Antimicrobial potential of Delphinium denudatum (Wall Ex Hook & Thom)

Abstract

Plants produce a diverse array of secondary metabolites, many of which are known to possess antimicrobial potential. The present study was carried out with an objective to investigate the antimicrobial potential of different plant parts of Delphinium denudatum. Antimicrobial potential of D.denudatum plant parts (stem, root, leaf) extracts were tested against three groups of microorganisms, bacteria(Gram positive and Gram negative bacteria), actinobacteria and fungi. Plant extracts were prepared in seven different solvents (methanol, ethanol, ethyl acetate, acetone, hexane, chloroform and water) according to their polarity. The qualitative estimation of antimicrobial activity was performed following plate assays through disk diffusion methodwhile the quantitative estimations were based on minimum inhibitory concentration (MIC). All the plant parts showed antibacterial activity, maximum being in case of stem. Among different solvents, ethanolic stem extract showed highest antibacterial activity(15.33±0.11mm) against S. marcescens and aqueous leaf extract showed highest antiactinobacterial activity (21.0 ± 0.07mm) against Nocardia tenirefensis. Antifungal activity, that was tested against 5 fungal species, was absent in all the plant parts extracts. Values obtained for MIC varied with respect to different solvents. This is a preliminary study showing the antibacterial potential of D. denudatum with respect to the importance of solvent selection in harvesting antimicrobial metabolites.

Full text

BEPLS Vol 8 [12] November 2019 152 | P a g e ©2019 AELS, INDIA
Bulletin of Environment, Pharmacology and Life Sciences
Bull. Env.Pharmacol. Life Sci., Vol 8 [12] November 2019 :152-158
©2019 Academy for Environment and Life Sciences, India
Online ISSN 2277-1808

CODEN: BEPLAD
Global Impact Factor 0.876
Universal Impact Factor 0.9804
NAAS Rating 4.95
ORIGINAL ARTICLE OPEN ACCESS
Antimicrobial potential of Delphinium denudatum (Wall Ex Hook
& Thom)
Kiran Kumari1*, Priyanka Adhikari2, Anita Pandey2, S.S. Samant1 and Veena Pande3
1G.B. Pant National Institute of Himalayan Environment and Sustainable Development,
Himachal Regional Centre, Mohal-Kullu Himachal Pradesh, 175126 India
2G.B. Pant National Institute of Himalayan Environment and Sustainable Development,
Center for Environmental Assessment and Climate Change,
Kosi-Katarmal, Almora, Uttarakhand-263 643, India
3Department of Biotechnology,
Kumaun University, Bhimtal campus, Bhimtal, Uttarakhand 263 136, India
*Correspondence: E mail: kiranminhans@gmail.com
ABSTRACT
Plants produce a diverse array of secondary metabolites, many of which are known to possess antimicrobial potential.
The present study was carried out with an objective to investigate the antimicrobial potential of different plant parts of
Delphinium denudatum. Antimicrobial potential of D.denudatum plant parts (stem, root, leaf) extracts were tested
against three groups of microorganisms, bacteria(Gram positive and Gram negative bacteria), actinobacteria and fungi.
Plant extracts were prepared in seven different solvents (methanol, ethanol, ethyl acetate, acetone, hexane, chloroform
and water) according to their polarity. The qualitative estimation of antimicrobial activity was performed following
plate assays through disk diffusion methodwhile the quantitative estimations were based on minimum inhibitory
concentration (MIC). All the plant parts showed antibacterial activity, maximum being in case of stem. Among different
solvents, ethanolic stem extract showed highest antibacterial activity(15.33±0.11mm) against S. marcescens and
aqueous leaf extract showed highest antiactinobacterial activity (21.0 ± 0.07mm) against Nocardia tenirefensis.
Antifungal activity, that was tested against 5 fungal species, was absent in all the plant parts extracts. Values obtained
for MIC varied with respect to different solvents. This is a preliminary study showing the antibacterial potential of D.
denudatum with respect to the importance of solvent selection in harvesting antimicrobial metabolites.
KEY WORDS: Delphinium denudatum, antibacterial, antiactinobacterial, antifungal, MIC
INTRODUCTION
ports on antibiotic resistance phenomenon and development of its side effects due to consumption of
microbe derived antibiotics are huge in literature. This suggests the need for alternate sources for
combating the infectious diseases[1]. In this perspective, plant based antimicrobials (derived from
medicinal plants, in particular) are increasingly receiving attention for harnessing their potential in
production of antimicrobial substances, as safer source of antibiotics. Crude extracts and essential oils of
medicinal plants possess bioactive compounds, often with antimicrobial and antioxidant
properties[2].Antimicrobial compounds are used in various areas such as, pharmaceutical,
neutraceuticals, textiles, dairy products, cosmetics and personal care products[3].
Delphinium denudatum is a critically endangered Himalayan perennial herb (Local name- Jadwar; Family-
Ranunculaceae). The generic name of this plant is derived from a Greek word, which means Dolphin. It is
found in the Westernand North Western Himalaya, from Kashmir to Kumaun at an altitudinal range from
1400-3600 m, amsl. It occurs in grassy and rocky moist slopes, open grasslands and margins of fields[4].
It has been recommended for treatment of paralysis, epilepsy, insanity, migraine, numbness, tremors,
infantile convulsions, aconite poisoning, snake bite, scorpion sting, opium addiction, arthritis, cardiac
weakness, palpitation, rheumatism, all kinds of pain, leucoderma and for improving skin complexion [5].
Selection of solvent system in obtaining the plant extracts is important indetermination of any biological
activity including production of antimicrobials.Reports are available on the importance of selection of

BEPLS Vol 8 [12] November 2019 153 | P a g e ©2019 AELS, INDIA
suitable solvents in assessing antimicrobial potential of medicinal plants [3,6,7]. The present study aims
on selection of solvent system in production of antimicrobial metabolites from various plant parts of D.
denudatum. The antimicrobial potential of D. denudatum plant parts is investigated with respect to three
major groups of microorganisms (bacteria, actinobacteria and fungi) using seven different solvents
according to their polarity.
MATERIAL AND METHODS
Study site and Sample collection
The fresh and healthy root, stem, and leaves of D. denudatum were collected from Sainj Valley of Kullu
          to  
longitudes) during the months May and June, brought to the laboratory, air dried, converted into fine
powder, and stored at 4-8 °C.
Extraction
Root, leaf, and stem were extracted (separately) in seven solvents (methanol, ethanol, acetone,
chloroform, ethyl acetate, hexane, and water separately) taking in a ratio of 1:5 (dry powder: solvent).
The mouth of conical flask was sealed with para-film. Samples were macerated in a rotary shaker (Remi)
at 160 rpm for 48 h.
Test microorganisms
Bacteria: 2 Gram +ve=Bacillus subtilis(NRRLB-30408) and B. megaterium(MCC3124); 3 Gram -
ve=Pseudomonas chlororaphis(MCC2693), Escherichia coli, and Serratia marcescens(MTCC4822).
Actinobacteria: Nocardiatenirefensis(MCC2012)
Fungi: Paecilomyces variotii(ITCC3710), Aspergillus niger(ITCC2546), Fusarium oxysporum (ITCC4219),
F.solani(ITCC 5017), and Trametes hirsuta(MTCC11397).
These test microorganisms were taken from the microbial culture collection, established in the
Microbiology Lab of the Institute (GBPNIHESD). Accession numbers shown in the parentheses have been
allocated by the National/International depositories: NRRL (Northern Regional Research Laboratory,
Agricultural Research Service Patent Culture Collection, United States Department of Agriculture,Illinois;
MCC (Microbial Culture Collection, National Centre for Cell Science, Pune, India); MTCC (Microbial Type
Culture Collection and GeneBank, Institute of Microbial Technology, Chandigarh, India); ITCC (Indian
Type Culture Collection, Indian Agricultural Research Institute, New Delhi, India).
Bioassays for determination of antimicrobial potential of D. denudatum extracts
Plate based bioassays
For qualitative estimation of antimicrobial potential of D. denudatum extracts, agar plate based bioassays
were performed using disc diffusion method. Bacterial and actinobacterial culture suspensions were
prepared in tryptone yeast extract (TYE) agar while fungal culture suspensions were prepared in potato
dextrose (PD) agar. 100 µl of all the test microorganisms (separately) were spread uniformlyon the
respective agar surface (TYE agar plates for bacteria andactinobacteria, and PD agar plates for fungus)
with the help of a glass spreader. Sterilized 5 mm filter paper (Whatman No. 1) discs were placed over the
agar surface with the help of sterile forceps.15 µl of extract was loaded over the agar disc. The plates were
then incubated at 25 ºC. The results were recorded measuring the zone of inhibition (mm) after 24 h for
bacteria and 120 h for actinobacteria and fungi. All the experiments were performed in triplicates.
Quantitative estimation/ Minimum inhibitory concentration (MIC)
MIC was determined following Clinical and Laboratory Standard Institute Methodology [8]. Bacterial
andactinobacterial culture suspensions were prepared in TYE broth. For determination of MIC, 1 ml
extract was diluted using different concentration ranging from 100 to 1000 µg/ml, 1 ml test
microorganism and 8 ml broth was taken in sterile test tube, and then incubated at 25 °C for 24 h for
bacteria and 120 h for actinobacteria. Control was prepared in two sets; one containing broth medium
and test microorganism while the other containing broth medium and extract. After 24 h, the MIC values
were recorded on the basis of the lowest concentration showing absence of growth in the tubes. The test
was further confirmed by plating on TYE agar medium.
Statistical analysis
Data was expressed as the means ± standard errors (SE) from experiments, performed in triplicates.
Statistical significance was determined using stude -test. A p value <0.05 was considered as
significant.
RESULTS AND DISCUSSION
Extract yield
Figure 1 shows the yield of different plant parts of D.denudatum (root, stem, and leaf) extracted in
different solvents according to their polarity (water> methanol> ethanol> acetone>ethyl acetate>

BEPLS Vol 8 [12] November 2019 154 | P a g e ©2019 AELS, INDIA
chloroform> hexane) following maceration. The extraction yield was recorded highest in root, followed
by leaf and stem, respectively. Methanol was found to be the best solvent for obtaining highest yield from
all the plant parts of D. denudatum. Extract yield was recorded lowest in hexane (root, stem) and
chloroform (leaf). Extract yields were found to be higher in polar solvents in comparison to nonpolar.
These results coincide with several previous reports[9,7]. Higher extract yield in polar solvents can be
attributed to their high di electric constant which is known to be responsible for solubility of bioactive
compounds[3]. The polarity of the solvent influence the extraction yield that, in turn, plays a crucial role
in increasing the solubility of phytochemical compounds[10]. This result indicates toward the role of
solvent system in obtaining the extract yield of D. denudatum.
Qualitative test (Plate based bioassays)
Root, stem, and leaf extracts of D. denudatum showed antimicrobial activity against two groups of
microorganisms, bacteria and actinobacteria. Zone developed due to inhibition of bacterial and
actinobacterial species due to production of antimicrobial metabolites of D. denudatum are shown in
Figure 2. All the extracts, exceptaqueous, inhibited the growth of bacteria, both Gram +ve and Gram -ve.
Antimicrobial activity, recorded in different plant parts, was in the order: stem> leaf> root. Maximum
inhibition was recorded in case of S. marcescens(15.33± 0.11 mm) in stem ethanolic extract, followed by
stem ethyl acetate extract (11.00 ± 0.28 mm) against P. chlororaphis(Table 1). In comparative assessment
with respect to the bacteria, E. coli, B. megaterium, and B. subtilis, leaf acetone extract (6.67 ± 0.20 mm),
root hexane extract (8.33±0.36mm) and stem ethanolic extract (7.00 ± 0.35 mm) were the most effective
extracts, respectively (Figure3). Similar results have been recorded in species of Delphinium namely D.
uncinatum[11].
Aqueous extracts of leaf, root, and stem showed strong inhibition of the actinobacterial species, N.
tenirefensis in an order: leaf (21.0 ± 0.07 mm)>root (17.33 ± 0.08mm)> stem (16.33 ± 0.04mm). None of
the extracts, prepared in different solvents, showed antiactinobacterial activity. This indicates toward
capability of water as potential solvent fordetection of actinobacterial compound(s) (Table 1 and Figure
3). This is likely to be the first reporton inhibition of an actinobacterial species by plant extracts of D.
denudatum.
None of the extracts showed antifungal activity against the test fungi namely Paecilomyces variotii,
Aspergillus niger, Fusarium oxysporum, F. solani and Trametes hirsuta. Similar observation (absence of
antifungal activity) was reported from another species of Delphinium, D.formosum[12].However,
D.denudatum has been reported for the presence of antifungal diterpenoid[13].
Quantitative estimation (MIC)
In the MIC experiments, out of seven solvents, acetone, ethanol, and methanol extracts showed strong
inhibition of bacterial species. The most affected group was actinobacteria which was supported by the
low MIC values of water extracts of leaf, stem, and root (Table 2). Significant variations in MIC recorded in
all the extracts demonstrated the role of selection of solvent as well as the type of test microorganisms.
Table 1. Antimicrobial activity of D. denudatumplant extracts
Zone of Inhibition (mm)
Root
Microorganism
Methanol
Ethanol
Acetone
Ethyl
acetate
Water
Hexane
Chlorofor
m
Gram(+)ve
B. subtilis
3.00±0.12
2.33±0.11
6.67±0.20
4.00±0.10
ND
5.33±0.11
2.33±0.11
B.
megaterium
3.33±0.23
3.23±0.14
6.63±0.15
4.33 ± 0.12
ND
8.33±0.36
2.67±0.04
Gram(-)ve
S. marcescens
4.67±0.11
9.33±0.18
3.00 ±
0.07
ND
ND
6.33±0.11
2.0±0.07
P.
chlororaphis
5.12±0.12
5.67±0.11
3.33± 0.11
5.67 ± 0.25
ND
7.33±0.11
11±0.28
E. coli
3.33±0.04
4.13±0.25
2.33± 0.04
2.33 ± 0.08
ND
3.33±0.04
4.67±0.15
Actinobacteri
a
N. tenirefensis
ND
ND
ND
ND
17.33 ±
0.08
ND
ND
Stem
Gram(+)ve
B. subtilis
2.33±0.11
7.00 ± 0.35
5.33 ±
0.15
1.33 ± 0.04
ND
6.12±0.19
2.00±0.07
B.
megaterium
ND
4.00± 0.14
ND
2.33 ± 0.08
ND
3.67±0.11
4.67±0.08
Gram(-)ve
S. marcescens
2.61±0.08
15.33±
0.11
2.67 ±
0.04
1.67 ± 0.11
ND
ND
1.33±0.04
P.
chlororaphis
3.53±0.09
6.00± 0.07
2.67 ±
0.11
11.00 ±
0.28
ND
5.67±0.11
3.33±0.11

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E. coli
1.67±0.10
5.33±0.08
1.33 ±
0.04
4.67 ± 0.15
ND
5.03±
0.19
ND
Actinobacteri
a
N. tenirefensis
ND
ND
ND
ND
16.33 ±
0.04
ND
ND
Leaf
Gram(+)ve
B. subtilis
1.67±
0.04
3.00± 0.07
6.00± 0.12
1.67 ± 0.04
ND
4.33±0.11
2.00±0.07
B.
megaterium
ND
5.67 ± 0.35
2.00± 0.07
1.67 ± 0.04
ND
1.67±0.08
3.00±0.07
Gram(-)ve
S. marcescens
8.00±0.51
6.33 ± 0.36
2.00± 0.12
2.33 ± 0.16
ND
3.33±0.04
3.67±0.18
P.
chlororaphis
9.00±
0.07
5.33 ± 0.20
3.00± 0.24
11.00 ±
0.07
ND
2.67±0.08
3.33±0.08
E. coli
3.33±
0.04
6.67 ± 0.08
6.67 ±
0.20
2.00± 0.07
ND
4.67±0.23
4.33±0.15
Actinobacteri
a
N. tenirefensis
ND
ND
ND
ND
21.0 ± 0.07
ND
ND
(ND = activity not detected)
Table 2. Minimum inhibitory concentration (MIC) of D.denudatum plant extracts
MIC ug/ml
Root
Microorganism
Methano
l
Ethano
l
Aceton
e
Ethyl
acetate
Wate
r
Hexan
e
Chlorofor
m
Gram(+)ve
B. subtilis
500
700
400
800
ND
700
900
B. megaterium
400
500
400
700
ND
600
800
Gram(-)ve
S. marcescens
400
300
500
ND
ND
600
900
P.
chlororaphis
300
500
600
500
ND
600
300
E. coli
500
600
900
900
ND
900
700
Actinobacteri
a
N. tenirefensis
ND
ND
ND
ND
300
ND
ND
Stem
Gram(+)ve
B. subtilis
700
400
400
800
ND
600
900
B. megaterium
ND
600
ND
700
ND
700
800
Gram(-)ve
S. marcescens
900
300
700
800
ND
ND
900
P.
chlororaphis
500
200
700
300
ND
600
900
E. coli
800
500
800
700
ND
600
ND
Actinobacteri
a
N. tenirefensis
ND
ND
ND
ND
300
ND
ND
Leaf
Gram(+)ve
B. subtilis
800
600
400
800
ND
800
900
B. megaterium
ND
500
700
800
ND
900
800
Gram(-)ve
S. marcescens
500
300
700
700
ND
800
800
P.
chlororaphis
500
500
700
300
ND
800
700
E. coli
700
500
500
700
ND
700
600
Actinobacteri
a
N. tenirefensis
ND
ND
ND
ND
200
ND
ND
(ND=activity not detected)

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Figure 1. Extract yield of root, stem, and leaf of D.denudatum
Figure 2.Antimicrobial activity of D.denudatum:(A) Antiactinobacterial activity (B) Antibacterial activity.
C= Control, EAE= Ethyl acetate extract, ME= Methanolic extract, EE= Ethanolicextract, AE= Acetone
extract, CE= Chloroform extract, HE= Hexane extract, LAE= Leaf aqueous extract, SAE=Stem aqueous
extract, and RAE= Root aqueous extract
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40 Root Stem Leaf
Solvents
Extract yield (g)
C
LAE
SAE
RAE
EE
ME
AE
EAE
HE
ME
CE
EE
EAE
ME
EE
AE

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Figure3. Antibacterial activity of leaf, stem, and root of D.denudatum:BS=B.subtilis,BM=B.megaterium,
EC=E.coli,SM=S. marcescens, PC=P.chlororaphis
CONCLUSION
On the basis of results obtained, it is concluded that the ethanolic extracts of stem, root, and leaf of
D.denudatum possessed good antibacterial activity. Besides, aqueous extracts of plant extracts were
inhibitory for actinobacteria only, without showing any inhibition of bacteria and fungi. The present study
justify the use of D. denudatum in the traditional system of the medicine in treating infectious diseases.
The experiments performed in the present study are at preliminary stage. These observations shall
provide a base for conducting detailed investigations in view of the effect of geographical and climatic
conditions on plants in production of secondary metabolites, including antimicrobials.
0
3
6
9
12
15
BS BM SM PC EC
Methanol Ethanol Acetone Ethyl acetate Hexane Chloroform
0
3
6
9
12
15
BS BM SM PC EC
0
3
6
9
12
15
BS BM SM PC EC
Root
Stem
Leaf
Zone of inhibition (mm)
Bacteria

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ACKNOWLEDGEMENT
Authors are grateful to Director GBPINHESD for extending the facilities and Funding agency Himachal
Pradesh Power Corporation limited, Sarabai, Distt-Kullu, H.P., for financial assistance.
CONFLICT OF INTEREST STATEMENT
We have no conflict of interest
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CITATION OF THIS ARTICLE
K Kumari, P Adhikari, A Pandey, S.S. Samant and Veena Pande. Antimicrobial potential of Delphinium denudatum
(Wall Ex Hook & Thom). Bull. Env.Pharmacol. Life Sci., Vol 8 [12] November 2019: 152-158