Use of Bletilla Formosana (Hayata) Schiltr. extract for the manufacture of a pharmaceutical composition for promoting chronic wound healing

Abstract

To provide the use of plant extract for the manufacture of a pharmaceutical composition for promoting chronic wound healing.SOLUTION: The present invention provides the use of a plant extract for the manufacture of a pharmaceutical composition for promoting chronic wound healing in a subject, wherein the plant extract comprises an effective amount of Bletilla formosana (Hayata) Schltr. extract.SELECTED DRAWING: Figure 3B

Description

[Technical Field]

[0001] The present invention relates to the use of a plant extract for the manufacture of a pharmaceutical composition for promoting chronic wound healing in a subject, in particular to the use of an extract of Bletilla formosana (hayata) schiltr. for the manufacture of a pharmaceutical composition for promoting chronic wound healing in a subject. [Background technology]

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

[0003] Impaired wound healing in diabetes is associated with a variety of factors, including vascular degeneration, neuropathy, and immune and biochemical factors. Hyperglycemia causes vascular stiffening, which leads to slowed circulation and microvascular dysfunction, resulting in reduced tissue oxygen supply. Hyperglycemia also reduces leukocyte migration to wounds, making DM patients more vulnerable to infection. Peripheral neuropathy in DM can cause numbness in the area and reduce the ability to sense pain, which can lead to wounds not being recognized immediately and not being properly treated. (Spampinato SF, Caruso GI, De Pasquale R, Sortino MA, Merlo S. The Treatment of Impaired Wound Healing in Diabetes: Looking among Old Drugs. Pharmaceuticals (Basel). 2020;13(4):60.)

[0004] Diabetic foot ulcers (DFUs) are a major complication of DM, occurring in 15% of diabetic patients. Risk factors associated with DFUs include neuropathy, vascular disease, and infection. Previous studies have also found that the risk of lower limb amputation in diabetic patients is 15 to 46 times higher than in non-diabetic patients.

[0005] Natural compounds, with their abundant supply and diverse backbone, are important bases for drug development. Between 1981 and 2019, nearly half of all new FDA-approved drugs were derived from natural products or their derivatives, including cocaine-derived opioids, morphine-derived painkillers, vincristine, doxorubicin, and paclitaxel for cancer treatment, and fungal-derived penicillin as an antibiotic. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Spampinato SF, Caruso GI, De Pasquale R, Sortino MA, Merlo S. The Treatment of Impaired Wound Healing in Diabetes: Looking among Old Drugs. Pharmaceuticals (Basel). 2020;13(4):60. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, the present invention actively pursues research to determine which natural compounds have the potential to be developed as novel agents for promoting chronic wound healing. [Means for solving the problem]

[0008] Amanita orchid is selected as the research target of the present invention.

[0009] The present invention relates to the use of an extract of Amanita orchid for the manufacture of a pharmaceutical composition for promoting chronic wound healing in a subject.

[0010] In the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier for the Amanita orchid extract.

[0011] In the present invention, the chronic wound includes a diabetic wound or a diabetic foot ulcer (DFU).

[0012] In the present invention, the effective amount of the Amanita alba extract is 0.01 mg / kg body weight to 100 mg / kg body weight.

[0013] Furthermore, the effective dose of Amanalan extract is 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, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 10 2, 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, or 100mg / kg body weight.

[0014] In a preferred embodiment, the effective amount of the Amanita orchid extract is 1 mg / kg body weight to 75 mg / kg body weight.

[0015] Preferably, the effective amount of the Amanita orchid extract is 25 mg / kg body weight to 50 mg / kg body weight.

[0016] In the present invention, the subject is a human or mammal.

[0017] When the Amanita orchid extract is formulated into a pharmaceutical composition, the pharmaceutical composition may contain pharmaceutically acceptable excipients, particularly a predetermined solvent or oil, a pH adjuster, and, if desired, a dispersant. 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, grapeseed oil, coconut oil, sunflower oil, shea butter, and any combination thereof.

[0018] The solvents and oils may be used alone or in any combination.

[0019] Examples of useful dispersing agents include, but are not limited to, lecithin, organic monoglycerides, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, sorbitan stearate, etc. These materials may also be used alone or in any combination thereof.

[0020] In preferred embodiments of the present invention, the pharmaceutical compositions are injectable or parenteral preparations and topical preparations, including but not limited to creams, ointments, gels, washes, patches, inhalants, aerosols, and suppositories.

[0021] When the pharmaceutical composition is used as an external preparation, a suitable external skin preparation may be used as a base, and an aqueous solution, non-aqueous solvent, suspension, emulsion, gel, cream, ointment or lyophilized preparation may be used according to known methods, and may be sterilized. Compositions in the form of gels, creams and ointments may be suitably prepared by using known methods and by adding known emollients, emulsifiers and thickeners or other materials known in the art, depending on the form of the composition.

[0022] A gel-type composition may be prepared by adding emollients such as trimethylolpropane, polyethylene glycol and glycerol, solvents such as propylene glycol, ethanol and isocetyl alcohol, and pure water.

[0023] As used herein, the term "effective amount" refers to an amount that, when administered to an individual, can achieve an effective result or have a desired activity in vivo or in vitro. In the case of promoting chronic wound healing, effective clinical outcomes include an improvement in the degree or severity of symptoms associated with a disease or condition, and / or an increase in the lifespan of the individual, and / or an improvement in the quality of life of the individual, when compared to an untreated or vehicle group. [Brief explanation of the drawings]

[0024] [Figure 1A] Figure 1 shows that the Amanita orchid extract "-W+EA" effectively stimulates cell migration of the human foreskin fibroblast cell line Hs68. Figure 1A shows staining images of cell migration. [Figure 1B] Figure 1 shows that the Amanita orchid extract "-W+EA" effectively stimulates cell migration of the human foreskin fibroblast cell line Hs68. Figure 1B shows a line graph of cell migration. [Figure 2] FIG. 2 shows a flow chart of the diabetic wound healing assay. [Figure 3A] Figure 3 shows the healing effect of the Amanita lily extract "-W+EA" on diabetic wounds. Figure 3A shows images of wound healing in the untreated group, vehicle group, and -W+EA treatment group. [Figure 3B]Figure 3 shows the healing effect of the Amanita japonica extract "-W+EA" on diabetic wounds. Figure 3B shows a line graph of the wound healing rate for the untreated group, vehicle group, and -W+EA-treated group. *: p<0.05 and **: p<0.01 indicate significant differences compared to the untreated group. Meanwhile, @: p<0.05 and @@: p<0.01 indicate significant differences compared to the vehicle group. [Figure 4] FIG. 4 shows that topical application of -W+EA only promotes healing of diabetic wounds and does not treat diabetes itself. [Figure 5] FIG. 5 shows the blood biochemistry assay of -W+EA on experimental animals. DETAILED DESCRIPTION OF THE INVENTION

[0025] This embodiment is only a record of the best example and is not intended to limit the present invention.

[0026] (Preparation of Amanita orchid extract) In the present invention, the Amanita orchid samples used to prepare the extract were selected from the roots, stems, or leaves of Amanita orchid, or a mixture of the roots, stems, and leaves. The Amanita orchid samples were dried, ground, and then stored at -20°C until ready for use.

[0027] In the present invention, the Amanita orchid sample was first added to double-distilled water (ddH2O) at room temperature, followed by ultrasonic shock to obtain the upper supernatant and residue. After freezing and drying, the residue was purified and extracted with ethyl acetate to obtain the Amanita orchid extract of the present invention, which was named "-W+EA."

[0028] (In vitro wound healing assay) In the present invention, -W+EA was used to evaluate the in vitro wound healing ability.

[0029] Human foreskin fibroblast cell line Hs68 (purchased from Bioresource Collection and Research Center; BCRC No. 60038) was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S). The cells were cultured in an incubator at 37°C with 5% CO.

[0030] Hs68 cells were cultured in 24-well culture plates and divided into three groups: control (basal), low-dose W+EA (0.1 μg / mL W+EA), and high-dose W+EA (1 μg / mL W+EA). IBIDI culture inserts (purchased from GmbH, Germany) were then used for in vitro wound healing assays according to the manufacturer's instructions.

[0031] The IBIDI culture insert was placed into a well of a 24-well culture plate and gently pressed down on top to ensure tight adhesion between the IBIDI culture insert and the well. The IBIDI culture insert consisted of two wells separated by a 500 μm thick wall. Equal amounts of Hs68 cells (4.5 × 10 4 Cells) were split into two wells of an IBIDI culture insert and cultured for 24 hours, the medium was replaced with FBS-free medium, the IBIDI culture insert was gently removed to create a 500 μm gap, and washed with phosphate-buffered saline (PBS).

[0032] Nuclei were stained with 2 μg / mL Hoechst 33342 for 10 minutes and then washed with 1x PBS. The nuclear-stained cells were treated with 0.1 μg / mL -W+EA and 1 μg / mL -W+EA, respectively, and observed 0, 6, 9, 12, and 24 hours after treatment using a Citation 5 image system (Biotek, UK).

[0033] As shown in Figures 1A and 1B, cell migration was observed in both the low-dose -W+EA group and the high-dose -W+EA group compared to the control group, demonstrating that -W+EA effectively promotes wound healing.

[0034] Diabetic wound healing assay All animal experiments in this invention were performed under the approval of the IACUC, Chang Gung University.

[0035] A flow chart of the diabetic wound healing assay of the present invention is shown in FIG.

[0036] To establish the diabetic animal model of the present invention, 8-9 week-old C57BL / 6 male mice weighing 20-25 grams were selected. After fasting for 4 hours, the mice were intraperitoneally injected with 55 mg / kg body weight of streptozotocin (STZ) dissolved in citrate buffer (pH 4.5) for 5 consecutive days. During the STZ injection, diabetes was induced by feeding the mice a normal diet and 3% glucose water for the first 4 days, and then on the final day, the mice were given normal water instead of glucose water.

[0037] Seventy-two hours after STZ injection, mice were fasted for 4 hours and then bled by tail prick. Fasting plasma glucose levels were measured using a Contour Plus portable blood glucose meter (Ascensia, UK). Mice with fasting plasma glucose levels between 150 and 250 mg / dL were considered prediabetic, and mice with fasting plasma glucose levels >250 mg / dL were considered diabetic and used in subsequent experiments. All mice had fasting plasma glucose levels >250 mg / dL.

[0038] After establishing a diabetic animal model, a diabetic wound healing animal model was established.

[0039] A total of 20 male C57BL / 6 diabetic mice were used in the experiment and were randomly divided into four groups, designated as: untreated group (5 mice), vehicle group (25% Pluronic F-127 and DMSO mixture, 5 mice), low dose-W+EA group (25 mg / kg-W+EA, 5 mice), and high dose-W+EA group (50 mg / kg-W+EA, 5 mice).

[0040] Under anesthesia, the hair on the dorsal region of diabetic mice was removed with a razor and depilatory cream, medical tape was applied to the dorsal skin to prevent stretching, and two full-thickness wounds, one on the left and one on the right, each 6 mm in diameter, were then created using a sterile biopsy punch.

[0041] During the experimental process, all wounds were fixed with medical tape with circular notches to maintain the wound gap. The medical tape was used to fix the skin around the wound to prevent skin contraction during wound healing, which may affect the calculation of wound area. The left wound was not treated with any treatment, while the right wound was treated with vehicle solution (25% Pluronic F-127 and DMSO mixture), 25 mg / kg -W+EA, and 50 mg / kg -W+EA every day for 2 weeks, and the wounds were photographed every day.

[0042] Wound area was measured by Image J analysis software according to photographs taken daily and expressed as percentage of wound healing compared to day 0.

[0043] The wound healing results are shown in Figures 3A and 3B. Figure 3A shows wound healing photographs for the untreated, vehicle, low-dose -W + EA, and high-dose -W + EA groups, and Figure 3B shows the quantified wound area illustrated in a line graph. Compared to the untreated and vehicle groups, mice treated with -W + EA were observed to exhibit significant wound healing efficacy.

[0044] To further confirm that topical application of -W+EA only promoted diabetic wound healing and did not treat diabetes itself, the fasting blood glucose levels of each mouse were measured during weeks 1 and 2. As shown in Figure 4, the fasting blood glucose levels of all mice continued to increase, confirming that topical application of -W+EA only promoted diabetic wound healing and did not treat diabetes.

[0045] (Blood Biochemistry Assay) As shown in Figure 5, it can be observed through the blood biochemistry values ​​that -W+EA did not cause hepatotoxicity (indicated by GOT and GPT levels) or nephrotoxicity (indicated by CRE and BUN levels) in experimental animals, indicating that -W+EA of the present invention has a high safety profile for experimental animals.

[0046] While 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.

[0047] Those skilled in the art will readily recognize that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The cells, animals, and processes, and methods for producing them, are representative of preferred embodiments and are exemplary and are not intended as limitations on the scope of the invention. Modifications and other uses of the present invention will occur to those skilled in the art. These modifications are included within the spirit of the invention and are defined by the scope of the claims.

Claims

1. 1. Use of a plant extract for the manufacture of a pharmaceutical composition for promoting chronic wound healing in a subject, the plant extract comprising an effective amount of Bletilla formosana (Hayata) Schiltr. extract; The above use, wherein the extract of Amanita orchid is obtained by extracting a mixture of the roots, stems and leaves of Amanita orchid with water, and then further extracting the residue with ethyl acetate.

2. The use of claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

3. The use of claim 1, wherein the pharmaceutical composition is a parenteral preparation or an external preparation.

4. 10. The use of claim 1, wherein the pharmaceutical composition is a topical skin preparation.

5. 2. The use of claim 1, wherein the effective amount is 0.01 mg / kg body weight to 100 mg / kg body weight.

6. 2. The use of claim 1, wherein the effective amount is 1 mg / kg body weight to 75 mg / kg body weight.

7. 2. The use of claim 1, wherein the effective amount is 25 mg / kg body weight to 50 mg / kg body weight.

8. 10. The use of claim 1, wherein the chronic wound comprises a diabetic wound.

9. 2. The use of claim 1, wherein the chronic wound comprises a diabetic foot ulcer.

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