Industrial Crops & Products 119 (2018) 218–225

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Industrial Crops & Products

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Optimization of ultrasonic-assisted extraction (UAE) of phenolics and T

antioxidant compounds from rhizomes of Rheum moorcroftianum using
response surface methodology (RSM)
⁎
Aseesh Pandeya,b, Tarun Belwala, K. Chandra Sekara, Indra D. Bhatta, , Ranbeer S. Rawala
a
G. B. Pant National Institute of Himalayan Environment and Sustainable Development, Kosi-Katarmal 263643, Almora, Uttarakhand, India
b
G. B. Pant National Institute of Himalayan Environment and Sustainable Development, Sikkim Unit, Pangthang, Gangtok 737101, Sikkim, India

A R T I C LE I N FO A B S T R A C T

Keywords: This study for the first time designed to optimize the extraction of polyphenolic compounds from rhizomes of
Antioxidants Rheum moorcroftianum using response surface methodology (RSM). Solvent was selected based on the pre-
Extraction optimization liminary experiments, and a four-factors-three-level, Box–Behnken Design (BBD) including 29 experimental
Himalaya runs. The polyphenolic content and antioxidant activity was significantly (p < 0.05) affected by vessel diameter
Polyphenols
– 6 cm (X1), sample to solvent ratio – 1:28.42 g/mL (X2) and extraction temperature – 37.11 °C (X3) under
Rheum
ultrasonic assisted extraction (UAE). The measured parameters were found in accordance with the predicted
UAE
values. High Performance Liquid Chromatography (HPLC) analysis in optimized condition revealed the presence
of 12 phenolic antioxidant compounds with the highest concentration of chlorogenic acid (26.68 mg/g). The
results indicate that optimization of extraction conditions in R. moorcroftianum is critical for precise quantifi-
cation of antioxidant phenolics and its further utilization in industry.

1. Introduction et al., 2012) and contribute to various pharmacological activities like

The genus Rheum consists of approximately sixty species (Rokaya
et al., 2012); cultivated for culinary, ornamental and medicinal pur-
poses across the world (Arvindekar and Laddha, 2016), and used in the
preparation of jams, jellies and wine (Clementi and Misiti, 2010). In
Indian Himalayan Region (IHR), the genus Rheum is represented by 10
species and distributed between 2800–4700 m asl (Uniyal et al., 2002;
Tabin et al., 2016). Rheum moorcroftianum Royle (Family – Poly-
gonaceae), is a Himalayan endemic species commonly known as rhu-
barb, grows in rocky, bouldery slopes and river banks between an
elevation range of 3200–4700 m asl (Uniyal et al., 2002; Rana and
Samant, 2010). The genus Rheum has been used in the traditional
Chinese medicine, Medieval Arabic and Ayurvedic system of medicine
(Xiao et al., 1984; Arvindekar and Laddha, 2016). Traditionally, the
roots and rhizomes of the Rheum are used as an astringent, purgative,
tonic and in the healing of ulcers (Anonymous, 2005). Rhubarb con-
tains a variety of bioactive compounds like flavonoids, anthraquinone
glycosides, tannins, volatile oils and saponins (Ye et al., 2007; Aslam
antifungal, antioxidant, hepatoprotective, nephroprotective and im-
mune modulatory activities (Zargar et al., 2011). Further, anti-can-
cerous properties of the Rheum rhizome in human breast carcinoma
(MDAMB-435S) and liver carcinoma (Hep3B) cell lines is reported
(Rajkumar et al., 2011a,b).
The medicinal properties of medicinal plants are attributed to the
presence of secondary metabolites, which are unique resources for
pharmaceuticals, nutraceuticals, food additives, and fine chemicals
(Zhao et al., 2005). These compounds exist in plants and enclosed by
insoluble structures such as the vacuoles of plant cells and lipoprotein’s
bilayers which complicate their extraction process (Corrales et al.,
2008). The nature of bioactive compounds and the presence of other
biomolecules along with several factors such as extraction methods,
type of solvent, pH, temperature, sample-solvent ratio and extraction
time are reported to affect yield (Cacace and Mazza, 2003; Chirinos
et al., 2007; Ng et al., 2012; Belwal et al., 2017b). However, there is no
universally standardized set of optimum conditions for the extraction of
bioactive compounds from different plants (Chirinos et al., 2007; Chen

Abbreviations: ABTS, 2, 2-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid); AAE, Ascorbic acid equivalent; BBD, Box–Behnken Design; CV, Coefficient of variation; DPPH, 2, 2-
Diphenyl-1-picryhydrazyl; FRAP, Ferric reducing antioxidant power; GA, Gallic acid; GAE, Gallic acid equivalent; HPLC, High performance liquid chromatography; Q, Quercetin; QE,
Quercetin equivalent; RSM, Response surface methodology; TAE, Tannic acid equivalent; TFC, Total flavonoid content; TPC, Total phenolic content; TPTZ, 2, 4, 6-Tripyridyl-s-triazine;
TTC, Total tannin content; UAE, Ultrasonic assisted extraction
⁎
Corresponding author.
E-mail address: idbhatt@gbpihed.nic.in (I.D. Bhatt).

https://doi.org/10.1016/j.indcrop.2018.04.019
Received 4 December 2017; Received in revised form 3 April 2018; Accepted 8 April 2018
Available online 24 April 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
A. Pandey et al.
et al., 2007) and Himalayan plants are no exception. Therefore, opti-
mization of species-specific optimal extraction conditions is essential.
As such, single factor at a time approach is labor-intensive, time-con-
suming and do not show any interactive effect. However, the statistical
model such as the Response Surface Methodology (RSM) is an effective
statistical tool for optimizing complex processes (Zhong and Wang,
2010). RSM is a multi-factors approach with a reduced number of ex-
perimental runs, and evaluate multiple parameters and their interac-
tions in a single experiment (Giovanni, 1983; Zhong and Wang, 2010;
Myers et al., 2016). It is widely used for optimizing the extraction of
polysaccharides, anthocyanins, vitamin E, phenolic compounds, protein
and polyphenolics from different plant materials (Cacace and Mazza,
2003; Chandrika and Fereidoon, 2005; Ge et al., 2002; Lee et al., 2005;
Li and Fu, 2005; Liyana-Pathirana and Shahidi, 2005; Qiao et al., 2009;
Zhong and Wang, 2010; Li et al., 2012, Alberti et al., 2014; Ilaiyaraja
et al., 2015; Belwal et al., 2016, 2017a,b). The Box–Behnken design
(BBD) is easy to perform experiments and interpret in comparison to
other models (Box and Behnken, 1960; Ferreira et al., 2007).
In recent times, the interest of consumers are growing towards
sustainability of extracting compounds in nutraceutical industries. This
include environmental friendly and low-cost raw materials and tech-
nologies (Corrales et al., 2008; Belwal et al., 2018). The traditional
extraction methods such as soxhlet, maceration, percolation, etc. are
reported to consume large amounts of solvents, time and energy (De
Castro and Garcıa-Ayuso, 1998), however, the new extraction tech-
nologies showed the higher recovery of valuable compounds in a lesser
solvents, lower extraction time and temperature (Barba et al., 2015;
Deng et al., 2015; Bouras et al., 2015). Ultrasonic-assisted-extraction
(UAE) method is reported to minimize extraction yield through mass
transfer phenomena in a number of plant extracts (Corrales et al., 2008;
Vilkhu et al., 2008). UAE increased mass transfer rate by cavitation
forces, where bubbles in the liquid/solid extraction can explosively
collapse and generate localized pressure causing plant tissue rupture
and improves the release of intracellular substances into the solvent
(Knorr et al., 2004). The feasibility of UAE for the improved extraction
of secondary metabolites compared with traditional methods have been
highlighted in many research (Mason and Zhao, 1994; Albu et al., 2004;
Jian-Bing et al., 2006; Corrales et al., 2008; Belwal et al., 2018) and
reviews (Vinatoru, 2001; Knorr et al., 2004; Zhang et al., 2003). Among
others, polyphenolics have been categorized under nutraceutically ac-
tive compound and their role has been justified in treating various
ailments. Thus, the present study was designed to determine the op-
timum UAE condition for maximizing polyphenolic antioxidant ex-
traction yield from R. moorcroftianum rhizome.
2. Material and methods
2.1. Plant material
The rhizome portions of R. moorcroftianum were collected from five
different plants growing near Parvati Lake, Jeolingkong (4500 m asl),
Byans valley, Uttarakhand, India. The rhizomes were brought to the
plant analytical laboratory of G. B. Pant National Institute of Himalayan
Environment and Sustainable Development, Almora India and dried in
shade at room temperature. Dried rhizomes were grounded into fine
powder using hammer mill (Model-WGM 197, UTS sales, Delhi, India).
The grounded powder was passed through a standard mesh size
of < 85 μm and stored in airtight bags at 4 °C, until further experi-
mental use.
2.2. Chemicals and phenolic standards
The analytical grade chemicals like 2,2-azinobis (3-ethylbenzthia-
zoline- 6-sulphonic acid) (ABTS), 2,4,6-tripyridyl-s-triazine (TPTZ),
acetone, ethanol methanol and propanol were procured from Merck
KGaA (Darmstadt, Germany). Ascorbic acid, 2,2-Diphenyl-1-
219
Industrial Crops & Products 119 (2018) 218–225
Fig. 1. Effect of different solvents on total polyphenolic content (TPC). Bars
capped with same letters are not significantly (p < 0.05) different to each
other.
picryhydrazyl (DPPH) radical, and HPLC grade phenolic standards:
rutin hydrate, phloridzindihydrate, p-coumaric acid, catechin hydrate,
gallic acid, quercetindihydrate, 3-hydroxybenzoic acid, vanillic, caffeic,
and chlorogenic acid were procured from Sigma-Aldrich (St. Louis,
Missouri, United States).
2.3. Selection of variables
The effects of different variables such as solvents type and con-
centration, extraction time, temperature, sample-to-solvent ratio, dia-
meter and shape of the extraction vessel, etc. are known to affect ex-
traction yield and phytochemical contents (Dai and Mumper, 2010). In
this context, different solvents such as acetone, ethanol, methanol,
isopropanol, and distilled water were tested in a preliminary experi-
ment for selecting suitable solvent (Fig. 1). For this, 2 g sample was
extracted in flat based 4 cm diameter vessel (Borosil, India) with 20 mL
of solvent (0.2 N) having concentration of 80% and subjected to ul-
trasonication (50 KHz) using an ultrasonic bath (Toshiba, India) under
a temperature of 50 °C for 15 min. Further, similar conditions were
applied for the optimization of solvent concentration. All the experi-
ments for each solvent type and concentrations were carried out in
triplicates. Selection of best solvent and solvent concentration was
based on the maximum value of total phenolic content (TPC).
2.4. Experimental design
Optimization experiment was carried out using response surface
methodology (RSM) for extraction of polyphenols from R. moor-
croftianum rhizomes. Using RSM one can predict the linear, quadratic
and interactive effect between the factors w.r.t. the responses of a
limited number of experiments. A three level, four-factor Box–Behnken
design (BBD) (Design Expert trial version 9.0 Stat-Ease Inc.,
Minneapolis, MN) was applied for the design of experiments, model
building and data interpretation. For BBD model, factor levels were
varied over 3 levels, and lesser experimental work is required as com-
pared to other models such as central composite design (CCD). In ad-
dition, BBD experimental runs allow testing factors to at least one at the
center point (0) (Box and Behnken, 1960).
Variables such as vessel diameter (X1), sample to solvent ratio (X2),
extraction temperature (X3), and sonication time (X4) were selected.
Keeping the independent variables at three levels (−1, 0, +1), a total
of 29 experimental runs were conducted to determine the TPC, TFC,
TTC and in vitro antioxidant activity (ABTS and DPPH) (Table 1). Fur-
ther, based on the BBD data, the selection of optimum condition and its
validation has been performed (Section 2.8). For each response vari-
ables, second order polynomial equation was determined as-
A. Pandey et al. Industrial Crops & Products 119 (2018) 218–225

Table 1
Box-Behnken design (BBD) with responses of the dependent variables to extraction conditions.
Independent variables Dependent Variables

Run X1 X2 X3 X4 TPC(mg GAE/g dw) TFC (mg QE/g dw) TTC(mg TAE/g dw) ABTS (mM AAE/g dw) DPPH (mM AAE/g dw)

1 2(−1) 20(0) 55(+1) 15(0) 92.99 120.71 50.13 0.96 25.31

2 6(+1) 20(0) 45(0) 20(+1) 65.89 112.31 58.60 1.93 30.05
3 4 (0) 10(−1) 45(0) 20(+1) 77.90 75.51 45.19 1.77 9.45
4 2 (−1) 20(0) 45(0) 20(+1) 70.99 101.92 46.93 0.99 25.58
5 6 (+1) 30(+1) 45(0) 15(0) 57.74 82.40 69.16 1.20 50.04
6 2 (−1) 20(0) 35(−1) 15(0) 83.53 135.58 46.08 0.96 22.17
7 4 (0) 10(−1) 45(0) 10(−1) 48.86 70.35 44.67 1.46 11.38
8 4 (0) 30(+1) 35(−1) 15(0) 86.78 114.05 63.80 0.94 41.00
9 4 (0) 30(+1) 45(0) 20(+1) 38.94 67.41 58.97 0.18 45.87
10 4 (0) 20(0) 45(0) 15(0) 50.95 72.50 72.30 1.40 33.52
11 2 (−1) 30(+1) 45(0) 15(0) 45.59 55.50 42.03 0.54 11.94
12 4 (0) 20(0) 35(−1) 10(−1) 83.53 98.01 53.64 0.59 29.66
13 4 (0) 20(0) 45(0) 15(0) 55.98 84.60 53.36 1.63 30.39
14 4 (0) 20(0) 45(0) 15(0) 40.21 49.34 51.92 1.80 20.31
15 4 (0) 20(0) 35(−1) 20(+1) 73.84 88.97 50.12 0.58 11.46
16 4 (0) 20(0) 55(+1) 10(−1) 70.70 85.26 46.14 0.96 23.69
17 4 (0) 20(0) 55(+1) 20(+1) 65.40 66.92 47.21 0.76 32.28
18 6 (+1) 20(0) 55(+1) 15(0) 81.20 84.60 58.83 1.84 31.07
19 4(0) 10(−1) 55(+1) 15(0) 60.35 45.10 28.37 0.98 17.21
20 4 (0) 30(+1) 45(0) 10(−1) 52.86 60.97 61.65 1.05 53.71
21 4 (0) 20(0) 45(0) 15(0) 46.08 64.57 52.73 1.44 34.03
22 6 (+1) 20(0) 35(−1) 15(0) 63.99 118.21 45.88 0.87 31.48
23 2 (−1) 20(0) 45(0) 10(−1) 75.93 115.19 47.37 1.04 16.48
24 4 (0) 30(+1) 55(+1) 15(0) 60.65 73.95 60.80 1.14 51.52
25 4 (0) 20(0) 45(0) 15(0) 47.74 63.53 54.05 0.92 26.41
26 2 (−1) 10(−1) 45(0) 15(0) 55.84 78.37 31.18 1.04 8.08
27 4(0) 10(−1) 35(−1) 15(0) 45.11 55.32 21.80 0.53 14.34
28 6 (+1) 20(0) 45(0) 10(−1) 49.09 60.80 82.42 1.67 31.91
29 6 (+1) 10(−1) 45(0) 15(0) 58.96 71.57 62.84 1.94 14.55

X1 = Vessel diameter (cm), X2 = Sample to solvent ratio (g/ml), X3 extraction temperature (°C), X4 sonication time (min) TPC = Total polyphenolic content,
TFC = Total flavonoids content, TT = Total tannin content, ABTS = 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) radical cation inhibition, DPPH = 2,2-
diphenyl-1-picrylhydrazyl radical scavenging ability.

k k k k−1
2.6. Phytochemical analysis
Y= β 0 + ∑ βiXi + ∑ βiiXi2 + ∑ ∑ βijXiXj
i=1 i=1 i=1 j=i+1
2.6.1. Total polyphenolic content (TPC)
Folin–Ciocalteu colorimetric method, based on oxidation/reduction
Where, Y is the response variable, β0 is intercept, βi is linear regression
reactions of phenols was used to determine the TPC (Singleton et al.,
coefficient for ith factor, βii for quadric and βij for the cross-product
1999). Diluted extract (0.5 mL) in 4.5 mL distilled water, was mixed to
term. Xi and Xj are the independent variables. k is the number of tested
0.5 mL of Folin–ciocalteu solution. After five min, sodium bicarbonate
variables (k = 4).
(Na2CO3; 7% w/v) was added to the mixture along with 2 mL of dis-
The three-dimensional response surface plots were generated for
tilled water. After stirring, the mixture was kept for 90 min in dark at
each response by keeping one response variable at its optimal value and
room temperature. The absorbance of resultant blue color was mea-
plotting the other two independent factors with the response. The ex-
sured at 765 nm using UV–vis spectrophotometer (Hitachi U-2001,
perimental data were fitted to second-order polynomial model to obtain
Japan) against the blank. Gallic acid was used for the calibration curve.
the regression coefficients (β), which determine the extent of effect of
The polyphenol content was then calculated from the gallic acid cali-
individual factor over response. The model F-value, lack of fitness and
bration curve in triplicate. The results were expressed as milligram
coefficient of determination (R2) were calculated for each response for
gallic acid equivalent per gram dry weight (mg GAE/g dw) of rhizome.
model fitness.

2.6.2. Total tannin content (TTC)

Folin-Denis method as described by Belwal et al. (2016) was used to
2.5. Extraction process
determine the TTC. Briefly, 0.5 mL of Folin-Denis reagent was added in
5 mL of sample extract. After proper mixing, 1 mL of sodium carbonate
The powdered sample (1 g) of R. moorcroftianum rhizome was mixed
(7%) was added to this mixture, followed by 3.5 mL of distilled water.
with different volumes (10–30 mL) of 40% acetone in flat base vessels
The mixture was kept in dark for 20 min at room temperature and the
(Borosil, India) having different diameters (10–40 cm). These samples
absorbance of resultant blue color was measured at 700 nm against
were mixed properly for 1–2 min using homogenizer and kept in ul-
blank (distilled water). The TTC was quantified using, tannic acid
trasonicator (Model- Toshiba, India) at various temperatures (35–55 °C)
standard curve and estimated as milligram of tannic acid equivalent per
for different time periods (10–20 min). The vessels were kept in the
gram of dry weight (mg TAE/g dw) of rhizome sample.
middle of the ultrasonicator bath for homogenous extraction and/or
homogenous distribution of ultrasonic waves inside the vessel and the
2.6.3. Total flavonoids content (TFC)
temperature inside the vessel was determined. The extract was filtered
Aluminum chloride method based on colorimetric reactions was
using Whatman filter paper No. 2 and stored at −20 °C during the
used to quantify the TFC (Chang et al., 2002). Extract (0.5 mL) was
experimental process. All assays were carried out in triplicates and the
diluted with 1.5 mL of distilled water, 0.5 mL of 10% (w/v) aluminum
results expressed as mean values ± standard error.
chloride and 0.1 mL of potassium acetate (1 M) and the whole mixture

220
A. Pandey et al.
Fig. 2. Effect of different concentrations of Acetone on total polyphenolic
content (TPC). Bars capped with same letters are not significantly (p < 0.05)
different to each other.
was diluted with 2.8 mL of distilled water. This final mixture was kept
at room temperature for 30 min and the absorbance was measured at
415 nm against blank (aluminum chloride solution). The TFC was
quantified using, quercetin standard curve and estimated as milligram
of quercetin equivalent per gram of dry weight (mg QE/g dw) of rhi-
zome sample.
2.7. Antioxidant activity
The radical scavenging activity was determined by means of (2, 2-
Diphenyl-1-picrylhydrazyl) assay and ABTS (2, 2-Azinobis-3-ethyl-
benzthiazoline-6-sulphonic acid) assay, following Rawat et al. (2011);
Belwal et al. (2016) with minor modifications. For the determination of
the DPPH radical scavenging activity, working solution of DPPH was
prepared in methanol (4 mL of stock DPPH solution in 96 mL of 80%
methanol). The 1 mL of extract was mixed with 3 mL of DPPH solution
and the mixture was kept in dark for 30 min at room temperature.
Absorbance was measured at the wavelength of 520 nm using UV–vis
spectrophotometer (Hitachi U-2001, Japan). The value of DPPH mea-
sures the antioxidant capacity of a given substance as compared to the
standard. The data were expressed in millimole of ascorbic acid
equivalent per gram dry weight (mM AAE/g dw) of rhizome sample.
This method is based on the decolorization of the stable free radical
(DPPH). The DPPH solution, upon mixing with a hydrogen atom do-
nating substance, gives rise to the reduced form with the loss of its
violet color to a pale yellow color.
For the determination of the ABTS radical scavenging activity,
working solution of ABTS was prepared by mixing potassium persulfate
(2.45 mM) and ABTS salt (7 mM) in equal proportions. This mixture
was kept at 4 °C in dark for at least 16 h. This working ABTS solution
was diluted with methanol 80% (v/v) till the absorbance of
0.7 ± 0.005 was obtained at 734 nm. Rhizome extract (0.1 mL) was
mixed in 3.9 mL of diluted ABTS solution and kept in dark for 15 min
for reaction at room temperature. The absorbance was measured at
734 nm against blank (diluted ABTS solution) using UV–vis spectro-
photometer (Hitachi U-2001, Japan). The data were expressed in mil-
limole of ascorbic acid equivalent per gram dry weight (mg AAE/g dw)
of rhizome sample.
2.8. Optimal condition and model validation
Experimental data of BBD was used to find out the optimal condi-
tion by the model. The entire response variables (TPC, TFC, TTC and
antioxidant activity) was kept at maximum and the independent vari-
ables (X1, X2, X3, X4) within the range (between lower and higher level).
The software generated the optimal conditions based on the BBD data.
Among all the optimum conditions, the one having higher desirability
index (0–1) was selected. The optimum condition also gives the model
221
Industrial Crops & Products 119 (2018) 218–225
predicted value for each response, to compare with the experimental
value. The experiment was performed on the suggested optimal con-
dition and the response values obtained were compared with the
model’s predicted values. The coefficient of variation (CV%) was de-
termined for validation of the model.
2.9. HPLC analysis
For high performance liquid chromatography analysis, 0.1 mL of the
filtered rhizome extract was injected into Purospher® (Merck, Damstadt,
Germany) RP-18 (250 mm × 4.6 mm id, × 5 μm) column for separation
of different compounds using Shimadzu LC-10AT HPLC system
(Shimadzu, Japan) equipped with diode array detector (DAD-MZOA)
and binary pumps. The mobile phase consists of a mixture of methanol
and 0.1% (v/v) ortho-phosphoric acid in ultrapure water at the ratio of
40:60 (v/v) and the flow rate was maintained at 0.8 mL/min for total
40 min. The detector wavelengths from 254 to 330 nm were selected
(Belwal et al., 2017a, 2017b). Calibration curves for fifteen standard
phenolics viz. caffeic acid, catechin, chlorogenic acid, ellagic acid,
ferulic acid, gallic acid, m- coumaric acid, p-coumaric acid, phloridzin,
quercetin, rutin, trans-cinnamic acid, vanillic acid, 3-hydroxybenzoic
acid and 4- hydroxybenzoic acid were plotted and estimated using
standard curve. Compounds from the sample were detected using re-
tention time of standard compounds and a linear equation was used for
quantification. The compounds in the sample were expressed as mg/g
dw.
3. Result and discussion
3.1. Solvent selection
Among all the tested solvents, acetone was found to recover sig-
nificantly (p < 0.05) higher concentration of total polyphenolics fol-
lowed by methanol, ethanol, isopropanol and distilled water (Fig. 1)
and therefore, acetone was selected and tested at various concentra-
tions from 20 to 80% in different experiments. Among different con-
centrations, 80% and 40% was found to be significantly superior as
compared with remaining concentrations (Fig. 2). As acetone has a
lower boiling point and can be easily vaporized during extraction,
acetone 40% concentration for further experiments was selected.
3.2. Model fitting
Box-Behnken design was used to optimize UAE factors for poly-
phenolic extraction. Analysis of variation (ANOVA) showed significant
(p < 0.05) model F-value with non-significant lack of fit for all re-
sponses. The coefficient of determination (R2) showed a good fit with
the experimental data and fewer variances around the mean value.
Factors such as vessel diameter, sample-to-solvent ratio, and extraction
temperature showed linear, quadratic and interactive, while sonication
time did not show any significant effect on the responses at its levels.
The regression coefficients (β) values of the identified variables are
shown in Table 2.
3.3. Effect of different UAE factors on responses
3.3.1. Effect of UAE factors on total polyphenolic content (TPC) extraction
Total phenolic content under ultrasonic extraction was significantly
(p < 0.05) affected by the quadratic effect of vessel diameter and ex-
traction temperature. Remaining factors did not show any significant
(p < 0.05) effect on TPC. Removing all non-significant terms, the
polynomial equation for TPC comes as:
YTPC = 48.19 + 10.84X12 + 19.13X32
The model was found significantly fit with model F-value as 2.51,
A. Pandey et al. Industrial Crops & Products 119 (2018) 218–225

Table 2 increases up to 6 cm. Increasing sonication temperature increases the

Regression coefficient (β), coefficient of determination (R2) and F-test value of polyphenolic content and this might be due to the fact that increasing
the predicted second order polynomial models (BBD) for polyphenolics and temperature decreases the surface tension at the solvent while it in-
antioxidant activities. creases vapor pressure, which allows the cavitation bubble formation at
Regression Coefficients (β) lower acoustic intensity and subsequently increases extraction yield
(Geng and Thagard, 2013; Zhang et al., 2016).
TPC TFC TTC ABTS DPPH

Intercept, X0 48.19* 66.91* 56.87* 1.44* 28.93** 3.3.3. Effect of UAE factors on total tannin content (TTC) extraction
Linear
Vessel diameter and sample-to-solvent ratio have shown the sig-
X1 −4.00 −6.45 9.50* 0.33** 6.63**
X2 −0.37 4.84 10.20** −0.22* 14.92*** nificant effect over TTC extraction. As such, the polynomial equation
X3 −0.46 −11.13* 0.85 0.18 2.58 for TTC comes as:
X4 1.00 1.87 −2.41 −0.05 −1.01
YTTC = 56.87 + 9.50X1 + 10.20X2
Quadratic
X12 10.84* 23.73** 0.75 0.07 −3.81 The model is well fitted (p < 0.05, F-value = 2.53) and also
X22 −3.18 −12.10 −5.62 −0.20 0.00
showed non-significant lack of fit. With increasing vessel diameter and
X32 19.13*** 17.42* −7.83 −0.41* 0.26
X42 7.37 7.04 0.94 −0.18 −0.95 sample-to-solvent ratio, the TTC extraction showed significant
(p < 0.05) linear increase (Fig. 3c). Ultrasonic extraction of TTC was
Cross Product
X12 2.25 8.42 −1.13 −0.06 7.91* affected by the sample-to-solvent ratio (X2) and vessel diameter (X1).
X13 1.94 −4.68 2.22 0.24 −0.89 Results revealed a significant (p < 0.05) linear effect of sample-to-
X14 5.44 16.19 −5.84 0.08 −2.74 solvent ratio and vessel diameter. Increasing solvent volume with re-
X23 −10.34 −7.47 −2.39 −0.06 1.91 spect to sample provides more surface area for the acoustic wave to
X24 −10.74 0.32 −0.80 −0.30 −1.48
X34 1.1 −2.32 1.15 −0.05 6.70
form cavitation bubbles and thus increases mass transfer between the
R2 0.71 0.71 0.71 0.72 0.84 solvent and sample (Vinatoru, 2001). Also, increasing temperature in-
F value (model) 2.51* 2.49* 2.53* 2.57* 5.48** creases mass transfer and thus affect extraction was observed.
F value (lack of fit) 4.86 2.29 1.3 1.18 1.78

X1 = Vessel diameter (cm), X2 = Sample to solvent ratio (g/ml),
X3 = Extraction temperature (°C), X4=Sonication time (min) TPC = Total
polyphenolic content, TFC = Total flavonoids content, TTC = Total tannin
content, ABTS = 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) ra-
dical cation inhibition, DPPH = 2,2-diphenyl-1- picrylhydrazyl radical
scavenging ability; Level of significance * p < 0.05, ** p < 0.01, ***
p < 0.001.
and among the significant terms, vessel diameter produces the max-
imum influence on TPC extraction as compared to extraction tem-
perature (Table 2). With increasing vessel diameter from 2 to 4 cm the
TPC decreases, but a two-fold increase in extraction was recorded as
vessel diameter increases upto 6 cm (Fig. 3a). Similarly, with increasing
temperature from 35 °C to 45 °C the TPC decreases, however, further
increase was observed upon increasing the temperature from 45 °C to
55 °C (Fig. 3a). With the increasing temperature the solubility of
polyphenolic increases due to penetration of solvent into the plant
matrix and higher mass transfer rate (Al-Farsi and Lee, 2008, Wang
et al., 2008) as also reported in many studies (Tan et al., 2013; Wang
et al., 2008). Increasing vessel diameter provides more horizontal sur-
face area for cavitation effect due to increasing vessel size diameter,
decreasing liquid height and more exposure of the sample to ultrasound
(Kulkarni and Rathod, 2014). This might results higher extraction of
polyphenolic compounds.
3.3.2. Effect of UAE factors on total flavonoids content (TFC) extraction
Similar to TPC, TFC extraction was significantly (p < 0.05) influ-
enced by vessel diameter and extraction temperature. The polynomial
equation showed significant linear and quadratic effect as:
YTFC = 69.91 − 11.13X3 + 23.73X12 + 17.42X32
The model was significantly (p < 0.05) fitted with modal F-value
as 2.49 and any significant terms. Quadratic effect of vessel diameter
influence TFC extraction followed by temperature (Table 2). With in-
creasing extraction temperature (X3) from 35 °C to 45 °C a significant
(p < 0.05) decrease in TFC was recorded. With further increase in
temperature, the positive quadratic effect (p < 0.05) dominates and
thus increase in TFC extraction was noticed (Fig. 3b). Similarly, with
respect to (X1) vessel diameter (from 2 to 4 cm) a decrease in TFC was
observed, which increased significantly, as the vessel diameter further
3.3.4. Effect of UAE factors on antioxidant activity
Antioxidant activity was determined by using ABTS and DPPH as-
says. For ABTS, the antioxidant activity was significantly (p < 0.05)
depends on vessel diameter, sample-to-solvent ratio and extraction
temperature. While DPPH activity was depended on vessel diameter
and sample-to-solvent ratio. The polynomial equation for antioxidant
activity comes as:
YABTS = 1.44 + 0.33X1 − 0.22X2 − 0.41X32
YDPPH = 28.93 + 6.63X1 + 14.092X2 + 7.91X1X2
Both assays showed significant (p < 0.05) model F-value with non-
significant lack of fit (Table 2). With increasing vessel diameter (X1)
from 2 to 6 cm the ABTS antioxidant activity increases linearly. How-
ever, increasing sample-to-solvent ratio from 1:20 to 1:30 showed de-
creasing trends (Fig. 3d). Similarly, increasing extraction temperature
above 45 °C resulted in a quadratic decrease in ABTS activity (Fig. 3e).
In case of DPPH antioxidant activity, both vessel diameter (X1) and
sample-to-solvent ratio (X2) showed significant (p < 0.05) linear ef-
fect. Moreover, increasing both X1 and X2 resulted in significant posi-
tive interactive effect on DPPH activity (Fig. 3f). Different responses of
the factors were seen for different assays. This could be due to different
polyphenolic compounds, which are responsible for different anti-
oxidant activity. Also, the mechanism of these antioxidant assays are
altogether different (Rice-Evans et al., 1997; Tian et al., 2012).
3.4. Optimum UAE condition and its validation
Under optimal UAE condition, 6 cm vessel diameter was used for
extraction. Sample was extracted in 1:28.42 g/mL sample to solvent
ratio having 40% of acetone concentration with 0.2N HCl concentration
and sonicated for 10 min under frequency of 50 KHz at 37 °C. The ex-
perimental values for the responses under this condition were in close
context with predicted values with a CV ranges from 0.72 to 3.71%
(Table 3). This reveals that the model is well fitted for the extraction of
polyphenolics from R. moorcroftianum rhizome under optimal UAE
condition and the designed model is good for predicting the optimal
extraction condition (Table 3).

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A. Pandey et al. Industrial Crops & Products 119 (2018) 218–225

Fig. 3. Three dimensional response surface graphs for (a) Total polyphenolic content (TPC); (b) Total flavonoid content (TFC); (c) Total tannin content (TTC); (d, e)
ABTS; and (g) DPPH antioxidant activity.

Table 3 Table 4
Experimental data of the validation of predicted values at optimal extraction Polyphenolic antioxidant profile of Rheum moorcroftianum rhizome extract
condition. under optimal UAE condition.
Dependent Variables Predicted value Experimental Value % Difference (CV) Polyphenolic antioxidant Retention Time Concentration mg/g
compounds (min)
TPC (mg GAE/g) 83.46 81.86 ± 2.31 2.61
TFC (mg QE/g) 102.73 103.95 ± 4.17 1.17 Gallic acid 3.1 0.587
TTC (mg TAE/g) 75.62 72.81 ± 3.62 3.71 Catechin 3.8 2.95
ABTS (mM AAE/g) 0.90 0.92 ± 0.07 2.17 Chlorogenic acid 4.3 26.68
DPPH (mM AAE/g) 57.9 58.32 ± 1.94 0.72 4-hydroxy benzoic acid 5.2 0.0072
Vanillic acid 5.7 0.05
TPC = Total polyphenolic content, TFC = Total flavonoids content, Caffeic acid 6.2 0.005
TTC = Total tannin content, ABTS = 2,2′- azinobis (3-ethylbenzothiazoline-6- 3-Hydroxy Benzoic acid 7.2 0.223
sulphonic acid) radical cation inhibition, DPPH = 2,2-diphenyl-1- picrylhy- Ferulic acid 9.3 0.04
p-coumaric acid 10.1 0.0051
drazyl radical scavenging ability, CV = coefficient of variation, ± standard
m-coumaric acid 12.1 0.15
error.
Rutin 16.4 0.064
trans cinnamic acid 33.7 0.0094
3.5. Polyphenolics screening

HPLC-DAD analysis detected 12 polyphenolic antioxidant com-
pounds from R. moorcroftianum rhizome extract under optimum UAE
condition. Among all detected compounds, chlorogenic acid was found
in higher concentrations (26.68 mg/g rhizome dw) followed by ca-
techin (2.95 mg/g rhizome dw) and gallic acid (0.58 mg/g rhizome dw)
(Table 4, Supplementary Fig. S1). This is the first report on extraction of
large number of polyphenolic compounds in a single extraction from R.
moorcroftianum. These large number and good concentration of poly-
phenolic compounds under optimum UAE condition is useful for in-
dustrial application, especially for pharmaceutical and nutraceutical
product formulations.
The quantified polyphenolic compounds such as rutin, phloridzin, p-
coumaric acid, catechin, gallic acid, quercetin, 3-hydroxybenzoic acid,
vanillic, caffeic, and chlorogenic acid have shown significant biological
activities including bactericidal, inhibition of HIV replication, free ra-
dical scavenging, cytotoxic, gastric protective action and anti-in-
flammatory activity (Dhalwal et al., 2008). Also, these natural anti-
oxidants were found to be helpful in the prevention of lipid oxidation
(Maqsood et al., 2014); antimicrobial, antifungal, and anti-allergic
agent, and treatment of various chronic diseases, such as cancer, dia-
betes, hypertension, and hypercholesterolemia (Sharma et al., 2013).
4. Conclusion
In the present study, an optimized and efficient ultrasonic assisted
extraction method was developed for polyphenolic antioxidants from
Rheum moorcroftianum rhizomes. A three-step process was optimized
and validated using Box-Behnken design. The experimental values for

223
A. Pandey et al.
the responses were found in close agreement with predicted values.
Further, it is concluded that the extraction of these polyphenolic anti-
oxidants was highly depended on vessel diameter (X1), sample-to-sol-
vent ratio (X2) and extraction time (X3). These conditions can be used
for the effective harnessing of natural antioxidants. This method can be
utilized for further screening of active fraction/compounds from the
Rheum moorcroftianum and other high-value medicinal plants of the
Himalaya. Further, up-scaling of these advanced extraction process
needs to be tested for mass production of compounds for industrial use.
Acknowledgements
The authors are thankful to the Director GBPNIHESD for providing
facilities during experimental work. Funding support from CSIR, Govt.
of India project (38-1346-12-EMR-II) is highly acknowledged. We are
grateful to Uttarakhand Forest Department and Indo Tibetan Border
Police for logistics and field support, respectively. Inputs received from
the anonymous reviewers helped to improve the manuscript sub-
stantially, we thanks them.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.indcrop.2018.04.019.
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