Isolation, chromatographic evaluation……..
Chapter 4
Isolation, chromatographic evaluation and application of phenolic rich
colored compounds/fractions from plants
Chemistry affords two general methods of determining the constituent principles of
bodies, the method of analysis, and that of synthesis. It ought to be considered as a
principle in chemical science, never to rest satisfied without both these species of proofs
………………………………………………………………………...Antoine-Laurent Lavoisier
Phenolic compounds are typical representatives of botanical gifts from nature having a long
history of scientific investigation and represent the most abundant and widely represented
class of plant natural products. Their occurrence in high concentration in large number of
plants exhibit a protective role against several oxidative damage diseases [Benavente-García
et al. (1997); Samman (1998); Lampe (1999); Middleton et al. (2000); Puupponen-Pimiä et
al. (2001); Kondratyuk and Pezzuto (2004); Manach et al. (2005); Rawat et al. (2011)].
In addition to the pharmacological role, phenolics act as pollinator attractants and also
contribute towards the color and sensory characteristics of flowers, fruits and vegetables
[Alasalvar et al. (2001)]; thus signifying great potential as a source of valuable natural
colors. Among the various groups of phenolic compounds which are important in
contributing to the color of plant parts are flavonoids (flavones, isoflavones, chalcones,
anthocyanins,
xanthones
etc)
and
quinones
(benzoquinones,
naphthoquinones,
anthraquinones etc) [Harborne (1976); Bhat et al. (2005)]. Table 1 compiles some of natural
color compounds based on the carbon skeleton, chromophore, source part and application.
Table 1: Major groups along with representative examples of some natural colored
compounds [Bhat et al. (2005)]
Group/ Class
Representive examples/ Color
Source part/ Application
Flavonoids
OH
1. Flavone
OH
HO
O
OH
2. Flavonol
HO
O
Luteolin
(yellow)
OH
OH
O
Rutin
(yellow)
O-R utin ose
OH
Reseda luteola leaves, seeds
Dyeing silk, wool and textiles
O
231
Saphora japonica flower buds
Dyeing silk threads for embroidery
Isolation, chromatographic evaluation……..
Group/ Class
Chapter 4
Representive examples/ Color
3. Flavanone
O-Glu
OH
Glu-O
O
OH O
4. Dihydroflavonol
Reseda luteola leaves, seeds
Dyeing silk, wool and textiles
OH
OH
HO
Butrin
(yellow-orange)
Source part/ Application
O
OH
Myricetin
(yellow)
Myrica rubra roots
Tanning
Diadzein
(yellow)
Glyciene max berries
Food supplement
OH
OH O
5. Isoflovone
HO
O
6. Anhydro base
OH
O
O
OH
O
Carajurin
(red)
HO
Bignonia chika
Dyeing textiles
OMe
7. Xanthone
CH3
O
OMe
OH O
8. Chalcone
COC H3
HO
Garcinia mangostana
Dyeing textiles
CO-CH=CH-C6H5
HO
O
OH
H3C
OH
Mangostin
(yellow)
O
Rottlerin
(salmon)
Mallotas phillippinensis seeds, flowers
Dyeing textiles
OH
Quinones
1. Benzoquinone
O
HO
OH
Polyporic acid
(bronze)
Polyporus fries
Dyeing textiles
Alkanin
(red)
Alkanna spp. roots
Cosmetic and food
O
2. Naphthaquinone
OH
OH
3. Anthraquinone
O
OH
O
O
OH
OH
Alizarin
(red)
Rubia cordifolia
Dyeing cloth
O
Plant colors have been used since ancient times as dye [Bhuyan and Saikia (2005)],
cosmetics, food color [Sarojini et al. (1995); Shah (1997)], in paintings [Nayar et al. (1999)]
232
Isolation, chromatographic evaluation……..
Chapter 4
and textiles [Singh (1985)]. However, their use declined globally after the synthesis of
‘Mauveine’ by William Henry Perkin in 1856 [Garfield (2001)]. Afterwards, synthetic dyes
received faster acceptability in various fields [Calnan (1976); Sinha and Mandal (1995);
Savarino et al. (1999); Lindong and Xuhong (2002)] due to ease in dyeing, reproducibility
in shades and overall cost factor [Kumar and Sinha (2004)]. Nevertheless, there has been an
increasing trend towards replacement of synthetic dyes by natural colors because of
increased environmental awareness, safety & health concerns and strong consumer demand
for more natural products during the last few decades [Kang et al. (1996); Kamel et al.
(2005)]. Moreover, ban on the use of some synthetic dyes (e.g. azodyes) in many countries
of European Economic Community (EEC), Germany, USA and India has triggered active
research and development to revive world heritage and traditional wisdom of employing
safer natural dyes [Vankar et al. (2007)].
The advantages of using natural colorants are manifolds as they are ecofriendly, safe for
body contact, unsophisticated and harmonized with nature [Glover (1998)], obtained from
renewable sources and also their preparation involves a minimum possibility of chemical
reactions [Kumar and Sinha (2004)]. Generally natural colors do not cause health hazards;
on the contrary, they sometimes act as a health cure like shikonin derivatives [Khatoon and
Shome (1993)], curcumin [Shah (1997)] and annatto [Kanjilal and Singh (1995)] (Figure 1)
etc.
OH
O
O
O
H3CO
OH
O
HO
OH
H
OCH3
OH
Curcumin
Shikonin
O
OH
O
OCH3
Bixin
Figure 1
These color rich preparation from plants are derived in variety of ways, as crude extracts,
standardized and partially purified extracts having few defined colored components and as
purified single colored chemical entity. However, many of the available natural colors have
their own limitations like yield, stability, complexity of dyeing process and reproduction of
shades [Kumar and Sinha (2004)]. With sustainably increasing markets for natural
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Isolation, chromatographic evaluation……..
Chapter 4
colorants, it is worthwhile to search for and develop new or alternative sources of natural
colorants.
Though, India is richly endowed with vast variety of flora that can yield natural colors
[Jahan and Gupta (1991); Gupta (1993)], yet only a few sources have been exploited
commercially. Recently biotechnological methods like tissue cultures [Sasson (1993);
Domenburg and Khorr (1996)] are in pace to produce natural dyes for use in food,
pharmaceutical and cosmetic applications. Keeping in view of the changing trends towards
accepting safer and eco-friendly natural colorants, it is the high time to explore new sources
for their isolation. Thus, in the above scenario, it was rational to investigate some plants
from Western Himalayan region, used by rural folks in dyeing, for the isolation &
chromatographic evaluation of phenolic rich colored compounds/fractions from them and
their subsequent application. For convenience, we have divided this objective into three
parts:
a) Isolation and investigation of phenolic rich colored compounds/fractions from
Rhododendron species
b) Isolation and chromatographic evaluation of colored compounds from Arnebia
species
c) Investigations of colored compounds/fractions from Juglans regia
4.1.
Isolation
and
investigation
of
phenolic
rich
colored
compounds/fractions from Rhododendron species
4.1.1. Introduction
The
genus
Rhododendron
(family
Ericaceae) consists of around 250 species
all over the world, ranging in size from few
centimeters to giant trees. Fifty species are
reported in India [Prakash et al. (2007)],
out of which three major species found in
Western Himalayas are Rhododendron
arboreum Smith, R. anthopogon D. Don
and R. campanulatum D. Don. The flowers
Figure 2: Rhododendron arboreum
and leaves of these Rhododendron species
possess nutritional and medicinal properties besides having significant commercial
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Isolation, chromatographic evaluation……..
Chapter 4
importance. R. arboreum (Figure 2) is the national flower of Nepal. Its beautiful red flowers
have a sweetish-sour taste and are consumed both raw and cooked in the form of salads,
pickles, chutney or sour jelly [Facciola (1990); Manandhar (2002)], and some sources claim
that the tender leaves may be used as a cooked vegetable [Tanaka (1976); Facciola (1990)].
In addition, the leaves and flowers are used for treating many illnesses, from diabetes
[Bhandary and Kawabata (2008)] to rheumatism [Skidel (1980)]. The fresh leaves and
flowers of R. anthopogan are made into a health-promoting tea by Himalayan healers
[Sharma et al. (2004); Kunwar et al. (2006)] and the leaves are used for preparation of a
non-alcoholic beverage [Gairola and Biswas (2008)]. The leaves and flowers of R.
campanulatum are used to treat some diseases, but are largely considered to be toxic
[Chauhan (1999)]. Further, several studies have accounted for the use of crude extracts of R.
arboreum flowers for dyeing cloth and as natural food coloring agent [Sati et al. (2003);
Rawat et al. (2006)]. However, the problems concerning variability in coloration and
reproducibility are encountered. Also, the process is laborious and time-consuming.
Rhododendron genus is considered to be a rich source of secondary metabolites like simple
phenols, flavonoids, flavanol-3-O-glycosides, phenolic acids, terpenoids, resins, etc.
[Harborne and Williams (1971); Cao et al. (2004); Feng et al. (2005)] and several
compounds have been isolated from R. dauricum, R. ponticum, R. molle, R. ferrugineum,R.
lepidotum [Chosson et al. (1998); Kashiwada et al. (2001); Cao et al. (2004); Zhong et al.
(2005) Ahmad et al. (2010)] nonetheless, very few reports are available on the chemical
screening of the western Himalayan Rhododendron species [Shafiullah et al. (1991); Kamil
et al. (1995); Swaroop et al. (2005)]. Thus, considering the indigenous knowledge and
future prospects of natural dyes as well as the significant nutritional, medicinal and
commercial value of R. arboreum, R. anthopogon and R. campanulatum, these species
deserves an investigation into their chemical composition, particularly for the presence of
phenolic compounds since these compounds are known to exhibit a wide range of biological
effects [Evans (1996); Haslam (1996)].
4.1.2. Result and Discussion
4.1.2.1. Isolation of phenolic rich colored fraction from R. arboreum: Application for
textile dyeing and identification of some phenolic constituents by HPLC
The relevance of natural products derived from plants for the well being of global society
and environment has been widely recognized. In the colors and dyes arena, there has been a
global concern regarding the harmful effects of synthetic dyes and colors, due to several
235
Isolation, chromatographic evaluation……..
Chapter 4
associated drawbacks like carcinogenicity, allergenicity and their non-biodegradability. A
number of studies have been reported for the extraction of dyes/colors from the natural
resources such as vegetables [Pifferi and Ortolani (1979); Grollier et al. (1982)], marine
organisms [Goswami and Ganguly (2006)], fungus [Bell et al. (1998)] and other plants
parts. In most of the reports, generally water or hydroalcoholic mixture is used for the
extraction which on lyophilization or spray drying at high temperatures gives color yielding
products. However, a major problem encountered with above methods is that the colors
generally become viscous/hygroscopic after some air exposure as extracts are usually
prepared using water or hydroalcoholic mixture as a solvent leading to presence of various
types of sugar moieties, fatty acids or other primary metabolites along with required color
product. Ultimately the colored product becomes highly susceptibility towards microbial
attack. So, such colors require additives such as preservatives and pest protective agents so
as to increase shelf life [Agrawal and Gulrajani (1997)]. The use of acids or bases during
extraction of colors from natural sources is also widespread [Pifferi and Ortolani (1979);
Agrawal and Gulrajani (1997)]. Such harsh extraction procedure might result in degradation
of some active components resulting in decreased therapeutic activity and lower interest
among consumers to use them in food items and cosmetics.
Moreover, the content of color active compounds in plants generally varies with
environmental variations, storage conditions and type of extraction resulting in the
variations in color yielding properties of extracts. Further, it is pertinent to mention that
isolation of single molecule based natural colors is usually tedious and hence not costeffective due to their presence in traces. At the same time, chances of degradation of heat,
light and air sensitive molecule will further deteriorate the quality of single molecule based
natural dye. To overcome the above problems, it is desirable to have color rich fractions,
instead of single molecule based colors, which might have better therapeutic properties and
stability due to synergistic effects.
In this regard, a green technology has been developed for the isolation of natural colors/dyes
from flowers of R. arboreum – a commercially important plant of Western Himalayas
(Figure 3). The same process has been successfully extended to various other plants
including fruits and vegetables (Figure 4). The process involves extraction (microwave,
ultrasound, soxhlet, percolation and reflux) with green solvent water, followed by passing
through a bed of polymeric XAD resin and eluting first with water and then with alcohol.
Concentration of alcohol part in vacuo provided non-hygroscopic and phenolic rich reddish
236
Isolation, chromatographic evaluation……..
Chapter 4
brown colored fraction with yield up to 10%. The developed protocol utilizes green
extraction tools like microwave and ultrasound which enhance its ecofriendly nature [Sinha
et al. WO/2010/109286 A1].
Solubility of dye
In H2O
In alcohol
Crystalline nonhygroscopic dye
Viscous R.
arboreum extract
Figure 4: Natural dyes isolated from
various other plants
Figure 3: Natural dye isolated from
R. arboreum
The isolated colors are found to possess several beneficial features like:
•
Crystalline and non-hygroscopic in nature
•
Free from usage of harsh chemicals like acid/base treatment during extraction
•
Stable towards heat, light and against microbial attacks, thus improved shelf life
•
Possess enhanced bioactivity including antioxidant activity (measured in terms of %
inhibition by DPPH assay)
•
Readily soluble both in water and alcohol
Further, the dyeing capability of the isolated crystalline fraction was evaluated with wool
fabric using both conventional as well as ultrasonic dyeing methods. Ultrasonication
furnishes dyeing comparable to conventional dyeing whilst offering savings in time and
energy. Colors obtained in the dyeing are shown in Figure 5; a slight variation in the color
was obtained with different mordants such as ferrous sulfate, alum, copper sulfate
1
5
9
2
3
4
6
7
8
10
11
12
Figure 5: Colors of the dyeing samples obtained
After recognizing the coloring potential of above crystalline natural dye, some of its
chemical constituents (Figure 6) were identified with the help of RP-HPLC which showed it
to be a mixture of flavonoids and phenolic acids (Figure 7).
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Isolation, chromatographic evaluation……..
Chapter 4
OH
OH
HO
HO
O
O
OH
OH
OH
O
Quercitrin
HO
O
O-Rhamnose
OH
OH
OH
OH
OH
O
Kaempferol
Quercetin
O
O
OH
O
O
COOH
OH
HO
OH
HO
OH
HO
p-Coumaric acid
Chlorogenic acid
Q UE RCITRIN
Figure 6: Structures of compounds identified in the dye
2000
1750
1500
CA
750
SA
Q UE RCE TIN
500
p-CA
1000
Q -3-O -G A L
m AU
1250
K MP
250
0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
Minut es
Figure 7: HPLC chromatogram of color rich fraction from R. arboreum
4.1.2.2. Simultaneous determination of epicatechin, syringic acid, quercetin-3-Ogalactoside and quercitrin in the leaves of Rhododendron species by using a validated
HPTLC method
In addition to above work, owing to the significant nutritional, medicinal and commercial
value of leaves of Rhododendron species (R. arboreum, R. campanulatum and R.
anthopogon) of Western Himalayas, four bioactive phenolics viz. epicatechin (1), syringic
acid (2), quercetin-3-O-galactoside (3) and quercitrin (4) (Figure 8) were simultaneously
quantified for the first time using a rapid and sensitive reverse phase high-performance thinlayer chromatographic (RP-HPTLC) method.
238
Isolation, chromatographic evaluation……..
COOH
OH
HO
O
Chapter 4
In order to develop an effective
solvent system for the separation of
OH
H3CO
OH
OH
OCH 3
OH
2. Syringic acid
1. Epicatechin
O
O
HPTLC
plates
acetate-acetic
OH
OR
OH
was first tried on normal phase
using
various
combinations like chloroform-ethyl
OH
HO
phenolic compounds, the analysis
acid,
chloroform-
methanol, chloroform-ethyl acetate-
3. R=galactose, Quercetin-3-O-galactoside
4. R=rhamnose, Quercitrin
Figure 8: Compounds identified in leaves of
Rhododendron species
methanol, chloroform-ethyl acetatemethanol-isopropyl alcohol and ethyl
acetate-hexane
etc.
in
different
proportions but good separation of
the compounds could not be achieved. This may be due to the wide polarity and
functionality differences among these phenolic compounds. Therefore, our attention shifted
towards the use of RP-TLC plate. Here, for the optimization of HPTLC system various
solvent systems like methanol-water-acetic acid, acetonitrile-water-acetic acid, acetonitrilewater-formic acid and methanol-water-formic acid in different ratios were tried. Out of
these, methanol-5% formic acid in water (50:50, v/v) gave the best resolution, with
symmetrical and reproducible peaks, of epicatechin (Rf = 0.63), syringic acid (Rf = 0.47),
quercetin-3-O-galactoside (Rf = 0.28) and quercitrin (Rf = 0.21) from the other components
of the sample extracts and enabled their simultaneous quantification. The plates were
visualized at two different wavelengths– 254 nm and 366 nm (Figure 9) as the compounds
were found to absorb at variable spectrum range.
In addition, this helped in the generating a better fingerprint data whereby species could be
well differentiated on enhanced visual identification of individual compounds. The method
developed was found to be quite selective with good baseline resolution of each compound
(Figure 10).
239
Isolation, chromatographic evaluation……..
Chapter 4
a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
b
Figure 9: CCD image of TLC plate, Lane 1-5: standard tracks, epicatechin (1), syringic
acid (2), quercetin-3-O-galactoside (3) quercitrin (4); 6–14: methanolic extract of
Rhododendron spp. (6-8 R. arboreum; 9-11 R. campanulatum; 12-14 R. anthopogon). (a) at
254 nm (b) at 366 nm
(Published in Journal of Food Composition and Analysis 2010, 23: 214–219)
D
C
4
3
B
2
1
A
Figure 10: HPTLC chromatogram of (A) standard track (B) R. arboreum methanolic extract
(C) R. campanulatum methanolic extract (D) R. anthopogon methanolic extract (at 290 nm)
(Published in Journal of Food Composition and Analysis 2010, 23: 214–219)
The identity of bands of compounds 1-4 in the sample extracts was confirmed by overlaying
their UV absorption spectra with those of the standards (Figure 11).
240
Isolation, chromatographic evaluation……..
Chapter 4
A
B
C
D
Figure 11: Overlay of UV absorption spectra of compounds in sample track with respective
standards (A) epicatechin (B) syringic acid (C) quercetin-3-O-galactoside (D) quercitrin
(Published in Journal of Food Composition and Analysis 2010, 23: 214–219)
The developed HPTLC method was validated for different parameters like specificity,
linearity, accuracy, precision, robustness, LOD and LOQ. The specificity of the method was
ascertained by analyzing the standard compounds and samples for the interference of other
components. The bands for compounds 1-4 were confirmed by comparing the Rf and spectra
of the bands with that of standards. Absence of any interfering peak indicated that the
method was specific. The purity of bands was confirmed by overlaying the absorption
spectra at the start, middle, and end position of the bands.
Linearity of compounds 1-4 was validated by the linear regression equation and
correlation coefficient. The six-point calibration curves for compounds 1, 3 and 4 were
found to be linear in the range of 200-1200 ng/spot and for compound 2 in the range of 4002400 ng/spot. Regression equation and correlation coefficient for the reference compound
were; Y= 610.217 + 3.5183x (0.9985) for 1, Y= 587.0926 + 6.9580x (0.9996) for 2, Y=
540.5656 + 5.0337x (0.9993) for 3, and Y= 568.0204 + 3.5209x (0.9991) for 4 which
revealed a good linearity response for developed method and are presented in Table 2.
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Isolation, chromatographic evaluation……..
Chapter 4
Table 2: Rf and method validation parameters for the quantitative determination of (1),
syringic acid (2), quercetin-3-O-galactoside (3) and quercitrin (4)
Parameters
1
2
3
4
Rf
Linearity range (ng/spot)
r2
Repeatability of application (n=6)
Repeatability of measurement (n=6)
LOD (ng)
LOQ (ng)
0.63
200-1200
0.9985
0.86
0.72
20
50
0.47
200-2400
0.9996
0.74
0.59
40
115
0.28
200-1200
0.9993
0.52
0.58
25
75
0.21
200-1200
0.9991
0.64
0.43
25
70
(Published in Journal of Food Composition and Analysis 2010, 23: 214–219)
The instrumental precision which was represented as repeatability of sample application and
measurement of peak area was found to be 0.86 and 0.72 for epicatechin, 0.74 and 0.59 for
syringic acid, 0.52 and 0.58 for quercetin-3-O-galactoside, 0.64 and 0.43 for quercitrin,
respectively (Table 2). To study the variability of method, intra-day precision and inter-day
precision (expressed in terms of % RSD) were calculated for three different concentration
(200, 400, and 600 ng/spot) by measurement of peak area for the compounds 1-4 and were
observed in the range of 0.41-1.37% and 0.67-2.04%, respectively, which demonstrated the
good precision of proposed method.
Lower limits of detection obtained for compounds 1–4 were 20, 40, 25 and 25 ng
respectively while the limit of quantification obtained were 50, 115, 75 and 70 ng
respectively (Table 2). This indicated that the proposed method exhibits a good sensitivity
for the quantification of above compounds.
RSD of the samples varied from 1.43 to 2.60% for all the compounds under analysis. Good
recoveries were obtained by the fortification of the sample at three concentration levels for
compounds 1-4. It is evident from the results that the percent recoveries for all the four
compounds after sample processing and applying were in the range of 95.45 - 98.50% as
shown in Table 3.
The mobile phase with a slight differences in their composition i.e. methanol-5% formic
acid in water with three different ratios 45:55, 50:50 and 55:45 (v/v) were used and
developing distance was checked varying between 7 to 9 cm and no considerable effect on
the analysis was recorded. Also different TLC plate lots of the same manufacturer had no
influence on the chromatographic separation.
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Isolation, chromatographic evaluation……..
Chapter 4
Table 3: Recovery study of epicatechin (1), syringic acid (2), quercetin-3-O-galactoside (3)
and quercitrin (4) by proposed HPTLC method
Compounds
Amount of
compound present
in plant material
(ng/spot)
Amount (ng/
spot) of
standard
added
Observed amount
(ng/spot)
% Recovery
%RSD
1
735.85
50
100
200
771.85
821.16
919.76
98.22
98.24
98.28
1.38
1.22
1.17
2
765.57
3
291.77
4
610.12
100
200
400
50
100
200
50
100
200
852.59
949.06
1138.98
327.12
379.28
472.48
631.26
678.03
773.26
98.50
98.29
97.71
95.71
96.81
96.07
95.63
95.48
95.45
1.67
1.08
1.11
1.20
1.52
1.29
1.66
1.32
1.59
(Published in Journal of Food Composition and Analysis 2010, 23: 214–219)
The content of epicatechin, syringic acid, quercetin-3-O-galactoside and quercitrin was
estimated in the methanolic extract of Rhododendron sp. by the proposed method and the
results obtained are summarized in Table 4.
Table 4: Content of epicatechin, syringic acid, quercetin-3-O-galactoside and quercitrin
found in Rhododendron species
Species
R. arboreum
R. campanulatum
R. anthopogon
Epc*
SA*
Q-3-O-gal*
Qrc*
__________________________________________________________
Av (n=3) ng/spot Av (n=3) ng/spot
%RSD
%RSD
Av (n=3) ng/spot
%RSD
735.85
2.65
211.56
2.36
220.44
2.23
291.77
2.36
446.71
2.43
384.73
2.02
765.57
2.77
535.93
1.89
1718.23
2.13
Av (n=3) ng/spot
%RSD
610.12
0.84
239.48
1.14
281.06
0.90
* Epc = epicatechin, SA = syringic acid, Q-3-O-gal = quercetin-3-O-galactoside, Qrc = quercitrin
The results showed interesting differences in the amounts of these derivatives present in the
same genus. It is for the first time, a simple, accurate and rapid HPTLC method has been
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Isolation, chromatographic evaluation……..
Chapter 4
developed for the simultaneous quantification of four bioactive compounds (1–4) in leaves
of R. arboreum, R. campanulatum and R. anthopogon.
4.2.
Isolation and chromatographic evaluation of colored compounds
from Arnebia species
4.2.1. Introduction
Naphthoquinones are the red colored pigments
present abundantly in the members of Boraginaceae
family including genus Arnebia. These compounds
show
a
wide
spectrum
of
significant
pharmacological activities such as antimicrobial,
anti-inflammatory, antioxidant, anticancer, and
antithrombotic activities [Chen et al. (2002);
Nigorikawa et al. (2006); Ishida and Sakaguchi
Figure 12: Arnebia euchroma
(2007)]. 2-substituted napthaquinones, represented by shikonin and its derivatives, are
important red coloured pigments found in a large number of Arnebia species that are
responsible for the various pharmacological activities exhibited by the plant [Chen et al.
(2002)]. Out of the numerous Arnebia spp. found all over the world, Arnebia euchroma
(Royle) John (Figure 12), A. benthamii (Wall. ex G. Don) John, A. guttata Bunge and A.
hispidissima (Lehm.) DC are reported from the Western Himalayan region of India. Out of
these four species, A. euchroma make the most of production of shikonin and its derivatives.
The plant is reported to possess a plethora of bioactivities like anti-inflammatory [Lin et al.
(1980); Tanaka et al. (1986); Wang et al. (1994); Kaith et al. (1996)], antimicrobial [Tabata
et al. (1975); Shen et al. (2002)], antitumor [Sankawa et al. (1977); You et al. (2000)],
antifungal [Sasaki et al. (2002)], antiviral [Choi (1988); Kashiwada et al. (1995)],
antiplatelet [Chang et al. (1993); Ko et al. (1995)] and contraceptive properties [Findley
(1981)]. It is widely used in Indian and Chinese traditional medicinal system for the
treatment of burns, skin ulcers, gynecological inflammations [Hsu and Hsu (1980); Fu and
Lu (1999)], in promoting blood circulation, and facilitating the movement of stool [Chang et
al. (1993)]. These compounds have also shown promising activity against human
immunodeficiency virus [Ko et al. (1995)]. In addition to this, A. euchroma is widely used
as source of natural dye in food additives and cosmetics as the shikonin derivatives present
in it demonstrate variable colors with change of pH [Srivastava et al. (1999)].
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Isolation, chromatographic evaluation……..
Chapter 4
Due to the above-reported important biological actions, it is crucial to have a large and easy
availability of these molecules. Yet, in spite of immense efforts by numerous researchers
around the world, the methods developed for the total synthesis of the molecules are very
complex, uneconomical and commercially unviable. Hence, cultivated/wild plants still
remain the major source for the production of these molecules. However, attempts for the
isolation and purification of naphthoquinone derivatives from the crude plant extract
through conventional methods like column chromatography is cumbersome and time
consuming [Ikeda et al. (1991); Papageorgiou et al. (1999); Yamamoto et al. (2000); Lay et
al. (2000); Lu et al. (2004)]. One reason is that these naphthoquinones have almost similar
retention factors and secondly, have low stability as they are susceptible to several
transformations e.g. photochemical decomposition [Chen et al. (1996)], thermal degradation
[Cho et al. (1999)] and polymerization [Papageorgiou (1976)] and are not available in the
market easily. Therefore, for meeting the demand of shikonin derivatives simplified
isolation and purification methodologies are desired.
4.2.2. Result and Discussion
4.2.2.1. Development of a preparative HPLC method for the rapid isolation of shikonin
derivatives from root extract of A. euchroma
For the isolation of shikonin derivatives, hexane extract of A. euchroma in acetonitrile was
subjected to prep-HPLC using a reverse phase C18 column. Solvent system consisting of
water and acetonitrile (20:80, v/v) was isocratically pumped at a flow rate of 3 mL/min. 1
mL of the extract (final conc. 5 mg/mL) was loaded on a Rheodyne injector.
Naphthoquinone derivatives are known to absorb around 214, 275 and 520 nm [Bozan et al.
(1997); Papageorgiou et al. (1999)]. Considerable noise was found around 214 and 275 nm;
therefore 520 nm was chosen as the optimized detection wavelength in this experiment.
Shikonin
derivative
solutions
were
rich
collected
2: 520 nm, 8 nm
A hispidissima CHCl3 ext-070808
A hispidissima CHCl3 ext-070808
60
in
Compound 2, Rt =5.7
50
clean pre-weighed flasks from
40
to
8.0
min
at
room
temperature (Figure 13). The
Compound 1, Rt =4.6
mAU
4.2
above experiment was repeated
till the desired amount (for
spectroscopic
compounds
analysis)
of
interest
of
was
30
Compound 3, Rt =7.8
20
10
0
0
1
2
3
4
5
6
7
8
9
10
Minutes
11
12
13
14
Figure 13: Chromatogram of A. euchroma hexane
extract on Prep-HPLC
245
15
Isolation, chromatographic evaluation……..
Chapter 4
obtained. The solvent was evaporated to dryness in each case under reduced pressure,
resulting in complete removal of organic phase and appearance of deep red viscous
compounds sticking to the walls of the flasks.
Purity of the isolated shikonin derivatives was analyzed by analytical HPLC using
water/acetonitrile (20:80, v/v) at a flow rate of 1 mL/min at 520 nm on a C 18 column and
was found to be more than 97%; which confirmed the validity of our developed method.
HPLC chromatograms of the purified fraction of isolated naphthoquinone derivatives along
with their UV spectra are shown in Figure 14.
Spectrum at time 5.98 min.
2: 520 nm, 8 nm
Shikonin-220808
Shikonin-220808
5.98 min
150
276
490
513
493
485
497
150
100
50
mAU
50
mAU
193
500
mAU
60
214
201
100
a
70
Lambda max :
Lambda min :
50
80
40
0
0
30
200
20
400
600
800
nm
10
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Minutes
Spectrum at time 9.10 min.
2: 520 nm, 8 nm
AKMSBR-09-7-Hexane-250309
AKMSBR-09-7-Hexane-250309
16
9.10 min
Lambda max :
Lambda min :
214
201
192
489
273
498
517
242
490
320
100
14
100
50
b
50
mAU
mAU
12
8
0
0
mAU
10
6
200
400
600
4
800
nm
2
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Minutes
Spectrum at time 13.48 min.
140
2: 520 nm, 8 nm
Arnebia column F 9-250808
Arnebia column F 9-250808
1000
510
487
485
245
c
489
373 1000
750
276
494
750
500
mAU
100
214
204
500
250
120
250
mAU
13.48 min
Lambda max :
Lambda min :
mAU
80
60
0
0
200
40
400
600
800
nm
20
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Minutes
Figure 14: HPLC chromatograms of peaks collected from prep-HPLC (a) at Rt 4.6; (b) at
Rt 5.7 and (c) at Rt 7.8 to analyze purity
246
Isolation, chromatographic evaluation……..
Chapter 4
Structures of the isolated compounds was confirmed by NMR spectra data which came out
to be shikonin, acetylshikonin and β-acetoxyisovalerylshikonin (Figure 15).
OH
O
OH
O
Shikonin
OH
OH
O
OH
O
OH
O
OH
O
O
Acetylshikonin
O
O
O
O
O
beta-Acetoxyisovalerylshikonin
Figure 15: Structures of the isolated compounds
Given that all the three isolated compounds have significant pharmacological potencies
[Guo et al. (1991); Pietrosiuk et al. (2004); Marcus et al. (2005); Wang et al. (2005); Han et
al. (2007); Lu et al. (2008)], the developed method would surely find applications at the
commercial scale.
4.2.2.2. Simultaneous densitometric determination of shikonin, acetylshikonin, and βacetoxyisovalerylshikonin in ultrasonic-assisted extracts of four Arnebia species using
reversed-phase thin layer chromatography
Due to the immense pharmacological and commercial importance alongside tedious
synthesis of shikonin derivatives, Arnebia is one of the widely sought-after plants because
of the presence of these compounds in good amount. Among the four species (A. euchroma,
A. benthamii, A. guttata and A. hispidissima) found in Western Himalayan region of India,
the natural sources of A. euchroma are over-exploited which has put an undesirable pressure
on their sustainability and hence the natural supplies are dwindling. For finding new sources
of shikonin derivatives, investigation of other species of the genus seems rational. In this
context, a simple, rapid and precise HPTLC method has been developed for the
quantification of shikonin (1) and its derivatives; acetyl shikonin (2) and βacetoxyisovalerylshikonin (3) in the root extracts of aforementioned four Arnebia species.
247
Isolation, chromatographic evaluation……..
Chapter 4
4.2.2.2.1. Optimization of conditions
In order to optimize the extraction of
analytes, effect of different extraction
solvents were studied using ultrasound
assisted extraction as a rapid and efficient
extraction tool. Four solvent with varying
polarity such as hexane, chloroform, ethyl
acetate and methanol, that had been used
in prior studies were selected [Kaith et al.
(1996); Bozan et al. (1999); Ozgen et al.
(2004); Hu et al. (2006)]. The temperature
for extraction was kept low (40 ± 2◦C) as
Figure 16: Effect of solvents on the mass
yield of analytes in different Arnebia spp.;
hexane, chloroform (CHCl3), ethyl acetate
(EtOAc) and methanol (MeOH)
increase in temperature may lead to the degradation of compounds of interest due to their
thermal sensitivity [Cho et al. (1999)]. On the basis of mass yield (Figure 16) methanol
seemed to be better solvent. However, HPTLC analysis demonstrated that the amount of
desired naphthoquinones was higher in case of ethyl acetate extract followed by chloroform,
hexane and quite low in methanol extract.
In order to develop an effective TLC method that is able to resolve the compounds present
in the extract, initially, normal phase silica gel TLC plate was tried with a combination of
different developing solvents like chloroform-methanol, chloroform-methanol-acetic acid,
chloroform-ethyl acetate-acetic acid in varying polarity however, the compounds were
found to overlap on TLC plate. Finally, it was decided to use of RP-TLC silica plates. Since
water is used as one of the mobile phases in reverse phase separation, so we started with a
combination of water with methanol and/or with acetonitrile in 90:10 (v/v), but the
compounds remained at TLC plate base. After trying different combinations of these
solvents in varying ratio, it was found that higher concentration of organic solvent gives
better separation. The optimal separation of all the compounds 1–3 was achieved with
combination of acetonitrile-methanol-water (40:02:08, v/v/v), however the shape of the
bands was not satisfactory. In order to improve the selectivity of bands, effect of different
organic acids (in varying concentrations from 1-5%) on the above mentioned solvent system
was investigated. It was ascertained that with acetonitrile-methanol-5% formic acid in water
(40:2:8, v/v/v) not only selectivity, but the shape of bands was also improved. Thus, this
system was finalized for further studies.
248
Isolation, chromatographic evaluation……..
1
2
3
4
5
6
7
8
Chapter 4
9
10
11
12
13
14
15
16
17
Figure 17: CCD image of TLC plate, Lane 1: standard track, shikonin (1), acetylshikonin
(2), β-acetoxyisovalerylshikonin (3); Lane 2–17: extracts of Arnebia spp (2, 6 10, 14 hexane
extract; 3, 7, 11, 15, chloroform extract; 4, 8, 12, 16, ethyl acetate extract; 5, 9, 13, 17,
methanolic extract) (3a) At 366 nm (3b) visible region.
(Published in Journal of Separation Science 2009, 32: 3239–3245)
The three compounds 1–3 (Figure 17) were well separated with Rf values of 0.62, 0.49 and
0.40 respectively and were used as
references for the identification and
quantification
of
above
three
naphthoquinones (1–3) present in the
different root extracts of Arnebia spp.
collected
from
Western
Himalayan
region. The plates were visualized at
three different wavelengths 254 nm, 366
nm
and visible
(Figure
17) as
the
compounds were found to absorb at
variable spectrum range. In addition, this
Figure 18: HPTLC chromatogram of (a)
standard track (b) sample track; shikonin
(1),
acetylshikonin
(2)
and
βacetoxyisovalerylshikonin (3)
249
Isolation, chromatographic evaluation……..
Chapter 4
helped in the generating a better fingerprint data where by species could be well
differentiated on enhanced visual identification of individual compounds.
4.2.2.2.2. Method validation
The developed HPTLC method was validated for different parameters like specificity,
linearity range, accuracy, precision, robustness, LOD and LOQ. The specificity of an
analytical method is the ability to assess the analyte clearly in the presence of other
components that may be expected to present in analyzing sample. The bands for compounds
1, 2 and 3 were confirmed by comparing the Rf and spectra of the bands with that of
standards. Absence of any interfering peak indicated that the method was specific. The peak
purity was assessed by comparing the spectra at three different levels, i.e., peak start, peak
apex, and peak end (Figure 18).
The calibration curves were found to be linear in the range of 100-600 ng for shikonin (1)
and acetylshikonin (2), and 100-1800 ng for β-acetoxyisovalerylshikonin. Regression
equation and coefficient of correlation ranging from 0.9985 to 0.9997 revealed a good
linearity response for developed method and are presented in Table 5. RSD % of the linear
equation varied from 1.57 to 3.53% for all the compounds under analysis.
Table 5: Rf, Linearity range, Linear Regression, correlation coefficient, SD, LOD and LOQ
for shikonin derivatives
Compd.
Rf
Linearity
Regression
range (ng)
equation
r
SD (%)
LOD
LOQ
(ng/spot) (ng/spot)
1
0.62
100-600
Y= 9.184x -520.02
0.99976 1.57
18.0
60.0
2
0.49
100-600
Y= 11.52x -746
0.99855 2.21
15.0
45.0
3
0.40
100-1800
Y= 4.401x + 440.16 0.99929 3.53
12.0
40.0
For each curve the equation is y= ax+b, where y is the peak area, x is the concentration of
the analyte, a is the slope, b is the intercept, r the correlation coefficient, RSD the relative
standard deviation of peak area
(Published in Journal of Separation Science 2009, 32: 3239–3245)
Good recoveries in the range were obtained by the fortification of the samples at three
concentration levels for shikonin, acetylshikonin and β-acetoxyisovalerylshikonin. It is
evident from the results that the percent recoveries for all the three naphthoquinone
derivatives (1–3) were in the range of 95.21-98.37% as shown in Table 6.
250
Isolation, chromatographic evaluation……..
Chapter 4
Table 6: Recovery study of naphthoquinone derivatives
Compd.
Amount present
in plant material
(ng)
Amount of
standard
added (ng)
Observed amount
(ng)
% Recovery %RSD
1
31.40
50
150
200
77.50
173.38
221.49
95.21
95.57
95.72
1.37
1.22
1.68
2
155.52
50
100
200
199.68
248.86
345.27
97.16
97.39
97.11
1.43
0.88
0.76
3
663.84
100
751.42
98.37
1.34
200
849.21
98.31
1.29
300
946.36
98.19
1.55
__________________________________________________________________________
(Published in Journal of Separation Science 2009, 32: 3239–3245)
The repeatability and inter-day precisions (expressed in terms of % RSD) were calculated
by measurement of peak area for the naphthoquinone derivatives and were observed in the
range of 0.31-1.31% and 0.90-2.37%, respectively, which demonstrated the good precision
of proposed method. Three different mobile phases with a slight difference in their
composition were used and developing distance was checked varying between 7 to 9 cm
and no considerable effect on the analysis was recorded. Also different TLC plate lots of the
same manufacturer had no influence on the chromatographic separation.
4.2.2.2.3. Quantification of naphthoquinone derivatives
The compounds 1, 2 and 3 in different root extracts of Arnebia spp. were quantitated using
the developed HPTLC method and the results for each species are summarized in Table 7.
The results have shown interesting differences in the amounts of these derivatives present in
the same genus with different solvents. In general, ethyl acetate was found to be more
effective solvent for the extraction of these compounds. Among all, shikonin and acetyl
shikonin was found higher in A. guttata and least in A. hispidissima. However, βacetoxyisovalerylshikonin was found higher in A. euchroma while it was not detected in A.
benthamii.
251
Isolation, chromatographic evaluation……..
Chapter 4
Table 7: Amount of naphthoquinone derivatives present in different Arnebia spp. in
different solvents
Species
Shikonin
Acetylshikonin
β-acetoxyisovalerylshikonin
135.31 ± 0.26
149.42 ± 0.82
207.04 ± 0.55
104.80 ± 0.90
796.69 ± 0.83
815.43 ± 0.76
1023.77 ± 0.23
528.44 ± 0.72
3015.94 ± 0.67
3575.21 ± 0.34
4370.64 ± 1.02
1529.08 ± 0.45
146.03 ± 0.36
177.32 ± 0.27
223.94 ± 0.80
121.58 ± 0.17
742.21 ± 0.67
1128.53 ± 0.43
1186.24 ± 0.79
533.40 ± 1.34
1968.12 ± 0.27
2125.71 ± 0.54
2248.04 ± 0.65
1424.19 ± 1.56
122.61 ± 0.36
127.64 ± 0.49
131.42 ± 0.33
106.48 ± 0.89
113.79 ± 0.97
118.36 ± 0.70
134.51 ± 0.93
90.10 ± 1.23
< LOQ
< LOQ
< LOQ
< LOQ
68.53 ± 0.73
72.51 ± 0.38
82.91 ± 1.32
36.86 ± 1.67
______________________________________________________________
Av. (n=3) ± SD in μg/g*
__________________________________________________________________________
A. euchroma
Hexane
Chloroform
Ethyl acetate
Methanol
A. guttata
Hexane
Chloroform
Ethyl acetate
Methanol
A. benthamii
Hexane
Chloroform
Ethyl acetate
Methanol
A.
hispidissima
Hexane
Chloroform
Ethyl acetate
Methanol
nd
nd
nd
nd
59.24 ± 1.41
64.29 ± 1.09
80.13 ± 0.74
28.12 ± 1.12
nd = not detected, * Amount represented in μg/g of dry plant material ± SD
(Published in Journal of Separation Science 2009, 32: 3239–3245)
4.3.
Investigations of colored compounds/fractions from Juglans regia
4.3.1. Introduction
Common walnut (Juglans regia L; Figure 19) belongs
to the family Juglandaceae and is extensively used in
the Indian (Ayurvedic) and Greco-Arab traditional
systems of medicine for the treatment of various
common ailments including tuberculosis and cancer. It
is reported to be astringent, antifungal, diuretic,
anthelmintic, laxative, tonic, blood purifier and
detoxifier [Kirtikar and Basu (1975); Haque et al.
(2003); Bhatia et al. (2006); Stamper et al. (2006)]. All
Figure 19: Juglans regia
parts of J. regia such as dry fruit nuts, green walnuts, shells, kernels, bark, leaves are used is
used in many countries as a dye for coloring the lips [Alkhawajah (1997)] and in cosmetics
252
Isolation, chromatographic evaluation……..
Chapter 4
[Stamper et al. (2006)]. Common walnut contains naphthoquinones and flavonoids as major
phenolic compounds [Wichtl and Anton (1999)], out of which juglone (5-hydroxy-1,4naphthoquinone) is of great interest due to its chemical reactivity and bioactivity.
Traditionally, juglone has been used as a natural dye for clothing and fabrics, particularly
wool, and as ink. Moreover, due to its tendency to create dark orange-brown stains, juglone
also finds use as a coloring agent for foods and cosmetics, such as hair dyes [Ghosh and
Sinha (2008)]. However, juglone is known to unstable in solution form [Girzu et al. (1998)]
and undergo reversible oxido-reduction reactions with the simultaneous formation of free
radicals [Anderson et al. (2001)]. Therefore, the choice of an extraction technique,
including appropriate solvent, extraction time, and temperature is crucial for the efficient
extraction of the analytes from J. regia.
4.3.2. Result and Discussion
4.3.2.1.
Microwave-assisted efficient extraction and stability of juglone in different
solvents from Juglans regia: Quantification of six phenolic constituents by validated
RP-HPLC and evaluation of antimicrobial activity
In order to reduce or eliminate the use of organic solvent and to improve the extraction
process, newer sample preparation methods, such as microwave-assisted extraction (MAE),
ultrasound-assisted extraction (UAE), accelerated solvent extraction etc. have been
introduced for the efficient extraction of bioactive compounds from plants to increase their
therapeutic functionality [Proestos and Komaitis (2006); Sharma et al. (2006); Proestos and
Komaitis (2008)]. Among these, MAE is the simplest and most economical technique in
terms of lesser solvent consumption and considerable reduction in extraction time [Pan et
al. (2003); Cos et al. (2006); Martino et al. (2006); Sharma et al. (2006); Proestos and
Komaitis (2008)]. In recent years, many papers have been published on the applicability of
MAE for the extraction of bioactive compounds from plants [Sharma et al. (2008)].
Nevertheless, there is no report available that could illustrate the feasibility of MAE as a
rapid and efficient tool for the extraction of juglone and other phenolic compounds from J.
regia. Hence, evidently, our objective was to explore MAE as an alternate and effective
approach for the rapid and efficient extraction of juglone from J. regia and its comparison
with other extraction techniques i.e. maceration and ultrasound-assisted extraction (UAE).
Further, a validated RP-HPLC method was developed for the quantification of some
phenolics in the extracts of J. regia bark. Simultaneously, the obtained extracts were
subjected to antimicrobial activity against certain bacteria and fungi.
253
Isolation, chromatographic evaluation……..
Chapter 4
4.3.2.1.1. Extraction optimization
For optimizing the conditions for the efficient extraction of juglone from J. regia bark,
maceration, MAE and UAE was performed in solvents ranging from non-polar to polar such
as chloroform, ethyl acetate, methanol, and water. In case of maceration, maximum yield of
extract was obtained in methanol (12.21%) followed by ethyl acetate (5.53%), water
(4.40%), and chloroform (2.12%); however, analysis by HPLC showed that the content of
juglone was more in chloroform extract (0.0146%) in comparison to methanol (0.006%) and
ethyl acetate extract (0.009%) while it was not detected in water extract. Based upon these
observations, chloroform seems to be the solvent of choice for extraction of juglone under
maceration. This result is in concurrence with the previous studies for extraction of juglone
from other plant matrices [Girzu et al. (1998); Hadjmohammadi and Kamel (2006)].
However, with MAE and UAE, content of juglone was found higher in ethyl acetate extract
(0.0147% with MAE and 0.0105% with UAE) instead of chloroform (0.002% with MAE
and 0.003% with UAE) though yield of the extract was again higher in methanol (10.8%
and 9.87%, respectively). This anomalism with chloroform in microwave irradiation may be
due to its low dielectric constant ultimately leading to poor yields of analytes [Hayes
(2002)] whereas lower content of juglone in methanol may be attributed to its
decomposition in it, as evident from the earlier report [Girzu et al. (1998)]. Even though
water has the highest dielectric constant, it did not yield any amount of juglone under
microwave irradiation as it may not be able to sufficiently solubilize juglone. Thus, ethyl
acetate was optimized as the best solvent for the extraction of juglone from the bark of J.
regia because of its good heating capacity under microwave and its ability to solubilize
juglone and, as a result, inevitably used in further studies.
4.3.2.1.2. HPLC method development, validation and quantification studies
Only few HPLC methods have been reported for the determination of napthoquinones
[Stensen and Jensen (1994), Girzu et al. (1998), Colaric et al. (2005); Babula et al. (2006);
Hadjmohammadi and Kamel (2006)] however, no RP-HPLC method is available for the
determination of phenolic compounds in walnut bark. In this context, we have proposed a
simple, rapid, and specific RP-HPLC (Figure 20), method for the determination six
important phenolic compounds i.e. gallic acid (1), caffeic acid (2), quercitrin (3), myricetin
(4), quercetin (5) and juglone (6) (Figure 21) in J. regia bark.
254
Isolation, chromatographic evaluation……..
Chapter 4
3
0 .6 0
1
0 .5 0
A
6
0 .4 0
U
A
4
0 .3 0
0 .2 0
5
2
0 .1 0
0 .0 0
2 .0 0
4 .0 0
6 .0 0
8 .0 0
1 0. 0 0
Min u te s
1 2 .0 0
1 4 .0 0
1 6 .0 0
1 8 .0 0
B
3
0 .9 0
2 0. 0 0
0 .8 0
0 .7 0
0 .6 0
0 .5 0
U
A
0 .4 0
1
0 .3 0
4
0 .2 0
0 .1 0
5
2
6
0 .0 0
2 .0 0
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
M in u t e s
1 2 .0 0
1 4 .0 0
1 6 .0 0
1 8 .0 0
2 0. 0 0
Figure 20: (A) HPLC chromatogram of standards: gallic acid (1), caffeic acid (2),
quercitrin (3), myricetin (4), quercetin (5) and juglone (6); (B) HPLC chromatogram of
ethyl acetate extract
(Published in Analytical Letters 2009, 42: 2592–2609)
COO H
HO
O
COO H
HO
OH
OH
OH
Gallic acid (1)
OH
Caffeic acid (2)
O
Juglone (6)
R1
R2
HO
O
R3
OR4
OH
Compound
R1
R2
R3
R4
Quercitrin (3)
OH
OH
OH
H
rhamnose
Myricetin (4)
OH
Quercetin (5)
OH
OH
OH
H
H
H
O
Figure 21: Chemical structures of the quantified phenolic compounds
(Published in Analytical Letters 2009, 42: 2592–2609)
255
Isolation, chromatographic evaluation……..
Chapter 4
The proposed chromatographic method was validated to determine the linearity, LOD,
LOQ, accuracy, and precision of each compound. The linearity, LOD, and LOQ for six
compounds (1–6) were investigated and results are presented in Table 8.
Table 8: Parameters of the linearity, detection limit and quantitation limit for chemical
compounds present in the bark of J. regia
________________________________________________________________________
Linearity
Linear equation
r2
LOD
LOQ
range (µg/mL)
(µg/mL)
(µg/mL)
________________________________________________________________________
Compound
Gallic acid (1)
1-60
Y=31998.65x-37025
0.997
0.16
0.50
Caffeic acid (2)
1-60
Y=22267.58x-6887
0.998
0.08
0.25
Quercitrin (3)
30-900
Y=46825x-539923
0.999
0.08
0.25
Myricetin (4)
0.312-28
Y=58325x+11358
0.997
0.05
0.15
Quercetin (5)
0.25-16
Y=112824x-23172
0.998
0.04
0.12
Juglone (6)
1.6-50
Y=63623x-30697
0.997
0.13
0.40
________________________________________________________________________
(Published in Analytical Letters 2009, 42: 2592–2609)
A good linearity was achieved in the range 0.997–0.999 for all the compounds. Detection
limit (LOD) is the lowest amount of analyte in a sample that can be detected, but not
necessarily quantified. LOD for all the compounds (1–6) was in the range 0.04–0.16 mg/mL
(Table 8). The LOQ, which is defined as the lowest concentration that can be determined
with acceptable accuracy and precision for all the compounds (1–6), was experimentally
verified by six injections and found in the range 0.12–0.50 mg/mL (Table 8).
The intra-day and inter-day precision (repeatability) of the method was calculated by six
replicate injections of three different concentrations of each compound (1–6), respectively.
Precision was expressed as % RSD. The intraday and interday % RSD of chromatographic
determination was observed in the range of 0.25–0.98 and 0.08–0.97%, respectively (Table
9). Hence, the results showed good precision of the developed RP-HPLC method.
Recoveries of the experiment were performed in order to study the accuracy of the method
which was found in the range between 95 and 105%. Overall, the validated method was
found to be suitable for quantification of juglone and other phenolic compounds.
256
Isolation, chromatographic evaluation……..
Chapter 4
Table 9: Repeatability of intra-day and inter-day analysis
Compound
Concentration
RSD %
Intraday (n=6)
Gallic Acid (1)
Caffeic acid (2)
Quercitrin (3)
Myricetin (4)
Quercetin (5)
Juglone (6)
60
30
15
60
30
15
64
32
16
28
14
7
16
8
4
52
26
13
0.36
0.60
0.28
0.44
0.83
0.98
0.76
0.50
0.80
0.76
0.50
0.38
0.38
0.39
0.78
0.25
0.43
0.93
Interday (n=6)
0.44
0.08
0.70
0.19
0.34
0.93
0.55
0.74
0.80
0.56
0.92
0.97
0.80
0.92
0.75
0.35
0.22
0.73
(Published in Analytical Letters 2009, 42: 2592–2609)
4.3.2.1.3. Quantitative determination of compounds (1–6) in bark extracts of J. regia
Juglone and other phenolic compounds (1–6) were quantitated using the developed and
validated RP-HPLC method in the microwave-assisted extracts of bark of J. regia. The
calculated amount of each compound (1–6) is shown in Table 10.
Table 10: Contents of compounds present in microwave-assisted extracts of J. regia bark
_____________________________________________________________________
Compound
Ethyl acetate
Methanol
Water
_____________________________________________________________________
Gallic acid (1)
0.1250%
0.2030%
0.3440%
Caffeic acid (2)
0.0048%
0.0398%
0.0050%
Quercitrin (3)
0.8620%
0.7398%
0.6144%
Myricetin (4)
0.0531%
0.0515%
0.0310%
Quercetin (5)
0.0208%
0.0202%
0.0111%
Juglone (6)
0.0147%
0.0029%
nd
____________________________________________________________________
nd= not detected
(Published in Analytical Letters 2009, 42: 2592–2609)
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Isolation, chromatographic evaluation……..
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4.3.2.1.4. Stability of juglone
Juglone is reported to undergo degradation in solvents such as acetonitrile, methanol, or in
acidic medium as well as saline water [Girzu et al. (1998); Hadjmohammadi and Kamel
(2006); Wright et al. (2007)]. In this study, we have examined the decomposition of juglone
in methanol and ethyl acetate solution at dark and at 4 oC over a period 7 days. The
concentration of juglone in these solutions was calculated from a standard curve derived
from a freshly prepared solution and degradation % is summarized in Table 11. From the
studies, it is evident that at 24 h juglone does not undergo decomposition in ethyl acetate
solution whereas a 6% loss was observed in methanol. Thus, ethyl acetate was optimized as
a better solvent in terms of stability and higher extraction of juglone.
Table 11: Degradation (%) of juglone in methanol and ethyl acetate at 4oC
Time→
24 h
48 h
72 h
96 h
120 h
144 h
Solvent↓
168
h
Methanol
6
12
17
20
21
25
33
Ethyl acetate
No
degradation
4
8
12
13
15
17
4.3.2.1.5. Antimicrobial Activity
The antimicrobial activity of synthetic and natural compounds including plant extracts has
been recognized for many years and has formed the basis of many applications including
food preservation, pharmaceuticals, and medicine [Narad et al. (1995); Tzoris et al. (2003);
Murthy et al. (2006)]. J. regia contains napthaquinones as major phenolic compounds
[Wichtl and Anton (1999)] which are reported to possess very interesting spectrum of
antimicrobial activities [Babula et al. (2009)]. In this direction, extracts of J. regia bark and
marker compound juglone were tested for antimicrobial activity against 16 microorganisms
and the results are given in Table 12. The extracts showed antimicrobial activity against
most of the microorganisms whereas juglone was active only against Pseudomonas
aeruginosa and Burkholderia cepacia. Enhanced activity of extracts may be due to the
synergistic effect of other compounds present in them. Most of the fungal test organisms
were resistant to the aforementioned plant extracts, except Trichophyton rubrum.
Methanolic extract showed broad spectrum antimicrobial activity against bacteria and
filamentous fungi under study while Bacillus subtilis appears to be most susceptible among
the test organisms. Earlier, leaf extracts of J. regia have been reported to inhibit the growth
258
Isolation, chromatographic evaluation……..
Chapter 4
of Gram positive bacteria but not Gram Negative bacteria and fungi [Preira et al. (2007)].
Moreover, MIC and MMC values of standard antibiotics used (Ampicillin and Nystatin)
were found to be relatively higher than the extracts against Pseudomonas aeruginosa and
Burkholderia cepacia (Table 12). Further fractionation of methanolic and ethyl acetate
extracts did not lead to any improvement in antimicrobial activity.
Table 12: Antimicrobial activity of the J. regia extracts (µg/mL) by broth micro dilution
method
Microorganism
Candida
albicans (3017)
Issatchenkia
orientalis (231)
Aspergillus
flavus (277)
Aspergillus
niger (404)
Aspergillus
parasiticus
(2797)
Aspergillus
sydowii (4335)
Trichophyton
rubrum (296)
Staphylococcus
aureus (3160)
Bacillus
subtilis (121)
Micrococcus
luteus (2470)
Burkholderia
cepacia (438)
Escherichia
coli (43)
Enterobacter
cloacae (509)
Klebseilla
pneumoniae
(109)
Pseudomonas
aeruginosa
(424)
Methanolic
extract
MIC MMC
-
Ethyl acetate
extract
MIC MMC
-
Water extract
Juglone
MIC
-
MMC
-
MIC
-
MMC
-
Ampicillina/
Nystatinb
MIC
MMC
7.8
7.8
-
-
-
-
-
-
-
31.3
31.3
1000
1000
-
-
-
-
-
-
62.5
62.5
500
500
-
-
-
-
-
-
15.6
62.5
-
-
-
-
-
-
-
62.5
125
500
500
-
-
-
-
-
-
3.9
3.9
1000
2000
250
500
2000
2000
-
-
31.3
62.5
500
1000
500
1000
1000
2000
-
-
2.0
3.9
250
500
125
250
-
-
-
-
3.9
7.8
1000
2000
1000
2000
2000
-
-
-
2.0
3.9
500
500
1000
1000
500
500
250
500
2000
2000
-
-
-
-
-
-
-
-
31.3
62.5
-
-
-
-
-
-
-
-
2000
2000
-
-
-
-
-
-
-
-
2000
2000
500
250
500
250
500
125
250
1000
1000
-
250
a
antibiotics for bacteria; b antibiotics for fungi
MTCC No. in parenthesis.
(Published in Analytical Letters 2009, 42: 2592–2609)
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Isolation, chromatographic evaluation……..
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4.3.2.2. Antioxidant potential and color value evaluation of extract/fractions of walnut
(Juglans regia L.) bark
It is well established that highly reactive free radicals, especially oxygenated radicals are
formed as a result of numerous physiological and biochemical processes in the human body.
Overproduction of these free radicals can cause oxidative damage to biomolecules such as
carbohydrates, proteins, lipids, and DNA, ultimately leading to many chronic diseases, such
as atherosclerosis, hypertension, cancer, diabetes, ageing, and other degenerative diseases
[Halliwell and Grootvelt (1987); Wang et al. (2010)].
In order to counteract the excess oxygenated radicals, dietary antioxidants or specific
pharmaceutics are essential because endogenous antioxidants are inadequate for this
purpose. Synthetic antioxidants like butylated hydroxyl anisole (BHA), butylated hydroxyl
toluene (BHT) and tertiary butyl hydroquinone (TBHQ) are commercially available and
currently used for lower cost. However, strict legislation on the use of synthetic antioxidants
owing to their toxic nature and side effects, as well as consumer’s preferences for natural
antioxidants have shifted the attention of manufacturers from synthetic to natural
antioxidants, which are relatively less damaging toward the human health and environment
[Ito et al. (1985); Kaur and Arora (2011)]. Various studies have reported that vegetables,
fruits, leaves, tree barks, roots, oilseeds, cereal crops, spices, and herbs possess wide variety
of antioxidants in the form of polyphenolic compounds [Rababah et al. (2004); Sharma et
al. (2008)]. Though, reports are available on several biological activities of J. regia bark
e.g., anti-inflammatory, blood purifying, anticancer, depurative, antimycobacterial, diuretic
and antimicrobial activities [Alkhawajah (1997); Bhatia et al. (2006); Cruz-Vega et al.
(2008); Sharma et al. (2009)], not much information exists about the antioxidant activity.
To the best of our knowledge, only one study dealing with antioxidant property of a
commercial water extract [Bhatia et al. (2006)] has been reported, and comparative study is
missing.
Keeping this in view, in vitro antioxidant potential of walnut bark methanolic extract
(MeOH) and its fractions i.e. hexane (HEX), chloroform (CHL), ethyl acetate (EA) and nbutanol (BU) was investigated using three different methods: 2,2′-diphenyl-1-picrylhydrazyl
(DPPH), 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)
radical-scavenging and ferric reducing/antioxidant power assays. The antioxidant properties
of synthetic antioxidant BHT, gallic acid, quercetin and marker compound juglone were
also determined in similar way for comparison. In addition, total phenolic content was of
260
Isolation, chromatographic evaluation……..
Chapter 4
the extract/fractions was determined using Folin-Ciocalteu reagent. Further, in context of
color applications of J. regia, color properties of above extract/fractions were determined to
enhance their importance.
4.3.2.2.1. Yield and total phenol content
The yield and total phenolic content of MeOH and its fractions are presented in Table 13.
From the results, it is evident that methanol is able to extract out a significant amount of
compounds from the walnut bark. The yield of fractions obtained after the partitioning of
methanol extract follows the order: EA> BU> CHL> HEX; suggesting that ethyl acetate is
more effective solvent for fractionation. Some phenolic compounds also remained in the
water insoluble part (INS). The total phenol content determined using Folin-Ciocalteau
reagent was highest in the EA (470.3 mg/g dry extract) followed by BU, MeOH, INS, CHL
and HEX.
Table 13: Yield and total phenol content of the J. regia bark extract/fractions
___________________________________________________________________
Extract/fraction
Yield (%)a
Total Phenol contentb
___________________________________________________________________
MeOH
11.36
343.5 ± 5.5 r
HEX
0.11
32.3 ± 2.3 u
CHL
0.21
100.2 ± 3.5 t
EA
3.18
470.3 ± 8.5 p
BU
1.87
360.5 ± 4.8 q
INS
1.49
193.4 ± 2.8 s
____________________________________________________________________
a
% weight of dry plant material.
Total phenol content (mg GAE g-1 dry extract) expressed as mean ± standard deviation (n=3).
p-u
values with different letters within a column are significantly different (P ≤ 0.05).
b
4.3.2.2.2. DPPH radical scavenging activity
At a dose of 0.5 mg/mL, all the extract/fractions were capable of scavenging DPPH · with
EA being the more potent scavenger followed by BU, MeOH, INS, CHL and HEX (Table
14). Also, it was observed that both EA and BU were better scavengers of DPPH · than the
positive control quercetin (P ≤ 0.05); however, none of the extracts were as effective as
BHT or gallic acid. The free radical scavenging activity of marker compound juglone was
found comparatively lower than MeOH, EA and BU.
4.3.2.2.3. ABTS radical scavenging activity
ABTS·+ is commonly used where problems of solubility or interference come up and use of
DPPH· assay becomes unsuitable [Arnao (2000)]. The results of ABTS ·+ scavenging are
presented in Table 14. The J. regia extract/fractions demonstrated a wide range of ABTS ·+
261
Isolation, chromatographic evaluation……..
Chapter 4
scavenging activities and could be ranked according to their calculated TEAC values (Table
14). Again, the EA fraction was found more efficient ABTS ·+ scavenger followed by
MeOH, BU, INS, CHL and HEX. However, none was found as effective as the positive
controls used i.e., gallic acid, BHT and quercetin.
Table 14: The effect of the positive controls and walnut bark extract/fractions upon DPPH
and ABTS radical scavenging as well as FRAP activities a
________________________________________________________________________
Sample
DPPH activity
ABTS activity
FRAP activity
(% Inhibition)
(mmol trolox/mg)
(mM AA/g)
________________________________________________________________________
Gallic acid
Quercetin
BHT
Juglone
MeOH
HEX
CHL
EA
BU
INS
94.7 ± 1.13 b, c
78.8 ± 0.57 f
95.3 ±1.08 b
64.1 ± 0.83 h
77.6 ± 1.58 g
5.4 ± 1.03 j
23.5 ± 1.05 i
90.4 ± 0.28 d
83.0 ± 1.01 e
42.6 ± 1.50 I
15.70 ± 1.05 b
7.23 ± 0.25 c
5.47 ± 0.15 d
2.86 ± 0.14 e
1.35 ± 0.03 g
0.09 ± 0.01 i
0.25 ± 0.04 h,i
1.95 ± 0.20 f
0.78 ± 0.04 g,h
0.61 ± 0.02 h,I
8.5 ± 0.10 b
4.5 ± 0.20 c, d
4.7 ± 0.30 c
0.4 ± 0.03 h
3.5 ± 0.03 f
0.4 ± 0.20 h
0.6 ± 0.05 h
4.3 ± 0.20 d
3.8 ± 0.20 e
1.3 ± 0.20 g
________________________________________________________________________
a
Values are mean ± standard deviation of three different assays.
Values with the same lower case letters within a column are not significantly (P ≤ 0.05) different.
AA: Ascorbic acid
b-j
Also, the effect of concentration of the extract/fractions on % inhibition of ABTS ·+ showed
a linear correlation at lower concentration (Figure 22). These results substantiate the
potential of ethyl acetate fraction as a good scavenger of free radicals.
Figure 22: Effect of concentration on ABTS radical scavenging (% Inhibition)
262
Isolation, chromatographic evaluation……..
Chapter 4
4.3.2.2.4. FRAP assay
Reducing power assay is often used to evaluate the ability of natural antioxidant to donate
electron [Dorman et al. (2003)] as a direct correlation between antioxidant activity and
reducing power of certain plant extracts has been reported [Yildirim et al. (2001)]. The
reducing power of all the samples was found to increase with the concentration used (Figure
23). At the same concentration, the reducing power of EA (4.3 mM AA/g) was more than
MeOH (3.45 mM AA/g) and other fractions and was comparable to positive controls
quercetin (4.5 mM AA/g) and BHT (4.7 mM AA/g) (Table 14). However, it was
significantly (P ≤ 0.05) lower than gallic acid (8.5 mM AA/g). The reducing power of
marker compound juglone (0.45 mM AA/g) was significantly lower than that exhibited by
the extracts and fractions except HEX and CHL (statistically same, P ≤ 0.05). From the
results, it is evident that these phenolic rich extract/fractions, particularly EA, possess good
reducing activities, and hence can protect oxidative damage by donating electron.
1.6
1.4
1.2
MeOH
OD
1
Hex
CHL
0.8
EA
BU
0.6
BHT
0.4
0.2
0
0
0.05
0.1
0.15
0.2
0.25
Concentration (mg/ml)
Figure 23: Dose dependant FRAP activity of extract/fractions of J. regia
It was also observed that the order of DPPH radical scavenging and ferric reducing
properties of the extract and fractions were similar to the order of TPC. It indicated that
there is significant correlation between antioxidant properties and TPC. The correlation
coefficients (r2) of total phenolic content, and DPPH, ABTS radical scavenging, and FRAP
activities were 0.975, 0.798 and 0.960, respectively.
4.3.2.2.5. Color characteristics
All the samples showed the desired light orange/yellow color as expected from photometry
of the solution of extract/fractions of J. regia bark. The MeOH showed more darkish color
(lower L value) compared to its fractions i.e. HEX, CHL, EA and BU except INS (Table
15). The higher color intensity of the INS may be due to presence of juglone which
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Isolation, chromatographic evaluation……..
Chapter 4
remained trapped in it at the time of fractionation due to its poor solubility in water. On the
other hand, the EA has higher chroma values than HEX, CHL, and BU fractions but lower
chroma values than MeOH and INS. In addition, all the extract and fractions exhibited
higher values of hue angle. The results suggest the possibility of the walnut bark for use as
natural coloring agents in pharmaceuticals.
Table 15: Color characteristics of walnut extract/fractions
Samples
MeOH
HEX
CHL
EA
BU
INS
4.4.
L
86.24
95.76
91.85
93.63
92.22
77.03
a
4.27
-0.83
-0.50
-0.81
0.99
9.27
b
C
h0
35.50
10.5
10.09
23.33
21.27
51.52
35.76
10.55
10.10
23.35
21.73
52.35
83.09
94.48
92.87
92.01
87.33
79.76
Conclusion
In the context of changing trends towards accepting safer and ecofriendly natural colorants,
three plants of Western Himalayan region were explored for the isolation of phenolic rich
colored compounds/fractions from them. In this direction, a green technology was
developed for the isolation of crystalline and non-hygroscopic natural colors from flowers
of R. arboreum which was extended to various other plants including fruits and vegetables.
Furthermore, a simple and rapid prep-HPLC method with single compound recovery has
been developed for the isolation of shikonin derivatives for commercial purpose from
Arnebia euchroma. Similarly, the feasibility of microwave-assisted extraction (MAE) as an
alternate and effective approach for the extraction of juglone from J. regia was investigated
along with the evaluation of antimicrobial activity, antioxidant activity and color properties
of various extracts/fractions of J. regia bark. In addition, simple and rapid analytical
procedures (RP-HPLC, RP-HPTLC) have been developed for quantification of some
phenolics present in these three plants.
264
Isolation, chromatographic evaluation……..
4.5.
Chapter 4
Experimental
4.5.1. Isolation and investigation of phenolic rich colored compounds/fractions from
Rhododendron species
4.5.1.1. Isolation of phenolic rich colored fraction from R. arboreum: Application for
textile dyeing and identification of some phenolic constituents by HPLC
4.5.1.1.1. Plant material and chemicals
Flowers of R. arboreum were collected from hilly regions of the Western Himalayas and
dried under a gentle stream of air in the laboratory (temp. 25±2°C) and powdered in an
electric grinder. Standards used for HPLC analysis i.e. chlorogenic acid, p-coumaric acid,
quercitrin, quercetin and kaempferol (Figure 6) was purchased from Sigma (India). HPLC
grade solvents (acetonitrile and methanol) were purchased from Merck (India). Milli-Q
water was obtained using ultra pure water purification system (Bio-Age, Punjab, India). All
samples and solvents were filtered through 0.45 µM membrane filters (Millipore, Germany)
and solvents were degassed prior to use.
4.5.1.1.2. Optimization of extraction conditions
Example 1: Isolation of colored crystalline fraction from Rhododendron arboreum with
Soxhlet extraction
The dried and powdered flowers of Rhododendron (100 g) were taken in Soxhlet apparatus
and extracted with water (500 mL; sample to solvent ratio of 1:5) for 8 hours at 60 oC. The
extract was further diluted with water (200 mL; extract to water ratio of 1:20) and filtered.
The filtered extract was then passed over XAD-7 resin column. The column was first
washed with water to remove the highly water soluble components which was spray dried
or concentrated separately. Then the column was eluted with ethanol. The organic layer was
concentrated in vacuo at 45oC to obtain crystalline, non-hygroscopic and phenolic rich
bioactive reddish brown colored fraction with yield of 9.72% (Figure 3).
Example 2: Isolation of colored crystalline fraction from Rhododendron arboreum with
percolation
The dried and powdered flowers of Rhododendron (100 g) were taken in percolator and
extracted with water (500 mL; sample to solvent ratio of 1:5) for 48 hours at room
temperature. Remaining procedure is similar to as described in example 1. Thus, a
crystalline, non-hygroscopic and phenolic rich bioactive reddish brown colored fraction was
obtained with yield of 8.56%.
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Isolation, chromatographic evaluation……..
Chapter 4
Example 3: Isolation of colored crystalline fraction from Rhododendron arboreum
employing ultrasound and microwave
For the extraction of Rhododendron flowers environmentally-benign techniques like
microwave assisted extraction (MAE), ultrasound assisted extraction (UAE) were also
employed. Kenstar domestic microwave (750 W, frequency 2450 MHz) and Vibracell
ultrasonicator (500-750 W) with frequency 20 KHz and system were used for extraction
purpose, respectively. The dried and powdered flowers of Rhododendron (100 g) were
taken in flask and extracted with water (500 mL; sample to solvent ratio of 1:5) employing
ultrasonic assisted extraction (for 60 min at room temperature at 100% ultrasonic power)
and microwave assisted extraction (for 5 min in small cycles of 30 sec, multimode
microwave with operating frequency 2450 MHz and operating wattage of 700 W).
Remaining procedure is similar to as described in example 1. Thus, crystalline, non
hygroscopic and phenolic rich bioactive reddish brown colored fractions was obtained with
a yield of 9.40 % in case of UAE and 9.89% in case of MAE.
Example 4: Isolation of colored crystalline fraction from Rhododendron arboreum
employing other resins
Extraction was performed under similar conditions as described in example 1 except for the
resin which was XAD-16 and XAD-4 instead of XAD-7 resin. In this case, reddish brown
color fractions were obtained with the yield of 0.82% in case of XAD-4 and 1.16% in case
XAD-16.
Example 5: Isolation of colored crystalline fraction from Rhododendron arboreum
employing used resins
Extraction was performed under similar conditions as described in example 1 except for the
resin which was previously used XAD-7 resin column (resin was regenerated by washing
with alcohol and water respectively) instead of fresh XAD-7 resin. In this case, reddish
brown color fraction was obtained with the yield of 8.46%.
4.5.1.1.3. HPLC analysis of colored fraction isolated from R. arboreum (Figures 6, 7)
HPLC analysis of the isolated colored fractions was performed on Shimadzu LC-20
instrument equipped with PDA detector (SPDM-20A) using reverse phase Phenomenex
Luna RP-18 column (4.6 mm i.d.  250 mm, 5 µm). A gradient elution comprising of
acidified water (0.1% acetic acid) and acetonitrile was run at a flow rate of 1 mL/min with
the following program, 10 to 40% acetonitrile in 0 to 10 min, 40 to 50% acetonitrile in 10 to
15 min, 50 to 70% acetonitrile 15 to 20 min and 70 to 100% acetonitrile in 20 to 25 min.
266
Isolation, chromatographic evaluation……..
Chapter 4
PDA detection was done at 290 nm. Following molecules were identified by matching their
retention time and UV spectra with reference standards: chlorogenic acid (Rt 9.86), pcoumaric acid (Rt 13.33), quercetin (Rt 17.28), quercitrin (Rt 13.63) and kaempferol (Rt
19.64).
4.5.1.2. Simultaneous determination of epicatechin, syringic acid, quercetin-3-Ogalactoside and quercitrin in the leaves of Rhododendron species by using a validated
HPTLC method
4.5.1.2.1. Plant material and chemicals
The plant material was collected from western Himalayas, India at an altitude ranging
between 1500 to 4000 meters above the mean sea level and processed as mentioned in
section 4.5.1.1.1. Standards epicatechin (1), syringic acid (2), quercetin-3-O-galactoside (3)
and quercitrin (4) (Figure 8) were purchased from Sigma (USA) and have purity more than
97%. The TLC plates RP-18 F254S (20 cm×10 cm) (E. Merck, Darmastadt, Germany) were
used without any pretreatment. Methanol and formic acid were of HPLC grade (E. Merck).
Deionised water was from J.T. Baker (USA).
4.5.1.2.2. Standard stock solution and sample preparation
Standard stock solutions containing 1 mg/10 mL of pure compounds 1–4 were prepared by
dissolving 5 mg of accurately weighed compound in 50 mL of methanol and filtered
through 0.45 µM (Millipore) filter. Samples were prepared from air-dried powdered leaves
(1 g each) of R. arboreum, R. campanulatum, R. anthopogon. The powder was defatted with
petroleum ether prior to extraction with methanol (3×50 mL) under ultrasonication at 40 ±
5oC for 30 min. The organic extracts were combined, filtered and concentrated in vacuo to
obtain a crude extract. Known amount of extracts were taken and dissolved in HPLC grade
methanol (final conc. 10 mg/mL) and filtered through 0.45 µm filter for HPTLC analysis.
4.5.1.2.3. HPTLC procedure
4.5.1.2.3.1.
Instrumentation and operating conditions
A Camag HPTLC system equipped with an automatic TLC sampler (ATS 4), TLC scanner
3 integrated with software (WinCATS version 1.4.2), UV cabinet and automatic developing
chamber ADC2 with humidity control facility was used for the analysis. The samples were
applied using automated TLC sampler in 10 mm bands at 10 mm from the bottom, 15 mm
from the sides and with 8 mm space between the two bands. Plates were developed in
software controlled Camag automatic developing chamber ADC2 pre-saturated with the 10
mL of developing solvent phase for 30 min at room temperature (25 ± 2 oC) and relative
267
Isolation, chromatographic evaluation……..
Chapter 4
humidity was maintained 45 ± 1%. The plates were developed to a height of about 8 cm
from the base in methanol-5% formic acid in water, 50:50 (v/v). After development, the
plate was removed, dried and spots were visualized under UV (254 and 366 nm) light.
Quantitative evaluation of the plate was performed in the reflectance/absorbance mode at
290 nm with following conditions: slit width 6 mm × 0.3 mm, scanning speed 20 mm/s and
data resolution 100 μm/step. To check the identity of the bands, UV absorption spectrum of
each standard was overlaid with the corresponding band in the sample track.
4.5.1.2.3.2.
Calibration and quantification
For the preparation of calibration curve, appropriate dilutions were made to get the desired
concentrations in the quantification range. The obtained working standard solutions were
then applied on the RP-TLC plate for preparing six point linear calibration curves.
Compound 2 was applied as 4, 8, 12, 16, 20, 24 µL while 1, 3 and 4 were spotted as 2, 4, 6,
8, 10, 12 µL. Sample solution (10 µL) was applied on RP-TLC plate in triplicate with
similar band pattern. The experimental parameters were identical for all the above analysis.
4.5.1.2.3.3.
Method validation (Tables 2-4)
4.5.1.2.3.3.1. Specificity
The specificity of the method was determined by analyzing the bands of different standards
and samples. The bands for compound 1-4 in sample solution were confirmed by comparing
the Rf and UV spectra with the reference standards. The peak purity of these compounds
was assessed by comparing the spectra at three different levels i.e. peak start, peak apex and
peak end positions.
4.5.1.2.3.3.2. Sensitivity
The sensitivity of the method was determined with respect to LOD and LOQ. The stock
solutions of the standards were serially diluted and applied on RP-TLC plates to plot the
calibration curves. LOD was determined based on the lowest concentration detected by the
instrument from the standard while the LOQ was determined based on the lowest
concentration quantified in the sample.
4.5.1.2.3.3.3.
Precision
Instrumental precision was checked by repeated scanning of the same spot of epicatechin,
syringic acid, quercetin-3-O-galactoside and quercitrin (400 ng) six times each.
The
repeatability of the sample application and measurements of peak area was expressed in
terms of percent relative standard deviation (% RSD). To study the intra-day precision
different concentration levels of 200, 400 and 600 ng/spot of reference compounds (1-4)
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Chapter 4
were spotted five times within 24 hours and expressed in terms of percent relative standard
deviation (% RSD). For intermediate precision six determinations was repeated at
concentration levels of 200, 400 and 600 ng/spot of reference compounds over a period of 5
days and expressed as % RSD.
4.5.1.2.3.3.4. Accuracy
The accuracy of the method was determined by analysing the percentage recovery of the
compounds in samples. For it, three sets were prepared from one of the species i.e. R.
arboreum. The samples were spiked with three different concentrations: 1, 3 and 4 (50, 100
and 200 ng) and 2 (100, 200 and 400 ng) before extraction. The spiked samples were
extracted in triplicate and then analyzed by proposed HPTLC method.
4.5.1.2.3.3.5. Robustness
Robustness is a measure of the method to remain unaffected by small but deliberate
variations in the method conditions, and is an indication of the reliability of the method. For
the robustness, different parameters such as mobile phase composition, developing TLC
distance and different TLC plate lots were checked.
4.5.2. Isolation and chromatographic evaluation of colored compounds from Arnebia
species
4.5.2.1. Development of a preparative HPLC method for the rapid isolation of shikonin
derivatives from root extract of A. euchroma
4.5.2.1.1.
Plant materials and chemicals
The roots of A. euchroma were procured from Kibber (Himalayas, 4200 m above mean sea
level) dried under a gentle stream of air in the laboratory (temp. 25±2°C) and powdered in
an electric grinder. HPLC grade solvents (acetonitrile and methanol) were purchased from
Merck (India). Milli-Q water was obtained using ultra pure water purification system (BioAge, Punjab, India). All samples and solvents were filtered through 0.45 µM membrane
filters (Millipore, Germany) and solvents were degassed prior to use.
4.5.2.1.2.
Preparation of sample solutions
The powdered roots of Arnebia euchroma (500 g) were macerated with hexane (1 L). The
organic layer was collected, filtered and dried in vacuo at temp 40oC using rotavapor.
Concentrated extract was dissolved in acetonitrile, filtered through 0.45 μM (Millipore)
filter and 1 mL of the extract (conc. 5 mg/mL) was injected in Prep-HPLC for the isolation
and purification of shikonin derivatives.
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4.5.2.1.3.
Chapter 4
Equipment and operating conditions
4.5.2.1.3.1.
Prep- HPLC (Figure 13)
Prep-HPLC (Waters Prep-LC system, Waters, USA) equipped with
reverse phase
Purospher®-Star RP-18e column (250 mm  10 mm I.D., 5 µM, Merck, Darmstadt,
Germany), a Waters 2487 Dual λ absorbance detector, an Waters temperature control
module (Waters), 10 mL loop manual injector and Waters empower software was used for
isolation of shikonin derivatives. Acetonitrile-water (80:20, v/v) was used as mobile phase
with a flow rate of 3 mL/min in an isocratic elution for 10 min. Column was equilibrated
under the starting conditions for 5 min. The detection wavelength was set at 520 nm. The
column temperature was 25oC, and the injection volume of samples was 1 mL. Pure
fractions were collected, dried in vacuo and finally lyophilized to obtain red colored
compounds.
4.5.2.1.3.2. Analytical HPLC (Figure 14)
HPLC analysis of the isolated shikonin derivatives compounds was performed on Shimadzu
LC-20 instrument equipped with PDA detector (SPD M20A) using reverse phase
Phenomenex Luna RP-18 column (4.6 mm i.d.  250 mm, 5 µM). An isocratic elution
comprising of water and acetonitrile (20:80, v/v) was run at a flow rate of 1 mL/min. PDA
detection was done at 520 nm. Column was equilibrated under the starting conditions for 5
min. The column temperature was 25oC, and the injection volume of samples was 20 µL.
4.5.2.1.3.3. NMR data (Figure 15)
1
H and
13
C spectra of isolated compounds were measured in CDCl3 at 300 MHz, using a
Bruker Avance-300 spectrophotometer (Switzerland).
1
H & 13C NMR data for peak at Rt = 4.6 (Figure 15, Shikonin)
OH
O
OH
O
OH
Red color viscous compound, 1H NMR (CDCl3, 300 MHz): δ
12.61 (1H, s); 12.5 (2H, s), 7.22 (2H, d, J = 2.4 Hz), 7.19 (1H, s), 5.23 (1H, d, J = 7.2 Hz),
4.91 (1H, dd, J = 4.1 and 7.7 Hz), 2.34-2.62 (2H, m), 1.75 (3H, s), 1.68 (3H, s);
13
C NMR
(CDCl3, 75.4 MHz): δ 180.4, 179.6, 165.3, 164.6, 151.1, 137.1, 132.3, 132.1, 131.4, 118.2,
112.0, 111.5, 68.2, 35.8, 25.7, 18.4. HRMS-ESI: m/z [M+H]+ for C16H16O5, calculated
289.1296; observed 289.1284. The spectral data matched well with the reported values [Cho
et al. (1999)].
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1
Chapter 4
H & 13C NMR data for peak at Rt = 5.7 (Figure 15, acetylshikonin)
OH
O
OH
O
O
Red color viscous compound, 1H NMR (CDCl3, 300 MHz): δ 12.5
O
(1H, s), 12.3 (1H, s), 7.12 (2H, s), 6.92 (1H, s), 5.97 (1H, t, J = 6.1 Hz), 5.07 (1H, t, J = 6.8
Hz), 2.55 (2H, m), 2.07 (3H, s), 1.62 (3H, s), 1.51 (3H, s); 13C NMR (CDCl3, 75.4 MHz): δ
178.2, 176.7, 169.8, 167.5, 167.0, 148.2, 136.1, 132.9, 132.7, 131.4, 116.6, 111.8, 111.6,
69.5, 32.8, 25.7, 21.0 and 17.9. HRMS-ESI: m/z [M+H]+ for C18O6H18, calculated
331.3463; observed 331.3447. The spectral data matched well with the reported values [Cho
et al. (1999)].
1
H & 13C NMR data for peak at Rt = 7.8 (Figure 15, β-acetoxyisovalerylshikonin)
OH
O
OH
O
O
O
O
O
Red color viscous compound, 1H NMR (CDCl3, 300 MHz): δ
12.52 (1H, s), 12.34 (1H, s), 7.11 (2H, s), 6.94 (1H, s), 5.99 (1H, t, J = 5.25 Hz), 5.08 (1H,
t, J = 7.67 Hz), 2.98 (2H, m), 2.55 (2H, m), 1.93 (3H, s), 1.61 (3H, s), 1.50 (3H, s), 1.49
(3H, s), 1.46 (3H, s);
13
C NMR (CDCl3, 75.4 MHz): δ 177.5, 176.0, 170.4, 169.0, 168.1,
167.6, 147.9, 136.1, 133.1, 132.9, 131.3, 117.7, 111.8, 111.5, 79.3, 69.7, 44.2, 32.9, 26.6,
26.5, 25.7, 22.3 and 17.9. HRMS-ESI: m/z [M+H]+ for C23H26O8, calculated 431.4640,
observed 431.4676.
4.5.2.2. Simultaneous densitometric determination of shikonin, acetylshikonin, and βacetoxyisovalerylshikonin in ultrasonic-assisted extracts of four Arnebia species using
reversed-phase thin layer chromatography
4.5.2.2.1. Plant materials and chemicals
The plant material was collected from various locations of western Himalaya, India. The
plants were identified and voucher specimens of each species studied i.e. A. benthamii
(PLP-8021), A. euchroma (PLP-11159), A. guttata (PLP-11137), and A. hispidissima (PLP10036), were deposited in the herbarium of the institute (IHBT) for the reference. The plant
material was processed as described in section 4.5.1.3.1.1. The TLC plates RP-18 F254S (20
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Isolation, chromatographic evaluation……..
Chapter 4
cm x 10 cm) (E. Merck, Darmstadt, Germany) were used without any pre-treatment. All the
chemicals and solvents used were of analytical grade (E. Merck, India).
4.5.2.2.2.
Isolation and characterization of standards
Standards shikonin (1), acetylshikonin (2) and β-acetoxyisovalerylshikonin (3) were
isolated using Prep-HPLC as described in section 4.5.2.1.3.1. The characterization of
compounds was established by 1H- and 13C-NMR spectral analysis.
4.5.2.2.3.
Standard stock solution and sample preparation
Standard stock solutions containing 1 mg/mL of pure compounds 1–3 were prepared by
dissolving 5 mg of accurately weighed compound in 5 mL of methanol and filtered through
0.45 µM (Millipore) filter. The stock solution is then further diluted to appropriate
concentration and applied on TLC plates. Samples were prepared from dried and powdered
roots of Arnebia species collected from different locations. About 2 g of powdered plant
material was sonicated with 50 mL of each extracting solvent (hexane, chloroform, ethyl
acetate and methanol) in an ultrasonicator bath (Elma ultrasonic, Germany). The finally
selected conditions were 80% (ultrasonic power) for 30 min. The temperature of the water
bath was maintained at 40 ± 2oC by changing the water at regular intervals. The extracts
were filtered and concentrated to dryness under vacuum and then subjected to lyophilization
till constant weight was obtained. A known amount of extracts were taken and dissolved in
methanol to obtain final conc. of 1 mg/mL for A. euchroma and A. guttata, 10 mg/mL for A.
benthamii and 20 mg/mL for A. hispidissima and filtered through 0.45 µM filter for HPTLC
analysis.
4.5.2.2.4.
HPTLC procedure
4.5.2.2.4.1.
Instrumentation and operating conditions
A Camag HPTLC system equipped with an automatic TLC sampler (ATS 4), TLC scanner
3 integrated with software (WinCATS version 1.4.2), UV cabinet and automatic developing
chamber ADC2 with humidity control facility was used for the analysis. The samples were
applied using automated TLC sampler in 6 mm bands at 10 mm from the bottom, 15 mm
from both sides and with 6 mm space between the two bands. Plates were developed in
software controlled Camag automatic developing chamber ADC2 pre-saturated with the 10
mL of developing solvent phase for 30 min at room temperature (25 ± 2oC) and relative
humidity was maintained 45 ± 1%. The plates were developed to a height of about 8 cm
from the base in acetonitrile: methanol: 5% formic acid in water (40:02:08, v/v/v). After
development, the plate was removed, dried again and spots were visualized under UV (254
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Isolation, chromatographic evaluation……..
Chapter 4
and 366 nm) and visible light. Quantitative evaluation of the plate was performed in the
reflectance/absorbance mode at 520 nm with following conditions: slit width 6 mm × 0.3
mm, scanning speed 20 mm/s and data resolution 100 μm/step.
4.5.2.2.4.2.
Method validation (Tables 5-7)
4.5.2.2.4.2.1.
Specificity
The specificity of the method was determined by analyzing the bands of different standards
and samples. The bands for compound 1-3 in sample solution were confirmed by comparing
the Rf and UV spectra with the reference standards. The peak purity of these compounds
was assessed by comparing the spectra at three different levels, i.e., peak start, peak apex
and peak end positions.
4.5.2.2.4.2.2.
Calibration and quantification
For the preparation of calibration curves, the diluted standard solutions of compounds 1–3
were taken and applied on the RP-TLC plate for preparing six points linear calibration
curves after fitting the corresponding peak areas to the amounts of analytes in nanograms
using least-square regression. Compounds 1, 2 were applied at 2.0, 4.0, 6.0, 8.0, 10.0, 12.0
µL while compound 3 was applied at 2.0, 4.0, 6.0, 12.0, 24.0, 36.0 µL. Sample solution (6
µL) from different species was taken and each one of them was applied on the TLC plate in
triplicate with similar band pattern. The experimental parameters were identical for all
above analysis.
4.5.2.2.4.2.3.
Precision
Precision was performed at two different levels- repeatability and intermediate precision.
The repeatability (intra-assay precision) of the sample application and measurements of
peak area was determined by six repeated assays of the different concentration levels of
100, 200 and 300 ng/spot of reference compounds and was expressed in terms of percent
relative standard deviation (% RSD). For intermediate precision six determinations was
repeated at concentration levels of 100, 200 and 300 ng/spot of reference compounds over a
period of 5 days and expressed as % RSD.
4.5.2.2.4.2.4.
Sensitivity
The sensitivity of the method was determined with respect to LOD, LOQ. The standards
from section 4.5.2.2.2 were serially diluted and applied on RP-TLC plates to plot the
calibration curves. LOD was determined based on the lowest concentration detected by the
instrument from the standard while the LOQ was determined based on the lowest
concentration quantified in the sample.
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Isolation, chromatographic evaluation……..
4.5.2.2.4.2.5.
Chapter 4
Accuracy
The accuracy of the method was determined by analyzing the percentage recovery of the
compounds in samples. For it, ethyl acetate extract of A. euchroma was spiked with three
different concentrations for each compound: 1 (50, 150 and 200 ng), 2 (50, 100 and 200 ng)
and 3 (100, 200 and 300 ng) before extraction. The spiked samples were extracted in
triplicate and then analyzed by proposed HPTLC method.
4.5.2.2.4.2.6.
Robustness
Robustness is a measure of the method to remain unaffected by small but deliberate
variations in the method conditions, and is an indication of the reliability of the method. For
the robustness, different parameters such as mobile phase composition, developing TLC
distance and different TLC plate lots were checked.
4.5.3.
Investigations of colored compounds/fractions from Juglans regia
4.5.3.1.
Microwave-assisted efficient extraction and stability of juglone in different
solvents from Juglans regia: Quantification of six phenolic constituents by validated
RP-HPLC and evaluation of antimicrobial activity
4.5.3.1.1.
Plant material and chemicals
J. regia bark and leaves were obtained from Kashmir and Palampur region of India and
plant material was confirmed at our biodiversity division. The bark was air-dried at room
temp (25 ± 5oC) and relative humidity of 50 ± 5%, powdered, and stored in air-tight plastic
bags. HPLC-grade acetonitrile, trifluroacetic acid (TFA) and methanol were purchased from
E. Merck (Merck, Darmstadt, Germany). HPLC-grade water was purchased from J.T. Baker
(USA). Gallic acid (1), caffeic acid (2), quercitrin (3), myricetin (4) and quercetin (5)
standards were purchased from Sigma (USA). Juglone (6) was from Acros Organics (USA).
All of the samples and solvents were filtered through a 0.45 mm membrane filter (Millipore,
Germany) and degassed prior to use.
4.5.3.1.2.
Extraction of plant material
Different solvent systems (chloroform, ethyl acetate, methanol, water) were used to
determine the effectiveness of solvent type on the extraction of juglone and other phenolic
compounds from the bark of J. regia.
4.5.3.1.2.1.
Microwave-Assisted Extraction (MAE)
About 2 g of powdered plant material was extracted with 20 mL of chloroform, ethyl
acetate, methanol, and water in a focused microwave (CEM Discover) for 10–40 min. On
274
Isolation, chromatographic evaluation……..
Chapter 4
mass yield basis, an extraction time of 20 min at 150W microwave power and 50 oC
temperature was taken as optimum. The extracts were filtered and concentrated to dryness
under vacuum (temperature, 45 ± 5oC) and then subjected to lyophilization until a constant
weight was obtained.
4.5.3.1.2.2.
Ultrasound-Assisted Extraction (UAE)
About 2 g each of powdered plant material was sonicated with 20 mL of chloroform, ethyl
acetate, methanol, and water in an ultrasonicator bath (Elma Ultrasonic, Germany) at a
controlled temperature 40 ± 5oC for 30–60 min. An extraction time of 40 min was taken as
optimum on mass yield basis. The extracts were filtered and concentrated to dryness under
vacuum (temperature 40 ± 5oC) and then subjected to lyophilization until a constant weight
was obtained.
4.5.3.1.2.3.
Maceration
About 2 g each of powdered plant material was macerated overnight in 20 mL of
chloroform, ethyl acetate, methanol, and water at room temperature. The extract obtained
was filtered and concentrated fully under vacuum (temperature 40 ± 5 oC) and lyophilized
until a constant weight was obtained.
All of the extractions were performed in triplicate. All samples were kept in a nitrogen
atmosphere and 4oC until further use. For the quantitative determination of compounds by
HPLC, concentrated extracts were dissolved in methanol (analytical grade) to obtain a
sample solution of 2mg/mL. The extracts were filtered through a 0.45 µm membrane filters
prior to use.
4.5.3.1.3.
Instrumentation and chromatographic conditions
HPLC was performed on Waters Model 600 pump system controlled by a Waters 600
automated gradient controller, a Waters 717 plus auto injector, and a Waters 2996
photodiode array detector (Waters Associates, Milford, MA, USA). Chromatographic
separations were performed on a LiChrospher RP-18 column (250 mm x 4.6 mm, 5µm)
(Merck, Darmstadt, Germany). The mobile phase was a mixture of A: 0.05% trifluoroacetic
acid in water and B: acetonitrile–methanol (70:30, v/v), with a gradient programmed as
follows: A: 80–0% in 0–10 min and back to 80% in 20 min with a flow-rate of 1mL/min.
The injected volume was 20 mL in each assay. The spectra of compounds were recorded
from 200 to 500 nm, and the detection wavelengths were 254 nm for gallic acid, myricetin,
quercitrin, quercetin, and juglone while caffeic acid was detected at 320 nm.
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Isolation, chromatographic evaluation……..
4.5.3.1.4.
Chapter 4
HPLC validation studies (Tables 8-10)
4.5.1.4.1.4.1. Identification of constituents and peak purity
Peaks were identified on the basis of retention times and by comparison with those of the
reference standard compounds. A peak purity test was performed using a photo diode array
detector coupled to the HPLC system and comparing the UV spectrum of each peak with
those of reference standards at the start, middle, and end of the peak.
4.5.3.1.4.2.
Calibration Curve
The stock solution (1 mg/mL) of each standard compound was freshly prepared in methanol
and desired concentrations were obtained by serial dilution for standard curve preparation.
The calibration graphs were plotted after linear regression of the peak area vs. concentration
and both detection limit (LOD) and quantification limit (LOQ) were measured following the
standard methods.
4.5.3.1.4.3.
Repeatability
The precision of the chromatographic determination for the proposed method, expressed as
a relative standard deviation (RSD %), was calculated by six replicate injections (n=6) of
each compound (intraday and inter-day). The standard solutions used for repeatability
experiments were the same as used in the calibration curve experiment.
4.5.3.1.4.4.
Recovery
For percent recovery, three sets of ethyl acetate extract of barks of J. regia were prepared
(conc. 2 mg/mL). Three different concentrations of standard compounds 1 (60, 30, and 15
mg/mL); 2 (60, 30, and 15 mg/mL); 3 (64, 32, and 16 mg/mL); 4 (28, 14, and 7 mg/mL); 5
(16, 8, and 4 mg/mL) and 6 (52, 26, and 13 mg/mL) were prepared. The three sets of
extracts were then individually spiked with 1 mL of each standard compounds (1–6) from
all the three spiking concentrations. The samples, to which standards were added, were
pretreated and analyzed using the developed HPLC method for measuring the percentage
recovery.
4.5.3.1.5.
Stability Study (Table 11)
The degradation rate (decrease of juglone concentration over time) was determined in
methanol and ethyl acetate. Juglone solutions were prepared in HPLC grade methanol and
ethyl acetate, followed by storage in the dark at 4oC in amber glass bottles. Initial
concentrations were 2 mg/mL. Actual concentrations were determined by the developed
reversed phase HPLC in triplicate. Samples were drawn at 24 h intervals respectively from
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Isolation, chromatographic evaluation……..
Chapter 4
each of methanol and ethyl acetate solution. Aliquots of 20 mL were injected and monitored
at 254 nm.
4.5.3.2.
Antioxidant potential and color value evaluation of extract/fractions of
walnut (Juglans regia L) bark
4.5.3.2.1. Materials and methods
Stem bark of J. regia was collected from Jammu and Kashmir, India and processed as
discussed in section 4.5.1.4.1.1. 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis(3ethylbenzothiazoline-6-sulphonate) diammonium salt (ABTS), Folin-Ciocalteu reagent,
butylated hydroxy toluene (BHT), and all standard compounds used in the work were from
Sigma (India). All chemicals and solvents were of analytical grade.
4.5.3.2.2. Preparation of extract/fractions
Walnut bark powder (50 g) was extracted with methanol (3 x 500 mL) for 6 h at room
temperature. The resulting extract was dried in vacuo to yield methanol extracted material
(MeOH). To this extract (5 g), distilled water (100 mL x 3) was added, ultrasonicated for 40
min at room temperature and filtered. The filtrate was sequentially partitioned with hexane,
chloroform, ethyl acetate and n-butanol in the ratio of 1:3 (v/v), three times each. The
resulting extracts were dried over sodium sulfate and evaporated in vacuo to yield the
hexane (HEX), chloroform (CHL), ethyl acetate (EA), and n-butanol (BU) fractions. The
water insoluble part was lyophilized to yield INS fraction.
4.5.3.2.3. Determination of total phenolic content (Table 13)
Total phenolic content was estimated as mg GAE (gallic acid equivalent) per gram dried
samples as described by Singleton et al. (1999). Absorbance was measured at 760 nm in a
spectrophotometer (Shimadzu, Kyoto, Japan) and compared with a gallic acid calibration
curve.
4.5.3.2.3.1. DPPH radical-scavenging activity (Table 14)
The ability of the extract/fractions to scavenge DPPH· radicals was assessed as described by
Brand-Williams et al. (1995) with slight modification. A 100 µL aliquot of methanolic
sample solution was mixed with 3.9 mL methanolic DPPH · (3 x 10-4 mol/L) solution. After
a 30 min reaction at 23oC, the absorbance was measured spectrophotometrically at 517 nm
and the capability to scavenge the DPPH. i.e. percent inhibition was calculated using the
following equation:
% Inhibition = [(AC - AS)/ AC] x 100
277
Isolation, chromatographic evaluation……..
Chapter 4
Where AC is the absorption of the control reaction and A S is the absorption of the sample
solution. The control consisted of 0.1 mL of methanol and 3.9 mL of DPPH · solution. All
readings were taken in triplicate.
4.5.3.2.3.2. ABTS radical-scavenging activity (Table 14, Figure 22)
The ABTS·+ scavenging activity was assessed as described by Re et al. (1999). The ABTS·+
was generated by reacting an ABTS aqueous solution (7 mmol/L) with K2S2O8
(2.45 mmol/L) in the dark for 12–16 h and adjusting the absorbance at 734 nm to 0.700 (±
0.030). Later, 25 µL of sample solution was added to 1.975 mL ABTS·+ and the absorbance
at 734 nm was recorded after 10 min. The trolox equivalent antioxidant capacity (TEAC)
was calculated against a trolox calibration curve.
4.5.3.2.3.3. Ferric reducing/antioxidant power (FRAP) assay (Table 14, Figure 23)
The iron (III) reductive capacity was assessed as described by Oyaizu (1986). Briefly, 100
µL of sample solution in methanol was mixed with 1 mL phosphate buffer (0.2 mol/L, pH
6.6) and 1 mL (1%, w/v) K3Fe(CN)6 solution. After 30 min incubation at 50°C, 1 mL (10%,
w/v) of trichloroacetic acid was added and mixed. Finally, 200 µL (0.1%, w/v) FeCl3 was
added to the upper layer, shaken thoroughly and the absorbance was recorded at 700 nm.
Values are presented as mM ascorbic acid (mM AA) equivalent.
4.5.3.2.4. Measurement of color properties (Table 15)
The color of all the extract/fractions were determined utilizing a CIELAB program for the
spectrophotometer (Premier Color Scan, India) that determined L, a, b, C and h0 for each
sample dissolved in methanol (1 mg/mL) in a 1 mm cuvette. L is the brightness ranging
from no reflection for black (L=0) to perfect diffuse reflection for white (L=100). The
values a and b indicate the degree of red (when a > 0), green (when a < 0), yellow (when b
> 0), and blue (when b < 0) color. Hue angle (h0) is defined as a color wheel, with redpurple at an angle of 0° and 360°, yellow at 90°, bluish green at 180° and blue at 270°.
Similarly, chroma stands for color saturation which varies from dull at low chroma values to
vivid at high chroma values [McGuire (1992)].
4.5.3.2.5. Statistical analysis
Results are expressed as mean ± SD of three different measurements. The data were
analyzed for statistical significance using ANOVA procedures. Significant differences
between mean were determined by Duncan’s multiple range test at a level of p ≤ 0.05
(Statistica 7, Statsoft, USA).
278
Isolation, chromatographic evaluation……..
4.6.
Chapter 4
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