Extraction and measurement of total lipids in fresh royal jelly samples: Comparison of several methods

Authors: Zhang, Weiqiang; Zeigler, Amy; Tundo, Giovanni; Lau, Pierre; Zhu, Yu Cheng

Submitted to: American Chemical Society Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 5/17/2023
Publication Date: 9/13/2023
Citation: Zhang, W., Ray, A.Z., Tundo, G., Lau, P.W., Zhu, Y. 2023. Extraction and measurement of total lipids in fresh royal jelly samples: Comparison of several methods. American Chemical Society Abstracts. https://doi.org/10.1021/scimeetings.3c10168.
DOI: https://doi.org/10.1021/scimeetings.3c10168

Interpretive Summary: Royal Jelly (RJ) is a honey bee dietary secretion for developing bees and has exceptional biological properties used in many industries. Several methods have been reported to isolate lipids from RJ; however, no standardized method is available. This study investigated the isolation efficiency of several organic solvent combinations, including chloroform/methanol (1:1, 2:1, 1:2, volume/volume %) and diethyl ether followed by methanol, and measured the purity of resulting lipid fractions using Folch and sulfo-phospho-vanillin (SPV) methods. We also tested the possibility of using SPV to measure total lipids content in RJ directly. Our data showed that (1) while RJ samples were difficult to mix with diethyl ether, chloroform/methanol combinations mixed well with RJ. Therefore, chloroform/methanol combinations were further investigated. (2) Increasing methanol levels in chloroform/methanol combinations produced lipid fractions with higher yield but less purity. Total lipids levels were 11±0.65%, 16.8±0.19%, and 17.5±0.14% using chloroform/methanol 2:1, 1:1, and 1:2, respectively, and, by the inclusion of a purification step with water washing, the levels were 4.2±0.8% and 1.7±0.6% using chloroform/methanol 1:1 and 1:2, respectively. It was difficult to separate organic and aqueous layers when chloroform/methanol 2:1 was used. (3) SPV direct measurement of RJ using olive oil as the standard gave total lipids level of 1.42% (±0.21), which seemed to under-estimate total lipids levels and indicated a need to identify more suitable standards which closely mimic the lipid compositions of RJ. Our study could help prepare samples for lipidomics studies of RJ and other biological samples and develop simpler methods to determine total lipids content in biological samples.

Technical Abstract: Royal Jelly (RJ) is a honey bee dietary secretion for developing bees and has exceptional biological properties used in many industries. Several methods have been reported to extract total lipids from RJ; however, no standardized method is available. This study investigated the extraction efficiency of several solvent combinations, including chloroform/methanol (C/M: 1:1, 2:1, 1:2, v/v%) and diethyl ether followed by methanol, and measured the purity of resulting lipid fractions using Folch and sulfo-phospho-vanillin (SPV) methods. We also tested the possibility of using SPV to measure total lipids content in RJ directly. Our data showed that (1) RJ samples were difficult to mix with diethyl ether. C/M combinations mixed well with RJ. (2) Increasing methanol levels in C/M combinations produced lipid fractions with higher yield but less purity. Total lipids levels were 11±0.65%, 16.8±0.19%, and 17.5±0.14% using C/M 2:1, 1:1, and 1:2, respectively (n =3), and, by the inclusion of a purification step with water washing, the levels were 4.2±0.8% and 1.7±0.6% using C/M 1:1 and 1:2, respectively (n =3). It was difficult to separate layers when C/M 2:1 was used. (3) SPV direct measurement of RJ using olive oil as the standard gave total lipids level of 1.42% (±0.21, n=4), which seemed to under-estimate total lipids levels and indicated a need to identify more suitable standards which closely mimic the lipid compositions of RJ. Our study could help prepare samples for lipidomics studies of RJ and other biological samples and develop simpler methods to determine total lipids content in biological samples.