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).
- RESULTS
- 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).
- In vitro antioxidant activities
- 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).
- Inhibition of human low density lipoprotein oxidation induced
by CuSO4
- 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).