1
SUPPLEMENTARY MATERIAL
2
Phytochemical composition, antioxidant activity and HPLC profiles of Swertia species from
3
Western Ghats
4
Parthraj Kshirsagar , Jaykumar Chavan , Mansingraj Nimbalkarb, Shrirang Yadava Ghansham Dixita,
5
Nikhil Gaikwada,*
a
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7
a
b
Department of Botany, Shivaji University, Kolhapur- 416 004, India
b
Sahyadri Group Enriching Nature, Environment and Science, Kawla Naka, Kolhapur- 416 003, India
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*Corresponding author: Dr. N. B. Gaikwad
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Laboratory of Cytogenetics and plant breeding,
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Department of Botany, Shivaji University, Kolhapur- 416004 (MS) India.
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E-mail – nbgaikwadsuk@gmail.com
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Tel: (+91 231) – 2609157 Fax: (+91 231) – 2692333
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Abstract
14
Swertia chirayita is one of the potential medicinal plants of family Gentianaceae in traditional medicine.
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Due to its high demand and scarcity, trade of chirayita is affected by adulterants. Swertia species from Western
16
Ghats were compared with S. chirayita for phytochemical characterization and antioxidant activities by using
17
different extracts. Present study revealed that acetone is the best extraction solvent of phenolic and flavonoid
18
compounds with antioxidant properties as compared with other extracts. Swertia chirayita showed better antioxidant
19
activity than other species with highest content of phenolics and flavonoids. Among the species from western Ghats
20
S. minor has better antioxidant properties with higher content of phenolics and flavonoids when compared with S.
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chirayita. Gallic acid was detected in all species under study by HPLC analysis. The Swertia species under study
22
show similar phytochemical properties and antioxidant potential and hence their use as substitute to S. chirayita
23
needs to be further investigated.
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Key Words: Swertia, Antioxidant activity, Phenolic compounds, HPLC, Flavonoids.
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1
1
2
Experimental
3
1. Plant material
4
The plants of Swertia species viz. S. densifolia, S. lawii and S. minor were collected during June-January
5
2011-12 from different localities of Western Ghats of Maharashtra. Swertia chirayita was collected from Darjeeling
6
which was used for further analysis. The plant material was authenticated by Prof. S. R. Yadav, Department of
7
Botany, Shivaji University, Kolhapur and voucher specimens of the plants have been deposited in the herbarium of
8
the Department of Botany, Shivaji University, Kolhapur which numbers are depicted in Table S2.
9
The plants were cleaned and air-dried at room temperature and grind to powder using a laboratory grinder,
10
passed through a sieve (fine sieve of pore size 100 µm), to obtain uniform powder for the analysis. 500 mg of the
11
powder of S. chirayita, S. densifolia, S. lawii and S. minor was extracted with different solvents (75 % ethanol, 75 %
12
methanol, 75 % acetone and water) in 50 mL, at room temperature for 24 h. The extracts were filtered and the
13
filtrate was used for further analysis. Preliminary analysis was carried out to standardize the best extraction solvent
14
percentage, in which 75 % solvent extracts compared to 25 %, 50 % and 100 % of various solvents showed best
15
result.
16
2. Determination of total phenol content
17
Total phenolic content of the extracts was quantified using folin-ciocalteu method described by Singleton
18
& Rossi (1965) with some modification. The amount of total phenol was calculated as mg/g dry powder (Gallic acid
19
equivalents) from calibration curve of Gallic acid standard solution.
20
3. Determination of total flavonoid content
21
The total flavonoid content of all the plant extracts were quantified by using the aluminium chloride
22
colorimetric method described by Chang et al. (2002). The content of the total flavonoids was expressed as mg/g dry
23
powder (Rutin equivalent) according to the calibration curve obtained from rutin standard solution.
24
4. DPPH radical scavenging activity
25
The antioxidant activities of all the plant extracts were evaluated by using DPPH assay described by Lee et
26
al. (2003). The absorbance was measured at 517 nm on UV-Visible spectrophotometer. A control (without extract)
27
also examined and the results were revealed as ascorbic acid corresponding antioxidant capacity (AEAC).
28
5. Ferric reducing antioxidant power (FRAP) activity
29
The FRAP assay was analyzed according to the described method of Benzie & Strain (1996) with minor
30
modifications. The plant extracts of various concentrations were reacted with 2.7 mL of the FRAP reagent and the
31
volume of the reaction was adjusted to 3 mL with distilled water, mixture was kept 30 min in dark and the
32
absorbance was recorded at 593 nm. The results were revealed as ascorbic acid corresponding antioxidant capacity
33
(AEAC).
34
6. Quantification of Phenolic compounds by HPLC Technique
35
For HPLC investigation of phenolic compounds the 75 % ethanolic extracts of all Swertia species under
36
study were used as a preliminary assessment of various compounds showed superior extraction in 75 % ethanol. The
37
HPLC apparatus used for analysis was composed of a waters 6785 multi solvent delivery system, equipped with a
2
1
UV dual detector and generated data were analyzed using Waters Empower software. For chromatographic
2
separation Waters C 18 column (Symmetry, 5 µm, 3.9 x 150 mm) was applied. For chromatographic analysis two
3
solvents were used i.e. A) methanol (25 %), B) methanol (30 %) both in 1 % acetic acid in the linear gradient
4
programme in a volumetric ratio ( 0-30 min, 100 A/0 B; 30-45 min, 82 A/18 B; 45-60 min, 72 A/28 B). The flow
5
rate of the mobile phase was 0.75 mL/min. The peaks were detected at 280 absorption spectrum accordance with the
6
Pai et al. (2010). Sample volume (20 uL) and analysis time was 60 min for both, standards and samples used for
7
analysis. An extensive range of phenolic compounds that are attributed to medicinal properties of various plants
8
were used as standards i.e Gallic acid, Cathechol, Caffeic acid, Vanillin and p-Coumaric acid.
9
7. Statistical analysis
10
The results obtained were statistically analyzed and were correlated for each parameter, i.e. phenolics to
11
flavonoid, DPPH and FRAP, flavonoids to phenolics, DPPH and FRAP in each species. The results are expressed as
12
the mean ± SE of three replications. Inhibition concentration of extracts that cause 50 % of inhibition (IC 50) were
13
also calculated.
14
15
16
17
Figure captions:
18
Figure S1 DPPH radical scavenging activity of different extracts of (a) S. chirayita, (b) S. minor, (c) S. densifolia
19
and (d) S. lawii
20
Figure S2 FRAP (Ferric Reducing Antioxidant Power) Assay of (a) S. chirayita (b) S. minor, (c) S. densifolia and
21
(d) S. lawii
22
Figure S3 HPLC profiles of Phenolic compounds in 75% ethanolic extracts (a) 5 standard phenolic compounds (50
23
ppm), (b) S. chirayita, (c) S. densifolia, (d) S. minor and (e) S. lawii
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25
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27
28
29
30
31
32
33
3
1
Fig. S1 DPPH radical scavenging activity of different extracts of (a) S. chirayita, (b) S. minor,
2
(c) S. densifolia and (d) S. lawii
mg AEAC/g of sample
(a)
3
2
Methanol
1
Ethanol
Acetone
0
200
400
600
800
1000
Water
Concentration mg/ml
3
mg AEAC/g of sample
(b)
1.5
1
Methanol
Ethanol
0.5
Acetone
0
200
400
600
800
1000
Water
Concentration mg/ml
4
mg AEAC/g of sample
(c)
3
2
Methanol
1
Ethanol
Acetone
0
200
400
600
800
1000
Water
Concentration mg/ml
5
mg AEAC/g of sample
(d)
2
1.5
Methanol
1
Ethanol
0.5
Acetone
0
200
400
600
800
1000
Water
Concentration mg/ml
6
7
4
Fig. S2 FRAP (Ferric Reducing Antioxidant Power) Assay of (a) S. chirayita (b) S. minor, (c) S.
2
densifolia and (d) S. lawii
mg AEAC/g of sample
1
6
5
4
3
2
1
0
(a)
Methanol
Ethanol
Acetone
water
200
400
600
Concentration mg/ml
800
1000
mg AEAC/g of sample
3
2.5
(b)
2
Methanol
1.5
1
Ethanol
0.5
Acetone
water
0
200
400
600
Concentration mg/ml
800
1000
mg AEAC/g of sample
4
3.5
3
2.5
2
1.5
1
0.5
0
(c)
Methanol
Ethanol
Acetone
water
200
400
600
Concentration mg/ml
800
1000
mg AEAC/g of sample
5
2.5
(d)
2
Methanol
1.5
1
Ethanol
0.5
Acetone
water
0
200
400
600
Concentration mg/ml
800
1000
6
7
8
5
Untitled
S A M P L E
std 50
Standard
1
1
20.00 ul
60.0 Minutes
Date Acquired:
Date Processed:
7/31/2012 12:08:25 PM IST
8/17/2012 2:09:19 PM IST
System
Phenolics_mth
phenolics_proc
2487Channel 1
280
Fig. S3 HPLC profiles of Phenolic compounds in 75% ethanolic extracts (a) 5 standard phenolic
compounds (50 ppm), (b) S. chirayita, (c) S. densifolia, (d) S. minor and (e) S. lawii
Gallic Acid - 6.609
0.14
S A M P L E
0.10
Sample
Name:
Sample Type:
0.08
Vial:
Injection #:
0.06
Injection Volume:
0.04
Run Time:
sample 1
Unknown
1
2
20.00 ul
60.0 Minutes
0.02
Date Acquired:
Processed:
5.00
10.00
0.35
25.00
30.00
35.00
Auto-Scale d Chromatogram
Minutes
Area
Height
Amount
45.00
50.00
6.609
6023845
204492
50.000
PPM
Cathechol
12.987
1235105
45141
50.000
PPM
3
Caf f ic Acid
24.241
2264722
22068
50.000
PPM
4
Vanillin
S29.686
A M 7566807
P L E
I N
F OPPR
M A T I O N
50.000
M
75616
By:
Sample Set Name:
Acq. Method Set:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
System
Phenolics_mth
phenolics_proc
2487Channel 1
280
40.00
Project Name:
DDYPTI
50.00
55.00 Printed:
60.00
Date
8/17/2012
2:16:11 PM Asia/Calcutta
45.00
Gallic Acid
20305
3792
4.720
2348581
330321
5.817
52912
2179
S A
M P67702
L E
6.876
Units
(c)
Untitled
I N0.562
F OPPR
M A T I O N
M
1961
42138
2691
34415
2390
3104
125
10260
413
21.033
7.948
Amount
2577
4.041
0.020
6
Sample
Name:
sample 2 7.871
Sample Type:
Unknown 8.526
7
0.015
Vial:
1
8
Cathechol
12.517
Injection #:
3
9
0.010
Injection
Volume:
20.00 ul 14.072
Run Time:
60.0 Minutes
2.608
3.345
4.810
5.557
Height
13032
Acquired By:
Sample Set Name:
Set:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
Acq.PMethod
0.126
PM
System
Phenolics_mth
phenolics_proc
2487Channel 1
280
Reported by User: System
Date Acquired:
7/31/2012 2:09:49 PM IST
Report
Untitled
0.000 Method:
Date
Processed:
8/17/2012 2:12:45 PM IST
Report Method ID: 125
125
0.00
6
5.00
10.00
15.00
20.00
25.00
30.00
35.00
Auto-Scale d Chromatogram
Minutes
40.00
45.00
56.576
5
Area
35.701
4
0.025
RT
3.564
27.137
Vanillin - 28.668
3
Gallic Acid - 6.424
2
0.030
Name
59.150
50.000
PPM
Acquired
54.750
69111
51.200
10186298
32.817
Cathechol - 12.517
14.072
3.564
4.041
5.817
Gallic Acid - 6.876
7.871
8.526
Untitled
Pe ak Re sults
0.040
1
60.00
(b)
Gallic Acid
2
0.035
55.00
Units
1
Date
Acquired:
7/31/2012 4:20:34 PM IST
0.00
Date Processed:
8/17/2012 2:14:48 PM IST
Reported
by User: System
0.00
10.00
15.00
20.00
25.00
30.00
35.00
Report
Method:5.00
Untitled
Minutes
Report Method ID: 125
125
Auto-Scale d Chromatogram
AU
Phenolics_mth
phenolics_proc
2487Channel 1
280
43.035
RT
5
p-Coumsample
aric Acid 447.188
Sample Name:
0.15
Sample
Type:
Unknown
Vial:
1
0.10
Injection
#:
5
Injection Volume:
20.00 ul
Run
60.0 Minutes
0.05 Time:
0.005
System
40.00
39.400
Name
AU
0.20
20.00
Caffic Acid - 24.779
26.566
0.25
Acquired By:
Sample Set Name:
Acq. Method Set:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
Pe ak Re sults
4.720
0.30
15.00
21.483
0.00
4
I N F O R M A T I O N
Untitled
7/31/2012 1:09:17 PM IST
8/17/2012 2:12:14 PM IST
Date
0.00
18.317
AU
0.12
(a)
Vanillin - 29.686
0.16
Caffic Acid - 24.241
0.18
p-Coumaric Acid - 47.188
Auto-Scale d Chromatogram
0.20
5
Acquired By:
Sample Set Name:
Acq. Method Set:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
Cathechol - 12.987
1
2
3
I N F O R M A T I O N
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume:
Run Time:
Project Name:
DDYPTI
Date Printed:
8/17/2012
50.00 PM Asia/Calcutta
55.00
60.00
2:16:44
0.12
2
3.345
62349
5787
3
4.810
59238
3968
Amount
Units
(d)
Untitled
830078
36543
69217
1320
27204
497
6.890Acquired
PPM
By:
Sample Set Name:
Acq. Method Set:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
35.550
14.026
Gallic Acid - 6.468
8.287
Caffic Acid - 24.300
S A
M P
L E 3420 I N F O R M A T I O N
5.557
47267
4
5
Gallic Acid
Sample Name:
sample 3 6.424
Sample
Type:
Unknown 7.948
6
0.04
Vial:
1
7
Cathechol
12.987
Injection #:
4
0.02
8
Injection
Volume:
20.00 ul 21.033
Run Time:
60.0
Minutes
9
Caf f ic Acid
24.241
3
3.589
234310
27972
4
3.854
1182072
126840
4.125
236053
16149
6.468
6791
520
8.287
30599
672
3.378
7
9
Project Name:
DDYPTI
Date Printed:
50.00
55.00
60.00
8/17/2012
2:18:17 PM Asia/Calcutta
Gallic Acid
Cathechol
Reported by User: System
-0.010
Report Method:
Untitled 10.00
0.00
5.00
Report Method ID: 125
125
0.056
12.987
14.026
0.000
15.00
40034
867
20.00
Units
(e)
25.00
PPM
30.00
Minutes
35.00
40.00
45.00
56.699
10334
5
45.00
43.735
Amount
12914
8
0.010
Height
180030
6
0.020
Area
188883
3.225
18.435
0.030
RT
2.894
2
15.916
AU
0.040
40.00
Pe ak Re sults
Name
Gallic Acid - 6.750
0.050
Phenolics_mth
phenolics_proc
2487Channel 1
280
34.898
1
28.817
Vanillin - 29.937
0.060
5.053
5.806
Date Acquired:
7/31/2012 3:10:23 PM IST
Date Processed:
8/17/2012 2:14:08 PM IST
Reported
by User: System
-0.02
Report0.00
Method:5.00
Untitled10.00
15.00
20.00
25.00
30.00
35.00
Auto-Scale d Chromatogram
Minutes
Report Method ID: 125
125
System
55.266
2373
p-Coumaric Acid - 46.767
Height
35203
0.070
8
9
Area
2.608
0.00
7
RT
1
2.894
3.225
4.1253.589
AU
0.06
Name
18.033
0.08
Pe ak Re sults
3.854
0.10
Project Name:
DDYPTI
Date
50.00
55.00 Printed:
60.00
8/17/2012
2:17:17 PM Asia/Calcutta
Pe ak Re sults
Name
RT
Area
Height
1
3.378
245778
3784
2
5.053
418174
53563
3
5.806
549833
63404
6.750
24306
993
4
Gallic Acid
5
Cathechol
12.987
6
15.916
9953
305
7
18.435
43342
614
53210
1552
8
9
Caf f ic Acid
24.241
28.817
Amount
0.202
Units
PPM
6
1
2
Extracts
Water
Methanol
Ethanol
Acetone
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Table S1: Total phenolic and total flavonoid content in different extracts of Swertia species
S. chirayita
TPC
TFC
(mg GAE/g)
(mg RE/g)
36.27±0.03
57.34±0.01
51.21±0.03
89.08±0.06
58.78±0.01
95.09±0.01
102.04±0.02
104.21±0.31
Data shown as mean ± SE, n=3
S. densifolia
TPC
TFC
(mg GAE/g)
(mg RE/g)
20.05±0.02
15.42±0.03
35.37±0.03
32.57±0.03
31.26±0.03
26.05±0.01
50.97±0.07
53.74±0.03
S. lawii
TPC
TFC
(mg GAE/g)
(mg RE/g)
23.06±0.03
14.07±0.06
25.06±0.02
25.45±0.01
24.64±0.02
26.90±0.01
40.83±0.01
43.97±0.05
S. minor
TPC
TFC
(mg GAE/g)
(mg RE/g)
28.32±0.03
46.16±0.01
33.77±0.02
68.66±0.30
40.25±0.03
70.23±0.06
58.82±0.01
85.84±0.01
7
1
Sr.
No.
1
2
3
4
Table S2: DPPH IC 50 values of different Swertia species.
Name of the
IC 50 (ug/ml)
species
Methanol Ethanol Acetone
Water
Swertia chirayita
551.26
557.61
551.96
559.05
Swertia minor
586.45
575.31
550.90
765.15
Swertia densifolia
602.50
571.64
539.21
1119.18
Swertia lawii
921.05
864.19
701.91
766.16
Voucher no.
PRK-20
PRK-05
PRK-07
PRK-11
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
8