Antimycobacterial activity of fractions
Nineteen fractions, from the ethyl acetate extract were collected and tested on M. tuberculosis. Sixteen fractions showed activity on both the pan sensitive strain and isoniazid resistant strains (Table 1). Fraction 18 was the most active on the pansensitive strain and the Isoniazid resistant strain with minimum inhibitory concentration values of 0.01mg/ml and 0.09mg/mlrespectively.Fraction 10 had MIC valueof 0.07mg/ml, 0.09mg/ml and 0.09 on H37Rv,TMC 301 and TMC331 respectively and was the most active on the rifampicin resistant strain.Only four of the fractions (3,10,13 and 18) had activity on the rifampicin resistant strainwith MIC values of 0.45, 0.09, 0.07 and 0.07mg/ml respectively.
Table1: Minimum inhibitory concentration values of fractions from C. citrinusagainst pan sensitive,
isoniazid resistant and rifampicin resistant M. tuberculosis
Minimum inhibitory concentration values (mg/ml)
|
Fraction
|
H37Rv
|
TMC 301
|
TMC331
|
1
|
>50
|
>50
|
na
|
2
|
0.12
|
1.17
|
>50
|
3
|
0.49
|
0.59
|
0.45
|
4
|
0.29
|
0.59
|
>50
|
5
|
0.78
|
0.09
|
>50
|
6
|
1.25
|
0.09
|
>50
|
7
|
0.15
|
0.24
|
>50
|
8
|
0.24
|
0.24
|
>50
|
9
|
>50
|
>50
|
>50
|
10
|
0.07
|
0.09
|
0.09
|
11
|
0.09
|
0.15
|
>50
|
12
|
0.78
|
1.18
|
>50
|
13
|
0.19
|
3.13
|
0.07
|
14
|
0.09
|
0.14
|
>50
|
15
|
0.24
|
0.09
|
>50
|
16
|
0.19
|
0.09
|
>50
|
17
|
>50
|
>50
|
>50
|
18
|
0.01
|
0.09
|
0.07
|
19
|
1.56
|
2.34
|
na
|
H37rv = pan sensitive strain, TMC 301= Isoniazid resistant strain, TMC 331= rifampicin resistant stain, >50 were considered in active.
Gas chromatography Mass Spectroscopy determination
From the oily fraction three, seven compounds were identified using GC-MS analysis (Table 2).Dodecanoic acid 1, 2, 3 propanenetriyl ester, hexadecane and octadecane were the most abundant with relative abundance of 38.2%, 18.5% and 16.1% respectively. Phthalic acid (1.8%), pentadecane (1.8%), aspidinol (9.1%)and tetradecane (14.5%)were also detected. The oily fraction mainly comprised of carboxylic acids and hydrocarbons.
Table 2: Compounds found from Gc Ms analysis of Callistemon citrinus fraction three
Compound name
|
Retention time (min)
|
Relative
composition %
|
Molecular
Formula
|
Molecular
Weight
|
Compound nature
|
Tetradecane
|
10
|
14.5
|
C14H30
|
196.39
|
Long chain alkane
|
Phthalic acid
|
23.0
|
1.8
|
C8H6O4
|
166.13
|
Carboxylic acid
|
Hexadecane
|
12.9
|
18.2
|
C16H34
|
226.44
|
hydrocarbon
|
Aspidinol
|
17.3
|
9.1
|
C12H16O4
|
224.25
|
Carboxylic ester
|
Pentadecane, 7 methyl
|
15.4
|
1.8
|
C6H34
|
226.44
|
Hydrocarbon
|
Dodecanoic acid 1,2,3 propanenetriyl ester
|
29.1
|
38.2
|
C39H74O6
|
639.00
|
Carboxylic ester
|
Octadecane
|
15.5
|
16.4
|
C18H38
|
254.5
|
hydrocarbon
|
Isolated compounds
Spectral data
Two pentacyclic triterpenoids including ursolic acid (1) and betulinic acid (2) plus a flavonoid epicatechin (3)(Fig 1) were isolated from the leaves of C. citrinus.Their proton and Carbon 13 spectral data is shown below.
Compound 1white powder1H NMR (300MHz, MeOH) δH (m, J in Hertz)0.87 (t, J=15Hz), 0.95(d, J=18Hz),2.17(d, J=9Hz),3.3brd,5.5(t, J=2.55Hz)
NMR (300MHz, MeOH) δC 38.4(CH2), 27.8(CH2), 78.3(CH), 38.4(C), 55.3(CH), 18.1(CH2), 32.9(CH2), 39.3(C), 48.3(CH), 36.7(C),22.9(C), 125.4(CH2), 138.2(CH2), 41.8(CH), 27.8(C), 22.9(CH2), 48.0(C), 52.9(CH), 39.0(CH), 38.4(C), 30.3(CH2), 36.7(CH2), 27.8(CH3), 14.9(CH3), 16.4(CH3), 18.0(CH3), 22.9(CH3), 180.1(C), 18.0(CH3), 27.3(CH3)ESI-MS ([M]+ at m/z 456.4) Calcd forC30H48O
Compound 2 white amorphous powder1H NMR (300MHz, CDCl3) δH(m, J in Hertz)4.428(d J=4.5Hz), 2.798 (d,J=3.6Hz),1.23(t, J=10.2), 1.05s, 1.05s, 0.94s, 1.05s, 1.23s, 0.94s, 1.0s.
NMR (300MHz, cdcl3) δC 38.2(CH2), 27.2(CH2), 82.4(CH), 38.22(C) 54.52(CH), 34.3(CH2), 40.9(C) 50.9(CH), 22.1(CH), 24.7(CH2), 38.2(CH2), 42.4(CH), 30.6(C), 31.5(CH2), 54.5(C), 46.2(CH), 50.9(CH), 150.4(C), 29.4(CH2), 34.0(CH2), 29.4(CH3), 27.9(CH3),15.3(CH3), 19.5(CH3) 19.5(CH3), 182.8(C), 113.1(CH3), 19.5(CH3) ESI-MS ([M]+ at m/z 438) Calcd for C30H50O
Compound 3 yellow solid 1H NMR (300MHz, CDCl3) δH(m, J in Hertz);4.7(br), 2.83(d, J=4.8Hz), 2.68(dd, 19.8Hz), 5.85(t, J=9Hz), 6.9(s), 6.72(dd, J=14.7)
NMR (300MHz, cdcl3) δC 80.2(CH), 67.62(CH), 29.38(CH), 158.1(C), 96.6(CH), 157.7(C), 96.61(CH), 157.4(C), 100.3(C), 132.4(CH), 115.5(CH), 145.9(CH), 145.8(CH), 115.5(CH),119.6(C)ESI-MS ([M]+ at m/z 290.5) Calcd forC15 H14 O6
Anti-mycobacterial activity of compounds
Isolated compounds (1-3) were tested on both pan sensitive and resistant strains of M. tuberculosisusing MABA. Among these, epicatechin (3) was found to be active on all the strains of M.tuberculosis.It had minimum inhibitory concentration values of 3.4μg/ml, 5.9μg/ml and 3.9μg/ml on the pan sensitive, rifampicin resistant and isoniazid resistant strains respectively (Table 3).
Table 3: Minimum Inhibitory Concentration values ofthe ursolic acid, betulinic acid and epicatechin on pan
sensitive, rifampicin resistant and isoniazid resistant M. tuberculosis
Compound
|
Minimum inhibitory concentration values (μg/ml)
|
|
Pan sensitive strain
(H37Rv)
|
Rifampicin resistant
strain (TMC 331)
|
Isoniazid resistant strain (TMC 301)
|
Ursolic acid (1)
|
30.1± 1.9
|
>32.5
|
>32.5
|
Betulinic acid (2)
|
29.3±2.0
|
>32.5
|
>32.5
|
Epicatechin (3)
|
3.4± 0.8
|
5.9±3.5
|
3.9±1.8
|
Isoniazid
|
2.0±0.0
|
4.0±2.5
|
>32.5
|
Rifampicin
|
4.0±0.0
|
-
|
2.0±0.0
|