Withania somnifera (root)

From AHPA Botanical Identity References Compendium
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(HPLC)
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           |caption1=Root transverse section (x400)
 
           |caption1=Root transverse section (x400)
 
           |source=Natural Remedies Pvt Ltd http://www.naturalremedy.com/}}
 
           |source=Natural Remedies Pvt Ltd http://www.naturalremedy.com/}}
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=High Performance Liquid Chromatographic Identification=
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{| border=1
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{{Botanical | source=Natural Remedies Pvt Ltd
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| companyURL=http://www.naturalremedy.com/
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| companyimage=Natural Remedies Logo.png
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|mainimage=Withania somnifera - NR - ashwagandha HPLC.png
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|caption1=
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| description=Ashwagandha (root) (''Withania somnifera'')
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{| border=1
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| Relative Retention Time || Peak
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|-
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|1.00 || Withanoside IV
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|-
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|1.07 || Physagulin D
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|-
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|1.15 || 27-hydroxywithanone
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|-
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|1.28 || Withanoside V and Withanoside VI
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|-
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|1.31 || Withaferin A
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|-
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|1.39 || Withastramonolide
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|-
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|1.45 || Withanolide A
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|-
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|1.47 || Withanone
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|-
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|1.68 || Withanolide B
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|}
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'''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.
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'''Column:''' 25-cm x 4.6-mm, 5 um, PhenomenexLuna C18(2)
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'''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)
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'''Elution:''' Gradient, see Table below
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'''Column Temperature:''' 25°C
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'''Flow rate:'''1.5 mL/min
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'''Detection:''' UV, 227 nm
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'''Injection volume:''' 20 uL}}
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'''Table: Gradient program'''
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{| border=1
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| Time (min) || Solution A (%) || Solution B (%)
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|-
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|0-18 || 95-55 || 5-45
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|-
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|18-25 || 55-20 || 45-80
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|-
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|25-28 || 20 || 80
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|-
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|28-35 || 55 || 45
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|-
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|35-40 || 55-95 || 45-5
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|-
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|40-45 || 95 || 5
 
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Revision as of 22:11, 6 November 2014

AHPA recognizes other valuable resources exist regarding the identity of Withania somnifera.

To submit a suggestion or contribution, please contact Merle Zimmermann.

Contents

Nomenclature

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

Botanical Voucher Specimen

bottomright

Withania somnifera Tropicos 100184298 (S).jpg
Source: MOBOT, Tropicos.org[1]

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 [2]
Natural Remedies Logo.png

Macroscopic Characteristics

bottomright bottomright bottomright

Withania somnifera - NR - Aerial parts.png
Aerial parts
Source: Natural Remedies Pvt Ltd http://www.naturalremedy.com/ [3]

Withania somnifera - EOL - Flower.jpg
Flower
Source: Encyclopedia of Life http://eol.org/data_objects/19246862 [4]

Withania somnifera - EOL - Fruit.jpg
Fruit
Source: Encyclopedia of Life http://eol.org/data_objects/19246863 [5]
bottomright bottomright

Withania somnifera - EOL - Ripe Fruit.jpg
Ripe Fruit
Source: Encyclopedia of Life http://eol.org/data_objects/19246864 [6]

Withania somnifera - NR - Dried Root.png
Dried roots
Source: Natural Remedies Pvt Ltd http://www.naturalremedy.com/ [7]

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 [8]
Natural Remedies Logo.png


bottomright bottomright bottomright

Withania somnifera - NR - Thick Root TS.png
Thick root transverse section (x100)
Source: Natural Remedies Pvt Ltd http://www.naturalremedy.com/ [9]

Withania somnifera - NR - Root TS.png
Root transverse section
Source: Natural Remedies Pvt Ltd http://www.naturalremedy.com/ [10]

Withania somnifera - NR - Root TS x400.png
Root transverse section (x400)
Source: Natural Remedies Pvt Ltd http://www.naturalremedy.com/ [11]

High Performance Liquid Chromatographic Identification

Natural Remedies Logo.png
Withania somnifera - NR - ashwagandha HPLC.png

Ashwagandha (root) (Withania somnifera)

{

Source: Natural Remedies Pvt Ltd [12]

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

HPTLC-assoc-Logo-farbig-Text-schwarz-300x47.png
Withania somnifera - HPTLC ASSOC.png

Ashwagandha (root) (Withania somnifera)

Lane Assignments Lanes, from left to right (Track, Volume, Sample):

  1. 5 µL Withania somnifera extract (20 mg/mL)
  2. 10 µL Withania somnifera root
  3. 10 µL Withania somnifera root
  4. 10 µL Withania somnifera root
  5. 2 µL β-Sitosterol
  6. 2 µL Withanoside IV
  7. 2 µL Withanolide A
  8. 2 µL Withanone
  9. 2 µL Withanolide D
  10. 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 [13]

Supplementary Information

Sources

  1. ↑ MOBOT, Tropicos.org http://www.tropicos.org/Image/100184298
  2. ↑ Natural Remedies Pvt Ltd http://www.naturalremedy.com/
  3. ↑ Natural Remedies Pvt Ltd http://www.naturalremedy.com/
  4. ↑ Encyclopedia of Life http://eol.org/data_objects/19246862
  5. ↑ Encyclopedia of Life http://eol.org/data_objects/19246863
  6. ↑ Encyclopedia of Life http://eol.org/data_objects/19246864
  7. ↑ Natural Remedies Pvt Ltd http://www.naturalremedy.com/
  8. ↑ Natural Remedies Pvt Ltd http://www.naturalremedy.com/
  9. ↑ Natural Remedies Pvt Ltd http://www.naturalremedy.com/
  10. ↑ Natural Remedies Pvt Ltd http://www.naturalremedy.com/
  11. ↑ Natural Remedies Pvt Ltd http://www.naturalremedy.com/
  12. ↑ Natural Remedies Pvt Ltd http://www.naturalremedy.com/
  13. ↑ HPTLC Association http://www.hptlc-association.org/
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