SUPPLEMENTARY MATERIAL
In vivo antinociceptive and anti-inflammatory activity of umbelliferone isolated from
Potentilla evestita
Abdur Rauf a,b, Rehan Khanc, Haroon Khand*, Samreen Perveze, Abdul Saboor Pirzadad
a
Institute of Chemical Sciences, University of Peshawar, Peshawar -25120, KPK, Pakistan,
b
Geology Department University of Peshawar, Peshawar, KPK, Pakistan, cHEJ Research
Institute, Institute of Chemical and Biological sciences, Karachi University Karachi, Pakistan.
d
Department of Pharmacy, Abdul Wali Khan University Mardan 23200, Pakistan, eDepartment
of Pharmacy, University of Peshawar, KPK, Pakistan
Corresponding author
Haroon Khan
hkdr2006@gmail.com
Abstract
The present study was designed to evaluate the antinociceptive and anti-inflammatory activities
of a compound; umbelliferone isolated the chloroform fraction of Potentilla evestita in animal
models. When tested against acetic acid induced noxious stimulus, it significantly prolonged pain
thresh hold and provided 38.38% and 60.95% reduction in abdominal constriction at 5 and 10
mg/kg i.p. respectively. Post umbelliferone injection evoked significant dose dependent
reduction in noxious stimulation with 33.65% and 58.89% % pain attenuation at 5 and 10 mg/kg
i.p. respectively in initial phase. In late phase, it illustrated more dominant effect with 37.65%
and 63.79% blockade of painful sensation. Similarly, it had significant anti-inflammatory activity
during various assessment times (1-5 h) with 46.28% and 66.13% amelioration after 4th of
administration against induced edema. In conclusion, umbelliferone had strong antinociceptive
and anti-inflammatory activities by inhibiting both peripheral and centrally acting pain
mediators.
Key words: Potentilla evestita, umbelliferone, antinociceptive and anti-inflammatory activity
3. Experimental
3.1. Plant specimen
The whole plant of P. evestita (15 kg) was collected from Gilgit, Pakistan. The plant was
identified by Taxonomist Department of Botany University of Karachi. Voucher specimen
(voucher No. 707) has been deposited at the herbarium of the Department of Botany, University
of Karachi, Karachi, Pakistan (Herbarium No. 71212).
3.2. Extraction and Isolation
Shade dried whole plant (15 kg) of P. evestita was ground into fine powder and soaked in 25 L
ethanol for 10 days at room temperature. The resulting extract was filtered and the filtrate
evaporated under reduced pressure at 45°C to yield 300 g dark brown residue. The residue was
suspended in water and subsequently extracted with solvents of increasing polarity, namely nhexane (3x10 L), chloroform (3x14 L), ethyl acetate (3x12 L) and methanol (1x3 L). Each
extract evaporated under reduced pressure to afford n-hexane fraction (70 g), chloroform (75 g)
EtOAc extract (8 g), and methanol (40 g). The chloroform fraction (60 g) was subjected to
Column chromatography on silica gel (Merck Silica gel 60 (0.063-0.200mm), 5  60 cm). The
column was first eluted with hexane-ethyl acetate (100:0 → 0:100) as solvent system. A total of
100 fractions, RF-1 to RF-100 were obtained based on TLC profiles. Elution of the
chromatogram with hexane- ethyl acetate (100:0 → 100:0) gave the isolation of three known
compound and umbelliferone (60 mg). The structures (Fig 1) of compounds were confirmed by
comparing their NMR and physical data with the reported data in literature (Campbell et al.,
1990).
3.3. Experimental animals
BALB/c mice (20-28 g) and Wistar rats (189-222 g) of either sex were used. Animals were kept
under standard laboratory condition at 25 ± 2 0C. They were fed laboratory diet ad libitum and
allowed free access to drinking water under standard environmental conditions of temperature
(25 0C) in 12 h dark/12 h light control. All the experimental animals were treated according to
ethical principles established (Reference No: HEJ/003: Dated 23/03/2013).
3.4. Acetic acid induced writhing test
BALB/c mice of either sex (n= 6) weighing 20-28 g were used. All animals were withdrawn
from food 2 h before the start of experiment and were divided in various groups. Group I was
injected with normal saline (10 ml/kg) as control, Group II received standard drug diclofenac
sodium (10 mg/ kg) while the remaining groups were injected with test compound, at the dose of
5 and 10 mg/kg. After 30 min of the above treatment animals were treated i.p. with 1% acetic
acid. The number of abdominal constrictions (writhes) was counted after 5 min of acetic acid
injection for the period of 10 min (Muhammad et al., 2013, Khan et al., 2011).
3.5. Formalin test
Wistar rats (189-222 g) of either sex were used in the formalin test following our previously
reported method (Kaleem et al., 2013). Briefly, the prescreened animals were arranged into
groups (n = 6) which received either saline (10 ml/kg), umbelliferone (5 and 10 mg/kg i.p.). For
the induction of pain, 0.05 ml of formalin (2.5% formaldehyde) was injected into the plantar
surface of the right hind paw, 30 min after the treatment of all the animal groups, as described
above. Nociceptive response was considered as the time spent by rat walking or can stand on
injected paw; partially elevated paw; total elevation of injected paw, injected paw licking or
biting. The first 0-5 min was computed as the first phase (neurogenic) and 25-30 min as last
phase (anti-inflammatory) in the assay. TramadolR (30 mg/kg i.p.) was used as a standard drug.
3.6. Carrageenan-induced edema
The carrageenan-induced hind paw edema test of umbelliferone was conducted according to the
previously described method (Khan et al., 2013b, Khan et al., 2013a). The test animals were
divided into groups (n = 6). Group Ι received normal saline (10 mL/kg) as control. The rats of
group ΙΙ, and ΙΙΙ received test extract (5 and 10 mg/kg i.p.). Group ΙV received aspirin (100
mg/kg i.p.) as a positive control. Following 30 min of the treatments, acute inflammation was
induced by sub-plantar injection of 0.1 mL of 1% suspension of carrageenan with 2% gum acacia
in normal saline, in the right hind paw of the rats. The paw volume was estimated with the help
of plethysmometer (Ugo Basile, Italy) at 1st, 2nd, 3rd, 4th and 5th h after the carrageenan injection.
Statistics was applied on the raw data for the calculation of reduction in rat paw volume (mL) for
each group against saline, followed by the calculation of percent reduction in the rat paw.
3.7. Statistical analysis
Values are shown as mean values ± SEM of at least six animals. One-way analysis of variance
was used for comparison test of significant differences among groups followed by Dunnet’s
multiple-comparison posttest. P<0.05 was considered as significant from control.
References
Campbell, W. E., Davidowitz, B. & Jackson, G. E. (1990) Quinolinone alkaloids from an
Agathosma species. Phytochemistry, 29: 1303-1306.
Kaleem, W. A., Muhammad, N., Qayum, M., Khan, H., Khan, A., Aliberti, L. & De Feo, V.
(2013) Antinociceptive activity of cyclopeptide alkaloids isolated from Ziziphus
oxyphylla Edgew (Rhamnaceae). Fitoterapia, 91: 154-158.
Khan, H., Khan, M. A., Gul, F., Hussain, S. & Ashraf, N. (2013a) Anti-inflammatory activity of
Heliotropium strigosum in animal models. Toxicology and Industrial Health:
0748233713491813.
Khan, H., Saeed, M., Khan, M. A., Khan, I. & Ashraf, N. (2011) Antinociceptive activity of
aerial parts of Polygonatum verticillatum: Attenuation of both peripheral and central pain
mediators. Phytotherapy Research, 25: 1024-1030.
Khan, H., Saeed, M., Mehmood, M. H., Rehman, N.-u., Muhammad, N., Ikram-ul, H., Ashraf,
N., El-Tahir, K. E. & Gilani, A.-H. (2013b) Studies on tracheorelaxant and antiinflammatory activities of rhizomes of Polygonatum verticillatum. BMC Complementary
and Alternative Medicine, 13: 197.
Muhammad, N., Saeed, M., Gilani, S. N., Haq, I. u. & Khan, H. (2013) Analgesic and antiinflammatory profile of n-hexane fraction of viola betonicifolia. Tropical Journal of
Pharmaceutical Research, 11: 963-969.
Figure S1. The effect (%) of umbelliferone 1 in acetic acid induced writhing test (n=6). The data
were analyzed by analysis of variance followed by Dunnett’s test. Asterisks indicated
statistically significant values from control.*p<0.05,**p<0.01.
Figure S2. Protection (%) of the umbelliferone 1 in formalin induced flinching behaviour (n=6).
The data were analyzed by analysis of variance followed by Dunnett’s test. Asterisks indicated
statistically significant values from control.*p<0.05,**p<0.01.
Figure S3. Percent effect of umbelliferone 1 in carrageenan induced paw edema. Experimental data are
expressed as mean ± S.E.M. for group of at least six animals. One- way ANOVA was utilized as
judgment test of significant differences among groups followed by Dunnett’s multiple comparison posttest. A probability of *P < 0.05 or **P < 0.01 was considered significant from control.