We are aware of the fact that some type of solvent is required for isolating a compound from herbs. I personally believe that water is the best solvent since mostly decoction is prepared using water by the herbalist or medicine man. What are your suggestions?
It depends on what you want to do. If you want to pharmacologically test a traditionally used phytomedicine that is usually used as a water decoction (hot) or extraction (hot or cold), you should use water in the same manner as the traditional medicine is prepared. When testing this in an animal model, take care to apply it orally (as in human treatment).
If you want to identify and isolate secondary plant compounds you should use methanol (lower boiling point) or ethanol (somewhat higher boiling point), pure acetone or acetone/warer mixtures. This is for most of the polar and semipolar constituents. However, for lipophilic compounds you should use lipophilic solvents such as petrol or (bit mor polar) chloroform. for some of these solvents you should take care; acetone is highly fire sensitive and liver toxic; chloroform shows liver toxicity; thus take care for a well working hood. Don't use diethylether because of its high tendency for explosion.
The solvent selection for isolation of any phyto-constituent depend upon its solubility in that solvent.
water are used if nature of phyto-constituents is polar
Alcohol is used for if its slightly non-polar (used for both polar and non polar)
Non polar solvent like ether and acetone are used for highly non polar solvent.
Hope I have clear on it that on the basis of polarity of your compound of interest you have to choose best solvent which are best suited to solublize the compound in it as well as should be preferred nontoxic, environment friendly.
factors should be considered when selecting a solvent:
polarity;
boiling temperature - this should be low in order to facilitate removal of the solvent from the product;
reactivity - the solvent should not react chemically with the extract, nor should it readily decompose;
viscosity - must be low;
stability to heat, oxygen and light;
coast and safety
The organic solvents more frequently used are:
aliphatic hydrocarbons: propane, butane, hexane
alcohols: methanol, ethanol, 2-propanol
hydrocarbons with a carbonyl group: acetone, methyl acetate
halogen derivation: dichloromethane, dichloroethane, freons.
Thanks a lot to both of you for enhancing my knowledge. As per the discussion I think fractionation study (non-polar to polar either alone or in combination) must be done in order to select the suitable solvent as per our requirement.
For phytochemical constituents are depends of polarity and size , this factors are major role in individual compounds isolation but selection of solvents for extraction it is also vital part of for natural products isolation. as per my experience if you have extract thermosensitive use ethanol and water combination and otherwise use ethanol is best sovents for extraction (non toxic to cell or biology activity) in cold extraction (successive extraction method.)
all the best sir
It depends on what you want to do. If you want to pharmacologically test a traditionally used phytomedicine that is usually used as a water decoction (hot) or extraction (hot or cold), you should use water in the same manner as the traditional medicine is prepared. When testing this in an animal model, take care to apply it orally (as in human treatment).
If you want to identify and isolate secondary plant compounds you should use methanol (lower boiling point) or ethanol (somewhat higher boiling point), pure acetone or acetone/warer mixtures. This is for most of the polar and semipolar constituents. However, for lipophilic compounds you should use lipophilic solvents such as petrol or (bit mor polar) chloroform. for some of these solvents you should take care; acetone is highly fire sensitive and liver toxic; chloroform shows liver toxicity; thus take care for a well working hood. Don't use diethylether because of its high tendency for explosion.
Thanks a lot sir for your concise and precise suggestion. I will surely consider it.
The solvents which in common use are Ethanol or Methanol, while for further fractionation other solvents like water, ether, hexane, ethyl acetate, chloroform, acetone etc can be used depends upon solubility of your compound of interest.
Hallo,
As said before, the choice of solvent depends on the product that you want to get. But for me, i always used a mixture of two solvent (CH2Cl2/ / MeOH) so that you can extract the non-polar and polar compounds. or you can try also another mixture of two solvent
The choice of menstruum is dependent on end use. As a European-style herbalist, who uses plant extracts for clinical treatments, my view is that above 16-18% ethanol the fibre, complex carbohydrates, proteins etc are excluded. At 25% ethanol the polar substances are present but not the gums, resins or oils and not some alkaloids. At 45% ethanol both polar and non-polar substances are present if the extraction process is prolonged, more than 3 days, as the concentrated menstruum has a different solvency to original menstruum composed of purely ethanol and water. Higher ethanol levels 70 to 90% are required for the extraction of gums, resins and oils. These later products are not clinically elegant and so I prefer encapsulated herbs.
In my opinion the mixture of organic and inorganic solvents is good choice. You can use methanol/ethanol with water in equal ratio (i.e. 1:1) for the polar compounds including quaternary alkaloids. However, for nonpolar compounds, you can use chloroform/n-hexane
Dear sir it depends upon many factors as discused above. but for the extraction of broad array of compounds use of methanol, ethanol and ethyl acetate has been recomended by many research groups. rest it follow the rule of like disolve like. now a day for better extraction of secondary metabolites from herbs combination of solvents may be used i.e. methanol-water, chlorofom-hexane, chloroform-ethylacetate, etc.
I'll suggest ethanol as the optimum solvent rather than methanol as methanol sometimes shows toxic effects with the herbal medicine or plant constituents, moreover methanol in many cases needs to purify, which indeed makes a problem.
Not only is alcohol a good solvent for the herbal active components it is also an excellent preservative. Alcohol has been used to make herbal preparations for hundreds, perhaps thousands, of years. Old texts describe steeping herbs in wine for long periods and then using the resultant liquid. With our increased knowledge of plant constituents herbalists now choose the appropriate alcohol:water mix to optimise the effectiveness of the extract. When looking at extracts to purchase it is also important to recognise that not all alcohol is equal. There are of course different grades and is important that medicinal extracts are made using a good quality alcohol. The cheaper grades have too many impurities that are hard for the liver to process.
Alcohol also provides a particularly effective way of maximizing the bioavailability of the actives extracted from the plant. Results of a double blind, placebo-controlled, crossover study on children with chronic obstructed airways have been reported in the Industry News section of the Zeitschrift füür Phytotherapie. The therapeutic effects of alcoholic and alcohol-free extracts of Ivy leaves (Hedera helix) were compared. Spirometric testing showed a significant improvement in lung function for both products, which was superior to conventional bronchodilators. However, it was shown that the addition of alcohol to the preparation yielded an increase in bioavailability of active components, and the dose of the alcohol-free preparation needed to be adjusted to a higher level to obtain the same effect. The mode of action seems to be that alcohol acts to keep the active components in solution after ingestion, thus facilitating their absorption into the bloodstream.
It is depend on your approach. For best extraction recovery of all hydrophobic and hydrophilic components, firstly you may want to obtain four parts of fractionations with: Hexane, Ethyl Acetate, Ethanol, and Water. Then, depend on your study design and need, take the individual part to do biological scanning; you may want to combine the four fraction parts; and by animal study, you'll find out the different biological performance between each part.
The herbs from high environmental pollution areas / countries contain high levels of heavy metals and/ or toxic compounds, and the toxic effects might be much higher than the therapeutic effects, so it turns out the overall result of poison.
It depends of the active compounds in the plant which you want to extract. However the hydro-alcoholic extract is the most popular and suitable one.
It depends on which compound is to be extracted. If it is in general then water itself is the best solvent as many of the herbs gives out maximum extraction through water. But it the compound to be extracted is a non water soluble in nature then one can go into different solvents like chloroform, petroleum ether, acetone, ethanol, methanol, etc.
What will be extracted? polar or apolar? methanol or ethanol is good solvent. You can use water but you cant evaporated water easly. Methanol and ethanol is good solvent and easly can be eavoprated under 50-60 degrees.
Methanol and ethanol is good solvent and easily can be eavporated under 50-60 degrees. Furthermore,alcohol also provides a particularly effective way of maximizing the bioavailability of the actives extracted from the plant.
I would agree with most of these comments, but overall, I would suggest that it depends on the herb and what you are looking for. As an herbalist, I generally like the constituents of the whole plant, not an isolated one; most herbalists would agree.
Resins, myrrh for example, will only extract in 95% etoh. Lemon balm, on the other hand, is almost totally water soluble; or if you want it long term [tincture], 25% alcohol is best. It also depends on whether or not the herb is fresh or dry. Most herbs will generally extract dry in 40% etoh. Generally.
I've been making herbal preps for nearly 20 years and I usually consult Michael Moore [now deceased] and James Green, both herbalists; James Duke, the U.S. ethnobotanist; and Kerry Bone, chemist/herbalist, to name a few whose texts I consult when I prepare herbal medicines.
Arvind, you mentioned decoctions. Those - made with twigs, roots, hard berries - are generally water soluble, often cold water maceration overnight [like marshmallow root]; strained and pressed and preserved with a bit of alcohol [25%] or a concentrated syrup for preservation.
That's the 'medicine woman' way.
Diane
Choice of solvent for extraction of plant material depends on purpose of extraction and type of compounds you intend to isolate. A plant material contains different class of compounds with varying degree of solubility with solvent. In general for total extraction Aqueous acetone, ethanol or methnol is use for total extraction. Other solvent like pt. ether, chloroform, acetone, ethanol, methanol, etc are used to extract compounds in order of their affinity/solubility with specific solvent. .
I agree with this opinion,"it depends on what you want to do". The extraction methods are important, too. Hydrodistillation is a suitable method for essential oil extraction. n-hexane or petroleum ether are good solvent for nonpolar constituents such as long chain hydrocarbons,lipids, some mono and sesquiterpenes and so on, in suxhelet apparatues.
If you want to investigate the compounds in the therapeutically used application form, then you should use the solvent taken for this form, in most cases cold or hot water, sometimes boiling water (decoction). However you should be aware that a cold water extractis not stabilized; even in dry herbal material enzymes (e.g. glycosidases) can persist which alter the original compounds (e.g. glycosides). Further, most herbal material contains more or less microbes, which also influence the composition of a cold water extract. This, of course is not so important with hot water extracts, although one should be aware that there are enzymes which tolerate heat up to 90 °C and more. E.g. ß-Glycosidases often are stabile up to 60 °C.
So for phytochemical investigations organic solvents are the best choice, most of which inhibit enzymes and kill microbes.
The most common extraction method in herbal medicine is to boil the herb in hot water. You make the extract very concentrated by using large amounts of the herb and/or boiling the mixture for a long time. Another type of extract is made by using a mortar and pestle to grind the herb with a solvent such as alcohol or acetone. However, solvents such as acetone and alcohol are highly flammable. Use these only in well-ventilated areas and away from open flames. Boiling liquids can cause serious burns. Handle them carefully to avoid splashes.
i to agree with above opinions it depends upon the type of secondary metabolite and biological activity you are interested in for initial primary screening you can use methanol.
If you have access to it, dense phase CO2 will pull everything that water or alcohol can and the CO2 can be evaporated off
excellent information suggested by the experts. My doubt got clarified. Thank u so much
Good Discussion could be summaries as :
EXTRACTION- It is a technique to separate active substance form crude drug
Principle : 'Like dissolve like' User should known the substance to be extracted is it polar, semipolar or non polar.
FOR IN-VIVO INVESTIGATIONS: : Prepare & use the extract as it is used in traditional method (in most of cases WATER)
FOR PHYTO-CHEMICAL INVESTIGATIONS: Use Organic solvents (Methanol, ethanol which are relatively safe in terms of toxicity and flammability in compared acetone and diethylether)
Frequently Used solvents
1. WATER:
NATURE- polar solvent
ADVANTAGES- safest, economical, widely used
DISADVANTAGES - Higher boiling point (Need higher temperature and time to evaporate), heat sensitive components may degrade, prone to micobiologial growth and enzymatic reaction so can not be stored for longer time.
2. METHANOL/ ETHANOL:
NATURE- polar solvent
ADVANTAGES- safe among other organic solvents, self preservative, evaporates faster
DISADVANTAGES - flammable, less toxic
Other Solvents:
Acetone- Polar- Highly toxic and flammable
Chloroform, haxane- Non polar - Toxic
Options for solvents systems:
1. Single solvent : Polar or non polar solvent alone
2. Solvent Mixture : Polar: non polar (in different ratio)
Thanks to all Experts and correct me if i have miss concluded anything.
Alcohol is best for gross extraction, however, depending upon the type of compounds other solvents should be selected for better extraction like ether& acetone for flavonoids & polyphenols; water (hot or cold) for polysaccharides and so on.
What about the safety of n-butanol when we use it for extracting fatty materials from an extract of ginkgo biloba leaves
For natural product research and development, from herbs extraction to enzyme and/or cell biological screening, animal studies, clinical try, and commercial production, to extract middle or high hydrophobic compound(s), using EtAc is best; to extract low hydrophobic compound(s), use EtOH; to extract very high hydrophobic compound(s), use Hexane; to extract high hydrophilic compound(s), use H2O.
To extract Fatty Acid(s) as your approach, considering the possibility of esterification reaction to convert your target fatty acid(s) into Easter(s), it is not the best choice to use alcohol (such as MeOH); if use it to extract low carbon and high hydrophilic carboxylic acid(s), try add a little bit of water, and avoid high acidic condition, strong acid addition, and heating.
If use alcohol to extract herbs containing ester compounds, to prevent base catalyzed transesterification reaction, avoid base addition.
So, for general Fatty Acids and lipids extraction, EtAc is the best choice.
I am agree with other comment which highlighted the aim of extraction and mentioned to the type of polar and non-polar ingredients which are existed in the herb.Also I like to ask for another question in this subject, if the the aim of extraction is evaluation the antimicrobial effect which solvents should be more safe and can act better as a second diluent?
For the purpose of drug / medical study experiment using plant/ herb extraction, one of the keys is that all of extraction solvents / chemicals should NOT have toxic effect(s) or strong interfering efforts to living cells, animals, and human beings.
About organic solvents applied for extraction, some of them have high toxicity to living cells, such as Methanol, Acetone, Chloroform, Dichloromethane, etc.; some solvents in low boiling point have high risk in laboratory and manufactory safety issues; it is better to opt out these chemicals/solvents.
In addition, to the early study experiment design, cost effectiveness and working efficiency should be considered, too.
Using high toxic chemicals / solvents, you have to COMPLETELY dry out the extraction, and then you need to do solvent residue test to each batch of products; and if the residue in the extraction dryness is high, you have to do many more study experiments on all cell biological, animal, and human study tests with the toxic blank solvent/chemical A, B, C, D, … as negative comparison tests without herb/plant involved. Whereas on extraction using the solvents of Water, Ethanol, Ethyl Acetate, and Hexane, which have no toxic effect(s) or minor interfering efforts to the living cells, animals, and human beings, you only need to do one blank test on water base.
Water extraction product mainly contains metals, ions, high hydrophilic compounds, and water soluble proteins/enzymes, glycoproteins, peptides, amino acids, nucleotides, sugars, and polysaccharides.
Ethanol extraction product mainly contains high hydrophilic compounds (including very polar neutral, basic and acidic compounds, amino acids, nucleotides, sugars, and polysaccharides).
Ethyl Acetate extraction product mainly contains medium hydrophobic compounds (including medium and low polar neutral, basic and acidic compounds), of which, steroids, wax, fatty acids, alkaloids, and polar-chain carbonated polymers are expected.
Hexane extraction product mainly contains low or non-polar hydrophobic compounds with extremely high lipophilicity, of which low polar neutral compounds, steroids and high carbon fatty acids are expected).
Hope above points are helpful to you.
All comments stated above are of vital importance, true and I am in support of them. But I would like to know the solvents for conducting anti fungal activity considering their effects in living cells.
n-hexane for defatting and oil extraxtion , alcohos for all plant phytochemicals
DX to you
Nov 18, 2015
Could you tell me what kind(s) of compound(s) will be extracted using the solvents as follows respectively from plant and crude drug (extracts)? If there have the others solvents can be used to extraction?
1. Ethanol (EtOH)
2. Ethyl Acetate (EtAc)
3. Hexane
4. Water (aqueous)
5. Methanol (MeOH)
6. Ether
7. Acetone
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Ying Li to DX
Nov 19, 2015
Dear DX,
For the purpose of drug / medical study experiment using plant/ herb extraction, the key is that all of extraction solvents / chemicals should NOT have toxic effect(s) or strong interfering efforts to the cells, animals, and human beings.
About organic solvents applied for extraction, some of them have high toxicity to living cells, such as Methanol, Acetone, Chloroform, Dichloromethane, etc.; some solvents in low boiling point have high risk in laboratory and manufactory safety issues; it is better to opt out these chemicals/solvents.
In addition, to the early study experiment design, cost effectiveness and working efficiency should be considered, too.
Using high toxic chemicals / solvents, you have to COMPLETELY dry out the extraction, and then you need to do solvent residue test to each batch of products; and if the residue in the extraction dryness is high, you have to do many more study experiments on all cell biological, animal, and human study tests with the toxic blank solvent/chemical A, B, C, D, … as negative comparison tests without herb/plant involved. Whereas on extraction using the solvents of Water, Ethanol, Ethyl Acetate, and Hexane, which have no toxic effect(s) or minor interfering efforts to the cells, animals, and human beings, you only need to one blank test on water base.
Water extraction product mainly contains metals, ions, high hydrophilic compounds, and water soluble proteins/enzymes, glycoproteins, peptides, amino acids, nucleotides, sugars, and polysaccharides.
Ethanol extraction product mainly contains high hydrophilic compounds (including very polar neutral, basic and acidic compounds, amino acids, nucleotides, sugars, and polysaccharides).
Ethyl Acetate extraction product mainly contains medium hydrophobic compounds (including medium and low polar neutral, basic and acidic compounds), of which, steroids, wax, fatty acids, alkaloids, and polar-chain carbonated polymers are expected.
Hexane extraction product mainly contains low or non-polar hydrophobic compounds with extremely high lipophilicity, of which low polar neutral compounds, steroids and high carbon fatty acids are expected).
Hope above all is helpful to you.
Best Wishes,
Ying Li
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DX to you
Nov 19, 2015
Dear Ying,
Thank you for your reply and opinions, it is very helpful to me.
I would like to do in vitro antifungal activity assay of extract of 9 herbs and crude extract of Km against dmtp.
In the extraction part of experiment, I plan to use several solvents as follows：
1. Ethanol (EtOH)
2. Ethyl Acetate (EtAc)
3. Hexane
4. Water (aqueous)
5. Methanol (MeOH)
6. Ether
7. Acetone
Thank you for your time and opinions again.
Kind regards,
DX
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Ying Li to DX
Jan 6, 2016
Dear DX,
If I am you, remove the last three solvents in your list.
If use ethanol, you don’t have to use methanol; and on the cell toxic-issue, ethanol is much better than methanol. Using both doesn’t make sense.
If use Ethyl Acetate (EtAc), you don’t have to use Ether, because their extraction recoveries will be similar; and EtAc is safer than Ether. Listing both wouldn’t make great sense.
Acetone (ACT) is a proton accepter, and the polarity is between EtOH and EtAc, If use ACT to extract, the extraction recovery should be relatively close to that from EtAc. However, if you apply EtOH and EtAc, you don’t have to apply ACT.
Finally, to the concentrated or dried extraction, if you desire to dissolve it in water and further use it in medium culture for biological/cell-based screening and animal study, it is a good selection to apply very small amount of Ethylene glycol mono-tert-butyl ether, one of most efficient solubilizers for organic compounds, as a cosolvent, for making your extraction mixable with water, as well as your target easily act in experiment medium.
(If you want to try ionic surfactants, to prevent the chemicals from the affect to your experiment, the amount of ionic surfactants is a key. Same as ionic surfactants, many of non-ionic surfactants are cell killers.)
Best Wishes,
Ying Li
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DX to you
Jan 7, 2016
Dear Ying Li,
Thank you for your advice, email and kindly wish.
Wishing you and your family peace, joy and happiness throughout the new year!
Your opinion is very precious, but I'm very sorry, I have already started my study. Please let me introduce.
At first, I used 7 kinds of solvents for extraction, these are
Name (Relative polarity)
1. Hexane 0.009 (Min)
2. Ether 0.117
3. Acetone 0.355
4. Acetonitrile 0.46
5. Acetic acid 0.648
6. Ethanol 0.654
7. Methanol 0.762 (Max)
After extraction, the solvents were removed by vacuum spin dryer, and the precipitate were dissolved in DMSO to assay these antifungal activity using micro-broth dilution assay.
Among them, Hexane and Ether are very easy to evaporate, so have been abandoned. Acetone extracts showed very low antifungal activity. Acetonitrile,Ethanol and Methanol extracts showed similar and medium activity. Acetic acid extracts showed the most strong antifungal activity.
Thus, yesterday, I applied the Acetic acid extracts to A pur + F SYST and each fractions were collected. Today, I will concentrate each fractions.
My next step is check the antifungal activity of each fractions.
That is all! Please let me know your opinion when you have free time.
Do let me know if I can help you in any other way. I will be happy to help! Thanks again!
Kind regards,
DX
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Ying Li to DX
Jan 8, 2016
Dear DX,
Thank you for the message. And I am so glad seeing your progress on your project, but there are many big problems you could have.
Well, in my major Pharmaceutical Science, before I start my project, I need to know what are my targets; such as antifungal candidates, of which many are lipophilic fatty acids, so I can estimate these compounds’ chemical structures or their functional groups, as well as their chemical and physical properties, pH range, polarity, solubility, log P, log D, chemical stability, and what chemical solvents I need to extract the active compounds,…, etc.; and upon the bioactivity-and-structure relationship, I can estimate their pharmacological and toxicological effects, and their ADME, DMPK and TK properties. Furthermore, based on the early screening results, if their major parameters (such as t1/2, log P, chemical stability, DMPK, therapeutic index, toxicity, etc.) indicate the high shortage(s) for drug, I’ll decide to do chemical structure modification or make pro-drug, and computational medicinal chemistry is a super useful tool for me. Finally, on the stage of drug candidate selection / identification, I’ll consider formulation research and development with API R&D for the new drug.
To antifungal candidates, you may want to try lipophilic solvent, but not the pure solvent or high percentage solvent of Acetic Acid (HAc), because HAc has strong antifungal effect!! You definitely don’t want your experiment to be interference by HAc. Of course, you can add a negative control with Acetic Acid to compare with your Herb HAc-extraction; if there is no interference, the antifungal activity of Herb HAc-extraction should be significantly higher than that of HAc. Alternately, you can completely remove HAc by evaporating your extraction to dryness; however, the drying / blowing-out process might cause the loose some components in volatile oil which relate to the herb's antifungal activity.
As we know that it is rare to see using pure Acetic Acid (HAc) or high percentage of HAc as the extraction agent. If you want to use HAc for extraction, (1) you have to make sure that your antifungal result is not from HAc antifungal effect, (2) HAc does not cause the chemical reactions of R-OH compounds’ esterification and/or RCOOR’ compounds’ ester-substitution and/or compound’s degradation in the process of extraction and evaporation, if these R-OH, RCOOR, and parent compounds associate to the herb's antifungal effect.
In addition, you may know that some compounds containing nitrogen also have antifungal effect. To such kind of compounds, when you use low pH water buffer (pH 3-4) that is acidified with very small amount of HCl or Formic Acid or HAc, you can obtain high extraction recovery, since ammonium salt forms in acidified water and becomes high water-soluble; or you can apply middle/high polar organic solvent to extract.
From your described, you use Acetic acid, ACN, EtOH and MeOH to extract. As we know that all of the four solvents have antifungal effect. So, to the individual extracted solution containing the residue of the solvents, when you perform biological screening on antifungals, you have to add negative controls with these solvents if any remaining in sample. If the result from testing sample has no significant difference with the negative control, you may want to COMPLETELY dry you extraction, and then dissolve the residual in water with a suitable surfactant in proper amount, and make a comparison test.
It often happened that the single compound from the fractionation / isolation of the extracts could has low or very minor expected antifungal activity or has different therapeutic performance, even though the extraction sample has strong and repeatable positive-result. It tells the DDI (drug-drug interaction), drug additive or synergism effect from the multiple active-compounds in the extraction sample. So, it is not strange that single compound might not work.
Best Wishes,
Ying Li
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Ying Li to DX
Jan 14, 2016
Dear DX,
As your described, if the fractionation (eluted with x% H3PO4-H2O in MeOH (a:b) is from the dryness of HAc-extract, and has some antifungal activity, then possibly it contains Alkaloids (which consist of Nitrogen); then you may want to pay more attention to alcohol extraction and EtAc extraction (without adding any organic or inorganic acid). Also, don't forget to remove the extraction solvent(s) before use them in early antifungal screen.
After the early screen, you'll know which extraction material has stronger antifungal performance. Repeat the screening experiment to make sure the results are reproducible.
Then, you may want to do fractionation / isolation. Apply each part of the eluents on biological test. If one compound is targeted with the same antifungal activity as the test with the material from solvent extract, you are gifted.
As the result from every portions of anti-fungal herb extractions and isolation, if every individual part of eluents, (which, I mean, is pure compound), has lower antifungal activity, it means the effect of DDI (drug-drug interaction), drug additive or synergism from the multiple active-compounds in sample is existing. It's not rare that in same case, single compound doesn't work.
Good luck and have fun!
Best Wishes,
Ying Li
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DX to you
Jan 18, 2016
Dear Ying Li,
Thank you for your kindly email.
All fractions showed no any activities, all fractions were lost activities! I do not know the reason, I think about may some active compounds were lost when removing mobile phase.
I am considering changing spin dryer to nitrogen blowing method for dry down mobile phase of fractions.
Please let me know your advice (you are gifted too!). Thank you.
Kind regards,
DX
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Ying Li to DX
Jan 20, 2016
Dear DX,
To the drying process, I would suggest you to try lyophilization method; and don't forget the issue on the chemical stability problem including high photosensitivity!!! In our National Research Center, we never use any of the ways that you are using; no any of research staff, scientists, experts, professors even applied them.
To fix your problem, it can try following simple ways:
1. Read my initial / earlier messages.
2. Avoid using acid or base in your extraction. Only use neutral H2O, and neutral organic solvents. If you already applied the acid in your extraction, you had better to make sure that the testing compounds from your extracted product are back to original neutral status, then step-by-step to do ...
3. It is often happened that DDI (drug-drug interaction), drug additive and/or synergism effects from the multiple active-compounds in your extracted product. It is very common that low or almost no expected bioactivity on the isolated single-compound from your fractionation.
Best Blessings,
Ying Li
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Ying to DX
Dear DX,
Thank you for the message. And I am so glad seeing your progress on your project, but there are many big problems you could have.
Well, in my major Pharmaceutical Science, before I start my project, I need to know what are my targets; such as antifungal candidates, of which many are lipophilic fatty acids, so I can estimate these compounds’ chemical structures or their functional groups, as well as their chemical and physical properties, pH range, polarity, solubility, log P, log D, chemical stability, and what chemical solvents I need to extract the active compounds,…, etc.; and upon the bioactivity-and-structure relationship, I can estimate their pharmacological and toxicological effects, and their ADME, DMPK and TK properties. Furthermore, based on the early screening results, if their major parameters (such as t1/2, LogP, LogD, chemical stability, DMPK, therapeutic index, toxicity, etc.) indicate the high shortage(s) as drug, I’ll decide to do chemical structure modification or make pro-drug, and computational medicinal chemistry is a super useful tool for me. Finally, on the stage of drug candidate selection / identification, I’ll consider formulation research and development with API R&D for the new drug.
To antifungal candidates, you may want to try lipophilic solvent, but not the pure solvent or high percentage solvent of Acetic Acid (HAc), like what you did, because HAc has strong antifungal effect!! You definitely don’t want your experiment to be interference by HAc. Of course, you can add a negative control with Acetic Acid to compare with your Herb HAc-extraction; if there is no interference, the antifungal activity of Herb HAc-extraction should be significantly higher than that of HAc. Alternately, you can completely remove HAc by evaporating your extraction to dryness; however, the drying / blowing-out process might cause the loose some volatile-components which relate to the herb's antifungal activity.
As we know that it is rare to see using pure Acetic Acid (HAc) or high percentage of HAc as the extraction agent. If you want to use HAc to extract herbs, (1) you have to make sure that your antifungal result is not from HAc antifungal effect, (2) HAc does not cause the chemical reactions of R-OH compounds’ esterification and/or RCOOR’ compounds’ ester-substitution and/or compound’s degradation in the process of extraction and evaporation, if these R-OH, RCOOR, and parent compounds associate to the herb's antifungal effect.
In addition, you may know that some antifungal drugs are base (containing nitrogen). To such kind of compounds, when you use low pH water buffer (pH 3-4) that is acidified with very small amount of HCl or Formic Acid or HAc, you can obtain high extraction recovery, since ammonium salt forms in acidified water and becomes high water-soluble; or you can apply middle/high polar organic solvent to extract.
From your described, you use Acetic acid, ACN, EtOH and MeOH to extract. As we know that all of the four solvents have antifungal effect in cell screening. So, to the individual extracted solution containing the residue of the solvents, when you perform biological screening on antifungals, you have to add negative controls with these solvents if any remaining in sample. If the result from testing sample has no significant difference with the negative control, you may want to COMPLETELY dry you extraction, and then dissolve the residual in water with a suitable surfactant in proper amount, and make a comparison test.
It often happened that the single compound from the fractionation / isolation of the extracts has low or very minor expected antifungal activity or has different therapeutic performance, even though the extraction sample has strong and repeatable positive-result. It tells the DDI (drug-drug interaction), drug additive or synergism effect from the multiple active-compounds in the extraction sample. So, it is not strange that single compound might not work.
Best Wishes,
Ying Li
Methanol for Flavonoids and Poly Phenolic compound.
Chloroform for Alkaloids,
n-Hexane for Fatty acids and Lipids.
Hydro distillation Method for Essential oil.
Mixture of Water and Methanol for polar compound such as glyconic flavonoid and polyphenolic ,....
If the active metabolites in the herb is not well known, sequential method of extraction using n-hexane, DCM, EtOAC and MeOH is recommended most of the time
I think Adolf has hit the nail on the head. Your answer will depend on what you are hoping to extract and why.
Perhaps you could look at the work of Kerry Bone; there is a brief summation in "A clinical guide to blending liquid herbs".
Also, there is a section on this in "Fundamentals of Pharmacognosy and Phytotherapy" by Heinrich, Barnes, Gibbons and Williamson that has a good overview on what techniques are available and why you would choose one over the other.
Hope this help lead you to your answer.
It depends on what you want to get and of course you can use any type of solvent like Ethanol, hexane, petroleum ether, chloroform etc in addition to water (aqueous).
Water and hydro-alcoholic are good solvents for some polar or low polar extraction. But for extraction of the non-polar compounds, hexane and petroleum ether are suitable.
The best solvent set for herb extraction is:
Water
Ethanol (EtOH)
Ethyl Acetate (EtAc)
Hexane
Extract your material(s) with above listed solvents individually. Then the four sets of extractions need to be concentrated to dryness with lyophilize (frozen drying).
Finally, to the concentrated or dried extractions, if you desire to dissolve it in water and further use it in medium culture for biological/cell-based screening and animal study, it is a good selection to apply very small amount of Ethylene glycol mono-tert-butyl ether, one of most efficient solubilizers for organic compounds, as a cosolvent, for making your extraction mixable with water, as well as for your target to easily act in experiment medium.
If you want to try ionic surfactants, to prevent the chemicals from the affect to your experiment, the amount of ionic surfactants is a key. Same as ionic surfactants, many of non-ionic surfactants are cell killers.
Enjoy Holiday Season and Have A Wonderful New Year!
Best Wishes,
Ying Li
Good question!
My short answer is: water, methanol (70%), ethanol for polar substances and hexane to non-polar substances!
regards
Correct water first then Ethanol to cover two pole of polarity and then Hydro alcohol, Ethyl acetate and Hexane.
Always start from the lipid fraction, i.e. first extract with petroleum ether or something similar---you want to remove the fatty fraction before you go to the more polar compounds. If you start with water you will have all sorts of emulsion problems.
this depends on the polarity of phytochemical compound you want to extract from herbal source and its chemical structure
There is general concept like dissolves like, so if you want to isolate specific phyto constituent depending upon the nature of the constituent you will have to make use of polar or non polar solvent.
The selection of extraction solvents is depended on the polarity of phytochemical compounds you want to extract from herbal source and its chemical structure .
Therefore, it will good to start for the extraction with hexane then to use methanol or ethanol.
Try solvents using increasing order of solvent polarity viz, n-Hex, chloroform, MeOH etc...This will cover almost all the components/compounds present in ur herbs/plants and isolation can be performed accordingly. Rest also depends on the polarity of the compounds u r trying to isolate.
Actually i think it depends up to the compounds, but water,methanol, ethanol and DMSO may cover most of the compounds .So, according to the dissolution property of the compounds, it can be happened that which solvent will be suitable for that compounds.
For medicinal purposes, water stand first, than ethanol.
Utility of the other solvent required the prove of their absence in finish product.
It depends on what you are looking for and the compunds you want to extract.
i want to check the in-vitro antibacterial activity of Gum Arabic which is highly soluble in water. which is the best solvent ( Aqueous, Ethanol or methanol) for Gum Arabic extraction for testing in-vitro antibacterial activity?
It largely depends on your goal. for pharmacological tests, water could be the best solvent, it will leave most of your likings free. For isolation, methanol is the best.
You can as well consider doing both concurrently to report on your comparative observations, your recommendations could be of a great importance.
All the best
It depends on the objective pursued in the extraction of the compounds
For me, after publishing more than 31 papers on antimicrobial activity of natural products, the best solvents and methods are methanol for semi-polar, water for polar compounds and hydro-distillation for aromatic plants to study the essential oils. Unfortunately, in developing countries a researcher should adapt with the lab facilities. for example, it is difficult to get good aqueous extract without lyphilization using a freeze dryer. If you are in a poorly equipped lab, use maceration in 80-70% methanol.
However, if you are within a research group and there is a well-equipped lab, a systematic study is preferable which will lead to extract a single bioactive compound and or a group of compounds for new discovery or patent.
Best wishes,
If the compounds of interest are hydrophilic or have high polarity, use water, ethanol or methanol to extract. If the compounds of interest is non-polar or hydrophobic, hexane or ethyl acetate. If the compounds have both polar and non-polar groups, the mixture of chloroform and methanol works fine. In many situations, the extract contains the target compounds and unwanted compounds, purification of extract is necessary.
Its depends on your compound nature. if the compound are highly polar or dissolve in only organic solvent then we have to used only polar organic solvent like methanol, butanol. If the compound are non polar then we have to used non polar solvent like hexane, pentane. If the compounds are hydrophilic or have high polarity, use water as green solvent.
hydromethanolic solvent i.e use below 50% of methanol as solvent
Please references supercritical fluid extraction herbal use CO2 and 95% ethanol method:
Use supercritical fluid CO2 extraction, it does not classify water-soluble (polar ) or oil-soluble (non-polar) ingredients, CO2 is own volatile, CO2 not remain in the sample after supercritical fluid CO2 extraction, ( herbal extract temperature be 42 -46 degree )(CO2 is volatile for a short time)(sample will not be oxidized).
Final step get ethanol was the pure ethanol after supercritical fluid extraction herbal use CO2 and 95% ethanol, the pure ethanol contain many the water-soluble (polar ) and oil-soluble (non-polar) ingredient, GC/MS can direct analysis the pure ethanol, no need to dilution the pure ethanol, if you want to dry the sample, continue CO2 blow off the pure ethanol, CO2 can isolate air for damage.
If sample hasn't contain caffeine, sample can add known caffeine run GC/MS, can quantitative chemical analysis. (recommend revise experimental design).
Solvents play an important role in pigments and herbal extracts. The performance attributes of paints are multiplied manifold with the use of solvents and for herbal extracts solvents act as the medium through which the herbal properties are extracted from the plant material.
Choosing the right solvent for extracting from plant material is essential if you want to obtain the full benefits and who knows this better than the herbal extracts manufacturers. If the herbal extract has to be used orally for medicine formulations then the solvent will be different than the one used for isolating secondary plant compounds. Normally, in the first case vinegar, alcohol and vegetable glycerine are made use of while extracting the herbal compounds and in most cases, alcohol is the popularly chosen medium. It is highly effective in the breakdown of tough herbs such as berries and barks and helps in extraction of waxes, fats, resins, few volatile oils and many alkaloids from herbs. Food-grade vegetable glycerine is another solvent type that helps in the extraction of some alkaloids, tannins, acids and few minerals from plant material. Herbal extracts prepared using vinegar are called as herbal vinegars. However, secondary plant compounds are extracted by means of solvents other than those used above. Pure acetone, ethanol at a rather upper boiling point, methanol at a rather lower boiling point or water/acetone mixtures is normally used in these cases. For extraction of lipophilic compounds, solvents such as chloroform or petrol are resorted to.
In the paint industry, solvents play an important role in dispersing or dissolving the resin or formulations. It enables the paint to arrive at the required consistency so that it can be applied smoothly and evenly. Evaporation of the solvent takes place after application of the paint enabling the pigment and the resin to develop a coat of paint that dries rapidly. Solvent based paints are a preferred choice due to the performance advantages provided by them and account for a large percentage of industrial applications. Superior finishing and flexibility of use are the two major benefits offered by them and in some applications as in case of architectural coatings it is the best option due to high performance requirements. There are different solvents used in industrial applications depending upon the purposes. Mineral spirits, VM&P naphtha, denatured ethyl alcohol, lacquer thinner, toluene, xylene are just to name a few of them. However, the solvents in the paint industry are regulated by governments due to environmental concerns. But with advancements in science and technology, modern hydrocarbon and oxygenated solvents help to economically address environmental issues besides offering durability and high product performance.
As far as reactive dyes are concerned they form an important ingredient for the textile industry. With regard to textile processing, there are three types of solvents that are being used; namely Tri-chloro Ethylene, Methyl Chloroform and Per-chloro Ethylen. Among the three, however, tri-chloroethylene is most suited. Though several advantages are obtained by solvent dyeing reactive dye manufacturers however, attribute high production costs to solvent dyeing in textile processing.
Depends upon what you want to extract.
Mixture of solvents are use generally are: Chloroform(nonpolar):Ethanol/methanol/water(polar)
Or other polar and nonpolar also you can use.
Generally methanol used as a first solvent for extraction purpose to look for bioactives in medicinal plants
Sir I think on the base of nature of the plant but hydroalcholic solvent with methanol and distilled water is most suitable because it contains both polar and non polar component in it.so it is best solvent system.
Dear Sir,
Please also have a look at this useful ResearchGate link.
https://www.researchgate.net/post/Which_is_the_best_solvent_for_the_extraction_of_the_constituents_polar_and_non_polar_organic_compounds_of_dried_herbal_plant#post-comment-editor
Good luck!
this link is useful
https://www.herb-pharm.com/faq/herbal-manufacturing
regards
It has been reported that ethanol is good for herbal extraction. Current findings indicated that Vegetable Glycerin is a Better Solvent than Alcohol.
Alcohol preserves through a denaturing, sterilization action. Denaturing is a process that alters the protein structure of cells, and in the case of living cells, may kill them. Effectively, alcohol wipes out the biological usability of many constituents. whereas glycerin preserves herbal and botanical characteristics.
Alcohol has a sterilizing action that kills bacteria and contaminating organisms. The sterilization action of alcohol is not biased, however, and also affects the biological usability of what’s being isolated. Herbal products processed with alcohol will contain many extracted constituents and compounds that have been rendered useless by alcohol’s denaturing action. Glycerin, on the other hand, has a desiccant action that neutralizes contaminating organisms. In other words, glycerin deprives contaminants of water.
Glycerin is a safe and effective preservative and aseptic agent when produced according to stringent standards that use the highest quality ingredients available. Conversely, alcohol can be toxic to the body and has a number of potential, adverse side effects.
Advantages of Vegetable Glycerin Based Extracts
As a solvent, alcohol is mechanically conducive to processing herbs, fresh botanicals, and resinous compounds. However, the denaturing characteristic of alcohol, while natural and sometimes beneficial, is detrimental to a plant’s extracted aromatic components, and makes them unusable. This prevents a product from offering the full benefits of the plant matter in question.
Source: https://www.globalhealingcenter.com/natural-health/extracts-vegetable-glycerin-better-than-alcohol/
Depending on need. If for pharmacological testing, it is better to use water with the decoction method. If for isolation, can use ethanol, methanol, or ethyl acetate. Depending on the specific compounds that will be isolated.
Whilst my college is correct in saying that many herbs are taken traditionally in a decoction of water ie tea, all have components that are oil soluble. What you extract into makes a very big difference in what you constituents you get. Lisa Ganora has a nice little book with a consice section on extraction and solubility.
When studying herbal medicine it is extremely important to think about two things, is it pharmacologically fit for the purpose which I’m studying. That is to say what is the physiology and pathophysiology of the condition being studied and does the mechanism of action of the herb and constituents coincide with those. Secondly, is this method of studying the constituents and the effects transferable because of method of administration . Eg peopl don’t tend to take herbs intraoeritoneal!!
The short answer is to extract herbs into the appropriate percentage ethanol (as is done traditionally) as this contains water and oil soluble components to the degree that you have selected your ethanol is percentage. Most herb sit around the 40% mark but Calendula is traditionally 90% to get those specific actives.
Extractability of plant material and phytochemicals largely depend on extraction solvent used. The selection of extraction solvents is depended on physico-chemical nature, chemical structure and polarity of phytochemical compounds you want to extract from the herbal material. Therefore, it is advisable to start extraction with hexane and use other extractants (solvents) in the order of increasing polarity.
Solvents play an important role in pigments and herbal extracts. The performance attributes of paints are multiplied manifold with the use of solvents and for herbal extracts solvents act as the medium through which the herbal properties are extracted from the plant material
Choosing the right solvent for extracting from plant material is essential if you want to obtain the full benefits and who knows this better than the herbal extracts manufacturers. If the herbal extract has to be used orally for medicine formulations then the solvent will be different than the one used for isolating secondary plant compounds. Normally, in the first case vinegar, alcohol and vegetable glycerine are made use of while extracting the herbal compounds and in most cases, alcohol is the popularly chosen medium. It is highly effective in the breakdown of tough herbs such as berries and barks and helps in extraction of waxes, fats, resins, few volatile oils and many alkaloids from herbs. Food-grade vegetable glycerine is another solvent type that helps in the extraction of some alkaloids, tannins, acids and few minerals from plant material. Herbal extracts prepared using vinegar are called as herbal vinegars. However, secondary plant compounds are extracted by means of solvents other than those used above. Pure acetone, ethanol at a rather upper boiling point, methanol at a rather lower boiling point or water/acetone mixtures is normally used in these cases. For extraction of lipophilic compounds, solvents such as chloroform or petrol are resorted to.
What do you want to extract? Proteins are almost always extracted into aqueous solutions. With small molecules, it depends on their hydrophobicity. Very hydrophobic molecules are extracted into hydrophobic solvents like diethy ether (for safety, replace with Methyl tert-butyl ether (MTBE) which has a lower tendency to form explosive peroxides). This can be followed with ethanol and finally water.
Alkaloids are very hydrophobic in an alkaline, but hydrophilic in an acidic environment. Therefore, grind the sample with lime, then extract with ether, separate the organic from the aqueous phase and shake the extract with dilute sulphuric acid. The aqueous phase will contain almost pure alkaloids.
Selection of solvent is based on what you want to extract. However, Alcohol (Ethanol, Methanol) is popularly used to get maximum number of bioactive components. When you want to get glycosides or sugar part you may use water.
It completely depends on the Nature of the Raw material and the target active ingredient you want to get from herbs.
Best solvent for extraction is always which fulfill your requirement and should be cost effective.
I guess that you want to isolate active ingredient from herba. , therefore you can use solvents having different polarity using hexan, dichloromethan, ethylacetate, methanol, n- Butanol and water in order to get all secondary metabolites.
If you want to isolate the bioactive principles for drug/extract. Its better to use solvent of different polarities cz each of the solvent extract differently. For example n-hexane, ethyl acetate, n-butanol solvents exhaustively
Its depend upon you what kind of compounds you would like to isolate; actually different solvent system (from non-polar to polar) gives different type of compounds. To isolate the bio-active principles which are more likely water soluble may extracted via Alcohol or hydro-alcoholic solvent system. Pure water extraction is very difficult, required modern and sophisticated instrumentation & techniques and skills!
For bioactive compounds extraction or herbal formulation best solvents are aqueous and hydroalcohol (70 or 50 percent ethanol). Because in this solvents we can produce herbal products. And we can use or consume those products without fear of high toxicity.
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