Citation
Khaoula, Hellal
(2021)
Metabolite profiling and characterization of Lupinus albus L. and its correlation with antioxidant and alpha-glucosidase inhibitory activities.
Doctoral thesis, Universiti Putra Malaysia.
Abstract
Diabetes mellitus (DM) is the most widely recognized endocrine disorder in almost all
countries. Due to the adverse effects of modern treatment such as hypoglycemic coma,
renal complications, stomach upset, and weight gain, the use of medicinal plants as
antidiabetic agents has been gaining increasing attention due to their effectiveness. The
main objective of this study was to profile and characterize the metabolites presence in
the Algerian medicinal food plants that are traditionally used for the treatment of
hyperglycemia (Peganum harmala, Zygophyllum album, Anacyclus valentinus,
Ammodaucus leucotrichus, Lupinus albus, and Marrubium vulgare). To achieve that
aim, this study was carried out in several phases. In the first phase, we aimed to screen the DPPH free radical scavenging, α-glucosidase and nitric oxide inhibitory activities as
well as total phenolic content (TPC) of the six Algerian medicinal food plants extracted
using different percentage of ethanol (0, 50, 80, and 100%). The findings revealed that the highest TPC of 612.84µg GAE/mg extract was observed in M. vulgare extracted
with 80% ethanol. The 100% ethanol extract of A. leucotrichus exhibited the highest free
radical scavenging activity with an IC50 value of 26.26 µg/mL. However, the 100%
ethanol extract of L. albus seeds was found to be the most potent in in habiting α-glucosidase activity with the lowest IC50 value of 6.45µg/ml. Therefore, it was the best
candidate for further investigation in the next phase where the metabolite profiling was
conducted using proton nuclear magnetic resonance (1H NMR) spectroscopy. A partial
least square analysis (PLS) was used to assess the relationship between that α -glucosidase
inhibitory activity of L. albus and the metabolites identified in the extract. Based on the
PLS model, isoflavonoids, amino acids, and several fatty acids were identified as metabolites that contributed to the inhibition of α -glucosidase. The PLS model exhibited
a perfect goodness of fit where (R2Y (cum) > 0.9) and predictive quality (Q2(cum) >
0.9). For further investigation, the metabolite profile was carried out by using ultra-highperformance
liquid chromatography-electrospray ionization-tandem mass spectrometry
(UHPLC-ESI-MS/MS) technique in both negative and positive ion mode. A total of 45
metabolites were tentatively identified by means of MS data, together with the interpretation of the observed MS/MS spectra in comparison with those found in the
literature. Consequently, in the next part of the study, the 100% ethanolic extract of L.
albus seeds was fractionated with different solvent polarity using solid phase extraction
(SPE) and the DPPH free radical scavenging, and α-glucosidase inhibitory activities of
the resulted fractions were evaluated. This phase provides useful information for the
identification of compounds in L. albus seeds fractions by NMR based metabolomics.
In total, 35 metabolites were identified from L. albus seeds fractions. Principal
component analysis (PCA) is used as a multivariate projection method for visualizing
the different composition of four different fractions. Among the fractions studied, the
chloroform fraction (CF) exhibits a high free radical scavenging (DPPH) and α-glucosidase inhibitory activities with IC50 values of 4.08 and 20.08 µg/mL, respectively. A PLS model has been successfully performed to correlate the potential active metabolites with the corresponding biological activities. Metabolites such as
proline, caprate, asparagine, lupinoisolone C, hydroxyiso-lupalbigenin and some
unknown compounds showed high correlation with the bioactivities studied. Autofit of
SIMCA treatment exhibited a perfect goodness of fit with R2Y (cum) > 0.9 and a
predictive quality of Q2 (cum) > 0.9. Moreover, a total of 25 metabolites were tentatively
identified from CF using UHPLC-ESI-MS/MS analysis by comparison with previously
reported data. The last phase of this study has been undertaken with the aim to isolate
and characterize active metabolites from CF through chromatography techniques. Five
compounds including, triolein (2), lupeol (3) β -sitosterol (4), betulinic acid (5), and
ursolic acid 13) were isolated from CF. All compounds found to have both α - glucosidase inhibitory and DPPH free radical scavenging activities. Triolein (2) was found to possess the highest α-glucosidase inhibition activity with IC50 value of 3.32 µg/mL
, while ursolic acid (13) exhibited the strongest DPPH free radical scavenging
inhibitor with IC50 value of 13.97 µg/mL
In summary, the findings of this research suggest that L. albus seeds extracts exert antidiabetic activity through their effects as antioxidant and α- glucosidase inhibitors. Triolein, lupeol, β-sitosterol, betulinic acid,
and ursolic acid can be contributed to the antidiabetic activity exhibited by L. albus seeds
extracts. The outcomes obtained endorse the claims that L. albus seeds extract can be
beneficial in the prevention and treatment of diabetes and its associated disorders. To the
best of our knowledge, this fingerprint profile is the first extensive study of metabolites
from L. albus seeds extract fractions, which may explain the use of L. albus as
antidiabetic agent in the Algerian folk medicine and its application in future
phytomedicinal preparations for the treatment of diabetes.
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