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Journal of Pharmacy
Research Paper
And Pharmacology

Antipsoriatic activity of extracts and fractions obtained from

Memecylon malabaricum leaves
Sangai Palanisamy Dhanabala, Nithyanantham Murugananthama, Kabbur Hanumanthappa Basavarajc,
Asish Wadhwanib and Nonavinakere Mannar Shamasundard
a
Department of Phytopharmacy and Phytomedicine, (TIFAC CORE HD), bDepartment of Pharmaceutical Biotechnology, JSS College of Pharmacy,
Rocklands, Ooty, Departments of cDermatology and Venerology and dAnatomy, JSS Medical College, Mysore, India

Keywords
HaCaT cells; lipoxygenase; Memecylon
malabaricum; mouse tail test
Correspondence
Sangai Palanisamy Dhanabal, Department of
Phytopharmacy and Phytomedicine (TIFAC
CORE HD), JSS College of Pharmacy, Post box
no. 20, Rocklands, Ooty 643001, India.
E-mail: dhanabalsp@rediffmail.com
Received October 13, 2011
Accepted March 26, 2012
doi: 10.1111/j.2042-7158.2012.01528.x
Abstract
Objectives This study involves the evaluation of Memecylon malabaricum Cogn.
(Melastomataceae) leaves for antipsoriatic activity.
Methods Aqueous extract, hydroalcoholic extract and their fractions of M. mala-
baricum leaves were evaluated for in-vivo antipsoriatic activity by mouse tail test and
for in-vitro antipsoriatic activity using HaCaT cells, lipoxygenase inhibition and
thymidine phosphorylase inhibition assays. Extracts and fractions were evaluated
for total phenol and flavonoid contents. HPTLC was used for screening and finger-
print analysis of the extracts and active fraction.
Key findings M. malabaricum hydroalcoholic extract (MMHA) and water fraction
of MMHA (MMHAW) produced significant (P < 0.05) percent orthokeratosis in
the mouse tail test. All samples except MMHA showed a significant (P < 0.05) reduc-
tion in epidermal thickness in the mouse tail test when compared with control.
Maximum activity against HaCaT cells was shown by chloroform fraction of
MMHA (MMHAC). The M. malabaricum decoction (MMD) and water fraction of
MMD (MMDW) showed equally good inhibition of lipoxygenase. In thymidine
phosphorylase inhibition assay only MMD showed activity.
Conclusions The findings of this investigation reveal that the leaves of M. mala-
baricum have good antipsoriatic potential, which provides scope for further detailed
research in to this plant for psoriasis.

Introduction
Psoriasis is a chronic recurrent problem affecting skin, nails
and joints. It affects 0.64.8% of the general population and is
grouped under the papulosquamous disorders.[1,2] Intensive
research is ongoing globally for effective management of the
disease. Currently available therapeutic options include both
topical (emollients, moisturizers, tars, anthralins, topical cor-
ticosteroids, vitamin A analogues and vitamin D analogues)
and systemic treatments (corticosteroids, methotrexate,
ciclosporin, etretinate (retinoids) and other immunomodu-
lators, and hydroxyurea, among others).[3,4] Phototherapy
and photo-chemotherapy are also being used extensively.
New biological drugs have also been approved by the US Food
and Drug Administration (FDA). Affordability, availability
and side effects of prolonged use of the above therapies still
remain a challenge and concern.[5,6]
Psoriasis of human skin is characterized by focal to coalesc-
ing raised cutaneous plaques with consistent scaling and
variable erythema.[7] Typical histological features of psoriasis
include epidermal hyperplasia (acanthosis) with elon-
gated rete ridges, a less discrete epidermal granular layer
(hypogranulosis), parakeratosis and leucocyte infiltration of
the dermis and epidermis.[79]
Many experimental animal models have been developed
for testing drugs for antipsoriatic activity[10] but none of them
exactly reproduce the entire histopathological spectrum seen
in human psoriasis. A major problem in the development of
an antipsoriatic drug is the lack of a direct animal model. One
simple model that is widely used for the evaluation of drugs
for psoriasis is the mouse tail test.[1113] This is a morphometry
based, relatively sensitive and well reproducible method. It
allows quantitative evaluation of the effects of antipsoriatic
drugs on epidermal differentiation.[14,15] The parakeratotic
condition is seen in the adult mouse tail and is one of the hall-
marks of psoriasis.[16] The granular layer of the epidermis
is greatly reduced or absent in psoriatic lesions. Induction
of orthokeratosis in parts of the adult mouse tail, which nor-
mally has a parakeratotic differentiation, is the basis behind
the mouse tail test.[17]

2012 The Authors. JPP 2012

Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, , pp. 1
Antipsoriatic activity of M. malabaricum
In this study the mouse tail test was used for assessing
antipsoriatic activity. Normal mice display the parakeratotic
condition in their tails. Potential antipsoriatic drugs have
the capacity to convert the parakeratosis into orthokeratosis.
This method does not involve the induction of psoriasis
and is a simple method for evaluating antipsoriatic drug
candidates. Many drugs presently used in the treatment
of psoriasis have been evaluated by the mouse tail test
and showed good efficacies.[18] Antipsoriatic activity of the
extracts of Caesalpinia bonduc were reported using the
mouse tail test.[19]
Memecylon malabaricum (C. B. Clarke) Cogn. (Melasto-
mataceae) is a shrub or small tree, with clusters of blue
flowers, found on the lower slopes of Western Ghats in Kerala
and in the moist sholas of the Niligiri and Palani hills up to an
altitude of 2000 m. The root has ecbolic properties. A decoc-
tion of the flowers and twigs has been used to treat skin dis-
eases.[20] It was found from our field survey that the leaves of
M. malabaricum have been traditionally used in the treat-
ment of psoriasis by traditional Siddha healers in the Malabar
region of Karnataka, India. So far this plant has not been
scientifically evaluated for antipsoriatic activity. Scientific
evaluation of traditionally used plants for various diseases is
increasing worldwide. Evaluation of plants for psoriasis treat-
ment is of paramount importance due to the conditions
severity, occurrence and problems in existing therapies.
Hence, this study was carried out to evaluate the leaves of
M. malabaricum for antipsoriatic activity. This study is the
first of its kind for the leaves of M. malabaricum.
Materials and Methods
Plant material
Leaves of Memecylon malabaricum collected from the forest
areas in Malabar region, Karnataka between June and August
2007 was authenticated by Dr Rajan, Botanist, Survey of
Homoeopathic Medicinal Plants and Collection Unit, Dept.
of AYUSH, Ootacamund and a voucher specimen (No. JSSU/
OT/119) was deposited at the Department of Phytopharmacy
and Phytomedicine, JSS College of Pharmacy, Ootacamund,
Tamilnadu, India.
Extraction and fractionation
M. malabaricum decoction (MMD) was prepared by boiling
500 g of powdered leaves (twice) in 3000 ml of distilled water
for 30 min at 70C. The combined extract was filtered,
concentrated and evaporated to obtain the extract with
12.9% yield. Similarly, M. malabaricum hydroalcoholic
extract (MMHA) was prepared separately from another 500 g
of leaf powder using 50% ethanol by reflux and the extract
obtained with 26% yield. MMD was dissolved in distilled
water and fractionated with water-saturated n-butanol to
2 Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, , pp.
Sangai Palanisamy Dhanabal et al.
give the butanol fraction of MMD (MMDB, yield 9%) and
remaining water fraction of MMD (MMDW, yield 88%).
MMHA was dissolved in distilled water and successively
fractionated with chloroform and water-saturated n-butanol
to give the chloroform fraction of MMHA (MMHAC, yield
0.99%), butanol fraction of MMHA (MMHAB, yield 50%)
and water fraction of MMHA (MMHAW, 44.9%).
Phytochemical investigation
Total polyphenolic compounds of the extracts and fractions
were determined according to a protocol similar to that of
Chandler and Dodds.[21] Quantification was based on a stan-
dard curve, established with 20, 40, 60, 80 and 100 mg/ml of
gallic acid and the results were expressed as gallic acid equiva-
lent in milligrams per gram dry weight (mg GAE/g DW) of
the sample. Aluminum chloride colorimetric method was
used for determination of flavonoids.[22] The concentration
of total flavonoids was found by using a standard curve
established with quercetin and the results were expressed as
quercetin equivalents in milligrams per gram dry weight
(mg QE/g DW) of the sample.
HPTLC analysis was performed for M. malabaricum
powder, MMD, MMHA and MMHAW for developing finger-
prints and to find any constituents of biological importance.
Solutions of M. malabaricum powder (100 mg/ml, 20 ml),
MMD (50 mg/ml, 30 ml), MMHA (50 mg/ml, 25 ml) and
MMHAW (50 mg/ml, 30 ml) were used for the HPTLC finger-
print analysis and screening for quercetin. The mobile phase
used for fingerprint and screening was n-butanolacetic acid
water (10 : 4 : 3 : 2) and ethyl acetateformic acidglacial
acetic acidwater (100 : 11 : 11 : 26), respectively. All samples
were analysed as per the HPTLC method described below.
Pre-coated TLC silica gel 60 F254 plates (Merck, Darmstadt,
Germany) were used as stationary phase. Samples were
applied as an 8 mm band using a Camag Linomat IV applica-
tor (Camag, Muttenz, Switzerland). Application was made on
the plate at a distance of 15 mm from the bottom and 12 mm
from the left margin with a distance of 6 mm between the
tracks at a constant application rate of 10 s/ml using a nitro-
gen aspirator. Development was carried out in a Camag twin
trough development chamber which had been previously
saturated with the specified mobile phase. The length of chro-
matogram run was maintained at 6 cm from the applied posi-
tion. Subsequent to development, TLC plates were dried in a
current of air with the help of a hair-dryer. The slit dimension
setting of length 4 mm and width 0.30 mm, and a scanning
rate of 20 mm/s and data resolution of 100 mm/step were
used. A deuterium lamp, mercury lamp and tungsten lamp
were used for scanning at 225 nm, 366 nm and 400800 nm,
respectively. Peaks were identified by densitometric scanning
using a Camag TLC scanner III and the results were obtained
with the help of winCATS software version 1.4.3.
2012 The Authors. JPP 2012
Sangai Palanisamy Dhanabal et al.
Mouse tail test for psoriasis
Healthy albino mice, 2530 g, were obtained from the animal
house, JSS College of Pharmacy, Ootacamund, India with due
approval from the Institutional Animal Ethics Committee
(Registration No. JSSCP/IAEC/ICMR/01/2008/2009). They
were maintained under standard conditions (12-h light
dark cycle, 25 3C, 4565% humidity) and were given free
access to standard rat feed and water. The studies were per-
formed as per CPCSEA, India guidelines and the experimen-
tal protocol was approved by the Institutional Animal Ethics
Committee, JSS College of Pharmacy, Ootacamund, India.
The mice were divided in to nine groups of six each. Group
1 and 2 served as normal and positive controls, respectively.
Normal saline (10 ml/kg body weight) was given to group 1
and retinoic acid (0.5 mg/kg body weight) was given to group
2. the remaining groups received the test samples (extracts
at 500 mg/kg body weight and fractions at 250 mg/kg body
weight). All test solutions were prepared in water and admin-
istered orally. Insoluble samples were administered as a sus-
pension in water immediately after intimate mixing. Mice
received their treatment once daily for five days a week for two
weeks. Mice were sacrificed at the end of the experiment by
deep ether anaesthesia and the proximal parts of their tails cut
and stored in separate containers containing 10% formalin
in saline.
Longitudinal histological sections of the tail skin were
prepared from the tail of each mouse and stained with
hematoxylin-eosin (HE). The specimens were histometri-
cally analysed for the horizontal length of an individual scale
(1) and the horizontal length of fully developed granular layer
within an individual scale (2). For (1) and (2), measurements
were performed in 10 scales per mouse in six mice per treat-
ment group (i.e. a total of 60 measurements per treatment
were performed). The vertical epidermal thickness (3) of the
skin was measured (n = 5 measurements per scale, n = 10
scales per mouse and hence a total of 300 measurements
performed per treatment). From these raw data, (1) to (3), the
following parameters were calculated according to Bosman
et al.[11]: the degree of orthokeratosis (OK) of an individual
scale, which is the percentage ratio of (2)/(1), the control
related drug activity upon epidermal differentiation:
Drug activity = (OKs OKc) 100 (100 OKc) (1)
where OK is the mean of the parameter for a test substance (s)
and the control (c), respectively, and the relative epidermal
thickness of individual scale, which is the percentage ratio of
the measure under (3), for a given treatment in relation to the
mean of controls set to 100% (n = 300 data per treatment con-
dition). The three overall parameters, namely, the degree of
orthokeratosis, drug activity and relative epidermal thickness,
were eventually used for the evaluation of drug effects.[17]
2012 The Authors. JPP 2012
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, , pp.
Antipsoriatic activity of M. malabaricum
Antiproliferative activity against HaCaT cells
The HaCaT cell line contains human keratinocytes and
multiplies at a higher than normal rate similar to psoriatic
epidermal cells and has been used previously for the evalua-
tion of antipsoriatic drugs.[19,2326] Human HaCaT kerati-
nocytes were obtained from the National Centre for Cell
Sciences, Pune, India and seeded at the concentration of
1.0 105 cells/ml in a 96-well microtitre plate and grown
in Dulbeccos modified Eagles medium (DMEM; Gibco,
Carlsbad, CA, USA) containing 10% fetal bovine serum
(Biowest, Miami, FL, USA). After 24 h, the supernatant was
decanted and the monolayer was washed once. Then 100 ml
of test substance in various concentrations was added to
the cells in microtitre plates. Test compounds were prepared
in dimethyl sulfoxide (DMSO) and then diluted with
DMEM; the final concentration of DMSO was 0.2% in the
culture medium. Controls were performed with DMSO
or medium alone. Asiaticoside (Sigma, St. Louis, MO, USA)
was used as positive control. The plates were then incubated
at 37C for three days in 5% CO2 atmosphere. The anti-
proliferative activity was assessed by performing the
sulphorhodamine B (SRB) assay as described by Skehan
et al.[27] Cells were fixed by adding 25 ml of ice-cold 50%
trichloroacetic acid on top of the growth medium and the
plates were incubated at 4C for 1 h, after which plates were
washed with cold water and air dried. SRB stain (50 ml; 0.4%
in 1% acetic acid) (Sigma) was added to each well and left
in contact with the cells for 30 min, after which they were
washed with 1% acetic acid. The plates were then dried,
100 ml of 10 mm Tris buffer (Sigma) added and were shaken
gently for 5 min. Absorbance was read at 550 nm using a
micro plate reader. Results obtained at different concentra-
tion were used for calculating IC50 value, which may be
defined as the concentration of a substance at which 50%
inhibition is seen.
Lipoxygenase inhibition assay
Lipoxygenase (LOX) inhibitor screening assay kit (Cayman,
Ann Arbor, MI, USA) was used for evaluating the LOX inhi-
bitory activity of the extracts and fractions by following the
kit procedure (Catalog no. 760700). The reaction mixture
containing 10 ml of sample (dissolved in DMSO), 10 ml of
linoleic acid substrate and 90 ml of enzyme (15-lipoxygenase)
was mixed well for 5 min. Finally, 100 ml of chromogen was
added, mixed well for 5 min and the absorbance read at
490 nm using an ELISA reader (Biorad, Hercules, CA, USA).
Quercetin was used as positive control.
Thymidine phosphorylase inhibition assay
The thymidine phosphorylase (TPase) inhibition assay was
carried according to Sigma protocol with little modification.
3
Antipsoriatic activity of M. malabaricum Sangai Palanisamy Dhanabal et al.

In brief, 2.090 ml of 200 mm phosphate buffer, along with
test samples/positive control of various concentrations (in
buffer/DMSO), 10 ml of enzyme (0.4 Units/ml) and 900 ml
of thymidine (3 mm) were mixed well and incubated at 25C
for 60 min. Absorbance was measured at 290 nm in a UV-Vis
spectrophotometer. Gallic acid was used as positive control.
Statistical analysis
Statistical calculations were performed using GraphPad
prism software. Results obtained are presented as weighed
mean standard error. In the mouse tail test, KruskalWallis
test followed by Dunns test was used. P < 0.05 was con-
sidered significant.
Results
Phytochemical studies
The total phenol and flavonoid contents of the extracts and
fraction of M. malabaricum leaves are presented in Table 1.
All the extracts and fractions were found to contain a good
amount of phenols, except for MMHAC, with the highest
amount present in MMHA (115.78 2.02 mg GAE/g).
Appreciable amounts of flavonoids were also found to be
present in the extracts and fractions, except in MMDW,
with the maximum amount present in MMHAC
(44.22 0.14 mg QE/g). Quercetin was found to be present
in M. malabaricum leaf powder, MMD, MMHA and
MMHAW by HPTLC screening, while rutin was not detected
in these samples. Detection wavelengths of 254 nm and
366 nm were used for quercetin and rutin, respectively.
HPTLC fingerprints for these samples resulted in a good
number of peaks obtained at two different wavelengths of
225 nm and 366 nm. HPTLC fingerprints of M. malabari-
cum leaf powder, MMD, MMHA and MMHAW are shown
in Figure 1.
Mouse tail test for psoriasis
Data for the degree of orthokeratosis and the relative epider-
mal thickness resulting from the application of the extracts
and fractions in the mouse tail test are presented in Table 2.
Statistical analysis for the main parameter (i.e. degree of
orthokeratosis) showed that MMHA and MMHAW had effi-
cacies which were significant (P < 0.05) compared with the
control in the induction of epidermal differentiation, with
MMHA > MMHAW. These evaluations were re-emphasized
when considering drug activity, which may be defined as the
relative orthokeratotic activity of a sample with respect to
control. The drug activity was calculated using the formula
given in Equation 1. In detail, the drug activity was 31.01% for
MMHA and 22.00% for MMHAW (Table 2).
Compared with control, significant (P < 0.05) difference in
the epidermal thickness was found in groups treated with
MMDW, MMHAB and MMHAW (Table 2). Representative
photomicrographs of the histological sections of mouse tail
skin samples are shown in Figure 2. The granular layer is the
layer beneath the stratum corneum. Its formation is clearly
noticeable in the group treated with standard retinoic acid
(Figure 2b), which is marked by an arrow. Similarly, groups
treated with MMHA (Figure 2f) and MMHAW (Figure 2i)
showed significant granular layer formation whereas the
control group (Figure 2a) did not.
In-vitro studies
Potent antiproliferative activity against HaCaT cells was shown
by MMHAC (IC50, 57.4 3.11 mg/ml), followed by MMHAB,
MMDB and MMDW (Table 1). The other samples, MMD,
MMHA and MMHAW, did not show activity until a concen-
tration of 500 mg/ml and hence were considered inactive.
Maximum LOX inhibition activity was found for MMDW
with an IC50 value of 134.61 8.07 mg/ml (Table 1),
followed by MMD, MMHAW, MMDB and MMHA. Both

Table 1 Results of in-vitro studies and quantitative estimations

IC50 (mg/ml)

Samples TP (mg GAE/g) TF (mg QE/g) Cytotoxicity against HaCaT Cells LOX inhibition assay TPase inhibition assay

MMD 72.70 3.44 13.31 0.09 >500 138.63 6.75 289.5 0.71
MMDB 32.32 0.70 8.97 0.18 155.8 13.50 269.22 15.82 >500
MMDW 15.46 0.46 -0.84 0.01 185.8 14.70 134.61 8.07 >500
MMHA 115.78 2.02 23.43 0.49 >500 411.73 23.16 >500
MMHAC -10.52 2.10 44.22 0.14 57.4 03.11 >1000 >500
MMHAB 44.02 0.70 15.36 0.79 156.3 10.70 >1000 >500
MMHAW 22.57 0.56 4.12 0.01 >500 197.19 10.61 >500

GAE, gallic acid equivalent; IC50, concentration at which 50% inhibition is seen; LOX, lipoxygenase; MMD, Memecylon malabaricum decoction; MMDB,
butanol fraction of MMD; MMDW, water fraction of MMD; MMHA, M. malabaricum hydroalcoholic extract; MMHAB, butanol fraction of MMHA;
MMHAC, chloroform fraction of MMHA; MMHAW, water fraction of MMHA; QE, quercetin equivalent; TF, total flavonoid; TP, total phenol; TPase,
thymidine phosphorylase. Results are shown as the mean SE of three values.

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4 Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, , pp.
Sangai Palanisamy Dhanabal et al. Antipsoriatic activity of M. malabaricum

Track 1. ID: MM leaf powder Track 1. ID: MM leaf powder

1000 1000
AU AU
MM at 225 nm MM at 366 nm
900 900

800 800

700 700

600 600

500 6 500 6
4
400 400
3 3
7
300 2 300 89 12
5 9 2 10
1 1 7
8 5 11
200 4 200

100 100

0 0
0.10 0.01 0.21 0.41 0.61 0.81 1.01 0.10 0.01 0.21 0.41 0.61 0.81 1.01
R1 R1
Track 2. ID: MMD Track 2. ID: MMD
1000 1000
AU AU
MMD at 225 nm MMD at 366 nm
900 900

800 800

700 700

600 600

500 500
6
400 400 8
3 7
18
300 7 8 10 300 2
5 12 13
2 9 56 11 14 1617
1 4 9 10 15
200 3 200 1
4
100 100

0 0
0.10 0.01 0.21 0.41 0.61 0.81 1.01 0.10 0.01 0.21 0.41 0.61 0.81 1.01
R1 R1
Track 3. ID: MMHA Track 3. ID: MMHA
1000 1000
AU AU
MMHA at 225 nm MMHA at 366 nm
900 900

800 800

700 700

600 600

500 500
8
4 4 14
400 400
7
11
300 5 300 10 12
6 9 13
1 2 7
200 200 1
3 6
23 5
100 100

0 0
0.10 0.01 0.21 0.41 0.61 0.81 1.01 0.10 0.01 0.21 0.41 0.61 0.81 1.01
R1 R1
Track 4. ID: MMHAW Track 4. ID: MMHAW
1000 1000
AU AU
MMHAW at 225 nm MMHAW at 366 nm
900 900

800 800

700 700

600 600

500 500

400 10 400 3 16

300 1 300
6 8 9

200 200 1 7 1314 15

7 9 6 1112
23 4 5 8 2 45 10
100 100

0 0
0.10 0.01 0.21 0.41 0.61 0.81 1.01 0.10 0.01 0.21 0.41 0.61 0.81 1.01
R1 R1

Figure 1 HPTLC fingerprints of Memecylon malabaricum leaf, its extracts and fractions at 225 nm and 366 nm. MM, M. malabaricum leaf; MMD,
M. malabaricum decoction; MHA, M. malabaricum hydroalcoholic extract; MMHAW, water fraction of M. malabaricum hydroalcohoic extract.

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Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, , pp. 5
Antipsoriatic activity of M. malabaricum
Table 2 Effect of extracts and fractions on the degree of orthokeratosis and relative epidermal thickness as well as the drug activity in the mouse
tail test
Degree of
Treatment groups orthokeratosis (%)
Normal saline (10 ml/kg) 15.18 3.09
Retinoic acid (0.5 mg/kg) 65.23 3.09*
MMD (500 mg/kg) 28.17 2.34
MMDB (250 mg/kg) 14.90 2.17
MMDW (250 mg/kg) 14.88 1.50
MMHA (500 mg/kg) 41.48 1.60*
MMHAC (250 mg/kg) 19.99 1.91
MMHAB (250 mg/kg) 18.20 0.75
MMHAW (250 mg/kg) 35.84 1.48*
MMD, Memecylon malabaricum decoction; MMDB, butanol fraction of MMD; MMDW, water fraction of MMD; MMHA, M. malabaricum
hydroalcoholic extract; MMHAB, butanol fraction of MMHA; MMHAC, chloroform fraction of MMHA; MMHAW, water fraction of MMHA; NA, not
applicable. Results are shown as the mean SE. Significance: *P < 0.05 vs control group values.
MMHAB and MMHAC showed no LOX inhibition even at a
final concentration of 1000 mg/ml.
In the TPase inhibition assay, only MMD showed good
activity with an IC50 value of 289.5 0.71 mg/ml (Table 1).
Discussion
In this study, MMHA, which caused maximum orthokerato-
sis, produced relatively little change in the epidermal thick-
ness. However, MMHAW produced significant thinning of
the epidermis when compared with control. Production of
orthokeratosis is the main parameter that is related to antip-
soriatic activity and the change in epidermal thickness in the
mouse tail test is unclear. It is understood from previously
reported studies that topical testing of drugs in the mouse tail
test most often results in the thickening of the epidermis
despite the good orthokeratotic activity, except for topical
corticosteroids.[18] However, we did not find any correlation
between the orthokeraotosis and change in the epidermal
thickness, which may be due to the oral administration of
drugs in this study.
Hyperproliferation of epidermis is a notable condition
among the many complex alterations of various cell types in
psoriasis. Epidermal cells of the normal skin shed at a rate
such that the average life span of a cell is about 28 days.
However, in psoriasis the cells are produced and shed at an
increased rate such that the average life span of a cell is only
three or four days.[28] As a model of epidermal hyperprolifera-
tion in psoriasis, we used HaCaT cells, a rapidly multiplying
human keratinocyte cell line,[2326] to evaluate the antiprolif-
erative effect of Memecylon malabaricum. The soundness of
the assay was ascertained from the IC50 value of the standard
asiaticoside (10.34 0.34 mg/ml). MMHAC, having shown
potent antiproliferative activity in HaCaT cells, was found to
be inactive in the induction of orthokeratosis in the mouse
tail test. The activity of MMHAC may be attributed more
6 Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, , pp.
Sangai Palanisamy Dhanabal et al.
Drug Relative epidermal
activity (%) thickness (%)
NA 100.00 9.32
59.01 57.46 9.65
15.31 72.54 2.64
-0.33 57.20 5.31
-0.35 52.18 4.04*
31.01 98.13 9.19
05.67 56.86 4.08
03.56 52.41 0.81*
22.00 54.30 3.28*
to the control of epidermal cell proliferation than to the
inflammatory pathways of psoriasis.
Lipoxygenases (LOXs) are enzymes in the arachidonic acid
pathway that catalyse peroxidation of polyunsaturated fatty
acids in a selective way. The products of LOX-catalysed oxy-
genation are involved in the development and maintenance
of psoriasis.[29] Hence, inhibition of the LOX enzyme may
possibly help in controlling psoriasis. LOX inhibition studies
of the samples reveal that the enzyme inhibition activity
is attributed to the polar constituents of M. malabaricum,
which is evident from the decreasing activity of samples,
MMDW > MMD > MMHAW > MMDB > MMHA, where
the polarity of the samples are in the decreasing order from
MMDW to MMHA. The assay results were confirmed by the
use of quercetin as positive control, which showed an IC50
value of 11.56 1.47 mg/ml.
Platelet-derived endothelial cell growth factor/thymidine
phosphorylase catalyses the reversible phosphorolysis of
thymidine and related analogues to 2-deoxy-d-ribose-1-
phosphate and their respective bases. 2-Deoxy-d-ribose-1-
phosphate is quickly dephosphorylated to 2-deoxy-d-ribose,
which freely diffuses out of the cell. It has been proposed that
2-deoxy-d-ribose is essential for the angiogenic activity of
thymidine phosphorylase.[3032] High levels of the enzyme
thymidine phosphorylase are seen in psoriatic lesions and
this enzyme is reported to be involved in psoriasis.[33,34]
Thus inhibition of thymidine phosphorylase is likely to be
supportive in psoriasis. Gallic acid standard showed good
activity in the TPase inhibition assay with an IC50 value of
128.75 8.84 mg/ml. Except for MMD, all samples were
inactive in the assay.
M. malabaricum was found to be a rich source of phenols
and flavonoids as seen from Table 1. So the extracts and frac-
tions were analysed by HPTLC for the presence or absence of
quercetin and rutin (common plant flavonoids). Results of
the HPTLC screening revealed the presence of quercetin in
2012 The Authors. JPP 2012
Sangai Palanisamy Dhanabal et al. Antipsoriatic activity of M. malabaricum

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Figure 2 Longitudinal histological sections of mouse tail skin treated orally for two weeks , HE staining (original magnification 40). (a) Vehicle control;
(b) retinoic acid 0.5 mg/kg; (c) M. malabaricum decoction (MMD) 500 mg/kg; (d) butanol fraction of MMD 250 mg/kg; (e) water fraction of MMD
250 mg/kg; (f) M. malabaricum hydroalcoholic extract (MMHA) 500 mg/kg; (g) chloroform fraction of MMHA 250 mg/kg; (h) butanol fraction of
MMHA 250 mg/kg; and (i) water fraction of MMHA 250 mg/kg. Retinoic acid induced orthogranulosis (indicated by an arrow) is clearly seen over the
whole horizontal length of the scale (b), whereas a granular layer is missing in most parts of the control specimen (a).

M. malabaricum leaf powder extracts and fractions. Querce-
tin has been found to be an effective inhibitor of phospholi-
pase A2 (PLA2) from human and rabbit leucocytes. PLA2
catalyses the hydrolysis of phospholipids esterified at the
second carbon in the glycerol backbone. Arachidonic acid is
commonly esterified in this position and the action of PLA2
releases arachidonic acid for subsequent metabolism via
the cyclooxygenase and lipoxygenase pathways.[35] The anti-
inflammatory effects of phenolics are mainly attributable to
inhibition of leukotriene synthesis.[36] Psoriasis is a chronic
inflammatory skin disease where cytokines play a central role.
Cytokines are produced via the LOX pathway.[7] Hence it can
be said that the flavonoid and phenolic constituents present
in the extracts and fractions may be responsible for their
antipsoriatic activity.
Conclusions
The results of this study show that MMHA, which displayed
significant activity in the mouse tail test for psoriasis, did
not have good activity in any of the three in-vitro studies
performed. MMHAW, besides showing significant in vivo

2012 The Authors. JPP 2012

Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, , pp. 7
Antipsoriatic activity of M. malabaricum Sangai Palanisamy Dhanabal et al.

activity, showed moderate activity in the LOX inhibition Declarations

assay. The developed HPTLC fingerprints can be used for
assessing the quality of new batches of raw material
(M. malabaricum leaf powder), extracts (MMD and
MMHA) and fractions (MMHAW). This investigation
shows that M. malabaricum leaf, as a whole, has antipsoriatic
potential, supporting its traditional use by the Siddha healers.
However, detailed phytochemical analyses of the extracts
and fractions need to be performed for the identification
of the constituents responsible for the antipsoriatic activity
of M. malabaricum.
Conflict of interest
The Author(s) declare(s) that they have no conflicts of
interest to disclose.
Funding
This work was supported by a grant from the Depart-
ment of Biotechnolgy, New Delhi, India (SAN no. 102/IFD/
SAN?PR1174/20062007 dated 23-11-2006).

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