TW202410913A - Auricularia polytricha polyomycoid extract for preparing pharmaceutical components that promote wound healing - Google Patents
Auricularia polytricha polyomycoid extract for preparing pharmaceutical components that promote wound healing Download PDFInfo
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Abstract
Description
本發明有關於毛木耳多醣體萃取物，特別是指由毛木耳蒂頭製成的多醣體萃取物及其用於製備促進傷口癒合之醫藥組成物的用途。The present invention relates to a polysaccharide extract of Auricularia auricula, in particular to a polysaccharide extract prepared from the pedicel of Auricularia auricula and its use in preparing a pharmaceutical composition for promoting wound healing.
毛木耳（Auricularia polytricha）為台灣主要栽培的木耳，具有豐富的多醣體，且近年對於毛木耳的多醣功能性研究，包括活化巨噬細胞產生TNF-α 和IL-6 去抑制癌細胞生長、抑制人類肺癌細胞株A549 的細胞週期、誘導癌細胞凋亡與抑制腫瘤細胞的活性等。此外，研究還發現給予小鼠毛木耳粉末可增強自然殺手細胞活性，強化小鼠免疫功能。Auricularia polytricha is the main cultivated fungus in Taiwan. It is rich in polysaccharides. In recent years, the functional studies of Auricularia polytricha have shown that it can activate macrophages to produce TNF-α and IL-6 to inhibit cancer cell growth, inhibit the cell cycle of human lung cancer cell line A549, induce cancer cell apoptosis, and inhibit the activity of tumor cells. In addition, the study also found that giving Auricularia polytricha powder to mice can enhance the activity of natural killer cells and strengthen the immune function of mice.
值得注意的是，在毛木耳採收加工過程，子實體多以鮮食方式進入市場，或以日光曝曬或機械烘乾方式進行乾燥後保存；而蒂頭由於質地較硬，口感不佳，無法食用，屬農業副產物資材，業者多以人工方式去除含菇包木屑之蒂頭而未進一步使用。此外，前述研究也幾乎集中在毛木耳子實體的可食用部分或是菌絲體，忽略了連接毛木耳菌絲體以及毛木耳食用部分之間的蒂頭。It is worth noting that during the harvesting and processing of Auricularia auricula, the fruiting bodies are mostly eaten fresh or stored after being dried by sunlight or mechanical drying. The pedicles are hard in texture, have a bad taste, and are inedible. They are agricultural by-products and materials. The industry often removes the pedicles containing the sawdust from the mushroom packaging manually and does not use them further. In addition, the aforementioned studies have almost all focused on the edible part of the fruiting body of Auricularia auricula or the mycelium, ignoring the pedicles that connect the mycelium of Auricularia auricula and the edible part of Auricularia auricula.
然而，根據專利文獻第TWI744971B號公告的「毛木耳萃取物用於製備誘導未極化的免疫細胞分化成抗發炎型免疫細胞之組成物的用途」的記載可知，毛木耳蒂頭含有相當豐富的多醣體，卻未被加以利用。However, according to the patent document No. TWI744971B, "Use of Auricularia auricula extract for preparing a composition for inducing non-polarized immune cells to differentiate into anti-inflammatory immune cells", it is known that the Auricularia auricula pedicles contain a considerable amount of polysaccharides, but they have not been utilized.
有鑑於上述問題，本發明之目的在於提供一種毛木耳多醣體萃取物之用途，是用於製備促進傷口癒合之醫藥組成物；其中，該毛木耳多醣體萃取物是將毛木耳蒂頭進行萃取所製得的毛木耳蒂頭水萃液。In view of the above problems, the purpose of the present invention is to provide a use of a polysaccharide extract of Auricularia auricula for preparing a pharmaceutical composition for promoting wound healing; wherein the polysaccharide extract of Auricularia auricula is an aqueous extract of Auricularia auricula obtained by extracting the Auricularia auricula pedicles.
其中，該毛木耳多醣體萃取物透過提升精氨酸酶（arginase）基因表現量，以競爭利用精胺酸（arginine）造成一氧化氮合成酶（NO）減少，促使降低發炎反應。Among them, the Auricularia auricularia polysaccharide extract increases the expression of the arginase gene to compete with arginine to cause a decrease in nitric oxide synthase (NO), thereby reducing the inflammatory response.
其中，該毛木耳多醣體萃取物透過使淋巴細胞的TGF-β基因表現量增加，促進傷口癒合。Among them, the Auricularia auricula polysaccharide extract promotes wound healing by increasing the expression of TGF-β gene in lymphocytes.
其中，該毛木耳多醣體萃取物是該毛木耳蒂頭水萃液經冷凍乾燥後製成的毛木耳蒂頭水萃凍乾粉；該醫藥組成物包括該毛木耳蒂頭水萃凍乾粉及瓊崖海棠種子油。The polysaccharide extract of Auricularia auricula is a freeze-dried powder of Auricularia auricula pedicle water extract prepared by freeze-drying the Auricularia auricula pedicle water extract; the pharmaceutical composition comprises the freeze-dried powder of Auricularia auricula pedicle water extract and Malus chinensis seed oil.
其中，該毛木耳蒂頭水萃凍乾粉在傷口癒合速率的發炎期投予，以促進傷口癒合。The freeze-dried powder extracted from the pedicles of Auricularia auricula is administered during the inflammatory phase of wound healing to promote wound healing.
其中，該瓊崖海棠種子油在傷口癒合速率的增生期投予，以促進刺激纖維母細胞增生，並防止細菌感染。The Malus chinensis seed oil is administered during the proliferative phase of wound healing to promote and stimulate fibroblast proliferation and prevent bacterial infection.
其中，該毛木耳多醣體萃取物是該毛木耳蒂頭水萃液經冷凍乾燥後製成的毛木耳蒂頭水萃凍乾粉；該醫藥組成物包括該毛木耳蒂頭水萃凍乾粉及瓊崖海棠種子油。The polysaccharide extract of Auricularia auricula is a freeze-dried powder of Auricularia auricula pedicle water extract prepared by freeze-drying the Auricularia auricula pedicle water extract; the pharmaceutical composition comprises the freeze-dried powder of Auricularia auricula pedicle water extract and Malus chinensis seed oil.
其中，該毛木耳多醣體萃取物是該毛木耳蒂頭水萃液經冷凍乾燥後製成的毛木耳蒂頭水萃凍乾粉；該醫藥組成物包括該毛木耳蒂頭水萃凍乾粉及瓊崖海棠種子油色素萃取液。The polysaccharide extract of Auricularia auricula is a freeze-dried powder of Auricularia auricula pedicle water extract prepared by freeze-drying the Auricularia auricula pedicle water extract; the pharmaceutical composition comprises the freeze-dried powder of Auricularia auricula pedicle water extract and a pigment extract of Malus chinensis seed oil.
其中，該毛木耳多醣體萃取物是該毛木耳蒂頭水萃液經脫色分離並濃縮後再冷凍乾燥後製成的脫色毛木耳蒂頭水萃凍乾粉；該醫藥組成物包括脫色毛木耳蒂頭水萃凍乾粉及瓊崖海棠種子油。The polysaccharide extract of Auricularia auricula is a decolorized Auricularia auricula pedicle water extract freeze-dried powder prepared by decolorizing, separating, concentrating and then freeze-drying the Auricularia auricula pedicle water extract; the pharmaceutical composition comprises decolorized Auricularia auricula pedicle water extract freeze-dried powder and Malus chinensis seed oil.
其中，該毛木耳多醣體萃取物是該毛木耳蒂頭水萃液經脫色分離並濃縮後再冷凍乾燥後製成的脫色毛木耳蒂頭水萃凍乾粉；該醫藥組成物包括脫色毛木耳蒂頭水萃凍乾粉及瓊崖海棠種子油色素萃取液。The polysaccharide extract of Auricularia auricula is a decolorized Auricularia auricula pedicle water extract lyophilized powder prepared by decolorizing, separating, concentrating and then freeze-drying the Auricularia auricula pedicle water extract; the pharmaceutical composition comprises the decolorized Auricularia auricula pedicle water extract lyophilized powder and the pigment extract of Malus chinensis seed oil.
有關於本發明為達成上述目的，所採用之技術、手段及其他功效，茲舉較佳可行實施例並配合圖式詳細說明如後。With regard to the techniques, means and other effects adopted by the present invention to achieve the above-mentioned objects, the preferred feasible embodiments are described in detail with reference to the drawings as follows.
本發明特徵與優點的一些典型實施例將在以下說明中詳細敘述。應理解的是本發明能夠在不同的態樣上具有各種的變化，然其皆不脫離本發明的範圍，且其中說明及圖式在本質上係當作說明之用，而非用於限制本發明。Some typical embodiments of the features and advantages of the present invention will be described in detail in the following description. It should be understood that the present invention can have various changes in different aspects, but they all do not deviate from the scope of the present invention, and the descriptions and drawings are essentially used for illustration rather than for limiting the present invention.
本發明提供了毛木耳多醣體萃取物及其用於製備促進傷口癒合之醫藥組成物的新用途。以下將進一步說明毛木耳多醣體萃取物的提取方法及其功效試驗。The present invention provides a polysaccharide extract of Auricularia auricula and a novel use thereof for preparing a pharmaceutical composition for promoting wound healing. The following will further describe the extraction method of the polysaccharide extract of Auricularia auricula and its efficacy test.
本發明的毛木耳多醣體萃取物是指從毛木耳蒂頭萃取製成的毛木耳蒂頭水萃液及/或其加工製成的產品，所述產品包括但不限於毛木耳蒂頭水萃凍乾粉及脫色毛木耳蒂頭水萃凍乾粉。The Auricularia auricula polysaccharide extract of the present invention refers to the Auricularia auricula pedicle water extract obtained by extracting the Auricularia auricula pedicle and/or products processed therefrom, including but not limited to Auricularia auricula pedicle water-extracted lyophilized powder and decolorized Auricularia auricula pedicle water-extracted lyophilized powder.
其中，該毛木耳蒂頭水萃凍乾粉的製備方法，是先將毛木耳蒂頭進行萃取程序以製得毛木耳蒂頭水萃液，再將該毛木耳蒂頭水萃液冷凍乾燥後形成該毛木耳蒂頭水萃凍乾粉。The preparation method of the Auricularia auricula pedicle water-extracted freeze-dried powder comprises first subjecting the Auricularia auricula pedicle to an extraction process to obtain an Auricularia auricula pedicle water extract, and then freeze-drying the Auricularia auricula pedicle water extract to form the Auricularia auricula pedicle water-extracted freeze-dried powder.
其中，該脫色毛木耳蒂頭水萃液的製備方法，是先將毛木耳蒂頭進行萃取程序以製得毛木耳蒂頭水萃液，再將該毛木耳蒂頭水萃液脫色分離並濃縮後形成該脫色毛木耳蒂頭水萃液，最後再將該脫色毛木耳蒂頭水萃液冷凍乾燥後形成該脫色毛木耳蒂頭水萃凍乾粉。The preparation method of the decolorized Auricularia auricula pedicle water extract comprises the steps of first subjecting the Auricularia auricula pedicle to an extraction process to obtain the Auricularia auricula pedicle water extract, then decolorizing, separating and concentrating the Auricularia auricula pedicle water extract to form the decolorized Auricularia auricula pedicle water extract, and finally freeze-drying the decolorized Auricularia auricula pedicle water extract to form the decolorized Auricularia auricula pedicle water extract lyophilized powder.
具體地，該毛木耳蒂頭水萃液的製法步驟包括：Specifically, the preparation steps of the Auricularia auricula pedicle water extract include:
取材步驟，提供經過毛木耳蒂頭預處理程序製成的毛木耳蒂頭冷凍樣品，將毛木耳蒂頭冷凍樣品與無菌水以重量比為1：1至1：10的比例混合；The sampling step comprises providing a frozen sample of the pedicle of Auricularia auricula prepared through a pretreatment procedure of the pedicle of Auricularia auricula, and mixing the frozen sample of the pedicle of Auricularia auricula with sterile water at a weight ratio of 1:1 to 1:10;
熱萃步驟，將取材步驟中的材料配比，在1.0至1.2 psi的高壓力範圍以及95至121℃的高溫度範圍內進行高壓蒸煮10至60分鐘，形成毛木耳蒂頭初萃產品；In a hot extraction step, the material ratio in the material collection step is subjected to high-pressure steaming for 10 to 60 minutes at a high pressure range of 1.0 to 1.2 psi and a high temperature range of 95 to 121° C. to form a primary extraction product of the hairy fungus pedicles;
固液分離步驟，該毛木耳蒂頭初萃產品經離心以固液分離後，形成毛木耳蒂頭水萃液及剩餘品；其中，該毛木耳蒂頭水萃液包括離心後形成的最上方的上層液（solution layer）及中間的黏稠層（viscosity layer），該毛木耳蒂頭水萃液的多醣濃度為150.6至690.8 mg/mL；該剩餘品為離心後形成的最下層產品。In the solid-liquid separation step, the primary extraction product of the Auricularia auricula pedicles is centrifuged for solid-liquid separation to form an Auricularia auricula pedicles water extract and a residue; wherein the Auricularia auricula pedicles water extract includes the topmost solution layer and the middle viscosity layer formed after centrifugation, and the polysaccharide concentration of the Auricularia auricula pedicles water extract is 150.6 to 690.8 mg/mL; the residue is the bottommost product formed after centrifugation.
進一步地，該毛木耳蒂頭水萃凍乾粉是經由以下步驟製成：Furthermore, the Auricularia auricula pedicle water-extracted freeze-dried powder is prepared through the following steps:
冷凍乾燥步驟：將該毛木耳蒂頭水萃液進行冷凍乾燥，以獲得該毛木耳蒂頭水萃凍乾粉；以及Freeze-drying step: freeze-drying the Auricularia auricula pedicle water extract to obtain the Auricularia auricula pedicle water extract freeze-dried powder; and
真空包裝步驟：該毛木耳蒂頭水萃凍乾粉經真空包裝後保存。Vacuum packaging step: the Auricularia auricula-derived water-extracted freeze-dried powder is stored after vacuum packaging.
其中，該毛木耳蒂頭水萃凍乾粉較佳保存在零下20℃至25℃的溫度範圍內。The water-extracted freeze-dried powder of Auricularia auricula-sinensis stem is preferably stored in a temperature range of -20°C to 25°C.
進一步地，該脫色毛木耳蒂頭水萃凍乾粉是經由以下步驟製成：Furthermore, the decolorized Auricularia auricula pedicle water-extracted freeze-dried powder is prepared through the following steps:
管柱層析脫色步驟：利用管柱層析法對該毛木耳蒂頭水萃液進行脫色分離，獲得固定相的毛木耳色素以及流動相的毛木耳水萃脫色液；Column chromatography decolorization step: decolorizing and separating the Auricularia auricula pedicle water extract by column chromatography to obtain Auricularia auricula pigment as a stationary phase and Auricularia auricula water extract decolorization solution as a mobile phase;
濃縮步驟：將該毛木耳水萃脫色液進行濃縮，以獲得脫色毛木耳水萃液；Concentrating step: concentrating the Auricularia auricula water extract decolorizing solution to obtain a decolorized Auricularia auricula water extract solution;
冷凍乾燥步驟：將該脫色毛木耳水萃液進行冷凍乾燥，以獲得該脫色毛木耳蒂頭水萃凍乾粉；以及Freeze-drying step: freeze-drying the decolorized Auricularia auricula water extract to obtain the decolorized Auricularia auricula pedicle water extract freeze-dried powder; and
真空包裝步驟：該脫色毛木耳蒂頭水萃凍乾粉經真空包裝後保存。Vacuum packaging step: the decolorized Auricularia auricula-derived water-extracted freeze-dried powder is stored after vacuum packaging.
本發明的毛木耳蒂頭水萃液中含有(1,3)-β-D-葡聚糖（(1-3)-b-D-glucan）；其中，每公克該毛木耳蒂頭水萃液中的多醣體含量為164.2至662.1 mg；每公克該毛木耳蒂頭水萃液中的(1,3)-β-D-葡聚糖的含量為3.5至8.4 mg；該多醣體的分子量為3.6×10 4至8.1×10 5Da；該多醣體中的(1,3)-β-D-葡聚糖的含量為20.5至22.0 mg。 The water extract of Auricularia auricula pedicles of the present invention contains (1,3)-β-D-glucan ((1-3)-bD-glucan); wherein the polysaccharide content in each gram of the water extract of Auricularia auricula pedicles is 164.2 to 662.1 mg; the (1,3)-β-D-glucan content in each gram of the water extract of Auricularia auricula pedicles is 3.5 to 8.4 mg; the molecular weight of the polysaccharide is 3.6×10 4 to 8.1×10 5 Da; and the (1,3)-β-D-glucan content in the polysaccharide is 20.5 to 22.0 mg.
以前述毛木耳蒂頭水萃液的提取方法的步驟對毛木耳的子實體進行相同的製備步驟，以獲得毛木耳子實體水萃液。如表1比較可知，以毛木耳蒂頭製備的水萃液所含(1,3)-β-D-葡聚糖含量明顯高於毛木耳子實體製備的水萃液；此外，經分析可知，毛木耳蒂頭水萃液的上層液中具有較高的多醣體含量及(1,3)-β-D-葡聚糖含量。The same preparation steps were performed on the fruiting bodies of Auricularia auricula in the same manner as the steps of the aforementioned method for extracting the water extract of the Auricularia auricula pedicles to obtain the water extract of the fruiting bodies of Auricularia auricula. As shown in Table 1, the water extract prepared from the Auricularia auricula pedicles contained a significantly higher (1,3)-β-D-glucan content than the water extract prepared from the fruiting bodies of Auricularia auricula; in addition, analysis showed that the upper layer of the water extract of the Auricularia auricula pedicles had a higher polysaccharide content and (1,3)-β-D-glucan content.
表1：
於本發明實施例中，該毛木耳多醣體萃取物是包含葡萄糖（Glucose）及甘露糖（Mannose）聚合物之多醣體，其中，該上層液中的糖/醣%（w/w）為89%，且該上層液中每克多醣中單醣含量包括41.5%（w/w）的葡萄糖以及58.5%（w/w）的甘露糖；該黏稠層中的糖/醣%（w/w）為87%，且該黏稠層中每克多醣中單醣含量包括56.9%（w/w）的葡萄糖以及43.1%（w/w）的甘露糖。In an embodiment of the present invention, the Auricularia auricula polysaccharide extract is a polysaccharide comprising glucose and mannose polymers, wherein the sugar/sugar % (w/w) in the upper liquid is 89%, and the monosaccharide content per gram of polysaccharide in the upper liquid includes 41.5% (w/w) glucose and 58.5% (w/w) mannose; the sugar/sugar % (w/w) in the viscous layer is 87%, and the monosaccharide content per gram of polysaccharide in the viscous layer includes 56.9% (w/w) glucose and 43.1% (w/w) mannose.
如下表2，顯示前述毛木耳蒂頭水萃凍乾粉的儲存安定性試驗數據。該毛木耳蒂頭水萃凍乾粉是經分裝在塑膠袋後密封再裝入鋁箔袋密封後進行保存試驗。經試驗可知，毛木耳蒂頭水萃凍乾粉的保存環境溫度以低溫為佳，但本發明毛木耳蒂頭水萃凍乾粉在25℃與40℃溫度下保存後的多醣體濃度差異在±10%之內，顯示本發明製得的毛木耳蒂頭水萃凍乾粉具有相當的產品穩定性，適合應用在醫藥、食品、化妝品、動物飼料添加劑等多個領域。Table 2 below shows the storage stability test data of the aforementioned Auricularia auricula pedicle water-extracted freeze-dried powder. The Auricularia auricula pedicle water-extracted freeze-dried powder was packaged in plastic bags, sealed, and then sealed in aluminum foil bags for storage tests. The test showed that the best storage environment temperature for Auricularia auricula pedicle water-extracted freeze-dried powder is low temperature, but the polysaccharide concentration of the Auricularia auricula pedicle water-extracted freeze-dried powder after storage at 25°C and 40°C is within ±10%, indicating that the Auricularia auricula pedicle water-extracted freeze-dried powder produced by the present invention has considerable product stability and is suitable for application in multiple fields such as medicine, food, cosmetics, and animal feed additives.
表2：
以下說明本發明測試毛木耳多醣體萃取物用於促進傷口癒合之功效的動物和實驗設計。The following describes the animals and experimental design used in the present invention to test the efficacy of the Auricularia auriculariae polysaccharide extract in promoting wound healing.
實驗實施例1：毛木耳以熱水離心萃取半商品樣本之半體外免疫調節評估試驗。Experimental Example 1: Semi-in vitro immunomodulatory evaluation test of Auricularia auricula using hot water centrifugal extraction of semi-commercial samples.
圖1是本發明毛木耳蒂頭水萃液以進行豬隻免疫細胞毒性測試的結果；其中，PBMC表示外周血單個核細胞（peripheral blood mononuclear cell），PMN表示顆粒型或多核型白血球，SB表示毛木耳蒂頭水萃液之上層液，VB表示毛木耳蒂頭水萃液之黏稠層。圖1分為SB組和VB組，每組受測免疫細胞由左而右為PBMC、PMN，且每組免疫細胞的測試量由左而右依序為 0 μg/mL（NC組）、5μg/mL、15μg/mL、25μg/mL、50μg/mL圖1顯示本發明的毛木耳蒂頭水萃液在半體外免疫毒性試驗中對於豬免疫細胞不具有明顯毒性。FIG1 is the result of the toxicity test of the water extract of Auricularia auricula pedicles in the present invention on pig immune cells; wherein PBMC represents peripheral blood mononuclear cells, PMN represents granular or multinuclear leukocytes, SB represents the upper layer of the water extract of Auricularia auricula pedicles, and VB represents the viscous layer of the water extract of Auricularia auricula pedicles. FIG1 is divided into SB group and VB group, and the immune cells tested in each group are PBMC and PMN from left to right, and the test amount of each group of immune cells is 0 μg/mL (NC group), 5μg/mL, 15μg/mL, 25μg/mL, 50μg/mL from left to right. FIG1 shows that the water extract of Auricularia auricula pedicles in the present invention has no obvious toxicity to pig immune cells in the semi-in vitro immunotoxicity test.
如圖2所示，由左而右依序為LPS組、NC組、SA組、SB組、VA組、VB組，圖2顯示50μg/mL的毛木耳水萃液對健康小鼠腹腔巨噬細胞iNOS的基因表現；其中，LPS組表示添加脂多糖（Lipopolysaccharide）毛木耳水萃液，NC組表示對照組（Negative Control），SA組表示毛木耳子實體水萃液之上層液，SB組表示毛木耳蒂頭水萃液之上層液，VA組表示毛木耳子實體水萃液之黏稠層，VB組表示毛木耳蒂頭水萃液之黏稠層。As shown in Figure 2, from left to right they are LPS group, NC group, SA group, SB group, VA group, and VB group. Figure 2 shows the gene expression of iNOS in peritoneal macrophages of healthy mice treated with 50 μg/mL of Auricularia auricula water extract; wherein, the LPS group represents the Auricularia auricula water extract supplemented with lipopolysaccharide, the NC group represents the negative control group, the SA group represents the upper layer of the Auricularia auricula fruiting body water extract, the SB group represents the upper layer of the Auricularia auricula pedicle water extract, the VA group represents the viscous layer of the Auricularia auricula fruiting body water extract, and the VB group represents the viscous layer of the Auricularia auricula pedicle water extract.
由圖2可知，LPS組、SA組、SB組、VA組、VB組等毛木耳水萃液樣本均可活化小鼠腹腔巨噬細胞株iNOS基因表達。巨噬細胞活化可分類為典型巨噬細胞活化產生一氧化氮合成酶（NO）、腫瘤壞死因子（TNF）等促發炎反應，延後傷口癒合；而當巨噬細胞以另類路徑（非典型）活化時，提升精氨酸酶（arginase）的基因表現量，精胺酸（arginine）造成一氧化氮合成酶（NO），降低發炎反應，同時產生轉化生長因子（Transforming Growth Factor，TGF）則具有加速纖維母細胞增生與疤痕傷口癒合的功能。As shown in Figure 2, the Auricularia auricula water extract samples in the LPS group, SA group, SB group, VA group, and VB group can activate the iNOS gene expression of mouse peritoneal macrophage lines. Macrophage activation can be classified into typical macrophage activation, which produces nitric oxide synthase (NO), tumor necrosis factor (TNF), and other pro-inflammatory reactions, delaying wound healing; and when macrophages are activated in an alternative pathway (atypical), the gene expression of arginase is increased, arginine produces nitric oxide synthase (NO), reduces inflammatory reactions, and produces transforming growth factor (TGF), which has the function of accelerating fibroblast proliferation and scar wound healing.
實驗實施例2：毛木耳萃取液複方促進正常豬隻傷口癒合試驗。Experimental Example 2: Experiment on promoting wound healing of normal pigs with the compound extract of Auricularia auricula.
試驗設計：Experimental design:
動物品系為藍瑞斯豬。豬隻週齡/體重為2-3月齡/28~30 kg。豬隻性別/隻數為公豬3頭。動物模式為正常豬，無模式誘發。試驗分組為隨機分組，每隻背部開創6個傷口。傷口開創為全皮層的方形創傷 （2±0.5公分 × 2 ±0.5公分），左右各 3個；傷口編號，左邊自頭部至尾部依序標示為L1、L2、L3，右邊自頭部至尾部依序標示為R1、R2、R3。皮膚採樣的面積為3±0.5公分 × 3 ±0.5公分，並浸泡於10% （v/v）中性福馬林液中固定。統計方式為(1)傷口癒合速率：各試驗組樣本數不同，利用 One Way ANOVA 的 Scheffe事後檢定，以及(2)組織病理切片：因以程度比較各試驗組，利用無母數Mann-Whitney 進行檢定。其中，具有顯著差異表示為 p＜ 0.05、非常顯著差異表示為 p＜ 0.01、或者極顯著性差異表示為 p＜ 0.001。 Animal strains were Blue Ridge pigs. The age/weight of the pigs was 2-3 months/28-30 kg. The sex/number of pigs was 3 boars. The animal pattern was normal pigs, without pattern induction. The experimental groups were randomly divided, and 6 wounds were made on the back of each pig. The wounds were square wounds of full thickness (2±0.5 cm × 2±0.5 cm), 3 on each side; the wound numbers were marked L1, L2, L3 from the head to the tail on the left, and R1, R2, R3 from the head to the tail on the right. The skin samples were collected from an area of 3 ± 0.5 cm × 3 ± 0.5 cm and fixed in 10% (v/v) neutral formalin. The statistical methods were (1) wound healing rate: the number of samples in each test group was different, and the Scheffe post hoc test was used for One Way ANOVA, and (2) tissue pathological sections: because the test groups were compared in degree, the Mann-Whitney test was used without a matrix. Among them, a significant difference was expressed as p < 0.05, a very significant difference was expressed as p < 0.01, or an extremely significant difference was expressed as p < 0.001.
實驗實施例2的投藥方式如下表3所示。The administration method of Experimental Example 2 is shown in Table 3 below.
表3：
於本發明實驗實施例2中，正常豬傷口癒合試驗為期共28天。期間，在第0天進行切割傷開創、描繪傷口面積與拍照，在第1、3、5、7、10、14、21天進行換包紮、描繪傷口面積與拍照，在第28天進行描繪傷口面積、拍照、採樣全皮層皮膚組織。完成前述流程後進行實驗實施例2的傷口癒合速率計算及組織病理切片分析。In Experimental Example 2 of the present invention, the wound healing test of normal pigs lasted for a total of 28 days. During the period, wound incision, wound area drawing and photographing were performed on day 0, bandage change, wound area drawing and photographing were performed on days 1, 3, 5, 7, 10, 14 and 21, and wound area drawing, photographing and sampling of full-thickness skin tissue were performed on day 28. After completing the above process, the wound healing rate calculation and tissue pathological section analysis of Experimental Example 2 were performed.
其中，傷口癒合速率計算，是在各時間點描繪傷口面積後，以Image J軟體分析計算傷口癒合面積，再計算傷口癒合百分比（wound healing rate, %）。傷口癒合百分比% = [（第0天傷口面積－第N天傷口面積） / 第0天傷口面積] × 100%。The wound healing rate was calculated by plotting the wound area at each time point, analyzing and calculating the wound healing area using Image J software, and then calculating the wound healing rate (%). Wound healing rate (%) = [(wound area on day 0 - wound area on day N) / wound area on day 0] × 100%.
其中，組織病理切片進行蘇木精-伊紅染色（H&E stain）後，以光學顯微鏡進行組織病理學分析上皮化（Epithelization）、炎症反應（Inflammation）、血管新生（New blood capillary）、纖維母細胞（Fibroblast）、表皮層厚度（Epidermis thickness）、真皮層厚度（Dermis thickness）。Among them, the tissue pathological sections were stained with hematoxylin and eosin (H&E stain), and then the histopathological analysis of epithelialization, inflammation, new blood capillary, fibroblast, epidermis thickness, and dermis thickness was performed under an optical microscope.
圖3顯示了正常豬隻在試驗期間的體重變化，以開創第0天的數值點為準，由上往下依序表示豬隻編號064、065、066的體重曲線。由圖3可知，各組試驗物質皆不影響豬隻生長且無造成死亡情況。Figure 3 shows the weight changes of normal pigs during the trial period. Taking the value point on the 0th day as the reference, the weight curves of pigs No. 064, 065, and 066 are shown from top to bottom. As can be seen from Figure 3, all groups of test substances did not affect the growth of pigs and did not cause mortality.
圖4顯示了正常豬隻的傷口癒合試驗，其是利用one-way ANOVA進行統計分析，以Scheffe事後檢定法比較不同試驗物質於各時間點之傷口癒合速率，圖4在時點第0、1、3、5、7、10、14、21、28天進行檢測，每一時點的四個長條數據由左而右依序為對照組、毛木耳萃取液複方(A)組、毛木耳萃取液複方(B)組、正對照組。由圖4可知，在傷口癒合中期（第10、14、21天），對照組與正對照組的傷口癒合速率皆顯著高於毛木耳萃取液複方(A)組及毛木耳萃取液複方(B)組(p＜0.05)。Figure 4 shows the wound healing test of normal pigs, which was statistically analyzed using one-way ANOVA, and the wound healing rates of different test substances at different time points were compared using the Scheffe post hoc test. Figure 4 was tested at time points 0, 1, 3, 5, 7, 10, 14, 21, and 28 days. The four long bars of data at each time point are from left to right: control group, Auricularia auriculariae extract compound (A) group, Auricularia auriculariae extract compound (B) group, and positive control group. As shown in Figure 4, in the middle stage of wound healing (days 10, 14, and 21), the wound healing rates of the control group and the positive control group were significantly higher than those of the Auricularia auriculariae extract compound (A) group and the Auricularia auriculariae extract compound (B) group (p < 0.05).
於本發明實驗實施例2中進行正常豬隻的傷口分泌物記錄。在第10天時，可觀察到對照組的肉芽明顯隆起，未見組織液分泌；毛木耳萃取液複方(A)組的肉芽明顯隆起，但未將傷口完全閉合，有少量組織液蓄積；毛木耳萃取液複方(B)組的肉芽明顯隆起，但未將傷口完全閉合，有少量組織液蓄積；正對照組的肉芽已隆起並覆蓋傷口，未見組織液分泌。In Experimental Example 2 of the present invention, wound secretions of normal pigs were recorded. On the 10th day, it was observed that the granulation tissue of the control group was obviously raised, and no tissue fluid secretion was observed; the granulation tissue of the Auricularia auriculariae extract compound (A) group was obviously raised, but the wound was not completely closed, and a small amount of tissue fluid was accumulated; the granulation tissue of the Auricularia auriculariae extract compound (B) group was obviously raised, but the wound was not completely closed, and a small amount of tissue fluid was accumulated; the granulation tissue of the control group had raised and covered the wound, and no tissue fluid secretion was observed.
於本發明實驗實施例2中進行正常豬隻的傷口癒合變化記錄。在第28天時，可觀察到對照組的疤痕面積縮小，但顏色偏暗紅色；毛木耳萃取液複方(A)組的疤疤痕面積縮小，但顏色偏暗紅色，且有少許開放傷口；毛木耳萃取液複方(B)組的疤痕面積縮小，但顏色偏暗紅色，且有少許開放傷口；正對照組的疤痕面積與顏色較其他組小。In Experimental Example 2 of the present invention, wound healing changes of normal pigs were recorded. On the 28th day, it was observed that the scar area of the control group was reduced, but the color was dark red; the scar area of the Auricularia auricula extract compound (A) group was reduced, but the color was dark red, and there were a few open wounds; the scar area of the Auricularia auricula extract compound (B) group was reduced, but the color was dark red, and there were a few open wounds; the scar area and color of the control group were smaller than those of the other groups.
表4：
本發明實驗實施例2的統計分析結合表4，顯示各試驗組皮膚表皮層厚度：毛木耳萃取液複方(A)組＞毛木耳萃取液複方(B)組＞正對照組＞對照組，各試驗組間未達顯著差異(p＞0.05)，與正常皮膚相比，僅毛木耳萃取液複方(A)組表皮層厚度達顯著差異(p＜0.05)。各試驗組皮膚真皮層厚度：正對照組＞對照組＞毛木耳萃取液複方(A)組＞毛木耳萃取液複方(B)組，正對照組與各試驗組及正常皮膚相比具顯著差異(p＜0.05)，對照組、毛木耳萃取液複方(A)組與毛木耳萃取液複方(B)組彼此間未達顯著差異(p＞0.05)。The statistical analysis of Experimental Example 2 of the present invention combined with Table 4 shows that the thickness of the epidermis of each test group is: Auricularia auriculariae extract compound (A) group > Auricularia auriculariae extract compound (B) group > control group > control group. There was no significant difference between the test groups (p>0.05). Compared with normal skin, only the epidermis thickness of the Auricularia auriculariae extract compound (A) group showed a significant difference (p<0.05). The thickness of the dermis layer of each test group was as follows: control group > control group > Auricularia auriculariae extract compound (A) group > Auricularia auriculariae extract compound (B) group. The control group had significant differences compared with each test group and normal skin (p < 0.05). There was no significant difference between the control group, Auricularia auriculariae extract compound (A) group and Auricularia auriculariae extract compound (B) group (p > 0.05).
圖5A至圖5D依序顯示了實驗實施例2的對照組、毛木耳萃取液複方(A)組、毛木耳萃取液複方(B)組、正對照組在試驗第28天時的各組皮膚組織病理切片；其中，由圖5A至圖5D可知，各試驗組皆可觀察到新生上皮。毛木耳萃取液複方(A)組及正對照組可觀察到新生血管。對照組可觀察到上皮化。對照組、毛木耳萃取液複方(A)組及毛木耳萃取液複方(B)組皆可觀察到新生表皮層。Figures 5A to 5D show the pathological sections of the skin tissue of the control group, the Auricularia auriculariae extract compound (A) group, the Auricularia auriculariae extract compound (B) group, and the positive control group of Experimental Example 2 on the 28th day of the experiment; wherein, as can be seen from Figures 5A to 5D, new epithelium can be observed in each experimental group. New blood vessels can be observed in the Auricularia auriculariae extract compound (A) group and the positive control group. Epithelialization can be observed in the control group. New epidermis can be observed in the control group, the Auricularia auriculariae extract compound (A) group, and the Auricularia auriculariae extract compound (B) group.
本發明實驗實施例2於試驗第28天的上皮化（Epithelization）、血管新生（New blood capillary）、炎症反應（Inflammation）、纖維母細胞（Fibroblast）的皮膚組織病理切片的統計分析顯示，各試驗組皮膚再上皮化程度與血管新生程度無顯著差異。炎症細胞浸潤反應之病理評分，毛木耳萃取液複方(A)組與毛木耳萃取液複方(B)組顯著低於對照組與正對照組(p＜0.05)，毛木耳萃取液複方(A)組與毛木耳萃取液複方(B)組彼此間無顯著差異(p＞0.05)，正對照組與對照組彼此間無顯著差異(p＞0.05)。纖維母細胞病理評分，正對照組、毛木耳萃取液複方(A)組與毛木耳萃取液複方(B)組顯著較對照組高(p＜0.05)，毛木耳萃取液複方(A)組與毛木耳萃取液複方(B)組與正對照組彼此間無顯著差異(p＞0.05) 。Statistical analysis of the skin tissue pathological sections of epithelialization, new blood capillary, inflammation, and fibroblasts in Experimental Example 2 of the present invention on the 28th day of the experiment showed that there was no significant difference in the degree of skin re-epithelialization and new blood capillary among the experimental groups. The pathological scores of inflammatory cell infiltration reaction in the Auricularia auricula extract compound (A) group and the Auricularia auricula extract compound (B) group were significantly lower than those in the control group and the positive control group (p < 0.05), and there was no significant difference between the Auricularia auricula extract compound (A) group and the Auricularia auricula extract compound (B) group (p > 0.05), and there was no significant difference between the positive control group and the control group (p > 0.05). The fibroblast pathological scores of the control group, Auricularia auriculariae extract compound (A) group and Auricularia auriculariae extract compound (B) group were significantly higher than those of the control group (p＜0.05), while there was no significant difference between the Auricularia auriculariae extract compound (A) group and the Auricularia auriculariae extract compound (B) group and the control group (p＞0.05).
綜上，本發明實驗實施例2利用正常豬隻模式探討試驗物質對於全皮層傷口癒合功效，其癒合速率以正對照組組最佳、其次為對照組、毛木耳萃取液複方(A)組、最後為毛木耳萃取液複方(B)組。換言之，在實驗實施例2中，毛木耳蒂頭水萃液促進傷口癒合的效果較優於毛木耳子實體水萃液。In summary, Experimental Example 2 of the present invention uses a normal pig model to explore the efficacy of the test substance on the healing of full-thickness wounds. The healing rate is best in the positive control group, followed by the control group, the Auricularia auricula extract compound (A) group, and finally the Auricularia auricula extract compound (B) group. In other words, in Experimental Example 2, the effect of the Auricularia auricula pedicle water extract on promoting wound healing is better than that of the Auricularia auricula fruit body water extract.
實驗實施例3：毛木耳萃取液複方促進糖尿病豬隻傷口癒合之試驗。Experimental Example 3: Experiment on promoting wound healing in diabetic pigs with the compound extract of Auricularia auricula.
試驗設計：Experimental design:
動物品系為藍瑞斯豬。豬隻週齡/體重為2-3月齡/28~30 kg。豬隻性別/隻數為公豬3頭。動物模式為糖尿病豬：試驗豬隻以120 mg/kg 之鏈脲佐菌素（Streptozotocin）經耳靜脈注射後誘導為糖尿病；當試驗豬隻空腹血糖高於250 mg/dL，即為糖尿病豬。試驗分組為隨機分組，每隻背部開創6個傷口。傷口開創為全皮層的方形創傷 （2±0.5公分 × 2 ±0.5公分），左右各 3個；傷口編號，左邊自頭部至尾部依序標示為L1、L2、L3，右邊自頭部至尾部依序標示為R1、R2、R3。皮膚採樣的面積為3±0.5公分 × 3 ±0.5公分，並浸泡於10% （v/v）中性福馬林液中固定。統計方式為(1)傷口癒合速率：各試驗組樣本數不同，利用 One Way ANOVA 的 Scheffe事後檢定，以及(2)組織病理切片：因以程度比較各試驗組，利用無母數Mann-Whitney 進行檢定。其中，具有顯著差異表示為 p＜ 0.05、非常顯著差異表示為 p＜ 0.01、或者極顯著性差異表示為 p＜ 0.001。 Animal strains were Blue Ridge pigs. The age and weight of the pigs were 2-3 months old and 28-30 kg. The sex and number of pigs were 3 boars. The animal model was diabetic pigs: the experimental pigs were induced to have diabetes by injecting 120 mg/kg of streptozotocin through the ear vein; when the fasting blood glucose of the experimental pigs was higher than 250 mg/dL, they were diabetic pigs. The experimental groups were randomly divided, and 6 wounds were made on the back of each pig. The wounds were square wounds (2±0.5 cm × 2±0.5 cm) with three wounds on each side. The wound numbers were L1, L2, L3 from the head to the tail on the left side and R1, R2, R3 from the head to the tail on the right side. The skin samples were fixed in 10% (v/v) neutral formalin solution. The statistical methods were (1) wound healing rate: the number of samples in each test group was different, and the Scheffe post hoc test was used for one-way ANOVA, and (2) tissue pathological sections: the Mann-Whitney test was used to compare the degree of each test group. Here, a significant difference was indicated as p < 0.05, a very significant difference was indicated as p < 0.01, and an extremely significant difference was indicated as p < 0.001.
於本發明實驗實施例3中，糖尿病豬隻傷口癒合試驗為期共34天。其中，在試驗開始前進行豬隻誘導進入糖尿病模式；誘導後於第1、2、3天量測試驗豬隻空腹血糖值，以監控血糖值變化；接著，進行糖尿病模式誘導監控時間：共14天，於誘導後第7、14天量測一次空腹血糖值，監控血糖值變化。完成後糖尿病模式誘導後，在傷口癒合試驗的第0天進行切割傷開創、描繪傷口面積、拍照與量測空腹血糖，在第1、3、5、7、10、14、21天進行換包紮、描繪傷口面積與拍照，其中，第7、14、21、28天還包括量測空腹血糖，最後在第34天進行描繪傷口面積、拍照、採樣全皮層皮膚組織。完成前述流程後進行實驗實施例3的傷口癒合速率計算及組織病理切片分析。In Experimental Example 3 of the present invention, the diabetic pig wound healing test lasted for a total of 34 days. Before the start of the test, the pigs were induced to enter the diabetic mode; after induction, the fasting blood glucose levels of the test pigs were measured on the 1st, 2nd, and 3rd days to monitor the changes in blood glucose levels; then, the diabetic mode induction monitoring time was carried out for a total of 14 days, and the fasting blood glucose levels were measured once on the 7th and 14th days after induction to monitor the changes in blood glucose levels. After the induction of the post-diabetes model, the wound was cut, the wound area was drawn, photographed and fasting blood glucose was measured on day 0 of the wound healing test, the bandage was changed, the wound area was drawn and photographed on days 1, 3, 5, 7, 10, 14 and 21, and fasting blood glucose was measured on days 7, 14, 21 and 28. Finally, the wound area was drawn, photographed and the whole dermis skin tissue was sampled on day 34. After completing the above process, the wound healing rate calculation and tissue pathological section analysis of Experimental Example 3 were performed.
其中，其中，傷口癒合速率計算，是在各時間點描繪傷口面積後，以Image J軟體分析計算傷口癒合面積，再計算傷口癒合百分比（wound healing rate, %）。傷口癒合百分比% = [（第0天傷口面積－第N天傷口面積） / 第0天傷口面積] × 100%。Among them, the wound healing rate was calculated by drawing the wound area at each time point, analyzing and calculating the wound healing area with Image J software, and then calculating the wound healing rate (%). Wound healing rate % = [(wound area on day 0 - wound area on day N) / wound area on day 0] × 100%.
其中，組織病理切片進行蘇木精-伊紅染色（H&E stain）後，以光學顯微鏡進行組織病理學分析上皮化（Epithelization）、炎症反應（Inflammation）、血管新生（New blood capillary）、纖維母細胞（Fibroblast）、表皮層厚度（Epidermis thickness）、真皮層厚度（Dermis thickness）。Among them, the tissue pathological sections were stained with hematoxylin and eosin (H&E stain), and then the histopathological analysis of epithelialization, inflammation, new blood capillary, fibroblast, epidermis thickness, and dermis thickness was performed under an optical microscope.
圖6顯示了正常豬隻在試驗期間的體重變化，以開創第0天的數值點為準，由上往下依序表示豬隻編號061、062、063的體重曲線。由圖6可知，各組試驗物質皆不影響豬隻生長且無造成死亡情況。Figure 6 shows the weight changes of normal pigs during the trial period. Taking the value point on the 0th day as the reference, the weight curves of pigs No. 061, 062, and 063 are shown from top to bottom. As shown in Figure 6, all the test substances did not affect the growth of pigs and did not cause mortality.
圖7顯示了糖尿病豬隻的傷口癒合試驗，其是利用one-way ANOVA進行統計分析，以Scheffe事後檢定法比較不同試驗物質於各時間點之傷口癒合速率，圖7在時點第0、1、3、5、7、10、14、21、28、34天進行檢測，每一時點的四個長條數據由左而右依序為對照組、毛木耳萃取液複方(A)組、毛木耳萃取液複方(B)組、正對照組。由圖7可知，在傷口癒合中期（第7、10、14天），對照組與正對照組的傷口癒合速率皆顯著高於毛木耳萃取液複方(A)組及毛木耳萃取液複方(B)組(p＜0.05)。Figure 7 shows the wound healing test of diabetic pigs, which was statistically analyzed using one-way ANOVA, and the wound healing rates of different test substances at different time points were compared using the Scheffe post hoc test. Figure 7 was tested at time points 0, 1, 3, 5, 7, 10, 14, 21, 28, and 34 days. The four long bars of data at each time point are from left to right: control group, Auricularia auriculariae extract compound (A) group, Auricularia auriculariae extract compound (B) group, and positive control group. As shown in Figure 7, in the middle stage of wound healing (days 7, 10, and 14), the wound healing rates of the control group and the positive control group were significantly higher than those of the Auricularia auriculariae extract compound (A) group and the Auricularia auriculariae extract compound (B) group (p < 0.05).
於本發明實驗實施例3中進行糖尿病豬隻的傷口分泌物記錄。在第10天時，可觀察到對照組的肉芽明顯隆起，未見組織液分泌；毛木耳萃取液複方(A)組的肉芽明顯隆起，但未將傷口完全閉合，有少量組織液蓄積；毛木耳萃取液複方(B)組的肉芽明顯隆起，但未將傷口完全閉合，有少量組織液蓄積；正對照組的肉芽已隆起並覆蓋傷口，未見組織液分泌。In Experimental Example 3 of the present invention, wound secretions of diabetic pigs were recorded. On the 10th day, it was observed that the granulation tissue of the control group was obviously raised, and no tissue fluid secretion was observed; the granulation tissue of the Auricularia auriculariae extract compound (A) group was obviously raised, but the wound was not completely closed, and a small amount of tissue fluid was accumulated; the granulation tissue of the Auricularia auriculariae extract compound (B) group was obviously raised, but the wound was not completely closed, and a small amount of tissue fluid was accumulated; the granulation tissue of the control group had raised and covered the wound, and no tissue fluid secretion was observed.
於本發明實驗實施例3中進行糖尿病的傷口癒合變化記錄。在第34天時，可觀察到對照組的疤痕面積縮小，但顏色偏暗紅色；毛木耳萃取液複方(A)組的疤疤痕面積縮小，但顏色偏暗紅色，且有少許開放傷口；毛木耳萃取液複方(B)組的疤痕面積縮小，但顏色偏暗紅色，且有少許開放傷口；正對照組的疤痕面積與顏色較其他組小，顏色與正常皮膚相近。In Experimental Example 3 of the present invention, wound healing changes of diabetes were recorded. On the 34th day, it was observed that the scar area of the control group was reduced, but the color was dark red; the scar area of the Auricularia auricula extract compound (A) group was reduced, but the color was dark red, and there were a few open wounds; the scar area of the Auricularia auricula extract compound (B) group was reduced, but the color was dark red, and there were a few open wounds; the scar area and color of the control group were smaller than those of the other groups, and the color was similar to normal skin.
表5：
本發明實驗實施例3的統計分析結合表5，顯示各試驗組皮膚表皮層厚度：正對照組＞對照組＞毛木耳萃取液複方(A)組＞毛木耳萃取液複方(B)組，但各試驗組間未達顯著差異(p＞0.05)，與正常皮膚相比，僅正對照組表皮層厚度達顯著差異(p＜0.05)。各試驗組皮膚真皮層厚度：正對照組＞對照組＞毛木耳萃取液複方(A)組＞毛木耳萃取液複方(B)組，正對照組與各試驗組及正常皮膚相比具顯著差異(p＜0.05)，對照組、毛木耳萃取液複方(A)組與毛木耳萃取液複方(B)組彼此間未達顯著差異(p＞0.05)。The statistical analysis of Experimental Example 3 of the present invention combined with Table 5 shows that the thickness of the epidermis of each test group is: positive control group > control group > Auricularia auriculariae extract compound (A) group > Auricularia auriculariae extract compound (B) group, but there is no significant difference between the test groups (p>0.05). Compared with normal skin, only the epidermis thickness of the positive control group has a significant difference (p<0.05). The thickness of the dermis layer of each test group was as follows: control group > control group > Auricularia auriculariae extract compound (A) group > Auricularia auriculariae extract compound (B) group. The control group had significant differences compared with each test group and normal skin (p < 0.05). There was no significant difference between the control group, Auricularia auriculariae extract compound (A) group and Auricularia auriculariae extract compound (B) group (p > 0.05).
本發明實驗實施例3於試驗第34天的上皮化（Epithelization）、血管新生（New blood capillary）、炎症反應（Inflammation）、纖維母細胞（Fibroblast）的皮膚組織病理切片的統計分析顯示，各試驗組皮膚再上皮化程度與血管新生程度之病理評分，各試驗組間無顯著差異。各試驗組皮膚的炎症細胞浸潤反應之病理評分，毛木耳萃取液複方(A)組與毛木耳萃取液複方(B)組顯著低於正對照組( p＜0.05)，毛木耳萃取液複方B組顯著低於對照組( p＜0.05)毛木耳萃取液複方(A)組與毛木耳萃取液複方(B)組彼此間無顯著差異( p＞0.05)，正對照組與對照組彼此間無顯著差異( p＞0.05)。各試驗組皮膚的纖維母細胞病理評分，毛木耳萃取液複方(A)組顯著較對照組高( p＜0.05)，毛木耳萃取液複方(A)組、毛木耳萃取液複方(B)組與正對照組彼此間無顯著差異( p＞0.05) 。 The statistical analysis of the skin tissue pathological sections of epithelialization, new blood capillary, inflammation, and fibroblasts on the 34th day of the experiment in Experimental Example 3 of the present invention showed that there was no significant difference in the pathological scores of the skin re-epithelialization degree and new blood capillary degree among the experimental groups. The pathological scores of inflammatory cell infiltration reaction in the skin of each test group were significantly lower in the Auricularia auriculariae extract compound (A) group and the Auricularia auriculariae extract compound (B) group than in the control group ( p < 0.05), and significantly lower in the Auricularia auriculariae extract compound B group than in the control group ( p < 0.05). There was no significant difference between the Auricularia auriculariae extract compound (A) group and the Auricularia auriculariae extract compound (B) group ( p > 0.05), and there was no significant difference between the control group and the control group ( p > 0.05). The fibroblast pathological scores of the skin of each test group were significantly higher in the Auricularia auriculariae extract compound (A) group than in the control group ( p ＜0.05), and there was no significant difference between the Auricularia auriculariae extract compound (A) group, the Auricularia auriculariae extract compound (B) group and the control group ( p ＞0.05).
綜上，本發明實驗實施例3利用糖尿病豬隻模式探討試驗物質對於全皮層傷口癒合功效，其癒合速率以正對照組組最佳、其次為對照組、毛木耳萃取液複方(B)組、最後為毛木耳萃取液複方(A)組。本發明實驗實施例3中，毛木耳子實體水萃液促進傷口癒合的效果較優於毛木耳蒂頭水萃液，但由圖7可知，兩組在第34天時傷口癒合速率相差極小。In summary, Experimental Example 3 of the present invention uses a diabetic pig model to explore the efficacy of the test substance on full-thickness wound healing. The healing rate is best in the positive control group, followed by the control group, the Auricularia auricula extract compound (B) group, and finally the Auricularia auricula extract compound (A) group. In Experimental Example 3 of the present invention, the effect of the Auricularia auricula fruit body water extract on promoting wound healing is better than that of the Auricularia auricula pedicle water extract, but as shown in Figure 7, the wound healing rates of the two groups on the 34th day are very different.
實驗實施例4：毛木耳多醣凍乾物促進糖尿病豬隻傷口癒合試驗。Experimental Example 4: Experiment on promoting wound healing in diabetic pigs with Auricularia auricularia polysaccharide lyophilized product.
試驗設計：Experimental design:
動物品系為藍瑞斯豬。豬隻週齡/體重為2-3月齡/25-35 kg。豬隻性別/隻數為公豬共4頭，誘導糖尿病後，傷口開創後隨機分組。試驗豬隻每隻背部開創6個傷口（共24個），按照表6隨機分為5組，每組n=3~4；傷口開創為全皮層的方形創傷 （2±0.5公分 × 2 ±0.5公分），左右各 3個；傷口編號，左邊為L1、L2、L3，右邊為R1、R2、R3。皮膚採樣的面積為3±0.5公分 × 3 ±0.5公分，並浸泡於10% （v/v）中性福馬林液中固定。統計方式為(1)傷口癒合速率：各試驗組樣本數不同，利用 One Way ANOVA 的 Scheffe事後檢定，以及(2)組織病理切片：因以程度比較各試驗組，利用無母數Mann-Whitney 進行檢定。其中，具有顯著差異表示為 p＜ 0.05、非常顯著差異表示為 p＜ 0.01、或者極顯著性差異表示為 p＜ 0.001。本發明實驗實施例4的試驗分組包括正常豬隻的A組至E組以及糖尿病豬的A組至E組，A組至E組試驗物質投予方式如下表6所示。 Animal strains were Blue Race pigs. The age/weight of the pigs was 2-3 months/25-35 kg. The sex/number of pigs was 4 boars. After diabetes was induced, the wounds were made and randomly divided into groups. Six wounds were made on the back of each experimental pig (a total of 24 wounds), and they were randomly divided into 5 groups according to Table 6, with n=3~4 in each group; the wounds were square wounds of full skin thickness (2±0.5 cm×2±0.5 cm), 3 on each side; the wound numbers were L1, L2, L3 on the left and R1, R2, R3 on the right. The skin samples were collected from an area of 3 ± 0.5 cm × 3 ± 0.5 cm and fixed in 10% (v/v) neutral formalin. The statistical methods were (1) wound healing rate: the number of samples in each test group was different, and the Scheffe post hoc test was used for One Way ANOVA, and (2) tissue pathological sections: because the degree of comparison was between each test group, the Mann-Whitney test was used for the test. Among them, a significant difference was expressed as p < 0.05, a very significant difference was expressed as p < 0.01, or an extremely significant difference was expressed as p < 0.001. The test groups of Experimental Example 4 of the present invention include Groups A to E of normal pigs and Groups A to E of diabetic pigs. The administration methods of the test substances in Groups A to E are shown in Table 6 below.
表6：
於本發明實驗實施例4中，該毛木耳多醣凍乾物具體為毛木耳蒂頭水萃凍乾粉。該抗生素軟膏具體為安那膚軟膏，其係可用於人體的藥膏。其中，該瓊崖海棠種子油為成熟的瓊崖海棠果實種仁，經乾燥後通過物理或化學方法萃取而得。In Experimental Example 4 of the present invention, the Auricularia auricula polysaccharide lyophilized material is specifically Auricularia auricula pedicle water-extracted lyophilized powder. The antibiotic ointment is specifically Anafu ointment, which is an ointment that can be used on the human body. Among them, the Malus chinensis seed oil is obtained by drying the mature Malus chinensis fruit kernel and then extracting it by physical or chemical methods.
如圖8、圖9，分別顯示了正常豬隻、糖尿病豬隻在發炎期的傷口癒合速率。其中，圖8在時點第0、1、3、5、7天進行檢測，每一時點的四個長條數據由左而右依序為A-對照組、B-先投海棠萃取液/後投毛木耳多醣、C-先投毛木耳多醣/後投海棠萃取液、D-海棠萃取液與毛木耳多醣混合投予、E-正對照組，由圖8可知，正常豬隻的C組在發炎初期具有抗發炎症狀之能力，並有較低的發炎反應；在第7天時與正常豬隻的E組(正對照組)有類似之趨勢；正常豬隻的B組、D組的發炎反應較大，傷口觀察較為明顯腫脹。其中，圖9在時點第0、1、3、5、7、10天進行檢測，每一時點的四個長條數據由左而右依序為A-對照組、B-先投海棠萃取液/後投毛木耳多醣、C-先投毛木耳多醣/後投海棠萃取液、D-海棠萃取液與毛木耳多醣混合投予、E-正對照組，由圖9可知，糖尿病豬隻的C組於發炎初期具有抗發炎症狀之能力，並有較低的發炎反應，與糖尿病豬隻的E組(正對照組)有類似之趨勢；且糖尿病豬隻的D組在第7至10天時，傷口快速縮口(斜率為各組間最大)。As shown in Figures 8 and 9, the wound healing rates of normal pigs and diabetic pigs during the inflammatory period are shown respectively. Among them, Figure 8 was tested at time points 0, 1, 3, 5, and 7 days. The four long bars of data at each time point are A-control group, B-first administration of Malus extract/later administration of Auricularia auricula polysaccharide, C-first administration of Auricularia auricula polysaccharide/later administration of Malus extract, D-administration of Malus extract and Auricularia auricula polysaccharide mixed, and E-positive control group. As shown in Figure 8, group C of normal pigs has the ability to resist inflammatory symptoms in the early stage of inflammation and has a lower inflammatory response; on the 7th day, it has a similar trend to group E (positive control group) of normal pigs; the inflammatory response of groups B and D of normal pigs is larger, and the wound is more obviously swollen. Among them, Figure 9 was tested at time points 0, 1, 3, 5, 7, and 10 days. The four long bars of data at each time point are from left to right A-control group, B-first administration of Malus extract/later administration of Auricularia auricula polysaccharide, C-first administration of Auricularia auricula polysaccharide/later administration of Malus extract, D-mixed administration of Malus extract and Auricularia auricula polysaccharide, and E-positive control group. As shown in Figure 9, Group C of diabetic pigs has the ability to resist inflammatory symptoms at the early stage of inflammation and has a lower inflammatory response, which is similar to the trend of Group E of diabetic pigs (positive control group); and Group D of diabetic pigs had a rapid wound healing from 7 to 10 days (the slope was the largest among the groups).
於本發明實驗實施例4中進行所有試驗豬隻的傷口癒合變化記錄。觀察後可發現，糖尿病豬隻之傷口發炎期較正常豬隻更長，在第7天時各組傷口皆仍呈現腫脹的情形；從第10天開始各組炎症反應降低，且C組、D組、E組已開始快速增加癒合速度。此外，當投予之瓊崖海棠種子油體積減半，可有效的降低上次試驗出現之傷口肉芽組織過度增生（Overgranulation）的情形，使B組、D組的傷口較為平坦。In Experimental Example 4 of the present invention, wound healing changes of all test pigs were recorded. After observation, it was found that the wound inflammation period of diabetic pigs was longer than that of normal pigs. On the 7th day, the wounds of all groups were still swollen; from the 10th day, the inflammatory response of each group decreased, and the healing speed of Group C, Group D, and Group E began to increase rapidly. In addition, when the volume of the administered Malus chinensis seed oil was halved, the over-proliferation of wound granulation tissue that appeared in the previous test was effectively reduced, making the wounds of Group B and Group D flatter.
如圖10、圖11，分別顯示了正常豬隻、糖尿病豬隻在增生期的傷口癒合速率。其中，圖10在時點第0、1、3、5、7、10、14、18、21、24、28天進行檢測，每一時點的五個長條數據由左而右依序為A-對照組、B-先投海棠萃取液/後投毛木耳多醣、C-先投毛木耳多醣/後投海棠萃取液、D-海棠萃取液與毛木耳多醣混合投予、E-正對照組，由圖10可知，正常豬隻的C組於增生初期第10至18天時傷口快速癒合，肉芽較平整，其癒合速率亦為各組間最高；正常豬隻的B組、D組於第10天肉芽組織過度增生。其中，圖11在時點第0、1、3、5、7、10、14、18、21、24、28、34天進行檢測，每一時點的五個長條數據由左而右依序為A-對照組、B-先投海棠萃取液/後投毛木耳多醣、C-先投毛木耳多醣/後投海棠萃取液、D-海棠萃取液與毛木耳多醣混合投予、E-正對照組，由圖11可知，糖尿病豬隻的C組、D組於增生初期第14至18天傷口快速癒合，其癒合速率亦為各試驗組間最高；此外，糖尿病豬隻的E組於增生期，傷口快速縮口，為各組間最佳。As shown in Figures 10 and 11, the wound healing rates of normal pigs and diabetic pigs during the proliferative period are shown respectively. Among them, Figure 10 was tested at time points 0, 1, 3, 5, 7, 10, 14, 18, 21, 24, and 28 days. The five long bars of data at each time point are A-control group, B-first administration of Malus extract/then administration of Auricularia auricula polysaccharide, C-first administration of Auricularia auricula polysaccharide/then administration of Malus extract, D-administration of Malus extract and Auricularia auricula polysaccharide, and E-positive control group. As shown in Figure 10, the wounds of group C of normal pigs healed quickly from the 10th to the 18th day of the initial proliferation, the granulation tissue was relatively smooth, and its healing rate was also the highest among all groups; the granulation tissue of groups B and D of normal pigs overproliferated on the 10th day. Among them, Figure 11 was tested at time points 0, 1, 3, 5, 7, 10, 14, 18, 21, 24, 28, and 34 days. The five long bars of data at each time point are A-control group, B-first administration of Malus extract/then administration of Auricularia auricula polysaccharide, C-first administration of Auricularia auricula polysaccharide/then administration of Malus extract, D-administration of Malus extract and Auricularia auricula polysaccharide mixed, and E-positive control group. As shown in Figure 11, the wounds of diabetic pigs in groups C and D healed rapidly from the 14th to the 18th day of the early proliferation period, and their healing rate was also the highest among all the test groups. In addition, the wounds of diabetic pigs in group E healed rapidly during the proliferation period, which was the best among all the groups.
於本發明實驗實施例4中進行所有試驗豬隻的傷口癒合變化記錄。觀察後可發現，當D組減少一半的瓊崖海棠種子油投予體積，可有效降低肉芽組織過度增生之症狀，傷口癒合亦較為快速且平整，且癒合速率與C組類似，並於第24天優於C組。C組於增生期傷口修復較為平整，傷口快速增加癒合速率；且改投予瓊崖海棠種子油後，有助於傷口癒合。再者，A組（對照組）僅以赫麗敷水凝膠手術傷口敷料覆蓋，於第14至24天與各組間比較，顯示最低之傷口癒合速率之水平。In Experimental Example 4 of the present invention, wound healing changes of all test pigs were recorded. After observation, it was found that when the volume of the Malus chinensis seed oil administered to Group D was reduced by half, the symptoms of excessive granulation tissue proliferation were effectively reduced, and the wound healing was also faster and smoother, and the healing rate was similar to that of Group C, and was better than that of Group C on the 24th day. The wound repair of Group C was smoother during the proliferation period, and the wound healing rate increased rapidly; and after the administration of Malus chinensis seed oil, the wound healing was helpful. Furthermore, Group A (control group), which was only covered with Heli Hydrogel Surgical Wound Dressing, showed the lowest level of wound healing rate compared with other groups from day 14 to day 24.
如圖12、圖13，分別顯示了正常豬隻、糖尿病豬隻在重塑期的傷口癒合速率。其中，圖12在時點第0、1、3、5、7、10、14、18、21、24、28天進行檢測，每一時點的五個長條數據由左而右依序為A-對照組、B-先投海棠萃取液/後投毛木耳多醣、C-先投毛木耳多醣/後投海棠萃取液、D-海棠萃取液與毛木耳多醣混合投予、E-正對照組，由圖12可知，正常豬隻於重塑期第21至28天，除了D組與其他組具有統計差異，其他組間不具統計差異；各組傷口癒合速度趨於平緩，此期趨勢與組織增生期類似，依序為C組＞A組＞E組＞B組＞D組。其中，圖13在時點第0、1、3、5、7、10、14、18、21、24、28、34天進行檢測，每一時點的五個長條數據由左而右依序為A-對照組、B-先投海棠萃取液/後投毛木耳多醣、C-先投毛木耳多醣/後投海棠萃取液、D-海棠萃取液與毛木耳多醣混合投予、E-正對照組，由圖13可知，糖尿病豬隻各組縮口速度趨於平緩，傷口癒合速率趨勢與組織增生期類似，E組（正對照組）＞C組＞D組＞B組＞A組（對照組）。As shown in Figures 12 and 13, the wound healing rates of normal pigs and diabetic pigs during the remodeling period are shown respectively. Among them, Figure 12 was tested at time points 0, 1, 3, 5, 7, 10, 14, 18, 21, 24, and 28 days. The five long bars of data at each time point are A-control group, B-first administration of Malus extract/then administration of Auricularia auricula polysaccharide, C-first administration of Auricularia auricula polysaccharide/then administration of Malus extract, D-administration of Malus extract and Auricularia auricula polysaccharide mixed, and E-positive control group. As shown in Figure 12, in the remodeling period from 21 to 28 days, normal pigs had no statistical differences between the other groups except that the D group had statistical differences with the other groups; the wound healing speed of each group tended to be stable, and the trend of this period was similar to the tissue proliferation period, in the order of C group>A group>E group>B group>D group. Among them, Figure 13 was tested at time points 0, 1, 3, 5, 7, 10, 14, 18, 21, 24, 28, and 34 days. The five long bars of data at each time point are from left to right A-control group, B-first administration of Malus extract/later administration of Auricularia auricula polysaccharide, C-first administration of Auricularia auricula polysaccharide/later administration of Malus extract, D-mixed administration of Malus extract and Auricularia auricula polysaccharide, and E-positive control group. As shown in Figure 13, the mouth shrinkage rate of each group of diabetic pigs tends to be stable, and the trend of wound healing rate is similar to the tissue proliferation period, Group E (positive control group) > Group C > Group D > Group B > Group A (control group).
本發明實驗實施例4的試驗豬隻的傷口癒合變化記錄顯示，各組於重塑期第28至34天，仍有開放性傷口。其中，E組於重塑期，已有傷口完全閉口，第34天傷口癒合速率已達98.85±0.68%。但可能因乳膏劑型被赫麗敷水凝膠敷料封閉住，導致豬皮產生過敏反應。其中，C組、D組兩組較為接近，第34天傷口癒合速率分別為94.07±0.53、93.63 ±0.58。其中，A組、B組則為較差之兩組，第34天傷口癒合速率分別為89.94±0.78、90.84±1.81。The wound healing changes of the test pigs in Experimental Example 4 of the present invention showed that each group still had open wounds from the 28th to the 34th day of the remodeling period. Among them, the wound of Group E was completely closed during the remodeling period, and the wound healing rate on the 34th day reached 98.85±0.68%. However, it may be because the cream dosage form was sealed by the Heli hydrogel dressing, causing an allergic reaction in the pig skin. Among them, Group C and Group D were relatively close, and the wound healing rates on the 34th day were 94.07±0.53 and 93.63±0.58, respectively. Among them, Group A and Group B were the two poorer groups, and the wound healing rates on the 34th day were 89.94±0.78 and 90.84±1.81, respectively.
綜上，本發明實驗實施例4中，從B、D組傷口觀察顯示本次投予之瓊崖海棠種子油體積減半，可有效的降低上次試驗出現之傷口肉芽組織過度增生（Overgranulation）的情形。發炎期期間，C組單一投予木耳多醣凍乾物可有效降低發炎反應，且可達到與正對照組相似之水平，顯示木耳多醣凍乾物可能具有減緩發炎之功效。發炎期期間，C組單一投予木耳多醣凍乾物可有效降低發炎反應，且可達到與正對照組相似之水平，顯示木耳多醣凍乾物可能具有減緩發炎之功效。從重塑期結果顯示，傷口癒合速率E組(正對照組)＞C組＞D組＞B組＞A組(對照組)。In summary, in Experimental Example 4 of the present invention, observations from the wounds of Groups B and D showed that the volume of the Malus chinensis seed oil administered this time was halved, which can effectively reduce the over-proliferation of wound granulation tissue that occurred in the previous test. During the inflammatory period, the administration of auricularia polysaccharide lyophilized material to Group C alone can effectively reduce the inflammatory response, and can reach a level similar to that of the positive control group, indicating that auricularia polysaccharide lyophilized material may have the effect of alleviating inflammation. During the inflammatory period, the administration of auricularia polysaccharide lyophilized material to Group C alone can effectively reduce the inflammatory response, and can reach a level similar to that of the positive control group, indicating that auricularia polysaccharide lyophilized material may have the effect of alleviating inflammation. The results of the remodeling period showed that the wound healing rate was group E (positive control group) > group C > group D > group B > group A (control group).
實驗實施例5：Experimental Example 5:
試驗設計：Experimental design:
動物品系為藍瑞斯豬。豬隻週齡/體重為2-3月齡/25~35 kg。豬隻性別/隻數為公豬共20頭（每組4頭，共五組）。動物模式為正常豬，無模式誘發。試驗豬隻每隻背部開創6個傷口。傷口開創為全皮層的方形創傷 （2±0.5公分 × 2 ±0.5公分），左右各 3個；傷口編號，左邊為L1、L2、L3，右邊為R1、R2、R3。皮膚採樣的面積為3±0.5公分 × 3 ±0.5公分，並浸泡於10% （v/v）中性福馬林液中固定。統計方式為(1)傷口癒合速率：各試驗組樣本數不同，利用 One Way ANOVA 的 Scheffe事後檢定，以及(2)組織病理切片：因以程度比較各試驗組，利用無母數Mann-Whitney 進行檢定。其中，具有顯著差異表示為 p＜ 0.05、非常顯著差異表示為 p＜ 0.01、或者極顯著性差異表示為 p＜ 0.001。 Animals were Lanrui pigs. The age and weight of the pigs were 2-3 months old and 25-35 kg. The sex and number of pigs were 20 boars (4 in each group, a total of five groups). The animal model was normal pigs without model induction. Six wounds were made on the back of each experimental pig. The wounds were square wounds of full thickness (2±0.5 cm × 2 ±0.5 cm), 3 on each side; the wound numbers were L1, L2, L3 on the left and R1, R2, R3 on the right. The skin sampling area was 3±0.5 cm × 3 ±0.5 cm, and was fixed in 10% (v/v) neutral formalin solution. The statistical methods were (1) wound healing rate: Since the number of samples in each test group was different, the Scheffe post hoc test was used for One-Way ANOVA, and (2) tissue pathological sections: Since the degree of comparison was made between the test groups, the Mann-Whitney test was used. Among them, a significant difference was indicated as p < 0.05, a very significant difference was indicated as p < 0.01, or an extremely significant difference was indicated as p < 0.001.
如下表7，說明本發明實驗實施例5，各組試驗物質投予方式。Table 7 below illustrates the administration method of each group of test substances in Experimental Example 5 of the present invention.
表7：
於本發明實驗實施例5中，該真菌多醣凍乾物具體為毛木耳蒂頭水萃凍乾粉；該去色素真菌多醣凍乾物具體為脫色毛木耳蒂頭水萃凍乾粉。該抗生素軟膏具體為安那膚軟膏，其係可用於人體的藥膏。其中，該瓊崖海棠種子油為成熟的瓊崖海棠果實種仁，經乾燥後通過物理或化學方法萃取而得。物理方法可利用機械壓榨獲得種子油，化學方法可利用醇類萃取獲得種子油。其中，該瓊崖海棠種子油為成熟的瓊崖海棠果實種仁，經乾燥後通過物理或化學方法萃取而得；物理方法可利用機械壓榨獲得種子油，化學方法可利用醇類萃取獲得種子油。其中，該瓊崖海棠種子油色素萃取液是由前述瓊崖海棠種子油，以醇類進一步萃取色素後獲得種子油色素萃取液。In Experimental Example 5 of the present invention, the fungal polysaccharide lyophilized material is specifically a water-extracted lyophilized powder of Auricularia auricula-derived pedicles; the depigmented fungal polysaccharide lyophilized material is specifically a decolorized water-extracted lyophilized powder of Auricularia auricula-derived pedicles. The antibiotic ointment is specifically an anaphylactic ointment, which is an ointment that can be used on the human body. Among them, the Qiongya Begonia seed oil is obtained by extracting the mature Qiongya Begonia fruit kernel through physical or chemical methods after drying. The physical method can obtain the seed oil by mechanical pressing, and the chemical method can obtain the seed oil by alcohol extraction. The seed oil of the Chinese hollyhock crabapple is obtained by physical or chemical extraction of mature Chinese hollyhock crabapple kernels after drying; the physical method can obtain the seed oil by mechanical pressing, and the chemical method can obtain the seed oil by alcohol extraction. The pigment extract of the Chinese hollyhock crabapple seed oil is obtained by further extracting the pigment from the Chinese hollyhock crabapple seed oil with alcohol.
於本發明實驗實施例5中，透過B組與C組，釐清若同時投予真菌多醣凍乾物，比較加入瓊崖海棠種子油是否有助於傷口癒合。透過B組與D組，釐清若同時投予真菌多醣凍乾物，比較加入瓊崖海棠種子油色素萃取液是否有助於傷口癒合。透過C組與D組，釐清若同時投予真菌多醣凍乾物，比較加入瓊崖海棠種子油與瓊崖海棠種子油色素萃取液何者對傷口癒合幫助較大。透過C組與E組，釐清若同時投予瓊崖海棠種子油，比較真菌多醣凍乾物去除色素前後對傷口癒合功效是否有差異。透過D組與F組，釐清若同時投予瓊崖海棠種子油色素萃取液，比較真菌多醣凍乾物去除色素前後對傷口癒合功效是否有差異。透過E組與F組，釐清若同時投予去色真菌多醣凍乾物，比較加入瓊崖海棠種子油與瓊崖海棠種子油色素萃取液何者對傷口癒合幫助較大。In Experimental Example 5 of the present invention, it was clarified by using Group B and Group C whether the addition of Malus chinensis seed oil would help wound healing if fungal polysaccharide lyophilized products were administered at the same time. It was clarified by using Group B and Group D whether the addition of Malus chinensis seed oil pigment extract would help wound healing if fungal polysaccharide lyophilized products were administered at the same time. It was clarified by using Group C and Group D whether the addition of Malus chinensis seed oil and Malus chinensis seed oil pigment extract would help wound healing more if fungal polysaccharide lyophilized products were administered at the same time. Through groups C and E, it was clarified whether there was any difference in the wound healing effect before and after the pigment removal by fungal polysaccharide lyophilized product if the Malus chinensis seed oil was administered at the same time. Through groups D and F, it was clarified whether there was any difference in the wound healing effect before and after the pigment removal by fungal polysaccharide lyophilized product if the pigment extraction liquid of Malus chinensis seed oil was administered at the same time. Through groups E and F, it was clarified whether the depigmented fungal polysaccharide lyophilized product was administered at the same time, and whether the addition of Malus chinensis seed oil or the pigment extraction liquid of Malus chinensis seed oil was more helpful for wound healing.
圖14顯示了本發明實驗實施例5的豬隻在試驗期間的體重變化，其中，符號●、■、▲、 依序表示豬隻編號033、035、037、039的體重曲線。由圖可知，各組試驗物質皆不影響豬隻生長且無造成死亡情況。 FIG. 14 shows the weight changes of the pigs in Experimental Example 5 of the present invention during the test period, wherein the symbols ●, ■, ▲, The weight curves of pigs No. 033, 035, 037, and 039 are shown in order. It can be seen from the figure that the test substances in each group did not affect the growth of pigs and did not cause mortality.
圖15顯示了本發明實驗實施例5的豬隻在試驗期間的血糖紀錄，其中，符號●、■、▲、 依序表示豬隻編號033、035、037、039的血糖紀錄。由圖可知，試驗豬隻空腹血糖高於250 mg/dL，皆屬於糖尿病豬隻。 FIG. 15 shows the blood glucose records of pigs in Experimental Example 5 of the present invention during the test period, wherein the symbols ●, ■, ▲, The blood sugar records of pigs No. 033, 035, 037, and 039 are shown in sequence. As can be seen from the figure, the fasting blood sugar of the test pigs is higher than 250 mg/dL, and they are all diabetic pigs.
圖16至圖18顯示了本發明實驗實施例5中，豬隻在發炎期（圖16）、增生期（圖17）及重塑期（圖18）的傷口癒合速率（%）。其中，圖16在屬於發炎期的時點第0、1、3、5、7天進行傷口檢測，圖17在屬於增生期的時點第14、18、21天進行傷口檢測，圖18在屬於重塑期的時點第24、28、34天進行傷口檢測。圖16至圖18中，每一時點的七個長條數據由左而右依序為A-對照組、B-真菌多醣凍乾物組、C-真菌多醣凍乾物+種子萃取液組、D-真菌多醣凍乾物+種子色素萃取組、E-去色素真菌多醣凍乾物+種子萃取液組、F-去色素真菌多醣凍乾物+種子色素萃取組、G-正對照組。由圖可知，發炎期期間，添加去色素真菌多醣凍乾物組別（E組、F組）均減緩傷口發炎反應；增生期期間，第18天添加種子色素萃取組（D組、F組）對於傷口癒合效果較佳；重塑期期間，第28天時添加種子油色素萃取液組別（D、F組）較佳傷口癒合，第34天時F組傷口癒合率最佳。Figures 16 to 18 show the wound healing rates (%) of pigs in the inflammatory phase (Figure 16), the hyperplastic phase (Figure 17) and the remodeling phase (Figure 18) in Experimental Example 5 of the present invention. In particular, Figure 16 shows wound detection at the time points of 0, 1, 3, 5 and 7 days in the inflammatory phase, Figure 17 shows wound detection at the time points of 14, 18 and 21 days in the hyperplastic phase, and Figure 18 shows wound detection at the time points of 24, 28 and 34 days in the remodeling phase. In Figures 16 to 18, the seven bars of data at each time point are, from left to right, A-control group, B-fungal polysaccharide lyophilized material group, C-fungal polysaccharide lyophilized material + seed extract group, D-fungal polysaccharide lyophilized material + seed pigment extract group, E-depigmented fungal polysaccharide lyophilized material + seed extract group, F-depigmented fungal polysaccharide lyophilized material + seed pigment extract group, and G-positive control group. As can be seen from the figure, during the inflammatory phase, the groups that added depigmented fungal polysaccharide freeze-dried matter (Group E and Group F) both alleviated the wound inflammation response; during the proliferative phase, the groups that added seed pigment extract (Group D and Group F) on the 18th day had a better wound healing effect; during the remodeling phase, the groups that added seed oil pigment extract (Group D and Group F) on the 28th day had better wound healing, and the wound healing rate of Group F was the best on the 34th day.
以下請以表8、表9配合參閱圖19、圖20及圖21，說明本發明實驗實施例5的組織病理分析。The following Tables 8 and 9 are used in conjunction with Figures 19, 20 and 21 to illustrate the histopathological analysis of Experimental Example 5 of the present invention.
表8顯示了本發明實驗實施例5的表皮層及真皮層厚度，及表9顯示了組（A組至G組）的纖維母細胞評分。Table 8 shows the thickness of the epidermis and dermis of Experimental Example 5 of the present invention, and Table 9 shows the fibroblast scores of the groups (Group A to Group G).
表8：
由表8可知，添加種子色素萃液之組別(D組、F組)，表皮層厚度較其他組別厚，其真皮層厚度亦較厚，但組別間差異不大。As shown in Table 8, the epidermis thickness of the groups added with seed pigment extract (Group D and Group F) was thicker than that of the other groups, and the dermis thickness was also thicker, but the difference between the groups was not significant.
表9：
由表9可知，各組均有許多纖維母細胞之分佈。As shown in Table 9, there are many fibroblasts distributed in each group.
圖19顯示了表8、表9的A組至G組的傷口上皮化組織病理分析，圖19包括對應圖16至圖18的A組至G組的七個皮膚組織病理切片，以及A組至G組切片中表皮厚度的數據長條圖。由圖19可知，添加種子油色素萃取液的D組及F組有較明顯的皮根生長，顯示D組、F組表皮的生長較其他組別佳。Figure 19 shows the pathological analysis of wound epithelialization tissues of Groups A to G in Tables 8 and 9. Figure 19 includes seven skin tissue pathological sections of Groups A to G corresponding to Figures 16 to 18, and a bar graph of epidermal thickness in sections of Groups A to G. As shown in Figure 19, Groups D and F, which were added with seed oil pigment extract, had more obvious root growth, indicating that the growth of the epidermis in Groups D and F was better than that in other groups.
圖20顯示了表8、表9的A組至G組的炎症反應（多核巨細胞）組織病理分析，圖20包括對應圖16至圖18的A組至G組的七個組織病理切片。由圖20可知，添加去色素真菌多醣凍乾物之組別E組及F組有較少多核巨細胞，表示去色素真菌多醣凍乾物具延緩傷口發炎的功效。FIG20 shows the histopathological analysis of inflammatory response (multinucleated giant cells) of Groups A to G in Tables 8 and 9, and FIG20 includes seven histopathological sections of Groups A to G corresponding to FIG16 to FIG18. As shown in FIG20, Groups E and F, which were supplemented with depigmented fungal polysaccharide lyophilized material, had fewer multinucleated giant cells, indicating that depigmented fungal polysaccharide lyophilized material has the effect of delaying wound inflammation.
圖21顯示了表8、表9的A組至G組的血管新生狀況的組織病理分析，圖21包括對應圖16至圖18的A組至G組的七個組織病理切片。由圖21可知，各組血管新生無差異。Figure 21 shows the histopathological analysis of angiogenesis in Groups A to G in Tables 8 and 9, and Figure 21 includes seven histopathological sections corresponding to Groups A to G in Figures 16 to 18. As can be seen from Figure 21, there is no difference in angiogenesis among the groups.
無without
圖1是本發明實驗實施例1中毛木耳蒂頭水萃液對豬隻免疫細胞毒性測試數據圖； 圖2是本發明實驗實施例1中毛木耳蒂頭水萃液對健康小鼠腹腔巨噬細胞iNOS基因表現的影響數據圖； 圖3是本發明實驗實施例2的正常豬體重變化數據圖； 圖4是本發明實驗實施例2的正常豬傷口癒合試驗數據圖； 圖5A至圖5D是本發明驗實施例2在試驗第28天的皮膚組織病理切片； 圖6是本發明實驗實施例3的糖尿病豬體重變化數據圖； 圖7是本發明實驗實施例3的糖尿病豬傷口癒合試驗數據圖； 圖8是本發明實驗實施例4的正常豬在發炎期的傷口癒合速率； 圖9是本發明實驗實施例4的糖尿病豬在發炎期的傷口癒合速率； 圖10是本發明實驗實施例4的正常豬在增生期的傷口癒合速率； 圖11是本發明實驗實施例4的糖尿病豬在增生期的傷口癒合速率； 圖12是本發明實驗實施例4的正常豬在重塑期的傷口癒合速率； 圖13是本發明實驗實施例4的糖尿病豬在重塑期的傷口癒合速率； 圖14是本發明實驗實施例5的豬隻體重變化數據圖； 圖15是本發明實驗實施例5的豬隻血糖變化數據圖；； 圖16是本發明實驗實施例5的豬隻在發炎期的傷口癒合速率； 圖17是本發明實驗實施例5的豬隻在增生期的傷口癒合速率； 圖18是本發明實驗實施例5的豬隻在重塑期的傷口癒合速率； 圖19是本發明實驗實施例5的豬隻傷口再上皮化的組織病理分析圖； 圖20是本發明實驗實施例5的豬隻炎症反應（多核巨細胞）的組織病理分析圖； 圖21是本發明實驗實施例5的豬隻血管新生的組織病理分析圖。 Figure 1 is a data graph of the cytotoxicity test of Auricularia auricula water extract on pig immune cells in Experimental Example 1 of the present invention; Figure 2 is a data graph of the effect of Auricularia auricula water extract on the expression of iNOS gene in peritoneal macrophages of healthy mice in Experimental Example 1 of the present invention; Figure 3 is a data graph of the weight change of normal pigs in Experimental Example 2 of the present invention; Figure 4 is a data graph of the wound healing test of normal pigs in Experimental Example 2 of the present invention; Figures 5A to 5D are pathological sections of skin tissue on the 28th day of the test in Experimental Example 2 of the present invention; Figure 6 is a data graph of the weight change of diabetic pigs in Experimental Example 3 of the present invention; Figure 7 is a data graph of the wound healing test of diabetic pigs in Experimental Example 3 of the present invention; Figure 8 is the wound healing rate of normal pigs in the inflammatory period of Experimental Example 4 of the present invention; Figure 9 is the wound healing rate of diabetic pigs in the inflammatory period of Experimental Example 4 of the present invention; Figure 10 is the wound healing rate of normal pigs in the hyperplasia period of Experimental Example 4 of the present invention; Figure 11 is the wound healing rate of diabetic pigs in the hyperplasia period of Experimental Example 4 of the present invention; Figure 12 is the wound healing rate of normal pigs in the remodeling period of Experimental Example 4 of the present invention; Figure 13 is the wound healing rate of diabetic pigs in the remodeling period of Experimental Example 4 of the present invention; Figure 14 is a data graph of pig weight changes in Experimental Example 5 of the present invention; Figure 15 is a graph showing blood sugar changes in pigs in Experimental Example 5 of the present invention; Figure 16 is a graph showing the wound healing rate of pigs in Experimental Example 5 of the present invention during the inflammatory phase; Figure 17 is a graph showing the wound healing rate of pigs in Experimental Example 5 of the present invention during the hyperplasia phase; Figure 18 is a graph showing the wound healing rate of pigs in Experimental Example 5 of the present invention during the remodeling phase; Figure 19 is a graph showing the histopathological analysis of wound re-epithelialization in pigs in Experimental Example 5 of the present invention; Figure 20 is a graph showing the histopathological analysis of inflammatory response (multinucleated giant cells) in pigs in Experimental Example 5 of the present invention; Figure 21 is a histopathological analysis of pig angiogenesis in Experimental Example 5 of the present invention.
Claims (10)
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| Application Number | Priority Date | Filing Date | Title |
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| TW111133822A TWI846046B (en) | 2022-09-07 | Auricularia polytricha polyomycoid extract for preparing pharmaceutical components that promote wound healing |
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| TW111133822A TWI846046B (en) | 2022-09-07 | Auricularia polytricha polyomycoid extract for preparing pharmaceutical components that promote wound healing |
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| TW202410913A true TW202410913A (en) | 2024-03-16 |
| TWI846046B TWI846046B (en) | 2024-06-21 |
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