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Ways to Inhibit Elastase for More Elastin?

elastase wrinkles ecm elastin stearic

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#1 Nate-2004

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Posted 15 December 2018 - 06:05 PM


This study was posted over in another thread in ageless looks. It's from 2007 though, surely there's more progress since this effort to inhibit HNE began. 
 
It appears that HNE builds up over time. Its normal function is to break down foreign proteins but apparently due to a number of factors. For instance, there are endogenous inhibitors of HNE (alpha-1-antitrypsin, elafin, and the secretory leukocyte proteinase inhibitor) that in younger people act as a control on these enzymes. Here is why:
 

 

Large quantities of oxidants and proteases released by leukocytes that are recruited to the site of inflammation can inactivate these endogenous inhibitors. Moreover, tight adhesion of neutrophils to the ECM leads to the compartmentalization of the released proteases between the neutrophil and the ECM, thereby excluding the large, circulating protease inhibitors. Tight binding of extracellular HNE to the cell membrane can render it inaccessible to circulating endogenous inhibitors. Altogether, the imbalance between HNE and its inhibitors caused by these events provokes severe tissue injuries resulting in a variety of diseases.
 
HNE starts breaking down elastase in an out of control sort of way. Once again inflammation is at the root.
 

 

It has become clear that serine proteases, such as HNE, have an important regulatory role in the local inflammatory response. Thus, its dysregulation resulting in its accumulation can be involved in the development of chronic inflammatory diseases, such as rheumatoid arthritis, pulmonary emphysema, adult respiratory distress syndrome (ARDS), cystic fibrosis, COPD, asthma, and delayed wound healing.
 
Rather than find exogenous inhibitors, why not attempt to free and enable the endogenous ones by addressing the root of the problem?  If uncontrolled elastase leads to all these diseases from arthritis to emphysema to slower wound healing, and our bodies have a built in means of controlling elastase, it makes sense to try and re-enable these means somehow.
 
Many of the exogenous inhibitors they test are the various flavonoids we often talk about all over this forum. These flavonoids are notorious for their lack of bioavailability. Many people including myself have tried several different methods of improving this by both topical and oral means to no real avail. The study here mentions this:
 

 

However, it has to be kept in mind that flavonoids are highly metabolized during oral application, and that some possible metabolites (such as 4-methylcatechol, 4-hydroxyphenylacetic acid and 3,4-dihydroxyphenylacetic acid ) exhibited a very low activity in the assay (IC50 range from 135 qM to > 400 juM, see Fig. 2A and Fig. 2B). Therefore, the in vitro studies mentioned above may be of limited therapeutic relevance and it is questionable that flavonoids may be orally active principles in a Drosera extract used to treat cough.
 
They say a very low IC50 value is achieved with EGCG (this means it is a strong inhibitor) but I've been taking that for a long time and it's actually in a lot of topical creams. It doesn't seem to do much in terms of restoring elastase in my opinion. If reversing the loss of elastase is the goal, then this is not exactly the outcome I'm seeing with EGCG.
 
So looking at exogenous possibilities...
 
Funny thing, they say stearic acid in this study was a fatty acid with the highest level of inhibition among saturated fats. Stearic acid has lengthy discussions in terms of fusion and stem cell proliferation in Turnbuckle's threads in this forum.
 
Erucic acid, which I've looked into as a problem with consuming ground broccoli seeds for the sulforaphane content, had the most potent inhibitory effect of all.  An IC50 value of 450nM. The smallest amount will inhibit HNE by more than 50%. But what of the dangers of erucic acid? That's where oleic acid was mentioned which is also an omega 9 monounsaturated fatty acid capable of inhibiting HNE considerably, especially when they combined it with albumin.
 

 

Based on these results a formulation of oleic acid with albumin was developed for the treatment of chronic wounds. Albumin was used as a carrier for the hydrophobic oleic acid. Oleic acid/albumin formulations with mole ratios of 100:1, 50:1, and 25:1 showed a strong inhibition of HNE with IC50 values at 0.029-0.049 /iM. Albumin alone increased to a small extent the substrate conversion by HNE, which could be equalized by a higher concentration of inhibitor. The authors suppose that an increase of the albumin concentration may even have positive effects, since albumin level is decreased in chronic wounds. The formulation was still active (IC50 = 0.26-0.42 /iM), even after being bound to derivatized cotton.
 
So while albumin increased the substrate for HNE alone, more oleic canceled this out I assume. Not only that but the albumin might help heal wounds.
 
Mango butter is something I have on hand for the whole stem cell protocol. This is part largely stearic and oleic acid. I don't know about adding albumin but I may already have what I need, especially when I (hopefully) increase its bioavailability by adding phospholipids like soy lecithin and cocoa to the mix.
 
Resveratrol is also tested but of course, like all the flavonols, these are hardly bioavailable enough to work well in vivo. We've probably all been down this road.
 
So their conclusion in the study was that more study is needed of course. As I mentioned, addressing the root cause may be a more productive path, but there was not a complete description of how the endogenous inhibitors are thwarted from acting and moreover, how does the root cause affect any attempt to introduce exogenous inhibitors in vivo?
 

Edited by Nate-2004, 15 December 2018 - 06:06 PM.

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#2 Nate-2004

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Posted 15 December 2018 - 08:07 PM

I dunno about well written, already seeing two errors (break down elastin not elastase and also, "this is largely" not "this is part largely") but thanks.

 

Wondering if I can do anything topical with mango butter.

 



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#3 Nate-2004

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Posted 18 December 2018 - 09:17 PM

What references are missing??

 

Working on a formulation for a serum based on the above info. Here's what I'm going to try:

 

EGCG is soluble at 100g per liter of distilled water. That's a nice 1/10th. I wouldn't go that far with it though because I'd want to add other ingredients for ease of application.

 

Note: I haven't tried this yet so I don't know how well it will go.

 

Part 1:

 

350ml distilled water heated to boiling

Mix in around 20 to 25g EGCG and let it dissolve thoroughly as it cools

Optional: Add 3g niacinamide and stir

Add in 3.5g hyaluronic acid powder, stir and then refrigerate 12 to 24 hours

 

The result should be a gel or a slightly thickened brown liquid.

 

Part 2: Here's where I'm not so sure this is how to go about it.

 

Melt 50g mango butter in a double boiler

Pour the mango butter into the gel.

Mix thoroughly.

If it's too thick or not the consistency of thick liquid at room temperature it might need to be reheated in the double boiler and mixed with more of the hot distilled water and EGCG made in part 1

 

 

 


Edited by Nate-2004, 18 December 2018 - 09:18 PM.

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#4 Nate-2004

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Posted 19 December 2018 - 05:43 PM

Interesting article here: https://www.ncbi.nlm...les/PMC3189735/

 

EGCG apparently has a much, much better bioavailability if taken in a fasted state with ascorbic acid and fish oil.

 

Unfortunately for anyone taking this while fasting, EGCG also happens to suppress hepatic gluconeogenesis, so look forward to feeling pretty low energy.


Edited by Nate-2004, 19 December 2018 - 05:44 PM.

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#5 Nate-2004

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Posted 19 December 2018 - 06:09 PM

I'm still debating about how to create a transdermal gel for EGCG. There are companies that sell Lipoderm which has been tested with EGCG but I have no access to this.

 

I am also unsure about whether adding any amount of DMSO is a good idea, because sulfation is an issue with EGCG. If anyone knows an ideal way to improve transdermal absorption that would be great. Some transdermal drug delivery systems use ethanol, or "nanoethosomes", which is a bit vague. I worry that ethanol would dry out the skin though. 

 

Coincidentally I learned that oleic acid is a permeation enhancer, which means the mango butter may help more, plus I have soy lecithin already mixed in with the mango butter I have. Adding a small amount of ethanol probably wouldn't hurt much. I cannot find any information on how nanoethosomes are produced but it definitely involves ethanol, phospholipids and water. Possibly requires a scanning and tunneling microscope so that's out.


Edited by Nate-2004, 19 December 2018 - 06:26 PM.


#6 Nate-2004

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Posted 24 December 2018 - 01:04 AM

Here's something else I can't shake as what should be a really solid question. If an overrun of HSE is one of the major causes of loss in elastin production, and oleic acid is one of the strongest inhibitors, then why on earth aren't oleic acid rich foods like walnuts reversing atherosclerosis and wrinkles? 

 

Well according to this study walnuts do actually improve endothelial function... https://www.ahajourn...124477.91474.FF

 

Maybe this is why, considering that the unsaturated fatty acids are said to be the active ingredient. However this study highlights a big point that elastin loss isn't the ONLY issue affecting the stiffening of tissues. If it was, wouldn't we see reversal? Or maybe it's because it's just not as effective as it needs to be. Perhaps our endogenous elastase inhibitors trapped by the strong adhesion of neutrophil to the ECM is what needs to be addressed. Not only that, I'm sure it all ties into glycation and AGEs being the other big issue.

 

 



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#7 Nate-2004

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Posted 24 December 2018 - 01:50 AM

So in my experimentation with the first batch of topical EGCG and Oleic, I have the same problem as I had trying to just mix it into ready made CeraVe lotion. It doesn't dissolve in anything but water and the moment you put it in anything with lipids it begins to ball up into little balls of wet extract powder.  I'm not sure if there's a word for that. It seems ok with just the hyaluronic acid serum so long as I mix it with glycerin to keep it from just making a film on my skin. I'll have to test this more but I don't think it's going to carry through well. You're also missing out on the oleic acid.

 

Maybe I need a better EGCG extract than 50%

 

Also,

 

I don't know if anyone knows about sulfation, but wouldn't DMSO destabilize the EGCG?


Edited by Nate-2004, 24 December 2018 - 01:52 AM.

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#8 Nate-2004

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Posted 27 December 2018 - 04:34 AM

Know what else destabilizes EGCG? HOT WATER! WTF?? My first question was how the hell did they come to the conclusion that EGCG polyphenols are why green tea, which is brewed in HOT WATER, is good for you?

 

Well turns out it just needs to be cooled fast within 30 mins, or put on ice. Luckily I think I got it into the cold fridge pretty quick after mixing in the hyaluronic acid. So gotta remember to do that, or better yet put it on ice as they did here. Or perhaps heating it isn't even needed to mix it in thoroughly. 

 

http://www.vup.sk/en...d.php?bulID=786

 

I wonder if I'd solve the clumping issue if I drained it through a cheesecloth beforehand.

 

 


Edited by Nate-2004, 27 December 2018 - 04:35 AM.

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#9 Nate-2004

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Posted 03 January 2019 - 09:36 PM

Got some help with the formulation from someone who's obviously better at this:

 

There are a few issues or concerns. I will state the normal practices that the formulators will have.

Normally when we write out the formulation for troubleshooting, we will write it out in percentages. I've converted it for the sake of the process, which you can double check if required. I've also written the ingredients in different phases for easy preparation. We will also state the required ending pH as well.

Formulation:

Oil Phase

  • Lecithin - 14.78g | 4%
  • Mango Butter - 59.15g | 15%

Water Phase

  • Distilled Water - 300g | 74%
  • EGCG - 15g | 4%
  • HA - 3g | 1%
  • Glycerin - 14.78g | 4%

Preservative Phase

  • Potassium Sorbate

Few things to take note:

  • Lecithin may not be a strong emulsifier as it's normally used as a co-emulsifier in skin care. Some expert formulator will advice not using it at all as it increases the chances of bacteria and mould growth. You can choose a proper emulsifier (if you require it to be natural), such as Olivem 1000, Montanov 68, etc
  • For the EGCG, you can make an extract out of it, depending on the concentration that i had mentioned earlier. You can take a look at this video here. If you decide to make it with just Distilled Water, you will need to continue straining with a finer filter paper.
  • As you didn't measure the pH, Potassium Sorbate has a preservative will not be effective as it's most effective if the pH is below pH 5. Any pH above pH 5, the Potassium Sorbate does not have any anti-mould activity.
  • If you insist on using all natural or natural derived ingredients, then i will take it that you will use a natural type preservative.

Formulation that i will suggest based on your ingredients + natural derived ingredients only:

Oil Phase

  • Mango Butter - 15% (Emollient)
  • Olivem 1000 - 5% (Emulsifier)
  • Glyceryl Stearate - 1% (Co-emulsifier)
  • Lecithin - 2% (Co-emulsifier)

Water Phase

  • Distilled Water - 64.3%
  • HA - 1%
  • 1,3-Propanediol 3% (Humectant / Preservative Booster)
  • Sodium Phytate - 0.2% (Chelating Agent)

Cool Down Phase

  • EGCG Extract - 4%

Preservative

  • Leucidal Liquid - 4% (Anti bacterial preservative)
  • Potassium Sorbate - 0.25% (Anti mould preservative)
  • Sodium Benzoate - 0.25% (Anti mould preservative)

Targeted pH: pH 5 (For the anti mould preservatives to work)

Steps:

  1. Sift the HA into the Distilled Water in a heat proof container and let it hydrate for 1 hour
  2. Mix the oil phase into a heat proof container
  3. Mix the remaining water phase ingredients into the premixed HA liquid
  4. Use the heat and hold method, heat both phases till 65 - 75 degrees (until the emulsifier melts)
  5. Pour the water phase into the oil phase and blend using a electric blender (preferably a stick blender) till emulsified
  6. When below 40 degrees, add the Cool Down Phase & Preservatives, blend till well mixed
  7. Adjust using a citric acid (50%) solution or lactic acid solution to pH 5 using a pH strip or pH meter for measurement

Read more about formulation here:


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