Compounds from Sedum caeruleum with antioxidant, anticholinesterase, and antibacterial activities

Abstract

Context: This is the first study on the phytochemistry, antioxidant, anticholinesterase, and antibacterial activities of Sedum caeruleum L. (Crassulaceae).

Objective: The objective of this study is to isolate the secondary metabolites and determine the antioxidant, anticholinesterase, and antibacterial activities of S. caeruleum.

Materials and methods: Six compounds (1–6) were isolated from the extracts of S. caeruleum and elucidated using UV, 1D-, 2D-NMR, and MS techniques. Antioxidant activity was investigated using DPPH^•, CUPRAC, and ferrous-ions chelating assays. Anticholinesterase activity was determined against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes using the Ellman method. Antibacterial activity was performed according to disc diffusion and minimum inhibitory concentration (MIC) methods.

Results: Isolated compounds were elucidated as ursolic acid (1), daucosterol (2), β-sitosterol-3-O-β-d-galactopyranoside (3), apigenin (4), apigetrin (5), and apiin (6). The butanol extract exhibited highest antioxidant activity in all tests (IC₅₀ value: 28.35 ± 1.22 µg/mL in DPPH assay, IC₅₀ value: 40.83 ± 2.24 µg/L in metal chelating activity, and IC₅₀ value: 23.52 ± 0.44 µg/L in CUPRAC), and the highest BChE inhibitory activity (IC₅₀ value: 36.89 ± 0.15 µg/L). Moreover, the chloroform extract mildly inhibited (MIC value: 80 µg/mL) the growth of all the tested bacterial strains.

Discussion and conclusion: Ursolic acid (1), daucosterol (2), β-sitosterol-3-O-β-d-galactopyranoside (3), apigenin (4), apigetrin (5), and apiin (6) were isolated from Sedum caeruleum for the first time. In addition, a correlation was observed between antioxidant and anticholinesterase activities of bioactive ingredients of this plant.

• Flavonoids
• phytochemistry
• steroids
• structure elucidation
• ursolic acid

Introduction

The Crassulaceae family comprises 1250–1500 species divided into six subfamilies and about 33 genera (Berger, 1930). Sedum is the largest genus comprising 350–500 species. It is often regarded as the core genus of Crassulaceae containing the least advanced as well as some highly derived taxa (Hart, 1982). Sedum species have been used in folk medicine against various symptoms (Niemann et al., 1976). They have been reported for their anti-inflammatory (Altavilla et al., 2008; De-Melo et al., 2005), antinociceptive, antioxidant (Bonina et al., 2000; Thuong et al., 2007), hepatoprotective, and antitumor effects (Camargo et al., 2002; Jung et al., 2008; Kang et al., 2000; Torres et al., 2003). Phenolic acids and flavonoids (Korul’kin, 2001; Mulinacci et al., 1995; Stevens et al., 1994; Wolbis, 1987, 1989a,b), coumarins (Wolbis & Krolikowska, 1988; Zaitsev et al., 1983), terpenes (He et al., 1998; Wollenweber et al., 1999), and alkaloids (Colau & Hootele, 1983; Kim et al., 1996) have been isolated from Sedum species.

After publication of some research data on synthetic antioxidants that may cause cancer (Grice, 1988), there is a growing trend to find naturally occurring safe and effective antioxidants (Scalbert et al., 2005) to use in food and pharmaceutical industries. According to Atta-ur-Rahman and Choudhary (2001), regular antioxidants may stop or slow down the neuronal degeneration that can prevent Alzheimer’s disease progression. Alzheimer's disease is the severe form of dementia, and the acetylcholinesterase (AChE) inhibitor drugs are used to treat Alzheimer’s disease. Most of these drugs cause bradycardia and liver, stomach, and intestine toxicities (Dökmeci, 2000). For these reasons, the improvement and handling of safe anticholinesterases and antioxidants from nature are desired.

With regard to medicinal uses of some Sedum species, this study investigates the secondary metabolites of aerial parts of S. caeruleum L. as well as its in vitro antioxidant, anticholinesterase, and antibacterial activities. We report the first phytochemical investigations on Sedum caeruleum, also known as Sky Stone-crop, Baby-blue, and Red-leaf in Algeria.

Materials and methods

Chemicals and drugs

Melting points were determined on the Barnstead Electrothermal apparatus (Thermo Scientific, Waltham, MA) and are uncorrected. ¹H-NMR and ¹³C-NMR spectra (δ, ppm) were recorded on a Bruker ALS 400 MHz spectrometer using tetramethylsilane (E. Merck, Darmstadt, Germany) as the internal reference. Silica gel (35–70 mm), TLC reagents, and Sephadex LH-20 were purchased from E. Merck (Darmstadt, Germany) and Fluka (Newport News, VA).

Bioactivity measurements were carried out on a 96-well microplate reader, SpectraMax 340PC³⁸⁴, Molecular (Newark, DE), at the Department of Chemistry, Muğla Sıtkı Koçman University (Turkey). The measurements and calculations of the activity results were evaluated using Softmax PRO v5.2 software (Molecular, Newark, DE). Ethanol, ammonium acetate, copper (II) chloride, and potassium persulfate were obtained from E. Merck (Darmstadt, Germany). Polyoxyethylenesorbitan–monopalmitate (Tween 40), neocuproine, α-tocopherol, butylatedhydroxytoluene (BHT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), electric eel AChE (Type-VI-S, EC 3.1.1.7, 425.84 U/mg, Sigma, St. Louis, MO), horse serum butyrylcholinesterase (BChE, EC 3.1.1.8, 11.4 U/mg, Sigma, St. Louis, MO), 5,5′-dithiobis (2-nitrobenzoic) acid (DTNB), acetylthiocholine iodide, butyrylthiocholine chloride, and galantamine were obtained from Sigma-Aldrich GmbH (Sternheim, Germany). All other chemicals and solvents were of analytical grade.

Plant material

The aerial parts of Sedum caeruleum were collected on May 2010 at Constantine (North Eastern Algerian) and identified by Professor Gérard De Bélair, Faculty of Sciences University of Annaba, Algeria. A voucher specimen was deposited at the Herbarium of the Laboratory of Therapeutic Substances (LOST), University of Constantine (Herbarium number: LOST.Sc.05.10).

Extraction and isolation of compounds

Air-dried and powdered aerial parts (1500 g) of S. caeruleum were extracted with 80% MeOH. After evaporating the methanol under vacuum, the residue was dissolved in water and extracted with petroleum ether, chloroform, ethyl acetate, and butanol, successively. The chloroform extract (4.5 g) was subjected to silica gel column chromatography and was eluted with dichloromethane, followed by gradient of methanol up to 100%. The fractions eluted with CHCl₃–MeOH (9:1) and (8:2) were further rechromatographed on silica gel column. This afforded three compounds (1–3). Ethyl acetate and butanol extracts were combined because they were in less amount. The combined extract (9.64 g) was chromatographed on silica gel column, eluted with dichloromethane, followed by gradients of methanol. Three main fractions (1–3) were collected. Fraction 1 (600 mg) was further passed through silica gel column (CH₂Cl₂–MeOH 9.5:0.5) yielding a white precipitate (4, 25 mg), washed with dichloromethane. Fraction 2 (540 mg) was also chromatographed on silica gel, eluted with an isocratic system (EtOAc–MeOH 15:1) that afforded compound 5 (10 mg). Fraction 3 (790 mg) eluted over Sephadex LH-20 with MeOH using flash column chromatography that led to compound 6 (12 mg). Compounds 1–6 (Figure 1) were identified by ¹H-NMR,¹³C-NMR, DEPT, and 2D-NMR techniques such as COSY, HMQC, HMBC, and ROESY, respectively. In order to identify the flavonoids, the NaOCH₃, AlCl₃/HCl, and NaOAc/H₃BO₃ UV spectrum were also used (Mabry et al., 1970). All these data were in good agreement with the respective literature data (Ahmad et al., 1987; Ali et al., 2007; Lendl et al., 2005; Markham, 1982; Markham & Geiger, 1993; Mizushina et al., 2006; Wang et al., 2009).

Figure 1. Structures of compounds 1–6 isolated from Sedum caeruleum.

Acid hydrolysis of compounds 2, 3, 5, and 6

Each compound (5 mg) was refluxed with 5% H₂SO₄ (5 mL) in water for 1 h. The reaction mixture was diluted with water and extracted by ethyl acetate. Thin-layer chromatography (TLC) was used to compare the samples with authentic samples. Each remaining aqueous layer was adjusted to pH 7 with NaHCO₃ and filtered. The concentrated filtrate and authentic sugars were developed on a silica gel TLC using acetone:H₂O (90:10, V/V) solvent mixture.

Determination of antioxidant activity

DPPH free radical scavenging assay

The free radical-scavenging activity was determined spectrophotometrically by the DPPH assay (Tel et al., 2013a). BHT and α-tocopherol were used as antioxidant standards for comparison of the activity. The scavenging capability of DPPH radical was calculated using the following equation. The results were given as IC₅₀ value (µg/mL) corresponding the concentration of 50% inhibition:

Metal chelating activity assay

The chelating activity of S. caeruleum extracts on Fe²⁺ was measured spectrophotometrically (Tel et al., 2013a), and the activity was calculated using the following equation. The results were given as IC₅₀ value (µg/mL) corresponding the concentration indicating 50% inhibition:

Cupric reducing antioxidant capacity assay

The cupric reducing antioxidant capacity (CUPRAC) was determined according to the CUPRAC method (Apak et al., 2004). The reducing capacity of the extracts was compared with those of α-tocopherol and BHT. The results were given as A_0.5 (µg/mL) corresponding the concentration indicating 0.5000 absorbances.

Determination of the anticholinesterase activity

Acetylcholinesterase (AChE) and BChE inhibitory activities were measured by slightly modifying the spectrophotometric method (Ellman et al., 1961). AChE from electric eel and BChE from horse serum were used, while acetylthiocholine iodide and butyrylthiocholine chloride were employed as substrates of the reaction. 5,5′-Dithiobis(2-nitrobenzoic) (DTNB) acid was used for the measurement of the activity. Galantamine was used as a positive reference compound. The results were given as IC₅₀ value (µg/mL) corresponding to the concentration shows 50% inhibition.

Determination of antibacterial activity

The antibacterial activity of the chloroform extract was tested against a range of microorganisms, namely Escherichia coli ATCC 25922, E. coli (HS), Staphylococcus aureus ATCC 43300, S. aureus (HS), Pseudomonas aeruginosa ATCC 27853, P. aeruginosa (HS), Klebsiella pneumonia ATCC 700603, K. pneumonia (HS), and Streptococcus enterococcus (HS). The reference strains (ATCC, American type culture collection) were obtained from the Pasteur Institute (Algiers) whereas the others (HS, hospital strains) were obtained from the laboratory of bacteriology, Benbadis Hospital, Constantine, Algeria, using conventional methods by The National Committee For Clinical Laboratory Standards (NCCLS) and The Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2006; NCCLS, 1997).

Statistical analysis

All data on both antioxidant and anticholinesterase activity tests were the average of triplicate analyses. The data were recorded as mean ± standard error meaning (SEM). Significant differences between means were determined by Student’s t-test, and p values <0.05 were regarded as significant.

Results and discussion

Identification of compounds 1–6

Ursolic acid (1) (20 mg), daucosterol (2) (7.2 mg), and β-sitosterol-3-O-β-d-galactopyranoside (3) (8 mg) were isolated from the chloroform extract whereas apigenin (4) (25 mg), apigetrin (5) (10 mg), and apiin (6) (12 mg) were the combined from ethyl acetate and butanol extracts.

β-Sitosterol-3-O-β-d-glucopyranoside (Daucosterol) (2)

C₃₅H₆₀O₆, white powder, ¹H-NMR (400 MHz, CD₃OD, δ, ppm, J/Hz): 5.30 (1H, d, J = 4.64, H-6), 4.21 (1H, J = 7.8, H-3), 0.65 (3H, s, H-18), 0.95 (3H, s, H-19), 0.90 (3H, d, J = 6.4, H-21), 0.78 (3H, m, H-26), 0.81 (3H, d, J = 6.4, H-27), 0.84 (3H, m, J = 6.5, H-29), 4.84 (1H, d, H-1′), 4.41 (1H, m, H-2′), 3.64 (1H, m, H-3′). ¹³C-NMR (100 MHz, CD₃OD, δ): 141.67 (C-5), 121.66 (C-6), 76.66 (C-3), 50.0 (C-9), 44.16 (C-25), 42.50 (C-13), 38.34 (C-4), 37.50 (C-1), 11.66 (C-29), 20.0 (C-11), 23.33 (C-28), 30.0 (C-7), (C sugar) 100.83 (C-1′), 61.66 (C-6′), 70.0 (C-4′), 77.0 (C-3′), 73.68 (C-2′), 76.84 (C-5′). Acid hydrolysis afforded β-sitosterol and d-glucopyranose.

β-Sitosterol-3-O-β-d-galactopyranoside (3)

C₃₅H₆₀O₆, white powder, ¹H-NMR (400 MHz, CD₃OD, δ, ppm, J/Hz): 5.40 (1H, s H-6), 4.24 (1H, d, H-3), 0.75 (3H, s, H-18), 1.06 (3H, s, H-19), 0.98 (3H, d, J = 7.0, H-21), 0.84 (3H, m, J = 6.4, H-26), 0.87 (3H, d, J = 6.4, H-27), 0.90 (3H, m, J = 6.5, H-29), 4.42 (1H, d, J = 7.7, H-1′), ¹³C-NMR (100 MHz, CD₃OD, δ): (Me):11.0 (C-19), 11.20 (C-29), 17.9 (C-21), 18.2 (C-26), 18.5 (C-18), 19.0 (C-27), (CH₂): 20.1 (C-11), 22.1 (C-28), 23.4 (C-15), 25.0 (C-23), 28.8 (C-2), 30.9 (C-7), 33.0 (C-22), 35.8 (C-16), 36.4 (C-12), 37.9 (C-1), 38.6 (C-4), (CH): 31.0 (C-8), 44.8 (C-25), 49.2 (C-9), 55.0 (C-17), 55.8 (C-14), 75.1 (C-3), 121.0 (C-6), (C quaternary) 41.4 (C-13), 139.6 (C-5), 35.2 (C-10), (C sugar) 61.5 (C-6′), 70.1 (C-4′), 72.8 (C-2′), 76.24 (C-5′), 77.6 (C-3′), 100.4 (C-1′). Acid hydrolysis gave β-sitosterol and d-galactopyranose.

Antioxidant properties

Three methods were selected to determine the antioxidant capacity of the extracts. DPPH free radical scavenging activity measures the ability of electron transfer to the media. The CUPRAC method also measures electron transferring of the antioxidant. In case of bulky compounds, CUPRAC gives better and accurate results. Since transition metals can accelerate the lipid peroxidation via the Fenton reaction (Tel et al., 2013b), the metal chelating activity was selected to measure the binding capacity of iron of the extracts.

The free radical scavenging activity of the extracts is given in Table 1. Radical scavenging activity increased linearly with increasing amount of the extracts. The butanol extract exhibited highest activity (IC₅₀ value: 28.35 ± 1.22 µg/mL). It indicated higher free radical scavenging activity than that of BHT, but lower than α-tocopherol.

Table 1. Antioxidant activity of S. caeruleum by the DPPH^•, CUPRAC, and metal chelating assays^a.

	Antioxidant activity
	DPPH^• assay	CUPRAC assay	Metal chelating assay
Extracts	IC50 (µg/mL)	A0.50 (µg/mL)	IC50 (µg/mL)
Chloroform extract	129.23 ± 1.16	29.40 ± 1.10	132.23 ± 2.31
Ethyl acetate extract	195.36 ± 1.21	99.36 ± 1.32	161.43 ± 2.34
Butanol extract	28.35 ± 1.22	23.52 ± 0.44	40.83 ± 2.24
α-Tocopherol^b	12.06 ± 0.17	2.65 ± 0.01	NT
BHT^b,c	54.01 ± 1.07	3.90 ± 0.01	NT
EDTA^b,d	NT^e	NT^e	3.47 ± 0.35

^aIC₅₀ and A_0.50 values represent the means ± SEM of three parallel measurements (p < 0.05).

^bReference compounds.

^cButylatedhydroxyltoluene.

^dEthylenediaminetetraacetic acid.

^eNot tested.

The results of CUPRAC of the extracts are compared with those of α-tocopherol and BHT (Table 1). Activity (absorbance) increased linearly with the increasing amount of extracts. The butanol extract exhibited highest activity (A_0.50 value: 23.52 ± 0.44 µg/mL) among the extracts, followed by chloroform (A_0.50 value: 29.40 ± 1.10 µg/mL) and ethyl acetate extract (A_0.50 value: 99.36 ± 1.32 µg/mL). However, none of the extracts exhibited higher activity than those of antioxidant standards.

Table 1 also shows the chelating effects of extracts on ferrous ions, compared with EDTA. Metal chelating activity increased linearly with increasing concentration of the extracts. The butanol extract (IC₅₀ value: 40.83 ± 2.24 µg/mL) also showed the highest metal chelating activity, followed by chloroform (IC₅₀ value: 132.23 ± 2.31 µg/mL) and ethyl acetate extracts (IC₅₀ value: 161.43 ± 2.34 µg/mL).

Anticholinesterase activity

Table 2 shows the AChE and BChE inhibitory activities of the extracts, compared with that of galantamine used as a standard drug to treat mild Alzheimer’s disease. Chloroform (IC₅₀ value: 180.79 ± 0.47 μg/mL) and butanol extracts (IC₅₀ value: 184.59 ± 2.04 μg/mL) showed weak inhibitory activity against AChE. The ethyl acetate extract, however, was inactive at all concentrations. The butanol extract (IC₅₀ value: 36.89 ± 0.15 μg/mL) exhibited highest inhibitory activity against BChE, even higher than galantamine (IC₅₀ value: 50.81 ± 0.91 μg/mL). The chloroform extract exhibited moderate BChE inhibitory activity (IC₅₀ value: 84.32 ± 0.46 μg/mL). The ethyl acetate extract, however, had a weak inhibitory activity (IC₅₀ value: 174.04 ± 1.42 μg/mL). The butanol and chloroform extracts showed weak AChE and the good BChE inhibitory activities, respectively. The activity of chloroform extract may be due the presence of ursolic acid (Kolak et al., 2009) found in large amounts. The presence of flavonoids in the butanol extract may have caused BChE inhibitory activity.

Table 2. Acetylcholinesterase and butyrylcholinesterase inhibitory activities of various extracts of S. caeruleum^a.

	AChE assay	BChE assay
Extracts	IC50 (µg/mL)	IC50 (µg/mL)
Chloroform extract	180.79 ± 0.47	84.32 ± 0.46
Ethyl acetate extract	NA^c	174.04 ± 1.42
Butanol extract	184.59 ± 2.04	36.89 ± 0.15
Galantamine^b	5.01 ± 0.10	50.81 ± 0.91

^aIC₅₀ values represent the means ± SEM of three parallel measurements (p < 0.05).

^bReference compound.

^cNot active.

Antibacterial activity

Table 3 shows the antibacterial activity (inhibition zones and MIC) of the chloroform extract at 128 µg/mL against six Gram (−) pathogen bacteria; i.e., E. coli ATCC 25922, E. coli (HS), P. aeruginosa ATCC 27853, P. aeruginosa (HS), K. pneumonia (HS), K. pneumoniae (ATCC 700603), and against three Gram (+) pathogen bacteria, namely, S. aureus ATCC 43300, S. aureus (HS), and S. enterococcus (HS). According to the data the chloroform extract mildly inhibited the growth of all bacterial strains with 12–13 mm inhibition zone diameters. The MIC values were found to be 80 µg/mL against all tested bacteria.

Table 3. Antibacterial activity (inhibition zones and MIC) of the chloroform extract of S. caeruleum.

	Microorganism	Inhibition zone^a (mm)	MIC (μg/mL)
Reference strains (ATCC)	Escherichia coli ATCC 25922	12	80
	Pseudomonas aeruginosa ATCC 27853	13	80
	Klebsiella pneumoniae ATCC 700603	13	80
	Staphylococcus aureus ATCC 43300	13	80
Hospital strains (HS)^b	Escherichia coli (HS)^b	12	80
	Pseudomonas aeruginosa (HS)^b	12	80
	Klebsiella pneumoniae (HS)^b	11	80
	Staphylococcus aureus (HS)^b	12	80
	Streptococcus enterococcus (HS)^b	12	80

^a128 μg/mL.

^bHS, hospital strain; MIC, minimum inhibitory concentration (µg/mL).

Conclusions

Sedum caeruleum was studied phytochemically for the first time. Ursolic acid (1), daucosterol (2), β-sitosterol-3-O-β-d-galactopyranoside (3), apigenin (4), apigetrin (5), and apiin (6) were isolated from this specie. The butanol extract exhibited highest activity in all tests except AChE assay, followed by chloroform and ethyl acetate extracts. The highest activity of polar extracts may be due to the presence of the flavonoids 4–6. The extracts showing higher antioxidant activity also exhibited the higher BChE inhibitory activity. Similarly, the chloroform extract exhibited weak antioxidant activity; it showed weak activity against BChE and no activity against AChE. Thus it can be said that there is a correlation between antioxidant and AChE inhibitory activities. The difference between the extracts and the control was statistically significant in all antioxidant tests (p < 0.05). Moreover, the chloroform extract showed a mild antibacterial activity against all tested bacterial strains.

Acknowledgements

The authors thank The Muğla Sitki Koçman University, Department of Chemistry.

Declaration of interest

The authors report that they have no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The authors are grateful to ANDRS and DG-RSDT (MESRS. Algeria) for financial support.