two -...

TWO NEW 5-DEO LATONES FROM ALBIZZIA ODORATISSIMA (MIMOSACEAE)

The genus Albizzia belongs to the family Mimosaceae consists of 50

,species of which 8 species occur in India1. A few plants belonging to this

genus are extensively in the treatment of hemicrania, piles, excessive

perspiration, bronchites, asthma, snake-bite, leprosy, ulcers, cough, diarrhea

and ophthalmia.

A systematic phytochemical investigation of the Albizzia odoratissina

now carried out has resulted in the isolation and characterisation of two new

5-deoxyflavones. Hence it is felt worthwhile t o review briefly the chemistry

of 5-deoxyflavones covering their distribution, classification, structure

determination by physical and chemical methods, and synthesis.

I. Distribution

Flavones with 5-deoxygenation occur abundantly in Faba~eaell-~~,

family followed by ~h~melaeaceae~ , Premulaceae6, CCyperaceae4' and

Myris t ica~eae~~~"~. The occurrence of 5-deoxyflavones in Primulaceae is

restricted to Primula genus only and have the special feature of less

oxygenation. The 5-deoxyflavones with high oxygenation are confirmed to

Mimosaceae52*54~.56'57. ~ ~ ~ ~ ~ ~ i t a c e a e ~ ~ and Convolvulaceae53.

The 5-deoxyflavones are also found scarcely in few members of

Bignoniaceae5, sapindaceaeg, ~ u n c a c e a e ~ ~ and Vochy~iaceae~~.

II. Classification

Naturally occurring 5-deoxyflavones can be conveniently classified into

four types based on the oxygenation pattern present in ring-B. They are:

i. 5-Deoxyflavones devoid of oxygenation in ring-B

ii. 5-Deoxyflavones having monooxygenation in ring-B

iii. 5-Deoxyflavones having dioxygenation in ring-B

iv. 5-Deoxyflavones having trioxygenation in ring-B

Table - 1: 5-Deoxyflavones devoid of oxygenation in ring-B

Compound Plant source Family Ref.

1. 6-Methoxyflavone (1) Pimeleu decora Thymelaeaceae 4

2. 7-Hydroxyflavone (2) Pirneleu simplex Thymelaeaceae 4

3. 7-Methoxyflavone (3) Pirneleu simplex Thymelaeaceae 4

4. 7,B-Dirnethoxyflavone (4) Godmania Bignoniaceae 5 aesculifolia

- -

From Table - 1 it is clear that excepting 7,8-dimethoxyflavone reported

from Godmania of Bignoniaceae all the 5-deoxyflavones devoid of oxygenation

in ring-B were reported from Pimelea of Thymelaeaceae.

R R, R, 1 OMe H H

2 H O H H

3 H OMe H 4 H OMe OMe

Table - 2: 5-Deoxyflavones having monooxygenation in ring-B

Compound

2'-Hydroxy- flavone (5)

Plant source Family Ref.

Pimelea simplex Thymelaeaceae Primula farina Primulaceae Dephnopsis Thymelae aceae selloniana

2'-Methoxy- flavone 16)

Pimelea simplex Thymelaeaceae Primula kewensis Primulaceae


Pimelea decora Thymelaeaceae

3'-Methoxy- flavone (8)

P. decora Thymelaeaceae


Sapind us saponaria Sapindaceae

4'-Methoxy- flavone (10)

S. saponaria Sapindaceae

6,3'-Dihydroxy- flavone (11)

Pimelea decora Thyrnelaeaceae

P. decora Thymelaeaceae 6,3'-Dimethoxy- flavone (12)

Prirnula macrophylla Primulaceae 7,2'-Dihydroxy- flavone (13)

Trifolium repens T . subterraneurn Medicago sativa Baptisia lecontei Glycyrrhiza pal lid i flora C, squamulosa 6. inflata Lespedeza nakaii Sophora viciifolia Castanospermum australe Pterocrapus

Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae

7,4'-Dihydroxy- flavone (14)

Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae

Fabaceae marsupium Vicia faba Fabaceae 26


11. 7-Hydroxy-4'- Bauhinia manca Caesalpiniaceae 27

rnethoxyflavone

12. 7-Methoxy-4'- Bauhinia manca Caesalpiniaceae 27

hydroxyflavone Trifol ium hybridum Fabaceae 28

(16)

13. 7,4'-Dimethoxy- Virola venosa Myristicaceae 29

flavone (17) V. michelli Myris ticaceae 30

14. 8,2'-Dihydroxy- Primula pulverulenta Primulaceae 31

flavone (18)

15. 7,8,4'-Trihydroxy- Acacia nigrescens Mimosaceae 32

flavone (19)

From Table-2 it is evident that majority of 5-deoxyflavones with mono

oxygenation in ring-I3 are found in Pimelea of Thymelaeaceae, Primula of

Primulaceae, Glycerrhiza of Fabaceae and Virola of Myristicaceae. The

occurrence of compounds 5-10 constitute the rare report of flavones without

any substitution in ring-A. The isolation of compounds 11 and 12 with

monooxygenation at 6-position is of very rare occurrence. The occurrence of

2-oxygenated flavones with 5-deoxygenation (13 and 18) in Nature is also

very rare.

5 O H H H

6 OMe H H

7 H O H H

8 H OMe H

9 H H O H

R, 11 OH OH

1 2 OMe OMe

10 H H OMe

R R, R2 14 OH H OH

15 OH H OMe

16 OMe H OH

17 OMe H OMe

19 OH OH OH

Table - 3: 5-Deoxyflavones having dioxygenation in ring-B

Compound

1. 2',5'-Dihydroxy- flavone (20)

2. 2',5'-Dihydroxy- flavone 5'-acetate (21)

3. 3',4'-Dihydroxy- flavone (22)

4. 6,2',3'-Trirnethoxy- flavone (23)

5. 7,3',4'-Trihydroxy- flavone (24)

Plant source Familv Ref.

Primula spp. Primulaceae 33

P. pulverulenta Primulaceae 33

Primula spp. Primulaceae 34,35

Pimelea decora Thymelaeaceae 4

Trifolium repense Fabaceae Baptisia lecontei Fabaceae Sophora uiciifoliu Fabaceae Vicia faba Fabaceae Trifblium Fabaceae subterraneum Medicago sativa Fabaceae Juncus trifidus Juncaceae Luzula purpurea Juncaceae Salvertia Vochysiaceae convallariodora Vochysia Vochysiaceae cinnamonea V. tucanorurn Vochysiaceae Carex Cyperaceae longebrachiata Schoenus spp. Cyperaceae Uicinia spp. C yperace ae Medicago arborea Fabaceae Medicago strasseri Fabaceae Cyperus ref ixus Cyperaceae C. distans Cyperaceae C. laevigatus Cyperaceae C. betchei Cyperaceae C. lhotskyanus Cyperaceae C. rigidellus Cyperaceae Pinnati betchei Cyperaceae P, rigidellus Cyperaceae Albizzia julibrisson Mimosaceae


6. 7,3'-Dihydroxy-4'- Sophoraviciifolia Fabaceae 23 methoxyflavone Acacia farnesiana Mimosaceae 44 (Farnisin) (25)

7. 7,4'-Dihydroxy-3'- Salvertia Vochysiaceae 39 rnethoxyflavone convallariodora (Geraldone) (26) Vochysia Vochysiaceae 39

cinnamonea Trifol i um Fabaceae 45 subterraneum

8. 7,4'-Dimethoxy-3'- Virola venosa Myristicaceae 29 hydroxyflavone V. rnichelli Myristicaceae 30 (Tithonine) (27) Tithonia Compositaceae 46

tubaeformis Tithonia spp. Compositaceae 47 Virola surinamensis Myristicaceae 48

9. 7,3',4'-Trimethoxy- Virola venosa Myristicaceae 29 flavone (28) V. michelli Myristicaceae 30

Umtixa listerana Fabaceae 49

10. 7-Methoxy-3',4'- Virola uenosa Myristicaceae 29 methylenedioxyflavone (29)

11. 6,7,3',4'- Abrus precatorius Fabaceae 50 Tetrahydroxy- flavone (30)

12. 6,4'-Dimethoxy-7,3'- A. precatorius Fabaceae 50 dihydroxyflavone (Abrectorin) (31)

13. 6,7,8,3',4'- SaturejcL montana Cyperaceae 51 Pentamethoxy- flavone (32)

From Table - 3 it is clear that majority of flavones with dioxygenation

in ring-B possess high degree of oxygenation at 3/:4' followed by 2':5'

positions.

22

R, OM e

20 011 Of1

21 OH OAc

M e 0

0

2 3

R R, K, 24 OH OH OH

25 01.1 OH OMe

26 OH OMe OH

27 OMe OH OMe

28 OMe OMe OMe

R R, R, R3 30 OH OH OH OH

31 OMe OH OH OMe

OMe

Table - 4: 5-Deoxyflavones having trioxygenation in ring-B

Compound

1. 7,2',4',5'-Tetramethoxy- flavone (33)

2. 7,3',4',5'-Tetrahydroxy- flavone (34)

3. 6,7,3',4',St- Pent ahydroxy- flavone (35)

4. 6,7,4'-Trihydroxy- 3',5'-dimethoxyflavone (36)

5. 6,7-Dihydroxy-3',4',5'- trimethoxyflavone (Prosogerin E) (37)

6. 7-Hydroxy-6,3',4',5'- tetramethoxyflavone (Prosogerin D) (38)

7. 6,7,3',4',5'-Pentamethox - flavone (Prosogerin C) (39)

Plant source Family Ref.

Calliandra Mimosaceae 52 californica Evo Lvulus Convolvulaceae 53 nummularius Prosopis spicigera Mimosaceae 54

Artemisia giraldii Compositaceae 55

Prosopis spicigera Mimosaceae 56

P. spicigera Mimosaceae 54

P. spicigera Mimosaceae 57

From Table-4 it is clear that all the 5-deoxyflavones with

trioxygenation in ring-B have oxygenation at 2':4':5' or 3':4':5' only.

R R, 33 OMe H

34 E-I OMe

R R, R, R3 K4

35 OH OH OH OH OH

36 OH OEI OMe OH OMe

37 OH OH OMe OMe OMe

38 OMe OH OMe OMe OMe

39 OMe OMe OMe OMe OMe

IlI. Structure Determination

The various methods which are generally employed for the structure

determination of 5-deoxyflavones are:

i. Physical methods

ii. Chemical methods

iii. Synthesis

i, Physical methods

The physical methods widely employed in the identification and

structure elucidation of 5-deoxyflavones are:

a. Ultraviolet spectroscopy

b. IR spectroscopy

c. 'El-NMR spectroscopy

d. I3C-NMIt spectroscopy

e. Mass spectroscopy

a. Ultraviolet spectroscopy

UV spectroscopy, a major technique for the structure analysis of

flavonoids, widely is used to distinguish 5-deoxyflavones from 5-

hydroxyflavoness8. Flavones with bdeoxygenation usually appear as bright

yellow / bluish green fluorescent spots under W light. 5-Deoxyflavones

exhibit two absorption bonds in the region 340-350 and 240-280 nm in band

I and band 11, as in the case of &oxygenated flavones but intensity of band

I1 absorption maxima in 5-deoxyflavones in methanol are usually very weak

than those of 5-hydroxyflavones58.

b. IR speetroseopy

5-Deoxyflavones are readily distinguished from 5-oxygenated flavones

from their IR spectra", as they show carbonyl absorption band in the region

1620-1650 cm" as distinct from 5-hydroxyflavones which usually exhibit

carbonyl absorption in the region 1650-1660 crne1.

R spectroscopy

In the structure elucidation of 5-deoxflavones certain useful

information can be obtained by comparison of their proton NMR spectra with

&oxygenated flavones. The chemical shift of H-5 proton of 5-deoxyflavones is

strongly deshielded by the 4-keto group and appears between 7.8-8.1 ppm. In

5-deoxy-7-oxygenated flavones the signals for both H-6 and H-8 occur at lower

field than in the 5,7-dihydroxyflavonoids5g. Moreover in 5-deoxyflavones the

H-8 may absorb a t either lower or higher field than the H-6 proton as

evidenced from 5,7-dihydroxy flavones, in which H-6 proton always absorbs

at higher field than the H-8 proton60.

R speetroseopy

13 C-NMR spectroscopy provides an elegant method for structure

establishment of 5-deoxyflavones. Lack of oxygenation at C-5 position will

have marked influence on the carbonyl carbon (C-4) resonance due to the

absence of intramolecular hydrogen bonding with carbonyl carbon. An upfield

shift of about +5 ppm of the C-4 resonance is typical of 5-deoxygenated

flavones6I.

e. Mass spectroscopy

The fragmentation pattern of 5-deoxyflavones is similar to 5-

hydrox$avones. The molecular ion peak is generally the base peak with

other major peaks corresponding to [M-HI', [M-CO]", A,'", [A,-CO]" and B,"

fragments in Chart - 1.

CHART - 1

ii. Chemical methods

Alkaline degradation, widely employed for the structure determination

of 5-oxygenated flavones may be employed for structure determination of 5-

deoxyflavones also.

a. Alkaline degradation

When 5-deoxyflavones are subjected to alkaline degradation with 50%

potassium hydroxide in methanol under nitrogen atmosphere for 5-8 h afford

2'-hydroxyacetophenone and benzoic acid derivatives.

The structure of abrectorin isolated from Abrus precatorius49 was

established as 7,3'-dihydroxy-6,4'-dimethoxyflavone (31), based on the

formation of 2,4-dihydroxy-5-methoxyacetophenone (31a) and 3-hydroxy-4-

methoxybenzoic acid (31b) when abrectorin was treated with 50% potassium

hydroxide (Chart - 2).

CHART - 2

OH OMe

50% KOH HOqL ~ Q M ~

MeOH, N,, 8h M e 0 HOOC /

0 31a 31b

0 31

Alkaline degradation of Abrectorin

iii. Synthesis

5-Deoxyflavones are preferably synthesised by Baker-Venkataraman

rearrangement. In this rearrangement ortho-hydroxyacetophenone derivative

is condensed with a substituted benzoyl chloride in the presence of potassium

carbonate. The resulting ester on treatment with potassium hydroxide in

pyridine is converted into a diketone which was cyclised in glacial acetic acid

and anhydrous sodium acetate yielded the corresponding 5-deoxyflavone.

Bhardwaj et als2 have carried out the total synthesis of prosogerin D

(38) by condensing 4-benzyloxy-2-hydroxy-5-methoxyacetophenone (40) with

tri-0-methylgalloylchlorde (41) in the presence of dry pyridine to give an ester

(42) which undergoes isomerization in the presence of pyridine-potassium

hydroxide to give 4-benzyloxy-2-hydroxy-5,3',4',5'-tetramethoxy

dibenzoylmethane (43). Cyclization of this P-diketone with acetic acid-soidum

acetate yielded 7-benzyloxy-6,3',4',5'-tetramethoxflavone (44) which on

catalytic (PdC) debenzylation in acetic acid resulted prosogerin D (38)

(Chart - 3).

Present work

ALbizzia odoratissima Benth is a large tree widely distributed in the

tropical India and Sri Lanka'. In traditional medicine, the bark of this plant

is used as a remedy for leprosy, ulcers and the leaves for coughs2s3.

Earlier chemical investigation on A. odoratissima has resulted in the

isolation of triterpenic acids63 and saponins from seeds"$' and flavonoids from

h e a r t w o ~ d ~ ~ .

A systematic chemical examination of the root bark of A. odoratissima

has now been undertaken as this part of this species has not been examined

earlier.

Chemical investigation of the root bark of Aodoratissima

The shade dried and powdered root bark (2 Kg) ofA. odoratissima was

successively extracted with n-hexane, acetone and methanol. Further work

up of methanol extract didn't yield any crystalline principle.

Examination of Wexane Extract

The hexane extract on concentration yielded dark green syrupy mass

(20 g). It was column chromatographed over silica gel using hexane-ethyl

acetate step gradient. The hexane-ethyl acetate, 9:1, 8:2 eluates yielded two

yellow solids and were designated as AO-1 and AO-2, respectively.

AO-1 was obtained as yellow amorphous powder (20 mg) from

methanol, mp 252-254°C. It gave negative ferric chloride test and orange

colour with Mg-HCI. I t was bluish green flourescent under UV and UV/NH,.

AO-1 showed [M-tKl', [M+NaIt and [M+Hl+ ions at mlz 365.0547,

349.0788 and 327.0983, respectively in its positive ESIMS (Fig. 1)

corresponding to the molecular formula C,,K,,O,. This was corroborated by

the decoupled 13C-NMR DEPT spectrum (Fig. 2) which showed signals for all

the eighteen carbons of the molecule. The UV absorption maxima of AO-1

(Fig. 3) in MeOH (254, 325 nm) suggested AO-1 to be a flavoneG7. The

addition of aluminium chloride and sodium acetate caused no shifts in its UV

spectrum indicating the absence of free hydroxyl groups a t C-5 and C-7,

respectively. The IR spectrum (Fig. 4) showed a carbonyl absorption band at

1648 and a aromatic C=C band at 1620 crnm1, respectively.

The 'H-NMR spectrum of AO-1 (Fig. 5) showed two rnethoxyl singlets at 6

3.97 and 4.0 I, a two-proton singlet at 6 6.05 assigned to a methylenedioxy group

and a sharp one-proton singlet at 6 6.61 ascribed to H-3. The EIMS of AO-1

(Fig. 6) showed two retro-Diels Alder fragments (Chart-4) at mlz 181 [A,+H]'

and 146 [B,]+ indicating the presence of two rnethoxyl groups in ring-A and a

methylenedioxy group in ring-B, respectively. The 'H-NMR spectrum of AO-1

further showed two ortho-coupled aromatic doublets at 6 7.91 and 7.01 and were

assigned to H-5 and H-6, respectively as the former showed correlations

with C-6, C-7, C-4, and C-8a, and the latter with C-4a, C-5, C-7 and C-8 in

its HMBC spectrum (Fig. 7). It also displayed three aromatic proton signals

at 6 7.50 (IH, dd, J = 8.2, 1.6 HZ), 7.36 (lH, d, J = 1.6 Hz) and 6.91 (IH, d,

J = 8.2 Hz) assigned to H-6', H-2' and H-5' respectively, characteristic of 3',

4'-dioxygenated f l a v ~ n e ~ ~ . This fixes the attachment of a rnethylenedioxy

group at 6 6.05 to 3' and 4' positions, further evidenced by the 3J correlation

of the methylene protons with (2-3' (6 148.4) and C-4' (6 150.4). Of the two

rnethoxyl groups in ring-A, the one at 6 3.97 was placed at C-8 as it resonated

at 61.6 ppm in its 13C-NM~ spectrum which is characteristic of a di-ortho-

substituted methoxyl group6'. This fixes the placement of another methoxyl

group at (6 4.01) to C-7, further supported by its NOESY spectrum (Fig. 8).

The NMR spectral assignments for AO-1 were further confirmed by HSQC

(Fig. 9 and Table - 51, HMBC (Fig. 7 and Table - 6), 'H-'H COSY (Fig. 10 and

Table - 7) studies.

Significant HMBC (-+-) and

NOESY (4-------b) correlations for 45

Thus from the foregoing spectral studies the structure of AO-1 was

characterised as 7,8-dimethoxy-3',4'-methylenedioxyflavon (45).

Table - 5: COSY lJcH (HSQC) Date of AO-1

Proton chemical shift (6)

7.91 (H-5)

7.50 (H-6')

7.36 (H-2')

7.01 (H-6)

6.91 (H-5')

6.61 (H-3)

6.05 (0CE120)

4.011 (OMe-7)

3.97 (OMe-8)

Correlated carbon chemical shift (6) Assignment

Table - 6: HMBC (2-3J,,) Correlations of AO-1

Proton

H-5

H-6'

H-2'

H-6

H-5'

H-3

OCH,O

OMe-7

OMe-8

Chemical shift (6)

7.91

7.50

7.36

7.01

6.91

6.61

6.05

4.01

3.97

Correlated carbon(s)

C-6, C-7, C-4, C-8a

C-2, C-2', C-5', C-4'

C-l', C-2, C-3', C-4'

(3-5, C-4a, C-7, C-8

6-l', C-4', C-3'

C-2, C-l', 6-4, C-4a

C-3', C-4'

C-7

C-8

Fig. 1 : ESI Mass Spectrum of AO-1

200 250 300 350 400

Wave length (nm)

Fig. 3 : UV Spectrum (MeOH) of AO-1

Table - 7: 'H-lH COSY Data of AO-1

Chemical shift of coupled protons Type of coupling Assignment

7.91 and 7.01

7.50 and 7.36

7.50 and 6.91

ortho

meta

ortho

H-5 and H-6

H-6' and H-2'

H-6' and H-5'

AO-2 was obtained as yellow amorphous solid (16 mg) from methanol,

mp. 128-130°C. It gave negative ferric chloride test and pale pink colour with

Mg-HC1. It was bluish green flourescent under W and WNH, .

AO-2 showed [M+H]+ ion peak at mlz 313.1000 in its positive ESIMS

(Fig. l l ) , consistent with the molecular formula C,,H,,O,, and was

corroborated by decoupled 13C-NMR DEFT spectrum (Fig. 12), which showed

signals for all the eighteen carbons of the molecule. The W absorption

maxima of 2 in MeOH at 238 and 339 nm (Fig. 13) is typical of a flavone".

The addition of aluminium chloride and sodium acetate didn't cause any shift

in its UV spectrum indicating the absence of free hydroxyl groups at C-5 and

(2-7, respectively. The IR spectrum (Fig. 14) showed two strong absorption

bands a t 1640 and 1600 ern-', due to carbonyl and aromatic C=C bonds,

The 'H-NMR spectrum of AO-2 (Fig. 15) displayed three methoxyl singlets

at 6 3.83, 3.86 and 3-87 and a sharp one-proton singlet at F 7.03 characteristic

of H-3 of 2'-oxygenated flavone70. The EIMS of AO-2 (Fig. 16) showed the

[W' ion peak at mlz 312 and two retro-Diels Alder fragments (Chart-5) at

mlz 151 [A,+H]+ and 162 [BJ' consistent with the presence of one methoxyl

group in ring-A and two methoxyl groups in ring-B, respectively. The 'H-NMR

spectrum of AO-2, further revealed two aromatic ABX spin coupled systems,

at 6 8.07 (IH, d, J = 8.8 Hz), 6.90 (1H, dd, J = 8.8, 2.3 Hz) and 6.85 (lH, d,

J = 2.3 Hz) assigned to H-5, H-6 and H-8, and 6 7.82 ( lH, d, J = 8.1 Hz), 6.57

(IH, dd, J = 8.1, 2.3 Hz) and 6.50 (lH, d, J = 2.3 Hz) assigned to H-6', H-5'

and H-3', confirming the presence of mono-substitution in ring-A and

disubstitution in ring-B. The three methoxyl groups at 6 3.83, 3.86 and 3.87

were placed at C-4', C-2' and C-7 positions as these methoxyls protons showed

correlations with these carbons at 163.0, 159.4, 163.8 ppm respectively in its

HMBC spectrum (Fig. 17). The placement of methoxyl groups were further

supported by the NOE studies (Fig. 18). The NMR spectral assignments were

further confirmed by HSQC (Fig. 19 and Table - 8), HMBC (Fig. 17 and Table

- 9) and *H-'H COSY (Fig. 20 and Table - 10) studies.

Significant HMBC (+) and

NOESY (4------+) correlations for 46

Thus from the foregoing spectral studies compound AO-2 was

established as 7,2',4'-trimethoxyflavone (46). The isolation of AO-2 constitutes

the first report of the occurrence of a 2'-oxygenated flavone from Albizia

genus.

Table - 8: 'H-"C-COSY (HSQC) Data of AO-2

Proton chemical shift Correlated carbon (6) chemical shift (6)

8.07 (H-5) 126.8

7.82 (H-6') 130.2

7.03 (H-3) 111.1

6.90 (H-6) 113.9

6.85 (H-8) 100.2

6.57 (H-5') a

105.2

6.50 (H-3') 98.8

3.87 (OMe-7) 55.7

3.86 (OMe-2') 55.6

3.83 (OMe-4') 55.7

Assignment

Table - 9: HMBC (2-3~c,) Correlations of AO-2

Proton

H-5

H-6'

H-3

H-6

H-8

H-5'

H-3'

OMe-7

OMe-2'

OMe-4'

Chemical shift (6)

8.07

7.82

7.03

6.90

6.85

6.57

6.50

3.87

3.86

3.83

Correlated carbon(s)

C-4, C-6, C-7, C-8a

C-2, C-2', C-4'

C-l', C-2, 6-4, C-4a

C-4a, C-7, C-8

C-4a, (3-6, C-7, C-8a

C-4', C-3', C-1'

C-l', C-2', C-4', (3-5'

C-7

C-2'

C-4'

IZE'BLI Wdd

200 250 300 350 400

Wave length (nm)

Fig. 13 : UV Spectrum (MeOH) of AO-2

I-" -. .-

1 3 0

' ' I " ' ' r *

0

v,

' . . . , . . ,

. , , , , , , , , , , , , , , , , . , , I . . . I , ,

Table - 10: 'I3-lE-I COSY Data of AO-2

chemical shift of coupled protons ( 6 ) Type of coupling Assignment

8.07 and 6.90 ortho H-5 and H-6

6.90 and 6.85 meta H-6 and H-8

7.82 and 6.57 H-6' and H-5'

6.57 and 6.50 meta H-5' and H-3'

Examination of Acetone Extract

The acetone extract ofA. odoratissima was concentrated under reduced

pressure to give dark brown gummy mass (25 g). It was segregated into

n-hexane soluble (10 g) and insoluble fraction (15 g). The hexane soluble

fraction on further workup didn't yield any crystalline principle.

The hexane insoluble fraction (15 g) on purification over a silica gel

column using hexane-ethyl acetate (7:3) as eluent furnished yellow solid, and

crystallized from methanol to give yellow needle and it was designated as

AO-3.

(Tithonine, 27)

AO-3 was crystallized from methanol as yellow needles (20 mg) mp

190-192°C. I t gave dark green colour with alcoholic ferric chloride and orange-

red colour with Mg-HC1. It was analysed for C,,H,,O, which is consistant

with the molecular ion at mlz 298 in its EI mass spectrum (Fig. 21) and

was corroborated by the presence of seventeen carbon signals in its 13C NMR

spectrum (Fig- 22). Its W absorption maxima (Fig. 23) at 235 and 338 nm

closely resembled that of a 5-deoxyflavone derivative6?. Addition of sodium

acetate didn't cause any change in band I1 absorption maximum indicating

the absence of a free hydroxyl a t (2-7. The IR spectrum (Fig. 24) showed two

strong absorption bands at 3278 and 1643 cm-' due to hydroxyl and

conjugated carbonyl functions, respectively.

The 'H NMR spectrum (Fig. 25) of AO-3 showed a downfield signal at

6 9.44 assignable to a phenolic hydroxyl group. It also showed signals for two

methoxyl groups (6 3.84 and 3.88) and six aromatic protons (6 7.89, 7.51,

7.43, 7.2 1, 7.03 and 7.01). Six aromatic protons appearing as two sets of ABX

type s i w a l s a t 6 7.51 (IH, dd, J = 8.9, 1.5 Hz), 7.43 (IH, d, s, H-2', J = 1.5

Hz), and 7.03 (lH, d, J = 8.9 Hz); 7.89 (lH, d, J = 8.9 Hz), 7.01 (IH, d, J =

1.5 Hz) and 7.21 ( lH, d, J = 1.5 Hz) were assigned to H-6', H-2', H-5' and H-

5, H-6, H-8, respectively. A sharp one proton singlet at 6 6.70 was assigned

to H-3 proton of flavone. Two retro-Diels Alder fragments at mlz 150 and

148, in its EI mass spectrum (Fig. 26) indicated the presence of a rnethoxyl

group in ring-A, and a hydroxyl and a methoxyl group in ring-B, respectively.

The methoxyl groups at 6 3.88 and 3.84 were placed at C-7 and C-4'

positions, based on the absence of bathochromic shift of the band-I1 and band-

I W absorption maxima of AO-3 with sodium acetate and sodium methoxide,

respectively.

Thus the structure of AO-3 was elucidated as 7,4'-dimethoxy-3'-

hydroxyflavone (tithonine) (27) by comparison of its spectral data with

literature v a l ~ e s ~ ~ > ~ ~ ' ~ ~ ~ ~ ~ .

LLB'L L06 ' L

EXPERIMENTAL

The root bark ofA. odoratissima used in the present investigation was

collected from Tirupati, Andhra Pradesh, India.

The dried and ground root bark of A. odoratissima (2 Kg) was

successively extracted with n-hexane (3 x 51), Me, CO (3 x 51) and MeOH (3

x 51). The MeOH extract on further workup didn't yield any crystalline

principle.

Examination of Hexane Extract

The hexane extract on concentration under reduced pressure gave a

dark green syrupy mass (20 g). It was found to be a mixture 'of two

components on preliminary TLC examination and was subjected to column

chromatography over silica gel (200 g) using hexane-ethyl acetate as eluent.

A total of 25 fractions of 50 ml each were collected by slow elution and the

column fractions were monitored by TLC (silica gel) using hexane-ethyl

acetate 9:1, 8:2 as solvent systems and alcoholic ferric chloride as the

detecting agent (Table - 11).

Fraction No. Eluent Nature of the Eluate

1-5 Hexane Fatty material (negligible)

6- 11 Hexane-EtOAc (9: 1) Yellow solid (25 mg)

12-15 Hexane-Et OAc (9: 1) No residue

16-20 Hexane-EtOAc (8:2) Yellow solid (20 rng)

21-25 Hexane-EtOAc (1: 1) No residue

The yellow solid (25 mg) obtained from fractions 6-11 on crystallization

from methanol afforded yellow amorphous powder (20 mg), rnp 252-254OC. It

was designated as AO-1.

Fractions 16-20 on concentration yielded yellow solid (20 mg) which on

crystallization from MeOH gave yellow amorphous powder (16 mg), mp 128-

130°C. It was designated as AO-2.

AO-1 was crystallized from MeOH as yellow amorphous solid (20 mg);

mp 252-254'6. It gave negative ferric chloride test and orange colour with

Mg-HCl. It was bluish green flourescent under UV and UV/NH,.

UV: h max (MeOH) (log E) 254 (4.231, 325 (4.14) nm.

IR: v max (KBr) 1648 (>C=O), 1620, 1596, 1544,1490 cm-l.

R: (400 MHz, CDCl,) 6 7.91 (lH, d, J = 8.9 Hz, H-5), 7.50 (IH, dd, J

= 8.9, 1.6 HZ, H-60, 7.36 (lH, d, J = 1.6 HZ, H-29, 7.01 (lH, d, J = 8.9 HZ, H-

6), 6.91 (lH, d, J = 8.2 HZ, H-5'), 6.61 (lH, S, H-3), 6.05 (2H, S, -0-CH,-0-),

4.01 (3H, s, OMe-7), 3.97 (3H, s, OMe-8).

R: (75 MHz, CDC1,) 6 178.0 (C-4), 162.6 (C-2), 156.6 (C-7), 150.5 (C-

8a), 150.4 ((2-47, 148.4 (C-3'), 136.9 (C-8), 125.8 (C-1'1, 121.4 (C-6'), 120.9 (C-

51, 118.6 (C-4a), 109.8 (C-6), 108.8 (G-5'), 106.3 (C-2'1, 105.9 (C-3), 101.9

(OCH,O), 61.6 (OMe-8), 56.4 (OMe-7).

ESIMS: m/z (positive ion mode) [M+Hl+, 327.0983 (calc. for C,,H,,O,:

327.3015).

Finally AO-1 was characterized as 7,8-dimethoxy-3',4'-

methylenedioxyflavone (45).

AO-2 was crystallized from MeOH as yellow amorphous solid (16 mg),

mp 128-130°C. It gave negative ferric chloride and pale pink colour with Mg-

HC1. I t was bluish green flourescent under UV and UVINH,.

UV: h max (MeOH) (log E) 238 (4.18), 339 (4.03) nm.

XR: v max (KBr) 1640 (>C=O), 1600, 1509, 1440, 1376 crnml.

R: (400 MHz, CDC1,) 6 8.07 (IH, d, J = 8.8 HZ, H-5), 7.82 (lH, d, J =

8.7 HZ, H-6'), 7.03 (lH, S, H-3), 6.90 (lH, dd, J = 8.8, 2.3 HZ, H-6), 6.85 (IH,

d, J = 2.3 Hz, H-8), 6.57 (lH, dd, J = 8.7, 2.3 HZ, H-5'1, 6.50 (lH, d, J = 2 . 3

Hz, H-3'), 3.87 (3H, s, OMe-7), 3.86 (3H, s, OMe-27, 3.83 (3H, s, OMe-4').

R: (75 MHz, CDCI,) G 178.3 (C-4), 163.8 (C-7), 163.0 (C-4'), 160.4 (C-

21, 159.4 (C-2'), 158.0 (C-8a), 130.2 (C-6'), 126.8 (C-5), 117.6 (C-4a), 113.9 (C-

6), 113.5 (C-1'), 111.1 (C-3), 105.2 (C-5'), 100.2 (C-8), 98.8 (C-3% 55.7 (OMe-71,

55.6 (OMe-2'), 55.5 (OMe-4').

ESIMS: (positive ion mode) mlz [M+H]+ 313.1000 (calc. for C,,H,,O,:

313.3239).

Finally, AO-2 was characterized as 7,2',4'-trimethoxyflavone (46).

Examination of Acetone Extract

The acetone extract was concentrated to yield a dark brown gummy

mass (25 g). It was defatted with n-hexane. The residue left behind (15 g) was

column chromatographed over silica gel using step gradient of hexane and

ethyl acetate. A total of 20 fractions were collected and the column fractions

were monitored by TLC (silica gel) using hexane-EtOAc (7:3) as the solvent

system and alcoholic ferric chloride as detecting agent. The column

chromatographic details are shown in Table - 12.

TABLE - 12

Fraction No. Eluent Nature of Eluate

1-5 Hexane Fatty material

6-10 Hexane-E t OAc (8: 2) No residue

11-15 Hexane-EtOAc (7:3) Colourless solid (30 mg)

16-20 EtOAc No residue

The colourless solid (30 mg) obtained by evaporation of hexane-EtOAc

(7:3) eluates on crystallization from MeOH furnished colourless needles (25

mg) and was designated as AO-3.

A 0 -3

(Tithonine, 27)

AO-3 was crystallized from MeOH as colourless needles (25 mg), rnp

190-192°C. It gave dark green colour with alcoholic ferric chloride, orange

colour with Mg-HC1.

W: hmax (hTeOH) (log E): 235 (4.411, 314 sh, 338 (4.20) nm.

IR: v m a . ( D r ) 3278 (OH), 2972,2840,1643 (>C=O), 1602,1511,1440,1379

cm-l.

1: (300 MHz, DMSO-d,) 8 9.44 (IH, S, OH-3'), 7.89 (IH, d, J = 8.9 Hz,

H-5), 7.51 ( lH, dd, J = 8.9, 1.5 HZ, H-67, 7.43 (lH, d, J = 1.5 Hz, H-Z'), 7.21

(IH, d, J = 1.5 HZ, H-8), 7.03 (lH, d, J = 8.9 HZ, H-5'), 7.01 (lH, dd, J = 8.9,

1.5 Hz, H-6), 6.70 (IH, s, H-3), 3.88 (3H, s, OMe-7), 3.84 (3H, s, OMe-4').

EIMS: mlz (rel. int. %) 298 (loo), 283 (5), 270 (4), 255 ( l l ) , 165 (lo), 151

(32).

Finally the identity of AO-3 as 7,4'-dimethoxy-3'-hydroxyflavone

(tithonine) (27) was confirmed by comparing with literature values of

tithonine.

REFERENCES

J. S. Gamble, Flora of the Presidency ofMadras, Vol. I, pp. 305-307,

BSI, Calcutta (1956).

K. R. Kirtikar and B. D. Basu, Indian Medicinal Plants, Vol. 11,

pp.936-944, Periodical Experts, New Delhi (1935).

R. N. Chopra, S. L. Nayer and I. C. Chopra, Glossary of Indian

Medicinal Plants, pp. 10-11, CSIR, New Delhi (1956).

P. W. Freeman, S. T. Murphy, J. E. Nemorin and W. C. Taylor, Aust.

J. Clzern., 1779 (1981).

F. R. Stermitz, R. L. Arslanian and 0. Castro, Biochem. Syst. Ecol., 20,

481 (1992).

M. L. Bouillant, E. Wollenweber and J. Chopin, C. R. Acad. Sci. Ser.

D., 273, 1629 (1971).

6. Blaske, L. Xun and G. A. Cordell, J. Nat. Prod., 51, 60 (1988).

E. Wollenweber and K. Mann, Biochem. Plzysiol. Pflanzen, 181, 665

(1986).

S. M. A. Wahab and M. A. Selim, Fitoterapia, 56, 167 (1985).

V. U. Ahrnad, M. G. Shah, F. V. Mohammad, N. Ismail and M.

Noonvala, Phytochemistry, 30, 4206 (1991).

J. B. Harborne, Comparatiue Biochemistry of the Flavonoids, p. 43,

Academic Press, London (1967).

12. A. L. Livingston and E. M. Bickoff, J. Pharrn. Sci., 53, 1557 (1964).

E. M. Bickoff, A. L. Livingston and S. C. Watt, Phytochemistry, 4, 523

(1965).

C. A. Maxwell, U. A. Hartwing, C. M. Joseph and D. A. Philips, Plant

PhysioL., 91 842 (1989).

R. A. Eade, I. Salasoo and J. J. H. Simes, Aust. J. Chern., 19, 1717

(1966).

K. R. Markham and T. J. Mabry, Phytochemistry, 7, 791 (f 968).

J. Liu, S. Yang, Y. Fu, D. Xu, C. Jiang and F. Hou, Zhongcaoyao, 23,

349 (1992).

L. Hong and Z. Ruyi, Beijing Yike Daxue Xuebao, 24, 399 (1992).

K. Kiichiro, D. Sachio, H. Yukio, K. Kaoru, K. Kiyotaka, T. Kunio, Y.

Yukiyoshi, 0. Kenzo and K. Takeshi, J. Nat. Prod., 55, 1197 (1992).

Z. Kun, D. Feng, Z. Ruyi, Z. Lu and F. Naiwu, Tianran Chanwu Yanjiu

Yu Kaifa, 5, 1 (1993).

Z. Kun, and 2. Ruyi, J. Chin. Pharrn. Sci., 3, 90 (1994).

K. B. Hae and K. C. Min, Saengyak Hakhoechi, 26, 13 (1995).

W. Wang, J. Li, L. Wei, 0. Shigeru, Zhongguo Zhongyao Zazhi, 21,165

(1996).

K. R. Markham, Phytochemistry, 12, 1091 (1973).

25. R. Maurya, A. B. Ray, F. K. Dauh, D. J. Slatkin and P. L. Schiff, J. Nat. Prod., 49, 167 (1986).

F. T. Lorente, M. M. G. Grau and F. A. T. Barberan, 2. Naturforsch,

45C, 1070 (1990).

H. Achenbach, M. Stocker and M. A. Constenla, Phytochemistry, 27,

1835 (1988).

P. D. Fraishtat and N. S. Wulfson, Khim. Prir. Soed., 663 (1981).

M. J. &to, M. Y. Yoshida and 0. R. Gottlieb, Phytochemistry, 31,283

(1992).

L. S. Santos, M. J. C. Correa, L. M. 0. Campos and M, A. Andrade,

Fitoterapia, 67, 555 (1996).

E. Wollenweber, K. Mann, K. Iinuma, T. Tanaka and M. Mizuno,

Phytochemistry, 27, 1483 (1988).

M. Elfranco, Phytochemistry, 33, 733 (1993).

E. Wollenweber, K. Mann, M. Iinuma, T. Tanaka and M. Mizuno, 2.

Naturforsch, 436, 305 (1988).

J. B. Harborne, Phytochemistry, 7, 1215 (1968).

E. Wollenweber, J. F. Bonvin and M. Jay, 2. Naturforsch , 33C, 831

(1978).

W. Baker, J. Chem. Soc., 1387 (1933).

E. M. Bickoff, S. C. Witt and A. L. Livingston, J. Pharm. Sci., 54, 1555

(1965).

38. C. A. Williams and J. B. Harborne, Biochem. Syst. Ecol., 3,181 (1975).

J. L. C. Lopes, J. N. C. Lopes and F. H. F. Leitao, Phytochemistry, 18, 362 (1979).

J. B. Harborne, C. A. Williams and K. L. Wilson, Phytochemistry, 24,

751 (1985).

F. P. Garcia, J. L. Ceresuela, A. E. Gonzalez and I. Aguinagalde, J.

Basic Microbiol., 32, 241 (1992).

J. B. Harborne, C. A. Williams and K. L. Wilson, Phytochemistry, 26,

2491 (1982).

P. Chamsuksai, J. S. Choi and W. S. Woo, Arch. Pharmacal Res., 4,

129 (1981).

N. P. Sahu, B. Achari and S. Banerjee, Phytochemistry, 49, 1425

(1998).

E. Wong and C. M. Francis, Phytochemistry, 7, 2123 (1968).

J. Correa, and M. L. Cervera, Bull. Soc. Chern. (France), 2,475 (1971).

J. C. L. Duke, Am. J. Bot ., 69, 784 (1982).

E. E. D. A. Blumenthal, M.S. D. Silva and M. Y. Yoshida,


A. P. N. Burger, E. V. Brandt and D. G. R o w , Phytochernistry, 22,

2813 (1983).

D. K. Bhardwaj, M. S. Bisht and C. K. Mehta, Phytochemistry, 19,

2040 (1980).

51. E. Wollenweber and K. M. V. Vetschera, Fitoterapia, 62, 462 (1991).

R. D. Encarnacion, N. A. Ochoa, U. Anthoni, C. Christophersen and P.

H. Nielsen, J. Nat. Prod., 57, 1307 (1994).

D. R. Gupta, B. Ahmed and R. P. Dhirnani, Shoyakugaku zasshz, 38,

341 (1984).

D. K. Bhardwaj, M. S. Bisht, R. K. Jain and G. C. Sharma,


W. F. Zheng, R. X. Tan, L. Yang and Z. L. Liu, Planta Med., 62, 160

(1996).

D. K. Bhardwaj, A. K. Gupta, R. K. Jain and G. C . Sharma, J. Nat.

Prod., 44, 656 (1981).

D. K. Bhardwaj, S, C. Jain, G. C. Sharma and C. K. Mehta, Indian J.

Chem., 16B, 1133 (1978).

T. J. Mabry, K. R. Markham and M. B. Thomas, The Systematic

Identification of Flavonoids, pp. 44-48, Springer-Verlag, New York

(1970).

J. B. Harborne, T. J. Mabry and H. Mabry, The Flauonoids, p. 63,

Chapman and Hall, London (1975).


Identification of Flavonoids, p. 263, Springer-Verlag, New York (1970).

P. K. Agrawal, Carbon-13 NMR of Flauonoids, p. 123, Elsevier,

Amsterdam, (1989).

D. K. Bhardwaj, A. K. Gupta, R. K. Jain and A. Munjal, Indian J. Chem., 20B, 446 (1981).

I. P. Varshney and M. S. Y. Khan, J. Pharm. Sci., 50, 923 (1961).

I. P. Varshney and M. S. Y. Khan, J. Sci. Ind. Res., 21B, 30 (1962).

I. P. Varshney and M. S. Y. Khan, Bull. Chem. Soc. (Japan), 38, 1214

(1965).

L.R. Row and C.V.R. Sastry, Tetrahedron, 19, 1371 (1963).


Identification of Flavonoids, p. 41, Springer Verlag, New York (1970).

D. M. Tomazela, M. T. Pupo, E, A. P. Passador, M. F. G. F. Silva, I?. C.

Vieira, J. B. Pernandes, E. R. Fo, 6. Oliva and J. R. Pirani

Phytochernistry, 55, 643-651 (2000).

M. Inuma, S. Matsuura and K. Kusuda, Chern. Pharrn. Bull., 28,708

(1980).

T. Tanaka, M. Iinuma and M. Mizumo, Chem. Pharm. Bull., 34, 1667

(1986).

two -...

Documents