Composition comprising acemannan and method of use thereof

Abstract

To solve the problem in which although a low molecular weight acemannan may possess the potential to enhance efficacy, the acemannan generally available on the market has a molecular weight in the range of 1,000 to 2,000 kDa.SOLUTION: A composition of the present invention is a composition comprising acemannan, which is an acetylated mannose-based polysaccharide compound. The molecular weight of the acemannan is 853 kDa or less.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to compositions comprising acemannan and uses thereof, in particular to the use of the compositions for gut health, skin care, pulmonary care, and eye care. [Background technology]

[0002] Acemannan is an acetylated mannan-based polysaccharide compound found primarily in the mucilaginous gel of Aloe vera leaves. Acemannan consists mainly of β-(1,4) linked mannose residues, partially acetylated at the C-2 and C-3 positions, and may contain additional side chains such as galactose at C-6. Most acemannans contain a single-stranded backbone of β-(1,4) mannose with β-(1,4) glucose inserted into the backbone, and may further contain α-(1,6) galactose branched from the backbone.

[0003] The preparation of acemannan involves its extraction from the mucilaginous gel of Aloe vera leaves, and the composition containing acemannan can be prepared as described in US11654177B2. The purity of acemannan in the composition can affect its efficacy for different indications. In addition to adjusting the method of extracting acemannan from Aloe vera plants to obtain a composition containing acemannan of appropriate purity, formulating the extract with any kind of excipients can also be an alternative to control the purity of acemannan in the composition. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 1,165,4177 Summary of the Invention [Problem to be solved by the invention]

[0005] In addition, the degree of acetylation (e.g., the degree of acetylation) of acemannan or a composition containing acemannan may be another factor in its effectiveness for different indications. Generally, the degree of acetylation in a composition is positively correlated with the content of acemannan, but the degree of acetylation in different compositions may vary.

[0006] In addition, the molecular weight of acemannan also affects its efficacy. Acemannan with a small molecular weight may enhance its absorption by epithelial tissue and promote efficient repair of tight junctions between cells. However, the molecular weight of acemannan generally available on the market is 1,000-2,000 kDa. [Means for solving the problem]

[0007] The composition of the present invention is a composition containing acemannan, which is an acetylated mannan-based polysaccharide compound, and has a molecular weight of 853 kDa (kilodaltons) or less.

[0008] In the composition of the present invention, the molecular weight of the acemannan is 853 kDa (kilodaltons) or less and 5 kDa (kilodaltons) or more.

[0009] In the composition of the present invention, the degree of acetylation, which represents the ratio of the number of acetylated hydroxyl groups to the number of all hydroxyl groups in the acemannan, is 30% or more and 78% or less.

[0010] In the composition of the present invention, the degree of acetylation is 30% or more and 40% or less, or 60% or more and 78% or less.

[0011] A method of using the composition of the present invention is to use the composition comprising acemannan, an acetylated mannan-based polysaccharide compound, for intestinal health.

[0012] The method for using the composition of the present invention is a method for using the composition containing acemannan, an acetylated mannan-based polysaccharide compound, for skin care.

[0013] A method of using the composition of the present invention is to use said composition comprising acemannan, an acetylated mannan-based polysaccharide compound, for lung care.

[0014] A method of using the composition of the present invention is to use the composition containing acemannan, an acetylated mannan-based polysaccharide compound, for eye care. [Brief description of the drawings]

[0015] [Figure 1] 1 is an ATR-FTIR spectrum of composition A50 containing acemannan in an embodiment of the present invention. [Diagram 2] 1 is a 1H-NMR spectrum of composition A50 containing acemannan in an embodiment of the present invention. [Diagram 3] 1 is a 13C-NMR spectrum of composition A50 containing acemannan in an embodiment of the present invention. [Figure 4] FIG. 2 shows the results of HPSEC-RI analysis of composition A50 containing acemannan in an embodiment of the present invention. [Diagram 5] FIG. 2 shows the results of HPLC analysis of composition A50 containing acemannan in an embodiment of the present invention. [Figure 6] FIG. 2 shows the results of a quantitative analysis (UV-Vis spectroscopic analysis) of the O-acetyl content of composition A50 containing acemannan in an embodiment of the present invention. [Figure 7] 1H-NMR spectrum of composition I50 containing acemannan in an embodiment of the present invention. [Figure 8] FIG. 2 shows the results of HPSEC-RI analysis of composition I50 containing acemannan in an embodiment of the present invention. [Figure 9]FIG. 2 shows the results of HPLC analysis of composition I50 containing acemannan in an embodiment of the present invention. [Figure 10] FIG. 2 shows the results of a quantitative analysis (UV-Vis spectroscopic analysis) of the O-acetyl content of composition I50 containing acemannan in an embodiment of the present invention. [Figure 11A] 1 is a graph showing the relationship between the concentration of acemannan in a composition according to an embodiment of the present invention and cell viability (treatment for 24 hours). [Figure 11B] 1 is a graph showing the relationship between the concentration of acemannan in a composition according to an embodiment of the present invention and cell viability in cells (treatment for 48 hours). [Figure 12A] 1 is a graph showing the relationship between the concentration of a composition containing acemannan in an embodiment of the present invention and cell viability in RGM1 cells (24-hour treatment). [Figure 12B] 1 is a graph showing the relationship between the concentration of a composition containing acemannan in an embodiment of the present invention and cell viability in RGM1 cells (48 hour treatment). [Figure 12C] 1 is a graph showing the relationship between the concentration of a composition containing acemannan in an embodiment of the present invention and cell viability in RGM1 cells (treatment for 24 and 48 hours). [Figure 13A] 1 is a graph showing the relationship between the concentration of a composition containing acemannan in an embodiment of the present invention and cell viability in RGM1 cells (treatment for 24 and 48 hours). [Figure 13B] 1 is a graph showing the relationship between the concentration of a composition containing acemannan in an embodiment of the present invention and cell viability in RGM1 cells (treatment for 24 and 48 hours). [Figure 14] 1 is a graph showing the relationship between cytotoxicity in RGM1 cells and incubation time for composition A50 containing acemannan according to an embodiment of the present invention. [Figure 15] 1 is a graph showing the protective effect against cell damage caused by indomethacin in composition A50 containing acemannan in an embodiment of the present invention. [Figure 16]1 is a graph showing the protective effect against cell damage caused by indomethacin in composition A50 containing acemannan according to an embodiment of the present invention. [Figure 17A] 1 is a graph showing the protective effect against cell damage caused by indomethacin in composition A50 containing acemannan according to an embodiment of the present invention. [Figure 17B] 1 is a graph showing the protective effect against cell damage caused by indomethacin in composition A50 containing acemannan according to an embodiment of the present invention. [Figure 18] 1 is a graph showing the protective effect against cell damage caused by indomethacin in composition A50 containing acemannan according to an embodiment of the present invention. [Figure 19A] 1 is a graph showing significant differences in gastric juice weight between groups in a composition containing acemannan according to an embodiment of the present invention. [Figure 19B] 1 is a graph showing significant differences in pH between groups in a composition containing acemannan according to an embodiment of the present invention. [Figure 19C] 1 is a graph showing significant differences in total acidity between groups in a composition containing acemannan according to an embodiment of the present invention. [Figure 20A] FIG. 2 shows the results of pH levels when a composition containing acemannan according to an embodiment of the present invention is administered. [Figure 20B] FIG. 1 shows the results of total gastric acid levels when a composition containing acemannan according to an embodiment of the present invention was administered. [Figure 20C] FIG. 1 shows the results of gastric damage area when a composition containing acemannan according to an embodiment of the present invention was administered. [Figure 20D] FIG. 13 is a graph showing the results of gastric lesion index when a composition containing acemannan according to an embodiment of the present invention was administered. [Figure 20E] FIG. 1 is a graph showing the gastroprotective effect on the gastric mucosa of a composition containing acemannan in an embodiment of the present invention. [Figure 21A]FIG. 2 is a diagram showing the results of a cytotoxicity test using HaCaT cells for a composition containing acemannan in an embodiment of the present invention (dissolved in sterile water). [Figure 21B] FIG. 2 is a diagram showing the results of a cytotoxicity test using HaCaT cells for a composition containing acemannan in an embodiment of the present invention (dissolved in 0.2% ethanol). [Figure 22A] FIG. 2 shows the results of flow cytometry analysis of cell viability in a composition containing acemannan according to an embodiment of the present invention. [Figure 22B] FIG. 2 shows the results of flow cytometry analysis of cell viability in a composition containing acemannan according to an embodiment of the present invention. [Figure 23A] 1 is a graph showing the protective effect of a composition containing acemannan according to an embodiment of the present invention against UVB-induced cytotoxicity in HaCaT cells. [Figure 23B] FIG. 1 shows the results of the protective effect of a composition containing acemannan in an embodiment of the present invention against UVB-induced cytotoxicity in HaCaT cells. [Figure 24] 1 is a graph showing the time-dependent effect of a composition containing acemannan according to an embodiment of the present invention on UVB-induced inflammatory gene expression in HaCaT cells. [Diagram 25] 1 is a graph showing the effect of a composition containing acemannan according to an embodiment of the present invention on the expression of UVB-induced inflammatory genes in HaCaT cells at different irradiation doses. [Figure 26] 1 is a graph showing the effect of treatment with ACE, EGF, LPS, and poly(I:C) on inflammatory gene expression in HaCaT cells in a composition containing acemannan according to an embodiment of the present invention. [Figure 27] 1 is a graph showing the effect of a composition containing acemannan in an embodiment of the present invention not altering MMP-1 gene expression. [Figure 28A] 1 is a graph showing the effect of a composition containing acemannan in an embodiment of the present invention in promoting the proliferation of HaCaT cells. [Figure 28B] 1 is a graph showing the effect of a composition containing acemannan in an embodiment of the present invention in promoting the proliferation of HaCaT cells. [Figure 29A] 1 is a graph showing the effect of a composition containing acemannan in an embodiment of the present invention in accelerating the migration of HaCaT cells. [Figure 29B] 1 is a graph showing the effect of a composition containing acemannan in an embodiment of the present invention in accelerating the migration of HaCaT cells. [Figure 29C] FIG. 1 shows the effect of a composition containing acemannan in an embodiment of the present invention in accelerating the migration of HaCaT cells. [Figure 29D] 1 is a graph showing the effect of a composition containing acemannan in an embodiment of the present invention in accelerating the migration of HaCaT cells. [Figure 30A] FIG. 2 is a graph showing the effect of a composition containing acemannan according to an embodiment of the present invention in inducing keratinocyte differentiation and activating PKC and EGFR. [Figure 30B] FIG. 2 is a graph showing the effect of a composition containing acemannan according to an embodiment of the present invention in inducing keratinocyte differentiation and activating PKC and EGFR. [Figure 30C] FIG. 2 is a graph showing the effect of a composition containing acemannan according to an embodiment of the present invention in inducing keratinocyte differentiation and activating PKC and EGFR. [Diagram 31] FIG. 2 is a graph showing the effect of a composition containing acemannan according to an embodiment of the present invention in inducing keratinocyte differentiation and activating PKC and EGFR. [Diagram 32] FIG. 2 is a graph showing the effect of a composition containing acemannan according to an embodiment of the present invention in inducing keratinocyte differentiation and activating PKC and EGFR. [Diagram 33] FIG. 2 is a graph showing the effect of a composition containing acemannan according to an embodiment of the present invention in inducing keratinocyte differentiation and activating PKC and EGFR. [Diagram 34]FIG. 1 shows the results of cytotoxicity when human bronchial epithelial cells (NHBE) are treated with a composition containing acemannan according to an embodiment of the present invention. [Diagram 35] FIG. 1 shows an experimental design for measuring transepithelial electrical resistance (TEER) using a composition comprising acemannan in an embodiment of the present invention. [Figure 36A] 1 is a graph showing the results of TEER measurement for a composition containing acemannan in an embodiment of the present invention. [Figure 36B] 1 is a graph showing the results of TEER measurement for a composition containing acemannan in an embodiment of the present invention. [Figure 36C] 1 is a graph showing the results of TEER measurement for a composition containing acemannan in an embodiment of the present invention. [Figure 36D] 1 is a graph showing the results of TEER measurement for a composition containing acemannan in an embodiment of the present invention. [Figure 36E] 1 is a graph showing the results of TEER measurement for a composition containing acemannan in an embodiment of the present invention. [Figure 37] 1 is a graph showing the results of real-time PCR quantifying viruses released into NHBE cells in a composition containing acemannan according to an embodiment of the present invention. [Figure 38] 1 is a graph showing the results of an analysis of the effect on the eyes of composition A50 containing acemannan in an embodiment of the present invention. [Figure 39] 1 is a graph showing the results of an analysis of the effects on the eyes of composition I50 containing acemannan in an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The detailed description provided below in conjunction with the accompanying drawings is intended as a description of the present embodiment and is not intended to represent the only manner in which the present embodiment may be constructed or utilized. The specification sets forth the functions of the present embodiment and the sequence of steps for constructing and operating the embodiment. However, the same or equivalent functions and sequences may be accomplished by different embodiments.

[0017] For convenience, certain terms used in the specification, examples, and appended claims are collected here. Scientific and technical terms used in this specification shall have the meanings commonly understood and used by those skilled in the art unless otherwise defined. Also, singular terms shall be understood to include the plural of the same, and plural terms shall be understood to include the singular, unless the context requires otherwise. Specifically, as used in this specification and claims, the singular forms "a" and "an" include plural references, unless the context clearly indicates otherwise. Also, as used in this specification and claims, the terms "at least one" and "one or more" have the same meaning, including one, two, three, or more.

[0018] Terms such as "at least one embodiment," "one embodiment," "multiple embodiments," "different embodiments," "several embodiments," "the present embodiment," and the like may indicate that the embodiments of the present disclosure so described may include a particular feature, structure, or characteristic, but not all possible embodiments of the present disclosure must include the particular feature, structure, or characteristic. Furthermore, repeated use of phrases such as "in one embodiment," "in an embodiment," and the like does not necessarily refer to the same embodiment, although they may be the same. Furthermore, the use of phrases such as "embodiment" in connection with "the present disclosure" does not mean that all embodiments of the present disclosure necessarily include a particular feature, structure, or characteristic, but rather should be understood to include "at least some embodiments of the present disclosure" that include the particular feature, structure, or characteristic described.

[0019] Further, for purposes of explanation and not limitation, specific details of functional entities, techniques, protocols, standards, etc. are set forth in order to provide an understanding of the described technology. In other instances, detailed disclosure of well-known methods, techniques, systems, architectures, etc. is omitted so as not to obscure the disclosure with unnecessary detail.

[0020] The terms "first," "second," and "third" in the description of this disclosure and in the above-mentioned drawings are not used to describe a particular order but to distinguish different objects.

[0021] Additionally, the term "including" and variations thereof are intended to cover a non-exclusive inclusion and may refer to "including, but not necessarily limited to," which specifically indicates an open-ended inclusion or membership in such listed combinations, groups, series, and the like. For example, a process, method, system, product, or apparatus that includes a series of steps or modules is not limited to the recited steps or modules, but optionally also includes unrecited steps or modules, or optionally includes other steps or modules inherent to the process, method, product, or apparatus.

[0022] The specification is better understood from the following detailed description when read in light of the accompanying drawings.

[0023] 1. Compositions containing acemannan 1.1 Composition A50

[0024] Composition A50 contains acemannan with a single chain backbone of β-(1,4) mannose intercalated with β-(1,4) glucose, and the β-(1,4) mannose partially acetylated at the C-2 and / or C-3 positions. The structural unit of acemannan is shown below.

[0025] [ka]

[0026] Composition A50 can be prepared by extracting plants containing acemannan, such as Aloe vera leaves. A structural study of composition A50 containing acemannan was performed by ATR-FTIR. It was observed that four major functional groups (OH, CO, CH3, COO) predominated, and the observation conditions were as follows: 3000-3676 Cm -1 (hydroxyl stretching vibration); ~2924 Cm -1 (Aliphatic groups (-CH2 and -CH3)); ~1734 Cm -1 (C=O stretching vibration of the carbonyl group of polysaccharides); 1236 Cm -1 (CO stretching vibration of the acetyl group of polysaccharides). The results are shown in Figure 1.

[0027] Another structural study of composition A50, which contains acemannan, 1 H-NMR was used, and the results are shown in Figure 2. 1 The H NMR spectrum showed three signals at 2.10, 2.06, and 2.02 ppm corresponding to methyl groups, indicating that the acetyl groups were at different positions. The position at 2.0 ppm indicates O-acetyl, and the figure at the bottom indicates that the amount of O-acetyl in composition A50 is 0.75.

[0028] Another structural study of composition A50, which contains acemannan, 13 The results were analyzed by C-NMR, and are shown in Figure 3. 13 From the C-NMR spectrum, signals of methyl carbons corresponding to 20.54, 20.27, and 20.06, and carboxyl carbons of the acetyl group (173.93, 173.42, and 12.58) were detected, indicating that composition A50 contained acetylated mannan (e.g., acemannan).

[0029] HPSEC-RI Analysis: Composition A50, which contains acemannan, was analyzed for its relative size using high performance size exclusion chromatography-refractive index detection (HPSEC-RI). A trend line was plotted using pullulan standards, and the relative molecular weight of the sample was determined based on the highest peak point detected within the trend line range. The results are shown in Figure 4. Composition A50 showed a relative molecular weight of 180 kDa (which represents the average within the range shown by the first peak in Figure 4), and a second peak outside the trend line at 45-50 min indicating the presence of a component smaller than 5 kDa.

[0030] Composition A50, which contains acemannan, was also analyzed by HPLC, and the results are shown in FIG.

[0031] Quantification of the O-acetyl content of composition A50 containing acemannan was carried out based on the principle of UV-Vis spectroscopy. Acetyl groups from 1,4-acetylated polymannose reacted with hydroxylamine to form acetohydroxamic acid. The resulting compound then reacted with ferric chloride to form a reddish ferric-acetohydroxamic complex with an absorption peak at 540 nm, whose O-acetyl content was determined by its absorbance. The absorbance of the complex at 540 nm was detected and compared with a calibration curve using acetylcholine chloride >=99% (TLC) as a standard reference substance.

[0032] The detailed method is as follows. 0.5 mL of each of the standard linear solution, diluted sample, and water (blank group) were collected in a 20 mL glass vial. 0.5 mL of 4 M hydrochloric acid was added to the blank group and shaken well. Then, 1 mL of alkaline hydroxylamine hydrochloride solution was added, shaken well, and left to stand for 4 minutes. 0.5 mL of 4 M hydrochloric acid was added to the samples other than the blank group to make the pH 1.2 ± 0.2 and shaken well. 0.5 mL of 0.62 M ferric chloride was added and stirred for 5 seconds. At this point, the solution turns reddish orange. The completed sample was placed in an ultrasonic cleaner to remove air bubbles. The absorbance of the standard solution and the sample was measured at 540 nm, and the O-acetyl content of the sample was calculated from the absorbance value of the standard linear solution. The results are shown in Figure 6.

[0033] 1.2 Composition I50

[0034] Composition I50 comprises acemannan, which is a single-chain β-(1,4) mannose backbone with β-(1,4) glucose inserted therein and α-(1,6) galactose branched from C-6 of the backbone. The β-(1,4) mannose backbone is partially acetylated at the C-2 and / or C-3 positions. The structural unit of acemannan is as follows:

[0035] [ka]

[0036] Composition I50 can be prepared by extracting plants containing acemannan, such as the leaves of Aloe vera. A structural study of composition I50 containing acemannan was performed. 1 The results were measured by H-NMR, and are shown in Figure 7. The position of 2.0 ppm indicates O-acetyl, and the numerical value at the bottom of the figure indicates that the amount of O-acetyl in composition I50 is 1.62.

[0037] HPSEC-RI Analysis: Composition I50 containing acemannan was analyzed for its relative size using high performance size exclusion chromatography-refractive index detection (HPSEC-RI). A trend line was plotted using pullulan standard and the relative molecular weight of the sample was determined based on the highest peak point detected within the trend line range. The results are shown in Figure 8. Composition I50 showed a relative molecular weight of 37 kDa (which represents the average within the range shown by the first peak in Figure 8) and also showed a second peak outside the trend line at 48 minutes, indicating the presence of components less than 5 kDa.

[0038] Composition I50, which contains acemannan, was also analyzed by HPLC, and the results are shown in FIG.

[0039] Quantitation of the O-acetyl content of the acemannan-containing composition I50 was carried out as described above, and the results are shown in FIG.

[0040] 2. Use of compositions containing acemannan for intestinal health 2.1 IBD treatment and / or prevention (in vitro experiments)

[0041] RGM1 (rat gastric mucosa) cells were cultured in DMEM / F12 (Corning, USA) supplemented with 10% (v / v) fetal bovine serum (FBS, HyClone, USA) and 1% (v / v) antibiotic-antimycotic (Gibco, Thermo Fisher Scientific, USA). Cells were cultured at 37°C under a 5% CO2 atmosphere. Before each experiment, the cultures were washed with phosphate-buffered saline (PBS, pH 7.4, PROTECH, Taiwan) and detached with 0.05% trypsin-EDTA solution (Gibco, Thermo Fisher Scientific, USA), centrifuged at 1700 rpm for 5 min (Hermle Z 36 HK, Germany), and then resuspended in complete medium.

[0042] The detailed method of in vitro cytotoxicity evaluation is as follows. 1×10^4 cells / well were seeded in a 96-well plate and cultured for 12 h to allow the cells to attach. Stock solutions (50 mg / mL) of acemannan with different acetylation degrees (42% acetylation degree is called A42, 50% acetylation degree is called A50, 10% acetylation degree is called A10, and 10% acetylation degree is called Excp) were prepared directly in the medium. The concentrations of excipients were calculated corresponding to the formulation of A10. Cells were treated with different concentrations of acemannan (1000, 500, 200, 100, 50 mg / mL) and incubated for 24 and 48 h. After treatment, 10 mL of CCK-8 solution (Dojindo Laboratories, Japan) was added to each well and the plate was further incubated for 1 h. The absorbance at 460 nm was measured using a microplate reader (EPOCH2, BioTek Instruments, USA). Data are presented as mean ± SD, and statistical significance was analyzed by Student's t-test. A p value of 0.05 was considered statistically significant. Microscopic photographs of cells treated with different concentrations of acemannan were taken at 10× and 20× magnifications using a Zeiss Axio Vert.A1, Germany.

[0043] Samples A42, A50, and A10 were weighed (if necessary), dissolved directly in serum-free medium, and mixed thoroughly before use. Sample A10 consisted of ~40% acemannan or A50 (50% acetylated) and ~60% pure excipient (corn starch), and could be prepared simply by adding excipients and diluting A50. Thus, the acetylation degree of A10 was about 10-12%. Sample A10 for cytotoxicity testing was a powder (not a tablet), but the recommended intake of A10 tablets is 1-2 tablets / day for adults, with each tablet containing 100 mg of product (A10 + excipients = 100 mg tablet). The results are shown in Figure 11A and Figure 11B.

[0044] The results showed that significant cell proliferation was observed when cells were treated with A10 and vehicle after 48 h. However, A42 and A50 inhibited cell proliferation (dose-dependently) when incubated for 24 h, but promoted cell proliferation (up to 500 mg / mL) when incubated for 48 h. Morphology and viability of RGM1 cells under different treatments were also evaluated by microscopy (results not shown here). Micrographs showed no significant changes in cell morphology in all treatment conditions. Further results are shown in Figure 12A, Figure 12B, Figure 12C, Figure 13A, and Figure 13B.

[0045] The LDH release of composition A50 was also analyzed and the results are shown in Figure 14. Pre-treatment represents RGM1 treated with mannan pro (i.e. composition A50) for 1 hour, followed by washing and replenishing with fresh complete medium. Co-culture represents RGM1 treated with mannan pro for 24 or 48 hours. The results showed that RGM1 cells treated with mannan pro (1 μg / mL) had no LDH leakage at 48 hours, indicating no cytotoxicity.

[0046] The protective effect of mannan pro (ie, composition A50) against cell damage caused by indomethacin was carried out and the results are shown in FIG. 15, FIG. 16, FIG. 17A, FIG. 17B, and FIG.

[0047] The benefits of dietary acemannan in gut health include attenuating the digestive process, increasing satiety, and slowing the release of sugars from starch. In the large intestine, acemannan is digested by microorganisms into short-chain fatty acids, which are then absorbed and provide a prebiotic effect to the host. As a soluble dietary fiber, acemannan also enhances mineral absorption, inhibits obesity, inhibits inflammatory bowel disease and its associated complications (colon cancer and IBD), improves laxatives, and inhibits chronic systemic inflammation.

[0048] 2.2 Improvement of mucosal protection (in vivo experiment)

[0049] In order to evaluate the mucosal protective function, a small-scale preliminary study was conducted based on the gastrointestinal function improvement evaluation method announced by the government, and an evaluation model and a preliminary effective amount were established.

[0050] Eleven 6-week-old male BALB / cAnNCrlBltw mice weighing 19-22 g were used, including three in the placebo group, three in the vehicle group, and five in the acemannan group.

[0051] Mice were housed at 22 ± 3 °C, 50 ± 20 % relative humidity, and a 12-h light / 12-h dark cycle. Mice were housed in individually ventilated cages (IVCs) and kept with absorbent corncob bedding (10054, Andersons, USA), sterile food (MFG, Oriental Yeast Co., Ltd., Japan), and unlimited access to sterile RO water.

[0052] The experiment was conducted based on the Gastrointestinal Function Improvement Evaluation Method for Health Foods (Ministry of Health and Welfare Notification No. 0920401629). The drug was administered orally at a dose of 500 mg / kg (BID). The experimental design is shown in Table 1.

[0053] [Table 1]

[0054] A homogenous solution prepared with sterile water for injection was used for preparation. The route and method of administration were in accordance with the "Standard Operating Procedure for Sample Administration" (SHGLP-SOP-IA-020). On the day of administration, a sterile syringe was used to draw out an amount of substance according to the weight of the mouse, and the substance was orally administered through a plastic feeding needle. The amount administered to each mouse was calculated based on the weight of the mouse before administration of the substance on the first day. The day when the substance was first administered was defined as day 1. The weight of the animals was measured on days 1 and 8 according to the "Standard Operating Procedure for Laboratory Animal Care" (SHGLP-SOP-IA-003).

[0055] After dosing was started, the "Standard Operating Procedure for Animal Ward Rounds" (SHGLP-SOP-IA-009) was followed. Clinical signs of mice were observed and recorded at least once daily during the study period. Survival status of mice was monitored twice daily with an observation interval of at least 6 hours.

[0056] Shay ulcer method: Mice in sham, acemannan and vehicle groups were fasted for 24 hours. The gastric pylorus was ligated and sutured to allow recovery. After 4 hours, the mice were sacrificed and the stomach was removed. Gastric juice was collected, quantified and titrated with 0.01 NaOH solution. The change in total acidity between the experimental and control groups was calculated. p<0.05 indicated that acemannan had the function of protecting the gastric mucosa.

[0057] Data were presented as mean ± standard deviation (SD). Data were analyzed using t-test to determine whether there were statistically significant differences compared to the sham group, with a significance criterion of ≦0.05.

[0058] As a result, no animals died during the experiment. The body weight, clinical symptoms, and autopsy findings of the animals on the 1st and 8th days are summarized in Table 2. No significant difference in body weight was observed before and after administration. No adverse clinical symptoms were observed during the test period (days 1 to 8). No abnormal findings were observed in the autopsy. From the above results, even when acemannan (500 mg / kg, BID) was administered continuously for 8 days, there was no weight loss or observed clinical side effects in the mice. In addition, no macroscopic abnormalities were observed in the organs.

[0059] [Table 2]

[0060] Acemannan and vehicle were administered on the morning of the 8th day, followed by Shay ulcer assay. After fasting for 24 hours, the stomach was ligated at the pylorus-cardia junction. After 4 hours, the mice were sacrificed and gastric juice was collected, weighed, pH measured, and total acidity measured. The results are shown in Table 3, Figure 19A, Figure 19B, and Figure 19C.

[0061] [Table 3]

[0062] Gastric juice weight and pH were not significantly different among the groups (G1: Sham, G2: Sterile water injection, G3: Acemannan 500 mg / kg, BID). However, the total acidity of G3 (0.390 ± 0.122) was significantly lower than that of G1 (0.660 ± 0.181). The total acidity of G2 (0.620 ± 0.167) fed with sterile water did not show a significant difference from that of G1. From these data, it was concluded that the gastric juice weight and pH were not statistically significantly different compared to the Shay control group after 8 consecutive days of administration of acemannan (500 mg / kg, BID) using the Shay ulcer method. However, the total acidity showed a significant decrease.

[0063] The gastric mucosal protective effects of the A10 and A50 compositions were tested using male Sprague-Dawley rats (total of 50 rats, 8 weeks old). After the adaptation period, rats were randomly divided into 10 groups (n=5 / group): (1) NC1 group (sham-operated and receiving 10 mL / kg BW / day distilled water); (2) NC2 group (receiving 10 mL / kg BW / day distilled water); (3) NC3 group (335 mg vehicle); (4) PC group (clinical drug 5-aminosalicylic acid); (5) L-A10 group (A10, 100 mg / human daily dose); (6) M-A10 group (A10, 300 mg / human daily dose); (7) H-A10 group (A10, 500 mg / human daily dose); (8) L-A50 group (A50, 33 mg / human daily dose); (9) M-A50 group (A50, 100 mg / human daily dose); (10) H-A50 group (A50, 167 mg / human daily dose).

[0064] All animals were pre-bred for one week, and 50 SD rats were acclimatized to the environment and then divided into groups. The rats were placed in plastic cages, with one or two per cage. The animals were first given temporary identification numbers, weighed, and divided into groups according to a weight-balanced order. During the experimental period, reverse osmosis purified water was provided as drinking water, and the rats were allowed to drink water ad libitum. The rats had free access to standard animal chow (Chow 5001) and water. In this experiment, the animals were weighed regularly to monitor their health and determine the dosage of the test sample for each animal based on their weight. The test samples were weighed daily according to the dosage required for each group. The test samples were then dissolved in sterile water and orally administered for 7 days. Clinical observations were made daily during the experimental period, and abnormal clinical symptoms and deaths were recorded. The rats' weights were recorded every two days and changes were observed. In addition, the rats' food intake was recorded once every three days, and their water intake was recorded daily.

[0065] After 7 consecutive days of administration of A10 and A50 supplements, rats were fasted for 24 hours. The abdomen was shaved along the midline to the xiphoid process, and a 1 cm incision was made along the midline to perform a pylorus ligation. The incision was then sutured for recovery. After 4 hours, the abdomen was opened along the midline and an incision was made along the greater curvature to expose the stomach. The sacrificed stomach was removed, and gastric juice was collected and its volume was measured. The collected gastric juice was titrated with 0.1 N NaOH solution. The total gastric acid volume (mEq / L) was calculated as follows: total gastric acid volume = gastric acid volume (L) × sodium hydroxide (NaOH) volume added (L) × 0.1 N (NaOH solution titration) × 1000.

[0066] After irrigation with saline, the stomach was flattened, photographed, and analyzed using the image analysis software ImageJ. Gastric mucosa was quantified by converting the images to black and white and adjusting the image threshold appropriately and consistently. Damage areas were calculated in mm 2 The gastric lesion index was calculated using the following formula: gastric lesion index = gastric damage area (mm 2 ) / Total gastric mucosal area (mm 2 )×100. The results are shown in Figures 20A, 20B, 20C, 20D and 20E.

[0067] All values ​​are expressed as mean ± SD, with a sample size of 5 animals per group. One-way analysis of variance (ANOVA) was performed using the SAS statistical software package. Duncan's test was used for post-hoc comparisons of differences between groups, and a significance level of p < 0.05 was considered statistically significant.

[0068] As a result, no significant differences were observed in body weight, food intake, or water intake among the groups. The results showed that the administration of high, medium, and low doses of "A10 supplement" and "A50 supplement" for seven consecutive days significantly reduced the total gastric acid value, gastric damage area, and gastric lesion index (p < 0.05). Therefore, this experiment confirmed the gastroprotective effect of the test substances "A10 supplement" and "A50 supplement" on the gastric mucosa.

[0069] 3. Use of a composition containing acemannan in skin care

[0070] We analyzed the effects of acemannan on cellular stress responses, including inflammation and death of keratinocytes induced by UVB, on the growth and differentiation of keratinocytes, and on skin aging.The results showed that acemannan promoted the proliferation of HaCaT cells, promoted the migration of HaCaT cells, induced the expression of differentiation markers (involucrin, loricrin, and TGase1) in HaCaT cells and NHEK cells, and promoted the activation of EGFR and PKC.

[0071] Detailed materials and methods are listed below. Cell culture, antibodies, reagents, and test compounds: Human keratinocyte cell line HaCaT was cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 10% penicillin-streptomycin solution and incubated at 37 °C and 5% CO2. Primary antibodies against β-actin, involucrin, keratin 10 (K10), TGase 1, and PKC II were purchased from Santa Cruz Biotechnology (USA). Primary antibodies against p63-, phospho-EGFR Y1068, phospho-PKC II S660, and peroxidase-labeled anti-rabbit / mouse IgG secondary antibodies were purchased from Cell Signaling Technology (USA). Primary antibodies against EGFR were purchased from EMD Millipore Corp. Primary antibodies against loricrin were purchased from GeneTex (Irvine, CA, USA). PMA (phorbol-12-myristate-13-acetate), LPS (lipopolysaccharides), EGF (epidermal growth factor), and poly(I:C) were purchased from Sigma-Aldrich (Burlington, MA, USA). ACE dehydrate and GP 5-1-1 were prepared and supplied by Dazzeon Biotech Co. Ltd. Before use, ACE (i.e., compositions containing Acemannan, such as composition A50) dehydrate was dissolved in sterile H2O (50 mg / mL) or 0.2% ethanol and stored at 4°C, while GP 5-1-1, which is insoluble in H2O, was suspended in DMSO at 10 mg / mL and stored at room temperature.

[0072] 3.1 MTT assessment of cell viability

[0073] Skin irritation by in vitro skin irritation test according to the procedure of OECD Guidelines for Testing of Chemicals (Test No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method). The aim of this embodiment is to measure the cytotoxic effect of the test substance on a reconstructed human epidermis (RhE) model upon exposure to an acemannan sample. EpiDermTM Tissue (surface 0.6 cm 2 ) was used for sample testing.

[0074] Methods: On the day of receiving the Epiderm tissues, under sterile conditions, the 24-well plates containing the epidermal tissues were carefully inspected and the inserts of each tissue were transferred to 6-well plates pre-filled with growth medium. The plates were placed in an incubator (37 °C, 5% CO2, 90% RH) for 60 ± 5 min. After 60 min of pre-incubation, the inserts were transferred to fresh medium and further incubated overnight for 18 ± 3 h. On the day of chemical exposure, 30 μL of different concentrations of test substances, negative control (NC, sterile DPBS) and positive control (PC, 5% SDS in H2O solution) were added to three single tissue inserts. After dosing the last tissue, all plates were transferred to a humidified incubator (37 °C, 5% CO2, 90% RH) for 35 ± 1 min. After 35 min, the plates were removed from the incubator and placed in a sterile hood for a period of 60 min for the first tissue dosed. After 60 min of test substance exposure, the tissue inserts were rinsed with sterile DPBS and filled and emptied 15 times to remove residual test substance. A constant flow of DPBS was applied from a distance of 1.5 cm from the tissue surface. After all inserts were washed, the surface of each tissue was carefully dried with a sterile cotton swab. The tissues were then cultured in an incubator for 24 h. After 24 h of culture, the medium was replaced with fresh medium and cultured for another 18 h. The tissues were then transferred to a 24-well plate containing MTT medium (1 mg / mL) for MTT evaluation. After 3 h of MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 mL / tissue of isopropanol (extraction solution, product number MTT-100-EXT), and the optical density of the extracted formazan was measured at 570 nm using a spectrophotometer. The relative cell viability was calculated for each tissue compared to the average value of the negative control tissues. According to the EU and GHS classification (R38 / Category 2 or no label), irritation was predicted if the mean relative tissue viability of the three tissues exposed to the substance was reduced to 50% or less of the mean viability of the negative control.

[0075] Cells were seeded in 24-well plates and cultured in 500 μL of DMEM at 37 °C and 5% CO2. When the cells reached 60% confluence, they were treated with vehicle and different concentrations of ACE for the indicated times. A 1 mg / mL solution of MTT [3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, St. The medium was removed and replaced with 150 μL of DMSO to dissolve the purple crystals. Cell viability was determined by the OD value (OD550 nm-OD630 nm) of the DMSO solution containing the crystal product.

[0076] Solubility and cytotoxicity of ACE: First, the solubility of ACE in HO and 2% ethanol was analyzed, respectively. At a concentration of 50 mg / mL, ACE dehydrate was completely dissolved in these two vehicle solutions, and the liquid appearance was clear and light yellow. At a concentration of 100 mg / mL, it was also completely dissolved, but the viscosity was high. Finally, 50 mg / mL was the stock concentration of ACE, which was stored at 4°C before use. Next, we analyzed whether ACE exhibited cytotoxicity to HaCaT cells. Cells were treated with different concentrations of ACE dissolved in 2% ethanol (Figure 21A) and HO (Figure 21B), and then MTT evaluation was performed to analyze cell viability. The results showed that ACE at concentrations up to 1 mg / mL in both vehicles did not cause cytotoxicity after 48 hours of incubation. Moreover, cells treated with 5 mg / mL of ACE dissolved in HO for 24 and 48 hours also did not cause cytotoxicity indexed by MTT evaluation (Figure 21B). When the ACE concentration was 10 mg / mL, cell viability decreased to about 40% after 24 hours of treatment (Figure 21B). To avoid unnecessary interference from cell death, ACE dissolved in HO at a concentration not exceeding 5 mg / mL was used in the analysis in the subsequent experiments.

[0077] As shown in Figures 21A and 21B, a cytotoxicity test of ACE in HaCaT cells was performed. ACE dehydrate was dissolved in (Figure 21A) sterile water or (Figure 21B) 0.2% ethanol at a concentration of 50 mg / mL before use. Cells (2 x 10 4 ) were seeded into 24-well plates and incubated overnight at 37 °C, 5% CO2, and various concentrations of ACE or vehicle were added, followed by incubation for 24 or 48 hours.

[0078] 3.2 Flow cytometric analysis of cell viability

[0079] Cells were harvested and resuspended in staining buffer containing Annexin V-FITC / PI according to the manufacturer's protocol (BioLegend, San Diego, CA, USA). Data analysis was performed using FlowJo software. After 20 min of incubation at 37°C, cells were measured by flow cytometry (FACSCalibur, BD, Franklin Lakes, NJ, USA). Cell viability was measured by flow cytometry with Annexin V-FITC / PI staining after treatment with 5 mg / mL ACE (i.e., acemannan-containing composition) for 48 h. The results are shown in Figure 22A and Figure 22B.

[0080] 3.3 UVB irradiation

[0081] UVB was delivered by a spaced array of five T-8M UV tubes. The UVB energy output (312 nm) was measured with a UV Bio-Link (BLX-312; Vilber Lourmat, France). Cells in culture medium or phosphate-buffered saline (PBS) were irradiated with different doses of UVB. After UVB irradiation, cells were fed with fresh medium and continued to be cultured for the indicated periods.

[0082] ACE does not show protective effect against UVB-induced cytotoxicity in HaCaT cells. To examine whether ACE (i.e., a composition containing acemannan) can protect cells from death after UVB irradiation, ACE was administered at concentrations of 1 mg / mL and 5 mg / mL, respectively, for 30 min, followed by 90 mJ / cm2 irradiation. 2 After 24 h of incubation, the cells were irradiated with 1000 μg of UVB and the cell viability was analyzed by flow cytometry. The results showed that the cell viability of cells cultured in DMEM containing FBS was reduced by approximately 60% upon irradiation, whereas the cell viability after UV irradiation in PBS was reduced to 15%, suggesting that serum reduces the sensitivity of cells to UVB. However, when cells were pretreated with ACE, there was no protective effect against UVB irradiation, regardless of the presence or absence of FBS (Figure 23A). Furthermore, the cells pretreated with ACE in DMEM were able to withstand low doses of UVB (25 and 30 mJ / cm2). 2 As shown in Figure 23B, ACE also did not show any cytoprotective effect against UVB irradiation.

[0083] As a result, a 60-minute exposure in vitro test (OECD, TG 439) was performed to evaluate the skin irritation potential of the samples. In this test method, the blank OD was 0.035 ± 0.0003. The cell viability of the negative control was 100 ± 2.115%, and that of the positive control (5% SDS) was 3.406 ± 0.270%. Meanwhile, the cell viabilities of the test substance at 10 mg / mL and 1 mg / mL were 107.975 ± 1.788% and 121.320 ± 5.635%, respectively (Table 4). According to the EU and GHS classification (R38 / Category 2 or unlabeled), irritation is predicted when the average relative tissue viability of three individual tissues exposed to the test substance is reduced to 50% or less of the average viability of the negative control. Based on the experimental results of this project, the average cell viability is greater than 100% for the 1 mg / mL and 10 mg / mL samples. According to the EU and GHS classification (R38 / Category 2 or no label), the test article is classified as non-irritating to the skin.

[0084] [Table 4]

[0085] As shown in Figures 23A and 23B, ACE cannot protect HaCaT cells from UVB-induced cell death. (Figure 23A) Cells (1x10^5) were seeded in 12-well plates and treated with ACE (1 and 5 mg / mL) for 30 min. Then, UVB was applied at 90 mJ / cm under complete medium or PBS conditions. 2 After UVB irradiation, cells were continued to be cultured for 24 h in complete medium. Cell viability was determined by flow cytometry with Annexin V-FITC / PI staining (Figure 23B). Cells were irradiated with 25 and 30 mJ / cm, respectively, with or without pretreatment with ACE (3 mg / mL). 2 After 24 hours of incubation, the extent of cell death was recorded by taking photos under a microscope.

[0086] 3.4 RNA extraction, cDNA generation, and quantitative polymerase chain reaction (qPCR)

[0087] Total RNA extraction reagent (REzol TM Total RNA was extracted from HaCaT cells using a PCR kit (Protech Technology Enterprise Co., Ltd.). RNA (1 μg) was used as a template to perform reverse transcription using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT, Promega Corporation, USA) to create cDNA. qPCR was performed using FastStart universal SYBR® Green Master (Roche Diagnostics GmbH, Germany) and a qRT-PCR device (QuantStudio TM 5. Applied Biosystems TMThe transcription levels of the target genes were quantitatively measured using the 2-fold increase in the expression of the target genes. The transcription levels of all genes were normalized to the level of b-actin. The changes in mRNA expression were calculated using the 2-fold increase in the expression of the target genes. -ΔΔCt The sequences of the primer sets used in this project are shown in Table 5.

[0088] [Table 5]

[0089] The results showed that ACE induced the expression of IL-1, IL-1, IL-6, IL-8, COX-2, and TNF-α genes. To understand whether ACE (i.e., a composition containing acemannan) can control inflammation, cells were treated with ACE (1 and 5 mg / mL) for 30 min, followed by 60 mJ / cm 2 The cells were irradiated with 1000 μg of UVB. After 4 and 8 hours, the cells were harvested and subjected to qPCR analysis. As shown in Figure 24, UVB caused a significant upregulation of IL-6, TNF-α, and COX-2 gene expression, but had only a weak effect of approximately two-fold increase on IL-8, IL-1, and IL-1. ACE (5 mg / mL) treatment can enhance the effect of UVB on the expression of IL-8 and TNF-α. Next, cells were irradiated with different doses of UVB (30 and 60 mJ / cm) with or without ACE treatment. 2) to the cells. In Figure 25, it was found that after 8 hours of culture, ACE (5 and 10 mg / mL) alone increased the gene expression of these inflammatory factors, with less increase in TNF-a. Compared with UVB, ACE showed a higher effect than UVB on IL-8, IL-1, and IL-1 gene expression, and showed a similar effect to UVB on TNF-a and COX-2. In addition to UVB irradiation, the effects of ACE combined with EGF (EGFR agonist), LPS (TLR4 agonist), and poly(I:C) (TLR3 agonist) on the gene expression of inflammatory factors were also measured. In Figure 26, it was found that EGF significantly enhanced COX-2 gene expression and slightly enhanced IL-1 and IL-1 gene expression. An additive effect on COX-2 expression was observed between ACE and EGF. In contrast, EGF suppressed ACE-induced IL-6 and TNF-a gene expression. LPS and poly (I:C) themselves did not affect these gene expressions, but suppressed ACE-induced IL-6, TNF-a, and COX-2 gene expressions. Whether ACE promotes IL-1 gene expression is a matter of concern, as the experimental results were inconsistent in this study (Figure 25 vs. Figure 26), and further confirmation is required. In addition, we also investigated whether ACE can control MMP-1 gene expression, which is an indicator of the skin wrinkle formation process. As a result, UVB and EGFR increased MMP-1 gene expression, but ACE, LPS, and poly (I:C) did not, and their effects after 8 hours were not changed by ACE (Figure 27).

[0090] Figure 24 shows the time-dependent effect of ACE on UVB-induced inflammatory gene expression in HaCaT cells. Cells were treated with vehicle or ACE (1 and 5 mg / mL) for 30 min, followed by UVB at 60 mJ / cm. 2 After 4 and 8 hours, cells were harvested and lysed for total RNA extraction, cDNA preparation, and subsequent qPCR analysis.

[0091] Figure 25 shows the effect of ACE on the expression of inflammatory genes induced by different doses of UVB in HaCaT cells. Cells were treated with vehicle or ACE (5 and 10 mg / mL) for 30 min, followed by treatment with 30 or 60 mJ / cm 2 After 8 hours, qPCR was performed as described above to analyze the mRNA expression levels of the target genes.

[0092] Figure 26 shows the effects of ACE, EGF, LPS, and poly(I:C) on inflammatory gene expression in HaCaT cells. Cells were treated with vehicle or ACE (5 and 10 mg / mL) for 30 min, followed by treatment with EGF (50 ng / mL), LPS (1 mg / ml), and poly(I:C) (10 mg / mL) for 8 h, respectively. To analyze the mRNA expression levels of target genes, qPCR was performed as described above.

[0093] Figure 27 shows that ACE does not alter MMP-1 gene expression. Cells were treated with vehicle or different concentrations of ACE (1, 5, 10 mg / mL) for 30 min, followed by 60 mJ / cm 2 were UVB irradiated for 4 and 8 h (A) or treated with EGF (50 ng / ml), LPS (1 mg / ml), and poly(I:C) (10 mg / ml) for 8 h (B). To analyze the mRNA expression level of MMP-1 gene, qPCR was performed as described above.

[0094] 3.5 Cell proliferation assessment

[0095] cells (2×10 4 (1 × 10 cells) were seeded in 12-well plates in DMEM without FBS and cultured for 24 h in a CO2 incubator. The medium was removed and fresh DMEM containing 10% FBS was added in the presence or absence of ACE (0.5, 1, 3 mg / mL). Cell proliferation was determined by counting the number of cells every 24 h using trypan blue staining. For BrdU incorporation evaluation, cells (1 × 10 cells) were cultured ... 4) were seeded in black 96-well plates with clear flat bottom and cultured in DMEM without FBS for 24 h. The medium was removed and fresh DMEM containing 10% FBS in the presence or absence of ACE (1 and 3 mg / mL) was added for 46 h. Cells were fixed, incubated with anti-BrdU-peroxidase for 90 min, washed and then subjected to the substrate reaction according to the manufacturer's protocol (Cell Proliferation ELISA, BrdU, Roche, Switzerland). Chemiluminescence was measured using a Synergy H1 microplate reader (BioTeK, Winooski, VT, USA).

[0096] The results showed that ACE promoted the proliferation of HaCaT cells. We examined whether ACE had an effect on cell proliferation. After 24 h of starvation, cells were replaced with DMEM containing 10% FBS, with or without different concentrations of ACE. We found that cell proliferation was promoted by ACE in a concentration-dependent manner (Figure 28A). Data from BrdU assessment also showed that ACE showed higher BrdU incorporation, indicating that ACE promoted DNA synthesis in cells in the S phase of the cell cycle (Figure 28B).

[0097] Figure 28A and Figure 28B show that ACE promotes the proliferation of HaCaT cells in a concentration-dependent manner. Cells were seeded and starved for 24 h on 12-well plates for cell counting by trypan blue staining (Figure 28A), or cells were seeded on 96-well plates for BrdU incorporation assessment (Figure 28B). Then, the medium was replaced with fresh DMEM containing 10% FBS and the indicated concentrations of ACE. Cell numbers were counted every 24 h. For BrdU incorporation assessment, cells were harvested after 48 h of culture and BrdU signal intensity was measured according to the manufacturer's protocol. *p<0.05 compared to vehicle-treated cells.

[0098] 3.6 Cell migration assay

[0099] Place two silicon culture inserts into each well of a 24-well plate and place 2 × 10 cells in the wells of the silicon culture insert.4 Cells were seeded overnight. Before observing cell migration, the cells were treated with various concentrations of ACE. At the indicated time points, cell migration was observed under a microscope and photographed and recorded. The percentage of gap recovery area was analyzed and quantified using Image J software.

[0100] The results showed that ACE promoted the migration of HaCaT cells. Next, we investigated the effect of ACE on the migration of keratinocytes. Using a wound healing model, cells were seeded on two-well silicon culture inserts and gaps were created between the cells. As shown in Figure 28A and Figure 28B, ACE can increase cell proliferation, so to avoid cell proliferation interfering with the results of cell migration, the cells were treated with mitomycin C. As shown in Figure 29A, 2 μg / mL and 5 μg / mL mitomycin C indeed inhibited cell proliferation and reduced cell number. The reduction in cell number occurred at 48 hours and 24 hours by 2 μg / mL and 5 μg / mL mitomycin C treatment, respectively. This finding indicates that 2 μg / mL mitomycin C may be appropriate for migration evaluation within 24 hours. To further confirm this issue, the combined effect of mitomycin C (2 μg / mL) and ACE was tested. As a result, it was found that ACE (1 and 3 mg / mL)-induced cell proliferation, as seen in Figure 28A, was inhibited by mitomycin C (2 μg / mL) (Figure 29B). Under these findings, cell migration was measured under conditions of co-treatment with mitomycin C (2 μg / mL) and ACE (0.5 and 5 mg / mL). As shown in Figure 29C and Figure 29D, in the presence of ACE (5 mg / mL), cell migration was faster than that in the vehicle treatment, revealing the effect of ACE in promoting cell migration.

[0101] Figure 29A, Figure 29B, Figure 29C, and Figure 29D show that ACE accelerates the migration of HaCaT cells. (Figure 29A and Figure 29B) Cells were treated with the indicated concentrations of mitomycin C in the absence (Figure 29A) or presence (Figure 29B) of ACE (1 mg / mL or 3 mg / mL). Cell numbers were counted by trypan blue staining. (Figure 29C) Cells cultured in mitomycin C (2 μg / mL)-containing medium were treated with vehicle or ACE (0.5 and 5 mg / mL), and cell migration was determined at the indicated time points under a microscope. (Figure 29D) The effect of ACE on cell migration was evaluated using Image J software to quantify the gap recovery area, as shown in Figure 29C.

[0102] 3.7 Western Blot

[0103] Cells were lysed using RIPA buffer (50 mM Tris at pH 7.6, 150 mM NaCl, 0.1% SDS, 0.1% NaDOc, 1% Triton X-100, 2 mM NaF, 2 mM EDTA, 2 mM Na3VO4, 1 mM PMSF). After homogenization with a sonicator, the cell extract was diluted with 5X sample buffer (50 mM Tris at pH 6.8, 5% SDS, 37.5% glycerol, 36 mM β-mercaptoethanol, 0.25% bromophenol blue) and heated at 95°C for 10-15 min, followed by SDS-polyacrylamide gel electrophoresis (PAGE). The proteins on the gel were transferred to a polyvinylidene difluoride membrane (PVDF, GE Healthcare Life Sciences, USA) and blocked with TBS-T (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20) containing 5% skim milk for 30 min. The membrane was washed three times with TBS-T buffer for 15 min, and then incubated with horseradish peroxidase-conjugated anti-goat IgG (1:10000) or anti-rabbit / mouse IgG secondary antibody (1:10000) in TBS-T buffer for 1 h. Development was performed using an enhanced chemiluminescence solution (Western Lightning Plus-ECL, PerkinElmer, Inc., MA, USA). Protein signals were acquired using a ChemiDocTM MP Image System (Bio-Rad) and analyzed using Image Lab software (Bio-Rad).

[0104] The results showed that ACE induced the expression of keratinocyte differentiation markers in HaCaT cells. We also investigated whether ACE affects the differentiation of HaCaT cells. For this purpose, cells treated with different concentrations of ACE (i.e., compositions containing acemannan) were analyzed at the indicated time points for the expression levels of specific differentiation marker proteins. In addition, cells treated with the differentiation-inducing reagent PMA (100 nM) were used as a standard reaction. The results showed that, similar to the effect of PMA, ACE (3 mg / mL) increased involucrin, loricrin, and TGase1, but not K10 (Figure 30A and Figure 30B). p63-a is a key regulator of keratinocyte differentiation, and its downregulation has been shown to be a prerequisite. We found that both PMA and ACE transiently downregulated p63-a, and its expression was 12-24 h for PMA and 0-12 h for ACE. These results suggest that ACE may function to control the differentiation of HaCaT. As EGFR and PKC are important regulators of keratinocyte differentiation, we deciphered the mechanism of action of ACE on both pathways. Similar to the action of PMA, ACE can activate EGFR and PKC bII by promoting their phosphorylation (Figure 30C). However, ACE-induced EGFR phosphorylation was less responsive and shorter-lasting than PMA. Notably, ACE induced a more obvious activation of PKC bII than PMA. All these findings indicate that ACE may induce keratinocyte differentiation via PKC activation.

[0105] Figure 30A, Figure 30B and Figure 30C show that ACE induces keratinocyte differentiation and activates PKC and EGFR similarly to PMA. (Figure 30A) Cells were treated with PMA (100 nM) or ACE (3 mg / mL) for 6, 12, 24 and 36 hours, respectively. (Figure 30B) Cells were treated with PMA (100 nM) or ACE (1, 3 or 5 mg / mL) for 36 hours. (Figure 30C) Cells were treated with PMA (100 nM) or ACE (3 mg / mL) for 0.5, 1, 2 and 6 hours, respectively. Cell lysates were collected and prepared for immunoblotting evaluation with primary antibodies against target proteins. PMA means phorbol-12-myristate-13-acetate. Further results are shown in Figure 31, Figure 32 and Figure 33.

[0106] 4. Use of acemannan-containing compositions for lung care 4.1 Antiviral (treatment or prevention of disease)

[0107] Human bronchial epithelial cells (NHBE) were treated with different amounts of acemannan-containing compositions to confirm their cytotoxicity. The results are shown in Figure 34. The integrity of airway epithelial cells is a key component of the innate immune system by forming a barrier against pathogens and the like. Epithelial integrity relies on tight junctions (TJs) and adherens junctions (AJs) between adjacent cells. Therefore, to monitor whether treatments maintain epithelial integrity in 3D normal human bronchial epithelial cell (NHBE) cultures, a composition containing acemannan (e.g., aloe extract, composition I50 used herein) was added to the apical side of the epithelium prior to infection with wild-type SARS-CoV-2 and BQ1.1. Transepithelial electrical resistance (TEER) was measured 1-2-3 days after infection, and the experimental design is shown in Figure 35. Measurement of TEER is a convenient technique that provides information on the uniformity of the cell layer on the filter support, and the integrity of the tight junctions formed between polarized cells. Therefore, TEER measurements are often used to study epithelial barrier function. For TEER analysis, three independent experimental repeats were performed according to SOP_TEER (Zaderer, V., Dichtl, S., Posch, W. et al. GlyPerA TM ColdZyme effectively protects airway epithelium against SARS-CoV-2 infection and inflammatory events. Respir Res 24, 88 (2023); Zaderer, V., Dichtl, S., Bellmann Weiler, R. et al. TM protects airway epithelium from infection with BA.4 / 5. Respir Res 23, 300 (2022). The results are shown in Figure 36A, Figure 36B, Figure 36C, Figure 36D and Figure 36E.

[0108] Real-time RT-PCR: Virus released into the supernatant of uninfected or infected and untreated or treated NHBE cells is absolutely quantified using a standardized SARS-CoV-2 real-time RT-PCR. The results are shown in Figure 37.

[0109] 5. Use of compositions containing acemannan in eye care

[0110] To analyze the effect of compositions A50 and I50 on the eye, an eye irritation test (by ITRI) was carried out. 5000 retinal pigment epithelial cells were seeded overnight in DMEM+F12 containing glutamic acid and 10% FBS. Different concentrations of acemannan, ITRI, and Acyl-50 (50% acetylated acemannan, e.g. composition A50) were added for 48 hours. 1 / 10 MTT was added for 30 minutes, and OD460 was detected. The results were shown as in Figure 38. Further results of composition I50 from a similar experiment were shown as Figure 39.

[0111] SIRC (Statens Serumstitut Rabbit Cornea) cells were transformed into ATCC (CCL60) TM ) and cultured in Eagle's minimum essential medium (ATCC, Cat. No. 30-2003) supplemented with 10% fetal bovine serum (Thermo Fisher, Cat. No. 16000044), 2 mM L-glutamin e, 50-100 units / mL penicillin, and 50-100 μg / mL streptomycin (Thermo Fisher, Cat. No. 15140122) at 37°C under 5% CO2. The cells were cultured at a density of 6 × 10 3 Cells were seeded in 96-well plates at 100 μL / well and cultured for 4 days until confluent. The cells cultured in 96-well plates were exposed to 200 μL / well of test drug solutions at 5% and 0.05% concentrations for 5 min at room temperature. After exposure, the cells were washed twice with 200 μL of PBS and 200 μL of 0.5 mg / mL MTT solution (Thermo Fisher, product number M6494) was added. After incubation at 5% CO2 and 37 °C for 2 h, the MTT solution was decanted and 200 μL of 0.04 N hydrochloric acid-isopropanol was added to extract MTT formazan in the dark at room temperature for 1 h, and the absorbance at 570 nm was measured using a plate reader (Thermo Fisher, Varioskan LUX). The optical density (OD) values ​​obtained for each test drug were used to calculate the cell viability relative to the solvent control, which was set at 100%. The experiment was repeated three times independently with triplicate wells.

[0112] Cell viability cut-off values ​​for identifying test chemicals causing serious eye damage (UN GHS Category 1) and test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS no category) are summarized in Table 6.

[0113] [Table 6]

[0114] As a result, a short-term exposure in vitro test (OECD, TG 491) was conducted to evaluate the eye irritation potential of the test article provided by DAZZEON BIOTECH CO., LTD. The test results meet the following criteria: (1) The OD of the medium control is 0.3 or more after subtracting the blank OD. (2) The cell viability of the solvent control is 80% or more compared to the medium control. (3) The cell viability of the positive control (0.01% SLS) is within 2 standard deviations of the previous experiment. (4) The standard deviation (SD) of three independent cell viabilities is 15% or less at both the 5% and 0.05% concentrations of the test article. In this test procedure, the OD of the medium control is 0.538±0.048. The cell viability of the solvent control was 93.935 ± 07.140%, the cell viability of the positive control (0.01% SLS) was 36.540 ± 08.854%, and the SD of the cell viability of the papillary stripe was less than 15% (Table 7). All the pass criteria were met in three independent tests. The test article was prepared in two concentrations, 0.05% and 5%, in PBS, and the cell viabilities were 97.076 ± 08.439% and 102.988 ± 07.420%, respectively. According to the prediction model of OECD TG 491, the cell viability was more than 70% at all the tested concentrations, and the eye irritation was classified as "no category" (Table 8).

[0115] [Table 7]

[0116] [Table 8]

[0117] The above and following embodiments are merely illustrative. Many details are often found in the art. Therefore, such many details are not shown or described herein for the sake of brevity. Although many features and advantages of the present disclosure, together with the details of the structure and function of the present disclosure, have been shown in the foregoing description, the present disclosure is merely illustrative, and modifications may be made in details. Therefore, it is understood that the above and following embodiments may be modified within the scope of the claims.

Claims

1. A composition comprising acemannan, an acetylated mannan-based polysaccharide compound, The molecular weight of the acemannan is Less than 853 kDa (kilodaltons) composition.

2. 2. The composition of claim 1 , The molecular weight of the acemannan is 853 kDa (kilodaltons) or less and 5 kDa (kilodaltons) or more composition.

3. The composition according to claim 2, The acetylation degree, which represents the ratio of the number of acetylated hydroxyl groups to the total number of hydroxyl groups in the acemannan, is 30% or more and 78% or less composition.

4. The composition according to claim 3, The degree of acetylation is 30% or more and 40% or less, or 60% or more and 78% or less composition.

5. The composition according to any one of claims 1 to 4. Use for Gut Health Methods of using the compositions.

6. The composition according to any one of claims 1 to 4. Use for skin care Methods of using the compositions.

7. The composition according to any one of claims 1 to 4. Use for lung care Methods of using the compositions.

8. The composition according to any one of claims 1 to 4. Use for eye care Methods of using the compositions.

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