Transcript OTCM1

Potential Indian Medicinal Plants
Against Psoriasis
Dr. Sushil K. Singh
Department of Pharmaceutics
Institute of Technology
Banaras Hindu University
Varanasi – 221005, India
Psoriasis
A non-contagious, multi-factorial chronic inflammatory
skin disorder.
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
Characterized by
Patches of thick, red skin, covered with
silvery scales.
Hyper proliferation of Keratinocytes
Abnormal epidermal differentiation.
Infiltration of inflammatory cells
Psychological difficulties : behavioral avoidance, excessive
worrying, depression
Management of Psoriasis
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
REOCCURRENCE, TOXICITY, HIGH COST
Indian medicinal plants used in skin
disorder
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
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 Constituents
Aliphatic compounds : 28-Hydroxy pentatriacontan-7-one, 7–hydroxy
dotriacontan-2-one, 5-acetoxytriacon-tane, 1hydroxytetratriacontan-4-one,
oleic
acid,
32-methyltetratria-contan-8-ol, linoleic acid
Monoterpene lactone : Isololiolide
Triterpenoids
: Leucolactone, Maslinic acid,
Diterpenes
: Leucasperone A and B, Leucasperol A and B
Volatile compounds
Leucasperoside A, B and C, Linifolioside
: α-Farnasene, α-thujene, Menthol, Amylpropionate,
Isoamylpropionate
: β – Sitosterol, Stigmasterol-3-O-β-glucoside
Steroids
Alkaloid(s)
: Nicotine
Lignans and Flavonoids
Leucas aspera
(leaves,2kg)
Extrd. twice with MeOH (20L)
Evpn. in vaccuo (40 – 45 ºC)
Extract mass (67 g)
Diaion HP – 200 CC
Fraction (51g)
n-Hexane
Fraction (7g)
CC/ODS flash
CHCl3
Fraction (14g)
Leucosperoside A & C, Leucassperon A
Maslinic acid,
β – sitosterol, stigmasterol
Linoleic acid, oleic acid
m/z 437.43, m/z 795.77
EtOAc,
Fraction (9g)
n-Butanol
Fraction (12g)
Flavanoids, Alkaloids,
Lignans
Keratinocyte anti proliferent effect of Extractives
(in vitro)*
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 )
* Sampson et al., Phytomedicine, 8 (3), 230 (2001)
Preparation of cell culture
• 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)
Microtitre Assay
• 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
Sulphorhodamine (SRB) 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
Results and Discussion
Dose response curves
reveal that Chloroform fraction and
methanol extract show
a good anti-proliferate
keratinocyte
activity
than ethylacetate and
n- Butanol fraction
Mean cell ±SD as a
percent of control
100
80
60
40
20
0
0.1
0.2
0.3
0.4
0.5
1
1.5
Log Conc.of plant extractives
Methanol extract of L. aspera leaves
Chlorofom fraction
Ethylacetate fraction
n-Butanol fraction
Presence of terpenoids/steroids/fatty acids in
chloroform fraction may be responsible for the activity.
Determination of IC50
Plant Extractives/Standard IC50 (μg/ml)
Methanol extract
Chloroform fraction
Ethylacetate fraction
n-Butanol fraction
Dithronol
151.33 ± 0.7
93.33 ± 0.6
575.33 ± 0.7
912.01 ± 0.9
2.2 ± 0.2
Thank You