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Acetylcholinesterase inhibitory and antioxidant activity
The inhibition of the enzyme, AChE and scavenging of free radicals or ROS are very important in identifying new agents or potential leads which may be useful in the treatment of AD. The dichloromethane/methanol (1:1) and ethyl acetate extracts were observed to have higher inhibition of AChE than the methanol and water extracts. This may indicate that the plants investigated in the present study contain AChE inhibitory compounds with intermediate polarity.
Similar findings have been reported in literature where authors screened several extracts of different plants for AChE inhibition, and plants extracted with solvents of intermediate polarity had higher activity (Şenol et al., 2010a, b; Noridayu et al., 2011). However, in contrast to the AChE inhibitory activity, the ethyl acetate extracts showed poor radical scavenging activity, while the methanol extracts contained good antioxidant activity. A variety of bioactive compounds that could be responsible for the observed bioactivities have been reported in some of the screened medicinal plants or related genera.
Good inhibition of AChE was observed for the organic extracts of S. tiliifolia in the present study. It showed a 54.2% inhibition of the enzyme at the highest concentration tested, and an IC50 value of 1 mg/ml. Numerous species of the genus Salvia have been used since ancient times in folk medicine and have been subjected to extensive research intended to identify biologically active compounds. Systematic and mechanistic studies into the effects of these extracts have revealed multiple activities potentially relevant to brain function, aging and the preventive and symptomatic treatment of mild cognitive impairment and AD (Loizzo et al., 2010). The n-hexane extract of S. leriifolia has been reported to show good inhibition of AChE with an IC50 value of 0.59 mg/ml. This activity is attributed to the presence of monoterpenes, including sabinene, δ-3- carene and α-terpinene, different sesquiterpenes and three diterpenes (neophytadiene, phytol and vulgarol B). The essential oil and ethanol extract of S. officinalis as well as the essential oil of S. lavandulaefolia have been shown to possess anticholinesterase activity (Perry et al., 1996), as have the major components of the essential oil, α-pinene, 1, 8-cineole, and camphor (Perry et al., 2000). The AChE inhibitory activity of some of the terpenes present in Salvia species, have been reported. δ-3-Carene is reported to be a potent inhibitor of AChE with an IC50 value of 200 μM, sabinene inhibited AChE with an IC50 value of 176.5 μg/ml, while α-terpinene showed inhibition with IC50 value of 1000 μM (Miyazawa et al., 1997; Herholz et al., 2005; Miyazawa and Yamafuji 2005). Similar compounds may be present and responsible for the good AChE inhibition observed with the DCM/MeOH (1:1) extracts of S. tiliifolia. S. brachypetala roots showed dose-dependent inhibition of AChE with a 62.8% inhibition of the enzyme for the organic extracts at the highest concentration tested. The organic root extracts also contained good antioxidant activity. Its neurological activity is supported by Stafford et al. (2007), who reported good monoamine oxidase (MAO) B inhibitory activity in the aqueous and ethanol extracts of the bark of this plant species. S. brachypetala contains stilbenes and phenolics which have been shown to have good radical scavenging activity (Glasby, 1991).
The family Caesalpiniaceae has been shown to contain several diterpenes with biological activity. The clerodane diterpenes present in fruit pulp extract of Detarium microcarpum Guill. & Perr. showed antifungal activity and inhibition of acetylcholinesterase (Cavin et al., 2006).
The presence of clerodane or similar diterpenes in C. mimosoides may be responsible for the good AChE inhibitory activity seen for the organic root extracts. Several plants in the family Caesalpiniaceae, including Caesalpinia bonducella, Cassia auriculata, C. fistula, Bauhinia racemosa and B. rufescens have also been reported to contain good antioxidant activity (Kumar et al., 2005; Kumar et al., 2008; Motlhanka, 2008; Aliyu et al., 2009; Shukla et al., 2009), which supports the present finding for the organic root extracts of C. mimosoides.
The genus Buddleja has been reported to contain various terpenoids; monoterpenes, sesquiterpenes, diterpenes and triterpenoids which have been reported to show good inhibition of AChE (Houghton et al., 2003; Loizzo et al., 2010). Some of the sesquiterpenes have also been shown to contain anti-inflammatory activity, which make it relevant in the treatment of AD (Liao et al., 1999). Various species of Buddleja have been found to contain luteolin, and its glycosides have been shown to contain good antioxidant and anti-inflammatory activity (López-Lázaro, 2009). It is therefore postulated that the presence of these and related compounds in B. salviifolia may be responsible for the antioxidant and AChEI activity shown in this study.
Isolation and structural elucidation of compounds from Boophane disticha
Nuclear Magnetic Resonance (NMR) spectroscopy and Quadrupole Time-of-Flight (QTOF) are the techniques most often used in the structural elucidation of compounds. NMR exploits the magnetic properties of nuclei present in the atoms which results in a characteristic spectrum and the information obtained is useful in determining the structure of compounds isolated. Various 1D and 2D experiments were used (such as proton – 1H, carbon – 13C, Heteronuclear Multiple Bond Correlation – HMBC, Heteronuclear Single Quantum Coherence – HSQC, Correlation Spectroscopy – COSY and Distortionless Enhancement by Polarisation Transfer – DEPT) to determine the proton to carbon relation and the chemical environment they are in for structure elucidation (Silverstein et al., 2005). High resolution Time-of-Flight Mass Spectroscopy (HRTOFMS) can be used to confirm the molecular mass of the compound and together with QTOF, it provides information of the accurate mass of the compounds under investigation and the resulting fragmentation pattern obtained is used in conjunction with NMR data for structure elucidation. QTOF is a technique used to determine the mass of the compound under investigation using the principle that smaller (lighter) ions will travel faster through a flight tube than larger (heavier) ions. The velocity of an ion depends on its mass-to-charge ratio. The velocity that an ion obtains in the TOF analyser is therefore used to determine the mass of the ion and is a more accurate estimate (Silverstein et al., 2005; Pavia et al., 2009).
Chapter 1: Literature Review
1.1 Neurodegeneration
1.2 Alzheimer’s disease
1.3 Pathology of Alzheimer’s disease
1.4 Current therapeutic approaches for Alzheimer’s disease
1.5 Traditional Medicine
1.6 Plants used traditionally to treat age-related/neurological disorders
1.7 Study Aim
1.8 Objectives
Chapter 2: Materials and Methods
2.1 Reagents and Chemicals
2.2 Plant material
2.3 Determination of acetylcholinesterase inhibition
2.4 Antioxidant activity
2.5 Phytochemical screening
2.6 Compound isolation
2.7 Structural elucidation of compounds
2.8 Cytotoxicity studies
2.9 Statistical analysis
Chapter 3: Results
3.1 Acetylcholinesterase inhibitory activity
3.2 Antioxidant activity and polyphenolic content
3.3 Isolation of compounds from Boophane disticha
3.4 Cytotoxicity studies
Chapter 4: Discussion
4.1 Acetylcholinesterase inhibitory and antioxidant activity
4.2 Isolation and structural elucidation of compounds from Boophane disticha
4.3 Cytotoxicity studies
Chapter 5: Conclusion
References
