Many different mechanisms may contribute to oxidative processes in the body, for example Fenton reactions, where transition metal ions are involved in the generation of ROS which can cause damage to various body structures such as lipids, proteins and DNA. Therefore, a variety of different antioxidant assays were studied to determine by which mechanism(s) each herbal formulation exerts their antioxidant activity.
Compounds such as, phenols and flavonoids are considered to be major contributors to the antioxidant activity of medicinal plants. The ability for phenolics to scavenge free radicals may be due to the presence of many phenolic hydroxyl group (Siddique et al., 2010). The antioxidant activities may be attributed to the their redox properties, which allow them to act as singlet oxygen quenchers, hydrogen donators and reducing agents, as well as their metal chelating abilities and can also act via inhibition of some enzymes involved in radical generation, such as lipoxygenases and xanthine oxidase (Pereira et al., 2009). In the present study, five different assays were performed to determine and compare the antioxidant properties of the five different traditional herbal medicines.
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Phenolic compounds are known to possess antioxidant activity, hence the phenolic composition of the extracts was first evaluated. The contents of total phenolic compounds were calculated in a dose of the herbal medicines taken per day. It was found that F1 contained the highest phenolic content, followed by F2 and F4. F5 had the lowest level of phenolics (Table 4.2). For proanthocyanidin content, F1 contained the highest amount, followed by F2 and F4, but the least amount was found with F3. The same trend as total proanthocyanidins was observed with total flavonoids.
The antioxidant potential of the herbal medicines was first evaluated using the FRAP assay. This assay measures the ferric reducing ability of plasma (FRAP). In the presence of a reducing antioxidant, Fe (III)-TPTZ is reduced to Fe (II)-TPTZ at low pH, which gives an intense blue colour at 593 nm.
When the reducing potential of the herbal formulations was evaluated using the FRAP assay, the results obtained, expressed as Fe (II) equivalents and presented in Figure 4.1, showed that F2 showed the highest FRAP value (1857 ± 7.5) in a dose of the herbal medicine per day while the lowest value was demonstrated by F5 (114 ± 1.1). All the extracts possessed reducing property, but a dose of F2 was found to be more effective. A very high significant (P < 0.01) correlation was obtained when FRAP values were compared with the total phenolics (r = 1.00), total proanthocyanidins (r = 0.985) and total flavonoids (r = 1.00). Therefore, good correlation suggests that the phenolic compounds present play an important role as antioxidants for the FRAP assay. However, a higher phenolic content was found to be present in F1 (Table 4.2) rather than in F2. A possible explanation may be due to synergistic effect being more pronounced in F2 for its ability to act as reducing agent. Synergistic effect relies on the working together effect of a combination of plant components. The effect produced by the combination of constituents is greater than produced with individual constituents. F2 contains Ginko biloba, Salvia miltiorrhiza and Hypericum perforatum. Synergistic action may also occur between the different phytochemicals present in these three plants.
A harmful ROS is the hypochlorous acid. Neutrophil enzyme myeloperoxidase oxidizes Cl- ions at sites of inflammation, resulting in the production of HOCl. This ROS in turns inactivates catalase, an antioxidant enzyme (Hazra et al., 2010). Figure 4.3 shows the HOCl scavenging potential of the herbal medicines, where ascorbic acid served as a positive control. All samples possessed the ability to scavenge HOCl in a dose dependent manner. The highest percentage of scavenging activity observed with a normal dose concentration of herbal medicine was exhibited by F2, F3 and F4, although F1 and F5 did show a relatively high percentage scavenging potential. Considering the IC50 values of the formulations, F2 (IC50 = 0.00021 ± 0.00004 mL/mL) and F3 (IC50 = 0.0274 ± 0.002 mg/mL) were found to be more potent, with comparable result to ascorbic acid (IC50 = 0.0066 ± 0.0002 mg/mL) which was used as positive control (P > 0.05). Thus, it can b suggested that F2 and F3 possess important OCl- scavenging capacities, making the herbal medicines good antioxidants. The ability of the extract to react with HOCl showed satisfactory correlation (P < 0.05) with the total phenolics (r = 0.638), total proanthocyanidins (r = 0.625) and total flavonoids (r = 0.633). Thus, the phenolic content of the extracts are said to participate in the antioxidant property.
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Hydroxyl radicals are highly reactive and can react with a variety of molecules found in living cells, such as amino acids, sugars, lipids and nucleotides. It has been implicated in many pathologies, namely neurodegenerative diseases.
The hydroxyl radical scavenging capacity of the five herbal medicines was also evaluated via the deoxyribose assay. In the deoxyribose assay, there is the reduction of Fe3+ to Fe2+, thereby causing formation of Fe2+-EDTA complex. The decomposition of H2O2 by the complex causes the formation of hydroxyl radical. This in turn attack deoxyribose and causes its fragmentation. The oxidative degradation can be detected by the formation of a pink chromogen with TBA and TCA. If the extracts have hydroxyl radical scavenger capacity, they will compete with deoxyribose for hydroxyl radicals and decrease chromogen formation (Vladimir-Knezevic et al., 2009).
The extracts showed a positive effect on the protection of deoxyribose from degradation, as shown by the dose-dependent curves in Figure 4.5. The highest scavenger of hydroxyl radical was observed with F2 (77% inhibition of deoxyribose oxidation), as well as the smallest IC50 value (0.0006 ± 0.00009 mL/mL). The IC50 values of F2 and F3 were close to the positive control, which is gallic acid (P > 0.05), thus being effective in reducing damage to deoxyribose. A relatively good relationship (P < 0.05) was observed inhibition of deoxyribose damage and phenolic content (r = 0.599). Similar relationship was observed with proanthocyanidin content (r = 0.588) and flavonoid content (r = 0.594).
An important mechanism of antioxidant action is the chelation of iron (II) ions which serves as catalysis in Fenton reactions. During Fenton reaction, there is the formation of hydroxyl radicals and decomposition of hydroperoxide due to oxidative processes catalysed by bivalent transition metal ions. Iron chelation can delay these processes and the ability of the herbal medicines to chelate iron (II) ions was determined. Antioxidant activities of medicinal plants may also be attributed to their ability to chelate transition metal ions. They may act as catalysts for the formation of reactive oxygen species.
The iron (II) chelating potential is an antioxidant assay which allows an estimation of the chelating activity of existing chelators present in the extracts through measurement of colour reduction. This assay uses ferrozine, which can quantitatively form complexes with Fe2+, but in the presence of chelating agents there is a decrease in the colour of the complex. Therefore, measurement of the colour reduction allows an estimation of the chelating activity of the extracts. The transition metal ion Fe2+ allows the formation and propagation of many radicals due to its ability to move single electrons. In order to avoid the generation of ROS related to redox active metal catalysis, metal ions should be chelated (Ebrahimzadeh et al., 2008).
Figure 4.7 shows that all extracts can act as iron (II) chelators to a certain extent. All extracts demonstrated a dose-dependent activity, with the highest chelating activity observed with F5 (90%). The lowest activity was found with F1 (56%). As presented by Figure 4.8, F2 was found to have the lowest IC50 value (0.0027 ± 0.0002 mL/mL) and was 2.25 greater than the IC50 value of EDTA (0.0012 ± 0.0005 mg/mL), while F5 had the highest IC50 value of 1.00 ± 0.183 mg/mL, which is almost close to that of F1 (0.691 ± 0.147 mg/mL). Significant (P < 0.01) negative correlation was observed between the IC50 for iron (II) chelating activity and with phenolic content (r = 0.693), proanthocyanidin content (r = 0.682 and flavonoid content (r = 0.692).
Throughout this study, it has been observed that all the five herbal medicines studied possess antioxidant properties and this property may be largely responsible for their use in the treatment of ND. The results show that phenolic compounds contribute largely to the antioxidant behavior of the herbal medicines. F1, F2 and F4 contain more than one plant material while F3 and F5 contain only one plant present in the herbal formulation (Table..).
Verbena officinalis, which is present in F1, has been shown to exert its antioxidant action particularly through radical scavenging properties (Casanova et al., 2008), as well as Rosmarinus officinalis which is mainly due to the presence of RA (Al-Sereitia et al., 1999). This study has demonstrated that a mixture of Rosmarinus officinalis and Verbena officinalis in F1 can act as antioxidant through different mechanisms. However, in the deoxyribose assay, 50% inhibition was not reached with a dose of F1. This shows that F1 are not good scavengers of hydroxyl radical.
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F2 contains 3 plant material: Salvia miltiorrhiza, Hypericum perforatum and Gingko biloba. Antioxidant activity of Salvia miltiorrhiza has previously been reported. Tan IIA was found to inhibit NO production. Additionally, danshensu, tanshinone, Sal A and Sal B have demonstrated anti-lipopeorxidant activity via scavenging of superoxide anion radical (Wang, 2010). Hypericum perforatum has been the subject of a spectrum of antioxidant assays. Altun et al. (2013) found that H. perforatum extract did not possess metal-chelating activity, but did show antioxidant activity in the FRAP assay. Other antioxidant mechanism through which H. perforatum exerts its action is through DPPH, DMPD scavenging activity, nitric oxide inhibition and PRAP assay. Previous work carried out with Gingko biloba also demonstrated antioxidant activity, which may be due to its free radical scavenging action. It has also been shown to activate gene or protein expression of endogenous antioxidants, such as glutathione and manganese superoxide dismutase (Luo and Smith, 2004; Christen, 2004). Kaur et al. (2013) found that activity of antioxidant enzymes (such as catalase and superoxide dismutase) was significantly improved following treatment with Gingko biloba extract.
F2 and F3 were found to be very good scavengers of HOCl, as well as very good scavengers of hydroxyl radical, with comparable result to the positive control. However, F2 showed better iron (II) chelating activity compared to F3. The antioxidant activity of F2 was more pronounced that observed with F3 (the herbal product contains only Gingko biloba). The maximum % inhibition of free radical observed with a dose of the herbal medicine was found to be higher in F2 compared to F3. The higher activity of F2 may be due to synergistic effect of the different phytochemicals present in the mixture or due to the presence of plant components other than present in Gingko biloba.
The antioxidant results demonstrated with F4 showed that F4 can work via different antioxidant mechanisms, namely by its reducing potential in the FRAP assay, as a scavenger of hypochlorous acid, as a scavenger of hydroxyl radical and as a chelator of iron (II). However, its iron (II) chelating property was shown to be better, with a smaller IC50 value (0.255 ± 0.039 mg/mL), compared to HOCl scavenging capacity and hydroxyl scavenging property. Radix Angelicae sinensis, also present in F4, was reported to decrease malondialdehyde levels and increase activity of superoxide dismutase (Li et al., 2007; Kuang et al., 2008) and Radix ginseng, containing saponins, can reduce free radical damage and increase glutathione peroxidase and superoxide dismutase activities (Yan et al., 2007). Therefore, these two plant material can be responsible for the antioxidant property shown with F4.
Centella asiatica has been previously reported to inhibit the activity of ROS and decrease lipid peroxidation (Tiwari et al., 2011; Hashim et al., 2011). The antioxidant mechanism via lipid peroxidation assay was not assessed throughout this study. Hence, F5 might would have shown a better inhibition of lipid peroxidation compared to the antioxidant methods used in this study.
Despite the antioxidant property of phytophenolics, they can act as pro-oxidants, under certain conditions, such as in systems where redox-active metals are present or at high doses. In the presence of oxygen, ROS and other organic radicals can be formed through the redox cycling of phenolics by transition metals such as copper and iron. These ROS can cause damage to biological molecules, for example lipids, proteins and DNA (Sakihama et al., 2002; Yordi et al., 2012).
The copper-phenanthroline assay is a method for assessing the pro-oxidant activity. In this assay, the copper-phenanthroline complex can induce strand breakage in DNA. Damage to DNA by the copper-phenanthroline system involves hydroxyl radicals. If the herbal medicines used in the study possess pro-oxidant activity, they will generate hydroxyl radical, which will react with the copper-phenanthroline complex and caused damage to DNA. The assay measures the amount of DNA damage. If the extracts possess pro-oxidant activity, increasing the concentration of the extracts will increase the damage caused to DNA (Aruoma and Cuppett., 1997).
Table 4.6 shows that a normal dose concentration of the herbal medicine for F1, F4 and F5 did not promote DNA damage since the extent of damage were lower than that for ascorbic acid, which was used as reference. However, pro-oxidant activities were observed at normal dose concentration of F2 and F3, where their extents of damage were higher than the basal value for ascorbic acid. Ascorbate can generate hydroxyl radical through its ability to redox-recycle and sustain the supply of Fe2+, but no clear evidence has been shown in vivo (Luximon-Ramma et al., 2005). Thus, despite the pro-oxidant activity observed with F2 and F3, there may be changes that occur to the phytoconstituents of the herbal medicine, such as enzymatic action and pH changes that occurs in the digestive tract, which renders them harmless. Moreover, the antioxidant activities of these herbal medicines may compensate for their pro-oxidant activity, thus cancelling their ability to cause oxidation. No significant correlation was found with pro-oxidant activity of the herbal medicine extracts and phenolic compounds.
Another approach to the treatment of ND includes the improvement of brain cholinergic activity by inhibition of AChE, resulting in hydrolysis of ACh and termination of nerve impulse transmission at cholinergic synapses. Several drugs, such as tacrine, galantamine and donepezil, were proven to be effective AChE inhibitors and delayed disease progression, but several adverse reactions were also involved with their use. Thus, research has been carried out on plants to find potential natural products which can act as AChE inhibitors, with lesser side effects. A variety of plants have been found to possess AChE inhibitory activity. Phytochemicals which may be responsible for this action may be alkaloids, ursolic acid, lignans, flavonoids, terpenoids and coumarins (Zhou et al., 2011; Tavares et al., 2012). Several herbal formulations are now available for the treatment of various ND. In this present study, the activities of the five traditional herbal medicines were investigated for their inhibition of acetylcholinesterase.
The results (Figure 4.10) indicate that all the five formulations possess anticholinesterase activity. No significant difference was found with IC50 value of F2 and F3 compared to galantamine which was used as reference. This shows that these two herbal formulations have potent activity in inhibiting acetylcholinesterase enzyme, which is a major aim in the treatment of several neurological disorders, such as AD.
The strongest AChE activity was exhibited by F2 (IC50 = 0.0133 ± 0.004 mL/mL). F2 contains Ginkgo biloba, Hypericum perforatum and Salvia miltiorrhiza and F3 contains Ginkgo biloba leaves. Hypericum perforatum was reported to be responsible for neurochemical modulation, which may be due to the presence of flavonoids, namely hyperforin and hypericin (Gomes et al., 2009). Altun et al. (2013) reported that methanolic extract of Hypericum perforatum did possess anticholinesterase activity. The inhibitory activity of AChE with Salvia miltiorrhiza was found to be due to the presence of triterpenoids (Lin et al., 2008). Ginkgo biloba has been shown to have potential activity in reducing acetylcholinesterase activity, also reported by Das et al. (2002). Another mechanism of action which may be useful in the treatment of AD may be due to its ability to inhibit Aβ formation (Shi et al., 2010), which is a major pathological hallmark in AD.
F4 contains a variety of herbal material, as shown in Table 3.1, Radix Angelicae sinensis, which is made up of 12% F4, contains Z-Ligustilide which was reported to increase choline acetyl transferase activity and decrease AChE activity (Kuang et al., 2008). Inhibition of AChE activity may not be the only mechanism by which F4 can act in ND. F4 also contains Radix ginseng which has been shown to possess central cholinomimetic and catecholaminomimetic activity (Yan et al., 2007).
F5 showed an IC50 value of 27.81 ± 1.57 mg/mL. F5 contains Centella asiatica as active ingredient. Its AChE inhibitory activity may be due to the presence of triterpenes (Table 2.3) which were reported to possess cholinergic mechanisms (Gomes et al. 2009). Apart from AChE inhibition, other mechanisms may be responsible for the potential efficacy of F5 in treating ND (Table 2.4), such neuroprotective effect and sedative effect.
Among the five formulations, F1 was found to be the less potent in decreasing AChE activity. Moderate inhibition of acetylcholinesterase was found with F1 (Figure 4.10). F1 contains Rosmarinus officinalis and its anti-acetylcholinesterase activity may be due to the synergistic action between 1,8 cineole and 2-pinene (Faixova and Faix, 2008). F1 also contains Verbena officinalis. Lai et al. (2006) reported that this plant causes attenuation of Aβ- triggered DEVD- and VDVAD-cleavage, as well as phosphorylation of interferon-inducing protein kinase.
However, there was no correlation between anticholinesterase ability and the total phenolic compounds was found.