Journal of Ethnopharmacology 279 (2021) 114402

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Phytochemical analysis and biological activities of “Cherchoomoro”

(Nepeta adenophyta Hedge)
Iftikhar Ali a, b, *, Muhammad Ali b, Huma Shareef c, Sadaf Naeem c, Adeeba Khadim d,
Meher Ali b, Faiza Amber e, Hidayat Hussain f, **, Muhammad Ismail b, Syed Tasadaque A. Shah g,
Ali Noor e, Daijie Wang a
a
Shandong Analysis and Test Center, Qilu University of Technology (Shandong Academy of Sciences), Jinan, 250014, China
b
Department of Chemistry, Karakoram International University, Gilgit, 15100, Pakistan
c
Institute of Pharmaceutical Sciences, Jinnah Sindh Medical University, Karachi, 75510, Pakistan
d
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan
e
Department of Biological Sciences, Karakoram International University, Gilgit, 15100, Pakistan
f
Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Weinberg 3, D-06120 Halle, Germany
g
Department of Education, Sukkur IBA University, Sukkur, 65200, Sindh, Pakistan

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

Keywords: Ethnopharmacological relevance: Nepeta adenophyta Hedge (Lamiaceae) is an endemic therapeutic herb from
Nepeta adenophyta Astore, Gilgit (Pakistan). This plant species has been reported among the local communities, especially for
Analgesic treating abdominal pain, kidney pain, menstrual pain, headache, and controlling bleeding disorders. Therefore,
Anti-inflammatory
the scientific basis is provided for the relief of pain as it is used in various pain management among the natives,
Antioxidant
especially as ethnogynecological herbal remedy.
GC-MS
LC-MS Aim of the study: The present study investigates the analgesic and anti-inflammatory effects of the ethanolic
extract of N. adenophyta in animal models. Furthermore, the extract was also studied to determine their valuable
phytoconstituents.
Material and methods: The biological effects were determined via tail-flick, hot plate, and acetic-acid-induced
abdominal writhing methods. At the same time, anti-inflammatory activity was assesed via oxidative burst
and antioxidant DPPH assay. Gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass
spectrometry (LC-MS) techniques were employed to understand the phytochemicals present in the crude etha­
nolic extract of Nepeta adenophyta.
Results: In the current study, Nepeta adenophyta extract exhibited potent analgesic and anti-inflammatory effects
on different pain models and indicated that the analgesic effect of N. adenophyta extract is mediated both in
central and peripheral ways. Dose-dependent and significant (P < 0.05) increases were shown in pain threshold,
at 45 min post-treatment, with 20 and 40 mg/kg of the extract in the tail-flick model. The effects of the extract
were similar to aspirin but lower to those by morphine (2.5 mg/kg) in the same tests. The extract (20–40 mg/kg)
showed dose-dependent inhibition of writhing with a significant (P < 0.001) increase protection against thermal
stimuli in hot plate test as compared to control and similar to aspirin and morphine. Further, the anti-
inflammatory activity of the crude in oxidative burst and DPPH assays showed significant inhibitory activity.

Abbreviations: NAE, ethanol extract of Nepeta adenophyta Hedge; DPPH, 2,2-diphenyl-1-picrylhydrazyl; LD50, median lethal dose; i.p., intraperitoneally; MPA,
maximum possible analgesia; HBSS++, Hank’s balanced salt solution, containing calcium chloride and magnesium chloride; SOZ, serum opsonized zymosan; ROS,
reactive oxygen species; RLU, relative light units; GC-MS, gas chromatography-mass spectrometry; LC-MS, liquid chromatography-mass spectrometry; DNP, dic­
tionary of natural products; MoNA, massbank of North America; SIM, selected ion monitoring; EI, electron impact; NIST, national institute of standards and tech­
nology; SPSS, statistical product and service solutions; ANOVA, analysis of variance; LSD, least significant difference; SD, standard deviation; IC50, concentration of
sample required for 50% inhibition; PGE2, prostaglandin E2.
* Corresponding author. Shandong Analysis and Test Center, Qilu University of Technology (Shandong Academy of Sciences), Jinan, 250014, China.
** Corresponding author.
E-mail addresses: iftikhar.ali@kiu.edu.pk (I. Ali), chem.kiu@gmail.com (M. Ali), huma.shareef@jsmu.edu.pk (H. Shareef), sadaf.naeem@jsmu.edu.pk (S. Naeem),
adeeba.abbas@gmail.com (A. Khadim), meher.ali@kiu.edu.pk (M. Ali), famber.kiu@gmail.com (F. Amber), Hidayat.Hussain@ipb-halle.de (H. Hussain), dr.ismail@
kiu.edu.pk (M. Ismail), tasadaque.shah@iba-suk.edu.pk (S.T.A. Shah), dr.alinoor@kiu.edu.pk (A. Noor), wangdaijie@126.com (D. Wang).

https://doi.org/10.1016/j.jep.2021.114402
Received 29 April 2021; Received in revised form 1 July 2021; Accepted 6 July 2021
Available online 7 July 2021
0378-8741/© 2021 Elsevier B.V. All rights reserved.
I. Ali et al.
The chemical profile analysis showed major phytochemicals, including long chain derivatives of alkane and
alcohol, phenolics, naphthalene, naphthopyran, androsten phenanthrenone, nepetalactones, flavonoids etc.
Conclusions: Nepeta adenophyta Hedge is suggested as a natural alternative for mild pain relief. Our findings
endorse the folklore use of N. adenophyta in different pain managements which can be attributed to the presence
of polyphenolic compounds, naphthalene derivatives, flavanoids and nepetalactones etc.
1. Introduction
Nepeta adenophyta Hedge (Lamiaceae previously known as Labiatae)
is endemic to Astore, Gilgit, Pakistan (Hedge, 1989). N. adenophyta
Hedge is a perennial herb locally known as “Cherchoomoro”.
N. adenophyta has been reported in the folklore for the treatment of
abdominal pain, kidney pain, urine problems and in menstruation pain
(Nia et al., 2017). Most importantly, the decoction of the whole plant is
used for irregular menstruation, and it is very famous as ethno­
gynaecological herbal remedy. In addition, the plant species has also
been employed for the treatment of various ailments, including dysen­
tery, stomach problems and general weakness, and pain problems in
cattle (Noor et al., 2012).
Several Nepeta species or their active components have been reported
for antioxidant, anti-inflammatory, antimicrobial, and analgesic prop­
erties (Suntar et al., 2018; Ur Rehman et al., 2018). Moreover, N. cataria
is the most extensively studied species, which is used as a fortifier, a
disinfectant, and an analgesic plant (Grognet, 1990). The medicinal ef­
fects of Nepeta species are usually attributed to their chemical constit­
uents viz. iridoid moieties, diterpenes and sesquiterpenes, triterpenes,
and flavonoids, etc (Sharma et al., 2021).
However, in our present study, N. adenophyta is investigated for the
first time, to the best of our knowledge, for analgesic and anti-
inflammatory activities to provide evidence for pain relief. The chemi­
cal profile analysis is also reported for the first time on this plant.
2. Material and methods
2.1. Plant material
The plant material of N. adenophyta (Lamiaceae) was collected from
Astore valley (Gilgit, Pakistan) at 2400–3100 m elevation in July 2017
and was authenticated by plant taxonomists. The voucher specimen
(180-KUH, by Ali Noor) was deposited at the Department of Biological
Sciences, Karakoram International University. The plant material was
thoroughly washed and cleaned with water, dried in the shade, and
converted to powder after crusing.
2.2. Extraction, fractionation and sample preparation
The powdered plant material of N. adenophyta was percolated in
absolute EtOH (Merck, Germany) for a week at room temperature and it
was then extracted in triplicate. The ethanolic extractives were com­
bined and evaporated to yield a crude residue of 8.67% (w/w). The
ethanolic extract (NAE) was directly subjected to analgesic and anti-
inflammatory activities.
The plant powder material was weighed (1 g) and extracted in
ethanol (10 mL) by sonication for 20 min. The sample was centrifuged
for 15 min at 6000 rpm to settle large particles and a clear supernatant
was obtained. The supernatant was then filtered through a 0.22 μm PTFE
syringe-driven filter. The solvent was then evaporated by rotary evap­
orator and the crude was dissolved in methanol (10 mL). Finally, 50 μL
of the filtered extract was diluted to 1000 μL with methanol for LC-MS
and GC-MS analyses.
2.3. Animals
Male and female Swiss albino mice weighing 18–25 g and Wister rats
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Journal of Ethnopharmacology 279 (2021) 114402
weighing 150–250 g were used in this study. Mice were taken from the
animal house, Institute of Pharmaceutical Sciences, Jinnah Sindh Med­
ical University, Karachi, Pakistan. Animals were provided ambient
conditions at 22 ± 2 ◦ C, and alternate 12 h light/dark cycle. The animals
were fed food and water ad libitum. Before the test, mice were kept on
fasting for 12 h with only access to water. All experimental procedures
were reviewed and approved by the Independent Animal Ethics Com­
mittee protocol number ICCBS/IEC-026-HV/HP-2017/Protocol/2.0.
The experimental protocols and animal care were performed in accor­
dance with ethical principles established in 1979 for laboratory animals
in the services of mankind, Lyons, France (Saeed et al., 2016).
2.4. Chemicals
Aspirin (Reckitt and Colman, Pakistan), acetic acid and morphine
(Sigma Chemicals Company, St. Louis, USA), diclofenac (Abbott labo­
ratories Pakistan limited) were used. Sterile normal saline (control) was
used in all studies. Ethanol (Merck, Germany), and methanol (Merck,
Germany) were of analytical grade. Acetonitrile was of chromatographic
grade (Fisher Company, Fairlawn, NJ, USA), used for LC-MS. The water
used in the LC-MS was produced by reverse osmosis in Milli-Q system
(Millipore, Bedford, MA, USA).
2.5. Acute oral toxicity study (LD50)
LD50 determination was performed as per previous protocol (Lorke,
1983). Total 60 experimental animals categorized into two sets
comprising 6 male and female mice in each set were fasted overnight.
NAE was administrated to set I orally at doses 100, 400, and 1000 mg/kg
in the first stage, while set II was treated intraperitoneally (i.p.) at 10, 20,
30, 40, and 50 mg doses. The NAE-treated mice were fed ad libitum and
observed 24 h for acute toxicity or death. The geometric mean of the
highest non-lethal dose and the lowest lethal dose in the second stage
were determined by estimating the LD50. The results of this stage helped
us select the doses for the analgesic and anti-inflammatory activities.
2.6. Analgesic activity
To evaluate the analgesic activity of NAE, the hot plate method, tail-
flick method, and writhing reflex methods were employed. Two
different concentrations of ethanol extract 20 and 40 mg/kg body
weight were used intraperitoneally. For the 20 mg/kg sample prepara­
tion, the average weight of mice (24.13 mg eq. to 0.0241 kg) was taken.
Moreover, 20 mg ethanol extract dose for 24.13 mg mice was calculated
by the unitary method. The extract (10 mg) was dissolved in distilled
water (25 mL) to obtain a stock solution of 0.4 mg/mL of the extarct. The
extract solution of 0.12 mL was given to mice i.p. 40 mg/kg sample was
prepared by dissolving 10 mg of extract in 12.5 mL of distilled water
(0.8 mg/mL approx.). 0.12 mL extract was given to mice i.p. for anal­
gesic activity. Aspirin, diclofenac, and morphine were used as standard
analgesic solutions. Aspirin was given 150 mg/kg body weight, 100 mg
tablet dissolved in 5 mL of distilled water, and were given 0.15 mL i.p.
for writhing reflex and tail-flick activity. Morphine was also used as
standard and was given in the dose of 2.5 mg/kg. For 0.024 kg mice,
0.066 mg morphine dose was calculated and 2.5 mg morphine diluted in
25 mL of saline, and 0.1 mL was given to each mice. 20 mg/kg diclofenac
sodium was used as a standard for hot plate activity, i.e., 50 mg tablet
was dissolved in 10 ml normal saline (0.9% NaCl solution), and 0.1 mL
I. Ali et al. Journal of Ethnopharmacology 279 (2021) 114402

Table 1 calculated.
Analgesic effect of ethanol extract of N. adenophyta by
writhing reflex method in rats (Values are Mean ± SD, n
= 6; significance at *P < 0.05 compared to the control). 2.8. Hot-plate test in mice
Groups No of writhes
The reported method was followed (Turner, 1965). The mice were
Control 33.5 ± 6.41
20 mg 14.50 ± 4.72*** divided into five groups (n = 6 per group) and treated as follows: groups
40 mg 6.50 ± 4.13***## I (20 mg/kg) and II (40 mg/kg) were injected NAE intraperitoneally,
Aspirin 15.67 ± 3.14*** respectively; group III was injected morphine (2.5 mg/kg, as positive
Morphine 2.83 ± 1.16*** control) intraperitoneally, group IV (diclofenac sodium, 20 mg/kg as
Control: P < 0.00***, P < 0.01**, P < 0.05*. positive control) and group V (distilled water, as vehicle control). Ani­
Aspirin: P < 0.00###, P < 0.01##, P < 0.05# mals were then placed on a hotplate (Eddy’s hot plate, German) kept at a
Morphine: P < 0.00¶¶¶, P < 0.01¶¶P < 0.005¶ temperature of 53 ± 0.5 ◦ C. The reaction time was then observed for 90
min (0, 15, 30, 60, and 90 min) after extract or drug administration. The
period between placing the mouse on the hotplate and licking the hind
paw or jumping was considered the reaction time. The increase in re­
action time was determined (%) for each NAE- and drug-treated group.
The %age protection against thermal stimulus was calculated as follows:

% protection against thermal stimuli = Treatment – Control / Control x 100

2.9. Tail flick activity

The tail-flick method (Huong et al., 1996) was followed to determine

the antinociceptive (analgesic) activity. The rats were divided into five
groups (n = 6 per group), each injected intraperitoneally. They were
treated as follows: group I (20 mg/kg, NAE), group II (40 mg/kg, NAE),
Fig. 1. Percentage reduction of writhes in 15 min. group III (2.5 mg/kg, morphine as positive control), group IV (150
mg/kg, aspirin as positive control), and group V (distilled water as
vehicle control). In addition the tail of each rat was immersed in the
Table 2
water bath (50 ◦ C) after dosing about 5 cm from the distal end. The
Change in reaction time of different treatments after doses administration in the
reaction time (sec) was the time taken by the rat to flick its tail due to
course of time (hot plate) (Values are Mean ± SD, n = 6; significance at *P < 0.05
as compared to the control) pain. The average of the next to the first two readings was taken as the
reaction time, and the first reading was ignored. The reaction time was
Groups 30 min 60 min 120 min 180 min
recorded at 15, 30, 45, and 60 min after the treatments. If the reading
Control 2.16 ± 0.144 2.241 ± 0.129 1.95 ± 0.141 1.97 ± 0.091 exceeds 15 s, the maximum reaction time, it was considered as
20 mg/kg 2.33 ± 3.13 ± 3.25 ± 2.57 ± 0.23**
maximum analgesia. The maximum possible analgesia (MPA) was
0.137** 0.100*** 0.144***
40 mg/kg 2.54 ± 3.95 ± 4.44 ± 3.31 ±
calculated as follows:
0.112*** 0.159*** 0.435*** 0.309***
Aspirin 2.73 ± 4.26 ± 4.80 ± 3.08 ±
MPA = Postdrug latency (T) – Predrug latency (To)
0.107*** 0.071*** 0.335*** 0.400***
Morphine 2.60 ± 4.87 ± 5.39 ± 4.19 ±
0.246*** 0.346*** 0.418*** 0.365***

Control: P < 0.00***, P < 0.01**, P < 0.05*.

Aspirin: P < 0.00###, P < 0.01##, P < 0.05#
Morphine: P < 0.00¶¶¶, P < 0.01¶¶P < 0.005¶
was given to each mouse.
2.7. Acetic-acid induced abdominal writhing test
The writhing test (Gülçin et al., 2003, 2004; Koster, 1959) with
modifications in the timing of observations was followed. To induce
abdominal writhing, the animals were injected acetic acid (6%, 10
mL/kg) intraperitoneally. Thirty mice comprising five groups (n = 6 per
group) were pretreated 30 min before acetic acid injection as follows:
Groups I and II were injected NAE 20 mg/kg and 40 mg/kg intra­
peritoneally, respectively; group III was injected Aspirin 150 mg/kg (as
positive control) intraperitoneally. Group IV (as positive control) and
group V (vehicle control) were injected intraperitoneally morphine (2.5
mg/kg) and distilled water, respectively. The number of writhing was
counted twice (at 15 min intervals) for 5 min after a 5-min lag period
after the injection of acetic acid and expressed as % of constriction in­
hibition. The percentage of reduction of writhes in 15 min was
2.10. Antioxidant assays
The DPPH radical scavenging (Blois, 1958; Gulcin, 2012, 2020,
2020; Taslimi and Gulçin, 2018) of NAE was measured at 517 nm
(absorbance) on a UV-VIS spectrophotometer (Shimadzu UV-2200).
Ascorbic acid was used as standard, and IC50 values were calculated.
Furthermore, NAE extract was subjected to oxidative burst (lumino­
l-enhanced chemiluminescence) assay to determine the
anti-inflammatory activity. In brief, 25 μL of diluted whole blood
HBSS++ (Sigma, St. Louis, USA) was incubated with 25 μl of NAE con­
centrations (1, 10, and 100 μg/mL) each in triplicate. Control wells
contained HBSS++ and cells without NAE. The test was carried out in a
white half area 96 well plates (Costar, NY, USA), incubated (37 ◦ C) for
15 min in thermostat chamber of luminometer (Labsystems, Helsinki,
Finland). Then serum opsonized zymosan (SOZ) (Fluka, Buchs,
Switzerland) and intracellular ROS detecting probe, luminol (Research
Organics, Cleveland, OH, USA), each 25 μL, were added into each well,
except blank wells (containing only HBSS++). The ROS level was
recorded in the luminometer in terms of relative light units (RLU).
Ibuprofen (IC50 = 11.2 ± 1.9 μg/mL) served as standard (Helfand et al.,
1982).

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I. Ali et al. Journal of Ethnopharmacology 279 (2021) 114402

Fig. 2. A. Percentage protection against the thermal stimulus, B. Change in latency time in tail-flick activity, C. DPPH antioxidant assay, D. Oxidative burst assay.

Table 3
Analgesic effect of ethanol extract of N. adenophyta by the tail-flick method in rats (Values are Mean ± SD, n = 6; significance at *P < 0.05 compared to the control).
Reaction time in seconds (mean ± SD)

Groups 0 min 15 min 30 min 45 min 60 min 75min 90 min

Control 0.01 ± 0.004 0.018 ± 0.004 0.028 ± 0.007 0.04 ± 0.01 0.35 ± 0.02 0.03 ± 0.01 0.04 ± 0.01
20 mg/kg 0.015 ± 0.005 0.027 ± 0.004 0.041 ± 0.007 0.14 ± 0.05* 0.14 ± 0.04* 0.09 ± 0.01* 0.07 ± 0.007
40 mg/kg 0.013 ± 0.005 0.03 ± 0.006* 0.23 ± 0.07*** 0.38 ± .132***# 0.44 ± 0.11***### 0.18 ± 0.07***### 0.26 ± 0.35**
Aspirin 0.013 ± 0.05 0.04 ± 0.005*** 0.30 ± 0.08*** 0.248 ± 0.04*** 0.11 ± 0.03* 0.07 ± 0.01 0.07 ± 0.03
Morphine 0.13 ± 0.005 0.041 ± 0.007*** 0.47 ± 0.04*** 0.41 ± 0.06*** 0.52 ± 0.08*** 0.22 ± 0.05*** 0.16 ± 0.04

Control: P < 0.00***, P < 0.01**, P < 0.05*.

Aspirin: P < 0.00###, P < 0.01##, P < 0.05#
Morphine: P < 0.00¶¶¶, P < 0.01¶¶P < 0.005¶

2.11. Chemical profile analysis
NAE was subjected to gas chromatography-mass spectrometry (GC-
MS) analysis. Gas chromatograph 7890A system (Agilent, USA) equip­
ped with a 5973 Network mass selective detector mass spectrometer
(Agilent, USA) was used for analysis. Helium (99.999%) was employed
as a carrier gas with a flow rate of 1.0 ml/min for all samples. The other
conditions were; injector temperature 280 ◦ C; initial oven temperature
100 ◦ C, increased to 270 ◦ C at 10 ◦ C/min. The inlet was operated in
splitless mode with a splitless time of 1 min. The GC-MS analysis was
performed in selected ion monitoring (SIM) detection mode. The tem­
perature of the transfer line was maintained at 280 ◦ C. The energy of
electrons used was 70 eV. Other parameters included low m/z 29 (auto)
and high m/z 600, instrument scan Quad - High to Low. NIST library was
consulted for spectral comparison and identification.
The liquid chromatography-mass spectrometry (LC-MS) analysis was
performed on UHPLC system (Waters, H-Class, USA) coupled to QTOF
mass spectrometer (Bruker Impact II, Germany). For HPLC analysis a
mixture of acetonitrile and water was chosen as the mobile phase at a
wavelength of 390 nm applying the following solvent system at a flow
rate of 1 mL/min: 0–5 min, isocratic 75% A (water), 25% B
(acetonitrile); 5–17 min, linear from 75% to 25% A; 17–22 min, linear
from 25% to 75% A; 22–25 min, isocratic 25% B. The injection volume
was set at 10 μL. The compounds were detected both in positive and
negative modes. Ion source parameters used are mentioned as follows
(parameters for negative mode next to positive mode parameters):
capillary voltage at 3500 V (− 3000 V), end plate offset at 500 V,
nebulizer gas 2.0 bar, drying gas at 8.0 L/min and drying gas temper­
ature at 200 ◦ C. All spectra were recorded in the mass range from m/z 50
to 1500.
2.12. Statistical analysis
The data obtained were analyzed on SPSS (version 21) software
using One-way ANOVA followed by an LSD post hoc test. The data were
presented as Mean ± Standard Deviation (SD), and P < 0.05 indicates
statistical significance.

4
I. Ali et al.
Fig. 3. (A) GC-MS chromatogram of NAE, (B) Expended part 1, (C) Expended
part 2.
3. Results
3.1. Acute oral toxicity studies
A decreased mobility and respiratory distress (gasping) with even­
tual immobility were shown in acute oral toxicity. But before death in
animals, no convulsions or loss of righting reflex was found. The
calculated LD50 was found 56.3 ± 6.5 mg/kg i.p. 645.5 ± 34.5 mg/kg
oral. Two intra-peritoneal therapeutic doses were selected from lower
(20 mg/kg) and second higher (40 mg/kg) to determine the analgesic
effect.
3.2. Acetic-acid writhing test
NAE administration at 20 and 40 mg/kg showed a significant (P <
0.05) inhibitory, dose-dependent effect on the number of acetic acid-
induced abdominal constrictions in mice. The effect was time-
dependent. The inhibitory effect was 48.69% (20 mg/kg) after 15 min
of acetic acid treatment, and at 40 mg/kg, the inhibitory effect was
79.07% (Table 1, Fig. 1). Aspirin significantly (P < 0.05) inhibited the
abdominal constrictions at 20 mg/kg, but at 40 mg/kg, the extract and
morphine showed insignificant inhibition. Post hoc analysis by LSD
revealed a highly significant analgesic effect (P < 0.001) at 40 mg/kg of
NAE. However, similar or insignificant results were observed compared
5
Journal of Ethnopharmacology 279 (2021) 114402
with morphine.
3.3. Hot-plate test in mice
NAE-pretreated animals (20–40 mg/kg/i.p.) showed a significant
dose-dependent increase in reaction time to thermal stimulation
compared to the control group (Table 2, Fig. 2A). The 40 mg/kg dose at
120-min post-treatment (127.6%) resulted in the highest increase in
reaction time. The increased reaction time by diclofenac sodium after
120 min was 126.15%, and morphine (2.5 mg/kg) was 176.41% at 120
min. Post hoc analysis by LSD exhibited 40 mg/kg NAE gave highly
significant (P < 0.01) increase protection against thermal stimuli
compared to control. At the same time, 20 mg/kg of NAE showed similar
or insignificant results than diclofenac and morphine.
3.4. Tail-flick activity
As shown in Table 3 and Fig. 2B, rats treated with normal saline
(negative control) did not significantly differ in the reaction time on tail-
flick throughout the observation (90 min). The increase in reaction time
at different time points significantly varied, in the same treatment
group, for morphine sulfate. The reaction time in aspirin, morphine and
NAE-treated animals was considerably higher than saline-treated ani­
mals, except for the 20 mg/kg extract group at 45 min. On the other
hand, the highest reaction time for the 40 mg/kg NAE-treated group was
0.456 s at 60 min, while it was 0.305 s and 0.562 s for aspirin and
morphine treated groups, respectively. At all-time points, the tail-flick
latency time changed significantly.
Additionally, no significant difference in reaction time was observed
at 20 mg/kg NAE and aspirin, but significant results were seen at the 40
mg/kg NAE-treated group. Aspirin-treated rats did not give any signif­
icant analgesic effect compared to baseline values, saline, or NAE
(except for 30 min after treatment). The analgesic effect of morphine
was evident within 30 min following intraperitoneal administration.
The change in latency time reached its peak at 60 min (86.0%). Later it
was gradually decreased. Likewise, at 40 mg/kg NAE also showed
analgesic activity at 30 min (78%), and it gradually reduced at 75 min
(37.2%). Concerning the change in latency time value, the 40 mg/kg
NAE demonstrated a stronger analgesic effect than aspirin at all-time
points.
3.5. Free radical scavenging effect
As shown in Fig. 2C, the IC50 values indicate higher antioxidant ac­
tivity. Furthermore, NAE had greater IC50 value (10.96 ± 0.40 μg/mL)
than that of ascorbic acid (6.11 ± 0.44 μg/mL).
3.6. Oxidative burst assay
The NAE (1 mg) was subjected to oxidative burst anti-inflammatory
activity, and interestingly, it showed significant inhibitory activity on
Reactive Oxygen Species (ROS) with 10.9 ± 0.2, IC50 ±SD value.
Ibuprofen (IC50 = 11.2 ± 1.9) was used as standard drug (Fig. 2D).
3.7. Gas chromatography-mass spectrometry analysis
As shown in Fig. 3A, the GC-MS chromatogram represents total 30
peaks. The most abundant compound was peak 19, corresponding to
methandriol (19a), or 12α-hydroxy-5α-pregnane (19b). However, the
chromatogram was further divided into parts 1 and 2. Peaks 1–18
(Fig. 3B) correspond to compounds 1–18, respectively. While peaks
20–30 (Fig. 3C) represent compounds 20–30, respectively.
The results of the chemical profile analysis of NAE are presented in
Table 4. The table contains the data on retention time (min), name of
compound, molecular formula, molecular weight, and concentration i.
e., peak area (%) for every individual compound identified through GC-
I. Ali et al. Journal of Ethnopharmacology 279 (2021) 114402

Table 4
GC-MS spectral analysis of ethanolic crude extract (NAE) of N. adenophyta
Peak RT (min) Name of compound Molecular Molecular Peak area
No. formula weight (%)

1 11.6428 Kaur-16-ene, (8β,13β)- C20H32 272 0.44

2 12.5911 Hexadecanoic acid, 2-methyl-, methyl ester C18H36O2 284 0.20
3 12.7348 1H-Naphtho[2,1-b]pyran, 3-ethenyldodecahydro-3,4a,7,7,10a-pentamethyl-, [3R- C20H34O 290 1.38
(3α,4aβ,6aα,10aβ,10bα)]-
4 13.1180 1H-Naphtho[2,1-b]pyran, 3-ethenyldodecahydro-3,4a,7,7,10a-pentamethyl-, [3S- C20H34O 290 1.10
(3α,4aα,6aβ,10aα,10bβ)]-
5 13.7348 2,6,10,14-Hexadecatetraen-1-ol, 3,7,11,15-tetramethyl-, acetate, (E,E,E)- C22H36O2 332 0.20
6 14.0831 17-Methylandrosta-5,7,9(11)-trien-17-ol acetate C22H30O2 326 0.35
7 14.6786 (7a-Isopropenyl-4,5-dimethyloctahydroinden-4-yl)methanol C15H26O 222 1.51
8 14.8508 Phenol, 2-(1,1-dimethylethyl)-4-(1-methyl-1-phenylethyl)- C19H24O 268 0.09
9 15.6463 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- C18H30O2 278 0.12
10 15.8884 cis-p-Mentha-2,8-dien-1-ol C10H16O 152 0.35
11 16.2386 1-Naphthalenepropanol, α-ethenyldecahydro-α,5,5,8a-tetramethyl-2-methylene-, [1S-[1α C20H34O 290 2.48
(S*),4aβ,8aα]]-
12 16.3492 5-(7a-Isopropenyl-4,5-dimethyl-octahydroinden-4-yl)-3-methyl-pent-2-en-1-ol C20H34O 290 0.77
13 16.7111 1-Phenanthrenecarboxylic acid, 7-ethenyl-1,2,3,4,4a,4b,5,6,7,9,10,10a-dodecahydro-4a,7- C19H28O2 288 0.40
dimethyl-, [1R-(1α,4aβ
14 18.0251 9(11)-Dehydrotestosterone C19H26O2 286 0.89
15 18.1597 5-(1-Isopropenyl-4,5-dimethylbicyclo[4.3.0]nonan-5-yl)-3-methyl-2-pentenol acetate C22H36O2 332 0.35
16 18.5267 Spiro[5.5]undec-2-ene, 3,7,7-trimethyl-11-methylene-, (− )- C15H24 204 0.75
17 19.1190 Retinal, 9-cis- C20H28O 284 0.61
18 19.7572 3-Buten-1-one, 4-[2,6,6-trimethyl-1(or 2)-cyclohexen-1-yl]- C13H20O 192 9.77
19 20.4621 a. Methandriol C20H32O2 304 65.39
b. 12α-Hydroxy-5α-pregnane C21H36O 304
20 21.055 1H-Naphtho[2,1-b]pyran, 3-ethyldodecahydro-3,4a,7,7,10a-pentamethyl-, [3S- C20H36O 292 0.57
(3α,4aβ,6aα,10aβ,10bα)]-
21 21.1968 8β-Podocarp-12-ene-14-carboxylic acid, 8,13-dimethyl-, methyl ester C21H34O2 318 2.47
22 21.4156 1-Penten-3-one, 4-methyl-1-[2,6,6-trimethyl-2-cyclohexen-1-yl]- C15H24O 220 0.20
23 22.1076 5-(7a-Isopropenyl-4,5-dimethyl-octahydroinden-4-yl)-3-methyl-pent-2-enal C20H32O 288 1.06
24 22.4901 Podocarpa-8,11,13-triene-7β,13-diol, 14-isopropyl- C20H30O2 302 1.21
25 23.1296 9(1H)-Phenanthrenone, 2,3,4,4a,10,10a-hexahydro-6-hydroxy-1,1,4a-trimethyl-7-(1-methyl­ C20H28O2 300 0.28
ethyl)-, (4aS-trans)-
26 24.2274 2(1H)-Phenanthrenone, 3,4,4a,9,10,10a-hexahydro-6-hydroxy-1,1,4a-trimethyl-7-(1-methyl­ C20H28O2 300 0.02
ethyl)-, (4aS-trans)-
27 24.6585 Cholestane-3,5-diol, (3β,5α)- C27H48O2 404 0.03
28 25.0715 13-Docosenamide, (Z)- C22H43NO 337 0.59
29 30.0220 Vitamin E C29H50O2 430 0.31
30 33.3652 Stigmasterol, 22,23-dihydro- C29H50O 414 6.10

MS analysis. The present study revealed 30 compounds in ethanol crude
extract (NAE) of N. adenophyta. The structures of the compounds are
shown in Fig. 4.
The kaurene, naphthopyran, phenanthrenone type compounds are
shown in Fig. 4A, while Fig. 4B represents long chain methyl esters,
acids and amides. Androstatriene, dehydrotestosterone, pregnane, and
sterol type compounds are shown in Fig. 4C, and octahydro indene type
skeletons are shown in Fig. 4D. Other miscellaneous structures are
shown in Fig. 4E. However, the major compounds found in NAE were
1H-naphtho[2,1-b]pyran, 3-ethenyldodecahydro-3,4a,7,7,10a-pentam­
ethyl-, [3R-(3α,4aβ,6aα,10aβ,10bα)]- (3), 1H-naphtho[2,1-b]pyran, 3-
ethenyldodecahydro-3,4a,7,7,10a-pentamethyl-, [3S-(3α,4aα,6aβ,10aα,
10bβ)]- (4), (7a-isopropenyl-4,5-dimethyloctahydroinden-4-yl)meth­
anol (7), 1-naphthalenepropanol, α-ethenyldecahydro-α,5,5,8a-tetra­
methyl-2-methylene-, [1S-[1α(S*),4aβ,8aα]]- (11), 3-buten-1-one, 4-
[2,6,6-trimethyl-1(or 2)-cyclohexen-1-yl]- (18), podocarpa-8,11,13-
triene-7β,13-diol, 14-isopropyl- (24), 8β-podocarp-12-ene-14-carbox­
ylic acid, 8,13-dimethyl-, methyl ester (21), and stigmasterol, 22,23-
dihydro- (30) etc. However, the most abundant compound (65.39%)
was assumed to be related to methandriol (19a), or 12α-hydroxy-5α-
pregnane (19b). The fragmentation pattern was not 100% matched,
hence it was predicted that this compound (peak 19) might be respon­
sible for the pain management, and the structure of this component
might be, to some extent, related to 19a or 19b.
3.8. Liquid chromatography-mass spectrometry analysis
Fig. 5A represents the LC-MS chromatogram in positive ion mode
while Fig. 5B shows the LC-MS chromatogram of NAE in negative ion
mode. A total of 21 compounds were detected based on their exact
masses, mass error, and mSigma values. Maximum 4 compounds were
identified in the positive ionization mode and 17 were identified in the
negative ionization mode while 12 were identified in both modes.
Compound identification was based on the preparation of an in-
house library of compounds previously known to be isolated from the
genus Nepeta. The list of compounds was prepared after an extensive
search using the Dictionary of Natural Products on DVD (DNP ver. 26.2)
and literature sources describing phytochemical studies on the genus.
All obtained LC-MS data were screened against the list of compunds of
built library using TargetAnalysis for the generation of a list of candidate
natural products. The candidate compounds were then filtered using
their mass error and mSigma values. All natural products were identified
based on a comparison of acquired data with the reported data. The
NIST MS Search (ver 2.2) software was used for indentification. Two
main MS and MS/MS libraries MassBank and MassBank of North
America (MoNA) were used for this purpose. The list of compounds
identified in N. adenophyta is shown in Table 5.
It was observed that most analytes, under the positive ionization
mode, were observed as protonated molecules and under negative
ionization as deprotonated molecules. Extracted ion chromatograms
(shown in Supplementary data) and mass spectra of major peaks showed
the presence of total 21 compounds namely nepetaracemoside A (1), 8
(14)-abietene-7,18-diol (2), nepetacilicioside (3), caffeic acid (4), the­
anine (5), nepetariaside (6), 3-(3,4-dihydroxyphenyl)-2-hydrox­
ypropanoic acid (7), nepetalactone; (5R,8R,9R)-form (8), kaemferol (9),
naringenin (10), quercetrin (11), p-mentha-3,6-diene-2,5-dione (12),

6
I. Ali et al.
Fig. 4. (A) Structures of kaurene, naphthopyran, phenanthrenone type compounds, (B) Structures of long chain methyl esters, acids and amides, (C) Structures of
androstatriene, dehydrotestosterone, pregnane, and sterol type compounds, (D) Structures of octahydro indene type skeletons, (E) Structures of miscella­
neous skeletons.
nepetalactone; (5R,8S,9R)-form (13), quercetin (14), dihy­
dronepetalactone (15), nepetalactone; (5R,8R,9S)-form (16), 15,16-
epoxy-3-hydroxy-13(16),14-clerodadien-18,6-olide (17), apigenin
(18), 15,16-epoxy-3,6-dihydroxy-13(16),14-clerodadien-18-al (19),
7,14-dihydroxy-8,15-isopimaradien-18-oic acid (20), and 8,15-isopi­
maradiene-7,18-diol (21). The structures of the nepetalactones are
shown in Fig. 6A. The dodecahydro-phenanthren moieties, diterpenes,
are presented in Fig. 6B. While Fig. 6C shows flavonoids identified in
NAE. And the other miscellaneous skeletons are presented in Fig. 6D.
4. Discussion
In previous literature, various Nepeta species e.g., N. cataria,
N. clarkei, N. atlantica, N. tuberosa, N. parmiriensis, N. pogonosperma have
been reported to exhibit analgesic or anti-inflammatory activities
(Suntar et al., 2018). Natural products like flavonoids, terpenoids, and
phenolic compounds possess analgesic and anti-inflammatory effects
(Bachhav et al., 2009; Upadhyay, 2010). In the current study, the
analgesic effect of NAE was investigated. NAE elicited potent analgesic
effects, both centrally and peripherally, using different pain models. The
pain models used in this study were selected such that both centrally and
peripherally mediated outcomes were measurable (D’amour and Smith,
1941; Qnais et al., 2017). In the writhing test, acetic acid injection ac­
tivates the synthesis of prostaglandins PGE2 (Bentley et al., 1981).
7
Journal of Ethnopharmacology 279 (2021) 114402
Aspirin reduces prostaglandin levels, decreasing the nociceptors’
sensitivity to pain-inducing agents (Chen et al., 1995; Subedi et al.,
2016). NAE produced a significant dose-dependent decrease in the
number of acetic acid-induced writhes. Moreover, this observation
points out that NAE possesses peripherally mediated antinociceptive
property that may work via reducing the level of prostaglandin synthesis
or other inflammatory mediators. Nepeta species are widely used in folk
medicine for antioxidant and radical scavenging and anti-inflammatory
properties (Baser et al., 2000; Yazici et al., 2012). Prescott et al. (2011)
reported that N. cataria directly inhibited calcineurin by caffeoyl phe­
nylethanoid glycosides and act as an immunomodulatory agent by
inhibiting inflammatory cascade (Prescott et al., 2011).
The hotplate test and analgesic-meter test have been used to evaluate
centrally mediated antinociceptive responses, which focus mainly on
changes above the spinal cord level (Hewitt et al., 2009). The
dose-dependent antinociceptive effect seen in the hotplate test indicates
the central antinociceptive effect of NAE as 40 mg/kg extract showed
time-dependent analgesic effect similar to morphine, but results were
insignificant. In the tail-flick model, NAE exhibited significant analgesic
activity by increasing the reaction time of the rats compared to control
(saline-treated rats) at all-time points, except at 60 min. Compared to
control, morphine produced the most significant antinociceptive effect
during all observation times, followed by the extract, while no signifi­
cant analgesic effect was observed for aspirin. This method is also useful
I. Ali et al. Journal of Ethnopharmacology 279 (2021) 114402

Fig. 5. (A) LC-MS (positive ion mode) chromatogram of NAE, (B) LC-MS (negative ion mode) chromatogram of NAE.

Table 5
LC-MS (positive and negative ionization modes) spectral analysis of ethanolic crude extract (NAE) of N. adenophyta
S. No Compound Name Formula Ion Type RT (min) m/z calculated m/z measured Error (ppm) mSigma

1 Nepetaracemoside A C16H24O8 [M+H]+ 1.93 345.1544 345.1523 6.08 40

2 8(14)-Abietene-7,18-diol C20H34O2 [M-H]- 3.11 321.2435 321.2445 3.1 43
3 Nepetacilicioside C16H22O9 [M+H]+ 4.29 359.1337 359.132 4.7 32
4 Caffefic acid C4H8O2 [M-H]- 4.5 179.0385 179.0380 8.9 49
5 Theanine C7H14N2O3 [M+H]+ 10.18 175.1046 175.1036 5.71 35
6 Nepetariaside C16H28O8 [M+H]+ 11.11 349.1857 349.1833 6.8 42
7 3-(3,4-Dihydroxyphenyl)-2-hydroxypropanoic acid C20H20O8 [M-H]- 13.39 387.1085 387.1099 3.61 26
8 Nepetalactone; (5R,8R,9R)-form C10H14O2 [M-H]- 13.48 165.0921 165.0926 1.36 12
9 Kaemferol C15H10O6 [M-H]- 13.5 285.0405 285.0415 3.5 32
10 Naringenin C15H12O5 [M-H]- 13.8 271.0612 271.0621 3.3 21
11 Quercetrin C21H20O11 [M-H]- 14.03 447.0937 447.0969 7.15 34
12 p-Mentha-3,6-diene-2,5-dione C10H12O2 [M-H]- 14.07 163.0765 163.0771 4.29 39
13 Nepetalactone; (5R,8S,9R)-form C10H14O2 [M-H]- 14.28 165.0921 165.0928 4.24 36
14 Quercetin C15H10O7 [M-H]- 14.5 301.0354 301.0367 4.31 45
15 Dihydronepetalactone C10H16O2 [M-H]- 14.57 167.1085 167.1078 4.1 35
16 Nepetalactone; (5R,8R,9S)-form C10H14O2 [M-H]- 15.13 165.0921 165.033 5.1 28
17 15,16-Epoxy-3-hydroxy-13(16),14-clerodadien-18,6-olide C20H28O4 [M-H]- 15.36 331.1915 331.1926 3.32 31
18 Apigenin C15H10O5 [M-H]- 16.01 269.0444 269.0464 7.43 45
19 15,16-Epoxy-3,6-dihydroxy-13(16),14-clerodadien-18-al C20H30O4 [M-H]- 16.59 333.2071 333.2079 2.4 16
20 7,14-Dihydroxy-8,15-isopimaradien-18-oic acid C22H32O4 [M-H]- 18.65 359.2216 359.2228 2.7 10
21 8,15-Isopimaradiene-7,18-diol C20H32O2 [M-H]- 20.43 321.2435 321.2438 0.9 19

in differentiating central opioid-like analgesics from peripheral analge­
sics (Fan et al., 2014). Analgesic drugs which are centrally acting,
elevate the pain threshold of animals towards heat and pressure (Subedi
et al., 2016). Therefore, the analgesic effect of NAE on this pain-state
model indicates that it might be centrally acting. Concerning the MPA
value, the analgesic effects of both NAE and morphine sulfate were
evident within 15 min following intraperitoneal administration. How­
ever, the extract showed short-lived analgesia as the MPA gradually
decreased after 30 min compared to morphine sulfate. The tail-flick la­
tency of the extract at all-time points was lesser than that of the refer­
ence drug morphine sulfate, a slow onset opioid with a long duration of
action (Tomazetti et al., 2005; Turner, 1965). Although there was no

8
I. Ali et al. Journal of Ethnopharmacology 279 (2021) 114402

utilized to identify chemical constituents in extracts or fractions of plant

species (Chander et al., 2016; Sardar et al., 2017; Zeb et al., 2017) and it
is an important technique for the identification of active principles. GC
can separate volatile, and semi-volatile compounds with great resolu­
tion, and MS can identify the components individually (Hites, 1997).
The gas chromatography-mass spectrometry technique has especially
been used to determine essential oil components (Marriott et al., 2001).
However, this is also a good technique for identifying plant metabolites
(Lisec et al., 2006; Stein, 1999). In the present study, several compounds
have been sorted out through GC-MS analysis. This may help future
researchers to work on the phytochemical isolation of the important
moieties present in N. adenophyta Hedge. Especially the most abundant
compound (peak 19) that is assumed to be methandriol (19a), or
12α-hydroxy-5α-pregnane (19b) might be responsible for the pain
management properties. Similarly, the liquid chromatography-mass
spectrometry revealed the presence of nepetalactones, flavonoids and
terpenes etc. Future studies might be focused on these important
chemical constituents.

5. Conclusions

NAE exhibited peripherally mediated antinociceptive property that

may work via reducing the level of prostaglandin synthesis or other in­
flammatory mediators. The extract showed short-lived analgesia as the
maximum possible analgesia gradually decreased after 30 min
compared to morphine sulfate. The plant extract also showed inhibitory
activity against ROS in anti-inflammatory activity. In addition, NAE was
also employed for gas chromatography-mass spectrometry, and liquid
chromatography-mass spectrometry analyses. Total 30 compounds
including the most abundant methandriol or 12α-Hydroxy-5α-pregnane
followed by 3-buten-1-one, 4-[2,6,6-trimethyl-1(or 2)-cyclohexen-1-yl]-
; stigmasterol, 22,23-dihydro-; 1-naphthalenepropanol, α-ethenyldeca­
hydro-α,5,5,8a-tetramethyl-2-methylene-, [1S-[1α(S*),4aβ,8aα]]-; 8β-
podocarp-12-ene-14-carboxylic acid, 8,13-dimethyl-, methyl ester; and
(7a-isopropenyl-4,5-dimethyloctahydroinden-4-yl)methanol, etc. were
reported in GC-MS from the ethanol crude extract of the plant species.
While total 21 compounds were identified in LC-MS analysis. Nepeta­
Fig. 6. (A) Structures of nepetalactones, (B) Structures of diterpenes, (C)
Structures of flavonoids, (D) Structures of miscellaneous skeletons. lactones, terpenes and flavonoids were the major compounds identified
by LC-MS. N. adenophyta has exhibited the most potent analgesic and
anti-inflammatory properties, which can be attributed to the presence of
significant analgesic effect between the reaction time of NAE and
nepetalactones, terpenes, flanovnoids, polyphenolic compounds and
aspirin, NAE exhibited a non-significant trend of higher reaction time
naphthalene derivatives. Thus, the ethanolic extract of N. adenophyta
compared to aspirin. Both treatments produced comparable reaction
showed the most potent analgesic, and anti-inflammatory effects.
times, suggesting that the NAE could be a better natural alternative for
mild pain relief.
CRediT authorship contribution statement
Previously certain active principles from Nepeta species have been
reported to possess analgesic properties that support our present study
Iftikhar Ali: Conceptualization, Supervision, Funding acquisition,
(Aydin et al., 1998, 1999; Hussain et al., 2012, 2015; Ur Rehman et al.,
Writing – original draft. Muhammad Ali: Investigation. Huma Shareef:
2018). Similarly, the extracts of certain Nepeta species have also been
Investigation, Resources, Formal analysis. Sadaf Naeem: Investigation,
studied for analgesic activity. The dichloromethane and ethyl acetate
Formal analysis. Adeeba Khadim: Formal analysis. Meher Ali: Formal
extracts of N. atlantica and N. tuberosa have shown central analgesic
analysis, Writing – review & editing. Faiza Amber: Writing – review &
activity (Bouidida el et al., 2008). In addition, the extract or the pure
editing. Hidayat Hussain: Supervision, Writing – original draft, Project
compounds of the Nepeta origin have shown anti-inflammatory prop­
administration. Muhammad Ismail: Formal analysis, Writing – review
erties (Al-Taweel et al., 2017; Ali et al., 2012a, b; Hussain et al., 2015;
& editing. Syed Tasadaque A. Shah: Formal analysis, Writing – review
Hussain et al., 2012; Miceli et al., 2005; Ur Rehman et al., 2018) that
& editing. Ali Noor: Resources. Daijie Wang: Resources, Supervision.
support the present results of anti-inflammatory activity of NAE. ROS
production is central to the progression of many inflammatory diseases
Declaration of competing interest
and acts as both a signaling molecule and a mediator of inflammation
(Mittal et al., 2014). Overproduction of ROS produces oxidative stress
The authors declare that they have no known competing financial
and ultimately overproduction of cytokines (Beckman, 1996). In this
interests or personal relationships that could have appeared to influence
study, a decreased production of ROS (IC5010.9±0.2) in oxidative burst
the work reported in this paper.
assay and significantly raised IC50±SD (10.96 ± 0.40 μg/ml) values
compared to ascorbic acid in DPPH assay represents its powerful anti­
Acknowledgments
oxidant effects. Based on these findings, it can be postulated that the
anti-inflammatory effect of NAE is due to its antioxidant effect.
The authors wish to highly acknowledge the Higher Education
The gas chromatography-mass spectrometry technique has been
Commission of Pakistan for the partial financial support through

9
I. Ali et al.
National Support Program for Universities (Project No. NRPU-3466).
The author (I. Ali) is thankful to Qilu University of Technology (China)
for providing the Postdoctoral fellowship.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jep.2021.114402.
References
Al-Taweel, A.M., Raish, M., Perveen, S., Fawzy, G.A., Ahmad, A., Ansari, M.A.,
Mudassar, S., Ganaie, M.A., 2017. Nepeta deflersiana attenuates isoproterenol-
induced myocardial injuries in rats: possible involvement of oxidative stress,
apoptosis, inflammation through nuclear factor (NF)-kappaB downregulation.
Phytomedicine 34, 67–75.
Ali, T., Javan, M., Sonboli, A., Semnanian, S., 2012a. Antinociceptive and anti-
inflammatory activities of the essential oil of Nepeta crispa Willd. in experimental rat
models. Nat. Prod. Res. 26 (16), 1529–1534.
Ali, T., Javan, M., Sonboli, A., Semnanian, S., 2012b. Evaluation of the antinociceptive
and anti-inflammatory effects of essential oil of Nepeta pogonosperma Jamzad et
Assadi in rats. DARU J. Pharm. Sci. 20–48.
Aydin, S., Beis, R., Ozturk, Y., Baser, K.H., 1998. Nepetalactone: a new opioid analgesic
from Nepeta caesarea Boiss. J. Pharm. Pharmacol. 50 (7), 813–817.
Aydin, S., Demir, T., Ozturk, Y., Baser, K.H., 1999. Analgesic activity of Nepeta italica L.
Phytother Res. 13 (1), 20–23.
Bachhav, R.S., Gulecha, V.S., Upasani, C.D., 2009. Analgesic and anti-inflammatory
activity of Argyreia speciosa root. Indian J. Pharmacol. 41 (4), 158–161.
Baser, K., Kirimer, N., Kurkcuoglu, M., Demirci, B., 2000. Essential oils of Nepeta species
growing in Turkey. Chem. Nat. Compd. 36 (4), 356–359.
Beckman, J.S., 1996. Oxidative damage and tyrosine nitration from peroxynitrite. Chem.
Res. Toxicol. 9 (5), 836–844.
Bentley, G.A., Newton, S.H., Starr, J., 1981. Evidence for an action of morphine and the
enkephalins on sensory nerve endings in the mouse peritoneum. Br. J. Pharmacol. 73
(2), 325–332.
Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature
181, 1199.
Bouidida el, H., Alaoui, K., Cherrah, Y., Chammache, M., Il Idrissi, A., 2008. [Analgesic
activity of different nonvolatile extracts of Nepeta atlantica Ball and Nepeta tuberosa
L. ssp. reticulata (Desf.) Maire]. Therapie 63 (4), 333–338.
Chander, M.P., Vinod Kumar, K., Lall, C., Vimal Raj, R., Vijayachari, P., 2016. GC/MS
profiling, in vitro anti-leptospiral and haemolytic activities of Boesenbergia rotunda
(L.) Mansf. used as a medicinal plant by Nicobarese of Andaman and Nicobar Islands.
Nat. Prod. Res. 30 (10), 1190–1192.
Chen, Y.F., Tsai, H.Y., Wu, T.S., 1995. Anti-inflammatory and analgesic activities from
roots of Angelica pubescens. Planta Med. 61 (1), 2–8.
D’amour, F.E., Smith, D.L., 1941. A method for determining loss of pain sensation.
J. Pharmacol. Exp. Therapeut. 72 (1), 74–79.
Fan, S.H., Ali, N.A., Basri, D.F., 2014. Evaluation of analgesic activity of the methanol
extract from the galls of Quercus infectoria (Olivier) in rats. Evid. Based
Complementary Altern. Med. 2014, 976764.
Grognet, J., 1990. Catnip: its uses and effects, past and present. CVJ (Can. Vet. J.) 31 (6),
455–456.
Gulcin, I., 2012. Antioxidant activity of food constituents: an overview. Arch. Toxicol. 86
(3), 345–391.
Gulcin, I., 2020. Antioxidants and antioxidant methods: an updated overview. Arch.
Toxicol. 94 (3), 651–715.
Gülçin, İ., Büyükokuroǧlu, M.E., Oktay, M., Küfrevioǧlu, Ö.İ., 2003. Antioxidant and
analgesic activities of turpentine of Pinus nigra Arn. subsp. pallsiana (Lamb.)
Holmboe. J. Ethnopharmacol. 86 (1), 51–58.
Gülçin, İ., Küfrevioǧlu, Ö.İ., Oktay, M., Büyükokuroǧlu, M.E., 2004. Antioxidant,
antimicrobial, antiulcer and analgesic activities of nettle (Urtica dioica L.).
J. Ethnopharmacol. 90 (2), 205–215.
Hedge, I.C., 1989. Nepeta adenophyta Hedge (Labiatae), a new species from Pakistan.
Pakistan J. Bot. 21 (1), 195–196.
Helfand, S.L., Werkmeister, J., Roder, J.C., 1982. Chemiluminescence response of human
natural killer cells. I. The relationship between target cell binding,
chemiluminescence, and cytolysis. J. Exp. Med. 156 (2), 492–505.
Hewitt, D.J., Hargreaves, R.J., Curtis, S.P., Michelson, D., 2009. Challenges in analgesic
drug development. Pharmacol. Ther. Part C 86 (4), 447–450.
Hites, R.A., 1997. Gas chromatography mass spectrometry.
10
Journal of Ethnopharmacology 279 (2021) 114402
Huong, N., Matsumoto, K., Yamasaki, K., Duc, N., Nham, N., Watanabe, H., 1996. Effects
of Vietnamese ginseng on opioid agonist—and conditioned fear stress-induced
antinociception. Phytomedicine 3 (1), 33–39.
Hussain, J., Rehman, N.U., Al-Harrasi, A., Khan, A.L., Rizvi, T.S., Mohammad, F.V.,
Mehjabeen Ali, L., 2015. In vivo evaluation of analgesic, anti-inflammatory, and
neuropharmacological activities of the chemical constituent from Nepeta clarkei.
Arch Pharm. Res. (Seoul) 38 (6), 1188–1194.
Hussain, J., Ur Rehman, N., Hussain, H., Al-Harrasi, A., Ali, L., Rizvi, T.S., Ahmad, M.,
Mehjabeen, 2012. Analgesic, anti-inflammatory, and CNS depressant activities of
new constituents of Nepeta clarkei. Fitoterapia 83 (3), 593–598.
Koster, R., 1959. Acetic acid for analgesic screening. Fed. Proc. 412.
Lisec, J., Schauer, N., Kopka, J., Willmitzer, L., Fernie, A.R., 2006. Gas chromatography
mass spectrometry–based metabolite profiling in plants. Nat. Protoc. 1 (1), 387.
Lorke, D., 1983. A new approach to practical acute toxicity testing. Arch. Toxicol. 54 (4),
275–287.
Marriott, P.J., Shellie, R., Cornwell, C., 2001. Gas chromatographic technologies for the
analysis of essential oils. J. Chromatogr. A 936 (1), 1–22.
Miceli, N., Taviano, M.F., Giuffrida, D., Trovato, A., Tzakou, O., Galati, E.M., 2005. Anti-
inflammatory activity of extract and fractions from Nepeta sibthorpii Bentham.
J. Ethnopharmacol. 97 (2), 261–266.
Mittal, M., Siddiqui, M.R., Tran, K., Reddy, S.P., Malik, A.B., 2014. Reactive oxygen
species in inflammation and tissue injury. Antioxidants Redox Signal. 20 (7),
1126–1167.
Nia, A.M., Kalantaripour, T.P., Basiri, M., Vafaee, F., Asadi-Shekaari, M., Eslami, A.,
Zadeh, F.D., 2017. Nepeta Dschuparensis Bornm extract moderates COX-2 and IL-
1beta proteins in a rat model of cerebral ischemia. Iran. J. Med. Sci. 42 (2), 179–186.
Noor, A., Khatoon, S., Ahmed, M., 2012. Enumeration of the ethnobotanical uses of some
herbs in Astore valley, Gilgit-Baltistan, Pakistan with particular reference to health
cure purposes. FUUAST J. Biol. 2 (2), 31.
Prescott, T.A., Veitch, N.C., Simmonds, M.S., 2011. Direct inhibition of calcineurin by
caffeoyl phenylethanoid glycosides from Teucrium chamaedrys and Nepeta cataria.
J. Ethnopharmacol. 137 (3), 1306–1310.
Qnais, E., Bseiso, Y., Wedyan, M., Alkhateeb, H., 2017. Evaluation of analgesic activity of
the methanol extract from the leaves of Arum palaestinum in mice and rats. Biomed.
Pharmacol. J. 10 (3), 1159–1166.
Saeed, M., Gul, F., Zakiullah, Z., Gilani, S.N., Rehman, Y.U., Khan, I., Muhammad, N.,
Khan, H., 2016. Antinociceptive and anti-inflammatory properties of methanol fruit
extract of Quercus incana in rat and mice models. Trop. J. Pharm. Res. 15 (8),
1691–1696.
Sardar, A.A., Perveen, A., Khan, Z.U., Farid, S., Khan, I., 2017. Phytochemical screening,
GC-MS analysis and in vitro antioxidant activity of pollen of Centella asiatica (Linn)
urban a traditional medicinal plant. Pak. J. Pharm. Sci. 30 (6), 2239–2245.
Sharma, A., Cooper, R., Bhardwaj, G., Cannoo, D.S., 2021. The genus Nepeta: traditional
uses, phytochemicals and pharmacological properties. J. Ethnopharmacol. 268,
113679.
Stein, S.E., 1999. An integrated method for spectrum extraction and compound
identification from gas chromatography/mass spectrometry data. J. Am. Soc. Mass
Spectrom. 10 (8), 770–781.
Subedi, N.K., Rahman, S.M., Akbar, M.A., 2016. Analgesic and antipyretic activities of
methanol extract and its fraction from the root of Schoenoplectus grossus. Evid. Based
Complementary Altern. Med. 2016, 3820704.
Suntar, I., Nabavi, S.M., Barreca, D., Fischer, N., Efferth, T., 2018. Pharmacological and
chemical features of Nepeta L. genus: its importance as a therapeutic agent.
Phytother Res. 32 (2), 185–198.
Taslimi, P., Gulçin, İ., 2018. Antioxidant and anticholinergic properties of olivetol.
J. Food Biochem. 42 (3), e12516.
Tomazetti, J., Avila, D.S., Ferreira, A.P., Martins, J.S., Souza, F.R., Royer, C., Rubin, M.
A., Oliveira, M.R., Bonacorso, H.G., Martins, M.A., Zanatta, N., Mello, C.F., 2005.
Baker yeast-induced fever in young rats: characterization and validation of an
animal model for antipyretics screening. J. Neurosci. Methods 147 (1), 29–35.
Turner, R.A., 1965. Analgesics.
Upadhyay, K.A., 2010. Anti-inflammatory evaluation of ethanolic extract of leaves of
Holoptelea integrifolia, Planch. Ann. Biol. Res. 1 (2), 185–195.
Ur Rehman, T., Khan, A.U., Abbas, A., Hussain, J., Khan, F.U., Stieglitz, K., Ali, S., 2018.
Investigation of nepetolide as a novel lead compound: antioxidant, antimicrobial,
cytotoxic, anticancer, anti-inflammatory, analgesic activities and molecular docking
evaluation. Saudi Pharm. J. 26 (3), 422–429.
Yazici, S.O., Ozmen, I., Celikoglu, U., Ozcelik, H., Genc, H., 2012. In vitro antioxidant
activities of extracts from some Nepeta species. Int. J. Health. Nutr. 3 (1), 8–12.
Zeb, A., Ullah, F., Ayaz, M., Ahmad, S., Sadiq, A., 2017. Demonstration of biological
activities of extracts from Isodon rugosus Wall. Ex Benth: separation and
identification of bioactive phytoconstituents by GC-MS analysis in the ethyl acetate
extract. BMC Complement. Altern. Med. 17 (1), 284.