Distinctive bioactivities possessed by luteolin (3', 4', 5, 7-tetrahydroxy-flavone) are advantageous for sundry practical applications. This paper reports the in vitro selection and characterization of single stranded-DNA (ssDNA) aptamers, specific for luteolin (LUT). 76-mer library containing 1015 randomized ssDNA were screened via systematic evolution of ligands by exponential enrichment (SELEX). The recovered ssDNA pool from the 8th round was amplified with unlabeled primers and cloned into PSTBlue-1 vector prior to sequencing. 22 of LUT-binding aptamer variants were further classified into one of the seven groups based on their N40 random sequence regions, wherein one representative from each group was characterized. The dissociation constant of aptamers designated as LUT#28, LUT#20 and LUT#3 was discerned to be 107, 214 and 109 nM, respectively with high binding affinity towards LUT. Prediction analysis of the secondary structure suggested discrete features with typical loop and stem motifs. Furthermore, LUT#3 displayed higher specificity with insignificant binding toward kaempferol and quercetin despite its structural and functional similarity compared to LUT#28 and LUT#20. Further LUT#3 can detect free luteolin within 0.2-1 mM in solution. It was suggested that LUT#3 aptamer were the most suitable for LUT recognition tool at laboratory scale based on the condition tested.
The palm oil industry generates several byproducts, and more than half of the dry weight of the waste is of oil palm leaf whereby the tissue is underutilized. Recently, several research studies found promising potential of oil palm fronds as a source of nutraceutical due to its bioactive properties. However, the chemical composition of the tissue is still not deciphered. Using reversed-phase liquid chromatography (LC) electrospray mass spectrometry (ESI-MS), glycosylated apigenin and luteolin were separated and identified from oil palm (Elaeis guineensis Jacq.) leaf and structures of the constituents were elucidated by collision-induced dissociation (CID) tandem MS. From 28 derivatives of the flavones, 9 compounds were conjugated with hydroxymethylglutaric (HMG) acid. Improved knowledge on oil palm especially on bioactive component of the leaf tissue will allow correlation of its beneficial effects and further promotes efficient utilization of this agriculture byproduct.
Luteolin and apigenin derivatives present in oil palm (Elaeis guineensis) leaves (OPL) are reported to possess excellent antioxidant properties relating to numerous health benefits. To meet the global demand for flavonoids, OPL, which is plentifully generated as an agricultural by-product from oil palm plantations, can be further exploited as a new source of natural antioxidant compounds. However, to produce a standardized herbal preparation, validation of the quantification method for these compounds is required. Therefore, in this investigation, we developed and validated an improved and rapid analytical method, ultra-high-performance liquid chromatography equipped with ultraviolet/photodiode array (UHPLC-UV/PDA) for the quantification of 12 luteolin and apigenin derivatives, particularly focusing on flavonoid isomeric pairs: orientin/isoorientin and vitexin/isovitexin, present in various OPL extracts. Several validation parameters were assessed, resulting in the UHPLC-UV/PDA technique offering good specificity, linearity, accuracy, precision, and robustness, where the values were within acceptable limits. Subsequently, the validated method was employed to quantify luteolin and apigenin derivatives from OPL subjected to different drying treatments and extraction with various solvent systems, giving total luteolin (TLC) and apigenin content (TAC) in the range of 2.04-56.30 and 1.84-160.38 µg/mg extract, respectively. Additionally, partial least square (PLS) analysis disclosed the combination of freeze dry-aqueous methanol yielded OPL extracts with high TLC and TAC, which are strongly correlated with antioxidant activity. Therefore, we provide the first validation report of the UHPLC-UV/PDA method for quantification of luteolin and apigenin derivatives present in various OPL extracts, suggesting that this approach could be employed in standardized herbal preparations by adopting orientin, isoorientin, vitexin, and isovitexin as chemical markers.
Background: In silico characterization can help to explain the interaction between molecules and predict three-dimensional structures. Various studies have confirmed the glucose-lowering effects of plant extracts, ie, lupeol and iso-orientin, which enable them to be used as antidiabetic agents. Purpose: Aims of the present study were to evaluate the hypoglycemic activities of lupeol and iso-orientin in a rat model. The study proposed the effects of alloxan on blood glucose level, body weight, and oxidative stress. Materials and Methods: Thirty (n=30) Wistar albino rats were divided into six groups and were subjected to different combinations of the compounds. Levels of different stress markers, ie, malondialdehyde, superoxide dismutase, catalase, nitric oxide, glutathione, glutathione peroxide, glutathione reductase, and blood glucose levels were estimated with their respective methods. Whereas, for their in silico analysis, identified target proteins, GPR40, glucose-6-phosphatase, UCP2, glycogen phosphorylase, aldose reductase, and glucose transporter-4 were docked with lupeol and iso-orientin. Three-dimensional structures were predicted by ERRAT, Rampage, Verify3D, threading and homology approaches. Results: Blood glucose levels were significantly increased in rats receiving intraperitoneal injection of alloxan (208±6.94 mg/dL) as compared to controls (90±7.38 mg/dL). Infected rats were administered plant extracts; combined treatment of both extracts (lupeol+iso-orientin) significantly reduced the levels of blood glucose (129.06±6.29 mg/dL) and improved the antioxidant status. Fifteen structures of each selected protein were evaluated using various techniques. Consequently, satisfactory quality factors [GPR40 (96.41%), glucose-6-phosphatase (96.56%), UCP2 (72.56%), glycogen phosphorylase (87.24%), aldose reductase (82.46%), and glucose transporter-4 (94.29%)] were selected. Molecular docking revealed interacting residues, effective drug properties and their binding affinities (ie, -8.9 to -12.6 Kcal/mol). Conclusion: Results of the study affirmed the antidiabetic activities of lupeol and iso-orientin. Administration of these extracts (either individually or in combination) significantly reduced blood glucose levels and oxidative stress. Hence, it may be considered beneficial in the treatment of diabetes.
Clinacanthus nutans (family Acanthaceae) has been used for the treatment of inflammation and herpes viral infection. Currently, there has not been any report on the qualitative and quantitative determination of the chemical markers in the leaves of C. nutans. The C-glycosidic flavones such as shaftoside, isoorientin, orientin, isovitexin, and vitexin have been found to be major flavonoids in the leaves of this plant. Therefore, we had developed a two-step method using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC) for the rapid identification and quantification of the flavones C-glycosides in C. nutans leaves. The TLC separation of the chemical markers was achieved on silica gel 60 plate using ethyl acetate : formic acid : acetic acid : water (100 : 11 : 11 : 27 v/v/v/v) as the mobile phase. HPLC method was optimized and validated for the quantification of shaftoside, orientin, isovitexin, and vitexin and was shown to be linear in concentration range tested (0.4-200 μg/mL, r(2) ≥ 0.996), precise (RSD ≤ 4.54%), and accurate (95-105%). The concentration of shaftoside, orientin, vitexin, and isovitexin in C. nutans leave samples was 2.55-17.43, 0.00-0.86, 0.00-2.01, and 0.00-0.91 mmol/g, respectively.
In the current study, changes in secondary metabolite synthesis and the pharmaceutical quality of sabah snake grass leaves and buds were considered in relation to plant age (1 month, 6 months, and 1 year old). The activity of the enzyme chalcone synthase (CHS, EC 2.3.1.74) was measured, as it is a key enzyme for flavonoid production. Significant differences in total flavonoid (TF) production were observed between the three plant growth periods and the different plant parts. The highest contents of TF (6.32 mg/g dry weight [DW]) and total phenolic (TP) (18.21 mg/g DW) were recorded in 6-month-old buds. Among the flavonoids isolated in this study the most important ones based on concentration were from high to low as follows: catechin > quercetin > kaempferol > luteolin. Production of phenolic acids increased from 1 to 6 months, but after 6 months up to 1 year of age, they decreased significantly. The highest contents of caffeic acid (0.307 mg/g DW) and gallic acid (5.96 mg/g DW) were recorded in 1-year and 6-month-old buds, respectively. The lowest and highest activity of CHS was recorded in 1-month and 6-month-old buds with values of 3.6 and 9.5 nkat/mg protein, respectively. These results indicate that the increment in flavonoids and phenolic acids in 6-month-old buds can be attributed to an increase in CHS activity. The highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) activity was observed in the extract of 1-year-old buds followed by 6-month-old buds, with 50% of free radical scavenging (IC50) values of 64.6 and 73.5 µg/mL, respectively. Interestingly, a ferric reducing antioxidant power (FRAP) assay showed a higher activity in 6-month-old buds (488 μM of Fe(II)/g) than in 1-year-old buds (453 μM of Fe(II)/g), in contrast to the DPPH result. Significant correlations (p < 0.05) were observed between CHS enzyme activity and FRAP activity, TF, catechin, and kaempferol content. Extracts of 6-month-old bud exhibited a significant in vitro anticancer activity against HeLa cancer cells with IC50 value of 56.8 µg/mL. These results indicate that early harvesting of snake grass (6-month-old) may yield increased concentrations of secondary metabolites, which are potent antioxidant compounds.