TW202319061A - Use of cordyceps cicadae mycelia active substance for preventing or curing macular degeneration - Google Patents
Use of cordyceps cicadae mycelia active substance for preventing or curing macular degeneration Download PDFInfo
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Abstract
Description
本發明關於一種蟬花菌絲體活性物質之用途,特別是指以蟬花菌絲體製備用以預防或治療黃斑部病變的組合物。The invention relates to the use of an active substance of cicada flower mycelium, in particular to a composition prepared by using cicada flower mycelium to prevent or treat macular degeneration.
老年性黃斑部病變(Age-related macular degeneration, AMD)是負責視力和色覺的視網膜黃斑部漸進退化性之疾病,主要是因中央視網膜感光接受器死亡所引起。位於感光層和脈絡膜之間的視網膜色素上皮(retinal pigment epithelium, RPE)細胞也與早期AMD的發病機制有關。如其名稱「老年性」所述,AMD的患病率隨著年齡的增長而逐漸增加,成為老年人失明的主要原因。Age-related macular degeneration (AMD) is a progressive degenerative disease of the macular part of the retina responsible for vision and color vision, mainly caused by the death of central retinal photoreceptors. The retinal pigment epithelium (RPE) cells located between the photoreceptor layer and the choroid are also involved in the pathogenesis of early AMD. As its name "senile" suggests, the prevalence of AMD gradually increases with age, becoming the leading cause of blindness in the elderly.
AMD 的發病機制與許多因素有關,如代謝紊亂,免疫,炎症,活性氧物質(reactive oxygen species;ROS)等。近年的研究顯示ROS 所造成的氧化壓力是AMD 的主要致病因子。在過去的幾十年中,用來治療 AMD的方法包括:(1)光凝固雷射 (laserphotocoagulation)、(2)經瞳熱療雷射 (transpupillary thermotherapy TTT)、(3)光動力療法 (photodynamictherapy PDT)、(4) 抗血管生成療法 (antiangiogenic therapy)、(5)營養補充療法、(6)基因治療、(7)抗氧化劑與(8)合併上述療法等,但手術療法具副作用,且術後失明率仍在增加。因此,開發具有較低毒性的新型治療劑對於AMD的預防或治療相當重要。The pathogenesis of AMD is related to many factors, such as metabolic disorders, immunity, inflammation, reactive oxygen species (reactive oxygen species; ROS) and so on. Recent studies have shown that oxidative stress caused by ROS is the main pathogenic factor of AMD. In the past few decades, the methods used to treat AMD include: (1) photocoagulation laser (laserphotocoagulation), (2) transpupillary thermotherapy (TTT), (3) photodynamic therapy (photodynamictherapy) PDT), (4) antiangiogenic therapy (antiangiogenic therapy), (5) nutritional supplement therapy, (6) gene therapy, (7) antioxidants and (8) combining the above therapies, etc., but surgical therapy has side effects, and surgery Blindness rates are still increasing. Therefore, the development of novel therapeutic agents with less toxicity is quite important for the prevention or treatment of AMD.
蟬花 (Cordyceps cicadae)為一種蟲生真菌,又名土蟬花、蟲花、蟬草、胡蟬、蟬菌、蟬蛹草、金蟬花、蟬茸或蠶茸等。蟬花為子囊菌亞門 (Ascomycotina)、麥角菌目 (Claricipiyales)、麥角菌科 (Clavicipitaceae)、蟲草屬 (Cordyceps)真菌,感染蟬蛹或蟬科山蟬 (Cicada flammate)、蟪蛄 (Platypleura kaempferi)、黑蚱 (Crytotympana pustulata)及竹蟬 (Platylomia pieli)等幼蟲使其死亡後,於蟬蛹前端或蟲體頭部形成花蕾狀子座而形成,故名蟬花。天然野生蟬花多產於長江以南熱帶和亞熱帶地區,在台灣部分山區亦有野生蟬花子實體蹤跡。 Cicadae (Cordyceps cicadae) is a kind of entomogenous fungus, also known as soil cicadae, insect flower, cicadae, cicada, cicadae, cicada pupa, cicadae, cicada or silkworm etc. Cicada is a fungus of Ascomycotina , Claricipiyales , Clavicipitaceae , Cordyceps , which infects cicada pupae or Cicada flammate , cicada ( Platypleura kaempferi) , black grasshopper (Crytotympana pustulata) and bamboo cicada (Platylomia pieli) and other larvae make them die, and form flower bud-like sub-seats at the front of the cicada pupae or the head of the insect body, hence the name cicadae. Natural wild cicadas are mostly produced in the tropical and subtropical regions south of the Yangtze River, and there are also traces of wild cicadas in some mountainous areas of Taiwan.
蟬花為名貴傳統中藥材,入藥已有一千多年歷史。現代藥理學實驗表明,蟬花及其人工培養物具有明顯的調節免疫、神經系統調節、抗疲勞、鎮靜、鎮痛解熱、改善腎功能、降血糖、降低血壓、減慢心率、抑制動脈粥樣硬化形成、抗腫瘤、抗輻射和滋補強身等功效。然而,目前並無研究指出蟬花對黃斑部病變具有療效。Cicada is a precious traditional Chinese medicinal material, which has been used as medicine for more than one thousand years. Modern pharmacological experiments have shown that cicadae and its artificial culture have obvious immune-regulating, nervous system regulation, anti-fatigue, sedative, analgesic, antipyretic, improving kidney function, lowering blood sugar, lowering blood pressure, slowing heart rate, and inhibiting atherosclerosis. Formation, anti-tumor, anti-radiation and nourishing and strengthening. However, there is no research to date that cicadae has a curative effect on macular degeneration.
本發明提供一種蟬花( Cordyceps cicadae)菌絲體活性物質的用途,其係用於製備預防或治療黃斑部病變之組合物。該蟬花菌絲體活性物質的製備方法包括下列步驟: The invention provides an application of the mycelia active substance of cicada ( Cordyceps cicadae) , which is used for preparing a composition for preventing or treating macular degeneration. The preparation method of the cicada flower mycelium active substance comprises the following steps:
(a)取一蟬花菌絲體於平板培養基上,於15-35℃之溫度下培養2-10天;(a) Take a cicadae mycelium on a plate culture medium and cultivate it at a temperature of 15-35° C. for 2-10 days;
( b)將步驟(a)培養後之蟬花菌絲體接種至一燒瓶內,於15-35℃、pH 2-8之環境培養3-7天;(b) Inoculate the cicadae mycelium cultivated in step (a) into a flask, and cultivate in an environment of 15-35°C and pH 2-8 for 3-7 days;
(c)將步驟(b)培養後之蟬花菌絲體接種於一發酵槽內,於15-35℃、pH 2-8之環境下攪拌培養3-5天,形成含有該蟬花菌絲體活性物質之一蟬花菌絲體發酵液。(c) inoculate the cicadae mycelium after step (b) cultivation in a fermenter, stir and cultivate for 3-5 days under the environment of 15-35° C. and pH 2-8, and form the cicadae mycelium containing the One of the body active substances cicadae mycelium fermentation broth.
一實施例中,製備該蟬花菌絲體活性物質的步驟更包括步驟(d):將該蟬花菌絲體發酵液冷凍乾燥後磨粉,形成含有該蟬花菌絲體活性物質之一蟬花菌絲體凍乾粉。In one embodiment, the step of preparing the active substance of the cicadae mycelium further includes a step (d): freeze-drying the fermented liquid of the cicadae mycelium and then pulverizing it to form one of the active substances containing the cicadae mycelium Cicada mycelium freeze-dried powder.
一實施例中,製備該蟬花菌絲體活性物質的步驟更包括步驟(e):將該蟬花菌絲體凍乾粉以一溶劑萃取,形成含有該蟬花菌絲體活性物質之一蟬花菌絲體萃取液。In one embodiment, the step of preparing the active substance of the cicada flower mycelium further includes step (e): extracting the freeze-dried powder of the cicada flower mycelium with a solvent to form one of the active substances containing the cicada flower mycelium Cicada mycelium extract.
一實施例中,製備該蟬花菌絲體活性物質的步驟更包括步驟(f):將該蟬花菌絲體萃取液乾燥,以獲得該蟬花菌絲體活性物質。In one embodiment, the step of preparing the active substance of the cicada flower mycelium further includes a step (f): drying the extract of the cicada flower mycelium to obtain the active substance of the cicada flower mycelium.
一實施例中,在步驟(e)中的溶劑為水。In one embodiment, the solvent in step (e) is water.
一實施例中,步驟(b)之燒瓶培養為震盪培養,且轉速為10-250 rpm。In one embodiment, the flask culture in step (b) is shaking culture, and the rotation speed is 10-250 rpm.
一實施例中,步驟(c)中該發酵槽係進一步通入一氣體,該氣體包括空氣、氧氣、二氧化碳、氦氣或其組合,該發酵槽的槽壓為0.5-1.0 kg/cm 2且通氣速率為0.01-1.5 VVM。 In one embodiment, the fermentation tank in step (c) is further fed with a gas, the gas includes air, oxygen, carbon dioxide, helium or a combination thereof, the tank pressure of the fermentation tank is 0.5-1.0 kg/cm 2 and The ventilation rate was 0.01-1.5 VVM.
一實施例中,該組合物為醫藥組合物,且該醫藥組合物進一步包含藥學上可接受之載劑、賦形劑、稀釋劑或輔劑。In one embodiment, the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.
一實施例中,該組合物為食品添加劑。In one embodiment, the composition is a food additive.
一實施例中,該組合物的施用方式為口服。In one embodiment, the composition is administered orally.
為使本發明之上述及其他方面更為清楚,下文特舉實施例,並配合所附圖式進行說明。In order to make the above and other aspects of the present invention more clear, the following specific embodiments are described together with the accompanying drawings.
蟬花菌絲體來源Cicada mycelium source
本實施例使用之蟬花( Cordyceps cicadae)菌絲體係由採集而得之台灣野生蟬花子實體,而非果實。子實體經分離而得其菌絲,並繼代於平板培養基上。此菌種經台灣食品工業發展研究所做鑑定證實基因序列為蟬花( Cordyceps cicadae),並已寄存於財團法人食品工業發展研究所之生物資源研究中心(BCRC),寄存編號為MU30106。此菌株亦寄存於中國普通微生物保藏管理中心,保藏標號為CGMCC No.10486。但本發明所述之蟬花菌絲體活性物質不限於由此菌種所得,亦可使用其他種類的蟬花菌株。 The mycelium system of the cicadae ( Cordyceps cicadae ) used in this example is collected from the fruiting bodies of wild cicadae in Taiwan, not the fruit. The fruiting bodies are separated to obtain their hyphae, and are subcultured on plate medium. The gene sequence of this strain was identified as Cordyceps cicadae by the Taiwan Institute of Food Industry Development, and it has been deposited in the Bioresource Research Center (BCRC) of the Institute of Food Industry Development, Taiwan, with deposit number MU30106. This strain is also deposited in the China General Microorganisms Collection and Management Center, and the preservation number is CGMCC No.10486. But the active substance of the cicadae mycelium described in the present invention is not limited to be obtained from this strain, and other kinds of cicadae strains can also be used.
菌絲體液體培養Mycelium liquid culture
將蟬花菌絲體接種於平板培養基上,於適當溫度15-35℃(較佳為25℃)下培養2-10天,刮取菌絲接種於燒瓶內。在15-35℃(較佳為25℃),pH 2-8(較佳為pH 4-7,更佳為pH 5.5),震盪速率10-250 rpm的條件下培養3-7天,然後將燒瓶培養物接種於發酵槽培養基(同燒瓶培養基)內,在15-35℃(較佳為25℃),槽壓0.5-2.0 kg/cm 2,pH 2-8,10-150 rpm攪拌速度或不攪拌(air lift)情況,以0.01-1.5 VVM通氣速率通入空氣、氧氣、二氧化碳、氮氣或上述氣體之混合物(較佳者為空氣),培養時間為2-10天內,即得蟬花菌絲體發酵液。此發酵液包括菌絲體與澄清液。前述培養條件僅為例示,使用者可視情況調整。 Inoculate the mycelia of cicadae on a plate culture medium, culture at an appropriate temperature of 15-35° C. (preferably 25° C.) for 2-10 days, scrape the mycelium and inoculate it in a flask. Cultivate for 3-7 days at 15-35°C (preferably 25°C), pH 2-8 (preferably pH 4-7, more preferably pH 5.5), shaking rate 10-250 rpm, and then The flask culture is inoculated in the fermenter medium (same as the flask medium), at 15-35°C (preferably 25°C), tank pressure 0.5-2.0 kg/cm 2 , pH 2-8, stirring speed 10-150 rpm or Without stirring (air lift), feed air, oxygen, carbon dioxide, nitrogen or a mixture of the above gases (preferably air) with a ventilation rate of 0.01-1.5 VVM, and the cultivation time is within 2-10 days to obtain cicadae. Mycelium Fermentation Broth. This fermented liquid includes mycelia and clarified liquid. The aforementioned culture conditions are only examples, and users can adjust them according to the situation.
本發明使用的燒瓶培養基、發酵槽培養基配方可如下:
其中該綜合性碳氮源可為穀類(如:麥粉類)或豆類(如:黃豆粉、綠豆粉、大豆粉等)。該無機鹽類可為硫酸鎂、磷酸氫二鉀、磷酸二氫鉀、硫酸鐵等。該醣類可為葡萄糖、果糖、麥芽糖、蔗糖等。特別說明的是,本發明使用的培養基並不限制為上述成份或比例,使用者可視實際情況進行調整。Wherein the comprehensive carbon and nitrogen source can be cereals (such as: wheat flour) or beans (such as: soybean flour, mung bean flour, soybean flour, etc.). The inorganic salts can be magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, iron sulfate and the like. The sugars can be glucose, fructose, maltose, sucrose and the like. In particular, the culture medium used in the present invention is not limited to the above-mentioned composition or ratio, and the user can adjust it according to the actual situation.
發酵液乾燥fermentation broth drying
此蟬花菌絲體發酵液可進一步藉由乾燥步驟製備為凍乾粉等其他劑型。乾燥方法包括但不限於:噴霧乾燥、熱風乾燥、滾筒乾燥、冷凍乾燥或其他方式。The cicadae mycelium fermentation broth can be further prepared into other dosage forms such as freeze-dried powder through a drying step. Drying methods include, but are not limited to: spray drying, hot air drying, drum drying, freeze drying or other methods.
凍乾粉萃取-水萃取Freeze-dried powder extraction-water extraction
取蟬花菌絲體凍乾粉加入20倍體積之蒸餾水溶解,以溫度100℃加熱30分鐘,待冷卻後經冷凍乾燥法進行乾燥,得蟬花菌絲體水萃物。Take the freeze-dried powder of cicada mycelium and add 20 times the volume of distilled water to dissolve it, heat it at 100°C for 30 minutes, and dry it by freeze-drying after cooling to obtain the water extract of cicada mycelium.
上述蟬花菌絲體發酵液、蟬花菌絲體凍乾粉、蟬花菌絲體水萃物皆含有本發明之蟬花菌絲體活性物質。以下根據上述方法製備蟬花菌絲體活性物質,並進行生物實驗評估其功效。 實施例一 :蟬花菌絲體活性物質製備 The above-mentioned fermented liquid of cicada flower mycelium, freeze-dried powder of cicada flower mycelium and aqueous extract of cicada flower mycelium all contain the active substance of cicada flower mycelium of the present invention. The active substance of the cicadae mycelium is prepared according to the above-mentioned method, and a biological experiment is carried out to evaluate its efficacy. Embodiment one : preparation of cicada flower mycelium active substance
菌株:寄存於財團法人食品工業發展研究所之生物資源研究中心(BCRC),寄存編號為MU30106之蟬花菌絲體。此菌株可由寄存單位BCRC官方網站上購買取得,為所屬技術領域中具有通常知識者易於獲得。Bacterial strain: deposited in the Bioresource Research Center (BCRC) of the Food Industry Development Research Institute of the foundation, the registration number is MU30106 Cicada mycelium. This bacterial strain can be purchased from the official website of the depository unit BCRC, and is easy to obtain for those with common knowledge in the technical field.
平板培養:將菌絲體接種於平板培養基上,培養基為馬鈴薯糊精培養基(Potato Dextrose Agar, PDA),於25℃下培養5天。Plate culture: the mycelia were inoculated on a plate medium, and the medium was potato dextrin medium (Potato Dextrose Agar, PDA), and cultured at 25° C. for 5 days.
燒瓶培養:刮取平板上之菌絲接種於燒瓶內,用下列培養基配方,在25℃,pH 5.5下,於震盪機上以轉速120 rpm震盪培養三天;Flask culture: Scrape the mycelia on the plate and inoculate into the flask, use the following medium formula, at 25°C, pH 5.5, shake and culture on a shaker at a speed of 120 rpm for three days;
培養基配方:
發酵槽培養:培養基同上,將燒瓶培養物接種於發酵槽培養基內,在25℃,槽壓1.0 kg/cm
2,pH 5.5下,10 rpm攪拌速度,以1.0 VVM通氣速率通入空氣,培養5天,得蟬花菌絲體發酵液。該蟬花菌絲體發酵液經冷凍乾燥可得蟬花菌絲體凍乾粉。
Fermentation tank culture: the culture medium is the same as above, inoculate the flask culture in the fermentation tank medium, at 25°C, tank pressure 1.0 kg/cm 2 , pH 5.5, stirring
萃取物製備:取蟬花菌絲體凍乾粉加入20倍體積之蒸餾水溶解,以溫度100℃加熱30分鐘,待冷卻後經冷凍乾燥法進行乾燥,得蟬花菌絲體水萃物。Extract preparation: take the freeze-dried powder of cicada mycelium and add 20 times the volume of distilled water to dissolve it, heat it at 100°C for 30 minutes, and dry it by freeze-drying after cooling to obtain the water extract of cicada mycelium.
結果:20公噸發酵槽培養完畢之蟬花菌絲體發酵液經冷凍乾燥後,可得約320公斤凍乾粉,再經萃取步驟可得約20-30公斤水萃物。以下生物實驗係以蟬花菌絲體水萃物進行。 實施例二 黃斑部病變動物模式及相關指標之分析 Results: After lyophilization, about 320 kg of freeze-dried powder can be obtained from the fermented broth of cicadae mycelium cultured in 20 metric tons of fermentation tanks, and about 20-30 kg of water extract can be obtained through the extraction step. The following biological experiment was carried out with water extract of cicada flower mycelium. Example 2 Analysis of macular degeneration animal models and related indicators
黃斑部病變小鼠動物模式之建立Establishment of mouse model of macular degeneration
碘酸鈉(Sodium iodate, NaIO 3)為一種穩定的氧化劑,已被證明是一種誘導視網膜變性的有效方法。碘酸鈉引起的視網膜變性與視網膜色素上皮細胞(RPE)的區域性喪失相關,同時也會出現區域性萎縮的一些形態學特徵。已有許多研究利用不同的哺乳動物之物種證實了NaIO 3對生物視網膜具有毒性,包括綿羊、兔子、大鼠和小鼠。上述的研究指出在視網膜中,NaIO 3主要標靶為 RPE 細胞,可誘導其死亡,如壞死(necrosis) 、細胞凋亡(apoptosis)或細胞自噬(autophagy),其次是脈絡膜毛細血管萎縮(choriocapillaris atrophy) 和全視網膜變性 (panretinal degeneration)。 Sodium iodate (NaIO 3 ), a stable oxidant, has been proven to be an effective method for inducing retinal degeneration. Sodium iodate-induced retinal degeneration is associated with regional loss of the retinal pigment epithelium (RPE), along with some morphological features of regional atrophy. Numerous studies have demonstrated the toxicity of NaIO 3 to the biological retina using different mammalian species, including sheep, rabbits, rats and mice. The above studies pointed out that in the retina, the main target of NaIO 3 is RPE cells, which can induce their death, such as necrosis, apoptosis or autophagy, followed by choriocapillaris atrophy. atrophy) and panretinal degeneration.
近年來,研究學者以低劑量NaIO 3(15-35 mg/kg)來誘導小鼠產生老年性黃斑部病變(Age-related macular degeneration, AMD)模型,發現其與視功能下降以及局部性RPE流失和外部視網膜損傷有關,與乾性AMD的共同發病機制極為相似,因此近年來大多以此模式來探討RPE再生及AMD的預防。 In recent years, researchers have used low doses of NaIO 3 (15-35 mg/kg) to induce age-related macular degeneration (Age-related macular degeneration, AMD) models in mice, and found that it is associated with decreased visual function and local RPE loss It is related to external retinal damage and is very similar to the common pathogenesis of dry AMD. Therefore, in recent years, this model has mostly been used to explore the prevention of RPE regeneration and AMD.
本實施例使用40 mg/kg劑量NaIO 3誘發小鼠產生AMD,藉此評估蟬花菌絲體活性物質對於黃斑部病變的預防及治療效果。 In this example, 40 mg/kg dose of NaIO 3 was used to induce AMD in mice, so as to evaluate the preventive and therapeutic effects of active substances of cicadae mycelium on macular degeneration.
實驗動物:品系Balb/c雄性小鼠購自台灣國家實驗動物中心,年齡約6-8周,體重約26.21±1.76 g克。小鼠飼養於中山醫學大學實驗動物中心,提供正常潔淨飼料及飲水,飼養環境為12小時照光及12小時黑暗之循環光照,溫度控制在20±2℃,濕度控制在50±5 %。Experimental animals: Strain Balb/c male mice were purchased from Taiwan National Experimental Animal Center, aged about 6-8 weeks, weighing about 26.21±1.76 g. Mice were raised in the Experimental Animal Center of Sun Yat-Sen Medical University, provided with normal clean feed and drinking water. The feeding environment was 12-hour light and 12-hour dark cycle light, the temperature was controlled at 20±2°C, and the humidity was controlled at 50±5%.
餵食劑量與實驗步驟Feeding dosage and experimental procedures
本試驗共進行21天,試驗進行前將小鼠分為4組,每組6隻:The experiment was carried out for 21 days in total. Before the experiment, the mice were divided into 4 groups, 6 in each group:
(1) 空白對照組(Mock):以靜脈注射 (i.v.) 磷酸鹽緩衝生理鹽水(PBS)100 μL後,並以每日口餵方式給予PBS 200 μL。(1) Blank control group (Mock): 100 μL of phosphate-buffered saline (PBS) was injected intravenously (i.v.), and 200 μL of PBS was given orally every day.
(2) 負對照組:以靜脈注射 (i.v.)給予碘酸鈉NaIO
340 mg/kg,誘發AMD,並以每日口餵方式給予PBS 200 μL。
(2) Negative control group:
(3) 蟬花水萃物組(實驗組):先以每日口餵方式給予實施例一製得的蟬花水萃物100 mg/kg 14天後,再以靜脈注射給予碘酸鈉NaIO
340 mg/kg誘發AMD,之後再每日餵食蟬花水萃物100 mg/kg 7天。
(3) Cicada flower water extract group (experimental group): first give the cicada
(4) 正對照組:洋蔥萃取物已知可用於改善黃斑部病變,於此作為正對照組,先以每日口餵方式給予洋蔥萃取物1500 mg/kg 14天後,再以靜脈注射給予碘酸鈉(NaIO
3) 40 mg/kg誘發AMD,之後再每日餵食洋蔥萃取物1500 mg/kg 7天。
(4) Positive control group: Onion extract is known to be used to improve macular degeneration. Here, as a positive control group, 1500 mg/kg of onion extract was administered orally every day for 14 days, and then administered intravenously Sodium iodate (NaIO 3 ) 40 mg/kg induced AMD, and then fed
各組小鼠在實驗21天後犧牲進行視網膜損傷程度檢測及相關指標分析。The mice in each group were sacrificed 21 days after the experiment to detect the degree of retinal damage and analyze related indicators.
視網膜損傷程度檢測及相關指標分析Detection of retinal damage and analysis of related indicators
1. 眼底影像偵測1. Fundus image detection
使用Phoenix-Micro IV 眼底影像偵測系統,可檢測光學眼底鏡、螢光眼底造影以及視網膜斷層掃描儀,並以此依據評估小鼠視網膜組織完整性與平滑度改變。圖1(A)為各組小鼠注射碘酸鈉誘發黃斑部病變第7天後(實驗天數第21天)的眼底攝影圖,圖1(B)則為對應照片紅線標記位置的光學斷層掃描(Optical Coherence Tomography)圖。Using the Phoenix-Micro IV fundus image detection system, it can detect optical ophthalmoscope, fluorescein fundus angiography and retinal tomography scanner, and use this to evaluate the integrity and smoothness of mouse retinal tissue changes. Figure 1(A) is the fundus photography of mice in each group on the 7th day after injection of sodium iodate to induce macular degeneration (the 21st day of the experiment), and Figure 1(B) is the optical tomography of the position marked by the red line in the corresponding photo (Optical Coherence Tomography) diagram.
對圖1(B)小鼠以視神經為中心,鼻側與顳側 300 μm ~ 600 μm 的範圍(黃線示意處)進行6次厚度(紅線範圍)測量後,計算其平均值,可得小鼠的視網膜厚度,其結果列為圖2。The thickness (red line range) of the mouse in Figure 1(B) was measured 6 times in the range of 300 μm to 600 μm on the nasal side and temporal side (indicated by the yellow line) with the optic nerve as the center, and the average value was calculated to obtain the small The retinal thickness of the mouse is shown in Figure 2.
從圖1(A)的眼底攝影圖來看,與空白對照組(Mock)相比,單獨施打碘酸鈉的小鼠組別 (NaIO 3)在接受碘酸鈉注射後7天後的視網膜排列較不規則,且組織間的排序較不平滑、界線較為模糊,有明顯視網膜扭曲的情形發生。而經由洋蔥萃取物(Onion)及蟬花萃取物預先處理的小鼠組別與單獨施打碘酸鈉的小鼠組別(NaIO 3)相比,視網膜排列不規則與模糊的情形有明顯較少的現象。 From the fundus photography in Figure 1(A), compared with the blank control group (Mock), the retina of the mouse group (NaIO 3 ) administered sodium iodate alone 7 days after receiving sodium iodate injection The arrangement is irregular, and the ordering of tissues is not smooth, the boundaries are blurred, and there are obvious retinal distortions. However, compared with the mouse group (NaIO 3 ) pretreated with onion extract (Onion) and cicadae extract, the retinal arrangement irregularity and blurred situation were significantly different. less phenomenon.
圖2視網膜厚度測量的結果則顯示(其中*號標記表示與單獨加入NaIO 3組別相比,p < 0.05,有顯著差異),與空白對照組(Mock)相比,單獨施打碘酸鈉的小鼠組別(NaIO 3)在接受碘酸鈉注射7天後,視網膜厚度有明顯變薄情形。而經由洋蔥萃取物(Onion)預先處理的小鼠組別與單獨施打碘酸鈉的小鼠組別(NaIO 3)相比,可顯著減緩碘酸鈉誘導之小鼠視網膜變薄情形,而蟬花萃取物雖無顯著差異但厚度仍有回升。不過,使用洋蔥萃取物之正對照組減緩視網膜變薄的濃度需高達1500 mg/kg,而本實施例使用蟬花萃取物的濃度僅需100 mg/kg (1/15)。 The results of retinal thickness measurement in Fig. 2 show (wherein the * sign indicates that compared with adding NaIO 3 groups alone, p < 0.05, there is a significant difference), compared with the blank control group (Mock), administering sodium iodate alone The retinal thickness of the mouse group (NaIO 3 ) was significantly thinner after receiving sodium iodate injection for 7 days. Compared with the mouse group (NaIO 3 ) pre-treated with onion extract (Onion), it can significantly slow down the thinning of the mouse retina induced by sodium iodate, while Although there was no significant difference in cicada flower extract, the thickness still rebounded. However, the concentration of the positive control group using onion extract to slow down retinal thinning needs to be as high as 1500 mg/kg, while the concentration of cicada flower extract used in this example only needs 100 mg/kg (1/15).
據此,可得知本發明實施例的蟬花萃取物可減緩碘酸鈉NaIO 3所誘導之小鼠視網膜扭曲與變薄,進而改善AMD的情形。且跟洋蔥萃取物相比,以較低的濃度即能達成效果。 Accordingly, it can be known that the cicadae extract of the embodiment of the present invention can slow down the distortion and thinning of the mouse retina induced by sodium iodate NaIO 3 , thereby improving the situation of AMD. And compared with onion extract, the effect can be achieved at a lower concentration.
2. 組織病理分析 (H&E stainine)2. Histopathological analysis (H&E stainine)
將各組小鼠於注射碘酸鈉第7天後進行犧牲採血,眼睛做組織切片後,以蘇木精和伊紅染色,區別RPE的結構與型態,其照片如圖3(A)所示。圖3(B)為圖3(A)的紅色虛線框放大圖,用以觀察內核層(Inner nuclear layer, INL)與外核層(Outer nuclear layer, ONL)厚度,以及觀察發炎細胞的數量。The mice in each group were sacrificed for blood collection on the 7th day after the injection of sodium iodate, and the eyes were sliced and stained with hematoxylin and eosin to distinguish the structure and type of RPE. The photos are shown in Figure 3(A) Show. Figure 3(B) is an enlarged view of the red dotted line frame in Figure 3(A), which is used to observe the thickness of the inner nuclear layer (Inner nuclear layer, INL) and outer nuclear layer (Outer nuclear layer, ONL), and to observe the number of inflammatory cells.
INL及ONL層厚度測試皆是以視神經為中心,在其鼻側與顳側 300 μm ~ 600 μm 進行6次厚度(圖3(B)黃線標記)測量後,計算其平均值,其結果分別列於圖4(A)與圖4(B),其中*號標記表示與單獨加入NaIO 3組別相比,p < 0.05,有顯著差異。 Both INL and ONL layer thickness tests were centered on the optic nerve, and the thickness was measured 6 times (marked by the yellow line in Figure 3(B)) at the nasal and temporal sides of 300 μm to 600 μm, and the average value was calculated. The results were respectively Listed in Figure 4 (A) and Figure 4 (B), where the mark * indicates that compared with the NaIO 3 group alone, p < 0.05, there is a significant difference.
與空白對照組(Mock)相比,單獨施打碘酸鈉的小鼠組別(NaIO 3)在接受碘酸鈉注射7天後,視網膜ONL與INL層厚度皆有變薄的情形。而經由洋蔥萃取物 (Onion)與蟬花萃取物預先處理的小鼠組別與單獨施打碘酸鈉的小鼠組別 (NaIO 3)相比,可顯著減緩碘酸鈉誘導之小鼠視網膜ONL與INL層厚度變薄。 Compared with the blank control group (Mock), the mouse group (NaIO 3 ) injected with sodium iodate alone had thinner retinal ONL and INL layers 7 days after sodium iodate injection. Compared with the mouse group (NaIO 3 ) administered with sodium iodate alone, the group of mice pretreated with onion extract (Onion) and cicadae flower extract can significantly slow down the retinal damage induced by sodium iodate. The ONL and INL layers become thinner.
據此,可得知本發明實施例的蟬花萃取物可減緩碘酸鈉(NaIO 3)誘導之小鼠視網膜內核層與外核層變薄,進而改善AMD的情形。 實施例三 組合物製備 Accordingly, it can be known that the cicadae flower extract of the embodiment of the present invention can slow down the thinning of the inner inner layer and outer nuclear layer of the mouse retina induced by sodium iodate (NaIO 3 ), thereby improving the condition of AMD. Embodiment three composition preparation
本實施例之蟬花菌絲體活性物質若應用於醫藥用途,則以下組合物1之態樣作為例示性實例。If the active substance of the cicadae mycelium of this embodiment is used in medicine, the following composition 1 is used as an illustrative example.
組合物1:取實施例一之蟬花菌絲體活性物質的凍乾粉或水萃物(20 wt%),與作為潤滑劑的硬脂酸鎂(8wt%)、作為防腐劑的二氧化矽(7wt%)充分混合,並溶於純水(65wt%)中,存放於4℃備用。前述wt%係指各成分佔組合物總重之比例。Composition 1: get the lyophilized powder or the water extract (20 wt%) of the active substance of the cicadae mycelium of embodiment one, and magnesium stearate (8wt%) as lubricant, the carbon dioxide as preservative Silicon (7wt%) was mixed thoroughly, dissolved in pure water (65wt%), and stored at 4°C for use. The aforementioned wt% refers to the ratio of each component to the total weight of the composition.
不過,雖然實施例二中的蟬花菌絲體活性物質係以口服方式餵食小鼠,但實際應用上亦可採用如滴劑、栓劑等其他方式。However, although the active substance of the cicadae mycelium in Example 2 is fed to the mice orally, other methods such as drops and suppositories can also be used in practice.
本揭露之蟬花菌絲體活性物質若以液體劑型應用於食品用途,則以下組合物2之態樣作為例示性實例。If the active substance of the cicadae mycelium disclosed in the present disclosure is applied to food in a liquid dosage form, the following composition 2 is used as an illustrative example.
組合物2:取實施例一之蟬花菌絲體活性物質的凍乾粉或水萃物(20 wt%),與作為防腐劑之苯甲醇(8 wt%)、作為稀釋劑之甘油(7 wt%)、作為稀釋劑之蔗糖(10 wt%)充分混合,並溶於純水(55 wt%)中,存放於4℃備用。前述wt%係指各成分佔組合物總重之比例。Composition 2: get the freeze-dried powder or water extract (20 wt%) of the active substance of cicadae flower mycelium of embodiment one, and benzyl alcohol (8 wt%) as preservative, glycerol (7 wt%) as diluent wt%) and sucrose (10 wt%) as a diluent were thoroughly mixed, dissolved in pure water (55 wt%), and stored at 4°C for later use. The aforementioned wt% refers to the ratio of each component to the total weight of the composition.
圖1(A)為各組小鼠的眼底攝影圖,圖1(B)則為對應照片紅線標記位置的光學斷層掃描(Optical Coherence Tomography)圖;Fig. 1 (A) is the photographic fundus of each group of mice, and Fig. 1 (B) is the optical tomography (Optical Coherence Tomography) map corresponding to the position marked by the red line in the photo;
圖2為圖1(B)的視網膜厚度測量結果;Fig. 2 is the retinal thickness measurement result of Fig. 1 (B);
圖3(A)為各組小鼠眼睛的蘇木精&伊紅染色(H&E stain)照片,圖3(B)為圖3(A)的紅色虛線框放大圖;Fig. 3 (A) is the photo of hematoxylin & eosin staining (H&E stain) of mouse eyes in each group, and Fig. 3 (B) is the enlarged view of the red dotted line frame of Fig. 3 (A);
圖4(A)為圖3(A)的內核層INL厚度測量結果,圖4(B)為圖3(A)的外核層ONL厚度測量結果。Fig. 4(A) is the measurement result of the INL thickness of the inner inner layer in Fig. 3(A), and Fig. 4(B) is the measurement result of the ONL thickness of the outer nuclear layer in Fig. 3(A).
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