**Potential Use of** *I. suffruticosa* **in Treatment of Tuberculosis with Immune System Activation**

Camila Bernardes de Andrade Carli1, Marcela Bassi Quilles1, Danielle Cardoso Geraldo Maia1, Clarice Q. Fujimura Leite1, Wagner Vilegas2 and Iracilda Z. Carlos1 *1Departamento de Análises Clínicas e Departamento de Ciências, Unesp, R. Expedicionários do Brasil 1601, Araraquara, SP, 2Departamento de Ciências Biológicas, Unesp, Rodovia Araraquara-Jaú, Araraquara, SP, 3Departamento de Química Orgânica, Unesp, R. Prof. Francisco Degni, Araraquara, SP, Brazil* 

## **1. Introduction**

362 Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance

Silas, J.H., Ramsay, L.E. & Freestone, S. (1982). Hydralazine Once Daily in Hypertension, *British Medical Journal*, Vol.284, No.6329, pp. 1602-1604. ISSN 0959 8138 Tanachatchairatana, T., Bremner, J.B., Chokchaisiri, R. & Suksamrarn, A. (2008).

Thimann, K.V. (1969). The auxins. In: *Physiology of Plant Growth and Development*, M.B.

van Heijenoort, J. (2001). Formation of the Glycan Chains in the Synthesis of Bacterial Peptidoglycan, *Glycobiology*, Vol.11, No.3, pp. 25R-36R, ISSN 1460 2423 Velichka, D., Ivana, A., Haruaki, T., Katsumasa, S., Venkata, R., Nadadhur, G., Donna, D.,

Warbasse, J.P. (1894). Cinnamic Acid in the Treatment of Tuberculosis, *Annals of Surgery*,

Wendakoon, C.N. & Sakaguchi, M. (1995). Inhibition of Amino Acid Decarboxylase Activity

Whetten, R.W., Mackay, J.J. & Sederoff, R.R. (1998). Recent Advances in Understanding

Wong, S.Y.Y., Grant, I.R., Friedman, M., Elliot, C.T. & Chen, S. (2008). Antibacterial

Wu, W., Sil, D., Szostak, M.L., Malladi, S.S., Warshakoon, H.J., Kimbrell, M.R., Cromer, J.R.

Xu, Y. & Miller, M.J. (1998). Total Syntheses of Mycobactin Analogues as Potent

Yoya, G. K., Bedos-Belval, F., Constant, P., Duran, H., Daffé, M. & Baltas. M. (2009) Synthesis

Zhang, Y., Broser, M. & Rom, W.N. (1994). Activation of the Interleukin-6 Gene by

*Organic Chemistry*, Vol.63, No. 13, pp. 4314-4322, ISSN 1520 6904

*Discovery*, Vol.5, No.1, pp. 76-90, ISSN 1574 891X

*Chemistry*, Vol.17, No.2, pp. 709-715, ISSN 0968-0896

Vol.91, No.6, pp. 2225-2229, ISSN 0027 8424

Vol.19, pp. 102-117. ISSN 1528 1140

Vol.49, pp. 585-609. ISSN 0066-4294

ISSN 0099-2240

341-343, ISSN 0960-894X

Vol.58, No.3, pp. 280-283, ISSN 0362-028X

194-198. ISSN 1347 5223

McGrew-Hill, London.

Antimycobacterial Activity of Cinnamate-Based Esters of the Triterpenes Betulinic, Oleanolic and Ursolic Acids, *Chemical & Pharmaceutical Bulletin*, Vol.56, No.2, pp.

Wilkinson (ed.), pp 2-45, Accession Number 1970:51751 CAN: 72:51751 CAPLUS

Patisapu, G., Todor, K., Arvind, D., Yurii, F., Ljudmila, Y., Toumanov, A., Zvetana, Z. & Chiaki, S. (2010). Experimental and Clinical Studies on Rifacinna® - The New Effective Antituberculous Drug (Review). *Recent Patents on Anti-Infective Drug* 

of *Enterobacter aerogenes* by Active Components in Spices, *Journal of Food Protection*

Lignin Biosynthesis, *Annual Review of Plant Physiology & Plant Molecular Biology*,

Activities of Naturally Occurring Compounds against *Mycobacterium avium* subsp. *Paratuberculosis, Applied & Environmental Microbiology*, Vol.4, No.19, pp. 5986-5990,

& David, S.A. (2009) Structure-activity Relationships of Lipopolysaccharide Sequestration in Guanylhydrazone-bearing Lipopolyamines, *Bioorganic & Medicinal* 

Antimycobacterial Agents Using a Minimal Protecting Group Strategy, *Journal of* 

and Evaluation of a Novel Series of Pseudo-Cinnamic Derivatives as Antituberculosis Agents, *Bioorganic & Medicinal Chemistry Letters*, Vol.19, No.2, pp.

*Mycobacterium tuberculosis* or Lipopolysaccharide is Mediated by Nuclear Factors NF-IL6 and NF-kappa β, *Proceedings of the National Academy of Science of the USA*,

#### **1.1 Tuberculosis and immune system**

*Mycobacterium tuberculosis* is a serious threat to humankind, with over 8 million cases of tuberculosis (TB) annually, killing almost 3 millions of people per year around the world (WHO, 2008). Moreover, side effects from first-line anti-TB drugs can cause significant morbidity, and compromise treatment regimens for TB (Yee et al., 2003). Most healthy individuals are able to control TB infection with a vigorous immune response, halting the progression of the disease, but not necessarily eradicating the microorganism (McKinney, 2000).

The bacterium resides within macrophages, allowing them to resist the antimicrobial effector mechanisms of the host (Raupach & Kaufmann 2001). Macrophages constitute one of the main phagocyte cells of the immunological system and they are the first cells involved in an immunological response. Part of their effectiveness is due to the production of nitric oxide (NO), hydrogen peroxide (H2O2) and cytokines, as well as phagocytosis of strange particles (Allavena et al., 2008; Carlos et al., 2004; Keil, 1999). Thus, the elimination of tuberculosis bacillus is involved in the production of these effectors molecules from immune system.

The hydrogen peroxide, generated by macrophages in a reaction catalyzed by an NADPH oxidase, was the first identified effector molecule that mediated mycobacteriocidal effects of mononuclear phagocytes (Lopes et al., 2005; Walker & Lowrie, 1981). In spite of several studies have indicated significant *M. tuberculosis* resistance to oxidative stress *in vitro* and *in vivo,* a recent study showed that H2O2 induced the complete sterilization of the cultures of *M. tuberculosis* by 24 h, after the exposition to 50mM of H2O2 (Volskuill et al., 2011).

Potential Use of *I. suffruticosa* in Treatment of Tuberculosis with Immune System Activation 365

Terpenoids are known as natural insecticides. This class also includes limonoids, limonene and myrcene which plays an important role in the protection of the plants against insects. Some terpenoids have already been tested and have manifested an activity against mycobacterium (Cantrell et al., 2001). Terpenes are composed by basic units of active isoprene isopentenilpirofosfatou, and originate triterpenes and sesquiterpenes previously mentioned in literature as substances with bacterial features (Januario et al., 2002; Pietro et

Essential oils such as geraniol, citronellol, cineole and other genus *Eucalyptus L'Herit*, are recognized as bactericide (Hinou et al. 1989; Leite et al., 1998). The alkaloid obtained from extracts of leaves of *A. Vasic*, vasicine acetate and 2-acetyl benzyl amine showed promising

The endophytic fungi are microorganisms capable of producing potentially bioactive metabolites. These molecules may have hormonal, antibiotic or antitumor activities and other biological functions of enormous industrial and biotechnology interest. Tan and Zou (2001) examined the diversity of metabolites from isolated endophytic fungi and reported the isolation of substances belonging to different structural groups such as steroids, xanthones, phenols, isocoumarins, alkaloids, quinones, furandionas, terpenoids, peptides, cytochalasins and aliphatic compounds. 3-D citosporona, fomopsolida and the acid "*coletótricose*" stand out for their antibacterial activity shown in several studies (Brady et al.

Considering the importance of immunomodulation in the treatment of tuberculosis, the activation of some components of the immune system is a great advantage when it is associated with the bacterial/bacteriostatic activity of the plants. As examples of substances which have immunostimulant and antimicrobial actvities associated, the lectin derived from *Synadenium carinatum* has an important stimulatory activity of granulocytes and NK cells. It is also able to stimulate the expression of TNF-α, IL-1 and iNOS in murine peritoneal macrophages (Cardoso, 2006). This activity is due, partially, to the presence of tannins. This class of secondary metabolites can stimulate the production of IL-1 and TNF-α in macrophages as well as having a significant antimicrobial activity with MIC <100μg/mL

*Scutellaria baicalensis* has also an immunostimulant action combined with antibacterial activity. In addition to the activity against *M. tuberculosis, S. baicalensis* employs a toxic activity against cholera, typhoid, streptococcus, *E. coli, Pneumococcus, Klebsiella pneumoniae, Proteus vulgaris Mycobacterium tuberculosis, Pseudomonas aeruginosa* and *Corynebacterium diphtheria*. This plant induces the production of TNF-α possibly due to the effect of flavonoid wogonin (Chang et al. 1986; Franzblav & Cross 1986; Huang, 1993; Jen et al., 2002). This mediator was also investigated as TNF-α acts in the production of nitric oxide. The results showed that low concentrations of wogonin induce the production of nitric oxide and high

Similarly, *Glycyrrhiza glabra* evinced antimicrobacterial activity in the concentration of 0.5mg/mL. After a phytochemical analysis, the tuberculosis which is toxic to the bacilli at concentrations of 0.029 mg / mL (Gupta et al., 2008) was attributed to the activity against *M. glabridin*. Regarding the immunomodulatory activity, it has been observed that the extract of

*Glycyrrhiza glabra* intensely activated granulocytes and NK cells (Cheell et al., 2010).

against *M. tuberculosis* (Lawal et al., 2011; Miyamoto et al. 1993).

concentrations inhibit the production (Jen et al., 2002).

antimycobacterial activities in several strains of *M. tuberculosis* (Gupta et al., 2010).

al., 2000).

2000; Zou et al., 2000).

NO formed by the action of the inducible form of nitric oxide synthase (iNOS) reacts with oxygen radical forming RNI. NO and related RNI have been reported to possess antimycobacterial activity (Chan et al., 2004; Kwon, 1997). Although the role of NO in human tuberculosis remains unsettled evidence supporting its importance has come from a variety of areas (Nathan & Shiloh, 2000) including the demonstration that human granulomas contain iNOS, endothelial-NOS and nitrotyrosine, a compound whose accumulation indicates production of NO (Nathan, 2002). Additionally, the ability of human alveolar macrophages to kill *M. tuberculosis* is dependent on the activity of iNOS and the human macrophages taken from healthy subjects latently infected with *M. tuberculosis*  produce NO controlling the growth of the bacteria (Yang et al., 2009). The presence of NO within human granulomas could contribute to host resistance since *in vitro* experiments demonstrate direct RNS-mediated bacteriostatic (Firmani & Riley, 2002; Ouellet et al., 2002; Voskuil et al., 2003) and bactericidal activity (Nathan, 2002). Mice deficient in both *phox* and *iNOS* are much more susceptible to *M. tuberculosis* infection than either mutant alone which would indicate that RNS and ROS protect the host in a partially redundant fashion (Shiloh & Nathan, 2000; Volskuill et al., 2011).

TNF-α is a cytokine that plays multiple roles in immune and pathologic responses in tuberculosis, also required for acute infection control (Babbar et al., 2006; Flynn et al., 1995; Palladino et al., 2003). The pro-inflammatory cytokine TNF-α produced by activated macrophages is a central contributor to the immune response against *M. tuberculosis* (Flynn, 1986; Marino et al., 2007 ). The role of TNF is of clinical interest due to the association of anti-inflammatory TNF-α blocking drugs with reactivation of latent TB in humans (Keane et al., 2001; Wintrop,2006). This cytokine has multiple immunological functions during infection with *M. tuberculosis*: It has a direct role in immune cell recruitment via upregulation of endothelial adhesion molecules (Zhou, et al., 2007) facilitating transendothelial migration of immune cells to the site of infection. TNF-α regulates production of chemokines by macrophages (Algood et al., 2006; Roach et al., 2002); chemokines can further induce transendothelial migration and coordinate recruitment of immune cells within the tissues. TNF-α activates macrophages in conjunction with the cytokine IFN-γ (Flesch & Kaufmann 1986; Roock et al., 1986; Carlos et al., 2009) such activated macrophages can kill intracellular mycobacteria. TNF-α can also induce necrotic or apoptotic cell death in macrophages (Laster et al., 1988) that is promoted by TB infection (Keane, et al., 2001).

#### **1.2 Plant with antimycobacterial and immunostimulating activity**

With proposal to stimulate the immune system, some plants can be used in collaboration with the standards drugs for the treatment of tuberculosis. Moreover, there are a lot of plants that can be able to presenting an antimycobacterial activity.

It is possible to assign this effect to the substances contained in its structure which are responsible for protecting the plant structure from aggressive agents in what concerns the active ingredients of plants with an antimicrobial character. Most of these substances are part of the secondary metabolites which consist of substances produced by plants which are not vital and involved in metabolic mechanisms. Flavonoids, tannins, terpenes, alkaloids, phenolic compounds, etc are examples of secondary metabolites. Thereby, many of these compounds protect the vegetal structure against external aggression such as insects, solar radiation, fungi, bacteria and viruses (Heldt, 1997).

NO formed by the action of the inducible form of nitric oxide synthase (iNOS) reacts with oxygen radical forming RNI. NO and related RNI have been reported to possess antimycobacterial activity (Chan et al., 2004; Kwon, 1997). Although the role of NO in human tuberculosis remains unsettled evidence supporting its importance has come from a variety of areas (Nathan & Shiloh, 2000) including the demonstration that human granulomas contain iNOS, endothelial-NOS and nitrotyrosine, a compound whose accumulation indicates production of NO (Nathan, 2002). Additionally, the ability of human alveolar macrophages to kill *M. tuberculosis* is dependent on the activity of iNOS and the human macrophages taken from healthy subjects latently infected with *M. tuberculosis*  produce NO controlling the growth of the bacteria (Yang et al., 2009). The presence of NO within human granulomas could contribute to host resistance since *in vitro* experiments demonstrate direct RNS-mediated bacteriostatic (Firmani & Riley, 2002; Ouellet et al., 2002; Voskuil et al., 2003) and bactericidal activity (Nathan, 2002). Mice deficient in both *phox* and *iNOS* are much more susceptible to *M. tuberculosis* infection than either mutant alone which would indicate that RNS and ROS protect the host in a partially redundant fashion (Shiloh

TNF-α is a cytokine that plays multiple roles in immune and pathologic responses in tuberculosis, also required for acute infection control (Babbar et al., 2006; Flynn et al., 1995; Palladino et al., 2003). The pro-inflammatory cytokine TNF-α produced by activated macrophages is a central contributor to the immune response against *M. tuberculosis* (Flynn, 1986; Marino et al., 2007 ). The role of TNF is of clinical interest due to the association of anti-inflammatory TNF-α blocking drugs with reactivation of latent TB in humans (Keane et al., 2001; Wintrop,2006). This cytokine has multiple immunological functions during infection with *M. tuberculosis*: It has a direct role in immune cell recruitment via upregulation of endothelial adhesion molecules (Zhou, et al., 2007) facilitating transendothelial migration of immune cells to the site of infection. TNF-α regulates production of chemokines by macrophages (Algood et al., 2006; Roach et al., 2002); chemokines can further induce transendothelial migration and coordinate recruitment of immune cells within the tissues. TNF-α activates macrophages in conjunction with the cytokine IFN-γ (Flesch & Kaufmann 1986; Roock et al., 1986; Carlos et al., 2009) such activated macrophages can kill intracellular mycobacteria. TNF-α can also induce necrotic or apoptotic cell death in macrophages (Laster et al., 1988) that is promoted by TB infection (Keane, et al., 2001).

With proposal to stimulate the immune system, some plants can be used in collaboration with the standards drugs for the treatment of tuberculosis. Moreover, there are a lot of

It is possible to assign this effect to the substances contained in its structure which are responsible for protecting the plant structure from aggressive agents in what concerns the active ingredients of plants with an antimicrobial character. Most of these substances are part of the secondary metabolites which consist of substances produced by plants which are not vital and involved in metabolic mechanisms. Flavonoids, tannins, terpenes, alkaloids, phenolic compounds, etc are examples of secondary metabolites. Thereby, many of these compounds protect the vegetal structure against external aggression such as insects, solar

**1.2 Plant with antimycobacterial and immunostimulating activity** 

plants that can be able to presenting an antimycobacterial activity.

radiation, fungi, bacteria and viruses (Heldt, 1997).

& Nathan, 2000; Volskuill et al., 2011).

Terpenoids are known as natural insecticides. This class also includes limonoids, limonene and myrcene which plays an important role in the protection of the plants against insects. Some terpenoids have already been tested and have manifested an activity against mycobacterium (Cantrell et al., 2001). Terpenes are composed by basic units of active isoprene isopentenilpirofosfatou, and originate triterpenes and sesquiterpenes previously mentioned in literature as substances with bacterial features (Januario et al., 2002; Pietro et al., 2000).

Essential oils such as geraniol, citronellol, cineole and other genus *Eucalyptus L'Herit*, are recognized as bactericide (Hinou et al. 1989; Leite et al., 1998). The alkaloid obtained from extracts of leaves of *A. Vasic*, vasicine acetate and 2-acetyl benzyl amine showed promising antimycobacterial activities in several strains of *M. tuberculosis* (Gupta et al., 2010).

The endophytic fungi are microorganisms capable of producing potentially bioactive metabolites. These molecules may have hormonal, antibiotic or antitumor activities and other biological functions of enormous industrial and biotechnology interest. Tan and Zou (2001) examined the diversity of metabolites from isolated endophytic fungi and reported the isolation of substances belonging to different structural groups such as steroids, xanthones, phenols, isocoumarins, alkaloids, quinones, furandionas, terpenoids, peptides, cytochalasins and aliphatic compounds. 3-D citosporona, fomopsolida and the acid "*coletótricose*" stand out for their antibacterial activity shown in several studies (Brady et al. 2000; Zou et al., 2000).

Considering the importance of immunomodulation in the treatment of tuberculosis, the activation of some components of the immune system is a great advantage when it is associated with the bacterial/bacteriostatic activity of the plants. As examples of substances which have immunostimulant and antimicrobial actvities associated, the lectin derived from *Synadenium carinatum* has an important stimulatory activity of granulocytes and NK cells. It is also able to stimulate the expression of TNF-α, IL-1 and iNOS in murine peritoneal macrophages (Cardoso, 2006). This activity is due, partially, to the presence of tannins. This class of secondary metabolites can stimulate the production of IL-1 and TNF-α in macrophages as well as having a significant antimicrobial activity with MIC <100μg/mL against *M. tuberculosis* (Lawal et al., 2011; Miyamoto et al. 1993).

*Scutellaria baicalensis* has also an immunostimulant action combined with antibacterial activity. In addition to the activity against *M. tuberculosis, S. baicalensis* employs a toxic activity against cholera, typhoid, streptococcus, *E. coli, Pneumococcus, Klebsiella pneumoniae, Proteus vulgaris Mycobacterium tuberculosis, Pseudomonas aeruginosa* and *Corynebacterium diphtheria*. This plant induces the production of TNF-α possibly due to the effect of flavonoid wogonin (Chang et al. 1986; Franzblav & Cross 1986; Huang, 1993; Jen et al., 2002). This mediator was also investigated as TNF-α acts in the production of nitric oxide. The results showed that low concentrations of wogonin induce the production of nitric oxide and high concentrations inhibit the production (Jen et al., 2002).

Similarly, *Glycyrrhiza glabra* evinced antimicrobacterial activity in the concentration of 0.5mg/mL. After a phytochemical analysis, the tuberculosis which is toxic to the bacilli at concentrations of 0.029 mg / mL (Gupta et al., 2008) was attributed to the activity against *M. glabridin*. Regarding the immunomodulatory activity, it has been observed that the extract of *Glycyrrhiza glabra* intensely activated granulocytes and NK cells (Cheell et al., 2010).

Potential Use of *I. suffruticosa* in Treatment of Tuberculosis with Immune System Activation 367

(MTT) colorimetric assay was performed as described by Mosmann (1983) . Only cells and culture medium (RPMI- 1640) were used as a control that corresponds to 100% of

Hydrogen peroxide measurement the adherent cells of PEC (2x106cells/mL) was measured using the horseradish peroxidase-dependent phenol red oxidation microassay (Pick & Mizel, 1981). Phorbol myristate acetate (PMA, Sigma, St. Louis, MO) were used as a positive

NO measurement the adherent cells of PEC (5x106cells/mL) was mensured using Griess reagent (Green et al., 1982). E. coli O111B lipopolysaccharide (LPS – 1 μg/mL) solution were

The determination of TNF-α in the supernatants was based in its property to destroy L929 tumoral cell line (mouse tumour fibroblast) Carlos et al. (1994). LPS (1 μg/mL) was used as a

The minimum inhibitory concentration (MIC) of DECE was determined against M. tuberculosis H37Rv (American Type Culture Collection 27294) in Middlebrook 7H9 medium using the Microplate Alamar Blue Assay - MABA (Collins & Franzblau, 1997). For standard test, the MIC value of Isoniazid (Sigma) was determined each time. The acceptable MIC of

The results are expressed as means ± SD of five experiments. One-way ANOVA with Dunnett's post test was performed using GraphPad InStat (San Diego, California, US) with

Actually, TB multiple drug resistance has become a major threat worldwide and thus calls for an urgent search for new and effective treatments for this deadly disease. Naturally occurring compounds as extracts from plants have indicated that inhibitory activity against

The cytotoxicity effect of the extract was evaluated by the determination of MTT (a tetrazolium salt: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Mosmann, 1983) (Table 1). The index of cytotoxicity 50 (IC50) found was in the concentration of

macrophages viability.

used as positive control.

**2.8 Statistical analysis** 

200μg/mL.

positive control.

control.

**2.4 Measurement of H2O2 production** 

**2.5 Measurement of NO production** 

**2.6 Measurement of TNF-α production** 

Isoniazid ranged from 0.015 to 0.05 μg/mL.

the level of significance set at p < 0.05.

**3. Results and discussion** 

**2.7 Determination of antimycobacterial activity by MABA** 

*M. tuberculosis* is widespread in nature (Okunade et al., 2004).

Our research group studied *Indigofera suffruticosa* Miller (Fabacesae) with the aim to collaborate with the discovery of alternatives treatments for tuberculosis*.* Since at this moment there is no new drug generation able to eliminate the bacillus and simultaneously stimulate the immune system we investigated the antimycobacterial and immunological activity of methanol (METH) and dichloromethane (DCM) extracts of *I. suffruticosa*.

*I. suffruticosa* is found in tropical and subtropical areas and is well adapted to growth in semi-arid regions and soils of low fertility (Paiva, 1987). A chemical investigation of extracts of leaves of *I. suffruticosa* in Natural Products Alert (NAPRALERT) and Chemical Abstracts databases has revealed the presence of alkaloids, flavanoids, steroids, proteins, carbohydrates and indigo. Some recent reports have demonstrated the in vitro bioassay activity of plant-derived terpenoids against *M. tuberculosis* (Cantrell et al., 2001; Higuchi et al., 2008). The literature also reports the antimycobacterial activity of many classes of natural products: such as alkanes, phenolics, acetogenic quinines, flavonoids, triterpenes, flavonones and chalcones (Copp, 2003; Higuchi et al., 2008; Pavan et al., 2009). Previous results demonstrated that indigotin alkaloid can enhance macrophage functions and therefore contribute to the host defense against pathogens and tumors (Lopes et al., 2006).

## **2. Materials and methods**

## **2.1 Plant material and samples**

Aerial parts of *I. suffruticosa* were collected in Rubião Junior, Botucatu city, São Paulo State, Brazil, and identified by Prof. Dr. Jorge Yoshio Tamashiro. The immunological assays were performed as soon as the plant was collected. A voucher specimen (HUEC 129598) was deposited at the Herbarium of the Universidade Estadual de Campinas (Unicamp), Campinas, SP, Brazil. Aerial parts of *I. suffruticosa* (1.1 Kg) were dried Activity of the *I. suffruticosa* (40°C), powdered and extracted exhaustively at room temperature with dichloromethane and methanol, successively. Solvents were evaporated at 40°C under reduced pressure to afford the DCM (15.2 g) and METH (30.0 g) extracts. Each extract was first solubilized in dimethyl sulfoxide (DMSO) and then diluted in an appropriated culture medium, RPMI-1640 for the immunological assays and Middlebrook 7H9 for the determination of antimycobacterial activity (62.5-4000 μg/mL).

#### **2.2 Peritoneal macrophages**

Peritoneal macrophages Thioglycollate-elicited PEC were harvested from Swiss mice using 5.0 mL of sterile PBS, pH 7.4. The cells were washed twice by centrifugation at 200 g for 5 min at 4ºC and re-suspended in RPMI-1640 medium (Sigma). The adherent cells were obtained by incubation for 1 h at 37ºC in an atmosphere of air/CO2 (95:5, v/v) (Forma Scientific) and, incubated with LPS or RPMI-1640 medium. This protocol was in agree with the regulations of Research Ethics Committee (# 01/2005).

#### **2.3 MTT assay for cell viability**

PEC (5x106 cells/mL) was re-suspended in RPMI- 1640 medium. The suspension (100 μL) and the extracts (100 μL) were added to each well of a 96-well tissue culture plate and they were incubated for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric assay was performed as described by Mosmann (1983) . Only cells and culture medium (RPMI- 1640) were used as a control that corresponds to 100% of macrophages viability.

## **2.4 Measurement of H2O2 production**

366 Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance

Our research group studied *Indigofera suffruticosa* Miller (Fabacesae) with the aim to collaborate with the discovery of alternatives treatments for tuberculosis*.* Since at this moment there is no new drug generation able to eliminate the bacillus and simultaneously stimulate the immune system we investigated the antimycobacterial and immunological

*I. suffruticosa* is found in tropical and subtropical areas and is well adapted to growth in semi-arid regions and soils of low fertility (Paiva, 1987). A chemical investigation of extracts of leaves of *I. suffruticosa* in Natural Products Alert (NAPRALERT) and Chemical Abstracts databases has revealed the presence of alkaloids, flavanoids, steroids, proteins, carbohydrates and indigo. Some recent reports have demonstrated the in vitro bioassay activity of plant-derived terpenoids against *M. tuberculosis* (Cantrell et al., 2001; Higuchi et al., 2008). The literature also reports the antimycobacterial activity of many classes of natural products: such as alkanes, phenolics, acetogenic quinines, flavonoids, triterpenes, flavonones and chalcones (Copp, 2003; Higuchi et al., 2008; Pavan et al., 2009). Previous results demonstrated that indigotin alkaloid can enhance macrophage functions and therefore contribute to the host defense against pathogens and tumors (Lopes et al., 2006).

Aerial parts of *I. suffruticosa* were collected in Rubião Junior, Botucatu city, São Paulo State, Brazil, and identified by Prof. Dr. Jorge Yoshio Tamashiro. The immunological assays were performed as soon as the plant was collected. A voucher specimen (HUEC 129598) was deposited at the Herbarium of the Universidade Estadual de Campinas (Unicamp), Campinas, SP, Brazil. Aerial parts of *I. suffruticosa* (1.1 Kg) were dried Activity of the *I. suffruticosa* (40°C), powdered and extracted exhaustively at room temperature with dichloromethane and methanol, successively. Solvents were evaporated at 40°C under reduced pressure to afford the DCM (15.2 g) and METH (30.0 g) extracts. Each extract was first solubilized in dimethyl sulfoxide (DMSO) and then diluted in an appropriated culture medium, RPMI-1640 for the immunological assays and Middlebrook 7H9 for the

Peritoneal macrophages Thioglycollate-elicited PEC were harvested from Swiss mice using 5.0 mL of sterile PBS, pH 7.4. The cells were washed twice by centrifugation at 200 g for 5 min at 4ºC and re-suspended in RPMI-1640 medium (Sigma). The adherent cells were obtained by incubation for 1 h at 37ºC in an atmosphere of air/CO2 (95:5, v/v) (Forma Scientific) and, incubated with LPS or RPMI-1640 medium. This protocol was in agree with

PEC (5x106 cells/mL) was re-suspended in RPMI- 1640 medium. The suspension (100 μL) and the extracts (100 μL) were added to each well of a 96-well tissue culture plate and they were incubated for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide

determination of antimycobacterial activity (62.5-4000 μg/mL).

the regulations of Research Ethics Committee (# 01/2005).

activity of methanol (METH) and dichloromethane (DCM) extracts of *I. suffruticosa*.

**2. Materials and methods 2.1 Plant material and samples** 

**2.2 Peritoneal macrophages** 

**2.3 MTT assay for cell viability** 

Hydrogen peroxide measurement the adherent cells of PEC (2x106cells/mL) was measured using the horseradish peroxidase-dependent phenol red oxidation microassay (Pick & Mizel, 1981). Phorbol myristate acetate (PMA, Sigma, St. Louis, MO) were used as a positive control.

## **2.5 Measurement of NO production**

NO measurement the adherent cells of PEC (5x106cells/mL) was mensured using Griess reagent (Green et al., 1982). E. coli O111B lipopolysaccharide (LPS – 1 μg/mL) solution were used as positive control.

## **2.6 Measurement of TNF-α production**

The determination of TNF-α in the supernatants was based in its property to destroy L929 tumoral cell line (mouse tumour fibroblast) Carlos et al. (1994). LPS (1 μg/mL) was used as a positive control.

## **2.7 Determination of antimycobacterial activity by MABA**

The minimum inhibitory concentration (MIC) of DECE was determined against M. tuberculosis H37Rv (American Type Culture Collection 27294) in Middlebrook 7H9 medium using the Microplate Alamar Blue Assay - MABA (Collins & Franzblau, 1997). For standard test, the MIC value of Isoniazid (Sigma) was determined each time. The acceptable MIC of Isoniazid ranged from 0.015 to 0.05 μg/mL.

#### **2.8 Statistical analysis**

The results are expressed as means ± SD of five experiments. One-way ANOVA with Dunnett's post test was performed using GraphPad InStat (San Diego, California, US) with the level of significance set at p < 0.05.

## **3. Results and discussion**

Actually, TB multiple drug resistance has become a major threat worldwide and thus calls for an urgent search for new and effective treatments for this deadly disease. Naturally occurring compounds as extracts from plants have indicated that inhibitory activity against *M. tuberculosis* is widespread in nature (Okunade et al., 2004).

The cytotoxicity effect of the extract was evaluated by the determination of MTT (a tetrazolium salt: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Mosmann, 1983) (Table 1). The index of cytotoxicity 50 (IC50) found was in the concentration of 200μg/mL.

Potential Use of *I. suffruticosa* in Treatment of Tuberculosis with Immune System Activation 369

*I. suffruticosa* did not produce significant amounts of H2O2 when compared with control negative (p>0,05), METH (0,59 nmols/2.105cells) and DCM (3,3 nmols/2.105cells) (Fig. 3). This fact can be justified by the presence of tannins, such as gallic acid in extracts of *I. suffruticosa*. This class of substances has been showed an antioxidant potential being responsible for the scavenger of free radicals such as hydrogen peroxide. (Akira et al.,

Thus, this screening suggests that both extracts of the *I. suffruticosa* promoted the activation of the macrophages. The significant production of studied mediators (NO and TNF-α) by activated macrophages in presence of *I. suffruticosa* is very important, since macrophages produces several effector molecules that can enhance or restore the ability of the innate

Fig. 2. Induction of tumor necrosis factor-α.

Fig. 3. Induction of hydrogen peroxide.

immune system to fight against TB infection.

2002).


Table 1. Effect of methanolic and dichloromethane extracts of *Indigofera suffruticosa* on the viability of peritoneal macrophages. a- µg/mL

This study evaluated the antimycobacterial activity the extracts of *I. suffruticosa* and its action in innate immune system. The antimycobacterial activity of METH and DCM is presented in table 2. Gu et al. (2004) considered active plant extracts with MIC value ≤128 μg/mL. Thus we considered a promising result the MIC of 125 μg/mL found in METH crude extract.


Table 2. Minimal inhibitory concentration (MIC) in presence of methanolic and dichloromethane extracts of the plant *Indogofera suffruticosa.* aStandard drug, bg/mL.

The extracts presented a high production of nitric oxide with statistically significant values compared to the negative control (p<0,001). The amount of NO produced by the METH extract (105, 99 μmol/5.105 cells) was larger than the production of DCM extract (58, 9 μmol/5.105 cells) (Fig. 1).

Fig. 1. Induction of nitric oxide.

The results regarding of TNF-α confirmed a significant production of this cytokine at levels near the positive control (252,7 and 234,6 units/mL, METH and DCM extracts, respectively) confirming a correlation between the synthesis of TNF-α and NO (Fig. 3) (Bogdan et al., 1991; Carli et al., 2009).

Fig. 2. Induction of tumor necrosis factor-α.

Table 1. Effect of methanolic and dichloromethane extracts of *Indigofera suffruticosa* on the

 Methanolic Dichloromethane Isoniazida MIC 125b 1000b 0,05b

The extracts presented a high production of nitric oxide with statistically significant values compared to the negative control (p<0,001). The amount of NO produced by the METH extract (105, 99 μmol/5.105 cells) was larger than the production of DCM extract (58, 9

The results regarding of TNF-α confirmed a significant production of this cytokine at levels near the positive control (252,7 and 234,6 units/mL, METH and DCM extracts, respectively) confirming a correlation between the synthesis of TNF-α and NO (Fig. 3) (Bogdan et al.,

Table 2. Minimal inhibitory concentration (MIC) in presence of methanolic and dichloromethane extracts of the plant *Indogofera suffruticosa.* aStandard drug, bg/mL.

This study evaluated the antimycobacterial activity the extracts of *I. suffruticosa* and its action in innate immune system. The antimycobacterial activity of METH and DCM is presented in table 2. Gu et al. (2004) considered active plant extracts with MIC value ≤128 μg/mL. Thus we considered a promising result the MIC of 125 μg/mL found in METH

15,73 ± 1,90 30,29 ± 4,32 55, 29 ± 1,36 71, 57 ± 1, 82

Extracts Viability (%) Control 100 ± 0,00

viability of peritoneal macrophages. a- µg/mL

600a 400a 200a 100a

crude extract.

μmol/5.105 cells) (Fig. 1).

Fig. 1. Induction of nitric oxide.

1991; Carli et al., 2009).

*I. suffruticosa* did not produce significant amounts of H2O2 when compared with control negative (p>0,05), METH (0,59 nmols/2.105cells) and DCM (3,3 nmols/2.105cells) (Fig. 3). This fact can be justified by the presence of tannins, such as gallic acid in extracts of *I. suffruticosa*. This class of substances has been showed an antioxidant potential being responsible for the scavenger of free radicals such as hydrogen peroxide. (Akira et al., 2002).

Fig. 3. Induction of hydrogen peroxide.

Thus, this screening suggests that both extracts of the *I. suffruticosa* promoted the activation of the macrophages. The significant production of studied mediators (NO and TNF-α) by activated macrophages in presence of *I. suffruticosa* is very important, since macrophages produces several effector molecules that can enhance or restore the ability of the innate immune system to fight against TB infection.

Potential Use of *I. suffruticosa* in Treatment of Tuberculosis with Immune System Activation 371

Algood, H. M.; Lin, P. L. & Flynn,J. L. (2005). Tumor necrosis factor and chemokine

*Clinical Infectious Diseases,* Vol. 41, Suppl. 3, pp. S189–S193, ISSN1537-6591. Allavena, P.; Sica, A.; Solinas, G.; Porta, C. & Mantovani, A. (2008). The inflammatory

Barrar, N. R. A. & Casero, J. (2006). Tumor necrosis factor-alpha increases reactive oxygen

Bean, A.; Roach, D.; Briscoe, H.; France, M.; Korner, H.; Sedgwick, J. & Britton, W. (1999).

Bekker, L. G.; Freeman, S.; Murray, P. J.; Ryffel, B. & Kaplan G. (2001). TNF-alpha controls

Bogdan, C.; Vodovotz, Y. & Nathan, C. (1991). Macrophage deactivation by interleukin 10. *The Journal of Experimental Medicine*, Vol. 174, No. 6, pp.1549-1555, ISSN1540-9538. Brady, S.F. & Clardy, J. (2000). CR377, a new pentakide antifungal agent isolated from an

Cantrell, C.L.; Franzblau, S.G. & Fischer, N.H. (2001). Antimycobacterialplant terpenoids.

Cardoso, S.R.A. (2006). Efeitos *in vivo* e *in vitro* da lectina de *Synadenium carinatum* sobre a infecção murina por *Leishmania amazonensis,*Uberlandia, Minas Gerais, Brasil. Carli, C. B. A.; Matos, D. C.; Lopes, F. C. M.; Maia, D. C. G.; Dias, M. B.; Sannomiya, M.;

Carlos, I.Z.; Sgarbi, D.B.G.; Angluster, J.; Alviano, C.S. & Silva, C.L. (1994). Disturbances in

Carlos, I. Z.; Monnazzi, L. G. S.; Falcão, D. P.& De Medeiros, B. M. M. (2004). TNF , H2O2

Carlos, I. Z. ; Sassá, M.F. ; Placeres, M.C.P. ; Maia, D.C.G. (2009). Current research on the

Chan, E.D.; Chan, J. & Schluger, N. W. (2001). What is the role of nitric oxide in murine and

*Molecular Biology*, Vol. 25, No. 5, pp. 606-612, ISSN1535-4989.

*Immunology,* Vol.166, No. 11, pp. 6728–6734, ISSN1550-6606.

*Planta Medica,* Vol. 67, No. 1, pp1-10, ISSN1439-0221 .

*Naturforschung*, Vol. 67, No. 1, pp. 32-36, ISSN0932-0784.

ISSN1040-8428.

23, pp. 11125-11130, ISSN1538-7445 .

3504–3511, ISSN1550-6606.

ISSN1520-6025 .

ISSN1469-0691.

10, ISSN1573-0832.

interactions in the formation and maintenance of granulomas in tuberculosis.

microenvironment in tumor progression: the role of tumor-associated macrophages. *Critical Reviews in Oncology/Hematology*. Vol. 66, No. 1, pp. 1-9,

species by inducing spermine oxidase in human lung epithelial cells: a potential mechanism for inflammation-induced carcinogenesis. *Cancer Research*, Vol. 66, No.

Structural deficiencies in granuloma formation in TNF gene targeted mice underlie the heightened susceptibility to aerosol *Mycobacterium tuberculosis* infection, which is not compensated for by lymphotoxin. *Journal of Immunology*, Vol.162, No. 6, pp.

intracellular mycobacterial growth by both inducible nitric oxide synthasedependent and inducible nitric oxide synthase-independent pathways. *Journal of* 

endophytic fungus. *Journal of Naural Products*, Vol. 63, No. 10, pp.1447-1448,

Rodrigues, C. M.; Andreo, M. A.; Vilegas, W.; Colombo, L. L. & Carlos, I. Z. (2009). Isolated Flavonoids against Mammary Tumour Cells LM2. *Zeitschrift für* 

the production of interleukin-1-necrosis and tumor necrosis factor in disseminated murine sporotrichosis. *Mycopathologia*, Vol. 127, No.3 , pp. 189-194, ISSN1573-0832.

and NO response of peritoneal macrophages to *Yersinia enterocolitica* O:3 derivatives. *Clinical Microbiology and Infection*, Vol. 174, No. 6, pp. 207-212,

immune response to experimental sporothrichosis. *Mycopathologia*, Vol. 168, pp. 1-

human host defense against tuberculosis? *American Journal of Respiratory Cell and* 

Nitric oxide (NO) formed by the action of the inducible form of nitric oxide synthase (iNOS) reacts with oxygen radical forming RNI. NO and related RNI have been reported to possess antimycobacterial activity (Kwon, 1997). Phagocytes kill intracellular organisms during an initial oxidative phase dependent on NADPH oxidase followed by a prolonged nitrosative phase during which bacterial growth is inhibited by iNOS (Nathan & Shiloh, 2000).There are several potential mechanisms that can explain how NO may affect microbial life-cycle. NO and other RNI can modify bacterial DNA, protein and lipids at both the microbial surface and intracellularly. They can alter cytokine production and induce or prevent apoptosis of host cells by controlling caspase activity (Raupach & Kaufmann, 2001).

*M. tuberculosis* strongly induces the release of several cytokines during infection. Tumor necrosis factor-α (TNF-α) is a cytokine that plays multiple roles in immune and pathologic responses in tuberculosis, also required for acute infection control (Flynn et al., 1995). It plays a major role in the recruitment of inflammatory cells to the site of infection and in the formation and maintenance of granulomas (Gaemperli et al., 2006). This cytokine is necessary for optimal co-ordination of both the differentiation of specific T cells to secrete the appropriate T helper 1 cytokines and the development of granulomas in which activated macrophages restrict mycobacterial growth (Ehlers, 2003). TNF-α is required for control of latent TB and it is also a key element for activating macrophages to produce iNOS and thus in maintaining the pathway for generating NO and preventing reactivation of the disease (Adams et al., 1995).

#### **4. Conclusion**

We suggest that the extract may be an important bactericidal source against *M. tuberculosis* once the same has natural origin and do not present the toxic effects provoked by the drugs current used in the treatment of tuberculosis. Moreover, a possible association with traditional drugs can be suggested considering that the most of standard drugs do not present the same simultaneous effect microbiological and immunological of the extract here tested. These results described here highlight the importance of conducting an in-depth study of the species of the Brazilian biome, and show the great potential of it's biodiversity in the treatment of infection diseases.

#### **5. Acknowledgements**

The authors thank FAPESP for its financial support.

#### **6. References**


Nitric oxide (NO) formed by the action of the inducible form of nitric oxide synthase (iNOS) reacts with oxygen radical forming RNI. NO and related RNI have been reported to possess antimycobacterial activity (Kwon, 1997). Phagocytes kill intracellular organisms during an initial oxidative phase dependent on NADPH oxidase followed by a prolonged nitrosative phase during which bacterial growth is inhibited by iNOS (Nathan & Shiloh, 2000).There are several potential mechanisms that can explain how NO may affect microbial life-cycle. NO and other RNI can modify bacterial DNA, protein and lipids at both the microbial surface and intracellularly. They can alter cytokine production and induce or prevent apoptosis of

*M. tuberculosis* strongly induces the release of several cytokines during infection. Tumor necrosis factor-α (TNF-α) is a cytokine that plays multiple roles in immune and pathologic responses in tuberculosis, also required for acute infection control (Flynn et al., 1995). It plays a major role in the recruitment of inflammatory cells to the site of infection and in the formation and maintenance of granulomas (Gaemperli et al., 2006). This cytokine is necessary for optimal co-ordination of both the differentiation of specific T cells to secrete the appropriate T helper 1 cytokines and the development of granulomas in which activated macrophages restrict mycobacterial growth (Ehlers, 2003). TNF-α is required for control of latent TB and it is also a key element for activating macrophages to produce iNOS and thus in maintaining the pathway for generating NO and preventing reactivation of the disease

We suggest that the extract may be an important bactericidal source against *M. tuberculosis* once the same has natural origin and do not present the toxic effects provoked by the drugs current used in the treatment of tuberculosis. Moreover, a possible association with traditional drugs can be suggested considering that the most of standard drugs do not present the same simultaneous effect microbiological and immunological of the extract here tested. These results described here highlight the importance of conducting an in-depth study of the species of the Brazilian biome, and show the great potential of it's biodiversity

Adams, L.B.; Mason, C.M.; Kolls, J.K.; Scollard, D.; Krahenbuhl, J.L. & Nelson, S. (1995).

Akira, N.; Yuto, U.; Kunihiko, T. &, Keisuke, M. (2002). Effect of tannin compounds on

Exacerbation of acute and chronic murine tuberculosis by administration of a tumor necrosis factor receptor- expressing adenovirus. *Journal of Infectious Diseases*,

uranium-hydrogen peroxide system. *Nippon Kagakkai Koen Yokoshu*, Vol. 81, No. 1,

host cells by controlling caspase activity (Raupach & Kaufmann, 2001).

(Adams et al., 1995).

in the treatment of infection diseases.

pp.634, ISSN0285-7626.

The authors thank FAPESP for its financial support.

Vol. 171, No.2, pp. 400-405, ISSN 00221899.

**5. Acknowledgements** 

**6. References** 

**4. Conclusion** 


Potential Use of *I. suffruticosa* in Treatment of Tuberculosis with Immune System Activation 373

Gupta, R.; Thakur, B.; Singh, P.; Singh, H.B.; Sharma, V.D.; Katoch, V.M. & Chauhan, S.V.S.

Heldt, H. W. (1997). Pflanzenbiochemie. Heidelberg: Spektrum Akademischer Verlag, ISBN

Higuchi, C.T.; Pavan, F.R.; Sannomiya, M.; Vilegas, W. Leite, S.R.A.; Sacramento, L.V.S.;

*Byrsonima crassa*. *Quimica Nova*, Vol. 31, No. 7, pp.1719-1721, ISSN0100-4042. Higuchi, C.T.; Sannomiya, M.; Pavan, F.R.; Leite, S.R.A.; Sato, D.N.; Franzblau, S.G.;

Hinou, J.B.; Harvala, C.E.; Hinou, E.B. (1989). Antimicrobial activity screening of 32

Huang, K.C. (1993). *The Pharmacology of Chinese herbs*, CRC Press, ISBN 9780849316654,

Keane, J., Gershon, S.; Wise, R. P.; Mirabile-Levens, E.; Kasznica, J.; Schwieterman, W. D.;

Keane, J.; Balcewicz-Sablinska, M. K; Remold, H. G.; Chupp, G. L ; Meek, B. B.; Fenton, M. J.

Keil, D.E.; Luebke, R.W. & Pruett, B.S. (1999). Differences in the effects of dexamethasone on

Kwon, O.J. (1997). The role of nitric oxide in the immune response of tuberculosis. *Journal of* 

Laster, S. M.; Wood, J. G. & Gooding, L. R. (1988). Tumor necrosis factor can induce both

Lawal, T.O.; Adeniyi, B. A.; Wan, B.; Franzblau, S.G. & Mahyad, G.B. (2011). *In-vitro*

Leite, C.Q.F.; Moreira, R.R.D. & Jorge-Neto, J.(1998). Action of Eucaliptus oils against *Mycobactrieum avium* . *Fitoterapia,*Vol.1, No.2 , pp.282-3, ISSN1808-4532. Lopes, F. C. M.; Benzatti, F. P.; Jordão-Junior, C. M.; Moreira, R. R. D. & Carlos, I. Z. (2005).

*Pharmaceutical Sciences* , Vol.41, No.3 , pp. 401-405, ISSN 15169332.

*Korean Medical Science*, Vol. 12, No. 6, pp. 481-487, ISSN1598-6357 .

131, pp. 809-813, ISSN0971-5916 .

978-3-8274-1800-5, Berlim, Germany.

*Medicine*, doi:10.1093/ecam/nen077

No. 3 , pp.157-166, ISSN 0192-0561.

pp.2629–2634, ISSN1550-6606.

pp.265-272, ISSN1674-8301 .

Vol. 345, No. 15, pp. 1098–1104, ISSN1533-4406.

ISSN0031-7144 .

Kentucky, USA.

ISSN1098-5522.

(2010). Anti-tuberculosis activity of selected medicinal plants against multi-drug resistant *Mycobacterium tuberculosis* isolates. *Indian Journal of Medical Research*, Vol.

Sato, D.N. & Leite, C.Q.F. (2008). Triterpenes and antitubercular activity of

Sacramento, L.V.S.; Vilegas, W. & Leite, C.Q.F. (2008). *Byrsonima fagifolia* Niedenzu apolar compound with antitubercular activity. *Complementary and Alternative* 

commos constituents of essential oils. *Pharmazie*, Vol. 44, No. 4, pp.302-303,

Siegel, J. N. & Braun, M. M. (2001). Tuberculosis associated with infliximab, a tumor necrosis factor- alpha neutralizing agent. *New England Journal of Medicine*,

& Kornfeld, H. (1997). Infection by *Mycobacterium tuberculosis* promotes human alveolar macrophage apoptosis. *Infection & Immunity,* Vol. 65, No. 1 , pp. 298–304,

macrophage nitrite production: Dependence on exposure regimem (*in vivo* or *in vitro*) and activation stimuli. *International Journal of Immunopharmacology*, Vol. 17,

apoptic and necrotic forms of cell lysis. *Journal of Immunology,* Vol. 141, No. 8,

susceptibility of mycobacterium tuberculosis to extracts of *Uvaria afzelli Scott elliot* and *Tetracera alnifolia* willd african. *Journal of Biomedical Research*, Vol. 14, No. 1,

Effect of the essential oil of *Achillea millefolium L.* in the production of hydrogen peroxide and tumor necrosis factor-α in murine macrophages. *Brazilian J of* 


Chan, J.; Xing, Y.; Magliozzo, R. S.; & Bloom, B. R. (1992). Killing of virulent *Mycobacterium* 

Chang HM, But PPH. (1986). *Pharmacology and Applications of Chinese Materia Medica* vol 1.

Cheel, J.; Antwerpen, P.V.; Tumová, L.; Onofre, G.; Vokurková, D.; Zouaoui-Boudjeltia, K.;

Copp, B.R. (2003). Antimycobacterial natural products. *Natural Product Reports*, Vol.20, No.

Ehlers, S. (2003). Role of tumour necrosis factor (TNF) in host defence against tuberculosis:

Firmani, M. A. & Riley, L. W. (2002). Reactive nitrogen intermediates have a bacteriostatic

Flesch, I. & Kaufmann, S.(1987). Mycobacterial growth inhibition by interferon- \_-activated

Flesch, I. E. & Kaufmann, S. H. (1990). Activation of tuberculostatic macrophage functions

Flynn, J.L.; Goldstein, M.M.; Chan, J.; Triebold, K.J.; Pfeffer, K.; Lowenstein, C.J.; Schreiber,

Franzblav SG, Cross C*J.*(1986). Comparative in vitro antimicrobial activity of Chinese

Gaemperli, A. Hauser, T. & Speck, R.F. (2006). Risk of infection during treatment with tumor

Green, L.C.; Wagner, D.A.; Glogowski, J.; Skipper, P.L.; Wishnok, J.S. & Tannenbaum, S.R.

Gu, J.Q.; Wang, Y.; Franzblau, S.G.; Montenegro, G.; Yang, D. & Timmermann, B.N. (2004).

*Biochemistry*, Vol. 126, No. 1, pp.131-138, ISSN1096-0309 .

doi:10.1084/jem.175.4.1111 .

World Scientific Inc., ISBN 9810236948, Singapore.

41, No. , pp. 1004-1009, ISSN1098-6596.

6, pp. 535-557 doi:10.1039/B212154A.

ISSN1550-6606.

7573.

Vol.62, Suppl. 2, pp. ii37-ii42, ISSN1468-2060.

58, No. 8, pp. 2675–2677, ISSN1098-5522 .

6, pp. 561-572, ISSN10747-613.

31, doi:10.1007/s00393-005-0018-z.

pp.509-514, ISSN1439-0221.

No. 9, pp. 3162–3166, doi:10.1128/JCM.40.9.3162-3166.2002.

*tuberculosis* by reactive nitrogen intermediates produced by activated murine macrophages. *Journal of Experimental Medicine*., Vol. 175, No. 4, pp. 1111–1122,

Vanhaeverbeek, M. & Nève, J. (2010) Free radical-scavenging, antioxidant and immunostimulating effects of a licorice infusion (*Glycyrrhiza glabra L*.). Food Chemistry*,* Vol. 122, No. 3, pp. 508-517, doi:10.1016/j.foodchem.2010.02.060. Collins, L.S. & 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, Vol.

implications for immunotherapies targeting TNF. *Annals of the Rheumatic Diseases*,

effect on *Mycobacterium tuberculosis* in vitro. *Journal of Clinical Microbiology*, Vol. 40,

bone marrow macrophages and differential susceptibility among strains of *Mycobacterium tuberculosis. Journal of Immunology,* Vol. 138, No. 12, pp. 4408–4413,

by interferon, interleukin-4, and tumor necrosis factor. *Infection & Immunity,* Vol.

R.; Mak, T.W. &, Bloom, B.R. (1995). Tumor necrosis factor-α is required in the protective immune response against M. tuberculosis in mice. *Immunity*, Vol. 2, No.

medicinal herbs. *Journal of Ethnopharmacology,*Vol. 15, No. 3, pp. 279-288, ISSN1872-

necrosis factor-alpha inhibitors. *Zeitschrift fur Rheumatologie*, Vol. 65, No. 1, pp. 24-

(1982). Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. *Analytical* 

Antitubercular constituents of *Valeriana laxiflora*. *Planta Medica*, Vol. 70, No. 6,


Potential Use of *I. suffruticosa* in Treatment of Tuberculosis with Immune System Activation 375

Palladino, M. A.; Bahjat, F. R.; Theodorakis, E. A. & Moldawer, L. L. (2003). Anti TNF-α

Pavan, F.R.; Leite, C.Q.F.; Coelho, R.G.; Coutinho, I.D.; Honda, N.K.; Cardoso, C.A.L.;

Pick, E & Keisare, Y. (1980). Superoxide anion and hydrogen peroxide production by

Raupach, B. & Kaufmann, S.H.E. (2001). Immune responses to intracellular bacteria. *Current* 

Roach, D.; Bean, A.; Demangel, C.; France, M.; Briscoe, H. & Britton, W. (2002). TNF

Rook, G. A.; Steele, J.; Ainsworth, M. & Champion, B. R. (1986). Activation of macrophages

Stein, J. & Nombela-Arrieta, C. (2005). Chemokine control of lymphocyte trafficking: a general overview. *Immunology,* Vol. 116, No. 1, pp 1–12, ISSN1365-2567 . Tan, R.X. & Zou, W.X. (2001). Endophytes: a rich search of functional metabólites. *Natural* 

Van Buul, J. & Hordijk, P. (2004). Signaling in leukocyte transendothelial migration. *Arterioscler. Thromb. Vasc. Biol.* Vol. 24, No. 5, pp. 824–833, ISSN1524-4636. Voskuil, M. I.; Schnappinger, D.; Visconti, K. C.; Harrell, M. I.; Dolganov, G. M.; Sherman, D.

Voskuil1, M. I.; Bartek, I.L.; Visconti, K & Schoolnik, G. K. (2011).The response of

Walker, L. & Lowrie, D.B. (1981). Killing of *Mycobacterium microti* by immunologically activated macrophages. Nature, Vol. 293, pp. 69-70, doi:10.1038/293069a0. Who.int [http://www.who.int/tb/publications/global\_report/2008/chapter\_1/en/index3.

macrophages. *Immunology* Vol. 59, No. 3, pp. 333–338, ISSN1365-2567. Shiloh, M. U. & Nathan, C. F. (2000). Reactive nitrogen intermediates and the pathogenesis

*Product Reports,* Vol. 18, No. 4, pp.448-459, ISSN1460-4752 .

*Microbiology*, Vol. 2, No. 105, doi: 10.3389/fmicb.2011.00105.

198, no. 5, pp. 705–713, ISSN1540-9538.

from: http://www.who.int.

*opinion in mmunology,* Vol.13, No. 4, pp. 417-428, ISSN0952-7915.

stimuli. *Cellular Immunology*, Vol. 59, No. 2, pp.301-3118, ISSN1090-2163. Pietro, R.C.L.R.; Kashima, S.; Sato, D. N.; Januário, A.H. & França, S.C. (2000). *In vitro* 

746, ISSN1474-1784.

pp.335-338, ISSN1618-095X.

4620–4627, ISSN1550-6606 .

42, ISSN1879-0364.

Vol. 32, No. 5, pp. 1222-1226, ISSN0100-4042.

therapies: the next generation. *Nature Reviews Drug Discovery*, Vol. 2, No. 9, pp.736-

Vilegas, W.; Leite, S.R.A. & Sato, D.N. (2009). Evaluation of anti-*Mycobacterium tuberculosis* activity of *Campomanesia adamantium* (MYRTACEAE). *Quimica Nova*,

chemically elicited peritoneal macrophages induction by multiple nonphagocytic

antimycobacterial activities of *Physalis angulate* L. *Phytomedicine*, Vol.7, No. 4,

regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection. *Journal of Immunology,*Vol. 168, No. 9, pp.

to inhibit proliferation of *Mycobacterium tuberculosis*: comparison of the effects of recombinant \_-interferon on human monocytes and murine peritoneal

of *Salmonella* and *mycobacteria*. *Current Opinion in Microbiology*, Vol. 3, No. 1, pp. 35–

R.; & Schoolnik, G. K. (2003). Inhibition of respiration by nitric oxide induces a *Mycobacterium tuberculosis* dormancy program. *Journal of Experimental Medicine*, Vol.

*Mycobacterium tuberculosis* to reactive oxygen and nitrogen species. *Frontier in* 

html]. World Health Organization [updated 2008; cited 2008 December 3, Available


Lopes F C M, Calvo T R, Vilegas W, Carlos I.(2006) Indigotin alkaloid obtained from

McKinney, J.D. (2000). *In vivo* veritas: The search for TB drug targets goes live. Nature

Miyamoto, K.; Nomura, M.; Sasacura, M.; Matsui, E.; Koshiura, R.; Murayama, T.;

Mohan, V. P.; Scanga, C. A.; Yu, K.; Scott, H. M.; Tanaka, K. E.; Tsang, E.; Tsai, M. M.; Flynn,

Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application

NAPRALER In: Natural Products Alert, Illinois University, Chicago. 2003. Accessed at http://www.uic.edu/phamacy/depts/PCRPS/ NAPRALERT.htm. Nathan, C. & Shiloh, M. U. (2000). Reactive oxygen and nitrogen intermediates in the

Nathan, C. & Shiloh, M.U. (2000). Reactive oxygen and nitrogen intermediates in the

Nathan, C. (2002). Inducible nitric oxide synthase in the tuberculous human lung. *American* 

Okunade, A.L.; Elvin-Lewis, M.P. & Lewis, W.H. (2004). Natural antimycobacterial

Ouellet, H., Ouellet, Y., Richard, C., Labarre, M., Wittenberg, B., Wittenberg, J., and Guertin,

Paiva, M.A.S.; Barbosa, A.C.D. & Alves, H.L.J. (1987). *Indigofera suffruticosa* Mill

Botânica. São Paulo, Brasil: Sociedade Nacional de Botânica.

No. 10, pp. 1909–1924, ISSN 1553-7358.

No. 16 , pp. 55-63, ISSN0022-1759 .

Vol. 99, No. 9, pp.5902–5907, ISSN1540-9538.

8848, ISSN1091-6490.

8848, ISSN1091-6490 .

ISSN1535-4970.

9422.

Medicine, Vol. 6, pp.1330-1333, doi:10.1038/82142.

Research ,Vol. 84, No.1 , pp.99-103, ISSN0910-5050 .

*Immunity,*Vol.69, No. 3, pp. 1847–1855, ISSN1098-5522.

Indigofera suffruticosa Miller induces NO and TNF-α production by murine macrophages. Proceedings of the XXXI Meeting of the Brazilian Society of Immunology- Signaling in the Immune System, Buzios, RJ, Brazil, October 2006. Marino, S., Sud, D.; Plessner, H.; Lin, P. L.; Chan, J.; Flynn, J. L. & Kirschner, D. E. (2007).

Differences in reactivation of tuberculosis induced from anti-TNF treatments are based on bioavailability in granulomatous tissue. *PLoS Computational Biology,*Vol.3,

Furucawa, T.; Hatano T, Yoshida, T. & Okuda, T. (1993). Antitumor activity of oenothein B, a unique macrocyclic *ellagitannin*. Japanese Journal of Cancer

J. L. & Chan, J. (2001). Effects of tumor necrosis factor on host immune response in chronic persistent tuberculosis: possible role for limiting pathology. *Infection and* 

to proliferation and cytotoxicity assays. Journal of Immunology Methods, Vol. 65,

relationship between mammalian hosts and microbial pathogens. *Proceedings of the National Academy of Sciences of the United States of America*, Vol.97, No. 16, pp. 8841–

relationship between mammalian hosts and microbial pathogens. *Proceedings of the National Academy of Sciences of the United States of America*, Vol. 97, No. 16 , pp. 8841-

*Journal of Respiratory and Critical Care Medicine*,Vol. 166, No. 2, pp.130–131,

metabolites: current status. *Phytochemistry*, Vol. 65, No. 8 , pp. 1017-1032, ISSN0031-

M. (2002). Truncated hemoglobin HbN protects *Mycobacterium bovis* from nitric oxide. *Proceedings of the National Academy of Sciences of the United States of America* ,

(Leguminosae) com potencial forrageiro em uma região de Caatinga no Semi-árido de Pernambuco. (Alagoinha). Proceedings of the XXXVIII Congresso Nacional de


Winthrop, K. L. (2006). Risk and prevention of tuberculosis and other serious opportunistic

Yang, C. S.; Yuk, J. M.; & Jo, E. K. (2009). The role of nitric oxide in mycobacterial infections.

Yee, D.; Valiquette, C.; Pelletier, M.; Parisien, I.; Rocher, I. & Menzies, D. (2003). Incidence of

Zhou, Z.; Connell, M. C. & Macewan, D. J. (2007). TNFR1-induced NF-kappaB, but not ERK,

Zou, W.X.; Meng, J.C.; Lu, H.; Chen, G.X.; Shi, G.X.; Zhang, T.Y. & Tan, R. X. (2000).

*Rheumatology,*Vol. 2, No. 11, pp. 602–610, ISSN1745-8390.

Immune Network Vol. 9, No. 2, pp. 46–52, ISSN 2092-6685.

167, No. , pp. 1472-1477, ISSN1535-4970.

ISSN1873-3913.

infections associated with the inhibition of tumor necrosis factor. *Nature Reviews* 

serious side effects from first-line antituberculosis drugs among patients treated for active tuberculosis. *American Journal of Respiratory and Critical Care Medicine*, Vol.

p38MAPK or JNK activation, mediates TNF-induced ICAM-1 and VCAM-1 expression on endothelial cells. *Cellular Signalling,* Vol .19, No.6, pp. 1238–1248,

Metabolites of Colletotrichum gloeosporioides, an endophytic fungus in Artemisia mongolica. *Journal of Natural Products*, Vol. 63, No. 11, pp.1529-1530, ISSN1520-6025.

## *Edited by Pere-Joan Cardona*

In 1957, a Streptomyces strain, the ME/83 (S.mediterranei), was isolated in the Lepetit Research Laboratories from a soil sample collected at a pine arboretum near Saint Raphael, France. This drug was the base for the chemotherapy with Streptomicine. The euphoria generated by the success of this regimen lead to the idea that TB eradication would be possible by the year 2000. Thus, any further drug development against TB was stopped. Unfortunately, the lack of an accurate administration of these drugs originated the irruption of the drug resistance in Mycobacterium tuberculosis. Once the global emergency was declared in 1993, seeking out new drugs became urgent. In this book, diverse authors focus on the development and the activity of the new drug families.

Photo by KatarzynaBialasiewicz / iStock

Understanding Tuberculosis - New Approaches to Fighting Against Drug Resistance

Understanding Tuberculosis

New Approaches to Fighting Against

Drug Resistance

*Edited by Pere-Joan Cardona*