International Journal of General Medicine and

Pharmacy (IJGMP)
ISSN(P): 2319–3999; ISSN(E): 2319–4006
Vol. 9, Issue 2, Feb–Mar 2020; 1-14
© IASET

EVALUATION OF ANTIMICROBIAL AND ANTIOXIDANT ACTIVITY OF CRUDE

METHANOL EXTRACT AND ITS FRACTIONS OF MUSSAENDA PHILIPPICA LEAVES

Debashisa Mishra1, Susanta Kumar Rout2 & GB. Alaka Kar3

1,3
Research Scholar, IMT Pharmacy College, Puri, Odisha, India
2
Research Scholar, Department of Science & Technology, Patent Information Centre, Odisha, India

ABSTRACT

Aim: To evaluate the antimicrobial and antioxidant activities of crude methanol extract and its fractions of the leaves of
Mussaenda Philippica.

Methods: MIC through disc diffusion methods and MBC are two methods to evaluate the antimicrobial activities. For
studying the antioxidant activity the free radical scavenging was studied in vitro method by measuring DPPH, Hydrogen
peroxide scavengering activity, Superoxide free radical (O2) and Nitric oxide (NO) free radical scavenging activity
measured by considering standard antioxidant e.g. ascorbic acid.

Results: The test compounds like different fractions i.e. chloroform, methanol and ethyl acetate and crude methanol extract
produced significant effect both in antioxidant as well as antimicrobial activities. Different microorganisms namely. E.
faecalis, S. aureus, A. baumannii, E. coli, P.merabilis, P.aeruginosa are responded in higher concentration all the test
compounds. Different fungal strains like C.albicans and A. niger are not inhibited by different test compounds.

Conclusions: The results revealed that the crude methanolic extract and different fractions like methanol and ethyl acetate
are produces remarkable antimicrobial and antioxidant activities. It may assume that the activity of the test compound may
be due to the active compounds such as flavonoids, terpenes, alkaloids and saponins.

KEYWORDS: Musa Philippica, Antimicrobial, Antioxidant, MIC

Article History
Received: 03 Mar 2020 | Revised: 01 Feb 2020 | Accepted: 01 Apr 2020

INTRODUCTION

At present natural substances are considered as a source of motivation for discovering a new drug. Plant derived
1
compounds/ substances having a significant contribution towards human health and well being. It is known from
literatures that most of the traditional medicines obtained from natures specifically from plant materials. Plant materials are
easily available in rural areas. Due to readily available of resources i.e. plants/ herbs the medicines in rural belt are cheaper
than the alternative medicines like modern medicines. Medical plants and their by products (secondary metabolites) are the
oldest health-care products. The importance of the herbal medicines is highly demanded in health care systems not only in
developing countries but also in many developed countries 2. The herbal medicines obtained from natural sources having
no side effect along with better therapeutic effect. Herbal medicines have wide therapeutic actions because of the safety
profile. From statistical report it is known that approximately 80% of the population in world rely on traditional medicine

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2 Debashisa Mishra, Susanta Kumar Rout & GB. Alaka Kar

for their primary health care and play an important role in the health care system. World Health Organization (WHO) is
promoting, encouraging and facilitating the research area on herbal medicines because of the effective use of herbal
medicine in developing countries for health programs 3. Different biological activities like anti microbial, anti oxidant,
sedative and anxiolytic effects of the plant extracts may be due to presence of the active compounds. Consequently, due to
some other biological activities on the same time make excellent leads for new drug development 4.

Mussaenda philippica ‘Aurorae’ (named after Dona Aurora, wife of a former President of the Philippines). It is an
ornamental plant in tropical areas. It is reported that the plant produced Hepatoprotective activity of two iridoids from.
Mussaenda 'dona aurora' (sepals) has been investigated for its hepatoprotective and antioxidant activities. The highest
activity was observed in the ethyl acetate fraction. 5, 6. From different literature it was found that the methanol extract of M.
frondosa leave produced normalizing activity against cold immobilization stress, it also reported that some biochemical
transmitters also changed the transmitters are norepinephrine (NE), dopamine (DA), 5-hydroxy tryptamine (5-HT), 5-
hydroxy indole acetic acid (5-HIAA), and enzyme monoamine oxidase (MAO).

The presence of bioactive compounds like iridoid glucosides, mussaenoside and shanzhiside methyl ester has been
reported in different species of Mussaenda 7, 8,. However, as far as the activity is concerned, there are few reports available
regarding antibacterial, antifungal and antioxidant activity. Thus, the aim of the present study was to investigate the
antimicrobial and antioxidant effect of crude methanol extract and different fractions like chloroform, ethyl acetate and
methanol fraction of crude methanol extract of leaves of M. Philippica.

MATERIALS AND METHODS

Plant Material

M. Philippica leaves were collected from Puri district of Odisha, India. The leaves were authenticated by Dr. Chinmay
Pradhan, Department of Botany, Utkal University, Odisha. The leaves were collected in large quantity for further
processing. The raw leaves were processed step wise i.e. first the leaves are washed in running because of water may
remove adhering soil and dirt particles. In second step the leaves are kept for drying i.e. shade dry. In IMT Pharmacy
College, Puri, Odisha a voucher specimen was deposited for further correspondence. In third step the dried leaves were
powered through mechanical grinder and stored in airtight, non-toxic polyethylene bags until used.

Preparation of Extract and Fractions

The powdered M. Philippica leaves of was extracted with ether in 700 C for 3 days for to defeating the materials after that
the plant materials were macerated in different solvents based on the polarity with constant stirring. The solvent
incorporating the extractives were filtered and the marc pressed to squeeze out residual extractives. This process was
repeated at least thrice time for complete the extraction. For final products the extracts are evaporated generally it will be
reduced to 1/8th of its original volume by using rotary evaporator with a constant temperature i.e. 45 °C and obtained the
yield. Chloroform, ethyl acetate and methanol are used as solvent for extraction. 9 Different fractions were obtained after
using different solvents then the fractions are concentrated followed by dry and preserved. Chemical tests like glycosides,
alkaloids, favonoids, saponins, carbohydrates, tannins, phenolic compounds, protein, and fats are performed in the
compounds obtained through fraction and extract.10 The test substances were prepared by using 10% w/v in normal saline
along with 0.1% propylene glycol.

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Evaluation of Antimicrobial and Antioxidant Activity of Crude Methanol 3
Extract and its Fractions of Mussaenda Philippica Leaves

Evaluation of Antimicrobial Activity

Disc Diffusion Method

Disc diffusion test for the extracts was carried out for antimicrobial activity. The test compound for antibacterial activity
was carried out by cup-plate method. Cups or discs with standard diameters are made in the nutrient agar medium. The test
compounds were poured inside the discs and for result observations the diameter regarding zone of inhibition was
measured. For antibacterial activity different strains like some gram positive microorganisms S. aureus, B. subtilis and
11, 12
some gram negative E. coli, P. aeruginosa organism are selected following agar diffusion method . Different test
0
organisms were again cultured by using nutrient agar medium. 37±1 C for 24 hours is the procedure for incubation then
they were stored in refrigerator. A loopful stock culture is used for bacterial inoculum then transferring it to the nutrient
broth (100ml) in a control flask (250ml). The flasks were incubated at 37±10C for 18 hours before starting the
experimentation. Solution of the test compounds were prepared as per the guidelines i.e. dissolving 10mg of each in 10ml
of dimethyl formamide (analytical grade) (1000 mg/ml conc.). A reference standard from gram positive and gram-negative
bacteria was made by dissolving accurately weighed with quantity of Ampicillin (100 µg/ml) i.e. standard for Gram
positive and Gram negative bacteria. Griseofulvin an antifungal agent (20 µg/ml) is used for antifungal activity.

Minimum Inhibitory Concentration (MIC) Method

MIC method is another method to evaluate the antimicrobial activity on test compounds like extracts and fractions. It
proved the efficacy against different microorganisms strain. Highest plant extract dilution retains an inhibitory effect
against the growth of a microorganism is known as MIC. M7-T2 publication of the National Committee for Clinical
Laboratory Standards is followed for the experiment13. The plant extract and fractions were subjected to a serial dilution
using sterile nutrient broth medium as a diluents. The two different strength of the plant extract and fraction were
inoculated with 20 µl of an individual microorganism. 37°C for 24 h are inoculated for each dilution. The highest dilution
of the plant extract/ fraction retained its inhibitory effect resulting in no growth (absence of turbidity) of a microorganism.
At the same time a control experiment conducted in parallel to study the impact of the solvent itself (without plant
components) on growth of the nine test organisms. Each solvent those are tested was diluted in a similar pattern with sterile
nutrient broth.

Anti-Oxidant Activity

The antioxidant activity of the test compound those are obtained from methanol extract was determined by in vitro models.
The in vitro methods include Diphenyl-picryl-hydrazyl (DPPH) radical, Superoxide free radical (O2), Peroxide radical
(H2O2), and Nitric oxide (NO) free radical scavenging activity with reference to standard antioxidant ascorbic acid14.

DPPH Free-Radical Scavenging Activity

The free radical scavenging activity of the metabolite, based on the scavenging activity of the stable 1,1- diphenyl-2-
picrylhydrazyl (DPPH) free radical was assayed according to the protocol 15. Briefly, different concentrations of the extracts and
ascorbic acid were prepared. Various concentration of test solution in 0.1ml was added to 0.9 ml of 0.1 mM solution of DPPH in
methanol. Control was 100µl methanol and 100µl DPPH solution. After 30 minute of incubation at room temperature, the
reduction in the number of free radical was measured, reading the absorbance at 517nm. Ascorbic acid was used as reference
standard. The scavenging activity of the samples corresponded to the intensity of quenching DPPH. Ascorbic acid was used as
reference standard. The compounds with antiradical activity changed color as yellow from the purple-blue.

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4 Debashisa Mishra, Susanta Kumar Rout & GB. Alaka Kar

Quantitative Determination of the DPPH Radical Scavenging Activity

The antioxidant activity of the M. Philippica methanolic extract and different fractions were evaluated
spectrophotometrically following the DPPH method. Different concentrations of the extracts and fractions 100, 200 and
500 µg/ml) were prepared and mixed 1 ml of them with 2 ml of a freshly prepared DPPH solution (0.01mM); then, each
particular sample was mixed thoroughly and kept in the dark for 30 minutes, at room temperature. After that, each mixture
was tested for the DPPH radical scavenging activity by reading the absorbance at 517 nm on a UV-VIS spectrophotometer.
As blank was used a solution prepared by mixing 1 ml of methanol with 2 ml of the DPPH solution (0.01mM) and reading
at the same wavelength. In addition, to eliminate the absorbance of the crude extracts at this wavelength, blank samples
were prepared with 1 ml of each extract and 2 ml of methanol. The antioxidant activity percentage was calculated
following the formula:

Antioxidant activity (%) = [(AC -AE) /AC] x 100

Where AC is the absorbance of a DPPH solution without extract, AE is the absorbance of the tested extract and
fractions, which is equal to the absorbance of the plant extract plus the DPPH (0.01mM) minus the blank extract
absorbance. As standard ascorbic acid at different concentration 5-25 µg.ml-1 was used. The EC50 value, defined as the
concentration of the sample leading to 50% reduction of the initial DPPH concentration, was calculated from the linear
regression of plots of concentration of the test extracts against the mean percentage of the antioxidant activity16.

Superoxide (O2) Free-Radical Scavenging Activity

Measurement of superoxide anion (O2) scavenging activity of extracts and fractions was based on the method with slight
modification 24. O2- radicals are generated non-enzymatically in Phenazine methosulphate–Nicotinamide adenine
dinucleotide phosphate (PMS–NADH) systems by the oxidation of NADH and assayed by the reduction of NBT. In this
experiment, the superoxide radicals were generated in 1 mL of Tris- HCl buffer (16 mM, pH 8.0) containing nitro blue
tetrazolium (NBT) (50 µM) solution and NADH (78 µM) solution. The reaction was started by adding PMS solution (10
µM) to the mixture. The reaction mixture was incubated at 25°C for 5 min, and the absorbance at 560 nm in a
spectrophotometer was measured against blank samples. Decreased absorbance of the reaction mixture indicated increased
superoxide anion scavenging activity. The percentage inhibition of superoxide anion generation was calculated using the
following formula:

(%) I = (A0 – A1) / (A0) x 100

Where A0 was the absorbance of the control and A1 was the absorbance of extract and the standard compound.

Peroxide Free Radical (H2O2) Scavenging Activity

Scavenging of H2O2 by the extract and fractions was determined. One millilitre of C. siamea flower extract solution
[prepared in phosphate buffered saline (PBS)] was incubated with 0.6 ml of 4mM H2O2 solution (prepared in PBS) for 10
min. The absorbance of the solution was measured at 230 nm against a blank solution containing the extract without
H2O217. The concentration of H2O2 was spectrophotometrically determined from absorption at 230 nm using the molar
absorptivity.

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Evaluation of Antimicrobial and Antioxidant Activity of Crude Methanol 5
Extract and its Fractions of Mussaenda Philippica Leaves

Nitric Oxide Free Radical (No) Scavenging Activity

Nitric oxide (NO) was generated from sodium nitropruside (SNP) and measured by Greiss reaction. SNP in aqueous
solution at physiological pH instinctively generates NO, which intermingles with oxygen to generate nitrite ions that can be
anticipated by Greiss reagent (1% sulphanilamide, 2% phosphoric acid and 0.1% naphthyl ethylene diamine
dihydrochloride). Scavengers of NO with oxygen leading to reduced production of NO. SNP (5 mM) in phosphate buffer
saline (PBS) was mixed with different concentration of (100-500 µg/mL) drug dissolved in suitable solvent and incubated
at 25oC for 150 min. The above samples were reacted with Greiss reagent. The absorbance of chromophore created during
diazotization of nitrite with sulphanilamide and following coupling with naphthyl ethylene diamine was read at 546 nm and
referred to the absorbance of standard solution of potassium nitrite treated in the same way with Greiss reagent18.

Statistical Data Analysis

Results were expressed as mean ± SEM. All the results were analyzed by One-way Analysis of Variance (ANOVA)
followed by Dunnett’s test. The level of significance was set at P<0.05.

RESULTS

The phytochemical screening (Table. 1) of the different fractions and crude methanolic extact of M. Philippica revealed the
presence of glycosides, steroids, flavonoids, terenoids, saponins and reducing sugar. The crude methanolic extracts and
fractions of the plant was studied against both gram-negative, grampositive bacteria and fungus related to their zone of
inhibition, MIC and MBC. The results of the antimicrobial screening of the methanol extracts and different fractions of M.
Philippica was shown in (Tables 2 and 3). The results were recorded as presence or absence of zones of inhibition around
the well as well as MIC and MBC. The inhibitory zone around the well indicated the absence of bacterial growth and it was
reported as positive and absence of zone as negative. The crude methanolic extracts of leaves of M. Philippica Showed
moderate to high antimicrobial activity against all the tested microorganisms except A.niger and C.Albicans. Crude
methanolic extract and different fractions were used to assess the in-vitro antioxidant activity. The antioxidant activity of
chloroform fraction was determined by different in vitro methods such as, the DPPH free radical scavenging assay and
reducing power methods in (Tables 4). All the assays were carried out in triplicate and average values were considered.
Antioxidant scavengering activity was studied using 1, 1—diphenyl, 2-picrylhydrazyl free radical. Various concentration
of test solution in 0.1ml was added to 0.9 ml of 0.1 mM solution of DPPH in methanol. Methanol only (0.1ml) was used as
experimental control. After 30 minutes of incubation at room temperature, the reduction in the number of free radical was
measured, Ascorbic acid was used as reference standard. The crude methanolic extract along with chloroform, ethayl
acetate methanolic, and fractions showed a concentration dependent antiradical activity by scavengering DPPH radical.
Crude methanolic extract was found to be more potent compared to other fractions. The observations made in the present
study showed that the extract of M. Philippica leaves exhibited good scavengering of H2O2 in the biological system, thus
preventing the stress induced by progressive increase in malondialdehyde and other free radicals which cause oxidative
damages (Tables 5). All various fractions and crude methanolic extract of these plants were capable of reducing DNA
damage comparing to control. The percentage inhibition of nitric oxide generation by different fractions and extracts of M.
Philippica leaves at different concentration were compared with standard (Tables 6). All test compounds exhibited potential
inhibiting activity against NO generation. Nitric oxide is a potent pleiotropic mediator of physiological processes. The antioxidant
activity of crude methanolic extract of M. Philippica showed highest inhibition of nitric oxide generation which is compared with
standard ascorbic acid. All the data along with test standard and control compounds are present in the table i.e. ((Tables 7)

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6 Debashisa Mishra, Susanta Kumar Rout & GB. Alaka Kar

DISCUSSIONS

The different parts of the plant such as root, bark and leaves of M. Philippica has been used for thousands of years for its
medicinal properties. It is rich in a wide variety of secondary metabolites such as glycosides, phytosterols, proteins,
saponins and phytosterols. In this connection the present study on the methanolic extract and the different fractions of
crude methanolic extract was conducted to evaluate the antimicrobial activity of leaves. Phytomedicines can be used for
the treatment of diseases as is done in case of Unani and ayurvedic system of medicines, a natural blue print for the
19
development of new drugs. The MIC values of those extract and fractions which gave positive results during prelim .
Screenings were determined by Tube Dilution Method. The antimicrobial study was conducted using different micro
organisms such as Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa,
Proteus mirabilis, A. Baumannii, Candida albicans, Aspergilus Niger. The extract and fractions of M. Philippica showed
the higher zone of inhibition. Though the growth of Staphylococccus aureus, Pseudomonas aeruginosa, Staphylococcus
epidermidis, Pseudomonas aeruginosa are controlled by M. Philippica, it indicates that they could inhibit the activity of
bacteria which causes diarrhoea, polymixin and typhoid respectively. Polyphenols, including flavonoids, forms a large
group of naturally occurring components of the plant kingdom and are present in every part of the plants. These
compounds are of considerable interest in various fields such as food, pharmacy and medicine because of wide range of
biological activities including antioxidant activity20. The antioxidant efficacy of phenolic compounds is chiefly due to their
redox potential. These compounds are known to act as reducing agents (free radical terminators), hydrogen donors, metal
chelators and singlet oxygen quenchers. Since it has been shown that phenolic compounds of plant kingdom are one of the
most effective antioxidative constituents. Flavonoids are polyphenolic compounds and consist of flavones, flavonols,
flavanols, flavanone and flavanonols. These compounds represent the majority of plant secondary metabolites and have
shown to possess remarkable health promontory effects such as anti-inflammatory, antioxidant, antimicrobial, anticancer
and others. Interception of free radicals or other reactive species is mainly by radical scavenging and is caused by various
antioxidants like vitamins C and E, glutathione, other thiol compounds, carotenoids, flavonoids, etc. While at the repair and
reconstitution level, mainly repair enzymes are involved. In general, peroxyl radicals cause chain reactions in lipids,
proteins and DNA 22. The high reactivity of the representative peroxyl radical shows that the possible mechanism behind
the observed protection of these biomolecules by DA may be through scavenging of secondary radicals. The soluble free
radical DPPH is well known as a good hydrogen abstractor yielding DPPH-H as by product. Thus, the scavenging of
DPPH radicals by phenols are most of the time very effective. All the plant extracts used in this study were primarily
screened against the test microorganisms by the Different Methods like Disc diffusion and MIC methods. The relative
efficacy of some commonly used antibiotics were compared with plant extract discs 23. On the basis of the results finding
in the present investigation, it is concluded that the crude methanolic extract of M. Philippica produces highest wound
healing activity plant extract and Fractions. The present study suggests that the antimicrobial and antioxidant activity can
be enhanced by the use of crude methanolic extracts of M. Philippica.

CONCLUSIONS

Drugs from plants have a long history in both traditional and modern societies as herbal remedies or crude drugs and as
purified compounds. The present study revealed that the selected Plant extract and some fractions of the crude extract
produced antimicrobial and antioxidant activity with dose dependent manner. The observed activities of leave extract
might be attributed to the presence of secondary metabolites such as flavonoids and phenolic compounds. The leaves can

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Evaluation of Antimicrobial and Antioxidant Activity of Crude Methanol 7
Extract and its Fractions of Mussaenda Philippica Leaves

be used to prevent oxidative damage caused by free radicals and to treat infections caused by pathogenic bacteria not to
fungus. Further studies with purified constituents are needed to understand the complete mechanism of wound healing
activity of the test plant. However, it needs further evaluation in clinical settings before consideration for the treatment of
different disorders.

ACKNOWLEDGMENTS

The research project was supported by University Grant Commission. The authors wish to thank Dr. Susanta Kumar Rout,
Patent Information Centre, Science & Technology Department, Government of Odisha for providing support to conduct
work and moral support.

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11. Bauer AW, Kirby WMM, Sherries JC, Turck M, Antibiotic susceptibility testing by single disk method, American
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12. Misra SB, Dixit SN, Antifungal properties of leaf extract of Ranunculus sceleratus. L. Experientia, 1978, 34,
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13. Shohayeb M. et al., Antibacterial and Antifungal Activity of Rosa damascena MILL. Essential Oil, Different
Extracts of Rose Petals, Global Journal of Pharmacology, 2014, 8 (1), 01-07.

14. Blois MS, Antioxidant determinations by the use of a stable free radical, Nature, 1958, 181, 1199-150.

15. Williams WB, Cuvelier M, Berset C, Use of a free radical method to evaluate antioxidant activity, Lebensm-Wiss
Technol, 1995, 28, 25- 30.

16. Shirwaikar A, Prabhu K and Punitha ISR. In-vitro antioxidant studies of Sphaeranthus indicus, Indian Journal of
Experimental Biology. 2006, 44, 993- 998.

17. Kumaran A, Karunakaran RJ. In vitro antioxidant activities of methanol extracts of Phyllantus species from India.
Lebens-Wiss technologie, 2007, 40, 344–352.

18. Ojala T, Remes S, Haansuu P, Vuorela H, Hiltunen R, Haahtela K, Vuorela P. Antimicrobial activity of some
coumarin containing herbal plants growing in Finland. Journal of Ethnopharmacology. 2000; 73: 299–305.

19. Cowan MM. Plant products as antimicrobial agents. Clinical Microbiology Reviews. 1999; 12(4): 564-582.

20. Tilak JC, Adhikari S, Devasagayam TPA. Antioxidant properties of Plumbago zeylanica, and Indian medicinal
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Table 1: Preliminary Phytochemical Screening of

Fractions and Extract of M. philippica
Constituent CF EAF MF AQF MEMP
Alkaloids - - + + +
Tannins + + + + +
Triterpenoids + + + + +
Saponins + + + + +
Flavonoids - + + + +
Phenols + + + + +
Glycosides + + + + +
Steroids - - - - -
Carbohydrates - - - - -
(-) Absent, (+) Present

The results of the antimicrobial screening of the methanol extracts and different fractions of M. philippica were
shown in (Table 3 and 4). The results were recorded as presence or absence of zones of inhibition around the well as well
as MIC and MBC. The inhibitory zone around the well indicated the absence of bacterial growth and it was reported as
positive and absence of zone as negative. The crude methanolic extracts of leaves of M. philippica showed moderate to
high antimicrobial activity against all the tested microorganisms except A. niger and C. albicans.

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Evaluation of Antimicrobial and Antioxidant Activity of Crude Methanol 9
Extract and its Fractions of Mussaenda Philippica Leaves

Table 2: Antimicrobial Activity of M. Philippica by Agar Well Diffusion Method

Zone of inhibition in( mm)
n P. A. A.
Sample Conc E. P. B. E. C.
S.aureus S.Epidermidis Aerugino Bauman Nig
Coli Merabilis Subtilis Faecalis Albicans
sa nii er

DMS
100(µ 4.1 ± 5.1 ± 3.8 ± 3.4
O 4.3± 0.56 3.9 ± 4.1 ± 3.2 ± 4.2 ± 4.2 ±
g/ml) 0.72 0.7 0.7 ±
0.5 0.6 0.4 0.7 0.5
0.5
Grise
30.
ofulvi 20(µg 31.4±2
NA NA NA NA 2±
n /ml) NA NA NA NA .3c
2.1
c

Ampi 100
26.42± 23.5 29.12± 31.4±0.
cillin (µg/ 28.6±0.22c 30.4 ± 24.8±0. 30.2± NA
0.25c ± 0.7c 7c NA
ml) 0.6c 9c 0.6c
0.7c
100
(mg/ 6.3±0.6 6.42±0. 13.2± 8.6 ± 4.8
8.2±1.15 10.4 12.2±0. 4.6 ± 7.4 ±
Chlor ml) 7 64a 0.8 c 0.6 ±
±0.5b 8c 0.4 0.6a
oform 0.5
Fracti 200
on (mg/ 10.2±0. 16.24± 18.12± 14.24± 5.1
11.42±0.34c 14.2 15.6±0. 14.8±0 7.6
ml) 46c 0.67c 0.44c 0.68c ±
±0.8c 6c .58 c ±0.4b
0.4
100
10.2
Ethay (mg/ 10.4±0. 14.54± 12.4 ± 8.2 ± 13.4 ± 4.2
12.23±1.44c ± 12.4± 6.8
l ml) 64c 0.46c 0.8 c 0.5 0.8 c ±
0.6c 0.8 c ±0.6
Aceta 0.8
te 200
14.2
Fracti (mg/ 13.8±0. 16.26± 14.24± 11.89± 15.2±0 5.2
14.24±0.48c 4±0. 14.4±0. 7.2 ±
on ml) 66c 0.62c 0.68c 0.48c .68c ±
66c 9 0.6a
0.4
100
16.2 4.8
(mg/ 14.22± 16.8±0. 14.8±0. 18.26± 12.2 14.2±0
16.24±0.67c 4±2. 6.8 ± ±
ml) 0.68c 9c 6c 0.7c ±0.5 c .7c
Aque 2c 0.9a 0.4
a
ous
Fracti
200
on 19.2 6.2
(mg/ 16.4±0. 18.9±0. 17.4±0. 20.4±0. 14.6 17.6±0
18.8±0.6c 4±0. 6.3 ±
ml) 6c 6c 9c 6c ±0.6 c .6c
8c ±0.5b 0.4
b

100
4.7
(mg/ 16.8±0. 17.8 18.24± 17.8±0. 18.24± 12.4 17.4±0
17.6±0.9c 6.6 ± ±
Crude ml) 6c ±0.8c 0.66c 46c 0.5c ±0.7 c .6c
0.6a 0.8
metha a
nolic
extrac 200
7.8
t (mg/ 20.4±0. 19.5 20.4±0. 20.4±0. 21.2±0. 16.2 19.6±0
22.4±0.64c 7.5 ±
ml) 8c ±0.6c 54c 62c 6c ±0.6 c .6c
±0.2b 0.6
b

Values are mean ± S.E.M. of 3 replications. S.aureus:- Staphylococcus aureus, S.epidermidis-Staphylococcus

epidermidis, E.coli- Escherichia coli, P.aeruginosa- Pseudomonas aeruginosa, P. mirabilis:- Pseudomonas mirabilis, A.
baumannii:- C.albicans:-Candida albicans. A. Niger:- aspergilus Niger ( NA)- no measurable zone of inhibition. Aqueous

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10 Debashisa Mishra, Susanta Kumar Rout & GB. Alaka Kar

extract,methanol extract and Chloroform extracts respectively each having cocentration 300 µg per disc. significance at
*p<0.05, **p<0.01. t-value denotes significance at ap<0.05, bp<0.01, cp<0.001

Table 3: The MBC Regimes of the Extracts and Fractions of the Leave of
M. Philippica
M. Philippica Extract and Fractions
Strain CF EAF AF MEMP
MIC MBC MIC MBC MIC MBC MIC MBC
E. faecalis 4.1 4.1 3.8 10.2 4.1 22.4 4.1 10.2
S. aureus 4.2 8.8 4.1 5.1 10.2 20.5 10.2 22.9
A. baumannii 7.1 16.2 10.2 22.4 10.4 22.8 4.2 20.8
E. coli 4.1 13.5 10.4 20.5 4.1 16.2 8.8 22.2
P. merabilis 3.4 14.2 5.1 10.2 10.4 22.5 4.4 23.8
P.aeruginosa 3.1 10.2 10.2 22.4 4.2 5.1 4.2 10.4

Table 4: Determination of 1, 1-Diphenyl-2-Picrylhydrazyl (DPPH), Superoxide (O2), Hydrogen Peroxide

(H2O2) and Nitric Oxide (NO) Scavenging Activity of Chloroform Fraction of M. Philippica

DPPH O2 H 2O 2 NO
Groups &
% % % %
Treatments % of % of % of % of
Scavenging Scavenging Scavenging Scavenging
Control Control Control Control
Activity Activity Activity Activity
100.0 ± 00 100.0 ± 00 100.0 ± 00 100.0 00
Control
4.1 3.8 3.9 ±4.2
Ascorbic acid
100 µg/ml 30.2 ± 69.8 40.2 ± 58.6 ± 68.2 ±
3.8c 4.1b 59.8 4.6 41.4 3.4 31.8
200 µg/ml 16.8 ± 83.2 28.6 ± 48.6 ± 60.2 ± 4a
2.2c 3.6b 71.4 4.1 51.4 39.8
400 µg/ml 4.2 ± 95.8 3.4 ± 26.4 ± 46.4 ±
2.2c 2.8c 96.6 1.6c 73.6 3.2b 53.6
500 µg/ml 0.00 100 1.2 ± 16.2 ± 28.4 ±
2.5c 98.8 1.2c 83.8 2.5c 71.6
CF
100 µg/ml 67.7 ± 66.4 ± 70.4 ± 74.3 ±
2.6 32.3 2.8 33.6 3.6 29.6 3.1 25.7
200 µg/ml 58.4 ± 58.6 ± 66.2 ± 64.2 ±
2.2 41.6 3.2 41.4 3.2 33.8 2.8 35.8
400 µg/ml 56.8 ± 53.5 ± 56.4 ± 60.4 ±
1.8a 43.2 2.4a 46.5 2.9a 43.6 3.1a 39.6
500 µg/ml 49.5 ± 47.6 ± 47.8 ± 54.6 ±
2.8a 50.5 2.8a 52.4 2.2b 52.2 3.3a 45.4

Values are expressed in MEAN ± S.E.M (n =3). (t-value denotes statistical significance at ap<0.05, bp<0.01 and
c
p<0.001 respectively, in comparison to control group)

Table 5: Determination of 1, 1-Diphenyl-2-Picrylhydrazyl (DPPH), Superoxide (O2), Hydrogen Peroxide

(H2O2) and Nitric Oxide (NO) Scavenging Activity of Ethyl Acetate Fraction of M. Philippica

DPPH O2 H 2O 2 NO
Groups &
% % % %
Treatments % of % of % of % of
Scavenging Scavenging Scavenging Scavenging
Control Control Control Control
Activity Activity Activity Activity
100.0 ± 00 100.0 ± 00 100.0 ± 00 100.0 00
Control
4.1 3.8 3.9 ±4.2
Ascorbic acid

Impact Factor (JCC): 5.9089 NAAS Rating 3.99

Evaluation of Antimicrobial and Antioxidant Activity of Crude Methanol 11
Extract and its Fractions of Mussaenda Philippica Leaves

100 µg/ml 30.2 ± 69.8 40.2 ± 58.6 ± 68.2 ±

3.8c 4.1b 59.8 4.6 41.4 3.4 31.8
200 µg/ml 16.8 ± 83.2 28.6 ± 48.6 ± 60.2 ±
2.2c 3.6b 71.4 4.1 51.4 4a 39.8
400 µg/ml 4.2 ± 95.8 3.4 ± 26.4 ± 46.4 ±
2.2c 2.8c 96.6 1.6c 73.6 3.2b 53.6
500 µg/ml 0.00 100 1.2 ± 16.2 ± 28.4 ±
2.5c 98.8 1.2c 83.8 2.5c 71.6
EAF
100 µg/ml 66.8 ± 76.7 ± 76.2 ± 78.4 ±
3.4a 33.2 5.4 23.3 4.1 23.8 6.7 21.6
200 µg/ml 56.4 ± 62.6 ± 67.5 ± 73.4 ±
3.8a 43.6 6.4 37.4 3.8 32.5 5.2 26.6
400 µg/ml 52.4 ± 58.4 ± 60.6 ± 68.4 ±
3.8a 47.6 4.6a 41.6 4.1a 39.4 3.8a 31.6
500 µg/ml 50.2 ± 52.8 ± 57.6 ± 64.5 ±
2.4b 49.8 3.8b 47.2 3.8b 42.4 4.4b 35.5

Values are expressed in MEAN ± S.E.M (n =3). (t-value denotes statistical significance at ap<0.05, bp<0.01 and
c
p<0.001 respectively, in comparison to control group)

Table 6: Determination of 1, 1-Diphenyl-2-Picrylhydrazyl (DPPH), Superoxide (O2), Hydrogen Peroxide (H2O2)

and Nitric Oxide (NO) Scavenging Activity of M. Philippica
DPPH O2 H 2O 2 NO
Groups & % % % %
Treatments % of % of % of % of
Scavenging Scavenging Scavenging Scavenging
Control Control Control Control
Activity Activity Activity Activity
100.0 ± 100.0 ± 100.0 ± 100.0
Control 00 00 00 00
4.1 3.8 3.9 ±4.2
Ascorbic acid
30.2 ± 40.2 ± 58.6 ± 68.2 ±
100 µg/ml 69.8
3.8c 4.1b 59.8 4.6 41.4 3.4 31.8
16.8 ± 28.6 ± 48.6 ± 60.2 ±
200 µg/ml 83.2
2.2c 3.6b 71.4 4.1 51.4 4a 39.8
4.2 ± 3.4 ± 26.4 ± 46.4 ±
400 µg/ml 95.8
2.2c 2.8c 96.6 1.6c 73.6 3.2b 53.6
1.2 ± 16.2 ± 28.4 ±
500 µg/ml 0.00 100
2.5c 98.8 1.2c 83.8 2.5c 71.6
AF

100 µg/ml 78.4 ± 82.6 ± 77.4 ± 77.4 ±

4.6a 21.6 5.4 17.4 4.8 22.6 5.4a 22.6
69.6 ± 74.6 ± 68.2 ± 68.2 ±
200 µg/ml
5.1a 30.4 4.9 25.4 4.1 31.8 4.8a 31.8
55.6 66.4 ± 56.6 ± 58.4 ±
400 µg/ml
±4.2b 44.4 4.2b 33.6 4.2 43.4 4.2c 41.6
44.2 ± 44.2 ± 48.4 ± 46.5 ±
500 µg/ml
4.3c 55.8 4.8b 55.8 3.7b 51.6 3.3c 53.5

Values are expressed in MEAN ± S.E.M (n =3). (t-value denotes statistical significance at ap<0.05, bp<0.01 and
c
p<0.001 respectively, in comparison to control group)

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12 Debashisa Mishra, Susanta Kumar Rout & GB. Alaka Kar

Table 7: Determination of 1, 1-Diphenyl-2-Picrylhydrazyl (DPPH), Superoxide (O2), Hydrogen Peroxide (H2O2)

and Nitric Oxide (NO) Scavenging Activity of Aqueous Fraction of M. Philippica

DPPH O2 H 2O 2 NO
Groups &
% % % %
Treatments % of % of % of % of
Scavenging Scavenging Scavenging Scavenging
Control Control Control Control
Activity Activity Activity Activity
100.0 ± 00 100.0 ± 00 100.0 ± 00 100.0 00
Control
4.1 3.8 3.9 ±4.2
Ascorbic acid
100 µg/ml 30.2 ± 69.8 40.2 ± 58.6 ± 68.2 ±
3.8c 4.1b 59.8 4.6 41.4 3.4 31.8
200 µg/ml 16.8 ± 83.2 28.6 ± 48.6 ± 60.2 ±
2.2c 3.6b 71.4 4.1 51.4 4a 39.8
400 µg/ml 4.2 ± 95.8 3.4 ± 26.4 ± 46.4 ±
2.2c 2.8c 96.6 1.6c 73.6 3.2b 53.6
500 µg/ml 0.00 100 1.2 ± 16.2 ± 28.4 ±
2.5c 98.8 1.2c 83.8 2.5c 71.6
MEMP
100 µg/ml 64.6 ± 31.9 72.2 ± 10.8 84.2 ± 8.6 68.6 ± 21.5
5.8a 4.8 4.6 3.4a
200 µg/ml 54.5 ± 46.5 67.4 ± 28.7 79.6 ± 12.8 56.4 ± 28.2
4.7a 3.9 3.9 3.6a
400 µg/ml 34.1 64.2 58.8 ± 46.4 45.7 ± 53.8 45.8 ± 42.8
±3.2b 3.2b 3.5 4.6c
500 µg/ml 12.5 ± 79.6 43.8 ± 56.2 34.6 ± 65.4 38.8 ± 56.8
2.3c 2.8b 2.6b 2.4c

Values are expressed in MEAN ± S.E.M (n =3). (t-value denotes statistical significance at ap<0.05, bp<0.01 and
c
p<0.001 respectively, in comparison to control group)

Table 8: Determination of 1, 1-Diphenyl-2-Picrylhydrazyl (DPPH), Superoxide (O2), Hydrogen Peroxide

(H2O2) and Nitric Oxide (NO) Scavenging Activity of Different Efractions and Crude Extract of M. Philippica
DPPH O2 H 2O 2 NO
Groups &
% % % %
Treatments % of % of % of % of
Scavenging Scavenging Scavenging Scavenging
Control Control Control Control
Activity Activity Activity Activity
100.0 ± 00 100.0 ± 00 100.0 ± 00 100.0 00
Control
4.1 3.8 3.9 ±4.2
Ascorbic acid
30.2 ± 40.2 ± 58.6 ± 68.2 ±
100 µg/ml 69.8
3.8c 4.1b 59.8 4.6 41.4 3.4 31.8
16.8 ± 28.6 ± 48.6 ± 60.2 ±
200 µg/ml 83.2
2.2c 3.6b 71.4 4.1 51.4 4a 39.8
4.2 ± 3.4 ± 26.4 ± 46.4 ±
400 µg/ml 95.8
2.2c 2.8c 96.6 1.6c 73.6 3.2b 53.6
1.2 ± 16.2 ± 28.4 ±
500 µg/ml 0.00 100
2.5c 98.8 1.2c 83.8 2.5c 71.6
CF
67.7 ± 66.4 ± 70.4 ± 74.3 ±
100 µg/ml
2.6 32.3 2.8 33.6 3.6 29.6 3.1 25.7
58.4 ± 58.6 ± 66.2 ± 64.2 ±
200 µg/ml
2.2 41.6 3.2 41.4 3.2 33.8 2.8 35.8
56.8 ± 53.5 ± 56.4 ± 60.4 ±
400 µg/ml
1.8a 43.2 2.4a 46.5 2.9a 43.6 3.1a 39.6

Impact Factor (JCC): 5.9089 NAAS Rating 3.99

Evaluation of Antimicrobial and Antioxidant Activity of Crude Methanol 13
Extract and its Fractions of Mussaenda Philippica Leaves

49.5 ± 47.6 ± 47.8 ± 54.6 ±

500 µg/ml
2.8a 50.5 2.8a 52.4 2.2b 52.2 3.3a 45.4
EAF
66.8 ± 76.7 ± 76.2 ± 78.4 ±
100 µg/ml
3.4a 33.2 5.4 23.3 4.1 23.8 6.7 21.6
56.4 ± 62.6 ± 67.5 ± 73.4 ±
200 µg/ml
3.8a 43.6 6.4 37.4 3.8 32.5 5.2 26.6
52.4 ± 58.4 ± 60.6 ± 68.4 ±
400 µg/ml
3.8a 47.6 4.6a 41.6 4.1a 39.4 3.8a 31.6
50.2 ± 52.8 ± 57.6 ± 64.5 ±
500 µg/ml
2.4b 49.8 3.8b 47.2 3.8b 42.4 4.4b 35.5
AF
78.4 ± 82.6 ± 77.4 ± 77.4 ±
100 µg/ml
4.6a 21.6 5.4 17.4 4.8 22.6 5.4a 22.6
69.6 ± 74.6 ± 68.2 ± 68.2 ±
200 µg/ml
5.1a 30.4 4.9 25.4 4.1 31.8 4.8a 31.8
55.6 66.4 ± 56.6 ± 58.4 ±
400 µg/ml
±4.2b 44.4 4.2b 33.6 4.2 43.4 4.2c 41.6
44.2 ± 44.2 ± 48.4 ± 46.5 ±
500 µg/ml
4.3c 55.8 4.8b 55.8 3.7b 51.6 3.3c 53.5
MEMP
44.2 ± 66.8 ± 56.4 ± 65.2 ±
100 µg/ml
3.8a 55.8 4.8 33.2 3.9 43.6 4.8a 34.8
39.4 ± 55.6 ± 48.4 ± 57.4 ±
200 µg/ml
4.2a 60.6 3.8 44.4 5.1 51.6 4.2a 42.6
35.4 47.2 ± 38.6 ± 47.6 ±
400 µg/ml
±3.8b 64.6 4.6b 52.8 4.8 61.4 5.2c 52.4
26.8 ± 38.4 ± 32.9 ± 38.4 ±
500 µg/ml
4.4c 73.2 4.2b 61.6 4.6b 67.1 4.4c 61.6

Values are expressed in MEAN ± S.E.M (n =3). (t-value denotes statistical significance at ap<0.05, bp<0.01 and
c
p<0.001 respectively, in comparison to control group)

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