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Acetylcholinesterase inhibitory activity and mutagenic effects of Croton penduliflorus leaf extract constituents
South African Journal of Botany 87 (2013) 48–51
Contents lists available at SciVerse ScienceDirect
South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Short communication
Acetylcholinesterase inhibitory activity and mutagenic effects of Croton penduliflorus leaf extract constituents M.A. Aderogba, A.R. Ndhlala, J. Van Staden ⁎ Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa
a r t i c l e
i n f o
Article history: Received 19 November 2012 Received in revised form 13 March 2013 Accepted 13 March 2013 Available online 15 April 2013 Edited by LJ McGaw Keywords: Acetylcholinesterase Croton penduliflorus Euphorbiaceae Mutagenicity
a b s t r a c t Croton penduliflorus is a medicinal plant widely employed in the management of inflammatory conditions, infections and oxidative stress related diseases. The activities demonstrated by leaf extracts indicate that they possess the ability to reduce oxidative damage to cells. Repeated column fractionation of the ethyl acetate fraction of a 20% aqueous methanol leaf extract of C. penduliflorus on Sephadex LH-20 afforded four phenolic compounds: quercetin-3-O-rhamnoside (1), kaempferol-3-O-rhamnoside (2), protocatechualdehyde (3A) and its solvent derived dimer (3B) along with p-hydroxybenzoic acid (4). Compound 3B is described for the first time and its significance in bioassay is briefly outlined. Structure elucidation of the isolated compounds was carried out using spectroscopic techniques. The inhibitory properties of the four compounds against acetylcholinesterase were determined using the microplate assay. The IC50 values of the isolated compounds ranged from 87.9 to 1231.9 μM, with compound 2 having the best inhibitory activity (IC50 = 87.9 μM). The four isolated compounds showed no mutagenic effects against Salmonella typhimurium tester strains TA98 and TA100. The moderate activity demonstrated by these compounds suggests that they could be helpful in the management of neurodegenerative disorders. © 2013 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Generation of excess reactive oxygen radicals and oxidative damage are believed to be involved in the pathogenesis of many neurodegenerative disorders. Accumulated studies have shown that plants used in traditional medicine have preventive and protective effects in oxidative stress conditions (Lee et al., 2003; Vijayakumar et al., 2006; George and Chaturvedi, 2008). Amelioration of oxidative stress conditions by higher plants has been associated with phenolic compounds such as flavonoids and other dietary polyphenols. These bioactive molecules exhibit multiple biological effects such as antithrombotic, anti-inflammatory, anticarcinogenic and antimutagenic effects as a result of their antioxidant properties (Urquiaga and Leighton, 2000). Croton penduliflorus Hutch belongs to the family Euphorbiaceae. It is widely employed in the management of inflammatory conditions, infections and oxidative stress related diseases (Anika and Shetty, 1983). The activities demonstrated by the plant extract support that its constituents possess the ability to reduce oxidative damage to cells. Previous phytochemical investigation of the root bark afforded penduliflaworosin, a clerodane-type diterpene ent-(1 2R)-methyl-15, 16-epoxy-9,10-friedolabda-5(10),13(16),14-trien-19-oate 20, l2-lactone (Adesogan, 1981).
⁎ Corresponding author. Tel.: +27 33 2605130. E-mail address: [email protected] (J. Van Staden).
Excess free radicals generated in the body attack most cellular macromolecules such as proteins (enzymes), lipids and DNA leading to oxidative stress and neurodegenerative disorders. Antioxidant compounds are useful in ameliorating these conditions (Reynolds et al., 2007). Acetylcholinesterase (AChE) catalyses the degradation of the neurotransmitter acetylcholine, resulting in choline and an acetate group (Ranga et al., 2002). Insufficient cholinergic function and neurotransmitter disturbances are considered as prominent among the main pathological features in central nervous system disorders (Eldeen et al., 2005). In this study, we have investigated C. penduliflorus leaf extracts in our efforts to find phytochemical agents that could be effective in the prevention and management of neurodegenerative conditions. 2. Material and methods 2.1. Plant material, collection and extraction The leaves of C. penduliflorus were collected at Obafemi Awolowo University (OAU), Ile-Ife, Nigeria, in June 2011. It was identified by Mr. G. Ibhanesebhor of the Herbarium Section, Department of Botany (OAU). A voucher specimen (IFE 16401) was deposited at the University Herbarium at the Department of Botany. The powdered leaves (700 g) were extracted with 8 L of 20% aqueous methanol at room temperature for 24 h and filtered. The crude extract was concentrated in vacuo at 40 °C to about ⅓ of the original volume. This was sequentially extracted
0254-6299/$ – see front matter © 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sajb.2013.03.013
M.A. Aderogba et al. / South African Journal of Botany 87 (2013) 48–51
with n-hexane (3 × 800 mL), dichloromethane (3 × 800 mL), ethyl acetate (3 × 1 L) and finally n-butanol (2 × 500 mL). The solvent fractions were individually concentrated to dryness in vacuo to afford four solvent extracts. 2.2. Isolation of compounds from C. penduliflorus extract The EtOAc fraction (1.10 g) was fractionated on a Sephadex LH-20 column using 95% DCM/MeOH as eluent. This was followed by an increasing gradient of MeOH up to 30%. The fractions collected were analysed by TLC using DCM/MeOH (9:1) as solvent system. This afforded four fractions (A1–A4). Fraction A4 (25 mg) gave a single spot (compound 1). Fraction A2 purified on Sephadex LH-20 using EtOAc/MeOH (9:1) as eluent followed by an increasing gradient of MeOH up to 15%. Analysis of the fractions collected on TLC plates using EtOAc/MeOH (9:1) yielded compound 2 (7 mg). Purification of fraction A1 on Sephadex LH-20 using EtOAc/MeOH (9:1) and analysis of the fractions by TLC using 100% EtOAc yielded compound 3 (18 mg). Further fractionation of fraction A3 on Sephadex LH-20 using EtOAc/MeOH (9:1) followed by an increasing gradient of MeOH up to 25% yielded compound 4 (5 mg) on analysis of the fractions collected on TLC plates using EtOAc/MeOH (5:1) as solvent system. 2.3. Acetylcholinesterase (AChE) inhibitory assay The microplate assay for AChE inhibition activity was performed based on Ellman's method as detailed by Moyo et al. (2010). 2.4. Ames test for mutagenic properties Mutagenicity was tested using the Salmonella microsome assay based on the plate-incorporation procedure with Salmonella typhimurium tester strains TA98 and TA100 without metabolic activation (Maron and Ames, 1983). 3. Results and discussion Structure elucidation of the compounds was carried out using spectroscopic techniques: Mass spectrometry (TOF MS ES) and NMR (1D and 2D, 500 MHz). The compounds were identified as: quercetin-3-Orhamnoside (1, Aderogba et al., 2012), kaempferol-3-O-rhamnoside (2, Gohar et al., 2009) and p-hydroxybenzoic acid (4, Martin et al., 2000). Compound (3A) was obtained as brown powder. Its mass spectrum showed the exact molecular ion peak as the base peak, m/z = 137.0237, [M-H] −, consistent with the molecular formula C7H6O3. The 1H NMR (acetone-d6) of 3A showed signals representing a trisubstituted phenyl ring at δ: 7.36 (1H, d, J = 1.9 Hz, H-2) meta-related coupled protons, 7.34 (1H, dd, J = 1.9, 8.0 Hz, H-6) meta and orthorelated protons and 7.00 (1H, d, J = 8.0 Hz, H-5) ortho-related protons. An aldehydic proton appeared at δ 9.77. The 13C NMR (acetone-d6) of 3A showed signals as follows: δ 131.0 (C-1), 115.2 (C-2), 146.7 (C-3), 152.6 (C-4), 116.2 (C-5), 125.5 (C-6) and 191.3 (CHO). Compound 3A was identified as 3,4-dihydroxybenzaldehyde, protocatechualdehyde (Kolehmainen et al., 1995). Further NMR studies of 3A in MeOD showed that in this solvent, 3A existed in a different form 3B (solvent derived artifact). The 1H NMR of 3B (Table 1) are characterized by broadness of the resonance lines between δ 7.31–6.73. 13C NMR and DEPT spectra of this compound revealed the presence of 14 carbons, (Table 1). DEPT showed the presence of 8 CH carbons. These were assigned as 6 aromatic CH, 1 aldehydic CH and the other one CH in the non-aromatic region. The remaining 6 carbons were assigned as quaternary carbons out of which four were oxygenated. The positions of attachment of the two monomers and the oxygenated carbons were established by HMBC correlations (Fig. 1). Compound 3B was identified as a dimer of 3,4dihydroxybenzaldehyde. Formation of compound 3B (artifact) in this work was as a result of MeOD used for the NMR of compound 3A.
Table 1 1 H (500 MHz) and
13
49
C (125 MHz) NMR chemical shifts of compound 3B in MeOD.
1
No
H
1 2 3 4 5 6 CHO
7.30 (1H, b d, J = 1.9 Hz)
6.92 (1H, d, J = 8.7 Hz) 7.31 (1H,b d, J = 2.0 Hz) 9.69 (1H, S)
13
C
No
131.2 115.7 147.5 154.0 116.2 126.7 193.4
1′ 2′ 3′ 4′ 5′ 6′ 7′
1
13
H
6.74 (1H, b d, 1.8 Hz)
6.73 (1H, b s) 6.85 (1H, b d, 1.5 Hz) 5.21 (1H, s)
C
131.5 115.2 146.6 147.0 116.6 119.8 105.2
There is no previous report of existence of 3,4-dihydroxybenzaldehyde in this dimeric form. The structures of the isolated compounds are presented in Fig. 2. Organic solvents are critical to extraction and purification of secondary metabolites from medicinal plants. Our observation calls into question the stability of secondary metabolites in bioassay media. There are apparent chemical reactions that took place in MeOD, by activation of the compound (3A) aldehydic function to form a dimer (3B). However, after solvent removal the sample remains unchanged as demonstrated by the NMR spectra in acetone-d6. Changes like this may alter the activity of secondary metabolites in the bioassay medium. This could lead to loss of activity, formation of toxic compounds or difficulty in reproducibility of bioassay results. The main objective of phytochemical investigation of medicinal plants to offer a unique opportunity for identifying novel therapeutic compounds and providing useful lead compounds will be greatly affected by occurrences like this. It is important that stability tests are conducted on secondary metabolites from medicinal plants before experimentation (Maltese et al., 2009). The results of AChE inhibitory activity of the four compounds are shown in Table 2. The IC50 values ranged from 87.9 μM (compound 2) to 1231.9 μM (compound 3A). The percentage inhibition of galanthamine (positive control) at a concentration of 20 μM was 93.5% and the IC50 value was 1.8 μM. Isolated compounds demonstrated moderate AChE inhibitory properties. Table 3 presents the results of the mutagenicity test as the spontaneous reversion response of the S. typhimurium tester strains TA98 and TA100 to the different dilutions of the four compounds. The results revealed that all four compounds were non-mutagenic towards the S. typhimurium strains TA98 and TA100. These compounds, therefore do not have the capacity to cause a base pair substitution.
O
H9.68 C
H 7.31 126.7 6 5 116.2 H 6.92
193.4
131.2 1
H 7.30
H 6.74
2 115.7 H 5.21
3 147.5 154.0
OH
2'
105.2
4
O
7' OH
OH
115.2
131.5 1'
3'
146.6 4'
147.0 OH
6' 5' 116.6 119.8 H 6.85
H 6.73
Fig. 1. HMBC correlations of protocatechualdehyde dimer (3B).
50
M.A. Aderogba et al. / South African Journal of Botany 87 (2013) 48–51
R OH
HO
O
O OH
OH
O
O
OH
OH
R
Compound 1
OH
2
H
O
O
O
CH
OH C
CH
OH H O
OH
OH OH
OH
OH
OH 3B
3A
4
Fig. 2. Structures of isolated compounds from Croton penduliflorus leaf extract.
4. Conclusions Investigation of C. penduliflorus for possible active constituents against neurodegenerative disorders afforded four phenolic compounds identified as: quercetin-3-O-rhamnoside (1), kaempferol-3-O-rhamnoside (2), protocatechualdehyde (3A) and its solvent derived dimer (3B) and p-hydroxybenzoic acid (4). These compounds are reported from the leaf extracts of C. penduliflorus for the first time. Phenolic compounds are known to exhibit several pharmacological and biological activities. It was therefore not surprising to observe the activities exhibited by these four compounds against AChE enzyme. All compounds showed a dose dependent increase in the percentage inhibition of the enzyme. Structural transformation of compound 3A in two different solvents
Table 2 AChE inhibitory activity (IC50 mg/mL) of four isolated compounds from Croton penduliflorus leaf extract. Compound (1) (2) (3A) (4)
Quercetin-3-O-rhamnoside Kaempferol-3-O-rhamnoside Protocatechualdehyde p-Hydroxybenzoic acid Galanthamine
% inhibition (1 mg/mL)
IC50 (μM)
60.3 68.3 54.6 51.6 93.5
133.9 87.9 1231.9 971.0 1.8
± 3.1 ± 5.5 ± 0.6 ± 4.9 (20 μM)
± ± ± ± ±
7.9 6.0 7.2 7.2 0.5
used during analysis had a significant effect on their bioactivity. Compound 2 exhibited highest AChE inhibitory activity and all four compounds showed no potential mutagenic effects towards the S. typhimurium strains TA98 and TA100.
Table 3 Number of revertant colonies of Salmonella typhimurium strains TA98 and TA100 induced by the four isolated compounds from Croton penduliflorus leaf extract. Compound
Concentration (μg/mL)
Number of His + revertants
(1)
Quercetin-3-O-rhamnoside
(2)
Kaempferol-3-O-rhamnoside
(3A)
Protocatechualdehyde
(4)
p-Hydroxybenzoic acid
1000 100 10 1000 100 10 1000 100 10 1000 100 10 2
23.4 22.1 19.4 29.4 21.4 23.9 22.4 23.1 23.0 20.1 19.9 21.3 208.0 22.0
4-NQO 10% DMSO (in water)
TA98
4-NQO; 4-nitroquinoline-oxide was used as a positive control. 10% DMSO (in water) was used as a negative control.
TA100 ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.0 1.1 2.2 3.9 1.3 2.2 0.1 1.3 1.0 3.5 2.1 1.1 0.6 1.6
183.2 197.3 167.8 145.4 176.0 164.6 133.9 166.3 158.3 157.5 137.6 144.9 992.3 174.5
± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.2 3.1 6.2 1.1 2.1 3.3 5.2 2.1 7.0 2.2 10.1 5.2 9.3 4.1
M.A. Aderogba et al. / South African Journal of Botany 87 (2013) 48–51
Acknowledgements This work was supported by the Claude Leon Foundation (ARN) and the University of KwaZulu-Natal, South Africa (MAA) in the form of Postdoctoral Fellowships.
References Aderogba, M.A., Kgatle, D.T., McGaw, L.J., Eloff, J.N., 2012. Isolation of antioxidant constituents from Combretum apiculatum subsp. Apiculatum. South African Journal of Botany 79, 125–131. Adesogan, E.K., 1981. The structure of penduliflaworosin, a new furanoid diterpene from Croton penduliflorus. Journal of the Chemical Society Perkin Transactions 1, 1151–1153. Anika, S.M., Shetty, S.N., 1983. Investigations on Croton penduliflorus Hutch.: II. A study on the mechanism of the hypotensive activity in pentobarbital-anesthetized dogs. Pharmaceutical Biology 21, 59–65. Eldeen, I.M.S., Elgorashi, E.E., Van Staden, J., 2005. Antibacterial, anti-inflammatory, anti-cholinesterase and mutagenic effects of extracts obtained from some trees used in South African traditional medicine. Journal of Ethnopharmacology 102, 457–464. George, S., Chaturvedi, P., 2008. Protective role of Ocimum canum plant extract in alcohol-induced oxidative stress in albino rats. British Journal of Biomedical Science 65, 80–85. Gohar, A., Gedara, S.R., Baraka, H.N., 2009. New acylated flavonol glycoside from Ceratonia siliqua L. seeds. Journal of Medicinal Plants Research 3, 424–428.
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Kolehmainen, E.T., Laihia, K.P., Hyötyläinen, J.M.I., Kauppinen, R.T., 1995. 1H, 13C and 17O NMR spectra study of chlorinated-3,4-dihydroxybenzaldehydes (protocatechualdehydes). Spectrochimica Acta 51A, 419–427. Lee, S.E., Hwang, H.J., Ha, J.S., Jeong, H.S., Kim, J.H., 2003. Screening of medicinal plant extracts for antioxidant activity. Life Sciences 73, 167–179. Maltese, F., Van der Kooy, F., Verpoorte, R., 2009. Solvent derived artifacts in natural products chemistry. Natural Product Communications 4, 447–454. Maron, D.M., Ames, B.N., 1983. Revised methods for the Salmonella mutagenicity test. Mutation Research 113, 173–215. Martin, T.S., Kikuzaki, H., Hisamoto, M., Nakatani, N., 2000. Constituents of Amomum tsao-ko and their radical scavenging and antioxidant activities. Journal of the American Oil Chemists' Society 77, 667–673. Moyo, M., Ndhlala, A.R., Finnie, J.F., Van Staden, J., 2010. Phenolic composition, antioxidant and acetylcholinesterase inhibitory activities of Sclerocarya birrea and Harpephyllum caffrum (Anacardiaceae) extracts. Food Chemistry 123, 69–76. Ranga, K., Krishnan, R., Delong, M., Kraemer, H., Carney, R., Spiegel, D., Gordon, C., McDonald, W., Dew, M., Alexopoulos, G., Buckwalter, K., Cohen, P.D., Evans, D., Kaufmann, P.G., Olin, J., Otey, E., Wainscott, C., 2002. Comorbidity of depression with other medical diseases in the elderly. Society of Biology and Psychiatry 52, 559–588. Reynolds, A., Laurie, C., Mosley, R.L., Gendelman, H.E., 2007. Oxidative stress and the pathogenesis of neurodegenerative disorders. International Review of Neurobiology 82, 297–325. Urquiaga, I., Leighton, F., 2000. Plant polyphenols, antioxidants and oxidative stress. Biological Research 33, 55–64. Vijayakumar, M., Govindarajan, R., Rao, G.M.M., Rao, C.V., Shirwaikar, A., Mehrotra, S., Pushpangadan, P., 2006. Action of Hygrophila auriculata against streptozotocininduced oxidative stress. Journal of Ethnopharmacology 104, 356–361.
Contents lists available at SciVerse ScienceDirect
South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Short communication
Acetylcholinesterase inhibitory activity and mutagenic effects of Croton penduliflorus leaf extract constituents M.A. Aderogba, A.R. Ndhlala, J. Van Staden ⁎ Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa
a r t i c l e
i n f o
Article history: Received 19 November 2012 Received in revised form 13 March 2013 Accepted 13 March 2013 Available online 15 April 2013 Edited by LJ McGaw Keywords: Acetylcholinesterase Croton penduliflorus Euphorbiaceae Mutagenicity
a b s t r a c t Croton penduliflorus is a medicinal plant widely employed in the management of inflammatory conditions, infections and oxidative stress related diseases. The activities demonstrated by leaf extracts indicate that they possess the ability to reduce oxidative damage to cells. Repeated column fractionation of the ethyl acetate fraction of a 20% aqueous methanol leaf extract of C. penduliflorus on Sephadex LH-20 afforded four phenolic compounds: quercetin-3-O-rhamnoside (1), kaempferol-3-O-rhamnoside (2), protocatechualdehyde (3A) and its solvent derived dimer (3B) along with p-hydroxybenzoic acid (4). Compound 3B is described for the first time and its significance in bioassay is briefly outlined. Structure elucidation of the isolated compounds was carried out using spectroscopic techniques. The inhibitory properties of the four compounds against acetylcholinesterase were determined using the microplate assay. The IC50 values of the isolated compounds ranged from 87.9 to 1231.9 μM, with compound 2 having the best inhibitory activity (IC50 = 87.9 μM). The four isolated compounds showed no mutagenic effects against Salmonella typhimurium tester strains TA98 and TA100. The moderate activity demonstrated by these compounds suggests that they could be helpful in the management of neurodegenerative disorders. © 2013 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Generation of excess reactive oxygen radicals and oxidative damage are believed to be involved in the pathogenesis of many neurodegenerative disorders. Accumulated studies have shown that plants used in traditional medicine have preventive and protective effects in oxidative stress conditions (Lee et al., 2003; Vijayakumar et al., 2006; George and Chaturvedi, 2008). Amelioration of oxidative stress conditions by higher plants has been associated with phenolic compounds such as flavonoids and other dietary polyphenols. These bioactive molecules exhibit multiple biological effects such as antithrombotic, anti-inflammatory, anticarcinogenic and antimutagenic effects as a result of their antioxidant properties (Urquiaga and Leighton, 2000). Croton penduliflorus Hutch belongs to the family Euphorbiaceae. It is widely employed in the management of inflammatory conditions, infections and oxidative stress related diseases (Anika and Shetty, 1983). The activities demonstrated by the plant extract support that its constituents possess the ability to reduce oxidative damage to cells. Previous phytochemical investigation of the root bark afforded penduliflaworosin, a clerodane-type diterpene ent-(1 2R)-methyl-15, 16-epoxy-9,10-friedolabda-5(10),13(16),14-trien-19-oate 20, l2-lactone (Adesogan, 1981).
⁎ Corresponding author. Tel.: +27 33 2605130. E-mail address: [email protected] (J. Van Staden).
Excess free radicals generated in the body attack most cellular macromolecules such as proteins (enzymes), lipids and DNA leading to oxidative stress and neurodegenerative disorders. Antioxidant compounds are useful in ameliorating these conditions (Reynolds et al., 2007). Acetylcholinesterase (AChE) catalyses the degradation of the neurotransmitter acetylcholine, resulting in choline and an acetate group (Ranga et al., 2002). Insufficient cholinergic function and neurotransmitter disturbances are considered as prominent among the main pathological features in central nervous system disorders (Eldeen et al., 2005). In this study, we have investigated C. penduliflorus leaf extracts in our efforts to find phytochemical agents that could be effective in the prevention and management of neurodegenerative conditions. 2. Material and methods 2.1. Plant material, collection and extraction The leaves of C. penduliflorus were collected at Obafemi Awolowo University (OAU), Ile-Ife, Nigeria, in June 2011. It was identified by Mr. G. Ibhanesebhor of the Herbarium Section, Department of Botany (OAU). A voucher specimen (IFE 16401) was deposited at the University Herbarium at the Department of Botany. The powdered leaves (700 g) were extracted with 8 L of 20% aqueous methanol at room temperature for 24 h and filtered. The crude extract was concentrated in vacuo at 40 °C to about ⅓ of the original volume. This was sequentially extracted
0254-6299/$ – see front matter © 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sajb.2013.03.013
M.A. Aderogba et al. / South African Journal of Botany 87 (2013) 48–51
with n-hexane (3 × 800 mL), dichloromethane (3 × 800 mL), ethyl acetate (3 × 1 L) and finally n-butanol (2 × 500 mL). The solvent fractions were individually concentrated to dryness in vacuo to afford four solvent extracts. 2.2. Isolation of compounds from C. penduliflorus extract The EtOAc fraction (1.10 g) was fractionated on a Sephadex LH-20 column using 95% DCM/MeOH as eluent. This was followed by an increasing gradient of MeOH up to 30%. The fractions collected were analysed by TLC using DCM/MeOH (9:1) as solvent system. This afforded four fractions (A1–A4). Fraction A4 (25 mg) gave a single spot (compound 1). Fraction A2 purified on Sephadex LH-20 using EtOAc/MeOH (9:1) as eluent followed by an increasing gradient of MeOH up to 15%. Analysis of the fractions collected on TLC plates using EtOAc/MeOH (9:1) yielded compound 2 (7 mg). Purification of fraction A1 on Sephadex LH-20 using EtOAc/MeOH (9:1) and analysis of the fractions by TLC using 100% EtOAc yielded compound 3 (18 mg). Further fractionation of fraction A3 on Sephadex LH-20 using EtOAc/MeOH (9:1) followed by an increasing gradient of MeOH up to 25% yielded compound 4 (5 mg) on analysis of the fractions collected on TLC plates using EtOAc/MeOH (5:1) as solvent system. 2.3. Acetylcholinesterase (AChE) inhibitory assay The microplate assay for AChE inhibition activity was performed based on Ellman's method as detailed by Moyo et al. (2010). 2.4. Ames test for mutagenic properties Mutagenicity was tested using the Salmonella microsome assay based on the plate-incorporation procedure with Salmonella typhimurium tester strains TA98 and TA100 without metabolic activation (Maron and Ames, 1983). 3. Results and discussion Structure elucidation of the compounds was carried out using spectroscopic techniques: Mass spectrometry (TOF MS ES) and NMR (1D and 2D, 500 MHz). The compounds were identified as: quercetin-3-Orhamnoside (1, Aderogba et al., 2012), kaempferol-3-O-rhamnoside (2, Gohar et al., 2009) and p-hydroxybenzoic acid (4, Martin et al., 2000). Compound (3A) was obtained as brown powder. Its mass spectrum showed the exact molecular ion peak as the base peak, m/z = 137.0237, [M-H] −, consistent with the molecular formula C7H6O3. The 1H NMR (acetone-d6) of 3A showed signals representing a trisubstituted phenyl ring at δ: 7.36 (1H, d, J = 1.9 Hz, H-2) meta-related coupled protons, 7.34 (1H, dd, J = 1.9, 8.0 Hz, H-6) meta and orthorelated protons and 7.00 (1H, d, J = 8.0 Hz, H-5) ortho-related protons. An aldehydic proton appeared at δ 9.77. The 13C NMR (acetone-d6) of 3A showed signals as follows: δ 131.0 (C-1), 115.2 (C-2), 146.7 (C-3), 152.6 (C-4), 116.2 (C-5), 125.5 (C-6) and 191.3 (CHO). Compound 3A was identified as 3,4-dihydroxybenzaldehyde, protocatechualdehyde (Kolehmainen et al., 1995). Further NMR studies of 3A in MeOD showed that in this solvent, 3A existed in a different form 3B (solvent derived artifact). The 1H NMR of 3B (Table 1) are characterized by broadness of the resonance lines between δ 7.31–6.73. 13C NMR and DEPT spectra of this compound revealed the presence of 14 carbons, (Table 1). DEPT showed the presence of 8 CH carbons. These were assigned as 6 aromatic CH, 1 aldehydic CH and the other one CH in the non-aromatic region. The remaining 6 carbons were assigned as quaternary carbons out of which four were oxygenated. The positions of attachment of the two monomers and the oxygenated carbons were established by HMBC correlations (Fig. 1). Compound 3B was identified as a dimer of 3,4dihydroxybenzaldehyde. Formation of compound 3B (artifact) in this work was as a result of MeOD used for the NMR of compound 3A.
Table 1 1 H (500 MHz) and
13
49
C (125 MHz) NMR chemical shifts of compound 3B in MeOD.
1
No
H
1 2 3 4 5 6 CHO
7.30 (1H, b d, J = 1.9 Hz)
6.92 (1H, d, J = 8.7 Hz) 7.31 (1H,b d, J = 2.0 Hz) 9.69 (1H, S)
13
C
No
131.2 115.7 147.5 154.0 116.2 126.7 193.4
1′ 2′ 3′ 4′ 5′ 6′ 7′
1
13
H
6.74 (1H, b d, 1.8 Hz)
6.73 (1H, b s) 6.85 (1H, b d, 1.5 Hz) 5.21 (1H, s)
C
131.5 115.2 146.6 147.0 116.6 119.8 105.2
There is no previous report of existence of 3,4-dihydroxybenzaldehyde in this dimeric form. The structures of the isolated compounds are presented in Fig. 2. Organic solvents are critical to extraction and purification of secondary metabolites from medicinal plants. Our observation calls into question the stability of secondary metabolites in bioassay media. There are apparent chemical reactions that took place in MeOD, by activation of the compound (3A) aldehydic function to form a dimer (3B). However, after solvent removal the sample remains unchanged as demonstrated by the NMR spectra in acetone-d6. Changes like this may alter the activity of secondary metabolites in the bioassay medium. This could lead to loss of activity, formation of toxic compounds or difficulty in reproducibility of bioassay results. The main objective of phytochemical investigation of medicinal plants to offer a unique opportunity for identifying novel therapeutic compounds and providing useful lead compounds will be greatly affected by occurrences like this. It is important that stability tests are conducted on secondary metabolites from medicinal plants before experimentation (Maltese et al., 2009). The results of AChE inhibitory activity of the four compounds are shown in Table 2. The IC50 values ranged from 87.9 μM (compound 2) to 1231.9 μM (compound 3A). The percentage inhibition of galanthamine (positive control) at a concentration of 20 μM was 93.5% and the IC50 value was 1.8 μM. Isolated compounds demonstrated moderate AChE inhibitory properties. Table 3 presents the results of the mutagenicity test as the spontaneous reversion response of the S. typhimurium tester strains TA98 and TA100 to the different dilutions of the four compounds. The results revealed that all four compounds were non-mutagenic towards the S. typhimurium strains TA98 and TA100. These compounds, therefore do not have the capacity to cause a base pair substitution.
O
H9.68 C
H 7.31 126.7 6 5 116.2 H 6.92
193.4
131.2 1
H 7.30
H 6.74
2 115.7 H 5.21
3 147.5 154.0
OH
2'
105.2
4
O
7' OH
OH
115.2
131.5 1'
3'
146.6 4'
147.0 OH
6' 5' 116.6 119.8 H 6.85
H 6.73
Fig. 1. HMBC correlations of protocatechualdehyde dimer (3B).
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M.A. Aderogba et al. / South African Journal of Botany 87 (2013) 48–51
R OH
HO
O
O OH
OH
O
O
OH
OH
R
Compound 1
OH
2
H
O
O
O
CH
OH C
CH
OH H O
OH
OH OH
OH
OH
OH 3B
3A
4
Fig. 2. Structures of isolated compounds from Croton penduliflorus leaf extract.
4. Conclusions Investigation of C. penduliflorus for possible active constituents against neurodegenerative disorders afforded four phenolic compounds identified as: quercetin-3-O-rhamnoside (1), kaempferol-3-O-rhamnoside (2), protocatechualdehyde (3A) and its solvent derived dimer (3B) and p-hydroxybenzoic acid (4). These compounds are reported from the leaf extracts of C. penduliflorus for the first time. Phenolic compounds are known to exhibit several pharmacological and biological activities. It was therefore not surprising to observe the activities exhibited by these four compounds against AChE enzyme. All compounds showed a dose dependent increase in the percentage inhibition of the enzyme. Structural transformation of compound 3A in two different solvents
Table 2 AChE inhibitory activity (IC50 mg/mL) of four isolated compounds from Croton penduliflorus leaf extract. Compound (1) (2) (3A) (4)
Quercetin-3-O-rhamnoside Kaempferol-3-O-rhamnoside Protocatechualdehyde p-Hydroxybenzoic acid Galanthamine
% inhibition (1 mg/mL)
IC50 (μM)
60.3 68.3 54.6 51.6 93.5
133.9 87.9 1231.9 971.0 1.8
± 3.1 ± 5.5 ± 0.6 ± 4.9 (20 μM)
± ± ± ± ±
7.9 6.0 7.2 7.2 0.5
used during analysis had a significant effect on their bioactivity. Compound 2 exhibited highest AChE inhibitory activity and all four compounds showed no potential mutagenic effects towards the S. typhimurium strains TA98 and TA100.
Table 3 Number of revertant colonies of Salmonella typhimurium strains TA98 and TA100 induced by the four isolated compounds from Croton penduliflorus leaf extract. Compound
Concentration (μg/mL)
Number of His + revertants
(1)
Quercetin-3-O-rhamnoside
(2)
Kaempferol-3-O-rhamnoside
(3A)
Protocatechualdehyde
(4)
p-Hydroxybenzoic acid
1000 100 10 1000 100 10 1000 100 10 1000 100 10 2
23.4 22.1 19.4 29.4 21.4 23.9 22.4 23.1 23.0 20.1 19.9 21.3 208.0 22.0
4-NQO 10% DMSO (in water)
TA98
4-NQO; 4-nitroquinoline-oxide was used as a positive control. 10% DMSO (in water) was used as a negative control.
TA100 ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.0 1.1 2.2 3.9 1.3 2.2 0.1 1.3 1.0 3.5 2.1 1.1 0.6 1.6
183.2 197.3 167.8 145.4 176.0 164.6 133.9 166.3 158.3 157.5 137.6 144.9 992.3 174.5
± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.2 3.1 6.2 1.1 2.1 3.3 5.2 2.1 7.0 2.2 10.1 5.2 9.3 4.1
M.A. Aderogba et al. / South African Journal of Botany 87 (2013) 48–51
Acknowledgements This work was supported by the Claude Leon Foundation (ARN) and the University of KwaZulu-Natal, South Africa (MAA) in the form of Postdoctoral Fellowships.
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