1. STASTISTICAL ANALYSIS
The statistical analysis was carried out using SPSS 16.0 for window in which One-Way ANOVA was used and P < 0.05 were set as significant. IC50 was calculated using GraphPad PRISM, version 5.00 for windows (GraphPad software).
  1. RESULTS
  2. Quantitative phytochemical contents of Plectranthus glandulosus leaves
Quantitative phytochemical screening of P. glandulosus leaves revealed the presence of saponins, flavonoids, and terpenoids. However, the plant leaves is highly concentrated in flavonoids (36.2%), compared to terpenoids (25.6%) and saponins (18.3%) (Table 1).
  1. In vitro antioxidant activities
  2. Hydrogen peroxide scavenging activity
Hydro ethanolic extract is a better scavenger of hydrogen peroxide than aqueous and ethanolic extracts. The scavenging activity induced with the hydro ethanolic extract varied significantly (F(4, 10)=4871; P˂0.001) from 57.35% (at 25 μg/ml) to 91.78% (at 400 μg/ml) (table 2) while among fraction ethyl acetate exhibited the most potent significant (F(4, 10) =4035; P˂0.001) hydrogen peroxide scavenging activity ranging from 63.72% (at 25 μg/ml) to 89.58 (at 400 μg/ml) (Table 3) . The positive control ascorbic exhibited a significant scavenging activitiy ranging from 63.72 to 93.25% when applied at 25 μg/ml and 400 μg/ml respectively. The inhibition concentration (IC50) value was 18.33μg/ml for hydro ethanolic extract at correlation coefficient value (r) of 0.9756. The IC50value of the ethyle acetate fraction was found to be 13.63 μg/ml at the correlation (r) of r=0.9976 13.63 μg/ml at the correlation (r) of r=0.9976 and 15.39 μg/ml for ascorbic acid at correlation coefficient value (r) of 0.9876 (table 2).
Nitric oxide scavenging activities
Tested with the aqueous, ethanolic and hydro ethanolic extract, a moderate nitric oxide scavenging activity was observed by the hydro ethanolic extract which varying significantly (F (4, 10)=3268; P˂0.001) from 22.58% (at 25 μg/ml) to 51.10% (at 400 μg/ml) (Table 4). Among fraction the most potent fraction ethyl acetate exhibited a moderate nitric oxide scavenging activity ranging significantly (F (4, 10) =3148; P˂0.001) from 29.75% (at 25 μg/ml) to 56.81% (at 400 μg/ml) (Table 5). The positive control (ascorbic acid) exhibited a high nitric oxide scavenging activity varying significantly (F (4, 10) = 614.971; P˂0.001) from 34.77% (at 25 μg/ml) to 59.14% (at 400 μg/ml). The calculated IC50 of the hydro ethanolic extract was found to be 27.42 μg/ml at the correlation (r) 0.9805. Ethyl acetate fraction exhibited an IC50 of 24.59 μg/ml at the correlation (r) 0.9838. The positive control (ascorbic acid) exhibited an IC50 value of 22.96 μg/ml (r=0.9612).
Total antioxidant capacity (TAOC)
Generally, the total antioxidant capacity of the extracts and fractions as well as ascorbic acid increased with the increasing concentration. Hydro ethanolic extract showed a total antioxidant capacity varying (F(4,10) =337600; P˂0.001) from 0.098A (at 25 μg/ml) to 0.949A (at 400 μg/ml) better than ethanolic and aqueous extract (table 6). Among fraction ethyl acetate showed the most potent total antioxidant capacity ranging (F (4,10) =343300; P˂0.001) from 0.186A (at 25 μg/ml) to 1.026 A (at 400 μg/ml). Ascorbic acid exhibited a higher total antioxidant capacity varying significantly (F(4,10) =2403000; P˂0.001) from 0.162A (at 25 μg/ml) to 2.298 A (at 400 μg/ml) (Table 7).
  1. Inhibition of human low density lipoprotein oxidation induced by CuSO4
  2. Continuous monitoring of formation of conjugated dienes in LDL and kinetics of CuSO4 induced LDL oxidation
A dose-dependent decrease of conjugated dienes formation and the increase in lag time period of LDL oxidation was observed in samples containing aqueous, ethanolic, hydroethanolic extract and quercetin at the concentration of 0.25, 0.5 and 1mg/ml. At the concentration 1mg/ml, hydroethanolic extract lengthened the lag time of conjugated diene (CD) formation the most (110 min) (Figure 1c). At the same concentration, ethyl acetate fraction exhibited the best lag time period (150 min) followed by n-butanol fraction (130mins) (Figure 2c). The absorbance of conjugated diene formed in quercetin sample at 1mg/ml increased insignificantly from 0.134 to 0.155 between 0 to 24hours at this concentration. At the concentration of 0.25mg/ml ethyl acetate fraction and n-butanol were found to lengthened the lag time of conjugated diene (CD) formation up to 60mins and 70mins respectively better than that of hexane and residual fraction (30 min and 20 min respectively) (Figure 2a).
Formation of Thiobarbituric Acid Reactant Substances (TBARS)
Hydroethanolic extract most highly inhibited the formation of TBARS than ethanolic and aqueous extract (Table 8) at the concentration of 0.25 and 0.5mg/ml, there were no significant difference between hydro ethanolic extract and quercetine percentage of inhibition (56.75% and 66.67% respectively for hydro ethanolic extract; 60.34% and 67.69% respectively for quercetine respectively). Ethyl acetate and n-butanol fractions also most highly inhibited the formation of TBARS than hexane and residual fractions (Table 8). No significant difference was observed betwen n-butanol and ethyl acetate fraction as well as quercetine at the concentration of 0.25 and 0.5 mg/ml (61.71% and 68.55% for n-butanol fraction; 56.75 and 68.21% for ethyle acetqte fraction; 60.34% and 67.69% for quercetine respectively). Quercetin exhibited an IC50 of 0.9003 mg/ml at correlation coefficient value (r) of 0.0006) while ethyle acetate fraction exhibited 0.9902mg/ml at correlation coefficient value (r) of 0.027).
DISCUSSION
Production of cardiac reactive oxygen species has been associated with atherosclerosis development (Agbor et al ., 2012; Heymes et al .,2003). Free radicals and the oxidation of low density lipoprotein (LDL) are the preliminary steps in the apparition of this disease. It is of paramount importance to search for agents capable with antioxidant capacity with a view to combating atherosclerosis. The present study was designed to evaluate the hydrogen peroxide, nitric oxide scavenging activity, total antioxidant capacity and inhibitory effects of extracts and fractions of P glandulosus on copper sulfate (CuSO4)-induced oxidation in human low density lipoprotein by in vitro method.
Phytochemical screening on P glandulosus leaves confirmed the presence of saponins (18,3%), flavonoids (36,2%) and terpenoids (25,6%). All phenolic compounds including flavonoids have been studied mainly for their properties against oxidative damage by scavenging dangerous free radicals like super oxide anion, hydrogen peroxide, hydroxyl radical and nitric oxide generated during normal metabolic processes which can lead to inflammation, allergie, bacterial infection, cancer, tumor, viral infection, atherosclerosis (Suja et al .,2016; Battisti et al .,2008; Balasundram et al .,2006).Terpenoids are known to have antimicrobial, antiviral, anti- inflammatory, antittumor activities and protective effects on the cardiovascular system (Alves-Silva et al .,2016). Tsopmejioet al (2019) isolated one new methoxylated flavonoid derivative, plectranmicin and one new monoterpene derivative, plectranmicinol, together with seven known compounds from the whole plant of P glandulosus. Saponins have been associated with hypoglycemic activity, accelerating metabolism of cholesterol in the liver, antifungal, antimicrobial, veridical and anti-inflammatory activities (Sapnaet al .,2009). These results confirmed that P glandulosushas pharmacologically active components which can act against many diseases and specially atherosclerosis.
From the in vitro antioxidant tests results, it appeared thatP glandulosus leaves extracts and fractions effectively reduced the generation of hydrogen peroxide. Hydrogen peroxide is a weak oxidizing agent that inactivates a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. It can cross cell membranes rapidly and once inside the cell, it can probably react with Fe2+ and possibly Cu2+ ions to form hydroxyl radicals and this may be the origin of many of its toxic effects (Hazra et al ., 2008; Miller et al ., 2013). Hydro ethanolic extract was the better scavenger among extracts. Among fractions, the better scavenger activity was observed with ethyl acetate fraction.
Nitric oxide is an unstable free radical involved in many biological processes which are associated with several diseases. It reacts with oxygen to produce stable product nitrate and nitrite through intermediates and high concentration of nitric oxide can be toxic and inhibition of over production is an important goal (Roghini et al .,2018; Menaga et al .,2013). Hydro ethanolic extract was the most active extract and ethyle acetate the most active fraction and showed a moderate nitric oxide scavenging activity which is not negligible compared to that of the standard ascorbic acid at the concentration of 400μg/ml.
The total antioxidant capacity (TAOC) of samples with higher electron donating activity can terminate the radical chain and turn free radicals into more stable products (Nedamani et al .,2015; Pan et al . 2011). Starting to concentration 100­­µg/ml to 400µg/ml each sample acted differently from the other no doubt due to differents concentrations and extraction solvent. Also their differents components can act by synergistic, antagonistic or additive effects and produce new physiological properties (Nedamani et al ., 2015).
The unsaturated portions of lipids especially the double bonds of fatty acids present in lipid molecules are most vulnerable to oxidative stress by free radicals and ions that lead to altered lipid structures resulting in the proatherosclerotic breakdown products (Rahman et al ., 2014; Morgan et al .,1995). High level of iron and copper ions (Cu2+) was indicated in the arterial walls of the atherosclerotic individuals by epidemiologic studies and thus, these redox-active metal ions have been implicated in playing a very important role in oxidizing the native LDL molecule both in vivo andin vitro (Rahman et al .,2014; Lynch et al .,1993).The modification of the polyunsaturated fatty acids presents in the LDL molecule and their molecular rearrangement by iron and copper ions are responsible in the formation of conjugated dienes (CD) (Rahman et al .,2014). P glandulosus leaves extracts (aqueous, ethanolic, hydrothanolic extract) and fractions (hexane, ethyle acetate, n-butanol and residual fraction) increased the lag time of the conjugated diene (CD) formation compared to the negative control sample (LDL+0.150µg/ml of CuSO4) which proved the evidence of oxidation by a gradual increase in absorbance which is proportional to the formation of conjugated diene (CDs). For CD formation, there has been a consensus that increase in lag time indicates the inhibition of LDL oxidation by the antioxidant compounds (Rahman et al .,2014; Yoshida et al ., 2010). There was a gradual decrease in absorbance with quercetin after 24 hours indicating the decreased oxidation of native LDL compounds preventing LDL oxidation and ox-LDL-mediated atherogenesis. The mechanism responsible for the inhibition of LDL oxidation mihgh be attribute to flavonoids which enable them to bind to the LDL molecule, subsequently offering protection against oxidation through their radical-scavenging capacity (Furhman et al., 1999).
During the course of LDL oxidation, the lag phase is followed by rapid oxidation (propagation phase) when lipid peroxides are formed. Then comes the breakdown of the double bonds (decomposition phase), and aldehydes, especially malondialdehydes (MDA), are formed (Rahmanet al .,2014). different extracts and fractions of P glandulosus leaves inhibit the formation of TBARS. This inhibitory effect might be attributed to the antioxidants capacity of extracts and fractions. Antioxidants may act as electron donors to the free radicals (here, Cu2+) to make them stable molecules, thus interfering the oxidation of LDL molecules (Rahman et al .,2014).