Standardization of Some Plant-Based Formulations By Modern...

Standardization of formulations containing Withania somnifera 5

Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 95

5.1. INTRODUCTION

5.1.1 Plant profile of Withania somnifera

Withania somnifera (L.) Dunal. (Ashwagandha) is a valued herb in Ayurveda, an

ancient system of medicine, and as such was used and cultivated for centuries in

India. Ashwagandha in sanskrit means "horse's smell," probably originating from the

odor of its root which resembles that of a sweaty horse. The plant is sometimes

referred to as Indian ginseng1-4

.

Withania somnifera Dunal.

(a) Classification:

Kingdom : Plantae

Division : Angiosperma

Class : Dicotyledoneae

Order : Tubiflorae

Family : Solanaceae

Genus : Withania

Species : somnifera Dunal

(b) Vernacular names:

Sanskrit : Asvagandha

Hindi : Asgandh

Marathi : Askandha

Kannada : Viremaddinagaddi

Malayalam : Amukkuram

Tamil : Amukkira

Telugu : Vajigandha

English : Winter cherry

(c) Part used: Roots



(d) Geographical distribution: This plant grows wildly in all drier parts of

subtropical India. It occurs in Madhya pradesh, Uttar pradesh, Punjab plains

and northwestern parts of India like Gujarat and Rajasthan. It is also found in

Congo, South Africa, Egypt, Morocco, Jordan, Pakistan and Afganistan197

.

(e) Botanical description: An erect branching undershrub reaching about 150

cm in height, usually clothed with minutely stellate tomentum; leaves ovate

upto 10 cm long, densely hairy beneath and sparsely above, flowers greenish

or lurid yellow in axillary fascicles, bisexual, pedicel long, fruits globose

berries which are orange coloured when mature, enclosed in a persistent calyx.

The fleshy roots when dry are cylindrical, gradually tapering down with a

brownish white surface and pure white inside when broken196

.

(f) Traditional uses: Withania somnifera popularly known as

‘ASHWAGANDHA’ is one of the major herbal components of geriatric tonics

mentioned in Indian systems of medicine. In the traditional system of

medicine Ayurveda, this plant is claimed to have potent aphrodisiac

rejuvenative and life prolonging properties. It has general animating and

regenerative qualities and is used among others for the treatment of nervous

exhaustion, memory related conditions, insomnia, tiredness potency issues,

skin problems and coughing. It improves learning ability and memory

capacity. The traditional use of ‘Ashwagandha’ was to increase energy,

youthful vigour, endurance, strength, health, nurture the time elements of the

body, increase vital fluids, muscle fat, blood, lymph, semen and cell

production. It helps counteract chronic fatigue, weakness, dehydration, bone

weakness, loose teeth, thirst, impotency, premature aging emaciation, debility,

convalescence and muscle tension. It helps invigorate the body by



rejuvenating the reproductive organs, just as a tree is invigorated by feeding

the roots198-202

.

(g) Anatomy of the Root: The roots show buff to grey yellow outer colour

with longitudinal wrinkles. They are unbranched, straight conical and some of

them bear a crown. The root crown possesses a number of bud scars. The

fracture is smooth and powdery. The transverse section of root shows

exfoliated cork which is nonlignified with, 2–4 layers of phellogen and about

15 – 20 rows of phelloderm. It prominently shows parts of vascular tissue like

cambium, consisting of 3–5 layers of tangentially elongated cells. Secondary

xylem is hard which forms continuos vascular ring interrupted by medullary

rays. The transverse section of stem base shows pith, pericyclic fibres, xylem

with tracheids, fibres and starch grains197

.

Fig. 5.1: Roots of Withania somnifera

(h) Pharmacology and Clinical Studies: Withania root extract 100mg/kg

orally for 4-8 weeks showed enhanced open field behaviour and emotional

stability along with a moderate but significant enhancement in the functional

sensitivity of 5-HT2 receptors in the brain and a reciprocal sub-sensitivity of

the 5- HT1A receptors. Chronic Withania treatment prophylacticaly was

effective in preventing the behavioural deficit in open field activity in an

animal model of depression.



The active principles of Withania somnifera consisting of equimolar amounts

of Sitoindosides VIIX and withaferin A were investigated for putative

nootropic activity in a experimentally validated Alzheimer’s disease model.

The syndrome was induced by ibotenic acid (IA) lesioning of the nucleus

basalis magnocellularis in rats. Withania somnifera significantly reversed

both IA induced cognitive deficit and the reduction in cholenergic markers

after 2 weeks of treatment. These findings validate the Medharasayan

(promoter of learning and memory) effect of Withania somnifera as has been

reported in Ayurveda. Studies of Ashwagandha showed that an acetone

extract of alkaloids caused mild CNS depression in dogs and mice and

protected against supramaximal electroshock seizures in rats. The extract

caused hypothermia in mice and potentiated hypnosis induced by barbiturates,

ethanol and urethane203-208

.

(i) Toxicity and Adverse Reactions209

: The LD50 of alcoholic extract of

Withania root (in mice) was found to be 1260 mg/kg. No acute mortality was

observed at 1100mg/kg, but with a further 100mg increment in the dose, there

was a sharp increase in the death rate. These data suggest that mice showed

relatively high tolerance to Ashwagandha.

(j) Phytochemistry209-214

: Majority of the constituents are withanolides

(steroidal lactones with ergostane skeleton) glycowithanolides and alkaloids.

These include withaferin A, withanolides G&D, sitoindosides IX&X &

withasomnine. Total alkaloids is about 0.2%1-5

.

(k) Active Principles: Withanolides, Glycowithanolides



Fig. 5.2: Structure of Withanolide G

Fig. 5.3: Structure of Sitoindosides IX (withaferin-A-C22-O-β-D-glucoside)

Sitoindosides X (6’-O-palmitoyl-A- C22-O- β-D-glucoside)



5.1.2 Withaferin A as marker compound for Standardization

Withaferin A is one of the withanolides isolated from Withania somnifera.

Withaferin A is well known constituent among the various bioactive

compounds in Ashwagandha. It has been proved that withaferin A has

different therapeutic activities such as anti-inflammatory, antiarthritic,

antibiotic, antitumour immunomodulatory and central nervous system

effects215,216

.

High Performance liquid chromatography (HPLC) is considered as one of the

most sensitive reliable methods of analysis. There are number of analytical

methods reported for analysis of withaferin A by HPLC and HPTLC 217-221

.

But, the reported methods are very time consuming with very much advanced

instrumentation like photodiode and evaporative light scattering detector and

with gradient solvent system. Due to which herbal manufacturers show their

relectance in adopting these methods for the routine analysis of their products.

C28H38O6 Mol. wt. 470.61

Fig.5.4 Structure of Withaferin A

Thus, we aim to develop a simple, rapid, accurate and precise HPLC

method for standardization of commercial formulations using withaferin

A as a potential marker which can be easily employed in quality control

testing of Ashwagandha formulations.



5.2 ISOLATION OF WITHAFERIN A

5.2.1 Procurement of commercial dry powder extract of Withania

somnifera

The commercial Methanol extract of Withania somnifera was reported to have

high yield of withaferin A, thus commercial extract was selected for isolation

of the marker. Withania somnifera extract was provided as gift sample by

Amsar Pvt. Ltd.

5.2.2 Procedure for isolation of withaferin A by column chromatography

a) Preparation of column:

350 gm of silica gel (60-120 mesh) was activated for 30 mins at 105ºC. Slurry

of silica gel was prepared in 1000 ml hexane and was transferred to glass

column; precaution was taken to avoid air entrapment. The length of the

column was approximately kept to 70 cm.

b) Preparation of sample:

The methanol extract (100g) was refluxed with 500ml of ethanol: water (3:1)

for 4 hours at 60oC. Each 100ml of the liquid extractive so obtained was

successively extracted with 3 X 50 ml portions of chloroform. The chloroform

fraction of Withania somnifera was dried and weighed. The chloroform

fraction was adsorbed on silica (approximately 10 gm) and loaded on the

column in form of thin band. Hexane was added slowly to the column,

precaution was taken to avoid air entrapment.

c) Elution:

Following combinations of solvents were used to elute the column (Table

5.1). 100 ml of each mobile phase was used at a time to run the column.



Table 5.1: Pattern of column chromatographic elution for withaferin A

Sr.No. Solvent

composition (%) TLC studies of

fractions Inference

Hexane Ethyl

acetate

1. 100 0 No spot --

2. 95 5 No spot ---

3. 90 10 No spot --

4. 85 15

One blue spot detected

under 254 nm

(Rf =0.95)

The spot does not

match with reference

standard of withaferin

A (Rf =0.82)

5. 80 20 No spot ---

6. 75 25 No spot ---

7. 70 30

Two spots were

observed. One blue

spot detected under

254 nm (Rf =0.90)

A blue spot with high

intensity after spraying

with anisaldehyde

sulphuric acid reagent

(ASR) at Rf =0.82.

(Component I)

The spot does not

match with reference

standard of withaferin

A (Rf =0.82)

The Rf and color of

the spot matched with

the reference

standard. (Rf =0.82)

8. 65 35 No spot ---

9. 60 40 No spot ---

50 ml aliquots were collected, concentrated and subjected to TLC studies. 7th

fraction [eluted with Hexane: Ethyl acetate (70:30)] gave an intense spot at

Rf 0.82 after spraying with ASR and a spot of less intensity at Rf 0.90 was

observed with mobile phase Toluene: Ethyl acetate :formic acid (10:5:0.5).



Since 7th

fraction showed mixture of spots, it was subjected to further

purification by crystallization process. Crystallization was carried out with hot

water and then the solution was refrigerated to allow the impurity to

precipitate.

The TLC chromatogram of chloroform fraction and isolated compound

component I was sprayed with Anisaldehyde Sulphuric acid (ASR) and

Vanillin Sulphuric acid (VSR) spraying reagents to check the presence of

withaferin A. (Table 5.2, Fig.5.5)

Table 5.2: Photodocumentation of methanolic extract and of the

Component I after derivatisation

Sr. No. Spray reagent At 540 nm

Possible

Phytoconstituent

present

1

Anisaldehyde

Sulphuric acid

(ASR)

Violet- brown Withanolides

2 Vanillin Sulphuric

acid (VSR) Blue Withaferin A

Standardization of Some Plant

Derivatization with ASR

Fig. 5.5:Video

Track 1: Component I

Track 2: Chloroform fraction of

1

Standardization of formulations containing Withania

Standardization of Some Plant-Based Formulations By Modern Analytical Techniques

Derivatization with ASR Derivatization with VSR

Video-images of TLC plates showing Component I and

Chloroform fraction of Ashwagandha

Track 1: Component I

Track 2: Chloroform fraction of Ashwagandha

1 2 1

Withania somnifera 5

Analytical Techniques 104

Derivatization with VSR

images of TLC plates showing Component I and

Ashwagandha

2



5.2.3. Physicochemical analysis of component I studies

The component I was subjected to different physicochemical parameters such

as melting point, solubility elemental analysis. The parameters were

compared with that of the reference standard. The melting point was found to

be 239-243° C which was matching with the standard (241-245°C). Solubility

was tested in different solvents such as chloroform, methanol and water. It

was observed that component I was readily soluble in chloroform and

methanol. The Lassaigne’s sodium fusion test was carried out for detection of

elements. Carbon, hydrogen were present and nitrogen, halides and sulphur

were found to be absent. The yield obtained was 0.087% from the commercial

methanol extract. The summarized data is mentioned in Table 5.3.

Table 5.3: Physicochemical analysis of component I

Sr. No. Parameters Component I Standard

1. Color Off white White

2. Melting point (°C) 239-243 241-245

3. Solubility Methanol Methanol

4. Elements present C, H, (O) C,H, (O)

5. Yield (%w/w) 0.087 -

5.2.4 Confirmation of identity of isolated compound as withaferin A

The isolated component I was confirmed to be withaferin A by comparing

with reference standard by HPTLC, HPLC and UV studies.



• HPTLC Studies: Standard withaferin A and component I was

dissolved in chloroform and the HPTLC analysis was carried out

using the following densitometric conditions:

Stationary phase : Precoated plates of Silica Gel 60 GF254 (Merck)

Mobile phase : Toluene: ethyl acetate: methanol (10:5:0.5)

Saturation time : 20 min

Development time :20 min

Wavelength :215 nm

Lamp :Deuterium

Band width :7 mm

Length of chromatogram :8 cm

• HPLC Studies: Standard withaferin A and component I of were

analyzed by HPLC technique using the following conditions:

Column: C18 Phenomenex (250 x 4.60mm) - 5µ

Mobile phase (Gradient): Acetonitrile (B): Water(A)

Time (min) Function Value

0.01 B. Conc 20.0

22.00 B.Conc 85.0

27.00 B.Conc 85.0

28.00 B.Conc 20.0

32.00 STOP

Flow rate: 1.6 ml/min

Wavelength : 215nm

Injection loop capacity: 20µl

Concentration of Samples: 1mg/ml of standard and isolated

compound.


HPTLC fingerprints of standard withaferin A and of the Component I

Fig. 5.6: HPTLC

withaferin A

Fig. 5.7: HPTLC




HPTLC chromatogram of reference standard

withaferin A (Rf 0.85)

Fig. 5.7: HPTLC chromatogram of Component I (Rf 0.86)




0.86)



HPLC profile of Reference standard and of the Component I

Fig. 5.8: HPLC profile of reference standard: RT 6.367 min

Fig. 5.9: HPLC profile of Component I: RT 6.367 min



� UV Spectroscopy: The UV spectrum of the standard withaferin A and

component I was recorded in methanol. The UV λ max of standard

withaferin A was obtained at 215nm and of component I was obtained at

216 nm.

Component I gave a single band of violet-brown color with ASR and blue

color with VSR (Table 5.2 and Fig. 5.5) matching with the reference standard

indicating presence of withaferin A. The HPTLC chromatogram (Fig.5.6, Fig

5.7) and HPLC profile (Fig. 5.8, Fig. 5.9) of component I matched with

reference standard of withaferin A. Thus with the acquired chemical and

spectral data and comparison with reference standard, it was confirmed that

Component I is withaferin A.



5.3 METHOD DEVELOPMENT

5.3.1 Preparation of Standard solutions

10.0 mg of withaferin A was dissolved in 100 ml methanol, yielding a stock

solution concentration of 0.1 mg ml−1

. Working standard solution of

concentration of 1.0 µgml-1

was prepared by diluting 1ml of stock solution to

100 ml with mobile phase. Series of dilutions were prepared by transferring

aliquots of 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 ml of the

working standard solution and diluting with the mobile phase to yield 10 ml

of standard solutions containing 100, 200, 300, 400, 500, 600, 700, 800, 900

and 1000 ng ml−1

, respectively.

5.3.2 Chromatographic conditions

The different parameters of RP-HPLC were tried and were optimized.

Detection wavelength was selected based on UV spectrum of the compound.

Various combinations of acetonitrile and methanol with water were tried.

Various combination of methanol with water and acetonitrile with water were

tried and their effects on peak symmetry and peak shape were observed. Flow

rate was also varied from 0.75 to 1.5 ml/min. The flow rate giving acceptable

retention time (RT) of the compound with a good peak symmetry was

selected.

Analysis was carried out with C18 reverse-phase analytical column. Mobile

phase was Acetonitrile–water 4:6 (v/v) with flow rate 1ml/min and detection

wavelength 215nm. A representative chromatograms indicating standard

withaferin A and methanol extract of Ashwagandha are shown in Fig 5.10

and Fig 5.11..



Acetonitrile: water in ratio of 40:60 with flow rate 1ml/min gave peak

symmetry of 1.701 and optimum retention time (RT = 6.358 min) with better

peak shape than other mobile phase combinations. (Table 5.4)

Table 5.4: Effect of mobile phase composition on retention time and peak

symmetry

Mobile phase

composition (v/v)

Flow

Rate

ml/min

Detection

Wavelength

(nm)

Retention

Time(RT)

min

Peak

Symmetry

Methanol/water (65/35) 1 215 6.91 1.587

Methanol/water (25/75) 1 215 6.812 1.496

Methanol/water (80/20) 1 215 6.019 1.467

Acetonitrile/water (50/50) 1 215 6.400 1.571





The optimized parameters for withaferin A by HPLC analysis are as below

mentioned:

• Column: C18 Phenomenex (250 x 4.60mm) - 5µ

• Mobile phase : Acetonitrile–water, 4:6 (v/v)

• Flow rate: 1ml/min

• Wavelength : 215nm


Fig.5.10: HPLC chromatogram of standard withaferin A using the

optimized parameters

Fig. 5.11: HPLC chromatogram of methanol extract of

using the optimized parameters



HPLC chromatogram of standard withaferin A using the

optimized parameters

HPLC chromatogram of methanol extract of Ashwagandha

using the optimized parameters



HPLC chromatogram of standard withaferin A using the

Ashwagandha



5.4 METHOD VALIDATION

Validation of HPLC method as per ICH guidelines

Analytical method validation is a process of performing several tests designed

to verify that an analytical test system is suitable for its intended purpose and

is capable of providing useful and valid analytical data. The developed

method was validated for various parameters like linearity, limit of detection

(LOD), limit of quantitation (LOQ), accuracy, precision, robustness and

system suitability as per ICH guidelines170,171

.

5.4.1 Linearity:

Linearity was evaluated in the range 50–1000 ng ml−1

. Peak area versus

concentration was subjected to least square linear regression analysis and the

slope, intercept and correlation coefficient for the calibration curve were

determined.

Under the above described experimental conditions, linear correlation

between the peak area and applied concentration was obtained in the

concentration range of 100–1000 ng ml−1

, as confirmed by the correlation

coefficient of 0.9988. The peak area (y) is proportional to the concentration of

withaferin A (x) following the regression equation y = 4019x as shown in

(Fig. 5.12).


Fig. 5.12:

5.4.2 Limit of detection (LOD) and limit of quantitation (LOQ)

LOD and LOQ were determined from the calibration curve using the

following expressions: 3σ/

S is the slope of the calibration curve.

quantitation was determined by using sta

calibration curve was prepared using concentrations in the range of 10

ng/ml.

Standard deviation of residuals was measured and kept in the following

equation for determination of detection limit and quantitation limit. Detecti

limit =3.3σ /S and quantitation limit=10 σ /S where σ is the residual standard

deviation of a regression line and S is the slope of the calibration curve.

The experimentally derived LOD and LOQ for withaferin A were determined

to be 30 and 90 ng ml



Fig. 5.12: Linearity graph of withaferin A (r2= 0.9988)

5.4.2 Limit of detection (LOD) and limit of quantitation (LOQ)


following expressions: 3σ/S and 10σ/S, where σ is the standard deviation and

is the slope of the calibration curve. Limit of detection and limit of

quantitation was determined by using standard deviation method. A

calibration curve was prepared using concentrations in the range of 10


equation for determination of detection limit and quantitation limit. Detecti




to be 30 and 90 ng ml−1

, respectively.



= 0.9988)

5.4.2 Limit of detection (LOD) and limit of quantitation (LOQ):


, where σ is the standard deviation and

Limit of detection and limit of

ndard deviation method. A

calibration curve was prepared using concentrations in the range of 10-18


equation for determination of detection limit and quantitation limit. Detection






5.4.3 Precision studies

Precision of the developed method was evaluated by repeatability (intra-day)

and intermediate precision (inter-day). Each level of precision was

investigated by three sequential replicates of injections of withaferin A at

concentrations of 200, 400 and 600 ng ml−1

. Repeatability was evaluated on

the same day, while intermediate precision was determined by comparing the

assays for 2 days.

Precision data on the intra-and inter-day variation for three different

concentration levels are summarized in (Table 5.5). Both inter-and intra-day

R.S.D. were less than 2%, indicating a sufficient precision.

Table 5.5: Results of precision studies withaferin A

Type of

Precision

Intra-day Inter-day

AUC for concentration of

withaferin A (ng/ml)

AUC for concentration of

withaferin A (ng/ml)

Sr. No 200 400 600 200 400 600

1. 803800 1607600 2411400 803850 1607673 2411518

2. 800888 1600027 2421031 809401 1615506 2432579

3. 807491 1615192 2439121 801155 1623507 2411618

Mean 804059.7 1607606 2423851 804802 1615562 2418572

% RSD 0.41 0.47 0.58 0.52 0.49 0.50

5.4.4 Accuracy studies

Accuracy studies were carried out by determining the percentage recoveries

of known amounts of withaferin A added to solutions of extracts of

commercial products. The analyzed samples were spiked with 80, 100 and



120 % of 400 ng of standard solution. For sample preparation of formulations

please refer section 5.5.

Accuracy was calculated from the following equation:

[(spiked concentration − mean concentration)/spiked concentration] × 100.

As per the ICH guidelines standard recovery range for reference standard is

from 80% to 120 %. The recoveries were in the range of 96.43–106.57 %

withaferin A. Thus, the proposed HPTLC method is accurate and reliable for

the quantification of withaferin A in seven Ashwagandha formulations (Table

5.6).

5.4.5 Robustness

Intraday variability was studied for the sample, by injecting the same

concentration (0.1mg/ml) of the sample in triplicate and the standard error

mean was calculated. The standard mean deviation was found to be < 2

(Table 5.7.1).

For the determination of the robustness of method, chromatographic

parameters, such as mobile phase composition, flow rate and detection

wavelength, were intentionally varied to determine their influence on the

retention time, AUC and quantitative analysis.

The mobile phase composition was altered by ± 5 % changes in the ratio of

acetonitrile – water, 4:6 (v/v). The chromatograms were studied at different

detection wavelength viz., 211nm, 213 nm, 215nm and 217nm. No changes

were observed in retention time and peak shape for these mobile phases and

detection wavelength. (Table 5.7.2 and Table 5.7.3)



Among the studied factors, the flow rate in the range of 0.8 – 1.2 mlmin−1

has

influenced the retention time but there was no change in AUC and peak

shape. (Table 5.7.4)

5.4.6 Stability studies

Stability of the sample solutions was tested after 24, 48 and 72 hours after

preparation and storage at 4.0°C and 25.0°C separately. Stability was assessed

by comparing the chromatographic parameters of the solutions after storage

with the same characteristics of freshly prepared solutions.

Stability of withaferin A in the sample solutions was evaluated at 4°C and

25°C for 3 days to verify whether spontaneous degradation occurred. The

results were calculated as the percentage of non-degraded withaferin A at the

specified time intervals. All formulations showed less than 4% degradation

indicating that the samples were stable at 4° C and 25°C for 3 days (Table

5.10)



Table 5.6: Results of recovery studies for withaferin A

40 µg of each

Formulations

Amount

added

in ng

AUC Recovery ±

S.D. (%) Formulations Standard

Standard

spiked

formulations

Ashwagandha

Vati

320 430257 1286080 1788080 104.18 ±0.11

400 430257

1607600 2135470

104.79 ±0.32

480 430257 1929120 2466965 104.56 ±0.09

Ashwagandha

Churna

(Brand A)

320 502100 1286080 1875443 104.88 ±0.13

400 502100 1607600 2197674 104.17 ±0.73

480 502100 1929120 2539166

104.44 ±0.34

Ashwagandha

Churna

(Brand B)

320 542109 1286080 1901134 103.99 ±0.22

400 542109 1607600 2235912 104.01 ±0.54

480 542109 1929120 2576009 104.24 ±0.55

Ashwagandha

Arishta

(Brand I)

320 410717 1286080 1692894

99.77 ±0.10

400 410717 1607600 2006005 99.39 ±0.23

480 410717 1929120 2319246 99.12 ±0.12

Ashwagandha

Arishta

(Brand II )

320 401991 1286080 1627807 96.43 ±0.09

400 401991

1607600 1957141

97.39 ±0.13

480 401991 1929120 2258147 96.87 ±0.17

Ashwagandha

Capsules

320 510212 1286080 1815512

101.07 ±0.20

400 510212 1607600 2142167 101.15 ±0.41

480 510212

1929120 2466409

101.11 ±0.05

Ashwagandha

Tablets

320 520437 1286080 1920147 106.29 ±0.31

400 520437

1607600 2255932

106.01 ±0.17

480 520437 1929120 2610493 106.57 ±0.07



Table 5.7.1: Robustness (Intraday variability) studies of withaferin A

Sr.No AUC

1. 370231

2. 370344

3. 370209

S.D ± 0.97

Table 5.7.2: Robustness (Mobile phase variation) studies of withaferin A

Sr. No

Mobile phase composition

(v/v) RT AUC

Acetonitrile Water

1. 4 6 6.31 370222.18±0.40

2. 4.2 5.8 6.31 370223.08±0.15

3. 3.8 6.2 6.31 370221.09±0.21

4. 3.7 6.3 6.31 370221.24

5. 4.3 5.7 6.31 370222.11±0.05

6. 4.2 5.8 6.31 370223.01±0.14

S.D. - 0.0 0.84

Table 5.7.3: Robustness (Detection wavelength variation) studies of

withaferin A

Sr.

No

Detection

Wavelength (nm) RT AUC

1. 215 6.31 370222.18±0.40

2. 211 6.31 370221.97±0.27

3. 213 6.31 370222.69±0.04

4. 217 6.31 370221.59±0.12

S.D - 0.46



Table 5.7.4: Robustness (Flow rate variation) studies of withaferin A

Sr.

No Flow rate (ml/min) RT AUC

1. 1.0 6.31 370222.18±0.40

2. 0.8 6.31 370222.10±0.16

3. 0.9 6.31 370221.93±0.05

4. 1.2 6.31 370222.65±0.28

S.D - 0.31

Table 5.8: Stability studies of withaferin A in formulations containing

Ashwagandha

Extracts &

Formulations

Temperature

4°C 25°C

24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs

Ashwagandha

Vati 99.71 99.47 98.97 99.59 98.73 98.07

Ashwagandha

Tablet 99.89 99.12 98.81 99.56 98.92 98.01

Ashwagandha

Capsule 99.93 99.53 98.16 98.73 98.24 97.91

Ashwagandha

Churna (Brand A) 99.88 99.71 98.90 99.66 99.13 98.65

Ashwagandha

Churna (Brand B) 99.77 99.03 98.69 98.69 98.19 97.97

Ashwagandha

Arishta (Brand I) 99.82 99.06 98.56 99.22 98.76 98.19

Ashwagandha

Arishta (Brand II) 99.86 99.46 98.80 99.59 98.49 98.07



5.5 Quantitative analysis of formulations containing Withania Somnifera

for content of Withaferin A by HPLC

5.5.1 Preparation of Sample solutions

5.5.1.1 For Ashwagandha vati (tablet formulation)

2 g of powdered vati was transferred to 100 ml volumetric flask containing 50

ml of methanol and the mixture was macerated on a shaker for 24 hrs at room

temperature and then the volume was made up to 100 ml with methanol. Then

1.0 ml of this extract was diluted to a 10 ml with mobile phase.

5.5.1.2 For Ashwagandha churna (powder formulation) (Two brands)

2 g of each brand of powder churna was transferred to 100 ml volumetric

flask containing 50 ml of methanol and the mixture was macerated on a

shaker for 24 hrs at room temperature and then the volume was made up to

100 ml with methanol. Then 1.0 ml of this extract was diluted to a 10 ml with

mobile phase.

5.5.1.3 For Ashwagandharishta (liquid preparation containing self

generated alcohol) (Two brands)

10 ml of each brand of Ashwagandharishta were evaporated to dryness. 2 g of

residue was dissolved in 50 ml methanol in a volumetric flask. 2.5 ml of this

extract was diluted to 10 ml with mobile phase.

5.5.1.4 For Ashwagandha Capsule

Sample solutions of capsule formulation were prepared same as that of vati by

transferring 2 g of capsule contents.

5.5.1.5 For Ashwagandha tablet

2 g of powdered tablets was transferred to 100 ml volumetric flask containing

50 ml of methanol and the mixture was macerated on a shaker for 24 hrs at

room temperature and then the volume was made up to 100 ml with methanol.



Then 1.0 ml of this extract was diluted to a 10 ml with mobile phase.

The developed and validated method was used to ascertain the content of

withaferin A in seven marketed formulations of Ashwagandha. The shapes of

the peaks were not altered by substances present in the matrix. The percent

content of withaferin A was found to be 0.511 ± 0.5 %, 0.759 ± 0.4% and

0.789 ± 0.25% in Ashwagandha Vati, capsule and tablet respectively. The two

different brands of churna showed 0.770 ± 0.37% and 0.811 ± 0.4% percent

content of withaferin A. The amount of withaferin A was found to be 0.150%

± 0.3 and 0.144 % ± 0.4 in two different brands of Ashwagandharishta (Table

5.9).

Table 5.9: Content of withaferin A in formulations containing

Ashwagandha

Extracts & Formulations

Percent

Content

± S.D. (%)

Weight of

unit dose

Content per

unit dosage

form (mg)

Ashwagandha Vati 0.511 ± 0.5 , 250 mg 1.2775/ vati

Ashwagandha Tablet 0.789 ± 0.25 250 mg 1.0079/tablet

Ashwagandha Capsule 0.759 ± 0.4 200 mg 1.518/capsule

Ashwagandha Churna

(Brand A) 0.770 ± 0.37

3 g

(1tbs) 23.10/tbs

Ashwagandha Churna

(Brand B) 0.811 ± 0.4

3 g

(1tbs) 24.30/tbs

Ashwagandha Arishta

(Brand I) 0.150 ± 0.3 2 g 3.0/10ml

Ashwagandha Arishta

(Brand II) 0.144 ± 0.4 2 g 2.9/10ml



A simple, rapid, accurate and convenient HPLC method was developed using

the isolated withaferin A. As per ICH guideline the developed HPLC method

was validated. The content of withaferin A was estimated in seven

Ashwagandha formulations.

The total time for analysis is more in the reported HPLC method and even

mobile phase composition was gradient217-221

. Whereas, the developed and

validated method in this chapter is less time consuming, precise validated

method can be very well used to determine batch to batch variations and

routine analysis by herbal manufacturers of Ashwagandha medicines.

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