Chapter 5: Isolation and characterisation of antimicrobial compounds from Terminalia
5.4. Results and discussion
5.4.2. Antimicrobial assays results
The results of the MIC values demonstrated by the fractions and isolated compounds from Terminalia phanerophlebia against Staphylococcus aureus, Klebsiella pneumoniae, Mycobacterium aurum A+ and Mycobacterium tuberculosis H37Ra are presented in Table 5.3. The results were interpreted as those MIC values less than 1 mg/ml had good antimicrobial activity (YORK et al., 2012). The extracts showed a broad spectrum of activity against the selected bacteria with MIC values ranging from 0.008 to ˃1 mg/ml. All four solvent fractions (hexane, DCM, EtOAc and butanol) obtained from the MeOH extract showed good antimicrobial activity, however, the EtOAc fraction demonstrated the lowest inhibitory values when compared to the others against all four bacterial strains tested, with MIC values ranging from 0.008 to 0.125 mg/ml. The best antimicrobial activity in this study was demonstrated by the EtOAc fraction at a MIC value of 0.008 mg/ml against
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Staphylococcus aureus. This led to the selection of the EtOAc fraction of Terminalia phanerophlebia for isolation of compounds responsible for good antimicrobial activity observed. Out of the 10 test tube fractions obtained from the EtOAc column fractionation, fractions 1 and 5 exhibited good antimicrobial activity against all four bacterial strains tested, and that led to their purification and isolation of compounds. The two isolated compounds in this study showed good antimicrobial activity against all tested bacterial strains at MIC values ranging from 0.063 to 0.250 mg/ml. Biological activity of 1,6-di-O-coumaroyl glucopyranoside (2), are reported for the first time. However, closely related compounds in the class phenylpropanoid glycoside exhibited significant antimicrobial activity against both Gram-positive and Gram-negative bacterial strains (DIDRY et al., 1999;
KYRIAKOPOULOU et al., 2001; LIMA et al., 2003; SI et al., 2007). Good MIC values ranging from 0.063 to 0.125 mg/ml exhibited by 1,6-di-O-coumaroyl glucopyranoside against the tested bacterial strains were noteworthy. When compared to that of the crude extracts 1,6- di-O-coumaroyl glucopyranoside exhibited activity which was three times better than that of the crude extracts against a strain of Mycobacterium tuberculosis. Inhibition of Mycobacterium tuberculosis by 1,6-di-O-coumaroyl glucopyranoside was noteworthy, as this bacterial strain is reported to be the leading cause of tuberculosis worldwide. Compound (2), 1,6-di-O-coumaroyl glucopyranoside could serve as a lead compound for tuberculosis drug discovery. Methyl-3,4,5-trihydroxybenzoate (1) is a biochemically available medicinally important compound derived from numerous plant species and possesses several biological activities including antibacterial, antifungal, antioxidant, antiasthmatic, anti-Herpes simplex virus and antineoplastic properties (KANG et al., 2008; CHOI et al., 2009; TEKE et al., 2011; MADIKIZELA et al., 2013b). Methyl-3,4,5-trihydroxybenzoate demonstrated high activity against Herpes simplex virus type 1 and 2 (KANE et al., 1988). This compound is considered as a useful antimicrobial agent. Therefore, the lower MIC values demonstrated by methyl-3,4,5-trihydroxybenzoate in this study were not surprising as it has demonstrated good antibacterial activities against both Gram-negative and Gram-positive bacterial strains.
In in vitro studies by KANG et al. (2008), CHOI et al. (2009), and TEKE et al. (2011), methyl-3,4,5-trihydroxybenzoate demonstrated significant antimicrobial activity. KIM et al.
(2008) did a study determining the effect of methyl-3,4,5-trihydroxybenzoate on intracellular survival of Mycobacterium fortuitum and Mycobacterium tuberculosis 5 h after phagocytosis, this compound significantly increased the rate of intracellular killing of both bacterial strains demonstrating significant efficacy. According to TEKE et al. (2011), the presence of
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hydroxyl groups on methyl-3,4,5-trihydroxybenzoate could be responsible for the antimicrobial activity observed from this compound.
Good antimicrobial activity exhibited by the compounds isolated from Terminalia phanerophlebia authenticates the traditional use of this plant in treating tuberculosis and its related symptoms. Further pharmacological studies on antimicrobial evaluation of these compounds against drug-resistant strains of bacteria as well as toxicology testing are required. Antimicrobial compounds isolated from plants may have better antimicrobial properties when combined with conventional drugs, therefore, further studies on the synergy between isolated compounds and antibiotics is required to confirm if there could be any synergism between the two.
124 Figure 5.2(a): 1H NMR spectrum of Compound 1
125 Figure 5.2(b):1H NMR spectrum of Compound 1
126 Figure 5.3:13C NMR spectrum of Compound 1
127 Figure 5.4: DEPT NMR spectrum of Compound 1
128 Figure 5.5: HSQC NMR spectrum of Compound 1
129 Figure 5.6: HMBC NMR spectrum of Compound 1
130 Figure 5.7(a): 1H NMR spectrum of Compound 2
131 Figure 5.7(b): 1H NMR spectrum of Compound 2
132 Figure 5.7(c): 1H NMR spectrum of Compound 2
133 Figure 5.8(a): 13C NMR spectrum of Compound 2
134 Figure 5.8(b): 13C NMR spectrum of Compound 2
135 Figure 5.8(c): 13C NMR spectrum of Compound 2
136 Figure 5.9: DEPT NMR spectrum of Compound 2
137 Figure 5.10(a): COSY NMR spectrum of Compound 2
138 Figure 5.10(b):COSY NMR spectrum of Compound 2
139 Figure 5.11(a): HSQC NMR spectrum of Compound 2
140 Figure 5.11(b): HSQC NMR spectrum of Compound 2
141 Figure 5.11(c): HSQC NMR spectrum of Compound 2
142 Figure 5.12 (a): HMBC NMR spectrum of Compound 2
143 Figure 5.12(b): HMBC NMR spectrum of Compound 2
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Table 5.2: 1H (500 MHz) and 13C (125 MHz) NMR chemical shifts of compound 2 (MeOD)
No 1H 13C No 1H 13C
1 5.61 (1H, d, J= 7.7 Hz) 95.8 7' 7.74 (1H,d, J= 15.9 Hz) 148.2
2 7.91 (3H, m, H-2, H-3, H-4) 74.1 8' 6.38 (1H, d, J= 15.9 Hz ) 114.3
3 78.0 9' C=O 167.7
4 71.5 1'' 127.0c
5 3.68 (1H, m,) 76.4 2'', 6'' 7.46a (2H, d, J= 8.7 Hz) 131.3d
6
1' 2', 6' 3', 5'
4'
4.50 (1H, dd, J= 12.1, 2.1Hz, H-6a) 4.32 (1H, dd, J= 12.1, 5.6 Hz, H-6b)
7.47a (2H, d, J= 8.7 Hz)
6.81b (2H, d, J= 8.6 Hz)
64.5
127.2c 131.5d 117.1e 162.1
3'', 5'' 4''
7'' 8'' 9''
6.79b (2H, d, J=8.6 Hz)
7.65 (1H, d, J=15.9 Hz) 6.36 (1H, d, J=15.9 Hz) C=O
117.0e 161.7f 147.0 114.9 169.3
a – f
Interchangeable NMR data
145 O
H
OH
HO H H
H
HO H O
O
OH H
H O
HO
H
H O
167.7
169.3
148.2
147.0
114.3
114.9
95.6
64.5 7.74 (d, J=15.9 Hz)
6.36 (d, J=15.9 Hz)
6.38 (d, J=15.9 Hz)
7.65 (d, J=15.9 Hz)
5.61 (d, J= 7.7 Hz) 1
2 3
4 5
6
1' 2'
3' 4' 6' 5' 8'
9'
7'
3'' 1'' 4''
9''
5''
7''
6'' 2'' 8''
Figure 5.13: Some definitive HMBC correlations of compound 2
O
H
OH
HO H H
H
HO H O
O
OH H
H O
HO
H
H O
H3CO O OH OH HO
1 3
2 4
5
6
1 2
Figure 5.14: Structures of isolated compounds from Terminalia phanerophlebia
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Table 5.3: Antibacterial activity of the crude extracts, solvent fractions, column fractions from ethyl acetate sample and isolated compounds from the leaves of Terminalia phanerophlebia
Sample Bacterial strains tested and minimum inhibitory concentration (MIC) values (mg/ml) K. pneumoniae S. aureus M. aurum A+ M. tuberculosis H37Ra
Crude 0.125 0.125 0.125 0.125
Dichloromethane 0.250 0.016 0.250 0.250
Hexane 0.250 0.031 0.250 0.250
Ethyl acetate 0.125 0.008 0.125 0.125
Butanol 0.250 0.031 0.250 0.250
Fraction 1 0.125 0.125 0.250 0.250
Fraction 2 0.250 0.250 > 1 > 1
Fraction 3 0.125 0.125 > 1 > 1
Fraction 4 0.125 0.125 > 1 > 1
Fraction 5 0.125 0.125 0.125 0.250
Fraction 6 0.063 0.125 > 1 > 1
Fraction 7 0.063 0.125 > 1 > 1
Fraction 8 0.063 0.250 > 1 > 1
Fraction 9 0.125 0.250 > 1 > 1
Fraction 10 0.125 0.250 > 1 > 1
Methyl-3,4,5- trihydroxybenzo ate
0.125 0.125 0.250 0.250
1,6-di-O- coumaroyl glucopyranoside
0.125 0.125 0.125 0.063
Streptomycin - - 1.95 ×10-1 -
Rifampicin - - - 2.4 ×10-2
Neomycin - 3.063 ×10-3 - -
K. pneumoniae: Klebsiella pneumoniae, S. aureus: Staphylococcus aureus, M. aurum: Mycobacterium aurum A+, M. tuberculosis: Mycobacterium tuberculosis H37Ra. The values highlighted in bold are considered very active, -: not determined. Streptomycin, rifampicin and neomycin: positive controls
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