potential indian medicinal plants against psoriasis dr. sushil k. singh department of pharmaceutics...
TRANSCRIPT
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