Presented By- Ruchi SaharanM.Pharm (Pharmacognosy)Goa College of PharmacyDepartment of PhytochemistryNRPL

Isolation & Characterization of Phytoconstituents fromWithania somnifera Roots

List of Isolated Compounds

Sr.no Isolated Compounds Quantity Purity(Area

normalisation)

1. 27- deoxywithaferin -A 54mg 97%

2. Withanolide-B 316mg 97%

3. Withanolide-A 1.0g 95%

4. Withaferin-A 1.0g 95%

5. 12-Deoxywithastramonolide 49.5 mg 95%

List of Enriched fractionsSr.no Enriched fractions Quantity

1. C4F8 (Withanolide-A + Withanolide-B) 439.8mg

2. C4F10 (Withanolide-A + 12-Deoxywithastramonolide) 3.24g

3. C4F10 (MLR) (Withanoside IV) 3.6g

4. C4F9(1,2) & C4F9 MLR (Withanolide A) 3.28g

5. C4F12 (MLR) (Withaferin –A) 1.9g

6. C11F5 (12-Deoxywithastramonolide + Withanolide –A) 2.9g

7. (C-8,9,10) Prep-HPLC Peak 3 & 4 (27-dehydroxywithaferin-A)

170mg & 26mg

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Partitioning of Withania somnifera methanolic extract – Pilot scale

Present Work

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Withania somnifera methanolic extract 3Kg

Ethyl acetate layer35.33% (1060g)

Butanol layer37.0%

(1110g)

Water layer20.67% (620.5g)

Mixed layer2.0%

(60.1g)

Total quantity= 2.85Kg

HPLC analysis was carried out on all the above fractions

Partition with 45L of solvents

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Partition Quantification

Sl. No. Sample Withanoside- IV Withanoside -V Withaferin- A 12Deoxy-

Withastramonolide Withanolide - A Withanolide- B Total Assay of WSR-03

1 Initial ME ext 0.42 0.45 0.26 0.26 0.41 0.12 2.42 0.016

2 EA layer 0.43 0.31 3.04 3.04 2.94 0.86 15.23 0.154

3 Butanol layer

4.69 3.73 1.19 1.19 1.80 0.36 15.62 0.025

4 Water layer 0.41 0.68 0.17 0.17 0.27 0.02 2.05 ND

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Partition overlay

Withanolide overlay

Ethyl acetate fraction1Kg

C-11Kg

C-2(300g)Fractions:C1F9,10,11,12

C-3(21g)Fraction:C2F8

C-4 (27g)Fractions:C2F9+ C2F10

C-5(6g)Fraction:C2F6

C-6(56g)Fraction:C2F11

C-11 (75g)Fraction:C1F14

C-10 (80g)Fraction:C1F8 + C1F9

C-7 (7g)Fraction:C6F8(1)

C-12 (13g)Fraction:C11F9

C-9 (20g)Fraction:C1F6

C-8 (30g)Fraction:C1F7

12-Deoxywithastra-monolide Withanolide-A, Withanolide-B

27-deoxywithaferin-APrep-HPLC

Withaferin-A

Withanolide-B

27-deoxywithaferin-A

HPLC Chromatogram

Prep-HPLC,PEAK-2 PPT

27-deoxywithaferin-A54 mg, Purity 97%

Mass data

27-deoxywithaferin-ALiterature reported M/Z-454.605

13C-NMR Data

O

OH

O

O

O

27-deoxy withaferin -A

C28H38O5Exact Mass: 454.27

Mol. Wt.: 454.6m/e: 454.27 (100.0%), 455.28 (30.9%), 456.28 (5.6%)

C, 73.98; H, 8.43; O, 17.60

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