HPTLC Analysis of Flavonoids in Bulb Extracts of Crinum woodrowii

Journal of Academia and Industrial Research (JAIR)
Volume 3, Issue 11 April 2015
567
ISSN: 2278-5213
RESEARCH ARTICLE
HPTLC Analysis of Flavonoids in Bulb Extracts of Crinum woodrowii Baker,
a Critically Endangered Plant
Sanjay Jagtap
Dept. of Botany, Elphinstone College, Mumbai (MS), India
[email protected]; +91 9969421282
______________________________________________________________________________________________
Abstract
Crinum woodrowii Baker is a critically endangered bulbous plant belonging to family Amaryllidaceae found
only at Kates point, Mahabaleshwar. It was considered as Crinum brachynema Herb but G.M. Woodrow first
collected this species from Mahabaleshwar in 1899 and confirmed as a new species by Baker as Crinum
woodrowii. It is also called as Woodrow’s Crinum lily. It was rediscovered by Dr. Punekar from same location
in 2004. There were only 150 individuals growing on hill slopes at Kates Points, Mahabaleshwar.
It is endemic to Kates Points, Mahabaleshwar in Satara district of Maharashtra. In 2013, Pethe and Tillu
reported this as second location after Mahabaleshwar at river bank of Vaitarana at Vahigaon. There was a
third new location of Crinum woodrowii at Khandus plateau of Bhimashankar Wild Life Sanctuary part of
Sahyadris of Western Ghats of Maharashtra in Pune district. In the present investigation, flavonoid analysis
of the bulb extracts of Crinum woodrowii Baker by HPTLC was evaluated. Our findings showed flavonoids
like Rutin, Isoquercetin, Astrangalin, Phenolic acid and Kaempferol. This study highlights the biochemical
and ethnopharmacological significance of Crinum woodrowii Baker.
Keywords: Crinum woodrowii Baker, antioxidants, critically endangered, flavonoids, Western Ghats.
Introduction
The distribution of Crinum woodrowii Baker is restricted
to the North-Western Ghats of Western India, where it
occurs in three areas: in the Dharmapur forest range of
the Balsar district in Gujarat State at about 700 m above
sea level; Mahabaleshwar; Kas Plateau and Kates point
Mahabaleshwar from Satara, Vihigaon from Thane,
Khandus plateau (Bhimashankar) from Pune districts of
Maharashtra (Cooke, 1958; Hooker, 1967; Bachulkar,
1993; Punekar et al., 2004; Pethe and Tillu, 2013;
Jagtap and Satpute, 2015). Crinum woodrowii Baker is a
critically endangered plant belonging to family
Amaryllidaceae
found
only
at
Kates
point,
Mahabaleshwar and was considered as Crinum
brachynema Herb but G.M. Woodrow first collected this
species from Mahabaleshwar in 1899 and confirmed as a
new species by Baker as Crinum woodrowii. It is also
called as Woodrow’s Crinum lily. It was rediscovered by
Dr. Punekar from same location in 2004. There were only
150 individuals growing on hill slopes at Kates Points,
Mahabaleshwar. It is endemic to Kates Points,
Mahabaleshwar in Satara district of Maharashtra.
There was a second location after Mahabaleshwar at
river bank of Vaitarana at Vahigaon with only
50 individuals (Pethe et al., 2013). There was a third new
location of Crinum woodrowii at Khandus plateau of
Bhimashankar Wild Life Sanctuary part of Sahyadris of
Western Ghats of Maharashtra in Pune district. There
were about 200-250 individuals along the hill slope and
water spring (Jagtap and Satpute, 2015).
©Youth Education and Research Trust (YERT)
A tall herbs; bulbs 8.6-16.2 cm in dia., globose
spheroidal, outer tunics brown membranous. Leaves
contemporary with the flowers, sometimes appear after
flowering, many (8-17), 45.5-80 cm x 4.5-14 cm, uniform,
flat, bright green, slightly glaucous beneath, glabrous,
apex acute, white waxy, scabrous along margin; leaf
sheaths forming a pseudostem. Scapes one, rarely two,
arising from bulb outside the tuft of leaves, stout,
compressed, 53.5-82.5 cm x 1-3 cm, green at base and
apex, purple in middle, faintly channeled. Flowers 10-20
in umbel, fragrant; pedicels 1-3 cm long, green with
purple tinge. Spathe valves (involucral bracts) two,
opposite, 8.7-10 cm x 2.7-3.9 cm, deltoid, obtuse or
acute at apex, margin inflexed, often green, purple
tinged, nervate, coriaceous. Bracteoles many, 3-8 cm
long, filiform, pale yellow or green. Perianth
hypocrateriform (salver-shaped); tube 4-8 cm long,
terete, curved, green with purple tinge in flowers, purple
in buds; segments spreading equally, white, lanceolate,
acute at apex, longer than perianth tube, 8.6-10 cm
x 1-1.8 cm, purple tinged on dorsal median line, shining.
Stamens 6; filaments 6-7.2 cm long, filiform, white in
lower half and at tip, red in upper half, shorter than
perianth lobes; anther lobes versatile, linear, crescent,
1.2-1.5 cm long, yellow, grey when wet. Ovary oblong,
8-10 mm x 3-4 mm, three-celled, with numerous ovules
in axile placentation; ovules sessile; style terete, filiform
overtopping the stamens, 15-15.6 cm long, white in lower
half, red in upper half; stigma lobed.
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Jagtap, 2015
Journal of Academia and Industrial Research (JAIR)
Volume 3, Issue 11 April 2015
568
Fig. 1. Habit of Crinum woodrowii.
The plants were identified and authenticated using
herbarium collection at Botany research laboratory,
DST-FIST School of Life Science, SRTM University,
Nanded (MS) and Dept. of Botany, Walchand College,
Solapur (MS). Fresh bulbs were washed thoroughly
under running tap water followed by sterile distilled water
and dried under shade. The material was ground into
coarse powder using mechanical grinder. This coarse
powder was sieved by 1 mm pore size sieve. The
powder was stored in airtight containers at room
temperature till further phytochemical screening of
secondary metabolites.
Soxhlet extraction: Exhaustive Soxhlet extraction was
performed using a classical Soxhlet apparatus with
accurately weighed 10 g of the crude powder of plant
material for 18-40 h. Extraction was performed with
water, methanol, chloroform and acetone as the
extracting solvent. The extraction was conducted for
6-8 h/d and finally all the extracts were evaporated under
vacuum. The water, methanol, chloroform and acetone
extracts of tubers of the plant were prepared according to
standard methods (Harbone, 1998). These extracts were
sealed in airtight containers and stored at -4C.
Fig. 2. Bulbs of Crinum woodrowii Baker.
Fruits irregular in shape, 3-7 cm across, trilocular, finally
bursting, edunclec, 3 cm long. Seeds large, rounded,
testa thick, albumencopious (Cooke, 1903). Crinum
woodrowii is used as ornamental as well as medicinal
herb. The scented flowers may be used in perfume and
pharmaceutical industry. Phytochemical screening of the
bulb extracts of Crinum woodrowii revealed the presence
of different phytochemicals. Indeed phytochemical
investigations have resulted in occurrences of
carbohydrates,
alkaloids,
glycosides,
saponins,
flavonoids, tannins, phenols, Vitamin E and C (Jagtap
and Satpute, 2015). Keeping the above facts in view, in
the present study, chemical fingerprinting of flavonoids
by HPTLC in bulb extracts of Crinum woodrowii is
evaluated.
Materials and methods
Sampling: Fresh samples of bulbs of Crinum woodrowii
were collected from Khandus plateau of Bhimashankar
Wildlife Sanctuary, District: Pune, region of Western
Ghats of Maharashtra (Fig. 1 and 2).
©Youth Education and Research Trust (YERT)
Flavonoids analysis by HPTLC: The standards
Quercetin, Kaemferol, Catechin gallate, Rutin hydrate
and Hesperdin were procured from Sigma Aldrich USA.
All the standard solutions were prepared in ethanol
whereas hesperdin in water. Chromatography was
performed on silica gel 60F254 (10 cm X 10 cm; 25 mm
layer thickness; Merk) with aqueous, methanolic,
chloroform and acetone extracts of Crinum woodrowii
bulb. The fraction residues were collected and (10 µL)
subjected for HPTLC (CAMAG, Switzerland) analysis.
The fractions were impregnated on silica gel 60F254 TLC
plate. The plate was air-dried and then inserted in
CAMAG-twin through lass chamber containing solvent
system of composition with ethyl acetate, acetic acid,
formic acid and water (100:11:11:27) as a gradient
mobile phase for 20 min. The well eluted TLC plate was
then dried at 105C for 15 min and scanned using
Scanner 3 (CAMAG, Switzerland) at 254 and 366 nm
using Win Cat 4 software.
Results and discussion
Flavonoids are important group of polyphenols widely
distributed among the plant flora. Over 4000 flavonoids
are known to exist and some of them are pigments in
higher plants. Quercetin, kaempferol and quercitrin are
common flavonoids present in nearly 70% of plants.
Other
group
of
flavonoids
include
flavones,
dihydroflavons, flavans, flavonols, anthocyanidins,
proanthocyanidins,
calchones
and
catechin
and leucoanthocyanidins (Godstime et al., 2014).
The hydrophilic flavonoids were detected in aqueous
extracts of all the plants. Additionally, the hydrophobic
flavonoids were found in rest of the organic extracts.
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Jagtap, 2015
Journal of Academia and Industrial Research (JAIR)
Volume 3, Issue 11 April 2015
569
Fig. 3. HPTLC chromatogram for Rutin.
Fig. 4. HPTLC chromatogram for Hesperidin.
Fig. 5. HPTLC chromatogram for Kaempferol.
Fig. 6. HPTLC chromatogram for Catechin.
Fig. 7. HPTLC chromatogram for Quercetin.
©Youth Education and Research Trust (YERT)
S. No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Table 1. Rf values of standard flavonoids.
Name of flavonoid
Rf value
Reference
Kaempferol
0.87
Rutin
0.46
Quercetin
0.98
STD Rf values
Hesperian
0.58
Catechin
0.97
Luteolin
0.34
Pavel et al.
(2011)
Epigenin
0.50
Saponanin
0.20
Apiin
0.39
Joseph
Diosmin
0.31
and Bernard
(2003)
Astrangnlin
0.65
Isoquericitrin
0.53
Caffeic acid
0.79
Coumaric acid
0.92
Gordana et al.
(2003)
Chlorogenic acid
0.64
Vanilic acid
0.99
The significant test was observed in methanolic extracts
of all the plants. These results prompted us to investigate
and identify the various types of flavonoids by HPTLC
method. Identification of flavonoids by HPTLC method
has been explained in a separate section. Flavonoids
were found profusely in all the extracts, which impelled
us to evaluate the antioxidant activity of all the plant
extracts. Flavoniods and saponins are known to be
antioxidant. They prevent the damage caused by free
radicals to cells. They can mediate in most cases of
chronic diseases such as cancer and diabetes. They are
also slow or even can stop the proliferation of cancer
cells (Bruneton, 1994; Trease and Evans, 2009; Ubwa,
2011).
Determination of flavonoids by HPTLC: HPTLC is the
most recent evolution of planar chromatography, whose
mission is to change the weakness of TLC into strength.
HPTLC rose from a need for major separation capacity,
obtained by the use of precoated plates with smaller
particles (2 μm vs. 15 μm), i.e. a more active surface in
order to obtain the efficacy needed for plant mixtures.
In the modern HPTLC, the plate is the central tool of a
complex automatic instrumentation system developed to
control analysis conditions, to optimize reproducible
results and to allow a complete comparison between
different laboratories. Being a multistep process, HPTLC
performance requires a separated device for each step
of the sequence: sample application, chromatogram
development,
derivatization,
visualization
and
documentation. HPTLC chromatograms of standard
compounds (quercetin, rutin, hesperidin, kaempferol and
catechin) are presented in Figs. 3-7. Water, methanol,
chloroform and acetone extracts, as well as the spots
were characterized by Rf values and colour under
UV light before (UV) and after spraying with
2-aminoethyl diphenylborinate (UV-NA). The results of
two-dimensional HPTLC analyses showed that different
flavonoids, phenolic compounds, and phenolic acids are
present in the investigated plant extracts. A large number
of flavonoids (Rutin, Quercetin, Epigenin, Hesperidin,
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Jagtap, 2015
Journal of Academia and Industrial Research (JAIR)
Volume 3, Issue 11 April 2015
570
Diosmin, Kaempferol, Catechin, Astrangnlin, Luteolin,
Isoquericitrin)
and
some
unidentified flavonoid
glycosides, phenolic acids (Chlorogenic, Caffeic acid,
Coumaric and Vanillic acid) and Saponins were identified
by Rf values (Table 1).
Among 3 peaks, three unknown flavonoids have been
located and a new peak of Vanilic acid (Rf=0.99) was
observed. The results are depicted in Table 2 and Figs.
9-10.
Fig. 9. HPTLC peaks at 254 nm and 366 nm after derivatization
of bulb extracts of Crinum woodrowii.
Fig. 8. HPTLC-Chemical profiling of flavonoids in the bulb
extracts of Crinum woodrowii.
After derivatization 254 nm
After derivatization 366 nm
A: Water, methanolic, chloroform and acetone extracts of C. woodrowii under UV 254 BD.
B. Water, methanolic, chloroform and acetone extracts of C. woodrowii under UV 366 BD.
C. Water, methanolic, chloroform and acetone extracts of C. woodrowii under UV 254 BD.
D. Water, methanolic, chloroform and acetone extracts of C. woodrowii under UV 366 AD.
E. Water, methanolic, chloroform and acetone extracts of C. woodrowii under visible light AD.
Flavonoid analysis by HPTLC: In the chromatogram of
Crinum woodrowii using water extract, total 9 and 7
peaks were obtained at 254 nm and 366 nm in water
extract respectively as shown in the Fig. 8. Two unknown
flavonoids were observed whereas, Rutin (Rf=0.47),
Isoquercetin (Rf=0.53), Astrangalin (Rf=0.64), Phenolic
acid (Rf=0.69), Kaempferol (Rf=0.89), Coumaric acid
(Rf=0.92), Vanilic acid (Rf=0.99) were identified at 254
nm. At 366 nm, 4 peaks of unknown flavonoids whereas
peaks of Rutin (Rf=0.47), Coumaric acid (Rf=0.92),
Vanilic
acid
(Rf=0.99)
were
located
and
peaks of Isoquercetin, Astrangalin, Phenolic acid,
Kaempferol were disappeared in the chromatogram.
The chromatogram of acetone plant extract was
investigated under UV light. Total 7and 9 peaks were
observed at 254 nm and 366 nm respectively.
One unknown flavonoid was observed, whereas Diosmin
(Rf=0.30), Luteolin (Rf=0.33), Vanilic acid (Rf=0.99),
Chlorogenic acid (Rf=0.63), Rutin (Rf=0.42), Kaempferol
(Rf=0.86) were identified under UV light at 254 nm and at
366 nm, additional spot of Epigenin (Rf=0.52) was
observed. The 2 unknown peaks were located and peak
of Luteolin was disappeared in the chromatogram.
Four peaks were observed in the chromatogram of
chloroform plant extract at 254 nm. Among 4 peaks,
1 peak was unidentified and peaks of saponin
(Rf=0.23), Rutin (Rf=0.42), Hesperidin (Rf=0.55) were
located in the chromatogram. Additional 2 peaks of
unknown flavonoids were located at 366 nm.
In methanolic plant extract, the chromatogram displayed
12 peaks, 5 remained unidentified, while 7 were
identified as flavonoids and phenolic acids. Saponanin
(Rf=0.18), Diosmin (Rf=0.29), Luteolin (Rf=0.34), Rutin
(Rf=0.45), Epigenin (Rf=0.49), Hesperidin (Rf=0.58),
Phenolic acid (Rf=0.73), Caffeic acid (Rf=0.76) were
identified at 254 nm. At 366 nm, 11 peaks were obtained.
©Youth Education and Research Trust (YERT)
M=Methanol, C=Chloroform, A=Acetone, W=Water.
Fig. 10. HPTLC peaks of standard bulb extracts
of Crinum woodrowii.
S3
STD-Q
S2
S4
S1
All peaks at 366 AD (Q-STD Quercetin, S1-Water, S2-Acetone,
S3-Chloroform and S4-Methanol).
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Jagtap, 2015
Journal of Academia and Industrial Research (JAIR)
Volume 3, Issue 11 April 2015
571
Table 2. Chemical profiling of bulb extracts of Crinum woodrowii at 254 nm and 366 nm after derivatization (AD).
254 nm AD
366 nm AD
Plant extract
Height
Area
Assigned
Rf
Height
Area
Assigned
Rf value
(mm)
(AU)
substances
value
(mm)
(AU)
substances
0.28
1.0
58933.2
Unknown
0.28
6.6
58526.8
Unknown
0.47
12.1
12613.8
Rutin
0.47
6.7
13417.6
Rutin
0.53
35.0
1948.4
Isoquercetin
0.53
35.8
2055.7
Unknown
0.64
6.2
8181.0
Astrangalin
0.64
0.0
8962.9
Unknown
Water
0.69
21.0
670.0
Phenolic acid
0.77
3.4
2102.9
Unknown
0.77
1.1
1064.9
Unknown
0.93
31.4
1583.6
Coumaric acid
0.89
25.4
721.1
Kaempferol
0.99
2.6
903.3
Vanilic acid
0.92
28.9
576.3
Coumaric acid
0.99
2.7
887.2
Vanilic acid
0.09
17.6
719.1
Unknown
0.09
19.6
775.5
Unknown
0.30
12.6
1130.9
Diosmin
0.12
23.9
347.3
Unknown
0.33
17.7
178.9
Luteolin
0.24
24.3
260.8
0.42
8.6
2366.0
Rutin
0.30
11.7
1004.1
Diosmin
Acetone
0.63
12.0
2076.9
Chlorogenic acid
0.41
18.2
2337.2
Rutin
0.86
15.3
433.8
Kaempferol
0.50
10.1
198.1
Epigenin
0.99
0.2
1011.3
Vanillic acid
0.63
14.2
2919.5
Chlorogenic acid
0.86
15.9
480.5
Kaempferol
0.99
0.1
1058.6
Vanillic Acid
0.01
30.5
9209.6
Unknown
0.02
2.1
5032.5
Unknown
0.23
30.3
4414.7
Saponin
0.16
5.0
465.2
Unknown
Chloroform
0.42
11.6
623.5
Rutin
0.57
2.0
456.7
Hesperidin
0.55
10.7
480.0
Hesperidin
-0.02
40.9
409.3
Unknown
0.05
43.5
3361.5
Unknown
0.04
23.4
1628.8
Unknown
0.19
24.2
8411.7
Saponin
0.11
75.9
2115.8
Unknown
0.25
14.8
723.6
Unknown
0.18
16.4
2560.4
Saponin
0.29
8.0
752.9
Diosmin
0.25
10.1
655.5
Unknown
0.34
0.6
405.7
Luteolin
0.29
6.5
602.6
Diosmin
0.52
19.9
6871.3
Epigenin
Methanol
0.34
0.6
423.1
Luteolin
0.58
75.8
2144.2
Hesperidin
0.52
15.1
5431.9
Epigenin
0.67
20.1
6105.8
Unknown
0.58
56.6
2014.3
Hesperidin
0.72
34.2
1062.1
Phenolic acid
0.67
17.2
4815.3
Unknown
0.80
4.2
2832.6
Caffeic acid
0.73
20.9
1023.3
Phenolic acid
0.99
0.3
140.4
Vanillic acid
0.79
13.5
1589.1
Caffeic acid
Conclusion
Crinum woodrowii is a critically endangered medicinal
plant newly located at Khandus plateau of Bhimashankar
Wild Life Sanctuary; Bhimashankar has an ancient
history of the multiple indigenous uses of medicines from
India. Investigations of the phytochemicals and their
biological activity have provided scientific support for
many of its traditional uses. The HPTLC technique
expressed for the determination of flavonoid from Crinum
woodrowii bulb is simple, precise and can be used for
standardization of biological compounds in the plant
extracts. This HPTLC technique is highly adaptable,
because of the precision and repeatability of compound
analysis in plant extracts. The detection of flavonoids like
Diosmin, Rutin, Epigenin, Saponanin, Hesperidin,
Phenolic acid, Chlorogenic acid, Quercetin, Isoquercetin
and Kaempferol by HPTLC revels strong medicinal value
in all the tuber extracts. The structural characterisations
(FTIR, NMR studies) of isolated flavonoids from various
bulb extracts of Crinum woodrowii are in progress.
©Youth Education and Research Trust (YERT)
The conservation practices for critically endangered
Crinum woodrowii is needed for its existence and its
utility in ethnopharmacology.
Acknowledgements
Author sincerely acknowledges the valuable support
provided by Institute of Science, Mumbai and THINQ
Pharma Inc., Nagpur.
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