Current Research in Biotechnology 5 (2023) 100118

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Current Research in Biotechnology

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Antioxidant capacity and combinatorial antimicrobial effects of Nardostachys

jatamansi essential oil with conventional antibiotics against some drug
resistant bacteria
Aasiya Majeed a, Sanjay Guleria a,⇑, Neha Sharma a, Khalid Hussain Salaria a, Faiqa Aiman a, Bikarma Singh b,
Vijai Kumar Gupta c
a
Natural Product-cum-Nano Lab, Division of Biochemistry, Faculty of Basic Sciences,Sher-e- Kashmir University of Agricultural Sciences and Technology of Jammu, Main Campus
Chatha, Jammu- 180009, Jammu and Kashmir, India
b
Botanic Garden Division, CSIR-National Botanical Research Institute, Lucknow 226001, Uttar Pradesh, India
c
Biorefining and Advanced Materials Research Center, SRUC, Kings Building, West Mains Road, Edinburgh EH9 3JG, UK

A R T I C L E I N F O A B S T R A C T

Keywords: The antibacterial and antioxidant properties of essential oils (EOs) have long been recognized. The present
Nardostachys jatamansi study was conducted to investigate the antioxidant capability of Nardostachys jatamansi essential oil and to
Antibiotics see if it has a synergistic antimicrobial effect with antibiotics against two Gram negative (Klebsiella pneumoniae
Synergistic antimicrobial activity and Escherichia coli) and three Gram positive (Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus) bac-
Antioxidant activity
terial strains. Guaia‐6,9‐diene (11.96 %), calarene (10.44 %), jatamansone (8.11 %), α‐gurjunene (7.42 %),
valencene (6.46 %), α‐maaliene (5.24 %), sprojatamol (5.06 %), and caratol (5.06 %) were found to be the pri-
mary components of N. jatamansi EO. According to antioxidant studies, N. jatamansi EO has moderate DPPH
radical scavenging activity, reducing power, and ferric reducing antioxidant power. Similarly, N. jatamansi
EO also showed significant antibacterial activity, with inhibition zones, MIC, and MBC values ranging from
10.5 ± 0.5 to 14.0 ± 0.4 mm, 1.5 to 3.1 mg/mL, and 1.8 to 3.5 mg/mL respectively. The results of N. jatamansi
EO interactions with conventional antibiotics revealed that amoxicillin, erythromycin, chloramphenicol, and
ampicillin MICs were reduced by 5 to 10 fold, 4 to 9.09 fold, 4 to 10.5 fold, and 4 to 8.0 fold, respectively.
The findings of this study are noteworthy because no previous reports of N. jatamansi EO's synergistic interac-
tion with conventional antibiotics have been published, and therefore may constitute an important strategy for
addressing problem of drug resistant bacteria.

1. Introduction oils and plant extracts are natural sources of physiologically active sub-
stances (Celiktas et al., 2007). Essential oils are complex mixtures of
The dynamic evolution and ubiquitous occurrence of multi‐drug substances that represent the most regal part of the plant, appearing
resistant bacterial strains is presenting a global challenge to disease as tiny droplets in the petals of flowers, the skin of fruits, the resin
management (Lambert, 2000). Antimicrobial resistance is most com- and bark of trees, and the roots of herbs and aromatic plants (Carson
monly caused by uncontrolled antibiotic use, self‐medication, inability and Hammer, 2011). These extracts are generally volatile elements
to follow an antibiotic course, acquired infections, inadequate biomed- that are soluble in alcohol and oil but not in water. Each essential
ical waste management, and antibiotic use in veterinary clinics oil can contain over 100 different chemical components, including
(Srivastava et al., 2014). Safe natural ingredients, such as herbal prod- alcohols, aldehydes, ketones, esters, phenols, sesquiterpenes, and ter-
ucts, are increasingly in demand to replace synthetic preservatives in penes. Each of these classes is distinguished by a homologous group
food and maintain antibacterial effectiveness (Fowler, 2006). Essential of substances that differ solely in elemental composition, structural

Abbreviations: GC/MS, Gas chromatography/mass spectrometry; DPPH, 2, 2‐diphenyl‐1‐picrylhydrazyl; EO, Essential oil; MIC, Minimum inhibitory concentration; MBC, Minimum
bactericidal concentration.
⇑ Corresponding author.
E-mail address: [email protected] (S. Guleria).

https://doi.org/10.1016/j.crbiot.2022.100118
Received 2 June 2022; Revised 12 November 2022; Accepted 19 November 2022

2590-2628/© 2022 The Authors. Published by Elsevier B.V.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
A. Majeed et al. Current Research in Biotechnology 5 (2023) 100118

(isomers), or spatial arrangement (enantiomers). Various studies have recorded with the following conditions: mass range 40–500 m/z; elec-
shown that essential oils have antibacterial properties (Lu et al., 2014; tron impact ionization voltage, 70 eV. Volatile components of N. jata-
Thielmann et al., 2019). The capacity of EOs to enhance bacterial cell mansi EO by comparing their spectra and retention indices with
permeability, coagulate cytoplasm, decrease intracellular ATP pool, authentic reference compounds (Adams, 2007).
and cause cytoplasmic and membrane protein molecules to malfunc-
tion is responsible for their antibacterial effect (Nazzaro et al., 2013; 2.2. Screening of antibacterial activity
Prasch and Bucar, 2015). The use of EOs in combination with conven-
tional antibiotics has recently piqued scientific interest (Sharma et al., The antibacterial activity of N. jatamansi EO was determined using
2020). In addition, EOs in combination with antibiotics may have var- the disc diffusion method (NCCLS, 1997). The selected test bacterial
ious antibacterial mechanisms of action, and so may play an essential strains were inoculated in Muller Hinton broth (MHB) and incubated
role in discovering novel ways to combat bacterial drug resistance for 24 h at 37 °C. Turbidity of the resulting bacterial suspension of
(Yap et al., 2013). As a result, combinatorial antibiotic therapy incor- active cultures was measured by taking absorbance at 600 nm using
porating EOs and conventional antibiotics could be a viable alternative spectrophotometer (Labomed, USA) and the optical density was
in which the combined antibacterial activity exceeds the antimicrobial adjusted equivalent to 0.5 Mcfarland standard by dilution with
activity of the individual components (Yahiaoui et al., 2017). MHB. Twenty milliliter of sterilized nutrient agar (NA) medium was
Indian spikenard, or Nardostachys jatamansi, is a Valerianaceae poured into the petri plates and allowed to solidify. Then 100 µL of
perennial herb that grows to a height of 10–60 cm. It can be found bacterial suspension (108 cfu/mL) was uniformly spread over the NA
at heights of 3000–5000 m above mean sea level in the alpine Hima- plates which were then kept to dry for five minutes. Whatman No. 1
layas, which span from Punjab to Sikkim and Bhutan. It has antide- sterile filter paper discs (6 mm diameter) impregnated with 3 µL of
pressant, anticonvulsant, anti‐ parkinson's, hepato‐protective, essential oil were placed on the media. The plates were left for thirty
antibacterial, and cardio‐protective characteristics, among others minutes and then they were incubated at 37 °C for 24 h. After this,
(Rao et al., 2005; Ahmad et al., 2006; Subashini et al., 2006; Joshi inhibition zone was measured using transparent ruler. Discs impreg-
and Parle, 2006, Kumar et al., 2006; Dandagi et al., 2008; Khan nated with chloramphenicol were used as positive control.
et al., 2012; Razack et al., 2015). Furthermore, there are just a few
studies on the antioxidant, anti‐inflammatory and antibacterial effects 2.3. Determination of antioxidant activity
of Nardostachys jatamansi EO in the literature (Parveen et al., 2011;
Singh et al., 2014). There is currently no data available on the antibac- 2.3.1. DPPH radical scavenging assay
terial activity of N. jatamansi EO in combination with conventional The DPPH radical is a stable molecule that is soluble in methanol
antibiotics against drug‐resistant Bacillus subtilis, Escherichia coli, Sta- and distinguished by its deep violet colour with a maximum absorp-
phylococcus aureus, Klebsiella pneumoniae and Micrococcus luteus. As a tion at 515 nm. By donating this stable radical an electron or hydrogen
result, the goal of this study was to investigate the antibacterial and atom, antioxidants are able to reduce it to 2,2‐diphenyl‐1‐
antioxidant effects of N. jatamansi EO and to see if it may work in syn- picrylhydrazine (DPPH‐H), which has a pale yellow colour and can
ergy with conventional antibiotics like amoxicillin, erythromycin, be easily measured using a spectrophotometer. DPPH radical scaveng-
chloramphenicol, and ampicillin. ing activity of N. jatamansi EO was determined according to Bozin
et al. (2006). Briefly, one milliliter of a 90 µM DPPH methanolic solu-
tion was mixed with 1 mL of various dilutions of EO (1 mg/ mL stock
2. Materials and methods
solution) and final volume was made to 4 mL with methanol. After 1 h
incubation in the dark at 25 °C, the absorbance was recorded as Asample
2.1. Experimental materials and chemicals
at 517 nm using a UV/VIS spectrophotometer (Labomed, USA). Solu-
tion without the test material constituted the blank and the absorbance
Natural Biotech Products, Baggi, Himachal Pradesh, India, pro-
was recorded as Ablank. The free radical scavenging activity was calcu-
vided the essential oil of Nardostachys jatamansi for this study. The bac-
lated as percent inhibition according to the following equation:
terial strains employed in this investigation were E. coli MTCC 2127, K.
% inhibition = 100 × (Ablank ‐ Asample)/Ablank.
pneumoniae MTCC 7172, M. leuteus MTCC 4821, S. aureus MTCC 7443,
Test compounds radical scavenging activity was expressed as IC50,
and B. subtilis MTCC 2389, all purchased from IMTECH, Chandigarh,
or the amount of test substance required to result in a 50 % reduction
India. Antibiotics (amoxicillin, erythromycin, chloramphenicol, and
in the initial DPPH concentration.
ampicillin) were procured from Hi‐Media Laboratories, Mumbai,
India.
2.3.2. Reducing power assay
Reducing power of N. jatamansi EO was determined according to
2.2. Determination of chemical composition by GC–MS analysis Oyaizu (1986). 2.5 mL of 200 mM sodium phosphate buffer (pH
6.6) and 2.5 mL of 1 % potassium ferricyanide were mixed with each
Separation and chemical profiling of essential oils is usually carried test sample, and the mixture was then incubated for 30 min at room
out by gas chromatography mass spectrometry (GC/MS) technique temperature. Then, 2.5 mL of 10 % trichloroacetic acid was added,
(Rubiolo et al., 2010). GC/MS generates representative chro- and the mixture was centrifuged at 1036 g for 10 min. The upper layer
matograms of volatile compounds, together with the associated mass (2.5 mL) was mixed with 2.5 mL of 0.1 % ferric chloride. The absor-
spectra for each separated component peak, which are then used for bance was then measured at 700 nm against a blank. From the graph
identification of metabolites present in essential oils. The gas chro- of absorbance against concentration, the test sample concentration
matography mass spectrometry analysis of N. jatamansi EO was per- that provided 0.5 of absorbance (IC50) was determined.
formed on a Varian GC (Varian Inc., Palo Alto, California, USA)
equipped with HP‐5 MS column (30 m × 0.25 mm × 0.25 μm) (Agi- 2.3.3. Ferric reducing antioxidant power assay
lent Technologies, Santa Clara, California, USA); carrier gas was Method of Benzie and Strain (1996) was used to measure the ferric
helium (1.0 mL/min). Working conditions were: the injector tempera- reducing antioxidant power of N. jatamansi EO. FRAP reagent was pre-
ture was set at 280 °C; initial temperature of column held at 50 °C for pared by mixing 100 mL of 300 mM sodium acetate buffer (pH 3.6),
5 min and then steadily increased by 3 °C/min to 300 °C; finally held 10 mL of 10 mM TPTZ [2,4,6‐tri‐(2‐pyridyl)‐1,3,5‐triazine] solution
constant at 280 °C for seven minute. 0.2 μL of N. jatamansi EO solution in 40 mM HCl plus 10 mL of 20 mM FeCl3 and 12 mL of distilled water.
in ethyl acetate was injected into the column. The MS scan was Test solutions were mixed with 3 mL of FRAP reagent and the absor-

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A. Majeed et al. Current Research in Biotechnology 5 (2023) 100118

bance was measured at 593 nm against blank after incubating the reac- that is compared to literature (Hu et al., 2006). Therefore, GC/MS
tion mixture for 10 min. The FRAP activity was calculated from the has become a part of the routine testing for essential oils and com-
calibration curve of ferrous sulphate (FeSO4 2H2O) and expressed as monly used for detecting adulterations. The qualitative and quantita-
mM Fe2+ eq./100 mg of the essential oil. tive profile of Nardostachys jatamansi EO determined by gas
chromatography mass spectrometry is depicted in Table 1. Nineteen
2.3. Determination of MIC and MBC of N. Jatamansi EO compounds were determined in N. jatamansi essential oil with guaia‐
6,9‐diene (11.96 %), calarene (10.44 %), jatamansone (8.11 %), α‐
MIC and MBC of EO were determined according to Wayne, 2003 gurjunene (7.42 %), valencene (6.46 %), α‐maaliene (5.24 %), spro-
with minor modifications. Mueller Hinton Broth (MHB) was used in jatamol (5.06 %) and caratol (5.06 %) as dominant compounds. Previ-
all the tests. N. jatamansi EO was dissolved in dimethyl sulfoxide ous studies have also reported chemical composition of N. jatamansi
(DMSO) in a 1.0:1.0 (v/v) ratio for stock solution preparation, fol- EO from different geographical locations. Chouhan et al. (2017)
lowed by filtration with a 0.22 µm filter disc before usage. MHB was reported that N. jatamansi EO from north and south facing slopes of
used to make the final volume to 1 mL. Similarly, MHB was also used Tungnath, Uttrakhand, India contained patchouli alcohol (40 to
to dilute the essential oil in the same way. In sterile microfuge tubes 52 %) as major component. In addition, caryophyllene oxide, cubeb‐
containing 90 µL of MHB, EO dilutions (100 µL) were added. Following 11‐ene, α‐patchoulene, seychellene, pogostol and carotol were also
that, 10 µL of working bacterial solution (106 CFU/mL) was added, present in appreciable amount. Similarly, Singh et al. (2018) demon-
resulting in a total volume of 200 µL in the microfuge tubes. Positive strated that EO extracted from air dried rhizomes of N. jatamansi from
control comprised of broth and inoculum but no EO or antibiotic, Haridwar, Uttrakhand, India was dominated by calarene (20.4 %),
whereas negative control was devoid of inoculum. After 24 h incuba- vardiflorene (12.3 %), α‐panasinsen (9.7 %), α‐santalene (4.6 %), resi-
tion period at 37 °C, each tube was added 40 µL of 0.4 mg/mL p‐ bufogenin (8.4 %) and epiglobulol (1.9 %). One report on N. jatamansi
iodonitrotetrazolium violet (INT) solution and incubated for another EO from India showed jatamansone (36.7 %); α‐cadinol (22.7 %) as
30 min at 37 °C. The development of a pink colour indicated bacterial the major constituents (Naquvi et al., 2013), while another reported
growth. The MIC was defined as the lowest concentration of essential β‐gurjunene (20.6 %); maaliol (8.2 %); patchouli alcohol (5.9 %)
oil or antibiotic that prevented bacterial growth. After 24 h of incuba- and 9‐aristolan‐12‐ol (5.8 %) (Vaze, 2003). α‐pinene (6.00 –
tion, 50 µL of broth was taken from the dilutions with no obvious bac- 8.53 %), 2‐ β‐pinene (0.51 – 19.26 %), α – terpinolene (1.32 –
terial growth and sub‐cultured on nutrient agar plates, then incubated 2.73 %) and myrtenyl acetate (0.54 – 8.89 %) were found as predom-
for another 24 h at 37 °C for MBC determination. MBC was the lowest inant compounds in N. jatamansi EO from five different regions of
dose of EO or antibiotic required to kill 99.9 % of inoculated bacteria. Nepal (Sharma et al., 2016). Similarly, ledene oxide [II] (13.021 %),
and sesquiterpine patchouli alcohol (9.582 %) were reported as major
components of the N. jatamansi EO from Pakistan (Parveen et al.,
3.4. Interaction studies of N. Jatamansi EO and antibiotics
2011). These data showed that several intrinsic and extrinsic factors
such as plant age, collection time, geographical location, and genetic
The broth dilution method was used to assess synergistic interac-
factors affect composition of essential oil (Hajdari et al., 2016; Nafis
tions between N. jatamansi EO and antibiotics (amoxicillin, ery-
et al., 2019).
thromycin, chloramphenicol, and ampicillin). In microfuge tubes
containing 50 μL of antibiotic dilutions and 100 μL of microbial cell
3.2. Antioxidant activity of N. Jatamansi EO
suspension, aliquots (50 μL) of N. jatamansi EO were introduced and
incubated at 37 °C for 24 h. After that, the tubes were incubated for
Findings of antioxidant assays are displayed in Table 2. N. jatamansi
another 30 min at 37 °C with 40 μL of 0.4 mg/mL
EO had moderate DPPH radical scavenging activity (IC50 of 0.95 ± 0.
p‐iodonitrotetrazolium violet (INT) solution to check for bacterial
008 mg/mL), reducing power (IC50 of 1.70 ± 0.082 mg/mL), and fer-
growth. The emergence of a pink colour indicated bacterial growth.
ric reducing antioxidant power (154 ± 6.13 mM Fe2+ eq./100 mg)
The FIC Index was used to determine the interaction between two
according to the results. Previous studies on the antioxidant activity
drugs (Pei et al., 2009). Didry et al. (1993) method as used to calculate
the fractional inhibitory concentration index (FICI) and gain. Frac-
tional inhibitory concentration indices (FICI) were determined as Table 1
follows: Chemical composition of volatile oil from Nardostachys jatamansi roots.
FIC of A = MIC of A in combination with B/ MIC of A alone.
RT Compound Relative
FIC of B = MIC of B in combination with A/ MIC of B alone. concentration (%)
FICI = FIC of A + FIC of B.
7.74 β- Patchoulene 4.83
Where A and B are two different essential oils/antibiotics.
8.34 Calarene 10.44
FICI ≤ 0.5, total synergism; 8.49 Guaia-6,9-diene 11.96
0.5 < FICI ≤ 0.75, partial synergism; 0.75 < FICI ≤ 2, no effect; 8.60 α-Gurjunene 7.42
FICI > 2, antagonism. 8.67 Seychellene 4.83
8.74 γ-Vetivenene 1.78
8.80 Kessane 4.68
3. Results and discussion 9.03 epi-Bicyclosesquiphellandrene 3.11
9.15 Valencene 6.46
9.22 2-Methylene-5-(1-methylvinyl)-8-methyl-bicyclo 3.88
3.1. Composition of Nardostachys jatamansi EO
[5.3.0]decane
9.44 Dehydroaromadendrene 3.12
Essential oils are widely used in the food and cosmetics industry 9.51 α-Maaliene 5.24
and also in the medical and pharmaceutical fields for various purposes. 9.98 4,5,9,10-dehydroisolongifolene 2.41
The semi‐volatile and volatile components that make up essential oils 10.29 Spirojatamol 5.06
10.56 Carotol 5.06
can be easily separated, identified, and quantified by gas chromatogra- 11.45 Jatamansone 8.11
phy and mass spectrometry making them excellent tools for essential 12.53 Methyl zizanoate 1.64
oil analysis. Gas chromatography and mass spectrometry are often 12.79 Octahydro-4,7-methano-1H-indenol 4.44
used in combination and is commonly referred to as GC/MS analysis. 13.28 Vetiselinenol 1.74
Total (%) 96.21
Each type of essential oil has a unique GC/MS qualitative fingerprint

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A. Majeed et al. Current Research in Biotechnology 5 (2023) 100118

Table 2 tions 0.1 %, 0.5 %, and 1 % v/v were reported to be effective against S.
Antioxidant activities of Nardostachys jatamansi essential oil. aureus and E. coli with inhibition zone of about 12.8 mm and 12.4 mm
Component DPPH radical Reducing Ferric reducing respectively, which were comparable with chloramphenicol at concen-
scavenging activity power antioxidant power tration of 0.1 mg/mL (Singh et al., 2018).
IC50 (mg/mL) IC50 (mg/mL) (mM Fe2+ eq./ Thielmann et al. (2019) found that commercial N. jatamansi EO
100 mg) had a significant inhibitory effect against S. aureus (MIC of 50 µg/
mL), but was ineffective against E. coli. The diffrences in antibacterial
Nardostachys 0.95 ± 0.008 1.70 ± 0.082 154 ± 6.13 activity of N. jatamansi EO observed in several studies may be due to
jatamansi quantitative and qualitative differences in chemical composition of N.
essential oil
BHT 0.02 ± 0.002 0.16 ± 0.004 –
jatamansi EO from different geographical regions and altitudes (Nafis
BHA 0.015 ± 0.001 0.12 ± 0.01 – et al., 2019).
It has been suggested that the antibacterial activity of EOs is mostly
due to their terpene content. Increased permeability, which leads to
of N. jatamansi EO has been limited. Antioxidant capacity of N. jata- breakdown of the plasma membrane and cytoplasmic leakage, is the
mansi root EO utilizing DPPH radical scavenging assay was reported principal antibacterial action mechanism observed in EOs (Álvarez‐
in one such study from Pakistan (Parveen et al., 2011). Similarly, Martínez et al., 2021). Furthermore, the molecular mechanisms of
Chaudhary et al. (2015) found that extract and fractions of N. jata- action of different terpenes are influenced by their chemical structure.
mansi had antioxidant and anticancer effects in breast carcinoma. In Terpenes containing phenolic OH groups, for example, can breach the
the same way, the ethanol fraction from N. jatamansi rhizomes has bacterial plasma membrane and disturb membrane potential and
been found to have significant antioxidant capacity, with an IC50 value homeostasis by binding monovalent cations like K+ and transporting
of 58.39 µg/mL (Mishra et al., 2014). The antioxidant potential of our them out of the bacterial cell, disrupting membrane potential and
N. jatamansi EO may be largely due to presence of calarene, seychel- homeostasis (Yang et al., 2015).
lene and maaliene in a higher amount. Furthermore, the presence of
an allyl structure in these terpenes, which has a lower bond‐ 3.4. Interaction of N. Jatamansi EO and antibiotics
breaking energy of CAH bonds than alkylic CAH or vinylic CAH
bonds, and has been reported to provide them higher scavenging According to the results obtained from interaction studies of N.
capacities. As a result, the CAH bond of methyl in allyl is easily bro- jatamansi EO with conventional antibiotics (Tables 4, 5, 6 and 7), of
ken, resulting in the loss of hydrogen atoms for DPPH free radical neu- the 70 combinations tested between N. jatamansi EO and amoxicillin,
tralization (Wu et al., 2020; Wojtunik et al., 2014). erythromycin, chloramphenicol and ampicillin 37 presented total syn-
ergism (52.85 %), 23 showed partially synergistic effect (32.85 %) and
3.3. Antibacterial activity of EO 10 showed no effect (14.30 %). Interaction of N. jatamansi EO and ery-
thromycin (Table 4) presented synergistic interaction against E. coli,
The results of the antibacterial activity of the N. jatamansi EO and M. luteus, S. aureus and K. pneumoniae (FICI values ranging from 0.44
conventional antibiotics against two Gram negative (Klebsiella pneumo- to 0.50), and partial synergy against B. subtilis (FICI = 0.62). Similarly,
niae and Escherichia coli) and three Gram positive (Bacillus subtilis, the interaction between N. jatamansi EO and ampicillin (Table 5) dis-
Micrococcus luteus and Staphylococcus aureus) bacterial strains are played complete synergism against E. coli, M. luteus and S. aureus (FICI
depicted in Table 3. It is evident from the above results that N. jata- ranging from 0.37 to 0.50) and partial synergy against B. subtilis and K.
mansi EO showed antibacterial activity against all tested strains with pneumoniae (FICI = 0.58). Also, the combination of N. jatamansi EO
inhibition zone diameter (IZD) ranging from 10.5 ± 0.5 mm to 14.0 and chloramphenicol (Table 6) showed synergism against E. coli, M.
± 0.4 mm. EO's MIC and MBC values against all of the investigated luteus, B. subtilis and S. aureus and FICI values ranged from 0.32 to
bacterial strains ranged from 1500 to 3100 µg/mL and 1800 to 0.45 and partial synergy against K. pneumoniae (FICI = 0.6). Interest-
3500 µg/mL, respectively. ingly, the combination of N. jatamansi EO and amoxicillin (Table 7,
Intriguingly, E. coli, an antibiotic‐resistant Gram‐negative bacteria, Fig. 1 & Fig. 2) demonstrated excellent synergism against all of the
was most sensitive to N. jatamansi EO of all the bacterial strains tested bacteria tested, with FICI values ranging from 0.32 to 0.45. In the pres-
(MIC = 1500 µg/mL; MBC = 1800 g/mL), whereas, S. aureus, an ence of N. jatamansi EO (1/4 MIC), the gain in MIC was also assessed
antibiotic‐resistant Gram‐positive bacteria, was least sensitive and expressed as MIC of antibiotics. Antibiotic MICs were lowered by 4
(MIC = 3100 g/mL; MBC = 3500 g/mL). N. jatamansi EO concentra- to 10.5 times in the presence of N. jatamansi EO against the tested spe-

Table 3
Inhibition zone diameters, MIC and MBC of Nardostachys jatamansi essential oil and conventional antibiotics.

Microorganisms Essential oil Amoxicillin Erythromycin Chloramphenicol Ampicillin

IZD MIC MBC IZD MIC MBC IZD MIC MBC IZD MIC MBC IZD MIC MBC

M. luteus 14.0 ± 0.4 2000 2400 14.2 ± 0.2 75 80 14.2 ± 0.5 1.5 1.5 15.3 ± 0.6 2.5 2.5 24.3 ± 0.6 0.8 0.9
E. coli 10.7 ± 0.4 1500 1800 12.2 ± 1.3 75 80 23.5 ± 0.8 1.0 1.8 15.2 ± 0.2 2.0 2.0 12.0 ± 0.4 0.5 0.8
S. aureus 12.5 ± 1.0 3100 3500 13.7 ± 1.0 70 79 24.7 ± 0.6 1.0 1.5 16.0 ± 1.0 1.5 2.0 12.0 ± 0.4 0.4 0.8
B. subtilis 10.5 ± 0.5 1600 2000 19.2 ± 0.6 70 75 22.3 ± 1.8 1.5 1.5 13.5 ± 0.7 2.0 2.5 12.6 ± 0.5 0.5 0.8
K. pneumnae 11.5 ± 0.9 2500 2900 18.3 ± 0.6 75 75 24.5 ± 0.8 1.0 2.0 15.6 ± 1.0 1.5 2.5 17.2 ± 0.6 0.4 1.0

IZD: Inhibition zone diameter (mm).

MIC: Minimum inhibitory concentration (µg/mL).
MBC: Minimum bactericidal concentration (µg/mL).
Concertation of Nardostachys jatamansi essential oil: 2.5 mg/disc.
Concentration of antibiotics: 3 µg/disc.

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A. Majeed et al. Current Research in Biotechnology 5 (2023) 100118

Table 4
Synergistic interaction of Nardostachys jatamansi essential oil and erythromycin.

Component E. coli M. luteus B. subtilis S. aureus K. pneumoniae

FIC FICI Gain FIC FICI Gain FIC FICI Gain FIC FICI Gain FIC FICI Gain

Nardostachys jatamansi 0.33 – – 0.25 – – 0.5 – – 0.25 – – 0.33 – –

(NJEO)
Erythromyin (Ery) 0.16 0.49* 6.0 0.25 0.50* 4.0 0.12 0.62** 8.33 0.25 0.50* 4.0 0.11 0.44* 9.09

FICNJEO = MIC of NJEO in combination with Ery/MIC of NJEO alone.

FICery = MIC of Ery in combination with NJEO/MIC of Ery alone.
FIC index = FICNJEO + FICEry.
* Synergism.
**Partial synergism.

Table 5
Synergistic interaction of Nardostachys jatamansi essential oil and ampicillin.

Component E. coli M. luteus B. subtilis S. aureus K. pneumoniae

FIC FICI Gain FIC FICI Gain FIC FICI Gain FIC FICI Gain FIC FICI Gain

Nardostachys jatamansi 0.25 – – 0.25 – – 0.33 – – 0.25 – – 0.33 – –

(NJEO)
Ampicillin (Amp) 0.16 0.41 6.02 0.25 0.50 4.0 0.25 0.58** 4.16 0.12 0.37 8.0 0.25 0.58** 4.0

FICNJEO = MIC of NJEO in combination with Amp/MIC of NJEO alone.

FICamp = MIC of Amp in combination with NJEO/MIC of Amp alone.
FIC index = FICNJEO + FICAmp.
* Synergism.
**Partial synergism.

Table 6
Synergistic interaction of Nardostachys jatamansi essential oil and chloramphenicol.

Component E. coli M. luteus B. subtilis S. aureus K. pneumoniae

FIC FICI Gain FIC FICI Gain FIC FICI Gain FIC FICI Gain FIC FICI Gain

Nardostachys jatamansi 0.20 – – 0.16 – – 0.25 – – 0.33 – – 0.5 – –

(NJEO)
Chloramphenicol (Chl) 0.25 0.45* 4.0 0.16 0.32* 6.0 0.12 0.37* 8.0 0.10 0.43* 10.0 0.1 0.6** 10.5

FICNJEO = MIC of NJEO in combination with Chl/MIC of NJEO alone.

FICCh l = MIC of Chl in combination with NJEO/MIC of Chl alone.
FIC index = FICNJEO + FICChl.
* Synergism.
**Partial synergism.

Table 7
Synergistic interaction of Nardostachys jatamansi essential oil and amoxicillin.

Component E. coli M. luteus B. subtilis S. aureus K. pneumoniae

FIC FICI Gain FIC FICI Gain FIC FICI Gain FIC FICI Gain FIC FICI Gain

Nardostachys jatamansi 0.33 – – 0.25 – – 0.33 0.33 – – 0.33 – –

(NJEO)
Amoxicillin (Amx) 0.12 0.45* 8.0 0.20 0.45* 5.0 0.16 0.49* 6.0 0.10 0.43* 7.0 0.1 0.43* 10

FICNJEO = MIC of NJEO in combination with Amx/MIC of NJEO alone.

FICamx = MIC of Amx in combination with NJEO/MIC of Amx alone.
FIC index = FICNJEO + FICAmx.
* Synergism.

5
A. Majeed et al. Current Research in Biotechnology 5 (2023) 100118

25 20
E. coli M. luteus
20
15

AMX (µg ml-1)

AMX (µg ml-1)
15
10
10

5 5

0 0
0.3 0.4 0.5 0.6 0.7 0.8 0.4 0.5 0.6 0.7 0.8 0.9 1
NJEO (mg ml-1) NJEO (mg ml-1)

25 20

20 S. aureus
B. sublis 15
AMX (µg ml-1)

AMX (µg ml-1)

15
10
10

5 5

0 0
0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6
NJEO (mg ml-1) NJEO (mg ml-1)

30

25 K. pneumoniae
ml-1)

20
AMX (µg

15

10

0
0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3
NJEO (mg ml-1)

Fig. 1. Synergistic antibacterial activity of Nardostachys jatamansi essential oil (NJEO) in combination with amoxicillin (AMX).

cies. With a gain of 10.5 times, K. pneumoniae was found to have the 2018; El Atki et al., 2019; Sharma et al., 2020). EOs from Cinnam-
highest reduction. N. jatamansi EO also increased the Gram‐negative monum zeylanicum, Mentha piperita, Origanum vulgare, and Thymus vul-
bacterium K. pneumoniae's susceptibility to the medicines amoxicillin, garis have been demonstrated to interact synergistically with
erythromycin, and chloramphenicol, with gains in MICs of 10, 9.09, norfloxacin (Rosato et al., 2020). Pelargonium endlicherianum EO in
and 10.5 fold, respectively. In fact, combinatorial antibiotic therapy combination with cefepime and gentamicin demonstrated promising
with natural products is one of the novel strategy against drug resistant synergism against K. pneumoniae (Dumlupinar et al., 2020). The most
microorganisms. This is the first study on N. Jatamansi EO's synergistic common mechanisms of synergistic antibacterial action are efflux
action with conventional antibiotics that we are aware of. EOs and pump inhibition, β‐lactamase inhibition, inhibition of shared meta-
antibiotics have been demonstrated to interact synergistically in vari- bolic pathways, and membrane permeabilization (Aleksic and
ous studies (Mahadlek et al., 2012; Yap et al., 2013; Aghraz et al., Knezevic, 2014; Álvarez‐Martínez et al., 2021).

6
A. Majeed et al. Current Research in Biotechnology 5 (2023) 100118

4 2 3
1
3
4 Synergy
Synergy 3 Synergy 2
4
2 1 1 2
S. aureus
M. luteus B. sublis

K. pneumonea Synergy

1 4
2 2

3
3 1
4
Synergy
E. coli

Fig. 2. Antimicrobial effect of NJEO and AMX alone and in combination on five drug resistant microbes. 1. DMSO (3 µL); 2. AMX (0.017 µg/mL); 3. NJEO
(187.5 µg/mL); 4. NJEO + AMX (187.5 µg/mL + 0.017 µg/mL). NJEO = Nardostachys jatamansi EO; AMX = Amoxicillin.

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