Withania somnifera (root)

Nomenclature

Withania somnifera (L.) Dunal   Solanaceae  
Standardized common name (English): ashwagandha  
Ayurvedic name(s): ashvagandha

Botanical Voucher Specimen

Source: MOBOT, Tropicos.org

Organoleptic Characteristics

Color: Yellowish brown or light brown. Outer surface is buff to gray-yellow with longitudinal wrinkles and in the center soft, solid mass with scattered pores. Odor: Characteristic. Taste: Bitter and acrid. Source: Natural Remedies Pvt Ltd

Macroscopic Characteristics

Aerial parts Source: Natural Remedies Pvt Ltd http://www.naturalremedy.com/ Flower Source: Encyclopedia of Life http://eol.org/data_objects/19246862 Fruit Source: Encyclopedia of Life http://eol.org/data_objects/19246863 Ripe Fruit Source: Encyclopedia of Life http://eol.org/data_objects/19246864 Dried roots Source: Natural Remedies Pvt Ltd http://www.naturalremedy.com/

Microscopic Characteristics

Transverse section: The young roots have less secondary tissue and prominent cortex. Primary xylem is tetrarch. In matured roots, the cork cells are isodiametric and non-lignified. As the roots increases in diameter it produces more secondary tissue which has more of ray parenchyma cells. The cells are nearly square in shape and in rows. Cells are filled with starch grains, small vascular bundles with one or two vessels and few fibers found in the secondary xylem. Intercellular spaces are present in phloem parenchyma while it is absent in xylem parenchyma. Fibers absent in phloem and present in xylem. Starch grains, simple, reniform and oval, normally found in parenchyma of the cortex andvascular region. Powder: Nearly white in color, xylem vessels with scaleriform and pitted thickenings. Xylem parenchyma is having bordered pits. Plenty of starch grains varying in size, circular or oval in shape, singular and sometimes compound, 2-3 grains. Source: Natural Remedies Pvt Ltd Thick root transverse section (x100) Source: Natural Remedies Pvt Ltd http://www.naturalremedy.com/ Root transverse section Source: Natural Remedies Pvt Ltd http://www.naturalremedy.com/ Root transverse section (x400) Source: Natural Remedies Pvt Ltd http://www.naturalremedy.com/ Root powder: 2-4 compound starch granules showing irregular hilum Source: Elan M. Sudberg, Alkemist Laboratories Root powder: microcrystals of calcium oxalate Source: Elan M. Sudberg, Alkemist Laboratories

High Performance Liquid Chromatographic Identification

Ashwagandha (root) (Withania somnifera) Test Sample Preparation: Reflux for 20 minabout 5 g of coarse powdered plant material in 50 mL of methanol. Repeat the extraction 4-5times till the extract is nearly colorless. Combine the extracts and concentrate to about 100 mL.Before injection, filter through a 0.45-ummembrane filter. Column: 25-cm x 4.6-mm, 5 um, PhenomenexLuna C18(2) Mobile Phase: Dissolve 0.14 g of anhydrous potassium dihydrogen phosphate in 900 mL of water, add 0.5 mL phosphoric acid, mix, complete to volume with water, and mix (Solution A); and acetonitrile (Solution B) Elution: Gradient, see Table below Column Temperature: 25°C Flow rate:1.5 mL/min Detection: UV, 227 nm Injection volume: 20 uL Source: Natural Remedies Pvt Ltd Table: Relative retention time Relative Retention Time Peak 1.00 Withanoside IV 1.07 Physagulin D 1.15 27-hydroxywithanone 1.28 Withanoside V and Withanoside VI 1.31 Withaferin A 1.39 Withastramonolide 1.45 Withanolide A 1.47 Withanone 1.68 Withanolide B Table: Gradient program Time (min) Solution A (%) Solution B (%) 0-18 95-55 5-45 18-25 55-20 45-80 25-28 20 80 28-35 55 45 35-40 55-95 45-5 40-45 95 5

High Performance Thin Layer Chromatographic Identification

Ashwagandha (root) (Withania somnifera) Lane Assignments Lanes, from left to right (Track, Volume, Sample): 5 µL Withania somnifera extract (20 mg/mL) 10 µL Withania somnifera root 10 µL Withania somnifera root 10 µL Withania somnifera root 2 µL β-Sitosterol 2 µL Withanoside IV 2 µL Withanolide A 2 µL Withanone 2 µL Withanolide D 2 µL Withaferin A  Other Notes Reference Standard Solution:0.2 mg/mL β-Sitosterolin methanol and 0.2 mg/mL withanolide A in methanol. Optional: 0.2 mg/mL withaferin A in methanol. Reference Sample Preparations: Sonicate 0.5g of powdered sample in 5mL of methanol for 15 minutes, centrifuge or filter the solution, and use the supernatant / filtrate. Stationary Phase: HPTLC, Silica gel 60 F254 Mobile Phase: Toluene, ethyl acetate, acetic acid (55:45:3) Development: Saturated chamber, developing distance 70 mm from lower edge of the plate; relative humidity 33%, temperature 25°C. Derivatization reagent: Sulfuric Acid Reagent– 10% sulfuric acid in methanol (prepare fresh) Detection: Dip (time 0, speed 5) in Derivatization reagent, heat plate at 100°C for 5 min,and examine under visible light. Procedure: Reference Standard Solutions, Stationary Phase, Mobile Phase, Derivatization reagent, and Detection, as described above. Test Sample Preparation: Prepare test sample as described under Reference Sample Preparations andapply 10uL. Identification: Compare Test Sample Preparation chromatogram with chromatograms of Reference Sample Preparations. The Test SamplePreparation chromatogram is similar to that of the Reference Sample Preparations chromatograms. Additional weak zones may be present. The Test Sample Preparation chromatogram exhibits a grey-violet zoneand a brownish-violet zone at Rf corresponding to those of β-Sitosterol and withanolide A, respectively, in Reference Standard Solutionchromatogram, and two browninsh-violet zones in the lower third of the chromatogram at Rf lower than that corresponding to withanolide A in Reference Standard Solution chromatogram. Source: HPTLC Association

Supplementary Information

Detection of Aerial Parts in Extracts of Withania somnifera Roots

Deepak Mundkinajeddu, Laxman P. Sawant, Rojison Koshy, Praneetha Akunuri, Vineet Kumar Singh,1 Anand Mayachari, Maged H. M. Sharaf, Murali Balasubramanian, and Amit Agarwal

Introduction:

Withania somnifera root is considered to be the medicinally important part of the plant according to classical Ayurveda texts. The aerial parts, being less expensive, are sometimes mixed with roots to prepare extracts of W. somnifera. Both aerial parts and root show similar qualitative chromatographic fingerprints for the withanolides’ content, which tend to be the criterion to standardize these extracts.Unlike the withanolides content, flavonoid glycosides are unique constituents of the aerial parts and are absent in roots of the plant. These flavonoids include quercetin 3-O-robinobioside-7-O-glucoside,quercetin 3-O-rutinoside-7-O-glucoside, and kaempferol3-O-robinobioside-7-O-glucoside.

Procedure: Test Sample Preparation: Extract about 2 g of finely powdered plant material in 50 mL of methanol under reflux in a water bath for 20 min. Repeat the extraction 3 times. Combine the extracts and concentrate to about 100 mL. Before injection, filter through a 0.45-ummembrane filter.

Column: 25-cm x 4.6-mm, 5 um, PhenomenexLuna C18

Mobile Phase: Dissolve 0.14 g of anhydrous potassium dihydrogen phosphate in 900 mL of water, add 0.5 mL phosphoric acid, mix, complete to volume with water, and mix (Solution A); and acetonitrile (Solution B)

Elution: Gradient, see Table below

Column Temperature: 25°C

Flow rate: 1.5 mL/min

Detection: UV, 350 nm

Injection volume: 20 uL

Detection of peaks corresponding to quercetin 3-O-robinobioside-7-O-glucoside (1), quercetin 3-O-rutinoside-7-O-glucoside (2), and kaempferol3-O-robinobioside-7-O-glucoside (3) indicates the presence of aerial parts.

Table: Gradient program

Time (min)	Solution A (%)	Solution B (%)
0.0-12	90-80	10-20
12-18	80-55	20-45
18-25	55-20	45-80
25-28	20	80
28-35	20-55	80-45
35-40	55-90	45-10
40-45	90	10

Reference:

Mundkinajeddu D, Sawant LP, Koshy R, Akunuri P, Singh VK, Anand Mayachari, Sharaf MHM, Balasubramanian M, Agarwal A. Development and Validation of High Performance Liquid Chromatography Method for Simultaneous Estimation of Flavonoid Glycosides in Withania somnifera Aerial Parts. ISRN Analytical Chemistry 2014. http://dx.doi.org/10.1155/2014/351547 (accessed July 9, 2014)