Use of Amanita orchid extract for the treatment of diseases associated with dysregulation of neutrophil activation

Abstract

Disclosed herein is a method for treating diseases and / or disorders associated with dysregulation of neutrophil activation and recruitment. The method comprises administering an effective amount of an extract of Amanita albicans to a subject in need thereof. The extract of Amanita albicans contains at least the compounds 3,3'-dihydroxy-5-methoxybibenzyl (Batatacin III), 9,10-dihydro-1-[(4-hydroxyphenyl)methyl]-4-methoxy-2,7-phenanthrenediol (Orchidble), 3',5-dimethoxy-3-hydroxybibenzyl (BF8-4-2), 3,5-dimethoxy-3'-hydroxybibenzyl (BF8-4-3) and 3-hydroxy-5-methoxybibenzyl (BF8-4-4).

Description

[Technical Field]

[0001] The present disclosure generally relates to novel uses of extracts of Bletilla formosana (hereinafter sometimes referred to as "Amanalan") in treating diseases and / or disorders associated with dysregulation of neutrophil activation, such as acute respiratory distress syndrome (ARDS). [Background technology]

[0002] Neutrophils are the most abundant granulocytes in the circulation and are responsible for eliminating pathogens through degranulation, neutrophil elastase (NE) release, superoxide production, and the respiratory burst, as well as neutrophil extracellular trap (NET) formation. Thus, neutrophils are key effectors of both the adaptive and innate immune systems. During inflammation, adhesion and migration are both crucial steps in neutrophil recruitment, which are regulated by conformational changes in the macrophage-1 antigen (Mac-1, also known as αMβ2 and CD11b-CD18) on the neutrophil surface. Dysregulated neutrophil activation and recruitment results in damage to host tissues through the release of excessive amounts of proteolytic enzymes, reactive oxygen species (ROS), and NETs, ​​leading to various pathological conditions such as autoimmune diseases (e.g., systemic lupus erythematosus, rheumatoid arthritis, and psoriasis), infectious diseases (e.g., sepsis), inflammatory diseases (e.g., ARDS, chronic obstructive pulmonary disease, and asthma), atherosclerosis, and other major diseases (e.g., diabetes and cancer).

[0003] In traditional Chinese medicine, Bletilla tuber has been used for thousands of years to treat pulmonary diseases, digestive diseases, skin inflammatory diseases, and hemorrhagic diseases. In the present application, the inventors unexpectedly discovered that Amanita orchid extract regulates the inflammatory state of activated human neutrophils, and therefore may serve as a candidate drug for the development of medicines for treating diseases and / or disorders associated with dysregulation of neutrophil activation and recruitment, such as ARDS, diabetes, psoriasis, liver damage, etc. Summary of the Invention

[0004] The present disclosure provides a novel use of Amanhalan extract, which inhibits dysregulation of activated neutrophils, and therefore, Amanhalan extract may act as a candidate drug for the development of pharmaceuticals to treat diseases and / or disorders associated with dysregulation of neutrophil activation and recruitment, such as ARDS, acute liver injury (ALI), diabetes, or psoriasis.

[0005] Thus, a first aspect of the present disclosure is directed to a method of treating a subject with ARDS, ALI, diabetes, or psoriasis, comprising administering to the subject an effective amount of an extract of Amanita albicans.

[0006] According to certain embodiments of the present disclosure, the Amanita orchid extract comprises: (i) extracting the bulbs, leaves, stems, or a mixture thereof of Amanita orchid with water to produce a first extract and a first residue; (ii) extracting the first residue of step (i) with ethyl acetate to produce an Amaranth extract; It is prepared by

[0007] According to another embodiment of the present disclosure, the Amanita orchid extract is prepared by extracting the bulbs, leaves, stems, or a mixture thereof of Amanita orchid with ethyl acetate.

[0008] According to an embodiment of the present disclosure, the Amanhalan extract contains at least the compounds 3,3'-dihydroxy-5-methoxybibenzyl (Batatacin III), 9,10-dihydro-1-[(4-hydroxyphenyl)methyl]-4-methoxy-2,7-phenanthrenediol (Orchidble), 3',5-dimethoxy-3-hydroxybibenzyl (BF8-4-2), 3,5-dimethoxy-3'-hydroxybibenzyl (BF8-4-3), and 3-hydroxy-5-methoxybibenzyl (BF8-4-4).

[0009] Exemplary ARDS that can be treated by the present methods include, but are not limited to, transfusion-related lung injury, ventilator-induced lung injury, bacterial-induced lung injury, viral-induced lung injury, and the like.

[0010] According to an embodiment of the present disclosure, the Amanita ulmoides extract is administered to a subject in an amount of 0.01 to 1000 mg / Kg, preferably 0.1 to 800 mg / Kg.

[0011] According to embodiments of the present disclosure, subjects suitable for treatment by the present methods are mammals, preferably humans.

[0012] The details of one or more embodiments of this disclosure are set forth in the accompanying description below. Other features and advantages of the invention will be apparent from the detailed description and claims.

[0013] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various exemplary systems, methods and other exemplary embodiments of various aspects of the present invention, and will be better understood from the following detailed description read in light of the accompanying drawings. [Brief explanation of the drawings]

[0015] [Figure 1] Amanita orchid extract inhibited elastase release in stimulated human neutrophils. Neutrophils (6 × 10 cells / mL) were incubated with 0.1% DMSO or 0.3–10 μg / mL of the indicated Amanita orchid extract for 5 minutes, followed by activation with fMLF (0.1 μM) or CB (1 μg / mL) for an additional 10 minutes. Amanita orchid extracts were individually prepared by extracting mashed Amanita orchid bulbs with (A) H (n-hexane), (B) 3H1E (n-hexane / ethyl acetate = 3 / 1), (C) 1H1E (n-hexane / ethyl acetate = 1 / 1), (D) EA (ethyl acetate), (E) EtOH (95% alcohol), and (F) HO (ddHO). Elastase release was measured spectrophotometrically at 405 nm. All data are presented as mean ± SEM (n = 6–7). Compared to control, **p<0.01, ***p<0.001. [Figure 2]Amanita orchid extract inhibited superoxide anion production in stimulated human neutrophils. Neutrophils (6 × 10 cells / mL) were incubated with 0.1% DMSO or 0.3–10 μg / mL of the indicated Amanita orchid extract for 5 min, followed by activation with fMLF (0.1 μM) / CB (1 μg / mL) for an additional 10 min. Amanita orchid extracts were individually prepared by extracting mashed Amanita orchid bulbs with (A) H (n-hexane), (B) 3H1E (n-hexane / ethyl acetate = 3 / 1), (C) 1H1E (n-hexane / ethyl acetate = 1 / 1), (D) EA (ethyl acetate), (E) EtOH (95% alcohol), and (F) HO (ddHO). Superoxide anion production was measured using the ferritintochrome C reduction method by spectrophotometry at 550 nm. All data are presented as mean ± SEM (n = 6–7). *p<0.05, **p<0.01, ***p<0.001 compared to control. [Figure 3] Effects of various Amaranth extracts on elastase release in stimulated human neutrophils. Human neutrophils were pretreated with 0.1% DMSO or Amaranth extract (1, 3, and 10 μg / mL) for 5 minutes and then stimulated for an additional 10 minutes with or without fMLF (0.1 μM) / CB (1 μg / mL). Amaranth extract was individually extracted by extracting mashed Amaranth bulbs with (A) EA and (B) -W+EA. Elastase release was measured spectrophotometrically at 405 nm. All data are presented as mean ± SEM (n = 6–8). **p<0.01, ***p<0.001 compared to control. [Figure 4]Effects of various Amanita orchid extracts on superoxide anion production in stimulated human neutrophils. Neutrophils (6 × 10 cells / mL) were incubated with 0.1% DMSO or 0.3–10 μg / mL of the indicated Amanita orchid extract for 5 minutes, followed by activation with fMLF (0.1 μM) / CB (1 μg / mL) for an additional 10 minutes. Amanita orchid extracts included (A) EA and (B) -W+EA extracts. Superoxide anion production was measured using the ferritintochrome C reduction method by spectrophotometry at 550 nm. All data are presented as mean ± SEM (n = 6–8). *p<0.05, **p<0.01, ***p<0.001 compared to control. [Figure 5] The lily of the valley extract did not exhibit cytotoxicity to human neutrophils. Neutrophils (6 × 10 cells / mL) were incubated with 0.1% DMSO or 0.3–10 μg / mL of the indicated lily of the valley extract for 15 minutes. The lily of the valley extracts were individually prepared by extracting mashed lily of the valley bulbs with n-hexane ("H"), n-hexane / ethyl acetate (3 / 1) ("3H1E"), n-hexane / ethyl acetate (1 / 1) ("1H1E"), ethyl acetate ("EA"), 95% alcohol ("EtOH"), ddH2O ("H2O"), and a combination of ddH2O and EA ("-W+EA"). Total LDH release was determined by treating cells with 0.1% TX-100 for 30 minutes, followed by detection of LDH via enzyme-linked immunosorbent assay at 490 nm. All data are presented as mean ± SEM (n = 5). [Figure 6] Amanita asiatica EA extract reduced NET formation in PMA-stimulated neutrophils. Human neutrophils were pretreated with 0.1% DMSO or EA extract (1, 3, and 10 μg / mL) for 10 minutes and then incubated with or without 10 nM PMA for 3 hours. SYTOX Green was then added to the plate for 15 minutes. All data are presented as mean ± SEM (n = 3). [Figure 7]Amanita asiatica EA extract reduced ROS production in neutrophils stimulated with fMLF. (A) Human neutrophils were pretreated with 0.1% DMSO or EA extract (0.1, 0.3, and 1 μg / mL) for 5 minutes and then stimulated with or without 0.1 μM fMLF for an additional 6 minutes. (B) Peak chemiluminescence and (C) area under the curve (AUC) of chemiluminescence are expressed as mean ± SEM (n = 7). *p<0.05, **p<0.01, ***p<0.001 compared to control. [Figure 8] Amanhalan extract reduced IMQ-induced psoriasis in mice. (A) The upper panel shows photographs from one representative mouse treated with vehicle (left), IMQ (center), and IMQ plus Amanhalan extract (right). (B) The lower panel shows microscopic images of skin samples taken from vehicle control, IMQ-treated mice, and mice treated with IMQ plus Amanhalan extract. [Figure 9] Amanita orchid extract reduced LPS-induced acute lung injury in mice. BALB / c mice (n = 2-3 per group) were treated intraperitoneally with vehicle (10% DMSO) or 50 mg / kg Amanita orchid extract, followed by intratracheal nebulization of LPS (2 mg / kg) for 5 hours. Light microscope images of H&E-stained lung sections from mice treated with or without Amanita orchid extract. [Figure 10] Amanita orchid extract attenuated D-GalN / LPS-induced acute liver injury (ALI) in mice. BALB / c mice (n = 2-3 per group) were treated intraperitoneally with vehicle (10% DMSO) or D-GalN (400 mg / kg) + LPS (40 μg / mL) for 1 hour, followed by intraperitoneal injection of Amanita orchid extract (50 or 100 mg / kg) for 5 hours. Blood samples were collected, and plasma levels of (A) GOP and (B) GPT were determined. [Figure 11]Amanita orchid extract alleviated D-GalN / LPS-induced acute liver injury (ALI) in mice. BALB / c mice (n = 2-3 per group) were treated intraperitoneally with vehicle (10% DMSO) or D-GalN (400 mg / kg) plus LPS (40 μg / mL) for 1 hour, followed by intravenous injection of Amanita orchid extract (50 or 100 mg / kg) for 5 hours. The mice were sacrificed, and their liver tissues were collected and analyzed by H&E staining. [Figure 12] Amanita japonica extract reduced elevated blood glucose levels in STZ-induced diabetic mice. C57BL / 6 mice (n = 2-3 per group) were treated intraperitoneally with vehicle (saline) or STZ (50 mg / kg) for 5 days. Mice that showed elevated blood glucose levels were selected for subsequent treatment, which involved intraperitoneal injection of Amanita japonica extract (25 mg / kg) for 5 consecutive days from days 9 to 13. The blood glucose levels (A) and body weights (B) of the mice were measured from days 9 to 13. DETAILED DESCRIPTION OF THE INVENTION

[0016] The detailed description given below in connection with the accompanying drawings is intended as an illustration of the disclosure and is not intended to represent the only form in which the disclosure may be constructed or utilized.

[0017] I. Definition The terms "administered," "administering," or "administration" are used interchangeably herein and refer to a mode of delivery, including, without limitation, intravenous, intramuscular, intraperitoneal, intraarterial, intracranial, or subcutaneous administration of an agent (e.g., a compound or composition) of the present invention.

[0018] An "effective amount" of the linden orchid extract described herein (taken either alone or in combination with other agents) refers to an amount sufficient to elicit a desired biological response, such as inhibiting inflammatory activity or alleviating a target disease or symptoms associated with the disease described herein. As will be appreciated by those skilled in the art, the effective amount of the linden orchid extract described herein can vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the extract, the condition being treated, the mode of administration, and the age and health of the subject. In some instances, the effective amount may be a therapeutically effective amount. A therapeutically effective amount refers to an amount of a therapeutic agent, alone or in combination with other therapies, sufficient to provide a therapeutic benefit in the treatment of a condition, delay the onset of the condition, or minimize one or more symptoms associated with the condition. A therapeutically effective amount refers to an amount that improves overall treatment, reduces or avoids the symptoms, signs, or causes of the condition, and / or enhances the therapeutic effectiveness of other therapeutic agents. In other instances, an effective amount may be a prophylactically effective amount. A prophylactically effective amount of linden orchid extract refers to an amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in the prevention of the condition. For example, a prophylactically effective amount of Amanita orchid extract may be an amount sufficient to prevent or delay the onset of a condition or one or more symptoms associated with that condition, or to prevent its recurrence. It may also be an amount that improves overall preventative measures or enhances the preventative effectiveness of other preventative agents. Furthermore, an effective amount may be a human equivalent dose (HED) converted from the animal dose used in the examples of this disclosure in accordance with industry guidance issued by the U.S. Food and Drug Administration (Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, USDapartment of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research (CDER), July 2005).

[0019] A "subject" as used herein can be a human subject (e.g., a pediatric subject, such as an infant, toddler, or adolescent, or an adult subject, such as a young adult, middle-aged adult, or elderly person) or a non-human animal, such as a dog, cat, cow, pig, horse, sheep, goat, rodent (e.g., mouse, rat), or non-human primate (e.g., cynomolgus monkey, rhesus monkey). The non-human mammal can be a transgenic or genetically engineered animal. In some embodiments, the subject is a human patient having, suspected of having, or at risk for a disease of interest as described herein (e.g., ARDS, diabetes, psoriasis, liver injury, etc.). In other embodiments, the subject is a human or non-human mammal having or suspected of having a condition secondary to dysregulated neutrophil activation (e.g., ARDS, diabetes, psoriasis, acute liver injury, etc.).

[0020] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within the acceptable standard error of the mean as considered by one of ordinary skill in the art. Other than in the working examples / examples, or unless expressly stated otherwise, all numerical ranges, amounts, values, and ratios, such as those relating to amounts of materials thereof, durations, temperatures, operating conditions, ratios of amounts, and the like, disclosed herein should be understood in all instances to be modified by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in this disclosure and the appended claims are approximations that may be varied as desired. At the very least, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0021] The singular forms "a," "an," and "the" are used herein to include the plural forms as well, unless the context clearly dictates otherwise.

[0022] 2. Use of the Amanita orchid extract of the present invention The present disclosure resides in the unexpected discovery that the Amanita orchid extract prepared according to the process described herein has a therapeutic effect on the dysregulation of activated human neutrophils. Thus, the Amanita orchid extract can be used as a candidate drug for the development of medicines suitable for treating diseases or disorders associated with the dysregulation of neutrophil activation and recruitment, such as ARDS, ALI, diabetes, psoriasis, etc.

[0023] Accordingly, a first aspect of the present disclosure provides a method for treating a subject suffering from ARDS, ALI, diabetes, or psoriasis, comprising administering to the subject an effective amount of an extract of Amanita alba.

[0024] Preferably, the lily of the valley extract of the present invention is prepared by a method comprising the steps of (i) extracting the bulbs, leaves, stems, or a mixture thereof with water to produce a first extract and a first residue, and (ii) extracting the first residue of step (i) with ethyl acetate to produce the lily of the valley extract, and therefore the lily of the valley extract produced is referred to as a "-W+EA" extract in the present disclosure. Alternatively, the lily of the valley extract of the present invention is prepared by extracting the bulbs, leaves, stems, or a mixture thereof with ethyl acetate, and therefore the lily of the valley extract produced is referred to as an "EA" extract in the present disclosure.

[0025] Bioactivity analysis of this amaranth extract indicates that it is a potent inhibitor of superoxide anion production, elastase release, reactive oxygen species (ROS) production, and degranulation in stimulated human neutrophils. Furthermore, the amaranth extract does not affect cell viability. HPLC / MS analysis of the amaranth extract indicates that it contains at least the following compounds: 3,3'-dihydroxy-5-methoxybibenzyl (Batatacin III), 9,10-dihydro-1-[(4-hydroxyphenyl)methyl]-4-methoxy-2,7-phenanthrenediol (Orchidble), 3',5-dimethoxy-3-hydroxybibenzyl (BF8-4-2), 3,5-dimethoxy-3'-hydroxybibenzyl (BF8-4-3), and 3-hydroxy-5-methoxybibenzyl (BF8-4-4). Bioactivity analysis of the compounds identified in the present Amanita orchid extract confirmed that each of them can suppress superoxide anion production from activated neutrophils, and three of them, except for BF8-4-2, can suppress elastase release from activated neutrophils. The findings of the present disclosure confirm that the present Amanita orchid extract (e.g., "EA" or "-W+EA" extract) can serve as a candidate drug for the development of medicines suitable for treating diseases associated with dysregulation of neutrophil activation and recruitment, such as ARDS, ALI, diabetes, psoriasis, etc.

[0026] According to embodiments of the present disclosure, ARDS can result from transfusion-related lung injury, ventilator-induced lung injury, bacterial-induced lung injury, viral-induced lung injury, and the like.

[0027] According to embodiments of the present disclosure, the present Amanhalan extract (e.g., "EA" or "-W+EA" extract) may have the following concentrations: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 1, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 1 50, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, The Amanita orchid extract is administered to the subject in an amount of 0.01 to 1000 mg / kg, such as 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg / kg, preferably 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, or 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 0, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680 , 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790 and 800 mg / kg, more preferably the Amanhalan extract is administered to the subject in an amount of 0.1 to 800 mg / kg, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 51.0, 52.0, 53.0, 54.0, 55.0, 56.0, 57.0, 58.0, 59.0, 60.0, 61.0, 62.0, 63.0,0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 12 The compound is administered to a subject in an amount of 1 to 100 mg / kg, such as 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 mg / kg. In one preferred embodiment, the lily of the valley extract is administered to a subject in an amount of 4 mg / kg. In another preferred embodiment, the lily of the valley extract is administered to a subject in an amount of 8 mg / kg. An effective amount of the compound may be administered in one or more doses over one or more days (depending on the mode of administration).

[0028] The present Amanhalan extract may be formulated with suitable carriers or excipients for any suitable route of administration, for example, oral, parenteral, by inhalation spray, topical, rectal, nasal, buccal, vaginal, or via an implanted reservoir. The term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrathoracic, intrathecal, and intracranial injection or infusion techniques.

[0029] Sterile injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as TWEEN® 80) and suspending agents. The sterile injectable preparations may also be sterile injectable solutions or suspensions in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally employed as solvents or suspending media (e.g., synthetic mono- or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives, particularly their polyoxyethylated versions, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, carboxymethylcellulose, or similar dispersing agents. Other commonly used surfactants, such as Tween or Span, or similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms, may also be used for formulation purposes.

[0030] Formulations suitable for oral administration can be any orally acceptable dosage form, including, but not limited to, capsules, tablets, emulsions, and aqueous suspensions, dispersions, and solutions. For tablets for oral use, commonly used carriers include lactose and cornstarch. Lubricants such as magnesium stearate are also commonly added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions or emulsions are administered orally, the amaranth extract of the present disclosure can be suspended or dissolved in an oil phase in combination with an emulsifier or suspending agent. If desired, specific sweeteners, flavors, or colorants can be added. Nasal aerosol or inhalation formulations can be prepared according to techniques well known in the pharmaceutical formulation art, and can be prepared as solutions in saline employing benzyl alcohol or other appropriate preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizers or dispersants well known in the art. The amaranth extract of the present disclosure can also be administered in the form of suppositories for rectal administration.

[0031] Pharmaceutically acceptable carriers or excipients that may be included in the formulations containing the Amanita orchid extract of the present disclosure include inert diluents, solubilizers, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavors, and fragrances may also be present in the pharmaceutical compositions.

[0032] The excipients present in the formulations of the invention must be "pharmaceutically acceptable" in the sense that they are compatible with the active ingredients of the pharmaceutical composition (preferably capable of stabilizing the pharmaceutical composition) and are not harmful to the subject to which the pharmaceutical composition is administered. For example, solubilizing agents such as cyclodextrins, which can form highly soluble specific complexes with the lily of the valley extract of the present invention, can be used as pharmaceutically acceptable excipients for delivering the lily of the valley extract of the present invention to a subject. Examples of other pharmaceutically acceptable excipients include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, etc.

[0033] Also disclosed herein are kits (e.g., pharmaceutical packs) comprising the amaranth extract described herein and a container (e.g., a vial, ampoule, bottle, syringe and / or dispenser package, or other suitable container). In some embodiments, the kit may also include a second container comprising a pharmaceutically acceptable excipient for diluting or suspending the inventive formulation. In some embodiments, the amaranth extract provided in the first container and the pharmaceutically acceptable excipient (e.g., saline) provided in the second container are combined to form a single unit dosage form.

[0034] In certain embodiments, the kits described herein are for use in inhibiting dysregulation of neutrophil activation and recruitment. In certain embodiments, the kits described herein are for use in treating any of the diseases described herein (e.g., ARDS, ALI, diabetes, psoriasis) in a subject in need thereof. Accordingly, any of the kits described herein may also include instructions for administering the included Amanita orchid extract. The kits of the present invention may also include information required by regulatory authorities, such as the FDA. In certain embodiments, the kits and instructions are for treating the diseases described therein. The kits of the present invention may include one or more additional pharmaceutical agents described herein as separate compositions.

[0035] It should also be understood that the extracts or formulations described herein can be used in any of the methods described herein in combination with one or more additional agents (e.g., therapeutically and / or prophylactically effective agents). The Amanita lily extracts or formulations can be administered in combination with additional agents that enhance their activity (e.g., activity (e.g., efficacy and / or effectiveness) in treating a disease described herein in a subject in need thereof, preventing a disease described herein in a subject in need thereof, or inhibiting neutrophil activation in a subject). It should also be understood that the treatments employed can achieve the desired effect for the same disorder and / or they can achieve different effects.

[0036] The present invention will now be more particularly described with reference to the following embodiments, which are given for purposes of illustration and not limitation, and which are standard in nature and may alternatively be used by those skilled in the art, including other procedures, methods, or techniques well known to those skilled in the art. [Example]

[0037] Materials and Methods Preparation of Amanita orchid extract The bulbs, leaves, stems, or a mixture thereof of Amanita orchid were dried and ground, then stored at -20°C until use. The ground Amanita orchid powder (10 g) was extracted at room temperature with one of the following solvents: n-hexane, n-hexane / ethyl acetate (1:1), n-hexane / ethyl acetate (3:1), ethyl acetate, 95% ethanol, and double-distilled water (ddH2O) to obtain the corresponding crude extracts. A total of six crude extracts were produced, designated "H," "1H1E," "3H1E," "EA," "EtOH," and "H2O" extracts. The residue of the H2O extract was further extracted with ethyl acetate to obtain the extract designated "-W+EA" extract.

[0038] Each of the six crude extracts or "-W+EA extracts" was sonicated (30 min), filtered, and concentrated to obtain the corresponding B. formosana extract. Each B. formosana extract was subjected to liquid chromatography / mass spectrometry (LC-QTOF-MS / MS) analysis to identify the active ingredients therein.

[0039] Isolation of human neutrophils The study was conducted under the approval of the Institutional Review Board of Chang Gung Memorial Hospital in accordance with the Declaration of Helsinki. After obtaining written informed consent, whole blood samples were obtained from healthy individuals aged 20–30 years who had not taken any medication within the preceding 5 weeks. Neutrophils were then isolated using standard procedures for dextran sedimentation, Ficoll-Hypaque gradient centrifugation, and hypotonic hemolysis of red blood cells. The isolated neutrophils were then analyzed by Ca ion transporter. 2+ The cells were suspended in HBSS (pH 7.4) containing no hydroxybenzoates and stored at 4°C until use.

[0040] Analysis of neutrophil elastase (NE) release Human neutrophils (6 × 10 5 Cells (cells / mL) were treated with 1 mM CaCl2 and 100 μM NE substrate (Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide) and then incubated with DMSO or the Amanita orchid extract for 5 min at 37°C. Cells were stimulated with fMLF (0.1 μM) / cytochalasin B (CB) (0.5 μg / mL) for 10 min, after which NE release was determined by measuring the change in absorbance at 405 nm using a spectrophotometer.

[0041] Analysis of neutrophil extracellular trap (NET) formation Neutrophils (2×10 6Neutrophils (cells / mL) were incubated with DMSO or Amanita orchid extract for 5 minutes and activated with 10 nM PMA for 3 hours, followed by the addition of deoxyribonuclease (DNase) (2 U / mL) for 10 minutes. The reaction was terminated by the addition of EDTA (2 mM) at 4°C, and the resulting mixture was then centrifuged at 4°C for 5 minutes. The supernatant was collected and mixed with SYTOX Green (5 μM) in a 96-well plate. NET formation of activated neutrophils was assessed by the fluorescence emitted in each 96-well using a fluorescence imaging system.

[0042] Measurement of extracellular superoxide anion production Extracellular superoxide anion production in activated neutrophils was assessed by the reduction of ferricytochrome c. 2+ After incubation with 1 mM erythrocytes (1 mM) and ferricytochrome c (0.6 mg / mL) at 37°C, isolated human neutrophils (6 × 10 5 Cells (cells / mL) were incubated with DMSO or the Amanita orchid extract for 5 minutes. Cells were pretreated with cytochalasin B (CB, 1 or 2 μg / mL) for 3 minutes and then stimulated with fMLF. The change in absorbance at 550 nm was continuously detected using a spectrophotometer (U-3010, Hitachi, Tokyo, Japan), and the superoxide anion level was calculated using a previously described method (Hwang et al., 2003 Mol. Pharmacol. 64(6), 1419-1427).

[0043] Analysis of total ROS generation Human neutrophils (2 × 10 6 Cells (cells / mL) were preincubated with 6 U / mL horseradish peroxidase (HRP) and 37.5 μM luminol in a 96-well plate at 37°C for 5 minutes. Cells were incubated with DMSO or Amanita orchid extract for 5 minutes and then stimulated with 0.1 μM fMLF. Chemiluminescence was then detected and analyzed in real time in a 96-well plate chemiluminometer (Tecan Infinite F200 Pro; Mannedorf, Switzerland).

[0044] Cell viability assay The cytotoxicity of the Amanita orchid extract on neutrophils was assessed by measuring lactate dehydrogenase (LDH) levels using a commercially available kit (Promega). LDH is a cytoplasmic enzyme that is released only when the cell membrane is disrupted (i.e., upon cell death) and can therefore be used as an indicator of cell viability. The kit measures the color change (at λmax = 450 nm) that occurs when LDH reduces NAD to NADH. Cytotoxicity was expressed as the percent LDH activity obtained in cell-free medium relative to total LDH activity. Total LDH activity was determined by lysing cells with 0.1% Triton X-100 at 37°C for 30 minutes.

[0045] animal Animal care and experimental protocols were approved by the Chang Gung University Animal Care and Use Committee (Taiwan). Furthermore, animal studies were reported in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals (National Research Council Committee for the Update of the Guide for the Care and Use of Laboratory, 2011). Specific pathogen-free (SPF) 8-week-old male BALB / c mice (weight: 20 ± 1 g) were purchased from BioLASCO (Taiwan). Five mice shared a ventilated cage with standard bedding and had free access to water and standard laboratory chow. All mice were housed in an SPF animal facility under a 12-h light-dark cycle. Mice were allowed to acclimate for at least 1 week before use in experiments.

[0046] Imiquimod (IMQ)-induced psoriasis in mice BALB / c mice were pretreated with IMQ (62.5 mg) by topical application to the back of the mice for 60 minutes every day for 5 days (i.e., from day 0 to day 4). The present Amanita japonica extract (50 mg / kg) was also topically administered to the IMQ-treated skin area for 60 minutes before the daily IMQ treatment for days 2 to 4 (i.e., from day 1 to day 4). The mice were sacrificed on day 5.

[0047] LPS-induced acute respiratory distress syndrome (ARDS) in mice Male BALB / c mice were randomly assigned to four groups (2–3 mice per group): vehicle only, lily of the valley extract only, LPS control, and lily of the valley extract treatment (lily of the valley extract + LPS). Mice were fasted overnight and then intraperitoneally injected with 50 μL of lily of the valley extract (50 mg / kg) or 50 μL of vehicle (10% DMSO). Under general anesthesia with xylazine (6 mg / kg) and Zoletil 50 (30 mg / kg), ARDS was induced by intratracheal instillation of 50 μL of LPS (E. coli O111:B4; 2 mg / kg) or 50 μL of 0.9% saline (vehicle-only and lily of the valley extract only groups). Five hours later, mice were sacrificed, and lungs were harvested and fixed in 10% paraformaldehyde for histological sectioning.

[0048] Histological sectioning and staining The collected lung tissue was washed with phosphate-buffered saline (PBS) and fixed in 10% formalin for 24 hours. The samples were then dehydrated, embedded in paraffin, and sliced ​​into 3-μm-thick sections using a microtome and mounted on glass slides. These sections were stained with hematoxylin and eosin (H&E). Images were then captured by light microscopy.

[0049] Combined treatment of D-GalN and LPS-induced acute liver injury (ALI) in mice Male BALB / c mice were randomly assigned to four groups (2–3 mice per group): vehicle only, LPS + D-GalN (LPS, 40 μg / mL; D-galactosamine, 400 mg / kg) control, LPS + D-GalN + "-W + EA" extract (50 mg / kg) in group A, and LPS + D-GalN + "-W + EA" extract (100 mg / kg) in group B. Each mouse was intraperitoneally injected with 10% DMSO or LPS + D-GalN for 1 h, followed by intraperitoneal injection of "-W + EA" extract for another 5 h. Blood samples were collected and analyzed for glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) levels. The mice were then sacrificed, and their liver tissues were collected and analyzed by H&E staining.

[0050] Streptozotocin (STZ)-induced diabetic mice and treatments Wild-type male C57BL / 6 mice were made diabetic by intraperitoneal injection of STZ (50 mg / kg / day) for 5 days from day 1 to day 5. Mice that showed an average blood glucose level higher than 230 mg / dL on day 6 were selected for further studies. Vehicle or "-W+EA" extract (25 mg / Kg) was intraperitoneally injected into each animal daily from day 9, and continued for 5 days from day 9 to day 13. The fasting blood glucose (i.e., blood glucose level after 8 hours of fasting) and body weight of each mouse were measured from day 9 to day 13.

[0051] Example 1 In vitro characterization of the present Amanita orchid extract 1.1 Amanita orchid extract inhibited superoxide anion production and elastase release in stimulated neutrophils Amanhalan extract was prepared according to the procedure described in the "Materials and Methods" section. A total of seven Amanhalan extracts were generated and individually tested for their effects on inflammatory responses by monitoring superoxide anion production and elastase release in human neutrophils stimulated with formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF) / cytochalasin B (CB). The results are presented in Table 1 and Figures 1 and 2. Table 1. Effect of Amanita ulmoides extract on superoxide anion and elastase release in fMLF / CB-activated human neutrophils. [Table 1] a : Concentration required for 50% inhibition. b Percentage of inhibition at 10 μg / mL. Results are expressed as mean ± SEM (n = 6-7). *p<0.05, **p<0.01, ***p<0.001 compared to control (DMSO).

[0052] According to Table 1, solvents with higher polarity showed higher extraction power for Amanita orchid, with water showing an extraction rate of 31.81% ("H2O extract"), followed by 95% ethanol with an extraction rate of 12.29% ("EtOH extract"). In contrast, non-polar solvents such as n-hexane had the lowest extraction power with an extraction rate of only 0.68% ("H extract").

[0053] Regarding the regulatory effects of each extract, except for the water extract ("HO extract"), all other extracts, including n-hexane / ethyl acetate (1:1) ("1H1E extract"), n-hexane / ethyl acetate (3:1) ("3H1E extract"), ethyl acetate ("EA extract"), and 95% alcohol ("EtOH extract"), were found to be able to inhibit superoxide anion production and elastase release in human neutrophils activated by fMLF / CB (Figures 1 and 2). Among these extracts, the "EA extract" showed an IC of 0.79 μg / mL on superoxide anion production. 50and an IC of 1.71 μg / mL for elastase release 50 showed the strongest inhibitory effect (Table 1).

[0054] Since water showed the highest extraction power of Amanita orchid, and the highest anti-inflammatory components were found in the "EA extract," the residue of the "H2O extract" was further extracted with EA to produce the "-W + EA extract." The regulatory effects of the "EA extract" and "-W + EA extract" on human neutrophils activated by fMLF / CB are presented in Table 2 and Figures 3 and 4. Table 2. Effects of various Amanita extracts on superoxide anion and elastase release in human neutrophils activated by fMLF / CB. [Table 2] a : Concentration required for 50% inhibition. b Percentage of inhibition at 10 μg / mL. Results are expressed as mean ± SEM (n = 6-7). *p<0.05, **p<0.01, ***p<0.001 compared to control (DMSO).

[0055] It was found that "-W+EA extract" exhibited a regulatory effect equivalent to that of "EA extract" in terms of inhibiting superoxide anion production and elastase release (Table 2 and Figures 3 and 4).

[0056] 1.2 Amanita orchid extract does not affect cell viability In this example, we investigated whether the Amanita orchid extract of Example 1.1 affected cell viability by monitoring the level of lactate dehydrogenase (LDH) released from cells. LDH is a cytoplasmic enzyme that is released only when the cell membrane is disrupted (i.e., in the event of cell death), and therefore can be used as an indicator of cell viability. The results are shown in Figure 5.

[0057] Since the level of LDH released from human neutrophils treated with the Amanita orchid extract was negligible, it was clear that none of the seven Amanita orchid extracts in Example 1.1 had cytotoxicity.

[0058] 1.3 Amanita japonica extract EA attenuated neutrophil extracellular trap (NET) formation NETs, ​​which consist primarily of chromatin filaments coated with granule proteins, proteases, and histones, are important in inflammatory and autoimmune disorders. To investigate the effect of Amanita orchid extract on NET formation, neutrophils were stained with Sytox green after activation with PMA (10 nM).

[0059] As shown in Figure 6, PMS-induced NET formation was significantly attenuated by Amanita orchid EA extract.

[0060] 1.4 Amanita japonica EA extract improved ROS production in stimulated neutrophils In this example, we investigated whether Amanita japonica EA extract affected ROS production in stimulated neutrophils by flow cytometry and chemiluminescence assay. The results are shown in Figure 7.

[0061] Quantitative results from flow cytometry and luminol-amplified chemiluminescence assays revealed that Amanita asiaticum EA extract significantly inhibited intracellular ROS production in fMLF-activated neutrophils in a dose-dependent manner (Figure 7).

[0062] 1.5 Chemical constituents of Amanita orchid extract Each of the Amanita orchid extracts from Example 1.1 was subjected to LC / MS analysis to identify its chemical components and further investigate the regulatory effects of each identified component on superoxide anion production and elastase release in activated neutrophils. The results are summarized in Tables 3 and 4.

[0063] Chemical components, including Batatacin III, Orchidble, BF8-4-2, BF8-4-3, and BF8-4-4, were present in the "3H1H," "1H1E," "EA," and "-W+EA" extracts (Table 3), and each component was found to inhibit superoxide anion production and elastase release in activated human neutrophils (Table 4). Table 3. Contents of chemical components of Amanita orchid in various extracts [Table 3] a : Not detected Table 4. Effects of chemical components of Amanita orchid extract on superoxide anion and elastase release in human neutrophils activated by fMLF / CB. [Table 4]

[0064] Example 2: Amanita orchid extract alleviated imiquimod (IMQ)-induced psoriasis in mice In this example, the effect of Amanita orchid extract on the development of psoriasis was investigated using an established animal model of psoriasis. Imiquimod (IMQ) was used to induce psoriasis-like skin inflammation. The results are shown in Figure 8.

[0065] Figure 8(A) shows photographs taken from a representative mouse treated with IMQ (center) and IMQ + "-W+EA" extract (right). Figure 8(B) shows microscopic images of skin samples taken from a control IMQ-treated mouse and a mouse treated with IMQ + "-W+EA" extract. It is clear that the Amanita ulmoides "-W+EA" extract significantly reduced the severity or progression of IMQ-induced psoriasis in the test animals.

[0066] Example 3: Amanita orchid extract alleviated lipopolysaccharide (LPS)-induced acute respiratory distress syndrome in mice In this example, we investigated the effect of Amanita japonica extract on LPS-induced acute respiratory distress syndrome. To this end, BALB / c mice were treated with "-W+EA" extract (50 mg / kg) or DMSO administered intraperitoneally, followed by intratracheal LPS nebulization for 5 hours. Photographs of the lungs and histopathological characteristics by HE staining revealed that LPS induced hemorrhage and erythema, alveolar interstitial thickening, and pulmonary interstitial edema formation (Figure 9). Lung structural distortion was significantly suppressed in the "-W+EA" extract-treated group.

[0067] Example 4: Amanita orchid extract improved D-GalN / LPS-induced acute liver injury in mice In this example, the effect of Amanita orchid extract on D-GalN / LPS-induced acute liver injury was investigated. To this end, BALB / c mice were intraperitoneally injected with D-GalN (400 mg / kg) and LPS (40 μg / mL) or saline (220 μL) for 1 hour, followed by intraperitoneal injection of "-W+EA" extract (50 or 100 mg / kg) for another 5 hours. Blood samples were collected, and plasma was analyzed for GOT and GPT enzyme levels. Meanwhile, liver tissues were collected and subjected to H&E staining. The results are shown in Figures 10 and 11.

[0068] Figure 10 is a bar graph showing the effect of the "-W+EA" extract on the levels of GOT and GPT, and Figure 11 is a photograph of liver tissue in control, D-GalN / LPS-treated, and D-GalN / LPS+"-W+EA" extract-treated mice.

[0069] It was revealed that D-GalN / LPS caused significant damage to liver tissue, resulting in elevated levels of GOT and GPT in serum (Fig. 11), and these elevated levels of GOT and GPT were effectively reduced by treatment with the "-W+EA" extract (Fig. 10).

[0070] Example 5: Amanita orchid extract reduced STZ-induced blood glucose elevation in mice In this example, the effect of Amanita orchid extract on STZ-induced blood glucose elevation was investigated. To this end, C57B6 mice were treated with STZ (50 mg / kg) to induce an increase in blood glucose levels, according to the procedure described in the "Materials and Methods" section. The diabetic-like animals were then treated with "-W+EA" extract (25 mg / kg) or saline for an additional 5 days. The results are shown in Figure 12.

[0071] The data in Figure 12 reveal that the "-W+EA" extract at a daily dose of 25 mg / kg can effectively reduce the STZ-induced increase in blood glucose levels without adversely affecting the body weight of the test animals.

[0072] It should be understood that the above description of the embodiments is given by way of example only, and that various modifications may be made by those skilled in the art. The above specification, examples, and data provide a complete description of the structure and use of example embodiments of the invention. While various embodiments of the invention have been described above with a certain degree of particularity or with reference to one or more specific embodiments, those skilled in the art could make numerous modifications to the disclosed embodiments without departing from the scope of the disclosure.

Claims

1. 1. Use of an extract of Amanhalan for the manufacture of a medicament for treating acute respiratory distress syndrome (ARDS), acute liver injury (ALI), diabetes, or psoriasis, wherein the extract of Amanhalan contains 3,3'-dihydroxy-5-methoxybibenzyl (Batacin III), 9,10-dihydro-1-[(4-hydroxyphenyl)methyl]-4-methoxy-2,7-phenanthrenediol (Orchidble), 3',5-dimethoxy-3-hydroxybibenzyl (BF8-4-2), 3,5-dimethoxy-3'-hydroxybibenzyl (BF8-4-3), and 3-hydroxy-5-methoxybibenzyl (BF8-4-4); The Amanita orchid extract is (i) extracting the bulbs, leaves, stems, or a mixture thereof of Amanita orchid with water to produce a first extract and a first residue; (ii) extracting the first residue of step (i) with ethyl acetate to produce the Amanita orchid extract; Prepared by, use.

2. 2. The use according to claim 1, wherein the ARDS is transfusion-associated lung injury, ventilator-induced lung injury, bacterial-induced lung injury or viral-induced lung injury.

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