Hello!
I am looking for an isolation and purification protocol for 10-HDA (Royal Jelly’s main fatty acid). I have searched the internet, but did not find any possible method to extract this bioactive compound and further use it in various experiments, for example on cell cultures. I only found methods on how to determine the quantity of 10-HDA but I don’t want to determine it for this particular experiment I want to do.
I came up with an idea for 10-HDA extraction from RJ, but I’m not sure if it would work:
1. Extraction of total lipids from RJ using the Soxhlet extractor.
2. Separation of the (total) RJ lipids using electrophoresis.
3. Obtaining 10-HDA (isolation from total lipids).
4. Purification of the obtained 10-HDA (and lyophilization for better preservation).
5. Use of the fatty acid in experiments.
Does anyone know if this idea would work and how I could practically apply the idea in the lab? What reactives and equipment are needed to fulfill my goal? Any suggestions on how to do this extraction and obtain postive results?
Thank you!
Extracting and purifying 10-HDA from Royal Jelly can be challenging as it is present in small quantities and can easily degrade during extraction and purification steps. Here's a protocol that can be used to extract and purify 10-HDA from Royal Jelly:
Materials required:
- Royal Jelly
- Chloroform
- Methanol
- Hexane
- Sodium hydroxide
- Hydrochloric acid
- Silica gel
- TLC plates
- Column chromatography equipment
- Rotary evaporator
- HPLC system
Procedure:
1. Collect fresh Royal Jelly and store it at -80°C until use.
2. Thaw the Royal Jelly at room temperature and weigh the desired amount of sample.
3. Add chloroform to the Royal Jelly at a ratio of 1:10 (w/v) and stir the mixture for 30 minutes.
4. Filter the mixture using filter paper and collect the filtrate in a round bottom flask.
5. Add methanol to the filtrate at a ratio of 1:5 (v/v) and stir for 30 minutes to obtain a two-phase system.
6. Separate the two phases and collect the upper phase containing the lipid fraction.
7. Add hexane to the upper phase at a ratio of 1:5 (v/v) and stir for 30 minutes to obtain a two-phase system.
8. Collect the upper phase containing the free fatty acids, including 10-HDA.
9. Neutralize the upper phase using 0.1 M sodium hydroxide solution.
10. Acidify the neutralized solution using 1 M hydrochloric acid solution to pH 3-4.
11. Extract the acidified solution with chloroform to remove impurities.
12. Dry the chloroform layer using anhydrous sodium sulfate and evaporate the solvent using a rotary evaporator.
13. Dissolve the residue in a minimum amount of chloroform and apply the sample on a silica gel column.
14. Elute the column using a stepwise gradient of hexane and ethyl acetate (e.g., 100% hexane, 95:5 hexane/ethyl acetate, 90:10 hexane/ethyl acetate, etc.).
15. Collect the fractions and analyze them using thin-layer chromatography (TLC).
16. Combine the fractions containing 10-HDA and concentrate them using a rotary evaporator.
17. Purify the concentrate using an HPLC system equipped with a C18 column and a UV detector (280 nm).
18. Collect the purified 10-HDA and confirm its purity using mass spectrometry and/or nuclear magnetic resonance (NMR) spectroscopy.
19. Lyophilize the purified 10-HDA and store it at -80°C until further use.
Overall, this protocol involves extracting the lipid fraction from Royal Jelly, isolating the free fatty acids using solvent extraction, and purifying 10-HDA using chromatographic techniques. However, the success of this protocol depends on various factors such as the quality and quantity of Royal Jelly, the choice of solvents and chromatographic conditions, and the expertise of the researcher. Therefore, it is important to optimize the protocol for each specific case to obtain the best results.
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