Department of Chemistry, Kwame Nkrumah University of Science and Technology (KNUST), Ghana
Submission: May 13, 2020; Published: May 27, 2020
*Corresponding author: Clement Osei Akoto, Department of Chemistry, Faculty of Physical and Computational Sciences, College of Science, Kwame Nkrumah University of Science and Technology (KNUST), Ghana
How to cite this article: Clement O A, Akwasi A, Osei K, Bartholomew A B. An Assessment of the Anti-Inflammatory, Antimicrobial, and Antioxidant
Activities of Ficus sur Stem-Bark. Organic & Medicinal Chem IJ. 2020; 9(4): 555766. DOI: 10.19080/OMCIJ.2019.09.555766
Ficus sur (Moraceae), is a plant that has found use in traditional African medicine in the treatment of sickle cell disease, epilepsy, pain and inflammations. The present study was aimed at investigating hexane and methanol stem-bark extracts of Ficus sur for their phytoconstituents, anti-inflammatory, antimicrobial and antioxidant activities. Phytochemical screenings were performed using standard protocols. In-vitro anti-inflammatory activities were assessed using egg albumin denaturation method. In-vitro antimicrobial (agar and broth dilution method) and antioxidant [total antioxidant capacity (TAC), DPPH and H2O2 scavenging] assays were carried out on the extracts. Thin layer chromatography was employed in the separation of the components of both extracts. The phytochemical investigation revealed the presence of secondary metabolites such as anthraquinones, terpenoids, flavonoids, steroids, saponins, phenols and tannins. The extracts showed anti-inflammatory activity comparable to that of diclofenac sodium. The extracts showed antimicrobial activity against test organisms with MICs ranging from 2.5- 40 mg/mL. The IC50 values for methanol and hexane extracts in the DPPH and H2O2 assays were 89.95 ± 0.30 and 350.70 ± 0.72 μg/mL and 708.51 ± 0.28 and 682.76 ± 0.20 μg/mL, respectively. The TAC (gAAE/100 g) for methanol and hexane extracts were 23.560 ± 0.014 and 17.863 ± 0.037 g, respectively. The results suggest that the stem bark of Ficus sur could be exploited as potential therapeutic candidate for the treatment of bacterial infections, inflammations and diseases associated with oxidative-stress.
Medicinal plants are known to have antioxidant, antimicrobial, anthelminthic, anti-inflammatory and wound healing activities amongst others [1-3]. They are classified as the richest bio-resource, because they are the source of modern medicines, nutraceuticals, functional foods, food supplements, folk medicines, pharmaceutical intermediates and chemical entities for synthetic drugs . Without specific knowledge of their cellular actions or mechanisms, various parts of plants have been used in traditional medicine to treat a variety of diseases and even as poisons (phytotoxins) .
Ficus is a genus of woody plants that comprises about 850 plant species and belongs to the family Moraceae. They are collectively called Figure trees or Figures and are distributed throughout the tropics and temperate zones, including most African countries such as Ghana, Burkina Faso, Nigeria and Cameroon. Ficus sur, a medicinal species of the family Moraceae has many therapeutic applications. A powdered preparation of the bark has been used to treat skin rashes and mouth sores in most parts of Africa.
A leaf preparation by maceration is used to cure chest problems. A decoction of the leaf has been used as a disinfectant wash and as a cure to ophthalmia . Studies have shown antispasmodic and antiplasmodial activities from aqueous extracts of bark and leaves .
Other parts of F. sur have proven to be potent against a wide variety of ailments including gonorrhoea, sore throat, toothache, eye problems and many more . F. sur is used in folk medicine for the treatment of sickle cell disease in Burkina Faso . Traditional medicine practitioners in Nigeria use F. sur for effective management of epilepsy . F. sur is used in the treatment of leprosy, infertility, gonorrhea, rickets, circumcision, oedema, respiratory disorders and many more . Both root and bark decoctions of F. sur are recorded to have caused death, due to toxic substances . The application of this plant species in the treatment of microbial diseases (example gonorrhoea), and in the treatment of sickle cell disease and management of epilepsy portrays it (Ficus sur) as a candidate for research.
Most research studies conducted on the pharmacological potential
of Ficus sur were mainly focused on crude extracts of the
leaves, roots, barks [12,13] and fruits . Nevertheless, it is also
important to identify the bioactive compounds responsible for
each one of the ascribed bioactivities, especially for the stem-bark.
At the time of carrying out this research, next to nothing had been
reported on the anti-inflammatory activities of the stem-bark. The
aim of this study was to examine the efficacy of Ficus sur methanol
and hexane extracts as an anti-inflammatory, antimicrobial and
antioxidant using in vitro assays.
The stem-barks of Ficus sur were collected in the month of October,
2018 at Kwahu-Asakraka, (Latitude: 6°37’44” N and Longitude:
0°41’29” W) in the Eastern Region of Ghana with the help of
a local herbalist. They were taxonomically identified and authenticated
by Mr. Clifford Asare at the Department of Herbal Medicine,
Faculty of Pharmacy and Pharmaceutical Sciences, KNUST,
Kumasi, Ghana. A voucher specimen number (KNUST/HMI/2019/
S042) was deposited in the Herbarium of Faculty of Pharmacy and
Pharmaceutical Sciences for reference purposes.
The stem-barks of Ficus sur were thoroughly washed, first under
running water to remove any form of debris and subsequently
rinsed in distilled water to exclude dissolve heavy metals in tap
water [1,2]. The stem-barks were chopped into smaller pieces, air
dried under shade for two weeks, pulverized into coarse powder,
and stored in a desiccator until analysis.
Maceration was used for the extraction of the phytoconstituents
of the pulverized sample. A mass of 100 g of the pulverized
sample of F. sur was soaked separately in 500 mL of methanol and
hexane and macerated with gentle stirring for 72 hours at ambient
temperature. The methanol and hexane extracts were condensed
and evaporated to dryness using the rotary evaporator at 50 oC
(BUCHI Rota vapor R-114). The extracts were dried and the percentage
yield of extracts with respect to powdered plant material
determined. The extracts were then stored at 4 oC in a refrigerator
Anti-inflammatory assay was carried out according to a modification
of the standard methods by Kumari . Stock solutions
of 1000 μg/mL of both extracts were prepared by using sterile
distilled water as a solvent. From the stock solutions, various concentrations
of 800, 600, 200 and 100 μg/mL were prepared using
sterile distilled water as a solvent.
The reaction mixtures of total volume 5 mL were prepared
by dissolving 0.2 mL of egg albumin (fresh egg of a hen), 2.8 mL
of phosphate buffer saline (PBS, pH of 6.4) and 2 mL of the various
concentrations of extract solutions. A volume of 2 mL of 200
μg/mL of diclofenac sodium was used as the standard reference
drug and 2 mL of double distilled water solution served as negative
control. The mixtures were incubated at 37 °C in Bio-Oxygen
Demand (BOD) incubator for 15 minutes.
The mixtures were then heated in a water bath at 70 oC for 5
minutes to induce denaturation. The absorbance of the solutions
was measured in triplicate at 660 nm using UV-vis spectrophotometer.
The procedure was independently repeated to obtain
three independent sets of data for the analysis in triplicate. The
percentage inhibition of protein denaturation was calculated as
Where, A0 = absorbance of negative control; A = absorbance
of test solution
Four bacteria and one fungus were used as test organisms.
There were two Gram positive bacteria (Staphylococcus aureus
and Enterococcus faecalis) and two Gram negative bacteria (Escherichia
coli, Pseudomonas aeruginosa). The fungus was Candida
albicans. The microbial strains were provided by the Pharmaceutical
Microbiology Section of the Department of Pharmaceutics,
Faculty of Pharmacy and Pharmaceutical Science, KNUST, Kumasi.
The microbial strains were sub-cultured on nutrient agar slants
and incubated at 37 °C for 24 hours.
Bacterial isolates were streaked onto nutrient agar (Oxoid,
United Kingdom) plates and incubated for 18–24 hours at 37 oC.
Using the direct colony suspension method, suspensions of the organisms
were made in nutrient broth and incubated overnight at
37 oC. For the tests, colony suspensions in sterile saline was adjusted
to 0.5 McFarland standard and further diluted in sterile double
strength nutrient broth (∼2 × 105 CFU/mL ) .
The antimicrobial activities of the different extracts were determined
using a modification of the agar well diffusion standard
method previously described [1,18]. Ciprofloxacin (0.05 mg/mL)
and clotrimazole (0.05 mg/mL) were used as the standard reference
antimicrobial drug. The extracts and antibiotics were tested
in triplicates and mean zones of inhibition were calculated for
each extract and the standard antibiotic.
In the determination of the minimum inhibitory concentration
(MIC), the method used was a modification of micro-well dilution
standard method previously described [1,18]. Ciprofloxacin and
clotrimazole were used as positive control. The experiment was
carried out in triplicate.
Three main assays were employed for the antioxidant activity
determination. They were the 1,1-diphenyl-2-picryl-hydrazyl
(DPPH) free radicals scavenging, Hydrogen Peroxide scavenging
(H2O2) and the Total Antioxidant Capacity (TAC) assays.
The DPPH free radical scavenging activity of the two extracts
were examined using the standard methods previously described
[1,19]. Ascorbic acid was used as reference standard. The experiment
was independently repeated to obtain three independent
sets of data for the analysis. The absorbance was measured at 517
nm. DPPH radical scavenging (%) was calculated using the formula:
Where, A0 = absorbance of control; A = absorbance of test solution
Determination of hydrogen peroxide scavenging potential of
the extracts were carried out employing the standard methods
previously described [1, 20]. Gallic acid was used as reference
standard. Absorbance was taken at 510 nm using a UV-vis spectrophotometer.
The experiment was independently repeated to obtain
three independent sets of data for the analysis. The percentage
scavenging activity was calculated using the formula below
Where Atest is absorbance of the test samples and Acontrol is the
absorbance of the negative control. The results were further reported
A methodology previously described was used to study the
total antioxidant capacity of the extracts of F. sur [1,21]. Ascorbic
acid was used as the reference standard antioxidant and distilled
water was used as the blank. The absorbance of the solutions was
measured in triplicates using a UV-visible spectrophotometer at
695 nm. The experiment was independently repeated to obtain
three independent sets of data for the analysis. From the linear
equation of the ascorbic acid concentration-absorbance plot, the
corresponding independent variables as ascorbic acid equivalents
(AAE) were determined, and the results expressed as gAAE/100g
The number of components present in the extracts were determined
by the analytical TLC method. The pre-coated silica gel
plates (0.25 mm) with a fluorescent indicator (F254) were spotted
with the extracts about 1 cm from the bottom edge of plates,
with the aid of capillary tubes and allowed to dry [1,22]. Various
Solvent systems of petroleum ether/ethyl acetate and hexane/
ethyl acetate in the ratio of 9:1 and 8:2 respectively were used.
The ratio of 8:2 (hexane/ethyl acetate) gave the best separation of
components for all the extracts. The plates were dried and visualized
by a 254 nm UV lamp. The separated spots were then marked
and their sample and solvent fronts were measured.
The retardation factor (Rf) of the eluted spots was calculated
The therapeutic activities of plants are as a result of the presence
of complex chemical constituents in different parts . The
phytochemical screening revealed the presence of seven secondary
metabolites out of the nine tested for in the pulverized sample
and the methanol extract, with alkaloids and carotenoids being
absent. Alkaloids, carotenoids and phenols were absent in the
hexane extract (Table 1).
The methanol and hexane extracts had six phytochemicals in
common, that is anthraquinones flavonoids, saponins, steroids,
tannins and terpenoids. The absence of alkaloids in the stem-bark
of F. sur corroborates the work of Adebayo  who investigated
haematinic properties of methanolic stem-bark and fruit extracts
of Ficus sur in rats pre-exposed to phenylhydrazine induced
haemolytic anaemia . Secondary metabolites of plants which
include phenolics and flavonoids, have been shown to exhibit several biological activities such as antioxidant, antiaging, antidiabetic,
antimutagenic, anticarcinogenic, anti-inflammatory and antimicrobial
. Saponins have a wide range of pharmacological
properties, including antifungal, antiparasitic, molluscicidal and
anti-inflammatory . The presence of these phytochemicals in
the extracts of F. sur stem-bark indicate that they will play a key
role in the prevention of various bacterial infections, inflammations
and diseases associated with oxidative-stress.
Denaturation of proteins is a well-documented cause of inflammation
and rheumatoid arthritis. Several anti-inflammatory
drugs have shown concentration-dose-dependent ability to inhibit
thermally induced protein denaturation. The denaturation
of albumin protein leads to formation of antigens which initiate
type III hypersensitive reaction leading to inflammation . The
ability of plant extract to inhibit thermal denaturation of protein
(egg albumin) is a reflection of its anti-inflammatory activity .
At the concentration of 200 μg/mL, percentage inhibition of
the standard, methanol and hexane extracts were 73.870, 47.176
and 20.500% respectively as shown in Table 2. The anti-inflammatory
activity shown by the extracts could be attributed to the
presence of saponins, terpenoids and steroids in the methanol and
hexane extracts of F. sur, which have been reported to exhibit anti-
inflammatory activity . The presence of polyphenols including
tannins and flavonoids in F. sur have been reported to reduce
inflammation and suppress several stages of angiogenesis, including
endothelial cell migration, invasion, matrix metalloproteinase
activity, and tube formation .
The antimicrobial activities of the extracts were determined
at two concentration levels of 50 and 100 mg/mL for the agar
well diffusion assay as shown in Table 3. The agar well diffusion
is carried out to test for the sensitivity of the organisms to the
antimicrobial agent (plant extract). The diameter of the zone of
inhibition determines the effectiveness of the extract against the
microorganism. The larger the diameter, the greater the sensitivity
of the microorganism to the extract. The sizes of the zone of
inhibition are compared to standards to determine if the microorganism
is sensitive or resistant to the plant extract.
From the results obtained, the methanol and hexane extracts
recorded zones of inhibition at the lower concentration of 50 mg/
mL. At this concentration, the methanol extract recorded inhibition
against E. faecalis, S. aureus and P. aeruginosa but showed no
inhibition against E. coli and C. albicans. However, the hexane extract
showed inhibition against only S. aureus at this concentration.
At a concentration of 100 mg/mL, all the tested organisms
were susceptible to both the methanol and hexane extracts.
Generally, susceptibility increased with the increased concentration
of extract as the zones of inhibition increased for all organisms.
P. aeruginosa was the most susceptible to the methanol extract
at 100 mg/mL with C. albicans being the least susceptible. At
the same concentration, S. aureus was the most susceptible to the
hexane extract with E. faecalis being the least susceptible. All the
four tested bacteria were susceptible to the ciprofloxacin (standard
drug) with the gram-positive bacteria S. aureus showing the
highest susceptibility. Both extracts and clotrimazole (standard
drug) showed activity against the fungus C. albicans.
The extracts showed broad spectrum antimicrobial activity
against the tested organisms. The methanol extract showed a
better antimicrobial activity (at MIC of 2.5 mg/mL to 10.00 mg/
mL) against the test organisms than the hexane extract (at MIC of
20.00 to 40.00 mg/mL). The results are shown in Table 4. The results
from the antimicrobial assay performed showed that the two
extracts of F. sur stem-bark exhibited varying inhibitory effects
against the five selected microorganisms (two Gram-positive, two
Gram-negative and one fungus). The best results were observed
with the use of the methanol extract against all the selected microorganisms.
The minimum inhibitory concentrations (MICs)
were between the range of 2.5 mg/mL to 10.0 mg/mL. The highest
activity observed with the use of methanol extract was against
P. aeruginosa with MIC of 2.5 mg/mL. The antimicrobial activity
shown by the extracts could be attributed to the presence of terpenoids,
saponins and polyphenols such as flavonoids and tannins
in the methanol and hexane extracts of F. sur which have been reported
to exhibit antimicrobial activity [31,32].
The total antioxidant potential of a plant extract depends
largely on both the constituent of the extract and the test system.
Different factors can also influence the activity of the extract,
therefore when carrying out a study related to the antioxidant and
antiradical properties of plant products, more than one method
is usually used to evaluate the antioxidant capacity/activity .
Considering the various mechanisms of antioxidant actions, the
antioxidant properties of the extracts were evaluated by (DPPH)
free radicals scavenging, Hydrogen Peroxide scavenging and the
Total Antioxidant Capacity assays.
The DPPH radical scavenging activity of the extracts was used
to determine and study the ability of the extracts of F. sur to mop
up free radicals that may be found in animals and humans. Methanol
and hexane extracts of F. sur and ascorbic acid (reference standard) scavenged DPPH radical in a dose dependent manner
(Figure 1). The reference antioxidant (ascorbic acid), hexane and
methanol extracts of F. sur showed antioxidant activity in the
DPPH free radical scavenging assay with IC50 of ascorbic acid,
hexane and methanol ranged from 3.17 ± 0.32 to 350.70 ± 0.72
μg/mL, as shown in Table 5.
The results implied that the potency of the tested samples
of extracts as antioxidants decreased in the order: ascorbic acid
> methanol > hexane (Figure 1). Methanol extract showed better
antioxidant activity compared to the hexane probably due to the
presence of the polyphenols that act as anti-aging agent by neutralizing
the effect of free radicals . Polyphenols including
tannins and flavonoids have many favourable effects on human
health, such as the inhibition of the low density proteins oxidization
. Though hexane and methanol extracts which comprise
of a mixture of compounds were not as potent as the ascorbic
acid, F. sur stem-bark extracts may be useful in the manufacture
of drugs to help prevent or cure health problems that could arise
from the systemic actions of oxidative agents, thus its usage in folk
medicine for the treatment of sickle cell diseases in Burkina Faso
Non-radical oxidizing agents scavenging potential of the hexane
and methanol extracts of F. sur were evaluated by the use of
hydrogen peroxide (H2O2) scavenging method. The results are
shown in Table 6. Methanol and hexane extracts of F. sur and gallic
acid (reference standard) exhibited H2O2 scavenging capacity in a
dose dependent manner (Figure 2).
The IC50 of a sample is the concentration of the sample required
to scavenge 50% of the peroxide in a system. It is used to evaluate
the antioxidant capacity of a sample. The lower the IC50, the better
the antioxidant potential of the sample under examination [1,36].
Results showed that, hexane and methanol extracts demonstrated
a significant antioxidant activity in concentration-dose dependent
manner. The IC50 values of gallic acid (standard drug), hexane and
methanol extracts ranged from 204.40 ± 0.01 to 708.51 ± 0.28
μg/mL as shown in Table 6.
From the results, both methanol and hexane extracts which
comprise of a mixture of compounds showed slightly lower activity
than gallic acid (reference standard), even though they are all
good antioxidants. Bioactive isolates from these extracts responsible
for antioxidant activity could be attributed to the terpenoids
and polyphenols, such as tannins and flavanoids in F. sur and could
be exploited for the treatment of oxidative-stress diseases .
Ascorbic acid also known as Vitamin C is an electron donor
antioxidant and this property is responsible for all its known functions.
Vitamin C is a potent reducing agent and scavenger of free
radicals in biological systems. It is a cofactor for enzymes involved
in regulating photosynthesis, hormone biosynthesis, and regenerating
other antioxidants .
Concentrations of ascorbic acid ranging between 6.125 to 100
μg/mL showed antioxidant activity and mean absorbances between
0.059 ± 0.003 to 0.932 ± 0.002 at wavelength of 695 nm
(Figure 3). The TAC was found to be proportional to the concentration
of extract. TAC of the extracts were examined by Phosphomolybdenum
method and the results were expressed as gram
ascorbic acid equivalent per 100 grams (gAAE/100g) . The
gAAE/100g, represents the fraction of the plant extract that can
act as ascorbic acid in 100 g of the extract. The hexane and methanol
extracts had 17.863 ± 0.037 and 23.560 ± 0.014 gAAE/100g,
respectively, (Table 7).
Generally, the TAC increased with increasing concentration,
thus the higher the TAC, the better the activity of the sample. Polyphenols
including flavonoids are important natural antioxidants,
which are basically associated with curing of various diseases and
disorders including cancer, diabetes, gout, urolithiasis, obesity,
and other diseases associated with ageing [38,39]. Both extracts
demonstrated appreciable antioxidant activities due to the presence
of the various phytochemicals such as flavonoids, phenols,
tannins, terpenoids among others in F. sur.
The number of components present in the extracts were determined
by the analytical TLC method. The chromatographic spots
which were representative of compounds in the various extracts were observed and their Rf values determined. Table 8 gives the
results of the TLC analysis. The hexane extract showed four spots
and methanol five spots with Rf values between 0.113 to 0.950
and 0.050 to 0.900, respectively. The number of spots indicating
the separated components in the two extracts were less for both
extracts when compared to the phytoconstituents identified to be
present in each stem-bark extract. This means that some of the
components existed as isomers, did not elute due to the polarity of
the mobile phase or co-eluted in mixtures and it may be necessary
to employ two dimensional TLC, HPLC or column chromatography
to achieve complete separation of the components .
The hexane and methanol extracts of F. sur showed the presence
of varying secondary metabolites including saponins, tannins,
terpenoids, steroids, flavonoids, phenols and anthraquinones. The
study demonstrated that the hexane and methanol extracts of F.
sur possess a variety of anti-inflammatory, antibacterial, antifungal
and antioxidant activities. This implies the extracts could be
effective against inflammations, infectious and diseases associated
with oxidative-stress, and could become a potential therapeutic
agent for their treatment. Further studies are ongoing in our
laboratory towards isolation, characterization, identification and
determination of biological activities present in the stem-barks of
Part of this work was presented as a poster at the “8th Ghana
Science Association, Research Seminar and Poster Presentations”
held at the Kwame Nkrumah University of Science and Technology,
Kumasi, Ghana, in May 2019.
The authors declare no competing financial, professional, or
personal interests that might have influenced the performance or
presentation of the work described in this manuscript. The authors
declare that there is no conflict of interests regarding the
publication of this paper.
The authors are grateful to the Departments of Chemistry
and Pharmaceutical Microbiology as well as the Central
Laboratory of KNUST for the use of their facilities for this study.
The authors appreciate Dr. Yaw Duah Boakye of the Department
of Pharmaceutics, KNUST for helpful discussions. The authors
are also grateful to Mr. Kennedy Ameyaw Baah, a PhD student
of the Department of Chemistry for his assistance. We will also
like to acknowledge Mr. Francis Amankwaah of the Department
of Pharmaceutical Microbiology , Faculty of Pharmacy and
Pharmaceutical Sciences, KNUST, for technical support.