Phytochemical Analysis - Science topic

Yes I am interested

Hadia Hemmami Ilham Ben Amor Soumeia Zeghoud

Identifying the class of phytochemicals and determining which compounds to include in your manuscript after GC-MS and NIST identification involves a systematic approach. Here are the steps to address your queries:

1. Identifying the Class of Phytochemicals

To identify the class of phytochemicals or secondary metabolites, you can follow these steps:

a. Database Searches:

- PlantaeDB and IMPPAT are good starting points. Ensure you are using the correct chemical names and synonyms.

- PubChem and ChemSpider can also be useful for finding information about the chemical structure and its known classes.

b. Literature Search:

- Use databases like Google Scholar, PubMed, and ScienceDirect to search for scientific articles related to the identified compounds. Keywords like "phytochemical class" or "secondary metabolite class" along with the compound name can help.

c. Chemical Structure Analysis:

- Analyze the chemical structure using software tools like ChemDraw or MarvinSketch to predict the class based on known structural features of flavonoids, carotenoids, phenols, etc.

d. Expert Consultation:

- If certain compounds are hard to classify, consider consulting with a phytochemist or a researcher specializing in natural products chemistry.

2. Handling Repeated Compounds

When you have repeated compounds with different retention times and similarity indices, consider the following:

a. Consistency Check:

- Check the consistency of identification across different runs. If the same compound is identified consistently with a high similarity index, it is likely accurate.

b. Retention Time Comparison:

- Retention time differences can sometimes indicate different isomers or derivatives. Compare the retention times and the similarity indices to choose the most reliable identification.

c. Reporting:

- If the similarity index is significantly different, consider reporting both and discussing the possible reasons (isomerism, different sources of the compound within the plant, etc.).

3. Selection of Compounds for the Manuscript

Including all identified compounds versus selecting important ones depends on the focus of your manuscript:

a. Relevance and Impact:

- Include compounds that are known to have significant biological or pharmacological activity relevant to your study.

b. Novelty:

- Highlight compounds that are newly identified or have unique properties not commonly reported.

c. Focused Analysis:

- For a more focused analysis, you can prioritize compounds based on their relative abundance (area percentage), biological significance, and the objectives of your study.

d. Supplementary Data:

- Consider including the complete list of compounds in supplementary materials while discussing the key bioactive compounds in the main text.

Practical Steps:

1. Data Organization:

- Create a comprehensive table of all identified compounds with their retention times, area percentages, molecular formulas, and similarity indices.

2. Class Identification:

- Use a combination of database searches, literature reviews, and chemical structure analysis to classify each compound.

3. Selection Criteria:

- Define clear criteria for selecting compounds (e.g., similarity index threshold, area percentage, known bioactivity).

4. Documentation:

- Clearly document the methods used for identification and classification to ensure reproducibility and transparency.

By following these steps, you can systematically identify the classes of phytochemicals, choose which repeated compounds to include, and decide on the most relevant compounds for your manuscript.

You can try search and match those compounds in Adam's Database, based on the RI, KI value. I think, this will help you to clarify.

Dear Jonathan,

Generally yes, however other drying strategies are equally valid (e.g., drying in an oven with air circulation). The idea is to eliminate moisture for proper concentration calculation.

Yours sincerely.

As you will be aware, plants are a very rich source of phytochemicals and this will be reflected in your aqueous extract. The IR spectra of your extract will be complex, containing the overlapping spectra of every compound. You will not be able to identify any individual compound with any degree of certainty.

If you want to identify the components of your extract then you need to resort to a Metabolomics type experiment. You need to add in several chromatographic steps and then resort to GC-MS (headspace for volatiles), after appropriate derivatisation, and to LC-MS. The spectra can be searched against various libraries. Even then you will be left with many unknowns.

It is useful to include the information about water content in fresh weight, so one can see how much the measured substances were concentrated by drying and whether they were destroyed by this procedure.

What are the phytochemicals?

Phenolic compounds have a wide range of polarities. Some are more polar and would not be well solubilized in non-polar hexane. Flavonoids tend to be more non-polar, so they can dissolve better in hexane. The more polar phenolics are likely to dissolve better in your other more polar solvents (ethyl acetate, methanol, and water). It is also possible that something in the hexane extract is interfering with the chemistry of the assay you are using to detect total phenols. Flavonoids use a different detection method, so they are unaffected. You may need to try a different phenol quantification method that is less prone to interference.

It depends on the type of your study and the factors under consideration. Usually, the best time to study for a primary response is 24 to 72 hours after using the phytohormone. However, if you want to examine secondary responses and factors related to biochemical responses, such as antioxidant enzyme activity, it is better to do it by 7 days. Also, if you intend to investigate the effect of phytohormone on plant stress and investigate their effects, it is better to evaluate the target factor in different organs of the plant within 14 to 21 days after the application of phytohormone. Of course, keep in mind that in the third case, you must repeat the phytohormone treatment every 5-7 days.

If the Rf value is similar, If the normal phase column is used, try to use the reverse phase for separation. If it is the reverse phase, use a column with a small particle size and high column efficiency for separation.

If the molecular weight difference is large, it can be filtered by gel, but the molecular weight difference should be more than 2 times.

For those with charges, we can try using ion exchange.

If there is a specific adsorption phenomenon, Protein A can be used.

Carbon isotopes or deuterated version of any phytochemical that is amenable to analysis using GC is applicable. You should consider both the extraction efficiency and detection capability of your selected internal prior to metabolomic analysis.

Before a convenient suggestion, we should know your extraction protocol/methodology, applied derivatization or not, and the target subset of the phytochemical (e.g. alkaloids, phenolics/flavonoids, tannins, saponins, terpenoids, sterols, organosulfur compounds, etc......)

I suggest several IS (3-4) holding varying polarities and belonging to different compound classes instead of a single one to represent comprehensive metabolome coverage...

Having tested a lot of essential oils for this specific purpose, I can ensure you that different essential oils give very different results. There is also a possibility that fumigation doesn't kill the adults but stops the developements of the eggs inside the grains.

Way of applying the product (fumigation, direct spraying...) also gives different results, so there is no easy direct answer to your question

It is possible in such case one should repeat the process for final confirmation .

DOI: 10.2174/1573401314666181015111724

Regards

which type of you are using mobile phase polar or non polar solvent. you can use MEG (Mono Ethylene Glycol) 50% mix with water 50%.

Linalool is a colourless or pale yellow liquid, and I think the reddish brown precipitation could be as a result of oxidation/reduction by the sulphuric acid presence

PubMed ID 30216794

see

1-High Performance Liquid Chromatography in Phytochemical Analysis

CRC Press

Joseph Sherma

Year:

2010

2-Biotechnological Advances, Phytochemical Analysis and Ethnomedical Implications of Sapindus species

Springer Singapore

Reetika Singh, Bechan Sharma

Year:

2019.

Good Luck

What do you mean by qualitative,

Check https://www.sciencedirect.com/science/article/pii/S1878535213000476

Also check https://www.sciencedirect.com/science/article/pii/S1011134414003728

If we don't dry our essential oil by Na2SO4 then it may slowly hydrolyzed by water slowly on storage.

It must be considered that the hydrodistillation extractions are quite long and at a temperature that allows us to evaporate the part we want to separate, so it is very likely that the other compounds of interest are also in the plant have been degraded. To know if there are compounds of interest in that waste you must analyze it.

Drying of the solvent should be careful as the compounds under investigation may degrade in the temperature we set. If you are studying about phenol (b.p = 181.7 degree) or falvanoid (b.p = 273 degree) then I believe it won't harm much if the extract dissolved in Methanol (b.p = 64.7 degree) is dried at 40 degree or even closer to 60 degree. But for investigating the presence of any novel compounds, drying either by rotary evaporator or at room temperature would be the best choice.

Your 'V' value will be 25 ml. Thanks

Check https://onlinelibrary.wiley.com/doi/pdf/10.1111/1541-4337.12006

your experiment results will answer your question. to get better results you should use a specific plant that used in folk medicine for the treatment of a specific disease, then you can make your study on it

Dear Ali Vafaei

The work of Manish Rai, K. N. Ramachandran and V. K. Gupta* contains good information. You can read (attached)

Ladies and Gentlemen

Solvent Extracts are never monomolecular pure, except if the solvents are used for crystal-growth (takes a lot of time, and many bio-molecule can’t be crystalized. However, if you are concerned about pure compounds e.g. for serious research results or for commercialization of compounds you must use Chromatography preferably HPLC or SFC

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you can use LC Mass for qualitative and quantitative measurement.

if your only choice is HPLC, you can use standards.

also, the following papers can help u

Perfumers World in Bangkok.

Just go to

Elsevier journal finder. Then, type your paper tittle and paper abstract. You could find huge options.

Adam B Shapiro thank you very much for your reply and the link.

Phytochemical screening may be carried out by using standard procedure as suggested by Brindha et.al

DearThe mobile phase is determined by the components to be examined The mobile phase of phenols is different, for example, from alkaloids and fats This is why sometimes several mobile phases of solvents are used with different volumetric ratios and placed in small containers, in principle, to examine small sheets of TLC ..... to obtain a clear separation of the compounds to be separated.

best regards

Gunawan Indrayanto Shilpa Varma Mini Shobi O. Aldulaimi Henry Bachmann Jack Silver

According your goal of study in my work some phytochemical were detected from part of plant freashly collected in methanol then GC. while other required fresh or dry grinding material followed by organic or aqeous extraction then phytochemical detection according to phytocemicals

Cinnamon is used for many purposes (1). There is no single solvent or extraction procedure that is "best" for all of them. Which indication interests you? There will probably be a class of compounds that can use an optimized extraction.

(1)

Test the raw extract on cancer cell lines in vitro and chemically induced cancer in vivo, if the extract. If it showed anticancer effect, try to separate and purificate the effective ingredients and test them separately. Determine the LD50 of the effective compound in two animal species. Perform preclinical study. Then clinical study

To be used for in vivo (e.g Wistar rats), What is the percentage of minimum DMSO that can be use to dissolve thymoquinone?

@Teferi Damto

You can use UV-Vis spectrophotometer only to quantify the phytochemicals. You cannot use it for qualitative analysis.

Yes

It depends on the compounds you are studying. Acetone will dissolve less polar compounds better than methanol. Conversely, you may not see as many polar compounds.

I see GC is being used for chromatography, suggesting more volatile, and possibly less polar, compounds are being analyzed so acetone may work fine.

The best thing to do is to compare the extraction from a homogeneous sample divided into two portions. One is extracted by methanol, and the other with acetone. You can then see if acetone is a useful substitute.

In Salkowski reaction red color is due to formation of bi-sulphonic acid of bi-cholestadiene.In this reaction concentrate sulphuric acid removes two molecules of water from two molecules of cholesterol and bicholestadien is formed. Sulphuric acid sulphonates this so due to formation of bi- sulphonic acid of bi-cholestadiene red color is produced,if cholesterol is present.

thanks to all

Dear Carlos,

Stanley Benedict was an American chemist that discovered a solution to detect the presence of reducing sugar. This reagent (Benedict's reagent) is a complex mixture of sodium carbonate, sodium citrate, and Copper (2) Sulfate. I attached Benedict's original paper for your reference.

Another method for this purpose is the Nelson-Somogyi method that is performing by a mixture of sodium sulfate, potassium sodium tartrate, sodium carbonate, copper (2) sulfate, and sodium bicarbonate that I attached Michal Kaczmarek's handout for you.

Use of dinitrosalicylic acid reagent in another method for determination of reducing sugar that I shared the protocol of this method with you.

I hope that the papers will be useful to you.

Kind regards, Homayoon.

It is not right. Hexane, gasoline extraction and benzene are used to separate a specific class of compounds. I use the listed solvents to isolate and separate terpenoids as system

The most good HPLC method If you have a standard, the most classical method is extraction, fractionation and chromatography, there is a chance to find a new natural compound

The concentration is selected by the target of which compound class you are isolating. For example, for mono and sesquiterpenoids 96% ethanol, for flavonoids 40% and 70% ethanol for alkaloids, 80% ethanol is ideal.

if you have another reagent such as plumbum acetate or aluminum chloride for spectrophotometric analysis of flavonoid determination

Dear Abbasi,

In addition answer above mentioned by Dadwal, you can use it to evaporate solvent at low temperature protecting thermal unstable compound. However, If you want evaporate water using a rotary evaporation, you have to cool the solvent collecting vessels (using Ice) while maintaining, temperature of plant extract around 30-40 degrees of Celsius (but you have to be careful about the temperature). Since water is a polar solvent, it will take relative longer time to evaporate it

However if, your target compounds are highly volatile , you have to think about other extraction method.

Piyal Aravinna

Hello, You can use from Subcritical Water or Super Critical CO2.

You can apply the threshold proposed by Suffness & Pezzuto. It stablishes that crude extracts showing an GI50 ≤ 100 mg/mL can be considered to be cytotoxic and selected for further studies, whereas the most promising ones are those with an GI50 lower than 30 mg/mL.

Cite it as :

M. Suffness and J. Pezzuto, Eds., Assays related to cancer drug discovery. London: Academic Press, 1999.

NOW A PERSONAL REFLECTION ON NATURAL PRODUCTS SUCH EXTRACTS:

This threshold is just an arbitrary convention, and still some non promising extracts may contain extraordinary compounds just in too low concentration to be seen. Disguising these numbers with physiological considerations makes it nice, but these are huge approximations and there are too many unknowns on ADME parameters.And for natural products such as extracts, 20 or 30 are the same order of magnitude to me.

I prefer to find out if the 50% effect is roughly above or below fixed conc such as 100, 50, 25 or 12.5. The linear trends in most studies are just horrible as complex natural products rarely fit to any line due to too many compounds acting independently (or in combination) ... there is people that finds pleasure in reporting an IC50 down to two decimals believing it makes them better scientists and the paper more credible... I believe that it is rather the contrary ...

I will refer you to the work of ANKIT GUPTA, MADHU NARANIWAL & VIJAY KOTHARI titled "MODERN EXTRACTION METHODS FOR PREPARATION OF BIOACTIVE PLANT EXTRACTS"

International Journal of Applied and

Natural Sciences (IJANS)

Vol.1, Issue 1

use any of the conventional methods as stated.

or the work of Odey et al. (2012) on Preparation of Plant Extracts from Indigenous Medicinal Plants.

International Journal of Science and Technology, 1(12); 2012

Hello Neelesh,

The suitable method for phytochemical analysis depends on the compound nature. The phytochemical analysis classified in two way such as qualitative and quantitative phytochemical analysis. In qualitative phytochemical analysis were used for screening sample to check for the presence of Alkaloids, Carbohydrates, Cardiac glycosides, Flavonoids, Phenols, Saponins, Tannins, Terpenoids, Quinones, and Proteins.

On the other hand, quantitative phytochemical analysis done by Gas Chromatography-Mass Spectroscopy (GC-MS), High-Performance Liquid Chromatography (HPLC) and High-Performance Thin Layer Chromatography (HPTLC). GC-MS applied to determine solid, liquid and gaseous samples. Desire samples first converted into gaseous state then analysis carried out on the basis of mass to charge ratio. HPLC suitable for the compounds soluble in solvents. HPTLC applicable for the separation, detection, and quantitative analysis of phytochemicals.

Most often spectroscopy is used in the detection of phytochemicals. UV Spectroscopy (UV), IR Spectroscopy (IR), Mass Spectroscopy (MS), Nuclear Magnetic Resonance Spectroscopy: (NMR) and X-Ray Crystallography are frequently used in the study of phytochemicals. UV applied to check whether the system is conjugated or not. IR commonly identify the functional groups that are present in the compound. MS determines the molecular weight of the compound and to identify the presence of isotopes patterns. NMR identify how many types of atoms presents in the compound.

you could use https://secure.megazyme.com/files/Booklet/K-PHYT_DATA.pdf

Dear Sir. Concerning your issue about the images of phytochemical screening bioactive compounds in nigella sativa cuminum cyminum and carum carvi seeds. The detection of the bioactive Black cumin which is one of the miraculous plant having multifarious roles in its phytochemical constituents and nutritional values, treating digestive tract conditions including gas, colic, diarrhea, dysentery, constipation, and hemorrhoids. Sopowdered Nigella sativa seed was used for crude oil extracts by using different solvents. In this manner, the results of investigation of qualitative phytochemical analysis conducted on thecrude cumin seeds extract revealed the presence of bioactive compounds in the petroleum ether, ethyl acetate and methanol extracts which areknown to exhibit medicinal as well as physiological activities. Identification and separations were taken by TLC and CC. Finally four potentially active phytochemicals have been obtained from methanol extracts; are alkaloids, phenol, flavonoids and steroids. I think the following below links may help you in your analysis:

http://shodhganga.inflibnet.ac.in/bitstream/10603/27678/8/08_chapter%202.pdf

http://ijpsr.com/bft-article/screening-of-phenolic-content-antioxidant-and-in-vitro-eye-irritation-activities-from-apiaceae-family-dry-seeds-for-potential-cosmetic-applications/?view=fulltext

Thanks

Thank you all of you

Dear Sir. Concerning your issue about the calculation for the determination of the total phenolic content (TPC) in Plant Extract.

First you need to use equation y=0.0109x- 0.022 to calculate the value of x

Where y = Absorbance

x = concentration from the calibration curve

This implies that x = 0.541 + 0.022/0.0109

x = 51.65 mg/mL

Therefore, TPC can be calculated as follows

TPC = cV/m

Where c= concentration from calibration curve

V= Volume of the extract used

m= Mass of the extract used

Therefore, TPC = cV/m

TPC = 51.65x50/5.0

TPC = 516.5 mg /g

May I know the type of standard and its concentration. I think the following below links may help you in your analysis:

Thanks



You can ask researchers working on soluble CD83. Here is the link for one of their publications. I hope it helps. Good Luck!

Yes, Sunlight / Moonlight spectrum affects the % of Phyto Chemicals in plants. The harvesting of Turmeric is done in Night then the % of an important active Curcumin is observed more, hence the Turmeric is also called Rajani=Night in Sanskrit.

You can find an answer in this article. 

Good Luck

Thank u sirs. Finally I reached the institution which is doing research in malaria. Thank u all for the answers and guidance..

Your question makes no sense if we don't know the purpose of the analysis.  Are you looking to analyse for a particular target compound or class of compounds? Are these compounds you are analysing for stable during the drying process? How are you preparing the dried plant material - frozen rapidly in liquid nitrogen and freeze dried?

You can use DPPH assay and MTT assay

from lagerstroemia speciosa. My goal is to compare the glucose uptake modulatory effects between the extract, the tannins, and the mixture of hydrolyzed products.

I recommend Rahul comment. Good luck

In case of herbal extraction it is very much essential to know about the actual amount of an active ingredient present in the raw material. For this purpose soxhlet extraction plays vital role to find out actual amount since the raw material is being continuously heated at the boiling point of solvent for longer period of time which gives highest extraction among all techniques of extraction.  

Even if you used the same solvent, there is a good chance that your mobility (migration) values will be slightly different from those published by someone else.  Variations in temperature, thickness of the plate coating, etc., can all influence mobility on TLC. That is why the recommended procedure is to compare mobility with a standard compound. With carbohydrates, we often use glucose, and report mobility as Rglc, that is, mobility relative to glucose.  Even more useful would be comparing your spots to those of authentic compound, if you can get some from the authors of the paper. With these things considered, then slight variations, or even major changes, in solvent conditions will not matter as much.

No if the calibration curve was constructed considering 1600 uL of standard. 

To convert:   25 mg/L * 0.005 L / 0.5 g

In my study I want to measure free phosphate (orthophosphate) in leaf only, therefore I do not perform ashing step, and I also cannot use any extracting solvent that may hydrolize organic phosphate compounds. Free phosphate level in tomato leaf, according to Bozzo et al. (2006), is only around 10 ppm FW (probably around 100 ppm DW), so I think molypdenum blue method which has higher sensitivity would be better.

Dear Salim

in any case you should try to define is the insoluble you got are organic or inorganic substance. Some time during the concentration process some interesting compounds could be isolated.

by other side you should follow the advices to the former researchers. Good luck.

I agree with Javad, you don't need to prepare standard in different solvents, to do it , i. e. in methanol is ok. Good luck. 

Yes, I will collect different leaves each month from the same trees (edit: I have 12 trees). Leaf samples will be used to look for changes in foliar compounds as the plant ages.

Dear Anil

as Vishwanath said, at first you have to know some chemical reference about your plant or the genus it belongs to have some idea about the metabolites they probably they have. Other way is to make a qualitative analysis to determine that. According that use the common standard , i. e. Quercetine for flavonoids, sitosterol for steroids and so on. Good luck

Hi, lyophilisation is the same as freeze drying. Freeze drying is really simple, and the vaccuum pressure and the temperature is not so majorly important if you only want to dry your sample. Put your liquid into a container with a lid and punch some holes into the lid. Through these holes the water will then "freeze dry" and your sample will remain inside. When you turn on the vaccuum of your freeze dryer, increase the vaccuum slowly, otherwise your sample "bubbles up" i.e. dries too fast and boils under the vaccuum. You will see from the outside if your sample has dried overnight or whether it needs longer. Once it is dry you can grind it up to a fine powder. You will see, it is simple! Greetings, Brig