CN103898186A - Method for preparing secondary saponins by enzymatic conversion of sea cucumber total saponins - Google Patents
Method for preparing secondary saponins by enzymatic conversion of sea cucumber total saponins Download PDFInfo
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- CN103898186A CN103898186A CN201410057554.5A CN201410057554A CN103898186A CN 103898186 A CN103898186 A CN 103898186A CN 201410057554 A CN201410057554 A CN 201410057554A CN 103898186 A CN103898186 A CN 103898186A
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Abstract
The invention discloses a method for preparing secondary saponins by enzymatic conversion of sea cucumber total saponins. The method comprises the following steps: (a) removing internal organs from fresh sea cucumbers, and boiling in boiling water to obtain sea cucumber boiling liquid; (b) adjusting the pH value of the sea cucumber boiling liquid, and centrifuging to obtain supernatant liquid; (c) separating the supernatant liquid by a column, flushing resin by using de-ionized water and a 20% ethanol solution respectively, eluting by using a 70% ethanol solution, and collecting 70% ethanol solution elution fractions; (d) performing reduced pressure concentration on the collected elution fractions until an extract is formed, re-dissolving by using de-ionized water, centrifuging to obtain supernatant liquid, extracting and degreasing the supernatant liquid through petroleum ether, extracting a water layer by using water-saturated butanol, performing reduced pressure concentration until an extract is formed, and freeze-drying to obtain sea cucumber total saponins; (e) enzymatically hydrolyzing sea cucumber total saponins by adopting snailase; (f) performing reduced pressure concentration and freeze-drying to obtain sea cucumber secondary saponins. The conditions are mild, and the preparation method is simple.
Description
Technical field
The present invention relates to a kind of enzymatic conversion method sea cucumber total saponins and prepare the method for secondary saponin(e.
Background technology
In ocean, containing very rich in natural resources and the energy, and the species that China sea area has are also very various, along with marine industries are constantly fast-developing, increasing entrepreneur has invested Zhe Kuai market, ocean sight, therefore China ocean pharmaceutical industry develops very rapidly in recent ten years, and domestic be also the field of a big hot topic to the research of sea cucumber in Echinodermata.
Sea cucumber is Echinodermata (Echinodenmata) Holothuroidea (Holothunoider) biology, and global sea cucumber has kind more than 1200, and approximately there is kind more than 140 in China marine site.In wall of sea cucumber Stichopus japonicus and internal organ, can secrete the material that some active functions are very strong, i.e. selenka, it is the main secondary metabolite of sea cucumber, is also its basic substance that carries out chemical defence.Pharmacological evaluation shows, selenka mostly has strong physiological activity, as antitumor, antimycotic, anti-inflammatory, antiviral, improve immunizing power, hemolytic and cholinolytic effect etc.
At present, saponin extraction technique in wall of sea cucumber Stichopus japonicus, Holothurian machining waste liquid, sea cucumber coelomic fluid is studied for example disclosed CN102150851A application for a patent for invention on August 17th, 2011 disclosed " selenka and preparation method thereof, application " in food and medicine both at home and abroad; And for example: disclosed CN102060905A application for a patent for invention on May 18th, 2011 disclosed " utilizing fresh Holothurian machining waste liquid to prepare the processing method of selenka Holotoxin A1 reference substance "; Further, disclosed CN102512452A application for a patent for invention on June 27th, 2012 disclosed " utilizing sea cucumber coelomic fluid to prepare the processing method of selenka ".
Up to now from sea cucumber, isolated, the selenka of more than at least 140 kinds.Selenka is made up of aglycon and oligonucleotide chain two portions, contains 5 angular methyl(group)s, has on 3 of aglycon hydroxyl to replace, and is combined into glycosides with sugar by β-O glycosidic link.The chemical structure of selenka is very complicated various, variation on substituting group on the position of the structure of side chain, the variation of lactonic ring, two keys and 12,16,17 all can cause the variation of aglycon structure, and the structure of oligonucleotide chain can change because of link position and the number etc. of the composition of the link position of monose and order, monose and number, sulfate group.Pharmacology activity research shows, the structure of the activity of selenka and aglycon, oligonucleotide chain has relation.The material that glycosyl side chain is different for aglycon structure is identical, their pharmacologically active also has marked difference.Therefore, change the structure of selenka glycosyl side chain, likely obtain the secondary saponin(e that pharmaceutical use is higher.
The modification of Structures of Natural Products is mainly contained to chemical method, microbial method and enzyme process.Chemical method mainly comprises acid-base method, although simple to operate, specificity is poor, and in a large number with an organic solvent can be to environment; And the bio-transformation of a lot of natural products adopts microbe fermentation method, with the metabolic enzyme of microorganisms, substrate is hydrolyzed, microbial method specificity is stronger, mild condition, environmental pollution is little, but microbe fermentation method culture condition harshness, and fermentation need to be found efficient bacterial strain to meet the needs of suitability for industrialized production.Therefore, than chemical method and microbe fermentation method, external enzyme process specificity is strong, and enzymolysis efficiency is high, simple to operate, as long as determine the optimum condition of enzyme reaction.About the enzymatic conversion method research of saponins obtains larger progress at ginsenoside, arasaponin, soybean saponin etc. at present.Disclosed " the enzymically hydrolyse ginsenoside Rb of disclosed CN101481725A application for a patent for invention on July 15th, 2009
1prepare ginseng saponin F
2method " in just by the ginsenoside Rb higher content, pharmaceutical use is lower
1change into the ginseng saponin F that content is lower, pharmaceutical use is higher
2.And for the bio-transformation of selenka, all also do not have relevant research report at present.
Summary of the invention
The object of the present invention is to provide a kind of enzymatic conversion method sea cucumber total saponins to prepare the method for secondary saponin(e.
Technical scheme of the present invention is as follows:
Enzymatic conversion method sea cucumber total saponins is prepared a method for secondary saponin(e, comprises the following steps:
A) fresh sea cucumber is gilled, put into boiling water and boil 20-30min, what obtain is sea cucumber cooking liquor;
B) sea cucumber cooking liquor regulates pH to 9.5-10.5, and 6000-10000r/min is centrifugal, and 20-60min obtains supernatant liquor;
C) supernatant liquor is crossed AB-8 type macroporous resin column, rinses resin respectively with the deionized water of 3-4 times of column volume and 20% ethanolic soln, then carries out wash-out with 70% ethanolic soln of 5-15 times of column volume, collects 70% ethanolic soln elution fraction;
D) the collected elution fraction of concentrating under reduced pressure, to medicinal extract shape, redissolve with deionized water, 6000-10000r/min is centrifugal, and 20-60min gets supernatant, supernatant liquor is through petroleum ether extraction degreasing, and water intaking layer is used water saturation n-butanol extraction 2-4 time again, merges n-butanol layer, be evaporated to medicinal extract shape, lyophilize obtains sea cucumber total saponins;
E) adopt helicase enzymolysis sea cucumber total saponins, damping fluid is the phosphoric acid citrate buffer solution of pH3.5-4.50.005-0.02 μ mol/L, the part by weight of the thick enzyme of helicase and sea cucumber total saponins is 1:1-3, in water-bath, react 24-48h, use methyl alcohol termination reaction, and with the alcohol extraction lixiviate 12-36h of 0.5-2 times of volume;
F) after concentrating under reduced pressure, with deionized water redissolution, then use water saturation n-butanol extraction 2-4 time, merge n-butanol layer, after concentrating under reduced pressure, carry out lyophilize, obtain the secondary saponin(e of sea cucumber.
Wherein, the present invention sea cucumber used is preferably stichopus japonicus.
Wherein, the boiling water volume that step a) of the present invention adds is preferably: boil after 20-30min, every kg sea cucumber produces 400-600ml times of cooking liquor.
Wherein, when step d) deionized water redissolves, the volume adding is preferably 1-5 times of medicinal extract volume.
Wherein, step e) the thick enzyme of helicase preferably in 45 ℃ of water-baths, activate 1h.
Wherein, step e) water-bath temperature of reaction can be 40-50 ℃, is preferably 45 ℃.
The present invention makes full use of the cooking liquor producing in fresh Holothurian machining boiling process, thereby can separation and Extraction go out sea cucumber total saponins, and sea cucumber total saponins is transformed with the thick enzyme of helicase, obtains secondary saponin(e.The invention provides and a kind ofly utilize the thick enzyme of helicase catalytic hydrolysis sea cucumber total saponins optionally under gentle condition, thereby obtain the method for more potential, rarer secondary saponin(e.Enzyme has the feature such as high efficiency, specificity, and for the bio-transformation of selenka, up to the present all yet there are no relevant research report.Therefore, the present invention contributes to selenka further developing at medical science, pharmaceutical industry.
Accompanying drawing explanation
Fig. 1 is sea cucumber total saponins dried frozen aquatic products.The sea cucumber total saponins that is that Fig. 1 shows is crossed the freeze-drying sample obtaining after AB-8 type macroporous resin.
Fig. 2 is the thin layer chromatogram before and after sea cucumber total saponins enzymolysis.As can be seen from Figure 2, react 36h at material ratio 1:2, pH4.0, at 45 ℃, sea cucumber total saponins is successfully converted into secondary saponin(e by helicase, and the mobility of enzymolysis component is 0.44 ± 0.01.
Fig. 3 is the thin layer chromatogram that sea cucumber cooking liquor is crossed AB-8 type macroporous resin column.As can be seen from Figure 3, the selenka extracting mainly contains two types.
Thin layer chromatogram before and after the selenka enzymolysis that Fig. 4 is two types.A, B represent two kinds of dissimilar selenkas.
Embodiment
Embodiment 1
(1) the fresh sea cucumber of 100kg is taken out after internal organ, the boiling water of putting into boiling boils 20-30min, and the sea cucumber of 100kg produces the sea cucumber cooking liquor of about 50kg.Get sea cucumber cooking liquor 5L, regulating pH value is that the centrifugal 40min of 10,8000r/min obtains supernatant liquor.
(2) supernatant liquor of gained is passed through to AB-8 type macroporous resin column, flow velocity is 2BV/h, adsorb saturated after, with the deionized water rinsing of 2L, rinse with the ethanolic soln of 2L20% again, finally use the ethanolic soln wash-out of 6L70%, collect elutriant, be evaporated to medicinal extract shape, with the deionized water redissolution of 500mL, 8000r/min is centrifugal, and 40min gets supernatant, supernatant liquor is through petroleum ether extraction degreasing, and water intaking layer is used water saturation n-butanol extraction three times again, merges n-butanol layer, be evaporated to medicinal extract shape, lyophilize obtains sea cucumber total saponins.
(3) preparation 500mL pH4.0, the phosphoric acid citrate buffer of 0.01 μ mol/L.First, take the thick enzyme of helicase of 125mg, add a small amount of damping fluid, at 45 ℃ of activation 1h.In addition, take the sea cucumber total saponins 250mg that lyophilize obtains, with remaining damping fluid dissolving.Thick the helicase of activation enzyme is joined in sea cucumber total saponins to water bath with thermostatic control reaction 36h at 45 ℃.
(4) carry out after termination reaction with methyl alcohol, add ethanol to extract lixiviate 24h, after concentrating under reduced pressure, with deionized water redissolution, use water saturation n-butanol extraction three times, merge n-butanol layer, after concentrating under reduced pressure, carry out lyophilize, whether obtain the secondary saponin(e of sea cucumber, and sample is detected with thin layer chromatography, there is enzyme digestion reaction in sea cucumber total saponins.
The sea cucumber total saponins that is that Fig. 1 shows is crossed the freeze-drying sample obtaining after AB-8 type macroporous resin; And as can be seen from Figure 2, react 36h at material ratio 1:2, pH4.0, at 45 ℃, and sea cucumber total saponins is successfully converted into secondary saponin(e by helicase, and the mobility of enzymolysis component is 0.44 ± 0.01.
Embodiment 2
(1), by sea cucumber cooking liquor 500ml described above, adjust pH is that the centrifugal 40min of 10,8000r/min obtains supernatant liquor.
(2) supernatant liquor is splined on to pretreated AB-8 type macroporous resin column, flow velocity is 2BV/h, adsorb saturated after, rinse with deionized water and 20% ethanolic soln of 300ml respectively again, then use the ethanolic soln wash-out of 600ml70%, elution fraction detects through thin-layer chromatography, collects different types of selenka.Be evaporated to medicinal extract shape, after redissolving with deionized water, 8000r/min is centrifugal, and 40min gets supernatant, supernatant liquor is through petroleum ether extraction degreasing, and water intaking layer is used water saturation n-butanol extraction three times again, merges n-butanol layer, be evaporated to medicinal extract shape, lyophilize obtains diverse selenka.
From thin layer chromatogram, (Fig. 3) can find out, the selenka extracting mainly contains two types.
(3) collection is obtained to the main selenka of two types, respectively it is carried out to enzyme digestion reaction.Each preparation 250mLpH4.0, the phosphoric acid citrate buffer of 0.01 μ mol/L, takes the thick enzyme of helicase of 40mg, adds a small amount of damping fluid, at 45 ℃ of activation 1h.In addition, get respectively cryodesiccated two kinds of each 100mg of selenka, with remaining damping fluid dissolving.Thick the helicase of activation enzyme is joined in sea cucumber total saponins to water bath with thermostatic control reaction 36h at 45 ℃.
(4) carry out after termination reaction with methyl alcohol, add ethanol to extract lixiviate 24h, after concentrating under reduced pressure, redissolve with deionized water, with water saturation n-butanol extraction three times, merge n-butanol layer, concentrating under reduced pressure also detects the selenka of two types with thin layer chromatography whether enzymolysis has all occurred.
At material ratio 1: 2, pH4.0,45 ℃, react 36h and carry out enzyme digestion reaction, as can be seen from Figure 4, only have the selenka of a type enzymolysis has occurred, the selenka that to have produced Rf value be 0.44, and as can be seen from Figure 4 not (Fig. 4-B) completely of enzyme digestion reaction.By after the sample concentrating under reduced pressure after enzymolysis, with deionized water redissolution, leave standstill for some time, there is floss, and added dehydrated alcohol, floss disappears, and showing has the alcohol of being dissolved in and water-fast material in solution.Remove after ethanol, enzymolysis sample is carried out centrifugal, precipitation detects with thin-layer chromatography (TLC), as shown in Fig. 4-B, illustrate in white floss and have a large amount of saponin(es, also show originally after water miscible selenka enzymolysis, produced water insoluble and be dissolved in the secondary saponin(e of alcohol.
Claims (6)
1. enzymatic conversion method sea cucumber total saponins is prepared a method for secondary saponin(e, comprises the following steps:
A) fresh sea cucumber is gilled, put into boiling water and boil 20-30min, what obtain is sea cucumber cooking liquor;
B) sea cucumber cooking liquor regulates pH to 9.5-10.5, and 6000-10000r/min is centrifugal, and 20-60min obtains supernatant liquor;
C) supernatant liquor is crossed AB-8 type macroporous resin column, rinses resin respectively with the deionized water of 3-4 times of column volume and 20% ethanolic soln, then carries out wash-out with 70% ethanolic soln of 5-15 times of column volume, collects 70% ethanolic soln elution fraction;
D) the collected elution fraction of concentrating under reduced pressure, to medicinal extract shape, redissolve with deionized water, 6000-10000r/min is centrifugal, and 20-60min gets supernatant, supernatant liquor is through petroleum ether extraction degreasing, and water intaking layer is used water saturation n-butanol extraction 2-4 time again, merges n-butanol layer, be evaporated to medicinal extract shape, lyophilize obtains sea cucumber total saponins;
E) adopt helicase enzymolysis sea cucumber total saponins, damping fluid is the phosphoric acid citrate buffer solution of pH3.5-4.50.005-0.02 μ mol/L, the part by weight of the thick enzyme of helicase and sea cucumber total saponins is 1:1-3, in water-bath, react 24-48h, use methyl alcohol termination reaction, and with the alcohol extraction lixiviate 12-36h of 0.5-2 times of volume;
F) after concentrating under reduced pressure, with deionized water redissolution, then use water saturation n-butanol extraction 2-4 time, merge n-butanol layer, after concentrating under reduced pressure, carry out lyophilize, obtain the secondary saponin(e of sea cucumber.
2. a kind of enzymatic conversion method sea cucumber total saponins as claimed in claim 1 is prepared the method for secondary saponin(e, it is characterized in that: described sea cucumber is stichopus japonicus.
3. a kind of enzymatic conversion method sea cucumber total saponins as claimed in claim 1 is prepared the method for secondary saponin(e, it is characterized in that: the boiling water volume that step a) adds is: boil after 20-30min, every kg sea cucumber produces 400-600ml times of cooking liquor.
4. a kind of enzymatic conversion method sea cucumber total saponins as claimed in claim 1 is prepared the method for secondary saponin(e, it is characterized in that: when step d) deionized water redissolves, the volume adding is 1-5 times of medicinal extract volume.
5. a kind of enzymatic conversion method sea cucumber total saponins as claimed in claim 1 is prepared the method for secondary saponin(e, it is characterized in that: step e) the thick enzyme of helicase activates 1h in 45 ℃ of water-baths.
6. a kind of enzymatic conversion method sea cucumber total saponins as claimed in claim 1 is prepared the method for secondary saponin(e, it is characterized in that: step is water-bath temperature of reaction 40-50 ℃ e).
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104585798A (en) * | 2015-01-15 | 2015-05-06 | 大连卓尔高科技有限公司 | Method for preparing nutrients from sea cucumber water decoction |
| CN105085368A (en) * | 2015-04-23 | 2015-11-25 | 华侨大学 | Sea cucumber alkaloid and preparation method and application thereof |
| CN106636286A (en) * | 2016-12-28 | 2017-05-10 | 中国海洋大学 | Deglycosylated holothurian secondary saponin and preparation method thereof |
| CN108997473A (en) * | 2018-08-31 | 2018-12-14 | 山东省科学院生物研究所 | A kind of non-sea cucumber alkane type selenka and the preparation method and application thereof |
| CN109125381A (en) * | 2018-09-05 | 2019-01-04 | 湖南中茂生物科技有限公司 | A method of promoting astragalus root total saponin conversion |
| CN111333695A (en) * | 2018-12-19 | 2020-06-26 | 扬州蓝色生物医药科技有限公司 | Preparation process of sea cucumber saponin |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104585798A (en) * | 2015-01-15 | 2015-05-06 | 大连卓尔高科技有限公司 | Method for preparing nutrients from sea cucumber water decoction |
| CN105085368A (en) * | 2015-04-23 | 2015-11-25 | 华侨大学 | Sea cucumber alkaloid and preparation method and application thereof |
| CN105085368B (en) * | 2015-04-23 | 2017-10-20 | 华侨大学 | A kind of sea cucumber bio alkali and its preparation method and application |
| CN106636286A (en) * | 2016-12-28 | 2017-05-10 | 中国海洋大学 | Deglycosylated holothurian secondary saponin and preparation method thereof |
| CN108997473A (en) * | 2018-08-31 | 2018-12-14 | 山东省科学院生物研究所 | A kind of non-sea cucumber alkane type selenka and the preparation method and application thereof |
| CN108997473B (en) * | 2018-08-31 | 2020-01-07 | 山东省科学院生物研究所 | Non-holothurian alkane type holothurin and preparation method and application thereof |
| CN109125381A (en) * | 2018-09-05 | 2019-01-04 | 湖南中茂生物科技有限公司 | A method of promoting astragalus root total saponin conversion |
| CN111333695A (en) * | 2018-12-19 | 2020-06-26 | 扬州蓝色生物医药科技有限公司 | Preparation process of sea cucumber saponin |
| CN111333695B (en) * | 2018-12-19 | 2022-08-09 | 扬州蓝色生物医药科技有限公司 | Preparation process of sea cucumber saponin |
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