The Antiinflammatory and Antiarthritic Properties of Ethanol Extract of Hedera helix

Abstract

The ethanol Hedera helix plant extract was tested for its antiinflammatory properties. Intraperitoneal injections of 7.5 ml/kg wt ethanol extract showed antiinflammatory activity with 88.89% inhibition as compared to reference drug diclofenac, which showed 94.44% inhibition in formalin-induced paw oedema. As formalin-induced paw oedema closely resembles human arthritis, the antiarthritic property of ethanol extract of Hedera helix was also investigated. The visible reduction in arthritic symptoms by extract of Hedera helix suggests the potential of the plant extract against inflammation and arthritis.

Full text

January - February 2013 Indian Journal of Pharmaceutical Sciences 99
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*Address for correspondence
E-mail: anuradha_62@hotmail.com
The Antiinflammatory and Antiarthritic Properties of
Ethanol Extract of
Hedera helix
ANURADHA RAI*
Department of Zoology, St. Joseph’s College, Darjeeling‑734 104, India
Rai: The Antiinflammatory and Antiarthritic Properties of Hedera helix
The ethanol
Hedera helix
plant extract was tested for its antiinflammatory properties. Intraperitoneal injections of
7.5 ml/kg wt ethanol extract showed antiinflammatory activity with 88.89% inhibition as compared to reference
drug diclofenac, which showed 94.44% inhibition in formalin‑induced paw oedema. As formalin‑induced paw
oedema closely resembles human arthritis, the antiarthritic property of ethanol extract of
Hedera helix
was also
investigated. The visible reduction in arthritic symptoms by extract of
Hedera helix
suggests the potential of the
plant extract against inflammation and arthritis.
Key words: Arthritis, diclofenac,
Hedera helix
, inflammatory, intraperitoneal injections
Hedera helix (Linn) is common ivy of family
Araliaceae found growing in the Darjeeling hills. It
is an evergreen woody climber scaling the walls and
covering the walls with a canopy of leaves. It is also
grown as an ornamental plant. In folklore medicine,
it is used for the cure of benign warts. Inammation
is a fundamental protective response. However,
inflammation can be harmful in conditions such as
life-threatening hypersensitive reactions to insect
bites, drugs, toxins and in chronic diseases such as
rheumatic arthritis, atherosclerosis, lung brosis and
cancer[1]. Inflammation has been seen to accelerate
cancer and chronic inflammation is regarded as an
essential factor for the progression of neoplastic
processes[2]. Hedera helix extract has been reported
to have antioxidant properties[3-6], antispasmodic
properties[7], antiallergic effects[8]. The effect of dry
extracts on respiratory functions of children with
chronic bronchial asthma[9,10] and its antitumour
activities has been reported[11-16]. In the quest for
seeking new herbal sources for the treatment of
inammation and cancer, in the present study, Hedera
helix was tested for its antiinflammatory properties
using 2% formalin for induction of inflammation.
Antiinammatory experiments were carried out using
diclofenac as the reference drug. The antiarthritic
property of Hedera helix extract has also been
investigated.
Swiss Albino mice (6-8 week old) of both sexes
were used in the study. Animals of approximately
equal age and weight were used for experimental and
control groups. All animal experiments were carried
out according to the guidelines of the Animal Ethics
Committee (Reg no. 840/ac/04/CPCSEA).
For the preparation of Hedera extract, leaves of the
plant were collected and was identied at the Botany
Department of St. Joseph’s College, Darjeeling. The
leaves were washed with water. After soaking away
excess water, 10 g of the leaves were taken and
crushed to a paste in a mortar and pestle. Fifteen
millilitres of absolute alcohol were added and kept
in a refrigerator at 4° for 12 h. The extract was then
ltered through Whatman lter paper no. 1; then the
ltrate was ltered through Millipore lter and the
nal solution obtained was stored at 4° for further
use[17].
Animals were divided into ve groups of eight mice
each (Group A, B, C, D and E). Twenty minutes
before induction of inammation, Group A mice were
used as control and each animal of Group A received
phosphate buffered saline (PBS) intraperitoneally (i.p.)
only (3 ml/kg). Group B, C and D test mice received
ethanol Hedera extract 2.5-7.5 ml/kg wt (25 ml,
50 ml and 75 ml per animal) i.p., respectively.
Group E animals received diclofenac as reference
drug 100 mg/kg. Diclofenac was used because it
is a nonsteriodal antiinammatory drug. Diclofenac
has been reported to suppress inammation induced
by various phlogistic agents in experimental animal
models. Diclofenac is commonly employed in the

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treatment or management of rheumatoid arthritis,
osteoarthritis and ankylosing spondylitis and for its
antiinflammatory and analgesic effects. Diclofenac
reduces inammation, swelling and arthritic pain by
inhibiting prostaglandin synthesis and/or production.
The drug also affects polymorphonuclear leukocyte
functions in vitro, thereby reducing chemotaxis,
superoxide toxic radical formation, oxygen-derived
free radical generation and neutral protease
production[18].
Freshly prepared 2% formalin was used as the
oedematogenic agent. Twenty minutes after
administration of PBS in control animals and
after injection of various doses of plant extract
and reference drug in experimental animals, each
animal was injected with 20 ml, of formalin
directly on subplantar region of left hind paw by
exmire microsyringe (ITO Corporation, Tokyo,
Japan) [19]. Formalin-induced paw oedema is one of
the most suitable test procedures to screen chronic
antiinflammatory agents, as it closely resembles
human arthritis[20]. The nociceptive effect of formalin
is also biphasic as it acts as an early neurogenic
component followed by a later tissue-mediated
response[21]. The antiinflammatory response of
Hedera extract on formalin-induced paw oedema
suggests the usefulness of Hedera extract in the
treatment of inammation-associated diseases like
arthritis.
Percent inhibition were calculated as, increase in
paw thickness in control and experimental animals
is Pc=Pt−Po and PT=Pt−Po, respectively and %
inhibition=([Pc−PT]/Pc)×100, where Pt is paw thickness
at time t, Po is initial paw thickness, Pc is increase in
paw thickness of control animals, PT is the increase in
paw thickness of the treatment animals[19].
Pedal inflammation was evident 5-8 min after
formalin injection. The paw thickness was measured
using Vernier callipers before and after formalin
treatment. The thickness of paw was recorded
at 30, 60, 90, 120, 150 and 180 min after formalin
injection. The data were statistically analysed using
Student’s t test and P values less than 0.001 were
considered signicant. All data were represented as
mean±SD[22,23].
After induction, paw thickness increased upto 90 min
of induction and then decreased steadily in treatment
animals as compared to control animals where the
paw thickness increased steadily. Within 180 min,
the paw thickness came back to nearly normal
size in diclofenac and 75 ml Hedera extract treated
animal (Table 1).
Percent inhibition was highest with 88.89%
inhibition with 75 ml Hedera extract at 180 min
after induction of inflammation as compared to
reference drug diclofenac, which was 94.44%
inhibition at 180 min after induction. Lower doses
of 25 ml and 50 ml Hedera extract also showed
61.11% and 77.7% inhibition, respectively. The
antiinammatory effect of 75 ml alcoholic Hedera
extract nearing the effect of diclofenac suggests its
strong antiinammatory property. However, the mice
could not tolerate peritoneal injections of more than
75 ml ethanol Hedera extract. Further works using
concentrated strengths of Hedera extract in ethanol
tolerable by the animals needs to be carried out in
future.
Control animals with oedema induced with formalin
were observed for 7 days. Arthritic properties with
swelling of joints of left leg and tail were observed.
These animals with swollen joints were taken for
further arthritic treatment with Hedera extract.
Erythrocyte sedimentation rate (ESR) of blood
was carried out in these animals with swollen
joints. Blood was collected by tail vein puncture
of the animals with a 1 ml syringe. The blood was
TABLE 1: EFFECT OF ETHANOL EXTRACT OF HEDERA HELIX AND DICLOFENAC ON LEFT PAW OEDEMA INDUCED
BY FORMALIN
Treatment Paw thickness (mm)
30 min 60 min 90 min 120 min 150 min 180 min
Control (PBS) 0.36±0.08 (0) 0.41±0.02 (0) 0.48±0.07 (0) 0.47±0.03 (0) 0.46±0.01 (0) 0.45±0.05 (0)
25 ml Hedera extract 0.33±0.04 (33.33) 0.35±0.01 (42.86) 0.37±0.03 (52.38) 0.36±0.03 (55.00) 0.35±0.03 (57.89) 0.35±0.04 (61.11)
50 ml Hedera extract 0.32±0.03 (44.40) 0.33±0.02 (57.14) 0.34±0.01 (66.7) 0.33±0.01 (70.00) 0.33±0.03 (73.68) 0.31±0.03 (77.78)
75 ml Hedera extract 0.31±0.04 (55.60) 0.32±0.04 (64.29) 0.33±0.07 (71.4) 0.32±0.01*(75.00) 0.33±0.01*(84.21) 0.29±0.01*(88.89)
Diclofenac 0.30±0.07 (66.67) 0.31±0.06 (71.43) 0.31±0.07*(80.95) 0.30±0.02*(85.00) 0.29±0.14*(89.47) 0.28±0.10*(94.44)
All values are mean±SD (n=8), *P<0.001 with respect to control, Values in parenthesis are % inhibition, PBS=Phosphate buffered saline

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anticoagulated with 12% ethylenediaminetetraacetic
acid and diluted 1:1 with sodium citrate. ESR was
measured using Westergren’s method with pipette
calibrated in mm from 0 to 200. ESR of normal
animals was regarded as control. ESR acts as a guide
to the progress of a disease. It is useful as an aid in
differential diagnosis.
For the treatment of arthritic symptoms, 75 ml Hedera
extract was injected at paw once daily for next
7 days. After 7 days treatment with Hedera extract,
blood was again collected by tail vein puncture and
whole trunk collection and ESR carried out.
Injection of 75 ml Hedera extract to the paws
with swollen joints once daily for 7 days showed
marked improvement on the swelling suggesting
that Hedera helix is a potent herb for the treatment
of arthritis in animals. ESR studies show that
ESR values before Hedera extract treatment is
3.4 mm/h as compared to normal and after Hedera
treatment (2.5 mm/h) (g. 1) indicating progression
towards recovery and decrease in inflammation in
the body.
Though, some work in plant extraction and
identication and synthesis of the various components
of the plant is being carried out[24,25] especially, for its
antitumour activity, the exact component that brings
about the antiinammatory, analgesic and antiarthritic
property needs to be isolated and tested. A new
horizon for herbal treatment has opened up and
Hedera helix would be a cost-effective and a potent
herbal medicine for the treatment of inflammation
and arthritis.
ACKNOWLEDGEMENTS
The author would like to thank Prof. Ashim K. Chakravarty,
Emeritus Professor, Centre for Life Sciences, NBU and Mr.
Tamal Mazumdar for extending their help in the course of
the study. The author would also like to thank the UGC for
granting the Minor Project and nancial help under which
this work was carried out.
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Accepted 11 January 2013
Revised 06 January 2013
Received 25 July 2011
Indian J Pharm Sci 2013;75(1):99-102
Viability of Human Melanocytes HEMa‑LP Exposed to
Amikacin and Kanamycin
D. WRZEŚNIOK, M. OTRĘBA, A. BEBEROK AND E. BUSZMAN*
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of Silesia, Jagiellońska 4, PL‑41‑200
Sosnowiec, Poland
Wrze śniok, et al.: Impact of Aminoglycosides on Melanocytes Viability
Aminoglycosides, such as amikacin and kanamycin, are powerful broad‑spectrum antibiotics used for the treatment
of many bacterial infections. The widely used aminoglycosides have the unfortunate side effects of targeting sensory
hair cells of the inner ear, so that treatment often results in permanent hair cell loss. The aim of the study was
to evaluate the influence of incubation time and drug concentration on viability of melanocytes cultured in the
presence of amikacin or kanamycin. The normal human melanocytes HEMa‑LP and the different concentrations
of amikacin (0.075, 0.75 and 7.5 mmol/l) and kanamycin (0.06, 0.6 and 6.0 mmol/l), were used. The estimations
were performed after 24, 48 and 72 h. The observed decrease in melanocytes viability may be an explanation for the
mechanisms involved in aminoglycosides toxicity on pigmented tissues during high‑dose and/or long‑term therapy.
Key words: Amikacin, cell viability, kanamycin, melanocytes
*Address for correspondence
E-mail: ebuszman@sum.edu.pl
Amikacin and kanamycin are recommended for the
treatment of Gram-negative and some Gram-positive
microorganisms infections, especially in severe and
bacteremic infections. However, an excessive dosage
of the aminoglycosides may cause the side effects of
ototoxicity and nephrotoxicity. In addition, a long-term
usage of these antibiotics may result in high and
persistent tissue residues due to their tissue afnity[1,2].
Many drugs are extensively accumulated in
melanin-containing tissues[3]. Antibiotics, psychotropic,
antirheumatic, anaesthetic and antitumour agents
are reported to possess high affinity to melanin
biopolymers[3-9]. However, a physiological function of
this binding is not fully understood.
It has been earlier demonstrated that amikacin[8] and
kanamycin[10] form stable complexes with model
synthetic melanin in vitro. Drug-melanin interactions
are still not fully characterised, partly because
melanins are not well-dened chemical entities but
rather mixtures of more or less similar polymers
apparently made up of different structural units linked
by nonhydrolysable bonds.
Melanin is synthesised in the melanosomes in
melanocytes and produced by a process that