Characteristics of immobilized bacterial D-hydantoinase on alginate
PPT-2 MODIFIED(1)
1. Presented By- Ruchi Saharan
S.Y M.Pharm( Pharmacognosy),GCP
Department of Phytochemistry
NRPL
Isolation & characterisation
of Phytoconstituents from
Withania somnifera Root
2. Introduction
• Withania somnifera has been an important herb in the ayurvedic & indigenous medicinal
system for centuries in India.[1]
• It is described as an herbal tonic & health food in Vedas and considered as ‘Indian Ginseng’ in
traditional Indian system of medicine.[2]
• Medicinally important plant known for variety of pharmacologically important withanolides
& their glycosides.
• Ashwagandha plant is especially attractive for studying the enzymes involved in steriodal
transformation like glycosylation because it is a rich source for variety of glycosylated
steroidal lactones called withanosides present in roots & leaves.[3]
• Withanosides mainly comprises of withanolides with one or more glucose units attached to C-
3 or C-27 position.
2
5. Macroscopic[5],[6]
• The sout fleshy roots when dry are cylindrical, gradually tapering down, straight,
unbranched, 10-17.5cm long & 6-12mm in diameter.
• The main roots bear fibre-like secondary roots.
• The outer surface of the roots is brownish white and interior in creamy white when
broken.
• They have a short and uneven fracture, a strong odour and mucilaginous bitter &
acrid taste.
5
6. Microscopic [5],[6]
• The young roots has a single layered epidermis followed by parenchymatous cortex
of 4-5 layers of cells, the endodermis being conspicuous by the presence of
casparian stipes.
• The cork cambium arises in the outermost layer of the cortex.
• The endodermis persists even after the secondary growth has been taken place.
6
7. Geographical Distribution[7]
• Withania somnifera is widely distributed around the world from Southern
Mediterranean regions to the canary island and from south to East Africa, from
Palestine to north India.
• India the plant can be seen growing wild in the North Western regions
extending to the mountains regions of Punjab, Himachal Pradesh and Jammu
up to an altitude of 1,500m.
7
9. WITHANOLIDES [8]
• These are group of naturally occuring steroids built on an ergostane skeleton in
which C-22 & C-26 are appropriably oxidised to form a α- lactone ring, occuring
almost exclusively in the solanaceae family.
• Two new and seven known withanolides along with β-sitosterol, stigmasterol, β-
sitosterol glucoside, stigmasterol glucoside, α + β glucose were isolated from the
roots of Withania somnifera.
• Among the known compounds, Viscosa lactone B, stigmasterol, stigmasterol
glucoside and α + β glucose are being reported from the roots of W. somnifera for
the first time.
• One of the new compounds contained the rare 16β-acetoxy-17(20)-ene the other
contained unusual 6α-hydroxy-5,7α-epoxy functional groups in the withasteroid
skeleton.
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12. Traditional and medicinal uses[11]
Roots of the plant withania somnifera reportedly exhibits:
• Anti-inflammatory
• Antitumor
• Antistress
• Antioxidant
• Immunomodulatory
• Haematopoietic
• Aphrodisiac
• Dyspepsia
• Sedative
Carminative
• Diuretic
• Bactericide 12
13. Objectives
• To isolate Root specific marker which are absent in leaves /aerial part.
• Isolation of Withanolide-B & other Withanolides from enriched fractions.
• Isolation of new marker apart from our regular compounds.
• Publication on Root specific markers & others.
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14. Previous work done
• Yogendra et.al-PLT-129
• The compound isolated was a derivative of withanolide which is having conjugated
diene ring system. The proposed structure was
• The compound isolated is specific to roots only (Confirmed by HPLC).
14
O
O
O
H3C
CH3
CH3
CH3
CH3
OH
16. 16
Withania somnifera
methanolic extract 3Kg
Ethyl acetate layer
35.33%
(1060g)
Butanol layer
37.0%
(1110g)
Water layer
20.67%
(620.5g)
Mixed layer
2.0%
(60.1g)
Total quantity= 2.85Kg
HPLC analysis was carried out on all the above fractions
Partition with 45L of solvents
20. Column chromatography on Ethyl acetate fraction... Column -1
1Kg of Ethyl acetate fraction + 2Kg of silica (120- 200) mixed adsorbed dried it.
Column charged with silica (60-120) 3Kg(1:3) Using wet packing.
sample is packed & 100% PE was elulated to deffat drug
Subsequently the mobile phase was (PE : EA) in the ratio of
90 : 10, 80 :20, 70 : 30, 60 : 40.......
20
26. Future Plan of work
• Fractions-10,11,12,13 from Column -1 were selected for next Silica column to
isolate Withanolide B & to generate enriched fractions for WSR-03, 01 & other
withanolides.
26
63. References
1. Indian Herbal Pharmacopoeia A Joint Publication of Regional Research institute (Jammu Tawai) and
Indian Drug Manufacturer’s Association (Mumbai); 1999.
2. Owais M, Sharad KS, Shehbaz A, Saleemuddin M. Antibacterial efficacy of withania somnifera an
indigenous medicinal plant against experimental murine salmonellosis. Phytomedicine. 2005;12:229-
235.
3. Bhaskara RM, Lokendra KS, Pankaj C, Rajender SS, Rakesh T. Purification & characterization of a
novel glucosyltransferase specific to 27- hydroxyl steriodal lactose from withania somnifera and its role
in stress responses Biochimica Biophysica Acta.2007; 1774: 1199-1207.
4. Withania somnifera Master Document; Natural Remedies Private Ltd.Bengaluru.
5. The Unani Pharmacopoeia of India. Part I, Vol. I, Depat. of AYUSH, Ministry of Health & Family
Welfare, Govt. of India, New Delhi (2007) 7-8.
6. Standardisation of Single Drugs of Unani Medicine. Part III, 1st ed. Central Council for Research in
Unani Medicine (CCRUM), New Delhi (2007) 9-14.
7. Shah CS and Qadri JS. Textbook of Pharmacognosy. 9th ed. BS Shah Prakashan, New Delhi (1993).
8. Singh S, Kumar S, withania somnifera: The Indian Ginseng, Ashwagandha. Central Institute of
Medical an Aromatic Plants, Lucknow, India; 1998
63
64. 9. Wilk, S Orlowski, M wilk, Pearce. Novel in vitro and in vivo inhibitors of Prolyendo peptidase. Journal of
Neurochemistry life sci. 1983; 33: 2149- 2157.
10. Steroids-Chemical constituents of Withania somnifera Dunal through TLC & HPLC. Monika christian
et.al. IRJP.ISSN 2321-2845,2321,3299.
11. Mark E , Allan W, Nicholas, John PR. High presssure liquid chromatography of derivatives & microbial
metabolites of withaferin- A. J . Chrom. 1977; 137; 465-467.
12. Traditional And Medicinal Uses of Withania Somnifera. M. Umadevi1 et.al. The Pharma Innovation .
2012, vol.1 No.9 : 102-110
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![Introduction
• Withania somnifera has been an important herb in the ayurvedic & indigenous medicinal
system for centuries in India.[1]
• It is described as an herbal tonic & health food in Vedas and considered as ‘Indian Ginseng’ in
traditional Indian system of medicine.[2]
• Medicinally important plant known for variety of pharmacologically important withanolides
& their glycosides.
• Ashwagandha plant is especially attractive for studying the enzymes involved in steriodal
transformation like glycosylation because it is a rich source for variety of glycosylated
steroidal lactones called withanosides present in roots & leaves.[3]
• Withanosides mainly comprises of withanolides with one or more glucose units attached to C-
3 or C-27 position.
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