TW202321434A - Use of hericium erinaceus mycelia active substance for preventing or curing retinopathy - Google Patents
Use of hericium erinaceus mycelia active substance for preventing or curing retinopathy Download PDFInfo
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- TW202321434A TW202321434A TW110142437A TW110142437A TW202321434A TW 202321434 A TW202321434 A TW 202321434A TW 110142437 A TW110142437 A TW 110142437A TW 110142437 A TW110142437 A TW 110142437A TW 202321434 A TW202321434 A TW 202321434A
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- hericium erinaceus
- mycelium
- erinaceus mycelium
- active substance
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- 208000017442 Retinal disease Diseases 0.000 title claims abstract description 11
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- 240000000588 Hericium erinaceus Species 0.000 title claims description 81
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
Description
本發明關於一種猴頭菇菌絲體活性物質之用途,特別是指以猴頭菇菌絲體製備用以預防或治療視網膜病變的組合物。The present invention relates to the application of a kind of active substance of Hericium erinaceus mycelium, in particular to a composition prepared by using Hericium erinaceus mycelium for preventing or treating retinopathy.
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.
猴頭菇 Hericium erinaceus (Bull.) Pers是一種珍貴的藥膳兼用真菌,主要分布於緯度較高的溫帶地區。《中國野菜食譜大全》認為其可治消化不良、胃潰瘍、胃炎、胃痛、胃脹及神經衰弱等病症,顯示猴頭菇除美味以外,具有胃部保護功能,可作為食藥兩用菇。1994 年起Kawagishi從猴頭菇菌絲體分離純化多種猴頭素(A、B、C等) ,猴頭素可刺激老鼠星狀細胞,增加神經生長因子分泌量(NGF),而NGF可治療智力衰退、神經衰弱等疾病,故猴頭菇亦被認為可促進神經及大腦健康。然而,目前並無研究指出猴頭菇對視網膜病變具有療效。 Hericium erinaceus (Bull.) Pers is a precious medicinal and dietary fungus, mainly distributed in temperate regions with higher latitudes. "Chinese Wild Vegetable Recipes" believes that it can cure indigestion, gastric ulcer, gastritis, stomach pain, bloating and neurasthenia. Since 1994, Kawagishi has isolated and purified a variety of Hericium erinaceus (A, B, C, etc.) from Hericium erinaceus mycelium. Hericium can stimulate mouse stellate cells and increase the secretion of nerve growth factor (NGF), and NGF can treat Mental decline, neurasthenia and other diseases, so Hericium erinaceus is also believed to promote nerve and brain health. However, there is currently no research showing that Hericium erinaceus has a curative effect on retinopathy.
本發明提供一種猴頭菇( Hericium erinaceus )菌絲體活性物質的用途,其係用於製備預防或治療視網膜病變之組合物。該猴頭菇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 active substance of Hericium erinaceus mycelium , which is used for preparing a composition for preventing or treating retinopathy. The preparation method of the Hericium erinaceus mycelium active substance comprises the following steps:
(a)取一猴頭菇菌絲體於平板培養基上,於15-35℃之溫度下培養7-15天;(a) Get a Hericium erinaceus mycelium on a plate medium, and cultivate it at a temperature of 15-35° C. for 7-15 days;
( b)將步驟(a)培養後之猴頭菇菌絲體接種至一燒瓶內,於15-35℃、pH 2-8之環境培養3-5天;(b) Inoculate the Hericium erinaceus mycelium cultured in step (a) into a flask, and cultivate for 3-5 days in an environment of 15-35° C. and pH 2-8;
(c)將步驟(b)培養後之猴頭菇菌絲體接種於一發酵槽內,於15-35℃、pH 4.5-5.5之環境下攪拌培養7-15天,形成含有該猴頭菇菌絲體活性物質之一猴頭菇菌絲體發酵液。(c) Inoculate the Hericium erinaceus mycelium after step (b) cultivation in a fermentation tank, stir and cultivate it for 7-15 days under the environment of 15-35 ℃ and pH 4.5-5.5, and form the Hericium erinaceus mycelium containing the Hericium erinaceus mycelium fermentation broth, one of the mycelium active substances.
一實施例中,製備該猴頭菇菌絲體活性物質的步驟更包括步驟(d):將該猴頭菇菌絲體發酵液冷凍乾燥後磨粉,形成含有該猴頭菇菌絲體活性物質之一猴頭菇菌絲體凍乾粉。In one embodiment, the step of preparing the active substance of the Hericium erinaceus mycelium further includes a step (d): freeze-drying the fermentation liquid of the Hericium erinaceus mycelium and grinding it into powder to form a powder containing the active substance of the Hericium erinaceus mycelium. One of the substances is freeze-dried powder of Hericium erinaceus mycelium.
一實施例中,製備該猴頭菇菌絲體活性物質的步驟更包括步驟(e):將該猴頭菇菌絲體凍乾粉以一溶劑萃取,形成含有該猴頭菇菌絲體活性物質之一猴頭菇菌絲體萃取液。In one embodiment, the step of preparing the active substance of the Hericium erinaceus mycelium further includes step (e): extracting the freeze-dried powder of the Hericium erinaceus mycelium with a solvent to form an active substance containing the Hericium erinaceus mycelium. One of the substances is Hericium erinaceus mycelium extract.
一實施例中,製備該猴頭菇菌絲體活性物質的步驟更包括步驟(f):將該猴頭菇菌絲體萃取液乾燥,以獲得該猴頭菇菌絲體活性物質。In one embodiment, the step of preparing the active substance of Hericium erinaceus mycelium further includes step (f): drying the extract of Hericium erinaceus mycelium to obtain the active substance of Hericium erinaceus mycelium.
一實施例中,在步驟(e)中的溶劑為乙醇,且該猴頭菇菌絲體凍乾粉以乙醇重複萃取二次。In one embodiment, the solvent in step (e) is ethanol, and the lyophilized powder of Hericium erinaceus mycelia is repeatedly extracted twice with ethanol.
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,該視網膜病變為黃斑部病變。In one embodiment, the retinopathy is macular degeneration.
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0.5-2.0 kg/cm2 且通氣速率為0.5-1 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 0.5-2.0 kg/cm2 and ventilation The rate is 0.5-1 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.
猴頭菇菌絲體來源Hericium erinaceus mycelium source
本發明之實施例所用之猴頭菇(Hericium erinaceus)菌種,購自於台灣財團法人食品工業研究所,寄存編號為BCRC 35669。該菌株係可由BCRC之官方網站(https://catalog.bcrc.firdi.org.tw/)直接購得,為公眾可輕易取得之菌株。但本發明所述之猴頭菇活性物質不限於由此菌種所得。The strain of Hericium erinaceus used in the examples of the present invention was purchased from Taiwan Institute of Food Industry, and the registration number is BCRC 35669. This strain can be directly purchased from the official website of BCRC (https://catalog.bcrc.firdi.org.tw/), and it is a strain that is easily available to the public. However, the active substances of Hericium erinaceus described in the present invention are not limited to those obtained from such strains.
菌絲體液體培養Mycelium liquid culture
將猴頭菇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7-15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100-250 rpm的條件下培養3-5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 4.5-5.5,10-150 rpm攪拌速度或不攪拌(air lift)情況,以0.5-1 VVM通氣速率通入空氣,或空氣、氧氣、二氧化碳、氮氣及上述氣體之混合物(較佳者為空氣),培養時間為7-15天內,即得猴頭菇菌絲體發酵液。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 mycelium of Hericium erinaceus on a plate medium, culture it at an appropriate temperature of 15-35° C. (preferably 25° C.) for 7-15 days, scrape the mycelium and inoculate it in a flask. Cultivate for 3-5 days at 15-35°C (preferably 25°C), pH 2-8 (preferably pH 4-7, more preferably pH 5.5), shaking rate 100-250 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 4.5-5.5, stirring speed 10-150 rpm or In the case of no stirring (air lift), air is introduced at a ventilation rate of 0.5-1 VVM, or a mixture of air, oxygen, carbon dioxide, nitrogen and the above gases (preferably air), and the cultivation time is within 7-15 days, that is, Obtain Hericium erinaceus mycelium fermentation liquid. 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
此猴頭菇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 Hericium erinaceus mycelium fermentation liquid 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-乙醇萃取Freeze-dried powder extraction-ethanol extraction
取猴頭菇菌絲體凍乾粉加入25倍體積之乙醇進行第1次萃取,利用超音波震盪萃取1小時,取懸浮液進行離心。離心後取上清液,將殘渣重複上述萃取步驟,以乙醇進行第二次萃取。第二次萃取獲得的上清液與第一次萃取的上清液混合,經減壓濃縮,得膏狀的猴頭菇菌絲體乙醇萃取物,其中包含猴頭菇菌絲體活性物質。重複萃取兩次的原因係為了獲得較高的產率。Take the freeze-dried powder of Hericium erinaceus mycelium and add 25 times the volume of ethanol for the first extraction, use ultrasonic vibration to extract for 1 hour, and take the suspension for centrifugation. After centrifugation, the supernatant was taken, and the residue was subjected to the above extraction step, and the second extraction was performed with ethanol. The supernatant obtained in the second extraction is mixed with the supernatant obtained in the first extraction, concentrated under reduced pressure to obtain a creamy ethanol extract of Hericium erinaceus mycelium, which contains active substances of Hericium erinaceus mycelium. The reason for repeating the extraction twice is to obtain a higher yield.
上述猴頭菇菌絲體發酵液、猴頭菇菌絲體凍乾粉、猴頭菇菌絲體乙醇萃取物皆含有本發明之猴頭菇菌絲體活性物質。以下根據上述方法製備猴頭菇菌絲體活性物質,並進行生物實驗評估其功效。 實施例一 :猴頭菇菌絲體活性物質製備 The above-mentioned Hericium erinaceus mycelium fermentation liquid, Hericium erinaceus mycelium freeze-dried powder, and Hericium erinaceus mycelium ethanol extract all contain the Hericium erinaceus mycelium active substance of the present invention. The active substance of Hericium erinaceus mycelium is prepared according to the above method, and the biological experiment is carried out to evaluate its efficacy. Embodiment one : Hericium erinaceus mycelium active substance preparation
菌株:本發明之實施例所用之猴頭菇(Hericium erinaceus)菌種係購自於財團法人食品工業研究所,寄存編號為BCRC 35669。Bacterial strains: Hericium erinaceus strains used in the examples of the present invention were purchased from the Institute of Food Industry, and the registration number is BCRC 35669.
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7天。Plate culture: the mycelium was inoculated on a plate medium, and the medium was potato dextrin medium (Potato Dextrose Agar, PDA), and cultured at 25° C. for 7 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 5.0下,於震盪機上以轉速120 rpm震盪培養5天;Flask culture: Scrape the mycelium on the plate and inoculate it in the flask, use the following medium formula, at 25°C, pH 5.0, shake and culture on a shaker at a speed of 120 rpm for 5 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 5.0下,50 rpm攪拌速度,以1.0 VVM通氣速率通入空氣,培養12天,得猴頭菇菌絲體發酵液。該猴頭菇菌絲體發酵液經冷凍乾燥可得猴頭菇菌絲體凍乾粉。 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 5.0, stirring speed at 50 rpm, and air at 1.0 VVM aeration rate, culture 12 One day, I got Hericium erinaceus mycelium fermentation liquid. The Hericium erinaceus mycelium fermented liquid can be freeze-dried to obtain Hericium erinaceus mycelium freeze-dried powder.
萃取物製備: 將猴頭菇菌絲體凍乾粉加入為凍乾粉25倍重量的95 v/v%乙醇進行第一次萃取,接著利用超音波震盪以震盪速率120rpm萃取一小時,取懸浮液進行離心,離心後取上清液。將第一次萃取之殘渣重複上述萃取步驟以85v/v%乙醇進行第二次萃取。兩次萃取離心得到的上清液混合,經減壓濃縮,得膏狀的猴頭菇菌絲體乙醇萃取物(簡稱醇萃物)。Extract preparation: Add the freeze-dried powder of Hericium erinaceus mycelium to 95 v/v% ethanol that is 25 times the weight of the freeze-dried powder for the first extraction, and then use ultrasonic vibration to extract at a shaking rate of 120rpm for one hour, and take the suspension The solution was centrifuged, and the supernatant was collected after centrifugation. The residue of the first extraction was repeated for the second extraction step with 85v/v% ethanol. The supernatants obtained by two extractions and centrifugation were mixed, concentrated under reduced pressure to obtain a creamy ethanol extract of Hericium erinaceus mycelium (referred to as alcohol extract).
結果:20公噸發酵槽培養完畢之猴頭菇菌絲體發酵液經冷凍乾燥後,可得約320公斤凍乾粉,再經二次萃取步驟,可得約20公斤乙醇萃取物。以下生物實驗係以猴頭菇菌絲體乙醇萃物進行。 實施例二 視網膜病變動物模式及相關指標之分析 Results: About 320 kg of freeze-dried powder can be obtained after freeze-drying the fermented broth of Hericium erinaceus mycelia cultured in 20 metric tons of fermentation tanks, and about 20 kg of ethanol extract can be obtained through a second extraction step. The following biological experiments were carried out with the ethanol extract of Hericium erinaceus mycelium. Embodiment 2 Retinopathy Animal Model and Analysis of Related Indexes
視網膜病變小鼠動物模式之建立Establishment of animal model of retinopathy in mice
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,藉此評估猴頭菇菌絲體活性物質對於視網膜病變(特別是黃斑部病變)的預防及治療效果。 In this example, 40 mg/kg dose of NaIO3 was used to induce AMD in mice, so as to evaluate the preventive and therapeutic effects of Hericium erinaceus mycelium active substances on retinopathy (especially 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) 猴頭菇乙醇萃取物組(實驗組):先以每日口餵方式給予實施例一製得的猴頭菇乙醇萃取物100 mg/kg 14天後,再以靜脈注射給予碘酸鈉NaIO
340 mg/kg誘發AMD,之後再每日餵食猴頭菇乙醇萃取物100 mg/kg 7天。
(3) Hericium erinaceus ethanol extract group (experimental group): first give 100 mg/kg of the ethanol extract of Hericium erinaceus prepared in Example 1 for 14 days, and then give iodic acid by intravenous
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為各組小鼠注射碘酸鈉第7天後的光學斷層掃描(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. Fig. 1 is the optical tomography (Optical Coherence Tomography) figure of each group of mice injected with sodium iodate on the 7th day.
對圖1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。Take the optic nerve as the center of the mouse in Figure 1, and measure the thickness (red line range) 6 times in the range of 300 μm to 600 μm on the nasal side and temporal side (indicated by the yellow line), and calculate the average value to obtain the retina of the mouse thickness, the results are listed in Figure 2.
請參圖1(A)的光學斷層掃描圖,Mock組紅線標記部分可看出視網膜結構完整。至於單獨施打碘酸鈉的小鼠組別 (NaIO 3)的視網膜排列較不規則、鬆散,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。而經由猴頭菇菌絲體萃取物預先處理的小鼠組別(HE)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。 Please refer to the optical tomography image in Figure 1(A), the part marked by the red line in the Mock group can be seen to have a complete retinal structure. As for the group of mice administered with sodium iodate alone (NaIO 3 ), the arrangement of the retina was irregular and loose, and the arrangement of the tissues was not smooth, the boundaries were blurred, and the retina was obviously distorted. Compared with the mouse group (NaIO 3 ) pre-treated with Hericium erinaceus mycelium extract, the irregular and blurred retinal arrangement was 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。而經由猴頭菇菌絲體萃取物(HE)預先處理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,雖然厚度仍有變薄,但仍有顯著回升。 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. However, compared with the mouse group (NaIO 3 ) that was pretreated with Hericium erinaceus mycelium extract (HE) pre-treated, the thickness was still thinner, but still There has been a significant recovery.
據此,可得知本發明實施例的猴頭菇菌絲體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。 Accordingly, it can be known that the Hericium erinaceus mycelium 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 condition of AMD.
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。而經由猴頭菇菌絲體萃取物(HE)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 ) pre-treated with Hericium erinaceus mycelium extract (HE), sodium iodate-induced retinal damage could be significantly slowed down. The ONL and INL layers become thinner.
據此,可得知本發明實施例的猴頭菇菌絲體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 Hericium erinaceus mycelium 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
本實施例之猴頭菇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 Hericium erinaceus mycelium in this embodiment is used in medicine, the following composition 1 is used as an illustrative example.
組合物1:取實施例一之猴頭菇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 Hericium erinaceus mycelium active substance of embodiment one, and magnesium stearate (8wt%) as lubricant, as preservative two Silicon oxide (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,雖然實施例二中的猴頭菇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 Hericium erinaceus mycelium in Example 2 is fed to mice orally, other methods such as drops and suppositories can also be used in practice.
本揭露之菌種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 bacterial species 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:取實施例一之猴頭菇菌絲體活性物質的凍乾粉或水萃物(20 wt%),與作為防腐劑之苯乙醇(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 lyophilized powder or water extract (20 wt%) of the active substance of Hericium erinaceus Hericium erinaceus in Example 1, and phenylethyl alcohol (8 wt%) as preservative, glycerin as diluent ( 7 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)為各組小鼠的光學斷層掃描(Optical Coherence Tomography)圖;Fig. 1 (A) is the optical tomography (Optical Coherence Tomography) figure of mice of each group;
圖2為圖1的視網膜厚度測量結果;Fig. 2 is the measurement result of retinal thickness in Fig. 1;
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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