Oncidium plant extract and its use for manufacture of drug for treating diabetes mellitus, protecting liver, and preventing acute respiratory distress syndrome (ARDS)

Abstract

To provide a natural compound and its use for the manufacture of a drug for treating diabetes mellitus, protecting the liver, and preventing acute respiratory distress syndrome (ARDS).SOLUTION: An Oncidium plant extract is provided. The preparation steps therefor include the steps of: taking an Oncidium plant sample; drying and grinding the sample to obtain a ground Oncidium plant sample; and extracting the ground Oncidium plant sample in a solvent through accelerated solvent extraction or ultrasonic extraction to obtain an Oncidium plant extract.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an Oncidium plant extract and its use for the manufacture of a medicament for treating diabetes, protecting the liver and preventing acute respiratory distress syndrome (ARDS). [Background technology]

[0002] Diabetes mellitus (DM) is a metabolic disease caused by impaired insulin and / or insulin production, leading to hyperglycemia. DM patients often have many complications, one of which is the failure of the body to heal itself.

[0003] Impaired diabetic wound healing is the major and most intractable complication in diabetic patients and is associated with diverse factors such as vascular, neuropathic, immune and biochemical components. Hyperglycemia hardens arteries, subsequently leading to circulatory delay and microvascular dysfunction, as well as reduced tissue oxygen supply; hyperglycemia also reduces leukocyte migration to wounds, making humans more susceptible to infection. Peripheral neuropathy in DM can numb the area and reduce the ability to feel pain, which may lead to wounds not being noticed immediately and appropriate treatment not being undertaken.

[0004] The liver is one of the most important organs in the human body, and can be damaged by drugs, alcohol, diet, poor quality work and rest. Liver injury can lead to hepatocyte degeneration, necrosis, fibrous tissue hyperplasia and other lesions, which will subsequently lead to a series of liver pathological changes and secondary diseases, such as hepatitis, liver cancer, and liver cirrhosis.

[0005] Glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) are mainly involved in the metabolism of amino acids and proteins in the human body and are highly concentrated in liver cells, so once the liver is damaged, GOT and GPT flow into the blood, and therefore they are used as biological indicators to identify liver damage in blood tests.

[0006] Acute respiratory distress syndrome (ARDS) is a potentially fatal acute disease that is caused by acute inflammatory damage to the lungs, affecting the normal functioning of the alveoli and respiratory exchange, resulting in insufficient oxygen levels in the blood and subsequent damage to various organs of the body. Treatment of ARDS is based on supportive care, including mechanical ventilation.

[0007] Natural compounds have the advantage of abundant supply and diverse backbones, and are important bases for drug development. From 1981 to 2019, nearly half of the new FDA-approved drugs were derived from natural products or their derivatives, such as cocaine-derived narcotics for cancer treatment, morphine-derived painkillers, vincristine, doxorubicin and paclitaxel, and fungus-derived penicillin as antibiotics. Therefore, the present invention actively studies to determine which natural compounds have the potential to be developed as novel drugs for treating DM, protecting the liver, or preventing ARDS. Summary of the Invention

[0008] The present invention is an Oncidium plant extract, the preparation steps of which are: harvesting an Oncidium plant sample; drying and grinding the sample to obtain a ground Oncidium plant sample; and then extracting the ground Oncidium plant sample in a solvent by accelerated solvent extraction or ultrasonic extraction to obtain an Oncidium plant extract.

[0009] In the present invention, the solvent is preferably an alcohol, an ester, or water, and the Oncidium plant extract is preferably an alcohol extract, an ester extract, or an extract extracted with water and then extracted with an ester of the Oncidium plant.

[0010] Alcohols of the present invention include, but are not limited to, most preferably methanol, ethanol, propanol, isopropyl alcohol, butanol, isobutanol, hexanol, and esters include, but are not limited to, most preferably methyl acetate, ethyl acetate, butyl acetate.

[0011] The Oncidium plant extract of the present invention is preferably extracted from the roots, stems or leaves of the Oncidium plant, or a mixture composed of the roots, stems and leaves of the Oncidium plant.

[0012] The Oncidium plant extract of the present invention is most preferably extracted from the root of the Oncidium plant.

[0013] In the present invention, Oncidium species include Oncidium stacyi, Oncidium splendidum, Oncidium pusillum, Oncidium jonesianum, Oncidium hawkesiana, Oncidium ascendens, Oncidium ampliatum, or Oncidium flexuosum.

[0014] The present invention relates to the use of an Oncidium plant extract for the manufacture of a medicament for treating diabetes in a subject in need thereof.

[0015] The present invention also relates to the use of an Oncidium plant extract for the manufacture of a medicament for protecting the liver in a subject in need thereof.

[0016] As used herein, the term "liver protection" in the present invention refers to protecting subject from liver injury, reducing the possibility of liver injury, or reducing the degree of liver injury.The term "liver injury" in the present invention includes, but is not limited to, liver injury, liver disease, such as hepatitis (including, but is not limited to, alcoholic hepatitis, drug-induced hepatitis, fatty hepatitis, chronic hepatitis), liver cancer, liver cirrhosis, liver fibrosis, fatty liver, hepatic decline, and other liver diseases caused by liver injury (including, but is not limited to, acute liver injury or chronic liver injury).

[0017] The present invention further relates to the use of an Oncidium plant extract for the manufacture of a medicament for preventing Acute Respiratory Distress Syndrome (ARDS) in a subject in need thereof.

[0018] As used herein, the term "acute respiratory distress syndrome (ARDS)" of the present invention includes transfusion-associated lung injury, ventilator-induced lung injury, bacterial-induced lung injury, or viral-induced lung injury.

[0019] In the present invention, the medicament may further comprise a pharma- ceutically acceptable carrier for the Oncidium plant extract.

[0020] The term "effective amount" refers to an amount that can achieve an effective result when administered to an individual, or has a desired activity in vivo or in vitro. In the case of inflammatory disorders, effective clinical outcomes include reducing the degree or severity of symptoms associated with the disease or condition, and / or extending the life of the individual, and / or improving the quality of life of the individual, when compared to no treatment. The exact amount of the compound administered to an individual depends on the type and severity of the disease or condition, and on individual characteristics such as the individual's general health, the individual's age, sex, weight, and drug tolerance. Such amounts are also governed by the condition, severity and type of inflammatory, autoimmune and allergic disorders, or the desired immunosuppressive effect. Those skilled in the art will be able to determine the appropriate dosage according to these and other factors based on the disclosed technical features of the present invention.

[0021] In the present invention, the effective amount of the Oncidium plant extract of the present invention is 0.001 mg / kg body weight to 100 mg / kg body weight.

[0022] Further, an effective amount of the Oncidium plant extract of the present invention is 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 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 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 100 mg / kg body weight.

[0023] The effective amounts of the present invention may be converted to equivalent effective amounts for subjects of different species according to the formulas and safety factors provided in Guidance for Industry: Establishing the Maximum Safe Starting Doses in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, US Department of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research (CDER), published by the FDA in July 2005.

[0024] In the use for the manufacture of a medicament for treating diabetes in a subject, the subject is a human or mammal, and the effective amount of the present invention is 0.007 mg / kg body weight to 1.5 mg / kg body weight; the effective amount of the present invention is preferably 0.035 mg / kg body weight to 1.3 mg / kg body weight; the effective amount of the present invention is most preferably 0.07 mg / kg body weight to 0.75 mg / kg body weight.

[0025] In particular, in the use for the manufacture of a medicament for treating diabetes in a subject, the effective amount of the present invention in a mouse having an average body weight of 25 grams is 1 mg / kg body weight to 100 mg / kg body weight, the equivalent effective amount in a dog or cat having an average body weight of 10 kilograms is 0.01 mg / kg body weight to 1.5 mg / kg body weight, the equivalent effective amount in an adult having an average body weight of 60 kilograms is 0.007 mg / kg body weight to 0.8 mg / kg body weight, and the equivalent effective amount in a child having an average body weight of 20 kilograms is 0.01 mg / kg body weight to 1.2 mg / kg body weight; preferably, the effective amount of the present invention in a mouse having an average body weight of 25 grams is 5 mg / kg body weight to 75 mg / kg body weight, and the equivalent effective amount in a dog or cat having an average body weight of 10 kilograms is 0.05 mg / kg body weight to 1.2 mg / kg body weight. most preferably, the effective amount of the present invention in a mouse with an average body weight of 25 grams is 10 mg / kg body weight to 50 mg / kg body weight, the equivalent effective amount in a dog or cat with an average body weight of 10 kilograms is 0.13 mg / kg body weight to 0.75 mg / kg body weight, the equivalent effective amount in an adult with an average body weight of 60 kilograms is 0.07 mg / kg body weight to 0.45 mg / kg body weight, and the equivalent effective amount in a child with an average body weight of 20 kilograms is 0.1 mg / kg body weight to 0.6 mg / kg body weight.

[0026] In use for the manufacture of a medicament for protecting the liver in a subject, the subject is a human or mammal, and the effective amount of the present invention is 0.001 mg / kg body weight to 1.75 mg / kg body weight; the effective amount of the present invention is preferably 0.009 mg / kg body weight to 1 mg / kg body weight; the effective amount of the present invention is most preferably 0.015 mg / kg body weight to 0.4 mg / kg body weight.

[0027] In particular, in the use for the manufacture of a medicament for protecting the liver in a subject, the effective amount of the present invention in a rat having an average body weight of 350 grams is 0.1 mg / kg body weight to 50 mg / kg body weight, the equivalent effective amount in a dog or cat having an average body weight of 10 kilograms is 0.003 mg / kg body weight to 1.75 mg / kg body weight, the equivalent effective amount in an adult having an average body weight of 60 kilograms is 0.001 mg / kg body weight to 1.25 mg / kg body weight, and the equivalent effective amount in a child having an average body weight of 20 kilograms is 0.002 mg / kg body weight to 1.5 mg / kg body weight; preferably, the effective amount of the present invention in a rat having an average body weight of 350 grams is 0.5 mg / kg body weight to 25 mg / kg body weight, and the equivalent effective amount in a dog or cat having an average body weight of 10 kilograms is 0.01 mg / kg body weight to 1.5 mg / kg body weight. 5mg / kg body weight to 1mg / kg body weight, the equivalent effective amount in an adult with an average body weight of 60 kilograms is 0.009mg / kg body weight to 0.5mg / kg body weight, and the equivalent effective amount in a child with an average body weight of 20 kilograms is 0.013mg / kg body weight to 0.7mg / kg body weight; most preferably, the effective amount of the present invention in a rat with an average body weight of 350 grams is 1mg / kg body weight to 10mg / kg body weight, the equivalent effective amount in a dog or cat with an average body weight of 10 kilograms is 0.03mg / kg body weight to 0.4mg / kg body weight, the equivalent effective amount in an adult with an average body weight of 60 kilograms is 0.015mg / kg body weight to 0.2mg / kg body weight, and the equivalent effective amount in a child with an average body weight of 20 kilograms is 0.025mg / kg body weight to 0.3mg / kg body weight.

[0028] In the use for the manufacture of a medicament for preventing acute respiratory distress syndrome (ARDS) in a subject, the subject is a human or mammal, and the effective amount of the present invention is 0.007 mg / kg body weight to 0.75 mg / kg body weight; the effective amount of the present invention is preferably 0.02 mg / kg body weight to 0.4 mg / kg body weight; and the effective amount of the present invention is most preferably 0.035 mg / kg body weight to 0.35 mg / kg body weight.

[0029] In particular, in the use for the manufacture of a medicament for preventing acute respiratory distress syndrome (ARDS) in a subject, the effective amount of the present invention in a mouse having an average body weight of 25 grams is 1 mg / kg body weight to 50 mg / kg body weight, the equivalent effective amount in a dog or cat having an average body weight of 10 kilograms is 0.01 mg / kg body weight to 0.75 mg / kg body weight, the equivalent effective amount in an adult having an average body weight of 60 kilograms is 0.007 mg / kg body weight to 0.45 mg / kg body weight, and the equivalent effective amount in a child having an average body weight of 20 kilograms is 0.01 mg / kg body weight to 0.65 mg / kg body weight; preferably, the effective amount of the present invention in a mouse having an average body weight of 25 grams is 3 mg / kg body weight to 25 mg / kg body weight, and the equivalent effective amount in a dog or cat having an average body weight of 10 kilograms is 0.005 mg / kg body weight to 0.005 mg / kg body weight. and the equivalent effective amount in an adult of average body weight 60 kilograms is 0.02 mg / kg body weight to 0.25 mg / kg body weight, and the equivalent effective amount in a child of average body weight 20 kilograms is 0.03 mg / kg body weight to 0.3 mg / kg body weight; most preferably, the effective amount of the present invention in a mouse of average body weight 25 grams is 5 mg / kg body weight to 20 mg / kg body weight, and the equivalent effective amount in a dog or cat of average body weight 10 kilograms is 0.06 mg / kg body weight to 0.35 mg / kg body weight, and the equivalent effective amount in an adult of average body weight 60 kilograms is 0.035 mg / kg body weight to 0.2 mg / kg body weight, and the equivalent effective amount in a child of average body weight 20 kilograms is 0.05 mg / kg body weight to 0.25 mg / kg body weight.

[0030] In the present invention, the subject of the present invention is a human (e.g., an infant, a toddler, a teenager, a young adult, a middle-aged adult or an elderly person), a rodent (e.g., a mouse, a rat) or a mammal (e.g., a companion animal such as a dog, a cat, a rabbit, a marten; an economic animal such as a horse, a sheep, a cow, a pig). In a preferred mode, the subject of the present invention is a human or a mammal. The subject of the present invention is most preferably a human.

[0031] The present invention may also include a pharma- ceutically acceptable carrier, particularly a pre-determined solvent or oil, and may further include a pH adjuster, and may further include a dispersant, if desired. Examples of solvents that may be used in the present invention include, but are not limited to, water, ethanol, isopropanol, 1,3-butanediol, propylene glycol, glycerin, etc. Examples of oils that may be used in the present invention include, but are not limited to, corn oil, sesame oil, linseed oil, cottonseed oil, soybean oil, peanut oil, monoglycerides, diglycerides, triglycerides, mineral oil, squalene, jojoba oil, olive oil, evening primrose oil, borage oil, grape seed oil, coconut oil, sunflower oil, shea butter, and any combination thereof.

[0032] Solvents and oils may be used alone or in any combination thereof.

[0033] Examples of useful dispersing agents beneficial in the present invention may include, but are not limited to, lecithin, organic monoglycerides, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, sorbitan stearate, etc. These raw materials may also be used alone or in any combination thereof.

[0034] In the present invention, the agent is preferably administered orally or by injection.

[0035] In the present invention, the agent of the present invention may also be prepared as an external preparation, and examples of the external preparation may include, but are not limited to, a cream, an ointment, a gel, a cleansing lotion, a patch, etc., or may be an inhalant, an aerosol, a suppository, etc.

[0036] When the drug is used as an external preparation, a suitable external skin preparation may be used as the base material, and may be sterilized according to a known method using an aqueous solution, a non-aqueous solvent, a suspension, an emulsion, or a freeze-dried preparation, etc. By using a known method and adding a known softener, an emulsifier, and a thickener or other materials known in the art, a composition in the form of a gel, a cream, and an ointment may be prepared.

[0037] For example, a gel-type composition may be prepared by adding softening agents such as trimethylolpropane, polyethylene glycol and glycerol, solvents such as propylene glycol, ethanol and isocetyl alcohol, and pure water. [Brief description of the drawings]

[0038] [Figure 1] Figure 1 illustrates the effectiveness of each Oncidium plant extract in inhibiting the nitrogen free radical DPPH. ** / *** means significantly different. [Diagram 2] Figure 2 illustrates the effectiveness of each Oncidium plant extract in inhibiting the oxygen free radical AAPH. ΔAUC means area under the curve of the receiver operating characteristic curve; ** / *** means significantly different. [Diagram 3] Figure 3 shows a flow diagram of the animal experiment to evaluate the efficacy of Oncidium plant extract in the treatment of type I diabetes. Legend of symbols: * indicates induction of diabetes by streptozotocin; $ indicates check of non-fasting blood glucose level; # indicates check of 18-hour fasting blood glucose level; → indicates the period of administration of Oncidium plant extract; ! indicates sacrifice; % indicates blood parameters for determining liver function index (glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT)) and kidney function index (creatinine (CRE), urea nitrogen (BUN)). [Figure 4]Figure 4 illustrates blood glucose levels before and after 18 hours of fasting in healthy mice, diabetic mice, and diabetic mice administered Oncidium plant extract. * indicates significant difference (P<0.05) when comparing treated group with untreated group; + indicates treatment; - indicates untreated. [Diagram 5] Figure 5 illustrates glucose tolerance tests in healthy mice, diabetic mice, and diabetic mice administered Oncidium plant extract. * indicates significant difference (P<0.05) when comparing low-dose ORC001R-U-EtOH group with non-treatment group; # indicates significant difference (P<0.05) when comparing medium-dose ORC001R-U-EtOH group with non-treatment group. [Figure 6] Figure 6 illustrates blood parameters of healthy mice, diabetic mice, and diabetic mice administered Oncidium plant extract. * indicates significant difference when comparing treated group to non-treated group; + indicates treatment; - indicates non-treatment. [Figure 7] Figure 7 illustrates staining of liver and kidney sections from healthy mice, diabetic mice, and diabetic mice administered Oncidium plant extract. + indicates treatment; - indicates non-treatment. [Figure 8] Figure 8 illustrates the weight change of healthy mice, diabetic mice, and diabetic mice administered Oncidium plant extract. * indicates significant difference (P<0.05) when comparing low-dose ORC001R-U-EtOH group with non-treatment group; # indicates significant difference (P<0.05) when comparing medium-dose ORC001R-U-EtOH group with non-treatment group; @ indicates significant difference (P<0.05) when comparing high-dose ORC001R-U-EtOH group with non-treatment group. [Figure 9]Figure 9 shows a flow diagram of the animal experiment to evaluate the efficacy of Oncidium plant extract in treating type 2 diabetes mellitus (T2DM). Legend: * indicates non-fasting blood glucose level check; $ indicates 8-hour fasting blood glucose level check; # indicates intraperitoneal (IP) insulin tolerance test; @ indicates IP glucose tolerance test; → indicates the period of administration of Oncidium plant extract; ! indicates sacrifice; % indicates blood parameters for determining liver function index (glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT)) and kidney function index (creatinine (CRE), urea nitrogen (BUN)). [Figure 10] Figure 10 shows blood glucose levels after 8 hours fasting in T2DM mice and T2DM mice administered Oncidium plant extract for 4 weeks. ** indicates significant difference (P<0.01) when comparing treated group with untreated group. [Figure 11] Figure 11 shows glucose tolerance test in T2DM mice and T2DM mice administered Oncidium plant extract for 4 weeks. * indicates significant difference (P<0.01) when comparing 50 mg / kg treatment group with non-treatment group. [Figure 12] Figure 12 shows insulin tolerance test in T2DM mice and T2DM mice administered Oncidium plant extract for 4 weeks. # indicates significant difference (p<0.01) when comparing 10mg / kg treatment group with non-treatment group. @ indicates significant difference (p<0.01) when comparing 25mg / kg treatment group with non-treatment group. * indicates significant difference (p<0.01) when comparing 50mg / kg treatment group with non-treatment group. [Figure 13] Figure 13 shows blood parameters of T2DM mice and T2DM mice administered with Oncidium plant extract for 4 weeks. - indicates non-administration. [Figure 14] Figure 14 shows the flow diagram of the animal experiment to evaluate the effectiveness of Oncidium plant extract in liver protection. Legend: * indicates induction of liver fibrosis by CCl4; % indicates blood collection; → indicates the period of administration of Oncidium plant extract; ! indicates sacrifice. [Figure 15] Figure 15 illustrates the blood biochemistry analysis results (GOT, GPT) of liver fibrosis rats in different groups at week 7. + indicates treatment; - indicates non-treatment. [Figure 16] FIG. 16 illustrates the weekly changes in blood biochemistry analysis of liver fibrosis rats in different groups. [Figure 17] FIG. 17 illustrates rat weight tracing results of liver fibrosis rats in different groups. [Figure 18] Figure 18 shows that Oncidium plant extract alleviated D-GalN / LPS-induced acute liver injury (ALI) in mice. + indicates treatment; - indicates no treatment; * indicates significant difference (p<0.05) between treated and no treatment; *** indicates significant difference (p<0.005) between treated and no treatment. [Figure 19] Figure 19 shows a flow diagram of an animal study to evaluate the efficacy of Oncidium plant extract in preventing acute respiratory distress syndrome (ARDS). Legend: * indicates induction of ARDS by lipopolysaccharide (LPS); % indicates administration of Oncidium plant extract; ! indicates sacrifice. [Figure 20] Figure 20 illustrates the staining results of lung tissue sections in different groups of mice. + indicates treatment; - indicates non-treatment. [Figure 21] Figure 21 illustrates the blood analysis results in the different groups of mice. + indicates treatment; - indicates non-treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present embodiment is merely a description of the best mode for carrying out the present invention, and is not intended to limit the present invention.

[0040] Preparation of Oncidium plant extract

[0041] In the present invention, the Oncidium plant samples used for the extraction preparation were either the roots (R), stems (B) or leaves (L) of the Oncidium plant, or a mixture of the roots, stems and leaves. The species of Oncidium plant may be selected from Oncidium staci, Oncidium splendinum, Oncidium psilum, Oncidium jonesianum, Oncidium haukesiana, Oncidium ascendens, Oncidium amphriatum, or Oncidium flexuosum. The Oncidium plant samples were dried, ground and stored at -20°C until ready for use.

[0042] In the present invention, Oncidium samples were first extracted by accelerated solvent extraction (A) and ultrasonic extraction (U).

[0043] In accelerated solvent extraction, the residue after extraction with ethyl acetate (A-EA), 95% ethanol (A-EtOH), HO (AW) and HO was then re-extracted by using ethyl acetate as the solvent for purification and extraction respectively, the extraction temperature was 30-70°C (preferably 50°C), the extraction time was 10-30 minutes (preferably 15 minutes), and the extraction pressure was 1300-1700 psi (due to slight pressure fluctuation during the extraction process, the average pressure is preferably 1500 psi), and then the Oncidium plant extract of the present invention (ORC001) was obtained. According to the different extraction parts and different solvents, the extractions were named: ORC001R-A-EA, ORC001R-A-EtOH, ORC001R-AW, ORC001R-A-W+EA, ORC001B-A-EA, ORC001B-A-EtOH, ORC001B-AW, ORC001B-A-W+EA, ORC001L-A-EA, ORC001L-A-EtOH, ORC001L-AW, and ORC001L-A-W+EA.

[0044] In ultrasonic extraction, the residue after extraction with ethyl acetate (U-EA), 95% ethanol (U-EtOH), HO (UW), and HO was then re-extracted by using ethyl acetate as the solvent for purification and extraction at room temperature, respectively, the extraction temperature is 30-70°C (preferably 50°C), the extraction time is 15-60 minutes (preferably 30 minutes), the power is 200-600 watts (preferably 400 watts), and the frequency is 20-60 Hz (preferably 40 Hz), and then the Oncidium plant extract of the present invention (ORC001) was obtained. According to the extraction part and solvent, the extractions were named: ORC001R-U-EA, ORC001R-U-EtOH, ORC001R-UW, ORC001R-U-W+EA, ORC001B-U-EA, ORC001B-U-EtOH, ORC001B-UW, ORC001B-U-W+EA, ORC001L-U-EA, ORC001L-U-EtOH, ORC001L-UW, and ORC001L-U-W+EA.

[0045] The determination of the constituent profiles of different species of Oncidium plant extracts (same parts, same solvents, same extraction methods) was performed by high performance liquid chromatography (HPLC) fingerprinting, and the results revealed that similar fingerprints were observed in different species of Oncidium plant extracts, which indicated that the same compounds were present in these different species of Oncidium plant extracts. Therefore, Oncidium flexuosum species was selected as the Oncidium plant sample of the present invention.

[0046] Inhibition assay of superoxide anion and elastase release

[0047] The anti-inflammatory potential of various Oncidium plant extracts mentioned above was evaluated in the present invention through inhibition tests of superoxide anion and elastase release.

[0048] First, neutrophils were prepared.

[0049] Approximately 50 mL of blood from healthy donors (20-35 years old; performing normal work and rest, and refraining from taking medication for more than one week) was collected from an antecubital vein into a vacuum sterile blood collection tube, which was added to an equal volume of 3% dextran solution (dextran), mixed uniformly, and allowed to stand to allow red blood cells to sediment. The supernatant containing neutrophils was collected and covered on a Ficoll-Paque solution contained in a centrifuge tube, and the centrifuge tube was then centrifuged to separate different blood cells by density gradient, resulting in neutrophils that settled at the bottom and a few red blood cells. The residual red blood cells were expanded with NaCl solutions of different concentrations, and the necessary neutrophils were retained by utilizing the difference in osmotic resistance between the two cells.

[0050] The inflammatory response of the various Oncidium plant extracts described above on neutrophils was assessed by respiratory blasting experiments and evaluation of the degranulation function.

[0051] Respiratory blasting is primarily caused by superoxide anion (O2 ·- ) and reactive oxygen species (ROS), and the above Oncidium plant extract was added to a neutrophil suspension (6x10 5 Superoxide anion (O2) was added at 1000x the concentration of 0.01% erythrocytes / mL for 2 min at 37 °C. ·- ) was detected, and then treated with 1 μg / mL cytochalasin B (CB) for 3 minutes. The cells were then activated for about 10 minutes using N-formyl-L-methionyl-L-leucinoyl-L-phenylalanine (fMLF) as a stimulant. The absorbance values ​​were then measured using a UV spectrophotometer at a wavelength of 550 nm.

[0052] In the elastase release experiment, 100 μM elastase acceptor, methionine Neutrophil suspensions (6x10) containing cis-ala-ala-prolinate-valine-p-nitroanilide (MeOSuc-Ala-Ala-Pro-Val-p-nitroanilide) were 5The cells were pre-warmed to reach 37°C for 5 min and then various Oncidium plant extracts were added for 2 min. The cells were treated with 0.5 μg / mL cytochalasin B (CB) for 3 min, neutrophils were activated by stimulation with 0.1 μM fMLF for approximately 10 min, and finally, absorbance values ​​were measured at 405 nm using a UV spectrophotometer. Experimental results were expressed as percent inhibition.

[0053] The results are shown in Table 1.

[0054] [Table 1] Percent inhibition (% inhibition) at 10 μg / ml. Results are presented as mean ± SEM (n=3-5). Compared to controls (DMSO or HO), * P < 0.05, ** P < 0.01, *** P<0.001. a Concentration required for 50% inhibition (IC 50 ) b Dissolve in H2O. c Before the experiment, ORC001R-A-EA (10 μg / ml) was filtered through a 0.22 μm filter.

[0055] The cytotoxicity of the Oncidium plant extract of the present invention is shown in Table 2.

[0056] [Table 2] Percentage (%) of cell viability at 10 μg / ml. Results are presented as mean±SEM (n=3). a Before the experiment, ORC001R-A-EA (10 μg / ml) was filtered through a 0.22 μm filter.

[0057] Free radical scavenging ability test

[0058] Nitrogen radical: 1,1-diphenyl-p-picrylhydrazyl (DPPH), and oxygen radical: 2,2'-azobis(2-aminopropane) hydrochloride (AAPH) are stable free radicals related to inflammation. In the present invention, 10μg / mL of each Oncidium plant extract was further tested for its ability to scavenge free radicals, where DMSO or H2O was used as the control group, and α-tocopherol was used as the comparative reagent, and each Oncidium plant extract was mixed with ethanol-set DPPH (100μM) or AAPH (25mM) for 30 minutes and 2 hours, respectively, as the activity index of free radical scavenging, if it has the ability to scavenge free radicals.

[0059] As shown in Figure 1, the organic solvent extracts (EA, EtOH, W+EA) of Oncidium root (ORC001R) were more significant in inhibiting the nitrogen radical DPPH.

[0060] Furthermore, as shown in Figure 2, the organic solvent extracts (EA, EtOH, W+EA) of Oncidium root (ORC001R) and Oncidium stem (ORC001B), as well as the EtOH and water extracts of Oncidium leaf (ORC001L) showed more significant in oxygen radical inhibition.

[0061] Establishment of a type 1 diabetes mellitus (T1DM) model

[0062] All experiments were approved by the Chang Gung University Animal Care and Use Committee (IACUC).

[0063] First, an animal model of diabetes was established. Diabetes was induced in 7-week-old C57BL / 6 mice weighing 20-25 grams by intraperitoneal injection of 55 mg / kg streptozotocin (STZ) (Sigma-Aldrich) in citrate buffer (pH 4.5) after 4-h fasting for 5 consecutive days. For induction of type 1 diabetes, normal chow and 1% glucose water were provided after STZ injection on days 1-4. From day 5 onwards, normal drinking water was provided instead of glucose water.

[0064] 72 hours after STZ injection, mice were fasted for 4 hours. They were bled by tail prick and fasting blood glucose levels were measured using a Contour Plus portable blood glucose meter (Ascensia, United Kingdom). Animals with fasting blood glucose levels of 150-250 mg / dL were considered prediabetic, whereas those >250 mg / dL were considered diabetic and used in subsequent experiments. All mice had fasting blood levels >250 mg / dL.

[0065] Evaluation of T1DM treatment efficacy

[0066] The therapeutic effect of Oncidium root extract (ORC001R) in diabetes was evaluated by monitoring blood glucose levels with or without extract administration. Oncidium root ultrasonic extract (ORC001R-U-EtOH) with EtOH as the solvent was used in the present invention. The experimental flow diagram is shown in Figure 3.

[0067] A total of 25 C57BL / 6 male mice were used in the experiment. These mice were randomly divided into 5 groups: 4 mice in the treatment control group (10% ethanol and 10% Tween-20 in saline; NC), 7 mice in the non-treatment group (STZ; T1DM), 7 mice in the low-dose ORC001R-U-EtOH group (received 10 mg / kg ORC001R-U-EtOH; T1DM+ORC001R-U-EtOH (10 mg / kg)), 4 mice in the medium-dose ORC001R-U-EtOH group (received 25 mg / kg ORC001R-U-EtOH; T1DM+ORC001R-U-EtOH (25 mg / kg)), and 3 mice in the high-dose ORC001R-U-EtOH group (received 50 mg / kg ORC001R-U-EtOH (25 mg / kg)). Administered ORC001R-U-EtOH; T1DM+ORC001R-U-EtOH (50 mg / kg)).

[0068] Before drug administration, the animals were weighed and their non-fasting blood glucose levels were checked by tail-prick and blood collection using a Contour Plus portable blood glucose meter (Ascensia, United Kingdom). The animals were then fasted for 18 hours to obtain fasting blood glucose levels. After drug administration, these steps were repeated on days 7 and 14 to observe the trends in blood glucose levels.

[0069] As shown in FIG. 4, the results of 18-hour fasting blood levels in animals 14 days after administration showed that the low-dose ORC001R-U-EtOH group showed a tendency to reduce blood glucose, a favorable effect of reducing blood glucose was observed in the medium-dose ORC001R-U-EtOH group, and a significant effect of reducing blood glucose was observed in the high-dose ORC001R-U-EtOH group.

[0070] The results of the tendency of blood glucose level of animals were examined by intraperitoneal glucose tolerance test (IPGTT). After fasting for 18 hours, glucose (purchased from Sigma) was intraperitoneally administered to mice at a dose of 1 g / kg body weight, and blood glucose level was detected at 0, 15, 30, 60, 120, and 180 minutes after injection by using a blood glucose meter. The results are shown in Figure 5, where it was demonstrated that low dosage ORC001R-U-EtOH group, medium dosage ORC001R-U-EtOH group and high dosage ORC001R-U-EtOH group all had tendency to decrease blood glucose level after oral administration for 14 consecutive days, and especially, the best tendency to decrease blood glucose level was shown by both medium dosage ORC001R-U-EtOH group and high dosage ORC001R-U-EtOH group.

[0071] After 14 consecutive days of ORC001R-U-EtOH treatment, blood was drawn from the heart of the animal model, then centrifuged at 2000xg for 15 minutes at room temperature to separate serum from whole blood, and liver function indexes (glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT)) and kidney function indexes (creatinine (CRE), urea nitrogen (BUN)) were tested in the blood of the animals by using a Fujifilm DRI-CHEM NX500i device, and liver and kidney tissues were also sectioned and stained with hematoxylin-eosin (H&E staining), and the results were shown in Figures 6 and 7, and ORC001R-U-EtOH showed no toxicity in liver or kidney, and also had the effect of repairing liver and kidney damage caused by diabetes.

[0072] The changes in animal body weight are shown in FIG. 8, and the administration of ORC001R-U-EtOH to animals showed a tendency for body weight to increase again from the 9th day onwards.

[0073] Establishment of a type 2 diabetes mellitus (T2DM) model

[0074] All experiments were approved by the Chang Gung University Animal Care and Use Committee (IACUC).

[0075] First, a type 2 diabetes animal model was established. In this study, 5-week-old male BKS.Cg-Dock7m+ / +Leprdb / Jnarl mice weighing 31-35 grams were selected. This strain is a genetically engineered spontaneous type 2 diabetes mouse that has been established for many years and is widely accepted as a model of metabolic syndrome disease.

[0076] Fasting blood glucose levels were measured by tail prick bleeding using a Contour Plus portable blood glucose meter (Ascensia, United Kingdom). Animals with fasting blood glucose levels between 150 and 250 mg / dL were considered to be in a prediabetic state. As the weeks of the experiment progressed, the mice's fasting blood glucose levels gradually increased, and those >250 mg / dL were classified as diabetic.

[0077] Evaluation of T2DM treatment efficacy

[0078] The therapeutic effect of Oncidium root extract (ORC001R) in diabetes was evaluated by monitoring blood glucose levels with or without extract administration. Oncidium root ultrasonic extract (ORC001R-U-EtOH) with EtOH as the solvent was used in the present invention. The experimental flow diagram is shown in Figure 9.

[0079] A total of 12 BKS.Cg-Dock7m+ / +Leprdb / Jnarl male mice were used in the experiment. These mice were randomly divided into 4 groups: 3 mice in the non-treated group (T2DM) (1% Tween20 and 99% CMC solution (0.5% w / v); T2DM), 3 mice in the low-dose ORC001R-U-EtOH group (10 mg / kg ORC001R-U-EtOH administered; T2DM+ORC001R-U-EtOH (10 mg / kg)), 3 mice in the medium-dose ORC001R-U-EtOH group (25 mg / kg ORC001R-U-EtOH administered; T2DM+ORC001R-U-EtOH (25 mg / kg)), and three mice in the high-dosage ORC001R-U-EtOH group (received 50 mg / kg ORC001R-U-EtOH; T2DM+ORC001R-U-EtOH (50 mg / kg));

[0080] Before drug administration, the animals were weighed and their non-fasting blood glucose levels were checked by tail-prick and blood collection using a Contour Plus portable blood glucose meter (Ascensia, United Kingdom).The animals were then fasted for 8 hours to obtain fasting blood glucose levels.These steps were repeated on days 1, 7, 14, 21 and 28 after drug administration to observe the trends in blood glucose levels.

[0081] Fasting blood glucose levels of the experimental animals after 28 days of treatment are shown in Figure 10. The results show that ORC001R-U-EtOH root extract shows a tendency to lower blood glucose at low doses. Medium doses showed a more pronounced blood glucose lowering effect, and high doses showed a significant blood glucose lowering effect, showing a dose-dependent response.

[0082] The results of the tendency of blood glucose level of animals were examined by intraperitoneal glucose tolerance test (IPGTT). After fasting for 18 hours, glucose (purchased from Sigma) was intraperitoneally injected into mice at a dose of 1 g / kg body weight, and blood glucose level was detected at 0, 15, 30, 60, 120, and 180 minutes after injection by using a blood glucose meter. The results are shown in FIG. 11, where it was demonstrated that low dosage ORC001R-U-EtOH group, medium dosage ORC001R-U-EtOH group, and high dosage ORC001R-U-EtOH group all had a tendency to decrease blood glucose level after oral administration for 28 consecutive days, and in particular, the best tendency to decrease blood glucose level was shown by both low dosage ORC001R-U-EtOH group and high dosage ORC001R-U-EtOH group. Only the high dose shows a significant improvement compared to the untreated group.

[0083] For IP insulin tolerance test (IPITT), mice were injected with 0.75 U / kg body weight of insulin (Humulin R U-500, Lilly) after 5 hours of fasting, and glucose concentrations were determined by glucometer at 0, 15, 30, 60, 120, and 180 minutes. The results are shown in FIG. 12, where it was demonstrated that the low-dose ORC001R-U-EtOH group, the medium-dose ORC001R-U-EtOH group, and the high-dose ORC001R-U-EtOH group all had a trend to reduce blood glucose levels after 28 consecutive days of oral administration. This trend demonstrated a significant improvement in improving insulin sensitivity, indicating a dose-dependent response.

[0084] After 28 consecutive days of ORC001R-U-EtOH treatment, blood was drawn from the heart of the animal model, then centrifuged at 2000xg for 15 minutes at room temperature to separate serum from whole blood, and liver function indexes (glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT)) and kidney function indexes (creatinine (CRE), urea nitrogen (BUN)) were tested in the blood of the animals by using a Fujifilm DRI-CHEM NX500i device. The results are shown in Figure 13. ORC001R-U-EtOH did not show toxicity in the liver or kidney.

[0085] Evaluation of liver protection effects

[0086] All experiments involving animals conformed to the Chang Gung University IACUC guidelines.

[0087] The experimental method was designed in accordance with the Ministry of Health and Welfare of Taiwan's Method for Evaluating the Liver Protection and Healthcare Efficacy of Health Foods (revised version of Announcement Food Characters No. 1031304063 of the Department of Health dated April 25, 2016).

[0088] First, CCl4 solution and ORC001R-U-EtOH suspension were prepared. To induce liver fibrosis in rats, CCl4 was dissolved in corn oil at a concentration of 40%. The ORC001R-U-EtOH extract was dissolved in a mixed solution consisting of 1% Tween 80 + 99% by volume of 0.5% CMC (carboxymethylcellulose) to produce a suspension.

[0089] Ten SD male rats aged 8–10 weeks were obtained from Lasco and randomly divided into the following groups: vehicle group (1% Tween 80 + 0.5% CMC in 99% by volume, no CCl4 induction; n = 3), CCl4 control (n = 3), ORC001R-U-EtOH 1 mg / kg-BW (kg body weight) (n = 2), and ORC001R-U-EtOH 10 mg / kg-BW (n = 2).

[0090] After daily oral administration of 0.5% CMC (vehicle group) in accordance with each treatment or 1% Tween80 + 99% by volume (vehicle group) for 1 week, liver fibrosis was induced by 40% CCl4, and then the same administration steps were maintained for 8 weeks. Liver fibrosis was induced in animals through intraperitoneal (IP) injection twice a week for a total period of 8 weeks. On the day of liver fibrosis induction, animals were administered 1 hour after intraperitoneal injection of CCl4.

[0091] Blood samples were collected at weeks 0, 1, 2, 4, 7, 8 and 9 for biochemical analysis of GOT, GPT, albumin, triglycerides and cholesterol. At the end of week 9, all rats were humanely sacrificed; blood, liver, kidney and spleen samples were collected. The experimental timeline is shown in Figure 14.

[0092] The results of the blood biochemistry analysis at week 7, the weekly changes in the blood biochemistry analysis, and the rat weight tracking are shown in Figures 15 to 17.

[0093] The results of blood biochemistry analysis at week 7 (Figure 15) and the results of the weekly changes of blood biochemistry analysis (Figure 16) showed that both low dose (1 mg / kg-BW) and high dose (10 mg / kg-BW) ORC001R-U-EtOH significantly reduced serum GOT and GPT levels, indicating that ORC001R-U-EtOH has liver protection potential. The results of rat weight tracking (Figure 17) showed that ORC001R-U-EtOH of the present invention did not cause side effects in rats.

[0094] Previous studies have shown that Oncidium sotoanum, a type of Oncidium plant, contains chrysin (5,7-dihydroxyflavone), which has the effect of liver protection or improving diabetes. Therefore, the present invention analyzed whether the Oncidium plant extract of the present invention contains chrysin. Interestingly, no chemical signal of chrysin was detected in the Oncidium plant extract of the present invention (data not shown).

[0095] In another experiment, combined treatment with D-GalN (D-galactosamine) and LPS induced acute liver injury (ALI) in mice.

[0096] Male C57BL / 6 mice were randomly divided into three groups (6–8 mice / group): vehicle only (DMSO, no LPS+D-GalN, no ORC001R-U-EtOH), LPS+D-GalN control (LPS, 40 μg / mL; D-galactosamine, 500 mg / kg, no ORC001R-U-EtOH), and LPS+D-GalN+ORC001R-U-EtOH extract (25 mg / kg body weight) group. Each mouse was intraperitoneally injected with 10% DMSO or LPS+D-GalN for 1 h, and then the corresponding group was intravenously injected with ORC001R-U-EtOH extract for an additional 5 h. Blood samples were collected and analyzed for glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), creatinine (CRE), and blood urea nitrogen (BUN) levels.

[0097] The results showed that D-GalN / LPS caused significant liver tissue damage, as evidenced by elevated serum levels of GOT, GPT, CRE, and BUN. However, treatment with Oncidium plant extract effectively reduced the elevated levels of GOT and GPT (Figure 18).

[0098] Evaluating the effectiveness of preventing acute respiratory distress syndrome (ARDS)

[0099] All animal experiments conformed to the Chang Gung University IACUC guidelines.

[0100] Fifteen 7-10 week old BALB / c male mice were selected and randomly divided into the following groups: negative control group (untreated, uninduced; n=3), lipopolysaccharide (LPS)-induced control (untreated, ARDS induced by LPS; n=3), low dosage group (5 mg / kg body weight ORC001R-U-EtOH administered, ARDS induced by LPS; n=3), medium dosage group (10 mg / kg body weight ORC001R-U-EtOH administered, ARDS induced by LPS; n=3), and high dosage group (20 mg / kg body weight ORC001R-U-EtOH administered, ARDS induced by LPS; n=3).

[0101] As shown in Figure 19, at the beginning of the experiment, all experimental animals except the negative control group and the LPS control group were first given an intravenous injection (IV) of ORC001R-U-EtOH of the present invention according to their groups. One hour after administration of ORC001R-U-EtOH, all experimental animals except the negative control group were administered 5mg / kg body weight of LPS using a pulmonary administration nebulizer (purchased from Shanghai Yuyan Instruments Co.,Ltd) to induce ARDS in mice. All experimental animals were euthanized 24 hours after induction of ARDS, and blood was collected and centrifuged, and the supernatant was taken for subsequent biochemical value detection, and lungs were collected under 21cm H2O column pressure, half of which was used for hematoxylin-eosin (H&E) tissue section staining analysis, and the other half was frozen at -80°C for later use.

[0102] The results are shown in Figures 20 and 21, and staining of lung tissue sections demonstrated that administration of ORC001R-U-EtOH of the present invention can effectively prevent the occurrence of ARDS or reduce the severity of ARDS, and this is dose-dependent. The blood analysis results also show that the present invention ORC001R-U-EtOH showed no obvious harm to the liver and kidneys of the experimental animals.

[0103] Although the invention has been described and exemplified in sufficient detail to enable those skilled in the art to make and use it, various alternatives, modifications, and improvements will become apparent without departing from the spirit and scope of the invention.

[0104] Those skilled in the art will readily recognize that the present invention is well adapted to carry out the objects and obtain the goals and advantages mentioned and inherent therein. The cells, animals, and processes and methods for producing them are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications thereof and other uses will occur to those skilled in the art. These modifications are within the spirit of the invention and are defined by the scope of the claims.

Claims

1. 1. An Oncidium plant extract comprising the steps of: collecting an Oncidium plant sample; drying and grinding the sample to obtain a ground Oncidium plant sample; extracting the ground Oncidium plant sample in a solvent by accelerated solvent extraction or ultrasonic extraction to obtain an Oncidium plant extract; The Oncidium plant extract is prepared by

2. 2. The Oncidium plant extract of claim 1, wherein the solvent is an alcohol, an ester, or water, and the Oncidium plant extract is an alcohol extract, an ester extract, or an extract extracted with water and then extracted with an ester of the Oncidium plant.

3. 2. The Oncidium plant extract of claim 1, wherein said Oncidium plant extract is extracted from the roots, stems or leaves of said Oncidium plant, or is a mixture comprised of the roots, stems and leaves of said Oncidium plant.

4. 4. The Oncidium plant extract of claim 3, wherein said Oncidium plant extract is extracted from the root of said Oncidium plant.

5. 2. The Oncidium plant extract of claim 1, wherein the Oncidium species comprises Oncidium stacyi, Oncidium splendidum, Oncidium pusillum, Oncidium jonesianum, Oncidium hawkesiana, Oncidium ascendens, Oncidium ampuliatum, or Oncidium flexuosum.

6. 10. Use of the Oncidium plant extract according to any of claims 1 to 5 for the manufacture of a medicament for treating diabetes in a subject in need thereof.

7. 7. The use according to claim 6, wherein the effective amount of the Oncidium plant extract is from 0.007 mg / kg body weight to 100 mg / kg body weight.

8. 7. The use according to claim 6, wherein the effective amount of the Oncidium plant extract is from 0.035 mg / kg body weight to 75 mg / kg body weight.

9. 7. The use according to claim 6, wherein the effective amount of the Oncidium plant extract in humans is 0.07 mg / kg body weight to 50 mg / kg body weight.

10. The use according to claim 6, wherein the subject is a mammal or a human.

11. 10. Use of the Oncidium plant extract according to any of claims 1 to 5 for the manufacture of a medicament for liver protection in a subject in need thereof.

12. 12. The use according to claim 11, wherein the effective amount of the Oncidium plant extract is from 0.001 mg / kg body weight to 50 mg / kg body weight.

13. 12. The use according to claim 11, wherein the effective amount of the Oncidium plant extract is from 0.009 mg / kg body weight to 25 mg / kg body weight.

14. 12. The use according to claim 11, wherein the effective amount of the Oncidium plant extract is from 0.015 mg / kg body weight to 10 mg / kg body weight.

15. The use according to claim 11, wherein the subject is a mammal or a human.

16. 10. Use of the Oncidium plant extract according to any of claims 1 to 5 for the manufacture of a medicament for preventing Acute Respiratory Distress Syndrome (ARDS) in a subject in need thereof.

17. 17. The use according to claim 16, wherein the effective amount of the Oncidium plant extract in humans is 0.007 mg / kg body weight to 50 mg / kg body weight.

18. 17. The use according to claim 16, wherein the effective amount of the Oncidium plant extract in humans is 0.02 mg / kg body weight to 25 mg / kg body weight.

19. 17. The use according to claim 16, wherein the effective amount of the Oncidium plant extract in humans is 0.035 mg / kg body weight to 20 mg / kg body weight.

20. 17. The use according to claim 16, wherein the subject is a mammal or a human.

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