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Pharmaceutical Biology

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Pharmacological and toxicological evaluation ofUrtica 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) Pharmacologicaland 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.

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170

Introduction

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

prescribed widely even when their biologically active 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

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

1Limnology 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

abstractContext: 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

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)

Pharmaceutical Biology, 2013; 51(2): 170–180© 2013 Informa Healthcare USA, Inc.ISSN 1388-0209 print/ISSN 1744-5116 onlineDOI: 10.3109/13880209.2012.715172

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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 materialThe 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 preparationThe 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 studiesAnimalsWistar rats, without any sexual discrimination, weighing 150–200 g were used in the experiments. The animals had

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 testRats 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 = Difference 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 scheduleHypoglycemic 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 assayThe 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

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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 salinaThe 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. LC

50 of frac-

tions and extracts above 250 μg/mL were considered as low toxic, LC

50 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 LC

50, Probit analysis was used.

Acute toxicity test in Wistar ratsToxicity 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 studiesMicrobiologic materialAll 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

25922) and Enterococcus faecalis (ATCC 29212) obtained from the National Institute of Immunology, New Delhi, India.

Antibacterial activityThe 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 PF

254 (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 concentrationThe 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

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(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 analysisHPLC–DAD analysisHigh-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 RP

18 (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 H

2O (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 analysisHigh-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 analysisIdentification 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 analysisGas 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

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 analysisResults 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 studiesHypoglycemic activityThe 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 assayThe 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

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(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 salinaThe 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 (LC

50 > 1000 μg/mL).

Acute toxicity test in Wistar ratsIn 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 studiesAntibacterial activityAll 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).

Table 2. Effective dose (ED) determination of aqueous extract of Urtica dioica leaves by GTT in streptozotocin-induced diabetic rats.

Group (n) FBGBlood glucose (mg/dL)

% fall between 0 h and 1 h0 h 1 h 2 h 3 hGroup I (Control) 270 ± 8.2 264 ± 7.2 362 ± 5.7 344 ± 7.2 356 ± 6.4Group 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.

Table 1. Hypoglycemic activity of leaf extracts of Urtica dioica in Wistar rats.Extract treated Blood glucose (mg/dL)

% fall between 0 h and 1 hGroup (n) FBG 0 h 1 h 2 h 3 hC 77 ± 7.3 75 ± 6.9 128 ± 5.9 107 ± 6.0 120 ± 6.8Aqueous 67.92 ↓T 77 ± 6.5 74 ± 6.8 91 ± 6.3*** 94 ± 5.4 82 ± 7.1C 87 ± 6.3 85 ± 7.2 138 ± 4.8 110 ± 6.7 130 ± 8.6Methanol 28.30 ↓T 86 ± 5.8 99 ± 5.6 137 ± 5.5 114 ± 7.5 123 ± 6.1C 83 ± 4.9 71 ± 5.3 126 ± 6.1 85 ± 9.0 94 ± 5.9Et-O-Ac 9.09 ↓T 85 ± 7.3 83 ± 6.0 133 ± 6.8 92 ± 8.3 93 ± 4.8C 79 ± 6.5 73 ± 4.9 111 ± 4.8 99 ± 8.5 105 ± 7.7Chloroform 15.78 ↓T 76 ± 5.4 83 ± 6.1 127 ± 7.0 113 ± 7.2 102 ± 8.1C 73 ± 6.8 70 ± 6.6 112 ± 6.5 97 ± 5.8 100 ± 6.9Hexane 11.90 ↑T 69 ± 5.3 81 ± 7.2 118 ± 6.1 102 ± 6.4 90 ± 5.1The 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.

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Bioassay guided isolation of active compound(s)Since HEUD showed good antibacterial activity, it was further fractionated using CC into 30 subfractions of 250 mL each which were pooled together accord-ing to their TLC profile to give five subfractions: HF1, HF2, HF3, HF4, and HF5. The results of antibacte-rial activity of the subfractions of HEUD are shown in Table 6. The antibacterial activity produced by the sub-fraction HF2 was comparable with that of the standard antibiotic gentamycin.

Minimum inhibitory concentrationThe MIC values of the most active subfraction (HF2) of HEUD were 31.25, 125, 7.81, 250, 31.25, 15.62 and 125 µg/mL against Staphylococcus aureus, Shigella flexneri, Salmonella typhi, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Enterococcus faeca-lis respectively. The MIC values of the standard drug gentamycin against all the tested strains were less than 7.81 µg/mL.

Phytochemical analysisHPLC–DAD analysisHPLC–DAD analysis was developed to characterize the compounds in crude AEUD (Figure 1). It was very abun-dant in hydroxycinnamic acid derivatives (main com-pounds being 2-O-caffeoylmalic and chlorogenic acid); flavonoids (rutin, quercetin p-coumaroyl glucoside, isor-hamnetin 3-O-rutinoside) and anthocyanins (peonidin 3-O-rutinoside, rosinidin 3-O-rutinoside) (Table 7).

GC–MS analysisIn order to find out the bioactive compounds respon-sible for antiinflammatory and antibacterial activity. HF2 was subjected to GC–MS analysis which identified a total of 23 compounds. HF2 showed six major peaks (Figure 2) which along with their retention time, molecu-lar formula and structure are given in Table 8, constituting

Table 3. Effect of leaf extracts and hexane subfractions of Urtica dioica on carrageenin-induced hind paw edema in Wistar rats.

Treatment and doseTime interval (h) and edema volume (mL)

0 h 1 h 2 h 3 h 24 hControl 0.20 ± 0.008 0.35 ± 0.006 0.39 ± 0.004 0.43 ± 0.003 0.39 ± 0.003Hexane (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 5. Antibacterial activity of different solvent extract of Urtica dioica leaves.Extract treated He Ch Ea Mt Aq C SdMicroorganisms DIZ (mm)Staphylococcus aureus ++ + − − − − +++Shigella flexneri ++ + − − − +++Salmonella typhi ++ + − − − − +++Pseudomonas aeruginosa

++ − − − − − +++

Klebsiella pneumoniae ++ + − − − − +++Escherichia coli ++ + − − − − +++Enterococcus faecalis ++ − − − − − +++The results are represented as inactive (−, inhibition zone

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).

Aq, aqueous extract; C, control (dimethyl sulphoxide, DMSO); Ch, chloroform extract; DIZ, diameter inhibition zone; Ea, ethyl acetate extract; He, hexane extract; Mt, methanol extract; Sd, standard (gentamycin).

Table 4. Lethal concentration 50% (LC50

) of leaf extracts and hexane subfractions of Urtica dioica.

Component LC50

(μg/mL)

Hexane extract >1000CH

3Cl exract >1000

EtOAc extract >1000Methanol extract >1000Aqueous >1000HF1 909.05 ± 7.5HF2 >1000HF3 877.15 ± 6.4HF4 386.07 ± 12.41HF5 609.15 ± 6

176 S. A. Dar et al.

Pharmaceutical Biology

67.96% of the total peak area percentage. The minor frac-tions include dodecane (2.35%), 2-hydroxy-5-bromo-benzoic acid (0.71%), 2-methyl-5-propylnonane (2.32%), n-eicosane (1.69%), nonadecane (0.84%), 2,4-ditert-butylphenol (4.30%), 8-hexylpentadecane (0.79%), 2-isopropyl-5-methyl-1-hexanol (0.90%), 2,3,8-trimeth-yldecane (1.26%), octadecan-1-ol (4.45%), 2,7,10-tri-methyldodecane (1.07%), n-cetane (2.43%), benzoic acid (0.80%), 4,6-di-tert-butyl-m-cresol (4.32%), 9-eicosene (0.68%), n-heneicosane (1.41%) and cis-9-tricosene (1.72%) constituting a total of 32.04%.

Discussion

Natural products are considered an important source of new bioactive agents. Medicinal plants continue to be used worldwide for the treatment of various diseases and have a great potential for providing novel drug leads with novel mechanism of action (Singh et al., 2012).

The maximum hypoglycemic activity of the plant was observed with AEUD in normoglycemic rats. This obser-vation suggests that the compounds responsible for the hypoglycemic activity of U. dioica are polar in nature and more soluble in water as compared to methanol. It is known that in diabetes mellitus, the sites and mecha-nism of pharmacological intervention in the attendant biochemical processes are diverse (Akah & Okafor, 1992; Marles & Farnsworth, 1995). It is likely that this possibil-ity of diversity in the hypoglycemic mechanism of the action of drugs can also apply to the AEUD (Cheng & Fantus, 2005; Figure 3). The major bioactive molecules of AEUD characterized by HPLC–DAD analysis are hydroxycinnamic acid derivatives and flavonoids which may probably possess insulin like effect or stimulate the pancreatic β-cells to produce insulin which in turn low-ers the blood glucose level (Farzami et al., 2003; Michael et al., 2009). Studies with different doses (100 to 400 mg/kg bw) of the AEUD in streptozotocin-induced diabetic rats indicated that 300 mg/kg bw was the most effective dose while 200 mg/kg bw has also got reasonably good activity. Higher dose of 400 mg/kg bw showed a lesser fall. Such a phenomenon of less hypoglycemic effect at higher dose is not uncommon with indigenous plants and has been observed with Aegle marmelose (Rao et 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.

Table 6. Antibacterial activity of different column eluted fractions of hexane extract of Urtica dioica.Extract treated HF1 HF2 HF3 HF4 HF5 C SdMicroorganisms DIZ (mm)Staphylococcus aureus

− +++ + + + − +++

Shigella flexneri − +++ + + + − +++Salmonella typhi − +++ + + + − +++Pseudomonas aeruginosa

− ++ + − − − +++

Klebsiella pneumoniae

− +++ + + + − +++

Escherichia coli − +++ + + + − +++Enterococcus faecalis

− +++ + + − − +++

HF1, combined fractions 1–4, HF2, combined fractions 5–11, HF3, combined fractions 12–17, HF4, combined fractions 18–24, HF5, combined fractions 25–30; C, control; DIZ, diameter inhibition zone; Sd, standard (gentamycin).

The results are represented as inactive (−, inhibition zone diameter ≤7 mm), moderate actvity (+, inhibition zone diameter ≤10 mm), good activity (++, inhibition zone diameter ≤15 mm) and very good activity (+++, inhibition zone diameter >15 mm).

Activity of Urtica dioica 177

© 2013 Informa Healthcare USA, Inc.

Brassica nigra (Anand et al., 2007). Like the aqueous extract, glibenclamide also produced significant reduc-tion in the blood glucose level. The present findings appear to be in consonance with the earlier suggestion (Jackson & Bressler, 1981) that sulphonylureas such as glibenclamide have an extra-pancreatic hypoglycemic mechanism of action secondary to their causing insulin secretion and the attendant glucose uptake into and uti-lization by the tissues.

The data also showed that HEUD contained ingre-dients that were active against all the tested pathogens. There are a number of reports of hexane extracts of plant possessing antibacterial activity (Elzaawely et al., 2005). These results indicate that the extracting solvent has a definite effect on bioactive principles. CC eluted HF2 of HEUD exhibited more antibacterial activity as compared to other subfractions in disc diffusion assay. The extracts and fractions can be considered active when the MIC is less than 100 μg/mL (Punitha et al., 2008).

The characteristic swelling that occurs in the rat paw model of inflammation is due to edema formation. In accordance with Marsha-Lyn et al. (2002), inflammation occurs throughout in three distinct phases: an initial phase mediated by histamine and 5-hydroxytryptamine

(up to 2 h), an intermediate phase involving the activity of bradikinin, and a third phase (3–6 h) with prostanoid syn-thesis induced by cyclooxygenase (COX) (Di Rosa, 1972; Pérez-Guerrero et al., 2001). Indomethacin is a non-ste-roidal antiinflammatory drug (NSAID) with strong anti-inflammatory activity that is effective in the treatment of rheumatic and nonrheumatic conditions. In experimental animals, indomethacin is able to totally inhibit acute and chronic inflammatory processes (erytema, edema, hyper-algesia, and glaucoma) (Litter, 1992). It is also accepted that indomethacin inhibits COX, limiting therefore the biosynthesis of PGs, although it also has other activities that contribute to its therapeutic effects (Insel, 1996). In the carrageenan test, initially the crude HEUD and then the subfraction HF2 significantly reduced the inflamma-tion showing activity right from the first (1 h) to last (24 h) measurement. According to these results, it can be sug-gested that the mechanism of antiinflammatory action of this extract occurs by interfering with the synthesis or liberation of histamine and PG mediators.

GC–MS analyzed six major bioactive constituents in HF2. All these compounds are likely to possess potent antibacterial activity. Essential oils rich in terpenes 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 could explain its antibacterial activity. Neophytadiene is already reported to possess antibacterial activity as well as helping in the treatment of headache, rheumatism and some skin disease (Suresh et al., 2010). Many fatty acids are known to have antibacterial and antifungal properties (Russel, 1991). Our findings showed 24.86% fatty acid esters, namely heptadecyl ester, hexyl octyl ester, butyl tetradecyl ester, and 1,2 benzenedicarboxylic acid, which are reported to exhibit both antiinflamma-tory (Li et al., 2004) and antibacterial activities (Modupe et al., 2010). In addition, the minor components might be responsible for both antiinflammatory and antibac-terial activity, possibly by producing a synergistic effect between other components. However, the composition

Table 7.  HPLC–DAD quantitative analysis of aqueous extract of Urtica dioica.Compound Contenta

Caffeic acid derivative 0.061 ± 0.021p-Coumaric acid 0.050 ± 0.071Chlorogenic acid 2.039 ± 0.073Caffeoylquinic acid 0.049 ± 0.0142-O-Caffeoylmalic acid 2.059 ± 1.015Peonidin 3-O-rutinoside 0.034 ± 0.009Rosinidin 3-O-rutinoside 0.531 ± 0.312Rutin 3.209 ± 1.151Quercetin p-coumaroyl glucoside 0.407 ± 0.177Isorhamnetin 3-O-rutinoside 2.675 ± 0.312a Data are the mean ± SD of three determinations and are expressed as mg g−1.

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).

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Pharmaceutical Biology

Table 8. The major compounds identified by GC–MS in the fraction-2 (HF2) of hexane extract of Urtica dioica.T

R (min) Compound Molecular formula Structure MW Peak area %

20.23 Hexyl octyl ester C14

H30

O3S 278 6.31

24.69 Heptadecyl ester C19

H36

Cl2O

2366 9.45

25.91 Neophytadiene C20

H38

278 19.96

26.65 Butyl etradecyl ester C26

H42

O4

418 9.53

30.37 2,6,10,15-Tetra-methylheptade-cane

C21

H44

296 12.82

40.62 1,2-Benzenedicarboxy-lic acid

C24

H38

O4

390 9.89

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.

Activity of Urtica dioica 179

© 2013 Informa Healthcare USA, Inc.

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 (LC

50>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 LC

50 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.

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