluated through the MTT experiment. When treated with MCE at concentrations of 20, 100, and 200 μg / mL, cell viability was 93.41%, 91.91%, and 76.93%, respectively. For FMCE, at the same concentrations, viability was 97.67%, 99.38%, and 77.93%, respectively. For DAA, at concentrations of 20 μg / mL and 100 μg / mL, viability was 99.40% and 77.88%, respectively. Cell viability after treatment with 200 μg / mL FMCE, MCE, and 100 μg / mL DAA was significantly reduced compared to the control group, indicating a cytotoxic effect at those concentrations.

[0188]

[0189] In subsequent experiments, FMCE and MCE were tested at non-toxic concentrations of 20 μg / mL and 100 μg / mL to observe their effects at low and high concentrations. DAA was evaluated at a concentration of 20 μg / mL, which was used as a reference point to compare its effects with the other two extracts.

[0190]

[0191] Experimental Example 5. Measurement of Nitric Oxide (NO)

[0192] 1.5 × 10 per well in a 6-well plate 5HUVECs were inoculated at a cell density and cultured for 24 hours. MCE and FMCE were treated at concentrations of 20 μg / mL and 100 μg / mL, respectively, for 24 hours. Subsequently, the cells were cultured for an additional 24 hours. PicoSens containing 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride and 1% sulfanilamide dissolved in 2.5% phosphoric acid TM 100 μL of the Griess reagent analysis kit (Biomax, Seoul, South Korea) was mixed with 100 μL of the culture supernatant for analysis. After incubating at room temperature for 10 minutes, the absorbance was measured at 540 nm. Standard curves were constructed using NaNO2 at various concentrations.

[0193]

[0194] Nitric oxide homeostasis deficiency contributes to the development of cardiovascular diseases such as hypertension and stroke.

[0195] Homeostasis significantly influences vascular tone. This institute aimed to investigate the physiological activity of noni extracts, MCE and FMCE, in relation to vascular pathophysiology.

[0196] Figure 5 is a graph showing the results of analyzing the effects of fermented noni extract and noni extract on nitric oxide production in HUVECs. MCE and FMCE (20 μg / mL and 100 μg / mL), respectively, were cultured for 24 hours, and nitric oxide production was measured using the Griess assay. Data are presented as mean ± standard deviation (n=3). ** p < 0.01 and **** p < 0.0001 represent the significance level compared to the control group. The effects of MCE and FMCE on nitric oxide release, which is important for vasodilation, were evaluated using DAA as a positive control. Referring to Figure 5, the evaluation of nitric oxide production in HUVECs treated with MCE and FMCE showed that when HUVECs were treated with MCE, FMCE, and DAA for 24 hours, nitric oxide production significantly increased by 9–12%, 19–35.5%, and 41.3%, respectively, compared to the control group. MCE was shown to have limited activation of nitric oxide synthesis, suggesting that its effect on vasodilation is minimal.

[0197] In contrast, FMCE was shown to significantly increase nitric oxide production in a dose-dependent manner. This suggests that FMCE can substantially increase nitric oxide production through mechanisms such as eNOS activation or antioxidant functions that maintain nitric oxide bioavailability. These actions can promote vasodilation and improve vascular flexibility. These results appear to be attributed to an increase in bioactive compounds generated during the fermentation process, particularly those influenced by DAA.

[0198] Vascular tone is regulated by intrinsic mechanisms such as nitric oxide and central regulatory mechanisms including neurotransmitters.

[0199]

[0200] Experimental Example 6. Western Blot Analysis

[0201] 1.5 × 10⁶ vascular endothelial cells in each well of a 6-well culture plate 5Cells were seeded at cell density and cultured for 24 hours. MCE, FMCE, and DAA were performed for 24 hours. After washing twice with HEPES buffer, cells were lysed using RIPA lysis buffer (Dynebio, Seongnam, Korea) and Xpert Duo Inhibitor Cocktail Solution (100×) (GenDEPOT, Barker, TX, USA) diluted 1:100. Pierce TM Protein samples were quantified using a BCA protein quantification kit (Thermo Fisher Scientific, Waltham, MA, USA). After separation on a 10% SDS-PAGE gel, 15 μL of the sample was transferred to a methanol-activated PVDF membrane (Roche, Mannheim, Germany). After blocking the membrane with 5% BSA, primary antibodies diluted 1:1000 or 1:500 in TBST were added and incubated overnight. After washing with TBST, the membrane was incubated for 1 hour with HRP (Horseradish Peroxidase)-conjugated anti-rabbit IgG secondary antibody (Cell Signaling Technology, Danvers, MA, USA). This antibody was diluted 1:10,000 for p-AKT and p-eNOS, and 1:5,000 for PI3K, AKT, and eNOS. Protein bands on the membrane were observed and captured using Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) and the ChemiDoc system (Bio-Rad, Hercules, CA, USA). The major antibodies used are listed in Table 2 below.

[0202]

[0203] Nitric oxide synthesis in endothelial cells is regulated primarily by Ser1177 phosphorylation of eNOS, which is activated through various signaling pathways, particularly the PI3K-AKT pathway.

[0204] In this study, we evaluated whether MCE and FMCE induce the activation of key signaling molecules associated with nitric oxide production, particularly PI3K, AKT, and eNOS.

[0205]

[0206] Figures 6 to 8 are graphs showing the results of analyzing the effects of fermented noni extract and noni extract on the expression of PI3K, p-AKT / AKT, and p-eNOS / eNOS. Data are expressed as mean ± standard deviation (n=3). (* p < 0.05, ** p < 0.01 compared to the control group) Here, PI3K means Phosphoinositide 3-kinase AKT; AKT means serine / threonine kinase; p-AKT means phosphorylated AKT; eNOS means endothelial Nitric Oxide Synthase; and p-eNOS means phosphorylated eNOS.

[0207] HUVECs were treated with MCE and FMCE at concentrations of 20 μg / mL and 100 μg / mL, respectively, and DAA (20 μg / mL) was used as a positive control. After 24 hours of treatment, the activation levels of PI3K, p-AKT / AKT, and p-eNOS / eNOS were evaluated by Western blot analysis.

[0208]

[0209] Analysis of PI3K, AKT, p-AKT, eNOS, and p-eNOS expression in HUVECs using Western blot revealed no statistically significant differences between the MCE-treated group and the control group. FMCE treatment significantly increased PI3K activation, increasing 1.018-fold at a concentration of 20 μg / mL and 1.021-fold at a concentration of 100 μg / mL (p < 0.05). These results suggest that FMCE specifically activates the PI3K pathway, which is essential for the initial signaling phase of nitric oxide production.

[0210] AKT phosphorylation increased in a dose-dependent manner in both MCE and FMCE treatment groups. In the MCE treatment group, the p-AKT / AKT ratio increased 1.7-fold at a concentration of 20 μg / mL (p < 0.001) and 2.0-fold at a concentration of 100 μg / mL (p < 0.001). FMCE treatment significantly increased PI3K-AKT pathway activation, with the ratio increasing approximately 2.1-fold at 20 μg / mL and 2.8-fold at 100 μg / mL (p < 0.0001). These results suggest that FMCE is more effective in activating the PI3K-AKT pathway compared to MCE.

[0211] eNOS phosphorylation was significantly increased only in the FMCE-treated group, while the MCE-treated group showed no statistically significant difference compared to the control group. The p-eNOS / eNOS ratio in the FMCE-treated group increased approximately 5.2-fold (p < 0.001) at 20 μg / mL and approximately 5.7-fold (p < 0.0001) at 100 μg / mL, which is similar to the 6.9-fold increase observed in the positive control (DAA). The results demonstrate that FMCE promotes eNOS phosphorylation through AKT activation, thereby increasing nitric oxide production.

[0212] FMCE significantly increased the phosphorylation levels of PI3K, AKT, and eNOS, thereby activating the nitric oxide production pathway as shown in Figure 1.

[0213]

[0214] Experimental Example 7. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Analysis

[0215] Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the effects of MCE, FMCE, and DAA on mRNA expression levels associated with variables contributing to the improvement of arterial relaxation. After removing the culture medium, HUVECs were washed twice with HEPES solution. 1 mL of TRIzol® was used per well for total RNA extraction. 200 μL of chloroform was added, and the cells were incubated for 15 minutes to disrupt the cell membranes. The samples were centrifuged at 13,200 rpm at 4°C for 15 minutes. To precipitate RNA, an equal volume of isopropanol was added to the aqueous layer, and the samples were stored at -20°C for 12 hours. After centrifugation at 13,200 rpm at 4°C for 15 minutes, the RNA pellet was purified by adding 1 ml of 75% ethanol to DEPC distilled water. After centrifuging again at 13,200 rpm for 15 minutes, the ethanol was removed. The RNA pellet was treated with DEPC distilled water containing DNase and incubated at 37°C for 10 minutes. The amount of RNA was measured using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, USA). To synthesize cDNA, 2 μg of RNA, 1 μL of 50 μM Oligo d(T)20 primer, and 1 μL of a 10 mM dNTP mixture were mixed to a total reaction volume of 13 μL. This mixture was incubated in an iCycler Thermal Cycler (Bio-Rad, Hercules, CA, USA) at 65°C for 5 minutes. Next, RNaseOUT was added to the reaction mixture. TM1 μL of recombinant RNase inhibitor, 1 μL of SuperScript® IV reverse transcriptase, 4 μL of 5x SSIV buffer, and 1 μL of 100 mM DTT were added. Sample reactions were conducted at 23°C, 55°C, and 80°C for 10 minutes each. Real-Time PCR Master Mix (Biofact, Daejeon, Korea) was used for qRT-PCR amplification. Each reaction was prepared with a final volume of 20 μL, containing 10 μL of 2x qPCR Master Mix, 1 μL of forward primer, 1 μL of reverse primer (10 pM / μL), 1 μL of cDNA, and DEPC distilled water. Reactions on the PCR plates were analyzed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The PCR setup consisted of initial denaturation at 95°C for 15 minutes, followed by 39 cycles of 15 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C.

[0216] For melting curve analysis, the temperature was increased stepwise from 65°C to 95°C in 0.5°C increments. Relative gene expression levels were evaluated using the 2-△△CT method with β-actin as an internal control (Livak & Schmittgen, 2001).

[0217] The primer sequences used for quantitative real-time PCR and reverse transcription polymerase chain reaction (qRT-PCR) are listed in Table 3 below.

[0218]

[0219] All data were measured independently at least three times and analyzed using GraphPad Prism 8.0.

[0220] All data were subjected to one-way ANOVA followed by a Dunnett test. A P value < 0.05 was considered statistically significant.

[0221]

[0222] Figures 9 and 10 are graphs showing the results of analyzing the effects of fermented noni extract and noni extract on AKT mRNA and eNOS mRNA expression levels in HUVEC. Values ​​are expressed as mean ± standard deviation. (** p < 0.0001 (compared to control group))

[0223] The present invention demonstrates that noni extract promotes nitric oxide release through the PI3K / AKT-eNOS pathway. This was confirmed by evaluating the levels of AKT and eNOS mRNA expression in HUVECs treated with MCE, FMCE, and DAA. Human umbilical vein endothelial cells (HUVECs) were treated with MCE (0 μg / mL, 20 μg / mL, and 100 μg / mL), FMCE (0 μg / mL, 20 μg / mL, and 100 μg / mL), and DAA (20 μg / mL) for 24 hours. After extracting total mRNA, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed.

[0224]

[0225] qRT-PCR results showed a strong correlation between AKT mRNA expression and nitric oxide production, which increased in a dose-dependent manner in the MCE and FMCE treatment groups.

[0226] Referring to Figure 9, compared to the control group, AKT expression in the MCE-treated group did not show a significant change at a concentration of 20 μg / mL, but increased significantly by approximately 1.4 times at a concentration of 100 μg / mL (p < 0.01). FMCE treatment showed an even more enhanced effect, with AKT expression increasing 1.5 times (p < 0.001) at 20 μg / mL and 1.8 times (p < 0.0001) at 100 μg / mL. The positive control, DAA (20 μg / mL), showed the highest expression level, increasing by approximately 2.0 times. The results demonstrate that FMCE significantly increases AKT mRNA expression compared to MCE, suggesting that FMCE may play a potential role in promoting nitric oxide production through the PI3K / AKT pathway.

[0227] Referring to Figure 10, eNOS mRNA expression similarly increased in a dose-dependent manner in both the MCE and FMCE treatment groups. Compared to the control group, eNOS expression in the MCE treatment group increased 1.4-fold at 20 μg / mL and 1.5-fold at 100 μg / mL (p < 0.0001). In the FMCE treatment group, expression levels increased significantly, rising 1.8-fold at 20 μg / mL and 2.1-fold at 100 μg / mL (p < 0.0001). The positive control, DAA (20 μg / mL), showed the highest eNOS expression, which is a 2.3-fold increase. These data demonstrate that FMCE promotes nitric oxide production through the upregulation of eNOS expression, thereby inducing arterial relaxation and improving endothelial function.

[0228] Increased AKT and eNOS mRNA expression indicates the activation of vasodilation signaling pathways associated with nitric oxide production. Figure 1 illustrates the mechanism of the PI3K / AKT-eNOS pathway. This study suggests that FMCE has a superior ability to promote nitric oxide production compared to MCE by increasing the expression levels of AKT and eNOS. FMCE significantly increases the expression of vasodilation-related genes to levels equivalent to those observed in the positive control, DAA. These findings suggest that FMCE can act as a potential bioactive substance that promotes vasodilation.

[0229] As described above, in Experimental Examples 1 to 7, the vasodilating and antioxidant properties of MCE and FMCE, which are functional materials for improving endothelial function, were analyzed and the mechanism of action was elucidated.

[0230] 1) HPLC analysis results showed that FMCE had a higher concentration of the active compound deacetylasperulosidic acid (DAA) compared to MCE, which was expected to be related to the activation of the PI3K / Akt-eNOS pathway associated with nitric oxide production.

[0231] 2) Analysis of total polyphenol content (TPC), total flavonoid content (TFC), and radical scavenging activity (ABTS, DPPH) showed that FMCE exhibited higher antioxidant activity than MCE. This suggests that FMCE may help alleviate impaired vasodilation caused by oxidative stress.

[0232] 3) As a result of nitric oxide analysis, it was confirmed that FMCE significantly increased nitric oxide production and exhibited a vasodilating effect through the activation of the PI3K / Akt-eNOS pathway.

[0233] 4) Western blot analysis of endothelial cells treated with MCE and FMCE showed that AKT phosphorylation levels significantly increased in both groups. eNOS phosphorylation significantly increased only in the FMCE-treated group.

[0234] 5) Quantitative reverse transcription PCR analysis showed that FMCE significantly increased the mRNA expression levels of AKT and eNOS, suggesting that FMCE is involved in improving endothelial cell function.

[0235] These results indicate that FMCE induces vasodilation in HUVECs by promoting nitric oxide production through the PI3K / Akt-eNOS signaling pathway. FMCE appears to be a promising functional food ingredient that enhances endothelial vasodilation compared to MCE.

[0236]

[0237] Experimental Example 8. Evaluation Test of Nitric Oxide Production Capacity in Raw 264.7 Cell

[0238] Figure 11 is a graph showing the effect of fermented noni extract on nitric oxide production ability by Raw 264.7 cells.

[0239] Referring to Fig. 11, inflammation was induced using LPS (Lipopolysaccharide), and then fermented red ginseng (FRE, commercially available), Comparative Example 1 (Noni extract, MCE), and Example 1 (fermented noni extract, FMCE) were treated to confirm the anti-inflammatory effect in LPS-treated cells. As a result of the experiment, it was confirmed that Example 1 of the present invention exhibited superior nitric oxide production ability compared to fermented red ginseng and unfermented noni extract.

[0240]

[0241] Experimental Example 9. Evaluation of efficacy in SHR rat, an animal model of essential hypertension

[0242] To confirm the physiological effects and long-term safety of FMCE, an efficacy evaluation test was conducted in SHR rats, an animal model of essential hypertension.

[0243] The goal was to evaluate changes in systolic / diastolic blood pressure and blood Angiotensin II and Renin concentrations by repeatedly oral administration of lactic acid bacteria-fermented noni extract (FMCE) to male SHR rats, and to confirm safety indicators through organ weight (liver, kidney, heart), body weight, and clinical observation.

[0244] The test substances are as shown in Tables 4 and 5 below.

[0245]

[0246]

[0247] During the acclimatization and experimental period, two animals were housed in individually ventilated cages in a rearing room equipped with environmental conditions of 23±3 ℃, 50±20% relative humidity, 10–15 ventilation cycles / hour, 12 hours of lighting, and 150–200 Lux illuminance. Sterilized feed and drinking water were provided via free feeding. Water bottles were replaced three times a week.

[0248] The test group was composed as shown in Table 6 below.

[0249]

[0250] The test substance was prepared and administered immediately before use on the day of administration.

[0251] Reflecting the solid content (56.2%) of the lactic acid bacteria fermented noni extract (FMCE), a homogeneous suspension was prepared using sterile distilled water as an excipient, in accordance with the dosage in Table 4.5 above. After adding sterile distilled water, a vortex was performed for 5 minutes, and additional vortexing was performed if necessary.

[0252] Potassium losartan was prepared by dissolving it in sterile distilled water at a concentration of 2 mg / mL. If the solution was not colorless and clear after vortexing for 5 minutes following the addition of sterile distilled water, additional vortexing was performed. If necessary, the pH was checked and fine-tuned.

[0253] The test substance was administered intragastrically using an oral gavage according to the set dosage (mg / kg) and volume (10 mL / kg) for each group.

[0254] For the analysis of blood markers (Angiotensin II, Renin), blood concentrations of Angiotensin II and Renin were measured using collected plasma. Measurements were performed using a rat Angiotensin II (Ang II) ELISA Kit (Elabscience®, E-EL-R1430) and a rat REN (Renin) ELISA Kit (Elabscience®, E-EL-R3075), with the plasma diluted by half for each measurement. The analysis was conducted in compliance with the manufacturer's manual for each kit.

[0255] Blood pressure data was analyzed using a RM two-way ANOVA (mixed effects) on the Time × Group factor followed by a Dunnett post-hoc test. Angiotensin II and Renin were analyzed using a one-way ANOVA followed by a Dunnett post-hoc test. Organ weights were analyzed using a Kruskal-Wallis test followed by a Dunn post-hoc test because normality and homogeneity of variances were not satisfied. The significance level was set at p<0.05. The analysis was performed using GraphPad Prism 10.0 with a statistical significance level of p<0.05.

[0256]

[0257] The antihypertensive efficacy and changes in blood Renin and Angiotensin II concentrations following repeated oral administration of lactic acid bacteria-fermented noni extract (FMCE) for 4 weeks were evaluated using male SHR rats and Wistar rats.

[0258] No mortality or specific clinical abnormalities were observed in any group during the study period, and since the FMCE administration group did not show significant changes in body weight and relative body weight compared to the Vehicle, it is concluded that FMCE did not affect toxicity or metabolic activity even under high-concentration administration conditions.

[0259] 9-1. Weight Measurement Results

[0260] Figures 12 and 13 are graphs showing changes in absolute and relative body weight of experimental animals upon oral administration of fermented noni extract (FMCE) according to one embodiment of the present invention. Referring to Figures 12 and 13, Table 7, and Table 8, the results of body weight measurements showed that FMCE administration did not induce a significant change in body weight among SHR rats in the comparison of absolute body weight, and no significant difference in body weight was observed in any of the test groups in the comparison of relative body weight, so it was determined that the test substance did not have a negative effect on metabolic activity, including feeding. In the following, G1 refers to Wistar_Vehicle, G2 to SHR_Vehicle, G3 to SHR_Losartan potassium, G4 to SHR_FMCE_300mg / kg, and G5 to SHR_FMCE_600mg / kg.

[0261] Data were expressed as mean ± standard deviation (SD). Two-way RM ANOVA (mixed-effects) was used, and Dunnett's multiple comparisons were performed in comparison with G2_SHR_Vehicle (control group). A p < 0.05 value was considered significant. ** indicates p < 0.01 and *** indicates p < 0.001, and all results are compared to G2_SHR_Vehicle (control group).

[0262]

[0263]

[0264] 9-2. Blood Pressure and Pulse Rate Measurement Results

[0265] FIGS. 14 to 17 are graphs showing changes in blood pressure and heart rate of experimental animals when a fermented noni extract (FMCE) according to one embodiment of the present invention is administered orally.

[0266] Blood pressure and pulse rate measurements were performed using a non-invasive tail-cuff method, and the deviation between measurements was minimized for each individual through 5 days of prior adaptation training. Blood pressure and pulse rate measurements were performed at weeks 0, 1, 2, 3, and 4, and valid systolic blood pressure (SBP), mean blood pressure (MBP), diastolic blood pressure (DBP), and pulse rate were measured three times for each individual and reflected in the average and statistics.

[0267] 9-2-1. Systolic Blood Pressure

[0268] Referring to Figure 14, the G1_Wistar_Vehicle group showed significantly lower blood pressure compared to the G2_SHR_Vehicle group at all time points (p < 0.001).

[0269] In the G3_SHR_Losartan potassium group (179.07±10.86 mmHg), blood pressure was significantly reduced from week 1 of administration compared to the G2_SHR_Vehicle group (214.27±17.85 mmHg) (p < 0.001) and continued until week 4 (163.47 ± 8.52 mmHg, p < 0.001).

[0270] In the G4_SHR_FMCE_300mg / kg group (191.80±7.66 mmHg), blood pressure was significantly reduced from week 3 of administration compared to the G2_SHR_Vehicle group (218.73±13.28 mmHg) (p < 0.001) and continued until week 4 (195.60 ±3.62 mmHg, p < 0.01).

[0271] In the G5_SHR_FMCE_600mg / kg group (194.60±6.75 mmHg), blood pressure was significantly reduced compared to the G2_SHR_Vehicle group starting from week 3 of administration (p < 0.001) and continued until week 4 (193.87±4.31 mmHg, p < 0.001).

[0272] Data were expressed as mean ± standard deviation (SD). Two-way RM ANOVA (mixed-effects) was used, and Dunnett's multiple comparisons were performed in comparison with G2_SHR_Vehicle (control group). A p < 0.05 value was considered significant. ** indicates p < 0.01 and *** indicates p < 0.001, and all results are compared to G2_SHR_Vehicle (control group).

[0273]

[0274] 9-2-2. Average Blood Pressure

[0275] Referring to Figure 15 and Table 10, the G1_Wistar_Vehicle group showed a statistically significantly lower mean blood pressure compared to the G2_SHR_Vehicle group at all time points (p < 0.001).

[0276] The G3_SHR_Losartan potassium group (157.42±11.31 mmHg) was significantly reduced compared to the G2_SHR_Vehicle group (180.18±19.42 mmHg) starting from week 1 of administration (p < 0.01) and continued until week 4 (142.02±7.96 mmHg, p < 0.001).

[0277] The G4_SHR_FMCE_300mg / kg group (165.16±10.51 mmHg) and the G5_SHR_FMCE_600mg / kg group (164.42±4.31 mmHg) showed a significant decrease compared to the G2_SHR_Vehicle group (185.73±13.79 mmHg) starting from week 3 of administration (p < 0.001), and the blood pressure lowering effect persisted in the G4_SHR_FMCE_300mg / kg (165.11±5.14 mmHg) and G5_SHR_FMCE_600mg / kg (162.13±3.53 mmHg) groups compared to the G2_SHR_Vehicle group (179.31±11.22 mmHg) at week 4, respectively (p < 0.001).

[0278] Data were expressed as mean ± standard deviation (SD). Two-way RM ANOVA (mixed-effects) was used, and Dunnett's multiple comparisons were performed against G2_SHR_Vehicle (control group). A p < 0.05 value was considered significant.

[0279] ** indicates p < 0.01 and *** indicates p < 0.001, and all are results compared to G2_SHR_Vehicle (control group).

[0280]

[0281] 9-2-3. Diastolic blood pressure

[0282] Referring to Figure 16, the G1_Wistar_Vehicle group showed significantly lower diastolic blood pressure compared to the G2_SHR_Vehicle group throughout the entire period (p < 0.001).

[0283] Compared to the G2_SHR_Vehicle group (163.13±20.64 mmHg), the G3_SHR_Losartan potassium group (146.60±12.52 mmHg) began to show a decrease (p < 0.05), followed by a continuous decrease at week 2 (143.33±13.21 mmHg, p < 0.01), week 3 (137.73±7.14 mmHg, p < 0.001), and week 4 (131.33±8.67 mmHg, p < 0.001).

[0284] A statistically significant decrease was observed in the G4_SHR_FMCE_300 mg / kg group (151.87±13.74 mmHg, p < 0.05) and the G5_SHR_FMCE_600 mg / kg group (149.33±5.56 mmHg, p < 0.001) compared to G2_SHR_Vehicle (169.27±15.38 mmHg) starting from week 3, and the reduction effect continued in the G4_SHR_FMCE_300 mg / kg group (149.87±6.31 mmHg, p < 0.01) and the G5_SHR_FMCE_600 mg / kg group (146.27±3.90 mmHg, p < 0.001) compared to G2_SHR_Vehicle (164.33±11.64 mmHg) at week 4, respectively.

[0285] Data were expressed as mean ± standard deviation (SD). Two-way RM ANOVA (mixed-effects) was used, and Dunnett's multiple comparisons were performed in comparison with G2_SHR_Vehicle (control group). A p value of < 0.05 was considered significant. ** indicates p < 0.01 and *** indicates p < 0.001, and all results are compared to G2_SHR_Vehicle (control group).

[0286]

[0287] 9-2-4. Pulse rate

[0288] Referring to Figure 17 and Table 12, the pulse rate of the G1_Wistar_Vehicle group was significantly higher than that of the G2_SHR_Vehicle group until week 3 (p < 0.05 to p < 0.001), but the average pulse rate remained within the generally reported normal range (approx. 330-480 bpm).

[0289] The G3_SHR_Losartan potassium, G4_SHR_FMCE_300mg / kg, and G5_SHR_FMCE_600mg / kg groups did not show a statistically significant difference compared to the G2_SHR_Vehicle group at any time point.

[0290] Therefore, as a result of repeated oral administration of FMCE for 4 weeks, systolic, mean, and diastolic blood pressure in SHR rats all showed a decreasing trend, and this effect was significantly reduced starting from week 3 (p < 0.05~0.001) and continued until week 4.

[0291] In the Losartan positive control group as well, a significant blood pressure-lowering effect was confirmed starting from week 1 of administration, confirming the adequacy of the study system. Although there were groups with lower average heart rates at some early time points (particularly weeks 0-2), this may be due to variations caused by adaptation to repeated measurements and tail-cuff characteristics, and by week 4, all groups converged to similar levels.

[0292] Data were expressed as mean ± standard deviation (SD). Two-way RM ANOVA (mixed-effects) was used, and Dunnett's multiple comparisons were performed in comparison with G2_SHR_Vehicle (control group). A p < 0.05 value was considered significant. ** indicates p < 0.01 and *** indicates p < 0.001, and all results are compared to G2_SHR_Vehicle (control group).

[0293]

[0294] 9-3. Results of Autopsy Organ Weight Measurement

[0295] FIGS. 18 and 19 are graphs showing changes in absolute organ weight and relative organ weight (liver, kidney, heart) of experimental animals when a fermented noni extract (FMCE) according to one embodiment of the present invention is administered orally.

[0296] In this study, toxicological changes were evaluated by measuring the weight of major organs, such as the liver, kidneys, and heart, following repeated oral administration of FMCE. Changes in organ weight caused by the test substance were assessed by calculating both absolute organ weight and body weight correction values ​​(relative organ weight).

[0297] 9-3-1. Absolute Organ Weight

[0298] Referring to Figure 18 and Table 13, the liver, left and right kidneys, and heart weights of the G1_Wistar_Vehicle group were 11.23±1.62 g, 1.37±0.09 g, 1.36±0.07 g, and 1.25±0.06 g, respectively.

[0299] The liver, left and right kidneys, and heart weights of the G2_SHR_Vehicle group were confirmed to be 12.02±1.17 g, 1.26±0.10 g, 1.22±0.12 g, and 1.29±0.07 g, respectively.

[0300] The G3_SHR_Losartan potassium group did not show a statistically significant difference in all organs compared to the G2_SHR_Vehicle group.

[0301] The weight of the right kidney (1.38±0.10 g) in the G4_SHR_FMCE_300mg / kg group was statistically significantly increased compared to the G2_SHR_Vehicle group (1.22±0.12 g) (p < 0.05), but no significant difference was found in the weight of the liver (11.98±0.33 g), left kidney (1.39±0.09 g), and heart (1.41±0.09 g).

[0302] The liver, left and right kidneys, and heart weights of the G5_SHR_FMCE_600mg / kg group were 10.85±0.50 g, 1.28±0.09 g, 1.29±0.04 g, and 1.35±0.06 g, respectively, with no statistically significant difference observed compared to the G2_SHR_Vehicle group.

[0303] Data were expressed as mean ± standard deviation (SD). Dunn's multiple comparison test (multiplicity-corrected p-value) was performed after the Kruskal-Wallis test (α=0.05). * indicates p < 0.05.

[0304]

[0305] 9-3-2. Opponent's Janggi Weight

[0306] Referring to Figure 19 and Table 14, relative organ weight was calculated by dividing the measured organ weight (g) by the individual's final body weight, so relative organ weight (%) = (organ weight / body weight) × 100.

[0307] The liver, left and right kidney, and heart ratios in the G2_SHR_Vehicle group were 3.73±0.32%, 0.39±0.03%, 0.38±0.03%, and 0.40±0.02%, respectively.

[0308] The liver, left and right kidney, and heart ratios of the G3_SHR_Losartan potassium group were 3.52±0.19%, 0.40±0.02%, 0.38±0.01%, and 0.39±0.02%, respectively, which were similar to those of the G2_SHR_Vehicle group.

[0309] The liver, left and right kidney, and heart ratios of the G4_SHR_FMCE_300mg / kg group were 3.62±0.06%, 0.42±0.03%, 0.42±0.03%, and 0.43±0.02%, respectively, and no statistically significant difference was found compared to the G2_SHR_Vehicle group.

[0310] The liver, left and right kidney, and heart ratios of the G5_SHR_FMCE_600mg / kg group were 3.33±0.10%, 0.39±0.02%, 0.40±0.02%, and 0.42±0.02%, respectively, showing no significant difference compared to the G2_SHR_Vehicle group.

[0311] Therefore, as a result of repeated oral administration of FMCE for 4 weeks, no statistically significant changes were observed in the absolute and relative weights of major organs such as the liver, kidneys, and heart. A slight increase in absolute weight (p < 0.05) was observed only in the right kidney of the G4_SHR_FMCE_300 mg / kg group, but no consistent changes were observed in relative weight or other organs.

[0312] Data were expressed as mean ± standard deviation (SD). Dunn's multiple comparison test (multiplicity-corrected p-value) was performed after the Kruskal-Wallis test (α=0.05). * indicates p < 0.05.

[0313]

[0314] 9-4. Results of Blood Angiotensin II and Renin Concentrations

[0315] FIGS. 20 and 21 are graphs showing changes in blood Angiotensin II concentration and blood Renin concentration in experimental animals when a fermented noni extract (FMCE) according to one embodiment of the present invention is orally administered.

[0316] To evaluate the antihypertensive efficacy of FMCE at the protein level, changes in Angiotensin II (Ang II) and Renin concentrations were measured by analyzing plasma obtained at autopsy after administering the test substance for 4 weeks.

[0317] 9-4-1. Blood Angiotensin II Concentration

[0318] Referring to Figure 20 and Table 15, the G1_Wistar_Vehicle group (19.91±5.54 pg / mL) showed a significantly lower concentration compared to the G2_SHR_Vehicle group (95.98±35.32 pg / mL) (p < 0.0001).

[0319] The G3_SHR_Losartan potassium group (128.71±14.37 pg / mL) showed a significantly higher concentration compared to the G2_SHR_Vehicle group (p < 0.05).

[0320] No significant difference was observed between the G4_SHR_FMCE_300mg / kg group (97.64±18.66 pg / mL) and the G2_SHR_Vehicle group.

[0321] The G5_SHR_FMCE_600mg / kg group (38.27±5.73 pg / mL) showed a statistically significantly lower concentration compared to the G2_SHR_Vehicle group (95.98±35.32 pg / mL) (p < 0.001).

[0322] Data were expressed as mean ± standard deviation (SD). Dunnett's multiple comparisons were performed after one-way ANOVA compared with G2_SHR_Vehicle (control group). * indicates p < 0.05, *** indicates p < 0.001, and **** indicates p < 0.0001, all representing results compared to G2_SHR_Vehicle (control group).

[0323]

[0324] 9-4-2. Blood Renin Concentration

[0325] Referring to Figure 21 and Table 16, the G1_Wistar_Vehicle group (74.84±5.14 pg / mL) was statistically significantly higher than the G2_SHR_Vehicle group (60.06±2.25 pg / mL) (p < 0.001).

[0326] G3_SHR_Losartan potassium group (10 4.13±4.51 pg / mL) was statistically significantly increased compared to the G2_SHR_Vehicle group (p < 0.0001).

[0327] On the other hand, no significant difference was observed between the G4_SHR_FMCE_300mg / kg group (60.03±6.16 pg / mL) and the G5_SHR_FMCE_600mg / kg group (57.48±3.98 pg / mL) and the G2_SHR_Vehicle group.

[0328] Data were expressed as mean ± standard deviation (SD). After one-way ANOVA, Dunnett's multiple comparisons were performed in comparison with G2_SHR_Vehicle (control group). *** indicates p < 0.001 and **** indicates p < 0.0001, and all results are compared to G2_SHR_Vehicle (control group).

[0329]

[0330] Therefore, in the high-dose FMCE group, blood Angiotensin II concentrations significantly decreased (p<0.001), while Renin levels remained at control levels. In contrast, an increase in blood Angiotensin II and Renin concentrations was observed in the Losartan group, showing a different trend from FMCE.

[0331]

[0332] Therefore, the efficacy evaluation of lactic acid bacteria-fermented noni extract (FMCE) in the SHR hypertension model conducted in this study demonstrated a significant reduction in blood pressure and angiotensin II, while having no effect on renin concentration. Furthermore, as no significant abnormalities were observed in systemic toxicity indicators such as clinical results, body weight, and organ weight measurements, it is evaluated as a safe candidate antihypertensive agent.

[0333]

[0334] Experimental Example 10. Analysis of Nitric Oxide Production Capacity of Spinach, Onion, Carrot, Garlic, Celery, and Beet by Fermentation Strain

[0335] Figure 22 is a graph showing the nitric oxide production ability of spinach, onion, carrot, garlic, celery, and beet by fermentation strain.

[0336] Spinach, onion, carrot, garlic, celery, and beet were each crushed and mixed with purified water to a concentration of 3 to 10% (w / v), then sterilized, and then inoculated with a pre-prepared Staphylococcus and Bacillus culture solution to a concentration of 1 to 5% (v / v), and fermented and aged at 30 to 40°C for 15 to 20 hours.

[0337] After fermentation, the filtrate was extracted and filtered, concentrated to a Brix level of 10° to 20°, and then freeze-dried to obtain beet fermentation powder, and the nitric oxide production capacity was analyzed.

[0338] Referring to Figure 22, it was confirmed that the nitric oxide production ability of celery and beet fermentation products is excellent, and that the nitric oxide production ability is excellent when using Bacillus culture medium, and that celery shows consistently excellent nitric oxide production ability when fermented for 3 to 5 days, and beet shows excellent nitric oxide production ability for 1 to 6 days.

[0339]

[0340] Experimental Example 11. Analysis of Nitric Oxide Production Ability of a Mixture of Fermented Noni Extract and Fermented Beet Extract

[0341] 11-1. Cell viability of a mixture of fermented noni extract and fermented beet extract

[0342] 1) Experimental Method

[0343] The cell viability test (MTT assay) was based on the activity of the succinate dehydrogenase enzyme within the mitochondria of living cells. Living cells reduce MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) from a water-soluble inactive salt (yellow) to insoluble formazan crystals (purple). Therefore, the intensity of the measured absorbance is proportional to the number of living cells.

[0344] 2) Cell viability measurement

[0345] 1×10 HUVEC 4 Cells were seeded in a 96-well plate at a concentration of cells / well and pre-cultured in an incubator (37 ℃, 5% CO₂) for 24 hours. Subsequently, fermented noni / fermented beet mixtures (Control, 1:1, 1:2, 2:1) were added to each well, and main culture was performed for 24 hours. After the culture was completed, MTT solution (20 μl in 5 mg / ml stock) was added to each well and reacted for 4 hours. After the reaction, the supernatant was removed, and the resulting formazan crystals were dissolved in 200 μl of DMSO and mixed for 10 minutes. Absorbance was measured at 570 nm using a microplate reader. Cell viability (%) was calculated by comparing the absorbance values ​​of the control group and the experimental group.

[0346]

[0347] To investigate the cell viability of HUVECs, various ratios of fermented noni / fermented beet mixtures (Control, 1:1, 1:2, 2:1) were used. After treating HUVECs with the fermented noni / fermented beet mixture for 24 hours, MTT solution was applied. The results are shown in Figure 23.

[0348] FIG. 23 is a graph showing the cell viability of HUVECs treated with a mixture of fermented noni extract (FMCE) and fermented beet extract according to one embodiment of the present invention. Here, HUVEC (1 x 10 6 After treating HUVEC cells (in cells / ml) with various ratios of fermented noni / fermented beet mixtures for 24 hours, cell viability values ​​were expressed as mean ± standard deviation, and statistically significant differences between groups (p < 0.005) were determined using one-way ANOVA and Dunnett's multiple range test. (Compared to the control group, **p < 0.005, ***p < 0.001, **** p < 0.0001)

[0349] Referring to Fig. 23, the experimental results showed that the cell viability of HUVECs with various ratios of fermented noni / fermented beet mixtures was 90% or higher compared to the control group for all mixture ratios, and it was determined that there was no serious toxicity. Therefore, in the present invention, the ratio of the fermented noni and fermented beet mixture was set to Control / 1:1 / 1:2 / 2:1.

[0350]

[0351] 11-2. Analysis of eNOS Expression Levels in Fermented Noni / Fermented Beet Extracts

[0352] 1) Experimental Method

[0353] Proteins are separated by size using an SDS-PAGE gel, and then specific proteins are detected from the total protein using an antigen-antibody reaction.

[0354] 2) Measure eNOS protein expression levels using western blot

[0355] 1 x 10 HUVEC 6After seeding into a 6-well plate at a concentration of cells / ml, pre-culture was performed in an incubator (37℃, 5% CO2) for 24 hours. After incubation, cold-fermented noni / fermented beet mixtures (Control, 1:1, 1:2, 2:1) were added, and main culture was performed for 24 hours. The 6-well plate was kept cold, and lysis was performed using lysis buffer. Pierce TM Protein content of each sample was quantified using the BCA Protein kit (Thermo Fisher Scientific, Waltham, MA, USA). 20 µL of sample was loaded onto 10% SDS-polyacrylamide gels and electrophoresis was performed at 80V for 2 hours. After transferring to a PVDF membrane (Sigma-Aldrich Co., St. Louis, MO, USA) at 80V for 2 hours, the membrane was blocked with 5% skim milk for 1 hour. Antibodies for eNOS (cell signaling, #19615) and β-actin (cell signaling, #4967) were conjugated (4℃ overnight). The membrane was incubated with horseradish peroxidase-conjugated secondary anti-mouse / rabbit IgG (Cell Signaling Tech., Danvers, MA, USA) for 1 hour. Clarity TM Detection was performed using a western ECL Substrate (Bio-Rad, Hercules, CA, USA). Protein bands were imaged using ChemiDoc (Bio-Rad, Hercules, CA, USA), and the expression levels of e-NOS protein relative to β-actin were calculated using the Image J program and are shown in Figure 24.

[0356] FIG. 24 is a figure showing the results of analyzing the effect on eNOS protein expression after treating HUVEC with a mixture of fermented noni extract (FMCE) and fermented beet extract according to one embodiment of the present invention. Here, HUVEC (1 x 10 6 After treating cells (cells / ml) with various ratios of fermented noni / fermented beet mixtures for 24 hours, the eNOS protein expression level was measured.

[0357] Referring to Figure 24, the experimental results showed that the expression of eNOS protein increased significantly starting from the 1:1 ratio of fermented noni to fermented beet compared to the control group. It was also confirmed that eNOS was expressed at higher levels than the control group in the 1:2 and 2:1 mixing ratio groups. In particular, the strongest eNOS expression was observed in the 2:1 mixing ratio, and since β-actin expression was similar in all experimental groups, the increase in eNOS expression can be attributed to the effect of the sample treatment. Therefore, it was confirmed that the mixed treatment of fermented noni extract and fermented beet extract has a synergistic effect in promoting eNOS protein expression.

[0358]

[0359] 11-3. Measurement of Nitric Oxide Production of Fermented Noni / Fermented Beet Juice Mixture via NO Assay

[0360] 1) Experimental Method

[0361] When NO2-(nitrate) reacts with sulfanilamide, it undergoes a Griess diazotization reaction to form diazonium salt, and when this diazonium salt combines with N-(1-naphthyl)ethylenediamine, it undergoes an azo coupling reaction to produce an azo dye. This azo dye exhibits a red-pink color, and becomes redder as the amount of nitric oxide produced in the sample increases.

[0362] 2) Measure nitric oxide production using an NO assay

[0363] 1 x 10 HUVEC 6 After seeding into a 6-well plate at a concentration of cell / ml, pre-culture was performed in an incubator (37℃, 5% CO2) for 24 hours. After culture, diluted frozen / fermented noni samples (25, 50, 100 µg / ml) were added, and main culture was performed for 24 hours. For each sample, 100 µl of the supernatant was dispensed into a 96-well plate. NO 2- 100 µl of the standard curve (STD) solution for concentration was dispensed into 96 wells. 100 µl of Griess reagent was dispensed into the sample and STD, respectively.

[0364] The samples were incubated in an incubator for 15 minutes, and the absorbance was measured at 540 nm. The results are shown in Figure 25.

[0365] FIG. 25 is a graph showing the results of analyzing the effect of nitric oxide production in HUVECs treated with a mixture of fermented noni extract (FMCE) and fermented beet extract according to one embodiment of the present invention. Here, HUVECs (1×10 6 Nitric oxide production was measured after treating cells (in cells / ml) with various ratios of fermented noni / fermented beet mixtures for 24 hours. Values ​​were expressed as mean ± standard deviation. Statistically significant differences between groups (p < 0.005) were determined using one-way analysis of variance and Dunnett's multiple comparison test (compared to the control group, *p < 0.01, **p < 0.005, ***p < 0.001).

[0366] Referring to Figure 25, the analysis of the effect of treatment with a mixture of fermented noni and fermented beet on nitric oxide production showed that nitric oxide production significantly increased in all 1:1, 1:2, and 2:1 groups compared to the control group. In particular, at the 1:2 and 2:1 mixing ratios, the nitric oxide concentration increased most significantly to over 6 μM. This indicates that a statistically significant difference was observed in the groups marked with an asterisk (*). These results suggest that fermented noni and beet may be effective in promoting nitric oxide production depending on the mixing ratio.

[0367] Therefore, through Experimental Example 11, a scientific basis for evaluating the blood circulation improvement of a mixture of fermented noni extract and fermented beet extract was established, scientific data on the blood circulation improvement effect of a complex extract mixture of fermented noni and fermented beet was secured, and it was confirmed that the blood circulation improvement effect is good when the ratio of fermented noni to fermented beet is 2:1.

[0368]

[0369] Although an embodiment of the present invention has been described above, those skilled in the art may modify and change the present invention in various ways by adding, changing, deleting, or adding components, etc., without departing from the spirit of the present invention as described in the claims, and such modifications and changes are also to be included within the scope of the rights of the present invention.

[0370]

[0371] [Correction pursuant to Rule 91 27.07.2026][Entrustment Number]

[0372] [Correction pursuant to Rule 91 27.07.2026] Name of depositing institution: Korean Culture Collection of Microorganisms (International)

[0373] [Correction pursuant to Rule 91 27.07.2026] Trustee No.: KCCM12833P

[0374] [Correction pursuant to Rule 91 27.07.2026] Date of Consignment: 20201117

[0375] [Correction pursuant to Rule 91 July 27, 2026]

Claims

1. A food composition for preventing and / or improving vascular diseases, comprising a fermented noni extract fermented with lactic acid bacteria including Lactobacillus plantarum.

2. In Paragraph 1, A food composition for preventing and / or improving vascular diseases, wherein the above lactic acid bacteria further comprises one or more of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus reuteri, or Lactococcus lactis.

3. In Paragraph 1, The above-mentioned fermented noni extract is a food composition for preventing and / or improving vascular diseases, which increases the production of nitric oxide in the body.

4. In Paragraph 1, The above-mentioned fermented noni extract is a food composition for the prevention and / or improvement of vascular diseases, which activates the signaling pathway mechanism to increase nitric oxide production by activating PI3K to phosphorylate AKT and activating eNOS.

5. In Paragraph 1, The above-mentioned fermented noni extract is a food composition for preventing and / or improving vascular diseases, which relaxes blood vessels.

6. In Paragraph 1, A food composition for preventing and / or improving vascular diseases, further comprising one or more of fermented celery extract or fermented beet extract.

7. A pharmaceutical composition for the prevention and / or treatment of vascular disease comprising a fermented noni extract fermented with lactic acid bacteria including Lactobacillus plantarum.

8. In Paragraph 7, A pharmaceutical composition for the prevention and / or treatment of vascular disease, wherein the above lactic acid bacteria further comprises one or more of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus reuteri, or Lactococcus lactis.

9. A step of inoculating noni with lactic acid bacteria including Lactobacillus plantarum to ferment and mature it; and A method for preparing a composition for preventing and / or improving vascular diseases, comprising the step of preparing a fermented noni extract from the fermented noni obtained by the above fermentation and aging.

10. In Paragraph 8, The above fermentation and aging steps are, The above lactic acid bacteria are inoculated at 1 to 5 weight percent relative to the above noni, and A method for preparing a composition for preventing and / or improving vascular diseases, comprising fermenting and aging at 35 to 40°C for 30 to 90 days.

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