Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: https://www.tandfonline.com/loi/iphb20

Pharmacological and toxicological evaluation of

Urtica dioica

Sabzar Ahmad Dar, Farooq Ahmad Ganai, Abdul Rehman Yousuf, Masood-ul-
Hassan Balkhi, Towseef Mohsin Bhat & Poonam Sharma

To cite this article: Sabzar Ahmad Dar, Farooq Ahmad Ganai, Abdul Rehman Yousuf,
Masood-ul-Hassan Balkhi, Towseef Mohsin Bhat & Poonam Sharma (2013) Pharmacological
and toxicological evaluation of Urtica�dioica, Pharmaceutical Biology, 51:2, 170-180, DOI:
10.3109/13880209.2012.715172

To link to this article: https://doi.org/10.3109/13880209.2012.715172

Published online: 05 Oct 2012.

Submit your article to this journal

Article views: 3687

View related articles

Citing articles: 17 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=iphb20
Pharmaceutical Biology, 2013; 51(2): 170–180
© 2013 Informa Healthcare USA, Inc.
ISSN 1388-0209 print/ISSN 1744-5116 online
DOI: 10.3109/13880209.2012.715172

Research article

Pharmacological and toxicological evaluation of Urtica dioica

Sabzar Ahmad Dar1, Farooq Ahmad Ganai1, Abdul Rehman Yousuf1, Masood-ul-Hassan Balkhi2,
Towseef Mohsin Bhat3, and Poonam Sharma4
1
Limnology and Fisheries Laboratory, Centre of Research for Development (CORD), University of Kashmir,
Srinagar, J & K, India, 2Faculty of Fisheries, sher-e-Kashmir University of Agricultural Sciences and Technology
of Kashmir (SKUAST-K), J & K, India, 3Department of Botany, Mutation Breeding Laboratory, Aligarh Muslim
University, Uttar Pradesh, India, and 4Department of Zoology, Bundelkhand University, Jhansi, Uttar Pradesh, India

Abstract
Context: Medicinal plants are a largely unexplored source of drug repository. Urtica dioica L. (Urticaceae) is used in
traditional medicine to treat diverse conditions.
Objective: The present study describes the antidiabetic, antiinflammatory, antibacterial activity, and toxicological
studies of Urtica dioica.
Materials and methods: U. dioica leaves were subjected to solvent extraction with hexane, chloroform, ethyl acetate,
methanol, and aqueous, respectively, and screened for antidiabetic (300 mg/kg bw by glucose tolerance test;
GTT), antiinflammatory (200 mg/kg bw by rat paw edema assay) and antibacterial activities [by disc-diffusion and
minimum inhibitory concentration (MIC) assays]. Toxicological studies were carried on Artemia salina and Wistar rats;
phytochemical analyses were carried out, using chromatographic and spectroscopic techniques.
Results: The aqueous extract of U. dioica (AEUD) significantly (p < 0.001; 67.92%) reduced the blood glucose level
during GTT in Wistar rats with an effective dose of 300 mg/kg bw in dose-dependent studies. High-performance liquid
chromatography with photodiode-array detection (HPLC-DAD) analysis showed the presence of hydroxycinnamic
acid derivatives and flavonoids in AEUD. Hexane Fraction-2 (HF2) exhibited both antiinflammatory activity (48.83%
after 3 h), comparable to that of indomethacin (53.48%), and potent antibacterial activity with MIC values ranging
from 31.25–250 µg/mL against all the tested strains. Gas chromatography–mass spectrometry (GC–MS) analysis
showed fatty acid esters and terpenes as the major constituents of HF2. Toxicity tests showed higher safety margin of
all the solvent extracts with LC50 > 1000 μg/mL each on A. salina.
Discussion and conclusion: Our results showed that the U. dioica leaves are an interesting source of bioactive
compounds, justifying their use in folk medicine, to treat various diseases.
Keywords:  Antidiabetic, antiinflammatory, antibacterial, Artemia salina, toxicity

Introduction prescribed widely even when their biologically active

Medicinal plants have been a repository of a wide variety
of biologically active compounds for many centuries but
are still largely unexplored (Singh et al., 2012). According
to the WHO, >80% of the world’s population relies on
traditional medicine for their primary healthcare needs
(Anon., 1993). It is estimated that today, plant materials
are present in, or have provided the models for 50% of
the Western drugs (Baker et al., 1995). Because of their
perceived effectiveness, minimal side effects in clini-
cal experience and relatively low cost herbal drugs are
compounds are unknown (Valiathan, 1998).
Diabetes is a complex and multifarious group of
disorders characterized by hyperglycemia and reaching
epidemic proportions in the present century (Chen
et al., 2005). Approximately 5% of the world’s population
suffers from diabetes. Independent forecasters have
suggested that the global prevalence of the disease will
increase from 150 million in 2000 to 300 million by 2025
(Chakrabarti & Rajagopalan, 2002). Today infectious
and inflammatory diseases are also the main cause of

Address for Correspondence:  Sabzar Ahmad Dar, Research Scholar, Limnology and Fisheries Laboratory, Centre of Research for
Development (CORD), University of Kashmir, Srinagar, J & K 190006, India. Tel: +91 097 9797 0976. E-mail: sabzar.cord@gmail.com.
(Received 14 March 2012; revised 04 July 2012; accepted 11 July 2012)

170

death in developing countries and worldwide they hold
the second position after heart diseases. The problem
is worsened by antibiotic resistance coupled with the
emergence of new pathogens with the potential for
rapid global spread (Walsh, 2003), boosting the search
for new bioactive agents. No doubt, there are so many
drugs available today against all these diseases, but
unfortunately all of them have significant negative side
effects, reducing their use in certain segments of the
population (Jüni et al., 2005; Pathak et al., 2005).
Urtica dioica L. (Urticaceae), commonly known as
stinging nettle or common nettle or “Soii” in Kashmiri
language, is widely distributed between the geographical
coordinates 34° 02′ N-75° 20′ E at an altitude of 2400 m
(above sea level) with an annual rainfall of 1075.5 mm.
The whole plant is used in folk medicine to treat allergies,
kidney stones, burns, anemia, rashes, internal bleeding,
diabetes, etc. (Singh et al., 2012). However, only a few of
these pharmacological activities have been experimen-
tally proved (Lourdes et al., 2008). Therefore, the aim of
our study was to evaluate the antidiabetic, antiinflamma-
tory and antibacterial activity of leaf extracts of U. dioica
along with their toxicological evaluation.
Materials and methods
Plant material
The fresh leaves of U. dioica were collected from the
undisturbed fields of Anantnag (Aishmuqam) district of
Kashmir valley, India in the month of September, 2010.
The plant material was authenticated by the Centre of
Biodiversity & Taxonomy (CBT), Department of Botany,
University of Kashmir and a voucher specimen (KASH
28100) has been deposited.
Extract preparation
The leaves of U. dioica were shade-dried at 25°C for 7
days. After being macerated to fine powder, 1 kg of leaves
were extracted successively with hexane, chloroform,
ethyl acetate and methanol for 16 h using Soxhlet appara-
tus (Singh et al., 2012). The extracts were filtered through a
Buchner funnel using Whatman No. 1 filter paper, and all
the extracts were concentrated to dryness under vacuum
using a Heidolph rotary evaporator, yielding hexane, chlo-
roform, ethyl acetate and methanol crude extracts of 68,
73, 44, and 79 g, respectively. However, 200 g of the residue
after methanol extract was soaked overnight in 400 mL of
distilled water at room temperature with constant stirring.
The next morning the extract was filtered over muslin cloth
and the filtrate was centrifuged at 5000 rpm for 10 min at
room temperature. The supernatant was further lyophi-
lized (Mac-Flow, India) to complete dryness to obtain
powder (11 g). All the extracts were stored at 4°C in air tight
glass bottles before use.
In vivo studies
Animals
Wistar rats, without any sexual discrimination, weighing
150–200 g were used in the experiments. The animals had
© 2013 Informa Healthcare USA, Inc.
Activity of Urtica dioica 171
free access to a standard commercial diet and water ad
libitum and were kept in the animal house at an ambient
temperature of 25°C and 45–55% relative humidity, with
a 12 h light/dark cycle. Animal experimental protocols
were in accordance with the recommendations of an
institutional animal ethical committee concerning the
care and use of laboratory animals.
Hypoglycemic activity by glucose tolerance test
Rats were divided into five groups. Twelve rats were used
in each group in which six rats serve as the control for
each group.
Group (I), received hexane extract (300 mg/kg bw).
Group (II), received chloroform extract (300 mg/kg bw).
Group (III), received ethyl acetate extract (300 mg/kg bw).
Group (IV), received methanol extract (300 mg/kg bw).
Group (V), received aqueous extract (300 mg/kg bw).
Animals were fasted overnight and fasting blood
glucose was checked with a glucometer (Murali et al.,
2007). All the extracts at a dose of 300 mg/kg bw were
administered by gavaging and after 90 min the glucose
level in blood was estimated. This was taken as 0 h value
for GTT. Following this, glucose (2 g/kg bw) was admin-
istered as an aqueous solution and blood glucose level
was estimated at intervals of 1, 2, and 3 h, respectively.
Improvement in glucose tolerance was assessed by com-
paring reduction in peak blood glucose levels seen at
1, 2, and 3 h, values. Fall in blood glucose level at peak
1 h value was calculated with the formula given below
(Murali et al., 2007):
Percent fall = (A − B)/A × 100
Where,
A=D  ifference between the 1 h and 90 min value in
control group of animals.
B = Difference between the 1 h and 90 min value in
test group of animals.
Experimental schedule
Hypoglycemic activity was completed in two phases
(Murali et al., 2007). Phase I: The best hypoglycemic
extract was selected by orally administering 300 mg/
kg bw dose of each extract, i.e., hexane, chloroform,
ethyl acetate, methanol, and aqueous in 12 h fasted nor-
moglycemic rats. Phase II: The most active hypoglycemic
extract was further studied for dose-dependent (100, 200,
300 and 400 mg/kg bw) effects in streptozotocin-induced
diabetic rats (35 mg/kg ip), prepared freshly by dissolving
in 0.05 M of citrate buffer with pH 4.5. Aqueous dimethyl
sulphoxide (DMSO) (50%) was used as vehicle for hex-
ane, chloroform, ethyl acetate, and methanol extracts,
while the aqueous extract was dissolved in distilled water.
Antiinflammatory assay
The antiinflammatory activity of all the solvent extracts
and hexane subfractions were screened by the rat paw
edema antiinflammatory assay (Winter et al., 1962). Rats of
172  S. A. Dar et al.
both sexes were divided into seven groups of six rats each.
Group I received normal saline and served as control.
Group II, III, IV, V, and VI were orally gavaged with 200 mg/
kg bw of the hexane, chloroform, ethyl acetate, methanol,
and aqueous extracts of U. dioica, respectively. Group
(VII), received the standard drug indomethacin (10 mg/kg
bw). Paw edemas were induced by subcutaneous injection
of 100 µL of 1% carrageenan solution (Sigma; w/v solution
in saline, 0.9% NaCl) in the plantar aponeurosis of the
right hind paw. One hour after carrageenan injection, ede-
mas were measured first at 0 and then at 1, 2, 3, and 24 h
after administration of drugs by a volume displacement
method using a digital plethysmometer (Ugo Basile, Italy).
The percentage inhibition of edema compared with that of
the control was taken as antiinflammatory activity (Perez,
1996). The percentage inhibition of edema was calculated
by the formula (Ahmed et al., 1993; Perez, 1996):
Percentage inhibition of edema = (A − B)/A × 100
where A represents the paw volume of the control group
at corresponding time and B represents the paw volume
of the test group at the same time.
Toxicity test with Artemia salina
The extract toxicity tests of U. dioica were held accord-
ing to the standard method (Meyer et al., 1982) using
Artemia salina. All the extracts and fractions of U. dioica
were dissolved in 50% aqueous DMSO solution and sub-
sequently in simulated seawater. The concentration of
the extracts and fractions ranged from 1000 to 2000 μg/
mL and they were placed in a plate containing between 6
and 12 naupli, followed by further incubation in a water
bath (20–25°C) for 24 h. A solution of potassium dichro-
mate at concentrations of 400, 600, and 800 μg/mL was
used as a positive control and the solvent used to dissolve
the samples was taken as negative control. LC50 of frac-
tions and extracts above 250 μg/mL were considered as
low toxic, LC50 ranging from 80 to 250 μg/mL were con-
sidered moderately toxic and LC50 of less than 80 μg/mL
were considered toxic (Parra et al., 2001). For the final
calculation of LC50, Probit analysis was used.
Acute toxicity test in Wistar rats
Toxicity and gross behavioral studies were carried out
in Wistar rats, comprising of both sexes, by gavaging the
active extracts at different doses (250, 500, 1000, and
2000 mg/kg bw). After the extracts were administered,
the animals were kept under observation for 24 h to note
behavioral changes (Turner, 1965).
In vitro studies
Microbiologic material
All the solvent extracts were screened against a
total of seven bacterial strains. The test strains were
Staphylococcus aureus (ATCC 25923), Shigella flex-
neri (ATCC 29903), Salmonella typhi (ATCC 19430),
Pseudomonas aeruginosa (ATCC 27853), Klebsiella-
pneumoniae (ATCC 15380), Escherichia coli (ATCC
 Pharmaceutical Biology
25922) and Enterococcus faecalis (ATCC 29212) obtained
from the National Institute of Immunology, New Delhi,
India.
Antibacterial activity
The agar diffusion assay was performed according to
European Pharmacopoe. One loopful of each test organ-
ism was suspended in 3 mL of 0.9% NaCl solution sepa-
rately. Nutrient agar (Difco™ Tryptic Soy Agar, Becton
Dickinson and Company, USA) was inoculated with this
suspension of the respective organism and poured into a
sterile Petri dish. Sterile filter paper discs with 6 mm diam-
eter impregnated with 2000 µg (dissolved in 8 µL of 50%
aqueous DMSO) of each extract, were transferred onto
these prepared Petri dishes as per standard procedures
(Chandrasekaran & Venkatesalu, 2004). Gentamycin (30
μg/disc, Merck, USA) was used as a positive control and
the vehicle of each extract served as a negative control. A
pre-diffusion for 3 h was guaranteed. After pre-diffusion
and incubation, the Petri dishes were sprayed with a
coloring solution (p-iodo nitrotetrazolium violet, 5% in
50% aqueous ethanol). Living bacteria on Petri dishes
produce a red colored compound; the inhibition zone
appears colorless (Brantner, 1997). All experiments were
carried out five times and results were recorded by mea-
suring the zones of growth inhibition around the discs
after 18 h incubation at 26°C.
Bioassay-guided isolation of active compound(s)
The active hexane extract of U. dioica (HEUD, 50 g) was
fractionated using silica gel 60 (0.063–0.200 mm) column
chromatography (CC). Solvents were distilled prior to
use. The column was eluted with a solvent gradient of
hexane-ethyl acetate (Et-O-Ac) in 100:0 and 0:100 ratios
to give 30 fractions (each of 250 mL). Thirty fractions
were collected, analyzed by thin layer chromatography
(TLC) on silica gel 60 PF254 (Merck) aluminum sheets
and pooled together due to similarity in TLC profile to
give overall five subfractions: HF1, HF2, HF3, HF4, and
HF5. The subfractions were tested for antibacterial activ-
ity against the pathogenic strains by standard assays
(European Pharmacopoe, 1997).
Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) values
were determined by standard serial broth microdilution
assays (European Pharmacopoe, 1997). Ten serial dilu-
tions of stock, ranging from 1000 to 0.97 µg/mL, were pre-
pared in test tubes. The tubes were inoculated with 100
µL of bacterial strain inoculums at a concentration of 106
cell/mL. Standard antibiotic gentamycin was included
in the assay for comparison. Nutrient broth with the
inoculums only was used as growth control. All experi-
ments were carried out in triplicates. The tubes were
incubated aerobically at 37°C for 12–18 h, after which 50
µL of 0.2 mg/mL 2-(4-iodophenyl)3-(4-nitrophenyl)-5
phenyltetrazolium chloride (INT) solution was added to
each test tube and the tubes were tested for color change

(Johnsen et al., 2002). The concentration at which a
decrease in red color is apparent compared with the next
dilution was taken as the MIC value. Bacterial growth is
indicated by the red color of INT reduced to formazan.
Phytochemical analysis
HPLC–DAD analysis
High-performance liquid chromatography with photo-
diode-array detection (HPLC-DAD) analyses were oper-
ated on a HP 1100 L liquid chromatograph equipped with
a DAD detector, operated by a HP Chemstation (Agilent
Technologies, Palo Alto, CA, USA). Compounds were sep-
arated using a 4.6 × 250 mm Polaris E RP18 (5 μm) column
(Germany) at 27 ± 0.5°C. The eluent was H2O (adjusted to
pH 3.2 with HCOOH/CH3CN). A four-step linear gradient
solvent system was used, starting from 100% H2O to 100%
CH3CN over a 53-min period, at a flow rate of 0.8 mL min−1
(Saracini et al., 2005). The anthocyanins were separated
using a RP-80 C12 column (Phenomenex Synergi Max,
Torrance, CA, USA), 150 × 3 mm, 4 μm (Phenomenex), at
27 ± 0.5°C. The eluent was H2O (adjusted to pH 2.0 with
HCOOH/CH3CN). A four-step linear gradient solvent sys-
tem, at a flow rate of 0.4 mL min-1 for 28 min, was used (Ieri
et al., 2006).
HPLC–MS analysis
High-performance liquid chromatography-mass spec-
trometry (HPLC-MS) analyses were performed using the
same analytical conditions as the HPLC–DAD analysis.
In detail, the HPLC–DAD was interfaced with a HP 1100
MSD API-electrospray (Agilent Technologies) operat-
ing in negative and positive ionization mode under the
following conditions: nitrogen gas temperature 350°C;
nitrogen flow rate 10 L min−1; nebulizer pressure 30 psi;
quadrupole temperature 30°C; capillary voltage 3500 V.
The mass spectrometer was operated at 120 eV of nega-
tive fragmentor for flavonoid and caffeic derivatives and
at 120 eV of positive fragmentor for anthocyanins.
Quantitative analysis
Identification of individual compounds was carried out
using retention times and both UV-Vis MS, and MS/MS
spectra. Quantitation of single compounds was directly
performed by HPLC–DAD using a four-point regres-
sion curve built with the available standards. Curves
with a correlation factor of r2 > 0.998 were considered.
In particular, caffeoyl acid amounts were calculated
at 330 nm using chlorogenic as reference. Quercetin
and isorhamnetin glycosides were calibrated at 350 nm
using isorhamnetin 3-O-rutinoside as reference. Finally,
anthocyanin glycosides were calibrated at 520 nm using
peonidin 3-O-glucoside as reference.
GC–MS analysis
Gas chromatography-mass spectrometry (GC-MS)
analysis was carried out on an Agilent 6890N Network
GC system combined with Agilent 5973 Network Mass
Selective Detector (GC–MS). The capillary column used
© 2013 Informa Healthcare USA, Inc.
Activity of Urtica dioica 173
was an Agilent 19091N-136 (HP Innowax Capillary; 60.0
m × 0.25 mm × 0.25 μm). Helium was used as carrier gas
at a flow rate of 3.3 mL/min with 1 μL injection volume.
Samples were analyzed with the column held initially
at 100°C for 1 min after injection, then increased to
170°C with a 10°C/min heating ramp without hold and
increased to 215°C with 5°C/min heating ramp for 7 min.
Then the final temperature was increased to 240°C with
a 10°C/min heating ramp for 15 min. The injections were
performed in split mode (30:1) at 250°C. Detector and
injector temperatures were 260°C and 250°C, respec-
tively. Pressure was established as 50.0 psi. Run time was
60 min. Temperature and nominal initial flow for flame
ionization detector were set as 250°C and 3.1 mL/min,
respectively. MS parameters were as follows: scan range
(m/z): 35–450 atomic mass units under electron impact
ionization (70 eV). The constituent compounds were
determined by comparing their retention times and mass
weights with those of authentic samples obtained by GC
and as well as the mass spectra from the Wiley and Nist
database.
Statistical analysis
Results were expressed as mean ± SEM. One-way analy-
sis of variance (ANOVA) followed by Dunnett’s t-test was
applied for statistical analysis.
Results
In vivo studies
Hypoglycemic activity
The results of hypoglycemic activity of leaf extracts of U.
dioica in normoglycemic rats are shown in Table 1. It is
clear that only the aqueous extract of U. dioica (AEUD)
produced a significant (p < 0.001) fall of 67.92% between
0 and 1 h during GTT. The methanol extract produced
only a 28.30 and 40% percentage fall at 1 and 2 h, respec-
tively. The ethyl acetate and hexane extracts were least
effective when compared with control values. Since the
aqueous extract was more active than the other solvent
extracts, it was further studied for its dose-dependent
effect in streptozotocin-induced diabetic rats (Table
2). In this case, the highest fall (p < 0.001) of 63.26%
in the peak blood glucose at 1 h during GTT was seen
with 300 mg/kg bw of AEUD when compared with the
untreated control group. Gavaging an extract at a dose
of 200 mg/kg bw also produced a significant fall of
61.22% (p < 0.01) whereas 100, 400 mg/kg bw produced
a fall of 48.97% (p < 0.05) and 56.12% (p < 0.01), respec-
tively. Thus, a dose of 300 mg/kg bw proved to be the
most effective dose.
Antiinflammatory assay
The results of antiinflammatory activity of all the five sol-
vent extracts along the five column eluted subfractions
of HEUD were evaluated (Table 3). There was a gradual
increase in edema paw volume of rats in the control
group. However, in the test groups, hexane extract
174  S. A. Dar et al.

Table 1.  Hypoglycemic activity of leaf extracts of Urtica dioica in Wistar rats.
Extract treated Blood glucose (mg/dL)
Group (n) FBG 0h 1h 2h 3h % fall between 0 h and 1 h
C 77 ± 7.3 75 ± 6.9 128 ± 5.9 107 ± 6.0 120 ± 6.8
Aqueous 67.92 ↓
T 77 ± 6.5 74 ± 6.8 91 ± 6.3*** 94 ± 5.4 82 ± 7.1
C 87 ± 6.3 85 ± 7.2 138 ± 4.8 110 ± 6.7 130 ± 8.6
Methanol 28.30 ↓
T 86 ± 5.8 99 ± 5.6 137 ± 5.5 114 ± 7.5 123 ± 6.1
C 83 ± 4.9 71 ± 5.3 126 ± 6.1 85 ± 9.0 94 ± 5.9
Et-O-Ac 9.09 ↓
T 85 ± 7.3 83 ± 6.0 133 ± 6.8 92 ± 8.3 93 ± 4.8
C 79 ± 6.5 73 ± 4.9 111 ± 4.8 99 ± 8.5 105 ± 7.7
Chloroform 15.78 ↓
T 76 ± 5.4 83 ± 6.1 127 ± 7.0 113 ± 7.2 102 ± 8.1
C 73 ± 6.8 70 ± 6.6 112 ± 6.5 97 ± 5.8 100 ± 6.9
Hexane 11.90 ↑
T 69 ± 5.3 81 ± 7.2 118 ± 6.1 102 ± 6.4 90 ± 5.1
The values represent the mean ± SD.
C, control; Et-O-Ac, ethyl acetate; FBG, fasting blood glucose; n, number of animals per group (6); T, treated.
*p < 0.05 (significant), **p < 0.01 (highly significant), ***p < 0.001 (extremely significant) as compared with control values at the same time.

Table 2.  Effective dose (ED) determination of aqueous extract of Urtica dioica leaves by GTT in streptozotocin-induced diabetic rats.
Blood glucose (mg/dL)
Group (n) FBG 0h 1h 2h 3h % fall between 0 h and 1 h
Group I (Control) 270 ± 8.2 264 ± 7.2 362 ± 5.7 344 ± 7.2 356 ± 6.4
Group II (Glibenclamide) 276 ± 7.2 260 ± 5.3 283 ± 6.1*** 279 ± 5.2 285 ± 6.2 76.53 ↓
Group III (100 mg/kg) 272 ± 7.1 265 ± 4.9 315 ± 6.8* 304 ± 7.4 311 ± 7.4 48.97 ↓
Group IV (200 mg/kg) 286 ± 5.6 279 ± 6.4 317 ± 6.6** 310 ± 6.9 313 ± 6.7 61.22 ↓
Group V (300 mg/kg) 267 ± 6.4 255 ± 6.8 291 ± 7.3*** 287 ± 7.0 290 ± 8.1 63.26 ↓
Group VI (400 mg/kg) 263 ± 6.3 251 ± 6.3 294 ± 5.9** 283 ± 6.5 288 ± 4.9 56.12 ↓
The values represent the mean ± SD.
FBG, fasting blood glucose; GTT, glucose tolerance test; n, Number of animals per group (6).
*p < 0.05, **p < 0.01, ***p < 0.001 as compared with control values at the same time.

(200 mg/kg bw) showed a significant inhibition of 46.51%
(p < 0.01) in the edema paw volume after 3 h of treatment
and was comparable to that of standard indomethacin
(10 mg/kg bw) which showed a significant inhibition of
53.48% (p < 0.01). There was no significant inhibition of
inflammation (p > 0.05) in case of test groups treated
with chloroform, ethyl acetate, methanol, and aqueous
extract of U. dioica. Among the five column eluted frac-
tions of HEUD, HF2 showed a significant inhibition of
48.83% (p < 0.01) after 3 h and 51.28% (p < 0.01) after 24 h
of treatment.
Toxicity test with Artemia salina
The extract and fraction toxicities of U. dioica were evalu-
ated using the A. salina test. The results are shown in
Table 4. All the extracts, viz., hexane, chloroform, ethyl
acetate, methanol, and aqueous of U. dioica along with
the most active subfraction (HF2) showed reduced toxic-
ity (LC50 > 1000 μg/mL).
Acute toxicity test in Wistar rats
In the acute toxicity study, no mortality was observed
during the 24 h period at the tested doses and the ani-
mals showed no stereotypical toxic symptoms such as
convulsion, atoxia, diarrhea or increased diuresis, except
the fourth group (2000 mg/kg bw), which exhibited
symptoms like diarrhea and diuresis.
In vitro studies
Antibacterial activity
All the solvent extracts of U. dioica, namely, hexane, chlo-
roform, ethyl acetate, methanol, and aqueous were tested
for antibacterial activity against seven strains compris-
ing of both Gram positive and Gram negative bacteria,
viz., Staphylococcus aureus, Shigella flexneri, Salmonella
typhi, Pseudomonas aeruginosa, Klebsiella pneumoniae,
Escherichia coli, and Enterococcus faecalis. The antibacte-
rial activity produced by HEUD was comparable with that
of the standard antibiotic gentamycin (Table 5).

 Pharmaceutical Biology
 Activity of Urtica dioica 175

Table 3. Effect of leaf extracts and hexane subfractions of Urtica dioica on carrageenin-induced hind paw edema in Wistar rats.
Time interval (h) and edema volume (mL)
Treatment and dose 0h 1h 2h 3h 24 h
Control 0.20 ± 0.008 0.35 ± 0.006 0.39 ± 0.004 0.43 ± 0.003 0.39 ± 0.003
Hexane (200 mg/kg) 0.20 ± 0.004 0.24 ± 0.009 (31.42)* 0.23 ± 0.011 (41.02)* 0.23 ± 0.007 (46.51)** 0.20 ± 0.008 (48.71)**
Chloroform (200 mg/kg) 0.22 ± 0.005 0.30 ± 0.004 (14.28) 0.35 ± 0.005 (10.25) 0.40 ± 0.009 (6.97) 0.42 ± 0.009 (−7.69)
Ethyl acetate (200 mg/kg) 0.21 ± 0.009 0.31 ± 0.005 (11.42) 0.37 ± 0.005 (5.12) 0.44 ± 0.008 (−2.32) 0.45 ± 0.005 (−15.38)
Methanol (200 mg/kg) 0.21 ± 0.006 0.27 ± 0.008 (22.85) 0.29 ± 0.011 (25.64) 0.32 ± 0.009 (25.58) 0.26 ± 0.010(33.33)*
Aqueous (200 mg/kg) 0.22 ± 0.009 0.30 ± 0.004 (14.28) 0.35 ± 0.005 (10.25) 0.27 ± 0.008 (22.85) 0.29 ± 0.011 (25.64)
Indomethacin (10 mg/kg) 0.17 ± 0.007 0.21 ± 0.009 (40.00)* 0.23 ± 0.011 (41.02)* 0.20 ± 0.010 (53.48)** 0.17 ± 0.007 (56.41)**
Subfractions of HEUD
  HF1 (200 mg/kg) 0.23 ± 0.011 0.25 ± 0.005 (28.57) 0.28 ± 0.007 (28.20) 0.30 ± 0.012 (30.23)* 0.30 ± 0.009 (23.07)
  HF2 (200 mg/kg) 0.19 ± 0.014 0.22 ± 0.008 (37.14)* 0.24 ± 0.009 (38.46)* 0.22 ± 0.010 (48.83)** 0.19 ± 0.011 (51.28)**
  HF3 (200 mg/kg) 0.21 ± 0.011 0.26 ± 0.013 (25.71) 0.31 ± 0.010 (20.51) 0.34 ± 0.008 (20.93) 0.33 ± 0.010 (15.38)
  HF4 (200 mg/kg) 0.18 ± 0.010 0.23 ± 0.009 (34.28)* 0.28 ± 0.007 (28.20) 0.30 ± 0.011 (30.23)* 0.25 ± 0.009 (35.89)*
  HF5 (200 mg/kg) 0.20 ± 0.010 0.24 ± 0.007 (31.42)* 0.27 ± 0.013 (30.76)* 0.29 ± 0.011 (32.55)* 0.24 ± 0.007 (38.46)*
Values of edema are mean ± SEM from six animals in each group, while those in parenthesis represent percent inhibition of edema.
One-Way ANOVA followed by Dunnett’s t-test is applied for statistical analysis.
HEUD, hexane extract of U. dioica.
*p < 0.05; **p < 0.01; ***p < 0.001.

Table 4.  Lethal concentration 50% (LC50) of leaf extracts and Table 5. Antibacterial activity of different solvent extract of
hexane subfractions of Urtica dioica. Urtica dioica leaves.
Component LC50 (μg/mL) Extract treated He Ch Ea Mt Aq C Sd
Hexane extract >1000 Microorganisms DIZ (mm)
CH3Cl exract >1000 Staphylococcus aureus ++ + − − − − +++
EtOAc extract >1000 Shigella flexneri ++ + − − − +++
Methanol extract >1000 Salmonella typhi ++ + − − − − +++
Aqueous >1000 Pseudomonas ++ − − − − − +++
aeruginosa
HF1 909.05 ± 7.5
Klebsiella pneumoniae ++ + − − − − +++
HF2 >1000 Escherichia coli ++ + − − − − +++
HF3 877.15 ± 6.4 Enterococcus faecalis ++ − − − − − +++
HF4 386.07 ± 12.41 The results are represented as inactive (−, inhibition zone
HF5 609.15 ± 6 diameter ≤7 mm), moderate activity (+, inhibition zone diameter
≤10 mm), good activity (++, inhibition zone diameter ≤15 mm),
and very good activity (+++, inhibition zone diameter >15 mm).
Bioassay guided isolation of active compound(s) Aq, aqueous extract; C, control (dimethyl sulphoxide, DMSO);
Ch, chloroform extract; DIZ, diameter inhibition zone; Ea, ethyl
Since HEUD showed good antibacterial activity, it acetate extract; He, hexane extract; Mt, methanol extract; Sd,
was further fractionated using CC into 30 subfractions standard (gentamycin).
of 250 mL each which were pooled together accord-
ing to their TLC profile to give five subfractions: HF1, Phytochemical analysis
HF2, HF3, HF4, and HF5. The results of antibacte- HPLC–DAD analysis
rial activity of the subfractions of HEUD are shown in HPLC–DAD analysis was developed to characterize the
Table 6. The antibacterial activity produced by the sub- compounds in crude AEUD (Figure 1). It was very abun-
fraction HF2 was comparable with that of the standard dant in hydroxycinnamic acid derivatives (main com-
antibiotic gentamycin. pounds being 2-O-caffeoylmalic and chlorogenic acid);
flavonoids (rutin, quercetin p-coumaroyl glucoside, isor-
hamnetin 3-O-rutinoside) and anthocyanins (peonidin
Minimum inhibitory concentration 3-O-rutinoside, rosinidin 3-O-rutinoside) (Table 7).
The MIC values of the most active subfraction (HF2) of
HEUD were 31.25, 125, 7.81, 250, 31.25, 15.62 and 125 GC–MS analysis
µg/mL against Staphylococcus aureus, Shigella flexneri, In order to find out the bioactive compounds respon-
Salmonella typhi, Pseudomonas aeruginosa, Klebsiella sible for antiinflammatory and antibacterial activity.
pneumoniae, Escherichia coli and Enterococcus faeca- HF2 was subjected to GC–MS analysis which identified
lis respectively. The MIC values of the standard drug a total of 23 compounds. HF2 showed six major peaks
gentamycin against all the tested strains were less than (Figure 2) which along with their retention time, molecu-
7.81 µg/mL. lar formula and structure are given in Table 8, constituting

© 2013 Informa Healthcare USA, Inc.

176  S. A. Dar et al.
67.96% of the total peak area percentage. The minor frac- Discussion
tions include dodecane (2.35%), 2-hydroxy-5-bromo-
benzoic acid (0.71%), 2-methyl-5-propylnonane (2.32%), Natural products are considered an important source of
n-eicosane (1.69%), nonadecane (0.84%), 2,4-ditert- new bioactive agents. Medicinal plants continue to be
butylphenol (4.30%), 8-hexylpentadecane (0.79%), used worldwide for the treatment of various diseases and
2-isopropyl-5-methyl-1-hexanol (0.90%), 2,3,8-trimeth- have a great potential for providing novel drug leads with
yldecane (1.26%), octadecan-1-ol (4.45%), 2,7,10-tri- novel mechanism of action (Singh et al., 2012).
methyldodecane (1.07%), n-cetane (2.43%), benzoic acid The maximum hypoglycemic activity of the plant was
(0.80%), 4,6-di-tert-butyl-m-cresol (4.32%), 9-eicosene observed with AEUD in normoglycemic rats. This obser-
(0.68%), n-heneicosane (1.41%) and cis-9-tricosene vation suggests that the compounds responsible for the
(1.72%) constituting a total of 32.04%. hypoglycemic activity of U. dioica are polar in nature
and more soluble in water as compared to methanol. It
Table 6.  Antibacterial activity of different column eluted fractions is known that in diabetes mellitus, the sites and mecha-
of hexane extract of Urtica dioica. nism of pharmacological intervention in the attendant
Extract treated HF1 HF2 HF3 HF4 HF5 C Sd biochemical processes are diverse (Akah & Okafor, 1992;
Microorganisms DIZ (mm) Marles & Farnsworth, 1995). It is likely that this possibil-
Staphylococcus − +++ + + + − +++ ity of diversity in the hypoglycemic mechanism of the
aureus action of drugs can also apply to the AEUD (Cheng &
Shigella flexneri − +++ + + + − +++ Fantus, 2005; Figure 3). The major bioactive molecules
Salmonella typhi − +++ + + + − +++ of AEUD characterized by HPLC–DAD analysis are
Pseudomonas − ++ + − − − +++ hydroxycinnamic acid derivatives and flavonoids which
aeruginosa
may probably possess insulin like effect or stimulate the
Klebsiella − +++ + + + − +++
pneumoniae pancreatic β-cells to produce insulin which in turn low-
Escherichia coli − +++ + + + − +++ ers the blood glucose level (Farzami et al., 2003; Michael
Enterococcus − +++ + + − − +++ et al., 2009). Studies with different doses (100 to 400 mg/
faecalis kg bw) of the AEUD in streptozotocin-induced diabetic
HF1, combined fractions 1–4, HF2, combined fractions 5–11, HF3, rats indicated that 300 mg/kg bw was the most effective
combined fractions 12–17, HF4, combined fractions 18–24, HF5, dose while 200 mg/kg bw has also got reasonably good
combined fractions 25–30; C, control; DIZ, diameter inhibition activity. Higher dose of 400 mg/kg bw showed a lesser
zone; Sd, standard (gentamycin). fall. Such a phenomenon of less hypoglycemic effect at
The results are represented as inactive (−, inhibition zone
diameter ≤7 mm), moderate actvity (+, inhibition zone diameter higher dose is not uncommon with indigenous plants
≤10 mm), good activity (++, inhibition zone diameter ≤15 mm) and has been observed with Aegle marmelose (Rao et
and very good activity (+++, inhibition zone diameter >15 mm). al., 1995), Murraya koenigii (Achyut et al., 2005) and

Figure 1.   HPLC–DAD profiles acquired at two different wavelengths (280 and 520 nm) of AEUD. (A) Peaks: 1: gallic acid (internal standard);
2: caffeic acid derivative; 3: p-coumaric acid; 4: chlorogenic acid; 5: caffeoylquinic acid; 6: 2-O-caffeoylmalic acid; X: anthocyanin compounds
[detailed in (B), as 7: peonidin 3-O-rutinoside; 8: rosinidin 3-O-rutinoside]; 9: rutin; 10: quercetin p-coumaroyl glucoside; 11: isorhamnetin
3-O-rutinoside.

 Pharmaceutical Biology
 Activity of Urtica dioica 177
Brassica nigra (Anand et al., 2007). Like the aqueous (up to 2 h), an intermediate phase involving the activity of
extract, glibenclamide also produced significant reduc- bradikinin, and a third phase (3–6 h) with prostanoid syn-
tion in the blood glucose level. The present findings thesis induced by cyclooxygenase (COX) (Di Rosa, 1972;
appear to be in consonance with the earlier suggestion Pérez-Guerrero et al., 2001). Indomethacin is a non-ste-
(Jackson & Bressler, 1981) that sulphonylureas such as roidal antiinflammatory drug (NSAID) with strong anti-
glibenclamide have an extra-pancreatic hypoglycemic inflammatory activity that is effective in the treatment of
mechanism of action secondary to their causing insulin rheumatic and nonrheumatic conditions. In experimental
secretion and the attendant glucose uptake into and uti- animals, indomethacin is able to totally inhibit acute and
lization by the tissues. chronic inflammatory processes (erytema, edema, hyper-
The data also showed that HEUD contained ingre- algesia, and glaucoma) (Litter, 1992). It is also accepted
dients that were active against all the tested pathogens. that indomethacin inhibits COX, limiting therefore the
There are a number of reports of hexane extracts of plant biosynthesis of PGs, although it also has other activities
possessing antibacterial activity (Elzaawely et al., 2005). that contribute to its therapeutic effects (Insel, 1996). In
These results indicate that the extracting solvent has a the carrageenan test, initially the crude HEUD and then
definite effect on bioactive principles. CC eluted HF2 of the subfraction HF2 significantly reduced the inflamma-
HEUD exhibited more antibacterial activity as compared tion showing activity right from the first (1 h) to last (24 h)
to other subfractions in disc diffusion assay. The extracts measurement. According to these results, it can be sug-
and fractions can be considered active when the MIC is gested that the mechanism of antiinflammatory action
less than 100 μg/mL (Punitha et al., 2008). of this extract occurs by interfering with the synthesis or
The characteristic swelling that occurs in the rat paw liberation of histamine and PG mediators.
model of inflammation is due to edema formation. In GC–MS analyzed six major bioactive constituents in
accordance with Marsha-Lyn et al. (2002), inflammation HF2. All these compounds are likely to possess potent
occurs throughout in three distinct phases: an initial antibacterial activity. Essential oils rich in terpenes
phase mediated by histamine and 5-hydroxytryptamine have been shown to possess good antibacterial activity
(Taylor et al., 1996). HF2 showed the appreciable pres-
ence of the terpene (neophytadiene; 19.96%) which
Table 7.   HPLC–DAD quantitative analysis of aqueous extract of could explain its antibacterial activity. Neophytadiene is
Urtica dioica.
already reported to possess antibacterial activity as well
Compound Contenta
as helping in the treatment of headache, rheumatism
Caffeic acid derivative 0.061 ± 0.021
and some skin disease (Suresh et al., 2010). Many fatty
p-Coumaric acid 0.050 ± 0.071
acids are known to have antibacterial and antifungal
Chlorogenic acid 2.039 ± 0.073
properties (Russel, 1991). Our findings showed 24.86%
Caffeoylquinic acid 0.049 ± 0.014
fatty acid esters, namely heptadecyl ester, hexyl octyl
2-O-Caffeoylmalic acid 2.059 ± 1.015
ester, butyl tetradecyl ester, and 1,2 benzenedicarboxylic
Peonidin 3-O-rutinoside 0.034 ± 0.009
acid, which are reported to exhibit both antiinflamma-
Rosinidin 3-O-rutinoside 0.531 ± 0.312
tory (Li et al., 2004) and antibacterial activities (Modupe
Rutin 3.209 ± 1.151
et al., 2010). In addition, the minor components might
Quercetin p-coumaroyl glucoside 0.407 ± 0.177
be responsible for both antiinflammatory and antibac-
Isorhamnetin 3-O-rutinoside 2.675 ± 0.312
terial activity, possibly by producing a synergistic effect
a
Data are the mean ± SD of three determinations and are
expressed as mg g−1. between other components. However, the composition

Figure 2.  TIC chromatogram of the fraction-2 (HF2) of hexane extract of Urtica dioica. Peaks: 1: hexyl octyl ester; 2: heptadecyl ester; 3:
neophytadiene; 4: butyl tetradecyl ester; 5: 2,6,10,15-tetramethylheptadecane; 6: 1,2 benzenedicarboxylic acid; X: minor fractions (detailed
in results).

© 2013 Informa Healthcare USA, Inc.

178  S. A. Dar et al.

Table 8.  The major compounds identified by GC–MS in the fraction-2 (HF2) of hexane extract of Urtica dioica.
TR (min) Compound Molecular formula Structure MW Peak area %
20.23 Hexyl octyl ester C14H30O3S 278 6.31

24.69 Heptadecyl ester C19H36Cl2O2 366 9.45

25.91 Neophytadiene C20H38 278 19.96

26.65 Butyl etradecyl ester C26H42O4 418 9.53

30.37 2,6,10,15-Tetra- C21H44 296 12.82

methylheptade-cane

40.62 1,2-Benzenedicarboxy-lic C24H38O4 390 9.89

acid

MW, molecular weight; TR, retention time.

Figure 3.   Major target organs and mechanism of action of orally administered antihyperglycemic agents in type II diabetes mellitus.

 Pharmaceutical Biology

of U. dioica in this study differs from that described by
other authors (Singh et al., 2012) because the composi-
tion of essential oil of any plant is influenced by several
factors such as planting, climatic, seasonal and experi-
mental conditions (Daferera et al., 2000).
Increase in popularity and scarcity of scientific stud-
ies on safety have raised concern regarding toxicity and
adverse effect of herbal remedies (Saad et al., 2006). The
toxicity results showed that both the aqueous extract and
HF2 of HEUD had a higher margin of safety (LC50>1000
μg/mL) against A. salina larvae. The test is used to evalu-
ate, in a preliminary way, natural product toxicities. It is
fast, easy and inexpensive, and can also be used to verify
the antitumor activity, since this cytotoxicity assessment
is related to possible activity against tumor cells, when
the LC50 values are less than 250 μg/mL (Siqueira et al.,
1998). In the acute toxicity studies with rats, no mortality
was observed during the 24 h period at the tested doses
which also indicates the nonlethality of both the hexane
and the aqueous extracts of U. dioica.
Conclusions
The findings showed that the leaves of U. dioica are an
interesting source of biologically active compounds that
may be applied for prophylaxis and therapy in humans,
justifying their traditional use, to treat diabetes, arthritis,
and infectious diseases. The results reinforce the impor-
tance of the ethnobotanical approach as a potential
source of bioactive substances.
Acknowledgments
The authors are grateful to Dr Rambir Singh (Department
of Biomedical Science), Bundelkhand University, Jhansi,
UP, India for providing laboratory facilities in the course
of this research. The authors are also highly grateful to
Mr Naresh Kumar Shah, Plasma Bioscience Research
Centre, Kwangwoon University, South Korea for provid-
ing technical assistance.
Declaration of interest
The authors report no conflicts of interest.
References
Achyut NK, Rahesh KG, Geeta W. (2005). Hypoglycemic effects of
Murraya koenigii on normal and alloxan diabetic rabbits. J
Ethnopharmacol, 97, 245–251.
Ahmed MM, Qureshi S, Al-bekairi AM, Shah AH, Rao RM. (1993). Anti-
inflammation activity of Caralluma tuberculata alcoholic extract.
Fitoterapia, 64, 359–362.
Akah PA, Okafo CL. (1992). Blood sugar lowering effect of Vernonia
amygdalina (Del) in an experimental rabbit model. Phytother Res,
6, 171–173.
Anand P, Murali KY, Tandon V, Chandra R, Murthy PS. (2007). Preliminary
studies on antihyperglycemic effect of aqueous extract of Brassica
© 2013 Informa Healthcare USA, Inc.
Activity of Urtica dioica 179
nigra (L.) Koch in streptozotocin induced diabetic rats. Indian J Exp
Biol, 45, 696–701.
Anonynous. (1993). World Health Organisation Summary of WHO
guidelines for the assesment of herbal medicines. Gram, 28, 13–14.
Baker JT, Borris RP, Carté B, Cordell GA, Soejarto DD, Cragg GM, Gupta
MP, Iwu MM, Madulid DR, Tyler VE. (1995). Natural product drug
discovery and development: New perspectives on international
collaboration. J Nat Prod, 58, 1325–1357.
Brantner A. (1997). Influence of various parameters on the evaluation
of antibacterial compounds by the bioautographic TLC assay.
Pharm Pharmacol Lett, 7, 152–154.
Chakrabarti R, Rajagopalan R. (2002). Diabetes and insulin resistance
associated disorders: Diasease and therapy. Curr Sci, 83, 12–19.
Chandrasekaran M, Venkatesalu V. (2004). Antibacterial and antifungal
activity of Syzygium jambolanum seeds. J Ethnopharmacol, 91,
105–108.
Chen H, Brahmbhatt S, Gupta A, Sharma AC. (2005). Duration of
streptozotocin-induced diabetes differentially affects p38-
mitogen-activated protein kinase (MAPK) phosphorylation in
renal and vascular dysfunction. Cardiovasc Diabetol, 4, 3.
Cheng AY, Fantus IG. (2005). Oral antihyperglycemic therapy for type 2
diabetes mellitus. CMAJ, 172, 213–226.
Daferera DJ, Ziogas BN, Polissiou MG. (2000). GC-MS analysis of essential
oils from some Greek aromatic plants and their fungitoxicity on
Penicillium digitatum. J Agric Food Chem, 48, 2576–2581.
Di Rosa M. (1972). Biological properties of carrageenan. J Pharm
Pharmacol, 24, 89–102.
Elzaawely AA, Xuan TD, Tawata S. (2005). Antioxidant and antibacterial
activities of Rumex japonicus HOUTT. Aerial parts. Biol Pharm
Bull, 28, 2225–2230.
European Pharmacopoe (1997). Microbiological assay of antibiotics.
Strasbourg, France 2.7.2.
Farzami B, Ahmadvand D, Vardasbi S, Majin FJ, Khaghani Sh. (2003).
Induction of insulin secretion by a component of Urtica dioica
leave extract in perifused Islets of Langerhans and its in vivo effects
in normal and streptozotocin diabetic rats. J Ethnopharmacol, 89,
47–53.
Ieri F, Giaccherini C, Zipoli A, Innocenti M, Vincieri FF, Mulinacci
N. (2006). Rapid HPLC/DAD method to analyze anthocyanins,
chlorogenic acids and glycoalkaloids in pigmented potato
cultivars. XXIII International Conference on Polyphenols,
Winnipeg, Manitoba, Canada, 2006, 179–180.
Insel AP. (1996). Analgésicos-antipiéticos y antiinflamatorios, y
fármacos antigotosos. In: Hardman JG, Limbird LE, Molinoff PB,
Ruddon RW, Goodman GA, eds. Las bases farmacológicas de
la terapéutica, 9th edn. México: McGraw-Hill Interamericana,
661–701.
Jackson JE, Bressler R. (1981). Clinical pharmacology of sulphonylurea
hypoglycaemic agents: part 1. Drugs, 22, 211–245.
Johnsen AR, Bendixen K, Karlson U. (2002). Detection of microbial
growth on polycyclic aromatic hydrocarbons in microtiter plates
by using the respiration indicator WST-1. Appl Environ Microbiol,
68, 2683–2689.
Jüni P, Reichenbach S, Egger M. (2005). COX 2 inhibitors, traditional
NSAIDs, and the heart. BMJ, 330, 1342–1343.
Li RW, Leach DN, Myers SP, Leach GJ, Lin GD, Brushett DJ, Waterman
PG. (2004). Anti-inflammatory activity, cytotoxicity and active
compounds of Tinospora smilacina Benth. Phytother Res, 18,
78–83.
Litter M. (1992). Compendio de Farmacología, 4th Edn. Argentina: El
Ateneo, 608–638.
Lourdes RF, Jorge RE, Scott B, Dea HR, Eliseo T. (2008). Risks and
benefits of commonly used herbal medicines in México. Toxicol
Appl Pharmacol, 227, 125–135.
Marles RJ, Farnsworth NR. (1995). Antidiabetic plants and their active
constituents. Phytomedicine, 2, 137–189.
Marsha-Lyn M, Mckoy G, Everton T, Oswald S. (2002). Preliminary
investigation of the anti-inflammatory properties of an aqueous
180  S. A. Dar et al.
extract from Morinda citrifolia (Noni). Proceed Western Pharm
Soc, 45, 76–78.
Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE,
McLaughlin JL. (1982). Brine shrimp: A convenient general
bioassay for active plant constituents. Planta Med, 45, 31–34.
Michael BW, Masoud SD, Vivian V, Christine A, Robson D, Gary S.
(2009). Insulin memetics in U. dioica: structural and computational
analysis of U. dioica extracts. Phytother Res, 10, 1002–1015.
Modupe O, Wesley O, Morufu A, Elizabeth AO. (2010). Analysis of
essential oil from the stem of Chansmanthera dependens. J Nat
Prod, 3, 47–53.
Murali YK, Anand P, Tandon V, Singh R, Chandra R, Murthy PS. (2007).
Long-term effects of Terminalia chebula Retz. on hyperglycemia
and associated hyperlipidemia, tissue glycogen content and in
vitro release of insulin in streptozotocin induced diabetic rats. Exp
Clin Endocrinol Diabetes, 115, 641–646.
Parra AL, Silva RY, Sardiñas IG, Buela IL. (2001). Comparative study
of the assay of Artemia salina L. and the estimate of the medium
lethal dose (LD50 value) in mice, to determine oral acute toxicity
of plant extracts. Phytomedicine, 8, 395–400.
Pathak SK, Sharma RA, Steward WP, Mellon JK, Griffiths TR, Gescher
AJ. (2005). Oxidative stress and cyclooxygenase activity in prostate
carcinogenesis: Targets for chemopreventive strategies. Eur J
Cancer, 41, 61–70.
Pérez-Guerrero C, Herrera MD, Ortiz R, Alvarez de Sotomayor M,
Fernández MA. (2001). A pharmacological study of Cecropia
obtusifolia Bertol aqueous extract. J Ethnopharmacol, 76, 279–284.
Perez GRM. (1996). Anti-inflammatory activity of Ambrosia
artemisiaefolia and Rheo spathaceae. Phytomedicine, 3, 163–164.
Punitha SMJ, Babu MM, Sivaram V, Shankar VS, Dhas SA, Mahesh TC,
Immanuel G, Citarasu T. (2008). Immunostimulating influence
of herbal biomedicines on nonspecific immunity in Grouper
Epinephelus tauvina juvenile against Vibrio harveyi infection.
Aquacult Int, 16, 511–523.
 Pharmaceutical Biology
Rao VV, Dwivedi SK, Swarup D, Sharma SR. (1995). Hypoglycaemic and
anti-hyperglycemic effects of Aeegle marmelose leaves in rabbits.
Curr Sci India, 69, 932–933.
Russel AD. (1991). Mechanisms of bacterial resistance of nonantibiotics:
Food additives and food pharmaceutical preservatives. J Appl
Bacteriol, 71, 191–201.
Saad B, Azaizeh H, Abu-Hijleh G, Said O. (2006). Safety of traditional
arab herbal medicine. Evid Based Complement Alternat Med, 3,
433–439.
Saracini E, Tattini M, Traversi ML, Pinelli P. (2005). Simultaneous
determination of ellagitannins, flavonoid glycosides and
acylglycosyl flavonoids in Cistus salvifolius L. leaves by HPLC-DAD
and HPLC-MS. Chromatographia, 62, 245–249.
Singh R, Dar SA, Sharma P. (2012). Antibacterial activity and
toxicological evaluation of semipurified hexane extract of Urtica
dioica leaves. Res J Med Plants, 6, 123–135.
Siqueira MJ, Bomm MD, Pereira NFG. (1998). Estudo fitoquímico de
Unonopsis lindmanii – Annonaceae, biomonitorado pelo ensaio
de toxicidade sobre a Artemia salina Leach. Química Nova, 21,
557–559.
Suresh L, Veerabahu RM, Gnanasingh SR. (2010). GC-MS analysis
of ethanolic extract of Zanthoxylum rhetsa (Roxb.) DC spines. J
Herbal Med Toxicol, 4, 191–192.
Taylor RS, Edel F, Manandhar NP, Towers GH. (1996). Antimicrobial
activities of southern Nepalese Medicinal Plants. J Ethnopharmacol,
50, 97–102.
Turner RA. (1965). Screening Methods of Pharmacology. New York:
Academic Press, 22–41.
Valiathan MS. (1998). Healing plants. Curr Sci, 75, 1122–1126.
Walsh C. (2003). Where will new antibiotics come from? Nat Rev
Microbiol, 1, 65–70.
Winter CA, Risley EA, Nuss GW. (1962). Carrageenin-induced edema in
hind paw of the rat as an assay for antiiflammatory drugs. Proc Soc
Exp Biol Med, 111, 544–547.