Potential Indian Medicinal Plants Against Psoriasis

1. Potential Indian Medicinal Plants Against Psoriasis • Dr. Sushil K. Singh • Department of Pharmaceutics • Institute of Technology • Banaras Hindu University • Varanasi – 221005, India

2. Environmental Immunological Genetic 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

3. 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

4. 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

5. 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

6. 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

7. Chemical Constituents Aliphatic 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 Triterpenoids : Leucolactone, Maslinic acid, Diterpenes : Leucasperone A and B, Leucasperol A and B Leucasperoside A, B and C, Linifolioside Volatile compounds : α-Farnasene, α-thujene, Menthol, Amylpropionate, Isoamylpropionate Steroids : β – Sitosterol, Stigmasterol-3-O-β-glucoside Alkaloid(s) : Nicotine Lignans and Flavonoids

8. 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) EtOAc, Fraction (9g) CHCl3 Fraction (14g) n-Butanol Fraction (12g) CC/ODS flash Leucosperoside A & C, Leucassperon A Maslinic acid, β – sitosterol, stigmasterol Linoleic acid, oleic acid Flavanoids, Alkaloids, Lignans m/z 437.43, m/z 795.77

9. 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)

10. 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)

11. 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

12. 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

13. Results and Discussion 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.

14. Determination of IC50

15. Thank You