1. Anti-Tubercular Glycolipids from the leaves of Sterculia setigera, Del
Anti-Tubercular Glycolipids from the leaves of Sterculia setigera, Del .(Sterculiaceae)
Ibrahim T. Babalola1,2, Esther A. Adelakun2* and Scott G.Franzblau3
1. Dept. of Chemistry, Faculty of Natural Sciences, Yobe State University, PMB 1144 Damaturu, Nigeria, +234(0)
2. Dept. of Chemistry, Faculty of Natural Sciences, University of Jos, Jos, Nigeria , +234(0)
3. Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833South Wood Street Chicago, IL 60612, United States
UNIJOS 40th Celebration – Book Fair/Research Exhibition, 2015.
Introduction
Aims & Objectives
The goal of this study is to identify phytochemicals that posses in vitro cidal effect or potent inhibitory activity against Mycobacterium tuberculosis in the study plant.
A long term goal of this research is to identify novel natural products that have the potential of becoming new drug leads for anti-Tb drug development.
Fractionation Tree on Sterculia setigera Leaves
Pulverized dried leaves
(0.7 kg)
Percolation
Hexane, DCM, &EtoAc extracts combined.
40.0g on silica gel
NP VLC eluted with a gradient of Hexane-EtOAc-MeOH
(5% stepwise increase)
40 fractions combined based on activity distribution
Fr(1-6)
E1.1
Fr(7-12) E1.2
Fr(13-16) E1.3
Fr(17)
E1.4
Fr(18-23) E1.5
Fr(1-4) A
Fr(5-7) B
Fr(8-12) C
Fr(13-23) D
Fr(28-40) F
NP VLC, eluted with a gradient of Hex-EtOAc-MeOH (10% stepwise increase) to afford 23 fractions.
MIC=128ug/ml
MIC=55.52ug/ml
MIC=28.65ug/ml
MIC=54.84ug/ml
MIC=63.01ug/ml
Fr(60%)
E.1.3.2
Fr(50%)
E.1.3
Fr(70%)
E1.3.3
Fr(80%) E1.3.4
Fr(85%)
E1.3.5
Fr (90%) E.1.3.6
Fr (98%) E.1.3.7
RP C18, gradient of MeOH-Water(50%,60%,70%,80%,90%,98%) at 1.6ml/min.
300mg
RP MPLC
Historically tuberculosis (TB) is one of the oldest and most pervasive diseases in history. Worldwide, TB is caused by Mycobacterium tuberculosis (MTB) and to a lesser degree M. bovis and M. africanum, and continues to be a major disease of global importance (Okunade et al .,2004).
Mycobacterium tuberculosis, the bacterium that causes this disease, is protected from the host by a unique cell wall. It is a highly infective airborne and chronic disease usually infecting the lungs, although other organs are also involved.
Although the number of tuberculosis (TB)-related deaths appears to have stabilized at around 2million per annum, the incidence of new infections is rising, largely owing to the HIV epidemic (Gutierrez-Lugo and Bewley , 2008).
There are many challenges to eradicating TB, not least of which are the complexity associated with the disease, such as latency and drug resistance (Gutierrez-Lugo and Bewley , 2008).
Methodology
The Pulverized leaves of Sterculia Setigera was extracted using percolation method. The anti-TB activity of the leaves extract was investigated by bioactivity-guided fractionation against virulent strains of Mycobacterium tuberculosis (H37Rv (ATCC27294)) in vitro, using the Alamar Blue Assay (MABA). The method has the advantage of being simple and do not require radioactive substrates and less risk of contamination as it is carried out in a containment lab (Collins and Franzblau,1997).
Repeated purifications of the active fractions using combination of normal and reverse phase chromatography led to the isolation of a pure fraction with an interesting anti-TB activity.
Fr(24-27) E
1000mg loaded dry on to silica gel
Proposed Structure of the major Glycolipids
(2S)-1-O-(monodecane)-glyceryl-β-D-galactopyranoside)
E1.3.4
E1.3.5
Gradient of MeOH-Water (75%,77%.80%.85%,90% &98% 306 fractions generated, like fractions combined.1.0 mg each sampled for anti-TB assay
160 mg On
RP C18
RP MPLC
Fraction 7&8
NP VLC ,Silica gel, eluted with a gradient solvent system of hexane - Eto Ac-TFA(5:5:0.1-2:8:0.1) cv
Fr E1.3.4&5. (12+13). (11-14)
coded Ibe_13 Wt = 0.41 mg
Fr E1.3.4&5. (12+13). (15-17)
coded IBe_14 Wt= 0.48mg
Conclusion
Natural products continue to play a most significant role in the drug discovery and development process (Newman and Cragg, 2007), and plants are recognised as useful source of highly active antimycobacterial metabolites(Gibbons, 2005). Repeated purifications of the active fractions from S. setigera extract using combination of normal and reverse phase chromatography led to the isolation of a pure fraction with an interesting anti-TB activity (100 % inhibition against the virulent strain of Mycobacterium tuberculosis, MICof µg/ml). The cytotoxicity of this compound was also evaluated against African green monkey kidney cells (Vero cells) and human hepatocellular carcinoma (HepG2) cell lines and were found non-toxic to both cell lines (IC , µg/ml respectively).
Spectroscopic studies shows that the most active fraction is a mixture of two isomeric glycolipids (1H, 13C nmr). The major component of the mixture is proposed to be ((2S)-1-O-(monodecane)-glyceryl-β-D-galactospyranoside) by simulation using ChemDraw software. Effort is ongoing to separate the mixture for complete individual structure elucidation. This report confirms the anti-TB activity of Sterculia setigera as claimed in ethnomedicine and establish the fact that the anti-tubercular agents in S. setigera leaves are glycolipids . This is the first report on the occurrence of biologically active glycolipids from S. setigera leaves.
The Global Challenge
The drug used in the present day six-month combination therapy for treating tuberculosis were discovered 40 years ago. New drugs for tuberculosis are urgently needed to shorten the duration of therapy and to effectively treat drug-resistant TB and to eliminate the latent state Such new anti-mycobacterium compound must posses suitable pharmacological targets that could affect cell wall synthesis, bacterial survival and be able to inhibit the mechanism by which these organisms overcome the early immune response of the host (Mc Kinney et al.,2000).
The WHO estimates that if efforts do not change, by 2020, nearly 1 billion additional people will be newly infected with tuberculosis, 200 million people will become sick, and 35 million will die of the disease. (WHO, 2002)
MIC=>128, MIC=>128 , MIC=>128, MIC=>128, MIC=38.9, MIC=53.2, MIC=15.1, MIC=29.7, MIC=63.7, MIC=118, MIC=>128, MIC=>128, MIC=>128, MIC=>128, MIC=31.4, MIC=>128, MIC=>128
Results of Anti-TB Assay of sub-fractions E1.3.4&5 of Sterculia setigera
Fraction Code
Percentage Inhibition at Tested Concentrations
MIC
µg/ml
IC50
Vero
HepG2
8 µg/ml
16 µg/ml
32 µg/ml
64 µg/ml
128 µg/ml
E1.3.4&5.1 -6 6 54
>128 NT
E1.3.4&5.2
-21 7 8 61
E1.3.4&5.3
-14
-15 25 65 55
E1.3.4&5.4 -1 -7 37 45 72
E1.3.4&5.5 2 87 99
38.99
101.2
E1.3.4&5.6 -3 9 71
100
53.21
87.21
44.1
E1.3.4&5.7 20 98
15.13
102.4
81.08
E1.3.4&5.8 15 32
29.68
99.35
81.69
E1.3.4&5.9
-11
-22 22 91
63.66
108.2
80.71
E1.3.4&5.10 -5
-16 29
118.27
35.46
24.56
E1.3.4&5.11
-29 27 33
E1.3.4&5.12
-50 13
E1.3.4&5.13
-24 14 18
E1.3.4&5.14
-25 23 10 17
E1.3.4&5.15 1 36 24
E1.3.4&5.16 60 93
31.43
25.44
30.22
E1.3.4&5.17 41 70 86 90
128.00
31.75
28.44
E1.3.4&5.18
-10 28 44
RMP 95
0.05
185.3
97.85
Antimycobacterial Natural Products
Natural products form one avenue in the search for new antitubercular agents with many groups undertaken screening of natural products from higher plants, fungi and marine organisms as the preliminary step to finding new lead compounds (Okunade et al., 2004, Copp, 2003).
Naturally occurring compounds from higher plants, fungi and marine organisms that demostrated significant inhibitory activity in the in vitro bioassays against mycobacterium tuberculosis and other mycobacterial species have been reported (Copp, 2003, Newton et al., 2000).
Acknowledgements
Institute for Tuberculosis Research, University of Illinois at Chicago, USA.
Natural Products Group, Department of Pharmacy and Pharmacognosy, University of Illinois at Chicago, USA.
Tertiary Education Fund (TETFund), Abuja, Nigeria.
It calls for celebration! Its time to celebrate!! Our Alma Mater is 40 !!!
Strain: H37Rv; Stock conc.:12.8 mg/ml; Test conc.:128-8 µg/ml ; NT- Not tested.
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