Arabian Journal of Chemistry (2017) 10, S1193–S1199

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Effect of extraction methods on yield,

phytochemical constituents and antioxidant activity
of Withania somnifera
Tushar Dhanani, Sonal Shah, N.A. Gajbhiye, Satyanshu Kumar *

Directorate of Medicinal and Aromatic Plants Research, Boriavi, Anand 378310, Gujarat, India

Received 11 July 2012; accepted 17 February 2013

Available online 5 March 2013

KEYWORDS Abstract Withnaia somnifera (L.) is a wonder herb with multiple medicinal properties. Its root is used
Withania somnifera; in the preparation of many Ayurvedic medicines. Withanolides, steroidal lactones, present in the root
Ultrasound; are active chemical markers, however, phenolics, and flavonoids have also been reported in the root of
Microwave; this plant. In most of the herbal preparations, water extraction is carried out using the infusion or
Antioxidant activity decoction preparation process. In the present study, extract yield, phytochemical constituents such
as total phenol and withanolide content of water and water-alcohol extracts prepared using two most
commonly used extraction techniques, also known as ‘‘Green Extraction’’ techniques, ultrasound and
microwave assisted solvent extraction were compared with the conventional extraction method. Anti-
oxidant activity of the extracts was also determined using DPPH and ABTS methods of antioxidant
assay. Extract yield, chemical composition of the extracts (total phenol and withanolide content) and
antioxidant activity of the extracts varied with the extraction process as well as solvent composition.
ª 2013 King Saud University. Production and hosting by Elsevier B.V. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction tain their activities (Aziz et al., 2003). Extraction is an important

step in the itinerary of phytochemical processing for the discov-
Plants are an important source of bioactive molecules for drug ery of bioactive constituents from plant materials. Selection of a
discovery. Isolated bioactive molecules serve as starting materi- suitable extraction technique is also important for the standard-
als for laboratory synthesis of drugs as well as a model for the ization of herbal products as it is utilized in the removal of desir-
production of biologically active compounds. Phytochemical able soluble constituents, leaving out those not required with the
processing of raw plant materials is essentially required to opti- aid of the solvents. Further, selection of suitable extraction pro-
mize the concentration of known constituents and also to main- cess and optimization of various parameters are critical for
upscaling purposes i.e. from bench scale to pilot plant level. Var-
* Corresponding author. Tel.: +91 2692 271605x208. ious extraction techniques most commonly used include conven-
E-mail address: [email protected] (S. Kumar). tional techniques such as maceration, percolation, infusion,
Peer review under responsibility of King Saud University. decoction, hot continuous extraction etc. Recently, alternative
methods like ultrasound assisted solvent extraction (UASE),
microwave assisted solvent extraction (MASE) and supercritical
fluid extractions (SFE) have gained increasing interest during
Production and hosting by Elsevier the last three decades (Co et al., 2012). Use of green extraction
http://dx.doi.org/10.1016/j.arabjc.2013.02.015
1878-5352 ª 2013 King Saud University. Production and hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
S1194 T. Dhanani et al.

techniques such as UASE (Wu et al., 2001; Ahh et al., 2007), in shade. The air dried roots were finely powdered using an elec-
MASE (Ganzler et al., 1986a,b) and SFE (Ollanketo et al., tric grinder. For conventional extraction (refluxing), 5 g of pow-
2002) has been rapidly and continuously increasing globally dered plant material was mixed with 50 ml of distilled water in a
for phytochemical processing of medicinal plants as these tech- round bottom flask and refluxed for about 5 h at 100 C. Simi-
niques are fast as compared to traditional methods. Also these larly, 5 g of powder was mixed with ethanol and water–ethanol
techniques are environmentally friendly in terms of solvent (9:1) separately in round bottom flasks and refluxed for 5 h. Li-
and energy consumption. Yield is also comparable to conven- quid extracts obtained were separated from the solid residue by
tional extraction and in some cases it is even higher. However, vacuum filtration, concentrated using a rotary evaporator.
extract yield as well as the bioactivities of the extract prepared For UASE, 5 g of powdered plant material was mixed with
using different extraction methods have been reported to vary 50 ml of distilled water, ethanol and water–ethanol (9:1) sepa-
in several studies (Hayouni et al., 2007). rately in beakers. Extraction was carried out by placing the
Withania somnifera (L.) Dunal., (Solanaceae) commonly three beakers in an ultrasonic bath (Bandelin Sonorex, Ger-
known as Ashwagandha, is a green shrub found throughout many, 480 W, 35 kHz) for 5, 10 and 20 min. Water in the ultra-
the drier parts of India, Pakistan, Afghanistan, Sri Lanka, sonic bath was circulated at room temperature (25 C) to avoid
Congo, South Africa and Egypt. In India, it is cultivated overheating caused by ultrasound. The supernatant was simi-
widely in Madhya Pradesh, Uttar Pradesh, Punjab, Gujarat larly processed as described in conventional extraction to get
and Haryana (Bhatia et al., 1987). It is categorized as a rasa- dried UASE extract of Withania somnifera. Similarly, for
yana in Ayurveda and traditional Indian systems of medicine. MASE, 5 g of powdered plant material was mixed with
The rasayanas, apart from their use for promoting physical 50 ml of distilled water, ethanol and water–ethanol (9:1) sepa-
and mental health also provide defence against diseases and ar- rately in Pyrex beakers. Extraction was carried out by placing
rest ageing process (Singh et al., 2001; Bhattacharya et al., the beakers in the middle of the oven over a rotating dish and
2002). W. somnifera is used as dietary supplement and the exposed to microwave radiation (LG Electronics, India, Model
decoction of its root is used as nutrient and health restorative MG-556 P, 1350 W, 2450 MHZ) for 5, 10 and 20 min. At the
to pregnant and old people. The extract of W. somnifera is a end of heating, the beaker was left for temperature stabiliza-
complex mixture of a large number of phytochemicals includ- tion (1 min).The supernatants were re-centrifuged and concen-
ing phenolic compounds and flavonoids. However, the phar- trated as described earlier. Dried extract samples were kept in
macological effect of the roots of W. somnifera is attributed an airtight container at 4 C.
to withanolides (Rahman et al., 1988; Chen et al., 1987; Uda-
yakumar et al., 2010). Withanolides are a series of naturally 2.2. Determination of total phenolics in extracts
occurring steroids containing a lactone with a side chain of
nine carbons, generally attached to C-17. Variation of lactone Total phenolic contents in the extracts were determined spec-
moiety is found in various classes of withanolides. Although, trophotometrically by Folin–Ciocalteau method (Singleton
withanolides also have been reported from the plants of fami- et al. 1999). Dried extracts were reconstituted in distilled water
lies such as Solanaceae, Taccaceae and Leguminose as well as (1 mg/ml). Folin–Ciocalteau reagent (0.5 ml) was added to the
from some marine organisms, genus withania is among the extract solution (0.5 ml) and the total volume was adjusted to
richest sources of steroidal lactones in nature (Srivastava 8.5 ml with distilled water. The tubes were kept at room tem-
et al., 1992; Ksebati and Schmitz, 1988). W. somnifera extract perature for 10 min and thereafter 1.5 ml of sodium carbonate
promoted learning and memory in animal models (Dhuley, (20%) was added. The tubes were incubated in a water bath at
2001), reversed the memory loss induced by oxidative damage 40 C for 20 min The intensity of the blue colour developed
caused by streptozotocin (Parihar et al., 2004). In vitro studies was measured by recording the absorbance at 755 nm using
have also established that an aqueous extract of W. somnifera a UV–visible spectrophotometer (Varian, CARY-300 Bio).The
root inhibited amyloid b (A b) fibril formation. Cerebral extra- reagent blank was also prepared using distilled water. For
cellular deposition of protein fibrils formed by A b, mainly quantification of the total phenolic in the extract, a standard
from the 40 or 42 amino acid A b peptide has been described calibration curve was prepared using gallic acid. Total pheno-
as the characteristics of Alzheimer’s disease in elderly people lic content of the extract samples was expressed as gallic acid
(Kumar et al., 2012). Keeping in view to find correlation be- equivalent (GAE) milligrams per gram of the extract.
tween the extraction methods, yield, total phenolics and wit-
hanolides content, the present investigation was carried out. 2.3. Identification and quantification of withanolides and
Extraction was done with water and water-alcohol as solvent withaferin in extracts using high performance liquid
using UASE and MASE since in most of the herbal prepara- chromatography (HPLC)
tions water or water-alcohol is used as a solvent. Conventional
extraction using refluxing was also carried out for the compar-
Withanolides (withanolide A and 12-deoxy withastramono-
ison of extract yield and phytochemical qualities of the extract.
lide) and withaferin A were quantified in the extracts using
HPLC (Shimazdu Prominence UFLC) on RP- C18 column
2. Materials and methods
(Lichrocart, Merck, 250 · 4.6 mm, 5 lm). Mobile phase con-
sisted of acetonitrile (40%) and 0.1% acetic acid in water
2.1. Plant material and extract preparation (60%) in an isocratic elution mode with the flow rate of
1.0 ml/min. Injection volume was 20 ll. The solvents were de-
Roots of W. somnifera were collected from the research farm of gassed using vacuum. Samples for HPLC analysis were also fil-
the Directorate of Medicinal and Aromatic Plants Research, tered through a 0.45 lm membrane filter. The peaks were
Boriavi, Anand, Gujarat, India in the year 2011 and air dried monitored at 230 nm using a UV–visible detector (UV–visible
Effect of extraction methods on yield, phytochemical constituents and antioxidant activity S1195

SPD 20 A).The peaks were identified on the basis of reten- dissolved in water to a 7 lM concentration and radical cation
tion time with the comparison of the retention time of stan- (ABTS+) was produced by reacting ABTS solution with
dard withanolide A, 12-deoxy withastramonolide and 2.45 lM potassium persulphate at room temperature in dark
withaferin A, their contents in the extracts were quantified (12–16 h) before use. For assay, ABTS+ solution was diluted
on the basis of area of the peak. with water to an absorbance value of 0.700 ± 0.02 at
734 nm. After addition of 3.0 ml of diluted ABTS+ solution
2.4. Antioxidant assay to 100 ll of extracts solutions, absorbance was recorded after
6 min.
2.4.1. Radical scavenging activity using DPPH method
The radical scavenging activity of extracts of W. somnifera pre- 2.4.3. Calculation of IC50 concentration
pared by refluxing, UASE and MASE was evaluated using The extract concentration corresponding to 50 percent inhibi-
DPPH assay. Different concentrations (equivalent to 200, tion (IC50) was calculated from the curve of RSA percentage
400, 600, 800 and 1000 ppm) of the extracts were taken in test against extract concentration. Ascorbic acid and trolox were
tubes. The total volume was adjusted to 8.5 ml by the addition used as standards. Each sample was assayed in triplicate for
of methanol. 5.0 ml of 0.1 mM methanolic solution of DPPH each concentration.
was added to these tubes and mixed well with a vortex mixer.
The tubes were kept at room temperature for 20 min. The
blank was prepared as above without the extract and methanol 3. Results and discussion
was used for the baseline correction. Changes in the absor-
bance of the extract samples were measured at 517 nm using Biologically active compounds usually occur in low concentra-
the UV–visible spectrophotometer. Radical scavenging activity tion in plants. An extraction technique is that which is able to
(RSA) was expressed as the inhibition percentage and was cal- obtain extracts with high yield and with minimal changes to
culated using the following formula the functional properties of the extract required (Quispe Can-
%Radical scavenging activity dori et al., 2008). Several studies have reported variations in
ðAbsorbance of blank  absorbance of sampleÞ the biological activities of extracts prepared using different
¼  100 extraction techniques. Therefore, it is necessary to select the
Absorbance of blank
suitable extraction method as well as solvent based on sample
matrix properties, chemical properties of the analytes, matrix-
2.4.2. Free radical scavenging ability by the use of a stable analyte interaction, efficiency and desired properties (Hayouni
ABTS radical cation et al., 2007; Ishida et al., 2001). In conventional extraction,
Free radical scavenging activity was determined by ABTS rad- heat is transferred through convection and conduction from
ical cation decolorization assay (Re et al., 1999). ABTS was the surface, here, the extractability of solvents depends mainly

Table 1 Yield, total phenolics, withanolide A, 12-deoxy withastramonolide, and IC50 values of the extracts of W. somnifera root
prepared using different extraction methods+.
Extraction Treatment Yield (%) Total phenolics Withanolide A 12 deoxy Total withanolides IC50
method (mg/g GAE) (lg/mg) withastramonolide (lg/mg) (mg/ml)
(lg/mg)
DPPH ABTS
Reflux (Soxhlet) Ethanol 9.08 35.93 3.57 1.22 4.79 0.18 1.05
Water: ethanol 9.43 21.15 1.25 0.40 1.65 0.39 2.04
Water 9.51 17.63 1.14 0.36 1.50 0.40 2.14
UASE Ethanol* 2.85 24.72 6.04 2.04 8.09 0.21 1.24
Ethanol** 2.96 25.27 6.58 2.08 8.66 0.20 1.21
Ethanol*** 3.17 29.15 6.08 1.94 8.02 0.18 1.20
Water: ethanol* 9.74 21.15 1.21 0.41 1.62 1.03 2.54
Water: ethanol** 8.96 21.39 1.48 0.47 1.95 0.99 2.53
Water:ethanol*** 9.08 22.12 1.84 0.62 2.46 0.97 2.51
Water* 9.90 14.90 0.91 0.26 1.16 1.20 2.68
Water** 11.85 15.81 0.81 0.23 1.04 1.19 2.64
Water*** 10.27 18.18 0.67 0.20 0.87 1.17 2.62
MASE Ethanol* 10.01 40.96 4.35 1.39 5.73 0.15 0.83
Water: ethanol* 11.39 20.84 1.09 0.28 1.36 0.73 2.11
Water: ethanol** 12.81 21.81 0.97 0.29 1.26 0.66 2.09
Water:ethanol*** 13.75 22.90 1.00 0.30 1.31 0.62 2.07
Water* 11.18 17.15 0.59 0.20 0.79 0.70 2.42
Water** 12.74 17.99 0.67 0.23 0.90 0.69 2.39
Water*** 13.02 18.72 0.69 0.19 0.89 0.68 2.36
+
Mean of three replications.
*
5 min.
**
10 min.
***
20 min.
S1196 T. Dhanani et al.

H 3C
CH 3
OH
H 3C
CH 3 O O
H
O
CH 3

O
OH

Withanolide-A

H 3C
OH
H
H3 C
CH 3 O O
H
O
CH 3

O
OH

12-deoxywithastramonolide

H 3C
OH
H
H 3C
CH3 O O
H
O
CH 3

Figure 2 IC50 values of DPPH and ABTS assays for free radical
O
scavenging activity of extracts prepared using (A) refluxing (B)
OH withaferin-A
UASE (C) MASE.
Figure 1 Structure of withanolide A, 12-deoxywithastromono-
lide and withaferin A.
migration of targeted compounds from the matrices to the sur-
roundings at a more rapid rate. Recoveries are similar or better
on the solubility of the compound in the solvent, the mass in both UASE and MASE as compared to conventional
transfer kinetics of the product and the strength of solute/ma- extraction. However, reduced extraction time and solvent con-
trix interaction with corresponding limitations on heat and sumption are the main advantages of UASE and MASE.
mass diffusion rate. Ultrasound assisted solvent extraction is Although, extraction of bioactive compounds from the plants
a process that uses high intensity, high frequency sound waves has been extensively investigated using conventional solvent
and solvents to extract targeted compounds from various extraction, the present investigation was undertaken to study
matrices. Physical and chemical properties of the materials the effect of extraction methods on yield and phytochemical
subjected to ultrasound are altered due to the propagation qualities of W. somnifera.
and interaction of sound waves as they disrupt the plant cell Extract yield of W. somnifera root prepared by refluxing,
walls, thereby, facilitating release of extractable compounds UASE and MASE methods using water, ethanol and
and enhancing mass transport of solvent from the continuous water–ethanol is summarized in Table 1. Extraction yield
phase into plant cells. Microwave energy and solvents are used (mass of extract/mass of dry matter) was used as an indicator
for the extraction of targeted compounds from plant matrices of the effects of the extraction conditions. In reflux, water ex-
in the case of microwave assisted solvent extraction. tract yield (9.51%) was maximum followed by water–ethanol
Highly localized temperature and pressure can cause selective and ethanol. For UASE and MASE, three time periods 5, 10
Effect of extraction methods on yield, phytochemical constituents and antioxidant activity S1197

(A) 1.00 230nm

0.75

0.50

14.323
0.25

11.330
0.220

0.00

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0

(B) 230nm

1.50

1.25

1.00

0.75

0.50

14.268
11.288
0.25

0.00

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0

(C) 1.50 230nm

1.25

1.00

0.75

0.50
14.267
11.287

0.25

0.00
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0

(D) 6.0
230nm
11.374

5.0

4.0
14.330
9.340

3.0

2.0

1.0

0.0

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0

Figure 3 HPLC chromatogram of extracts of W. somnifera root prepared using (A) refluxing (B) UASE and (C) MASE extraction (D)
standard withaferin A, (Rt = 9.32 min) 12-deoxy withastramonoidie (Rt = 11.46 min), withanolide A (Rt = 14.26 min).

and 20 min were used. For UASE also, the trend was similar as mum extract yield (11.85%) obtained with water at 15 min.
observed in the case of refluxing. But extract yield with ethanol Possibly, this could be due to the higher polarity of water
(3.17%, 20 min) was about 3.74 times lower than the maxi- and also at elevated extraction temperature as in the case of
S1198 T. Dhanani et al.

UASE and MASE, water has a similar dielectric constant as for constant support and encouragement for undertaking the
organic solvents like methanol and acetonitrile. In case of present research work. Technical help provided by Shri B.K.
MASE, the extract yield increased with the time of exposure Mishra is also acknowledged.
to microwave radiation and water (13.02%, 20 min) and
water–ethanol (13.75%, 20 min) were found to be comparable. References
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