extraction and analytical procedures for botanical...
TRANSCRIPT
Extraction and analytical procedures for botanical compounds
Vittal Mallya Scientific Research Foundation
# 94/3 & 94/5, 23rd Cross, 29th Main, BTM II Stage, Bengaluru – 560 076 Email: [email protected]; Web site: www.vmsrf.org
CONTENTS
Sl. No. Botanical Name Page No. 1. Acacia concinna 1 2. Acorus calamus 2 3. Adhatoda vasica 4 4. Annona squamosa 5 5. Argemone Mexicana 6 6. Azadirachta indica 7 7. Basella alba 8 8. Bougainvillea spp. 9 9. Butea monosperma 10 10. Catharanthus roseus 11 11. Clerodendron inerme 12 12. Clerodendrum phlomidis 13 13. Curcuma longa 14 14. Justicia gendarussa 15 15. Lantana camera 16 16. Momordica charantia 17 17. Ocimum sanctum 18 18. Parthenium hysterophorus 19 19. Piper nigrum 20 20. Polyalthia longifolia 21 21. Pongamia pinnata 22 22. Ricinus communis 25 23. Stevia spp 26 24. Tridax procumbens 27 25. Vitex negundo 28 26. Zingiber officinale 29
1
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24
mA
U
0
500
1000
1500
2000
2500
3000
mA
U
0
500
1000
1500
2000
2500
3000
0.93
9 9
7.99
1.92
0 0
.59
4.45
9 0
.85
9.10
9 0
.27
11.0
51 0
.30
1: 206 nm, 8 nmTRC/JS/35/Da102
Retention TimeArea Percent
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
mA
U
0
50
100
150
200
250
300
350
mA
U
0
50
100
150
200
250
300
350
1.02
4 2
7.48
1.48
3 8
.07
1.65
3 5
.95
1.92
0 3
.72
2.21
9 0
.74
2.34
7 0
.60
2.49
6 1
.25
2.80
5 1
.33
3.91
5 1
7.78
4.42
7 8
.73
5.65
3 3
.70
15.9
47 2
0.65
1: 206 nm, 8 nmTRC/JS/35/Aa104
Retention TimeArea Percent
Crude extract obtained from 25 g of pods Ethyl acetate
extract Water extract
0.16 g 7.46 g
1. Acacia concinna
Title: Extraction of pesticidal principles from the pods of Shikakai (Acasia concinna)
Introduction: Shikakai (Acacia concinna) is a tree native to Asia, common in the warm plains of central and south India. Alkaloids from shikakai fruit are used traditionally for hair care in the Indian Subcontinent. The plant parts used for the dry powder or the extract are the bark, leaves or pods. Extract contains high levels of saponins, to use as mild cleaning agents. Extracts from shikakai pods were suggested to have pesticidal properties.
Protocol:
1. Powder 25gms of fruits. 2. Charge powder into 500 ml RB flask. 3. Add 5 volume of Ethyl acetate/water. 4. Stir the mixture at 60° C for 3 hrs. 5. Filter to get clear layer. 6. Distil the filtrate under vacuum to get crude material.
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: Acetonitrile:Water (70:30) Flow rate: 1 ml/min, Wave length: 206 nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
2
Crude extract obtained from 25 g of roots Ethyl acetate
extract Water extract
2.15 g 3.69 g
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
mA
U
-25
0
25
50
75
100
125
150
175
200
225
mA
U
-25
0
25
50
75
100
125
150
175
200
225
1.02
4 0
.02
1.38
7 8
5.84
2.14
4 1
0.23
2.33
6 0
.44
2.92
3 0
.09
3.39
2 0
.04
3.75
5 0
.12
4.02
1 0
.50
4.72
5 0
.10
17.1
31 0
.08
19.5
84 2
.14
22.9
01 0
.38
2: 254 nm, 8 nmJS/167/1/10Bs 273
Retention TimeArea Percent
2. Acorus calamus
Introduction
Sweet flag (Acorus calamus) is a widespread, semi-aquatic plant of temperate to sub-temperate regions. Herbaceous perennial sweet flag has long, erect, narrow, aromatic leaves ascending from a branched,
underground rhizome. β-Asarone (cis-2, 4, 5-trimethoxy-1-propenylbenzene) is a major active principle found in sweet flag leaves and rhizomes and used medicinally against different ailments like, fever, asthma, bronchitis and cough and mainly for digestive problems such as gas, bloating, colic, and poor digestive function.
Antifungal activity of sweet flag is well documented, however, not much work related plant pathogens.
Extraction of Acorus calamus
1. Powder the dry 25g of rhizome using grinder 2. Charge the powder into 1l RB flask with mechanical stirrer 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60°C for 10-12hrs 5. Filter to get clear layer, dispose the insoluble mass 6. Distil the filtrate under vacuum to get pale yellow liquid
Analytical information
Column: C18, 15cmlength, 4.6mm diameter, Atlantis.
Mobile phase: 30mM Ammonium acetate (in 0.1% acetic acid): Acetonitrile (60:40)
Flow rate: 1ml/min
Wave length: 254nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mrs Suvarna Shenvi, Mr. Karunakar A.C.,
Incharge Scientist: Mrs. Suvarna
3
NMR of Beta-asarone NMR of alpha-asarone(std)
Mass spectrum of beta asarone
4
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
1.25 g 6.96 g
Minutes
0 5 10 15 20 25 30 35 40 45 50 55 60
mA
U
-20
0
20
40
60
80
100
120
140
160
180
200
mA
U
-20
0
20
40
60
80
100
120
140
160
180
200
1 3
.573
22.
542
4.5
33 1
.33
3 5
.077
1.4
84
5.3
97 0
.93
5 5
.781
13.
296
6.3
25 3
.06
7 7
.211
8.9
7
8 9
.749
1.6
99
9.9
41 1
.02
10 1
5.23
2 0
.98
11 1
8.12
3 0
.70
12 1
8.63
5 0
.45
13 2
1.66
4 4
.47
14 2
3.15
7 0
.53
15 2
4.78
9 0
.91
16 2
6.63
5 2
.63
17 3
3.23
7 2
.34
18 3
6.67
2 4
.01
19 4
2.25
1 2
3.11
20 4
5.18
4 2
.45
21 4
7.75
5 0
.86
22 5
1.56
3 2
.27
1: 300 nm, 8 nmJS/167/13/10 BTRC 110
Pk #Retention TimeArea Percent
3. Adathoda vasica
Introduction: Malabar Nut (Adathoda vasica) is a medicinal plant native to Asia. This is a shrub with lance-shaped leaves. The leaves are bitter-tasting. From preliminary phytochemical analysis it was found that the extracts showed positive response for the presence of flavonoids, tannins, alkaloids, reducing sugars and saponins. The leaf extract is found to have very good fungicidal properties.
Protocol
1. Powder 25 g of leaves 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer, dispose the insoluble mass 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: 0.1M KH2PO4:Acetonitrile:Acetic acid(85:15:1)
Flow rate: 0.7ml/min
Wave length: 300nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
5
Crude extract obtained from 25 g of seeds Ethyl acetate
extract Water extract
2.19 g 1.22 g
Minutes
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80
mA
U
-250
0
250
500
750
1000
1250
1500
1750
2000
2250
mA
U
-250
0
250
500
750
1000
1250
1500
1750
2000
2250
1 2
.261
3.0
4 1
2345
72
2.6
99 1
1.90
483
359
3 5
.824
5.6
3 2
2871
74
6.2
93 2
.73
110
962
5 7
.179
8.8
0 3
5755
46
7.7
97 2
.14
869
397
8.8
32 1
8.45
749
303
8 9
.109
5.5
3 2
2452
7
9 1
3.20
5 3
.91
158
698
10 1
4.02
7 5
.28
214
564
11 2
2.57
1 5
.15
209
009
12 2
3.60
5 4
.52
183
468
13 2
4.80
0 1
6.69
677
945
14 2
5.52
5 6
.22
252
633
1: 220 nm, 8 nmJS/167/9/10 Sample aTRC 117
Pk #Retention TimeArea PercentArea
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
mA
U
0
5
10
15
20
25
30
35
mA
U
0
5
10
15
20
25
30
35
1 2
.432
38.
24 8
5724
12
2.6
99 6
.15
137
967
3 3
.051
13.
02 2
9195
94
3.3
28 2
8.51
639
116
5 4
.523
14.
07 3
1541
6
1: 220 nm, 8 nmJS/167/9/10 Sample bTRC 118
Pk #Retention TimeArea PercentArea
4. Annona squamosa
Introduction: Seetaphal (Annona squamosa) is a small well-branched tree or shrub that bears edible fruits called sugar-apple. The seeds of annona are known to have insecticidal compounds and were used traditionally for pest control. Many compounds were isolated and identified as annonaceous acetogenins: squamocenin , annotemoyin-2 , reticulatain-2 , squamocin-I , squamocin-B , squamocin , motrilin , squamostatin-D , squamostatin-E , cherimolin-1 , cherimolin-2 from the ethyl acetate and water extract of A. squamosa L. Squamocenin is a new acetogenin. Annotemoyin-2 and reticulatain-2 .
Protocol
1. Powder 25g of seeds 2. Charge powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer, dispose the insoluble mass 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: Methanol:Water(90:10)
Flow rate: 1. ml/min
Wave length: 220nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
6
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
1.55 g 11.01 g
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mA
U
0
20
40
60
80
100
1 2
.283
54.
172
2.6
35 5
.29
3 2
.837
33.
41
4 3
.904
2.1
55
4.4
05 3
.19
6 4
.683
0.3
9
7 6
.411
1.4
1
1: 280 nm, 8 nmTRC/SS/32TRC 121
Pk #Retention TimeArea Percent
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mAU
0
50
100
150
200
250
300
1 1
.184
86.
572
1.7
92 1
0.35
3 2
.389
0.3
54
2.9
12 0
.02
5 3
.147
0.5
36
3.7
23 1
.92
7 5
.056
0.2
5
1: 280 nm, 8 nmA 125.dat
Pk #Retention TimeArea Percent
5. Argemone Mexicana
Introduction: Mexican prickly poppy (Argemone Mexicana) is a native of Mexico and now widely naturalized in the United States, India and Ethiopia. An annual herb with bright yellow sap, it is poisonous, but has been used medicinally by many people including those in its native area. The seeds contain 22–36% of a pale yellow non-edible oil, called argemone oil or katkar oil, which contains the toxic alkaloids sanguinarine and dihydrosanguinarine. Four quaternary isoquinoline alkaloids, dehydrocorydalmine, jatrorrhizine, columbamine, and oxyberberine. The pesticidal property of the leaf extract has been reported.
Protocol:
1. Powder 25g of leaves 2. Charge the powder into 250 ml RB flask. 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer, dispose the insoluble mass 6. Distil under vacuum to get crude material 7. Check the weight of the crude material.
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: A-50mM KH2PO4:K2HPO4, (pH7.7)
B-Acetonitrile A: B (50:50)
Flow rate: 1ml/min
Wave length: 280nm
ETHYL ACETATE EXTRACT WATER EXTRACT
7
Crude extract obtained from 25 g of seeds Ethyl acetate
extract Water extract
1.59 g 2.49 g
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mA
U
-250
0
250
500
750
1000
1250
1500
1750
2000
2250
mA
U
-250
0
250
500
750
1000
1250
1500
1750
2000
2250
1 1
.749
23.
682
2.4
75 1
.73
3 2
.816
2.1
4
4 4
.192
1.6
35
4.5
33 2
.60
6 4
.693
9.4
4
7 8
.309
2.0
1
8 9
.643
2.2
49
10.
507
3.9
3
10 1
1.68
0 4
.45
11 1
3.10
9 2
3.67
12 1
4.28
3 1
2.20
13 2
2.38
9 1
.13
14 2
4.26
7 3
.80
15 2
9.33
3 1
.74
16 3
3.67
5 3
.61
1: 215 nm, 8 nmJS/167/6/10 Aa 273
Pk #Retention TimeArea Percent
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24
mA
U
0
200
400
600
800
1000
mA
U
0
200
400
600
800
1000
1 1
.760
53.
42 8
8633
712
1.8
88 3
2.10
532
6327
3 2
.187
5.8
8 9
7590
2
4 2
.827
5.7
8 9
5917
85
3.0
29 1
.88
312
624
6 4
.683
0.1
8 2
9248
7 6
.219
0.2
6 4
2314
8 7
.147
0.5
0 8
2297
1: 215 nm, 8 nmJS/167/6/10 Aa 276
Pk #Retention TimeArea PercentArea
6. Azadirachta indica
Introduction: The Indian Neem (Azadirachtin Indica) is the most exploited botanical getting the attention of chemists and biologists all over the world because of its biologically active compounds. Azadirachtin is one of the most interesting constituents of neem due to its insect growth regulatory activity. Many Azadirachtin based Eco-Friendly insecticides are now available. Protocol
1. Powder 25g of neem seeds 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer and dispose the insoluble mass 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: Acetonitrile:Water(35:65)
Flow rate: 1.2ml/min
Wave length: 215nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
8
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
2.25 g 4.40 g
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24
mAU
-100
0
100
200
300
400
500
600
700
800
mAU
-100
0
100
200
300
400
500
600
700
800
2.38
9 7
7.49 7.
605
2.3
3
11.2
85 2
0.18
3: 205 nm, 8 nmTRC/194/47 B(Water Extract)s 100
Retention TimeArea Percent
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24
mAU
0
100
200
300
400
500
600
mAU
0
100
200
300
400
500
600
1.74
9 1
.90
2.36
8 5
4.31
2.85
9 1
7.51
4.43
7 0
.62
4.75
7 0
.28
5.68
5 2
.50
6.29
3 0
.11
7.59
5 0
.97 8.
725
21.
19
11.2
11 0
.60
3: 205 nm, 8 nmTRC/194/47 B(Ethanol Extract)s 101
Retention TimeArea Percent
7. Basella alba
Introduction: Indian Spinach (Basella alba) is native to central and southwestern Asia. Various flvonoids are reported to be present are (querecetin; myricetin; kampeferol; apigenin; luteolin; patuletin; spinacetin; jaceidin etc). The leaf extract is found to have both fungicidal and bactericidal activity. Its specific use against plant pests are to be tested.
Protocol
1. Powder 25g of leaves 2. Charge the powder into 500ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer, dispose the insoluble mass 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: 0.1% Orthophosphoric acid:Acetonitrile (20:80)
Flow rate: 1 ml/min
Wave length: 205 nm
ETHYL ACETATE EXTRACT WATER EXTRACT
9
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
1.48 g 4.78 g
Minutes
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
mAU
-10
0
10
20
30
40
50
60
70
80
mAU
-10
0
10
20
30
40
50
60
70
80
4.59
7 3
1.74
5.23
7 2
.71
5.46
1 2
.05
5.89
9 6
.15 6.
368
15.
827.
221
1.5
3 7.97
9 1
2.37
8.41
6 1
.17
8.93
9 1
.83
9.68
5 1
.03
11.0
19 1
.29
11.6
69 3
.08
17.2
05 3
.58
22.2
08 3
.95
25.2
91 1
.16
29.1
84 1
0.56
1: 254 nm, 8 nmJS/167/14/10-ATRC 119
Retention TimeArea Percent
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
mAU
-20
0
20
40
60
80
100
120
140
160
180
mAU
-20
0
20
40
60
80
100
120
140
160
180
2.22
9 0
.35
2.61
3 2
2.44
2.73
1 2
2.26
2.91
2 5
3.20
19.3
28 1
.75
1: 254 nm, 8 nmJS/167/14/10-BTRC 120
Retention TimeArea Percent
8. Bougainvillea spp.
Introduction: Bougainvillea (Bougainvillea glabra) is a flowering plant native to South America from Brazil west to Peru and south to southern Argentina. The plant is sometimes referred to as "paper flower" because the bracts are thin and papery. Preliminary qualitative chemical test done for both extracts show the presence of alkaloids, glycosides, carbohydrates, anthraquinone, flavonoids, terpenoids, saponins, steroids, proteins, fixed oils, fats and tannins.
Extraction of Bougainvillea using ethyl acetate:
1. Powder 25g of leaves 2. Charge powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: Methanol: Water (90:10)
Flow rate: 1ml/min
Wave length: 254nm&280nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mrs. Suvarna, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mrs. Suvarna
10
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
1.31 g 4.12 g
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
mAU
0
20
40
60
80
100
120
mAU
0
20
40
60
80
100
120
1 1
.995
486
02
2 2
.453
297
07
3 2
.997
336
39
4 3
.125
108
148
5
3.4
24 4
5890
6 5
.536
650
863
7 7
.040
125
87
8 7
.755
521
0
1: 254 nm, 8 nmJS/167/4/10 ATRC 133
Pk #Retention TimeAreaName
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
mA
U
-100
0
100
200
300
400
500
600
700
800
mA
U-100
0
100
200
300
400
500
600
700
800
1 2
.080
562
2136
2 2
.485
684
023
3
2.7
84 2
3718
0
4 3
.072
113
7674
5 6
.400
322
701
1: 254 nm, 8 nmJS/167/4/10BTRC 134
Pk #Retention TimeAreaName
9. Butea monosperma
Introduction: This plant is native to tropical and sub-tropical parts of the Indian Subcontinent and Southeast Asia. Besides stigmasterol, stigmasterol-ßD-glucopyranoside and nonacosanoic acid, two new compounds isolated from the leaves of Butea monosperma have been characterised as 3α-hydroxyeuph-25-ene and 2,14-dihydroxy-11,12-dimethyl-8-oxo-octadec-11-enylcyclohexane by spectral data and chemical studies. Glucoside, Kino-oil containing oleic and linoleic acid, palmitic and lignoceric acid are also present in butea leaves.
Protocol:
1. Powder 25g of leaves 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: Acetonitrile:Water (60:40)
Flow rate: 1 ml/min
Wave length: 254nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mrs. Suvarna, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mrs. Suvarna
11
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
4.10 g 5.13 g
Minutes
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
mA
U
0
20
40
60
80
100
120
140
mA
U
0
20
40
60
80
100
120
140
0.27
7 0
.00
2.34
7 1
.40
3.21
1 0
.59
3.33
9 0
.65
3.57
3 0
.18
3.94
7 0
.79
4.28
8 0
.16
4.54
4 0
.39
4.81
1 0
.10
5.47
2 1
.49
14.5
39 5
.89
15.8
83 0
.00
22.8
48 7
1.70
39.1
15 1
6.66
1: 254 nm, 8 nmJs/167/2/10 ATRC 102
Retention TimeArea Percent
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
mA
U
0
100
200
300
400
500
mA
U
0
100
200
300
400
500
1 1
.952
105
1696
7 2 3
.115
543
190
3 3
.200
364
581
4 3
.605
269
12
5 4
.299
119
406
4.9
28 9
757
7 5
.899
112
342
8 6
.784
584
568
9 8
.779
248
44
10 9
.525
131
2911
9.9
52 1
696
12 1
0.82
7 1
3767
13 1
1.62
7 4
4512
14 1
3.88
8 9
375
15 1
5.12
5 3
7606
16 2
0.94
9 6
746
1: 254 nm, 8 nmJs/167/3/10 BTRC 106
Pk #Retention TimeArea
10. Catharanthus roseus
Introduction: Periwinkle (Catharanthus roseus) is a native of Madagascar. Synonyms include Vinca rosea (the basionym), Ammocallis rosea, and Lochnera rosea; other English names occasionally used include Cape Periwinkle, Rose Periwinkle, and Rosy Periwinkle. In the wild, it is considered as endangered due to habitat destruction by slash and burn agriculture. It is also however widely cultivated and is naturalized in subtropical and tropical areas of the world. Main chemical components are ajmaline, catharanthaine, leurosidine, vincristine, vinblastine, vinorelbine, vindesine, vincamine & vinorelbine. The extracts of the leaves and flowers are supposed to be active specially against sucking pests
Protocol:
1. Powder 25g of leaves 2. Charge powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: 1% Ammonium acetate: Acetonitrile (60:40)
Flow rate: 1ml/min
Wave length: 254nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mrs. Suvarna, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mrs. Suvarna
12
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
2.07 g 2.93 g
Minutes
0 5 10 15 20 25 30 35 40 45 50
mA
U
0
10
20
30
40
50
60
70
80
1 1
.824
0.3
42
1.9
63 0
.33
3 2
.283
0.2
24
2.5
81 1
5.05
5 3
.296
5.1
96
3.6
05 4
.30
7 4
.779
0.6
58
5.6
64 1
.51
9 6
.592
0.6
610
7.1
25 1
4.76
11 7
.808
2.2
012
8.7
25 1
0.19
13 9
.899
0.7
114
10.
421
3.3
615
11.
061
1.8
0
16 1
2.85
3 0
.38
17 1
4.34
7 2
2.80
18 1
6.20
3 2
.44
19 1
8.01
6 9
.57
20 2
5.68
5 0
.55
21 3
2.78
9 0
.70
22 3
6.10
7 2
.29
1: 270 nm, 8 nmjs/167/10/10 sample aTRC 154
Pk #Retention TimeArea Percent
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
mA
U
-50
0
50
100
150
200
250
300
350
400
450
1 2
.187
4.9
52
2.3
36 3
.04
3 2
.464
2.5
34
2.9
65 3
4.91
5 3
.307
7.6
06
3.6
69 9
.97
7 4
.096
2.1
88
4.3
41 4
.46
9 4
.640
5.7
410
5.0
24 6
.54
11 5
.760
1.5
312
6.1
55 5
.98
13 7
.061
4.4
214
7.4
45 6
.15
1: 270 nm, 8 nmjs/167/10/10 sample bTRC 155
Pk #Retention TimeArea Percent
11. Clerodendron inerme
Introduction: Clerodendron species are landscaping plants valued for their unique flowers. The major chemical constituents present in the Clerodendrum genus are steroids such as β-sitosterol, γ-sitosterol octacosanol, clerosterol, bungein A, acteoside, betulinic acid, etc. The leaf extract is known to have pesticidal properties
Protocol:
1. Powder 25g of leaves 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil ethyl under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: Methanol: Water:Acetic acid 70:30:1.5
Flow rate: 1ml/min, Wave length: 270nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mrs. Suvarna, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mrs. Suvarna
13
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
2.65 g 6.29 g
Minutes
0 5 10 15 20 25 30 35 40 45 50
mA
U
0
50
100
150
200
250
300
1 3
.029
0.4
2 2 3
.328
4.0
43
3.6
59 4
.28
4 5
.621
2.6
95
6.6
99 0
.81
6 7
.275
16.
01
7 8
.587
0.9
0
8 1
0.68
8 1
.19
9 1
5.09
3 6
5.01 10
18.
581
3.0
4
11 3
3.94
1 0
.50
12 3
7.13
1 0
.53
13 3
9.68
0 0
.59
1: 270 nm, 8 nmjs/167/18/10 sample a methTRC 152
Pk #Retention TimeArea Percent
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mA
U
-20
0
20
40
60
80
100
120
140
160
180
1 2
.197
6.7
82
2.3
47 7
.07
3 2
.453
7.9
84
2.9
44 3
9.15
5 3
.328
9.6
06
3.6
80 1
5.66
7 4
.832
8.8
2
8 6
.507
0.5
19
7.1
68 0
.84
10 1
4.81
6 3
.59
1: 270 nm, 8 nmjs/167/18/10 sample bTRC 153
Pk #Retention TimeArea Percent
12. Clerodendrum phlomidis
Introduction: Arni (Clerodendrum phlomidis ) is a fairly common shrub of arid plains, low hills, deserts of Sind, Punjab and Baluchistan. Root is bitter tonic and given in convalescence of measles. Juice of leaves is alterative and given in neglected syphilitic complaints. The major chemical constituents present in the Clerodendrum are steroids such as β-sitosterol, γ-sitosterol octacosanol, clerosterol, bungein A, acteoside, betulinic acid, clerosterol 3-O-β-Dglucopyranoside, etc.
Protocol:
1. Powder 25g of leaves 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: Methanol: Water:Acetic acid 70:30:1.5
Flow rate: 1ml/min
Wave length: 220nm&270nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mrs. Suvarna, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mrs. Suvarna
14
Crude extract obtained from 25 g of rhizomes Ethyl acetate
extract Water extract
1.80 g 3.81 g
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mA
U
0
5
10
15
20
25
30
mA
U
0
5
10
15
20
25
30
3.08
3 5
5.71
3.41
3 8
.95
3.79
7 1
2.22
5.22
7 1
1.75
6.13
3 8
.26
6.65
6 3
.11
1: 254 nm, 8 nmJS/167/20/10 Sample ATRC 119
Retention TimeArea Percent
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mA
U
0
100
200
300
400
500
600
700
mA
U
0
100
200
300
400
500
600
700
5.86
7 6
8.95 6.
891
0.3
37.
445
0.6
78.
128
0.1
1
11.3
39 0
.44
11.7
97 1
.60 14
.432
7.7
0
17.5
15 0
.14
19.0
51 0
.24 22
.144
8.3
422
.837
10.
05
28.0
21 0
.25
30.8
16 1
.19
1: 254 nm, 8 nmJS/167/20/10 Sample ATRC 118
Retention TimeArea Percent
13. Curcuma longa
Introduction: Turmeric is native to tropical South Asia. Plants are gathered annually for their rhizomes, and propagated from some of those rhizomes in the following season. Its active ingredient is curcumin and it has a distinctly earthy, slightly bitter, slightly hot peppery flavor and a mustardy smell. About 73 constituents are identified in rhizomes comprising 95.2% of the oil, of which the major ones were ar-turmerone (31.7%), α-turmerone (12.9%), β-turmerone (12.0%) and (Z) β-ocimene (5.5%). In the oils, 75 constituents comprising 77.5% of the oils were identified, the major ones were α-phellantrene (9.1%), terpinolene (8.8%), 1,8-cinceole (7.3%), undecanol (7.1) and p-cymene (5.5%).
Protocol:
1. Powder 25g of rhizomes 2. Charge powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60°C for 3 hrs 5. Filter to get clear layer, 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: A-10Mm KH2PO4, pHadjusted to 2.8 using H3PO4
B-Methanol, A: B (25:75)
Flow rate: 1.5ml/min
Wave length: 254nm, 345&280nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mrs. Suvarna, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mrs. Suvarna
15
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
2.54 g 6.71 g
Minutes
0 5 10 15 20 25 30 35 40 45 50
mA
U
-2
0
2
4
6
8
10
12
14
16
18
20
mA
U
-2
0
2
4
6
8
10
12
14
16
18
20
1 4
.117
2.0
52
4.6
61 0
.20
3 4
.853
0.6
24
5.4
72 0
.37
5 6
.187
0.5
36
6.5
49 0
.43
7 7
.211
0.8
28
7.5
20 0
.28
9 8
.555
2.0
7
10 2
0.13
9 5
5.55
11 2
5.84
5 9
.51
12 3
6.45
9 2
7.58
1: 254 nm, 8 nmJS/167/19/10 ATRC 136
Pk #Retention TimeArea Percent
Minutes
0 5 10 15 20 25 30 35 40 45 50
mA
U
0
20
40
60
80
100
120
mA
U
0
20
40
60
80
100
120
1 2
.443
4.8
82
3.0
19 0
.21
3 3
.221
0.2
34
3.4
56 1
.42
5 4
.032
0.4
16
4.2
56 1
.55
7 4
.704
0.2
98
4.8
85 0
.32
9 5
.504
0.4
510
6.2
29 0
.51
11 6
.592
0.5
112
7.3
07 0
.44
13 3
3.49
3 6
5.40
14 4
1.64
3 2
3.38
1: 254 nm, 8 nmJS/167/19/10 bTRC 137
Pk #Retention TimeArea Percent
14. Justicia gendarussa
Introduction: Karilakki (Justicia gendarussa) is a small erect, branched shrub, endemic to India. It is useful in asthma, rheumatism and colics of children. Many di-substituted aromatic amines have been isolated from the leaves and characterized as 2-amino benzyl alcohol, 2-(2′-amino-benzylamino) benzyl alcohol. Analysis of ethyl acetate infusions of leaves showed that J. pectoralis contained 1,2-benzopyrene and umbelliferone as the main constituents, and quercetin and kaempferol as the main aglycones, while J. gendarussa contained coumarins, and the flavonoids were C-glycosides, with α-amyrin as the main triterpenoid, followed by simiarenol, β-amyrin and β-sitosterol.
Protocol
1. Powder 25g of leaves 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: Methanol: Water pH 70:30
Flow rate: 1ml/min
Wave length: 254nm
ETHYL ACETATE EXTRACT WATER EXTRACT
16
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
2.90 g 6.66 g
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mAU
-250
0
250
500
750
1000
1250
1500
1750
2000
2250
1 2
.261
4.0
1
2 3
.872
4.0
2
3 5
.205
5.3
1
4 6
.571
3.0
85
6.8
27 0
.45
6 8
.213
27.
527
8.8
43 5
3.47
8 1
5.76
5 0
.83
9 1
6.57
6 0
.76
10 2
1.48
3 0
.55
1: 210 nm, 8 nmJS/167/12/10 Sample ATRC 107
Pk #Retention TimeArea Percent
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mA
U
-20
0
20
40
60
80
100
120
140
160
1 0
.981
5.1
92
1.3
33 1
4.63
3 1
.536
41.
974
2.1
87 5
.56
5 3
.616
2.5
56
4.0
64 0
.68
7 4
.203
2.1
38
4.8
64 1
.17
9 5
.984
0.2
3 10 6
.635
5.0
211
7.4
35 2
.04
12 7
.904
14.
90
13 9
.451
0.1
7
14 1
2.44
8 0
.26
15 2
7.68
0 3
.48
1: 210 nm, 8 nmJS/167/12/10 Sample bTRC 108
Pk #Retention TimeArea Percent
15. Lantana camera
Introduction: Spanish Flag (Lantana camara) is native to the American tropics. It has been introduced into other parts of the world as an ornamental plant and is considered an invasive species in many tropical and sub-tropical areas. The plant contains pentacyclic triterpenoids. The extract is reported to have multiple types of pesticidal activities and sometimes beneficial insect growth regulatory activities like in silkworms.
Extraction of Lantana camara using ethyl acetate:
1. Powder 25g of leaves 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: A-95:5 Water:Acetonitrile, pH adjusted to 2.8 using H3PO4
B-Methanol A: B (10:90)
Flow rate: 2ml/min
Wave length: 210nm&245nm
ETHYL ACETATE EXTRACT WATER EXTRACT
IR spectrum of 3-keto-24-formyl –olean-12-en-28-oic acid
Scientists involved: Dr.G.C.Reddy, Mrs. Suvarna, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mrs. Suvarna
17
Crude extract obtained from 25 g of fruits Ethyl acetate
extract Water extract
5.24 g 4.98 g
Minutes
0 2 4 6 8 10 12 14 16 18 20
mA
U
-200
0
200
400
600
800
1000
1200
1400
1600
mA
U
-200
0
200
400
600
800
1000
1200
1400
1600
3.0
61
91.1
1
4.1
39
1.0
6
8.7
89
2.4
0
1: 204 nm, 8 nmTRC/JS/25 Aa101
Retention TimeArea Percent
16. Momordica charantia
Introduction: The bitter gourd (Momordica charantia) is tropical plant and the fruits are used traditionally as both food and medicine. Photochemical present in bitter melon are Alkaloids, charantin, charine, cryptoxanthin, cucurbitins, cucurbitacins, cucurbitanes, cycloartenols, diosgenin, elaeostearic acids, erythrodiol, galacturonic acids, gentisic acid, goyaglycosides, and so on.
Protocol:
1. Powder 25g of fruits 2. Charge the powder into 500 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs. 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cm length, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: Methanol: Water (100:2)
Flow rate: 1 ml/min
Wave length:204 nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
18
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
2.14 g 3.19 g
Minutes
0 5 10 15 20 25 30 35 40 45 50
mA
U
-10
0
10
20
30
40
50
60
70
80
90
mA
U
-10
0
10
20
30
40
50
60
70
80
90
1 0
.171
131
2 3
.531
336
31
3 3
.744
776
2
4 4
.171
121
513
5
4.5
44 1
4559
0
6 4
.587
795
139
7 6
.720
508
8
8 7
.307
283
13
9 7
.755
180
68
10 9
.600
761
59
11 1
0.50
7 2
3096
7
12 1
4.65
6 2
1039
13 1
7.24
8 1
8808
0
14 1
9.24
3 1
2315
5
15 2
6.36
8 5
6334
1: 254 nm, 8 nmJS/167/7/10 Sample ATRC 111
Pk #Retention TimeAreaName
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
mA
U
0
100
200
300
400
500
600
mA
U
0
100
200
300
400
500
600
1 1
.931
305
1465
2 6
.965
446
465
3
7.4
45 5
1263
3
4 1
1.44
5 2
0446
1: 254 nm, 8 nmJS/167/7/10 Sample BTRC 112
Pk #Retention TimeAreaName
17. Ocimum sanctum
Introduction: Tulasi (Ocimum sanctum) is an aromatic plant and widespread as a cultivated plant or as an escaped weed. It is cultivated for religious and medicinal purposes, and for its essential oil. It is widely known across South Asia as a medicinal plant and an herbal tea, commonly used in Ayurveda. This is suggested to contain high eugenol (40-66%) and high thymol (31%).
Protocol :
1. Powder 25g of leaves 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: Acetonitrile:Water(50:50)
Flow rate: 1 ml/min
Wave length:254 nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
19
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
1.65 g 3.73 g
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24
mA
U
-250
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
mA
U
-250
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
2.36
8 0
.71
3.39
2 0
.41
3.66
9 0
.32
3.88
3 4
.06
4.07
5 4
.27
4.43
7 0
.96
4.81
1 7
2.53
5.04
5 1
5.01
6.02
7 0
.34
6.37
9 0
.47
14.0
05 0
.93
1: 210 nm, 8 nmJS/167/11/2010 aTRC 152
Retention TimeArea Percent
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24
mA
U
0
200
400
600
800
1000
1200
mA
U
0
200
400
600
800
1000
1200
1.52
5 2
5.88
1.70
7 4
.45
1.85
6 2
3.49
2.15
5 4
1.36
2.64
5 3
.06
4.69
3 0
.29
15.2
75 0
.61
17.5
36 0
.85
1: 210 nm, 8 nmJS/167/11/2010 BTRC 153
Retention TimeArea Percent
18. Parthenium hysterophorus
Introduction: The Congress weed (Parthenium hysterophorus) is a native to the American tropics. Parthenin, hexacosanol, myricyl alcohol, β-sitosterol, campesterol, stigmasterol, betulin, ursolic acid, β-D-glucoside of β-sitosterol and saponin are found in leaves. The aqueous extract is found to have pesticidal properties.
Protocol:
1. Powder 25g of leaves 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: A-10mM KH2PO4,pH adjusted to 3.0 using H3PO4
B-Acetonitrile
Flow rate: 1.5ml/min, Linear gradient, Wave length: 210nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
20
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
2.35 g 2.85 g
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mA
U
0
200
400
600
800
1000
1200
mA
U
0
200
400
600
800
1000
1200
5.69
6 1
.58
6.56
0 7
1.52 8.
832
0.5
4
9.82
4 5
.53
10.3
79 1
0.35
12.7
25 5
.27
14.4
53 0
.55
15.0
19 0
.58
17.2
91 2
.20
19.1
04 1
.10
24.5
87 0
.78
1: 254 nm, 8 nmJS/167/21/10 Sample ATRC 116
Retention TimeArea Percent
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mAU
-20
0
20
40
60
80
100
120
140
160
1 1
.941
57.
012
2.1
87 9
.77
3 2
.347
25.
314
2.7
09 4
.29
5 4
.853
0.2
76
5.2
59 0
.47
7 6
.133
0.1
98
6.6
88 2
.20
9 8
.928
0.4
9
1: 254 nm, 8 nmJS/167/21/10 Sample BTRC 117
Pk #Retention TimeArea Percent
19. Piper nigrum
Introduction: Black pepper (Piper nigrum) is a flowering vine, cultivated for its fruit, which is usually dried and used as a spice and seasoning. Black pepper has been found to contain piperine, alkamides, piptigrine, wisanine, dipiperamide D, and dipiperamide E .The essential oil is composed of various chemical constituents and includes the following; a-thujone, a-pinene, camphene, sabinene, b-pinene, a-phellandrene, myrcene, limonene, caryophyllene, b-farnesene, b-bisabolene, linalool and terpinen-4-ol.
Protocol:
1. Powder 25g of seeds 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: A-10Mm KH2PO4, pHadjusted to 2.8 using H3PO4
B-Methanol
A: B (25:75)
Flow rate: 1.5ml/min, Wave length: 254nm, 345&280nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
21
Crude extract obtained from 25 g of roots Ethyl acetate
extract Water extract
3.71 g 3.18 g
Minutes
0 5 10 15 20 25 30 35 40 45 50
mA
U
0
100
200
300
400
500
600
mA
U
0
100
200
300
400
500
600
2.40
0 6
.41
3.18
9 6
.59
3.57
3 5
.07
3.72
3 2
.17
4.04
3 4
.02
4.32
0 2
.80
4.77
9 1
.71
5.00
3 0
.23
5.25
9 0
.24
5.61
1 4
.25
10.1
12
3.70
11.4
45
1.01
14.8
37
2.42
15.6
91
2.57
16.5
97
0.90
37.9
73
30.8
2
40.2
03
25.0
9
1: 254 nm, 8 nmJs/167/3/10 ATRC 104
Retention TimeArea Percent
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mA
U
0
100
200
300
400
500
mA
U
0
100
200
300
400
500
1.95
2 8
5.45
3.11
5 4
.44
3.20
0 3
.01
3.60
5 0
.27
4.92
8 0
.08
5.89
9 0
.91
6.78
4 4
.53
8.77
9 0
.25
9.16
3 0
.03
9.52
5 0
.17
9.95
2 0
.04
10.8
27
0.14
11.6
27
0.48
13.0
13
0.02
13.8
88
0.05
15.1
25
0.08
20.9
49
0.05
1: 254 nm, 8 nmJs/167/3/10 BTRC 106
Retention TimeArea Percent
20. Polyalthia longifolia
Introduction: The False Ashoka tree (Polyalthia longifolia) is a lofty evergreen tree, native to India, commonly planted due to its effectiveness in alleviating noise pollution. It exhibits symmetrical pyramidal growth with willowy weeping pendulous branches and long narrow lanceolate leaves with undulate margins. The tree is known to grow over 30 ft in height. The leaf extract is known to have mainly di terpenoids, alkaloids, tannins, and mucilage and to have pesticidal properties.
Extraction of Polyalthia longifolia using ethyl acetate:
1. Powder 25g of leaves 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear filtrate, dispose the insoluble mass 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: 1% Ammonium acetate: Acetonitrile (60:40)
Flow rate: 1ml/min Wave length: 254nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
22
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
1.48 g 3.15 g
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
mAU
0
50
100
150
200
250
300
350
400
mAU
0
50
100
150
200
250
300
350
400
1 1
.845
515
0019
2
2.2
19 1
3232
79
1: 300 nm, 8 nmJS-167-5-10 SAMPLE BTRC 101
Pk #Retention TimeAreaName
21. Pongamia pinnata
Introduction: Honge (Pongamia pinnata or Millettia pinnata) is a tree, native to tropical and temperate Asia including parts of India. The qualitative chemical test performed revealed the presence of alkaloids, glycosides, flavonoids, saponins, carbohydrates, phenolic compounds, tannins and fats. The metabolites, beta-sitosteryl acetate and galactoside, stigma sterol, its galactoside and sucrose are being reported for the first time from this plant. The saturated and unsaturated fatty acids (two monoenoic, one dienoic and two trienoic) were present in exactly the same amount. Oleic acid occurred in highest amount (44.24%), stearic (29.64%) and palmitic (18.58%) acids were the next in quantity.
Protocol:
Extraction of Pongamia pinnata using ethyl acetate:
1. Powder 25g of leaves 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: Methanol:Water:Acetic acid(70:30:1.5)
Flow rate: 1 ml/min
Wave length: 300nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
23
NMR of Isolanchocarpin compound
Mass spectrum of Karanjin
Mass spectrum of Pongamol
24
Mass spectrum of Pongapin
Mass spectrum of Isolonchocarpin
25
Crude extract obtained from 25 g of seeds Ethyl acetate
extract Water extract
1.0 g 2.95 g
Minutes
0 2 4 6 8 10 12 14 16 18 20
mA
U
0
50
100
150
200
250
300
mA
U
0
50
100
150
200
250
300
12.3
0
33.0
8
5.44
4.
79
16.7
9
6.27
16.2
1
1.58
2.
39
0.23
0.26
0.65
1: 254 nm, 8 nmcastor Seed extractc 109
Area PercentName
22. Ricinus communis
Introduction: The castor oil plant (Ricinus communis) is a indigenous to the southeastern Mediterranean Basin, Eastern Africa, and India, but is widespread throughout tropical regions. Castor seed is the source of castor oil, which has a wide variety of uses. A study was carried out to investigate the chemical composition of castor plant seed. The chemical composition include: the proximate analysis and the mineral analysis. The parameters under proximate analysis include crude protein, crude fat, crude fiber, total ash, nitrogen free extract, and dry matter while calcium, potassium, sodium, magnesium, and phosphorus were parameters under the mineral analysis. The proximate analysis revealed that the seed contain valuable nutrients including protein, fat . it is the only source of an 18-carbon hydroxylated fatty acid with one double bond ricinoleic acid (12-Hydroxyoleic Acid) comprises approximately 90% of the fatty acid composition.
Protocol:
1. Powder 25g of castor seeds 2. Charge the powder into 500 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get ethyl acetate layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: 1% Orthophosphoric acid: Organic solvents (70:30)
Flow rate: 1 ml/min
Wave length: 254 nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
26
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
2.15 g 3.69 g
Minutes
0 2 4 6 8 10 12 14 16 18 20
mA
U
0
200
400
600
800
1000
1200
1400m
AU
0
200
400
600
800
1000
1200
1400
2.20
8 2
0.35
2.40
0 7
3.95
3.47
7 3
.03
3.74
4 1
.66
8.62
9 0
.38
8.71
5 0
.63
1: 210 nm, 8 nmtrc/44/52 water extracts 111
Retention TimeArea Percent
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24
mA
U
0
500
1000
1500
2000
2500
mA
U
0
500
1000
1500
2000
2500
2.40
0 8
0.06
2.85
9 5
.67
3.05
1 1
.25
5.60
0 4
.88
6.83
7 1
.86
8.13
9 1
.30
8.60
8 2
.38
14.0
91 2
.60
1: 210 nm, 8 nmtrc/44/52 ethyl acetate extracts 115
Retention TimeArea Percent
23. Stevia rebaudiana
Introduction: Stevia (Stevia rebaudiana) is a genus of about 240 species of herbs and shrubs, native to subtropical and tropical regions from western North America to South America. With its steviol glycoside extracts having up to 300 times the sweetness of sugar; stevia has garnered attention with the rise in demand for low-carbohydrate, low-sugar food alternatives. Because stevia has a negligible effect on blood glucose, it is attractive as a natural sweetener to people on carbohydrate-controlled diets. The Stevia rebaudiana contains a complex mixture of labdane diterpenes, triterpenes, stigmasterol, tannins, volatile oils, and eight diterpenenic glycosides: stevioside, steviobioside, dulcoside, and rebaudiosides A, B, C, D, and E. the most abundant substances are stevioside and rebaudioside A. Of the stevia glycosides rebaudioside A is the sweetest and the most stable, and it is less bitter than stevioside. Rebaudioside E is as sweet as stevioside, and rebaudioside D is as sweet as rebaudioside A, while the other glycosides are less sweet than stevioside.
Protocol:
1. Powder 25g of leaves 2. Charge the powder into 500ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: 0.1% Orthophosphoric acid: Acetonitrile (20:80)
Flow rate: 1 ml/min
Wave length: 210 nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
27
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
1.63 g 3.76 g
Minutes
0 5 10 15 20 25 30 35 40 45 50
mAU
-25
0
25
50
75
100
125
150
175
200
225
1 1
1.07
2 0
.35
2 1
1.55
2 0
.58
3 1
2.13
9 1
.62
4 1
3.25
9 0
.47
5 1
5.69
1 0
.50
6 1
6.13
9 1
4.08
7 1
6.73
6 1
.71 8 1
7.17
3 6
.65
9 1
7.35
5 2
.54
10 1
7.66
4 2
2.32
11 1
8.03
7 3
3.58
12 1
9.00
8 1
.99
13 1
9.93
6 3
.02
14 2
0.57
6 5
.33
15 3
1.60
5 2
.09
16 3
4.37
9 2
.03
17 3
5.79
7 0
.77
18 3
9.08
3 0
.35
2: 254 nm, 8 nmjs/167/8/10 sample a methTRC 151
Pk #Retention TimeArea Percent
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mAU
-20
0
20
40
60
80
100
120
140
160
180
200
1 2
.453
2.9
12
2.8
91 1
4.96
3 3
.819
0.5
74
4.2
13 0
.99
5 4
.363
5.3
4
6 5
.483
12.
87
7 7
.200
15.
058
7.7
12 1
6.65
9 8
.128
5.1
710
8.6
51 1
.29
11 8
.917
6.3
212
9.5
15 1
.29
13 1
1.06
1 1
.50
14 1
1.34
9 3
.15
15 1
1.53
1 5
.41
16 1
1.96
8 0
.83
17 1
2.58
7 0
.56
18 1
2.65
1 2
.24
19 1
3.17
3 1
.67
20 1
6.83
2 1
.25
24. Tridax procumbens
Introduction: Tridax (Tridax procumbens) is a flowering plant, best known as a widespread weed. It is native to the tropical Americas but it has been introduced to tropical, subtropical, and mild temperate regions worldwide. The number of compounds, including three flavones: 8,3′-dihydroxy-3,7,4′-trimethoxy-6-O-β-D-glucopyranosyl flavone ,6,8,3′-trihydroxy-3,7,4′-trimethoxyflavone ,and puerarin ,one coumarin, esculetin , and two triterpenoids: oleanolic acid andbetulinic acid are reported from this plants.
Protocol:
1. Powder 25g of leaves 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: A-Water, B-methanol
Gradient elution
Flow rate: 1ml/min,Wave length: 254 nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
28
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
6.40 g 5.54 g
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mA
U
-2
0
2
4
6
8
10
1 2
.112
0.8
0
2 3
.872
1.8
83
4.3
84 0
.22
4 4
.821
0.3
75
5.4
08 0
.60
6 5
.611
0.2
97
6.1
65 0
.74
8 7
.029
0.3
39
7.8
72 0
.93
10 1
5.05
1 2
0.97
11 1
8.82
7 6
4.25
12 2
4.05
3 8
.63
1: 258 nm, 8 nmJS/167/16/10 aTRC 132
Pk #Retention TimeArea Percent
Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mAU
0
10
20
30
40
50
1 2
.293
1.4
72
2.6
77 0
.33
3 2
.976
0.7
54
3.1
36 0
.61
5 3
.285
1.0
86
3.9
68 1
.86
7 4
.309
0.2
18
4.5
12 0
.22
9 4
.992
0.4
610
5.5
89 1
.19
11 5
.835
0.7
412
6.4
21 1
.07
13 6
.912
0.3
5
14 1
2.10
7 1
.28
15 1
3.93
1 1
.26
16 1
5.95
7 1
.81
17 1
8.19
7 1
0.40
18 2
1.98
4 4
0.67
19 2
4.91
7 3
4.27
1: 258 nm, 8 nmJS/167/16/10 bTRC 131
Pk #Retention TimeArea Percent
25. Vitex negundo
Introduction: The chaste tree (Vitex negundo) is a large aromatic shrub, used in folk medicine, particularly in South and Southeast Asia. From the leaves, seven compounds were isolated and identified, by the use of various spectroscopic methods, to be mixture of the flavonoids luteolin, luteolin-3’-O-glucuronide, and isoorientin, the iridoid glycosides 2’-p-hydroxybenzoylmussaenosidic acid and agnuside, and phydroxyl benzoic acid as well as stigmasterol and β-sitosterol. They were identified as negundoside, agnuside, vitegnoside, 7,8 dimethyl herbacetin 3-rhamnoside,5,3'-dihydroxy—7,8,4'-trimethoxy flavanone, 5-hydroxy-3,6,7,3',4'-pentamethoxy flavone, 5,7 dihydroxy- 6,4' dimethoxy flavonone, and 5 hydroxy-7,4' dimethoxy
flavone.
Protocol:
1. Powder 25g of leaves 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: Methanol: 2%Acetonitrile (30:70)
Flow rate: 1ml/min
Wave length: 254nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash
29
Crude extract obtained from 25 g of leaves Ethyl acetate
extract Water extract
1.48 g 5.45g
Minutes
0 5 10 15 20 25 30 35 40 45 50
mA
U
0
5
10
15
20
25
mA
U
0
5
10
15
20
25
1 2
.016
210
27
2 2
.187
183
11
3 2
.325
259
71
4 2
.624
119
87
5 3
.392
792
0
6 3
.744
212
3
7 4
.181
270
8
8 4
.309
365
9
9 7
.296
332
24
10 1
1.48
8 1
9056
11
11.
893
937
3
12 1
7.22
7 3
9902
13 2
6.33
6 4
2696
14 2
9.92
0 1
5120
15 3
6.16
0 6
4152
1: 254 nm, 8 nmJS/167/17/10 Sample ATRC 113
Pk #Retention TimeAreaName
Minutes
0 5 10 15 20 25 30 35 40 45 50
mA
U
0
20
40
60
80
100
120
140
160
mA
U
0
20
40
60
80
100
120
140
160
1 2
.197
162
720
2
2.3
25 1
0562
0
3 3
.733
174
12
4 4
.171
318
18
5 4
.597
992
081
6 5
.184
124
240
7
5.5
79 1
8963
1
8 6
.507
29
9 1
1.55
2 1
2538
3
10 1
2.03
2 8
6052
11 1
3.53
6 1
7361
12 1
7.22
7 1
1804
6
13 2
9.85
6 1
1689
4
14 3
6.14
9 8
7544
3
1: 254 nm, 8 nmJS/167/17/10 Sample ATRC 114
Pk #Retention TimeAreaName
26. Zingiber officinale
Introduction: Ginger (Zingiber officinale) is consumed as a delicacy, medicine, or spice. A number of compounds present in ginger like gingerdione dimers, bisgingerdiones A and B ; two new gingerol derivatives, (5R)-5-acetoxy-1,7-bis(4-hydroxy-3-methoxyphenyl)heptan-3-one and methyl (Z)-neral acetal-[6]-gingerdiol and present number of alkaloid, steroids,phlobotannins, flavanoids, glycosides, saponins, tannin and terpenoids .
Protocol:
1. Powder 25g of rhizomes. 2. Charge the powder into 250 ml RB flask 3. Add 5 volume of Ethyl acetate/water 4. Stir the mixture at 60° C for 3 hrs 5. Filter to get clear layer 6. Distil under vacuum to get crude material
Analytical information
Column: C18, 25cmlength, 4.6mm diameter, Inertsil ODS 3V
Mobile phase: Acetonitrile:Water(50:50)
Flow rate: 1 ml/min
Wave length:254 nm
ETHYL ACETATE EXTRACT WATER EXTRACT
Scientists involved: Dr.G.C.Reddy, Mr. Shivaprakash, Mr. Karunakar A.C., Mr.Jasbir Singh
Incharge Scientist: Mr. Shivaprakash