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In my opinion, you have first to define what you want to extract. In the case of the articles you mention, they are working with a polysaccharide, and alcohol is used to precipitate that polysaccharide. According to what I read, you want to obtain an extract from medicinal mushrooms, is there any compound of interest that you want to isolate? According to the characteristics of that compound, such as its polarity, you can define which solvent to use. You can even use mixtures of solvents.

I agree with Gabriela, the compounds of interest can be found in previously published literature

Moses Mwajar Ngeiywa Gabriela Barragan Many thanks for your input in this discussion.

However, my confusion remains regarding this question.

I understand that the extraction solvent type and order is determined by what compounds we want to extract from the material, as Gabriela Barragan mentioned.

However, the question remains - why do some studies use ethanol before water and some vice versa? I cannot find any logical explanation for this.

What is the reason to use ethanol prior to hot water extraction (most studies mention "pre-treatment" or "precipitation")? If ethanol use is indeed solely to precipitate polysaccharides, it makes sense only to use it after a round of extraction first. For pre-treatment purposes, I assume ethanol before water extraction would impact the effectiveness of extraction, correct?

These details still seem to be unclear and the literature I have come across seems to confirm the lack of detailed reasoning as to the order of extraction solvents and the reason for certain "pretreatments" or "post-treatments". As linked above, these two studies clearly show water use first (Svagelj et al 2012) or ethanol use first (Li et al 2019) for the same extraction objective of heteropolysaccharides

If I zoom out, I can postulate that a high % ethanol pretreatment may "unlock"/"soften" water-insoluble materials prior to a water extraction, hence, allowing for optimised extraction.

I am just surprised that such common-place methodology is not standardised among scientists for the same objective of extracting the same compounds.

The order of such processes seems to be randomly conducted in literature and the reasoning for these detailed methodology choices are rarely described in literature I have come across.

The purpose of these articles may not be the same. To isolate the heteropolysaccharide it is more logical to use the method in which first they make an extraction with water and later they use the ethanol to precipitate the polysaccharide of that aqueous extract. By using first ethanol and then water, it seems like a fractionated extraction; where I first want to separate the medium polarity compounds, maybe phenols, flavonoids, etc. and then the compounds more related to water, like sugars. Therefore it should be analyzed what was is the aim of each investigation to understand why they used these methodologies. If you want you can share with me the article by Li et al (2019).

Gabriela Barragan the link is as posted originally (and here for you: Article The Positive Effects of Grifola frondosa Heteropolysaccharid...

To reply to your statement, both papers aimed to observed the effect of heteropolysaccharide. Li et al 2019 observed in vivo effect on rat gut microbiota and the effects of ameliorating fat-related diseases. Svagelj et al 2012 looked at in vitro immune-modulatory activities of heteropolysaccharide on human blood cells.

The only difference being that Svagelj et al 2012 described the extraction methodology in reference to the well-studied/patented extract, MD-fraction (also known as D or MX-fraction), methodology, which is as far as I am aware, a general Maitake mushroom polysaccharide fraction.

Apart from one paper citing the extraction methodology as similar to a previous extraction methodology, Li et al 2019 do otherwise without explanation, which is common-place in literature (either the extraction methodology is cited as conducted in previous studies or not).

I find it interesting not to be able to find a clear scientific consensus/explanation.

The full extraction methodology they employed is as follows:

Dried Grifola frondosa was chopped by an electric grinder and sieved through an 80 mesh to prepare dry Grifola frondosa powder. Grifola frondosa powder (100 g) was pretreated 5 times with (v/w) 95% ethanol using ultrasonic extraction (300 W, 50 °C) for 60 min. The precipitate was extracted 5 times with (v/w) 55% ethanol using ultrasonic extraction (300 W, 50 °C) for 60 min. Subsequently, the precipitate was extracted 10 times with hot water (v/w) using ultrasonic extraction (300 W, 80 °C) for 60 min. The supernatant was again precipitated with the addition of cold absolute ethanol to a final concentration of 80% (v/v) and kept at 4 °C overnight. The resulting precipitation was vacuum concentrated and freeze dried, yielding Grifola frondosa heteropolysaccharide (GFP).

As I understand the pre-treatment with ethanol is used to "soften" the cellular matrix before the extraction with water, as you said. Because at the last step it uses that supernatant aqueous and precipitates from there to the heteropolysaccharide with alcohol to 80% and low temperature during the whole night.

Gabriela Barragan

"As I understand the pre-treatment with ethanol is used to "soften" the cellular matrix before the extraction with water, as you said. Because at the last step it uses that supernatant aqueous and precipitates from there to the heteropolysaccharide with alcohol to 80% and low temperature during the whole night."

correct :)

The conclusion, I assume from this discussion, is that there is no standardised method in this regard. Although pre-treatment with ethanol to "soften" the constituents makes sense prior to extraction.

Yes, there is no standardized method for the extraction of a polysaccharide. Different pre-treatments can be tested as well as different extraction conditions.