Volume 6, Issue 6, June – 2021 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Pharmacological Activities of Essential Oil Extracted

from Viola patrinii against Some Gram Positive and
Gram Negative Bacteria as Well Fungal Strains
Sajad Yousuf1, Tahira Yousuf2, Chhaya Deshmukh*1, R.K. Bachheti3
1
Department of Biochemistry, Maharishi University of Information Technology, Lucknow, India
2
Department of Immunology & Molecular Medicine, SKIMS, J&K, India
3
College of Natural and Computational Science, Haramaya University Ethiopia

*Corresponding Authors:

Dr. Chhaya Deshmukh Sajad Yousuf

Departmentof Biochemistry Department of Biochemistry
Maharishi University of Information Technology Maharishi University of Information Technology
Lucknow (UP), India Lucknow (UP), India

Abstract:- The rapid alarming drug resistance against I. INTRODUCTION

pathogenic microorganisms is a medical catastrophe for
the world. The possible deferring force is to concise the The mixture of terpenoids makes essential oil that is
use of antimicrobials synthesised at industrial level and obtained from aromatic and medicinal plants. The phytotype
investigate for natural medicinal plant derived composition present in the essential oil is specific for a
compounds having properties of fighting specific plant species [1]. Plants and plant derived
microorganisms. In this regard, the aim of this study was compounds have played essential roles for the synthesis of
to find the antimicrobial properties of Essential oil new pharmaceutical products and many known drugs. From
obtained from Viola patrinii. The essential oil of Viola that time higher plants have become main source for the
patrinii showed lowest inhibition zone about 6.62 mm discovery of many drugs and among the discovered drugs a
against S.aureus at the concentration of 250 μg/ml and handful of 100 drugs with well known structure are
showed highest zone of inhibition about 16.37 mm commonly used [2]. The great number of chemicals have
against E.coli at the concentration of 1000 μg/ml for been derived from plants as medicine by scientific
bacterial strains. The essential oil of Viola patrinii evaluation, and these plants are used traditionally for the
showed lowest inhibition zone about 8.02 mm against treatment of bacterial infection and hence exploiting the
A.niger at the concentration of 250 μg/ml and showed power of plant derived compounds for the discovery of anti-
highest zone of inhibition about 15.23 mm against microbials with a great therapeutic potential [3].
C.albicans at the concentration of 1000 μg/ml for fungal
strains. The essential oil Viola patriii showed high Medicinal plants are to be screened from recent years
antimicrobial activity for both fungal strains and to develop noble compounds for treating the Multi-drug
bacterial strains. The order of antimibacterial activity resistant (MDR) bacterial diseases. Some natural and
for these strains were E.coli>M.luteus>P.aeruginosa> synthetic compounds are also used for the purpose which
S.aureus>S.typhi>K.rhizophila>S.epidermis. This includes screening for bioactive compounds or plant derived
investigation lead to the findings that described the extracts against MDR efflux pump inhibition, α-lactamase
antimicrobial potential of this plant essential oil and inhibitors, synergistic approaches such as antibiotic–
usefulness of oil against antibiotic resistance. However, phytocompound interaction, and targeting the virulence and
further studies are needed to evaluate active compounds pathogenicity of bacteria and use of quorum-sensing
and probable medicinal benefits in chemotherapy among inhibitors [4].
humans.

Keywords:- Viola Patrinii, Violaceae, Essential Oil,

Antibiotic Resistance, Antimicrobials, Inhibition Zone.

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Volume 6, Issue 6, June – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
The herb namely as V. patrinii (China violet) is having Extraction of essential oil
short and thin stem, as the thickening of its stem is inhibited Clevenger type apparatus was used to extract essential
by a secondary woody growth. This plant contains leaves oil from Viola patrinii. The extraction of oil is based on
which are glabrous and triangular in shape and are narrow, hydro-distillation that was carried for four hours. After this
elongated, and superficially chordate. The flowers of this procedure, anhydrous sodium sulphate was used to remove
plant are having lilac colour. This plant is found in the moisture from extracted plant oil. The amount of extracted
temperate regions of Himalaya, they are also found at high oil was determined by following formula:-
altitudes (900-2400 m) of Manipur, Meghalaya and
Arunachal Pradesh. The plant is expanded from the east
south wards to the hills of the Eastern and Western Ghats
[5]. Viola patrinii is a plant belongs to the family Violaceae,
it is a medicinal herb also known as China violet, it is a
therapeutic agent of traditional medicine used for the
treatment of bruises, ulcers and purification of blood. In the
Chinese system, it is also recommended for treating cancer Test Microorganisms used
and disorders related to it [6]. For cough and cold, its dried Gram positive bacterial species: Staphylococcus
flowers are used and these dried flowers are also used as aureus (MTCC-3160), Kocuria rhizophila (1541),
purgative. Different recipes are also made from it like Staphylococcus epidermis (MTCC 3615), Micrococcus
Joshanda and Rogan Banafshah [5]. luteus (MTCC 1541), Gram negative bacterial species:
E.coli (MTCC-614, Pseudomonas aeruginosa (MTCC-424),
All these findings observed that Viola patrinii is Salmonella typhi (NCTC 786) and Fungal species :Candida
having potent antibacterial activity. Escherichia coli, albicans (MTCC-227), Aspergillus niger (MTCC 1344),
Staphylococcus aureus, Streptococcus pyogenes, Bacillus were for the screening of antimicrobial properties of
subtilis, Klebsiella pneumoniae and lactococcus were tested essential oil of Viola patrinii.
against extracts from Viola patrinii by well diffusion
method, each extract were determined by their Minimum
Determination of diameter of zone of inhibition by well
inhibitory concentration (MIC) and Minimum lethal
diffusion method.
concentration(MLC) values. Ethanolic extract is having By using clinical isolates disc /well diffusion method
significant antimicrobial activity, when extracted from [Marmonier] and MIC by microdilution were the two
whole plant i.e from Viola patrinii [7]. Gelatinase (A and methods used for the determination of antimicrobial activity
B) and collagenase are inhibited by this plant [8]. Oxidative of plant essential oils. Nutrient agar/broth (pH 7.2) and
stress and inflammation in the brain is the reason for the Saboured dextrose agar/broth were used for bacterial culture
diseases caused in central nervous system. In murine and fungal culture respectively. The media was autoclaved
hippocampal HT22 cells and BV2 microglia, ethanolic and 500µl of inoculum was mixed with it. The bacteria
extracts of Viola patrinii showed anti-inflamatory and inoculated in the media was checked to provide 105 CFU/ml.
antioxidative activities [9]. In vitro grown callus of Viola The media having inoculum was then poured in sterilized
patrinii showed more antioxidant activity in comparison to petridishes, paper discs containing plant essential oil (size
wild plant, when this activity was determined by DPPH (α, 6mm) were placed on the smooth and dry surface of the
α-diphenyl-β-picrylhydrazyl) method [10]. This plant can be poured media in the petriplates. The essential oil was further
used for treating complications like skin disorders, upper serially diluted in 5% of DMSO (dimethylsulfoxide) to the
respiratory tract infections and abdominal pain. Different different concentrations like 1, 0.50, 0.250 and 0.125 mg/ml.
activities like anti-inflammatory, purgative properties and Wells were filled with sample dilution (0.1µl) when well
diuretic properties were shown by this plant. [11-13]. diffusion method was performed. These petriplates were left
Therefore, the objective of our study is to find the in Laminar air flow for half an hour for the diffusion of plant
antimicrobial activities of essential oil from whole plant of essential oil in the media. Then these petriplates were
Viola patrinii using Agar well diffusion method. transferred to B.O.D incubator and were incubated at
37±1ºC for 24 hrs for bacterial growth and at 22-25ºC for 5
II. MATERIALS AND METHODS days for fungal growth. After proper incubation period
zones of inhibition were measured with the help of zone
Collection and preparation of plant materials reader, these samples were run along with positive and
Fresh plants of Viola patrinii are collected from negative controls. Ciprofloxacin and Fluconazole (1mg/ml)
Rishiganga Valley, District Chamoli located at high altitude, were used as antibacterial and antifungal positive controls
Uttarakhand, India. The plants were authenticated at Forest respectively. These inhibition zones of plant essential oil
Research India (FRI), India. showed bactericidal and fungicidal activity. The terms
bactericidal and fungicidal means that the plant essential oils
kills the bacteria and fungi respectively by showing the clear
zone around the discs. The negative controls were used and
showed no inhibitory zones around the discs used for
bacterial and fungal strains.

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Volume 6, Issue 6, June – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
III. RESULTS AND DISCUSSION [7]. Yousuf S, Bachheti RK, Joshi A. Comparative analysis
of in vitro antibacterial activity of extracts of Viola
When inhibition zones were observed, it was analyzed patrinii on pathogenic microorganisms. Int. J. Res.
that Plant essential oil showed antimicrobial activity against Pharm. Sci. 2012; 3 (3): 432-435.
Gram positive, Gram negative and Fungal strains. The [8]. Svangard, Goransson U, Hocaoglu Z, Gullbo J. and
essential oil of Viola patrinii showed lowest inhibition zone Larsson R., Cytotoxic cyclotides from Viola tricolor, J.
about 6.62 mm against S.aureus at the concentration of Nat. Prod., 2004, 67,144-147.
0.250 mg/ml and showed highest zone of inhibition about [9]. Li B, Lee DS, Choi HG. and Kim KS., Involvement
16.37 mm against E.coli at the concentration of 1 mg/ml for of Heme Oxygenase-1 Induction in the Cytoprotective
bacterial strains. The essential oil of Viola patrinii showed and Immunomodulatory Activities of Viola patrinii in
lowest inhibition zone about 8.02 mm against A.niger at the Murine Hippocampal and Microglia Cells, Evidence-
concentration of 0.250 mg/ml and showed highest zone of Based Complementary and Alternative Medicine.,
inhibition about 15.23 mm against C.albicans at the 2012, 5, 12-20.
concentration of 1 mg/ml for fungal strains. The essential oil [10]. Muhammad N, Ishrat N, Syed M, Naqvi S. and
Viola patriii showed high antimicrobial activity for both Mahmood T., Standardization of tissue culture
fungal strains and bacterial strains. The order of conditions and estimation of free scavenging activity
antimibacterial activity for these strains were in Viola odorata, Pak. J. Bot., 2013, 45(1),197-202.
E.coli>M.luteus>P.aeruginosa> [11]. Witkowska BE, Byka W, Matlawska I, Goslinska O
S.aureus>S.typhi>K.rhizophila>S.epidermis. The essential and Muszynski Z., Antimicrobial activity of Viola
oil Viola patriii showed high antimicrobial activity against tricolor herb, Fitoterapia., 2005, 76,458-461.
fungal strains in comparison to the bacterial strains. This [12]. Marmonier AA, “Bacteriologie medicale. Techniques
may indicate that the Viola partinii essential oil has broad usuelles,” Chap. 4. Antibiotiques, Technique de
inhibitory activities to pathogenic microorganisms and diffusion en gelose method des disque.@ SIMEP SA-
promising to act as potential antibacterial as well as PARIS, France, pp238-244, 1987.
antifungal from natural plant sources. The experiments [13]. Walter C, Shinwari ZK, Afzal I. and Malik RN.,
were performed in triplicates. The results of antibacterial Antibacterial Activity in Herbal Products Used in
activities of essential oil are summarized in Table 1 and Pakistan, Pak. J. Bot., 2011, 43,155-162.
Figure 1, while as the results of antifungal activities are
depicted by Table 1 and Figure 2.

Conflict of Interest
The authors hereby declare that there is no conflict of
interest as per the publication of this paper.

Acknowledgments
The authors are very thankful to the Department of
Biological Sciences, Maharishi University of Information
technology, Lucknow (UP), India and JNU, Delhi, India for
their support and coordination to carry this research work.

REFERENCES

[1]. Adams RP, “Identification of Essential Oil

Components by Gas Chromatography-Mass
Spectroscopy, Allured, Carol Stream, IL, USA (1995).
[2]. Farnsworth NO. The role of medicinal plants in drug
development. Natural Products & Drug Development.
Ballerey Tindall & Cox, London.1984; 8-98.
[3]. Cowan M.,Plant products as antimicrobial agents,
Clinical Microbiol Rev., 1999, 12(4),564-582.
[4]. Usha G, Chunderika M, Prashini M, Willem SA,
Yusuf ES. Characterization of extended-spectrum
beta-lactamases in Salmonella spp. at a tertiary
hospital in Durban, South Africa. Diagn. Microbiol.
Infect. Dis. 2008; 62: 86–91.
[5]. Wealth Of India,Vol: x,1976,514,515,516.
[6]. Kirtikar KR, Basu BD. 1991. Indian Medicinal Plants.
Vol.3, Singh B & Singh MP. Publishers, India, 2032.

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Volume 6, Issue 6, June – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
FIGURE AND TABLE LEGENDS:

Figure 1: Antibacterial activity of Viola patrinii Essential Oil

*SA,Staphylococcus aureus., KR, Kocuria rhizophila., SE, Staphylococcus epidermis., ML, Micrococcus luteus., EC, E.coli, PA,
Pseudomonas aeruginosa., ST, Salmonella typhi.
*X-axis, Concentration of essential oil, Y-axis, Zone of inhibition

Figure 2: Antifungal activity of Viola patrinii Essential Oil

*CA, Candida albicans., AN, Aspergillus niger, *X-axis, Concentration of essential oil, Y-axis, Zone of inhibition

Table 1: Antibacterial And Antifungal activity of Viola patrinii Essential Oil

*Postive control for antibacterial activity is Ciprofloxacin, Fluconazole is positive control for antifungal activity.

Figure 1:

Figure: 2

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Volume 6, Issue 6, June – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
Table 1:
S. No. Gram Positive Bacteria Concentration Zone of Inhibition Disc size
(μg/ml) (mm) (mm)
1. Staphylococcus aureus 10 (Ciprofloxacin) 26.25 6
(MTCC 3160)
Negative Control 0 6
1000 (Sample) 7.52 6
500 (Sample) 6.96 6
250 (Sample) 6.62 6
125(Sample) 0 6
2. Kocuria rhizophila 10 (Ciprofloxacin) 28.43 6
(MTCC 1541)
Negative Control 0 6
1000 (Sample) 8.80 6
500 (Sample) 0 6
250 (Sample) 0 6
125(Sample) 0 6
3. Staphlococus epidermis 10 (Ciprofloxacin) 38.76 6
(MTCC 3615)
Negative Control 0 6
1000 (Sample) 0 6
500 (Sample) 0 6
250 (Sample) 0 6
125(Sample) 0 6
4. Micrococcus luteus 10 (Ciprofloxacin) 35.34 6
(MTCC 1541)
Negative Control 0 6
1000 (Sample) 9.98 6
500 (Sample) 8.12 6
250 (Sample) 0 6
125(Sample) 0 6
Gram Negative Bacteria
1. Escherchia Coli 10 (Ciprofloxacin) 30.32 6
(MTCC 614)
Negative Control 0 6
1000 (Sample) 16.37 6
500 (Sample) 13.17 6
250 (Sample) 11.7 6
125(Sample) 11.5 6
2. Pseudomonas aeruginosa 10 (Ciprofloxacin) 43.36 6
(MTCC 424)
Negative Control 0 6
1000 (Sample) 8.79 6
500 (Sample) 7.77 6
250 (Sample) 7.64 6
125(Sample) 0 6
3. Salmonella typhi 10 (Ciprofloxacin) 43.02 6
(NCTC 786)
Negative Control 0 6
1000 (Sample) 9.45 6
500 (Sample) 0 6
250 (Sample) 0 6
125(Sample) 0 6
Fungal Strains
1. Candida albicans 10 (Fluconazole) 25.52 6
(MTCC 227)
Negative Control 0 6
1000 (Sample) 15.23 6
500 (Sample) 15.15 6

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Volume 6, Issue 6, June – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
250 (Sample) 11.64 6
125(Sample) 11.21 6
2. Aspergilus niger 10 (Fluconazole) 17.72 6
(MTCC 1344)
Negative Control 0 6
1000 (Sample) 12.03 6
500 (Sample) 8.23 6
250 (Sample) 8.02 6
125(Sample) 0 6

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