|Year : 2020 | Volume
| Issue : 2 | Page : 195-199
Antitubercular compounds isolated and characterized in Tithonia diversifolia and Couroupita guianensis
Nivedita Priyadarshini, Aranganathan Veeramani
Department of Biochemistry, School of Sciences, Jain (Deemed-to-be-University), Bengaluru, Karnataka, India
|Date of Web Publication||29-May-2020|
Department of Biochemistry, School of Sciences, Jain (Deemed-to-be-University), Jayanagar 3rd Block, Bengaluru - 560 011, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Tuberculosis (TB) has become a public health challenge in the current scenario with a single causative agent, Mycobacterium tuberculosis (MTB) causing the highest morbidity and mortality affecting almost 1.7 million of the population. Furthermore, there has been no novel drug discovery for the past five decades, and the emergence of latent, multiple drug-resistant, and extensively drug-resistant species has given rise to an alarming necessity for a novel compound/s for treating this highly untamable microbe. In developing countries, plant-based drugs have shown promising results in combating TB or its symptoms; naturally occurring secondary metabolites can act as lead-drug molecules or can be co-administered with conventional drugs. Therefore, the present study was focused to identify and characterize potential antimycobacterial compounds found in the screened ethnobotanical plants, Tithonia diversifolia (TD) and Couroupita guianensis (CG). These plants are used for treating respiratory disorders and allergies in the traditional medicinal systems. Methods: These plant leaf extracts were detected and purified using chromatographic techniques for potent antitubercular phytochemicals, and the purified eluents were tested on Mycobacterium smegmatis (MSM) as a surrogate for MTB; further, the fractions inhibiting growth of MSM were characterized through gas chromatography–mass spectrometry. A toxicity test of the purified samples was also assessed by an in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and hemolytic assays. Results: The analyzed plant extracts showed the presence of a C-15 sesquiterpene, zingiberene in TD, and a phthalate ester, bis (2-ethylhexyl) phthalate, in CG leaf extracts. The toxicity assessment proved the purified fractions to be moderately toxic at higher concentrations (≥100 μg/mL). Conclusion: Therefore, the identified compounds can be promising antitubercular agents; however, further in vivo investigations will add substantial value to the compounds being pharmacologically useful.
Keywords: Antimycobacterial, bis (2-ethylhexyl) phthalate, Couroupita guianensis, Tithonia diversifolia, zingiberene
|How to cite this article:|
Priyadarshini N, Veeramani A. Antitubercular compounds isolated and characterized in Tithonia diversifolia and Couroupita guianensis. Int J Mycobacteriol 2020;9:195-9
|How to cite this URL:|
Priyadarshini N, Veeramani A. Antitubercular compounds isolated and characterized in Tithonia diversifolia and Couroupita guianensis. Int J Mycobacteriol [serial online] 2020 [cited 2020 Jul 9];9:195-9. Available from: http://www.ijmyco.org/text.asp?2020/9/2/195/285222
| Introduction|| |
Tuberculosis (TB) is a major health hazard, also one of the highest causes of death worldwide.Mycobacterium tuberculosis, a causative agent, remains an untamed microorganism with ever-challenging multiple and drug-resistant forms. Incidences of TB are seen mostly in underdeveloped and developing countries due to inadequate health-care facilities for diagnosis and treatment. Rampant development of drug-resistant species of mycobacterium since the 1980s is largely due to genetic mutations by human immunodeficiency virus (HIV) and diabetes mellitus co-infections and also noncompliance of the patients to the stringent and long-term drug regimen. Another kind of TB acquired by humans, especially those who are exposed to infected live stocks by Mycobacterium bovis, a zoonotic species, spread through inhalation of aerosols, ingestion of unpasteurized milk, and poorly cooked meat. Apart from drug resistance and other opportunistic mycobacteria, latent TB is another challenge as it has no clinical manifestation of the disease as it remains dormant for long period and reactivates in immune-suppressed conditions.
Antitubercular drugs such as ethambutol, isoniazid, pyrazinamide, rifampicin, and streptomycin were discovered between 1950 and 1970, but there have been no anti-TB drugs introduced in the past five decades. Hence, there is an urgent need for a potent drug molecule with a new mode of action, and the current trend of TB drug development involves in the optimization of existing drugs and development of novel chemical entities. Plants and its secondary metabolites act as a potential source for alternative lead chemicals for drug development and also synthetic drugs that mimic the action of natural molecules. Natural products cover wider chemical space than combinatorial library compounds, and they also have biologically accepted structures due to their co-existence with natural proteins. With progress in TB genomics and proteomics, target identification has become easier and also helpful to look for better and safer drug regimens, thereby shortening the duration of treatment in achieving the goal to reduce 95% of human deaths by 2035.
Since plants have a history of ethnobotanical use because of its plethora of curative properties, there are a number of diseases treated using various parts of plants such as leaf, bark, stem, root, and inflorescence in Ayurveda and Unani. In the present study, two such ethnobotanically important plants, Tithonia diversifolia (TD) Hemsl. A. Gray and Couroupita guianensis (CG), that are widely used in Indian medicinal system to cure TB and its related symptoms were selected for isolation and characterization of antimycobacterial components.
| Methods|| |
Chemicals and strains
Chemicals and solvents were procured from HiMedia and Fisher scientific. LB agar, acetone, hexane, methanol, ethyl acetate (EtOAc), chloroform, silica gel (60–120 mesh size), dimethyl sulfoxide (DMSO), Tween 80, Mycobacterium smegmatis (MSM) (ATCC-MC2 155), MCF7 cell line (ATCC HTB-22™), phosphate-buffered saline (PBS), and Triton X-100 were used in this study.
Screening and extraction of antimycobacterial compounds
Plant leaves of TD (Hemsl.) A. Gray and CG Aubl. were collected from Chikkamagaluru district in Karnataka, India, and identified by Regional Ayurveda Research Institute for Metabolic Disorders, Bangalore. The matured healthy leaves of the plants were powdered and subjected to Soxhlet extraction using methanol as a solvent. These extracts were concentrated and tested for antimycobacterial activity in terms of zone of inhibition (ZOI) by agar well diffusion assay. The extract that showed a significant antimycobacterial activity was subjected to purification and characterization.
Purification of compounds
Detection of compounds
The active EtOAc extract of TD and chloroform extract of CG leaves were subjected to thin-layer chromatography (TLC) to detect the presence of secondary metabolite (s). Precoated alumina with silica sheets was used as a stationary phase while hexane/acetone (increasing polarity from 0 to 100% with 10% rise in acetone) for TD leaves and benzene/EtOAc (5:1) for CG leaves as solvent systems.,
Isolation of compounds
Size exclusion column chromatography was performed to purify the active leaf extracts with minor modifications in the methodology., The glass column of 15 cm length and 2 cm diameter was packed with 15 g of preactivated silica gel (mesh size 60–120) and left overnight. 50 μL of TD and CG extracts was added to the column by mixing in activated silica. A total of 10 fractions of 30 mL each were collected using the same TLC solvent systems for elution. The fractions were evaporated at room temperature and dissolved in DMSO for antimycobacterial activity, and active fractions were further characterized.
Characterization of compounds
Active fraction from the chromatogram eluents was analyzed using gas chromatography (Thermo Scientific model system number Trace 1310) and mass spectrometer (triple-quadrupole mass spectrometer TSQ8000) having a column length of 30 m (DB-5MS, 0.250 mm ID). The initial column temperature was held at 40°C for 2 min, whereas injection temperature was maintained at 250°C having a volume and mode of 1 μL and 30, respectively, with a total run time of 47 min. Helium was used as carrier gas with ion source temperature of 230°C and MS transfer temperature of 300°C. Scanning of the eluent analysis was set at a range of 50–600 Da for the total run time. Data analysis was done using X-Calibur 4.0 software and matched with NIST library 2.2.
The cell toxicity was tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay as explained by Gupta et al., with slight modifications. Cells of MCF7 (100 μL) were seeded on the 96-well microtiter plates with 1 × 105 density each at 37°C and 5% CO2 environment for 24 h. These adherent MCF7 cells were treated with different concentrations of active compounds (10, 50, 100, 150, 200, 250, and 300 μg/mL) for 48 h in 5% CO2 atmosphere, using DMSO and puromycin as negative and positive controls, respectively.
A solution of MTT (5.0 mg/mL) was added to the wells and placed in the CO2 chamber for 4 h of incubation. Later, DMSO was added to all the wells to dissolve the formazan dye, optical density was read at 570 nm, and cell toxicity was expressed as a 50% decline in cell viability as IC50, calculated as mean ± standard deviation (SD) of triplicates.
In vitro hemolytic assay
The toxicity of the two purified compounds was determined as previously described by Kumar et al., with minor modifications. A red blood cell (RBC) suspension was prepared by washing (3000 rpm, 3 min) whole blood three times with 10% chilled PBS (v/v). RBC suspension was incubated for 1 h with varied concentrations (6, 12, 25, 50, and 100 μg/mL) of purified compounds, and 1% Triton X-100 and PBS were taken as positive and negative controls, respectively. 200 μL of centrifuged (5000 rpm, 5 min) supernatant of each dilution was read at 546 nm spectroscopically, and the percentage of hemolysis was calculated as mean ± SD of triplicates using the following formula:
| Results|| |
Detection and isolation of antimycobacterial compounds
[Figure 1]a and [Figure 1]b represents the presence of antimycobacterial compounds of EtOAc and chloroform extracts of TD and CG, respectively. The resolved TD leaf extract was viewed under the ultraviolet light, whereas the CG extract was viewed under visible light. The detection showed the presence of 5 and 7 compounds in TD and CG partially purified extracts, respectively. Further, the purified TD and CG leaf extract of size exclusion gel chromatography shows 8 mm ZOI (8th eluent) and 6 mm ZOI (5th eluent) against MSM, respectively [Figure 2]a and [Figure 2]b.
|Figure 1: (a) Thin-layer chromatography showing the compounds present in Tithonia diversifolia ethyl acetate extract in ultraviolet light. (b) Thin-layer chromatography showing the compounds present in Couroupita guianensis chloroform extract in visible light|
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|Figure 2: (a) Tithonia diversifolia plate assay of size exclusion gel chromatographic eluents 1 – control (dimethyl sulfoxide), 2 – eluent 7, 3 – eluent 9, 4 – eluent 10, 5 – eluent 8 (zone of inhibition – 8 mm). (b) Couroupita guianensis plate assay of size exclusion gel chromatographic eluents 1 – control (dimethyl sulfoxide), 2 – eluent 2, 3 – eluent 3, 4 – eluent 4, 5 – eluent 5 (zone of inhibition – 6 mm), 6 – eluent 6, 7 – eluent 7|
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Identification of compounds
The gas chromatography–mass spectrometry analyzed that chromatographic eluent of TD and CG leaves showed the presence of potential bioactive compounds. In TD extract, zingiberene (ZGB) was the major component with ~40% of abundance and retention time (rt) of 25.22 and also other sesquiterpenes such as α-curcumene (rt: 24.86), β-sesquiphellandrene (rt: 25.92), α-farnesene (rt: 25.41), β-bisabolene (rt: 25.52), and β-selinene (rt: 25.09) found in trace quantities [Figure 3]. Similarly, in CG extract, bis (2-ethylhexyl) phthalate or di (2-ethylhexyl) phthalate (DEHP) was found as a major component with 94% of abundance and rt of 44.84 [Figure 4].
|Figure 3: Gas chromatography mass spectrometry chromatogram profile representing the highest intensity of Zingiberene (retention time: 25.22)|
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|Figure 4: Gas chromatography mass spectrometry chromatogram profile representing the highest intensity of bis (2-ethylhexyl)phthalate (retention time: 44.84)|
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MTT assay [Figure 5] and hemolytic test [Figure 6] results of bioactive components in TD and CG purified leaf extracts are concluded as a percentage of cell viability. MTT assay showed an IC50 value of 100 and 300 μg/ml concentration for TD and CG leaves, respectively, both moderately toxic at higher concentration. Similarly, hemolytic assay results also showed a moderate amount of toxicity at 100 μg/ml concentration by annihilating 30% and 0.028% erythrocytes in TD and CG extract, respectively.
|Figure 5: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay performed with purified plants extracts|
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| Discussion|| |
Previous studies show active metabolites having intermediate polarity such as terpenes are known to get extracted in EtOAc layer, whereas lipophilic compounds such as esters get extracted in chloroform., There are several bioactive compounds such as tagitinin C, essential oils, sesquiterpene lactones, benzoates, indirubin, and indigo isolated from TD and CG leaves.,,, Essential oils of TD leaves from Nigerian origin have shown α-pinene, caryophyllene, and β-pinene as major constituents, whereas TD leaves of Cameroon origin had trans-β-ocimene, α-pinene, and limonene as main constituents. Although ZGB was the major constituent of the present study, very low concentrations of α-pinene, trans-β-ocimene, limonene, and caryophyllene were also observed. These variations in percentage of components may be due to changes in climatic conditions, soil type, and other environmental factors.
The major antimycobacterial component of TD plant was ZGB (C15H24), a monocyclic sesquiterpene which is also a predominant constituent of the ginger (Zingiber officinale) oil. ZGB is known to exhibit antioxidant, anticarcinogenic, antiulcer, antibacterial, antiviral activities and attenuates cytotoxicity and oxidative DNA damage. Elevated levels of ZGB along with other sesquiterpenes act as a defense metabolite against emerald ash borers, a tree-dwelling pest in the United States. It is also used as a flavoring agent in combination with shogaol and gingerol in food products in the Asiatic region. Invariably, terpenoids and essential oils derived from the Asteraceae family are known to be resistant against multidrug-resistant strain of bacteria.
Whereas DEHP, a phthalate ester major component of CG plant, is widely used as a plasticizer in plastic industry, it is also produced by natural resources such as plants (Aloe vera Linne, Nigella glandulifera, and Nauclea latifolia), animals, and microorganisms (Streptomyces bangladeshensis, Bacillus pumilus MB40, Penicillium olsonii, and Cladosporium sp.) as secondary metabolites. Anthropogenic DEHP is absorbed from the environment and gets hydrolyzed in natural system to produce monoalkyl phthalates (MPEs) as intermediates. Previous studies state MPEs and DEHP as important drug candidates in cancer, retroviral, fungal, tumor, and antimicrobials.,, An in silico study conducted by Rajiniraja et al. showed that MPEs exhibit antimycobacterial activity by binding to PKnB gene which is necessary for the sustainability of Mycobacterium in host. CG plants can also be used for phytoremediation for removal anthropogenic DEHP around the agricultural areas as a study conducted by Wu et al. suggests intercropping of (Benincasa hispida) trees with vegetable crops in farms.
Although plant-based drugs being natural show negligible side effects, it is always wise to ascertain safety at the preliminary stages for which in vitro toxicity assays are performed, which removes high-risk components in early stages of drug development. Aqueous extract of TD leaves is known to cause hepatic, renal, and hematological changes in Wister mice on repeated high doses. Although TD plants have several biologically active compounds, the toxic effect of plants at higher doses cannot be neglected; thus, the dosage should be fixed. However, in case of CG plant, previous studies pertaining to flower extract were proved to be nontoxic.,,, Furthermore, an acute toxicity study on albino mice with crude ethanolic leaf extract showed no sign of mortality or behavioral changes proving CG plant to be nontoxic and safe. All the parts (flower, leaves, roots, and fruit) of both plants are used in traditional medicine systems (Ayurveda, Unani, and Siddha) for curing broad spectrum of bacterial and fungal diseases.
The results of the present study also conclude that the selectivity index value of TD and CG extract, which includes the IC50 with MSM, was found above 1.0, which proves that the filaricidal and toxin components to be different and further elimination of these toxins may lead to develop potent phytochemical drug.,
| Conclusion|| |
The present investigation propagates the use of plants as a primary source of antitubercular compound and also as a significant source for the naturally occurring alternates for countering the mode of action. The isolated and characterized antitubercular components in this study show antitubercular activity in an acceptable range with moderate toxicity at higher concentrations. However, these potential components will also be further investigated for combinatorial effect and in vivo studies to ascertain their potency in pharmacological application.
Financial support and sponsorship
This study was financially supported by Jain (Deemed-to-be-University) Student Fellowship.
Conflicts of interest
There are no conflicts of interest.
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