Acetylation of SC:
Further purification of SC was achieved by acetylation using standard method to give an acetylated mixture. This mixture was purified by preparative TLC (silica gel 60 F254 Merck 5715) to give acetylated compound A’.
2.4.2 Results and Discussion
A. Structural characterization of Acetylated Compound A’:
13C-NMR (Figure 7.2). A literature search revealed compound A to be a flavanone glycoside. The fully assigned NMR data of compound A acetate is shown in Table 2.1.12,13,
14
Proposition of structure of Compound A peracetate
8
5
3 O
O 4' 2'
6'
OAc
OAc
O
2"
4"
O
OAc
OAc AcO
6"
O 1```
O AcO
C H3
OAc AcO
Figure 2.11 Structure of acetylated flavanone glycoside A.
It is therefore proposed tentatively that the structure of compound A is 7-[[2-O-(6-deoxy-α- L-mannopyranosyl)-β-D-glucopyranosyl]] oxy]-2,3-dihydro-5-hydroxy 2-(4-
hydroxyphényl)-4H-1-benzopyran-4-one as shown in Figure 2.12. Further chemical experimentation and semi-synthesis are necessary to confirm the proposed structure.
.
O O H HH H
H
O O
H O H
OH
O O H
OH
O OHOH H O H C H3
Figure 2.12 Proposed structure of compound A: Flavanone glycoside
7-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]] oxy]-2,3-dihydro-5-hydroxy 2-(4-hydroxyphényl)-4H-1-benzopyran-4-one
Table 2.1: 1H NMR and 13C-NMR (100.6 MHz) spectral data for compound A
Position δC δH COSY NOESY HMBC
C→H 2 79.2 (CH) 5.47 (1H,
dd)
H-3a, H- 3b
Ha-3; Hb-3; H- 5`
H-3 2J
3 45.1 (CH2) 2.75(1H, dd; 3.04 (1H, dd)
H-3b, H- 3a, H-2
Hb-3; Ha-3; H-2
4 188.6 (C) - - - Ha-3 2J; Hb-3 2J
5 151.9 (C) - - - H-6 2J
6 105.9 (CH) 6.32 (1H, s) - H-3``; H-2``` H- 8
H-8 3J
7 161.8 (C) - - - H-6 2J; H-8 2J; H-
1`` 3J
8 102.4 (CH) 6.53 (1H, s) - H-6 H-6 3J;
9 163.9(C) - - - H-8 2J
10 109.6 (C) - - - H-3 3J; H-6 3J; H-8
3J
1` 135.6 (C) - - - H-3a 3J; H-2` 2J
2` 122.1(CH) 7.15 (1H, s) H-3` H-3`; H-5` H-6` 3J 3` 127.43 (CH) 7.45 H-2` H-2`; H-6` H-2` 2J; H-5` 3J
4` 151.04 (C) - - H-2`3J; H-3`2J; H-
5` 2J; H-6` 3J 5` 127.4 (CH) 7.45 d;
J:1.72
H-6` H-2`; H-6`; H-2 H-3`3J; H-6` 2J
6` 122.1(CH) 7.15 (br d) H-5` H-3`; H-5` H-2` 3J 1`` 98.2 (CH) 5.13 d H-2`` H-3``; OC-CH3 H-5`` 3J;
2`` 70.1(CH) 5.03 dd H-1``; H- 3``
H-5``; H-3`` H-4`` 3J; H-1`` 2J
Table 2.1 Continued.
3`` 72.4(CH) 3.94 dd H-2``; H- 4``
H-1``; H-2``; H- 4``, H-6``, OC- CH3; H-6a; H-
6b
H-1`` 3J; H-2`` 2J
4`` 68.3(CH) 5.02 dd H-5`` H-3``; H-5``; H- 6``
H-5`` 2J; H-2`` 3J
5`` 74.1(CH) 5.33 ddd H-4`` H-1```; H-2``;
H-4``
H-1`` 3J; H-4`` 2J
6`` 61.9(CH2) 4.22 dd;
4.17 dd
OC-CH3; H-3`` H-4`` 3J
1``` 98.2(CH) 5.12 d H-2```; H-5``;
H-5```; H-6``
H-3``` 3J
2``` 70.9(CH) 5.09 dd H-3``` H-1```; H-6; H- 3```; H-6```
H-1``` 2J; H-3``` 2J
3``` 68.4(CH) 5.01 dd H-2``` H-2```; OC- CH3;
H-5```; H-6```
H-2``` 2J
4``` - (CH) dd H-6``` 3J
5``` 66.9(CH) 4.03 m H6``` H-1```; H-3```;
OC-CH3; H- 6```
H-6``` 2J; H-3``` 3J
6``` 17.6(CH3) 1.23 d H-5``` H-4``; H-1```;
H-2```; H-3```;
H-5```
H-5``` 2J
CH3-CO O=C-O-
20.51-21.12 170.5-169.2
2.5 Extractives from Syzygium guineense of South African (S.A.) origin
2.5.1 Extraction and isolation
The leaves of Syzygium guineense were collected on the campus of the University of KwaZulu Natal in South Africa. The fresh plant materials were blended using a laboratory blinder (Russell Hobbs). The homogenate materials were used for further studies.
S. guineense (756 g) of fresh homogenated leaves were extracted sequentially in n-hexane, dichloromethane, ethyl acetate, methanol and aqueous methanol 80% as shown in scheme 2.1. Each extraction was optimised by repeating the maceration twice. Each solvent extraction was concentrated under reduced pressure and allowed to dry at room temperature and weighed to give hexane-solubles (DS/8/A) (3.0g, 0.39%), dichloromethane-solubles (DS/8/B) (7.90g, 1.05%), ethyl acetate-solubles (DS/8/C) (0.60g, 0,08%), methanol- solubles (DS/8/D) (5.00g, 0.66%), and aqueous methanol-solubles (DS/8/E) (2.10g, 0,28%), respectively.
The dichloromethane extract was subjected to column chromatography using the mixture of hexane - ethyl acetate (9:1 to 6:4). A total of 90 fractions of 40 ml each were collected and 10 similar fractions (FA, FB, FC, FD, FE, FF, FG, FH, FI and FJ) were combined.
Combined fraction FA eluted with hexane 100% afforded one spot; a greenish oily liquid, in small amounts. Combined fractions FC (brownish oily) and FF (whitish solid) eluted with hexane-ethyl acetate 8:2 and 7:3 respectively, afforded two spots each. The combined fraction FF (110 mg) contained the major compound. Combined fraction FD (brownish oily) and FE (brownish oily) afforded three spots each, in small amounts; while the combined fraction FB afforded four spots, but in small quantities.
Preparative TLC
For further purification, 20 mg of the combined fraction FF were submitted to a preparative TLC analysis. Two compounds were obtained chromatographically pure in hexane- ethyl acetate 7:3.
The methanol extract was subjected to column chromatography using the mixture of hexane - ethyl acetate (9:1 to 3:7). A total of 120 fractions of 40 ml each were collected and 13 similar fractions labelled SD1, SD2, SD3, SD4, SD5, SD6, SD7, SD8, SD9, SD10, SD11, SD12 and SD13 were combined. Combined fraction SD1 eluted with hexane - ethyl acetate 9:1 afforded 6 spots of greenish oily liquid, in traces. Combined fraction SD3 eluted with hexane - ethyl acetate 9:1 afforded one spot of yellowish oily liquid, in traces. Combined fractions SD2, SD4, SD5, SD8 and SD9 eluted with hexane-ethyl acetate 9:1 for SD2, SD4, hexane-ethyl acetate 8.5-1.5 for SD5 and hexane-ethyl acetate 8:2 for SD8 and SD9 afforded two spots each. The combined fractions SD6, SD7, SD12 and SD13 afforded three spots each, while the combined fractions SD10 and SD11 afforded four spots each. The combined fractions SD1, SD5 and SD9 gave the products chromatographically pure, after precipitating them in hexane for the SD1 and SD5 and in dichloromethane for SD9.
2.5.2 Results and discussion
A. Structure elucidation of compound B from dichloromethane extract
Physical data:
Name: 3-Hydroxy-20(29)-lupen-28-oic acid (Betulinic acid) Yield: 0.110 g (0.015 %)
Experimental melting point: 315-316 °C (Literature: 316-318 °C)15, 16 Molecular formula: C30H48O3
Molecular mass (LC-MS): m/z: 455.2 (M-H)+ (Figure 7.14) FT-IR: νcm-1
(KBr): 2920.25 and 2851.42 (C-H stretching); 1724.37 (carboxylic acid) (Figure 7.13)
The 1H-NMR (CDCl3+DMSO) spectrum (Figure 7.10) showed a deshielded characteristic proton at δ 3.18 (1H, dd, H-3) assigned to the proton of C-3 which is bonded to the hydroxyl group. This compound shows also two one proton broad singlets at δ 4.58 (1H, br s, H-29) and δ 4.71(1H, br s, H-29), characteristic of the vinyl methylene group in a lup- 20(29)-ene skeleton. The other protons are assigned as follows: δ 3.1 (1H, t, H-19); δ 1.67 (3H, s, Me-30); δ 0.67 (3H, s, Me-23); δ 0.79 (3H, s, Me-24); δ 0.84 (3H, s, Me-25); δ 0.86 (3H, s, Me-26); and δ 0.88 (3H, s, Me-27). The remaining protons, CH and CH2 groups, 25H appear, between δ 0.99-2.26 ppm. The assigned values agreed with the literature values. The minimal differences are due to the different solvents used.17
13C-NMR: see Table 2.2 (Figure 7.11). A literature search revealed compound B to be betulinic acid.18
Structure of Compound B: Betulinic acid:
10 5 1
4 2
3
9 8
6 7 11
12
14 13
15 16 18
17
C H3
23
CH3
24
CH3
25
CH3
26
CH3
27
28
H
19
22 21
H
H
H
20
CH2
30
C H3
29
O OH
Figure 2.13 Structure of Compound B: Betulinic acid
Table 2.2: 13C-NMR (100.6 MHz) spectral data for compound B Carbon
Position
δC Lit. (18) δC CpdB Carbon Position
δC Lit.(18) δC CpdB
1 38.7 38.7(CH2) 16 32.1 32.2(CH2)
2 27.4 27.4(CH2) 17 56.3 56.2(C)
3 78.9 79.0(CH) 18 46.8 46.9(CH)
4 38.8 38.9(C) 19 49.2 49.3(CH)
5 55.3 55.4(CH) 20 150.3 150.4(C)
6 18.3 18.3(CH2) 21 29.7 29.7(CH2)
7 34.3 34.3(CH2) 22 37.0 37.0(CH2)
8 40.7 40.7(C) 23 27.9 28.0(CH3)
9 50.5 50.5(CH) 24 15.3 15.3(CH3)
10 37.2 37.2(C) 25 16.0 16.0(CH3)
11 20.8 20.9(CH2) 26 16.1 16.1(CH3)
12 25.5 25.5(CH2) 27 14.7 14.7(CH3)
13 38.4 38.4(CH) 28 180.5 179.1(C)
14 42.4 42.4(C) 29 109.6 109.7(CH2)
15 30.5 30.5(CH2) 30 19.1 19.4(CH3)