The method of catalyzing and hydrolyzing natural nucleoside compound by metal ion
Technical field:
The present invention relates to a kind of method for preparing the low secondary glycosides compound of glycosyl, especially a kind of simple to operate, cost is low, be fit to produce in enormous quantities, saponin(e is destroyed the method for little and free of contamination catalyzing and hydrolyzing natural nucleoside compound by metal ion.
Background technology:
Natural nucleoside compound is the important component part of activeconstituents in the natural drug, and source (plant is herbal medicine particularly) is extensive.Nature, no matter in plant or other natural product, glycosides compound all is first biomass (Biomass) sources at content with on distributing, and has consequence and meaning aspect medicine resource.
Studies show that many glycosides compounds all are that the form with natural prodrug or natural radioactivity compound exists, and have a plurality of glycosyls on the aglycon, show low activity even non-activity.And after entering human body as medication, through the effect of Digestive tract and entero-bacte, after conversion generated secondary low glycosyl glycosides or aglycon, it was just easier to absorb, and drug effect is bigger.As the higher dioscin of content in the Wild yam herbal medicine, two glucose saponin(e and trillenoside, all contain three glycosyls in each molecule, drug effect is low, have hemolytic again.But removing some or all of glycosyl, when changing into the dioscin of a glycosyl and aglycon, the drug effect height, hemolytic is low.Therefore, for improving the drug effect of natural nucleoside compound, existing method is to remove the part glycosyl of polysaccharide base glycosides, even complete " sloughing " sugared coat, is converted into active higher secondary low glycosyl glycosides or aglycon.With usual employing acid and alkali hydrolysis method, but acid and alkali hydrolysis method reaction is violent, and reaction preference is poor, to saponin(e destroy big and by product many, can pollute environment simultaneously.The microbial enzyme conversion method of Xing Qiing had obtained bigger development in recent years, but microorganism is strict to culture condition, and especially enzyme has specificity (a kind of enzyme can only hydrolysis one class glycosidic link), and range of application is restricted.
Summary of the invention:
The present invention is in order to solve the above-mentioned technical problem of existing in prior technology, provide a kind of simple to operate, cost is low, be fit to produce in enormous quantities, saponin(e is destroyed the method for little and free of contamination catalyzing and hydrolyzing natural nucleoside compound by metal ion.
Technical solution of the present invention is: a kind of method of catalyzing and hydrolyzing natural nucleoside compound by metal ion is characterized in that adopting metal ion catalysis, the glycosyl of hydrolyzing natural nucleoside compound, the active higher secondary glycosides compound of low glycosyl of preparation.
Described metal ion is Fe
3+, Fe
2+, Mg
2+, Cu
2+, Zn
2+, Co
2+, Al
3+, Ba
2+, Pb
2+, Ca
2+, Mn
2+, Li
+, Na
+Or K
+
Described natural nucleoside compound is saponins or flavonoid glycoside; Described saponins is protopanoxadiol saponins, Protopanaxatriol's saponins, gypenoside, Radix Astragali saponin, dioscin, pulchinenoside, Root of Coral Ardisia saponin(e, saikoside, ophiopogonin, Herba Phyllanthi Urinariae's saponin(e or glycyrrhizin; Described flavonoid glycoside is rutin, Quercetin 3-galactoside, naringin, Hesperidin, epimedium flavone glycosides, soybean isoflavones glycosides or baicalin.
Described metal ion catalysis hydrolysis reaction condition is: concentration of substrate is 0.4%~5.0%, and concentration of metal ions is 2mmol/L~4000mmol/L, 20 ℃~80 ℃ of temperature, and the reaction times is less than or equal to 24 hours.
Behind the hydrolysis reaction, reaction solution adopts absorption with macroporous adsorbent resin, and metal ion is removed in washing, again alcohol wash obtain the hydrolysate natural nucleoside compound the secondary glycosides of low glycosyl or/and aglycon.
The concentration of described alcohol is 20%~95%.
The present invention finds that metal ion can catalyzing and hydrolyzing natural nucleoside compound, set up the method for common metal ionic catalysis Polysaccharides base glycosides compound (saponins and flavonoid glycoside), the glycosyl part of glycosides compound be can make or conversion secondary low glycosyl glycosides of generation or aglycon all removed.The metal ion of catalytic hydrolysis is reusable, preparation cost is low, and whole process of preparation safety simple to operate is fit to produce in enormous quantities, saponin(e is destroyed little and pollution-free, product can be widely used in that medicine is clinical, the exploitation of healthcare products, makeup and foodstuff additive.
Common metal ion (Fe
3+, Fe
2+, Mg
2+, Cu
2+, Zn
2+, Co
2+, Al
3+, Ba
2+, Pb
2+, Ca
2+, Mn
2+, Li
+, Na
+Or K
+But) all catalytic hydrolysis saponins, flavonoid glycoside:
(1). saponins
(1) higher PPD saponin(e (Rb class, Rc, the Rd) hydrolysis of content generates rare saponin(e Rg in genseng, the Radix Notoginseng etc.
3With a spot of F
2, K
1, Rg
5, Rh
2, C-K; Higher main conversion of PPT saponin(e (Re) of content produced rare saponin(e Rg
2With a spot of Rg
1, Rg
4, Pg
6, Rh
1
The pulchinenoside of (2) five glycosyls transforms and generates 3-o-Ara-(28-o-Glc-) disaccharide pulchinenoside, 3-o-Ara-monose pulchinenoside, 28-o-Glc-monose pulchinenoside and aglycon;
(3) hydrolysis of polysaccharide base saikoside generates monosaccharide groups saikoside and aglycon;
(4) the main hydrolysis of polysaccharide base gypenoside generates the ginsenoside Rg
3
The Radix Astragali saponin hydrolysis of (5) two glycosyls generates Cyclosiversioside F;
The Root of Coral Ardisia saponin(e hydrolysis of (6) four glycosyls generates two glycosyl Root of Coral Ardisia saponin(es, monosaccharide groups Root of Coral Ardisia saponin(e and aglycons; (7) the dioscin hydrolysis generates monosaccharide groups dioscin and aglycon; (8) hydrolysis of polysaccharide base Herba Phyllanthi Urinariae saponin(e generates secondary low glycosyl Herba Phyllanthi Urinariae saponin(e.
(2). flavonoid glycoside:
(1) the rutin hydrolysis generates Quercetol 3-monoglucoside and Quercetin;
(2) the Quercetin 3-galactoside hydrolysis generates Quercetin;
(3) the Hesperidin hydrolysis generates 7-glucose-Hesperitin and Hesperitin;
(4) the naringin hydrolysis generates naringenin.
Embodiment:
Embodiment 1:Fe
3+Catalytic hydrolysis protopanoxadiol saponins
Get 270.3 gram Fecl
36H
2O is dissolved in 1000 ml waters fully, is mixed with Fe
3+Solution.The 14 gram protopanoxadiol saponin(e Rb classes that the macroporous adsorbent resin separation and purification obtains, the mixture of Rc, Rd are dissolved in 1000 ml waters, are mixed with saponin(e solution.Behind above-mentioned two kinds of solution thorough mixing, under 20~80 ℃ of temperature, stirring reaction 14 hours.After reaction finished, reaction solution was through the absorption with macroporous adsorbent resin saponin(e of 300 ml volumes, remove impurity such as salt ion and sugar with 2400 milliliters washing after, use 20%~95% ethanol elution saponin(e again.Collect ethanol eluate, obtain product 9.5 grams after the drying.Perhaps 1000 milliliters of reaction solutions add 400 milliliters of water-saturated n-butanols, shake up static 2 hours of back, separate n-butanol layer, and above-mentioned n-butanol extraction operation repeats 4 times, merges n-butanol layer, washes propyl carbinol with 600 ml deionized water and removes Fe
3+Deng impurity, above-mentioned washing operation repeats 4 times, and n-butanol layer obtains product 9 grams after the concentrating under reduced pressure drying.Through high-efficient liquid phase chromatogram technique analysis (document 1), Rb class, Rc almost completely transform in the substrate, and Rd is most of to be transformed, and total transformation efficiency reaches 80%~90%.Product mainly is Rg
3With a spot of F
2, K
1, Rg
5, Rh
2, C-K, wherein Rg
3Content is more than 70%.
Embodiment 2:Al
3+Catalytic hydrolysis Protopanaxatriol saponins
Get 666g gram Al
2(SO
4)
318H
2O is dissolved in 1000 ml waters fully, is mixed with Al
3+Solution.The 14 gram Protopanaxatriol saponin(e Re monomers that the silicagel column separation and purification obtains are dissolved in 1000 ml waters, are mixed with saponin(e solution.Behind above-mentioned two kinds of solution thorough mixing, under 40~60 ℃ of temperature, stirring reaction 14 hours.After reaction finished, reaction solution was through macroporous adsorbent resin, and the absorption saponin(e after impurity such as salt ion and sugar are removed in washing, is used 20%~95% ethanol elution saponin(e again.Collect ethanol eluate, obtain product 10.5 grams after the drying.Perhaps adopt the water-saturated n-butanol extraction process of embodiment 1 to obtain hydrolysate.Through high-efficient liquid phase chromatogram technique analysis (document 1), substrate 80%~90% main the conversion generates rare saponin(e Rg
2With a spot of Rg
1, Rg
4, Pg
6, Rh
1, Rg wherein
2Content is more than 50%.
Embodiment 3:Zn
2+The catalytic hydrolysis pulchinenoside
Get 143.5g gram ZnSO
4H
2O is dissolved in fully in 500 ml waters and prepares Zn
2+Solution.The pulchinenoside of five glycosyls of 7 grams that obtain through the silicagel column separation and purification is dissolved in 500 water, is mixed with saponin(e solution.Behind above-mentioned two kinds of solution thorough mixing, under 40 ℃ of temperature, stirring reaction 14 hours.After reaction finished, the absorption with macroporous adsorbent resin saponin(e after impurity such as salt ion and sugar are removed in washing, was used 20%~95% ethanol elution saponin(e again.Collect ethanol eluate, obtain 4.3 gram products after the drying.Perhaps adopt the water-saturated n-butanol extraction process of embodiment 1 to obtain hydrolysate.Detect (document 2) with thin layer chromatography, the pulchinenoside of 85% above five glycosyls transforms and generates two glycosyl pulchinenosides of 3-o-Ara-(28-o-Glc-), 3-o-Ara-monosaccharide groups pulchinenoside, 28-o-Glc-monosaccharide groups pulchinenoside and sapogenin as a result.
Embodiment 4:Cu
2+The catalytic hydrolysis saikoside
Get 125g gram CuSO
45H
2O is dissolved in fully in 500 ml waters and prepares Cu
2+Solution.Total saponins from radix bupleuri extract is through the silicagel column separation and purification, and the 7 gram polysaccharide base saikosides (mixture of three glycosyl saikosides, saikoside B, saikoside A) that obtain are dissolved in 500 ml waters, are mixed with saponin(e solution.Behind above-mentioned two kinds of solution thorough mixing, under 40~60 ℃ of temperature, stirring reaction 14 hours.After reaction finished, reaction solution after impurity such as salt ion and sugar are removed in washing, was used 20%~95% ethanol elution saponin(e through the absorption with macroporous adsorbent resin saponin(e again.Collect ethanol eluate, drying obtains product 3.9 grams.Perhaps adopt the water-saturated n-butanol extraction process of embodiment 1 to obtain hydrolysate.Detect (document 3) through thin-layer chromatography, the substrate major part is converted into monosaccharide groups saikoside and aglycon, and transformation efficiency reaches 70%~85%.
Embodiment 5:Mn
2+The catalytic hydrolysis gypenoside
Get 198g gram Mncl
24H
2O is dissolved in fully in 1000 ml waters and prepares Mn
2+Solution.The gynostemma total saponin extract is through the silicagel column separation and purification, and the 14 gram polysaccharide base gypenosides that obtain are dissolved in 1000 ml waters, are mixed with saponin(e solution.Behind above-mentioned two kinds of solution thorough mixing, under 40~60 ℃ of temperature, stirring reaction 14 hours.After reaction finished, reaction solution after impurity such as salt ion and sugar are removed in washing, was used 20%~95% ethanol elution saponin(e through the absorption with macroporous adsorbent resin saponin(e again.Collect ethanol eluate, drying obtains product 8.4 grams.Perhaps adopt the water-saturated n-butanol extraction process of embodiment 1 to obtain hydrolysate.Detect (document 1) through thin-layer chromatography, polysaccharide base gypenoside almost all transforms as a result, and transformation efficiency reaches more than 90%, and product is mainly the ginsenoside Rg
3
Embodiment 6:Li
+The catalytic hydrolysis Radix Astragali saponin
Get 64g gram Li
2SO
4H
2O is dissolved in fully in 500 ml waters and prepares Li
+Solution.Diglycosyl Radix Astragali saponin 7 grams through separation and purification obtains are dissolved in 500 ml waters, are mixed with saponin(e solution.Behind above-mentioned two kinds of solution thorough mixing, under 40~60 ℃ of temperature, stirring reaction 15 hours.After reaction finished, reaction solution was through the absorption with macroporous adsorbent resin saponin(e, after impurity such as salt ion and sugar are removed in washing, and 20%~95% ethanol elution saponin(e.Collect ethanol eluate, obtain product 4.6 grams after the drying.Perhaps adopt the water-saturated n-butanol extraction process of embodiment 1 to obtain hydrolysate.Detect (document 4) through thin-layer chromatography, the Radix Astragali saponin of two glycosyls obviously is converted into Cyclosiversioside F, and transformation efficiency is more than 70%.
Embodiment 7:Co
2+The catalytic hydrolysis dioscin
Get 119g gram Cocl
26H
2O is dissolved in fully in 500 ml waters and prepares Co
2+Solution.7 gram dioscins through the silicagel column separation and purification obtains are dissolved in 500 ml waters, are mixed with saponin(e solution.Behind above-mentioned two kinds of solution thorough mixing, under 40~60 ℃ of temperature, stirring reaction 15 hours.After reaction finished, reaction solution after impurity such as salt ion and sugar are removed in washing, was used 20%~95% ethanol elution saponin(e through the absorption with macroporous adsorbent resin saponin(e again.Collect ethanol eluate, obtain product 4.4 grams after the drying.Perhaps adopt the water-saturated n-butanol extraction process of embodiment 1 to obtain hydrolysate.Thin layer chromatography detects (document 5), and the dioscin more than 75% changes into monose dioscin and sapogenin as a result.
Embodiment 8:Mg
2+Catalytic hydrolysis Root of Coral Ardisia saponin(e
Get 246g gram MgSO
47H
2O is dissolved in fully in 1000 ml waters and prepares Mg
2+Solution.The Root of Coral Ardisia saponin(e of four glycosyls of 14 grams that obtain through the silicagel column separation and purification is dissolved in 1000 ml waters, is mixed with saponin(e solution.Behind above-mentioned two kinds of solution thorough mixing, under 40~60 ℃ of temperature, stirring reaction 15 hours.After reaction finished, reaction solution was through the absorption with macroporous adsorbent resin saponin(e, after impurity such as salt ion and sugar are removed in washing, and 20%~95% ethanol elution saponin(e.Collect ethanol eluate, drying obtains product 9.5 grams.Perhaps adopt the water-saturated n-butanol extraction process of embodiment 1 to obtain hydrolysate.Product detects (document 6) through thin layer chromatography, and display substrate transforms and generates diglycosyl Root of Coral Ardisia saponin(e, monosaccharide groups Root of Coral Ardisia saponin(e and saponin(e aglycon as a result, and transformation efficiency is 40%~60%.
Embodiment 9:K
+Catalytic hydrolysis Herba Phyllanthi Urinariae saponin(e
Get 74.5g gram KCl, be dissolved in fully in 1000 ml waters and prepare K
+Solution.14 gram Herba Phyllanthi Urinariae total saponins are dissolved in 1000 ml waters, are mixed with saponin(e solution.Behind above-mentioned two kinds of solution thorough mixing, under 40~60 ℃ of temperature, stirring reaction 17 hours.After reaction finished, reaction solution after impurity such as salt ion and sugar are removed in washing, was used 20%~95% ethanol elution saponin(e through the absorption with macroporous adsorbent resin saponin(e again.Collect ethanol eluate, drying obtains 9.8 gram products.Perhaps adopt the water-saturated n-butanol extraction process of embodiment 1 to obtain hydrolysate.Detect (document 7) with thin layer chromatography, the polysaccharide base Herba Phyllanthi Urinariae saponin(e more than 50% transforms and generates secondary low glycosyl Herba Phyllanthi Urinariae saponin(e as a result.
Embodiment 10:Ba
2+The catalytic hydrolysis ophiopogonin
Get 315.5g gram Ba (OH)
28H
2O is dissolved in fully in 1000 ml waters and prepares Ba
2+Solution.14 gram Radix Ophiopogonis total saponins are dissolved in 1000 ml waters, are mixed with saponin(e solution.Behind above-mentioned two kinds of solution thorough mixing, under 40~60 ℃ of temperature, stirring reaction 18 hours.After reaction finished, reaction solution after impurity such as salt ion and sugar are removed in washing, was used 20%~95% ethanol elution saponin(e through the absorption with macroporous adsorbent resin saponin(e again.Collect ethanol eluate, obtain 8.9 gram products after the drying.Perhaps adopt the water-saturated n-butanol extraction process of embodiment 1 to obtain hydrolysate.Polysaccharide base ophiopogonin with thin layer chromatography (document 8) detected result 45%~60% changes into secondary low glycosyl ophiopogonin.
Embodiment 11:Na
+The catalytic hydrolysis rutin
Get 58.5g gram NaCl, be dissolved in fully in 1000 ml waters and prepare Na
+Solution.Get 9 gram rutoside monomers and be dissolved in 1000 ml waters, preparation rutin solution.Above-mentioned two kinds of solution thorough mixing, under 40~60 ℃ of temperature, stirring reaction 15 hours.After reaction finished, reaction solution after impurity such as salt ion and sugar are removed in washing, was used 20%~95% ethanol elution flavonoid glycoside through the absorption with macroporous adsorbent resin flavonoid glycoside again.Collect ethanol eluate, drying obtains 6 gram products.Perhaps adopt the water-saturated n-butanol extraction process of embodiment 1 to obtain hydrolysate.Measure through high performance liquid chromatography (document 9), substrate conversion efficiency is more than 95%, and product is mainly Quercetol 3-monoglucoside and Quercetin, and wherein Quercetol 3-monoglucoside content is more than 80%.
Embodiment 12:Ca
2+The catalytic hydrolysis Quercetin 3-galactoside
Get 111g gram CaCl
2, be dissolved in fully in 1000 ml waters and prepare Ca
2+Solution.Get 10 gram Quercetin 3-galactoside monomers and be dissolved in 1000 ml waters preparation Quercetin 3-galactoside solution.Above-mentioned two kinds of solution thorough mixing, under 40~60 ℃ of temperature, stirring reaction 15 hours.After reaction finished, reaction solution after impurity such as salt ion and sugar are removed in washing, was used 20%~95% ethanol elution flavonoid glycoside through the absorption with macroporous adsorbent resin flavonoid glycoside again.Collect ethanol eluate, drying obtains 7.2 gram products.Perhaps adopt the water-saturated n-butanol extraction process of embodiment 1 to obtain hydrolysate.Measure through high performance liquid chromatography (document 9), substrate conversion efficiency reaches more than 90%, and quercetin content is more than 85%.
Embodiment 13:Fe
2+The catalytic hydrolysis Hesperidin
Get 199g gram Fecl
24H
2O is dissolved in fully in 1000 ml waters and prepares Fe
2+Solution.Get 10 gram Hesperidin monomers and be dissolved in 1000 ml waters preparation Hesperidin solution.Above-mentioned two kinds of solution thorough mixing, under 40~60 ℃ of temperature, stirring reaction 20 hours.After reaction finished, reaction solution after impurity such as salt ion and sugar are removed in washing, was used 20%~95% ethanol elution flavonoid glycoside through the absorption with macroporous adsorbent resin flavonoid glycoside again.Collect ethanol eluate, drying obtains 6.5 gram products.Perhaps adopt the water-saturated n-butanol extraction process of embodiment 1 to obtain hydrolysate.Measure through high performance liquid chromatography (document 10), substrate conversion is 7-glucose-Hesperitin and a small amount of Hesperitin, and transformation efficiency reaches 75%~90%.
Embodiment 14:Pb
2+The catalytic hydrolysis naringin
Get 379g gram Pb (CH3COO)
23H
2O is dissolved in fully in 1000 ml waters and prepares pb
2+Solution.Get 10 gram naringin monomers and be dissolved in the water preparation naringin solution.Above-mentioned two kinds of solution thorough mixing, under 40~60 ℃ of temperature, stirring reaction 24 hours.After reaction finished, reaction solution after impurity such as salt ion and sugar are removed in washing, was used 20%~95% ethanol elution flavonoid glycoside through the absorption with macroporous adsorbent resin flavonoid glycoside again.Collect ethanol eluate, drying obtains 6.1 gram naringenins.Perhaps adopt the water-saturated n-butanol extraction process of embodiment 1 to obtain hydrolysate.Measure through high performance liquid chromatography (document 10), substrate conversion efficiency reaches more than 80%.
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