European Journal of Integrative Medicine 36 (2020) 101140

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European Journal of Integrative Medicine

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Research paper

Anti-bacterial, anti-fungal and anti-oxidative properties of different extracts T

of Bruguiera gymnorrhiza L. (Mangrove)
Srinivas Acharyaa, Deepak Kumar Patrab,*, Chinmay Pradhana, Pradipta Kumar Mohapatrac
a
Department of Botany, Utkal University, Bhubaneswar, 751004, India
b
Department of Botany, Nimapara Autonomous College, Nimapara, Puri, 752106, India
c
Department of Botany, Ravenshaw University, Cuttack, 753003, India

A R T I C LE I N FO A B S T R A C T

Keywords: Introduction: Bruguiera gymnorrhiza L. a Rhizophoraceae is an intertidal marine plant found in mangroves.
Antimicrobial activity Previous studies suggest this plant may have antibacterial, antifungal and anti-oxidant activities. This study
Bruguieragymnorrhiza aimed to explore the potential of the leaf, root and bark extracts of this plant.
DPPH assay Methods: The successive methanol and combination solvent (Chloroform: Methanol: Ethanol in 60:20:20) ex-
Mangrove
traction of the plant materials were assayed against ten pathogenic bacteria and four fungal strains. The total
Minimum inhibitory concentration
phenolic content, antimicrobial and antioxidant assay were estimated by using well diffusion, DPPH (diphenyl-1-
Phenolic compounds
picryl-hydrazyl) assay and FRAP (Ferric-reducing Power) assay
Results: Dried root extract of B. gymnorrhiza obtained by a combination of solvents demonstrated maximum
effectiveness on tested microorganisms with an optimal zone of inhibition (ZOI) value of 22.31 ± 0.36 mm
followed by bark extract with ZOI 21.63 ± 0.49 against Penicillium chrysogenum. Different combinations of
solvent extracts of bark exhibited optimal minimum inhibitory concentration (MIC) (7.26 μg/mL) against
Micrococcus sp.followed by MIC (7.62 μg/mL) against Streptococcus mitis. Methanolic root extracts contained a
maximal quantity of phenolics of 25.36 μg Gallic Acid Equivalents and 69.64% of DPPH activity. A combination
extract of root obtained a maximum 96.07% of ferric-reducing antioxidant power.
Conclusions: Crude extracts of B. gymnorrhiza possess noteworthy toxicity against bacterial pathogens and
Candida albicans (a fungal pathogen). Combination solvent extracts showed more promising results with phar-
maceutical activities for developing natural compound based drugs.

1. Introduction of natural antimicrobial agents to treat common infectious diseases

More than 50,000 plant species and their secondary metabolites are
used in pharmaceutical and cosmetic industries [1–3]. Medicinal plants
have also been exploited for their therapeutic effect and used in drug
formulation for the treatment of various diseases [4–6]. There is great
potential in screening medicinal herbs for their antioxidant and anti-
bacterial properties in order to identify potent new molecules with
therapeutic value. Clinical trials are also needed to establish the safety
standards for patients, which will support the practice of traditional
systems of medicine [7]. Agricultural by-products constitutes many
metabolites like polyphenols, food packaging materials, pectin, dietary
fibers, fatty acids and other bioactive molecules of pharmaceutical
importance [8,9].
The growing problem of antibiotic resistance of pathogens has
emerged as a challenge across the globe and demands the development
[10,11]. Recently significant adverse effects like gastrointestinal ab-
scesses, blockages and haemorrhages have been observed with the use
of non-steroidal and synthetic anti-inflammatory drugs (NSAIDs) [12].
On the other hand, traditional herbal extracts of Andrographis paniculata
are being safely used for treating hepatitis in India [13]. Similarly
chronic environmental stress and genetic disorders induce hyper-ac-
cumulation of free radicals that cause various lethal diseases like Par-
kinson's, cardiovascular, Alzheimer's, cancer, etc. Therefore, DPPH and
FRAP assay are very credible methods to measure anti-oxidative ca-
pacity of natural organic molecules [14–17].
Mangroves constitute intertidal facultative halophytes pre-
dominantly distributed in inter-tropical coastlines. Different plant ex-
tracts from Rhizophoraceaemangroves have been shown to be effective
against different human pathogens [18]. A polysaccharide constituent
obtained from the leaf of Rhizophora apiculata has shown toxicity

⁎
Corresponding author.
E-mail address: deepakpatranayapali@gmail.com (D.K. Patra).

https://doi.org/10.1016/j.eujim.2020.101140
Received 4 April 2020; Received in revised form 9 May 2020; Accepted 9 May 2020
1876-3820/ © 2020 Elsevier GmbH. All rights reserved.
S. Acharya, et al.
against HIV-1/HIV-2 or SIV viral strains causing AIDS under controlled
conditions [19]. Published reports suggest that different extracts from
mangrove such as from Avicennia sp., Excoecaria agallocha and Acanthus
ilicifolius had antimicrobial, anti-inflammatory, utrine stimulant and
anti-leukemic activity against many human pathogens like Candida al-
bicans, Mycobacterium vaccae, M. aurum, M. smegmatis, M. fortuitum and
Staphylococcus aureus [20–25]. Effective anti-oxidative properties of
different phyto-extracts, evaluated for different plant extracts by using
DPPH free radical scavenging assay, have also been observed [26–29].
Antimicrobial activity, DPPH assay and phytochemical screening of
Bruguiera gymnorrhiza fruit and leaf plant parts by n-hexane have shown
the presence of natural antioxidants as well as bactericidal properties
[30,31]. However, no attempt has been made to measure application
potential of such extracts with combination of solvents. This study aims
at evaluating antibacterial, antifungal and anti-oxidant properties of
extracts from leaf, root and bark of Bruguiera gymnorrhiza L. (Rhizo-
phoraceae) with combination of organic solvents. Though bark and root
of mangroves are exposed to environmental stress and rich in secondary
metabolites. The solubility of these constituents depends on their po-
larity and these parts can be effectively extracted and screened.
2. Materials and methods
2.1. Plant materials
Bruguiera gymnorrhiza L. was identified taxonomically following
morphological features of plants following standard identification
protocols and a Herbarium specimen was submitted to the Department
of Botany, Utkal University bearing Herbarium Sheet Numbers
UU.BOT-791. The leaves, roots and barks of each plant were collected
from the buffer zone of Bhitarkanika National Park, Kendrapada,
Odisha and were kept in polythene zip packs.
2.2. Plant extraction process
Roots, leaves and barks of B. gymnorrhiza were thoroughly washed
and air-dried for 3–4 days. The dried materials were ground into a fine
powder and stored in air tight containers for repeated use. A mass of
100 g of air dried plant material was processed using Soxhelt extraction
unit containing 300 mL of 95% (v/v) methanol for 36−40 h. The plant
material was then methodically extracted with combination of solvents
- chloroform, ethanol and methanol in the ratio 6:2:2 for the same time
period. The ratio of the solvents was selected as per their polarity and
previously reported methods [32–34].
2.3. Test microorganisms
The pathogens used for the antimicrobial activity evaluation were
obtained from MTCC, IMTECH, Chandigarh, India and S.C.B. Medical
College, Cuttack, India (clinical isolates). The bacterial species taken
were Klebsiella pneumoniae (MTCC 4030), Micrococcus sp. (clinical iso-
lates), Staphylococcus aureus (MTCC 3160), E. coli (NX, AMP, OF re-
sistant strain (clinical isolates)), Staphylococcus aureus (LZ, BA resistant
strain), Shigella flexneri (MTCC 1457), Pseudomonas auregenosa (clinical
isolates), Streptococcus mitis (clinical isolates), Salmonella enterica
(MTCC 733) and Proteus mirabilis (MTCC 9493). The fungal species used
were Candida albicans (MTCC 227), Aspergillus niger (MTCC 9933), A.
fumigatus (MTCC 2544) and Penicillium chrysogenum (MTCC 161). For
bacteria, nutrient agar medium (Himedia India Ltd.) was used whereas
for fungi two kinds of media viz., potato dextrose agar (Himedia India
Ltd.) and Czapek eeast extract agar were used for culture.
2.4. Antimicrobial activity assay (well diffusion method)
One hundred milligram of powdered extract was dissolved with 2
mL DMSO (Dimethyl Sulphoxide) [35]. About 25 mL of melted nutrient
2
European Journal of Integrative Medicine 36 (2020) 101140
agar was poured into each petri-dish, air cooled to solidify inside a
laminar airflow (Klenzaids ltd., India) and swabbed with freshly cul-
tured (24 h) the test microorganisms. Wells were made gently using a
sterile cork borer (6 mm dia) and filled with 65 μL (50 mg/mL) of the
extract. After incubation of 24 h at 27 °C, diameters of the zone of
inhibition (ZOI) were recorded in HiMedia Antibiotic Scale. DMSO was
used as negative control and antibiotics viz. roxithromycin and azi-
thromycin and antifungals viz. clotrimazole and fluconazole were taken
as positive controls.
2.5. Determination of MIC
The minimum inhibitory concentration (MIC) for test organisms was
evaluated with graded concentration (5 to 0.0125 mg/l) of extracts
using DMSO as negative control. A volume of 65 μL of each con-
centration was added in each well of the solidified agar petri-dishes
with specified nutrient supplementations and swabbed with freshly
cultured test organisms. The plates were made in triplicate and in-
cubated at 27 °C for 24 h for bacteria and 37 °C for 48 h for fungal
pathogens. MICs were calculated by probit analysis for each treatment.
2.6. Estimation of total phenolics
Dried extracts (1 mg) of methanol and combination of solvents were
dissolved with 20% methanol and kept overnight. An aliquot of 100 μL
was taken with 0.5 N Folin–Ciocalteu reagent, incubated for 4 min
followed by addition of 1500 μL of NaCO3 solution (20%w/v) and fur-
ther incubated at 37 °C for 45 min. The absorbance of the assay solution
was recorded at 760 nm by a UV–vis spectrophotometer (Lambda 25,
Perkin Elmer, USA) against gallic acid standard [36].
2.7. Antioxidant activity assay
2.7.1. DPPH assay
One milligram of powdered extracts of B. gymnorrhiza was mixed
with 5 mL of 0.004% (v/v) 1, 1-diphenyl-1-picryl-hydrazyl (Sigma
Aldrich, India) and absorbance was measured at 517 nm taking DPPH
as standard. DPPH scavenging activity of the samples was estimated
and expressed in percentage (%) following standard methods [37,38].
2.7.2. FRAP assay
One millilitre of the extract was dissolved in 2.5 mL of phosphate
buffer (pH 6.6) and potassium ferricyanide and kept for 20 min (at 50
°C). A volume of 0.5 mL of the solution containing ferric chloride and
trichloroacetic acid in equal proportion was then added to the assay
mixture to terminate the reaction. The absorbance was recorded at 700
nm after 30 min of incubation at room temperature. The percentage of
FRAP was calculated using ascorbic acid (HiMedia Ltd, India) as stan-
dard [39].
2.8. Statistical analysis
For each measurement the samples were taken in triplicate and the
experiments were repeated twice. Mean of the replicates with their
standard deviation have been presented in the figures and tables. The
correlations between the parameters were determined by linear re-
gression analysis and MICs were calculated by probit analysis (using MS
Excel software). Comparison of the ZOI and MIC of different variables
(plant parts, solvent extracts and pathogens) was calculated using a
two-way ANOVA and F values were calculated at P ≤ 0.05 for n = 36
[40].
S. Acharya, et al. European Journal of Integrative Medicine 36 (2020) 101140

3. Results
3.1. Antimicrobial activity and MIC
Though both the solvent extracts for all the plant parts of B. gym-
norrhiza formed a ZOI with a mean value of greater than 10 mm, the
root extract was found to be most effective compared to other plant
parts with very limited deviation. The variation among different me-
thanolic extracts ranged from 13% (Staphylococcus aureus MTCC 3161)
to 56% (Salmonella enteric MTCC 733) while with combination solvents
the difference in toxicity ranged from 7% (E. coli NX, AMP, OF re-
sistant) to 40% (P. aureginosa). No significant toxicity of the extracts
was caused to E. coli (MTCC 42) whereas all other bacterial strains were
significantly affected at the 5% level of significance. From among the
four selected fungal strains, Penicillium chrysogenum (MTCC 161) was
found to be the most sensitive showing the maximum ZOI of
21.63 ± 0.49 mm on treatment with combination extract of B. gym-
norrhiza bark followed by Candida albicans (MTCC 227). The maximum
ZOI for fungi were measured to be 54.8% and 71.2% for B. gymnorrhiza
with respect to the activity of standard antibiotics as positive control in
both methanolic and combination solvent extracts, respectively. The
variation was significant (P ≤ 0.05) between two solvent extracts of
different plant parts against the selected pathogenic strains (Table 1).
The comparison between two extraction systems showed that the
MIC in case of combination extract was lower than of the methanolic
extract in most of the pathogenic strains. Nevertheless, there were some
variations with regard to the toxicity of extracts from different plant
parts but bark extract of B. gymnorrhiza was observed to be the most
toxic against most of the pathogens in this experiment. It was also
observed that the extracts of B. gymnorrhiza were more effective against
bacterial strains than against fungal pathogens. Further, comparison
among bacterial strains showed that Micrococcus sp., Staphylococcus
aureus, S. mitis and Proteus mirabilis were more susceptible to the ex-
tracts of B. gymnorrhiza, irrespective of the parts taken, with almost
similar MICs approximated to 7−10 μg/mL when compared with other
bacterial strains. Klebsiella pneumoniae, Salmonella enterica, and
Escherichia coli (resistant strain) were found to be next susceptible
group having a minimum MIC value of about 8 μg/mL for combination
solvent extracts. Pseudomonas aeruginosa was comparatively more re-
sistant against the plant extracts with a maximum MIC value of 206.5
μg/mL and Shigella flexneri was observed to be the most resistant pa-
thogen, irrespective of plant extracts tken, having the maximum MIC
value of 368.2 μg/mL (Table 2).
Among the fungal pathogens, the extracts of B. gymnorrhiza have the
highest toxicity to Candida albicans, having the lowest MICs
(9.04–10.87 μg/ml). Aspergillus fumigatus was the next susceptible strain
having MIC 11.77–33.43 μg/ml, while Aspergillus niger was found to be
the most resistant one to the extracts of B. gymnorrhiza showing MIC in
the range of 306.1–3292 μg/ml.
3.2. Total phenolics
Total phenolics content, measured as μg GAE/mg extracts is a
measure of the secondary metabolite level in the plants. The highest
phenolic content was observed in methanolic extract of B. gymnorrhiza
root (25.36 μg of GAE/mg of extract) followed by combination extract
of B. gymnorrhiza root (24.74 μg of GAE/mg of extract). Nevertheless,
methanolic bark extracts of the plant showed significant amounts of

Table 1
Zone of inhibition (mm) of various pathogenic strains of bacteria and fungi on treatment with antibiotics as positive control and active extracts of B. gymnorrhiza.
Values are the means ± SD at n = 6 of two different experiments.
Bacterial strains Extracts Leaf Bark Root Rox Azi

Klebsiella pneumoniae (MTCC 4030) Me 10.64 ± 0.43 8.38 ± 0.31 12.53 ± 0.39 38.61 ± 0.32 40.23 ± 0.39
Co 11.42 ± 0.43 10.56 ± 0.30 14.47 ± 0.41
Micrococcus sp. Me 10.67 ± 0.61 11.61 ± 0.41 14.23 ± 0.37 36.53 ± 1.23 38.52 ± 1.94
Co 11.26 ± 0.32 11.64 ± 0.44 16.21 ± 0.31
Staphylococcus aureus (MTCC 3160) Me 11.27 ± 0.30 12.38 ± 0.55 12.77 ± 0.43 38.21 ± 0.86 38.37 ± 1.17
Co 11.36 ± 0.23 14.29 ± 0.40 14.58 ± 0.26
Escherichia coli (NX, AMP, OF resistant strain) Me 11.61 ± 0.37 12.47 ± 0.53 13.29 ± 0.38 38.51 ± 1.38 39.62 ± 1.29
Co 13.51 ± 0.42 13.43 ± 0.63 14.53 ± 0.44
E.coli (MTCC 42) Me 0.49 ± 0.08 0.64 ± 0.20 0.52 ± 0.14 18.37 ± 0.35 34.77 ± 0.83
Co 0.62 ± 0.32 0.86 ± 0.10 0.58 ± 0.21
Staphylococcus aureus (LZ, BA resistant strain) Me 8.32 ± 0.36 10.87 ± 0.41 10.31 ± 0.29 39.23 ± 1.33 45.17 ± 1.58
Co 11.42 ± 0.52 11.43 ± 0.24 13.77 ± 0.37
Shigella flexneri (MTCC 1457) Me 10.53 ± 0.39 10.38 ± 0.43 13.61 ± 0.48 33.42 ± 0.98 34.64 ± 1.69
Co 11.32 ± 0.25 10.36 ± 0.38 13.27 ± 0.29
Pseudomonas aeruginosa Me 12.22 ± 0.58 12.43 ± 0.37 16.47 ± 0.47 31.67 ± 0.97 38.47 ± 1.28
Co 13.53 ± 0.39 12.61 ± 0.53 17.43 ± 0.36
Streptococcus mitis Me 11.27 ± 0.31 10.44 ± 0.42 13.32 ± 0.23 34.65 ± 0.92 36.52 ± 1.12
Co 12.22 ± 0.21 14.23 ± 0.43 15.27 ± 0.40
Salmonella enterica (MTCC 733) Me 12.34 ± 0.25 10.45 ± 0.43 16.31 ± 0.21 37.42 ± 1.39 37.87 ± 1.24
Co 13.29 ± 0.24 11.33 ± 0.41 15.57 ± 0.37
Proteus mirabilis (MTCC 9493) Me 10.43 ± 0.23 10.34 ± 0.42 13.41 ± 0.26 32.15 ± 1.19 37.23 ± 1.26
Co 11.36 ± 0.38 12.43 ± 0.23 15.19 ± 0.36
Fungal strains Flu Clo
Candida albicans (MTCC 227) Me 10.08 ± 0.28 11.14 ± 0.19 11.71 ± 0.23 1.58 ± 0.19 21.36 ± 0.90
Co 12.27 ± 0.35 14.32 ± 0.22 14.39 ± 0.30
Aspergillus niger (MTCC 9933) Me 7.37 ± 0.50 6.45 ± 0.61 8.29 ± 0.19 21.57 ± 0.33 21.68 ± 0.33
Co 10.39 ± 0.63 8.67 ± 0.43 11.62 ± 0.47
Aspergillus fumigatus (MTCC 2544) Me 12.04 ± 0.30 11.13 ± 0.29 13.22 ± 0.38 30.23 ± 0.58 na
Co 14.14 ± 0.27 13.35 ± 0.32 15.26 ± 0.24
Penicillium chrysogenum (MTCC 161) Me 17.53 ± 0.23 19.42 ± 0.58 20.47 ± 0.37 31.33 ± 0.33 32.33 ± 0.33
Co 19.32 ± 0.28 21.63 ± 0.49 22.31 ± 0.36

Abbreviation: na-no activity; Me-methanol; Co-combination solvents (methanol:ethanol:chloroform::60:20:20); Rox-roxithromycin; Azi-azithromycin; NX-nor-
floxacin; AMP-ampicilin; OF-ofloxacin; LZ-lysozyme; BA-bactericidal antibiotic; Flu-fluconazole; Clo-clotrimazole; MTCC-microbial type culture collection and gene
bank (Govt. of India). Note. 65 μL of extract with concentration (50 mg/mL) were poured into 6 mm dia wells into the pure cultured agar plate. Note. The values in the
table represent mean ± standard deviation of three replicates. Comparison of the zone of inhibition of different variables (plant parts, solvent extracts and pathogens)
was made by two-way ANOVA. All F values were significant at P ≤ 0.05.

3
S. Acharya, et al. European Journal of Integrative Medicine 36 (2020) 101140

Table 2
Minimum inhibitory concentration (μg/mL) for various strains of bacteria and fungi on treatment with active extracts of B. gymnorrhiza. Values are the means ± SD at
n = 6 of two different experiments. The MICs are calculated by probit analysis (using MS Excel software) for each treatment.
Pathogens Methanol Combination

Leaf Bark Root Leaf Bark Root

Klebsiella pneumoniae (MTCC 4030) 10.42 ± 0.51 11.13 ± 0.42 8.69 ± 0.31 9.88 ± 0.42 9.33 ± 0.32 8.24 ± 0.16
Micrococcus sp. 128.3 ± 2.77 8.94 ± 0.31 82.3 ± 9.96 103.2 ± 3.78 7.26 ± 0.18 79.5 ± 11.31
Staphylococcus aureus (MTCC 3160) 7.62 ± 0.17 11.35 ± 0.36 9.45 ± 0.39 7.33 ± 0.27 10.34 ± 0.41 8.41 ± 0.42
Escherichia coli (NX, AMP, OF resistant strain) 119.4 ± 3.53 11.45 ± 0.28 334.2 ± 13.17 10.83 ± 0.39 8.81 ± 0.26 10.74 ± 0.36
Staphylococcus aureus (LZ, BA resistant strain) 10.31 ± 0.36 397.3 ± 11.19 10.22 ± 0.43 9.83 ± 0.27 10.45 ± 0.47 9.34 ± 0.38
Shigella flexneri (MTCC 1457) 109.5 ± 4.08 384.2 ± 17.52 368.2 ± 16.7 10.64 ± 0.81 353.1 ± 14.18 184.5 ± 6.18
Pseudomonas aeruginosa 171.6 ± 5.53 206.5 ± 9.26 182.6 ± 6.13 124.7 ± 3.16 10.53 ± 0.53 8.27 ± 0.24
Streptococcus mitis 10.07 ± 0.39 8.49 ± 0.33 9.21 ± 0.38 10.06 ± 0.42 7.62 ± 0.28 8.71 ± 0.33
Salmonella enterica (MTCC 733) 162.4 ± 4.84 187.5 ± 4.86 13.41 ± 1.67 71.82 ± 2.85 182.5 ± 5.85 8.56 ± 0.41
Proteus mirabilis (MTCC 9493) 211.6 ± 8.16 7.31 ± 0.17 8.27 ± 0.18 113.7 ± 3.82 11.03 ± 0.51 6.37 ± 0.21
Candida albicans (MTCC 227) 9.82 ± 0.31 10.87 ± 0.36 10.04 ± 0.28 9.04 ± 0.31 9.14 ± 0.47 9.74 ± 0.37
Aspergillus niger (MTCC 9933) 2362 ± 213 381.45 ± 13.17 3292 ± 156 1424 ± 49.0 306.1 ± 10.36 2412 ± 192
Aspergillus fumigatus (MTCC 2544) 33.43 ± 0.86 26.41 ± 0.47 22.44 ± 0.95 11.77 ± 1.02 24.81 ± 0.79 18.41 ± 1.07
Penicillium chrysogenum (MTCC 161) 46.26 ± 1.79 32.57 ± 0.95 39.27 ± 1.19 19.39 ± 0.91 19.31 ± 0.83 21.37 ± 1.33

Abbreviation: NX-norfloxacin; AMP-ampicilin; OF-ofloxacin; LZ-lysozyme; BA-bactericidal antibiotic.

Note. Comparison of the MIC of different variables (plant parts, solvent extracts and pathogens) was made by two-way ANOVA. All F values were significant at P ≤
0.05.

Fig. 1. Total Phenolics content (μg GAE/mg of Extract) of different extracts of B. gymnorrhiza. Values in the figure indicate mean ± standard error of mean.

phenolics among other extracts (Fig. 1). FRAP activity (96.07%) followed by methanolic root extract (95.94%)
and combination bark extract (95.50%). Similar results were found in
3.3. Antioxidant activity other extracts i.e. around 88% (Fig. 2).

The antioxidant activity was estimated using the stable DPPH free
radical scavenging activity and ferrous reducing antioxidant power
assay.
3.4. Free radical scavenging activity (DPPH assay)
The DPPH free radical scavenging assay generally used to in-
vestigate the scavenging activities of several natural products such as
phenolic compound, and anthocyanin of crude extracts of plants. Fig. 2
showed that almost all extracts have a considerably high DPPH
scavenging activity compared to the standard ascorbic acid. Methanolic
root extract of B. gymnorrhiza has the highest DPPH radical scavenging
activity i.e. 69.31% followed by combination solvent bark extract
(67.08%). The methanolic root extract exhibited the minimum activity
among all extracts taken in this experiment (12.50%).
3.5. Ferric-Reducing antioxidant power assay (FRAP assay)
Combination root extract of B. gymnorrhiza has shown the maximum
4. Discussion
Protections from erosion, cyclones, storms surge, etc. are the main
role of the mangroves in the coastal zones [41]. Mangroves are also
traditionally used as medicines against many diseases. The present
study revealed that different plant parts extracted with different solvent
systems have varying antioxidant and antimicrobial activities. Such
variations have been reported with other plant materials earlier
[42,43]. The total phenolic content also significantly varied with the
solvents used and plant parts taken for extraction.
Some Rhizophoracaemangroves viz. Rhizophora mucronata, R. la-
markii and Bruguiera cylindrica were reported for their toxicity against
methicillin resistant Staphylococcus aureus [43]. In the present study the
antibacterial positive controls showed maximum ZOI, ranging from
18.37 mm to 45.17 mm, against different pathogenic microorganisms
whereas antifungal drugs showed the inhibition zones ranging between
1.58 mm and 32.33 mm. Among the tested microorganisms, S. enterica
(MTCC 733), K. pneumoniae, and S. aureus are more pathogenic causing
diseases like Salmonellosis, infection in urinary tract and meningitis,

4
S. Acharya, et al.
Fig. 2. DPPH activity and Ferric-Reducing Power Assay (% FRAP per g of Ascorbic Acid) in different mangrove plant extracts. Values in the figure indicate
mean ± standard error of mean.
respectively. The observations revealed the fact that, the combination
extract of B. gymnorrhiza leaf and root, when compared to bark, showed
more toxicity with inhibition zones ranging from 11.26 mm to 17.43
mm for different bacterial species like K. pneumonia (MTCC 4030),
Micrococcus sp. (Clinical Isolates), S. aureus (MTCC 3160), E. coli (MTCC
42), S. aureus (antibiotic resistant clinical isolates), S. flexneri (MTCC
1457), P. auregenosa (clinical isolates), S. mitis (clinical isolates)and P.
mirabilis (MTCC 9493). However, the methanolic and combination ex-
tracts of B. gymnorrhiza root showed more toxicity with inhibition zone
of 17.43 mm and 16.31 and having MIC values of 8.27 μg/mL and
13.41 μg/mL, against P. auregenosa and S.enteric, respectively. Never-
theless, all the other bacterial species were also more or less inhibited
by this extract. In case of B. gymnorrhiza root, combination of different
solvents such as methanol, ethanol and chloroform showed better
phytochemical extraction, vis-a-vis more toxicity to pathogenic strains.
Such improved efficacy of combination extracts (acetate + methanol +
water) for apple skins have previously been reported [44].
Apart from the bacterial pathogens, the fungal strains, Candida al-
bicans, Aspergillus sp. and Penicillium chrysogenum cause many harmful
diseases in human being like candidiasis, aspergillosis, and urinary tract
infection (UTI) respectively [45]. Candidiasis and UTIs are the most
common fungal diseases in human [46]. The results showed that me-
thanolic extract of B. gymnorrhiza leaf and combination extracts of B.
gymnorrhiza root, having lower MIC values (∼ 9 μg/mL), showed a
maximum ZOI of around 10−14 mm against Candida albicans. Com-
bination extract of B. gymnorrhiza root showed maximum ZOI of 22.31
mm against P. chrysogenum (MIC 21.37 μg/mL) followed by ZOI of
15.26 mm against A. fumigatus (MIC 18.41 μg/mL). A. niger was found
to be comparatively more resistant to both the extracts showing the
maximum MIC (2412 μg/mL) (Table 1 and 2). Hence these extracts
from B. gymnorrhiza exhibited significant toxicity against some patho-
gens showing their efficacy for the formulation of novel pharmaceutical
drugs.
Phenolic compounds, found in different plant parts, show anti-al-
lergenic, anti-apherogenic, anti-inflammatory, antimicrobial and anti-
oxidant properties [45–49]. Fig. 1 showed maximum value of total
phenolics of methanolic extract of B. gymnorrhiza root and combination
solvent extract of B. gymnorrhiza bark. Both the extracts also showed
positive correlations with ZOI for the pathogen C.albicans and P. chry-
sogenum (R2 = 0.71 & 0.25, respectively) (data not shown). Such an-
timicrobial properties of the constituent phenolic compounds are
known to be expressed by the disorganisation of cell peptidoglycan
5
European Journal of Integrative Medicine 36 (2020) 101140
leading to membrane disruption [50–54]. The mechanism may involve
change in membrane permeability that could lead to blockage of active
transport, uncoupling of oxidative phosphorylation, and loss of meta-
bolites [54]. The total phenolics, which may constitute these com-
pounds, exhibit antioxidant activity [55,56] and also found to be
maximum in B. gymnorrhiza root and bark extracts with combination
solvents (Fig. 2). These compounds also impede enzyme activities such
as tyrosinase, which plays a vital role in pigmentation, growth and
development [57].
Methanol extracts of B. gymnorrhiza root as well as combination
extract of bark were observed to have higher percentage of DPPH ac-
tivity than other extracts. The results showed a positive correlation
between phenolic content and DPPH activity (R2 = 0.89; slope = 0.37)
(data not shown). Phenolic compounds including flavonoids and an-
thocyanins could be a major determinant of antioxidant potency of the
samples. The free radical scavenging activity could be correlated to
their antimicrobial activities because the phenolic compounds are
known to induce antimicrobial activity by causing intracellular hyper-
acidification and thereby disrupting ATP synthesis in pathogens [58].
The antioxidant property of phenolic compounds is determined by
redox potential, stabilization and delocalization of the unpaired elec-
trons [59,60]. In the present study the order of ferric reducing power of
all the extracts was combination root > methanolic root >
combination bark > methanolic leaf > methanolic bark > combination
leaf extracts. The reducing potential also established a positive corre-
lation with DPPH scavenging activity (R2 = 0.83; slope = 1.34). This
reducing power of mangrove extracts might have higher reduction
potential which could react with free radicals to neutralise the chain
reactions.
But these preliminary tests are not enough to formulate therapeutic
compounds. An extensive study is required for separation, purification
and characterization of pharmaceutically active molecules.
5. Conclusion
Among the bacterial strains, Micrococcus sp., Staphylococcus aureus,
Proteus mirabilis and Streptococcus mitis were the most susceptible ones
to the extracts of B. gymnorrhiza with a minimum MIC of about 7 μg/
mL. Root extracts of B. gymnorrhiza were more toxic to bacterial strains
except E. coli than to fungal strains. However, irrespective of plant parts
taken, the extracts with combination of solvents exhibited significant
toxicity against Candida albicans and Penicillium chrysogenum. The DPPH
S. Acharya, et al.
activity and FRAP activity of root extracts of B. gymnorrhiza were re-
ported to be significantly higher (at P < 0.01) compared to the extracts
from other plant parts. A significant positive correlation was seen be-
tween total phenolic contents of B. gymnorrhiza root and the potency of
the extract to inhibit the microbial growth, which may be associated
with antioxidant activity. It was also evident that resistant strains of E.
coli, S. aureus and Urinary Tract Infections (UTI) bacterial pathogens
e.g. P. aeruginosa, K. pneumoniae and P. mirabilis were inhibited sig-
nificantly by the mangrove extracts. Though the extracts from both the
solvent systems are rich in hydrophilic compounds such as phenolics
and are known to be more effective against Gram negative bacteria
[59,60], B. gymnorrhiza extracts in this study effectively inhibited the
growth of both Gram positive and Gram negative bacteria. Our results
also emphasises that there is a need for development of new drugs
because of the resistance developed to existing antibiotics by patho-
gens. B. gymnorrhiza may be explored to derive bioactive molecules
against these bacteria and fungi.
Ethno-pharmaceutical surveys can be used as a possible strategy to
identify phyto-medicines for screening against human pathogens. After
investigating through these preliminary antimicrobial, antioxidant and
phyto-constituents screenings, it may be concluded that the plant has
potential medicinal properties. In addition to the above tested phyto-
chemicals, plants can be screened for other secondary metabolites like
polyphenols, coumarins, essential oils, tannins, flavenoids etc. This
study may help in the discovery of novel plant based drug formulations
with effective active principles to combat against deadly pathogens in
humans.
Authors contribution
Author contribution statement, Srinivas Acharya: literature search,
data analysis, data interpretation, writing, figures and tables. Deepak
Kumar Patra, Chinmay Pradhan and Pradipta Kumar Mohapatra: lit-
erature search, study design, data communication, data interpretation.
Author Agreement
We confirm that the manuscript has been read and approved by all
named authors and that there are no other persons who satisfied the
criteria for authorship but are not listed. We further confirm that the
order of authors listed in the manuscript has been approved by all of us.
Financial support
None.
Declaration of Competing Interest
There are no conflicts of interest to declare.
Acknowledgements
The authors are grateful to acknowledge DST FIST, UGC DRS (SAP
III), CoE (World Bank& RUSA 2.0) of Department of Botany, Utkal
University for upgrading the laboratory facility to carry out the work.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.eujim.2020.101140.
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