Ocidium extract and application thereof in preparation of medicine for treating diabetes, protecting liver and preventing acute respiratory distress syndrome

Abstract

The invention provides an oncidium extract and application of the oncidium extract in preparation of drugs for treating diabetes, protecting the liver and preventing acute respiratory distress syndrome, and the oncidium extract is prepared by the following steps: firstly, taking an oncidium sample, drying and grinding the oncidium sample to obtain a ground oncidium sample; and placing the ground oncidium sample in a solvent, and extracting by an accelerated solvent extraction method or an ultrasonic oscillation extraction method to obtain the oncidium extract.

Description

Background Art

[0002] Diabetes mellitus (DM) is a metabolic disease that results in high blood sugar levels due to impaired insulin and / or insulin production. DM patients are often accompanied by many complications, one of which is impaired self-healing ability.

[0003] Diabetic wound healing disorder is the most important and difficult complication of diabetes, which is related to multiple factors such as blood vessels, neuropathy, immunity and biochemical components. High blood sugar can cause vascular hardening, which in turn leads to slowed circulation and microvascular dysfunction, and reduced tissue oxygenation; high blood sugar can also reduce the migration of white blood cells to the wound, making patients more susceptible to infection. Peripheral neuropathy in diabetes mellitus (DM) may cause numbness in the area and reduced ability to feel pain, which may result in the wound not being immediately noticed and treated appropriately.

[0004] The liver is one of the most important organs in the human body. It can be damaged by drugs, alcohol, or poor diet and work and rest. Liver damage can lead to degeneration, necrosis, and fibrosis of liver cells, which in turn can cause a series of liver pathological changes and secondary diseases, such as hepatitis, liver cancer, and cirrhosis.

[0005] Aspartate aminotransferase (Glutamic Oxaloacetic Transaminase; GOT) and alanine aminotransferase (Glutamic Pyruvic Transaminase; GPT) are mainly responsible for the metabolism of amino acids and proteins in the human body. Due to their high concentrations in liver cells, once the liver is damaged, GOT and GPT will flow into the blood. Therefore, they are used as biological indicators to identify liver damage during blood tests.

[0006] Acute respiratory distress syndrome (ARDS) is an acute inflammatory injury to the lungs that affects the normal function of the alveoli and respiratory exchange, resulting in insufficient oxygen concentration in the blood and damage to various organs of the body. It is a fatal emergency. Its treatment mainly adopts supportive treatment such as artificial respirator.

[0007] Natural compounds have the advantages of rich sources and diverse skeletons, and are an important basis for drug development. From 1981 to 2019, nearly half of the new drugs approved by the U.S. Food and Drug Administration came from natural products or their derivatives, including analgesics derived from morphine, vincristine, doxorubicin and paclitaxel for self-treatment of cancer, and penicillin derived from fungi as an antibiotic, etc. Therefore, the present invention actively studies which natural compounds have the potential to develop into new drugs for treating diabetes, protecting the liver or preventing acute respiratory distress syndrome. Summary of the invention

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

[0009] Preferably, in the present invention, the solvent is alcohol, ester or water, and the Oncidium extract comprises an alcohol extract, an ester extract, or an extract obtained by subjecting the product extracted with water and then subjected to ester extraction.

[0010] More preferably, the alcohols of the present invention include but are not limited to methanol, ethanol, propanol, isopropanol, butanol, isobutanol, and hexanol; the esters of the present invention include but are not limited to methyl acetate, ethyl acetate, and n-butyl acetate.

[0011] Preferably, the Oncidium extract of the present invention is extracted from the roots, stems or leaves of Oncidium, or a mixture of the roots, stems and leaves thereof.

[0012] More preferably, the Oncidium extract of the present invention is extracted from the root of Oncidium.

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

[0014] The present invention provides a use of an Oncidium extract for preparing a medicine for treating diabetes mellitus in an individual.

[0015] The present invention also provides a use of an Oncidium extract in preparing a medicine for protecting an individual's liver.

[0016] Liver protection as referred to in the present invention refers to protecting an individual from liver damage, reducing the chance of suffering from liver damage or alleviating the degree of liver damage. Liver damage as referred to in the present invention includes but is not limited to liver damage, liver diseases caused by liver damage (including but not limited to acute liver damage or chronic liver damage) such as hepatitis (including but not limited to alcoholic hepatitis, drug-induced hepatitis, fatty liver hepatitis, chronic hepatitis), liver cancer, cirrhosis, liver fibrosis, fatty liver or liver failure.

[0017] The present invention also provides a use of an Oncidium extract in preparing a medicine for preventing an individual from suffering from acute respiratory distress syndrome, comprising an effective dose of the Oncidium extract.

[0018] The acute respiratory distress syndrome referred to in the present invention includes lung injury caused by transfusion-related lung injury, ventilator-induced lung injury, bacterial lung injury, or viral lung injury.

[0019] In the present invention, the drug may further include a medically acceptable carrier of the Oncidium extract.

[0020] As used herein, an "effective dose" is a dose that achieves an effective result when administered to an individual, or a dose that has the desired activity in vivo or in vitro. In the case of inflammatory conditions, effective clinical results include a reduction in the degree or severity of symptoms associated with the disease or condition, and / or a prolongation of the life span of the individual, and / or an improvement in the quality of life of the individual, compared to no treatment. The exact amount of compound administered to an individual will depend on the type and severity of the disease or symptom and individual characteristics, such as the individual's general health, age, sex, weight, and tolerance to drugs. It will also depend on the degree, severity, and type of inflammatory conditions, autoimmune conditions, allergic conditions, or the immunosuppressive effect sought. A person skilled in the art will be able to determine the appropriate dose within the scope disclosed herein based on these and other factors.

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

[0022] Further, the effective dose of the Oncidium extract of the present invention can be 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] In the present invention, the effective dose can be based on the formula provided in the guidelines published by the U.S. FDA (Guidance for Industry: Establishing the Maximum Safe Starting Doses 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), and the safety factor can be further considered to perform equivalent dose conversion between different species.

[0024] In the use of the present invention in preparing a drug for treating diabetes in an individual, the individual is a mammal or a human, and the effective dose is 0.007 mg / kg to 1.5 mg / kg body weight; preferably, the effective dose is 0.035 mg / kg to 1.3 mg / kg body weight; more preferably, the effective dose is 0.07 mg / kg to 0.75 mg / kg body weight.

[0025] Further, in the use of preparing a drug for treating diabetes in an individual, the effective dose of the present invention for mice (average weight 25 grams) is 1 mg / kg to 100 mg / kg, which is equivalent to an effective dose of 0.01 mg / kg to 1.5 mg / kg for dogs and cats (average weight 10 kg), which is equivalent to an effective dose of 0.007 mg / kg to 0.8 mg / kg for 60 kg adults, which is equivalent to an effective dose of 0.01 mg / kg to 1.2 mg / kg for 20 kg children; preferably, the effective dose of the present invention for mice (average weight 25 grams) is 5 mg / kg to 75 mg / kg, which is equivalent to an effective dose of 0.05 mg / kg for dogs and cats (average weight 10 kg). / kg body weight to 1.3 mg / kg body weight, equivalent to an effective dose of 0.035 mg / kg body weight to 0.6 mg / kg body weight for a 60 kg adult, and equivalent to an effective dose of 0.05 mg / kg body weight to 0.85 mg / kg body weight for a 20 kg child; more preferably, the effective dose for mice of the present invention (average body weight 25 grams) is 10 mg / kg body weight to 50 mg / kg body weight, equivalent to an effective dose of 0.13 mg / kg body weight to 0.75 mg / kg body weight for dogs and cats (average body weight 10 kg), equivalent to an effective dose of 0.07 mg / kg body weight to 0.45 mg / kg body weight for a 60 kg adult, and equivalent to an effective dose of 0.1 mg / kg body weight to 0.6 mg / kg body weight for a 20 kg child.

[0026] In the use of the present invention in preparing a drug for protecting an individual's liver, the individual is a mammal or a human, and the effective dose is 0.001 mg / kg to 1.75 mg / kg body weight; preferably, the effective dose is 0.009 mg / kg to 1 mg / kg body weight; more preferably, the effective dose is 0.015 mg / kg to 0.4 mg / kg body weight.

[0027] Further, in the use of preparing a drug for protecting an individual's liver, the effective dose of the present invention for rats (average weight 350 grams) is 0.1 mg / kg to 50 mg / kg, which is equivalent to an effective dose of 0.003 mg / kg to 1.75 mg / kg for dogs and cats (average weight 10 kilograms), equivalent to an effective dose of 0.001 mg / kg to 1.25 mg / kg for 60 kilograms of adults, and equivalent to an effective dose of 0.002 mg / kg to 1.5 mg / kg for 20 kilograms of children; preferably, the effective dose of the present invention for rats (average weight 350 grams) is 0.5 mg / kg to 25 mg / kg, which is equivalent to an effective dose of 0.001 mg / kg to 1.25 mg / kg for dogs and cats (average weight 10 kilograms). 15 mg / kg body weight to 1 mg / kg body weight, equivalent to an effective dose of 0.009 mg / kg body weight to 0.5 mg / kg body weight for a 60 kg adult, and equivalent to an effective dose of 0.013 mg / kg body weight to 0.7 mg / kg body weight for a 20 kg child; more preferably, the effective dose for rats (average body weight 350 grams) of the present invention is 1 mg / kg body weight to 10 mg / kg body weight, equivalent to an effective dose of 0.03 mg / kg body weight to 0.4 mg / kg body weight for dogs and cats (average body weight 10 kg), equivalent to an effective dose of 0.015 mg / kg body weight to 0.2 mg / kg body weight for a 60 kg adult, and equivalent to an effective dose of 0.025 mg / kg body weight to 0.3 mg / kg body weight for a 20 kg child.

[0028] In the use of the present invention in preparing a drug for preventing an individual from suffering from acute respiratory distress syndrome, the individual is a mammal or a human, and the effective dose is 0.007 mg / kg to 0.75 mg / kg body weight; preferably, the effective dose is 0.02 mg / kg to 0.4 mg / kg body weight; more preferably, the effective dose is 0.035 mg / kg to 0.35 mg / kg body weight.

[0029] Further, in the use of preparing a drug for preventing an individual from suffering from acute respiratory distress syndrome, the effective dose of the present invention for mice (average weight 25 grams) is 1 mg / kg to 50 mg / kg, which is equivalent to an effective dose of 0.01 mg / kg to 0.75 mg / kg for dogs and cats (average weight 10 kg), equivalent to an effective dose of 0.007 mg / kg to 0.45 mg / kg for 60 kg adults, and equivalent to an effective dose of 0.01 mg / kg to 0.65 mg / kg for 20 kg children; preferably, the effective dose of the present invention for mice (average weight 25 grams) is 3 mg / kg to 25 mg / kg, which is equivalent to an effective dose of 0. Preferably, the effective dose for mice (average weight 25 grams) of the present invention is 5 mg / kg to 20 mg / kg body weight, which is equivalent to an effective dose of 0.06 mg / kg to 0.35 mg / kg body weight for dogs and cats (average weight 10 kg), equivalent to an effective dose of 0.035 mg / kg to 0.2 mg / kg for 60 kg adults, and equivalent to an effective dose of 0.05 mg / kg to 0.25 mg / kg for 20 kg children.

[0030] In the present invention, the subject referred to in the present invention is a human (e.g., an infant, a toddler, a teenager, a young adult, a middle-aged or elderly individual), 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 mink; an economic animal such as a horse, a sheep, a cow, a pig). Preferably, the subject referred to in the present invention is a human or a mammal. Most preferably, the subject referred to in the present invention is a human.

[0031] The drug of the present invention may also contain a pharmaceutically acceptable excipient, in particular, it may further contain a predetermined solvent or oil, a pH regulator, and if necessary, it may further contain a dispersant. Examples of the solvent used include, but are not limited to, water, ethanol, isopropanol, 1,3-butylene glycol, propylene glycol, glycerol, etc. Examples of the oil used are selected from the group consisting of corn oil, sesame oil, linseed oil, cottonseed oil, soybean oil, peanut oil, mono-glyceride, di-glyceride, tri-glyceride, mineral oil, deep-sea fish oil squalene, jojoba oil, olive oil, evening primrose oil, borage oil, grape seed oil, coconut oil, sunflower seed oil, shea butter, and any combination thereof, but are not limited thereto.

[0032] The solvents and oils may be used alone or in any combination.

[0033] Examples of dispersants useful for the present invention include, but are not limited to, lecithin, organic monoglycerides, behenyl alcohol 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, preferably, the medicament of the present invention is administered orally or by injection.

[0035] In the present invention, the drug of the present invention can also be made into an external preparation, wherein examples of the external preparation include but are not limited to creams, ointments, gels, lotions, patches, etc., or inhalants, aerosols, suppositories, etc.

[0036] When the drug is used as an external preparation, an appropriate external preparation for skin can also be used as a basic raw material, and aqueous solutions, non-aqueous solvents, suspensions, emulsions, gels, creams, ointments or freeze-dried preparations can all be used and sterilized according to known methods. Compositions in the form of gels, creams and ointments can be appropriately prepared by adding known softeners, emulsifiers and thickeners or other raw materials known in the art according to the form of the composition using known methods.

[0037] The composition in the form of a gel can be prepared, for example, by adding a softener such as trimethylolpropane, polyethylene glycol, and glycerin, a solvent such as propylene glycol, ethanol, and isocetyl alcohol, and purified water. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The results show the inhibitory effects of various Oncidium extracts on the nitrogen free radical DPPH. ** / *** indicate significant differences.

[0039] Figure 2 The results show the efficacy of various Oncidium extracts in inhibiting the oxygen-containing free radical AAPH. ΔAUC represents the area under the receiver operating characteristic curve (ROC); ** / *** indicates a significant difference.

[0040] Figure 3Flow chart of animal experiment for evaluating the efficacy of Oncidium extract for treating diabetes. Explanation of symbols: *: Streptozotocin (STZ) induced diabetes; $: confirmed non-fasting blood glucose value; #: confirmed 18-hour fasting blood glucose value; →: period of administration of Oncidium extract; !: sacrifice; %: blood test (testing liver function indexes (aspartate aminotransferase (GOT), alanine aminotransferase (GPT)) and renal function indexes (creatinine (CRE), urea nitrogen (BUN)) in the blood).

[0041] Figure 4 The fasting blood glucose levels of healthy mice, diabetic mice and diabetic mice given Oncidium extract before and after 18 hours of fasting are shown. * indicates that the group given the Oncidium extract of the present invention has a significant difference compared with the non-treated group (P<0.05); + indicates that the Oncidium extract was given; - indicates that the Oncidium extract was not given.

[0042] Figure 5 The figure shows the glucose tolerance test of healthy mice, diabetic mice and diabetic mice given Oncidium extract. * indicates that the group given the low dose Oncidium extract of the present invention has a significant difference compared with the non-treatment group (P<0.05); # indicates that the group given the medium dose Oncidium extract of the present invention has a significant difference compared with the non-treatment group (P<0.05).

[0043] Figure 6 The blood parameters of healthy mice, diabetic mice and diabetic mice treated with Oncidium extract are shown. * indicates that the group treated with Oncidium extract of the present invention has significant difference compared with the non-treated group (P<0.05); + indicates treatment; - indicates no treatment.

[0044] Figure 7 Shown are the results of staining of liver and kidney tissue sections of healthy mice, diabetic mice, and diabetic mice treated with Oncidium extract. + indicates treatment; - indicates no treatment.

[0045] Figure 8 The figure shows the weight changes of healthy mice, diabetic mice and diabetic mice given Oncidium extract. * indicates that the group given the low dose Oncidium extract of the present invention has a significant difference compared with the non-treatment group (P<0.05); # indicates that the group given the medium dose Oncidium extract of the present invention has a significant difference compared with the non-treatment group (P<0.05); @ indicates that the group given the high dose Oncidium extract of the present invention has a significant difference compared with the non-treatment group (P<0.05).

[0046] Fig. 9Flow chart of animal experiment for evaluating the efficacy of Oncidium extract for treating type 2 diabetes. Explanation of symbols: *: confirmation of non-fasting blood glucose value; $: confirmation of 8-hour fasting blood glucose value; #: insulin tolerance test; @: intraperitoneal glucose tolerance test; →: period of administration of Oncidium extract; !: sacrifice; %: blood test (testing liver function indexes (aspartate aminotransferase (GOT), alanine aminotransferase (GPT)) and renal function indexes (creatinine (CRE), urea nitrogen (BUN)) in the blood).

[0047] Fig.10 The fasting blood glucose levels of type II diabetic mice and diabetic mice given the Oncidium extract for four weeks after fasting for 8 hours. ** indicates that the group treated with the Oncidium extract of the present invention has a significant difference compared with the non-treated group (P<0.01).

[0048] Fig.11 Shown is a glucose tolerance test in type 2 diabetic mice and diabetic mice administered with Oncidium extract.

[0049] Fig.12 Shown are insulin tolerance tests in type 2 diabetic mice and diabetic mice given Oncidium extract.

[0050] Fig.13 Shown are blood parameters of type 2 diabetic mice and diabetic mice administered with Oncidium extract. - indicates no administration.

[0051] Fig.14 Flow chart of animal experiment for evaluating the efficacy of Oncidium extract in protecting liver. Explanation of symbols: *: carbon tetrachloride (CCl4) induced liver fibrosis; %: blood collection; →: period of administration of Oncidium extract; !: sacrifice.

[0052] Fig.15 The results of blood tests (GOT, GPT) of rats with liver fibrosis in different groups at week 7 are shown. + indicates administration; - indicates no administration.

[0053] Fig.16 Shown are the results of weekly blood biochemical analysis changes in rats with liver fibrosis in different groups.

[0054] Fig.17 These are the body weight tracking results of rats with liver fibrosis in different groups.

[0055] Fig.18The extract of Oncidium candidum alleviates acute liver tissue injury induced by D-galactosamine / lipopolysaccharide (D-GalN / LPS) in mice. + indicates administration; - indicates no administration. * indicates that the group administered with a low dose of the Oncidium candidum extract of the present invention has a significant difference compared with the non-treatment group (P<0.05); *** indicates that the group administered with a low dose of the Oncidium candidum extract of the present invention has a significant difference compared with the non-treatment group (P<0.005).

[0056] Fig.19 This is a flow chart of animal experiments to evaluate the efficacy of Oncidium extract in preventing acute respiratory distress syndrome. Symbols: *: acute respiratory distress syndrome induced by lipopolysaccharides (LPS); %: administration of Oncidium extract; !: sacrifice.

[0057] Fig. 20 Shown are the staining results of lung tissue sections of mice in different groups. + indicates administration; - indicates no administration.

[0058] Fig.21 The blood analysis results of mice in different groups are shown. + indicates administration; - indicates no administration. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0060] It should be understood that the detailed description of the embodiments is intended to illustrate the preferred embodiments of the present invention, rather than to limit the present invention to certain embodiments. It should be noted that the present invention is intended to cover all alternative embodiments within the same spirit and scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0061] Preparation of Oncidium Extract

[0062] In the present invention, the Oncidium sample used for preparing the extract is the root (R), stem (B) or leaf (L) of Oncidium, or a mixture of the root, stem and leaf thereof. The species of the Oncidium can be selected from Oncidium stacyi, Oncidium splendidum, Oncidium pusillum, Oncidium jonesianum, Oncidium hawkesiana, Oncidium ascendens, Oncidium ampliatum, or Oncidium flexuosum. The Oncidium sample is dried, ground and stored at -20°C until it is used.

[0063] In the present invention, the Oncidium sample is firstly extracted by accelerated solvent extraction (A) and ultrasonic extraction (U).

[0064] In the accelerated solvent extraction method, ethyl acetate (A-EA), 95% ethanol (A-EtOH), water (H2O; AW), and water extraction followed by secondary extraction of the residue with ethyl acetate (H2O and Ethyl Acetate) were used. The oncidium extract (ORC001) of the present invention is obtained by using 1,300-1,700 psi (preferably 1,500 psi on average) of 2,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1,4-dimethoxy-1

[0065] In the ultrasonic extraction method, ethyl acetate (U-EA), 95% ethanol (U-EtOH), water (H2O; UW), and water extraction followed by secondary extraction of the residue with ethyl acetate (H2O+Ethyl acetate; U-W+EA) are used as solvents for purification extraction at room temperature, the temperature is 30-70°C (preferably 50°C), the time is 15-60 minutes (preferably 30 minutes), the output power is 200-600 watts (preferably 400 watts), the oscillation frequency is 20-60 Hz (preferably 40 Hz), and the Oncidium extract (ORC001) of the present invention is obtained, which is named as ORC001R according to the extraction part and the solvent. -U-EA, ORC001R-U-EtOH, ORC001R-UW, ORC001R-U-W+EA, ORC001B-U-EA, ORC001B-U-EtOH, OR C001B-UW, ORC001B-U-W+EA, ORC001L-U-EA, ORC001L-U-EtOH, ORC001L-UW and ORC001L-U-W+EA.

[0066] By using high performance liquid chromatography (HPLC) to identify the chemical component fingerprints of different varieties of Oncidium extracts (same part, same solvent, same extraction method), it can be found that the different varieties of Oncidium extracts have similar fingerprint distributions, indicating that the Oncidium extracts of these varieties have common compounds. Therefore, Oncidium flexuosum was selected as the Oncidium sample of the present invention.

[0067] Superoxide anion and elastase release inhibition test

[0068] The present invention uses the inhibition test of superoxide anion and elastase release to evaluate the anti-inflammatory ability of the above-mentioned various Oncidium extracts.

[0069] First, neutrophils are prepared.

[0070] Take healthy blood donors (20-35 years old; normal work and rest and abstain from medication for more than one week) who are willing to assist or are interested in this program, and collect about 50mL of blood from the elbow vein using a vacuum sterile blood collection tube. Add the blood to an equal volume of 3% dextran solution (Dextran) and mix evenly, and let it stand to precipitate red blood cells. Take the supernatant containing neutrophils, cover it in a centrifuge tube containing Ficoll-Paque solution, and centrifuge it. Use density gradient to separate different blood cells, and obtain neutrophils and a small amount of red blood cells precipitated at the bottom. Use different concentrations of NaCl solution to burst the remaining red blood cells, and use the difference in osmotic pressure tolerance between the two to retain the required neutrophils.

[0071] The evaluation of the anti-inflammatory response of the various Oncidium extracts of the present invention against neutrophils was carried out by respiratory burst test and degranulation function evaluation.

[0072] The respiratory burst mainly produces superoxide anions (O2 ·- ) and reactive oxygen species (ROS), among which superoxide anion (O2 ·- ) was detected by using a neutrophil suspension (6×10 5 cells / mL) were added with the above-mentioned Oncidium extracts at 37°C for 2 minutes, and then treated with 1μg / mL cytochalasin B (CB) for 3 minutes. Then, N-formyl-methionyl-leucyl-phenylalanine (fMLF) was used as a stimulator to activate the cells for about 10 minutes. The absorbance was measured at a wavelength of 550nm using an ultraviolet spectrophotometer.

[0073] In the elastase release experiment, a neutrophil suspension (6×10 5 cells / mL) for 5 minutes to 37°C, then add the above-mentioned Oncidium extracts for 2 minutes, treat the cells with 0.5μg / mL cytochalasin B (CB) for 3 minutes, and then stimulate neutrophil activation with 0.1μM fMLF for about 10 minutes. Finally, use a UV spectrophotometer to measure the absorbance at a wavelength of 405nm. The experimental results are expressed as inhibition percentage.

[0074] The results are shown in Table 1.

[0075] Table 1

[0076]

[0077]

[0078]

[0079] The inhibition rate (Inh%) of 10 μg / ml. The results are presented as mean ± standard error (SEM) (n=3-5), *P<0.05, **P<0.01, ***P<0.001 compared with the control group (DMSO or water).

[0080] a The half maximal inhibitory concentration (IC 50 ).

[0081] b Dissolve in water.

[0082] c ORC001R-A-EA (10 μg / ml) was filtered through a 0.22 μm filter before the experiment.

[0083] The cytotoxicity of the Oncidium extract of the present invention is shown in Table 2.

[0084] Table 2

[0085]

[0086]

[0087] The cell viability (%) results at 10 μg / ml are presented as mean ± standard error (SEM) (n=3).

[0088] a ORC001R-A-EA (10 μg / ml) was filtered through a 0.22 μm filter before the experiment.

[0089] Free radical scavenging ability test

[0090] Nitrogen free radicals: 1,1-diphenyl-2-trinitrophenylhydrazine (Diphenyl-p-picrylhydrazyl, DPPH) and oxygen free radicals: 2,2'-Azobis (2-amidinopropane) hydrochloride (AAPH) are stable free radicals related to inflammation. In the present invention, the ability of 10 μg / mL of each Oncidium extract to scavenge free radicals was further tested, wherein dimethyl sulfoxide (DMSO) or water (H2O) was used as a control group, and α-tocopherol (α-Tocopherol) was used as a comparative reagent. When each Oncidium extract had free radical scavenging ability, it was mixed with ethanol-prepared DPPH (100 μM) or AAPH (25 mM) reaction solution and reacted for 30 minutes and 2 hours respectively, and then used as an activity indicator for scavenging free radicals.

[0091] like Figure 1 As shown, the organic solvent extracts (EA, EtOH, W+EA) of Oncidium root extract (ORC001R) inhibited the nitrogen free radical DPPH more significantly.

[0092] In addition, if Figure 2 As shown, the organic solvent extracts (EA, EtOH, W+EA) of Oncidium root extract (ORC001R) and Oncidium stem extract (ORC001B) and the EtOH and water extracts of Oncidium leaf extract (ORC001L) were more effective in inhibiting oxygen-containing free radicals.

[0093] Establishment of animal model of type 1 diabetes mellitus (T1DM)

[0094] All animal experiments in the present invention were approved by the Institutional Animal Care and Use Committee (IACUC) of Chang Gung University.

[0095] First, a diabetic animal model was established. The present invention selected 7-week-old C57BL / 6 male mice weighing 20-25 grams, fasted the mice for 4 hours, and intraperitoneally injected 55 mg / kg body weight of streptozotocin (STZ) dissolved in pH 4.5 citrate buffer for five consecutive days. In the first 4 days after the injection of STZ, the mice were given normal food and 1% glucose water to induce diabetes. On the last day, glucose water was no longer given, and ordinary drinking water was given instead.

[0096] 72 hours after STZ injection, mice were fasted for 4 hours, and the tails of mice were pierced and blood was collected using a Contour Plus portable blood glucose meter (Ascensia, UK) to measure fasting blood glucose values. Animals with fasting blood glucose values ​​of 150-250 mg / dL were considered prediabetic, while those with >250 mg / dL were considered diabetic and used for subsequent experiments. All mice had fasting blood glucose values ​​>250 mg / dL.

[0097] Evaluating the effectiveness of treatments for type 1 diabetes

[0098] The present invention evaluates the effect of treating diabetes in mice given Oncidium root extract (ORC001R) or not given Oncidium root extract (ORC001R) by monitoring the blood glucose level of mice. The Oncidium root extract is an extract obtained by ultrasonically vibrating the roots of Oncidium and using 95% ethanol as a solvent (ORC001R-U-EtOH). The experimental process is as follows Figure 3 shown.

[0099] A total of 25 male C57BL / 6 mice were used in the experiment. The mice were randomly divided into five groups: 4 treated control groups (10% ethanol and 10% Tween-20 in saline; NC), 7 untreated groups (STZ; T1DM), 7 low-dose ORC001R-U-EtOH groups (administered 10 mg / kg of ORC001R-U-EtOH; T1DM+ORC001R-U-EtOH(10 mg / kg)), 4 medium-dose ORC001R-U-EtOH groups (administered 25 mg / kg of ORC001R-U-EtOH; T1DM+ORC001R-U-EtOH(25 mg / kg)), and 3 high-dose ORC001R-U-EtOH groups (administered 50 mg / kg of ORC001R-U-EtOH; T1DM+ORC001R-U-EtOH(50 mg / kg)).

[0100] Before administration, the weight of the experimental animals was confirmed, and blood was collected by pricking the tail end with a Contour Plus portable blood glucose meter (Ascensia, UK) to confirm the non-fasting blood glucose value of the experimental animals. Then, the experimental animals were fasted for 18 hours to obtain the fasting blood glucose value of the experimental animals. During the experiment, the experimental animals repeated the above steps on the 7th and 14th days after administration to confirm the trend of blood glucose value.

[0101] The fasting blood glucose levels of experimental animals at 14 days after administration were as follows Figure 4As shown, the Oncidium root extract showed a trend of lowering blood sugar at a low dose, produced a better blood sugar lowering effect at a medium dose, and produced a significant blood sugar lowering effect at a high dose.

[0102] The trend of blood glucose values ​​was measured by intraperitoneal glucose tolerance test (IPGTT). After fasting for 18 hours, mice were given 1 g / kg body weight of glucose (purchased from Sigma) by intraperitoneal injection, and blood samples were obtained using a glucometer at time points 0, 15, 30, 60, 120, and 180 minutes for blood glucose measurement. The results are shown in Figure 5 As shown, oral administration of low, medium and high doses of Oncidium extract for 14 consecutive days all showed a blood sugar lowering trend, and the blood sugar lowering trend of medium and high doses of Oncidium root extract was the best.

[0103] After the experimental animals were given the drug for 14 consecutive days, blood was collected from the heart and centrifuged at 2000xg for 15 minutes at room temperature to separate the serum. The liver function indexes (aspartate aminotransferase (GOT), alanine aminotransferase (GPT)) and renal function indexes (creatinine (CRE), urea nitrogen (BUN)) in the serum were measured using Fujifilm DRI-CHEM NX500i instrument. The liver and kidney tissues of the experimental animals were also sectioned and stained with hematoxylin-eosin (H&E stain). The results are shown in the figure. Figure 6 and Figure 7 As shown, Oncidium root extract showed no toxicity in the liver or kidneys and also had the effect of repairing liver and kidney damage caused by diabetes.

[0104] The weight changes of experimental animals Figure 8 As shown, the experimental animals showed a significant trend of weight recovery starting from the 9th day of administration.

[0105] Establishment of animal model of type 2 diabetes mellitus (T2DM)

[0106] All animal experiments in the present invention were approved by the Institutional Animal Care and Use Committee (IACUC) of Chang Gung University.

[0107] First, an animal model of type II diabetes was established. The present invention selected 5-week-old BKS.Cg-Dock7m+ / +Leprdb / Jnarl male mice weighing 20-25 grams. This strain is a genetically modified spontaneous type II diabetic mouse that has been established for many years and is widely accepted as a model mouse for metabolic syndrome disease.

[0108] The Contour Plus portable blood glucose meter (Ascensia, UK) was used to pierce the tail of the mice and collect blood to measure fasting blood glucose. Animals with fasting blood glucose values ​​of 150-250 mg / dL were considered prediabetic. As the number of experimental weeks increased, the fasting blood glucose values ​​of the mice gradually increased, and >250 mg / dL was considered diabetic. All mice had fasting blood glucose values ​​>250 mg / dL.

[0109] Evaluating the effectiveness of treatments for type 2 diabetes

[0110] The present invention evaluates the effect of treating diabetes in mice given Oncidium root extract (ORC001R) or not given Oncidium root extract (ORC001R) by monitoring the blood glucose level of mice. The Oncidium root extract is an extract obtained by ultrasonically vibrating the roots of Oncidium and using 95% ethanol as a solvent (ORC001R-U-EtOH). The experimental process is as follows Fig. 9 shown.

[0111] A total of 12 male BKS.Cg-Dock7m+ / +Leprdb / Jnarl mice were used in the experiment. The mice were randomly divided into 4 groups: 3 untreated mice (1% tween 80and 99% CMC solution (0.5% w / v); T2DM), 3 low-dose ORC001R-U-EtOH group (administered 10 mg / kg of ORC001R-U-EtOH; T2DM+ORC001R-U-EtOH(10 mg / kg)), 3 medium-dose ORC001R-U-EtOH group (administered 25 mg / kg of ORC001R-U-EtOH; T2DM+ORC001R-U-EtOH(25 mg / kg)), and 3 high-dose ORC001R-U-EtOH group (administered 50 mg / kg of ORC001R-U-EtOH; T2DM+ORC001R-U-EtOH(50 mg / kg)).

[0112] Before administration, the weight of the experimental animals was confirmed, and blood was collected by pricking the tail end with a Contour Plus portable blood glucose meter (Ascensia, UK) to confirm the non-fasting blood glucose value of the experimental animals. Then, the experimental animals were fasted for 18 hours to obtain the fasting blood glucose value of the experimental animals. During the experiment, the experimental animals repeated the above steps on the 1st, 7th, 14th, 21st and 28th days after administration to confirm the trend of blood glucose value.

[0113] The fasting blood glucose levels of experimental animals at 28 days after administration were as follows Fig.10As shown, the Oncidium root extract showed a trend of lowering blood sugar at low doses, produced better blood sugar lowering effect at medium doses, and produced significant blood sugar lowering effect at high doses, showing a dose-dependent manner.

[0114] The trend of blood glucose values ​​was measured by intraperitoneal glucose tolerance test (IPGTT). After fasting for 18 hours, mice were given 1 g / kg body weight of glucose (purchased from Sigma) by intraperitoneal injection, and blood samples were obtained using a glucometer at time points 0, 15, 30, 60, 120, and 180 minutes for blood glucose measurement. The results are shown in Fig.11 As shown, oral administration of low and high doses of Oncidium root extract for 28 consecutive days both showed a blood sugar lowering trend, and the high dose of Oncidium root extract had the best blood sugar lowering trend.

[0115] Insulin tolerance tests (IPITT) can be used to evaluate insulin sensitivity. Mice were fasted for 5 hours and injected with 0.75 U / kg body weight of insulin (Humulin R U-500, Eli Lilly). Blood glucose concentration was measured with a glucometer at 0, 15, 30, 60, and 120 minutes. The results are shown in Figure 2. Fig.12 As shown in the figure, the blood glucose concentrations of the low-dose ORC001R-U-EtOH group, the medium-dose ORC001R-U-EtOH group, and the high-dose ORC001R-U-EtOH group all showed a downward trend after continuous oral administration for 28 days. This trend shows that ORC001R-U-EtOH has a significant improvement in improving insulin sensitivity and is dose-dependent.

[0116] After the experimental animals were given the drug for 28 consecutive days, blood was collected from the heart and centrifuged at 2000xg for 15 minutes at room temperature to separate the serum. The liver function indexes (aspartate aminotransferase (GOT), alanine aminotransferase (GPT)) and kidney function indexes (creatinine (CRE), urea nitrogen (BUN)) in the serum were measured using Fujifilm DRI-CHEM NX500i instrument. The extract of Oncidium root showed no toxicity in the liver or kidney. The results are as follows Fig.13 shown.

[0117] Evaluating liver protection efficacy

[0118] All animal experiments in the present invention were approved by the Institutional Animal Care and Use Committee (IACUC) of Chang Gung University.

[0119] First, a carbon tetrachloride (CCl4) solution and an Oncidium root extract (ORC001R-U-EtOH) suspension were prepared. Carbon tetrachloride (CCl4) was dissolved in 40% corn oil to induce rat liver fibrosis. ORC001R-U-EtOH was dissolved in a mixture consisting of 1% Tween 80 and 99% volume of 0.5% carboxymethyl cellulose (CMC) to establish a suspension.

[0120] The present invention purchased 10 SD male rats aged 8 to 10 weeks from Lesco and randomly divided them into the following groups: a vehicle group (administered with a mixture consisting of 1% Tween 80 and 99% by volume of 0.5% CMC, which did not induce liver fibrosis; n=3), a carbon tetrachloride-induced control group (n=3), a 1 mg / kg body weight ORC001R-U-EtOH group (n=2) and a 10 mg / kg body weight ORC001R-U-EtOH group (n=2).

[0121] Before the administration of 40% carbon tetrachloride (CCl4) to induce liver fibrosis, all groups were orally administered with their respective extracts or solvents (administered with a mixture of 1% Tween 80 and 99% volume of 0.5% CMC) every day for one week, and then the same administration procedure was performed every day for eight weeks. After the first week, the experimental animals were intraperitoneally injected with 0.1 ml / 100 g body weight of 40% carbon tetrachloride (CCl4) twice a week to induce liver fibrosis for a total of 8 weeks. On the day of induction of liver fibrosis, the experimental animals were administered one hour after the intraperitoneal injection of 40% carbon tetrachloride (CCl4).

[0122] Blood samples were collected at 0, 1, 2, 4, 7, 8 and 9 weeks for biochemical analysis of GOT, GPT, albumin, triglycerides and cholesterol. At the end of the 9th week, all experimental animals were euthanized and blood, liver, kidney and spleen samples were collected from the experimental animals. The timeline of the experiment is as follows: Fig.14 shown.

[0123] Results of blood biochemical analysis at week 7, weekly changes in blood biochemical analysis, and rat body weight tracking are shown in Figure 2. Figure 15 to Figure 17 shown.

[0124] Blood biochemical analysis at week 7 ( Fig.15 ) and weekly changes in blood biochemical analysis ( Fig.16) showed that ORC001R-U-EtOH significantly reduced the levels of GOT and GPT in serum in rats in the 1 mg / kg body weight ORC001R-U-EtOH group and the 10 mg / kg body weight ORC001R-U-EtOH group, indicating that ORC001R-U-EtOH has the potential to prevent or treat liver damage. The results of rat weight tracking ( Fig.17 ) showed that the Oncidium extract of the present invention had no adverse effects on rats.

[0125] The present invention further provides an experiment for treating acute liver injury in mice induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS).

[0126] Male C57BL / 6 mice were randomly divided into 3 groups (6-8 mice / group): vehicle group (DMSO, without LPS+D-GalN, without ORC001R-U-EtOH), LPS+D-GalN control group (LPS, 40μg / mL; D-galactosamine, 500mg / Kg, without ORC001R-U-EtOH), LPS+D-GalN+ORC001R-U-EtOH (25mg / kg body weight) group. Each mouse was first intraperitoneally injected with 10% DMSO or LPS+D-GalN for 1 hour, and then intravenously injected with ORC001R-U-EtOH extract to the corresponding group for 5 hours. Blood samples were collected and analyzed for the expression of GPT, GOT, CRE and BUN.

[0127] The results showed that D-GalN / LPS induced significant acute liver tissue damage, as evidenced by increased levels of GOT, GPT, CRE, and BUN in serum. However, ORC001R-U-EtOH extract treatment effectively reduced the elevated levels of GOT and GPT (see Fig.18 ).

[0128] Previous studies have shown that one of the species of Oncidium, "Oncidium sotoanum", contains chrysin (5,7dihydroxyflavone), which has the effect of protecting the liver or improving diabetes. Therefore, further analysis was conducted to determine whether the Oncidium extract of the present invention contains chrysin. Interestingly, the chemical information of chrysin was not detected in the Oncidium extract of the present invention (the result is not shown in the figure).

[0129] To evaluate the efficacy of preventing acute respiratory distress syndrome (ARDS)

[0130] All animal experiments in the present invention were approved by the Institutional Animal Care and Use Committee (IACUC) of Chang Gung University.

[0131] The present invention uses 15 7-10 week old BALB / c male mice weighing 20-25 grams, and the mice are randomly divided into three groups, namely: 3 negative control groups (no medication, no induction), 3 lipopolysaccharide (LPS) control groups (no medication, LPS induced ARDS), 3 low-dose groups (administered 5 mg / kg body weight of Oncidium extract, LPS induced ARDS), 3 medium-dose groups (administered 10 mg / kg body weight of Oncidium extract, LPS induced ARDS), and 3 high-dose groups (administered 20 mg / kg body weight of Oncidium extract, LPS induced ARDS).

[0132] like Fig.19 As shown, at the beginning of the experiment, except for the negative control group and the LPS control group, all experimental animals were given the Oncidium extract of the present invention by intravenous injection according to their groups. One hour after the administration, all experimental animals except the negative control group were given 5 mg / kg body weight of LPS using a pulmonary administration nebulizer (purchased from Shanghai Yuyan Scientific Instrument Co., Ltd.) to induce acute respiratory distress syndrome in mice, and all experimental animals were euthanized 24 hours after induction, blood was collected, centrifuged, and the supernatant was used for subsequent biochemical numerical detection, and the lungs were collected under a pressure of 21 cm of water column, half of which was used for hematoxylin-eosin (H&E) tissue section staining analysis, and the other half was frozen at -80°C for future use.

[0133] The results are as follows Fig. 20 and Fig.21 As shown, lung tissue section staining showed that the administration of the Oncidium extract of the present invention can effectively prevent the occurrence of ARDS or alleviate the degree of ARDS, and is dose-dependent, while the blood analysis results also showed that the Oncidium extract of the present invention had no obvious damage to the liver and kidney of the experimental animals.

[0134] Although the present invention has been described and illustrated in sufficient detail for one skilled in the art to make and implement it, it should be apparent that various substitutions, modifications and improvements can be made without departing from the spirit and scope of the invention.

[0135] It will be readily appreciated by those skilled in the art that the present invention is well adapted to achieve the objects and advantages mentioned as well as those inherent therein. The cells, animals, and processes and methods for producing them represent only the best mode of implementation, are illustrative, and are not intended to limit the scope of the invention. Modifications and other uses thereof will occur to those skilled in the art, and such modifications are within the spirit of the invention and are limited by the scope of the claims.

Claims

1. An Oncidium extract, which is prepared by the following steps: Take samples of Oncidium; dry-grinding the Oncidium sample to obtain a ground Oncidium sample; and The ground Oncidium sample is extracted in a solvent by accelerated solvent extraction or ultrasonic extraction to obtain an Oncidium extract.

2. The Oncidium extract as claimed in claim 1, wherein the solvent is alcohol, ester or water, and the Oncidium extract comprises an alcohol extract, an ester extract, or an extract obtained by extracting the product with water and then subjecting it to ester extraction.

3. The Oncidium extract of claim 1, wherein the Oncidium extract is extracted from roots, stems or leaves of Oncidium, or a mixture of roots, stems and leaves of Oncidium. The Oncidium extract according to claim 3 , wherein the Oncidium extract is extracted from the root of Oncidium.

5. The Oncidium extract according to claim 1, wherein the Oncidium species include: Oncidium stacyi, Oncidium splendidum, Oncidium pusillum, Oncidium jonesianum, Oncidium hawkesiana, Oncidium ascendens, Oncidium ampliatum, or Oncidium flexuosum.

6. Use of the Oncidium extract according to any one of claims 1 to 5 for preparing a medicament for treating diabetes mellitus in an individual.

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

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

9. The use according to claim 6, wherein the effective dose of the Oncidium extract is 0.07 mg / kg to 50 mg / kg body weight.

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

11. Use of the Oncidium extract according to any one of claims 1 to 5 for preparing a medicament for protecting an individual's liver.

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

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

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

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

16. Use of the Oncidium extract according to any one of claims 1 to 5 for preparing a medicament for preventing acute respiratory distress syndrome in an individual.

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

18. The use according to claim 16, wherein the effective dose of the Oncidium extract is 0.02 mg / kg to 25 mg / kg body weight.

19. The use according to claim 16, wherein the effective dose of the Oncidium extract is 0.035 mg / kg to 20 mg / kg body weight.

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

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