Volume 8, Issue 11, November 2023 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Antioxidant Activities of Methanol and

Dichloromethane Blend Extracts of
Caesalpinia volkensii Harms. and
Carissa edulis (Forssk.) in Vitro
Kibiwott Scolar Jepkorir
MSc. Biotechnology, http://orcid.org/000-0002-2568-3026
Department of Biochemistry, Microbiology and Biotechnology, Kenyatta University

Ngugi Mathew Piero

Ph.D. (Biochemistry and Molecular Biology) https://orcid.org/0000-0002-0226-9092
Department of Biochemistry, Microbiology and Biotechnology, Kenyatta University
Ng’ang’a Margaret Mwihaki Ph.D. (Natural Product Chemistry)
http://orcid.org/000-0002-7870-8821 Department of Chemistry, Kenyatta University

Abstract:- Oxidative stress is the primary cause of many Free radicals are produced due to intense physical exercise,
human ailments, including aging. Synthetic antioxidants improper diet, long-term stress conditions and exposure to
are unaffordable and are associated with severe effects. ultraviolet radiation [1]. In a normal cell, an appropriate
This necessitates the need for alternative antioxidant balance between oxidants and antioxidants exists [3]. When
agents. This investigation aimed to determine the the oxidants levels increase and antioxidants decrease, this
antioxidant activities and qualitative phytochemical balance shifts, causing oxidative stress that causes
composition of DCM and MeOH blend extracts of root physiological disorders such as Parkinsons disease,
barks of Carissa edulis and leaves of Caesalpinia volkensii. Alzheimers disease, atherosclerosis, diabetes mellitus,
The antioxidant assays included ferric reducing premature aging and cancer, among others [4]. The harmful
antioxidant power (FRAP), H2O2 radical scavenging and action caused by ROS can be naturally controlled by
DPPH radical scavenging activities, as well as total enzymatic antioxidants such as glutathione, superoxide
flavonoid and total phenolic content tests. The extracts dismutase and catalase, among others [5]. Free radicals can
revealed potent FRAP and DPPH and H2O2 radical be inhibited, scavenged, or chelated by antioxidants.
scavenging activities, including a considerable amount of Antioxidants convert free radicals into safe molecules by
total flavonoids and phenolics contents. The C. edulis donating an electron or an active hydrogen atom [6].
extract noted better antioxidant activities than C.
volkensii extract. The antioxidant effects of the two Synthetic antioxidants like butylated hydroxytoluene,
studied extracts were concentration-dependent. In propylgallate and tertiary butyl-hydroquinone are known to
addition, the C. edulis extract had a considerably higher ameliorate oxidative stress. However, the use of these
amount of total phenolic and total flavonoid contents antioxidants is associated with cancer and liver toxicity [7].
relative to C. volkensii extract. Phenolics, terpenoids, For this reason, there has been a lot of interest in searching
alkaloids, cardiac glycosides, saponins, steroids, and for alternative agents that are efficacious, and non-toxic.
flavonoids were detected in the qualitative phytochemical Lately, the utilization of natural antioxidants in the treatment
analysis, except for alkaloids and steroids in C. volkensii of ailments and disorders has drawn more attention [8]. Many
extract and cardiac glycosides in C. edulis extract. In medicinal plants have long been used to relieve oxidative
conclusion, the two extracts have potent antioxidant stress [9]. Nevertheless, there is insufficient scientific
activities and are endowed with phytochemicals evidence to validate these claims [10].
associated with antioxidant activities. The two extracts,
therefore, may be used as alternative antioxidant agents. Medicinal plants possess phytochemicals (secondary
metabolites) that exert antioxidant activities. These secondary
Keywords:- Antioxidant; oxidative stress; phytochemicals; metabolites such as saponins, alkaloids, phenolic acids,
flavonoids; phonolics carotenoids, tocopherols, flavonoids and cinnamic acids have
been documented to possess potent antioxidant effects [11].
I. INTRODUCTION They ameliorate oxidative stress through scavenging or
mopping up free radicals [12]. The root barks of Carissa
Reactive oxygen species (ROS) refers to extremely edulis and leaves of Caesalpinia volkensii are used
reactive molecules with oxygen atoms and unpaired traditionally by Kenya communities to manage oxidative
electrons. These molecules can damage biomolecules, stress. However, no empirical scientific data have been
including proteins, lipids, and deoxyribonucleic acid, thereby documented to ascertain these claims. This investigation
causing many pathological conditions in the body [1]. Free aimed to assess in vitro antioxidant effects, including total
nitrogen radicals, singlet oxygen, hydroxyl radicals and phenolic and total flavonoid contents of C. edulis and C.
superoxide anion radicals are some examples of ROS [2]. volkensii, as well as qualitative phytochemical composition.

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II. MATERIALS AND METHODS 7.4) to form 40 Mm of H2O2 solution. Concentrations of 0.2,
0.4, 0.6, 0.8, and 1 mg/ml were prepared for the two extracts
A. Medicinal sample collection and preparation and ascorbic acid. Then, 1 ml of the sample and 2 ml of the
Fresh root barks of C. edulis and leaves of C. volkensii H2O2 solution were mixed for 10 minutes. The phosphate
were collected with the assistance of a traditional medical buffer solution served as the blank. The sample absorbances
herbalist from Mbeere North, Embu County, Kenya. The were detected at 560 nm by the use of a spectrophotometer.
medicinal samples were availed to a recognized taxonomist The H2O2 radical scavenging (%) effect was computed as
for botanical identification and voucher specimens (SJK 001 follows:
and SJK 002 for C. edulis and C. volkensii, respectively)
deposited in the herbarium of Kenyatta University. Samples
were then sorted out properly, cleaned, chopped, air-dried
and milled into a fine homogenous powder.
Where,
B. Extraction As = Sample absorbance
500 grams of each finely powdered medicinal sample was Ac = Blank absorbance (control)
soaked in two liters of MeOH and DCM in the ratio of 1:1
followed by regular shaking for two hours and then let to F. Evaluation of total flavonoid contents
stand for 48 hours. The mixture was passed through A method described by [16] was adopted to assess for
Whatman filter paper number 1, and then the filtrate total flavonoid content. 0.5 ml of the extract (1 mg/ml) was
concentrated using a rotary evaporator under reduced blended with 10% AlCl3 (0.1 ml), 1 M potassium acetate (0.1
pressure (40 °C). A semisolid extract was then dried using an ml), MeOH (1.5 ml), and distilled water (2.8 ml) to make a
oven at 38 °C. volume of 5 ml. It was then left standing for 30 min. Distilled
water was utilized as the blank and quercetin as the standard.
C. Determination of FRAP The samples' absorbances were measured at 415 nm using a
A method described by [13] was used to assess the FRAP spectrophotometer. Concentrations of 50, 100, 150, 200 and
of the two studied extracts. The extracts as well as the 250 mg/ml of quercetin were used to draw a standard curve.
standard used concentrations of 0.2, 0.4, 0.6, 0.8 and 1 Afterwards, the total flavonoid concentration was calculated
mg/ml. One milliliter of the extract was put into 2.5 ml of using the standard calibration curve equation, and the
phosphate buffer (pH 6.6, 0.2 M). The resultant mixture was findings were presented as milligrams of quercetin
added to 2.5 ml of potassium ferricyanide, incubated for 20 equivalence per gram of the extract.
minutes at 50 °C and consequently centrifuged at 3000 rpm
(revolutions per minute) for 10 minutes after adding 2.5 ml of G. Evaluation of total phenolic contents
10% trichloroacetic acid. 2.5 ml of the supernatant was A protocol by [17] was used to assess total phenolic
aspirated, and then blended with 2.5 ml of freshly made contents. A volume of 2.5 ml of Folin-Ciocalteau's reagent
FeCl3 solution and distilled water (2.5 ml). The assay used (diluted 1/10) and two milliliters of sodium bicarbonate
distilled water as the blank. A spectrophotometer was used to (7.5%, w/v) were added to each sample (0.5 ml) prior to
determine the optical densities of the samples at a wavelength incubation at 45°C for 15 minutes. Using a
of 700 nm. spectrophotometer, the sample absorbances were detected at
765 nm. Gallic acid (standard) values of 50, 100, 150, 200,
D. Determination of DPPH radical scavenging activity and 250 mg/ml were utilized to draw a standard curve. The
A protocol used by [14] was adopted to assess the DPPH gallic acid equivalence was computed using a standard curve
scavenging effects of the two extracts. 50 ml of MeOH was equation and results were expressed as milligrams of gallic
used to dissolve 2.66 mg of DPPH to prepare a 0.135 mM acid equivalence per gram of the extract.
DPPH radical solution. 1 ml of each extract or ascorbic acid
concentrations of 0.2, 0.4, 0.6, 0.8 and 1 mg/ml was mixed H. Qualitative phytochemical analysis
with 1 ml of the DPPH solution. The resultant mixture was The DCM and MeOH blend extracts of C. edulis and C.
vortexed and then allowed to stand for half an hour in a dark volkensii were subjected to several assays to determine their
room. MeOH served as the blank. The sample absorbances qualitative phytochemical analysis.
were determined at 517 nm by the use of a
 Alkaloids test
spectrophotometer. The samples’ percentage DPPH radical
Five milliliters of each extract was first acidified with 1 M
scavenging activities were calculated as follows:
HCl. After heating the acidic medium, Dragendroff's reagent
was added. Alkaloids were present when reddish-brown or
orange precipitate formed [18].
Where,
As = Sample absorbance  Flavonoids (Sodium hydroxide test)
Ac = Blank absorbance (control) Two milliliters of each extract was blended with two
milliliters of diluted NaOH. Positive outcomes were
E. Determination of hydrogen peroxide radical scavenging indicated by an intense/golden-yellow precipitate [19].
activity
A procedure described by [15] was used to assess the two
extracts' in vitro H2O2 radical scavenging abilities. 4.53ml of
H2O2 was diluted with 1 L of phosphate buffer (0.1 M; pH

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Volume 8, Issue 11, November 2023 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
 Terpenoids (Salkowski test)
After mixing 0.5 g of each extract with chloroform (2 ml) I. Statistical data analysis
and 1 ml of petroleum ether, 3 ml of concentrated H2SO4 was Raw data was tabulated in the Microsoft Excel
gradually added to form a layer. Terpenoids were identified Spreadsheet, cleaned and exported to GraphPad Prims
by a reddish-brown colored interface [20]. version 9 statistical software for analysis. The mean±standard
deviations (SD) were used to express descriptive statistics.
 Saponins (Froth test) One-way ANOVA (analysis of variance) was utilized to
Two milliliters of each extract was blended with a few perform inferential statistical analysis between distinct
drops of NaHCO3 solution, vigorously shaken, and let to treatment groups. Tukey's multiple comparisons were
stand for 15 to 20 minutes. The formation of foam exceeding computed to ascertain the group differences when one-way
1 cm was indicative of saponins [21]. ANOVA revealed a significant variation. An independent t-
test was computed to compare the total flavonoid content and
 Steroids test total phenolic content of the two extracts. The level of
Two milliliters of chloroform was used to dilute a mass of significance was set at p less than 0.05. Tables and graphs
0.5 g of the extract. The test tube's sides were carefully filled were used to present the study outcomes.
with 3 ml of concentrated H2SO4 to form a layer. The
steroidal ring formed a reddish-brown coloration at the III. RESULTS
interface [15].
A. Ferric reducing antioxidant power
 Phenolic test The DCM and MeOH blend extracts of C. edulis and C.
One milliliter of FeCl3 solution was put into 2 ml of the volkensii at 0.2, 0.4, 0.6, 0.8 and 1 mg/ml revealed FRAP
extract. Phenolics were present as evidenced by the formation (Figure 1). The two extracts at 0.2, 0.8 and 1 mg/ml noted a
of a blue to green coloration [22]. considerable variation (p>0.05) in FRAP. However, the
FRAP of C. edulis extract concentrations of 0.4 and 0.6
 Cardiac glycosides (Keller-Kilian test) mg/ml were statistically greater than those (p<0.05) recorded
A volume of two milliliters of glacial acetic acid and 2 in C. volkensii extract. The FRAP of ascorbic acid was
drops of a 10% FeCL3 solution were used to dissolve 0.5 g of substantially higher relative to those of the two extracts
the extract. Consequently, one milliliter of concentrated (p<0.05) at the same concentrations (Figure 1).
H2SO4 was added slowly. Cardiac glycosides were indicated
by a violet, brown, or greenish ring at the interphase [23].

1.5
a Ascorbic acid
a
C. edulis
a
Absorbance (nm)

C. volkensii
1.0 b
a b
b b
a b
c
b
0.5 b b c

0.0
0.2 0.4 0.6 0.8 1
Concentrations (mg/ml)

Fig. 1: FRAP of C. edulis and C. volkensii extracts. Bars with distinct letters differ statistically (p<0.05) at the same concentration
using one-way ANOVA and Tukey’s multiple comparisons.

B. DPPH radical scavenging activity statistically higher percentage of DPPH radical scavenging
The DCM and MeOH blend extracts of C. edulis and C. activity relative the effect (p<0.05) of the two extracts at all
volkensii noted potent in vitro DPPH scavenging effects the tested concentrations (Figure 2). The ascorbic acid as
(Figure 2). At the corresponding concentrations, the effect of well as C. edulis and C. volkensii extracts had half-maximal
C. edulis extract had a considerably higher percentage of inhibitory concentration (IC50) of 2.16, 3.15 and 3.52,
DPPH radical scavenging activity than the effect of C. respectively.
volkensii extract (p<0.05). The effect of the standard had a

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DPPH radical scavenging activity (%)

100 a
Ascorbic acid
b
80 a b C. edulis
b C. volkensii
a
60 c
b b
a
40 a
b
b c
20 c

0
0.2 0.4 0.6 0.8 1
Concentrations (mg/ml)

Fig. 2: DPPH radical scavenging effects of C. edulis and C. volkensii extracts. Bars with distinct letters differ statistically (p<0.05)
at the same concentration using one-way ANOVA and Tukey’s multiple comparisons.

C. Hydrogen peroxide radical scavenging activity radical scavenging activity when compared to the two extract
The DCM and MeOH blend extracts of C. edulis and C. concentrations of 0.2 and 0.4 mg/ml (p<0.05). Nevertheless,
volkensii showed in vitro H2O2 radical scavenging effects at the percentage of H2O2 scavenging activity of the standard
all the concentrations tested (Figure 3). At the concentrations and C. edulis extract showed no significant differences at the
of 0.2, 0.6, 0.8, and 1 mg/ml, the percentage of H2O2 radical concentrations of 0.6, 0.8, and 1 mg/ml (p>0.05; Figure 3).
scavenging activity of the C. edulis extract was significantly The IC50 of C. edulis and C. volkensii extracts as well as
higher than that of the C. volkensii extract (p<0.05). Ascorbic ascorbic acid were 1.45, 2.08 and 0.19, respectively.
acid exhibited a significantly higher percentage of H2O2

100
H2O2 radical scavenging activity (%)

a
90 a a Ascorbic acid
80 a a b C. edulis
a a b
70 C. volkensii
a c
60 b
b
50 b
c
40
30
20
10
0
0.2 0.4 0.6 0.8 1
Concentrations (mg/ml)

Fig. 3: Hydrogen peroxide radical scavenging effects of C. edulis and C. volkensii extracts. Bars with distinct letters differ
statistically (p<0.05) at the same concentration using ANOVA and Tukey’s multiple comparisons.

D. Total phenolic and total flavonoid contents

The C. volkensii extract had significantly lower total flavonoid and total phenolic contents than those of C. edulis extract
(p<0.05; Figure 4).

200
C. edulis
a C. volkensii
mg of equivalnce/g

150
b

100 a

b
50

0
Total phenolic Total flavonoid

Fig. 4: Total flavonoid content and total phenolic content of C. edulis and C. volkensii extracts. Bars with distinct lowercase letters
differ significantly using (p<0.05) an independent t-test.

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E. Qualitative phytochemical screening extract had saponins, terpenoids, cardiac glycosides,
The results demonstrated that C. edulis extract had flavonoids, and phenolics. However, cardiac glycosides were
saponins, terpenoids, alkaloids, steroids, flavonoids, cardiac absent in C. edulis extract, while steroids and alkaloids were
glycosides and phenolics. On the other hand, C. volkensii absent in C. volkensii extract (Table 1).

Table 1: Qualitative phytochemical composition of C. edulis and C. volkensii DCM and MeOH blend extracts
Phytochemicals C. edulis C. volkensii
Saponins + +
Alkaloids + -
Flavonoids + +
Terpenoids + +
Cardiac glycosides - +
Steroids + -
Phenolics + +
+ = present; - = absent

IV. DISCUSSION The study also showed that the FRAP of the two extracts was
concentration-dependent. Studies by [30-32] have reported
Oxidative stress causes numerous ailments like diabetes similar findings on the FRAP.
mellitus, arthritis, cancer, Alzheimer’s, liver cirrhosis,
including aging. This occurs as a result of an imbalance DPPH generates a free radical and is used in
between antioxidants and oxidants produced in the body [24]. determining radical scavenging activities [33]. The DPPH
Synthetic antioxidant agents are used to manage oxidative test is a popular choice for in vitro antioxidant screening due
stress. Nevertheless, these agents have been documented to to its ease of use, convenience, and simplicity in evaluating
possess severe effects [7], necessitating the need for antioxidant activity. The color of DPPH changes when an
alternative antioxidant agents. This study revealed that DCM antioxidant substance donates an electron, and the electron is
and MeOH blend extracts of C. edulis and C. volkensii had accepted by DPPH. The change of color from purple to
potent FRAP, H2O2 radical scavenging and DPPH radical yellow is an indication of the presence of a radical scavenger
scavenging activities, as well as a considerable quantity of [34].
total flavonoid and total phenolic contents, suggesting
antioxidant activity. The two extracts also showed the The results of this investigation show that the two
presence of major phytocompounds that are linked with extracts have the potential to scavenge DPPH radicals. The
antioxidant potential. percentage of DPPH radical scavenging activity in C. edulis
extract was significantly higher than that of C. volkensii
FRAP analysis assesses the ability of antioxidants to extract. The findings also showed that ascorbic acid noted a
donate electrons. It is an efficient and quick way to evaluate a considerably higher percentage of DPPH radical scavenging
substance's antioxidant impact [25]. A reducing agent activity than those of the two extracts. Numerous studies
contains atoms that can donate one or more of their electrons have shown a correlation between higher percentages of
to react with free radicals and convert them into more stable DPPH radical scavenging activities of medicinal plant
products. This, therefore, terminates or blocks the radical extracts and high total phenolic and total flavonoid contents
chain reactions [26]. The substances that have FRAP react [35]. The high levels of total flavonoid content and total
with potassium ferricyanide (Fe3+) to generate potassium phenolic content that were detected in the two extracts may
ferrocyanide (Fe2+), which then reacts with FeCl3 to generate be attributed to a greater percentage of DPPH radical
a ferrous complex. This complex is Perl’s Prussian blue in scavenging activities. As a result, the two examined extracts
color. The maximum absorption of the ferrous complex is may be regarded as proton donors and may be utilized as
700 nm. The FRAP of a compound is thus explained by the substitute medicinal agents for the treatment of oxidative
ability to donate an electron [27]. stress.

This study’s findings noted that the two extracts had The two extracts' abilities to scavenge DPPH radicals
significant FRAP, an indication of antioxidant activity. The followed a dose-dependent pattern. The percentage of DPPH
phytochemicals present in the two extracts were therefore radical scavenging activity was highest in the highest
attributed to the reduction of Fe3+ to Fe2+. The FRAP of the concentrations of the extracts. The findings on DPPH radical
two extracts suggested that the two extracts have reducers scavenging activity were consistent with those reported by
that serve as electron donors and can terminate radical chain [36-38].
reactions, hence minimizing tissue oxidative damage [28].
The standard (ascorbic acid) proved to have better ferric- The term "IC50" is used to describe the quantity of
reducing ability than the two extracts. Moreover, C. edulis extract required to scavenge 50% of the free radicals. The
extract had a considerably higher FRAP than C. volkensii scavenging effects of the extracts are inversely correlated
extract. This could be explained by the fact that C. edulis had with IC50. This is because the smaller the IC50, the greater the
additional alkaloids that have been documented to possess scavenging potential [39]. According to the results of this
antioxidant effects [29]. The increased absorbance at 700 nm study, C. edulis extract noted a considerably higher
indicated an increase in the extract’s ferric-reducing abilities. percentage of DPPH scavenging activity than that of C.

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volkensii extract. These results, therefore, suggest that C. DECLARATION
edulis extract had a better DPPH radical scavenging effect
than C. volkensii extract. A. Author Contributions:
 Scolar Jepkorir Kibiwott: Carried out experiments,
Hydrogen peroxide is produced in vivo from a reaction analyzed data and reported data, as well as developed the
of enzymes such as superoxide dismutase. They can rapidly manuscript.
cross the cell membrane and cause toxic effects by reacting  Mathew Piero Ngugi: Conceived the study idea,
with Cu2+ and Fe2+ ions, forming highly reactive hydroxyl developed the study design, interpreted data, and
radicals through Fenton reaction [40]. Polyphenols supervised the development of the manuscript.
(flavonoids and phenolic acids) can protect body cells from  Mary M. Ng’ang’a: Contributed reagents, provided
the cytotoxicity effect that is caused by H2O2. The technical support, interpreted data and proofread the
polyphenols have hydroxyl groups that have a remarkable manuscript.
potential to scavenge hydrogen peroxide [41]. Therefore, by
inhibiting H2O2, the production of these reactive radicals will B. Funding
be prevented and hence the body systems are protected. The This study did not secure external funding.
concentration of H2O2 is decreased by compounds that have
scavenging effects. These compounds accelerate the C. Conflicts of Interest
conversion of H2O2 to water and oxygen [40]. The authors affirm that they have no competing interests.

This study demonstrated that the two studied extracts ACKNOWLEDGEMENT

scavenged H2O2 in a concentration-dependent response.
Carissa edulis extract showed a better H2O2 scavenging The authors acknowledge Kenyatta University’s
ability than C. volkensii. The differences in the H2O2 radical Department of Chemistry and Department of Biochemistry,
scavenging abilities could be associated with the moieties of Microbiology and Biotechnology for granting us access to
their active compounds which determine their ability to their facilities. Daniel Gitonga Mwaniki deserves a mention
donate electrons or active hydrogen atoms. The percentage of for his technical support.
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