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Influence of phytochemical composition on in vitro antioxidant and reducing activities of Indian ginseng [Withania somnifera (L.) Dunal] root extracts
Accepted Manuscript Influence of phytochemical composition on in vitro antioxidant and reducing activities of Indian ginseng [Withania somnifera (L.) Dunal] root extracts Bhaskar Ganguly, Nirbhay Kumar, Abul H. Ahmad, Sunil K. Rastogi PII:
S1226-8453(17)30043-X
DOI:
10.1016/j.jgr.2017.05.002
Reference:
JGR 287
To appear in:
Journal of Ginseng Research
Received Date: 18 February 2017 Revised Date:
4 May 2017
Accepted Date: 8 May 2017
Please cite this article as: Ganguly B, Kumar N, Ahmad AH, Rastogi SK, Influence of phytochemical composition on in vitro antioxidant and reducing activities of Indian ginseng [Withania somnifera (L.) Dunal] root extracts, Journal of Ginseng Research (2017), doi: 10.1016/j.jgr.2017.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Influence of phytochemical composition on in vitro antioxidant and reducing activities
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of Indian ginseng [Withania somnifera (L.) Dunal] root extracts
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Author names and affiliations:
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Bhaskar GANGULY*1, Nirbhay KUMAR2, Abul H. AHMAD2, Sunil K. RASTOGI1
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Pharmacology and Toxicology; College of Veterinary and Animal Sciences, G. B. Pant
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University of Agriculture and Technology, Pantnagar, PIN – 263145. India. *Corresponding
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author, Tel: +91-9411159689, Fax: +91-5944233473, email: [email protected]
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Running title: Phytochemical influence on Indian ginseng
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Department of Veterinary
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Department of Veterinary Physiology and Biochemistry,
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ACCEPTED MANUSCRIPT ABSTRACT
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Background: Roots of Withania somnifera (WS) are a celebrated medicinal ingredient in
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Ayurvedic and many other indigenous systems of medicine. The present study investigates
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the effect of the phytochemical composition of the extracts on their antioxidant and reducing
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activities. Methods: WS roots were extracted with water, acetone, aqueous methanol (1:1)
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and methanol : chloroform : water (1:1:1) to obtain aqueous, acetone, hydro-methanolic and
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methanol-chloroform-water extracts. Thereafter, phytochemical constitution and antioxidant
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and reducing activities of the extracts were compared using different qualitative and
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quantitative tests. Results: Maximum extraction recovery was obtained with 50% aqueous
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methanol whereas extraction with acetone yielded the poorest recovery. Methanol-
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chloroform-water extract had the highest content of phytochemical constituents, except
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tannins, and also exhibited the highest antioxidant and reducing activities. Conclusion:
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Phytochemical composition and antioxidant and reducing activities of the extracts were
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positively associated with the use of organic solvents during the extraction process. Alkaloids
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and flavonoids were the most important contributors in the antioxidant and reducing activities
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of the extracts.
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Keywords: antioxidant; extracts; phytochemicals; roots; Withania somnifera
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ACCEPTED MANUSCRIPT 1. INTRODUCTION
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Withania somnifera (Linn.) Dunal, commonly known as Indian Ginseng or Indian winter
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cherry, is recognized as the Queen of Indian herbs and has received similar admiration in
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Unani, Siddha, and Chinese systems of medicine [1]. The roots are the most commonly used
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part and find enormous medicinal use; powder of the roots is the key ingredient of almost any
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antioxidant, anti-stress, and anti-aging formulation, human or veterinary, in India. The root
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powder and its preparations are consumed extensively as a functional food for promoting
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vitality and virility. The plant is cultivated in the states of Madhya Pradesh, Uttar Pradesh,
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Punjab, Gujarat, and Rajasthan. The plant also grows at large, wildly or commercially, in
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Congo, South Africa, Morocco, Egypt, Israel, Jordan, Pakistan and Afghanistan [2, 3]. The
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roots are reputed to promote health and longevity by augmenting defense against disease,
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arresting aging, revitalizing the body in debility, increasing resistance to adverse
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environmental factors and creating a sense of well-being [1, 4]. Extraction is an important
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step in the itinerary of phytochemical processing for the discovery of bioactive constituents
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from plant materials [5]. In the present study, qualitative and quantitative methods were used
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for comparing the phytochemical composition and in vitro antioxidant and reducing activities
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of different extracts of Withania somnifera roots. The effect of the varying concentration of
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phytochemicals in the extracts on their antioxidant and reducing activities was determined.
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ACCEPTED MANUSCRIPT 2. MATERIALS AND METHODS
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2.1. Withania somnifera roots
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Roots were procured through a professional herb vendor and got authenticated from the
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Medicinal Plant Research and Development Centre, Pantnagar (Supplementary File 1). They
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were cut into smaller pieces, dried under hot circulating air at 40ºC for 3-4 days, and ground
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to a fine powder. The powder was subjected to compositional tests for ascertaining
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pharmacognostic standards [6]. Total lipid, total protein, and total sugar were estimated
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according to standard methods [7]. Loss on drying, total ash, acid insoluble ash, water soluble
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ash, alcohol soluble extractive value and water soluble extractive value were determined as
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described in the Ayurvedic Pharmacopoeia of India [8].
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2.2. Extraction and Spectral analysis
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200 grams of root powder was left soaked in 1000 mL of either water, methanol : chloroform
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: water (12:5:3), acetone or aqueous methanol (1:1) for 48 hours at 40ºC in a shaking
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incubator to obtain aqueous (Aqu), methanol-chloroform-water (MCW), acetone and hydro-
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methanolic (HM) extracts, respectively; this step was repeated thrice. The resulting extracts
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were first filtered through double-layered muslin, and then through Whatman paper no. 42.
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The final extracts were obtained by drying the filtrate to a constant weight at 40ºC. The yield
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was expressed as the mass of extract obtained per 100 grams of roots. 0.1% solutions of the
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dried extracts were subjected to spectral analysis for evaluation of composition.
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2.3. Qualitative and Quantitative Phytochemical analysis
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The extremely low yield of acetone extract precluded its further use in the study. Small
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portions of each of the remaining three extracts viz. Aqu, MCW, and HM were subjected to
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qualitative tests for different phytochemical compounds as per standard methods [9, 10].
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Thereafter, alkaloids, total phenolic content (TPC), flavonoids, flavonols, proanthocyanidins,
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ACCEPTED MANUSCRIPT and tannins were quantitated as described earlier by Shabbir et al [10] whereas withanolide
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content was determined as per Pati et al [11].
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2.4. In vitro Antioxidant and Reducing assays
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Antioxidant assays were performed with minor modifications to standard methods as
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described elsewhere [10]. Further dilutions of the stocks were prepared in an appropriate
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medium/ buffer as per the requirements of the individual assay. Where applicable, the median
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inhibitory concentration, IC50, was also calculated.
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2.5. Statistical analyses
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Statistical inferences were drawn on the basis of analysis of variance (ANOVA) followed by
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post-hoc Tukey’s test performed with Daniel’s XL Toolbox 6.53. Multivariate analysis, viz.
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Partial Least Square (PLS) Regression Analysis and Principal Component Analysis (PCA),
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was performed in Multibase 2014.
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ACCEPTED MANUSCRIPT 3. RESULTS AND DISCUSSION
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According to the Ayurvedic Pharmacopoeia of India, the loss on drying, total ash, acid
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insoluble ash, and alcohol extractive values for WS roots should be ≤ 8%, ≤ 7%, ≤ 1% and ≥
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15%, respectively [6]. The WS roots used in the present study satisfied these standards of
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pharmacognostic quality (Table 1).
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Previously, comparison of six different solvents and solvent combinations found that the best
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extraction was achieved by acetone and MCW (Methanol : chloroform : water, 12:5:3) [12].
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This formed the basis for selection of acetone and MCW as solvents in the current study;
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water was also included as a third extraction solvent. Of these, acetone had a very poor yield
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(~0.162%); hence, it was excluded from further study on the basis of non-feasibility, and the
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HM extract was included. Mean yields of Aqu, HM and MCW extracts were 11.64, 16.82,
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and 14.39%, respectively. Maximum yield with aqueous methanol (1:1) may be due to
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optimum solvation of both hydrophilic and lipophilic components in the milieu. Previously, a
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mean yield of 15.40% was obtained upon hydro-ethanolic extraction of WS roots [13].
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Spectral analysis showed increased absorbance by the extracts in the 240-360 nm range (Fig.
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1) that can be attributed to a host of chemical constituents. Carbohydrates absorb strongly at
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360 nm, whereas absorbance at 200 nm, 240 nm, 270 nm and 300 nm reflect the presence of
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proteins and glycoproteins. Many alkaloids absorb strongly in the 250-260 nm wavelength
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range. Coumarins absorb strongly at 280 and 320 nm [14]. The MCW extract, though
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comparable to HM, exhibited the strongest absorbance of the three extracts. It must be noted
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that although spectral analysis gives a fair indication of the prominent phytochemical
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constituents, the results are not always reliable because the presence of many other
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compounds can also cause absorbance patterns. Thus, qualitative and quantitative estimation
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of the phytochemical constituents was performed. Qualitative phytochemical analyses (Table
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2) also suggest a higher concentration of phytochemical constituents in the MCW extract.
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ACCEPTED MANUSCRIPT Quantitative tests were performed to determine the concentration of the phytochemicals in the
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extracts. Alkaloids and withanolides were quantified using gravimetric methods. Both
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alkaloid and withanolide content of the extracts were in the order: MCW > HM > Aqu (Fig.
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2) and the differences were highly significant (p < 0.01). The concentration of withanolides in
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the roots usually ranges from 0.001 to 0.5% of the dry weight. Total alkaloid content in the
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roots of Indian W. somnifera varies between 0.13 and 0.31%, though yields up to 4.3% have
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been recorded in other regions [15].
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Total phenolic content (TPC) was determined on a gallic acid curve (y = 0.0072x + 0.0472;
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R² = 0.9897). TPC of the extracts was in the order: MCW > HM > Aqueous (Fig. 2); all
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differences being highly significant. Previously, the TPC of methanol, chloroform, and
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aqueous extracts have been reported at 42, 66.72 and 88.58 µg gallic acid equivalents
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(GAE)/mg extract, respectively. It was further suggested that most polyphenolics of WS were
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polar in nature [16]. Another study found the phenol content of the extracts in the order:
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chloroform > petroleum ether > methanol > ethanol > n-hexane; TPC of chloroform extract
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was 60.992 ± 0.367 µg GAE/mg [17]. In the present study, a higher TPC of MCW over HM
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and Aqu suggests a preferential extraction of phenolics in the order: chloroform > methanol >
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water, which appears to be in agreement with the latter study. Much lower TPC values for
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80% methanolic extracts of WS roots have been reported from Sri Lanka [18].
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Phenolic compounds are represented by flavonoids, tannins, and coumarins [14]. Flavonoid
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content was determined on a quercetin standard curve (y = 0.012x + 0.0604; R² = 0.9918).
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Flavonoid content of the extracts was in the order: MCW > HM > Aqu (Fig. 2), and
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differences between extracts were highly significant. Alam et al prepared WS fruit, root and
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leaf extracts by using 80% methanol; 17.80 ± 5.80 to 32.58 ± 3.16 mg GAE phenolics and
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15.49 ± 1.02 to 31.58 ± 5.07 mg catechin equivalent (CEQ) flavonoids were found per gram
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[19]. Subsequently, 15.49 ± 1.02 mg GAE phenolics and 17.80 ± 5.80 mg CEQ flavonoids
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ACCEPTED MANUSCRIPT have been reported per gram WS roots from Bangladesh [20]. Shahriar et al reported
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flavonoid content of methanol and chloroform extracts at 88.761 ± 1.032 and 122.094 ±1.351
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mg QE/g, respectively [17]. Udayakumar et al reported 28.26 mg/g total phenolic compounds
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and 17.32 mg/g flavonoids in WS root extracts [21]. Flavonol contents were determined on a
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quercetin standard curve (y = 0.0103x + 0.3781; R² = 0.9858); these were in the order: MCW
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> HM > Aqu (Fig. 3); the differences MCW vs. HM and MCW vs. Aqu were highly
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significant, whereas the difference HM vs. Aqu was non-significant.
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Tannins are chemically classified into hydrolysable tannins and condensed tannins,
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commonly called proanthocyanidins [14]. All differences in the proanthocyanidin content of
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the extracts (Fig. 2), determined on a gallic acid curve (y = 0.0051x + 0.059; R² = 0.9905),
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were non-significant. Tannin content was determined from a tannic acid curve (y = 0.0061x +
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0.0434; R² = 0.9895). Tannin content of the extracts was in the order: HM > MCW > Aqu
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(Fig. 2); the differences HM vs. Aqu and MCW vs. Aqu were highly significant whereas the
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difference HM vs. MCW was non-significant. The differences between the three extracts in
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their overall phytochemical composition were significant (0.01 < p < 0.05).
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DPPH scavenging activity of MCW matched that of HM closely. However, none of the
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extracts fared as well as the ascorbic acid standard (Fig. 3). The statistical differences
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between the extracts were highly significant. Previously, Alam et al. have reported much
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lower DPPH scavenging activities of 80% aqueous methanol extract of WS root (IC50 =
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801.93 ± 7.92 µg/mL) [20]. The ascorbic acid content of the roots remained indeterminate by
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the standard method as described by Alam et al. (results not shown); previously, the ascorbic
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acid content of WS roots has been reported to be as low as 0.02% [20].
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The superoxide radical scavenging activities of the three extracts and the standard (Fig. 3)
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closely matched at lower concentrations but differences became prominent at higher
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concentrations. Amongst the extracts, MCW showed the highest activity followed by HM and
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ACCEPTED MANUSCRIPT Aqu. Albeit graphical indifference, the statistical differences were highly significant. Our
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results are in partial agreement with former reports of IC50 of methanol, water, and
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chloroform extracts of WS root for superoxide radical at 94, 125, and 178 µg/mL,
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respectively [16].
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Hydroxyl radicals are the most reactive and predominant radicals generated during aerobic
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metabolism [16]. The hydroxyl ion scavenging activity of ascorbic acid was prominently
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higher than any of the extracts (Fig. 3). Again, the statistical differences between the extracts
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and between concentrations within extracts were highly significant. Previously, IC50 of
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aqueous extract of WS root for hydroxyl radical was reported at ~800 µg/mL [16], which is
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comparable to our results (IC50 = 999.82 µg/mL).
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Peroxide scavenging activities of the three extracts (Fig. 3) were similar at low concentrations
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but showed greater differences at higher concentrations. At a concentration of 100 µg/mL,
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both ascorbic acid and MCW exhibited nearly equal inhibition. The differences between the
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activities were highly significant. Although the IC50 of ascorbic acid for peroxide radical
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obtained by us in the present study is comparable to the previously reported value (164
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µg/mL), the IC50 of Aqu for peroxide radical obtained in the present study (1069.79 µg/mL)
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varied greatly from the previously reported value of 323 µg/mL [16].
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Nitric oxide scavenging activity of ascorbic acid was much higher than any of the extracts
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(Fig. 3). The activities of MCW and HM were similar at lower concentrations whereas, at
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highest tested concentration, the activities of all three extracts were similar. Yet, the
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differences between groups were highly significant. The performance of the different extracts
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in the different radical scavenging assays mostly showed good correlation with each other
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(Table 3).
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Total antioxidant capacity (ToAC) was determined from an ascorbic acid standard curve (y =
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0.171 ln(x) - 0.2106; R² = 0.9746). ToAC of MCW was highest (83.354 ± 1.828) followed by
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ACCEPTED MANUSCRIPT HM (76.978 ±2.210) and Aqu (68.439 ± 1.000). The difference between the ToAC of MCW
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and Aqu was highly significant whereas that between HM and Aqu was significant and that
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between MCW and HM was non-significant.
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The ability of the extracts to reduce Fe3+ to Fe2+ was compared with that of ascorbic acid
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(Fig. 4). A higher absorbance value indicates higher activity. Fe3+ reducing activity of
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ascorbic acid was much higher than that of the extracts. Aqu exhibited the poorest activity of
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the three extracts, all differences between activities being highly significant.
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PLS regression analysis (Fig. 5) revealed the importance of each phytochemical in the
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individual and overall antioxidant and reducing activities of the three extracts. The alkaloid
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content of an extract was the most important contributing variable in its hydroxyl radical
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scavenging, peroxide scavenging, total antioxidant and Fe3+ reducing activities. Flavonol
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content appeared the most important determinant for superoxide and nitric oxide scavenging
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activities. Tannin content of an extract was most decisive of its DPPH radical scavenging
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activity; however, tannin content was also the least important variable in the overall
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antioxidant and reducing activities. Alkaloid content was the leading contributor to the
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overall antioxidant and reducing activities of the extracts, closely followed by flavonoids and
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withanolides.
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PCA showed that MCW and HM were more similar to each other than to Aqu in terms of the
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phytochemical constitution as well as antioxidant and reducing activities. Also, the two
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extracts were more similar in terms of their Fe3+ reducing, hydrogen peroxide scavenging and
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superoxide radical scavenging activities as compared to other parameters studied (Table 4;
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Fig. 6). It follows that the phytochemical composition and antioxidant and reducing activities
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of the extracts are positively influenced by the use of an organic solvent during the extraction
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process.
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ACCEPTED MANUSCRIPT 4. CONCLUSIONS
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In the present study, a comparison of different solvent systems for extraction of the WS root
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powder showed maximum extraction recovery with aqueous methanol, whereas acetone
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extract had to be excluded from further studies due to exceptionally poor yield. Methanol-
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chloroform-water (MCW) extract exhibited the strongest absorbance and showed the highest
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content of all phytochemical constituents, except tannins. MCW also exhibited the highest
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antioxidant and reducing activities. The inclusion of an organic solvent in the extraction
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medium was found to positively influence the antioxidant and reducing activity of the extract.
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Multivariate analysis found alkaloids and flavonoids to be the most important contributors
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towards the overall antioxidant and reducing activities of WS root extracts. The MCW and
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HM extracts were more similar to each other than to aqueous extract, in terms of the
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phytochemical composition, and antioxidant and reducing activities, suggesting a favorable
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influence of organic solvents.
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ACKNOWLEDGMENTS
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The financial assistance received by BG in the form of a Senior Research Fellowship from
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Council of Scientific and Industrial Research (CSIR), India is gratefully acknowledged. CONFLICT OF INTERESTS
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The authors declare no conflicting interests.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS
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Fig. 1: Spectral analysis of WS root extracts. UV-Vis absorption characteristics of Aqu,
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HM, and MCW have been shown for comparison. Stronger absorbance suggests a higher
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concentration of phytochemicals.
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Fig. 2: Results of quantitative tests for phytochemicals in different extracts of WS roots;
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Mean ± S.E. values of different phytochemicals have been expressed as microgram
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equivalents of respective standards per milligram of extract; alkaloids and withanolides have
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been expressed as percent of dry weight.
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Fig. 3: Percent inhibition and median inhibitory concentration (IC50) of ascorbic acid
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and different extracts of WS roots in antioxidant and reducing assays; IC50, median
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inhibitory concentration; DPPH, DPPH radical scavenging assay; SRSA, Superoxide radical
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scavenging assay; HRSA, Hydroxyl radical scavenging assay; HPSA, Hydrogen peroxide
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scavenging assay; NOSA, Nitric oxide scavenging assay; AA, ascorbic acid.
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Fig. 4: Fe3+ reducing activities of ascorbic acid and different WS root extracts at
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varying concentrations; higher absorbance at 700 nm is indicative of greater reducing
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activity.
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Fig. 5: PLS regression analysis of phytochemical constituents on antioxidant and
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reducing properties of WS root extracts; values indicate the importance of a variable i.e.
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phytochemical concentrations in antioxidant/ reducing activity; total height of a column
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represents contribution in the overall antioxidant and reducing activities of extracts.
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Fig. 6: Principal Component Analysis of the effect of phytochemical constitution of WS
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root extracts, PC1-PC2 biplot; red ellipse shows the distribution of scavenging assays, blue
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ellipse shows the distribution of phenolics (except tannins), yellow ellipse shows the
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distribution of reducing assays; purple ellipse shows tannins and green ellipse shows the
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distribution of alkaloids and withanolides.
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ACCEPTED MANUSCRIPT TABLES Physicochemical composition Analyte Total lipid Total protein Total sugars Loss on drying Total ash Acid insoluble ash Water soluble ash Alcohol soluble extractive value Water soluble extractive value
Amount (%) 1.38 4.27 7.17 2.08 4.33 0.65 1.85 17.36 66.64
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Table 1. Physicochemical composition of WS roots; all values are expressed as per cent of
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air-dried weight.
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HM ++ +++ ++ +++ +++ +++ ++ ++
MCW +++ +++ +++ +++ +++ +++ ++ ++
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Phytochemical Alkaloids Anthraquinones Coumarins Flavonoids Phenolics Reducing sugars Glycosides Saponins Tannins Terpenoids
Table 2. Results of qualitative tests for phytochemical constituents of aqueous, hydro-
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methanolic and methanol-chloroform-water extracts; +++ reaction within 5 min; ++
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reaction in 5-30 min; + reaction in > 30 min; - no reaction up to 24 hours; ± undefined color
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change; HM, hydro-methanolic extract; MCW, methanol-chloroform-water extract.
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DPPH 1.000 0.907 0.980 0.976 0.871
SRSA 1.000 0.960 0.933 0.996
HRSA 1.000 0.995 0.932
HPSA 1.000 0.899
NOSA 1.000
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DPPH SRSA HRSA HPSA NOSA
Table 3. Correlation (R) between performance of different extracts in the radical
9
scavenging assays; DPPH, DPPH radical scavenging assay; SRSA, Superoxide radical
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scavenging assay; HRSA, Hydroxyl radical scavenging assay; HPSA, Hydrogen peroxide
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scavenging assay; NOSA, Nitric oxide scavenging assay.
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Component 2 2.24%
3 0.16%
97.60%
99.84%
100.00%
1.95
0.04
0.00
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Contribution Accumulation of Contribution Eigenvalue
1 97.60%
Table 4. Principal Component Analysis of the effect of phytochemical constitution on
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antioxidant and reducing activities of WS root extracts; contribution and eigenvalue of
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individual components.
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5 4.5 4 3.5
Absorbance
3 2.5 2
Aqu MCW HM
1 0.5 0 200
300
TE D
1.5
400
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λ(nm)
500
600
700
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109.65
0.377
0.230 97.38
0.197
AC C
Fig. 2
5.44
32.29
5.38
29.67
38.66
Aqueous
HM
36.00
37.75
63.49
53.9
EP
0.117
77.13
TE D
0.313
37.34
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0.250
4.20 18.19
MCW
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999.82
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326.54
278.77
251.95
193.39
178.08 121.74
111.31
32.80
5.60
0 AA
Aqu MCW DPPH
HM
AA
TE D
40
20
130.44
100 89.62
58.62
48.61
38.83
60
168.27
IC50 (µg/mL)
Per cent Inhibition
80
Aqu MCW
HM
AA
SRSA
10 µg/mL
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EP
5 µg/mL
Fig. 3
471.82
421.46
100
1000
1,069.79
Aqu MCW 50 µg/mL
10
1 HM
AA
HRSA
20 µg/mL
36.38
Aqu MCW
HM
AA
HPSA 100 µg/mL
500 µg/mL
Aqu MCW NOSA
IC50
HM
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2.5
O.D.700
2
1.5
0.5
0 Ascorbic acid
AC C
Fig. 4
Aqueous
31.25 µg/mL
EP
15.625 µg/mL
TE D
1
62.5 µg/mL
MCW 125 µg/mL
250 µg/mL
HM 500 µg/mL
0.998
0.978
0.996
0.972
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0.967
0.944
0.997
0.979
0.974
0.886
0.875
0.975
0.970
0.968
0.962
0.963
0.914
0.904
0.830
0.902
EP AC C
SRSA
0.864
0.965
0.936
0.924 0.867
1.000
0.784 0.698
0.995
0.917
0.970
0.991
0.977
0.957
0.912
DPPH
Fig. 5
0.997
0.843
HRSA
HPSA
0.858
0.904
0.842
0.822
0.743
0.967
0.992
0.999
TE D
0.994
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0.921
0.998
0.609
NOSA
ToAC
Fe3+
AC C
Fig. 6
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S1226-8453(17)30043-X
DOI:
10.1016/j.jgr.2017.05.002
Reference:
JGR 287
To appear in:
Journal of Ginseng Research
Received Date: 18 February 2017 Revised Date:
4 May 2017
Accepted Date: 8 May 2017
Please cite this article as: Ganguly B, Kumar N, Ahmad AH, Rastogi SK, Influence of phytochemical composition on in vitro antioxidant and reducing activities of Indian ginseng [Withania somnifera (L.) Dunal] root extracts, Journal of Ginseng Research (2017), doi: 10.1016/j.jgr.2017.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Influence of phytochemical composition on in vitro antioxidant and reducing activities
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of Indian ginseng [Withania somnifera (L.) Dunal] root extracts
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Author names and affiliations:
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Bhaskar GANGULY*1, Nirbhay KUMAR2, Abul H. AHMAD2, Sunil K. RASTOGI1
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1
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Pharmacology and Toxicology; College of Veterinary and Animal Sciences, G. B. Pant
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University of Agriculture and Technology, Pantnagar, PIN – 263145. India. *Corresponding
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author, Tel: +91-9411159689, Fax: +91-5944233473, email: [email protected]
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Running title: Phytochemical influence on Indian ginseng
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Department of Veterinary
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Department of Veterinary Physiology and Biochemistry,
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ACCEPTED MANUSCRIPT ABSTRACT
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Background: Roots of Withania somnifera (WS) are a celebrated medicinal ingredient in
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Ayurvedic and many other indigenous systems of medicine. The present study investigates
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the effect of the phytochemical composition of the extracts on their antioxidant and reducing
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activities. Methods: WS roots were extracted with water, acetone, aqueous methanol (1:1)
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and methanol : chloroform : water (1:1:1) to obtain aqueous, acetone, hydro-methanolic and
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methanol-chloroform-water extracts. Thereafter, phytochemical constitution and antioxidant
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and reducing activities of the extracts were compared using different qualitative and
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quantitative tests. Results: Maximum extraction recovery was obtained with 50% aqueous
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methanol whereas extraction with acetone yielded the poorest recovery. Methanol-
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chloroform-water extract had the highest content of phytochemical constituents, except
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tannins, and also exhibited the highest antioxidant and reducing activities. Conclusion:
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Phytochemical composition and antioxidant and reducing activities of the extracts were
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positively associated with the use of organic solvents during the extraction process. Alkaloids
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and flavonoids were the most important contributors in the antioxidant and reducing activities
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of the extracts.
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Keywords: antioxidant; extracts; phytochemicals; roots; Withania somnifera
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ACCEPTED MANUSCRIPT 1. INTRODUCTION
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Withania somnifera (Linn.) Dunal, commonly known as Indian Ginseng or Indian winter
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cherry, is recognized as the Queen of Indian herbs and has received similar admiration in
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Unani, Siddha, and Chinese systems of medicine [1]. The roots are the most commonly used
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part and find enormous medicinal use; powder of the roots is the key ingredient of almost any
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antioxidant, anti-stress, and anti-aging formulation, human or veterinary, in India. The root
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powder and its preparations are consumed extensively as a functional food for promoting
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vitality and virility. The plant is cultivated in the states of Madhya Pradesh, Uttar Pradesh,
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Punjab, Gujarat, and Rajasthan. The plant also grows at large, wildly or commercially, in
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Congo, South Africa, Morocco, Egypt, Israel, Jordan, Pakistan and Afghanistan [2, 3]. The
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roots are reputed to promote health and longevity by augmenting defense against disease,
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arresting aging, revitalizing the body in debility, increasing resistance to adverse
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environmental factors and creating a sense of well-being [1, 4]. Extraction is an important
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step in the itinerary of phytochemical processing for the discovery of bioactive constituents
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from plant materials [5]. In the present study, qualitative and quantitative methods were used
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for comparing the phytochemical composition and in vitro antioxidant and reducing activities
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of different extracts of Withania somnifera roots. The effect of the varying concentration of
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phytochemicals in the extracts on their antioxidant and reducing activities was determined.
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ACCEPTED MANUSCRIPT 2. MATERIALS AND METHODS
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2.1. Withania somnifera roots
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Roots were procured through a professional herb vendor and got authenticated from the
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Medicinal Plant Research and Development Centre, Pantnagar (Supplementary File 1). They
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were cut into smaller pieces, dried under hot circulating air at 40ºC for 3-4 days, and ground
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to a fine powder. The powder was subjected to compositional tests for ascertaining
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pharmacognostic standards [6]. Total lipid, total protein, and total sugar were estimated
52
according to standard methods [7]. Loss on drying, total ash, acid insoluble ash, water soluble
53
ash, alcohol soluble extractive value and water soluble extractive value were determined as
54
described in the Ayurvedic Pharmacopoeia of India [8].
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2.2. Extraction and Spectral analysis
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200 grams of root powder was left soaked in 1000 mL of either water, methanol : chloroform
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: water (12:5:3), acetone or aqueous methanol (1:1) for 48 hours at 40ºC in a shaking
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incubator to obtain aqueous (Aqu), methanol-chloroform-water (MCW), acetone and hydro-
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methanolic (HM) extracts, respectively; this step was repeated thrice. The resulting extracts
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were first filtered through double-layered muslin, and then through Whatman paper no. 42.
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The final extracts were obtained by drying the filtrate to a constant weight at 40ºC. The yield
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was expressed as the mass of extract obtained per 100 grams of roots. 0.1% solutions of the
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dried extracts were subjected to spectral analysis for evaluation of composition.
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2.3. Qualitative and Quantitative Phytochemical analysis
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The extremely low yield of acetone extract precluded its further use in the study. Small
66
portions of each of the remaining three extracts viz. Aqu, MCW, and HM were subjected to
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qualitative tests for different phytochemical compounds as per standard methods [9, 10].
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Thereafter, alkaloids, total phenolic content (TPC), flavonoids, flavonols, proanthocyanidins,
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ACCEPTED MANUSCRIPT and tannins were quantitated as described earlier by Shabbir et al [10] whereas withanolide
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content was determined as per Pati et al [11].
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2.4. In vitro Antioxidant and Reducing assays
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Antioxidant assays were performed with minor modifications to standard methods as
73
described elsewhere [10]. Further dilutions of the stocks were prepared in an appropriate
74
medium/ buffer as per the requirements of the individual assay. Where applicable, the median
75
inhibitory concentration, IC50, was also calculated.
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2.5. Statistical analyses
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Statistical inferences were drawn on the basis of analysis of variance (ANOVA) followed by
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post-hoc Tukey’s test performed with Daniel’s XL Toolbox 6.53. Multivariate analysis, viz.
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Partial Least Square (PLS) Regression Analysis and Principal Component Analysis (PCA),
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was performed in Multibase 2014.
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ACCEPTED MANUSCRIPT 3. RESULTS AND DISCUSSION
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According to the Ayurvedic Pharmacopoeia of India, the loss on drying, total ash, acid
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insoluble ash, and alcohol extractive values for WS roots should be ≤ 8%, ≤ 7%, ≤ 1% and ≥
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15%, respectively [6]. The WS roots used in the present study satisfied these standards of
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pharmacognostic quality (Table 1).
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Previously, comparison of six different solvents and solvent combinations found that the best
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extraction was achieved by acetone and MCW (Methanol : chloroform : water, 12:5:3) [12].
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This formed the basis for selection of acetone and MCW as solvents in the current study;
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water was also included as a third extraction solvent. Of these, acetone had a very poor yield
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(~0.162%); hence, it was excluded from further study on the basis of non-feasibility, and the
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HM extract was included. Mean yields of Aqu, HM and MCW extracts were 11.64, 16.82,
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and 14.39%, respectively. Maximum yield with aqueous methanol (1:1) may be due to
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optimum solvation of both hydrophilic and lipophilic components in the milieu. Previously, a
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mean yield of 15.40% was obtained upon hydro-ethanolic extraction of WS roots [13].
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Spectral analysis showed increased absorbance by the extracts in the 240-360 nm range (Fig.
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1) that can be attributed to a host of chemical constituents. Carbohydrates absorb strongly at
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360 nm, whereas absorbance at 200 nm, 240 nm, 270 nm and 300 nm reflect the presence of
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proteins and glycoproteins. Many alkaloids absorb strongly in the 250-260 nm wavelength
99
range. Coumarins absorb strongly at 280 and 320 nm [14]. The MCW extract, though
100
comparable to HM, exhibited the strongest absorbance of the three extracts. It must be noted
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that although spectral analysis gives a fair indication of the prominent phytochemical
102
constituents, the results are not always reliable because the presence of many other
103
compounds can also cause absorbance patterns. Thus, qualitative and quantitative estimation
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of the phytochemical constituents was performed. Qualitative phytochemical analyses (Table
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2) also suggest a higher concentration of phytochemical constituents in the MCW extract.
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ACCEPTED MANUSCRIPT Quantitative tests were performed to determine the concentration of the phytochemicals in the
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extracts. Alkaloids and withanolides were quantified using gravimetric methods. Both
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alkaloid and withanolide content of the extracts were in the order: MCW > HM > Aqu (Fig.
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2) and the differences were highly significant (p < 0.01). The concentration of withanolides in
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the roots usually ranges from 0.001 to 0.5% of the dry weight. Total alkaloid content in the
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roots of Indian W. somnifera varies between 0.13 and 0.31%, though yields up to 4.3% have
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been recorded in other regions [15].
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Total phenolic content (TPC) was determined on a gallic acid curve (y = 0.0072x + 0.0472;
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R² = 0.9897). TPC of the extracts was in the order: MCW > HM > Aqueous (Fig. 2); all
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differences being highly significant. Previously, the TPC of methanol, chloroform, and
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aqueous extracts have been reported at 42, 66.72 and 88.58 µg gallic acid equivalents
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(GAE)/mg extract, respectively. It was further suggested that most polyphenolics of WS were
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polar in nature [16]. Another study found the phenol content of the extracts in the order:
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chloroform > petroleum ether > methanol > ethanol > n-hexane; TPC of chloroform extract
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was 60.992 ± 0.367 µg GAE/mg [17]. In the present study, a higher TPC of MCW over HM
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and Aqu suggests a preferential extraction of phenolics in the order: chloroform > methanol >
122
water, which appears to be in agreement with the latter study. Much lower TPC values for
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80% methanolic extracts of WS roots have been reported from Sri Lanka [18].
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Phenolic compounds are represented by flavonoids, tannins, and coumarins [14]. Flavonoid
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content was determined on a quercetin standard curve (y = 0.012x + 0.0604; R² = 0.9918).
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Flavonoid content of the extracts was in the order: MCW > HM > Aqu (Fig. 2), and
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differences between extracts were highly significant. Alam et al prepared WS fruit, root and
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leaf extracts by using 80% methanol; 17.80 ± 5.80 to 32.58 ± 3.16 mg GAE phenolics and
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15.49 ± 1.02 to 31.58 ± 5.07 mg catechin equivalent (CEQ) flavonoids were found per gram
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[19]. Subsequently, 15.49 ± 1.02 mg GAE phenolics and 17.80 ± 5.80 mg CEQ flavonoids
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ACCEPTED MANUSCRIPT have been reported per gram WS roots from Bangladesh [20]. Shahriar et al reported
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flavonoid content of methanol and chloroform extracts at 88.761 ± 1.032 and 122.094 ±1.351
133
mg QE/g, respectively [17]. Udayakumar et al reported 28.26 mg/g total phenolic compounds
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and 17.32 mg/g flavonoids in WS root extracts [21]. Flavonol contents were determined on a
135
quercetin standard curve (y = 0.0103x + 0.3781; R² = 0.9858); these were in the order: MCW
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> HM > Aqu (Fig. 3); the differences MCW vs. HM and MCW vs. Aqu were highly
137
significant, whereas the difference HM vs. Aqu was non-significant.
138
Tannins are chemically classified into hydrolysable tannins and condensed tannins,
139
commonly called proanthocyanidins [14]. All differences in the proanthocyanidin content of
140
the extracts (Fig. 2), determined on a gallic acid curve (y = 0.0051x + 0.059; R² = 0.9905),
141
were non-significant. Tannin content was determined from a tannic acid curve (y = 0.0061x +
142
0.0434; R² = 0.9895). Tannin content of the extracts was in the order: HM > MCW > Aqu
143
(Fig. 2); the differences HM vs. Aqu and MCW vs. Aqu were highly significant whereas the
144
difference HM vs. MCW was non-significant. The differences between the three extracts in
145
their overall phytochemical composition were significant (0.01 < p < 0.05).
146
DPPH scavenging activity of MCW matched that of HM closely. However, none of the
147
extracts fared as well as the ascorbic acid standard (Fig. 3). The statistical differences
148
between the extracts were highly significant. Previously, Alam et al. have reported much
149
lower DPPH scavenging activities of 80% aqueous methanol extract of WS root (IC50 =
150
801.93 ± 7.92 µg/mL) [20]. The ascorbic acid content of the roots remained indeterminate by
151
the standard method as described by Alam et al. (results not shown); previously, the ascorbic
152
acid content of WS roots has been reported to be as low as 0.02% [20].
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The superoxide radical scavenging activities of the three extracts and the standard (Fig. 3)
154
closely matched at lower concentrations but differences became prominent at higher
155
concentrations. Amongst the extracts, MCW showed the highest activity followed by HM and
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ACCEPTED MANUSCRIPT Aqu. Albeit graphical indifference, the statistical differences were highly significant. Our
157
results are in partial agreement with former reports of IC50 of methanol, water, and
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chloroform extracts of WS root for superoxide radical at 94, 125, and 178 µg/mL,
159
respectively [16].
160
Hydroxyl radicals are the most reactive and predominant radicals generated during aerobic
161
metabolism [16]. The hydroxyl ion scavenging activity of ascorbic acid was prominently
162
higher than any of the extracts (Fig. 3). Again, the statistical differences between the extracts
163
and between concentrations within extracts were highly significant. Previously, IC50 of
164
aqueous extract of WS root for hydroxyl radical was reported at ~800 µg/mL [16], which is
165
comparable to our results (IC50 = 999.82 µg/mL).
166
Peroxide scavenging activities of the three extracts (Fig. 3) were similar at low concentrations
167
but showed greater differences at higher concentrations. At a concentration of 100 µg/mL,
168
both ascorbic acid and MCW exhibited nearly equal inhibition. The differences between the
169
activities were highly significant. Although the IC50 of ascorbic acid for peroxide radical
170
obtained by us in the present study is comparable to the previously reported value (164
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µg/mL), the IC50 of Aqu for peroxide radical obtained in the present study (1069.79 µg/mL)
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varied greatly from the previously reported value of 323 µg/mL [16].
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Nitric oxide scavenging activity of ascorbic acid was much higher than any of the extracts
174
(Fig. 3). The activities of MCW and HM were similar at lower concentrations whereas, at
175
highest tested concentration, the activities of all three extracts were similar. Yet, the
176
differences between groups were highly significant. The performance of the different extracts
177
in the different radical scavenging assays mostly showed good correlation with each other
178
(Table 3).
179
Total antioxidant capacity (ToAC) was determined from an ascorbic acid standard curve (y =
180
0.171 ln(x) - 0.2106; R² = 0.9746). ToAC of MCW was highest (83.354 ± 1.828) followed by
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ACCEPTED MANUSCRIPT HM (76.978 ±2.210) and Aqu (68.439 ± 1.000). The difference between the ToAC of MCW
182
and Aqu was highly significant whereas that between HM and Aqu was significant and that
183
between MCW and HM was non-significant.
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The ability of the extracts to reduce Fe3+ to Fe2+ was compared with that of ascorbic acid
185
(Fig. 4). A higher absorbance value indicates higher activity. Fe3+ reducing activity of
186
ascorbic acid was much higher than that of the extracts. Aqu exhibited the poorest activity of
187
the three extracts, all differences between activities being highly significant.
188
PLS regression analysis (Fig. 5) revealed the importance of each phytochemical in the
189
individual and overall antioxidant and reducing activities of the three extracts. The alkaloid
190
content of an extract was the most important contributing variable in its hydroxyl radical
191
scavenging, peroxide scavenging, total antioxidant and Fe3+ reducing activities. Flavonol
192
content appeared the most important determinant for superoxide and nitric oxide scavenging
193
activities. Tannin content of an extract was most decisive of its DPPH radical scavenging
194
activity; however, tannin content was also the least important variable in the overall
195
antioxidant and reducing activities. Alkaloid content was the leading contributor to the
196
overall antioxidant and reducing activities of the extracts, closely followed by flavonoids and
197
withanolides.
198
PCA showed that MCW and HM were more similar to each other than to Aqu in terms of the
199
phytochemical constitution as well as antioxidant and reducing activities. Also, the two
200
extracts were more similar in terms of their Fe3+ reducing, hydrogen peroxide scavenging and
201
superoxide radical scavenging activities as compared to other parameters studied (Table 4;
202
Fig. 6). It follows that the phytochemical composition and antioxidant and reducing activities
203
of the extracts are positively influenced by the use of an organic solvent during the extraction
204
process.
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ACCEPTED MANUSCRIPT 4. CONCLUSIONS
206
In the present study, a comparison of different solvent systems for extraction of the WS root
207
powder showed maximum extraction recovery with aqueous methanol, whereas acetone
208
extract had to be excluded from further studies due to exceptionally poor yield. Methanol-
209
chloroform-water (MCW) extract exhibited the strongest absorbance and showed the highest
210
content of all phytochemical constituents, except tannins. MCW also exhibited the highest
211
antioxidant and reducing activities. The inclusion of an organic solvent in the extraction
212
medium was found to positively influence the antioxidant and reducing activity of the extract.
213
Multivariate analysis found alkaloids and flavonoids to be the most important contributors
214
towards the overall antioxidant and reducing activities of WS root extracts. The MCW and
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HM extracts were more similar to each other than to aqueous extract, in terms of the
216
phytochemical composition, and antioxidant and reducing activities, suggesting a favorable
217
influence of organic solvents.
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ACKNOWLEDGMENTS
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The financial assistance received by BG in the form of a Senior Research Fellowship from
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Council of Scientific and Industrial Research (CSIR), India is gratefully acknowledged. CONFLICT OF INTERESTS
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royleanus leaves. BMC Comp Alt Med 2013; 13:143. 11. Pati PK, Sharma M, Salar RK, Sharma A, Gupta AP, Singh B. Studies on leaf spot
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disease of Withania somnifera and its impact on secondary metabolites. Ind J Micro 2008;
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48:432-437.
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12. Eloff JN. Which extractant should be used for the screening and isolation of antimicrobial components from plants? J Ethnopharmacol 1998; 60:1-8.
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13. Ganguly B. Effect of hydroalcoholic extract of Withania somnifera (Ashwagandha) on
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growth kinetics of Infectious Bursal Disease Virus in Chicken Embryo Fibroblast culture
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[Dissertation]. G. B. Pant University of Agriculture and Technology; 2009.
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14. Cavalcanti de Amorim EL, de Almeida de Castro VTN, de Melo JG, Corrêa AJC, da Silva
Peixoto
Sobrinho
TJ.
Standard
Operating
Procedures
(SOP)
for
the
259
Spectrophotometric Determination of Phenolic Compounds Contained in Plant Samples.
260
Available from: http://dx.doi.org/10.5772/51686
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15. Alam N, Hossain M, Khalil MI, Moniruzzaman M, Sulaiman SA, Gan SH. Recent
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advances in elucidating the biological properties of Withania somnifera and its potential
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role in health benefits. Phytochem Rev 2012; 11:97-112.
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16. Pal A, Naika M, Khanum F, Bawa AS. In-vitro studies on the antioxidant assay profiling
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of Withania somnifera L. (Ashwagandha) Dunal root: Part 1. Pharmacog J 2011;
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3(20):47-55.
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17. Shahriar M, Bahar ANM, Hossain MI, Akhter S, Haque MA, Bhuiyan MA. Preliminary
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phytochemical screening, in vitro antioxidant and cytotoxic activity of five different
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extracts of Withania somnifera root. Int J Pharmacy 2012; 2:450-453.
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18. Fernando IDNS, Abeysinghe DC, Dharmadasa, RM. Determination of phenolic contents
271
and antioxidant capacity of different parts of Withania somnifera (L.) Dunal from three
272
different growth stages. Ind Crops Products 2013; 50:537-539. 19. Alam N, Hossain M, Khalil MI, Moniruzzaman M, Sulaiman SA, Gan SH. High catechin
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concentrations detected in Withania somnifera (Ashwagandha) by high performance
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liquid chromatography analysis. BMC Comp Alt Med 2011; 11:65.
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20. Alam N, Hossain M, Mottalib MA, Sulaiman SA, Gan SH, Khalil MI. Methanolic
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extracts of Withania somnifera leaves, fruits and roots possess antioxidant properties and
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antibacterial activities. BMC Comp Alt Med 2012; 12:175.
SC
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21. Udayakumar R, Kasthurirengan S, Vasudevan A, Mariashibu TS, Rayan JJS, et al.
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Antioxidant effect of dietary supplement Withania somnifera L. Reduce blood glucose
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levels in alloxan-induced diabetic rats. Plant Foods Human Nutr 2010; 65:91-98.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS
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Fig. 1: Spectral analysis of WS root extracts. UV-Vis absorption characteristics of Aqu,
284
HM, and MCW have been shown for comparison. Stronger absorbance suggests a higher
285
concentration of phytochemicals.
286
Fig. 2: Results of quantitative tests for phytochemicals in different extracts of WS roots;
287
Mean ± S.E. values of different phytochemicals have been expressed as microgram
288
equivalents of respective standards per milligram of extract; alkaloids and withanolides have
289
been expressed as percent of dry weight.
290
Fig. 3: Percent inhibition and median inhibitory concentration (IC50) of ascorbic acid
291
and different extracts of WS roots in antioxidant and reducing assays; IC50, median
292
inhibitory concentration; DPPH, DPPH radical scavenging assay; SRSA, Superoxide radical
293
scavenging assay; HRSA, Hydroxyl radical scavenging assay; HPSA, Hydrogen peroxide
294
scavenging assay; NOSA, Nitric oxide scavenging assay; AA, ascorbic acid.
295
Fig. 4: Fe3+ reducing activities of ascorbic acid and different WS root extracts at
296
varying concentrations; higher absorbance at 700 nm is indicative of greater reducing
297
activity.
298
Fig. 5: PLS regression analysis of phytochemical constituents on antioxidant and
299
reducing properties of WS root extracts; values indicate the importance of a variable i.e.
300
phytochemical concentrations in antioxidant/ reducing activity; total height of a column
301
represents contribution in the overall antioxidant and reducing activities of extracts.
302
Fig. 6: Principal Component Analysis of the effect of phytochemical constitution of WS
303
root extracts, PC1-PC2 biplot; red ellipse shows the distribution of scavenging assays, blue
304
ellipse shows the distribution of phenolics (except tannins), yellow ellipse shows the
305
distribution of reducing assays; purple ellipse shows tannins and green ellipse shows the
306
distribution of alkaloids and withanolides.
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16
ACCEPTED MANUSCRIPT TABLES Physicochemical composition Analyte Total lipid Total protein Total sugars Loss on drying Total ash Acid insoluble ash Water soluble ash Alcohol soluble extractive value Water soluble extractive value
Amount (%) 1.38 4.27 7.17 2.08 4.33 0.65 1.85 17.36 66.64
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Table 1. Physicochemical composition of WS roots; all values are expressed as per cent of
3
air-dried weight.
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2
1
ACCEPTED MANUSCRIPT Aqueous ++ + + ++ +++ ± + +++ ++
HM ++ +++ ++ +++ +++ +++ ++ ++
MCW +++ +++ +++ +++ +++ +++ ++ ++
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Phytochemical Alkaloids Anthraquinones Coumarins Flavonoids Phenolics Reducing sugars Glycosides Saponins Tannins Terpenoids
Table 2. Results of qualitative tests for phytochemical constituents of aqueous, hydro-
5
methanolic and methanol-chloroform-water extracts; +++ reaction within 5 min; ++
6
reaction in 5-30 min; + reaction in > 30 min; - no reaction up to 24 hours; ± undefined color
7
change; HM, hydro-methanolic extract; MCW, methanol-chloroform-water extract.
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2
ACCEPTED MANUSCRIPT
DPPH 1.000 0.907 0.980 0.976 0.871
SRSA 1.000 0.960 0.933 0.996
HRSA 1.000 0.995 0.932
HPSA 1.000 0.899
NOSA 1.000
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DPPH SRSA HRSA HPSA NOSA
Table 3. Correlation (R) between performance of different extracts in the radical
9
scavenging assays; DPPH, DPPH radical scavenging assay; SRSA, Superoxide radical
10
scavenging assay; HRSA, Hydroxyl radical scavenging assay; HPSA, Hydrogen peroxide
11
scavenging assay; NOSA, Nitric oxide scavenging assay.
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3
ACCEPTED MANUSCRIPT
Component 2 2.24%
3 0.16%
97.60%
99.84%
100.00%
1.95
0.04
0.00
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Contribution Accumulation of Contribution Eigenvalue
1 97.60%
Table 4. Principal Component Analysis of the effect of phytochemical constitution on
13
antioxidant and reducing activities of WS root extracts; contribution and eigenvalue of
14
individual components.
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4
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5 4.5 4 3.5
Absorbance
3 2.5 2
Aqu MCW HM
1 0.5 0 200
300
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1.5
400
AC C
Fig. 1
EP
λ(nm)
500
600
700
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109.65
0.377
0.230 97.38
0.197
AC C
Fig. 2
5.44
32.29
5.38
29.67
38.66
Aqueous
HM
36.00
37.75
63.49
53.9
EP
0.117
77.13
TE D
0.313
37.34
M AN U
0.250
4.20 18.19
MCW
120
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999.82
M AN U
326.54
278.77
251.95
193.39
178.08 121.74
111.31
32.80
5.60
0 AA
Aqu MCW DPPH
HM
AA
TE D
40
20
130.44
100 89.62
58.62
48.61
38.83
60
168.27
IC50 (µg/mL)
Per cent Inhibition
80
Aqu MCW
HM
AA
SRSA
10 µg/mL
AC C
EP
5 µg/mL
Fig. 3
471.82
421.46
100
1000
1,069.79
Aqu MCW 50 µg/mL
10
1 HM
AA
HRSA
20 µg/mL
36.38
Aqu MCW
HM
AA
HPSA 100 µg/mL
500 µg/mL
Aqu MCW NOSA
IC50
HM
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3
2.5
O.D.700
2
1.5
0.5
0 Ascorbic acid
AC C
Fig. 4
Aqueous
31.25 µg/mL
EP
15.625 µg/mL
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1
62.5 µg/mL
MCW 125 µg/mL
250 µg/mL
HM 500 µg/mL
0.998
0.978
0.996
0.972
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0.967
0.944
0.997
0.979
0.974
0.886
0.875
0.975
0.970
0.968
0.962
0.963
0.914
0.904
0.830
0.902
EP AC C
SRSA
0.864
0.965
0.936
0.924 0.867
1.000
0.784 0.698
0.995
0.917
0.970
0.991
0.977
0.957
0.912
DPPH
Fig. 5
0.997
0.843
HRSA
HPSA
0.858
0.904
0.842
0.822
0.743
0.967
0.992
0.999
TE D
0.994
M AN U
0.921
0.998
0.609
NOSA
ToAC
Fe3+
AC C
Fig. 6
EP
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