extraction and analytical procedures for botanical compounds · 2016. 2. 22. · annona squamosa 5...

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


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

http://en.wikipedia.org/wiki/Asia

http://en.wikipedia.org/wiki/Central_India

http://en.wikipedia.org/wiki/Alkaloids

http://en.wikipedia.org/wiki/Indian_Subcontinent

http://en.wikipedia.org/wiki/Saponin

2

Crude extract obtained from 25 g of roots Ethyl acetate


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


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


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


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


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



Mobile phase: 0.1M KH2PO4:Acetonitrile:Acetic acid(85:15:1)

Flow rate: 0.7ml/min

Wave length: 300nm




http://en.wikipedia.org/wiki/Medicinal_plant

http://en.wikipedia.org/wiki/Shrub

5

Crude extract obtained from 25 g of seeds Ethyl acetate


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


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



Mobile phase: Methanol:Water(90:10)

Flow rate: 1. ml/min

Wave length: 220nm




http://en.wikipedia.org/wiki/Tree


http://en.wikipedia.org/wiki/Sugar-apple

http://en.wikipedia.org/wiki/Sugar-apple

6



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


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


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.



Mobile phase: A-50mM KH2PO4:K2HPO4, (pH7.7)

B-Acetonitrile A: B (50:50)

Flow rate: 1ml/min

Wave length: 280nm


http://en.wikipedia.org/wiki/Mexico

http://en.wikipedia.org/wiki/Naturalisation_%28biology%29

http://en.wikipedia.org/wiki/United_States

http://en.wikipedia.org/wiki/India

http://en.wikipedia.org/wiki/Ethiopia

http://en.wikipedia.org/wiki/Sap

http://en.wikipedia.org/wiki/Alkaloid

http://en.wikipedia.org/wiki/Sanguinarine

http://en.wikipedia.org/wiki/Dihydrosanguinarine

http://en.wikipedia.org/w/index.php?title=Dehydrocorydalmine&action=edit&redlink=1

http://en.wikipedia.org/wiki/Jatrorrhizine

http://en.wikipedia.org/w/index.php?title=Columbamine&action=edit&redlink=1

http://en.wikipedia.org/w/index.php?title=Oxyberberine&action=edit&redlink=1

7



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


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


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



Mobile phase: Acetonitrile:Water(35:65)


Wave length: 215nm




8



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


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


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



Mobile phase: 0.1% Orthophosphoric acid:Acetonitrile (20:80)

Flow rate: 1 ml/min

Wave length: 205 nm


http://en.wikipedia.org/wiki/Asia

9



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


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


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



Mobile phase: Methanol: Water (90:10)

Flow rate: 1ml/min

Wave length: 254nm&280nm


Scientists involved: Dr.G.C.Reddy, Mrs. Suvarna, Mr. Karunakar A.C., Mr.Jasbir Singh


http://en.wikipedia.org/wiki/Bougainvillea_glabra

http://en.wikipedia.org/wiki/Flowering_plant

http://en.wikipedia.org/wiki/South_America

http://en.wikipedia.org/wiki/Brazil

http://en.wikipedia.org/wiki/Peru

http://en.wikipedia.org/wiki/Argentina

10



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


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



Mobile phase: Acetonitrile:Water (60:40)

Flow rate: 1 ml/min

Wave length: 254nm




http://en.wikipedia.org/wiki/Indian_Subcontinent

http://en.wikipedia.org/wiki/Southeast_Asia


11



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


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



Mobile phase: 1% Ammonium acetate: Acetonitrile (60:40)

Flow rate: 1ml/min

Wave length: 254nm




http://en.wikipedia.org/wiki/Madagascar

http://en.wikipedia.org/wiki/Basionym

http://en.wikipedia.org/wiki/Slash_and_burn

http://en.wikipedia.org/wiki/Agriculture

http://en.wikipedia.org/wiki/Naturalisation_%28biology%29

12



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


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


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



Mobile phase: Methanol: Water:Acetic acid 70:30:1.5

Flow rate: 1ml/min, Wave length: 270nm




13



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


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


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:




Mobile phase: Methanol: Water:Acetic acid 70:30:1.5

Flow rate: 1ml/min





14

Crude extract obtained from 25 g of rhizomes Ethyl acetate


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


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



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



Mobile phase: A-10Mm KH2PO4, pHadjusted to 2.8 using H3PO4

B-Methanol, A: B (25:75)


Wave length: 254nm, 345&280nm




http://en.wikipedia.org/wiki/Tropical

http://en.wikipedia.org/wiki/South_Asia

http://en.wikipedia.org/wiki/Rhizome

http://en.wikipedia.org/wiki/Curcumin

http://en.wikipedia.org/wiki/Black_pepper

15



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


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


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




Mobile phase: Methanol: Water pH 70:30

Flow rate: 1ml/min

Wave length: 254nm



16



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



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


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:




Mobile phase: A-95:5 Water:Acetonitrile, pH adjusted to 2.8 using H3PO4

B-Methanol A: B (10:90)

Flow rate: 2ml/min



IR spectrum of 3-keto-24-formyl –olean-12-en-28-oic acid



http://en.wikipedia.org/wiki/Americas

http://en.wikipedia.org/wiki/Tropics

http://en.wikipedia.org/wiki/Introduced_species

http://en.wikipedia.org/wiki/Ornamental_plant

http://en.wikipedia.org/wiki/Invasive_species

17

Crude extract obtained from 25 g of fruits Ethyl acetate


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


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


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




18



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



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


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 :





Flow rate: 1 ml/min

Wave length:254 nm




http://en.wikipedia.org/wiki/Ayurveda

19



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


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


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:




Mobile phase: A-10mM KH2PO4,pH adjusted to 3.0 using H3PO4

B-Acetonitrile

Flow rate: 1.5ml/min, Linear gradient, Wave length: 210nm




http://en.wikipedia.org/wiki/Americas

http://en.wikipedia.org/wiki/Neotropic_ecozone

20



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



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


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



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




http://en.wikipedia.org/wiki/Flowering_plant

http://en.wikipedia.org/wiki/Vine

http://en.wikipedia.org/wiki/Fruit

http://en.wikipedia.org/wiki/Spice

http://en.wikipedia.org/wiki/Seasoning

21

Crude extract obtained from 25 g of roots Ethyl acetate


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


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


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



Mobile phase: 1% Ammonium acetate: Acetonitrile (60:40)

Flow rate: 1ml/min Wave length: 254nm




22



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


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:




Mobile phase: Methanol:Water:Acetic acid(70:30:1.5)

Flow rate: 1 ml/min

Wave length: 300nm




23

NMR of Isolanchocarpin compound

Mass spectrum of Karanjin

Mass spectrum of Pongamol

24

Mass spectrum of Pongapin

Mass spectrum of Isolonchocarpin

25



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



Mobile phase: 1% Orthophosphoric acid: Organic solvents (70:30)

Flow rate: 1 ml/min

Wave length: 254 nm




http://en.wikipedia.org/wiki/Mediterranean_Basin

http://en.wikipedia.org/wiki/Eastern_Africa


http://en.wikipedia.org/wiki/Castor_oil

26



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


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


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



Mobile phase: 0.1% Orthophosphoric acid: Acetonitrile (20:80)

Flow rate: 1 ml/min

Wave length: 210 nm




http://www.google.co.in/imgres?q=stevia&num=10&hl=en&biw=1272&bih=842&tbm=isch&tbnid=s9UP6RiJ7CdJkM:&imgrefurl=http://gardenoftomorrow.com/stevia/stevia-stevia-rebaudiana-the-all-natural-sugar-free-sweetener-234/attachment/stevia-common/&docid=c59e4ulpGojfnM&imgurl=http://gardenoftomorrow.com/wp-content/uploads/2011/04/Stevia-Common.jpg&w=2288&h=1712&ei=w4DtT-LUEYzIrQexn9i9DQ&zoom=1&iact=hc&vpx=193&vpy=474&dur=110&hovh=194&hovw=260&tx=159&ty=78&sig=117286847766368026227&page=2&tbnh=156&tbnw=210&start=24&ndsp=30&ved=1t:429,r:0,s:24,i:217

http://en.wikipedia.org/wiki/Genus

http://en.wikipedia.org/wiki/Species

http://en.wikipedia.org/wiki/Herbaceous_plant


http://en.wikipedia.org/wiki/Subtropical

http://en.wikipedia.org/wiki/Tropical

http://en.wikipedia.org/wiki/North_America

http://en.wikipedia.org/wiki/South_America

http://en.wikipedia.org/wiki/Steviol_glycoside

http://en.wikipedia.org/wiki/Sweetness

http://en.wikipedia.org/wiki/Blood_glucose

http://en.wikipedia.org/wiki/Low-carbohydrate_diet

http://en.wikipedia.org/wiki/Low-carbohydrate_diet

27



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


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:




Mobile phase: A-Water, B-methanol

Gradient elution

Flow rate: 1ml/min,Wave length: 254 nm




http://en.wikipedia.org/wiki/Noxious_weed

28



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


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


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:




Mobile phase: Methanol: 2%Acetonitrile (30:70)

Flow rate: 1ml/min

Wave length: 254nm




http://en.wikipedia.org/wiki/Essential_oil


http://en.wikipedia.org/wiki/Folk_medicine

http://en.wikipedia.org/wiki/South_Asia


29



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



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



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




Flow rate: 1 ml/min

Wave length:254 nm




http://www.google.co.in/imgres?q=ginger&num=10&hl=en&biw=1272&bih=842&tbm=isch&tbnid=1Vmc8vBL4jha2M:&imgrefurl=http://originalfilipinorecipes.blogspot.com/2012/04/salabat.html&docid=ErvG4AuhZZC7aM&imgurl=http://3.bp.blogspot.com/-z60AmaoBrS4/T4VtKHzzlwI/AAAAAAAAAIs/xJV0_rFFdYA/s1600/ginger.jpg&w=454&h=340&ei=ToDtT-7yE9DHrQfv05S-DQ&zoom=1&iact=hc&vpx=593&vpy=501&dur=906&hovh=194&hovw=259&tx=148&ty=136&sig=117286847766368026227&page=2&tbnh=145&tbnw=193&start=24&ndsp=25&ved=1t:429,r:7,s:24,i:252

http://en.wikipedia.org/wiki/Delicacy

http://en.wikipedia.org/wiki/Medicine

http://en.wikipedia.org/wiki/Spice

extraction and analytical procedures for botanical compounds · 2016. 2. 22. · annona squamosa 5...

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