JP2004517172A - Natural fluorescent dyes obtained from marine invertebrates, compositions containing said dyes, and uses thereof - Google Patents

Abstract

The present invention discloses steps for the extraction, purification and characterization of fluorescent dyes from the marine echinoderm sea cucumber Holothuria scabra, the dye-containing compositions and various applications of the dyes.

Description

【０１５７】
【表２】

【０１５８】
【表３】

【０１５９】
現在市販の染料に勝る利点
１．前記染料は、天然起源由来の染料でありかつ合成ではないので、非放射性である。
【０１６０】
２．この染料はその単一形状において、３種の異なる合成フルオロクロム類に匹敵し、同一蛍光色発光を示す。
【０１６１】
３．前記染料は迅速マイクロスコピイ染色剤として使用でき、マイクロスコープの相コントラストアクセサリーに全く余分な出費をすることなくかつ試料の固定および保存に長いプロトコールを実行することなく相コントラスト効果を付与する。特に、生試料の現場での定性検査。
【０１６２】
４．長期にわたり蛍光品質に劣化がないので、輸出時に冷蔵を必要としない。現在市販の蛍光染料類は、−２０℃に相当する冷蔵下で輸出されている。
【０１６３】
５．我々の染料は、前に公知となっている海洋性クラゲ由来緑色蛍光たんぱく質（ＧＦＰ）と異なり、レポーター遺伝子ではない。結果は直接的である。ＧＦＰは、３９５ｎｍの青色光を吸収し、４７０ｎｍに小ピークを有し、緑色光を発する。我々の染料は、３種の異なる蛍光波長で３種の蛍光色を発光する。前記染料は水に溶解性であり、したがって、水溶性染料類が必要な成分中で使用可能である。前記染料は、エタノール、メタノールおよびアセトンのような有機溶媒中で不溶性である。
【０１６４】
６．前記染料は、陰電荷を有している。
【０１６５】
７．前記染料はｐＨ６．５であり、ほとんど中性であり、したがって、組成物中において最終ｐＨ性に大きな影響をもたらすことはないであろう。
【０１６６】
８．前記染料は性質が非タンパク質性であり、したがって、天然条件下で分解しない。
【０１６７】
９．前記染料は、生物界面活性剤の性質を有しており、したがって、石鹸類およびトイレッタリ組成物中で使用できる。
【０１６８】
１０．前記染料は、抗菌性を有している。
【０１６９】
１１．前記染料は、１：４００００倍（すなわち、染料粉末１グラムを超純水４０リットルに溶解させた）の希釈範囲でもこれらの蛍光色を発光した。抽出物の蛍光は、室温で少なくとも１年後も持続していた。異なる励起波長におけるこれらの多色発光染料は、既に市販されているフルオロクロムマイクロスコピイ用染色剤類に匹敵する。
【０１７０】
１２．本染料の青色着色蛍光は、同一波長励起におけるＤＡＰＩフルオロクロムによる同一色発光に匹敵し、前記ＤＡＰＩフルオロクロムは、生化学、細胞生物学、免疫化学および分子生物学の非放射性標識キット類の成分として使用されている。
【０１７１】
１３．本染料の青色着色蛍光は、また、生化学、細胞生物学、免疫化学および分子生物学の非放射性標識および検出キット類の成分として使用されているＨｏｅｃｈｅｓｔ３３２５８による色発光に匹敵する。
【０１７２】
１４．本染料の青色着色蛍光は、また、生化学、細胞生物学、免疫化学および分子生物学の非放射性標識および検出キット類の成分として使用されているＨｏｅｃｈｅｓｔ３３３４２フルオロクロムの同一励起波長における色発光に匹敵する。
【０１７３】
１５．本染料の可視範囲における黄色着色蛍光は、また、生化学、細胞生物学、免疫化学および分子生物学の非放射性標識および検出キット類の成分として使用されているアクリジンオレンジの同一色発光に匹敵する。
【０１７４】
１６．本染料の可視範囲における黄色着色蛍光は、また、生化学、細胞生物学、免疫化学および分子生物学の非放射性標識および検出キット類の成分として使用されているオーラミンの同一色発光に匹敵する。
【０１７５】
１７．本染料の可視範囲における黄色着色蛍光は、また、生化学、細胞生物学、免疫化学および分子生物学の非放射性標識および検出キット類の成分として使用されているＦＩＴＣの同一色発光に匹敵する。
【０１７６】
１８．その橙色着色蛍光発光は、また、生化学、細胞生物学、免疫化学および分子生物学の非放射性標識および検出キット類の成分として使用されているヨウ化プロピジウムフルオロクロムの橙色蛍光色に匹敵する。
【０１７７】
１９．その橙色着色蛍光発光は、また、生化学、細胞生物学、免疫化学および分子生物学の非放射性標識および検出キット類の成分として使用されているローダミンフルオロクロムの橙色蛍光色に匹敵する。
【０１７８】
２０．その橙色着色蛍光発光は、また、生化学、細胞生物学、免疫化学および分子生物学の非放射性標識および検出キット類の成分として使用されているＴＲＩＴＣフルオロクロムの橙色蛍光色に匹敵する。
【０１７９】
２１．同一目的のために使用されている合成市販染料類と異なり、本染料は、室温で安定で長い保存期間を有している。前記染料の分子非放射性キット類は、室温で輸出できる。
【０１８０】
２２．前記の単一染料は、現在市場にある少なくとも１２３種の異なるフルオロクロム類（ＤＡＰＩ、Ｈｏｅｃｈｅｓｔ３３２５８、Ｈｏｅｃｈｅｓｔ３３３４２、ＦＩＴＣ，アクリジンオレンジ、オーラミン、ローダミン、ＴＲＩＴＣ、およびヨウ化プロピジウム等）の特徴を有している。通常のマイクロスコープの光線の下で、灰色色調は相コントラスト効果をもたらし、それは、細胞遺伝的、細胞学的、および組織化学的スライド類の迅速スクリーニングに有用でありマイクロスコープの余分な相コントラストアクセサリ部品に対する出費を節約させる。蛍光色発光は、蛍光のストークス則に従う。
【０１８１】
２３．コダックフィルムロールによるマイクロフォトグラフは、隣接色発光波長の色調を示す。マイクロフォトグラフにおいてエピフルオロセンスマイクロスコープの下で青色蛍光を観察するときのように、緑色色調も同様にでてくる。
【０１８２】
２４．コダックフィルムロールによるマイクロフォトグラフは、隣接色発光波長の色調を示す。マイクロフォトグラフにおいてエピフルオロセンスマイクロスコープの下で黄色蛍光を観察するときのように、緑色色調も同様にでてくる。マイクロフォトグラフにおいてエピフルオロセンスマイクロスコープの下で橙色蛍光色を観察するときのように、赤色色調も同様にでてくる。
【０１８３】
２５．全蛍光下で観察した細胞遺伝的スライド類は、試料がなく染料のみが存在するバックグランドにおいて細胞の逆染色効果をもたらす。
【０１８４】
２６．前記染料は、蛍光色を発する塩化ポリビニルの調製のために使用できる。それはまた、種々の塗料類、インク類およびテキスタイル類において蛍光色で使用できる。
【０１８５】
２７．前記染料は、ポリマーの漂白および増白のための蛍光染料組成物中で使用できる。前記染料は、全スペクトル蛍光染料によるもれ検出に使用できる。それはまた、自動化化学測定系でも使用できる。それはまた、墜落航空機、救命イカダ、およびロケットのような装備の位置を記録するために使用できる。さらに、それは、海中プローブ類中でも使用できる。前記染料は、皮膚がんの光化学療法において使用できる。
【０１８６】
２８．前記染料は、化粧品クリーム類および化粧液類のクロマトフォアサンスクリーン成分として使用できる。
【０１８７】
２９．前記染料の水混合性は、モイスチャライザー類と容易に混合できるようにしている。それは、分子診断学のための蛍光インサイチュハイブリッド形成適用キット成分として使用できる。それはまた、ＤＮＡ，ＲＮＡ，たんぱく質類および酵素類の標識用として生化学、細胞生物学、免疫化学、および分子生物学の非放射性標識および検出キット類、免疫蛍光検出類、ＤＩＧ標識オリゴヌクレオチドプローブ類および抗ＤＩＧ Ｆａｂフラグメント類の逆染色、フローサイトメトリにおける単一および複数細胞定量的蛍光、エピフルオロセンスマイクロスコピイのためのフルオロクロム染色剤類の成分として使用できる。
【０１８８】
３０．前記染料は、健康食品業界、化粧品業界、薬品および化学業界における生物汚染の迅速検査、実験室培養物中の生物汚染物の迅速推定、および屋外条件下における生物汚染物質の迅速検査のために使用できる。それはまた、コリンエステラーゼ類の競合的阻害剤でもある。
【０１８９】
３１．前記染料は、抗菌組成物中で使用できる。
【０１９０】
３２．前記染料は、トイレッタリ組成物中で生物界面活性剤として使用できる。
【０１９１】
３３．前記染料は、超純水中１：４００００比率で海洋性染料の天然着色剤Ａ生物活性組成物として使用でき、異なる３種の波長における３種の色の蛍光と透過光線下で相コントラスト効果を得ることができる。
【０１９２】
３４．前記染料の精製は、５０％エタノール粗抽出物２５０ｍｌで実施でき、８０℃の水浴で５分間蒸発させ精製し、粉末形状の精製染料２．５グラムを得ることができる。２ｍｇ／１０ｍｌ溶液組成物を、濃度１０ｍｇ／ｍｌを用いる化学的方法による前記染料の分光測光法構造解析のために用いる。前記染料の結合剤としての水による１：４００００倍希釈生物活性組成物は、３種の異なる波長における３種の色の蛍光を付与する。
【図面の簡単な説明】
【図１】
染料抽出前、屋外由来の新鮮ナマコＨｏｌｏｔｈｕｒｉａ ｓｃａｂｒａ。
【図２】
染料４回抽出後のナマコＨｏｌｏｔｈｕｒｉａ ｓｃａｂｒａ。
【図３】
ＵＶ可視領域におけるスキャニング。
【図４】
ＵＶ領域におけるスキャニング。
【図５】
蛍光分光測光。励起波長２７０ｎｍ。
【図６】
蛍光分光測光。励起波長４５０ｎｍ。
【図７】
蛍光分光測光。励起波長５４０ｎｍ。
【図８】
蛍光分光測光。励起波長２７０ｎｍ。
【図９】
ＷｈａｔｍａｎフィルタＮｏ．１を丸く切り、その先端を前記染料の希釈粗抽出物に浸漬し、Ｇｅｌ Ｄｏｃ．においてＵＶ放射線下で観察する。矢印は蛍光を示し、ＵＶフィルタを通して写真撮影した。下部は、染料を含まない対照である。
【図１０】
Ｗｈａｔｍａｎフィルタ類を前記抽出物のろ過のために使用し、ＵＶトランスイルミネータ（２６０ｎｍ−２８０ｎｍ波長範囲バルブ）の下で観察した。矢印は、蛍光を示す。下部フィルタペーパーは抽出物がなく、対照として保持した。
【図１１】
励起範囲３３０ｎｍ−３８５ｎｍを有するＷＵキューブによるエピフルオロセンスマイクロスコピイ青色蛍光発光であり、図１１は、試料を全く含まない染料の蛍光である。
【図１２】
励起範囲３３０ｎｍ−３８５ｎｍを有するＷＵキューブによるエピフルオロセンスマイクロスコピイ青色蛍光発光であり、図１２は、１０倍対物レンズで観察した細胞を含む場合である。
【図１３】
励起範囲３３０ｎｍ−３８５ｎｍを有するＷＵキューブによるエピフルオロセンスマイクロスコピイ青色蛍光発光であり、図１３は、同じ物を４０倍対物レンズで観察したものである。
【図１４】
励起範囲３３０ｎｍ−３８５ｎｍを有するＷＵキューブによるエピフルオロセンスマイクロスコピイ青色蛍光発光であり、図１４は、１００倍油浸対物レンズで観察した細胞である。
【図１５】
励起範囲４５０ｎｍ−４８０ｎｍを有するＷＢキューブによるエピフルオロセンスマイクロスコピイ緑黄色蛍光発光。図１５は、試料を全く含まない染料の蛍光である。
【図１６】
励起範囲４５０ｎｍ−４８０ｎｍを有するＷＢキューブによるエピフルオロセンスマイクロスコピイ緑黄色蛍光発光。図１６は、４０倍対物レンズで観察した細胞を含む場合である。
【図１７】
励起範囲４５０ｎｍ−４８０ｎｍを有するＷＢキューブによるエピフルオロセンスマイクロスコピイ緑黄色蛍光発光。図１７は、同一物を１００倍油浸対物レンズで観察したものである。
【図１８】
励起範囲４５０ｎｍ−４８０ｎｍを有するＷＢキューブによるエピフルオロセンスマイクロスコピイ緑黄色蛍光発光。図１８は、同一物を１００倍油浸対物レンズで観察したものである。
【図１９】
励起範囲５１０ｎｍ−５５０ｎｍを有するＷＧキューブによる橙色蛍光発光のエピフルオロセンスマイクロスコピイ色調。図１９は、試料を全く含まない染料の蛍光である。
【図２０】
励起範囲５１０ｎｍ−５５０ｎｍを有するＷＧキューブによる橙色蛍光発光のエピフルオロセンスマイクロスコピイ色調。図２０は、１０倍対物レンズで観察した細胞を含む場合の蛍光である。
【図２１】
励起範囲５１０ｎｍ−５５０ｎｍを有するＷＧキューブによる橙色蛍光発光のエピフルオロセンスマイクロスコピイ色調。図２１は、同一物を４０倍対物レンズで観察した場合である。
【図２２】
励起範囲５１０ｎｍ−５５０ｎｍを有するＷＧキューブによる橙色蛍光発光のエピフルオロセンスマイクロスコピイ色調。図２２は、１００倍油浸対物レンズで観察した細胞である。
【図２３】
１０倍対物レンズ下における明視野を通して観察した細胞。灰色色調−相コントラスト効果が見られた。
【図２４】
１００倍油浸対物レンズ下における明視野を通して観察した同一細胞。[0001]
(Technical field)
The present invention relates to a novel fluorescent dye obtained from the marine invertebrate sea cucumber (Holothuria scbra). The present invention also provides a process for the extraction, purification and characterization of this novel dye, a natural dye obtained from marine invertebrates, especially sea cucumber. Sea cucumbers are echinoderms that belong to the echinodermis group that also includes starfish and sea urchins. They occupy the following taxonomic positions:
[0002]
(Background technology)
The sea cucumber has the following taxonomic position:
Subworld: Metazoa
Gate: Echinoderm
Amon: Remnants
Class: Sea cucumber
Subclasses: Aspidocrotasea (Shielding Species: Aspidochirotacea), Dendrochirotasea (Trees: Dendrochirotacea), Apodacea (Apodacea)
Eyes: Dendrochirota, Aspidorota, Elasipoda, Molpodonia and Apoda
In these eyes, the sea cucumber Holothuria scbra,
Eyes: Aspidorota
Family: Holothuriidae
Genus: Holothuria
Species: scbra
Belongs to.
[0003]
Echinoderms are vertebrate invertebrates that do not exist on land but only in the ocean, are not divided by the five basic radial symmetries in adults, and have a head or brain. And all other animals are distinguished by their structural specificity of skeleton and body cavity. The genus sea cucumber Holothuroidea is an animal having a symmetrical body on both sides, usually extending along the mouth-antral axis, having a mouth at or near one end, and near or at the other end. Have an anus. The body surface is rough, the endoskeleton changes to a microscopically sized copulator or shield embedded in the body wall, and the mouth is surrounded by a set of tentacles connected to the aqueduct; The feet are usually present and motile; the vegetative lumen is long and coiled, and the rectal wall usually has a respiratory tree. The sexes are usually segregated and the gonads are single or paired tubular tufts. They are a stationary form that is bonded to a rigid substrate or burrow into a soft part, with protruding front and rear ends. They occur mainly in all seas in shallow waters, and few species appear deeper than 1000 meters. The species Holothuria scabra is also called, in part, Metriatila scabra Jaegea, and is a species of East Africa, the Red Sea, the Bay of Bengal, East India, Australia, Japan, the South Pacific, the Philippine Islands, It is widely distributed in the Indian Ocean and other Indo-Pacific regions. It is used for human / animal consumption in mackerel, Malaysia, and Indonesia and other Indo-Pacific countries.
[0004]
Pigments belong to the range of inorganic and organic types. The former are inorganic chemical compounds used for various decorative and painting purposes and the like. Organic pigments, such as organic dyes, date back to ancient times. The use of dyes from plants such as Brazilian wood, logwood, Persian berry indigo and madder has been reported in the Near East and Far Eastern countries from as far back as BC (George L. Clark, 1966 (Encyclopedia of chemistry, second edition). Debra K. Hobson and David S. Wales have secondary metabolism from several groups of organisms such as fungi, blue-green algae, sea urchins, starfish, and coral gut. (Journal of the Society of Dyers and Colorists (JSDC), 114, 42-44, 1998.) These are anthraquinone compounds, and their history is described. Was also of great importance in the dyes industry. Stainsfile-Dye A shows a die index for 264 dyes, of which only six are from all living species. Natural dyes.
HYPERLINK "http: // members"http: // members. pgonline. com /? bryand / dies / dyes. htm
[0005]
A few patents have recently been published on natural dyes, many of which are of plant origin. Wrolstad et al. Described a natural colorant from potato extract (US Pat. No. 6,180,154 issued Jan. 30, 2001). Shrinkhande, Anil J, reported in U.S. Pat. No. 4,452,822 issued Jun. 5, 1984, on the extraction and fortification of anthocyanins from grape pomas and other materials. U.S. Pat. No. 5,908,650, issued Jun. 1, 1999, Lenoble et al., Described a novel composition for enhancing the red color of anthocyanin dyes. Carotenoid-producing strains are described in Hirschberg et al., U.S. Pat. No. 5,935,808 issued Aug. 10, 1999 and in inventors Tsubokura et al., U.S. Pat. No. 5,858,761 issued Jan. 12, 1999. Collin; Peter Donald, in US Pat. No. 6,055,936, issued May 2, 2000, discloses sea cucumber carotenoid lipid fractions and methods in two US patents. I have.
[0006]
However, all of these colorants and dyes are not fluorescent. Fluorescent dyes, mostly synthetic, have been disclosed in a few U.S. and international patents. These have been used in various applications. The amount of patents in this field indicates the importance of these dyes.
[0007]
Synthetic paraazoantoxanthin A (molecular weight 214.2) fluoresces at λ (em) 420 nm and was found to be a pure competitive inhibitor of cholinesterase. Sepic K, Turk T, Macek P (Toxicon, 36 (6): 937-940, 1998). Welch; David Emanuel (US Patent No. 5,989,135, issued November 23, 1999) disclosed a chemiluminescent golf ball. White et al. (US Patent No. 6,110,566, issued August 29, 2000). And WO9920668) describe flexible polyvinyl chloride films that emit a durable fluorescent color.
[0008]
Dipitro Thomas C (International Patent No. WO9938916) disclosed the use of fluorescent polymeric dyes in various paints, inks and textiles. Cramer Randall J (Patent EP 0206718, issued December 30, 1986) described a fluorescent dye composition for bleaching and whitening of polymers.
[0009]
Leak detection is another application disclosed by others (Leighley; Kenneth C. U.S. Patent 6,056,162, May 2, 2000). Cooper et al., US Pat. No. 6,165,384, issued Dec. 26, 2000, disclosed a full spectrum fluorescent dye composition for the same purpose.
[0010]
Lichtwart et al., US Pat. No. 5,902,749, issued May 11, 1999, uses fluorescent dyes in an automated chemical assay system.
[0011]
There are few reports on marine animals. A green fluorescent protein GFP-new reporter gene from the Pacific jellyfish Aequora aeguora is described in Shimomura, O, Johnson, F. et al. H. And Saiga, Y (Journal of cellular and comparative physiology, 59, 223-239, 1962). GPF is characterized by the presence of highly fluorescent dye-containing cells (chromatophores). Purified GFP absorbs blue light maximally at 395 nm, with a minor peak at 470 nm and emits green light. Sepcic K, Turk T, and Macek P reported a fluorescent zoanthode dye, parazoantoxanthin A. Toxicon, 36 (6): 937-940, 1998.
[0012]
Marine dyes have several uses as dyes themselves and as part of compositions.
[0013]
Several authors have disclosed fluorescent dye mixtures for multiple purposes (Burns et al., US Pat. No. 5,920,429 issued Jul. 6, 1999; Burns; David M and Pavelka Lee A, (International Patent AU704112). Marine dye compositions have been used in some applications to document military positions such as crash aircraft, life raiders, and rockets. Different dye compositions are being tested for higher efficiency and long-lasting fluorescence in diluted form (Swinton; Robert J, U.S. Patent issued April 11, 1995). No. 5,405,416; International Patent WO9010044 disclosed on Jul. 7, 1990. Hyosu et al. April 5, U.S. Patent 4,016,133) is prepared fluorescent colored resin particles such.
[0014]
Another use of marine dyes as underwater probes has been reported by Crosby David A and Ekstrom Philip A in U.S. Pat. No. 5,321,268 issued June 14, 1994. The probe instrument includes a central optical fiber containing a fluorescent dye surrounded by a transparent or transparent, protective, and entanglement-resistant sheath. It can be attached to marine animals for data collection on light intensity and temperature in the area where the marine animals move around.
[0015]
Several authors have used UVA in photochemotherapy for skin cancer. Kowzick L; Ott A; Waldemann T; Suckow M; Ponnighaus J, M. Vogtlandklinikum Plauen discloses PUVA bath photochemotherapy in lymphomatous papulosis (a type of skin cancer), with UVA treatment showing improvement (Elsevier Science, BV, 2000). UV A sunbeds are widely used by psoriasis patients.
[0016]
Sabatelli, Anthony D, issued May 11, 1993, U.S. Pat. No. 5,210,275, disclosed a chromatophoric sunscreen composition for sun protection. This chromatophor has the ability to absorb UVA and UVB wavelength radiation.
[0017]
Fluorescent dyes are very useful in labeling molecular probes for fluorosense microscopy. Fluorosense microscopy is also known as reflected light fluorescence or epifluorosense microscopy and is very important for non-radioactive in situ hybridization, but it has a different sensitivity to its high sensitivity and spectrally separate emission Depends on the ability to excite the three immunofluorophores. It enables multiplex detection (Chapter II. Nonradioactive In Situ Hybridization Application Manual. Boehringer Mannheim GmbH, Biochemica, printed in Germany, 1992). The background principle is that, for a sample, the Stokes law (Fluorescence (by George L. Clark, 1996, in Encyclopedia of chemistry, 2), stating that the wavelength of the fluorescent radiation is always longer than the wavelength of the excitation radiation.^nd ed. Page 435-436). Chapter Vin: In Situ Hybridization Application Manual. Boehringer Mannheim GmbH, Biochemica, printed in Germany, 1992, page 23-62 and Olympus Optical Co. Ltd, Tokyo Japan. Catalog. (Instructions BX-FLA Reflected Light Fluorescence Attachment (Page 16. 1999) described various non-radioactive fluorochrome stains in recent applications.
[0018]
Different stains have been used for different excitation cubes of the fluorosense microscope. For example, DAPI (DNA stain, blue emission), fluorescein-dUTP; Hoechest 33258, 33342 are observed on excitation with 330-385 excitation cubes; FITC, acridine orange on 450-480 excitation cubes (for DNA, RNA, green / Rhodamine, TRITC and propidium iodide (DNA, emitting orange tones) under auramine and 510-550 excitation cubes.
[0019]
Rosenlum Barnett B and Surgeon Sandra L; Lee Linda G; Benson Scott c; Graham Ronald J are 4,7-dichlororhodamines useful as molecular probes in the international patent WO0058406 issued October 5, 2000 as fluorescent dyes. It was used.
[0020]
LaClair; James J .; (U.S. Pat. Nos. 6,140,041 and WO9938919, issued Oct. 31, 2000) disclosed the synthesis and fluorescence of fluorescent dyes and their application in protein labeling, DNA labeling, single molecule spectroscopy and fluorescence.
[0021]
Applicants have taken a different approach; the dyes reported in the present invention are natural dyes and not synthetic. It is derived from marine animals, not plants or fungi. Partially pure dyes are extracted directly from invertebrate skin cells. This is the first report on a natural dye derived from marine animals, and this dye is a fluorescent dye. The marine animal source, the echinoderm, sea cucumber called Holothuria scabra, is a novel source of material. Unlike most other known fluorescent synthetic dyes, our dyes do not need to be mixed with another dye to obtain different fluorescent tones at different wavelengths. It emits three different colored fluorescents that can have multiple tones at three different excitation wavelengths. Furthermore, among the known natural fluorescent dyes, such as the best known jellyfish-derived green fluorescent protein (GFP), our dyes are non-proteinaceous in nature and more stable at room temperature for months, And there is no contamination by fungi. It also has biosurfactant properties. Another important property of the dye is that, after excitation in the low UV wavelength spectral range (UVB), it fluoresces in the UVA wavelength range. Both these absorption and emission ranges can be subjected to selective applications depending on which UV spectrum is suitable for a particular situation.
[0022]
One important aspect of the dye is that it produces compositions and kits for non-radioactive labeling and reverse staining of molecular probes. It has effects comparable to DAPI, FITC and PI colors at different wavelength excitations. Despite being a single dye, one has three (3 in 1) effects. Although this dye is a single dye, it has three effects in one. In fact, this single dye covers the tones of the wavelength spectrum of the 123 fluorochromes currently known on the market. (Internet Bitplane Products (Fluorochrome) HYPERLINK "Http: // www. b " http: // www. bitplane. ne. ch / public / support / standard / Fluorochrome. htm.See
[0023]
Yet another aspect is its use as a fluorochrome stain in epifluorosense microscopy, which is the first report in the text of any marine natural dye. This application is useful for molecular diagnostics using fluorescence in situ hybridization, rapid diagnosis of biological contamination in tissue culture, cytogenetical preparation testing for multiple uses such as industrial preparation, water quality testing in laboratory and outdoor conditions. Provides a simple and quick method.
[0024]
Yet another aspect of the dye is its use as a component of non-radioactive labeling kits for advanced molecular biology applications.
[0025]
Purpose of the invention
The main object of the present invention is to provide a novel fluorescent dye obtained from sea cucumber Holothuria scabra.
[0026]
Another object of the present invention is to provide a process for the extraction, partial purification and characterization of said natural dye / pigment from the marine cucumber Holothuria scabra.
[0027]
It is a further object of the present invention to provide compositions using dyes obtained from the sea cucumber Holothuria scabra tissue.
[0028]
It is a further object of the present invention to investigate its insecticidal and insecticidal rodenticide effects.
[0029]
A further object of the invention is its application for veterinary formulations.
[0030]
It is a further object of the present invention to provide a dye which fluoresces at a particular excitation wavelength in three different wavelength ranges of the UV and visible spectrum.
[0031]
Yet another object of the present invention is to observe fluorescence and visible spectroscopy at the emission wavelength and its range.
[0032]
Yet another object is to observe three different fluorescent colored emissions of the dye in the UV and visible range of epifluorosense microscopic cubes.
[0033]
Yet another object of the present invention is to observe the fluorescent staining effect of cytogenetic slides and to screen chromosomes, cells and tissues using the dyes of the present invention.
[0034]
A further object of the present invention is a biosurfactant characterization.
[0035]
It is a further object of the present invention to develop kits containing said fluorescent dyes as non-radioactive labels for molecular probes.
[0036]
Summary of the Invention
Accordingly, the present invention provides a novel fluorescent dye obtained from the sea cucumber Holothuria scabra. The present invention also provides extraction, isolation and characterization steps of said dye. Further, the present invention provides the above-mentioned dye-containing compositions.
[0037]
DETAILED DESCRIPTION OF THE INVENTION
Applicants have identified, after extensive research, novel fluorescent dyes obtained from marine animals, particularly invertebrates, and more particularly from the sea cucumber Holothuria scabra.
Subworld: Metazoa
Gate: Echinoderm
Amon: Remnants
Class: Sea cucumber
Subclass: Aspidochirotacea
Eyes: Aspidorota
In these eyes, sea cucumber Hoothuria scbra is
Eyes: Aspidochirota
Family: Holothuriidae
Genus: Holothuria
Species: scabra
Belongs to.
[0038]
The present invention further provides a novel fluorescent dye obtained from the animal skin. It also describes the physicochemical properties of the dye and its stability in direct sunlight, high and low temperatures. The dye has three colored fluorescent emissions at three different excitation wavelengths in the UV and visible light spectrum. The invention also relates to cells screening under a fluorosense microscope for rapid inspection of contamination and cytogenetic screening. The invention also relates to the use of said dyes as non-radioactive labels for proteins, DNA and RNA molecular probes for advanced molecular diagnostics, epimicro for single and double staining of chromosomes, cells and tissues. Such as scopies, fluorescent in-situ hybridization applications, biosurfactants, biofouling and leak testing, photochemotherapy, new remote sensing devices, subsea probes, lifesaving equipment, crash aircraft, liferafts and rockets It relates to the recording of military positions, various fluorescent applications in sub-zero conditions and many others.
[0039]
The present invention describes a fluorescent dye obtained from marine animals that absorbs sunlight or is exposed to sunlight for an extended period of time due to its physiological function and likely to have a fluorescence evolution mechanism at different wavelengths. . The necessary wavelengths from the light spectrum are used in the chemical pathway so that phytoplankton, picoplankton, and photosynthetic fungi absorb sunlight for their photosynthetic functions, and the extra light is emitted according to Stokes' law.
[0040]
Invertebrates that do not have excess external equipment such as shells and distinct defense organs, have hard and sharp skin, and have a strong endoskeleton formed of pillars (ossicles), It is sticky, ie slow acting, prolonged exposure to direct sunlight, inhabits in sand or crevices and may show fluorescence.
[0041]
The present invention relates to highly efficient and selective methods for the extraction, purification and characterization of dyes from marine invertebrates, and the manufacture of kits for molecular diagnostics using non-radioactive labels, molecular markers By providing the components of novel instrumental devices for astronauts, epifluorosense microscopy, photochemotherapy, terrestrial and subsea probes, and their many uses in the cosmetics industry, the food industry and the military, etc. It aims to overcome its inherent disadvantages.
[0042]
Said marine invertebrates are echinoderms that are taxonomically referred to as sea cucumber Holothuria scabra belonging to the genus sea cucumber Holothuroidea. The product of the present invention is a novel dye, which will be reported for the first time. The animals were collected from the shores of central west coast of India at ebb tide, brought to the laboratory and kept in glass tanks containing 30-32% salinity per reference volume. The animals were adult and sexually mature. The classification positions were identified as described above. In fact, most of the available dyes are virtually synthetic. There are only six types of natural dyes. This includes dyes obtained from whole organisms. The fluorescent dyes reported in the present invention are the only species from marine organisms.
[0043]
In the present text, the term dye is used for pigments that are not decolorized with a reducing agent. The dye imparts color to fibers, cellulose and the like. It is called a natural dye because it is derived from marine animals that are virtually common on the world coast and is not a synthetic pigment. Fluorescent dyes emit light when excited at a specific wavelength during the transition from a high electronic state to a low electronic state in a very short time.
[0044]
Multicolored fluorescence means emitting different colored light rays when excited in different wavelength ranges. It emits blue, yellow and orange fluorescent tones upon excitation by different UV and visible light spectra. By biosurfactant is meant a dye solution which, when shaken, produces a soap-like foam and exhibits antimicrobial properties. As used herein, molecular diagnostics refers to the use of the dyes as non-radioactive labels of molecular probes for fluorescence in situ hybridization applications in molecular cytogenetics, and as markers in microarrays, and in molecular biology research. Means In this text, epifluorosense microscopy is a type of fluorescent dye that emits a specific colored fluorescence when excited by known fluorochromes when observed with different cube structures using this dye as a stain. It relates to a microscope study of cytogenetic preparations of slides by recording. The fluorochrome cubes WUB, WB, WG are designated filter cube structures for Olympus BX-FLA reflected light fluorescent attachments for different wavelengths.
[0045]
Accordingly, the present invention provides a method for the extraction, purification and characterization of a natural fluorescent dye, said method comprising:
(I) harvesting said material from the outdoors and maintaining it under laboratory conditions;
(Ii) extraction of pigment from the skin of the echinoderm sea cucumber Holothuria scbra, and
(Iii) partial purification of the dye,
including.
[0046]
The biologically active extract of the present invention is obtained from the sea cucumber Holothuria scabra. This extract is useful as a natural fluorescent dye and has the following characteristics:
i. Decolorization by reducing agent,
ii. Not a synthetic compound,
iii. The crude extract of the dye is yellow-green in color,
iv. When observed with the naked eye under sunlight, the partially purified dye is a reddish-brown powder,
v. Under the light of an electric light, a slightly green color is emitted,
vi. Amorphous in nature,
vii. Water soluble,
viii. Insoluble in organic solvents such as ethanol, methanol and acetone,
ix. Negative charge,
x. having a pH of 6.5,
xi. The presence of a phenolic group,
xii. Absence of a quinonoid ring,
xiii. Absence of aromatic amine groups,
xiv. Non-proteinaceous in nature,
xv. No reducing sugars,
xvi. The dye has the properties of a biosurfactant,
xvii. Also, the dye showed antimicrobial properties and showed an inhibition zone when the antimicrobial assay was performed.
xviii. The dye / dye is a fluorescent dye, which emits fluorescence when excited by a spectrophotometer at different wavelengths in the UV and visible light spectral ranges,
xix. 300 nm-700 nm UV, visible spectroscopy, and peaks are recorded at 379 nm and 439 nm wavelengths.
xx. UV, visible spectroscopy at 250 nm-350 nm, and peaks at 272 nm and 299 nm wavelengths.
xxi. Fluorescence spectroscopy in the UV and visible spectra shows that, when excited at a wavelength of 270 nm, it fluoresces in the 324 nm-380 nm range and falls into the UVA wavelength range of solar UV.
xxii. At an excitation wavelength of 450 nm in fluorescence spectroscopy, fluorescence emission occurred between 500 nm and 580 nm and had a maximum intensity.
xxiii. At an excitation wavelength of 540 nm in fluorescence spectroscopy, fluorescence emission occurred between 500 nm and 620 nm and had a maximum intensity.
xxiv. At an excitation wavelength of 555 nm in fluorescence spectroscopy, fluorescence emission occurred between 575 nm and 620 nm and had a maximum intensity.
xxv. Whatmann Filter No. 2 immersed in a 1: 40,000 dye concentration dilution under a UV transilluminator with a UV lamp in the range of 260 nm-280 nm and a Gel Documentation system. Physical examination according to 1 fluoresces blue-green tones,
xxvi. Emitting three different colored fluorescences at three different wavelengths in the UV and visible range of the fluorescent cube of the epifluorosense microscope,
xxvii. Fluorescent blue emission appears with UVA in the 380 nm-400 nm range when excited in the UV cube WU-330 nm-385 nm excitation range.
xxviii. Fluorescent yellow emission appears in the 500 nm-570 nm range when excited in the WB Cube-450 nm-480 nm excitation range.
xxix. Fluorescent orange emission appears in the 570 nm-650 nm range when excited in the WG Cube-510 nm-550 nm excitation range.
xxx. The dye takes on a gray tint under normal transmission light of an epifluorosense microscope when viewed with a 10 × objective lens,
xxxi. The dyes emit these fluorescent colors even at a dilution range of 1: 40000 times (ie, 1 gram of dye powder dissolved in 40 liters of ultrapure water),
xxxii. The fluorescence of the extract persisted after at least one year at room temperature,
xxxiii. The fluorescence of the dye is highly photostable, does not degrade upon prolonged exposure to direct light, and
xxxiv. The fluorescence of the dye does not change when frozen at 20 ° C., a temperature at which the molecules cannot obtain the energy required for activation, as in extracts from chemiluminescent organisms.
[0047]
The physical and other characteristics of the dye can also be assessed by the following steps:
(I) structural analysis of the dye,
(Ii) biosurfactant analysis,
(Iii) antibacterial test,
(Iv) visible spectroscopic analysis of the dye,
(V) fluorescence spectroscopy of the dye,
(Vi) Physical inspection of UV transilluminator emission in the 260-280 nm range
(Vii) Preparation of cytogenetic slides by air drying
(Viii) Dyeing slides with the dye
(Ix) Epifluorosense microscopic screening of cytogenetic slides in fluorochrome cubes WU, WB, WG and Bright Field
(X) microphotography of luminescence fluorescence in a slide area without any cytogenetic material,
(Xi) microphotography of the emission fluorescence of fluorogenic cubes WU, WB, WG and cytogenetic slides under Bright Field, and
(Xii) Examining the wavelength range of the emitted fluorescent color tone and the excitation range of fluorochrome cubes.
[0048]
Thus, the present invention provides a marine that emits three different colored fluorescence in blue, yellow and orange shades when excited in three different wavelength ranges in the UV and visible light spectrum cubes of an epifluorosense microscope. Provide a natural fluorescent dye of animal origin. The present invention further relates to emission peaks in almost the same range of excitation wavelengths by recording fluorescence and visible light spectrophotometer readings, respectively. The invention further relates to epifluorosense microscopy of cellular genetic material on air-dried preparations by using this dye as epifluorosense microscopy stain. The dye could also be used to produce non-radioactive labeling kits for molecular diagnostics by fluorescence in situ hybridization in various molecules, biomedical and engineering sciences.
[0049]
In one embodiment, the dye material source is an invertebrate marine animal belonging to the subdivision: metazoan, phylum: echinoderm, subphylum: migratory, genus: sea cucumber, name: sea cucumber Holothuria scabra.
[0050]
In yet another embodiment, the sea cucumber Holothuria scabra is selected from the group consisting of sea cucumber and is widely distributed around the world, especially on the coasts, shallow waters and deep waters of the Indo-Pacific. The best known related organisms to sea cucumber are sea urchins and starfish.
[0051]
In a further embodiment, the skin of the sea cucumber Holothuria scbra is separated and weighed. 250 ml of 50% alcohol is added to 15 g of skin wet weight and filtered in vacuo using a peristaltic pump operating at a speed of 200 rpm.
[0052]
In a further embodiment, the extract is kept on a water bath at 80 ° C. and evaporated to a third volume, ie concentrated from 250 ml to 80 ml. It takes about 3 hours to evaporate.
[0053]
In a further embodiment, 100 ml of 99.5% ethanol is added to 80 ml of the extract concentrate and allowed to precipitate overnight.
[0054]
In a further embodiment, the concentrate with the precipitate is centrifuged at 1500 rpm for 4-5 minutes and the top layer is discarded. The precipitate is evaporated to dryness on a water bath at 80 ° C. for 5 minutes. From 250 ml of 50% ethanol extract, 2.5 g of dye are obtained after evaporation.
[0055]
In a further embodiment, the partially pure dye is scraped out with a spatula and stored in a dry glass vial at room temperature.
[0056]
In one further embodiment, the physical properties of the dye are recorded. Pure dry dye is reddish brown in sunlight. Under light rays, a green tint is observed. The dye is water-soluble. Insoluble in organic solvents such as pure ethanol, methanol, chloroform and acetone. It is amorphous in nature. In an aqueous solution, it has a pH of 6.5.
[0057]
In one further aspect, structural analysis was performed by chemical methods. The dye was dissolved in distilled water at $ 2 mg / ml and examined for chemical properties.
[0058]
Furthermore, in one embodiment, neutral (Neutral) ferric chloride was added, and purple coloration was observed. It proved that phenolic groups were present.
[0059]
In yet another embodiment, a β-mercaptoethanol (beta-mercaptoethanol) reducing agent was added. There was no decolorization of the compound. This proved that the quinonoid ring was absent and the dye was a dye.
[0060]
In another embodiment, the diazotization is carried out with 0.1 N HCl and NaNO₂(Sodium nitrite) was added and an alkaline solution of beta (() naphthol was added to it, no precipitation was observed, demonstrating the absence of amine groups.
[0061]
In another embodiment, the concentrated dye solution at $ 10 mg / ml was heated, but no precipitation or coagulation was observed. This demonstrated that the compound was non-protein in nature. One drop of concentrated HCl was added to the same solution and Fehling's solution was added. The absence of any color change proved the absence of reducing sugars.
[0062]
In another embodiment, the biosurfactant nature of the dye was observed by adding it to water and forming a foam while shaking.
[0063]
In another embodiment, an antimicrobial disc test was performed and zones of inhibition were observed.
[0064]
Applicants have studied the properties of the dye and have found that it exhibits multicolor emission at different excitation wavelengths, comparable to fluorochrome microscopic dyes already on the market. The blue fluorescence of this dye is comparable to the same color emission by DAPI fluorochrome used as a component of non-radioactive labeling kits for biochemistry, cell biology, immunochemistry and molecular biology at the same wavelength excitation. The yellow fluorescence in the visible range of the dye is comparable to the same color emission of auramine used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. The yellow fluorescence in the visible range of the dye is comparable to the same color emission of FITC used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. The orange fluorescence emission is comparable to the orange fluorescence color of propidium iodide fluorochrome used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. 2. The dye of claim 1, wherein the orange fluorescence is orange fluorescence by TRITC fluorochrome used as a component of non-radioactive labels and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. Is comparable to The dyes are stable at room temperature and have a long shelf life. The dye molecular and radioactive kits can be exported at room temperature. The dyes are characterized by at least 123 different fluorochromes, i.e., DAPI, Hoechest 33258, Hoechest 33342, FITC, acridine orange, auramine, rhodamine, TRITC, and propidium iodide, which are currently It is commercially available. The dye takes on a gray shade under ordinary microscope light, which produces a phase contrast effect, which is useful for rapid screening of cytogenetic, cytological, and histochemical slides, No extra expense for microscope phase contrast accessory options. Fluorescent color emission follows Stokes' law of fluorescence. Microphotographs with Kodak film rolls show adjacent color emission wavelengths, such as blue fluorescence, under an epifluorosense microscope in green color microphotographs. The microphotograph with the Kodak film roll shows the tones of adjacent color emission wavelengths such that a green color appears when observing yellow fluorescence under an epifluorosense microscope in the microphotograph. Similarly, a red tint appears with an orange fluorescent color under an epifluorosense microscope in a micrograph. Cytogenetic slides examined for fluorescence show a counterstaining effect on the cells compared to a background where there is no sample and only the dye is present.
[0065]
In another aspect, UV-visible spectroscopy at 300 nm-700 nm wavelength (FIG. 3) was performed. Peaks are recorded at 379 nm and 439 nm wavelengths.
[0066]
In another embodiment, the UV spectroscopy at the 250 nm-350 nm wavelength (FIG. 4) was performed. Peaks are recorded at 272 nm and 299 nm wavelengths.
[0067]
In yet another aspect, fluorescence spectroscopy at an excitation wavelength of 270 nm. The fluorescence appeared at 324 nm-380 nm and had the maximum intensity (FIG. 5).
[0068]
In another embodiment, fluorescence spectroscopy with an excitation wavelength of 450 nm. The fluorescence appeared between 500 nm and 580 nm with maximum intensity (FIG. 6).
[0069]
In another embodiment, fluorescence spectroscopy with an excitation wavelength of 540 nm. The fluorescence appeared between 500 nm and 620 nm with maximum intensity (FIG. 7).
[0070]
In another embodiment, fluorescence spectroscopy with an excitation wavelength of 555 nm. The fluorescence appeared at 575 nm-620 nm with maximum intensity (FIG. 8).
[0071]
In another embodiment, Whatman No. One filter paper was dipped in the dye extract and observed under a UV transilluminator with a UV lamp in the 260 nm-280 nm wavelength range. It emitted blue fluorescence.
[0072]
In yet another aspect, the dye is used as a dye at a dilution of 1: 10000 (1 g per 10 liters) to record the light emission when excited by different cubes and compare the color to known fluorochromes. Perform epifluorsense microscopic studies.
[0073]
Different stains are used for different excitation cubes of the fluorosense microscope. For example, DAPI (DNA stain, blue emission), fluorescein-dUTP; Hoechest 33258, 33342 are observed under excitation by 330 nm-385 nm excitation cubes; FITC, acridine orange (DNA, RNA under 450 nm-480 nm excitation cubes) Emits a green / yellowish shade), auramine; and rhodamine, TRITC and propidium iodide (DNA emits an orange shade) under a 510 nm-550 nm excitation cube.
[0074]
In one embodiment for this epifluorescence, microscopic screening of cytogenetic slides is performed by placing a drop of diluted extract and exciting with a WU filter having a spectral range of 330-385 nm wavelength.
[0075]
In another embodiment, epifluorosense microscopic screening of said cytogenetic slides is performed by placing a drop of extract and exciting with a WB filter having a spectral range of 450-480 nm wavelength.
[0076]
In another embodiment, epifluorosense microscopic screening of said cytogenetic slides is performed by placing a drop of extract and exciting with a WG filter having a spectral range of 510-550 nm wavelength.
[0077]
In another embodiment, epifluorosense microscopy screening of said cytogenetic slides under a Bright Field objective is performed by transmitted light using this dye.
[0078]
In yet another embodiment, epifluorosense microscopic screening of cytogenetic slides stained with this dye is performed by observing the tone of the fluorescent color emitted by each excitation.
[0079]
In another embodiment, the fluorescence emitted by excitation by the WU 330 nm-385 nm range is in the 380 nm-400 nm range.
[0080]
In yet another aspect, excitation by a WB filter having a spectral range of 450 nm-480 nm emitted fluorescence in the 550 nm-570 nm range.
[0081]
In yet another aspect, excitation by a WG filter having a spectral range of 510 nm-550 nm emitted fluorescence in the 600 nm-650 nm range.
[0082]
In yet another embodiment, epifluorosense microscopy screening using transmitted light of the cytogenetic slides in a bright field, in a visible light spectrum in the entire white range depending on the concentration of the cellular components. It emitted light and produced a phase contrast effect.
[0083]
In yet another aspect of the invention, microphotography of the fluorescence emitted in the cell free slide area in the WU 330 nm-385 nm range is performed by changing the exposure from 50 seconds to 60 seconds with a 400 ASA speed Kodak film. A blue fluorescent tone was emitted.
[0084]
In another embodiment, microphotography of the fluorescence emitted in the slide area without cells in the WB 450 nm-480 nm range is performed by changing the exposure from 50 seconds to 60 seconds with a Kodak film at 400 ASA speed. A yellow fluorescent tone was emitted.
[0085]
In another embodiment, microphotography in the WG 510 nm-550 nm range of the fluorescence emitted in the slide area without cells is performed by changing the exposure from 50 seconds to 60 seconds with a 400 ASA speed Kodak film. An orange fluorescent tone was emitted.
[0086]
In another embodiment, microphotography of fluorescence emitted under a bright field in a slide area without cells exhibited a gray shade.
[0087]
In yet another embodiment, microphotography in the WU 330 nm-385 nm range of the fluorescence emitted in the slide area containing the cells is performed by changing the exposure from 50 seconds to 60 seconds with a 400 ASA speed Kodak film. A blue fluorescent tone was emitted.
[0088]
In another embodiment, microphotography of the fluorescence emitted in the slide area containing the cells in the WB 450 nm-480 nm range is performed by changing the exposure from 50 seconds to 60 seconds with a Kodak film at 400 ASA speed. A yellow fluorescent tone was emitted.
[0089]
In another embodiment, microphotography in the WG 510 nm-550 nm range of the fluorescence emitted in the slide area containing the cells is performed by changing the exposure from 50 seconds to 60 seconds with a Kodak film at 400 ASA speed. An orange fluorescent tone was emitted.
[0090]
In another embodiment, microphotography of fluorescence emitted in a slide area with cells under a bright field exhibited a gray shade.
[0091]
In yet another aspect of the invention, a 1: 10,000 dilution of the dye when prepared with distilled water and used as a stain resulted in colored fluorescent emission in the UV and visible ranges of an epifluorosense microscope.
[0092]
In yet another embodiment, the dye is diluted 1:40 000 with water, which produces three colors of fluorescence at three different wavelengths.
[0093]
In yet another aspect, the present invention provides an extract-containing bioactive composition obtained from marine cucumber Holothuria scbra in a ratio of 1: 40000 in ultrapure water, comprising three colors at three different wavelengths under transmitted light. Produces a fluorescent and phase contrast effect.
[0094]
The present invention also provides a composition containing a bioactive extract obtained from the marine cucumber Holothuria scabra with conventional additives, and is useful for preparing a flexible polyvinyl chloride film exhibiting a fluorescent color.
[0095]
In certain embodiments, the present invention provides compositions containing a bioactive extract obtained from the marine cucumber Holothuria scabra with conventional additives, and is useful in the preparation of coating compositions and inks.
[0096]
In another aspect, the present invention provides a composition comprising a biologically active extract obtained from the marine cucumber Holothuria scabra with conventional additives, and is useful for leak detection.
[0097]
In yet another aspect, the present invention provides a composition comprising a bioactive extract obtained from the marine cucumber Holothuria scabra with conventional additives, and is useful for underwater probes.
[0098]
In yet another aspect, the present invention provides a composition comprising a biologically active extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful for photochemotherapy of skin cancer.
[0099]
In yet another aspect, the present invention provides a cosmetic composition comprising a biologically active extract obtained from the marine cucumber Holothuria scabra with conventional additives.
[0100]
In yet another aspect, the present invention provides a composition comprising a biologically active extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful as fluorescent probe in situ hybridization kits for molecular diagnostics. .
[0101]
In yet another aspect, the present invention provides a composition comprising a biologically active extract obtained from the marine cucumber Holothuria scbra with conventional additives, comprising the non-radioactive of biochemistry, cell biology, immunochemistry and molecular biology. Useful as a component of labels and detection kits.
[0102]
In yet another aspect, the invention provides a composition comprising a bioactive extract obtained from the marine cucumber Holothuria scabra with conventional additives, and is useful in immunofluorescence detection.
[0103]
In yet another aspect, the present invention provides a composition comprising a biologically active extract obtained from the marine cucumber Holothuria scabra, together with conventional additives, comprising a counterstain for DIG-labeled oligonucleotide probes and anti-DIG Fab fragments. Useful as
[0104]
In yet another aspect, the present invention provides a composition comprising a biologically active extract obtained from the marine cucumber Holothuria scbra with conventional additives and is useful in single and multiple cell quantitative fluorescence in flow cytometry. .
[0105]
In yet another aspect, the present invention provides a composition comprising a bioactive extract obtained from the marine cucumber Holothuria scbra with conventional additives, useful as a fluorochrome stain for epifluorosense microscopy. is there.
[0106]
In yet another aspect, the present invention provides a composition comprising a bioactive extract obtained from the marine cucumber Holothuria scbra with conventional additives to provide rapid biocontamination in the health food, cosmetic, pharmaceutical and chemical industries. Useful for testing.
[0107]
In yet another aspect, the present invention provides a composition comprising a bioactive extract obtained from the marine cucumber Holothuria scbra with conventional additives, which is useful for rapid estimation of biological contamination in laboratory culture.
[0108]
In yet another aspect, the present invention provides a composition comprising a bioactive extract obtained from the marine cucumber Holothuria scbra with conventional additives, which is useful for rapid testing of biocontaminants under outdoor conditions.
[0109]
In yet another aspect, the present invention provides a composition comprising a bioactive extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful as a competitive inhibitor of cholinesterases.
[0110]
In yet another aspect, the present invention provides a composition comprising a bioactive extract obtained from the marine cucumber Holothuria scabra with conventional additives, and is useful in antimicrobial compositions.
[0111]
In yet another aspect, the present invention provides a composition comprising a bioactive extract obtained from the marine cucumber Holothuria scabra with conventional additives, and is useful as a biosurfactant in toiletry compositions. .
[0112]
In yet another aspect, the present invention provides a composition comprising a bioactive extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful as a natural colorant.
[0113]
Table description
Table 1 (a) (b) Structural analysis of the dye by chemical methods for the presence or absence of quinonoid ring, phenolic and amine groups.
Table 2 Structural analysis of the dye by chemical methods to determine the absence of quinonoid rings, proteinaceous / non-proteinaceous properties and the presence / absence of reducing sugars.
Table 3 Different tonal fluorescence colors of the dyes used as stains when excited with different wavelength cubes of an Olympus epifluorosense microscope.
[0114]
The present invention provides a process for the extraction, purification and characterization of a novel dye, a natural dye from echinoderms (Holothuroidea: Sea cucumber Holothuria scabra) widely distributed along the central west coast of India and the Indo-Pacific region throughout the world. About.
[0115]
The present invention further provides a novel fluorescent pure dye derived from the animal skin pigment, which can be repeatedly extracted from the same sample 3-4 times by storing at -20 ° C, thus stopping overfishing of natural resources. Can be applied.
[0116]
The present invention further provides that the dyes emit light by three different fluorochromes (DAPI, FITC and PI) currently marketed for fluorescent microscopy at three different excitation wavelengths of the UV and visible light spectra. It has the same three types of colored fluorescent emission.
[0117]
Thus, the dyes follow a simple protocol for single and double staining of chromosomes, cells and tissues, and are fluorochromes for three-in-one (one serving three) epifluorosense microscopy. Commercially available as a dye.
[0118]
The present invention also explores the use of dyes in the non-radioactive labeling of proteins, DNA and RNA probes for fluorescent in situ hybridization applications in molecular biology.
[0119]
Thus, in a preferred form of use, the dye can be a component of molecular labeling and detection kits, most of which are imported and sold at high prices.
[0120]
These labeling kits are widely sought for rapid molecular cytogenetics and molecular diagnostics using microarray technology.
[0121]
In yet another preferred form of use, dyes can be beneficial in making cosmetic compositions for UVB absorption from sunlight.
[0122]
Yet another advantage of the present dye is that its fluorescence is visible even in very dilute solutions (1: 40000).
[0123]
This property can be used in life equipment as a component of life jackets, can be used to record the location of armaments such as crashed aircraft, life rafts and rockets, and can be used for leak checking in industry.
[0124]
The invention will be useful for quantitative measurement of fluorescence in flow cytometers for single and multiple cells.
[0125]
The present invention will also be useful for rapid estimation of biological contaminants in natural and controlled environments, such as tissue contaminants, contamination, industrial contaminants in the health, food and cosmetic industries.
[0126]
The ability of the dye to fluoresce in the UVA range with low wavelength UV irradiation is useful for selective photochemotherapy of skin cancer. In another preferred form, the dye may be for use as a component of a sunscreen composition in the cosmetics industry.
[0127]
Yet another application is that the dye is a biosurfactant and can be used in antimicrobial toiletries and compositions.
[0128]
In yet another preferred form of use, the dye has a long shelf life at room temperature as determined by fluorescence spectrophotometry.
[0129]
In yet another preferred mode of use, the fluorochromes present will impart a natural color to the cosmetics and will save the expense of coloring additives.
[0130]
Another use for the fluorescent dyes is as a component of new remote sensing instruments and underwater probes, which require data based on light wavelength sensitivity.
[0131]
The invention is illustrated by the following examples, which should not be construed as limiting the scope of the invention in any way.
[0132]
【Example】
Details of methods for the extraction, partial purification and characterization of the dye and experiments performed to examine the fluorescent effect of the dye by spectroscopy and epifluorosense microscopy are disclosed.
[0133]
Example 1
Material extraction
Subworld: Metazoa
Gate: Echinoderm
Amon: Remnants
Class: Sea cucumber
Subclass: Trees (Dendrochirotacea)
Eyes: Dendrochirota
Genus: Holothuria
Species: scbra
The animals belonging to the above were collected on the coast of central west coast of India at ebb. These were brought back to the laboratory and kept in a glass tank containing 30-32 (30%) seawater per 100 salinity until taxonomic identification and subsequent use. The animals were adult and sexually mature.
[0134]
Example 2
Pigment extraction
Two methods were tried.
1) In our original experiments, the post-harvest animals were frozen at −20 ° C. and when thawed, partial dye appeared in the tray. This was processed and the dye was removed from one animal three to four times in this manner. Figures 1 and 2 show fresh animals and animals used four times for extraction.
2) In a second method, the animals were washed first with tap water and then with Milliq water (Millicu water; ultrapure water). The body was cut open with sharp scissors and the body wall was separated from other internal organs. The epidermis skin was peeled off from the body wall with a sharp razor and stored in a refrigerator at −20 ° C. if not immediately processed. Thereafter, the mixture was placed in a beaker, to which a 50% v / v ethanol alcohol and MQ water mixture was added in the following ratio.
Animal skin 15g: 50% ethanol alcohol 250ml
[0135]
Example 3
Filtration of dye solution
This step was performed to remove cell debris and some of the suspended and precipitated impurities. It was centrifuged to make a clear solution and to settle any suspension.
The colored solution was decanted and filtered through a microfiltration unit (Vensil make) glass filter with the aid of a peristaltic pump. The filtrate was placed in a parabolic flask and left at 200 rpm for 30 minutes.
[0136]
Example 4
Dye concentration
The coloring solution was then placed in a water bath at 80 ° C. and concentrated to a third volume. This also caused the alcohol present to evaporate. The concentrate was again subjected to filtration with the same filter device.
[0137]
Example 5
Dye purification
The dye solution is NaCl, MgCl₂, MgSO₄And other impurities such as water-soluble compounds. It has been found that when polar organic solvents such as alcohol (dehydrated), acetone are added to the dye concentrate solution, it will precipitate out rapidly.
It can then be separated from seawater salts. This is to purify the dye for spectroscopic analysis.
The concentrated solution prepared according to Example 5 described above was placed in a 500 ml separatory funnel, and ethanol (80 ml of concentrated supernatant + 100 ml of 99.5% ethanol) was added thereto. The separatory funnel was tilted, the contents were mixed slowly, and the precipitate was collected overnight. The concentrate containing the precipitate is centrifuged at 1500 rpm for 4-5 minutes and the upper layer is discarded. The precipitate was dissolved again in 5 ml of MQ water, and 100% ethanol was added again until the precipitation was completed. This was centrifuged once more, and the precipitate was collected. This step is repeated 3-4 times to purify the dye.
The precipitate is evaporated for 5 minutes in a water bath at 80 ° C. until dry. The pure dye is scraped off with a spatula and stored in a dry glass vial at room temperature.
250 ml of the 50% ethanol crude extract from Example 2 was evaporated to a powder form to give 2.5 g of partially purified dye.
[0138]
Example 6
Physical properties of the compound
The crude extract is yellow-green in color. The physical properties of a pure dry dye are reddish-brown under sunlight when viewed with the naked eye. In the light beam, a green tint is observed. The dye is water soluble and insoluble in organic solvents such as pure ethanol, methanol, and acetone. It is amorphous in nature. It has a pH of 6.5 and has a negative charge.
[0139]
Example 7
Structural analysis of the dye by chemical methods
Experiment 1: The dye was subjected to CHNS elemental analysis, and the results are shown in Tables 1 (a) and (b).
Experiment 2:
The dye is dissolved in MQ water at ＠ 2 mg / ml and examined for chemical properties. The presence and absence of certain groups were tested and the results are shown in Table 2. Whenever the test showed negative, the experiment was repeated with a high concentration of dye.
For example, beta-mercaptoethanol (reducing agent) was added to a 2 mg / ml dye solution. No decolorization of the compound occurred. This proved that the quinonoid ring was absent and the dye was a dye.
In experiment 3, the concentrated dye solution @ 10 mg / ml was heated, but no precipitation or aggregation was observed. This demonstrated that the compound was non-protein in nature.
In experiment 4, one drop of concentrated HCl was added to the same solution and Fehling's solution was added. No color change proved that the reducing sugar was absent.
[0140]
Example 8
Inspection of the charge of the dye:
The charge of the compound was determined by gel electrophoresis.
Dye samples (10 ml) are loaded on a 1% agarose gel prepared in 0.5X TBE. The gel was run at 65 volts for 1 hour. It was removed from the gel mold system and observed visually and with a UV transilluminator. It can be seen that the dye is migrating towards the positively charged electrode, the dye itself being a negatively charged compound. Therefore, it was being drawn towards the positively charged electrode.
[0141]
Example 9
Biosurfactant analysis:
The biosurfactant nature of the dye was observed by adding foam to the water while shaking. The solution had a foamy feel.
[0142]
Example 10
Antibacterial test:
Since the marine dyes are phenolic compounds and phenolic compounds generally have antimicrobial activity, an antimicrobial assay was performed with this compound and the zone of inhibition was observed.
E. FIG. The E. coli (wild type) culture was grown overnight in 50 ml of MacConkey culture in an Erlenmeyer flask (100 ml). Antibiotic assay agar medium was prepared and sterilized. Thereafter, the sample was placed at a temperature of 50 ° C. 1 ml of E. coli (wild type) culture was added to it. The culture was mixed with antibiotic assay agar and allowed to solidify. A 10 mg / ml sample was prepared and immersed in a filter paper disc. Then, E. E. coli and placed on the antibiotic assay agar medium.
Then, it incubated at 37 degreeC for 24 hours in the incubator. The zone of inhibition surrounding the filter disk was observed. This means that the dye is E.I. It has been shown to have antibacterial activity against Gram-negative organisms such as E. coli.
[0143]
Example 11
UV / visible spectroscopy analysis of the dye
Equipment used: Genesys2 UV spectrophotometer
A 2 mg / 10 ml solution was prepared in a volumetric flask, and spec readings were made in the UV visible range by adding 2 ml of the solution using a quartz cell. The control was ultrapure water.
UV and visible spectroscopy at 300 nm-700 nm wavelength (FIGS. 3 and 4) was performed. Peaks are recorded at wavelengths of 379 nm and 439 nm.
UV-visible spectroscopy at 250 nm-350 nm wavelength (FIGS. 3 and 4) was performed. Peaks are recorded at wavelengths 272 nm and 299 nm.
[0144]
Example 12
Fluorescence spectroscopy of the dye
Equipment used: Hitachi fluorescence spectrophotometer
Fluorescence spectroscopy was performed at different wavelength ranges of the visible and UV spectra to examine the emission range. It had an excitation wavelength of 270 nm. Fluorescence appeared at a maximum intensity of 324-380 (FIG. 5).
In fluorescence spectroscopy, at an excitation wavelength of 450 nm, fluorescence appeared at a maximum intensity of 500-580 (FIG. 6).
In fluorescence spectroscopy, at excitation wavelength 540 nm, fluorescence appeared at a maximum intensity of 500-620 (FIG. 7), and in fluorescence spectroscopy at excitation wavelength 555 nm, fluorescence appeared at a maximum intensity of 575-620 nm (FIG. 8). .
[0145]
Example 13
Physical inspection of luminescence by UV transilluminator and gel documentation system
Whatman No. One filter paper was cut, soaked in the diluted crude extract, and observed under a gel doc UV light. It can be clearly seen that as the dye penetration progressed, the fluorescent region further deepened (FIG. 9).
In another test, the filter paper used for filtration was observed with a UV transilluminator in a lamp having a UV range of 260-280 nm. Blueish greenish fluorescence was observed (FIG. 10).
[0146]
Example 14
Epifluorosense Microscopy
An epifluorosense microscopic study was performed using this dye as a stain at a dilution of 1: 40000 and recording the light emitted upon excitation with different cubes, comparing the color to known fluorochromes. A cytogenetic air-dried preparation of fixed tissue was made. In contrast, one drop of the dye was added and a cover slip was placed. Screening was performed with WLY, WB and WG cubes of Olympus reflected light, using excitation of UV and visible light spectra.
The wavelength range of the WU cube was 330 nm-385 nm.
The wavelength range of the WB cube was 450 nm-480 nm.
The wavelength range of the WG cube was 510 nm-550 nm.
[0147]
Example 15
Emission ranges in different excitation ranges were found. It was as follows:
Excitation in the WU 330 nm-385 nm range emitted fluorescence in the 380 nm-400 nm range. Excitation with a WB filter having a spectral range of 450 nm-480 nm emitted fluorescence in the 500 nm-570 nm range. Excitation by a WG filter having a spectral range of 510 nm-550 nm emitted fluorescence in the 570 nm-650 nm range.
Epifluorosense microscopy screening of cytogenetic slides in bright field using transmitted light emits in the entire white range of the visible spectrum and depends on the density of the cell components, gray shades, phase Provides a contrast-like effect.
[0148]
Example 16
Emission fluorescent color
The color tone of the emitted light was observed in areas where only the dye was present and where some samples were present. Excitation spectral range and emitted fluorescence strictly followed Stokes' law (Table 3).
[0149]
Example 17
Microphotography of slides with the dye used as epifluorosense microscopic dye
Microphotography of luminescence fluorescence in slide-like areas without cells or with sample cells, exposure from 50 seconds to 60 seconds with Kodak film 400 ASA speed under WU330-385 nm range, WB450 nm-480 nm range, WG510 nm-550 nm range and bright field. The test was performed by changing the time to seconds.
The results are shown in FIGS.
[0150]
Example 18
Stability test
The dye is stable, remains active at room temperature and remains in that state up to 120 ° C., demonstrating no change in spectral properties after such treatment. The compound retained its stability for about one year without any contamination or chemical degradation. The marine dye did not undergo photolysis after light treatment. Thus, the marine dye did not require a stabilizer.
[0151]
Example 19
Insecticidal effect
The compound was toxic to insects. It has shown toxicity to insects such as ants. The filter moistened with the dye was left on a laboratory bench as it was. The next day, it was observed that it was full of dead ants.
[0152]
Example 20
The dye was tested on cell lines to confirm activity.
[0153]
Example 21
Dyeing with dye
Fixed tissues from various sources with glacial acetic acid and methanol were taken on slides and the dye solution was added to it without pretreatment. Different cell parts were observed to take up dye solutions differently. The nucleus was stained due to arginine and lysine rich protein staining present in the nucleus (eg histone). Other cell organelles were also stained. As the marine dyes stain chromosomal proteins, their value in cell karyotype studies has increased.
[0154]
Example 22
The bioactive extract of the dye was placed in a microcentrifuge tube, stored at -20 ° C, and the frozen state was observed with UV light. In another experiment, what was later placed in the dye solution was kept at -20 ° C and observed with a UV transilluminator.
[0155]
Example 23
The extract was used as a veterinary formulation of dog mite / flea killing. The 1: 200 diluted crude extract killed mites and fleas in less than 40 seconds.
[0156]
[Table 1]

[0157]
[Table 2]

[0158]
[Table 3]

[0159]
Advantages over currently available dyes
1. The dye is non-radioactive because it is a dye of natural origin and not synthetic.
[0160]
2. This dye, in its single form, is comparable to three different synthetic fluorochromes and shows the same fluorescent emission.
[0161]
3. The dyes can be used as rapid microscopic stains and impart a phase contrast effect without any extra expense for microscope phase contrast accessories and without having to perform long protocols for sample fixation and storage. In particular, on-site qualitative inspection of raw samples.
[0162]
4. Since there is no deterioration in fluorescence quality for a long time, refrigeration is not required for export. Currently, commercially available fluorescent dyes are exported under refrigeration corresponding to -20 ° C.
[0163]
5. Our dye, unlike previously known marine jellyfish-derived green fluorescent protein (GFP), is not a reporter gene. The result is direct. GFP absorbs blue light at 395 nm, has a small peak at 470 nm, and emits green light. Our dye emits three fluorescent colors at three different fluorescent wavelengths. The dyes are soluble in water and can therefore be used in components where water-soluble dyes are required. The dye is insoluble in organic solvents such as ethanol, methanol and acetone.
[0164]
6. The dye has a negative charge.
[0165]
7. The dye has a pH of 6.5 and is almost neutral and will not have a significant effect on the final pH in the composition.
[0166]
8. The dyes are non-protein in nature and therefore do not degrade under natural conditions.
[0167]
9. Said dyes have the property of biosurfactants and can therefore be used in soaps and toilet compositions.
[0168]
10. The dye has antibacterial properties.
[0169]
11. The dyes emitted these fluorescent colors even in a dilution range of 1: 40000 times (that is, 1 gram of the dye powder was dissolved in 40 liters of ultrapure water). The fluorescence of the extract persisted for at least one year at room temperature. These multicolor emitting dyes at different excitation wavelengths are comparable to already commercially available fluorochrome microscopic dyes.
[0170]
12. The blue colored fluorescence of this dye is comparable to the same color emission by DAPI fluorochrome at the same wavelength excitation, said DAPI fluorochrome being a component of non-radioactive labeling kits for biochemistry, cell biology, immunochemistry and molecular biology. Has been used as
[0171]
13. The blue colored fluorescence of the dye is also comparable to the color emission by Hoechest 33258, which is used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.
[0172]
14． The blue-colored fluorescence of this dye is also comparable to the color emission at the same excitation wavelength of Hoechest 33342 fluorochrome used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. I do.
[0173]
15. The yellow-colored fluorescence of this dye in the visible range is also comparable to the same color emission of acridine orange, which is used as a component in non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology .
[0174]
16. The yellow-colored fluorescence in the visible range of this dye is also comparable to the same color emission of auramine, which is used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.
[0175]
17． The yellow-colored fluorescence of the present dye in the visible range is also comparable to the same color emission of FITC used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.
[0176]
18. Its orange-colored fluorescent emission is also comparable to the orange fluorescent color of propidium fluorochrome iodide used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.
[0177]
19. Its orange colored fluorescent emission is also comparable to the orange fluorescent color of rhodamine fluorochrome used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.
[0178]
20. Its orange-colored fluorescent emission is also comparable to the orange fluorescent color of TRITC fluorochrome used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.
[0179]
21. Unlike synthetic commercial dyes used for the same purpose, the dyes are stable at room temperature and have a long shelf life. The dye non-radioactive kits can be exported at room temperature.
[0180]
22. The single dye has the characteristics of at least 123 different fluorochromes currently on the market (such as DAPI, Hoechest 33258, Hoechest 33342, FITC, acridine orange, auramine, rhodamine, TRITC, and propidium iodide). . Under normal microscope light, the gray shade produces a phase contrast effect, which is useful for rapid screening of cytogenetic, cytological, and histochemical slides and extra phase contrast accessories for microscopes Save money on parts. Fluorescent color emission follows Stokes' law of fluorescence.
[0181]
23. The microphotograph with the Kodak film roll shows the color tone of the adjacent color emission wavelength. Green tones appear as well, such as when observing blue fluorescence under an epifluorosense microscope in a micrograph.
[0182]
24. The microphotograph with the Kodak film roll shows the color tone of the adjacent color emission wavelength. Green tones appear as well, such as when observing yellow fluorescence under an epifluorosense microscope in a microphotograph. Similar to observing an orange fluorescent color under an epifluorosense microscope in a microphotograph, a red color tone appears as well.
[0183]
25. Cytogenetic slides observed under total fluorescence provide a counterstaining effect on cells in a background where there is no sample and only dye is present.
[0184]
26. The dyes can be used for the preparation of polyvinyl chloride which emits a fluorescent color. It can also be used in fluorescent colors in various paints, inks and textiles.
[0185]
27. The dyes can be used in fluorescent dye compositions for bleaching and brightening of polymers. The dyes can be used for leak detection with full spectrum fluorescent dyes. It can also be used in automated chemical measurement systems. It can also be used to record the location of equipment such as crashed aircraft, life raiders, and rockets. In addition, it can be used in underwater probes. The dyes can be used in photochemotherapy for skin cancer.
[0186]
28. The dye can be used as a chromatophor sunscreen component in cosmetic creams and cosmetic liquids.
[0187]
29. The water miscibility of the dye allows it to be easily mixed with moisturizers. It can be used as a component of a fluorescence in situ hybridization application kit for molecular diagnostics. It is also used for labeling DNA, RNA, proteins and enzymes, non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry, and molecular biology, immunofluorescence detections, DIG-labeled oligonucleotide probes. And reverse staining of anti-DIG Fab fragments, single and multiple cell quantitative fluorescence in flow cytometry, can be used as a component of fluorochrome stains for epifluorosense microscopy.
[0188]
30. The dyes are used for rapid testing of biological contamination in the health food, cosmetics, pharmaceutical and chemical industries, for rapid estimation of biological contamination in laboratory cultures, and for rapid testing of biological contaminants under outdoor conditions it can. It is also a competitive inhibitor of cholinesterases.
[0189]
31. The dye can be used in an antimicrobial composition.
[0190]
32. The dye can be used as a biosurfactant in a toilet composition.
[0191]
33. The dye can be used as a natural colorant A bioactive composition of a marine dye at a ratio of 1: 40000 in ultrapure water, and has a phase contrast effect under three different colors of fluorescence and transmitted light at three different wavelengths. Obtainable.
[0192]
34. The dye can be purified with 250 ml of 50% ethanol crude extract, and purified by evaporating in a water bath at 80 ° C. for 5 minutes to obtain 2.5 g of the purified dye in powder form. A 2 mg / 10 ml solution composition is used for spectrophotometric structural analysis of the dye by chemical methods using a concentration of 10 mg / ml. A 1: 40,000 dilution of the bioactive composition with water as a binder for the dye confers fluorescence of three colors at three different wavelengths.
[Brief description of the drawings]
FIG.
Before dye extraction, fresh sea cucumber Holothuria scabra from the outdoors.
FIG. 2
Sea cucumber Holothuria scabra after four extractions of dye.
FIG. 3
Scanning in the UV visible range.
FIG. 4
Scanning in the UV region.
FIG. 5
Fluorescence spectrophotometry. Excitation wavelength 270 nm.
FIG. 6
Fluorescence spectrophotometry. Excitation wavelength 450 nm.
FIG. 7
Fluorescence spectrophotometry. Excitation wavelength 540 nm.
FIG. 8
Fluorescence spectrophotometry. Excitation wavelength 270 nm.
FIG. 9
Whatman filter no. 1 was cut into a circle, and the tip was immersed in a diluted crude extract of the dye, and the gel was extracted from Gel Doc. Under UV radiation. Arrows indicate fluorescence and were photographed through a UV filter. Bottom is control without dye.
FIG. 10
Whatman filters were used for filtration of the extract and observed under a UV transilluminator (260 nm-280 nm wavelength range bulb). Arrows indicate fluorescence. The lower filter paper was free of extract and was retained as a control.
FIG. 11
Epifluorescence microscopy blue fluorescence emission from a WU cube with an excitation range of 330 nm-385 nm, and FIG. 11 shows the fluorescence of the dye without any sample.
FIG.
Epifluorescence microscopy blue fluorescence emission from a WU cube having an excitation range of 330 nm to 385 nm. FIG. 12 shows a case including cells observed with a 10 × objective lens.
FIG. 13
Epifluorescence microscopic blue fluorescence emission from a WU cube having an excitation range of 330 nm to 385 nm, and FIG. 13 shows the same object observed with a 40 × objective lens.
FIG. 14
Epifluorescence microscopy blue fluorescence emission from a WU cube having an excitation range of 330 nm to 385 nm. FIG. 14 shows cells observed with a 100 × oil immersion objective lens.
FIG.
Epifluorosense microscopic green-yellow fluorescence emission by WB cube with excitation range 450nm-480nm. FIG. 15 shows the fluorescence of the dye without any sample.
FIG.
Epifluorosense microscopic green-yellow fluorescence emission by WB cube with excitation range 450nm-480nm. FIG. 16 shows a case including cells observed with a 40 × objective lens.
FIG.
Epifluorosense microscopic green-yellow fluorescence emission by WB cube with excitation range 450nm-480nm. FIG. 17 shows the same product observed with a 100 × oil immersion objective lens.
FIG.
Epifluorosense microscopic green-yellow fluorescence emission by WB cube with excitation range 450nm-480nm. FIG. 18 shows the same object observed with a 100 × oil immersion objective lens.
FIG.
Epifluorosense microscopic tone of orange fluorescence emission from a WG cube having an excitation range of 510 nm-550 nm. FIG. 19 shows the fluorescence of the dye without any sample.
FIG.
Epifluorosense microscopic tone of orange fluorescence emission from a WG cube having an excitation range of 510 nm-550 nm. FIG. 20 shows fluorescence in the case where cells observed with a 10 × objective lens are included.
FIG. 21
Epifluorosense microscopic tone of orange fluorescence emission from a WG cube having an excitation range of 510 nm-550 nm. FIG. 21 shows a case where the same object is observed with a 40 × objective lens.
FIG. 22
Epifluorosense microscopic tone of orange fluorescence emission from a WG cube having an excitation range of 510 nm-550 nm. FIG. 22 shows cells observed with a 100 × oil immersion objective lens.
FIG. 23
Cells observed through a bright field under a 10 × objective. A gray shade-phase contrast effect was observed.
FIG. 24
Identical cells observed through brightfield under a 100x oil immersion objective.

Claims (54)

Biologically active extracts obtained from marine organisms and useful as natural fluorescent dyes having the following characteristics:
i. Decolorization by reducing agent,
ii. Not a synthetic compound,
iii. The crude extract of the dye is yellow-green in color,
iv. When observed with the naked eye under sunlight, the purified dye is a reddish-brown powder,
v. Under the light of an electric light, a slightly green color is emitted,
vi. Amorphous in nature,
vii. Water soluble,
viii. Insoluble in organic solvents such as ethanol, methanol and acetone,
ix. Negative charge,
x. having a pH of 6.5,
xi. The presence of a phenolic group,
xii. Absence of a quinoid ring,
xiii. Absence of aromatic amine groups,
xiv. Non-proteinaceous in nature,
xv. No reducing sugars,
xvi. The dye has the properties of a biosurfactant,
xvii. Also, the dye showed antimicrobial properties and showed an inhibition zone when the antimicrobial assay was performed.
xviii. The dye / dye is a fluorescent dye, which emits fluorescence when excited by a spectrophotometer at different wavelengths in the UV and visible spectral ranges,
xix. 300 nm-700 nm UV, visible spectroscopy, and peaks are recorded at 379 nm and 439 nm wavelengths.
xx. UV, visible spectroscopy at 250 nm-350 nm, and peaks at 272 nm and 299 nm wavelengths.
xxi. Fluorescence spectroscopy in the UV and visible spectra shows that, when excited at UV 270 nm wavelength, it fluoresces in the 324 nm-380 nm range and falls into the UVA wavelength range of solar UV.
xxii. At an excitation wavelength of 450 nm in fluorescence spectroscopy, fluorescence emission occurred between 500 nm and 580 nm and had a maximum intensity.
xxiii. At an excitation wavelength of 540 nm in fluorescence spectroscopy, fluorescence emission occurred between 500 nm and 620 nm and had a maximum intensity.
xxiv. At an excitation wavelength of 555 nm in fluorescence spectroscopy, fluorescence emission occurred between 575 nm and 620 nm and had a maximum intensity.
xxv. Whatmann Filter No. 2 immersed in a 1: 400,000 dye concentration diluent under a UV transilluminator with a UV lamp in the range of 260 nm-280 nm and a Gel Documentation system. Physical examination according to 1 fluoresces blue-green tones,
xxvi. The fluorescent cubes of the epifluorosense microscope emit three different colored fluorescences at three different wavelengths in the UV and visible range,
xxvii. Fluorescent blue emission appears in UVA in the 380 nm-400 nm range when excited in the UV cube WU 330 nm-385 nm excitation range.
xxviii. Fluorescent yellow emission appears in the 500 nm-570 nm range when excited in the WB cube 450 nm-480 nm excitation range.
xxix. Fluorescent orange emission appears in the 570 nm-650 nm range when excited in the WG cube 510 nm-550 nm excitation range.
xxx. The dye exhibits a gray shade under a normal transmitted light of an epifluorosense microscope when viewed with a 10 × objective lens;
xxxi. The dyes emit these fluorescent colors even at a dilution range of 1: 40,000 (ie, 1 gram of dye powder dissolved in 40 liters of ultrapure water),
xxxii. The fluorescence of the extract persisted after at least one year at room temperature,
xxxiii. The fluorescence of the dye is highly photostable, does not degrade upon prolonged exposure to direct light, and xxxiv. The fluorescence of the dye does not change upon freezing at 20 ° C., as in extracts from chemiluminescent organisms, at temperatures at which the molecules cannot obtain the energy required for activation. The dyes appear in intertidal flooded shallow waters and deep waters, and are abundant in shades such as lobes, fissures, rock shelves, overhangs, rocky and sandy habitats, from dull colors. It is a sticking and fixed floating body that may or may not have exo- and endoskeletons, colored to a bright color, is a swimming organism with various swimming powers, and is usually nocturnal. The dye according to claim 1, obtained from a marine organism such as sea cucumber Holothuria scabra, which is susceptible to active prey. 2. A dye according to claim 1, wherein the multicolor emission of the dye at different excitation wavelengths is comparable to fluorochrome microscopic dyes already on the market. 2. The blue colored fluorescence of the present dye is comparable to the same color emission by DAPI fluorochrome used as a component of non-radioactive labeling kits for biochemistry, cell biology, immunochemistry and molecular biology at the same wavelength excitation. Dye. 2. The dye of claim 1, wherein the yellow color fluorescence of the present dye in the visible range is comparable to the same color emission of auramine used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. dye. 2. The yellow color fluorescence of the present dye in the visible range is comparable to the same color emission of FITC used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. dye. The orange-colored fluorescent emission is comparable to the orange fluorescent color of Propidium Iodide Fluorochrome used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. The dye according to claim 1. 2. The dye of claim 1, wherein the orange colored fluorescent emission is comparable to the orange fluorescent color of rhodamine fluorochrome used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. . The dye of claim 1, wherein the orange colored fluorescent emission is comparable to the orange fluorescent color of TRITC fluorochrome used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. . The dye of claim 1, wherein said dye has a stable and long shelf life at room temperature. The dye of claim 1 wherein the molecular and radioactive kits of the dye are exportable at room temperature. Said single dyes comprise at least 100 different fluorochromes currently marketed, such as (DAPI, Hoechest 33258, Hoechest 33342, FITC, acridine orange, auramine, rhodamine, TRITC, and propidium iodide). The dye according to claim 1, which has characteristics. 2. The dye of claim 1, wherein said dye can be used in any application where phycobiliproteins are currently used, unlike the dyes which do not undergo fluorescence loss upon freezing. When viewed under a 10x objective in the bright field of a fluorescent microscope, the bluish-gray shade provides a phase contrast effect useful for rapid screening of cytogenetic, cytological, and histochemical slides, The dye of claim 1 which saves money on extra phase contrast accessory parts. When observed under a 100-fold oil immersion objective with a normal transmitted light microscope, the yolk, nucleoplasm and chromatin proteins of actively dividing cleavage cells show different staining colors. 2. A dye according to claim 1, which has a yellow shade, a yellow shade for the latter and a dark blue shade for the last cellular component. This can be used in rapid bioassays of efficacy and can be observed in the various biochemical components of the cells. The dye according to claim 1, wherein the fluorescent color emission complies with Stokes' law of fluorescence. 2. A dye according to claim 1 wherein the microphotograph on a Kodak roll film shows adjacent color emission wavelengths such as blue fluorescence under epifluorosense. 2. The microphotographs by Kodak roll film exhibit adjacent color emission wavelengths such that green color also appears when observing yellow fluorescence in the microphotograph under an epifluorosense microscope. dye. 2. A dye according to claim 1, wherein a red tint also appears when observing the orange fluorescent color microscopically under the epifluorosense microsoup. 2. A dye according to claim 1, wherein the cytogenetic slides observed under total fluorescence show a counterstaining effect of the cells and cell components on the background color when there is no sample and only the dye is present. 2. A dye according to claim 1, wherein said dye is diluted with water in a ratio of 1: 10,000 and which gives three colors of fluorescence at three different wavelengths. 2. A dye according to claim 1, wherein said dye is diluted with water at a ratio of 1: 40,000 and this gives three colors of fluorescence at three different wavelengths. A composition comprising a biologically active extract obtained from the marine cucumber Holothuria scabra together with conventional additives, and is useful for preparing a flexible polyvinyl chloride film exhibiting a fluorescent color. A composition comprising a biologically active extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful in the preparation of coating compositions and inks. A composition containing a bioactive extract obtained from the marine cucumber Holothuria scabra with conventional additives, and is useful for leak detection. A composition comprising a biologically active extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful for underwater probes. A composition containing a biologically active extract obtained from the marine cucumber Holothuria scabra with conventional additives, and is useful for photochemotherapy of skin cancer. A cosmetic composition comprising a biologically active extract obtained from the marine cucumber Holothuria scbra with conventional additives. A composition comprising a biologically active extract obtained from the marine cucumber Holothuria scabra together with conventional additives, and is useful as fluorescent molecular probe in situ hybridization kits for molecular diagnostics. A composition comprising a biologically active extract from the marine cucumber Holothuria scabra with conventional additives, useful as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology . A composition comprising a biologically active extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful in immunofluorescence detection. A composition comprising a biologically active extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful as a counterstain for DIG-labeled oligonucleotide probes and anti-DIG Fab fragments. A composition comprising a bioactive extract obtained from the marine cucumber Holothuria scbra with conventional additives, useful in single and multiple cell quantitative fluorescence in single and multicolor flow cytometry applications. A composition comprising a bioactive extract obtained from the marine cucumber Holothuria scabra, which can be used in experiments that require the application of various fluorescent dyes to be performed at outdoor stations located in the sub-zero temperature range. A composition comprising a bioactive extract obtained from the marine cucumber Holothuria scabra with conventional additives, and is useful as fluorochrome stains for epifluorosense microscopy. A composition comprising a bioactive extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful for the rapid testing of biological contamination in the health food, cosmetic, pharmaceutical and chemical industries. A composition comprising a biologically active extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful for rapid estimation of biological contaminants in laboratory cultures. A composition comprising a biologically active extract obtained from the marine cucumber Holothuria scabra with conventional additives, and is useful for rapid testing of biological pollutants under outdoor conditions. A composition comprising a biologically active extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful as a competitive inhibitor of cholinesterases. A composition comprising a biologically active extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful in antimicrobial compositions. A composition comprising a bioactive extract obtained from the marine cucumber Holothuria scabra with conventional additives, and is useful as a biosurfactant in toilet compositions. A composition comprising a bioactive extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful in insecticidal and rodenticidal compositions. A composition comprising a biologically active extract obtained from the marine cucumber Holothuria scbra with conventional additives, and is useful as a natural colorant. A bioactive composition comprising an extract obtained from the marine cucumber Holothuria scbra in ultrapure water at a ratio of 1: 40000 can obtain three colors of fluorescence at three different wavelengths. A biologically active composition comprising an extract obtained from the marine cucumber Holothuria scabra, which can provide phase contrast and histochemical reverse staining effects for different biochemical components of cells under transmitted light. A skin care composition comprising the dye together with excipients such as a physiologically and cosmetically acceptable diluent, dispersant or carrier. The dye according to claim 1,
(A) preparation of a fluorescent or flexible polyvinyl chloride film;
(B) the use of fluorescent colors in various paints, inks and textiles;
(C) a fluorescent dye composition for bleaching and whitening of the polymer;
(D) leak detection with a full spectrum fluorescent dye;
(E) use in an automated chemical measurement system;
(F) to record the location of equipment such as crashed aircraft, life raiders, and rockets;
(G) underwater probes;
(H) UVA is used in photochemotherapy for skin cancer;
(I) chromatophore sunscreen components of cosmetic creams and cosmetic liquids;
(J) the water miscibility of the dye allows it to be easily mixed with moisturizers;
(K) fluorescence in situ hybridization application kit components for molecular diagnostics;
(L) components of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry, and molecular biology for labeling DNA, RNA, proteins and enzymes;
(M) immunofluorescence detections;
(N) reverse staining of DIG-labeled oligonucleotide probes and anti-DIG Fab fragments; and (o) single and multiple flow cytometry applications;
(P) fluorochrome stains for epifluorosense microscopy;
(Q) For quick inspection of biological contamination in health food industry and cosmetics industry;
(R) the pharmaceutical and chemical industries;
(S) for rapid estimation of biological contaminants in laboratory cultures;
(T) for rapid inspection of biological pollutants under outdoor conditions;
(U) competitive inhibitors of cholinesterases;
(V) in an antimicrobial composition;
(W) as a biosurfactant in a toilet composition;
(X) a natural colorant;
(Y) a bioactive composition of the marine dye in a 1: 40000 ratio in ultrapure water;
(Z) to obtain a phase contrast effect under three colors of fluorescence and transmitted light at three different wavelengths;
(Aa) dyes for various fluorescent applications performed in the sub-zero temperature range;
Useful for. A process for the extraction of natural fluorescent dyes from the marine sea cucumber Holothuria scbra,
a) harvesting said material from the shore, exchanging seawater and keeping it in a laboratory tank without any mechanical ventilation throughout the night;
b) freezing said animals at −20 ° C.
c) thawing the animal to obtain the dye; and d) repeating steps (b) to (c) 3-4 times for dye removal, and, if desired, purifying the dyes ,
Including the stage. In the process for the extraction of natural fluorescent dyes from the marine cucumber Holothuria scabra, the pigment is osmotically shocked from the skin of the marine cucumber Holothuria scabra, preferably by first adding 50% ethanol to ultrapure water and repeating this at least four times. Extraction to obtain the dye. 49. The process of claim 48, wherein the dye is diluted with water in a ratio of 1: 40000, which imparts three colors of fluorescence at three different wavelengths. 49. The process according to claim 48, wherein 250 ml of 50% ethanol crude extract is purified by evaporating in a water bath at 80 ° C for 5 minutes to obtain 2.5 g of partially purified dye in powder form. 49. The process of claim 48, wherein the dye is diluted with water at a ratio of 1: 10000, which provides three colors of fluorescence at three different wavelengths. 49. The process of claim 48, wherein the physical characteristics of the dye are assessed by taking a 2 mg / 10 ml solution for spectrophotometry. 49. The process of claim 48, wherein said dye was used at a concentration of 10 mg / ml for dye analysis using a chemical method.

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