isolation of novel bioactive diterpenoids from -...

CHAPTER-V

Isolation of novel bioactive diterpenoids from Jatropha multifida

5.1. AN INTRODUCTION TO JATROPHA

Jatropha is a non-edible oil seed plant from Euphorbiaceae family.

The word Euphorbiaceae is a Greek word, euphorbus, which means a

physician who was supposed to use the species’ milky latex for

medicinal purposes,1 which clearly indicates the medicinal importance

of this family.2-5

Classification

Division: Magnoliophyta

Class: Magnoliopsida

Subclass: Rosidae

Order: Euphorbiales

Family: Euphorbiaceae

Genus: Jatropha

Euphorbiaceae, also known as spurge family due to the use of

plants’ sap as a purgative (laxative), is a large family of flowering

plants with 321 genera and around 7550 species.6 The plants of this

family are an important source of medicines and toxins. One of the

major metabolites, diterpenoids, possess therapeutic potentiality as

antibacterial, antihypertensive, anti-inflammatory, antileukemic,

antioxidants, antiretroviral, antitumor, analgesic, cytotoxic,

hallucinogens, sweeteners and may stimulate contraction of the

uterus.2-5,7 Apart from diterpenoids, Euphorbiaceae family is a source

of alkaloids, coumarins, coumarino-lignoids, cyclic peptides,

flavonoids and steroids. Jatropha is one of the largest genuses

belonging to the Euphorbiaceae family.

The genus Jatropha is widespread in the tropical regions of the

world such as Americas, Africa and parts of the Asian subcontinent.

The word Jatropha is derived from a Greek word Jatras=Doctor,

Trophe=Nutrition/food. Jatropha is a genus of about 200 species that

are succulent plants, shrubs and trees. Since ages, the extracts from

different parts such as root, stem, bark and leaves of the Jatropha

plant have been used in ethno-medicines.8 It is a rich source of

phytochemicals such as alkaloids, terpenoids, lignoids and cyclic

peptides having a broad range of biopotency.9 The phytochemicals can

also be utilized in agricultural, nutritional and pharmaceutical

industries.10 Jatropha possess various metabolites of which terpenoids

are the major part.

5.1.1. TERPENOIDS

Terpenoids, a subclass of prenyllipids, are derived from five-carbon

isoprene units. These form a large and diverse class of naturally-

occurring organic chemicals. Terpenoids contribute to the flavors of

cinnamon, cloves, the scent of eucalyptus, and ginger, and the color of

yellow flowers. Well-known terpenoids include citral, menthol,

camphor etc. Based on the number of isoprene units present in the

basic molecular skeleton, terpenes can be classified as below:

Table 1.1: Classification of Terpenes

Jatropha species are a rich source of terpenoids among which

diterpenoids have dominated due to their novel chemical entities and

medicinal activities. There are around 69 diterpenoids which were

isolated from Jatropha species, most of them are tricyclic and

tetracyclic. The diterpenoids from Jatropha species can be categorized

as below.

Deoxypreussomerins

Rhamnofolane Lathyrane tigliane dinorditerpenepimarane

JatrophaDiterpenoids

Daphnane

Fig. 1.1

The structures for the above classified diterpenoids can be found from

Table 1.2

5.1.2. REPORTED BIOLOGICAL ACTIVITY OF JATROPHA SPECIES

The in-vitro studies of the Jatropha species revealed the biopotency

of secondary metabolites that play a vital role in the field of medicine

and biology. A few Jatropha species with their biopotency are

mentioned below.

S. No Terpenes Isoprene units

Carbon atoms

1 Monoterpenes 2 10

2 Sesquiterpenes 3 15

3 Diterpenes 4 20

4 Sesterpenes 5 25

5 Triterpenes 6 30

6 Carotenoids 8 40

7 Rubber >100 >500

5.1.2.1. Antibacterial Activity:

Solvent extracts of J. multifida from the root and bark effectively

inhibited the growth of Bacillus subtilis and Staphylococcus aureus at

a concentration of 200 mg/disk.11 Japodagrin, a macrocyclic

diterpenoid from the roots of J. podagrica exhibited antibacterial

activity against Bacillus subtilis (ATCC 6051) and Staphylococcus

aureus (ATCC25923) with an inhibitory zone of 16 and 12 mm at 20

lg/disk. (4Z)-Jatrogrossidentadione, another diterpenoid with

jatrophane skeleton, also from J. podagrica displayed antibacterial

activity against some Gram-positive bacteria.12

O

O

HO

15

HOO

Japodagrin

O

OHO

HO

15

Jatrogrossidentadione

5.1.2.2. Anticancer and Antileukemic Activity:

Out of several compounds isolated from Jatropha species, much of

the attention was paid to diterpenes because of their antitumor

activity. Jatrophone, isolated from J. gossypifolia was found to

possess antileukemic and antinasopharyngeal carcinoma activity.13

O

O

O

H

Me

Jatrophone

O

O

O

H

H

Jatrophatrione

Jatrophatrione, a tricyclic diterpene isolated from chloroform

extracts of J. macrorhiza roots has tumor inhibitory effect (0.5 mg/kg)

and is particularly active against the in-vitro P338 (3PS) lymphocytic

leukemia test system.14 Compounds citlalitrione and riolozatrione

showed a curing effect against the skin cancer.15

Jatropham, a lactam and acetylaleuritolic acid, a tritepane have

shown tumor inhibitory properties against the P-388 lymphocytic

leukemia test system.16, 17

NOHO

H AcO

H

COOH

Jatropham Acetylaleuritolic acid

5.1.2.3. Anti-Inflammatory Activity:

Topical application of J. curcas root powder in paste form in TPA-

induced ear inflammation confirmed anti-inflammatory activity in

albino mice. Such an activity might be due to effects on several

mediators and arachidonic acid metabolism involving cyclo-oxygenase

pathway resulting in prostaglandin formation anti-proliferative activity

leading to reduction in granular tissue formation and leukocyte

migration from the vessels.18

5.1.2.4. Anticoagulant and Coagulant Activities:

J. curcas is traditionally used as a haemostatic. Investigation of the

coagulant activity of the latex of J. curcas showed a significant

reduction in the clotting time of human blood.19 However, diluted latex

prolonged the clotting time, and at high dilutions the blood did not

clot at all. This result indicated that latex from J. curcas possesses

both procoagulant as well as anticoagulant activities. Solvent

partitioning of the latex with ethyl acetate and butanol led to a partial

separation of the two opposing activities: the ethyl acetate fraction

exhibited a procoagulant activity, whereas the butanol fraction had

the highest anticoagulant activity at low concentrations.

5.1.2.5. Antidiarrheal Activity:

J. curcas roots were subjected to pharmacognostic studies and

evaluation of antidiarrheal activity in albino mice.20 The MeOH extract

from the roots showed activity against castor oil-induced diarrhea and

intraluminal accumulation of fluid. It also had an effect on the

reduction of gastrointestinal motility after charcoal meal

administration in albino mice. These results indicated that the

mechanistic action of MeOH extract could be through a combination

of inhibition of elevated prostaglandin biosynthesis and reduced

propulsive movement of the small intestine.

5.1.2.6. Pregnancy-Terminating Effect:

The fruit of J. curcas was investigated for the fertility regulatory

effect by oral administration of different extracts to pregnant rats for

varying periods of time. The results showed fetal resorption with

hexane, MeOH, and CH2Cl2 extracts, indicating the abortifacient

properties of this fruit.21

5.1.2.7. Other Activities:

There are also other activities of Jatropha plants. The EtOH extract

from J. gossypiifolia L. in rats showed hypotensive and vasorelaxant

effects.22 Oral administration of EtOH extract (EE; 125 or 250 mg

kg/d) caused a significant reduction of the systolic blood pressure.

The expensive antimalarial drugs except chloroquine are not readily

accessible to most people in malaria-endemic countries. This created

an interest in the development of herbal medicines with the potential

to treat malaria with little or no side effects. AM-1, a formulation from

J. curcas, had shown to eliminate malaria parasites (Plasmodium

falciparum and P. malarie) from the peripheral blood of patients with

malaria without any side effects in the patients as well as in

laboratory rats. Further studies need to be carried out for possible

drug interactions.23

Jatrogrossidion, a diterpene from Jatropha grossidentata showed a

strong in vitro leishmanicidal and trypanocidal activity with IC100 0.75

and 1.5-5.0 µg/ml. when tested against Leshmania and Trypanosoma

cruzi strains in vitro as well as against Leishmania amazonensis in

vivo.24

O

HO

O

Jatrogrossidion

5.1.2.8. Antiviral Properties:

The H2O extract of the branches of J. curcas has shown to inhibit

strongly the HIV-induced cytopathic effects with low cytotoxicity.2

5.2. REPORTED COMPOUNDS FROM GENUS JATROPHA

The genus Jatropha witnessed the production of various types of

bioactive complex metabolites. Major chemical constituents from the

Jatropha species belong to the following class (Fig. 5.2):

major chemicalconstituents fromJatropha species

Diterpenoids

Alkaloids

Flavonoids

Coumarins and lignanes

Triterpenoids and sesquiterpenoids

Phytosterols

Cyclic peptides

Miscellaneous

_

Fig. 5.2 Diterpenoids form the major class of isolated phytochemicals from

Jatropha multifida. Below (Table 5.2) are the diterpenoids isolated

from various Jatropha species.

Table 5.2: Diterpenoids from Jatropha species S.No Species

Diterpenoid Str. Bioactivity Ref

1 J. curcas

acetoxyjatropholone 1 Antiproliferative activity

26

2 J. curcas Spirocurcasone 2 Practically antiproliferative inactive

26

3 J. curcas Curcusone-E 3 Practically antiproliferative inactive

26

4 J. curcas Curcasone-A 4 Antiinvasive effects in tumor cells

27

5 J. curcas Curcasone-B 5 Antiinvasive effects in tumor cells

27

6 J. curcas

Curcasone-C 6 Cytotoxic 27

7 J. curcas Curcasone-D 7 Cytotoxic 27

8 J. curcas Jatrophodione-A 8 NR 28

9 J. curcas Jatrophol 9 NR 29

10 J. curcas Curculathyrane-A 10 NR 30

11 J. curcas Curcalathyrane-B 11 NR 30

12 J. curcas 15-O-acetyl-15-epi-(4E) Jatrogrossidentadione

12 NR 31

13 J. curcas Jatrothrin 13 NR 31

14 J. curcas Podocarpane-I 14 NR 31

15 J. curcas Podocarpane-II 15 NR 31

16 J. curcas Jatromerin-I 16 NR 32

17 J. curcas Jatromerin-II 17 NR 32

18 J. curcas (14E)-14-O-acetyl-5,6- epoxyjatrogrossidentadion

18 NR 31

19 J. curcas 3β-acetoxy-12-methoxy-13-methyl-podocarpa- 8,11,13-trien-7-one

19 NR 31

20 J. curcas 3β,12-dihydroxy-13-methyl-podocarpane-8,10,13-triene

20 NR 31

21 J. curcas Jatropherol 21 Insecticidal, Rodenticidal

33

22 J. curcas Jatropha factor C1 Jatropha factor C2 Jatropha factor C3 Jatropha factor C4 Jatropha factor C5 Jatropha factor C6

22 23 24 25 26

27

Cytotoxic,

Molluscicidal,

Rodenticidal

34

23 J. curcas Heudolotinone 28 NR 35

24 J. curcas Jatropha lactam 29 cytotoxic 36

25 J. curcas Palmarumycin CP1 Palmarumycin JC1 Palmarumycin JC2

30 31 32

Antibacterial

32

26 J. dioica

Riolozatrione 33 Antibacterial 37

27 J .divaricata Cleistanthol 34 Antitumor 38

28

J. divaricata ent-3β,14α-hydroxy-pimara-7,9(11),15- triene-12-one

35 NR 38

29 J .divaricata ent-15(13→8)abeo- 8β (ethyl)pimarane

36 NR 38

30 J .divaricata Spruceanol 37 Cytotoxic Antitumor

38,39

31 J. elliptica Jatrophone 38 Antitumor, Cytotoxic Molluscicidal Leishmanicidal

40

32 J. gaumeri 2-epi-Jatrogrossidione 39 Antimicrobial 41

33 J. gaumeri 15-epi-4E-jatrogrossidentadione

40 NR

41

34 J. gossypifolia Jatrophone 38 Antitumor 13

35 J. gossypifolia 2α-OH Jatrophone 41 Cytotoxic 42

36 J. gossypifolia 2β-OH Jatrophone 42 Cytotoxic 42

37 J. gossypifolia 2β-OH-5,6-isoJatrophone

43 Cytotoxic 42

38 J. gossypifolia Citlalitrione 44 NR 43

39 J. gossypifolia Jatropholone-A 45 Gastroprotection Cytotoxic Molluscicidal Antiplasmodial

44

40 J. gossypifolia Jatropholone-B 46 Gastroprotective effect molluscicidal

44

41 J. gossypifolia Jatrophenone

47 Antibacterial 45

42 J. grossidentata Jatrogrossidione 48 Leishmanicidal Trypanocidal

46

44 J. grossidentata Isojatrogrossidion 49 NR 47

45 J. grossidentata 2-epi-isojatrogrossidion

50 NR 47

46 J. grossidentata 2-Hydroxyiso-jatrogrossidion

51 Antibacterial Antifungal

47

47 J. grossidentata 2-epi-hydroxyiso-jatrogrossidion


47

48 J. grossidentata 15-epi-4E-jatrogrossidentadione

40 NR 47

49 J. grossidentata (4Z)-Jatrogrossidentadion


47

50 J. grossidentata (4Z)- 15- Epijatrogrossidentadion


47

51 J. integerrima

Integerrimene 55 NR 48

52 J. integerrima 1, 11 bisepicaniojane 56 Antiplasmodial 48, 49

53 J. integerrima 2-epicaniojane 57 NR 48, 49

54 J. integerrima 2α-Hydroxyjatropholone

58 Antibacterial Antiplasmodial

49

55 J. integerrima 2β-hydroxyjatropholone

59 Antibacterial Cytotoxic

49

56 J. multifida 15-epi-(4E)-jatrogrossidentadione acetate

60 NR 50

57 J. multifida Multifidone 61 cytotoxic 51

58 J. multifida 15-O-Acetyl japodagrone

62 NR 52

59 J. multifida Cp 2 63 NR 52

60 J. multifida Multidione 64 NR 53

61 J. multifida Multifolone

65 NR 54

62 J. multifida 6-O-Acetyl-(4E)-jatrogrossidentadione

66 NR 54

63 J. podagrica Japodagrin 67 Antibacterial 12

64 J. podagrica Japodagrone 68 Antibacterial 12

65 J. podagrica Japodagrol 69 Antitumor 55

66 J. wedelliana Jatrowedione 70 NR 56

67 J. wedelliana Jatrowediol 71 NR 57

68 J. zeyheri Jaherin 72 Antibacterial 58

NR: Not Reported

5.2.1. Structures of Diterpenoids

OH

O O

OH

H

1

OH H

O

2

OHH

HOO

3

O

O

R'

R2

4. R1= Me, R2= H

5. R1= H, R2= Me

6. R1= Me, R2= OH

7. R1= OH, R2= Me

8

O

HO

O HO

H

9

OH

O

OH

10

O

OHO

HO

11

O

O O

HO

12

O

O

OH

AcO

13

OAc

OO

14

OMe

OAcO

15

HO

OH

O

O

O

OH

OR

H

16. R=H17. R=Ac 18

O O

H

AcOH

H

H

19

O

HH

AcOO

20

OH

HH

HO

21

OOH

OH

OH

OH

22

O

O

O

O

(C13)

(C16)

23

O

O

(C16)

O

O

(C13)

24

O

O

O

O

(C13)

(C16)

25, 26 (inseparable mixture)

O

O

O

O

(C13)

(C16)

27O

O(C13)

(C16)

O

O

28

OH

OH

29

HN

O

H

O

HO

30

O O

O OH

31

O O

OH OH

O

32

O O

O OH

HO

33

OH

O

O

H

H

34

HO

H

HO

OH

35

HO

OH

O

H

36

O

HO

H

37

OH

H

H

HO

O

O

O

R'

R2

38. R1= H, R2= Me

41. R1= Me, R2= OH

42. R1= OH, R2= Me

R1

O

R2

39. R1= H, R2= Me

48. R1= Me, R2= H

40

OH

H

O

HO

15

HO

43

OH

CH3

OO

H3C

O

O

O

O

H

H

O

44

OH

O

R'

R2

45. R1= H, R2 = Me

46. R1= Me, R2 = H

AcO

H

HO

O

H

47

O

OR1

R2

O

49. R1= H, R2= Me

50. R1= Me, R2= H

O

OR1

R2

O

51. R1=O H, R2= Me

52. R1= Me, R2= OH

O

OHO

HO

15

53. 15 β-OH 54. 15 α-OH

O

O

O

O

55 O

O

O

O

H

HH

H

HOH

56

O

O

O

O

H

HH

HOH

2

57

O

R1

R2

OH

H

H

58. R1=Me, R2=OH

59. R1=OH, R2=Me

O

OH

O

OAc

H

H

H

60

O

O

O

H

H

61

O

O HAcO

OH

15

62

O

O

15

HO

H

H

H

63

O

O

H

HHO

64

65. R=H66. R=OAc

H

H

HO

HO

O

OR

O

O

HO

15

HOO

67

O

O

HO

O

68

O

OOH

HO

69

O

O

H

O

70

O

O

HO

71

HO

O

O

HO

72

Apart from the above mentioned diterpenoids, other

phytochemicals, alkaloids,59-61 coumarins and lignanes,62-75

flavonoids,76-78 triterpenoids and sesquiterpenoids,79-83 cyclic

peptides,84-89 phytosterols,90-91 and some miscellaneous compounds92-

95 were also reported.

5.3. Present Work-Isolation and characterization of novel bioactive diterpenoids from Jatropha multifida

Jatropha multifida Linn, a non edible shrub grows in different parts

of India.96 The plant possesses several medicinal properties including

antibiotic activity.18 The earlier investigation on the latex of the plant

yielded cyclic peptides, phenolics and glucosides.95,97,98 Diterpenoids,

one of the major metabolites from this plant, were previously reported

from the plant.52-54 Presently, isolation and characterization of two

novel bioactive diterpenoids along with six known compounds is

reported. The structures of the new compounds were established from

their extensive spectroscopic (IR, 1D, 2D NMR and MS) and elemental

analysis studies. The known compounds were characterized by

comparison of their spectral data and physical properties with those

reported earlier in the literature and/or by direct comparison with

authentic samples available in the laboratory (Table 5.3).

Table 5.3: Compounds Isolated from the stems of Jatropha multifida#

Entry Compound Structure Status

A Tetradecyl-(E)-

ferulate

73 Known compound

B Fraxidin 74 Known compound

C Jatrothrin 13 Known compound

D Jatropholone-A 45 Known compound

E Jatrophenone 47 Known compound

F Japodagrone 68 Known compound

G Multifidanol 75 New compound

H Multifidenol 76 New compound

#The structure numbers of compounds C-F have been followed from

the Section-B, Table 5.2.

Compound A

Eluent: Hexane: ethyl acetate-90:10

Physical state: white solid

Melting point: 65 oC

Solubility: chloroform, ethyl acetate and methanol

m/z: 413 [M+Na]+.

Molecular formula: C24H38O4

Compound A was obtained as a white solid having m.p. 65 oC. It

was readily soluble in chloroform, ethyl acetate and methanol. From

the elemental analysis and mass spectrum (m/z 413 [M+Na]+.) (Fig.

5.3), the molecular formula of the compound was assigned as

C24H38O4.

The IR spectrum (Fig. 5.4) of the compound showed bands at νmax

3115, 1702, 1632 cm-1 and revealed the presence of hydroxyl and

conjugated carbonyl functionalities. The 1H-NMR spectrum (Fig. 5.5)

showed two AB pattern doublets at δ 7.62 and 6.30 with J = 16.0 Hz

for α,β-unsaturated conjugated trans double bond along with signals

for three aromatic protons. In addition to this, the spectrum also

showed signal for a -OMe group at δ 3.92 (3H, s). Foregoing spectral

studies along with its mass spectral data and literature survey

revealed the compound A as tetradecyl-(E) ferrulate (73).52,99

O

O

HO

MeO1

113

1

141

12

3

4

5

6

7

8

( )11

Tetradecyl-(E)-ferulate (73)

Table 5.4: 1H-NMR Spectral Data of Compound A

Position 1H-NMR

(δ)

Corresponding

number of protons

Multiplicity

(J in Hz)

1 - - -

2 7.04 1 d (2.0)

3 - - -

4 - - -

5 6.91 1 d (8.0)

6 7.08 1 dd (8.0, 2.0)

7 7.62 1 d (16.0)

8 6.30 1 d (16.0)

11 4.20 2 t (7.0)

21-121 1.41-1.17 22 m

131 1.75-1.64 2 m

141 0.89 3 t (7.0)

-OMe 3.92 3 s

-OH 5.86 1 brs

Compound B


Physical state: colorless crystals

Melting point: 196-197 oC

Solubility: DMSO

m/z: 222 [M]+.


Compound B was obtained as colorless crystals having m.p. 196-

197 oC. It was readily soluble in DMSO. From the mass spectrum (Fig.

5.6) which showed signal for molecular ion peak at m/z 222 [M]+. and

elemental analysis the molecular formula of the compound was

assigned as C11H10O5.

The IR spectrum (Fig. 5.7) of the compound showed characteristic

absorption band of hydroxyl group at υmax 3069 cm-1, penta

substituted aromatic moiety at νmax 1246 cm-1 and α, β-unsaturated

carbonyl moiety at νmax 1690 cm-1 revealing the presence of hydroxyl

group and conjugated carbonyl functionality in the molecule. The 1H-

NMR spectrum (Fig. 5.8) showed two AB pattern doublets resonated

at δ 7.72 (1H, d, J = 9.5 Hz) and 6.21 (1H, d, J = 9.5 Hz). The

spectrum showed the presence of two aromatic methoxy groups at δ

3.87 (3H, s) and 3.84 (3H, s). The signals at δ 9.57 and 6.59 indicated

the presence of a hydroxyl group and an aromatic proton. From the

spectral (IR, 1H-NMR and MS) studies and literature survey the

compound B was identified as fraxidin (74).100

O O

MeO

MeO

OH1

2

3

45

6

78

9

10

Fraxidin (74)

Table 5.5: 1H-NMR Spectral Data of Compound B

Position 1H-NMR

(δ)

Corresponding

number of protons

Multiplicity

(J in Hz)

1 - - -

2 - - -

3 6.21 1 d (9.5)

4 7.72 1 d (9.5)

5 6.59 1 s

6 - - -

7 - - -

-OMe (a) 3.87

(b) 3.84

3

3

s

s

-OH 9.57 1 brs

Compound C


Physical state: semi solid


[ ] 25

Dα : +68.2 (c 0.5, CHCl3)

m/z: 359 [M+H]+.


Compound C was isolated as semi solid having [ ] 25

Dα +68.2 (c 0.5,

CHCl3) and was readily soluble in chloroform, ethyl acetate and

methanol. The molecular formula C22H30O4 was assigned for the

compound from its elemental analysis and mass spectrum (Fig. 5.9)

which showed peak at m/z 359 [M+H]+. It was also supported by 13C-

NMR spectrum (Fig. 5.10) of the compound.

The IR spectrum (Fig. 5.11) showed absorption bands at νmax

1761, 1708 cm-1 indicating the presence of carbonyl functionalities in

the molecule. The 1H-NMR spectrum (Fig. 5.12) showed signals for an

olefinic proton at δ 7.37 (1H, s) and an acetoxy methyl group at δ 2.32

(3H, s). The signals at δ 1.90 (3H, s), 1.41 (3H, s), 1.02 (3H, d, J = 7.0

Hz), 1.00 (3H, s) and 0.87 (3H, s) were indicative for five methyl

groups present in the molecule. The signals at δ 0.11 (1H, t, J = 9.0

Hz) and 0.52 (1H, m) revealed the presence of cyclopropane ring in the

molecule. The 13C-NMR spectrum (Fig. 5.10) showed signals for

twenty-two carbons including a carbonyl (δ 203.5), two epoxy (δ 62.0

and 59.2) and four olefinic (δ 149.5, 147.6, 142.7 and 125.4) carbons.

It also indicated the presence of an acetoxy group at δ 169.0 and 20.6.

From all these spectral (IR, 1H-NMR, 13C-NMR and MS) data and

literature survey, the compound C was identified as Jatrothrin (13).31

OAc

OO

151

2

34

5

6

8

9

10

1112

13

14

16

17

18

19

20

7

Jatrothrin (13)

Table 5.6: NMR Spectral Data of Compound C

Position 1H-NMR

(δ)

Corresponding

number of protons

Multiplicity

(J in Hz)

13C-NMR

(δ)

1 7.37 1 s 149.5

2 - - - 142.7

3 - - - 203.5

4 2.79 1 d (9.6) 45.0

5 2.90 1 d (9.6) 62.0

6 - - - 59.2

7 (a) 2.34

(b) 1.19

1

1

m

m

40.9

8 (a) 1.73

(b) 0.98

1

1

m

m

19.2

9 0.11 1 t (9.6) 28.0

10 - - - 17.2

11 0.52 1 m 24.5

12 (a) 1.75

(b) 1.08

1

1

m

m

28.9

13 2.50 1 m 35.5

14 - - - 147.6

15 - - - 125.4

16 1.90 3 s 10.8

17 1.41 3 s 17.1

18 1.00 3 s 29.1

19 0.87 3 s 15.1

20 1.02 3 d (7.0) 18.7

-OAc 2.32 3 s 20.6, 169.0

Compound D





[ ] 25

Dα : +89.4 (c 1.25, CHCl3)

m/z: 296 [M]+.


Compound D was isolated as white solid having m.p. 213 oC and

][25

Dα +89.4 (c 1.25, CHCl3). The compound was readily soluble in

chloroform, ethyl acetate and methanol. From the mass spectrum

(Fig. 5.13) which showed signal for the molecular ion peak at m/z

296 and its elemental analysis the molecular formula of the

compound was assigned as C20H24O2.

The IR spectrum displayed signals at νmax 3220, 1675, 1565 cm-1

and revealed the presence of hydroxyl and carbonyl groups as well as

unsaturation in the molecule. The 1H-NMR spectrum (Fig. 5.14)

showed signals for four methyl groups resonated at δ 2.25 (3H, s),

1.30 (3H, s), 1.25 (3H, d, J = 7.0 Hz) and 0.82 (3H, s). It also showed

two broad singlets for exocyclic double bond protons at δ 5.16 and

4.64. D2O exchange experiment revealed the presence of one hydroxyl

group at δ 4.80. Based on the spectral (IR, 1H-NMR and MS) studies

and literature survey the compound D was identified as Jatropholone-

A (45).44,52

7

OH

O

1

2

34

5

6

8

9

10

11 12

13

14

15

16 17

18

19

20

Jatropholone-A (45)

Table 5.7: 1H-NMR Spectral Data of Compound D

Position 1H-NMR

(δ)

Corresponding number of protons

Multiplicity (J in Hz)

1 - - -

2 3.25 1 m

3 2.76-2.41 2 m

4 - - -

5 - - -

6 - - -

7 - - -

8 - - -

9 - - -

10 - - -

11 2.76-2.41 2 m

12 1.88-1.53 2 m

13 0.90 1 m

14 0.93 1 d (6.5)

15 (a) 5.16 (b) 4.64

1 1

brs brs

16 - - -

17 0.82 3 s

18 1.30 3 s

19 2.25 3 s

20 1.25 3 d (7.0)

-OH 4.80 1 brs

Compound E





[ ] 25

Dα : -4.5 (c 0.5, CHCl3)

m/z: 381 [M+Na]+.


Compound E was isolated as white solid having m.p. 204 oC and

[ ] 25

Dα -4.5 (c 0.5, CHCl3). It was readily soluble in chloroform, ethyl

acetate and methanol. The molecular formula of the compound was

deduced to be C22H30O4 from its elemental analysis and mass

spectrum (Fig. 5.15) which showed signal at m/z 381 [M+Na]+.

The IR spectrum (Fig. 5.16) of the compound exhibited bands at

νmax 1730, 1718, 1594 cm-1 indicating the presence of ester and

ketone carbonyl as well as unsaturation in the molecule. The 1H-NMR

spectrum (Fig. 5.17) showed signals for four methyl groups at δ 1.02

(3H, d, J = 6.0 Hz), 1.15 (6H, d, J = 4.5 Hz) and 1.70 (3H, s) along with

an acetoxy methyl group at δ 2.09 (3H, s). The spectrum also revealed

the presence of a disubstituted double bond (δ 5.58, 1H, dd, J = 16.0,

9.5 Hz and 5.10, 1H, dd, J = 16.0, 9.5 Hz), an exocyclic double bond (δ

4.80, 1H, s and 4.73, 1H, s) and a trisubstituted double bond (δ 5.79,

1H, d, J = 9.5 Hz). Based on these spectral (IR, 1H-NMR and MS) data

and literature survey the compound E was assigned as Jatrophenone

(47).45,52

HO

O

H

OAc

1

2

3 4

56

8

9

10

11

1213

14

1516

17

18

19

20

7

Jatrophenone (47)

Table 5.8: 1H-NMR Spectral Data of Compound E

Position 1H-NMR

(δ)

Corresponding

number of protons

Multiplicity

(J in Hz)

1 (a) 2.10

(b) 1.85

1

1

m

m

2 2.05 1 m

3 5.28 1 d (10.2)

4 - - -

5 5.79 1 d (9.5)

6 3.30 1 m

7 - - -

8 (a) 2.73

(b) 2.40

1

1

t (10.5)

dd (10.5, 3.5)

9 2.85 1 m

10 - - -

11 5.58 1 dd (16.0, 9.5)

12 5.10 1 dd (16.0, 9.5)

13 3.36 1 m

14 - - -

15 3.65 1 t (4.5)

16 1.02 3 d (6.0)

17 1.15 3 d (4.5)

18 1.70 3 s

19 (a) 4.80

(b) 4.73

1

1

s

s

20 1.15 3 d (4.5)

-OAc 2.09 3 s

Compound F


Physical state: colorless crystals

Melting point: 152-154 oC


[ ] 25

Dα : -261 (c 0.1, CHCl3).

m/z: 355 [M+Na]+.


Compound F was isolated as colorless crystals (CHCl3) having m.p.

152-154 oC and [ ] 25

Dα -261 (c 0.1, CHCl3). The compound was readily

soluble in chloroform, ethylacetate and methanol. The molecular

formula of the compound was assigned as C20H28O4 from its elemental

analysis and mass spectrum (Fig. 5.18) which showed the signal at

m/z 355 [M+Na]+. and was also supported by 13C-NMR spectrum (Fig.

5.19).

The IR spectrum (Fig. 5.20) displayed absorption bands at υmax

3437, 1704 and 1663 cm-1 indicated that hydroxyl and carbonyl

functionalities were present in the molecule. The 1H-NMR spectrum

(Fig. 5.21) showed signals for five methyl groups resonated at δ 1.90

(3H, s), 1.39 (3H, s), 0.94 (3H, d, J = 7.0 Hz), 0.89 (3H, s) and 0.76

(3H, s). The signal at δ 6.81 (2H, s) indicated the presence of two

trisubstituted double bond attached protons in the molecule. The

spectrum also showed the signal at δ 3.75 (1H, t, J = 6.5 Hz) indicative

of the presence of an oxygenated proton in the molecule. D2O

exchange experiment provided the evidence for presence of a hydroxyl

group at δ 4.86 (1H, brs). The signal for H-13 at δ 2.88 (1H, m)

revealed the structural similarity with that of 15-epi-(4E)-

jatrogrossidentadion (26).47

The 13C-NMR spectrum (Fig. 5.19) showed signals for twenty

carbons including two carbonyl, three oxygenated and four olefinic

carbons. From all of these spectral (IR, 1H-NMR, 13C-NMR and MS)

data and literature survey, the compound F was identified as

Japodagrone (68).12,52

1

2

3 4

56

78

9

10

11

12

1314

1516

17

18

19

20

HO

O

O

O

H

H

Japodagrone (68)

Table 5.9: NMR Spectral Data of Compound F

Position 1H-NMR

(δ)

Corresponding

number of protons

Multiplicity

(J in Hz)

13C-NMR

(δ)

1 6.81 1 s 154.0

2 - - - 143.9

3 - - - 196.7

4 - - - 136.0

5 6.81 1 s 139.8

6 - - - 83.0

7 (a) 1.87

(b) 1.84

1

1

m

m

37.8

8 (a) 1.89

(b) 1.70

1

1

m

m

26.0

9 3.75 1 t (6.5) 88.7

10 - - - 36.1

11 (a) 1.56

(b) 1.54

1

1

m

m

34.5

12 (a) 2.04 1 m 31.4

(b) 1.41 1 m

13 2.88 1 m 42.1

14 - - - 212.0

15 - - - 82.3

16 1.90 3 s 10.6

17 1.39 3 s 24.2

18 0.89 3 s 23.7

19 0.76 3 s 28.4

20 0.94 3 d (7.0) 20.6

-OH 4.86 1 brs -

5.3.1. Compound G (75) (New compound, Multifidanol)

Compound G was isolated as a white solid having m.p 203-205 oC

and [ ] 25

Dα -67.1 (c 0.21, CHCl3). The compound was readily soluble in

chloroform, methanol and ethyl acetate. The molecular formula of the

compound was assigned as C20H32O4 from its elemental analysis and

HRESIMS spectrum (Fig. 5.22), which showed the peak at m/z

359.2183. 13C-NMR spectrum also supported the molecular formula

(Fig. 5.23).

5.3.1.1. Spectral characterization:

The structure of the compound was assigned from detailed

analysis of its IR, 1H-NMR, 13C-NMR, 2D-NMR (1H-1H COSY, HSQC,

HMBC and NOESY) DEPT and MS spectral data. A comparison of the

spectral data with those of the reported constituents of Jatropha

species revealed that the structure of the new compound is closely

related to that of 15-epi-4(E)-Jatrogrossidentadione (40).29

HO

H

O

H

H

OH

O

1

2

3 4

56

7

8

9

1011

12

1314

15

16

17

18

1920

15-epi-4(E)-jatrogrossidentadione (40)

5.3.1.2. IR Spectrum

The IR spectrum (Fig. 5.24) displayed absorption bands at νmax

3353, 1706, 1457, 1377 cm-1 indicating the presence of hydroxyl and

carbonyl functionalities.

5.3.1.3. 1H-NMR Spectrum

The 1H-NMR spectrum (Fig. 5.25) displayed signals for five methyl

groups resonated at δ 1.28 (3H, s, Me-17), 1.21 (3H, d, J = 6.7 Hz, Me-

20), 1.20 (3H, s, Me-16), 0.99 (3H, s, Me-18) and 0.71 (3H, s, Me-19).

The signals at δ 5.78 (1H, s, H-5) indicated the presence of a proton

attached to trisubstituted double bond in the molecule. The spectrum

also showed signals for cyclopropane ring protons at δ 0.65 (1H, m, H-

11) and 0.44 (1H, m, H-9). D2O exchange experiment revealed the

presence of three tertiary hydroxyl groups at δ 5.28 (1H, brs, -OH),

3.80 (1H, brs, -OH) and 1.71-1.65 (1H, m, -OH). Thus 1H-NMR

spectrum confirmed that the compound belongs to lathyrane type

diterpenoid.29

5.3.1.4. 13C-NMR Spectrum

The 13C-NMR spectrum (Fig. 5.23) displayed signals for twenty

non equivalent carbons. The protonated carbon atoms were

characterized by HSQC (Fig. 5.26) and DEPT (Fig. 5.27) experiments

while non-protonated carbons by HMBC (Fig. 5.28) spectra. 13C-NMR

spectrum (Fig. 5.23) showed signals for a keto carbonyl at δ 212.8 (C-

14); two olefinic carbons at δ 145.1 (C-4) and 134.5 (C-5); five methyl’s

at δ 29.7 (C-17), 28.5 (C-18), 16.94 (C-16), 16.91 (C-20) and 14.91 (C-

19); four methylene’s at δ 42.0 (C-1), 41.8 (C-7), 28.1 (C-12) and 19.3

(C-8) and five methyne groups at δ 82.87 (C-3), 40.2 (C-2), 38.9 (C-13),

27.10 (C-9) and 19.0 (C-11). By comparison of the 13C-NMR values of

the double bonded carbons with those reported for corresponding

carbons 15-epi-4(E)-Jatrogrossidentadione (26), the configuration of

the double bonded carbons (C-4 and C-5) was assigned as trans-(E).29

Table 5.10: NMR Spectral Data of Compound G

Position 1H-NMR Multiplicity

(J in Hz)

13C-

NMR

1H-1H COSY

(Selected)

HMBC

(Selected)

NOESY

(Selected)

1 1.90(a)

1.63(b)

dd (14.0, 9.0)

dd (14.0, 2.0)

42.0 H-2 C-2, C-3, C-4

C-2, C-16

-

2 1.96 m 40.2 H-1, H-3,

H-16

C-1, C-3 -

3 4.01 d (8.0) 82.87 H-2 - H-13, Me-

16, Me-17

4 - - 145.1 - -

5 5.78 s 134.5 - C-1, C-3, C-4,

C-6, C -17

-

6 - - 74.5 - - -

7 1.80(a)

1.60(b)

m

m

41.8 H-8 C-5, C-6, C-9, C-11

-

8 1.58(a)

0.80(b)

m

m

19.3 H-7, H-9 C-6, C-7, C-9, C-11 -

9 0.44 m 27.10 H-8 C-18 -

10 - - 17.5 - - -

11 0.65 t (8.0) 19.0 H-12 C-13, C-18, C-20 H-9, H-20

12 1.71-

1.65(a)

1.44(b)

m

dd (14.0, 3.0)

28.1

H-11, H-13 C-9, C-11, C-13

C-14, C-20

H-11

13 3.04 m 38.9 H-12, H-20 C-11, C-20 H-12, H-16

14 - - 212.8 - - -

15 - - 83.02 - - -

16 1.20 d (7.0) 16.94 H-2 C-1, C-3, C-13 -

17 1.28 s 29.7 - C-5, C-6, C-7 -

18 0.99 s 28.5 - C-9, C-11 -

19 0.71 s 14.91 - C-9, C-11 -

20 1.21 d (7.0) 16.91 H-13 C-12, C-13 -

-OH 5.28

3.80

1.71-

1.65

brs

brs

m

The 1H-1H COSY spectrum (Fig. 5.29) showed the correlation

sequence: H-1 – H-2 – H-3 – H-7 – H-8 – H-9 – H-11 – H-12 – H-13 –

H-16 – H-20. The HMBC spectrum (Fig. 5.28) indicated that H-1a (δ

1.90) was correlated to C-2 (δ 40.2), C-3 (δ 82.87), and C-4 (δ 145.1),

H-1b to C-2 (δ 40.2) and C-16 (δ 16.94), H-2 (δ-1.96) to C-1 (δ 42.0)

and C-3 (δ 82.87), H-5 (δ 5.78) to C-1 (δ 42.0) to C-3 (δ 82.87), C-4 (δ

145.1) C-6 (δ 74.5) and C-17 (δ 29.7), H-7 and H-8 to C-6, (δ 74.5), C-

9 (δ 27.1), C-11 (δ 19.0), H-9 (δ 0.26) and H-11 (δ 0.53) to C-18 (δ

28.5), Me-16 to C-1 (δ 40.2), C-3 (δ 82.87), and C-13 (δ 38.9) and Me-

20 (δ 1.12) to C-12 (δ 28.1) and C-13 (δ 38.9).

HOH

HO

H

O

H

H

OH

1

2

3 4

5 67

8

9

1011

12

1314

15

16

17

18

1920

HOH

HO

H

O

H

H

OH

H—C →

Selected 1H-1H COSY Correlations of

Compound G (75)

Selected HMBC Correlations of

Compound G (75)

The relative stereochemistry of the new compound G was

established from the NOESY (Fig. 5.30) correlations and was found

to be similar to that of 15-epi-4(E)-jatrogrossidentadione (26).29 The

strong NOESY correlations between H-9, H-11 and H3-18 showed that

the gem-dimethyl cyclopropane ring was cis-fused. The α

stereochemistry of –OH group at C-6 was established by its correlation

with H-9. The strong correlations between –OH (C-6), –OH (C-16) and

–OH (C-3) indicated that they were cofacial and hence with α

stereochemistry while H-3 was related to Me-16 (δ 1.20, d), Me-17 (δ

1.28, s) and H-13 (δ 3.04, m) indicating their presence in another

plane (i.e., β). Thus the planarity of the asymmetric centers is

established and from all the above spectral conclusions, the structure

(75) was confirmed for the new compound, multifidanol (75).

HOH

HO

H

O

H

H

OH

Selected NOESY correlations of Compound G (75)

Table 5.11: Comparative 1H and 13C NMR Spectral Data of 15-epi-

(4E)-jatrogrossidentadion (40) and Compound G (75)

Position 15-epi-(4E)-jatrogrossidentadion (40) Compound G (75)

1H NMR Multiplicity (J in Hz)

13C NMR 1H NMR Multiplicity (J in Hz)

13C NMR

1 6.79 s 152.1

1.90(a)

1.63(b)

dd (14.0,

9.0)

dd (14.0,

2.0)

42.0

2 - - 147.0 1.96 m 40.2

3 - - 196.2 4.01 d (8.0) 82.87

4 - - 137.3 145.1

5 6.57 s 143.9 5.78 s 134.5

6 - - 75.4 74.5

7 1.94(a)

1.71(b)

m

m 42.3

1.80(a)

1.60(b)

m

m 41.8

8 1.68(a)

0.85(b)

m

m 17.6

1.58(a)

0.80(b)

m

m 19.3

9 0.44 m 27.0 0.44 m 27.10

10 - - 17.0 17.5

11 0.62 m 19.9 0.65 t 19.0

12 1.45(a)

1.36(b)

m

m 29.8

1.71-

1.65(a)

1.44(b)

m

dd (14.0,

3.0)

28.1

13 2.86 m 38.2 3.04 m 38.9

14 - - 211.8 212.8

15 - - 84.6 83.02

16 1.99 s 10.5 1.20 d (7.0) 16.94

17 1.32 s 29.0 1.28 s 29.7

18 1.01 s 28.1 0.99 s 28.5

19 0.68 s 14.9 0.71 s 14.91

20 1.10 d (7.0) 19.7 1.21 d (7.0) 16.91

-OH 5.12(a)

3.23(b)

brs

brs

-

-

5.28

3.80

1.71-

1.65

brs

brs

m

-

-

Though a difference was found in the spectral data of ring A of the

new compound with that of its relative (40), the remaining data was

very much similar. Once the structural elucidation was done, our

interest made us to probe for its bioactivity. Thus the compound was

tested for antimicrobial and cytotoxic assays. The results found were

impressive.

5.3.1.5. ANTIMICROBIAL ACTIVITY

The antimicrobial activity (Table 5.12) was determined using

Microtiter broth dilution method101 against different pathogenic

reference strains procured from the Microbial Type Culture Collection

(MTCC), Institute of Microbial Technology, Chandigarh, India. The test

pathogens used in sequence were, Bacillus subtilis MTCC 121,

Staphylococcus aureus MTCC 96, Micrococcus luteus MTCC 2470,

Escherichia coli MTCC 739, Klebsiella planticola MTCC 530,

Pseudomonas aeruginosa MTCC 2453 and Candida albicans MTCC

3018.

Table 5.12: Antimicrobial and cytotoxic activity of Multifidanol (75)

Neomycin was used as the positive control. As can be seen from

the Fig. 5.31 that the compound G showed prominent activity against

B. subtilis and E. coli and the activity against other strains was also

impressive.

Antimicrobial activity (MIC µg/ml) Cytotoxic activity

IC50 (50% in µM)

S. No Test pathogen Multifidanol

(75)

Neomycin

(control)

Test

cell

line

Multifidanol

(75)

Doxorubicin

(control)

1

Bacillus

subtilis

MTCC 121

4.68

18.75

A-549

6.27

1

2 Staphylococcus

aureus

MTCC 96

18.75 18.75 Neuro-

2a

6.35 1.2

3 Staphylococcus

aureus MLS16

MTCC 2940

18.75 18.75 HeLa 15.4 0.9

4 Escherichia

coli

MTCC 739

4.68 18.75 MDA-

231

7.04 0.9

5 Klebsiella

planticola

MTCC 530

9.37 18.75 MCF-

7

6.39 1

6 Pseudomonas

aeruginosa

MTCC 2453

9.37 18.75 - - -

Fig. 5.31

Fig. 5.32

5.3.1.6. CYTOTOXIC ACTIVITY

Cytotoxicity (Table 5.12) of the isolated compound G was assessed

on the basis of measurement of in vitro growth of tumor cell lines in

96 well plates by cell-mediated reduction of tetrazolium salt to water

insoluble formazan crystals using doxorubicin as a standard. The

compound was tested for cytotoxicity towards a panel of five different

tumor cell lines: A549 derived from human alveolar adenocarcinoma

epithelial cells (ATCC No. CCL-185), Neuro2a derived from mouse

neuroblastoma cells (ATCC No. CCL-131), HeLa derived from human

cervical cancer cells (ATCC No. CCL-2), MDA-MB-231 derived from

human breast adenocarcinoma cells (ATCC No. HTB-26) and MCF7

derived from human breast adenocarcinoma cells (ATCC No. HTB-22).

The MTT assay was followed according to the Mosmann method.102

The concentration of compounds at which 50% of cell growth inhibited

(IC50) was calculated and the values are summarized in Fig 5.32.

5.3.2. Compound H (76) (New compound, Multifidenol)

Compound H was isolated as a white solid having m.p 201-203 oC

and [ ] 25

Dα -142.5 (c 0.07, CHCl3). The compound was readily soluble in

chloroform, methanol and ethyl acetate. The molecular formula of the

compound was assigned as C20H30O4 from its elemental analysis and

HRESIMS spectrum (Fig. 5.33), which showed the peak at m/z

357.2034. This molecular formula indicated that the compound H

may be a dehydro derivative of compound G. 13C-NMR spectrum also

supported the molecular formula (Fig. 5.34).

5.3.2.1. Spectral characterization:

The structure of the compound was assigned from detailed

analysis of its IR, 1H-NMR, 13C-NMR, 2D-NMR (1H-1H COSY, HSQC,

HMBC and NOESY), DEPT and MS spectral data. A comparison of the

spectral data with those of the reported constituents of Jatropha

species revealed that the structure of the new compound is closely

related to that of 15-epi-4(E)-Jatrogrossidentadione (40).29

HO

H

O

H

H

OH

O

1

2

3 4

56 7

8

9

1011

12

1314

15

16

17

18

1920

15-epi-4(E)-jatrogrossidentadione (40)

5.3.2.2. IR Spectrum

The IR spectrum displayed absorption bands at νmax 3362, 1709,

1624, 1238 cm-1 indicating the presence of hydroxyl and carbonyl

functionalities. Its 1H and 13C NMR spectral data were very similar to

those of compound G. However, these data suggested that the ring A

of compound H was unsaturated having a double bond at C-1—C-2.

In the 1H NMR spectrum H-1 appeared at δ 5.33 (1H, s) while in the

13C NMR spectrum C-1 and C-2 at δ 128.6 and 148.5 respectively. The

H-3 in compound H resonated somewhat more downfield (δ 4.82, 1H,

brs) compared to the position of the corresponding proton (δ 4.01, 1H,

d, J = 8.0 Hz) in compound G. The HMBC experiment showed that H-1

was related to C-3 (δ 80.0) and C-4 (δ 145.2) while Me-16 (δ 1.94, s) to

C-1 and C-3. The NMR spectral data indicated that the remaining

protons and carbons of both the compounds 75 and 76 were similar.

5.3.2.3. 1H-NMR Spectrum

The 1H-NMR spectrum (Fig. 5.35) displayed signals for five methyl

groups resonated at δ 1.94 (3H, s, Me-16), 1.26 (3H, s, Me-17), 1.12

(3H, d, J = 7.0 Hz Me-20), 0.99 (3H, s, Me-18) and 0.72 (3H, s, Me-19).

The signals at δ 5.33 (1H, s, H-1) and 6.10 (1H, s, H-5) executed the

presence of two protons attached to two trisubstituted double bonds

in the molecule. The spectrum also showed signals for cyclopropane

ring protons at δ 0.64 (1H, m, H-11) and 0.41 (1H, m, H-9). D2O

exchange experiment revealed the presence of three tertiary hydroxyl

groups at δ 5.52 (1H, brs, -OH), 4.12 (1H, brs, -OH) and 1.69-1.53

(1H, m, -OH). Thus 1H-NMR spectrum confirmed that the compound

belongs to lathyrane type diterpenoid.29

5.3.2.4. 13C-NMR Spectrum

The 13C-NMR spectrum (Fig. 5.34) displayed signals for twenty

non equivalent carbons. The protonated carbon atoms were

characterized by HSQC (Fig. 5.36) and DEPT experiments while non-

protonated carbons by HMBC (Fig. 5.37) spectrum. 13C-NMR

spectrum (Fig. 5.34) showed signals for a keto carbonyl at δ 212.3 (C-

14); four olefinic carbons at δ 128.6 (C-1), 148.5 (C-2), 145.2 (C-4) and

137.5 (C-5); five methyl’s at δ 29.3 (C-17), 28.8 (C-18), 16.6 (C-20),

14.9 (C-19) and 13.9 (C-16); three methylene’s at δ 42.1 (C-7), 28.5 (C-

12) and 19.4 (C-8) and four methyne groups at δ 80.0 (C-3), 38.1 (C-

13), 27.3 (C-9) and 19.2 (C-11). By comparison of the 13C-NMR values

of the double bonded carbons with those reported for corresponding

carbons 15-epi-4(E)-Jatrogrossidentadione (26), the configuration of

the double bonded carbons (C-4 and C-5) was assigned as trans-(E).29

Table 5.13: NMR Spectral Data of Compound H

Position 1H-NMR Multiplicity

(J in Hz)

13C-NMR 1H-1H COSY

(Selected)

HMBC

(Selected)

NOESY

(Selected)

1 5.33 s 128.6 H-16 C-2, C-3, C-4

C-15, -

2 - - 148.5 - - -

3 4.82 brs 80.0 H-16 C-8 H-13, Me-

16, Me-17

4 - - 145.2 - - -

5 6.10 s 137.5 H-3 C-3, C-4, C-6, C-

7, C-15, C -17 -

6 - - 73.6 - - -

7 1.79(a)

1.69-1.53(b)

m

m 42.1 H-8

C-6, C-9, C-17

C-8 -

8 1.69-1.53(a)

0.80(b)

m

m 19.4 H-7

C-6, C-7, C-9,

C- 11, C-17 -

9 0.41 m 27.3 H-8 C-7, C-11, C-18 H-11, H3-18

10 - - 17.2 - - -

11 0.64 m 19.2 H-9, H-12 C-8, C-10, C-12,

C-13, C-19 H-9

12 1.69-1.53(a)

1.52-1.41(b)

m

m 28.5 H-11, H-13

C-8, C-10, C-13

-

13 2.82 m 38.1 H-12, H-20 C-11 -

14 - - 212.3 - - -

15 - - 87.8 - - -

16 1.94 s 13.9 H-1, H-3 C-1, C-3, C-4 -

17 1.26 s 29.3 H-16 C-5, C-6, C-7 -

18 0.99 s 28.8 - C-9, C-10, C-11,

C-19 -

19 0.72 s 14.9 H-8, H-12 C-9, C-10, C-11 -

20 1.12 d (7.0) 16.6 H-12, H-13 C-12, C-13 -

-OH

5.52

4.12

1.69- 1.53

brs

brs

m

- - -

The 1H-1H COSY spectrum (Fig. 5.38) showed the correlation

sequence: H-1 – H-3 – H-5 – H-7 – H-8 – H-9 – H-11 – H-12 – H-13 –

H-16 – H-17 – H-19 – H-20. The HMBC spectrum (Fig. 5.37) indicated

that H-1 (δ 5.33) was correlated to C-2 (δ 148.5), C-3 (δ 80.0), C-4 (δ

145.2), C-15 (δ 87.8) and C-16 (δ 13.9), H-3 (δ 4.82) to C-8 (δ 19.4), H-

5 (δ 6.10) to C-3 (δ 80.0), C-4 (δ 145.2), C-6 (δ 73.6), C-7 (δ 42.1), C-15

(δ 87.8) and C-17 (δ 29.3), H-9 (δ 0.41) to C-7 (δ 42.1), C-11 (δ 19.4)

and C-18 (δ 28.8), H-11 to C-8 (δ 19.2), C-10 (δ 17.2), C-12 (δ 28.5), C-

13 (δ 38.1) and C-19 (δ 14.9), Me-16 to C-1 (δ 128.6), C-3 (δ 80.0), and

C-4 (δ 145.2) and Me-20 (δ 1.12) to C-12 (δ 28.5) and C-13 (δ 38.1).

HOH

HO

H

O

H

H

OH

1

2

3 4

5 67

8

9

1011

12

1314

15

16

17

18

1920

HOH

HO

H

O

H

H

OH

H—C →

Selected 1H-1H COSY Correlations of

Compound H (76)

Selected HMBC Correlations of

Compound H (76)

Table 5.14: Comparative 1H and 13C NMR Spectral Data of 15-epi-

(4E)-jatrogrossidentadion (40) and Compound H (76)

Position 15-epi-(4E)-jatrogrossidentadion (40) Compound H (76)

1H NMR Multiplicity (J in Hz)

13C NMR 1H NMR Multiplicity (J in Hz)

13C NMR

1 6.79 s 152.1 5.33 s 128.6

2 - - 147.0 - - 148.5

3 - - 196.2 4.82 brs 80.0

4 - - 137.3 - - 145.2

5 6.57 s 143.9 6.10 s 137.5

6 - - 75.4 - - 73.6

7 1.94(a)

1.71(b)

m

m

42.3 1.79(a)

1.69-

1.53(b)

m

m

42.1

8 1.68(a)

0.85(b)

m

m

17.6 1.69-

1.53(a)

0.78(b)

m

m

19.4

9 0.44 m 27.0 0.41 m 27.3

10 - - 17.0 - - 17.2

11 0.62 m 19.9 0.64 m 19.2

12 1.45(a)

1.36(b)

m

m

29.8 1.69-

1.53(a)

1.52-

1.41(b)

m

m

28.5

13 2.86 m 38.2 2.82 m 38.1

14 - - 211.8 - - 212.3

15 - - 84.6 - - 87.8

16 1.99 s 10.5 1.94 s 13.9

17 1.32 s 29.0 1.26 s 29.3

18 1.01 s 28.1 0.99 s 28.8

19 0.68 s 14.9 0.72 s 14.9

20 1.10 d (7.0) 19.7 1.12 d (J = 7.0) 16.6

-OH 5.12(a)

3.23(b)

brs

brs

-

-

5.52

4.12

1.69-1.53

brs

brs

m

-

-

The relative stereochemistry of the new compound H was

established from the NOESY (Fig. 5.39) correlations and was found

to be similar to that of 15-epi-4(E)-jatrogrossidentadione (26).29 The

strong NOESY correlations between H-9, H-11 and H3-18 showed that

the gem-dimethyl cyclopropane ring was cis-fused. The α

stereochemistry of –OH group at C-6 was established by its correlation

with H-9. The strong correlations between –OH (C-6), –OH (C-16) and

–OH (C-3) indicated that they were cofacial and hence with α

stereochemistry while H-3 was related to Me-16 (δ 1.94, s), Me-17 (δ

1.26, s) and H-13 (δ 2.82, m) indicating their presence in another

plane (i.e., β). Thus the planarity of the asymmetric centers is

established and from all the above spectral conclusions, the structure

(76) was confirmed for the new compound, multifidenol (76).

HOH

HO

H

O

H

H

OH

Selected NOESY correlations of Compound G (76) 5.3.2.5. ANTIMICROBIAL ACTIVITY

The antimicrobial activity (Table 5.15) against different pathogenic

test strains was determined using the same method as described for

the previous compound G using neomycin as standard drug. The test

pathogens used in sequence were, Bacillus subtilis MTCC 121,

Staphylococcus aureus MTCC 96, Micrococcus luteus MTCC 2470,

Escherichia coli MTCC 739, Klebsiella planticola MTCC 530,

Pseudomonas aeruginosa MTCC 2453 and Candida albicans MTCC

3018.

Table 5.15: Antimicrobial and cytotoxic activity of Multifidenol (76)

A key point noted was the compound H exhibited selective activity

against S. aureus (Fig 5.40) and showed no activity against other

pathogenic strains.

Antimicrobial activity (MIC µg/ml)

Cytotoxic activity

IC50 (50% in µM)

S. No Test pathogen Multifidenol

(76)

Neomycin

(control)

Test

cell

line

Multifidenol

(76)

Doxorubicin

(control)

1

Bacillus

subtilis

MTCC 121

-

18.75

A-549

12.5

1

2 Staphylococcus

aureus

MTCC 96

4.68 18.75 Neuro

-2a

5.6 1.2

3 Staphylococcus

aureus MLS16

MTCC 2940

4.68 18.75 HeLa 7.56 0.9

4 Escherichia

coli

MTCC 739

- 18.75 MDA-

231

5.39 0.9

5 Klebsiella

planticola

MTCC 530

- 18.75 MCF-

7

8.57 1

6 Pseudomonas

aeruginosa

MTCC 2453

- 18.75 - - -

Fig. 5.40

Fig. 5.41

5.3.2.6. CYTOTOXIC ACTIVITY

Cytotoxicity of the isolated compound H was assessed using

doxorubicin as a standard. The compound was tested for cytotoxicity

towards a panel of five different tumor cell lines: A549 derived from

human alveolar adenocarcinoma epithelial cells (ATCC No. CCL-185),

Neuro2a derived from mouse neuroblastoma cells (ATCC No. CCL-

131), HeLa derived from human cervical cancer cells (ATCC No. CCL-

2), MDA-MB-231 derived from human breast adenocarcinoma cells

(ATCC No. HTB-26) and MCF7 derived from human breast

adenocarcinoma cells (ATCC No. HTB-22). The MTT assay was

followed according to the method of Mosmann.102 The concentration of

compounds at which 50% of cell growth inhibition (IC50) was

calculated and the values are summarized in Fig 5.41.

In conclusion, we accomplished the isolation of novel bioactive

diterpenoids from the stem of Jatropha multifida with considerable

antimicrobial and cytotoxic activities.

5.4. EXPERIMENTAL

5.4.1. General

Spectra were recorded with the following instruments: NMR:

Varian Gemini 200 MHz, Bruker 300 MHz, Unity 400 MHz, Inova 500

MHz, Avance 600 MHz; IR: Perkin Elmer RX1 FT-IR

spectrophotometer; LSIMS: Micromass Quattro LC; EIMS: Micromass

VG 7070 H (70 eV) and ESIMS: LC-MSD-Trap-SL. Melting points were

measured in Buchi-510 instrument and uncorrected. Optical rotations

were measured on a JASCO DIP-360 polarimeter. Column

chromatography was performed over silica gel (BDH 60-120 mesh)

and TLC with silica gel GF254. The spots over the TLC plate were

visualized either by using UV light or exposing the plates to iodine

vapors or charring with 5% sulfuric acid in methanol.

5.4.2. Plant material

The stems of Jatropha multifida were collected from the botanical

garden, Osmania University campus, Hyderabad in March 2010. A

voucher specimen with No.56112 is preserved in IICT herbarium.

5.4.2.1. Extraction from plant material and separation of

phytoconstituents

The plant material (2 kg) was shade dried, powdered and extracted

three times (72 h in each case) with a mixture of CHCl3/MeOH (1:1, 2

L) at room temperature. The total extract was concentrated to afford a

thick brown mass (51.4 g). The residue (51.0 g) was subjected to

column chromatography which was eluted with solvents of increasing

polarity using hexane and EtOAc. The eluents were collected in

fractions and concentrated. The eluents with similar profiles were

analyzed by TLC experiment and were combined and

rechromatographed (Table 5.16).

Table 5.16: Chromatographic Resolution of CHCl3:MeOH (1:1)

Extract of the Stems of Jatropha multifida

Eluent Hexane:EtOAc

Fractions collected Residue (g)

Remarks

100:00 1-24 3.6 Fatty oil

95:05 25-40 3.2 Waxy material

90:10 41-57 2.8 Fraction-I

80:20 58-78 3.5 Fraction-II

70:30 79-95 4.1 Fraction-III

60:40 96-112 2.6 Fraction-IV

50:50 113-130 2.1 Fraction-V

40:60 131-143 1.1 Fraction-VI

20:80 144-155 0.8 Brown mass

The fractions, 1-40 yielded only fatty oil and waxy material, which were not pursued further. Fraction-I

Fraction-I was rechromatographed over silica gel column using

hexane-EtOAc mixture by increasing polarities. Fractions (25 ml each)

were collected and the results were recorded (Table 5.17).

Table 5.17: Rechromatography of Fraction-I:

Eluent Hexane: EtOAc

Fractions Yield (g) Remarks

100:0 1-15 1.22 Fatty oil

95:05 16-27 0.87 Fatty oil

90:10 28-43 0.14 Fatty oil

80:20 44-58 0.32 Green matter

70:30 59-72 0.23 Green matter

Fraction-II

Fraction-II showed two spots along with fatty and green materials

on TLC and was rechromatographed over silica gel column using

hexane-EtOAc mixture by increasing polarities. Fractions (25 ml each)

were collected (Table 5.18).

Table 5.18: Rechromatography of Fraction-II



100:00 1-22 1.35 Fatty oil

95:05 23-31 0.21 Compound A

90:10 32-41 0.25 Compound A

80:20 41-54 0.27 Compound B

70:30 55-70 0.80 Green matter

60:40 71-79 0.61 Green matter

Fraction-III

Fraction-III showed two spots on TLC and was rechromatographed

over silica gel column using hexane-EtOAc mixture by increasing

polarities. Fractions (25 ml each) were collected (Table 5.19).

Table 5.19: Rechromatography of Fraction-III:



80:20 21-36 2.13 Green matter

75:25 37-43 0.52 Green matter

70:30 44-61 0.62 Compound D

60:40 62-70 0.33 Compound E

50:50 71-80 0.49 Green matter

Fraction-IV

Fraction-IV showed one spot along with green matter on TLC and

was carefully rechromatographed over silica gel column using hexane-

EtOAc mixture by increasing polarities. Fractions (25 ml each) were

collected (Table 5.20).

Table 5.20: Rechromatography of Fraction-IV:



90:10 1-12 0.87 Fatty solid

80:20 13-25 0.81 Green matter

75:25 26-34 0.14 Compound C

70:30 35-47 0.78 Green matter

Fraction-V

Fraction-V showed one spot along with green matter on TLC and

was rechromatographed over silica gel column using hexane-EtOAc

mixture by increasing polarities. Fractions (25 ml each) were collected

(Table 5.21).

Table 5.21: Rechromatography of Fraction-V



90:10 1-10 0.76 Fatty solid

80:20 11-18 0.49 Green matter

70:30 19-31 0.40 Green matter

60:40 32-41 0.01 Compound F

50:50 42-53 0.45 Green matter

Fraction-VI

Fraction-VI showed two spots those were very close along with fatty

solid and green matter on TLC and was rechromatographed over silica

gel column using hexane-EtOAc mixture by increasing polarities.

Fractions (25 ml each) were collected (Table 5.22).

Table 5.22: Rechromatography of Fraction-VI



90:10 1-12 0.36 Fatty solid

70:30 22-35 0.21 Green matter

50:50 36-44 0.19 Green matter

40:60 45-55 0.10 Compound G and H

30:70 56-65 0.24 Green matter

Fraction-VII

Fraction-VII contained only green matter and was

rechromatographed over silica gel column using hexane-EtOAc

mixture by increasing polarities. Fractions (25 ml each) were collected

(Table 5.23).

Table 5.23: Rechromatography of Fraction-VII



90:10 1-16 0.14 Fatty solid

70:30 17-31 0.27 Green matter

50:50 32-45 0.40 Gummy material


MeO

HO

O(CH2)13Me

O


Compound A : Tetradecyl-(E)-ferulate

Physical State : White solid

Melting Point : 65 oC

Molecular formula : C24H38O4

Elemental analysis : Anal. Calcd: C, 73.85; H, 9.74 %

Found: C, 73.18; H, 9.53 %

Yield : 0.46 g

Rf : 0.52 (solvent system EtOAc:hexane 1:9)

IR Spectrum : νmax (KBr) 3115, 1702, 1632 cm-1 (Fig. 5.4).

1H-NMR Spectrum : (200 MHz, CDCl3): δ 7.62 (1H, d, J = 16.0 Hz, H-

7), 7.08 (1H, dd, J = 8.0, 2.0 Hz, H-6), 7.04 (1H,

d, J = 2.0 Hz, H-2), 6.91 (1H, d, J = 8.0 Hz, H-5),

6.30 (1H, d, J = 16.0 Hz, H-8), 4.20 (2H, t, J =

7.0 Hz, H2-1'), 3.92 (3H, s, -OMe), 1.75-1.64 (2H,

m, H2-13'), 1.41-1.17 (22H, brs, H2-2'–H2-12'),

0.89 (3H, t, J = 7.0 Hz, Me-14'). (Fig. 5.5).

FAB-Mass Spectrum : m/z 413 [M+Na]+. (Fig. 5.3).

Fraxidin (74)

O O

MeO

MeO

OH1

2

3

45

6

78

9

10

Fraxidin (74)

Compound B

:

Fraxidin

Physical State : Colorless crystals

Melting Point : 196-197 oC



Found: C, 59.98; H, 4.36 %

Yield : 0.27 g

Rf : 0.23 (solvent system MeOH:CHCl3 1:9)

IR Spectrum : νmax (KBr) 3069, 1690, 1246 cm-1 (Fig. 5.7).

1H-NMR Spectrum : (400 MHz, DMSO-d6): δ 9.57 (1H, brs, -OH), 7.72

(1H, d, J = 9.5 Hz, H-4), 6.59 (1H, s, H-5), 6.21

(1H, d, J = 9.5 Hz, H-3), 3.87 (3H, s, -OMe), 3.84

(3H, s, -OMe). (Fig. 5.8).

EI-Mass Spectrum : m/z 222 [M]+. (Fig. 5.6).

Jatrothrin (13)

OAc

OO

151

2

34

5

6

8

9

10

1112

13

14

16

17

18

19

20

7 Jatrothrin (13)

Compound C : Jatrothrin

Physical State : Semi solid

Specific Rotation : ][25

Dα = +68.2 (c 0.5, CHCl3)



Found: C, 73.08; H, 8.51 %

Yield : 0.14 g


IR Spectrum : νmax (KBr) 1761, 1708, 1506, 1198 cm-1 (Fig.

5.11).

1H-NMR Spectrum : (500 MHz, CDCl3): δ 7.37 (1H, s, H-1), 2.90 (1H,

d, J = 9.6 Hz, H-5), 2.79 (1H, d, J = 9.6 Hz, H-4),

2.50 (1H, m, H-13), 2.34 (1H, m, H-7a), 2.32

(3H, s, -OAc), 1.90 (3H, s, Me-16), 1.75 (1H, m,

H-12a), 1.73 (1H, m, H-8a), 1.41 (3H, s, Me-17),

1.19 (1H, m, H-7b), 1.08 (1H, m, H-12b), 1.02

(3H, d, J = 7.0 Hz, Me-20), 1.00 (3H, s, Me-18),

0.98 (1H, m, H-8b), 0.87 (3H, s, Me-19), 0.52

(1H, m, H-11), 0.11 (1H, m, H-9). (Fig. 5.12).

13C-NMR Spectrum : (125 MHz, CDCl3): δ 203.5 (C-3), 169.0 (-CO-Me),

149.5 (C-1), 147.6 (C-14), 142.7 (C-2), 125.4 (C-

15), 62.0 (C-5), 59.2 (C-6), 45.0 (C-4), 40.9 (C-7),

35.5 (C-13), 29.1 (C-18), 28.9 (C-12), 28.0 (C-9),

24.5 (C-11), 20.6 (-CO-Me), 19.2 (C-8), 18.7 (C-

20), 17.2 (C-10), 17.1 (C-17), 15.1 (C-19), 10.8

(C-16). (Fig. 5.10).

FAB-Mass Spectrum : m/z 359 [M+H]+. (Fig. 5.9).

Jatropholone-A (45)

7

OH

O

1

2

34

5

6

8

9

10

11 12

13

14

15

16 17

18

19

20

Jatropholone-A (45)

Compound D : Jatropholone-A


Melting Point : 213 oC.


Dα = +89.4 (c 1.25, CHCl3)



Found: C, 81.76; H, 8.27 %

Yield : 0.62 g


IR Spectrum : νmax (KBr) 3220, 1675, 1565 cm-1

1H-NMR Spectrum : (200 MHz, CDCl3): δ 5.16 (1H, brs, H-15a), 4.80

(1H, brs, -OH), 4.64 (1H, brs, H-15b), 3.25 (1H,

dd, J = 14.0, 7.5 Hz, H-2), 2.76-2.41 (4H, m, H2-

3 and H2-11), 2.25 (3H, s, Me-19), 1.88-1.53

(2H, m, H2-12), 1.30 (3H, s, Me-18), 1.25 (3H, d,

J = 7.0 Hz, Me-20), 0.93 (1H, d, J = 6.5 Hz, H-

14), 0.90 (1H, m, H-13), 0.82 (3H, s, Me-17).

(Fig. 5.14).

FAB-Mass Spectrum : m/z (%) 319 [M+Na]+. (Fig. 5.13).

Jatrophenone (47)

H

O

O

H

OAc

1

2

3 4

56

8

9

10

11

1213

14

1516

17

18

19

20

7

Jatrophenone (47)

Compound E : Jatrophenone


Melting Point : 204 oC


Dα -4.5 (c 0.5, CHCl3)



Found: C, 73.09; H, 8.52 %

Yield : 0.33 g



5.16).

1H-NMR Spectrum : (500 MHz, CDCl3): δ 5.79 (1H, d, J = 9.5 Hz, H-

5), 5.58 (1H, dd, J = 16.0, 9.5 Hz, H-11), 5.28

(1H, d, J = 10.2 Hz, H-3), 5.10 (1H, dd, J = 16.0,

9.5 Hz, H-12), 4.80 (1H, s, H-19a), 4.73 (1H, s,

H-19b), 3.65 (1H, t, J = 4.5 Hz, H-15), 3.36 (1H,

m, H-13), 3.30 (1H, m, H-6), 2.85 (1H, m, H-9),

2.73 (1H, t, J = 10.5 Hz, H-8a), 2.40 (1H, dd, J =

10.5, 3.5 Hz, H-8b), 2.10 (1H, m, H-1a), 2.09

(3H, s, -OAc), 2.05 (1H, m, H-2), 1.85 (1H, m, H-

1b), 1.70 (3H, s, Me-18), 1.15 (6H, d, J = 4.5 Hz,

Me-17 and Me-20), 1.02 (3H, d, J = 6.0 Hz, Me-

16). (Fig. 5.17).

FAB-Mass Spectrum : m/z 381 [M+Na]+. (Fig. 5.15).

Japodagrone (68)

1

2

3 4

56

78

9

10

11

12

1314

1516

17

18

19

20

HO

O

O

O

H

H

Japodagrone (68)

Compound F : Japodagrone

Physical State : Colorless crystals



Dα -261.0 (c 0.001, CHCl3)



Found: C, 72.13; H, 8.51 %

Yield : 0.01 g



5.20).

1H-NMR Spectrum : (500 MHz, CDCl3): δ 6.81 (2H, s, H-1, H-5), 4.86

(1H, brs, -OH), 3.75 (1H, t, J = 6.5 Hz, H-9), 2.88

(1H, m, H-13), 2.04 (1H, m, H-12a), 1.90 (3H, s,

Me-16), 1.89 (1H, m, H-8a), 1.87 (1H, m, H-7a),

1.84 (1H, m, H-7b), 1.70 (1H, m, H-8b), 1.56

(1H, m, H-11a), 1.54 (1H, m, H-11b), 1.41 (1H,

m, H-12b), 1.39 (3H, s, Me-17), 0.94 (3H, d, J =

7.0 Hz, Me-20), 0.89 (3H, s, Me-18), 0.76 (3H, s,

Me-19). (Fig. 5.21).

13C-NMR Spectrum : (100 MHz, CDCl3): δ 212.0 (C-14), 196.2 (C-3),

154.0 (C-1), 143.9 (C-2), 139.8 (C-5), 136.0 (C-

4), 88.6 (C-9), 83.0 (C-6), 82.1 (C-15), 42.3 (C-

13), 37.9 (C-7), 36.2 (C-10), 34.5 (C-11), 31.9 (C-

12), 28.4 (C-18), 25.9 (C-8), 24.2 (C-17), 23.5 (C-

19), 20.7 (C-20), 10.6 (C-16). (Fig. 5.19).

LC-Mass Spectrum : m/z 355 [M+Na]+., 687 [M+Na]+ (Fig. 5.18).

Multifidanol (75)

HOH

HO

H

O

H

H

OH

Multifidanol (75)

Compound G : Multifidanol




Dα -67.1 (c 0.21, CHCl3)


Yield : 0.05 g



5.24).


brs, -OH), 4.01 (1H, d, J = 8.0 Hz, H-3), 3.80

(1H, brs, -OH), 3.04 (1H, m, H-13), 1.96 (1H, m,

H-2), 1.90 (1H, dd, H-1a, J = 14.0, 9.0 Hz), 1.80

(1H, m, H-7a), 1.71-1.65 (2H, m, H-12a, -OH),

1.63 (1H, dd, J = 14.0, 2.0 Hz H-1b), 1.60 (1H,

m, H-7b), 1.58 (1H, m, H-8a), 1.44 (1H, dd, J =

14.0, 3.0 Hz, H-12b), 1.28 (3H, s, Me-17), 1.21

(3H, d, J = 7.0 Hz, Me-20), 1.20 (3H, d, J = 7.0

Hz, Me-16), 0.99 (3H, s, Me-18), 0.80 (1H, m, H-

8b), 0.71 (3H, s, Me-19), 0.65 (1H, t, J = 8.0, H-

11), 0.44 (1H, m, H-9). (Fig. 5.25).


134.5 (C-5), 83.0 (C-15), 82.9 (C-3), 74.5 (C-6),

42.0 (C-1), 41.8 (C-7), 40.2 (C-2), 38.9 (C-13),

29.7 (C-17), 28.5 (C-18), 28.1 (C-12), 27.1 (C-9),

19.3 (C-8), 19.0 (C-11), 17.5 (C-10), 16.9 (C-16),

16.9 (C-20), 14.9 (C-19). (Fig. 5.23).

HRESI-Mass

Spectrum

: Calcd for C20H32O4Na: 359.2192; Found:

359.2183 (Fig. 5.22).

Multifidenol (76)

HOH

HO

H

O

H

H

OH

Multifidenol (76)

Compound H

:

Multifidenol




Dα = -142.5 (c 0.07, CHCl3)


Yield : 0.04 g


IR Spectrum : νmax (KBr) 3362, 1709, 1624, 1238 cm-1


brs, -OH), 5.33 (1H, s, H-1), 4.82 (1H, brs, H-3),

4.12 (1H, brs, -OH), 2.82 (1H, m, H-13), 1.94

(1H, s, Me-16), 1.79 (1H, m, H-7a), 1.69-1.53

(4H, m, H-7b, H-8a, H-12a, -OH), 1.52-1.41 (1H,

m, H-12b), 1.26 (3H, s, Me-17), 1.12 (3H, d, J =

7.0 Hz, Me-20), 0.99 (3H, s, Me-18), 0.78 (1H, m,

H-8b), 0.72 (3H, s, Me-19), 0.64 (1H, m, H-11),

0.41 (1H, m, H-9). (Fig. 5.35).


145.2 (C-4), 137.5 (C-5), 128.6 (C-1), 87.8 (C-

15), 80.0 (C-3), 73.6 (C-6), 42.1 (C-7), 38.1 (C-

13), 29.3 (C-17), 28.8 (C-18), 28.5 (C-12), 27.3

(C-9), 19.4 (C-8), 19.2 (C-11), 17.2 (C-10), 16.6

(C-20), 14.9 (C-19), 13.9 (C-16). (Fig. 5.34).

HRESI-Mass

Spectrum

: Calcd for C20H30O4Na: 357.2036; Found:

357.2034 (Fig. 5.33).

5.4.3. Antimicrobial activity

Sterile blank discs (6.0 mm diameter, HiMedia Laboratories Pvt.

Ltd., Mumbai, India) were impregnated with the crude extracts at a

dose of 200 µg disc-1 and allowed to dry at room temperature in

laminar air flow chamber. The prepared paper discs containing

different test compounds were placed individually on the surface of

the medium in petri plates, containing Muller-Hinton agar seeded with

0.1 ml of previously prepared microbial suspensions individually

containing 1.5 × 108 cfu mL-1 (equal to 0.5 McFarland). Standard

antibiotic discs of neomycin and fluconazole and the disc containing

methanol served as a positive and negative controls, respectively. The

assessment of antimicrobial activity is based on the measurement of

inhibition zones formed around the discs. The plates were incubated

for 24 h at 37 °C and the diameter of inhibition zones was recorded.

All experiments were carried out in duplicates and mean values were

considered.

5.4.4. In vitro cytotoxicity testing

All tumor cell lines were maintained in a Modified Eagle’s medium

(Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum

(Sigma), along with 1% non-essential amino acids without L-glutamine

(Sigma), 0.2% sodium bicarbonate, 1% sodium pyruvate (Sigma) and

1% of antibiotic mixture (10,000 units penicillin and 10 mg

streptomycin per ml, Sigma). The cells were washed and resuspended

in the above medium and 100 µl of this suspension was seeded in 96

well flat bottom plates. The cells were maintained at 37°C in a

humidified 5% CO2 incubator (Model 2406 Shellab CO2 incubator,

Sheldon manufacturing, Cornelius, OR, USA). After 24 h incubation,

the cells were treated for 2 days with 26 test compounds at

concentrations ranging from 0.1-100 µM in DMSO (1% final

concentration) and were assayed at the end of the 2nd day. Each assay

was performed with two internal controls: (1) an IC0 with cells only, (2)

an IC100 with media only. After 48 h incubation, the cells were

subjected to MTT colorimetric assay (5 mg ml-1 MTT). The effect of the

different test compounds on the viability of tumor cell lines was

measured at the wavelength of 540 nm on a multimode reader

(Infinite® M200, Tecan, Switzerland). Dose-response curves were

plotted for the test compounds and controls after correction by

subtracting the background absorbance from that of the blanks. The

IC50 values (50% inhibitory concentration) were calculated from the

plotted absorbance data for the dose-response curves. IC50 values (in

µM) are expressed as the average of two independent experiments.

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Selected IR, 1D & 2D NMR and Mass Spectra of Compounds Pertaining to Chapter-V

Fig. 5.3: Mass spectrum of compound 73

Fig. 5.4: IR spectrum of compound 73

Fig. 5.5: 1H NMR spectrum of compound 73



Fig. 5.10: 13C NMR spectrum of compound 13


Fig. 5.22: HRMS spectrum of compound 75

Fig. 5.26: HSQC spectrum of compound 75

Fig. 5.27: DEPT spectrum of compound 75

Fig. 5.28: HMBC spectrum of compound 75

Fig. 5.29: 1H-1H COSY spectrum of compound 75

Fig. 5.30: NOESY spectrum of compound 75

Fig. 5.33: HRMS spectrum of compound 76


Fig. 5.36: HSQC spectrum of compound 76

Fig. 5.37: HMBC spectrum of compound 76

Fig. 5.38: COSY spectrum of compound 76

Fig. 5.39: NOESY spectrum of compound 76

isolation of novel bioactive diterpenoids from -...

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