Antioxidant and Inhibitory Activities of Filipendula glaberrima Leaf Constituents against HMG-CoA Reductase and Macrophage Foam Cell Formation

In our search for bioactive components, various chromatographic separations of the organic fractions from Filipendula glaberrima leaves led to the isolation of a new ellagitannin and a triterpenoid, along with 26 known compounds. The structures of the isolates were determined based on their spectroscopic properties and chemical evidence, which were then evaluated for their antioxidant activities, inhibitory activities on 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and foam cell formation in THP-1 cells to prevent atherosclerosis. Rugosin B methyl ester (1) showed the best HMG-CoA reductase inhibition and significantly reduced ox-low-density lipoprotein-induced THP-1 macrophage-derived foam cell formation at 25 µM. In addition, no cytotoxicity was observed in THP-1 cells at 50 μg/mL of all extracts in the macrophage foam cell formation assay. Therefore, F. glaberrima extract containing 1 is promising in the development of dietary supplements due to its potential behavior as a novel source of nutrients for preventing and treating atherosclerosis.

Phytochemicals have attracted considerable attention as sources of functional ingredients in food formulations because of their numerous health benefits.In light of the growing demand for functional foods aimed at promoting a healthy lifestyle, there is a need for novel sources of raw materials that can offer consumers both desirable taste and beneficial functionality.The beneficial effects of these plant constituents have been partly linked to the abundance of various polyphenolic compounds, which exhibit antioxidant activity and/or free radical-scavenging properties in vitro [3].Polyphenols and tannins have gained significant attention due to their impact on the odor, flavor, and color of beverages and foods and health-promoting properties [4,5].The efficacy and safety of dietary supplements and herbal medicines sourced from purified natural compounds or extracted from edible plants have contributed to their widespread popularity [6].
Cholesterol is present in the human body and is an essential substance involved in various biochemical reactions as a component of the cell membrane.However, excess Molecules 2024, 29, 354 2 of 14 cholesterol may accumulate in vascular endothelial cells or the endothelium, causing vascular diseases, such as hyperlipidemia, and secondary diseases, such as arteriosclerosis, hypertension, obesity, and diabetes.Atherosclerosis is a chronic inflammatory process involving hypercholesterolemia, low-density lipoprotein (LDL) oxidation, hypertension, and platelet aggregation and is initiated by the accumulation of macrophage foam cells in the subendothelial arterial space [7,8].
Macrophages, integral cellular constituents of the host defense system, perform vital functions in both innate and adaptive immunity.Certain actions of macrophages can be beneficial, such as eliminating oxidized lipoproteins and facilitating the release of cholesterol derived from lipoproteins to high-density lipoprotein receptors, aiding in reverse cholesterol transport [9].Many cholesterol-lowering agents, including nicotinic acid, plant sterols, and statins, have been introduced for clinical use [10].Statins, such as pravastatin and lovastatin, decrease serum cholesterol levels by inhibiting hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis.HMG-CoA reductase is an enzyme that mediates the synthesis of mevalonic acid, an intermediate in the biosynthetic pathway of sterol or isoprenoid compounds.When the activity of HMG-CoA reductase is lowered, the lipid and cholesterol levels in the blood can be lowered by inhibiting cholesterol [10].
The oxidation of LDL potentially plays a significant role in the progression of atherosclerosis [11].Oxidative stress serves as a prominent risk factor in the oxidation of LDL.The internalization of oxidized LDL (ox-LDL) by endothelial cells and macrophages contributes to endothelial dysfunction and the formation of foam cells.Foam cells are a major cause of atherosclerosis, while increased blood cholesterol levels are one of the most critical risk factors for the pathogenesis of coronary heart disease [7].Therefore, lowering LDL oxidation is a useful strategy to prevent atherogenic diseases, for which plant-derived dietary components can play an important role.
Filipendula glaberrima Nakai (Rosaceae), also known as Korean meadowsweet, is a perennial plant found in the northern mountains of Central Korea.Its young leaves are wild herbs that are blanched and eaten as vegetables.F. glaberrima exhibits a notable abundance of phenolic compounds, including tannins and flavonoids, making it suitable for medicinal applications such as alleviating spasms, inducing sedation, addressing neuralgia and gout, providing analgesic relief, managing arthritis, and exerting anti-inflammatory effects [12].Therefore, this study reports the isolation and structural elucidation of the corresponding functional ingredients of F. glaberrima, the active constituents, and their biological effects on antioxidant activity and HMG-CoA reductase (HMGR) inhibition.Furthermore, the inhibitory effects on macrophage foam cell formation in THP-1 cells were assessed for two newly discovered compounds in conjunction with the organic extracts.These findings hold promise for the future development of valuable functional ingredients for nutraceuticals.
Additionally, two new compounds, together with the organic extracts, were evaluated for their inhibitory activities on macrophage foam cell formation in THP-1 cells.The obtained results are expected to play a role in the development of valuable functional ingredients for nutraceuticals.

Structure Elucidation
Based on the structural differences between the isolated compounds, they were divided into four types.Compounds 1-9 are hydrolyzable tannins, compounds 10-14 are flavonoid glycosides, compounds 15-20 are triterpenes, and compounds 21-28 are phenolic compounds (Figure 1).Among them, compounds 1 and 20 were identified as new compounds.The structural determination procedures for compounds 1 and 20 are described below: compounds.The structural determination procedures for compounds 1 and 20 are described below:  Rugosin B methyl ester (1) was obtained as a brownish amorphous powder with a molecular formula of C 42 H 32 O 27 as deduced from the HR-ESI-MS (Figure S1) measurement at m/z 967.1053 [M − H] − .The 1 H and 13 C NMR spectra of 1 indicated that the tannin formed an anomeric mixture (α-anomer: β-anomer = 4:3), which is a structural feature similar to that of rugosin B (2), except for the presence of a methyl ester signal at δ H 3.74 and δ C 52.5 in the valoneoyl moiety.The 1 H NMR spectrum of 1 (Figure S2) exhibited additional methyl ester signals at δ H 3.75 (β-anomer) and 3.74 (α-anomer), belonging to the ester of the valoneoyl group.The two proton singlet signals at δ H 7.04 and 6.95 and three singlet signals at δ H 7.06, 6.52, and 6.20 were assigned to two galloyl groups and a valoneoyl group in the α-anomer signals, respectively.Furthermore, the presence of two galloyl groups (δ H 7.02 and 6.91) and a valoneoyl group (δ H 6.95, 6.48, and 6. 19) was supported by the β-anomer signals in the 1 H NMR spectrum (Table 1).The 13 C NMR spectrum (Figure S3) indicated signals corresponding to two sets of four carbon signals and two carbonyl carbons for two symmetrical galloyl groups and a valoneoyl group from αand β-anomers, respectively (Table 1).Based on the 1 H-1 H COSY and HMQC spectral data (Figures S4 and S5), the proton signals of the methyl ester moiety in 1 were assigned.In the HMBC experiment (Figures S6 and S7), the protons at δ H 5.11 (H glc -2α) and 7.04 (H gal-I -2 and 6) were crossed with the signal at δ C 167.6 (C gal-I -7), which showed a cross-peak between the protons at δ H 5.82 (H glc -3α) and 6.95 (H gal-II -2 and 6) and the carbon signal at δ C 167.9 (C gal-II -7), indicating that the hydroxyl groups on glucose C-2 and -3 were acylated by galloyl groups.The HMBC correlations demonstrated the presence of connections of H glc -4 (δ H 5.09) and H-3 (δ H 6.52) with C-7 of the valoneoyl group (δ C 169.2).Additionally, we observed correlations of H glc -6 (δ H 5.24 and 3.76) and H-3 ′ (δ H 6.20) with C-7 ′ of the valoneoyl group (δ C 169.4).These findings indicate that the hexahydroxydiphenoyl component was positioned at O-4 and -6 of the glucose moiety, while the orientation of the valoneoyl group matched that of rugosin B.Moreover, the methoxy group located at δ H 3.74 and the H-6 ′′ signal at δ H 7.06 displayed correlations with the carbonyl group (C-7 ′′ ) at δ C 167.5 within the galloyl portion of the valoneoyl group.This correlation suggests that the carboxyl group of the galloyl component formed a methyl ester.In a previous study, methyl esters of rugosin B were synthesized as artefacts when prostratin B was treated with a mixture of MeOH and hot water [13].To test whether compound 1 is an artefact in this study, rugosin B in MeOH solution was kept at room temperature for 3 d in the presence of Sephadex LH-20, which is a condition similar to that of the isolation procedure.Rugosin B did not change with any formulation, indicating that compound 1 was not an artefact formed during the isolation process.Therefore, this finding confirmed that compound 1 is a new natural compound, a rugosin B methyl ester.
6 ′ -O-Galloylrosamultin (20) was obtained as an amorphous powder, and its molecular formula C 43 H 62 O 14 was determined by HR-ESI-MS (Figure S8) which showed a molecular ion peak [M − H] − at m/z 801.4069 (calculated for C 43 H 61 O 14 , 801.4061).The molecular formula mentioned above was validated through the analysis of the 13 C NMR spectroscopic data.The 1 H and 13 C NMR spectra (Figures S9 and S10) of compound 20 revealed a structural characteristic resembling that of rosamultin, which is derived from Rosa multiflora [14], with the exception of the inclusion of a galloyl group at the C-6 ′ -hydroxyl position of glucose.The 1 H NMR spectrum of 20 exhibited an additional singlet signal at δ H 7.09, corresponding to the aromatic protons in the galloyl moiety.The presence of this signal was additionally corroborated by the correlations observed between the methylene peaks at δ H 4.40 and 4.33 of glucose (C-6 ′ ) and the carbonyl peak at δ C 168.6 (C-7 ′′ ) of the galloyl group, as evidenced in the 1 H-1 H COSY, HMQC and HMBC spectra (Figures S11-S13).Thus, the structure of 20 was established as 6 ′ -O-galloylrosamultin.

Antioxidant Capacity
The DPPH and superoxide anion radical-scavenging activities are frequently utilized to evaluate the antiradical/antioxidant capability of isolated compounds for comparison with those of various reference standards.As shown in Table 2, the findings of DPPH radical scavenging revealed that the ethyl acetate and butanol fractions of F. glaberrima had the highest antioxidant activities with an IC 50 value of 4.62 and 5.25 µg/mL, respectively.Additionally, the ethyl acetate and butanol fractions had strong scavenging activities against the superoxide anion radical based on their low IC 50 values (4.07 and 4.64 µg/mL, respectively), indicating better activities than quercetin (IC 50 of 21.3 µM).The recorded IC 50 value of the methanol extract was lower than that of the standard, with a strong antioxidant potential near the standard.Among the active compounds, compounds 1-6 containing hexahydroxydiphenoyl (HHDP) or valoneoyl groups are a class of ellagitannins, and compounds 7-9 with only galloyl groups are a class of gallotannins (Figure 1).Rugosin B methyl ester (1) showed strong scavenging activity with an IC 50 value of 3.62 µM, which was 8-11-fold more potent than quercetin (IC 50 of 42.1 µM), ascorbic acid (IC 50 of 31.7 µM), and Trolox (IC 50 of 31.3 µM) and 4.7-fold more potent than quercetin (IC 50 of 17.3 µM).Compounds 2-9 exhibited substantial DPPH radical-scavenging activities with an IC 50 range of 3.19-4.70µM, which was better than the positive controls (Table 3).A comparison of ellagitannins 1-9 revealed that their radical-scavenging activities were significantly enhanced by increasing the number of galloyl units or HHDP moieties, including a valoneoyl group in the compounds, and the highest radical-scavenging activity was particularly observed for rugosin A (4) (IC 50 of 3.19 and 3.21 µM against DPPH and superoxide anion radical, respectively).In addition, the organic fractions and isolated compounds from F. glaberrima were tested for their lipid peroxidation activities (Table 3).Among the fractions, the ethyl acetate fraction showed the most potent inhibitory effect, with an IC 50 value of 9.67 µg/mL, followed by the butyl alcohol fraction (IC 50 = 18.8 µg/mL) and methanol extract (IC 50 = 26.3µg/mL).Ethanolic extracts (30% and 70% ethanol extracts) exhibited moderate inhibitory effects with an IC 50 of 40.1 and 34.7 µg/mL, respectively (Table 2).Among the isolates, hydrolyzable tannins 1-9 showed prominent inhibitory effects with an IC 50 range of 3.54-5.92µM.Quercetin 3-glucuronic acid (10) showed moderate inhibition of lipid peroxidation activity (IC 50 of 34.1 µM), comparable to that of Trolox (IC 50 of 33.2 µM), which was used as a positive control.Flavonols and hydrolyzable tannins containing multiple adjacent OH groups, particularly catechol groups, exhibited enhanced radical-scavenging activities against DPPH [32].It has been reported that glycosidation at C3 of the C-ring of flavonols leads to a decrease in their antioxidant activity [33], which aligns with the findings observed for compounds 11-13 (all derivatives of kaempferol glycosides) (Figure 1).These experimental data are in good agreement with the literature [32,33].

Inhibitory Effect of HMG-CoA Reductase
The methanol extract from the F. glaberrima leaves was sequentially fractionated with dichloromethane, ethyl acetate, and butanol.In Table 2, the butyl alcohol fraction had the highest HMGR inhibitory activity, with an IC 50 value of 0.74 µg/mL, followed by the ethyl acetate fraction (IC 50 = 1.73 µg/mL) and methanol extract (IC 50 = 2.86 µg/mL).

Inhibitory Effect of HMG-CoA Reductase
The methanol extract from the F. glaberrima leaves was sequentially fractionated with dichloromethane, ethyl acetate, and butanol.In Table 2, the butyl alcohol fraction had the highest HMGR inhibitory activity, with an IC50 value of 0.74 µg/mL, followed by the ethyl acetate fraction (IC50 = 1.73 µg/mL) and methanol extract (IC50 = 2.86 µg/mL).
Among the isolates, rugosin B methyl ester (1) showed the highest inhibitory activity against the HMGR enzyme with an IC50 value of 1.46 µM, the structure of which is different from that of rugosin A methyl ester (3) (IC50 = 8.40 µM) with respect to the presence of the galloyl group at the anomeric position of rugosin B methyl ester (1).In addition, 1,2,3.4.6-penta-O-galloyl-β-D-glucoside (7) showed reasonable inhibitory activity with an IC50 value of 4.98 µg/mL, whereas an analog of 7, 2,3.4.6-tetra-O-galloyl-D-glucoside (8) exhibited three times less activity (IC50 = 13.8)than 7 (Table 3).Moreover, 1,2,3,6-tetra-Ogalloyl-β-D-glucoside (9), without a galloyl unit at the C-4 position, resulted in a weak effect, indicating that the position of the galloyl unit on the C4 core could play an important role in the inhibition of HMGR, even for compounds with the same number of galloyl moieties.The analytical HPLC chromatogram (Figures 2 and S14) of the ethyl acetate fraction in this study showed relatively high contents of rugosin A methyl ester (3) and 1,2,3,4,6-penta-O-galloyl-β-D-glucoside (7).Hence, the notable inhibitory impact of F. glaberrima leaf extract on HMGR may be attributed to its significant ellagitannin content.However, to establish a clearer understanding of the connection between HMGR inhibitory activity and the structures of hydrolyzable tannins, further comprehensive analysis utilizing ellagitannin derivatives is warranted.

Inhibitory Effect of Foam Cell Formation in THP-1 Cells
The uptake of ox-LDL by macrophages is a critical factor in the formation of foam cells and the development of atherosclerosis.THP-1 cells have been widely employed as a convenient cellular model for investigating foam cell formation and studying macrophage behavior in vitro.THP-1 macrophages were exposed to 50 µg/mL of ox-LDL, with or without the inclusion of solvent extracts, two novel compounds (1 and 20), and pravastatin (used as a positive control), for 16 h.This approach aimed to examine the impact of these substances on lipid accumulation and the formation of foam cells.First, we observed the effects of different concentrations (20,40, and 60 µg/mL) of the solvent extracts on the THP-1 macrophage activity.While higher concentrations of the samples resulted in reduced viability of THP-1 macrophages, a maximum concentration of 50 µg/mL did not have any adverse effects on cell viability (Figure 3A).As a result, we selected and optimized the concentration of 50 µg/mL for the solvent extract to be used in the subsequent experiments.According to the findings presented in Figure 3B,C, the administration of ethanolic and organic solvent extracts resulted in a notable decrease in lipid accumulation within THP-1 macrophage foam cells when compared to the group treated with ox-LDL alone (model group).Following the extraction of the intracellular dye using isopropanol, a quantitative assessment of the intracellular lipid content was conducted.The ethanolic extracts and butanol fraction exhibited a reduction in lipid content within THP-1 macrophage foam cells, reaching approximately 70-72% at a concentration of 50 µg/mL.This reduction was significantly different from the lipid content of cells treated solely with ox-LDL.The observed results are comparable to the 77% inhibitory effect of pravastatin, which was used as a positive control at a concentration of 5 µM.The ethyl acetate fraction also showed an inhibitory effect on foam cell formation of 36% at a concentration of 50 µg/mL compared to the model group.Moreover, at a safer concentration of 25 µM of compound 1, both the number of intracellular red-stained particles and the clustering of foam cells were lower (54%) than those in the model group.The results showed that compound 1 significantly reduced lipid accumulation.Previous studies on the chemical composition of Filipendula species have revealed substantial quantities of polyphenolic compounds, with particular emphasis on notable constituents such as flavonol glycosides and ellagitannins [34].These plants are worthy of further extensive investigation in phytochemistry and in terms of their health-promoting effects.Furthermore, many studies have indicated that dietary antioxidant supplements and food plants, which are mainly rich in tannins and polyphenols, play a major role in controlling or preventing various diseases, such as cardiovascular diseases, diabetes, and even cancer, by slowing or reducing the level of oxidative stress, principally by protecting lipoproteins from lipid peroxidation without any obvious toxicity.

General Experimental Procedures
High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) data were obtained using an Agilent 6210 ESI/TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA).Semi-preparative chromatography was performed on a Waters 1525 pump and a 2996 photodiode detector equipped with a Luna C18 column (5 µm, 250 × 10 mm, Phenomenex, Torrance, CA, USA) and used for analytical high-performance liquid chromatography (HPLC; Waters Corporation, Milford, MA, USA) with a Luna C18 column (5 µm, 250 × 4.6 mm). 1 H and 13 C-nuclear magnetic resonance (NMR) spectra were recorded on a Bruker spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) at 400, 600, and 800 MHz for 1 H and 100, 150, and 200 MHz for 13 C. Previous studies on the chemical composition of Filipendula species have revealed substantial quantities of polyphenolic compounds, with particular emphasis on notable constituents such as flavonol glycosides and ellagitannins [34].These plants are worthy of further extensive investigation in phytochemistry and in terms of their health-promoting effects.Furthermore, many studies have indicated that dietary antioxidant supplements and food plants, which are mainly rich in tannins and polyphenols, play a major role in controlling or preventing various diseases, such as cardiovascular diseases, diabetes, and even cancer, by slowing or reducing the level of oxidative stress, principally by protecting lipoproteins from lipid peroxidation without any obvious toxicity.

General Experimental Procedures
High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) data were obtained using an Agilent 6210 ESI/TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA).Semi-preparative chromatography was performed on a Waters 1525 pump and a 2996 photodiode detector equipped with a Luna C18 column (5 µm, 250 × 10 mm, Phenomenex, Torrance, CA, USA) and used for analytical high-performance liquid chromatography (HPLC; Waters Corporation, Milford, MA, USA) with a Luna C18 column (5 µm, 250 × 4.6 mm). 1 H and 13 C-nuclear magnetic resonance (NMR) spectra were recorded on a Bruker spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) at 400, 600, and 800 MHz for 1 H and 100, 150, and 200 MHz for 13 C. Enzyme assays were performed using an Epoch Microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA) in transparent 96-well plates (Greiner Bio-One, Kremsmünster, Austria).All other reagents and chemicals were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and different commercial suppliers and were of analytical grade.

Plant Material
The leaves of F. glaberrima were collected from a wide-growing habitat in Yanggu County, Gangwon Province, Republic of Korea, in May 2018 and verified by Professor Emeritus, Chang-Soo Yook (Department of Pharmacognosy, Kyung Hee University).A voucher specimen (308-43A) was deposited in the herbarium of the Korea Institute of Science and Technology (KIST) in Seoul, Republic of Korea.

Measurement of Antioxidant Activity
To assess the antioxidant activity of the isolated compounds and organic extracts, various assays including 2,2-diphenylpicrylhydrazyl (DPPH) [35], superoxide anion radical scavenging [36], and lipid peroxidation [37] were conducted (see the Supporting Information).Lipid peroxidation was carried out following the guidelines for the handling and utilization of laboratory animals and received approval from the Animal Research Ethics Committee of the Korea Institute of Science and Technology (approval number: KISTIACUC-2018-081).As positive controls, ascorbic acid, quercetin, resveratrol, and Trolox were employed.Triplicate measurements were performed for each sample, and the mean values were determined.The results are presented as the mean ± standard deviation (SD).

HMG-CoA Reductase (HMGR) Inhibition Assay
The HMGR activity assay was optimized using a 96-well microplate reader to measure nicotinamide adenine dinucleotide phosphate (NADPH) oxidation during enzyme turnover.Each well was loaded with a mixture comprising 89 µL of 50 mM sodium phosphate buffer (pH 6.8), 0.8 mM NADPH, and 2 µg of the enzyme (2-8 units/mg).To initiate the reaction, 1 µL of the test sample and 10 µL of 0.8 mM HMG-CoA were introduced to the well.As a positive control, 10 µM pravastatin was utilized, while blank DMSO served as the negative control.The activity of HMGR was assessed by monitoring the reduction in NADPH absorbance at 340 nm for a duration of 900 s at 37 • C.

Figure 1 .
Figure 1.Structures of compounds 1-28 isolated from the leaves of F. glaberrima.Figure 1. Structures of compounds 1-28 isolated from the leaves of F. glaberrima.

Figure 1 .
Figure 1.Structures of compounds 1-28 isolated from the leaves of F. glaberrima.Figure 1. Structures of compounds 1-28 isolated from the leaves of F. glaberrima.

Figure 3 .
Figure 3. Effects on lipid accumulation in THP-1 macrophage foam cells.THP-1 macrophages were treated with 50 µg/mL ox-LDL alone or in combination with various samples from the leaves of F. glaberrima and pravastatin as a positive control.(A) Cells were treated with gradient dilutions of organic extracts (20-60 µg/mL) and subjected to cell viability experiments using the MTT method.(B) Quantitative analysis of intracellular lipid contents based on the absorbance of intracellular dye extracted with isopropanol at 450 nm.The results are reported as the means ± SD from three independent experiments performed in triplicate.## p < 0.01 compared with control; * p < 0.05 and ** p < 0.01 compared with treatment with ox-LDL alone.(C) Representative optical microscopy images of Oil Red O-stained lipid droplets at a magnification of ×40.Cells in the (a) control, (b) ox-LDL alone, (c) pravastatin (5 µM), (d) rugosin B methyl ester (25 µM), and 50 µg/mL concentration of (e) ethyl acetate fraction, (f) butanol fraction, (g) 30% ethanol extract and (h) 70% ethanol extract.Control cells were cultured for the same time without any treatments.

Figure 3 .
Figure 3. Effects on lipid accumulation in THP-1 macrophage foam cells.THP-1 macrophages were treated with 50 µg/mL ox-LDL alone or in combination with various samples from the leaves of F. glaberrima and pravastatin as a positive control.(A) Cells were treated with gradient dilutions of organic extracts (20-60 µg/mL) and subjected to cell viability experiments using the MTT method.(B) Quantitative analysis of intracellular lipid contents based on the absorbance of intracellular dye extracted with isopropanol at 450 nm.The results are reported as the means ± SD from three independent experiments performed in triplicate.## p < 0.01 compared with control; * p < 0.05 and ** p < 0.01 compared with treatment with ox-LDL alone.(C) Representative optical microscopy images of Oil Red O-stained lipid droplets at a magnification of ×40.Cells in the (a) control, (b) ox-LDL alone, (c) pravastatin (5 µM), (d) rugosin B methyl ester (25 µM), and 50 µg/mL concentration of (e) ethyl acetate fraction, (f) butanol fraction, (g) 30% ethanol extract and (h) 70% ethanol extract.Control cells were cultured for the same time without any treatments.

Table 2 .
Anti-oxidant and HMGR inhibition effects of organic fractions form the leaves of F. glaberrima.IC 50 data represent mean ± SD of n = 3. a

Table 3 .
Anti-oxidant and HMGR inhibition effects of isolates from the leaves of F. glaberrima.IC 50 data represent mean ± SD of n = 3. b n.d.: not detected. a