JPH0749365B2 - Method for producing propolis extract - Google Patents
Method for producing propolis extractInfo
- Publication number
- JPH0749365B2 JPH0749365B2 JP3162596A JP16259691A JPH0749365B2 JP H0749365 B2 JPH0749365 B2 JP H0749365B2 JP 3162596 A JP3162596 A JP 3162596A JP 16259691 A JP16259691 A JP 16259691A JP H0749365 B2 JPH0749365 B2 JP H0749365B2
- Authority
- JP
- Japan
- Prior art keywords
- propolis
- fraction
- concentration
- residue
- methyl alcohol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 229940069949 propolis Drugs 0.000 title claims description 53
- 241000241413 Propolis Species 0.000 title claims description 52
- 239000000284 extract Substances 0.000 title claims description 28
- 238000004519 manufacturing process Methods 0.000 title description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 99
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 58
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 57
- 230000002378 acidificating effect Effects 0.000 claims description 31
- 235000019441 ethanol Nutrition 0.000 claims description 28
- 239000002904 solvent Substances 0.000 claims description 20
- 238000004440 column chromatography Methods 0.000 claims description 14
- 238000010828 elution Methods 0.000 claims description 13
- 239000002024 ethyl acetate extract Substances 0.000 claims description 13
- 238000001704 evaporation Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 description 38
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 27
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 24
- 238000012360 testing method Methods 0.000 description 22
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 21
- 239000000243 solution Substances 0.000 description 20
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 16
- 238000000034 method Methods 0.000 description 16
- 239000002609 medium Substances 0.000 description 14
- 239000000725 suspension Substances 0.000 description 13
- 230000000844 anti-bacterial effect Effects 0.000 description 12
- 239000000126 substance Substances 0.000 description 12
- 230000009471 action Effects 0.000 description 11
- 238000004811 liquid chromatography Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 238000007796 conventional method Methods 0.000 description 7
- 235000011187 glycerol Nutrition 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 230000002476 tumorcidal effect Effects 0.000 description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- 230000018199 S phase Effects 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 5
- WOVKYSAHUYNSMH-UHFFFAOYSA-N BROMODEOXYURIDINE Natural products C1C(O)C(CO)OC1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-UHFFFAOYSA-N 0.000 description 5
- 230000006820 DNA synthesis Effects 0.000 description 5
- 229950004398 broxuridine Drugs 0.000 description 5
- 201000007270 liver cancer Diseases 0.000 description 5
- 208000014018 liver neoplasm Diseases 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- 230000008020 evaporation Effects 0.000 description 4
- 239000010410 layer Substances 0.000 description 4
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 4
- 239000004094 surface-active agent Substances 0.000 description 4
- NNJPGOLRFBJNIW-HNNXBMFYSA-N (-)-demecolcine Chemical compound C1=C(OC)C(=O)C=C2[C@@H](NC)CCC3=CC(OC)=C(OC)C(OC)=C3C2=C1 NNJPGOLRFBJNIW-HNNXBMFYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- NNJPGOLRFBJNIW-UHFFFAOYSA-N Demecolcine Natural products C1=C(OC)C(=O)C=C2C(NC)CCC3=CC(OC)=C(OC)C(OC)=C3C2=C1 NNJPGOLRFBJNIW-UHFFFAOYSA-N 0.000 description 3
- 241000204031 Mycoplasma Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000001569 carbon dioxide Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000002147 killing effect Effects 0.000 description 3
- 238000000464 low-speed centrifugation Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241000257303 Hymenoptera Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 230000022131 cell cycle Effects 0.000 description 2
- 238000011067 equilibration Methods 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 2
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000009876 antimalignant effect Effects 0.000 description 1
- 229960003589 arginine hydrochloride Drugs 0.000 description 1
- 235000013871 bee wax Nutrition 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000005880 cancer cell killing Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 229940018489 pronto Drugs 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000005909 tumor killing Effects 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/74—Recovery of fats, fatty oils, fatty acids or other fatty substances, e.g. lanolin or waxes
Landscapes
- Jellies, Jams, And Syrups (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Fats And Perfumes (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、プロポリスを原料とす
る殺腫瘍細胞作用および抗菌作用に優れた抽出物の製造
方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an extract of propolis which is excellent in tumoricidal cell activity and antibacterial activity.
【０００２】[0002]
【背景技術】プロポリスは、ミツバチにより採取された
樹脂類、ミツバチの唾液分泌物、蜂ロウ、花粉等が混合
された一種のにかわ質であり、抗菌作用、抗炎症作用等
を示す民間薬として古くから知られている。現在、プロ
ポリスは食品添加物として認可されており、プロポリス
のエチルアルコール溶液、界面活性剤でミセル化された
プロポリスのグリセリン懸濁液等、プロポリスを含有し
た種々の飲食物が製造されている。そして、プロポリス
が各種の炎症、潰瘍、アレルギー性疾患等に対して治癒
効果を有していることを示す臨床例や抗悪性腫瘍作用が
認められた症例が、近年、一般的にも知られるようにな
り、これに伴って、健康保持、疾病予防等のためにこの
プロポリスを飲用する人が増加している。BACKGROUND ART Propolis is a kind of glue that is a mixture of resins collected by bees, salivary secretions of bees, bee wax, pollen, etc. Known from. Currently, propolis is approved as a food additive, and various foods and drinks containing propolis such as an ethyl alcohol solution of propolis and a glycerin suspension of propolis micelle-ized with a surfactant are produced. In recent years, clinical cases showing that propolis has a curative effect on various inflammations, ulcers, allergic diseases, etc., and cases with an anti-malignant tumor effect are generally known. As a result, the number of people who take this propolis for the purpose of maintaining health and preventing diseases is increasing.
【０００３】[0003]
【発明の目的】プロポリスは、前述したエチルアルコー
ル溶液やグリセリン懸濁液等のかたちで飲食しても人体
にとって有用な物質であるが、本発明は、プロポリスが
有する生物活性作用、特に殺腫瘍細胞作用および抗菌作
用をより効率的に利用することができるプロポリス抽出
物の製造方法を提供することを目的とする。The object of the present invention is that propolis is a substance useful to the human body even if it is eaten and drinked in the form of the above-mentioned ethyl alcohol solution, glycerin suspension, etc. It is an object of the present invention to provide a method for producing a propolis extract that can more efficiently utilize its action and antibacterial action.
【０００４】[0004]
【目的を達成するための手段】本発明者は、プロポリス
に含まれる抗腫瘍物質の探索を進めていく過程で、プロ
ポリスに特定の処理を施すことにより殺腫瘍細胞作用お
よび抗菌作用に優れたプロポリス抽出物が得られること
を見出だし、本発明を完成するに至った。すなわち、本
発明のプロポリス抽出物の製造方法は、プロポリスの
酢酸エチル抽出液から溶媒を蒸散除去した後、得られた
残渣をメチルアルコール中に分散させ、不溶物を除去し
て、メチルアルコール抽出液を得る工程（以下、第１の
工程ということがある）、前記メチルアルコール抽出
液をカラムクロマトグラフィーにかけて、７０％から１
００％（Ｖ／Ｖ）までの酸性メチルアルコール濃度勾配
による溶出操作を行い、酸性メチルアルコール濃度が８
２〜８８％（Ｖ／Ｖ）の範囲内の画分を得る工程（以
下、第２の工程ということがある）、前記画分から溶
媒を蒸散除去した後、得られた残渣をカラムクロマトグ
ラフィーにかけて、７０％から８０％（Ｖ／Ｖ）までの
酸性アセトニトリル濃度勾配による溶出操作を行い、酸
性アセトニトリル濃度が７３〜７６％（Ｖ／Ｖ）の範囲
内の画分を得る工程（以下、第３の工程ということがあ
る）、前記画分から溶媒を蒸散除去した後、得られた
残渣をカラムクロマトグラフィーにかけて、７５％から
９０％（Ｖ／Ｖ）までのエチルアルコール濃度勾配によ
る溶出操作を行い、エチルアルコール濃度が８０〜８５
％（Ｖ／Ｖ）の範囲内の画分を得る工程（以下、第４の
工程ということがある）とを含むことを特徴とするもの
である。Means for Achieving the Purpose The present inventor has found that, in the process of searching for an antitumor substance contained in propolis, by applying a specific treatment to propolis, propolis is excellent in tumoricidal cell action and antibacterial action. It was found that an extract was obtained, and the present invention was completed. That is, the method for producing a propolis extract of the present invention is, after evaporating and removing the solvent from the ethyl acetate extract of propolis, dispersing the obtained residue in methyl alcohol, removing insoluble matter, and removing the methyl alcohol extract. (Hereinafter, sometimes referred to as the first step), the methyl alcohol extract is subjected to column chromatography to obtain 70% to 1
The elution operation was performed with an acidic methyl alcohol concentration gradient up to 00% (V / V), and the acidic methyl alcohol concentration was adjusted to 8%.
A step of obtaining a fraction within a range of 2 to 88% (V / V) (hereinafter sometimes referred to as a second step), the solvent is evaporated from the fraction, and the obtained residue is subjected to column chromatography. , 70% to 80% (V / V) acidic acetonitrile concentration gradient is performed to obtain a fraction having an acidic acetonitrile concentration in the range of 73 to 76% (V / V) (hereinafter referred to as the third After the solvent is removed from the fraction by evaporation, the obtained residue is subjected to column chromatography to perform an elution operation with an ethyl alcohol concentration gradient from 75% to 90% (V / V), Ethyl alcohol concentration is 80-85
% (V / V) in the range of obtaining a fraction (hereinafter sometimes referred to as a fourth step).
【０００５】以下、本発明を詳細に説明する。本発明の
プロポリス抽出物の製造方法では、まず、上述した第１
の工程を行う。この第１の工程での出発原料であるプロ
ポリスの酢酸抽出液は、プロポリス（塊状物、粉砕物、
乾燥粉末等）、プロポリスのエチルアルコール溶液、界
面活性剤でミセル化されたプロポリスのグリセリン懸濁
液等を用いて、例えば下記Ｉ、IIおよびIII のようにし
て調製することができる。The present invention will be described in detail below. In the method for producing a propolis extract of the present invention, first, the above-mentioned first
Process. The acetic acid extract of propolis, which is the starting material in the first step, is a propolis (lump, pulverized product,
(Eg dry powder), a solution of propolis in ethyl alcohol, and a suspension of glycerol in propolis that has been micelle-ized with a surfactant, and the like, for example, as described in I, II and III below.
【０００６】Ｉ．プロポリスを用いる場合には、プロポ
リスを酢酸エチルで直接抽出することにより、プロポリ
スの酢酸エチル抽出液を得てもよいが、下記ａ〜ｃの要
領で酢酸エチル抽出液を得た方がより好ましい。 ａ．まず、プロポリスを５〜１０倍量（Ｖ／Ｗ）のエチ
ルアルコール［濃度：９５〜９９．５％（Ｗ／Ｗ）］と
混合し、マグネチックスターラー等を用いて２０〜３０
℃で一昼夜以上撹拌して懸濁液を得た後に、この懸濁液
を常圧下ないし減圧下でろ過して、プロポリスのエチル
アルコール抽出液を得る。 ｂ．次いで、得られたエチルアルコール抽出液中の溶媒
をローターリーエバポレーター等を用いて蒸散除去す
る。 ｃ．この後、得られた残渣を、水と酢酸エチルとの１：
１〜１：２混液に分散させ、上層の酢酸エチル層を分取
することによりプロポリスの酢酸エチル抽出液を得る。I. When propolis is used, the ethyl acetate extract of propolis may be obtained by directly extracting the propolis with ethyl acetate, but it is more preferable to obtain the ethyl acetate extract according to the following procedures a to c. a. First, propolis is mixed with 5 to 10 times amount (V / W) of ethyl alcohol [concentration: 95 to 99.5% (W / W)], and 20 to 30 using a magnetic stirrer or the like.
After stirring at 0 ° C for one day or more to obtain a suspension, this suspension is filtered under normal pressure or reduced pressure to obtain an ethyl alcohol extract of propolis. b. Next, the solvent in the obtained ethyl alcohol extract is evaporated and removed using a rotary evaporator or the like. c. After this, the residue obtained is taken up in water and ethyl acetate 1:
The mixture is dispersed in a mixture of 1-1: 2, and the upper ethyl acetate layer is separated to obtain an ethyl acetate extract of propolis.
【０００７】II. プロポリスのエチルアルコール溶液を
用いる場合には、上記ａの操作は省略して、上記ｂおよ
びｃの操作を行うことによりプロポリスの酢酸エチル抽
出液を得る。 III.界面活性剤でミセル化されたプロポリスのグリセリ
ン懸濁液を用いる場合には、まず、この懸濁液を１〜２
倍量（Ｖ／Ｖ）のイソプロピルアルコールやＤＭＳＯ
（ジメチルスルホキシド）等と混合し、マグネチックス
ターラー等で撹拌しながら前記懸濁液に対して１０〜１
５倍量（Ｖ／Ｖ）の５〜１０％（Ｗ／Ｖ）食塩水と、同
じく２〜３倍量（Ｖ／Ｖ）の酢酸エチルとを加え、２０
〜３０℃で５〜１０分撹拌を続ける。撹拌後、静置また
は低速遠心して、上層の酢酸エチル層を分取することに
よりプロポリスの酢酸エチル抽出液を得る。II. When an ethyl alcohol solution of propolis is used, the above step a is omitted and the steps b and c are performed to obtain an ethyl acetate extract of propolis. III. When using a glycerin suspension of propolis that has been micellized with a surfactant, first add 1 to 2 of this suspension.
Double volume (V / V) of isopropyl alcohol and DMSO
(Dimethylsulfoxide) or the like and stirred with a magnetic stirrer or the like for 10 to 1 with respect to the suspension.
A 5-fold amount (V / V) of 5-10% (W / V) saline and a 2-3-fold amount (V / V) of ethyl acetate were added,
Continue stirring at -30 ° C for 5-10 minutes. After stirring, the mixture is left standing or centrifuged at low speed to separate the upper ethyl acetate layer to obtain an ethyl acetate extract of propolis.
【０００８】本発明の方法における第１の工程では、上
述のようにして得られるプロポリスの酢酸エチル抽出液
から溶媒を蒸散除去した後、得られた残渣をメチルアル
コール中に分散させ、不溶物を除去して、メチルアルコ
ール抽出液を得る。ここで、酢酸エチル抽出液からの溶
媒の蒸散除去は、常法に基づき、ローターリーエバポレ
ーター等により行うことができる。また、残渣を分散さ
せたメチルアルコール中からの不溶物の除去は、常法に
基づき、ろ過や低速遠心等により行うことができる。第
１の工程で用いるメチルアルコールの濃度は、９９．５
〜９９．７％（Ｗ／Ｗ）であることが好ましい。In the first step in the method of the present invention, the solvent is evaporated from the ethyl acetate extract of propolis obtained as described above, and the obtained residue is dispersed in methyl alcohol to remove insoluble matter. Removal to obtain a methyl alcohol extract. Here, the solvent can be evaporated and removed from the ethyl acetate extract by a rotary evaporator or the like based on a conventional method. In addition, the insoluble matter can be removed from the methyl alcohol in which the residue is dispersed by filtration, low-speed centrifugation or the like based on a conventional method. The concentration of methyl alcohol used in the first step is 99.5.
It is preferably ˜99.7% (W / W).
【０００９】本発明の方法では、第２の工程として、上
述した第１の工程で得られたメチルアルコール抽出液を
カラムクロマトグラフィーにかけて、７０％から１００
％（Ｖ／Ｖ）までの酸性メチルアルコール濃度勾配によ
る溶出操作を行い、酸性メチルアルコール濃度が８２〜
８８％（Ｖ／Ｖ）の範囲内の画分を得る。この第２の工
程で用いるカラムは、予め７０％（Ｖ／Ｖ）酸性メチル
アルコール（ｐＨは溶離剤のｐＨと同じ）で平衡化して
おくことが好ましい。また、カラムクロマトグラフィー
としては液体クロマトグラフィーが特に好ましく、液体
クロマトグラフィーを行う場合のカラムとしては、ＯＤ
Ｓ系カラム［例えば、東ソー（株）製のＯＤＳ ８０Ｔ
M ］等を用いることが好ましい。酸性メチルアルコール
濃度が８２〜８８％（Ｖ／Ｖ）の範囲内の画分は、例え
ば液体クロマトグラフィーの場合、オートマチックフラ
クションコレクター等を用いて常法により得ることがで
きる。また、以下のクロマト操作においては、紫外線
（例えば波長２５４ｎｍ）の吸収を自動的に測定、記録
することにより、画分の溶出を検出することができる。
なお、第２の工程で溶離剤として用いる７０〜１００％
（Ｖ／Ｖ）酸性メチアルコール、およびカラムの平衡化
のために第２の工程で用いられることがある７０％（Ｖ
／Ｖ）酸性メチルアルコールとは、それぞれ、当該濃度
（Ｖ／Ｖ）のメチルアルコールに酢酸等の揮発性酸を加
えてｐＨを３．４〜３．６に調整した液を意味する。In the method of the present invention, as a second step, the methyl alcohol extract obtained in the above-mentioned first step is subjected to column chromatography to obtain 70% to 100%.
The elution operation was performed with an acidic methyl alcohol concentration gradient up to 80% (V / V), and the acidic methyl alcohol concentration was 82-
Fractions within the range of 88% (V / V) are obtained. The column used in this second step is preferably equilibrated with 70% (V / V) acidic methyl alcohol (pH is the same as the pH of the eluent) in advance. Liquid chromatography is particularly preferable as the column chromatography, and OD is used as the column in the case of performing liquid chromatography.
S column [For example, ODS 80T manufactured by Tosoh Corporation
M ] and the like are preferably used. The fraction having an acidic methyl alcohol concentration in the range of 82 to 88% (V / V) can be obtained by a conventional method using an automatic fraction collector or the like in the case of liquid chromatography. In the following chromatographic operation, the elution of the fraction can be detected by automatically measuring and recording the absorption of ultraviolet rays (for example, wavelength 254 nm).
70 to 100% used as an eluent in the second step
(V / V) acidic methyl alcohol, and 70% (V / V) that may be used in the second step for column equilibration.
/ V) acidic methyl alcohol means a liquid in which a volatile acid such as acetic acid is added to methyl alcohol having the concentration (V / V) to adjust the pH to 3.4 to 3.6.
【００１０】本発明の方法では、第３の工程として、上
述した第２の工程で得られた酸性メチルアルコール濃度
が８２〜８８％（Ｖ／Ｖ）の範囲内の画分から溶媒を蒸
散除去した後、得られた残渣をカラムクロマトグラフィ
ーにかけて、７０％から８０％（Ｖ／Ｖ）までの酸性ア
セトニトリル濃度勾配による溶出操作を行い、酸性アセ
トニトリル濃度が７３〜７６％（Ｖ／Ｖ）の範囲内の画
分を得る。この第３の工程における前記画分からの溶媒
の蒸散除去は、常法に基づき、ローターリーエバポレー
ター等により行うことができる。得られた残渣をカラム
クロマトグラフィーにかけるにあたっては、残渣を７０
％（Ｖ／Ｖ）酸性アセトアニリルに溶解させてカラムに
注入することが好ましい。また、この第３の工程で用い
るカラムは、予め７０％（Ｖ／Ｖ）酸性アセトニトリル
（ｐＨは溶離剤のｐＨと同じ）で平衡化しておくことが
好ましい。カラムクロマトグラフィーとしては、液体ク
ロマトグラフィーが特に好ましく、液体クロマトグラフ
ィーを行う場合のカラムとしては、ＯＤＳ系カラム［例
えば、東ソー（株）製のＯＤＳ ８０ＴM ］等を用いる
ことが好ましい。酸性アセトアニリル濃度が７３〜７６
％（Ｖ／Ｖ）の範囲内の画分は、例えば液体クロマトグ
ラフィーの場合、オートマチックフラクションコレクタ
ー等を用いて常法により得ることができる。なお、第３
の工程で溶離剤として用いる７０〜８０％（Ｖ／Ｖ）酸
性メチアルコール、およびカラムの平衡化のために第３
の工程で用いられることがある７０％（Ｖ／Ｖ）酸性ア
セトニトリルとは、それぞれ、当該濃度（Ｖ／Ｖ）のア
セトニトリルに酢酸等の揮発性酸を加えてｐＨを３．４
〜３．６に調整した液を意味する。In the method of the present invention, in the third step, the solvent is evaporated from the fraction obtained in the above-mentioned second step and having a concentration of acidic methyl alcohol within the range of 82 to 88% (V / V). Then, the obtained residue is subjected to column chromatography to perform an elution operation with a gradient of acidic acetonitrile concentration from 70% to 80% (V / V), and the concentration of acidic acetonitrile is within the range of 73 to 76% (V / V). Get the fraction of. The evaporation of the solvent from the fraction in the third step can be carried out by a rotary evaporator or the like based on a conventional method. When the obtained residue is subjected to column chromatography, the residue is
% (V / V) Acidic acetanilyl is preferably dissolved and injected into the column. The column used in the third step is preferably equilibrated with 70% (V / V) acidic acetonitrile (pH is the same as the pH of the eluent) in advance. The column chromatography, particularly preferably liquid chromatography, the column for performing liquid chromatography, it is preferable to use an ODS-based column [e.g., Tosoh Corporation of ODS 80T M] or the like. Acid acetanilyl concentration is 73-76
The fraction within the range of% (V / V) can be obtained by a conventional method using, for example, an automatic fraction collector in the case of liquid chromatography. The third
70-80% (V / V) acidic methyl alcohol used as an eluent in the step of step 3, and a third column for equilibration of the column.
The 70% (V / V) acidic acetonitrile that may be used in the step of (3) is adjusted to pH 3.4 by adding a volatile acid such as acetic acid to acetonitrile at the concentration (V / V).
It means a solution adjusted to ~ 3.6.
【００１１】本発明の方法では、第４の工程として、上
述した第３の工程で得られた、酸性アセトニトリル濃度
が７３〜７６％（Ｖ／Ｖ）の範囲内の画分の溶媒を蒸散
除去した後、得られた残渣をカラムクロマトグラフィー
にかけて、７５％から９０％（Ｖ／Ｖ）までのエチルア
ルコール濃度勾配による溶出操作を行い、エチルアルコ
ール濃度が８０〜８５％（Ｖ／Ｖ）の範囲内の画分を得
る。この第４の工程における前記画分からの溶媒の蒸散
除去は、常法に基づき、ローターリーエバポレーター等
により行うことができる。得られた残渣をカラムクロマ
トグラフィーにかけるにあたっては、残渣を７０％（Ｖ
／Ｖ）アセトニトリルに溶解させてカラムに注入するこ
とが好ましい。また、この第４の工程で用いるカラム
は、予め７５％（Ｖ／Ｖ）エチルアルコールで平衡化し
ておくことが好ましい。カラムクロマトグラフィーとし
ては、液体クロマトグラフィーが特に好ましく、液体ク
ロマトグラフィーを行う場合のカラムとしては、ＯＤＳ
系カラム［例えば、東ソー（株）製のＯＤＳ ８０
ＴM ］等を用いることが好ましい。エチルアルコール濃
度が８０〜８５％（Ｖ／Ｖ）の範囲内の画分は、例えば
液体クロマトグラフィーの場合、オートマチックフラク
ションコレクター等を用いて常法により得ることができ
る。紫外線（例えば波長２５４ｎｍ）の吸収を自動的に
測定、記録することにより画分の溶出を確認する。この
際、主画分が２つ得られた場合は、両方の主画分を得て
もよいが、エタノール濃度にして８２〜８３％（Ｖ／
Ｖ）近辺で溶出される後方の画分を得た方がより好まし
い。In the method of the present invention, as the fourth step, the solvent of the fraction obtained in the above-mentioned third step and having a concentration of acidic acetonitrile within the range of 73 to 76% (V / V) is removed by evaporation. After that, the obtained residue was subjected to column chromatography to perform an elution operation with an ethyl alcohol concentration gradient from 75% to 90% (V / V), and the ethyl alcohol concentration was in the range of 80 to 85% (V / V). Get the fraction inside. The evaporation of the solvent from the fraction in the fourth step can be carried out by a rotary evaporator or the like based on a conventional method. When the obtained residue was subjected to column chromatography, the residue was 70% (V
/ V) It is preferable to dissolve in acetonitrile and inject it into the column. The column used in the fourth step is preferably equilibrated with 75% (V / V) ethyl alcohol in advance. Liquid chromatography is particularly preferable as column chromatography, and ODS is used as a column for liquid chromatography.
System column [eg, ODS 80 manufactured by Tosoh Corporation]
It is preferable to use T M ]. The fraction having an ethyl alcohol concentration in the range of 80 to 85% (V / V) can be obtained by a conventional method using an automatic fraction collector or the like in the case of liquid chromatography. The elution of the fraction is confirmed by automatically measuring and recording the absorption of ultraviolet rays (for example, wavelength 254 nm). At this time, when two main fractions are obtained, both main fractions may be obtained, but the ethanol concentration is 82 to 83% (V / V).
V) It is more preferable to obtain a rear fraction that is eluted in the vicinity.
【００１２】このようにして得られる、エチルアルコー
ル濃度が８０〜８５％（Ｖ／Ｖ）の範囲内の画分中に
は、プロポリス中に含まれていた殺腫瘍細胞作用および
抗菌作用に優れた物質が高濃度で含まれている。したが
って、本発明の方法に基づいて得られた、上記エチルア
ルコール濃度が８０〜８５％（Ｖ／Ｖ）の範囲内の画分
を水、生理食塩水等により希釈したものを食することに
より、前述したエチルアルコール溶液やグリセリン懸濁
液等のかたちでプロポリスを食する場合よりも、プロポ
リスが有する殺腫瘍細胞作用および抗菌作用をより効率
的に利用することが可能となる。また本発明の方法は、
上述のようにして得た、エチルアルコール濃度が８０〜
８５％（Ｖ／Ｖ）の範囲内の画分の溶媒を蒸散除去して
残渣を得る工程を含んでいてもよい。そしてこの残渣そ
のもの、あるいは得られた残渣を食物に添加したものを
食することにより、プロポリスが有する殺腫瘍細胞作用
および抗菌作用をより効率的に利用することが可能とな
る。さらに、本発明の方法に基づいて得られたプロポリ
ス抽出物は、医薬品製造原料等として利用することもで
きる。[0012] The thus obtained fraction having an ethyl alcohol concentration within the range of 80 to 85% (V / V) was excellent in the tumoricidal action and antibacterial action contained in propolis. High concentration of substance. Therefore, by eating a fraction obtained by the method of the present invention, wherein the ethyl alcohol concentration is within the range of 80 to 85% (V / V), diluted with water, physiological saline or the like, The tumor cell-killing action and antibacterial action of propolis can be utilized more efficiently than when propolis is eaten in the form of the above-mentioned ethyl alcohol solution or glycerin suspension. The method of the present invention also comprises
The ethyl alcohol concentration obtained as described above was 80 to
The method may include a step of evaporating off the solvent of the fraction within a range of 85% (V / V) to obtain a residue. By eating this residue itself or the one obtained by adding the obtained residue to food, it becomes possible to more efficiently utilize the tumoricidal cell action and antibacterial action of propolis. Furthermore, the propolis extract obtained by the method of the present invention can also be used as a raw material for producing pharmaceutical products and the like.
【００１３】[0013]
【実施例】以下、本発明の実施例について説明する。 実施例１ (1) 第１の工程 まず、ブラジル産プロポリス１００ｇを１０倍量（Ｖ／
Ｖ）の９９．５％（Ｗ／Ｗ）エチルアルコールと混合
し、マグネチックスターラーを用いて室温下で撹拌し
た。得られた懸濁液をろ紙を通して減圧ろ過して、プロ
ポリスのエチルアルコール抽出液を得た。次いで、得ら
れたエチルアルコール抽出液の溶媒をローターリーエバ
ポレーターを用いて蒸散除去し、得られた残渣３０ｇを
水と酢酸エチルとの１：１混液に分散させ、上層の酢酸
エチル層を分取することによりプロポリスの酢酸エチル
抽出液を得た。この後、得られた酢酸エチル抽出液の溶
媒をローターリーエバポレーターにより蒸散除去し、得
られた残渣２０ｇを９９．５％（Ｗ／Ｗ）メチルアルコ
ールに分散させた後に不溶物を低速遠心により取除い
て、メチルアルコール抽出液を得た。EXAMPLES Examples of the present invention will be described below. Example 1 (1) First Step First, 100 g of Brazilian propolis was added in a 10-fold amount (V /
V) was mixed with 99.5% (W / W) ethyl alcohol and stirred at room temperature using a magnetic stirrer. The obtained suspension was filtered under reduced pressure through a filter paper to obtain an ethyl alcohol extract of propolis. Then, the solvent of the obtained ethyl alcohol extract was evaporated and removed using a rotary evaporator, 30 g of the obtained residue was dispersed in a 1: 1 mixture of water and ethyl acetate, and the upper ethyl acetate layer was collected. By doing so, an ethyl acetate extract of propolis was obtained. After that, the solvent of the obtained ethyl acetate extract was evaporated and removed by a rotary evaporator, 20 g of the obtained residue was dispersed in 99.5% (W / W) methyl alcohol, and insoluble materials were collected by low-speed centrifugation. Then, a methyl alcohol extract was obtained.
【００１４】(2) 第２の工程 上記(1) で得られたメチルアルコール抽出液を、７０％
（Ｖ／Ｖ）酸性メチルアルコール［７０％（Ｖ／Ｖ）メ
チルアルコールのｐＨを酢酸で３．５に調整したもの］
で平衡化した高速液体クロマトグラフィー用ＯＤＳ系カ
ラム［商品名：東ソーＯＤＳ ８０ＴM 、東ソー（株）
製］に注入し、７０％から１００％（Ｖ／Ｖ）までの酸
性メチルアルコール（ｐＨは酢酸で３．５に調整）濃度
勾配による傾斜溶離を行った。この後、酸性メチルアル
コール濃度８２〜８８％の画分をオートマチックフラク
ションコレクターを用いて分取した。(2) Second step 70% of the methyl alcohol extract obtained in the above (1)
(V / V) acidic methyl alcohol [70% (V / V) methyl alcohol pH adjusted to 3.5 with acetic acid]
In equilibrated for high performance liquid chromatography ODS-based column [product name: TOSOH ODS 80T M, Tosoh Corporation
The product was injected into the column] and the gradient elution was performed with a concentration gradient of acidic methyl alcohol (pH adjusted to 3.5 with acetic acid) from 70% to 100% (V / V). Then, a fraction having an acidic methyl alcohol concentration of 82 to 88% was collected using an automatic fraction collector.
【００１５】(3) 第３の工程 まず、上記(2) で得られた酸性メチルアルコール濃度８
２〜８８％の画分を１つに集め、溶媒をローターリーエ
バポレーターにより蒸散除去して、残渣２００ｍｇを得
た。次いで、得られた残渣を７０％（Ｖ／Ｖ）酸性アセ
トニトリル［７０％（Ｖ／Ｖ）アセトニトリルのｐＨを
酢酸で３．５に調整したもの］に溶解させ、得られた溶
液を、７０％酸性アセトニトリル［７０％（Ｖ／Ｖ）ア
セトニトリルのｐＨを酢酸で３．５に調整したもの］で
平衡化した高速液体クロマトグラフィー用ＯＤＳ系カラ
ム［商品名：東ソーＯＤＳ ８０ＴM 、東ソー（株）
製］に注入し、７０〜８０％（Ｖ／Ｖ）の酸性アセトニ
トリル（ｐＨは酢酸で３．５に調整）濃度勾配による傾
斜溶離を行った。この後、酸性アセトニトリル濃度７３
〜７６％の画分をオートマチックフラクションコレクタ
ーを用いて分取した。(3) Third step First, the concentration of acidic methyl alcohol obtained in (2) above is 8
Fractions of 2 to 88% were collected together and the solvent was evaporated off by a rotary evaporator to obtain 200 mg of residue. Then, the obtained residue was dissolved in 70% (V / V) acidic acetonitrile [70% (V / V) acetonitrile whose pH was adjusted to 3.5 with acetic acid], and the obtained solution was adjusted to 70%. acidic acetonitrile high performance liquid chromatography ODS system column equilibrated with [70% (V / V) the pH of acetonitrile which was adjusted to 3.5 with acetic acid] [trade name: TOSOH ODS 80T M, Tosoh Corporation
[Preparation], and 70-80% (V / V) acidic acetonitrile (pH adjusted to 3.5 with acetic acid) concentration gradient elution was performed. After this, the concentration of acidic acetonitrile 73
Fractions of ˜76% were collected using an automatic fraction collector.
【００１６】(4) 第４の工程 まず、上記(3) で得られた酸性アセトニトリル濃度７３
〜７６％の画分を１つに集め、溶媒をローターリーエバ
ポレーターにより蒸散除去して、残渣５０ｍｇを得た。
次いで、得られた残渣を７０％（Ｖ／Ｖ）アセトニトリ
ルに溶解させ、得られた溶液を、７５％（Ｖ／Ｖ）エチ
ルアルコールで平衡化した高速液体クロマトグラフィー
用ＯＤＳ系カラム［商品名：東ソーＯＤＳ ８０ＴM 、
東ソー（株）製］に注入し、７５〜９０％（Ｖ／Ｖ）の
エチルアルコール濃度勾配による傾斜溶離を行った。こ
の後、エチルアルコール濃度８０〜８５％の画分をオー
トマチックフラクションコレクターを用いて分取した。
紫外線（波長２５４ｎｍ）を吸収する主画分が２つ得ら
れたので、後方の画分を得た。得られた画分を１つに集
めて、本発明の目的物であるプロポリス抽出物（Ａ）を
得た。(4) Fourth step First, the acidic acetonitrile concentration obtained in the above (3) is 73
Fractions of ˜76% were collected together and the solvent was evaporated off on a rotary evaporator to give a residue of 50 mg.
Next, the obtained residue was dissolved in 70% (V / V) acetonitrile, and the obtained solution was equilibrated with 75% (V / V) ethyl alcohol. An ODS column for high performance liquid chromatography [trade name: Tosoh ODS 80T M,
Tosoh Co., Ltd.] and gradient elution was performed with a 75-90% (V / V) ethyl alcohol concentration gradient. Then, a fraction having an ethyl alcohol concentration of 80 to 85% was collected using an automatic fraction collector.
Since two main fractions that absorb ultraviolet rays (wavelength 254 nm) were obtained, a rear fraction was obtained. The obtained fractions were collected to obtain a propolis extract (A), which was the object of the present invention.
【００１７】実施例２ まず、界面活性剤でミセル化されたプロポリスのグリセ
リン懸濁液［商品名：ＥＳＴＡ ＰＲＯＮＴＯ、日本プ
ロポリス（株）製］１００ｍｌと１．５倍量（Ｖ／Ｖ）
のイソプロピルアルコールとを混合し、マグネチックス
ターラーで撹拌しながら前記グリセリン懸濁液に対して
７倍量（Ｖ／Ｖ）の１０％（Ｗ／Ｖ）食塩水と同じく２
倍量（Ｖ／Ｖ）の酢酸エチルとを加え、室温下で約５分
撹拌を続けた。撹拌後、静置して、上層の酢酸エチル層
を分取することによりプロポリスの酢酸エチル抽出液を
得た。次いで、得られた酢酸エチル抽出液の溶媒をロー
ターリーエバポレーターにより蒸散除去し、得られた残
渣１０ｇを９９．５％（Ｗ／Ｗ）メチルアルコールに分
散させた後に不溶物を低速遠心により取除いて、メチル
アルコール抽出液を得た。この後は実施例１(2) 〜(4)
と同様の操作を行ってエチルアルコール濃度８０〜８５
％の画分を分取し、得られた画分を１つに集めて、本発
明の目的物であるプロポリス抽出物（Ｂ）を得た。Example 2 First, 100 ml of a glycerol suspension of propolis micellarized with a surfactant [trade name: ESTA PRONTO, manufactured by Nippon Propolis Co., Ltd.] and 1.5 times volume (V / V).
Isopropyl alcohol and mixed with a magnetic stirrer and mixed with 7 times amount (V / V) of 10% (W / V) saline solution with respect to the glycerin suspension in the same manner.
Double volume (V / V) of ethyl acetate was added, and stirring was continued at room temperature for about 5 minutes. After stirring, the mixture was allowed to stand and the upper ethyl acetate layer was separated to obtain an ethyl acetate extract of propolis. Then, the solvent of the obtained ethyl acetate extract was evaporated and removed by a rotary evaporator, 10 g of the obtained residue was dispersed in 99.5% (W / W) methyl alcohol, and then insoluble matter was removed by low-speed centrifugation. Thus, a methyl alcohol extract was obtained. After this, Example 1 (2) to (4)
Perform the same operation as above to obtain an ethyl alcohol concentration of 80-85.
% Fractions were collected and the obtained fractions were collected to obtain a propolis extract (B), which is the object of the present invention.
【００１８】殺腫瘍細胞作用試験 実施例１(4) で得られたプロポリス抽出物（Ａ）から溶
媒をローターリーエバポレーターにより蒸散除去して残
渣（以下、これを残渣Ａというが、これも本発明の目的
物であるプロポリス抽出物に相当する）を得、この残渣
Ａを被験物質として用いて、以下のようにして殺癌細胞
作用の試験を行った。まず、ヒト肝癌ＨｕＨ１３株を液
体培地（１０％仔ウシ血清を含むイーグルＭＥＭ培地）
で培養した後、１×１０5 細胞／ｍｌのＨｕＨ１３株浮
游液に調製した。次いで、この浮游液に、被験物質のＤ
ＭＳＯ溶液を被験物質の濃度が所定濃度となるように添
加した後、この液をプラスチックプレートのウエルに
０．１ｍｌ分注して、５％炭酸ガスと９５％空気との通
気雰囲気下、３７℃で４８時間培養した。次に、プレー
ト壁に付着した細胞をトリプシンとＥＤＴＡ（エチレン
ジアミンテトラ酢酸）とで処理して剥離し、剥離した細
胞を新しい別のプラスチックプレートに移して、５％炭
酸ガスと９５％空気との通気雰囲気下、３７℃で培養し
た。このときの培養液としては、同上の血清を含むイー
グル培地を用いた。 Tumor-killing cell activity test The solvent was evaporated from the propolis extract (A) obtained in Example 1 (4) by a rotary evaporator to remove a residue (hereinafter referred to as residue A, which is also referred to as the present invention. (Corresponding to the propolis extract, which is the desired product), and using this residue A as a test substance, a test for the action of cancer-killing cells was performed as follows. First, human hepatoma HuH13 strain is a liquid medium (Eagle MEM medium containing 10% calf serum)
After culturing in, a suspension of HuH13 strain containing 1 × 10 5 cells / ml was prepared. Then, add D of the test substance to this floating solution.
After adding the MSO solution so that the concentration of the test substance becomes a predetermined concentration, 0.1 ml of this solution was dispensed into the wells of the plastic plate, and the mixture was placed in an aeration atmosphere of 5% carbon dioxide gas and 95% air at 37 ° C. The cells were cultured for 48 hours. Next, the cells attached to the plate wall are treated with trypsin and EDTA (ethylenediaminetetraacetic acid) to detach them, and the detached cells are transferred to another new plastic plate and aerated with 5% carbon dioxide gas and 95% air. The culture was carried out at 37 ° C in an atmosphere. As the culture medium at this time, an Eagle medium containing the same serum as above was used.
【００１９】２４時間後、プレート壁に付着している生
細胞をＭＴＴ法｛Slater,T.F. ら、Biochim. Biophys.
Acta 77 (1963) 353〜393 。ＭＴＴ［３−（４，５−ジ
メチルチアゾール−２−イル）−２，５−ジフェニルテ
トラゾリウム ブロマイド］を細胞に取り込ませ、これ
を還元することにより生じるＭＴＴフォルマザンの呈色
反応を利用する方法｝により定量し、被験物質で処理し
た際の生細胞数と無処理対照生細胞数との割合を算定す
ることにより生存率を求めて、生存率の高低により殺癌
細胞作用の強弱を評価した。なお、コントロールでは被
験物質に代えて、被験物質を溶解するＤＭＳＯを用い
た。また、対照細胞としてはウサギ腎ＲＫ１３株と初代
ウサギ腎細胞とを用い、これらの細胞には、プラスチッ
クプレート中で単層培養して集密的になったときに、被
験物質のＤＭＳＯ溶液を被験物質の濃度が所定濃度とな
るように加え、以後はヒト肝癌ＨｕＨ１３株の場合と同
様にして、生存率を求めた。これらの結果を図１に示
す。After 24 hours, living cells attached to the plate wall were subjected to the MTT method {Slater, TF et al., Biochim. Biophys.
Acta 77 (1963) 353-393. MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] is incorporated into cells and reduced by reducing MTT formazan. The survival rate was determined by quantifying and calculating the ratio between the number of living cells when treated with the test substance and the number of untreated control living cells, and the strength of the cancer-killing cell action was evaluated based on the level of the survival rate. In the control, DMSO in which the test substance was dissolved was used instead of the test substance. In addition, rabbit kidney RK13 strain and primary rabbit kidney cells were used as control cells, and these cells were tested with DMSO solution of the test substance when they became confluent by monolayer culture in a plastic plate. The substance was added so as to have a predetermined concentration, and thereafter, the survival rate was calculated in the same manner as in the case of the human liver cancer HuH13 strain. The results are shown in FIG.
【００２０】図１から明らかなように、本発明の方法に
より得られるプロポリス抽出物の１つである残渣Ａは、
ヒト肝癌ＨｕＨ１３株に対しては優れた殺傷作用を示
し、ウサギ腎ＲＫ１３株や初代ウサギ腎細胞に対しては
比較的低い殺傷作用しか示さない。このことから、残渣
Ａは殺癌細胞作用に優れているということがわかる。As is clear from FIG. 1, the residue A, which is one of the propolis extracts obtained by the method of the present invention, is
It shows an excellent killing action against the human liver cancer HuH13 strain, and a relatively low killing action against the rabbit kidney RK13 strain and primary rabbit kidney cells. From this, it is understood that the residue A is excellent in the action of killing cancer cells.
【００２１】抗菌作用試験 上述した残渣Ａを被験物質として用いて、以下のように
して抗菌作用の試験を行った。まず、表１に示す被験菌
をそれぞれの培地で１００〜１０００個／ｍｌに希釈し
て、９６穴Ｕ底マイクロプレートのＡ列にはウエル当た
り１９６μl 、Ｂ列からＨ列までは１００μl 、それぞ
れ分注した。次いで、残渣Ａを２５mg／ｍl の濃度で含
有するＤＭＳＯ溶液４μl をＡ列ウエルに加え、撹拌
後、１００μl をＢ列ウエルに移して撹拌し、以下、Ｃ
〜Ｆ列まで順に希釈操作を繰返した。希釈操作終了後、
感圧フィルム（Pressure Sensitive Film 、商品名：Fa
lcon 3073 、Falcon社製）でＡ〜Ｆ列のウエルを封止
し、３７℃で培養して毎日観察した。抗菌作用の判定
は、残渣ＡのＤＭＳＯ溶液を添加しなかった培地中で被
験菌の増殖が認められた時点で行った。なお、被験菌の
うちのアルギニン分解マイコプラズマの培地としては
０．２５％アルギニン−ＨＣｌ添加ＳＰ−４培地（ｐＨ
７．２）を用い、グルコース分解マイコプラズマの培地
としては０．２５％グルコース添加ＳＰ−４培地（ｐＨ
７．８）を用い、培地の色調変化で増殖を判定した。ま
た、細菌の培地としてはBrain Heart Infusion培地（脳
肝浸出液培地、Difco 社製）を用い、菌塊の形成で増殖
を判定した。これらの結果を表１および表２に示す。 Antibacterial Action Test Using the above residue A as a test substance, an antibacterial action test was conducted as follows. First, the test bacteria shown in Table 1 were diluted to 100 to 1000 cells / ml with each medium, and 196 μl per well in the A column of the 96-well U-bottom microplate, 100 μl from the B column to the H column, respectively. I made a note. Then, 4 μl of DMSO solution containing the residue A at a concentration of 25 mg / ml was added to the wells in row A, and after stirring, 100 μl was transferred to the wells in row B and stirred.
The diluting operation was repeated in order up to column F. After the dilution operation,
Pressure Sensitive Film, product name: Fa
Wells in rows A to F were sealed with lcon 3073, manufactured by Falcon), cultured at 37 ° C., and observed daily. The antibacterial action was determined when the growth of the test bacterium was observed in the medium in which the DMSO solution of the residue A was not added. As a medium for arginine-degrading mycoplasma among the test bacteria, SP-4 medium (pH: 0.25% arginine-HCl) was added.
7.2) was used as a glucose-degrading mycoplasma medium containing 0.25% glucose-containing SP-4 medium (pH
7.8) was used to determine the growth based on the color change of the medium. A Brain Heart Infusion medium (brain liver exudate medium, manufactured by Difco) was used as a bacterial medium, and the growth was determined by the formation of bacterial mass. The results are shown in Tables 1 and 2.
【００２２】[0022]
【表１】 [Table 1]
【００２３】[0023]
【表２】 [Table 2]
【００２４】表１および表２から明らかなように、本発
明の方法により得られるプロポリス抽出物の１つである
残渣Ａは、特定のマイコプラズマおよび菌に対して優れ
た抗菌作用を示す。As is clear from Tables 1 and 2, the residue A, which is one of the propolis extracts obtained by the method of the present invention, exhibits an excellent antibacterial action against specific mycoplasma and fungi.
【００２５】細胞周期およびＤＮＡ合成に及ぼす影響試
験 前述した殺癌細胞作用試験の場合と同様にして得た残渣
Ａがヒト肝癌ＨｕＨ１３株の細胞周期およびＤＮＡ合成
に及ぼす影響を、以下のようにして解析した。まず、１
０％仔ウシ血清を含むイーグルＭＥＭ培地中、３７℃、
５％炭酸ガスと９５％空気との通気雰囲気下で所定期間
培養した細胞株を７０％（Ｖ／Ｖ）エタノールで処理し
て、各細胞を固定化すると共に細胞膜の透過性を高め
た。次いで、各細胞を塩酸で処理して、ＤＮＡを変性さ
せた。次に、各細胞を３０分間ＢｒｄＵ（ブロモデオキ
シウリジン）で処理した後、ＰＩ（ヨウ化プロピジュウ
ム）を各細胞に取り込ませてＤＮＡと結合させた。ま
た、ＰＩを取り込ませるのと同時に、ＦＩＴＣ（フルオ
レッセント イソチオシアネート）で標識した抗Ｂｒｄ
Ｕモノクローナル抗体を各細胞に取り込ませた。この
後、ＤＮＡフローサイトメーター［商品名：ＦＡＣＳｃ
ａｎ、Becton Dickinson Immunocytometry Systems社
製］により、ＰＩを取り込んだ各細胞内ＤＮＡを定量し
て細胞株におけるＤＮＡ量の分布パターンを調べ、同時
に、ＤＮＡ中に取り込まれたＢｒｄＵの有無を調べた。
なお、残渣ＡはＤＭＳＯに溶解させて、最終濃度が２μ
ｇ／ｍｌとなるように培養開始時に上記培養液中に添加
した（以下、このときの細胞群を試験群という）。ま
た、コントロールでは残渣ＡのＤＭＳＯ溶液に代えてＤ
ＭＳＯを添加した。そして、１つの対照群では、残渣Ａ
のＤＭＳＯ溶液に代えてコルセミドのＤＭＳＯ溶液を、
コルセミドの最終濃度が１μｇ／ｍｌとなるように添加
し（以下、このときの細胞群を対照群１という）、他の
対照群では前記コルセミドのＤＭＳＯ溶液の添加と同時
に、前記残渣ＡのＤＭＳＯ溶液を添加した（以下、この
ときの細胞群を対照群２という）。 Test for effects on cell cycle and DNA synthesis
Test The effects of residue A obtained in the same manner as in the above-mentioned cancer cell killing test on the cell cycle and DNA synthesis of human liver cancer HuH13 strain were analyzed as follows. First, 1
In Eagle MEM medium containing 0% calf serum at 37 ° C,
A cell line cultured for a predetermined period under an aeration atmosphere of 5% carbon dioxide and 95% air was treated with 70% (V / V) ethanol to immobilize each cell and enhance the permeability of the cell membrane. Each cell was then treated with hydrochloric acid to denature the DNA. Next, each cell was treated with BrdU (bromodeoxyuridine) for 30 minutes, and then PI (propidium iodide) was incorporated into each cell to bind to DNA. In addition, at the same time as incorporating PI, anti-Brd labeled with FITC (fluorescent isothiocyanate)
U monoclonal antibody was incorporated into each cell. After this, DNA flow cytometer [Product name: FACSc
An, manufactured by Becton Dickinson Immunocytometry Systems, Inc.] was used to quantify each intracellular DNA incorporating PI, and the distribution pattern of the amount of DNA in the cell line was examined, and at the same time, the presence or absence of BrdU incorporated in the DNA was examined.
The residue A was dissolved in DMSO to give a final concentration of 2μ.
It was added to the above-mentioned culture solution at the start of the culture so as to be g / ml (hereinafter, the cell group at this time is called a test group). In the control, D was used instead of the DMSO solution of residue A.
MSO was added. And in one control group, residue A
DMSO solution of colcemid instead of DMSO solution of
The final concentration of colcemid was added to be 1 μg / ml (hereinafter, the cell group at this time is referred to as control group 1), and in other control groups, the DMSO solution of residue A was added at the same time as the DMSO solution of colcemid was added. Was added (hereinafter, the cell group at this time is referred to as control group 2).
【００２６】この結果、残渣Ａを添加した試験群では、
Ｇ1 期（ＤＮＡ合成準備期）およびＭ期（分裂期）にあ
る細胞の割合よりもＳ期（ＤＮＡ合成期）にある細胞の
割合の方が相対的に高くなってた。一方、コントロール
では、Ｇ1 期にある細胞の割合が最も高く、次いで、Ｍ
期にある細胞、Ｓ期にある細胞が多く見られた。そし
て、対照群１ではＭ期にある細胞の割合が最も高く、対
照群２ではＳ期にある細胞の割合が圧倒的に高かった。
このことから、残渣Ａはヒト肝癌ＨｕＨ１３株の分裂を
Ｓ期で停止させる作用を有し、かつ、Ｇ1 期からＳ期へ
の移行およびＭ期からＧ1 期への移行に対しては特別な
影響は及ぼさないことがわかる。また、残渣Ａを添加し
た試験群および対照群２ではＤＮＡ中へのＢｒｄＵの取
り込みが確認されなかったのに対して、対照群１ではＤ
ＮＡ中へのＢｒｄＵの取り込みが確認された。このこと
から、残渣Ａは、ヒト肝癌ＨｕＨ１３株におけるＤＮＡ
合成を抑制していることがわかる。As a result, in the test group to which the residue A was added,
The proportion of cells in S phase (DNA synthesis phase) was relatively higher than the proportion of cells in G 1 phase (preparatory phase for DNA synthesis) and M phase (division phase). On the other hand, in the control, the ratio of cells in the G 1 phase was the highest, followed by M
Many cells in the S phase and cells in the S phase were found. The control group 1 had the highest proportion of cells in the M phase, and the control group 2 had an overwhelmingly high proportion of cells in the S phase.
Therefore, the residue A has an action of stopping the division of the human liver cancer HuH13 strain at the S phase, and has a special effect on the transition from the G 1 phase to the S phase and the M phase to the G 1 phase. It can be seen that this has no effect. Moreover, BrdU incorporation into the DNA was not confirmed in the test group and the control group 2 to which the residue A was added, whereas in the control group 1, D
BrdU incorporation into NA was confirmed. From this, the residue A is the DNA in the human liver cancer HuH13 strain.
It can be seen that the synthesis is suppressed.
【００２７】[0027]
【発明の効果】以上説明したように、本発明の方法によ
り得られるプロポリス抽出物は、殺腫瘍細胞作用および
抗菌作用に優れている。また、ＤＮＡ合成抑制作用等も
有している。したがって本発明によれば、プロポリスが
有する生物活性作用、特に殺腫瘍細胞作用および抗菌作
用を、飲食物、医薬品製造原料等のかたちでより効率的
に利用することが可能となる。As described above, the propolis extract obtained by the method of the present invention is excellent in tumoricidal cell action and antibacterial action. It also has a DNA synthesis inhibitory action and the like. Therefore, according to the present invention, it becomes possible to more efficiently utilize the bioactive action of propolis, in particular, the tumoricidal cell action and antibacterial action, in the form of foods and drinks, raw materials for producing pharmaceuticals and the like.
【図１】は、本発明の方法により得られたプロポリス抽
出物の殺腫瘍細胞作用の試験結果を示すグラフである。FIG. 1 is a graph showing the test results of the tumoricidal cell action of the propolis extract obtained by the method of the present invention.
Claims (1)
媒を蒸散除去した後、得られた残渣をメチルアルコール
中に分散させ、不溶物を除去して、メチルアルコール抽
出液を得る工程、 前記メチルアルコール抽出液をカラムクロマトグラ
フィーにかけて、７０％から１００％（Ｖ／Ｖ）までの
酸性メチルアルコール濃度勾配による溶出操作を行い、
酸性メチルアルコール濃度が８２〜８８％（Ｖ／Ｖ）の
範囲内の画分を得る工程、 前記画分から溶媒を蒸散除去した後、得られた残渣
をカラムクロマトグラフィーにかけて、７０％から８０
％（Ｖ／Ｖ）までの酸性アセトニトリル濃度勾配による
溶出操作を行い、酸性アセトニトリル濃度が７３〜７６
％（Ｖ／Ｖ）の範囲内の画分を得る工程、 前記画分から溶媒を蒸散除去した後、得られた残渣
をカラムクロマトグラフィーにかけて、７５％から９０
％（Ｖ／Ｖ）までのエチルアルコール濃度勾配による溶
出操作を行い、エチルアルコール濃度が８０〜８５％
（Ｖ／Ｖ）の範囲内の画分を得る工程とを含むことを特
徴とする、プロポリス抽出物の製造方法。1. A step of evaporating and removing a solvent from an ethyl acetate extract of propolis and then dispersing the obtained residue in methyl alcohol to remove insolubles to obtain a methyl alcohol extract. The liquid is subjected to column chromatography to perform an elution operation with an acidic methyl alcohol concentration gradient from 70% to 100% (V / V),
A step of obtaining a fraction in which the concentration of acidic methyl alcohol is within a range of 82 to 88% (V / V), the solvent is evaporated from the fraction, and the obtained residue is subjected to column chromatography to obtain 70% to 80%.
The elution operation was performed with an acidic acetonitrile concentration gradient up to 50% (V / V), and the acidic acetonitrile concentration was 73 to 76.
% (V / V) to obtain a fraction, after the solvent is evaporated from the fraction, the obtained residue is subjected to column chromatography to obtain 75% to 90%.
The elution operation was performed with an ethyl alcohol concentration gradient up to 50% (V / V), and the ethyl alcohol concentration was 80 to 85%.
And a step of obtaining a fraction within the range of (V / V).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3162596A JPH0749365B2 (en) | 1991-07-03 | 1991-07-03 | Method for producing propolis extract |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3162596A JPH0749365B2 (en) | 1991-07-03 | 1991-07-03 | Method for producing propolis extract |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH05271031A JPH05271031A (en) | 1993-10-19 |
| JPH0749365B2 true JPH0749365B2 (en) | 1995-05-31 |
Family
ID=15757603
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3162596A Expired - Lifetime JPH0749365B2 (en) | 1991-07-03 | 1991-07-03 | Method for producing propolis extract |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0749365B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109674730A (en) * | 2019-02-12 | 2019-04-26 | 广州小蜜蜂生物科技有限公司 | A kind of Mel extract and its preparation method and application |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09143179A (en) * | 1995-11-24 | 1997-06-03 | Eiken Chem Co Ltd | Benzopyran derivative derived from propolis |
| JPH09183724A (en) * | 1995-12-29 | 1997-07-15 | Eiken Chem Co Ltd | Naphthalenecarboxylic acid derivative |
| JP2002332257A (en) * | 2001-05-11 | 2002-11-22 | Biomedeikusu:Kk | Method for extracting and purifying 3-[4-hydroxy-3, 5-bis (3-methyl-2-butenyl) phenyl]-2-propenoic acid from propolis |
| KR100618497B1 (en) * | 2005-02-28 | 2006-08-31 | 거창군 (관리부서 : 거창군 농업기술센터) | How to extract quercetin from propolis |
| KR100841247B1 (en) * | 2006-05-25 | 2008-06-26 | 김영생 | Plant Nutrients Containing Bongyo Extract |
-
1991
- 1991-07-03 JP JP3162596A patent/JPH0749365B2/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109674730A (en) * | 2019-02-12 | 2019-04-26 | 广州小蜜蜂生物科技有限公司 | A kind of Mel extract and its preparation method and application |
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| Publication number | Publication date |
|---|---|
| JPH05271031A (en) | 1993-10-19 |
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| A01 | Written decision to grant a patent or to grant a registration (utility model) |
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