Difference between revisions of "Team:UST Beijing/Experiments"

 
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               <li><a href="https://2018.igem.org/Team:UST_Beijing/Background">Background</a ></li>
 
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           <li><a href="https://2018.igem.org/Team:UST_Beijing/Model">Model</a ></li>
 
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               <li><a href="https://2018.igem.org/Team:UST_Beijing/Attributions">Attribution</a ></li>
 
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         <h1 class="title">Experiments</h1>
 
         <h1 class="title">Experiments</h1>
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<h1><span>Our long-term goal is to improve the health-promoting effects of ginsenosides.  We believe that sterols （triterpenes) in the ginsenosides are responsible for their main benefits. Therefore in the past projects we engineered synthetic squalene cyclase for in situ production of ginseng-sterols in human cells and produced synthetic β-glucosidase in E.coli for removal of  sugar from ginsenosides. In the current strategy, in the wake of “No release” policy of the iGEM community, we are able to by-pass synthetic biology methods to achieve our goal by applying in vitro chemical reactions.  </span></h1>
 
 
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<section style="background-image:url(https://static.igem.org/mediawiki/2018/9/95/T--UST_Beijing--ep12.png);background-position:center center;background-repeat:no-repeat;-moz-background-size: cover;background-size: cover;background-attachment:fixed;">
 
<section style="background-image:url(https://static.igem.org/mediawiki/2018/9/95/T--UST_Beijing--ep12.png);background-position:center center;background-repeat:no-repeat;-moz-background-size: cover;background-size: cover;background-attachment:fixed;">
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<blockquote>
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<h3><span>Experimental Design</span><br></h3>
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<p>Ginseng products offer a unique opportunity to meet the atherosclerosis challenge. Herbs containing ginsenosides include: Ginseng, Western Ginseng, Notoginseng, Jiaogulan etc.  Current herb preparation and administration practice results in poor absorption profile limit its efficacy and cost-effectiveness.  Since the ginseno-sterols are responsible for their main pharmacological effects, how to achieve effective concentration of sterol in the human body becomes critical.  
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<h3>Ginseng products offer a unique opportunity to meet the atherosclerosis challenge. Herbs containing ginsenosides include: Ginseng, Western Ginseng, Notoginseng, Jiaogulan etc.  Common herb preparation and oral administration practice results in poor absorption profile thus limits its efficacy and cost-effectiveness.  Since the ginseno-sterols are responsible for their main pharmacological effects, how to achieve effective concentration of sterol in the human body become critical. <br> Our long-term goal is to improve the health-promoting effects of ginsenosides.  We believe that sterols (triterpenes)in the ginsenosides are responsible for their main benefits. Therefore in the past projects we engineered synthetic squalene cyclase for in situ production of ginseng-sterols in human cells and produced synthetic β-glucosidase in E.coli for removal of  sugar from ginsenosides. In the current strategy, in the wake of “No release” policy of the iGEM community, we are able to by-pass synthetic biology methods to achieve our goal by applying in vitro chemical reactions. </h3>
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        </p>
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</blockquote>
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<h3><span>In the past, two approaches have been tried to achieve this: </span></h3>
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<blockquote>
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<h3>(1) Synthesize ginseno-sterols in situ. Pro: no need to plant ginseng and harvest, continuous supply of ginseno-sterols; Con: interference with host physiology, lack of control in production. <br>
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    <p>In the past, two approaches have been tried to achieve this:   (1) Synthesize ginseno-sterols in situ. Pro: no need to plant ginseng and harvest, continuous supply of ginseno-sterols; Con: interference with host physiology, lack of control in production. (2) Produce beta-glucosides in the gut micro-organism. Pro: convenient to hydrolyze ginsenosides in the gut; Con: interference with host gut physiology and probiotics. <br>In the current third approach, we use chemical reaction to hydrolyze the conjugated sugars, to satisfy “No-release” policy if iGEM safety requirement.</p>
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(2) Produce beta-glucosides in the host gut micro-organism. Pro: convenient to hydrolyze ginsenosides in the gut; Con: interference with gut physiology and probiotics.<br></h3>
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</blockquote>
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                  <img src="https://static.igem.org/mediawiki/2018/0/05/T--UST_Beijing--ep13.png" alt="">
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  <div class="span4">
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<div><h3><span>In the current third approach</span>, we use chemical reaction to hydrolyze the conjugated sugars, to satisfy “No-release” policy if iGEM safety requirement.</h3>
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<img src="https://static.igem.org/mediawiki/2018/0/05/T--UST_Beijing--ep13.png" alt=""></div>
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</blockquote></div>
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<div class="span4"><blockquote><p>A synthetic beta-glucosidase gene is introduced into E.coli, along with PNPG as illustrated below. The enzyme (3D structure is displayed on the left) will make a yellow color product in the medium, which is measured by spectrometry</p></blockquote></div>
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  <div class="span5">
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<div class="span4" ><img src="https://static.igem.org/mediawiki/2018/3/32/T--UST_Beijing--ep14.png" heigth="70%" alt=""></div>
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<h2 class="title">1. Thin—layer chromatography</h2>
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<div class="span3"></div>
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<h3>Experimental purpose: Through parallel experiment of different developing methods, we identified a suitable condition in TLC system to separate ginsenosides.
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<div class="span4" ><img src="https://static.igem.org/mediawiki/2018/3/32/T--UST_Beijing--ep14.png" alt=""></div>
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          <br>Standarded samples: Rb1, Re1, Rg1 10mg/ml<br>After many preliminary experiments, we try to find out a general range of proportion. Here is the last TLC experiment.</h3>
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<img src="https://static.igem.org/mediawiki/2018/e/e0/T--UST_Beijing--ep16.png" alt="">
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            <img src="https://static.igem.org/mediawiki/2018/1/1c/T--UST_Beijing--ep15.png" alt=""></div>
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<h3>chromogenic agent: concentrated sulfuric acid: carbinol = 1:9<br>
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<div><span>A synthetic beta-glucosidase gene is introduced into E.coli, which is cultured along with PNPG as illustrated below. The enzyme (structure as illustrated) will produce a yellow color product which is secreted to the medium, and measured by spectrometry.<br> Experiment assignment：</span><br>
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standarded sample: extract ginsenosides from traditional Chinese medicine with n-butanol<br>
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Result: after change the ratio of chromogenic agent,the experimental phenomenon is much more clear for observing and measuring.
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</h3>
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<h3>Change the developing agent ratio(see table below)</h3>
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<img src="https://static.igem.org/mediawiki/2018/0/0c/T--UST_Beijing--ep17.png" alt="">
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<h3>Result: it turns out when the ratio of developing agent is 10:2.5:0.25 and the ratio of chromogenic agent is 9:1,the number of Rf is relatively ideal.</h3>
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<h2 class="title">2. A synthetic beta-glucosidase gene is introduced into E.coli, which is cultured along with PNPG as illustrated below. The enzyme (3D structure is displayed on the left) will make a yellow color product in the medium, which is measured by spectrometry.</h2>
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<div class="span3"></div><img src="https://static.igem.org/mediawiki/2018/1/1c/T--UST_Beijing--ep15.png" alt="">
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<h3><span>Experiment assignment：</span><br>
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We set three different concentrations of PNPG in 2.5%, 5%, 10% and chose ten different germs (including germ 1 without plasmids) to examine their OD (optional density) by spectrometer once hour.<br>
 
We set three different concentrations of PNPG in 2.5%, 5%, 10% and chose ten different germs (including germ 1 without plasmids) to examine their OD (optional density) by spectrometer once hour.<br>
 
<span>Specific experimental scheme：</span><br>
 
<span>Specific experimental scheme：</span><br>
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Plate E.coli (empty, without Beta glycosidase plasmid) and transformed E.coli on solid medium and culture overnight to obtain monoclonal bacteria and repeat the latter for three times.（temperature：37℃）<br>
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Plate E.coli (empty, without Beta glycosidase plasmid) and transformed E.coli on solid medium and culture overnight to obtain a single clone of bacteria and repeat the experiment for three times.（temperature：37℃）<br>
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Pick the monoclonal bacteria to 2ml liquid LB medium and culture overnight.（temperature：37℃）<br>
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Pick a single clone bacteria into 2ml liquid LB medium and culture overnight.（temperature：37℃）<br>
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Isolate E.coli(BL21-β) from 2 ml LB culture and grow E.coli(BL21-β) in M9 culture for four to six hours.（temperature：37℃）Repeat it for several times.<br>
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Isolate E.coli(BL21) from 2 ml LB culture and grow E.coli(BL21) in M9 culture for four to six hours.（temperature：37℃）Repeat it for several times.<br>
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Use PNPG to verify whether E.coli is modified by Beta glycosidase plasmid.Put samples（including E.coli without Beta glycosidase plasmid) at three different concentrations of 5，10，20% in a 96-well plate by micropipet.<br>
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Use PNPG to verify whether E.coli is transformed by beta-glucosidase expressing plasmid. Put samples（including untransformed E.coli as negative control) at three different concentrations 5，10，20% in a 96-well plate by micropipet.<br>
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Use ultraviolet spectrophotometer to measure OD value every other hour for 6 times to test whether the plasmid in E. coli was expressed. The color is getting yellow visibly.
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Use spectrophotometer to measure OD405nm and OD620nm value every 2 hours hour for 12 hours to test whether the plasmid in E. coli was expressed. The color is getting yellow visibly.
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</h3>
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</h3></div>
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<h3><span>Initial data：</span></h3>
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<h3><span>Time-dependent production of hydrolzyed color product by beta-glucosidase under different culture conditions. (The blue line: OD405nm, red line: OD620nm, green line: logistic model simulation.) </span></h3>
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<img src="https://static.igem.org/mediawiki/2018/0/06/T--UST_Beijing--ep18.png" alt="">
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    <div class="span2"></div><img src="https://static.igem.org/mediawiki/2018/4/4c/T--UST_Beijing--ep20.png" alt="">
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<img src="https://static.igem.org/mediawiki/2018/4/4c/T--UST_Beijing--ep20.png" alt="">
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<h3><span>Use chemical methods to hydrolyze ginsenoside:</span></h3>
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<h3><span>Time-dependent production of hydrolzyed color product by beta-glucosidase under different culture conditions:</span></h3>
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        <h3>Chemical hydrolysis of ginsenosides have been well studied and widely reported in the past.  Methods include hydrochloric acid, sodium hydroxide, lactic acid, acidic amino acids, acetic acid, etc. The published methods could generate partial hydrolyzed ginsenosides. However, strong acid hydrolyzation results in modification of sterol side chains, as examplified in the following reaction:</h3>
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<div class="span2"></div><img src="https://static.igem.org/mediawiki/2018/0/06/T--UST_Beijing--ep21.png" alt="">
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<h3>As you can see from the above picture, the trend of data curve continues <span>upward</span>. That is to say, PNPG is decomposed，which confirms plasmids are positively transferred.</h3>
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<h2 class="title">3. Use chemical method to hydrolyze ginsenoside.</h2>
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<div class="span6">
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<h3>Chemical hydrolysis of have been well studied and widely reported in the past.  Methods include hydrochloric acid, sodium hydroxide, lactic acid, acidic amino acids, acetic acid, etc. The published methods could generate partial hydrolyzed ginsenosides. Strong acid hydrolyzation results in modification of sterol side chains, as exemplified in the following.</h3>
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<img src="https://static.igem.org/mediawiki/2018/d/d8/T--UST_Beijing--ep001.png" alt="">
 
<img src="https://static.igem.org/mediawiki/2018/d/d8/T--UST_Beijing--ep001.png" alt="">
 
</div>
 
</div>
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<div class="span6">
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<h3>In the current approach,  we applied a combination of water, butanol, and acetic acid in a proprietary ratio to the ginsenoside substrate mixture to perform the hydrolysis:</h3>
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<h3>In the current approach,  we used a combination of water, butanol, and acetic acid with a proprietary ratio to the ginsenoside substrate mixture to perform the hydrolysis:</h3>
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<img src="https://static.igem.org/mediawiki/2018/3/3f/T--UST_Beijing--ep002.png" alt="">
 
 
<img src="https://static.igem.org/mediawiki/2018/3/3f/T--UST_Beijing--ep002.png" alt="">
 
<img src="https://static.igem.org/mediawiki/2018/3/3f/T--UST_Beijing--ep002.png" alt="">
 
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</div>
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<h3><span>Experimental procedure: </span></h3>
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            <div class="row">
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<div><h3><span>Experimental procedure: </span></h3>
 
<h3>①use different hydrolysis system to hydrolyze ginsenosides. <br>
 
<h3>①use different hydrolysis system to hydrolyze ginsenosides. <br>
 
②Separate hydrolyzed ginsenosides by standarded TLC system. <br>
 
②Separate hydrolyzed ginsenosides by standarded TLC system. <br>
 
③utilize Double Gene report Test system built in Laboratory to test the bioactivity of hydrolyzed ginsenosides.
 
③utilize Double Gene report Test system built in Laboratory to test the bioactivity of hydrolyzed ginsenosides.
 
</h3>
 
</h3>
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<div class="span3"></div><img src="https://static.igem.org/mediawiki/2018/3/3f/T--UST_Beijing--ep003.png" alt="">
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<h3><span>Cellular assay of Ginsenoside hydrolysate biological activity:</span></h3></div>
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<h2 class="title">Discussion:</h2>
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<div class="span1"></div>
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<h3>Through the chart, we can see that the hydrolyzed ginsenosides has better LXR expression intensity compared to unhydrolyzed LXR expression intensity as density arises. Meanwhile, the effect of hydrolyzed ginsenosides is relatively closed to positive contrast. That is to say, we demonstrate that our natural-re-lease’s method is valid.</h3>
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<img src="https://static.igem.org/mediawiki/2018/2/21/T--UST_Beijing--reporter.jpg" alt="">
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<h3><span>Conclusion:</span></h3>
 +
<h3>Through the chart, we can see that the hydrolyzed ginsenosides has better LXR tranactivation ability compared to the unhydrolyzed. Meanwhile, the effect of hydrolyzed ginsenosides is relatively closed to a positive control sample. To conclude, we demonstrate that our "natural RE-lease" approach is working.</h3>
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Latest revision as of 15:30, 16 October 2018

Team:UST_Beijing/Experiments

Experimental Design

Ginseng products offer a unique opportunity to meet the atherosclerosis challenge. Herbs containing ginsenosides include: Ginseng, Western Ginseng, Notoginseng, Jiaogulan etc. Common herb preparation and oral administration practice results in poor absorption profile thus limits its efficacy and cost-effectiveness. Since the ginseno-sterols are responsible for their main pharmacological effects, how to achieve effective concentration of sterol in the human body become critical.
Our long-term goal is to improve the health-promoting effects of ginsenosides. We believe that sterols (triterpenes)in the ginsenosides are responsible for their main benefits. Therefore in the past projects we engineered synthetic squalene cyclase for in situ production of ginseng-sterols in human cells and produced synthetic β-glucosidase in E.coli for removal of sugar from ginsenosides. In the current strategy, in the wake of “No release” policy of the iGEM community, we are able to by-pass synthetic biology methods to achieve our goal by applying in vitro chemical reactions.

In the past, two approaches have been tried to achieve this:

(1) Synthesize ginseno-sterols in situ. Pro: no need to plant ginseng and harvest, continuous supply of ginseno-sterols; Con: interference with host physiology, lack of control in production.
(2) Produce beta-glucosides in the host gut micro-organism. Pro: convenient to hydrolyze ginsenosides in the gut; Con: interference with gut physiology and probiotics.

In the current third approach, we use chemical reaction to hydrolyze the conjugated sugars, to satisfy “No-release” policy if iGEM safety requirement.

A synthetic beta-glucosidase gene is introduced into E.coli, which is cultured along with PNPG as illustrated below. The enzyme (structure as illustrated) will produce a yellow color product which is secreted to the medium, and measured by spectrometry.
Experiment assignment：
We set three different concentrations of PNPG in 2.5%, 5%, 10% and chose ten different germs (including germ 1 without plasmids) to examine their OD (optional density) by spectrometer once hour.
Specific experimental scheme：
Plate E.coli (empty, without Beta glycosidase plasmid) and transformed E.coli on solid medium and culture overnight to obtain a single clone of bacteria and repeat the experiment for three times.（temperature：37℃）
Pick a single clone bacteria into 2ml liquid LB medium and culture overnight.（temperature：37℃）
Isolate E.coli(BL21) from 2 ml LB culture and grow E.coli(BL21) in M9 culture for four to six hours.（temperature：37℃）Repeat it for several times.
Use PNPG to verify whether E.coli is transformed by beta-glucosidase expressing plasmid. Put samples（including untransformed E.coli as negative control) at three different concentrations 5，10，20% in a 96-well plate by micropipet.
Use spectrophotometer to measure OD405nm and OD620nm value every 2 hours hour for 12 hours to test whether the plasmid in E. coli was expressed. The color is getting yellow visibly.

Time-dependent production of hydrolzyed color product by beta-glucosidase under different culture conditions. (The blue line: OD405nm, red line: OD620nm, green line: logistic model simulation.)

Use chemical methods to hydrolyze ginsenoside:

Chemical hydrolysis of ginsenosides have been well studied and widely reported in the past. Methods include hydrochloric acid, sodium hydroxide, lactic acid, acidic amino acids, acetic acid, etc. The published methods could generate partial hydrolyzed ginsenosides. However, strong acid hydrolyzation results in modification of sterol side chains, as examplified in the following reaction:

In the current approach, we applied a combination of water, butanol, and acetic acid in a proprietary ratio to the ginsenoside substrate mixture to perform the hydrolysis:

Experimental procedure:

①use different hydrolysis system to hydrolyze ginsenosides.
②Separate hydrolyzed ginsenosides by standarded TLC system.
③utilize Double Gene report Test system built in Laboratory to test the bioactivity of hydrolyzed ginsenosides.

Cellular assay of Ginsenoside hydrolysate biological activity:

Conclusion:

Through the chart, we can see that the hydrolyzed ginsenosides has better LXR tranactivation ability compared to the unhydrolyzed. Meanwhile, the effect of hydrolyzed ginsenosides is relatively closed to a positive control sample. To conclude, we demonstrate that our "natural RE-lease" approach is working.