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. 2013 Feb 1;18(2):1916-32.
doi: 10.3390/molecules18021916.

Anti-oxidative and Cholinesterase Inhibitory Effects of Leaf Extracts and Their Isolated Compounds From Two Closely Related Croton Species

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Anti-oxidative and Cholinesterase Inhibitory Effects of Leaf Extracts and Their Isolated Compounds From Two Closely Related Croton Species

Ashwell R Ndhlala et al. Molecules. .
Free PMC article


A comparative evaluation of the antioxidant and acetylcholinesterase inhibitory activity of the leaf extracts of Croton gratissimus and Croton zambesicus (subgratissimus) and compounds isolated from the extracts was carried out to determine their potential and suitability or otherwise as a substitute for each other in the management of oxidative and neurodegenerative conditions. Different antioxidant assays (DPPH, FRAP, β-carotene-linoleic and the lipid peroxidation models) and the microplate assay for acetylcholinesterase (AChE) inhibition were carried out separately to study the activities of the crude leaf extracts and four solvent fractions from each of the two Croton species. Bioassay guided fractionation was used to target antioxidant constituents of the crude extracts and ethyl acetate fractions of 20% aqueous methanol extract of C. gratissimus on silica gel and Sephadex LH-20 columns resulted in the isolation of kaempferol-3-O-β-6''(p-coumaroyl) glucopyranoside (tiliroside, 2), apigenin-6-C-glucoside (isovitexin, 3) and kampferol (4). The extract of C. zambesicus yielded quercetin-3-O-β-6''(p-coumaroyl) glucopyranoside-3'-methyl ether (helichrysoside- 3'-methyl ether, 1), kaempferol-3-O-β-6''(p-coumaroyl) glucopyranoside (tiliroside, 2) and apigenin-6-C-glucoside (isovitexin, 3). Three of the isolated compounds and their different combinations were also included in the bioassays. In all the assays performed, the antioxidant capacity and AChE inhibitory effects of C. zambesicus extracts were weaker than those of C. gratissimus. This suggests that C. gratissimus may not be substituted by C. zambesicus, despite the similarity in some of their constituents. Generally, the combinations made from the isolated compounds showed better activities in most of the assays compared to the individual isolated compounds. This suggests mechanisms such as synergism and/or additive effects to be taking place. This study established low, moderate and high antioxidant activities as well as AChE inhibitory effects by the crude extracts, fractions, compounds and compound combinations. This means some of the extracts, isolated compounds and compound combinations could be useful in the management of neurodegenerative conditions and serve as sources of natural neurodegenerative agents.


Figure 1
Figure 1
Compounds isolated from Croton gratissimus and Croton zambesicus. Quercetin-3-O-β-6’’(p-coumaroyl) glucopyranoside-3’-methyl ether (helichrysoside-3’-methyl ether, 1), kaempferol-3-O-β-6’’(p-coumaroyl) glucopyranoside (tiliroside, 2), apigenin-6-C-glucoside (isovitexin, 3) and kampferol (4).
Figure 2
Figure 2
Ferric reducing activity of the compounds, crude extracts and fractions from C. gratissimus and C. zambesicus. (A); Helichrysoside-3’-methyl ether (1), tiliroside (2), isovitexin (3), combination 1 (helichrysoside-3’-methyl ether + tiliroside + isovitexin), combination 2 (helichrysoside-3’-methyl ether + tiliroside), combination 3 (helichrysoside-3’-methyl ether + isovitexin), combination 4 (tiliroside + isovitexin). (B); Crude extracts and fractions. Increase in absorbance of the reaction mixture indicates the increase in reducing power. Values represent mean ± standard error (n = 3).
Figure 3
Figure 3
Malondialdehyde (MDA) equivalents (nmol/mL) recovered from the peroxidation of phospholipid in the presence of test samples. Isolated compounds were tested at 350 µg/mL while the crude extracts, fractions and ascorbic acid were tested at 3.5 mg/mL. Helichrysoside-3’-methyl ether (1), tiliroside (2), isovitexin (3), combination 1 (helichrysoside-3’-methyl ether + tiliroside + isovitexin) (4), combination 2 (helichrysoside-3’-methyl ether + tiliroside) (5), combination 3 (helichrysoside-3’-methyl ether + isovitexin) (6), combination 4 (tiliroside + isovitexin) (7), C. gratissimus crude 20% methanol extract (8), C. gratissimus hexane fraction (9), C. gratissimus DCM fraction (10), C. gratissimus ethyl acetate fraction (11), C. gratissimus butanol fraction (12), C. zambesicus crude 20% methanol extract (13), C. zambesicus hexane fraction (14), C. zambesicus DCM fraction (15), C. zambesicus ethyl acetate fraction (16), C. zambesicus butanol fraction (17), ascorbic acid (positive control) (18) and water (negative control) (19). Lower amounts of MDA, signifies higher protective ability of extract against lipid peroxidation. Values represent mean ± standard error (n = 2).

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