the compound mf-2 was obtained from the ethanolic extract...
TRANSCRIPT
![Page 1: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/1.jpg)
[89]
The compound MF-2 was obtained from the ethanolic extract was
suspended in water and then consecutively partitioned with
trichloromethane, ethyl acetate and butanol.
The extract was purified by column chromatography. The compound
was isolated as yellow amorphous powder, mp 199-2000C, analysed for
C34H34O20.
The molecular formula of compound was established as C34H34O20 by
mass spectrometry. The UV spectrum exhibited absorption maxima at 267
and 355nm that are characteristic absorption bands of a flavone skeleton.36
In the IR spectrum, along with the signal for an ester carbonyl (1710cm-1
)
signals for hydroxyl group (3414cm-1
), a conjugated carbonyl (1638cm-1
),
and phenyl group (1618,1498cm-1
) were apparent.
The compound gave positive Molisch‟s test1 but did not reduce
fehling solution nor gave positive test with AHP2, indicating its glycosidic
nature, on acid hydrolysis it gave an aglycone, mp. 288-289 and sugars were
identified as D-galactose and D-xylose by tlc.
In order to study the molecular size of the aglycone, glycoside was
thus subjected to systematic analysis.
![Page 2: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/2.jpg)
[90]
AGLYCONE
The aglycone mp. 288-2890C was analysed for C16H12O7, [M
+316]. It
gave all the characteristic colour reaction of flavonoid.
1. The alc. solution of the compound gave white ppt. with zirconium
oxychloride10-11
in presence of citric acid confirmed free hydroxyl
group at position – 3.
2. The aglycone was treated with a solution of boric acid in the presence
of citric acid12-13
in acetone gave colour indicated its free hydroxyl
group at position-5.
3. The aglycone did not give any colour with vanilline hydrochloric
acid15-16
reagent indicating the 7- hydroxyl group was either substitute
or absence.
4. The compound performed purple colour with nitric acid showing the
methoxy (-OCH3) group at position-7.37
5. The compound also gave yellow colour with zirconium oxychloride14
indicating free hydroxyl group at position -5.
6. The alc. solution of the compound gave red brown ppt. with
ammonium molybdate and acetic acid confirming presence of ortho
diphenolic hydroxy group.38
![Page 3: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/3.jpg)
[91]
These reactions are characteristic of 3-dydroxy flavones (flavonols).
So the compound must have got a flavonol nucleus (I).
(I)
The flavonoid nature of the compound was further confirmed by the
UV absorption bands18-19
at 256 (band II) and 371 (band I).
When acetylated, it yielded a tetra acetate and there by indicating the
presence of four hydroxyl groups, in the compound. This was further
supported by broad peak at 3420 cm-1
in the IR- spectrum. The peak at 2850
and 1110 cm-1
showed the presence of methoxy group and analysis of
compound by Zeisel‟s method,54
which was supported by the signal at
3.91 ppm in proton magnetic spectrum.
![Page 4: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/4.jpg)
[92]
On the basis of above fundamental observation the skeleton structure
of the aglycone could be presented as II
The relative positions of these groups have been ascertained on the
basis of colour reactions, chemical degradation and spectral data.
Spectral studies:
The presence of hydroxyl group at the C-4` is evident by the
bathchromic shift of 62nm and increase in intensity of band I in the
NaOMe20
spectrum related to band I of the NaOMe spectrum. Absence of
bath chromic shift in short wavelength after addition of NaOMe in the
methanolic solution of the compound suggested that the C-7 hydroxy group
was either substituted or absence.
OH
OH
OH
OCH3
![Page 5: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/5.jpg)
[93]
Table – I
Ultraviolet absorption maxima of the aglycone
Medium of Spectra Band Position
Band II (nm) Band I (nm)
max
Shift max
Shift
MeOH 257 - 372 -
+NaOMe 245 - 432 62
+AlCl3 273 16 452 80
+AlCl3/HCl 268 - 424 53
+Na OAc 256 - 380 -
+ NaOAc/H3BO3 261 - 390 18
It exhibited a bathochromic shift of 18 nm on addition of sodium acetate
(NaOAc/H3BO3) boric acid to the ethanolic solution thus showing the
presence of vicinal hydroxyl groups of position 3`,4`. It was further
confirmed by the appearance of blue colour on addition of sodium carbonate
to the Shinoda reaction mixture of the aglycone5.
IR Spectrum showed hydroxyl absorption at 3700-3300 cm-1
band at
2920cm-1
due to methylene groups and peak at 1600 and 1660 cm-1
due to
the presence of an , unsaturated formation (C-4 of flovonoid), other
peak at 1500-1600 cm-1
due to an aromatic ring. A broad carbonyl striating
![Page 6: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/6.jpg)
[94]
band in region 1100-1600 cm-1
and other absorption frequency include 1360,
1060, 910 cm-1
.
ALKALYNE DEGRADATION OF THE AGLYCONE :
The aglycone on treatment with 50% ethanolic KOH forms
mono methyl ether of phloroglucinol and protocatechuic acid.
Aglycone 50%
KOH
when the methyl ether of the compound was oxidised with neutral KMnO4
veratric acid was obtained as one of the oxidation products.
[O]
methyl ether of aglycone KmnO4
Phlonroglucinol
mono methyl ether
Protocatechuic acid
+
Veratric acid
![Page 7: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/7.jpg)
[95]
The formation of veratric acid on oxidation of methyl ether and
protocatechuic acid on alkaline fusion place the two hydroxyl groups at
position 3` and 4` of ring. Further the formation of mono methyl ether of
phloroglucinol again confirms the presence of methoxyl group at C-7 of
ring.
1H NMR analysis of the compound showed the presence of five
aromatic protons. Two protons were meta coupled and appeared as doublets
at 6.20 (1H,d, J=2.5 Hz0 and 6.45 (1H,d, J= 2.6 Hz) ppm assignable to
H-6 and H-8 respectively. One meta coupled doublet at 6.8 ppm
(1H,d,J=8.0Hz) corresponds to the H-5` and a doublet integrating for two
protons at 7.60 ppm (J=2.7 Hz) and 7.65 ppm (J= 8.0 Hz 3.0 Hz) were
assigned at to the H-2` and H-6` protons A sharp singlet was obtained at
3.91 ppm corresponding to the three protons indicating the presence of
aromatic methoxyl group and their position at C-7 was ascertained by the
strong fragment at m/z 167 39
.
![Page 8: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/8.jpg)
[96]
Table – II
1HNMR- chemical shifts of the aglycone ppm from TMS
(CDCl3 300MHz)
Assignment Chemical Shifts ( ppm) J- Value (Hz)
H-6 6.20 (1H,d) 2.5
H-8` 6.45 (1H,d) 2.6
H-5` 6.80 (1H,d) 8.0
H-2`` 7.60 (1H,d) 2.7
H-6` 7.65 (1H,dd) 8.0, 3.0
-O CH3 3.91 (3H,s) -
13C NMR spectrum dissolved 16 signals of which could be attributed
to the aglycone unit. The 13
C NMR signals at 115.5 and 120.3 were
assigned to an oxygen free aromatic carbon C-2` and C-6`. C13
NMR of
aglycone C-2, C-3, C-4, C-5 and C-7 (methoxy group) signal appeared at
147.5, 136.2, 175,6, 160.5 and 164.7 respectively, further confirming of the
5,3`,4`, trihydroxy-7 methoxy flavonol (Rhamnetin).
![Page 9: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/9.jpg)
[97]
![Page 10: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/10.jpg)
[98]
![Page 11: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/11.jpg)
[99]
Table – III
13C NMR chemical shift of the aglycone ppm from TMS
(CDCl3-300MHz)
Assignments Chemical shift (ppm)
C-2 147.5
C-3 136.2
C-4 175.6
C-5 160.5
C-6 97.6
C-7 164.7
C-8 91.6
C-9 156.0
C-10 103.8
C-1` 121.8
C-2` 115.5
C-3` 147.4
C-4` 147.4
C-5` 115.8
C-6` 120.3
O CH3 54.5
The Mass fragmentation pattern of the compound was in good
agreement with the above structure. The fragmentation pattern is represented
as follows as scheme-I.
Mass : m/z 316 [M+ ], 315, 301, 287, 167, 137, 109.
![Page 12: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/12.jpg)
[100]
![Page 13: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/13.jpg)
[101]
![Page 14: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/14.jpg)
[102]
-CO
pathway II m/z-287
m/z-315
- CH3
m/z-301
- H - CHO
RDA
cleavage
pathway I
m/z-316 [M]+
m/z-167
m/z-109
m/z-137
Scheme - I
![Page 15: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/15.jpg)
[103]
![Page 16: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/16.jpg)
[104]
Upon the basis of the facts discussed above, the aglycone could
therefore, be represented as III.
This aglycone has already been investigated under the name
rhamnetin. The identity of the aglycone as rhamnetin has been family
confirmed by mp. and their spectral analysis.
(3,5,3`,4` tetrahydroxy 7- methoxy flavone)
![Page 17: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/17.jpg)
[105]
GLYCOSIDE
The glycoside C34H34O20 on hydrolysis gave the aglycone C16H12O7
and xylose and D- galactose as a sugar moieties and gallic acid.
C34H34O20 7%H2SO4 C16H12O7 + C5H10O5
glycoside Aglycone xylose
C6H12O6 + C7H6O5
galactose + gallic acid
Quantitative sugar estimation29-30
confirmed the presence of one moles
of sugar per mole of glycoside and gallic acid.
Position of sugar linkage:
New let us consider the following facts regarding the structural feature
of the glycoside.
3. It gave yellow colour with boric acid in presence of citric acid12-13
and
with zirconium oxychloride14
also, showing the presence of free
hydroxyl group of position – 5.
4. Appearance of red colour with sodium hydroxide and a bathochromic
shift upon the addition, sodium acetate boric acid32-33
reagent suggest
free 3` position.
![Page 18: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/18.jpg)
[106]
As the position – 7, is already blocked by methyl group hence the
position -3 and 4` ultimately is vacant for the site of glycolysation.
Formation of 5, 7, 3`, trimethoxy quercetin by the hydrolysis
permethylated glycoside confirmed the position -3 and 4` to be involved in
glycolysation.
This was further confirmed by the comparison of the glycoside and
glycone by subjecting them to test for flavonols. In this test the glycoside
failed to respond the test for flavonols (3-Hydroxyl group ) where as the
aglycone does well.
As there is only one possible site available for glycolysation. Hence it
might be expedient to conclude have that sugar and gallic acid are present as
bioside type and linked to be the aglycone at position 3` and 4`.
Nature of the sugar linkage:
The 1H NMR spectral shift of the glycoside displayed two aromatic
proton signals at 5.55 (1H,d, J=8 Hz) and 5.70. (1H,d, J=7.3) ppm, these
were attributed to H-1`` glucosyl and H-1``` xylosyl protons respectively. A
broad signal at 3.60-3.87 integrating for ten protons corresponds to the rest
of the sugar protons.
![Page 19: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/19.jpg)
[107]
The conclusion was further confirmed by the permethylation of the
glycoside by Hakomori‟s Method40
followed by hydrolysis. The partly
methylated sugar thus obtained were identified as 3,4, tri-O-methyl D-
glucose and 2,3,4, tri-o- methyl L- xylosyl by confirming their RG value with
2,3,4,6 tetra –O - methyl D- glucose as reference sugar (n BuOH: EtOH:
H2O, 5:1:4 RG = 0. 65 and 1.02 respectively spray AHP).
As the reducing groups of both the sugar were involved in glycosidic
linkage, the glycoside was non reducing in nature.
Easy hydrolysis eliminated the possibility of any C-C-type glycosidic
linkage and suggested the C-O-C type of linkage.
The IR spectrum of MF-2 showed strong absorption band at 3420
(OH), 2970 (C-H), 1650 (C=C, aromatic) 1620 (C=O), 1730-1715 (ester
C=O streaching); 2832 ( - O CH3 group) 1260 (C-O-C , vibration), 1050,
780 cm-1
Its reaction (florescent yellow in UV with AlCl3) and UV spectral date
with diagnostic shift reagents suggested the likely presence of a 3,7, 4`-
trisubstituted flavonol glycoside with hydroxyl group at 5 and 3` position.
The 1H-NMR spectrum of compound spectroscopic, showed
characteristics signals assignable to an anomeric proton at 5.55
![Page 20: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/20.jpg)
[108]
(1H,d, J=8.0 Hz) and 5.70 (1H,d, J= 7.3 Hz) ppm these uses attributed to H-
1`` galactosyl and H-1```` xylosyl proton and methylene proton adjacent to
am ester group at 4.24, (2H,dd, J=6.0, 11.20 Hz) and 4,43 (2H,dd, J= 7.1,
11.20 Hz). Attachment of galloyl group through an ester linkage at C-2`` in
galactose was suggested by the down field shift of 2`` (5.44) in 1H NMR
spectrum.
Three oxygenated methane protons at 3.84 (dd, J= 7.0 Hz) and 3.93
(J=3.5 Hz) together with anomeric proton at 6.93 (2H, ) group, suggested
the presence of a 2``` galloyl galactoside and 4`-xyloside in compound.
Table – IV
1H NMR chemical shift of the glycoside
(DMSO d6 – 300 MHz)
Assignments Chemical Shift ( :ppm) J-Value (Hz)
C-6 6.30 (1H,d, H-6) 2.10
C-8 6.60 (1H,d, H-8) 2.1
C-2` 8.10 (1H,d, H-2`) 9.0
C-5` 7.10 (1H,d, H-5`) 9.6
C-6` 8.10 (1H,d,H-6`) 9.0
gall-C-1`` 5.55 (1H,d, H-1``) 8.0
gall-C-2`` 3.44 (1H,d, H-2``) ---
C-3`` 3.28 (1H,d, H-3``) ---
C-4`` 3.12 (1H,d, H-4``) ---
![Page 21: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/21.jpg)
[109]
C-5`` 3.39 (1H,d, H-5``) ---
C-6`` 3.48, 3.54 (2H,dd, H-6``) 7.1,11.2
galloyl C-2``` 6.92 (1H,s H-2```) ---
galloyl C-6``` 6.90 (1H,s, H-6```) ---
xylose C-1```` 4.78 (1H,s,H-1```) 6.8
C-2```` 3.85 (1H,m, H-2````) -
C-3```` 3.87 (1H, m, H-3````) -
C-4```` 3.90 (1H,m H-4````) -
C-5```` 4.20, 4.23 (2H,H-5````) -
OCH3 3.80 (3H,s) -
The 13
C NMR spectrum of the glycoside the C-3 carbon shifted
upfield by 2.7 ppm and C-2 signal was shifted field by 9.2 ppm the C-4
resonance was also shifted to a down field direction by 1.5 ppm when
compound to rhamnetin the magnitude of these glycosylation. Moreover a
down field shift of C-4 in C13
NMR at 148.8 confirmed this result. The C13
NMR spectrum of compound indicted that carbon signal 9168.2) C -7```of
the galloyl carbonyl unit showed C13
long range. Shift are characterised of
the general pattern of the glycosilation shifts when glycosilation there was
a were able effect on the C-2 and C-3` thus confirming glycosilation at
position 3 and 4`.
![Page 22: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/22.jpg)
[110]
Table – V
13C NMR (DMSO - d6 – 300 MHz)
Assignments Chemical shift ( :ppm)
C-2 156.8
C-3 133.5
C-4 177.6
C-5 160.6
C-6 97.8
C-7 165.0
C-8 91.8
C-9 156.5
C-10 104.9
C-1` 121.0
C-2` 115.5
C-3` 144.6
C-4` 148.4
C-5` 116.3
C-6` 121.9
OCH3 55.7
![Page 23: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/23.jpg)
[111]
gal. C-1`` 101.3
C-2`` 73.6
C-3`` 76.5
C-4`` 69.8
C-5`` 76.5
C-6`` 68.4
gallosyl-C-1``` 103.2
C-2``` 73.6
C-3``` 76.5
C-4``` 75.9
C-5``` 76.6
C-6``` 60.8
gallosyl-C-7```
carbonyl (C=O) 168.2
xylose C-1```` 105.4
C-2```` 74.9
C-3```` 78.2
C-4```` 71.0
C-5```` 66.7
![Page 24: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/24.jpg)
[112]
On the basis of the fore going observation compound was assigned as
rhamnetin 3-O- (2``-galloyl) - –D- galactopyranosyl – 4`- -D-
xylopyranoside.
![Page 25: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/25.jpg)
[113]
EXPERIMENTAL
Chromatography :
TLC (Cellulose silica gel plate) :
1 Butanol – Benzene-Pyridine - H2O
(5:1:3:3, v/v) upper layer % Rf = 0.18
2 n BuOH - AcOH – H2O
(6:1:3, v/v) Rf = 0.47 (Ammonia vapours)
3 EtOAc – MeOH (9:1, v/v) Rf = 0.40
Test :
The alc. solution of the compound 4-5 drops of naphthol solution
was added, on addition of conc. H2SO4 a violet ring1 was formed, on boiling
with fehling solution no reduction was observed and no violet color, when
spot the compound on paper was sprayed with AHP reagent2
and heated at
1000C.
Acid hydrolysis :
50 mg of compound dissolved in minimum amount of ethanol was
refluxed with 70% H2SO4 (50ml) for 3-hrs. The reaction mixture was poured
![Page 26: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/26.jpg)
[114]
in excess of ice cold water and taken in ether. The compound was
crystallised from ethyl acetate, mp- 288-289o.
Sugar:
The remaining aqueous solution was neutralised with barium
carbonate and the filtrate was concentrated on a rotary evaporator. The
concentrate gave positive Molisch‟s test reduced fehling solution and on
cellulose tlc plate gave two spot.
Rf 0.35 and 0.43 (B.A.W., 4:1.5 v/v, spray –AHP) identical with
authentic sample of galactose and xylose respectively.
Aglycone :
Elemental Analysis:-
Found : C = 60.8%, H = 3.8%
Calculated for C16H12O7 : C = 60.81% H = 3.78%
Acetylation :
The aglycone (50mg) was dissolved in pyridine (1.0ml) and heated
with acetic anhydride (3.0 ml) on water both for 7-hours. The reaction
mixture was poured over crushed ice. The residue upon crystallisation with
![Page 27: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/27.jpg)
[115]
methanol-benzene yielded pure rhamnetin tetra acetate C24H20O11, mp.
1930C.
Found : C = 59.52% H = 4.14%
Calculated for : C = 59.50% H = 4.13%
C16H12O7 (COCH3)4
percentage of acetylation :
Found – 35.54 %
Calculated - 35.53%
Methylation :
A solution of aglycone (50mg) in acetone 150 ml was treated with
dimethyl sulphate (Me2SO4) and anhydrous potassium carbonate (15mg) for
four hrs. under reflux. The carbonate was filtered off and washed with
acetone. Acetone filtered and washing were mixed and concentrated under
reduced pressure to a small volume and poured in to ice cold water. The
methylated aglycone was crystallised from methanol as yellow crystalline
mass, mp.1450-147
0 and analysed for methoxyl groups (Zeisels method)
54.
Found : C = 64.50% H = 5.38%
Calculated for : C = 64.51% H= 5.37%
C16H8O3 (OCH3)4
![Page 28: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/28.jpg)
[116]
Percentage of methylation :
Found : 33.35%
Calculated : 33.33%
KMnO4 oxidation of methyl ether of aglycone:
Methyl ether of aglycone (30mg) in acetone solution was refluxed
with KMnO4 (50mg) on a water bath for five hours. The reaction mixture
was cooled, diluted with water and the solvent was removed by distillation.
A solution of sodium bisulphate was added to remove the excess of
manganese dioxide formed. The solution was extracted with ether and the
etherial extract was shaken with aqueous saturated solution of sodium
bicarbonate. This solution was acidified with HCl and repeatedly extract
with ether. The ether extract on concentration gave veratric acid, mp.182. It
was further identified by mmp and co-chromatography (n-butanol saturated
with ammonia spray-bromophenol blue, Rf = 0.42) with an authentic sample
of veratric acid.
Cleavage of the aglycone with KOH:
The aglycone (25mg) was dissolved in minimum amount of ethanol
and refluxed for 6 hours with KOH solution (25%, 3ml). It was acidified
![Page 29: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/29.jpg)
[117]
with dil. HCl and extracted with ether. The ether layer was washed several
times with water until it was free from mineral acid.
This was fractionated in to
1. 5% sodium bi carbonate solution fraction
2. 10% sodium bi carbonate solution fraction
3. 1% sodium hydroxide solution fraction
1. 5% sodium bi carbonate solution fraction:
The fraction was acidified with HCl and extracted with ether. The
ether layer was washed with distilled water to remove mineral acid. In the
solution protocatechuic acid (3,4-dihydroxy benzoic acid) was identified by
co-chromatography with an authentic sample (B:A:W, 4:1:5, v/v) Rf = 0.48,
spray- FeCl3) and mmp which was observed to be 1970.
2. 10% sodium bicarbonate soluble fraction :
This fraction was neutralised with dil. HCl and extracted with ether.
The solution was evaporated. Nothing could be obtained from it.
3. 1% sodium hydroxide soluble fraction :
This fraction was neutralised with dil. HCl and aqueous solution was
shaken with ether. The etherial layer was washed with sodium bi carbonate
![Page 30: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/30.jpg)
[118]
solution and then with water to remove traces of HCl. In the solution
phloroglucinol mono methyl ether was obtained and identified by its mp 720,
mmp and co-chromatography with an authentic sample (B:A:W, 4:1:5, Rf =
0.46,v/v, spray, 1% aq. FeCl3).
Spectral Studies :
UV :
max nm
MeOH : 257, 286 sh, 372
+NaOMe : 245, 432
+AlCl3 : 273, 302sh, 452
+AlCl3/HCl : 268, 424
+NaOAc : 256, 380
+NaOAc/H3Bo3 : 261, 390
IR :
KBr
max : 3700-3300, 3050, 2920, 2832, 1600, 1660,
1500-1600, 1100-1600, 1360, 1060, 910, cm-1
![Page 31: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/31.jpg)
[119]
1H NMR :
(CDCl3, 300MHz)
ppm:
Assignment Chemical Shifts ( ppm) J- Value (Hz)
H-6 6.20 (1H,d) 2.5
H-8 6.45 (1H,d) 2.6
H-5` 6.80 (1H,d) 8.0
H-2` 7.60 (1H,d) 2.7
H-6` 7.65 (1H,dd) 8.0, 3.0
-O CH3 3.91 (3H,s) -
13C NMR :
(CDCl3-300MHz)
ppm:
Assignments Chemical shift (ppm)
C-2 147.5
C-3 136.2
C-4 175.6
C-5 160.5
C-6 97.6
C-7 164.7
![Page 32: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/32.jpg)
[120]
C-8 91.6
C-9 156.0
C-10 103.8
C-1` 121.8
C-2` 115.5
C-3` 147.4
C-4` 147.4
C-5` 115.8
C-6` 120.3
O CH3 54.5
Mass : m/z 316 [M+], 315, 301, 287, 273,167, 137, 109.
Glycoside :
Elemental Analysis :
Found : C = 53.58%, H = 4.67 %
Calculated for C34H34O20 : C=53.54% H=4.46%
![Page 33: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/33.jpg)
[121]
Spectral Studies (glycoside) :
IR KBr
max : 3420, 2970, 2832, 1715-1730, 1650
1620, 1570, 1260, 1050, 845,780 cm-1
1H NMR :
(DMSO -d6 – 300 MHz)
ppm:
Assignments Chemical Shift ( :ppm) J-Value (Hz)
C-6 6.30 (1H,d, H-6) 2.10 Hz
C-8 6.60 (1H,d, H-8) 2.1 Hz
C-2` 8.10 (1H,d, H-2`) J=9.0, Hz
C-5` 7.10 (1H,d, H-5`) J=9.6, Hz
C-6` 8.10 (1H,d,H-6`) J=9.0, Hz
gall-C-1`` 5.55 (1H,d, H-2``) J=8.0, Hz
gall-C-2`` 3.44 (1H,d, H-2``) ---
C-3`` 3.28 (1H,d, H-3``) ---
C-4`` 3.12 (1H,d, H-4``) ---
C-5`` 3.39 (1H,d, H-5``) ---
C-6`` 3.48, 3.54 (2H,dd, H-6``) 7.1,11.2
gall C-2``` 6.92 (1H,s, dd, H-6```) ---
![Page 34: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/34.jpg)
[122]
gall C-6``` 6.90 (1H,s, H-6```) ---
xyl C-1```` 4.78 (1H,s,H-6```) 6.8 Hz
C-2```` 3.85 (1H,m, H-2````)
C-3```` 3.87 (1H, m, H-3````)
C-4```` 3.90 (1H,m H-4````)
C-5```` 4.20, 4.23 (2H,m 5````)
OCH3 3.80 (3H,)
13C NMR :
(DMSO -d6 – 300 Hz)
ppm:
Assignments Chemical Shift ( :ppm)
C-2 156.8
C-3 133.5
C-4 177.6
C-5 160.6
C-6 97.8
C-7 165.0
C-8 91.8
C-9 156.5
C-10 104.9
C-1` 121.0
C-2` 115.5
C-3` 144.6
![Page 35: The compound MF-2 was obtained from the ethanolic extract wasshodhganga.inflibnet.ac.in/bitstream/10603/18206/10/10_chapter 02… · [90] AGLYCONE The aglycone mp. 288-2890C was analysed](https://reader034.vdocuments.us/reader034/viewer/2022052001/60142ec330e13520b93e40b7/html5/thumbnails/35.jpg)
[123]
C-4` 148.4
C-5` 116.3
C-6` 121.9
OCH3 55.7
gal C-1`` 101.3
C-2`` 73.6
C-3`` 76.5
C-4`` 69.8
C-5`` 76.5
C-6`` 68.4
gallosyl-C-1``` 103.2
C-2``` 73.6
C-3``` 76.5
C-4``` 75.9
C-5``` 76.6
C-6``` 60.8
gallosyl-C-7```
carbonyl (C=O)
168.2
xylose C-1```` 105.4
C-2```` 74.9
C-3```` 78.2
C-4```` 71.0
C-5```` 66.7
Mass : 762 [M+], 617,437, 284, 273, 1`67, 137, 109
(GCMS m/z)