Potential Indian Medicinal Plants

Against Psoriasis

Dr. Sushil K. Singh

Department of Pharmaceutics

Institute of Technology

Banaras Hindu University

Varanasi – 221005, India

A non-contagious, multi-factorial chronic inflammatory

skin disorder.

Psoriasis

2 – 3 % population afflicted worldwide

10-30 % people with psoriasis develop psoriatic

arthritis

3 billion USD annual cost to the society

Can occur in any part of body including nails, scalp,

palms and soles, beard and pubic area and is triggered by

Environmental Genetic Immunological

Psoriasis

Patches of thick, red skin, covered with silvery scales.

Hyper proliferation of Keratinocytes

Abnormal epidermal differentiation.

Infiltration of inflammatory cells

Characterized by

Psychological difficulties : behavioral avoidance, excessive worrying, depression

ORAL : Methotrexate, Acitretin,

Cyclosporin,

TOPICAL : Corticosteroids, Coal tar,

Dithranol, Vitamin D analogues,

Retinoids

UV LIGHT : Phototherapy, Laser,

Photochemotherapy (PUVA)

BIOLOGICALS : Recombinant cytokines,

Anticytokines, Infliximab,

Etanercept, Alfacept

Management of Psoriasis

REOCCURRENCE, TOXICITY, HIGH COST

Aloe vera

Azadirachta indica

Argemone mexicana

Centella asiatica

Calendula officinalis

Coptis chinensis

Daucus carota

Glycyrrhiza glabra

Glycyrrhiza uralensis

Momordica charantia

Oenothera biennis

Psoralea corylifolia L

Podophyllum emodi

Rubia cordifolia

Withania somnifera

Crotalaria juncea

Zea mays

Leucas aspera

Indian medicinal plants used in skin disorder

Leucas aspera

Herbaceous, annual plant (30 – 35

cm.) Family: labiatae

Distributed throughout Asia, China

and tropical American countries.

Traditionally used in cold and cough, wound healing,

insecticide, snake bite, skin diseases, rheumatism

Chemical ConstituentsAliphatic compounds : 28-Hydroxy pentatriacontan-7-one, 7–hydroxy

dotriacontan-2-one, 5-acetoxytriacon-tane, 1- hydroxytetratriacontan-4-one, oleic acid,

32-methyltetratria-contan-8-ol, linoleic acid

Monoterpene lactone : Isololiolide

Diterpenes : Leucasperone A and B, Leucasperol A and B Leucasperoside A, B and C, Linifolioside

Steroids : β – Sitosterol, Stigmasterol-3-O-β-glucoside

Triterpenoids : Leucolactone, Maslinic acid,

Volatile compounds : α-Farnasene, α-thujene, Menthol, Amylpropionate, Isoamylpropionate

Alkaloid(s) : Nicotine

Lignans and Flavonoids

Extract mass (67 g)

Fraction (51g)

Leucas aspera (leaves,2kg)

n-Hexane Fraction (7g)

CHCl3

Fraction (14g)

EtOAc, Fraction (9g)

n-Butanol Fraction (12g)

Extrd. twice with MeOH (20L)

Evpn. in vaccuo (40 – 45 ºC)

Leucosperoside A & C, Leucassperon A

Maslinic acid,

β – sitosterol, stigmasterol

Linoleic acid, oleic acid

Flavanoids, Alkaloids, Lignans

Diaion HP – 200 CC

CC/ODS flash

m/z 437.43, m/z 795.77

Preparation of test substance (for all assay)

• Medium FDMEM: Dulbecco’s Modified Eagle

Medium with 10% fetal bovine serum (FBS), 1%

penicillin, 2% streptomycin• Extractives dissolved in MeOH, and Dithronol in

DMSO, sterilization by filtration (0.2 μm) & dilution

by sterilized FDMEM

( Conc. Dithronol 0.33–170 Extractives )

Keratinocyte anti proliferent effect of Extractives (in vitro)*

* Sampson et al., Phytomedicine, 8 (3), 230 (2001)

• SVK-14 Keratinocyte cells cultured in FDMEM, 10%

CO2, at 37 ºC

• Cells washed with Ca/Mg free Phosphate Buffer Saline

(PBS), decanted and cells were detached by 5 ml

Trypsin (0.05%) in EDTA (0.02%), vol. adjusted to

50ml with PBS• Cell plate obtained by centrifugation (1000 g, 5 min.) &

re-suspended 10 ml FDMEM• Cell density adjusted to 25000/ml with FDMEM (cells

counted by haemocytometer)

Preparation of cell culture

• Cells (5000 in 200 µl) inner wells & 200 µl of 10 % FBS

in PBS in outer wells, inoculated 24 h, at 37 ºC, 10

%CO2

• Plating medium replaced with FDMEM containing test

material /dithranol in 200 µl., each sample conc. tested

with 6 replicate• Cells exposed to FDMEM alone serves 100 % growth

control

• Cells inoculated for 7 days at 37 ºC; 10% CO2 prior to

Sulphorhodamine( SRB )assay

Microtitre Assay

• Cells fixed by layering 100 μl of ice-cold 50%

trichloroacetic acid on growth medium &

incubated at 4 ºC for 1 hour

• Plates washed with cold water + SRB strain (50 μl,

0.4% in 1% acetic acid ) in each well, washed with

1% acetic acid and dried

• 100 μl of 10 µM Tris buffer to solublize dye

• Absorbance read at 550 nm by plate reader

Sulphorhodamine (SRB) Assay

Dose response curves reveal that Chloro-form fraction and methanol extract show a good anti-proliferate keratinocyte activity than ethylacetate and n- Butanol fraction

Presence of terpenoids/steroids/fatty acids in chloroform fraction may be responsible for the activity.

Results and Discussion

0

20

40

60

80

100

0.1 0.2 0.3 0.4 0.5 1 1.5

Log Conc.of plant extractives

Me

an

ce

ll ±

SD

as

a

pe

rce

nt

of

co

ntr

ol

Methanol extract of L. aspera leaves

Chlorofom fraction

Ethylacetate fraction

n-Butanol fraction

Plant Extractives/Standard

IC50 (μg/ml)

Methanol extract 151.33 ± 0.7

Chloroform fraction 93.33 ± 0.6

Ethylacetate fraction 575.33 ± 0.7

n-Butanol fraction 912.01 ± 0.9

Dithronol 2.2 ± 0.2

Determination of IC50

Thank YouThank You