- Email: [email protected]
Evaluation of the antimycobacterium activity of the constituents from Ocimum basilicum against Mycobacterium tuberculosis
Journal of Ethnopharmacology 144 (2012) 220–222
Contents lists available at SciVerse ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Ethnopharmacological Communication
Evaluation of the antimycobacterium activity of the constituents from Ocimum basilicum against Mycobacterium tuberculosis Bina S. Siddiqui n, Huma A. Bhatti, Sabira Begum, Sobiya Perwaiz International Center for Chemical and Biological Sciences, HEJ Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2012 Received in revised form 17 July 2012 Accepted 2 August 2012 Available online 17 August 2012
Ethnopharmacological relevance: Ocimum basilicum belongs to the genus Ocimum (Lamiaceae). Many species of this genus including O. basilicum have been traditionally used for the treatment of a variety of diseases including respiratory diseases and symptoms of tuberculosis. Aim of study: The aim of the study was to evaluate the activity of pure constituents of O. basilicum, a plant used in traditional medicine to treat respiratory diseases including symptoms of tuberculosis, against Mycobacterium tuberculosis H37Rv. Materials and methods: The crude methanolic extract from the aerial parts (leaves, fruits and flowers) of the plant was separated into EtOAc and water subextracts and the EtOAc subextract was further divided into acidic and neutral fractions. Each of these was subjected to fractionation through column chromatography followed by prep. TLC. Several pure compounds were thus isolated and their activity was determined against M. tuberculosis H37Rv using MABA assay. Results: Nine compounds were assayed for antituberculosis activity which exhibited upto 49% inhibition of M. tuberculosis H37Rv at 6.25 mg/mL. These include one new compound bacilicin, the structure of which was elucidated based on 2D-NMR (HSQC, HMBC, COSY and NOESY) and Ms spectral analysis. The known compounds were identified on the basis of comparison of their spectral data with those reported earlier. Conclusions: Inhibition of M. tuberculosis by pure compounds from O. basilicum supports the use of this plant in ethnomedicine as a remedy for symptoms of tuberculosis. The results also suggest that the activity of the plant may be due to a synergistic effect of active compounds including those investigated in the present studies, and hence this plant is a potential candidate for obtaining further new antituberculosis natural products. & 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ocimum basilicum Mycobacterium tuberculosis H37Rv Antituberculosis activity
1. Introduction Tuberculosis is one of the oldest and most fatal, highly infective airborne chronic diseases infecting about one third of the world’s population annually resulting in three million deaths per year (WHO, 2010a, 2011). In Pakistan over 200 people in 100,000 are the victims of this fatal disease (WHO, 2010b). In the past decade a number of new possible antimycobacterial agents have been added into the literature including extracts as well as pure compounds from plants, microorganisms and marine organisms (Copp and Pearce, 2007; Mohamad et al., 2011). The present studies were aimed on one of the plants reputed in folklore for the treatment of tuberculosis or its symptoms, namely Ocimum basilicum to isolate and assay pure compounds for antituberculosis activity to validate the reputed folkloric activity and to identify active compounds.
O. basilicum (Lamiaceae) commonly known as sweet basil and tulsi is a herbaceous, perennial plant native to Asia, Africa and South America and widely cultivated world wide (Jayaweera, 1981). The oil has antibacterial and antifungal properties (Manitto et al., 1974) and thymol, a constituent of O. basilicum is well known as an antiseptic agent (Dikshit and Husain, 1984). The aqueous soxhlet extract of O. basilicum leaves has shown bronchodilatory activity (Boskabady et al., 2005) while the methanolic leaves extract has been reported to inhibit the growth of Mycobaterium smegmatis (Nag et al., 2008). The plant has yielded different types of constituents including phenol derivatives, flavonoids, phenyl propanoids, triterpenoids (Manitto et al., 1974; Siddiqui et al., 2007a), steroids and steroidal glycosides (Siddiqui et al., 2007b).
2. Materials and methods 2.1. General spectrometric conditions
n
Corresponding author. Tel.: þ92 21 99271716; fax: þ92 21 99261713. E-mail address: [email protected] (B.S. Siddiqui).
0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.08.003
The 1H NMR, and two-dimensional COSY, NOESY and J-resolved NMR spectra were obtained in CDCl3 on Bruker 400 MHz NMR
B.S. Siddiqui et al. / Journal of Ethnopharmacology 144 (2012) 220–222
spectrometer operating at 400 MHz and 13C-NMR, DEPT and two-dimensional HSQC, and HMBC NMR spectra were recorded in CDCl3 on Bruker Avance 400-A spectrometer operating at 100 MHz. The HREIMS were obtained from JEOL JMS-600H mass spectrometer. UV spectra (MeOH) were run on Hitachi-U-3200 spectrophotometer and IR spectra (CHCl3) were obtained using JASCO-A-302 spectrophotometer.
221
saturated with saline, acidified with 30% HCl and extracted out with ethyl acetate to obtain the acidic fraction.
2.4. Isolation and identification of compounds The neutral fraction was treated with pet. ether to give pet. ether soluble and insoluble fractions. The pet. ether insoluble fraction furnished basilimoside (1, 12 mg) (Siddiqui et al., 2007b), through repeated prep. TLC using pre-coated silica gel aluminium cards (Merck; pet. ether-EtOAc 8:2). The pet. ether soluble portion was subjected to gravity column chromatography (CC) (pet. ether, CHCl3, CHCl3–MeOH in increasing order of polarity). The fractions were combined on the basis of TLC to ultimately afford 14 fractions (A–N). Fraction J (2.7 g) was again subjected to CC (CHCI3–MeOH; 9.1:0.9). The fraction eluted with CHCl3–MeOH (9.5:0.5) was purified on pre-coated silica gel aluminium cards using CHCl3–MeOH, 9.6:0.4 to furnish a new compound bacilicin (2) as an amorphous powder (15 mg). The acidic fraction referred to above was subjected to column chromatography over silica gel and eluted with pet. ether, pet. etherEtOAc; 8:2 to 7:3 and then CHCl3, CHCl3–MeOH in increasing order of polarity. The fractions were combined on the basis of TLC to ultimately afford three major fractions (A–C). Fraction A was purified through prep. TLC (CHCl3–MeOH; 9.4:0.6) to furnish 40 -carbomethoxy-20 -hydroxyphenyl ferulate (3) (10 mg) (Siddiqui et al., 2007a) and fraction B was purified by passing through a pencil column eluted with CHCl3–MeOH; 9.9:0.1–8:2 to give
2.2. Plant material The aerial parts of O. basilicum were collected from Karachi, Pakistan and identified by Mr. Sherwali (Herbarium Incharge) Department of Botany, University of Karachi. A voucher specimen (GH. No. S.N. 68316) has been deposited in the Herbarium of the same Department. 2.3. Preparation of extract and fractions Fresh aerial parts (15 kg) of O. basilicum were chopped to small pieces and repeatedly ( 4) extracted with methanol (25 L; 48 h each time) at room temperature. The combined extract was concentrated under vacuum to a syrupy residue which was partitioned between EtOAc and H2O. The EtOAc phase was divided into acidic and neutral fractions by treatment with 4% aqueous Na2CO3. The EtOAc phase was washed, dried over anyhydrous Na2SO4 and freed of the solvent under reduced pressure to afford the neutral fraction. The aqueous basic phase was
CH3 CH3 CH3 CH3 RO
O
RO RO
O
OR
1
R= H
1a R = Ac O
O
C 2'''
O HO
C 1"
8
4 5
3
3a
3'''
O
CH3
O
H
6'
O
5'
1'
4'
OR
2
H
6
7
7a
O 1
3'
2'
OCH3
OCH3
OR
RO
3 OCH3
OCH3
2 R=H 2a R=Ac
R= H
3a R = Ac
OH OH
O O CH3O
4 O
O O
HOH2C
5
H
O HO
Fig. 1. Structures of compounds 1–6, 1a–3a.
OH
6
222
B.S. Siddiqui et al. / Journal of Ethnopharmacology 144 (2012) 220–222
Table 1 % Inhibition of M. tuberculosis by 1–6 and 1a–3a at 6.25 mg/mL. Compounds tested
% inhibition at 6.25 (mg/mL)
1 1a 2 2a 3 3a 4 5 6
8 30 35 38 28 30 49 22 24
Rifampin, MIC: 0.0031. Isoniazid, MIC: 0.063.
(E)-30 -hydroxy-40 -(100 -hydroxyethyl)-phenyl-4-methoxycinnamate (4) (4.5 mg) (Siddiqui et al., 2007a). Fraction C furnished two compounds 4-(hydroxymethyl)-benzoic acid (5) (3.8 mg) and methyl-3,4-dihydroxy benzoate (6) (4.2 mg) (Miyazawa et al., 2003) on prep. TLC (CHCl3–MeOH; 9.4:0.6). The acetyl derivatives of compounds 1–3 were also prepared and evaluated for their activity.
4. Conclusions The results of present studies support the use of this plant in ethnomedicine as a remedy for symptoms of tuberculosis and this plant may be a potential candidate for further phytochemical and pharmacological studies for obtaining compounds effective against M. tuberculosis. Additionally, its constituents may be used as lead compounds for obtaining more active analogues as can be inferred from the example of compound 1 in which case the inhibition significantly increased from 8% for 1 to 30% for the acetyl derivative 1a at 6.25 mg/mL
Acknowledgements This work was financially supported by the Pakistan Academy of Sciences through a Project no. 5-9/PAS. The authors are grateful to the TAACF screening facilities, Southern Research Institute Birmingham, AL, USA for providing antimycobacterial data through a research and development contract with the US National Institute of Allergy and Infectious Diseases.
Appendix A. Supporting information 2.5. Acetylation of compounds 1–3 Compounds 1–3 were acetylated with acetic anhydride in presence of pyridine (vide supplementary data, Appendix A) to afford the acetyl derivatives la–3a.
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2012.08.003.
2.6. Antituberculosis activity assay References The anti-mycobacterial assays were conducted by the courtesy of the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF), USA. In the established protocol, primary screening was conducted at 6.25 mg/mL against M. tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using a broth microdilution assay, the Microplate Alamar Blue Assay (MABA) (Collins and Franzblau, 1997).
3. Results and discussion 3.1. Antimycobacterial activity Compounds isolated in the present studies (Fig. 1) showed upto 49% inhibition of M. tuberculosis at 6.25 mg/mL concentration (Table 1) indicating that their MIC would be greater than 6.25 mg/mL. The results suggest that the activity of the plant may be due to a synergistic effect of active compounds including those obtained in the present studies. It would be interesting to note that in one of the studies undertaken in view of Ayurvedic properties attributed to O. basilicum, it was reported that its leaves and callus extracts inhibited the growth of M. smeguratis ATCC 14468 (Nag et al., 2008), whereas in another study (Mohamad et al., 2011), the methanolic extract of O. basilicum leaves was not found to show any inhibition of M. tuberculosis at 1600 mg/mL. 3.2. Characterisation of new compound The structure of new compound was elucidated by MS, IR and NMR data vide supplementary data Appendix A.
Boskabady, M.H., Kaini, S., Haghiri, B., 2005. Relaxant effects of Ocimum basilicum on guinea pig tracheal chains and its possible mechanism. DARU Journal of Pharmaceutical Sciences 13, 28–33. Collins, L.A., Franzblau, S.G., 1997. Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrobial Agents and Chemotherapy 41, 1004–1009. Copp, B.R., Pearce, A.N., 2007. Natural product growth inhibitors of Mycobacterium tuberculosis. Natural Product Reports 24, 278–297. Dikshit, A., Husain, A., 1984. Antifungal activities of some essential oils against animal pathogens. Fitoterapia 55, 171–176. Jayaweera, D.M.A., 1981. Medicinal Plants, Part 3. National Science Council of SriLanka Publication, Colombo, pp. 101. Manitto, P., Monti, D., Gramatica, P., 1974. Biosynthesis of phenylpropanoid compounds, Part I. Biosynthesis of eugenol in Ocimum basilicum L. Journal of the Chemical Society, Perkin Transaction 1, 1727–1731. Miyazawa, M., Oshima, T., Koshio, K., 2003. Tyrosinase inhibitor from black rice bran. Journal of Agriculture and Food Chemistry 51, 6953–6956. Mohamad, S., Mohd, N.Z., Wahab, H.A., Ibrahim, P., Sulaiman, S.F., Zahariluddin, A.S.M., Noor, S.S.M., 2011. Antituberculosis potential of some ethnobotanically selected Malaysian plants. Journal of Ethnopharmacology 133, 1021–1026. Nag, T.N., Mathur, K., Vyas, M., Chhangani, D., Ranaga, A., Choudhary, A., 2008. In Vitro Study of Cultivation and Antibacterial Activity of Ocimum basilicum Against Mycobacterium smegmatis ATCC 14468. Paper Presented at International Conference on Molecular Biology and Biotechnology held at Banasthali University, Tonk, Rajasthan, India. Siddiqui, B.S., Aslam, H., Begum, S., Ali, S.T., 2007a. New cinnamic acid ester from Ocimum basilicum. Natural Products Research 21, 736–741. Siddiqui, B.S., Aslam, H., Ali, S.T., Khan, S., Begum, S., Khatoon, N., 2007b. Two new triterpenoids and a steroidal glycoside from the aerial parts of Ocimum basilicum. Chemical and Pharmaceutical Bulletin 55, 516–519. WHO, 2010a. WHO Tuberculosis Fact sheet No. 104, Available at /http://www. who.int/mediacentre/factsheets/fs104/en/ (accessed 01.05.10.)S. WHO, 2010b. Multidrug and Extensively Drug-Resistant Tb (M/XDR-TB): 2010 Global Report on Surveillance and Response, Available at /http://www.whqlibdoc.who. int/publications/2010/9789241599191 eng.pdf (accessed 10.05.10.)S. WHO, 2011. WHO Global Tuberculosis Control Report 2011, Available at /http:// extranet.who.int/sree/ReportsS. (accessed 15.12.11.).
Contents lists available at SciVerse ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Ethnopharmacological Communication
Evaluation of the antimycobacterium activity of the constituents from Ocimum basilicum against Mycobacterium tuberculosis Bina S. Siddiqui n, Huma A. Bhatti, Sabira Begum, Sobiya Perwaiz International Center for Chemical and Biological Sciences, HEJ Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2012 Received in revised form 17 July 2012 Accepted 2 August 2012 Available online 17 August 2012
Ethnopharmacological relevance: Ocimum basilicum belongs to the genus Ocimum (Lamiaceae). Many species of this genus including O. basilicum have been traditionally used for the treatment of a variety of diseases including respiratory diseases and symptoms of tuberculosis. Aim of study: The aim of the study was to evaluate the activity of pure constituents of O. basilicum, a plant used in traditional medicine to treat respiratory diseases including symptoms of tuberculosis, against Mycobacterium tuberculosis H37Rv. Materials and methods: The crude methanolic extract from the aerial parts (leaves, fruits and flowers) of the plant was separated into EtOAc and water subextracts and the EtOAc subextract was further divided into acidic and neutral fractions. Each of these was subjected to fractionation through column chromatography followed by prep. TLC. Several pure compounds were thus isolated and their activity was determined against M. tuberculosis H37Rv using MABA assay. Results: Nine compounds were assayed for antituberculosis activity which exhibited upto 49% inhibition of M. tuberculosis H37Rv at 6.25 mg/mL. These include one new compound bacilicin, the structure of which was elucidated based on 2D-NMR (HSQC, HMBC, COSY and NOESY) and Ms spectral analysis. The known compounds were identified on the basis of comparison of their spectral data with those reported earlier. Conclusions: Inhibition of M. tuberculosis by pure compounds from O. basilicum supports the use of this plant in ethnomedicine as a remedy for symptoms of tuberculosis. The results also suggest that the activity of the plant may be due to a synergistic effect of active compounds including those investigated in the present studies, and hence this plant is a potential candidate for obtaining further new antituberculosis natural products. & 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ocimum basilicum Mycobacterium tuberculosis H37Rv Antituberculosis activity
1. Introduction Tuberculosis is one of the oldest and most fatal, highly infective airborne chronic diseases infecting about one third of the world’s population annually resulting in three million deaths per year (WHO, 2010a, 2011). In Pakistan over 200 people in 100,000 are the victims of this fatal disease (WHO, 2010b). In the past decade a number of new possible antimycobacterial agents have been added into the literature including extracts as well as pure compounds from plants, microorganisms and marine organisms (Copp and Pearce, 2007; Mohamad et al., 2011). The present studies were aimed on one of the plants reputed in folklore for the treatment of tuberculosis or its symptoms, namely Ocimum basilicum to isolate and assay pure compounds for antituberculosis activity to validate the reputed folkloric activity and to identify active compounds.
O. basilicum (Lamiaceae) commonly known as sweet basil and tulsi is a herbaceous, perennial plant native to Asia, Africa and South America and widely cultivated world wide (Jayaweera, 1981). The oil has antibacterial and antifungal properties (Manitto et al., 1974) and thymol, a constituent of O. basilicum is well known as an antiseptic agent (Dikshit and Husain, 1984). The aqueous soxhlet extract of O. basilicum leaves has shown bronchodilatory activity (Boskabady et al., 2005) while the methanolic leaves extract has been reported to inhibit the growth of Mycobaterium smegmatis (Nag et al., 2008). The plant has yielded different types of constituents including phenol derivatives, flavonoids, phenyl propanoids, triterpenoids (Manitto et al., 1974; Siddiqui et al., 2007a), steroids and steroidal glycosides (Siddiqui et al., 2007b).
2. Materials and methods 2.1. General spectrometric conditions
n
Corresponding author. Tel.: þ92 21 99271716; fax: þ92 21 99261713. E-mail address: [email protected] (B.S. Siddiqui).
0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.08.003
The 1H NMR, and two-dimensional COSY, NOESY and J-resolved NMR spectra were obtained in CDCl3 on Bruker 400 MHz NMR
B.S. Siddiqui et al. / Journal of Ethnopharmacology 144 (2012) 220–222
spectrometer operating at 400 MHz and 13C-NMR, DEPT and two-dimensional HSQC, and HMBC NMR spectra were recorded in CDCl3 on Bruker Avance 400-A spectrometer operating at 100 MHz. The HREIMS were obtained from JEOL JMS-600H mass spectrometer. UV spectra (MeOH) were run on Hitachi-U-3200 spectrophotometer and IR spectra (CHCl3) were obtained using JASCO-A-302 spectrophotometer.
221
saturated with saline, acidified with 30% HCl and extracted out with ethyl acetate to obtain the acidic fraction.
2.4. Isolation and identification of compounds The neutral fraction was treated with pet. ether to give pet. ether soluble and insoluble fractions. The pet. ether insoluble fraction furnished basilimoside (1, 12 mg) (Siddiqui et al., 2007b), through repeated prep. TLC using pre-coated silica gel aluminium cards (Merck; pet. ether-EtOAc 8:2). The pet. ether soluble portion was subjected to gravity column chromatography (CC) (pet. ether, CHCl3, CHCl3–MeOH in increasing order of polarity). The fractions were combined on the basis of TLC to ultimately afford 14 fractions (A–N). Fraction J (2.7 g) was again subjected to CC (CHCI3–MeOH; 9.1:0.9). The fraction eluted with CHCl3–MeOH (9.5:0.5) was purified on pre-coated silica gel aluminium cards using CHCl3–MeOH, 9.6:0.4 to furnish a new compound bacilicin (2) as an amorphous powder (15 mg). The acidic fraction referred to above was subjected to column chromatography over silica gel and eluted with pet. ether, pet. etherEtOAc; 8:2 to 7:3 and then CHCl3, CHCl3–MeOH in increasing order of polarity. The fractions were combined on the basis of TLC to ultimately afford three major fractions (A–C). Fraction A was purified through prep. TLC (CHCl3–MeOH; 9.4:0.6) to furnish 40 -carbomethoxy-20 -hydroxyphenyl ferulate (3) (10 mg) (Siddiqui et al., 2007a) and fraction B was purified by passing through a pencil column eluted with CHCl3–MeOH; 9.9:0.1–8:2 to give
2.2. Plant material The aerial parts of O. basilicum were collected from Karachi, Pakistan and identified by Mr. Sherwali (Herbarium Incharge) Department of Botany, University of Karachi. A voucher specimen (GH. No. S.N. 68316) has been deposited in the Herbarium of the same Department. 2.3. Preparation of extract and fractions Fresh aerial parts (15 kg) of O. basilicum were chopped to small pieces and repeatedly ( 4) extracted with methanol (25 L; 48 h each time) at room temperature. The combined extract was concentrated under vacuum to a syrupy residue which was partitioned between EtOAc and H2O. The EtOAc phase was divided into acidic and neutral fractions by treatment with 4% aqueous Na2CO3. The EtOAc phase was washed, dried over anyhydrous Na2SO4 and freed of the solvent under reduced pressure to afford the neutral fraction. The aqueous basic phase was
CH3 CH3 CH3 CH3 RO
O
RO RO
O
OR
1
R= H
1a R = Ac O
O
C 2'''
O HO
C 1"
8
4 5
3
3a
3'''
O
CH3
O
H
6'
O
5'
1'
4'
OR
2
H
6
7
7a
O 1
3'
2'
OCH3
OCH3
OR
RO
3 OCH3
OCH3
2 R=H 2a R=Ac
R= H
3a R = Ac
OH OH
O O CH3O
4 O
O O
HOH2C
5
H
O HO
Fig. 1. Structures of compounds 1–6, 1a–3a.
OH
6
222
B.S. Siddiqui et al. / Journal of Ethnopharmacology 144 (2012) 220–222
Table 1 % Inhibition of M. tuberculosis by 1–6 and 1a–3a at 6.25 mg/mL. Compounds tested
% inhibition at 6.25 (mg/mL)
1 1a 2 2a 3 3a 4 5 6
8 30 35 38 28 30 49 22 24
Rifampin, MIC: 0.0031. Isoniazid, MIC: 0.063.
(E)-30 -hydroxy-40 -(100 -hydroxyethyl)-phenyl-4-methoxycinnamate (4) (4.5 mg) (Siddiqui et al., 2007a). Fraction C furnished two compounds 4-(hydroxymethyl)-benzoic acid (5) (3.8 mg) and methyl-3,4-dihydroxy benzoate (6) (4.2 mg) (Miyazawa et al., 2003) on prep. TLC (CHCl3–MeOH; 9.4:0.6). The acetyl derivatives of compounds 1–3 were also prepared and evaluated for their activity.
4. Conclusions The results of present studies support the use of this plant in ethnomedicine as a remedy for symptoms of tuberculosis and this plant may be a potential candidate for further phytochemical and pharmacological studies for obtaining compounds effective against M. tuberculosis. Additionally, its constituents may be used as lead compounds for obtaining more active analogues as can be inferred from the example of compound 1 in which case the inhibition significantly increased from 8% for 1 to 30% for the acetyl derivative 1a at 6.25 mg/mL
Acknowledgements This work was financially supported by the Pakistan Academy of Sciences through a Project no. 5-9/PAS. The authors are grateful to the TAACF screening facilities, Southern Research Institute Birmingham, AL, USA for providing antimycobacterial data through a research and development contract with the US National Institute of Allergy and Infectious Diseases.
Appendix A. Supporting information 2.5. Acetylation of compounds 1–3 Compounds 1–3 were acetylated with acetic anhydride in presence of pyridine (vide supplementary data, Appendix A) to afford the acetyl derivatives la–3a.
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2012.08.003.
2.6. Antituberculosis activity assay References The anti-mycobacterial assays were conducted by the courtesy of the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF), USA. In the established protocol, primary screening was conducted at 6.25 mg/mL against M. tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using a broth microdilution assay, the Microplate Alamar Blue Assay (MABA) (Collins and Franzblau, 1997).
3. Results and discussion 3.1. Antimycobacterial activity Compounds isolated in the present studies (Fig. 1) showed upto 49% inhibition of M. tuberculosis at 6.25 mg/mL concentration (Table 1) indicating that their MIC would be greater than 6.25 mg/mL. The results suggest that the activity of the plant may be due to a synergistic effect of active compounds including those obtained in the present studies. It would be interesting to note that in one of the studies undertaken in view of Ayurvedic properties attributed to O. basilicum, it was reported that its leaves and callus extracts inhibited the growth of M. smeguratis ATCC 14468 (Nag et al., 2008), whereas in another study (Mohamad et al., 2011), the methanolic extract of O. basilicum leaves was not found to show any inhibition of M. tuberculosis at 1600 mg/mL. 3.2. Characterisation of new compound The structure of new compound was elucidated by MS, IR and NMR data vide supplementary data Appendix A.
Boskabady, M.H., Kaini, S., Haghiri, B., 2005. Relaxant effects of Ocimum basilicum on guinea pig tracheal chains and its possible mechanism. DARU Journal of Pharmaceutical Sciences 13, 28–33. Collins, L.A., Franzblau, S.G., 1997. Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrobial Agents and Chemotherapy 41, 1004–1009. Copp, B.R., Pearce, A.N., 2007. Natural product growth inhibitors of Mycobacterium tuberculosis. Natural Product Reports 24, 278–297. Dikshit, A., Husain, A., 1984. Antifungal activities of some essential oils against animal pathogens. Fitoterapia 55, 171–176. Jayaweera, D.M.A., 1981. Medicinal Plants, Part 3. National Science Council of SriLanka Publication, Colombo, pp. 101. Manitto, P., Monti, D., Gramatica, P., 1974. Biosynthesis of phenylpropanoid compounds, Part I. Biosynthesis of eugenol in Ocimum basilicum L. Journal of the Chemical Society, Perkin Transaction 1, 1727–1731. Miyazawa, M., Oshima, T., Koshio, K., 2003. Tyrosinase inhibitor from black rice bran. Journal of Agriculture and Food Chemistry 51, 6953–6956. Mohamad, S., Mohd, N.Z., Wahab, H.A., Ibrahim, P., Sulaiman, S.F., Zahariluddin, A.S.M., Noor, S.S.M., 2011. Antituberculosis potential of some ethnobotanically selected Malaysian plants. Journal of Ethnopharmacology 133, 1021–1026. Nag, T.N., Mathur, K., Vyas, M., Chhangani, D., Ranaga, A., Choudhary, A., 2008. In Vitro Study of Cultivation and Antibacterial Activity of Ocimum basilicum Against Mycobacterium smegmatis ATCC 14468. Paper Presented at International Conference on Molecular Biology and Biotechnology held at Banasthali University, Tonk, Rajasthan, India. Siddiqui, B.S., Aslam, H., Begum, S., Ali, S.T., 2007a. New cinnamic acid ester from Ocimum basilicum. Natural Products Research 21, 736–741. Siddiqui, B.S., Aslam, H., Ali, S.T., Khan, S., Begum, S., Khatoon, N., 2007b. Two new triterpenoids and a steroidal glycoside from the aerial parts of Ocimum basilicum. Chemical and Pharmaceutical Bulletin 55, 516–519. WHO, 2010a. WHO Tuberculosis Fact sheet No. 104, Available at /http://www. who.int/mediacentre/factsheets/fs104/en/ (accessed 01.05.10.)S. WHO, 2010b. Multidrug and Extensively Drug-Resistant Tb (M/XDR-TB): 2010 Global Report on Surveillance and Response, Available at /http://www.whqlibdoc.who. int/publications/2010/9789241599191 eng.pdf (accessed 10.05.10.)S. WHO, 2011. WHO Global Tuberculosis Control Report 2011, Available at /http:// extranet.who.int/sree/ReportsS. (accessed 15.12.11.).