**The Susceptibility of** *Staphylococcus aureus* **and** *Klebsiella pneumoniae* **to Naturally Derived Selected Classes of Flavonoids**

Johannes Bodenstein\* and Karen Du Toit *Discipline of Pharmaceutical Sciences, University of KwaZulu-Natal (Westville Campus), Durban, South Africa* 

### **1. Introduction**

72 Antimicrobial Agents

van Wetering, S., Mannesse-Lazeroms, S.P.G., van Sterkenburg, M.A.J.A. and Hiemstra, P.S.,

Wang, Z. and Wang, G., 2004. APD: the Antimicrobial Peptide Database. Nucleic Acids Res,

Wimley, W.C., 2010. Describing the mechanism of antimicrobial peptide action with the

Xiao, Y., Dai, H., Bommineni, Y.R., Soulages, J.L., Gong, Y.X., Prakash, O. and Zhang, G.,

Xie, C., Prahl, A., Ericksen, B., Wu, Z., Zeng, P., Li, X., Lu, W.Y., Lubkowski, J. and Lu, W.,

Yamashita, T. and Saito, K., 1989. Purification, primary structure, and biological activity of guinea pig neutrophil cationic peptides. Infect. Immun., 57:2405-2409. Yang, Y., Poncet, J., Garnier, J., Zatylny, C., Bachere, E. and Aumelas, A., 2003. Solution

Yang, Y.H., Zheng, G.G., Li, G., Zhang, X.J., Cao, Z.Y., Rao, Q. and Wu, K.F., 2004.

Yasin, B., Wang, W., Pang, M., Cheshenko, N., Hong, T., Waring, A.J., Herold, B.C., Wagar,

membrane by distinct mechanisms of action. J Clin Invest, 101:178-187. Yonezawa, A., Kuwahara, J., Fujii, N. and Sugiura, Y., 1992. Binding of tachyplesin I to DNA

Zhang, L., Dhillon, P., Yan, H., Farmer, S. and Hancock, R.E., 2000. Interactions of bacterial

Pseudomonas aeruginosa. Antimicrob Agents Chemother, 44:3317-3321.

peptide LL-37, in Escherichia coli. Protein Expr Purif, 37:229-235.

cells: modulation by dexamethasone. Inflammation Research, 51:8-15. VanderMeer, T.J., Menconi, M.J., Zhuang, J., Wang, H., Murtaugh, R., Bouza, C., Stevens, P.

fragment of CAP18 in endotoxemic pigs. Surgery, 117:656-662.

interfacial activity model. ACS Chem Biol, 5:905-917.

peptide in chicken. FEBS J, 273:2581-2593.

amino acids. J Biol Chem, 280:32921-32929.

Chem, 278:36859-36867.

Lancet, 360:1116-1117.

32:D590-592.

2002. Neutrophil defensins stimulate the release of cytokines by airway epithelial

and Fink, M.P., 1995. Protective effects of a novel 32-amino acid C-terminal

2006. Structure-activity relationships of fowlicidin-1, a cathelicidin antimicrobial

2005. Reconstruction of the conserved beta-bulge in mammalian defensins using D-

structure of the recombinant penaeidin-3, a shrimp antimicrobial peptide. J Biol

Expression of bioactive recombinant GSLL-39, a variant of human antimicrobial

E.A. and Lehrer, R.I., 2004. {theta} Defensins Protect Cells from Infection by Herpes Simplex Virus by Inhibiting Viral Adhesion and Entry. J. Virol., 78:5147-5156. Yeaman, M.R., Bayer, A.S., Koo, S.P., Foss, W. and Sullam, P.M., 1998. Platelet microbicidal

proteins and neutrophil defensin disrupt the Staphylococcus aureus cytoplasmic

revealed by footprinting analysis: significant contribution of secondary structure to DNA binding and implication for biological action. Biochemistry, 31:2998-3004. Zangger, K., Gossler, R., Khatai, L., Lohner, K. and Jilek, A., 2008. Structures of the glycinerich diastereomeric peptides bombinin H2 and H4. Toxicon, 52:246-254. Zasloff, M., 2002. Innate immunity, antimicrobial peptides, and protection of the oral cavity.

cationic peptide antibiotics with outer and cytoplasmic membranes of

The emergence of multi-drug resistant organisms has increasingly become a global public health issue. Rational and appropriate uses of antibiotics as well as strict infection control measurements are recommended in order to reduce the emergence of antibiotic resistant bacteria (Tseng et al., 2011). The complexity in treating multi-drug resistant infections has led to an increase in the search for novel and effective antibiotics, especially structures originating from natural products. Promising molecules could serve as lead compounds to be developed and researched further.

This chapter aims to review the susceptibility of two of the most common micro-organisms that are often implicated in antibiotic resistant infections, namely the Gram-positive *Staphylococcus aureus* and Gram-negative *Klebsiella pneumoniae* against natural products, specifically plants. Numerous researchers have investigated the susceptibility of these bacteria to plant extracts as well as to the individual components thereof. Flavonoids as a group of compounds originating from natural products have been investigated against these bacteria.

Flavonoids are diverse polyphenolic compounds which are widely distributed in the plant kingdom. They are abundantly found in natural sources like fruits, vegetables, seeds, nuts, flowers, tea, wine honey and propolis and therefore form part of the normal diet of humans (Cook & Samman, 1996). Many reports claim the usefulness of flavonoids in medical conditions, including anti-inflammatory, oestrogenic, antimicrobial, antioxidant and chelating, vascular and antitumour activities (Cook & Samman, 1996; Cushnie & Lamb, 2005). Flavonoids consist of a C15 skeleton composed of two phenolic rings, namely the A and B rings linked through a heterocyclic ring, C. They are classified according to their biosynthetic origin into major classes including flavones, flavonols, flavanones, chalcones, flavanols, anthocyanidins, isoflavones and dihydroflavonols. Substitution patterns vary and some flavonoids occur as glycosides which are hydrolysed in the human gut to the aglycones. Flavonoids also occur as monomers, dimers or oligomers (Cook & Samman, 1996; Cushnie & Lamb, 2005).

Many reports exist on the antimicrobial activity of flavonoids (Basile et al., 2010; Du Toit et al., 2009; Tanaka et al., 2011). Extracts as well as isolated compounds were tested against a

<sup>\*</sup> Corresponding Author

The Susceptibility of *Staphylococcus aureus* and

**4. Occurrence of flavonoids in plant sources** 

Table 1 and their properties reviewed (Tables 2-7).

*Erythrina costaricensis*

*Erythrina costaricensis*

*Erythrina poeppigiana*

*Erythrina poeppigiana*

Brazilian red propolis

*Erythrina variegata* (Leguminosae)

*Erythrina variegata* (Leguminosae)

*Erythrina variegata* (Leguminosae)

*Erythrina variegata* (Leguminosae)

*Erythrina variegata* (Leguminosae)

*Erythrina variegata* (Leguminosae)

*Erythrina variegata* (Leguminosae)

*Erythrina variegata* (Leguminosae)

*Klebsiella pneumoniae* to Naturally Derived Selected Classes of Flavonoids 75

Several studies have identified flavonoids in natural products. Many flavonoid-containing plants are used therapeutically for the treatment of a variety of non-microbial illnesses as well as microbial infections. Flavonoids were derived from different parts of the plant and tested against *S. aureus* and *K. pneumoniae*. The vast number of identified compounds in studies were limited to cases where antibacterial activity was measured by means of dilution methods and where susceptibility was up to 50 μg/ml, providing a workable approach. At least 44 different compounds were identified according to the criteria, listed in

Plant/Product Traditional Use Part Compound Reference

Micheli (Leguminosae) Microbial infections Stems 1 Tanaka et al., 2009

Micheli (Leguminosae) Microbial infections Stems 2 Tanaka et al., 2009

(Leguminosae) Microbial infections Roots 3 Tanaka et al., 2004

(Leguminosae Microbial infections Roots 4 Tanaka et al., 2004

5 Oldoni et al., 2011

Roots 6 Tanaka et al., 2002

Roots 7 Tanaka et al., 2002

Roots 8 Tanaka et al., 2002

Roots 9 Tanaka et al., 2002

Roots 10 Tanaka et al., 2002

Roots 11 Tanaka et al., 2002

Roots 12 Tanaka et al., 2002

Roots 13 Tanaka et al., 2002

Inflammation, heart disease, diabetes, cancer

Microbial infections, inflammation

Microbial infections, inflammation

Microbial infections, inflammation

Microbial infections, inflammation

Microbial infections, inflammation

Microbial infections, inflammation

Microbial infections, inflammation

Microbial infections, inflammation

comprehensive panel of micro-organisms. Methods of assessing the activity include different diffusion and dilution methods. However, many flavonoids are insoluble in water and will thus have a low rate of diffusion in an aqueous medium such as agar, leading to inaccurate results. Therefore, only results based on dilution methods will be considered and discussed.

Extracts are complex mixtures of many chemical compounds in different ratios and the results of such studies are not contributing to the understanding of the activity of specific flavonoids. Studies that investigated the antibacterial activity of individual flavonoids isolated from natural products will be reviewed instead.

Various parameters have been used to express the antimicrobial activity of flavonoids. The minimum inhibitory concentration (MIC) will be considered and values up to 50 μg/ml will be reported. It must be appreciated that varying laboratory conditions and technical skills will have an influence on published results generated by different research groups and used in this review. The question also arises whether flavonoids exhibit bactericidal or bacteriostatic activity. Although some studies suggest that flavonoids are capable of bactericidal activity, the interpretation of the results remains inconclusive and it has been suggested that bacterial aggregates may be formed, thereby reducing the number of colony forming units in viable counts (Cushnie & Lamb, 2005).

#### **2.** *Staphylococcus aureus*

*Staphylococcus aureus* has long been recognised as an important pathogen in many diseases, for example the toxic shock syndrome, vasculitis and glomerulonephritis. The bacterium is commonly found in the nose and upper respiratory tract, locations that play an important role in the epidemiology and pathogenesis of infection. Therapy of infection has become problematic due to an increasing number of methicillin-resistant strains (MRSA). The difference between MRSA and methicillin-susceptible strains is that MRSA is resistant to βlactamase stable β-lactam antibiotics. Often this is also associated with resistance to many other antibiotics, which limits the therapeutic options. The prevalence of MRSA has also increased world-wide and new therapeutic agents, optimisation of infection control measures and introduction of new medical devices with a reduced risk of infection are being investigated (Kluytmans et al., 1997).

#### **3.** *Klebsiella pneumoniae*

*Klebsiella pneumoniae* is being considered the most common causative pathogen for infections caused by antibiotic-resistant bacteria. The rate of resistance to carbapenems has increased to more than 25% in the European Union in 2009 (Tseng et al., 2011). The nasopharynx and gastrointestinal tract are commonly colonised by the bacterium and it is well known to cause community-acquired bacterial pneumonia, occurring particularly in chronic alcoholics and showing characteristic radiographic abnormalities due to severe pyogenic infection which has a high fatality rate if untreated. It is an opportunistic pathogen that would most likely attack immunocompromised patients who are hospitalised and suffer from severe underlying diseases such as diabetes mellitus and chronic pulmonary obstructive diseases. The three most common conditions caused by *Klebsiella* spp. are urinary tract infections, septicaemia and wound infections. Septicaemia is particularly problematic in premature infants and patients in intensive care units (Podschun & Ullmann, 1998).

#### **4. Occurrence of flavonoids in plant sources**

74 Antimicrobial Agents

comprehensive panel of micro-organisms. Methods of assessing the activity include different diffusion and dilution methods. However, many flavonoids are insoluble in water and will thus have a low rate of diffusion in an aqueous medium such as agar, leading to inaccurate results. Therefore, only results based on dilution methods will be considered and discussed. Extracts are complex mixtures of many chemical compounds in different ratios and the results of such studies are not contributing to the understanding of the activity of specific flavonoids. Studies that investigated the antibacterial activity of individual flavonoids

Various parameters have been used to express the antimicrobial activity of flavonoids. The minimum inhibitory concentration (MIC) will be considered and values up to 50 μg/ml will be reported. It must be appreciated that varying laboratory conditions and technical skills will have an influence on published results generated by different research groups and used in this review. The question also arises whether flavonoids exhibit bactericidal or bacteriostatic activity. Although some studies suggest that flavonoids are capable of bactericidal activity, the interpretation of the results remains inconclusive and it has been suggested that bacterial aggregates may be formed, thereby reducing the number of colony

*Staphylococcus aureus* has long been recognised as an important pathogen in many diseases, for example the toxic shock syndrome, vasculitis and glomerulonephritis. The bacterium is commonly found in the nose and upper respiratory tract, locations that play an important role in the epidemiology and pathogenesis of infection. Therapy of infection has become problematic due to an increasing number of methicillin-resistant strains (MRSA). The difference between MRSA and methicillin-susceptible strains is that MRSA is resistant to βlactamase stable β-lactam antibiotics. Often this is also associated with resistance to many other antibiotics, which limits the therapeutic options. The prevalence of MRSA has also increased world-wide and new therapeutic agents, optimisation of infection control measures and introduction of new medical devices with a reduced risk of infection are being

*Klebsiella pneumoniae* is being considered the most common causative pathogen for infections caused by antibiotic-resistant bacteria. The rate of resistance to carbapenems has increased to more than 25% in the European Union in 2009 (Tseng et al., 2011). The nasopharynx and gastrointestinal tract are commonly colonised by the bacterium and it is well known to cause community-acquired bacterial pneumonia, occurring particularly in chronic alcoholics and showing characteristic radiographic abnormalities due to severe pyogenic infection which has a high fatality rate if untreated. It is an opportunistic pathogen that would most likely attack immunocompromised patients who are hospitalised and suffer from severe underlying diseases such as diabetes mellitus and chronic pulmonary obstructive diseases. The three most common conditions caused by *Klebsiella* spp. are urinary tract infections, septicaemia and wound infections. Septicaemia is particularly problematic in premature

infants and patients in intensive care units (Podschun & Ullmann, 1998).

isolated from natural products will be reviewed instead.

forming units in viable counts (Cushnie & Lamb, 2005).

**2.** *Staphylococcus aureus* 

investigated (Kluytmans et al., 1997).

**3.** *Klebsiella pneumoniae* 

Several studies have identified flavonoids in natural products. Many flavonoid-containing plants are used therapeutically for the treatment of a variety of non-microbial illnesses as well as microbial infections. Flavonoids were derived from different parts of the plant and tested against *S. aureus* and *K. pneumoniae*. The vast number of identified compounds in studies were limited to cases where antibacterial activity was measured by means of dilution methods and where susceptibility was up to 50 μg/ml, providing a workable approach. At least 44 different compounds were identified according to the criteria, listed in Table 1 and their properties reviewed (Tables 2-7).


The Susceptibility of *Staphylococcus aureus* and

*Galium fissurense* Ehrend. & Schönb.-Tem. (Rubiaceae)

*Cirsium hypoleucum* DC. (Asteraceae)

*Cirsium hypoleucum* DC. (Asteraceae)

*Artocarpus sepicanus* (Moraceae)

*Erythrina zeyheri* (Leguminosae)

*Erythrina zeyheri* (Leguminosae)

*Erythrina zeyheri* (Leguminosae)

*Erythrina zeyheri* (Leguminosae)

*Erythrina zeyheri* (Leguminosae)

*Sophora exigua* Criab

*Sophora exigua* Criab

*Sophora exigua* Criab

*Sophora exigua* Criab

*Sophora exigua* Criab

*Sophora exigua* Criab

*Klebsiella pneumoniae* to Naturally Derived Selected Classes of Flavonoids 77

Plant/Product Traditional Use Part Compound Reference

Leaves

Aerial

Aerial

and stems 25 Orhan et al., 2010

parts 26 Orhan et al., 2010

parts 27 Orhan et al., 2010

Leaves 28 Radwan et al., 2009

Roots 29 Tanaka et al., 2003

Roots 30 Tanaka et al., 2003

Roots 31 Tanaka et al., 2003

Roots 32 Tanaka et al., 2003

Roots 33 Tanaka et al., 2003

Diuretic, astringent, gastrointestinal conditions, gout, epilepsy

Haemorrhoids, peptic ulcers, cough, bronchitis

Haemorrhoids, peptic ulcers, cough, bronchitis

Microbial infections, asthma, tuberculosis, rheumatic fever

Microbial infections, asthma, tuberculosis, rheumatic fever

Microbial infections, asthma, tuberculosis, rheumatic fever

Microbial infections, asthma, tuberculosis, rheumatic fever

Microbial infections, asthma, tuberculosis, rheumatic fever

Microbial infections, asthma, tuberculosis, rheumatic fever

(Leguminosae) Microbial infections Roots 34 Tsuchiya et al., 1996

(Leguminosae) Microbial infections Roots 35 Tsuchiya et al., 1996

(Leguminosae) Microbial infections Roots 36 Tsuchiya et al., 1996

(Leguminosae) Microbial infections Roots 37 Tsuchiya et al., 1996

(Leguminosae) Microbial infections Roots 38 Tsuchiya et al., 1996

(Leguminosae) Microbial infections Roots 39 Tsuchiya et al., 1996


Plant/Product Traditional Use Part Compound Reference

(Leguminosae) Microbial infections Roots 15 Tanaka et al., 2011

(Leguminosae) Microbial infections Roots 16 Tanaka et al., 2011

(Cycadaceae) Purgative Leaflets 17 Moawad et al., 2010

(Cycadaceae) Purgative Leaflets 18 Moawad et al., 2010

*minimiflorus* (Noctuidae) Microbial infections Bark 21 Salvatore et al., 1998

Leaves

Leaves

Leaves

Roots 14 Tanaka et al., 2002

Leaflets 19 Moawad et al., 2010

Fruits 20 Basile et al., 2010

and stems 22 Orhan et al., 2010

and stems 23 Orhan et al., 2010

and stems 24 Orhan et al., 2010

Microbial infections, inflammation

Rheumatic fever, expectorant, astringent, flatulence, vomiting, oestrogendependent cancer

Perfume, microbial infections, inflammation, cancer

Hypertension, epilepsy, exhaustion, anxiety, arthritis, vertigo, degenerative inflammation of the joints, cancer

Hypertension, epilepsy, exhaustion, anxiety, arthritis, vertigo, degenerative inflammation of the joints, cancer

Diuretic, astringent, gastrointestinal conditions, gout, epilepsy

*Erythrina variegata* (Leguminosae)

*Erythrina variegata*

*Erythrina variegata*

*Cycas circinalis*

*Cycas circinalis*

*Cycas revoluta* Thumb (Cycadaceae)

*Feijoa sellowiana* Berg (Myrtaceae)

*Lonchocarpus* 

*Viscum album* ssp. *album* (Loranthaceae)

*Viscum album* ssp. *album* (Loranthaceae)

*Galium fissurense* Ehrend. & Schönb.-Tem. (Rubiaceae)


The Susceptibility of *Staphylococcus aureus* and

<sup>5</sup>R2,R5=OH

<sup>8</sup>R2,R3=2",2" dimethylpyran

<sup>21</sup>R1,R3=γ,γ-dimethylallyl

<sup>22</sup>R2,R4=OCH3

<sup>24</sup>R2,R4,R5=OH

<sup>25</sup>R4,R6=OH

<sup>28</sup>R2,R4,R6=OH

<sup>29</sup>R1,R8=γ,γ-dimethylallyl

<sup>31</sup>R1,R3=γ,γ-dimethylallyl

*Klebsiella pneumoniae*. ND, not determined.

1

2

12

15

23

30

32

*Klebsiella pneumoniae* to Naturally Derived Selected Classes of Flavonoids 79

**Compound Structure Susceptibility (MIC,** μ**g/ml)** 

R7=OCH3 (3*S*-enantiomer) 31.2-62.5 ND

R5,R7=OH (3*R*-enantiomer) 12.5-25\* ND

R2,R4,R6=OH (2*S*-enantiomer) 0.78-1.56\* ND

R6=*O*-glc 4 16

R6=*O*-glc 4 16

R2=*O*-glc 4 16

R2,R7,R9=OH 12.5-25\* ND

R3=geranyl (2*S*-enantiomer) 1.23\* ND

R2,R5,R7=OH (3*R*-enantiomer) 3.13-6.25\* ND

R2,R4,R6=OH R3,R8=γ,γ-dimethylallyl R7=OCH3

R2,R3=2",2" dimethylpyran R4,R6=OH R7=OCH3 R8=γ,γ-dimethylallyl

R1,R2=2",2" dimethylpyran R3=γ,γ-dimethylallyl R7,R9=OH

R1=γ,γ-dimethylallyl R2,R3=2",2" dimethylpyran R7,R9,R10=OH

R2,R4=OCH3 R6=*O*-[2"-*O*-(5"'-*O*-*trans*-cinnamoyl)-β-*D*apiofuranosyl]-β-*D*-glucopyranoside

> R1,R3=γ,γ-dimethylallyl R2,R7=OH R5=OCH3 (3*R*-enantiomer)

R1=γ,γ-dimethylallyl R2,R3=2",2"-dimethylpyran R5,R7=OH

Table 2. Isoflavanones of the following structure isolated from plants and propolis

(compound 5) and their activities against *Staphylococcus aureus* (\*denotes MRSA strains) and

A study by Du Toit and co-workers reported little activity of the flavonoids luteolin, eriodictyol and quercetin against *S. aureus* and MIC-values could not be determined. These

*S. Aureus K. Pneumoniae* 

3.13-6.25\* ND

12.5- >50\* ND

3.13-12.5\* ND

12.5-25\* ND

4 16

25- >50 ND

6.25-12.5\* ND


Table 1. Compounds isolated from different parts of plants/products that occur world-wide and their traditional use. Only compounds tested against *Staphylococcus aureus* and *Klebsiella pneumoniae* by means of dilution methods where the MIC-values were up to 50 μg/ml are reported (refer to Tables 2-7 where the classification, chemistry and biological activity of each compound are explained in more detail).

#### **5. Flavonoids and bacterial susceptibility**

The flavonoids identified in different plants/products which were investigated for their antibacterial activity using dilution methods, were divided into 6 structural types and the susceptibility of *S. aureus* and *K. pneumoniae* reviewed. Some of the strains of *S. aureus* were MSRA.

Structural types were used in order to compare similar structures and to determine the influence of substituents on these structures. Susceptibility was also compared where the methods were similar to reduce the presence of too many variables.

Compounds 17-19 (Table 6), which are biflavonoids, exhibited the weakest activity against *S. aureus* in comparison with all the other structures. This may be attributed to the size and stereochemistry of the molecules. The compounds exhibiting the highest activity were compounds 16 (Table 3) and 21 (Table 2), which interestingly share the same substitution pattern at R1 and R2 (which were substituted with a γ,γ-dimethylallyl and hydroxyl group respectively). Comparison of compounds 3, 6 and 16 (Table 3), which were all substituted with a γ,γ-dimethylallyl group at R5, showed that the addition of an extra γ,γ-dimethylallyl group influences activity. Addition at R3 increases activity and addition at R1 has an even more pronounced effect in the structural group. Comparison of compounds 30 and 31 (Table 2) showed that substitution at R5 with a hydroxyl group leads to better activity than substitution with a methoxy group in the specific structural group.


The Susceptibility of *Staphylococcus aureus* and *Klebsiella pneumoniae* to Naturally Derived Selected Classes of Flavonoids 79

Plant/Product Traditional Use Part Compound Reference

Nakai (Leguminosae) Microbial infections Roots 40 Tsuchiya et al., 1996

Nakai (Leguminosae) Microbial infections Roots 41 Tsuchiya et al., 1996

Nakai (Leguminosae) Microbial infections Roots 42 Tsuchiya et al., 1996

Nakai (Leguminosae) Microbial infections Roots 43 Tsuchiya et al., 1996

Table 1. Compounds isolated from different parts of plants/products that occur world-wide and their traditional use. Only compounds tested against *Staphylococcus aureus* and *Klebsiella pneumoniae* by means of dilution methods where the MIC-values were up to 50 μg/ml are reported (refer to Tables 2-7 where the classification, chemistry and biological activity of

The flavonoids identified in different plants/products which were investigated for their antibacterial activity using dilution methods, were divided into 6 structural types and the susceptibility of *S. aureus* and *K. pneumoniae* reviewed. Some of the strains of *S. aureus* were

Structural types were used in order to compare similar structures and to determine the influence of substituents on these structures. Susceptibility was also compared where the

Compounds 17-19 (Table 6), which are biflavonoids, exhibited the weakest activity against *S. aureus* in comparison with all the other structures. This may be attributed to the size and stereochemistry of the molecules. The compounds exhibiting the highest activity were compounds 16 (Table 3) and 21 (Table 2), which interestingly share the same substitution pattern at R1 and R2 (which were substituted with a γ,γ-dimethylallyl and hydroxyl group respectively). Comparison of compounds 3, 6 and 16 (Table 3), which were all substituted with a γ,γ-dimethylallyl group at R5, showed that the addition of an extra γ,γ-dimethylallyl group influences activity. Addition at R3 increases activity and addition at R1 has an even more pronounced effect in the structural group. Comparison of compounds 30 and 31 (Table 2) showed that substitution at R5 with a hydroxyl group leads to better activity than

O

R10 R5

R8

R6

R7

R1

R4

O

R9

methods were similar to reduce the presence of too many variables.

substitution with a methoxy group in the specific structural group.

R2

R3

(Leguminosae) Microbial infections Roots 44 Tsuchiya et al., 1996

*Echinosophora koreensis*

*Echinosophora koreensis*

*Echinosophora koreensis*

*Echinosophora koreensis*

*Sophora leachiana* Peck

MSRA.

each compound are explained in more detail).

**5. Flavonoids and bacterial susceptibility** 

A study by Du Toit and co-workers reported little activity of the flavonoids luteolin, eriodictyol and quercetin against *S. aureus* and MIC-values could not be determined. These

Table 2. Isoflavanones of the following structure isolated from plants and propolis (compound 5) and their activities against *Staphylococcus aureus* (\*denotes MRSA strains) and *Klebsiella pneumoniae*. ND, not determined.

The Susceptibility of *Staphylococcus aureus* and

7

33

10

11

determined.

determined.

*Klebsiella pneumoniae* to Naturally Derived Selected Classes of Flavonoids 81

O

R2

R3

<sup>14</sup>R2,R4=OH

O

**Compound Structure Susceptibility (MIC,** μ**g/ml)** 

H

R1

R2=OH R3,R5=γ,γ-dimethylallyl R4=OCH3

R1,R5=γ,γ-dimethylallyl R2=OH R4=OCH3 (6a*S*, 11a*S*-enantiomer)

R1

R2

Table 4. Isoflavonoids of the following structure isolated from plants and their activities against *Staphylococcus aureus* (\*denotes MRSA strains) and *Klebsiella pneumoniae*. ND, not

O

O

**Compound Structure Susceptibility (MIC,** μ**g/ml)** 

H

R1=OCH3 R2=γ,γ-dimethylallyl R3=OH RR

R1,R2=2",2"-dimethylpyran R3=OH R4=γ,γ-dimethylallyl

Table 5. Isoflavonoids of the following structure isolated from plants and their activities against *Staphylococcus aureus* (\*denotes MRSA strains) and *Klebsiella pneumoniae*. ND, not

<sup>13</sup>R1=OH

<sup>R</sup> R3 <sup>4</sup>

R3,R4=2",2"-dimethylpyran 25\* ND

H

<sup>R</sup> R4 <sup>5</sup>

R3,R5=γ,γ-dimethylallyl 6.25-12.5\* ND

*S. Aureus K. Pneumoniae* 

12.5-25\* ND

6.25-25\* ND

*S. Aureus K. Pneumoniae* 

3.13-6.25\* ND

6.25-25\* ND

OH

flavonoids are commonly found in propolis (Du Toit et al., 2009). Combinations of flavonoids at different concentrations as well as other components present in the propolis could account for its antimicrobial activity.

Compared to *S. aureus*, it is noteworthy that significantly fewer compounds have been tested against *K. pneumoniae*. Compounds 22-25 (Table 2) and 20, 26-27 (Table 7) were the only compounds tested using dilution methods. Out of the few compounds tested, compound 20 showed the highest activity and it also has the least number of substituents. Future research should investigate the activity of more compounds against *K. pneumoniae*.

New drug targets in the bacterial structure are important. Drugs will be less susceptible to resistance if it has several modes of action. Pharmacokinetic parameters such as bioavailability and plasma protein binding are also important, since successful traditional use indicates that the drug has successfully reached a specific target.


Table 3. Isoflavonoids of the following structure isolated from plants and their activities against *Staphylococcus aureus* (\*denotes MRSA strains) and *Klebsiella pneumoniae*. ND, not determined.

flavonoids are commonly found in propolis (Du Toit et al., 2009). Combinations of flavonoids at different concentrations as well as other components present in the propolis

Compared to *S. aureus*, it is noteworthy that significantly fewer compounds have been tested against *K. pneumoniae*. Compounds 22-25 (Table 2) and 20, 26-27 (Table 7) were the only compounds tested using dilution methods. Out of the few compounds tested, compound 20 showed the highest activity and it also has the least number of substituents. Future research should investigate the activity of more compounds against *K. pneumoniae*.

New drug targets in the bacterial structure are important. Drugs will be less susceptible to resistance if it has several modes of action. Pharmacokinetic parameters such as bioavailability and plasma protein binding are also important, since successful traditional

O

R1

O

**Compound Structure Susceptibility (MIC,** μ**g/ml)** 

R2=OH R4=OCH3 R5=γ,γ-dimethylallyl

R2=OH R4=OCH3 R5=γ,γ-dimethylallyl

Table 3. Isoflavonoids of the following structure isolated from plants and their activities against *Staphylococcus aureus* (\*denotes MRSA strains) and *Klebsiella pneumoniae*. ND, not

R5

R5=γ,γ-dimethylallyl 12.5\* ND

R3,R5=γ,γ-dimethylallyl 3.13-6.25\* ND

R2,R4=OH 1.56-3.13\* ND

R4

*S. Aureus K. Pneumoniae* 

12.5-25 ND

12.5-25\* ND

use indicates that the drug has successfully reached a specific target.

R2

R3

<sup>3</sup>R2,R4=OH

<sup>6</sup>R2,R4=OH

<sup>16</sup>R1,R5=γ,γ-dimethylallyl

4

9

determined.

could account for its antimicrobial activity.


Table 4. Isoflavonoids of the following structure isolated from plants and their activities against *Staphylococcus aureus* (\*denotes MRSA strains) and *Klebsiella pneumoniae*. ND, not determined.


Table 5. Isoflavonoids of the following structure isolated from plants and their activities against *Staphylococcus aureus* (\*denotes MRSA strains) and *Klebsiella pneumoniae*. ND, not determined.

The Susceptibility of *Staphylococcus aureus* and

*Klebsiella pneumoniae* to Naturally Derived Selected Classes of Flavonoids 83

**Compound Structure Susceptibility (MIC,** μ**g/ml)** 

R2,R4,R5,R8=OH 12.5\* ND

R3=geranyl 12.5\* ND

R6=geranyl 3.13-12.5\* ND

R3=lavandulyl 3.13-12.5\* ND

R2,R4,R5,R7,R8=OH 6.25-12.5\* ND

R1=prenyl 6.25-12.5\* ND

R2,R4,R8=OH 12.5\* ND

Table 7. Flavones of the following structure isolated from plants and their activities against *Staphylococcus aureus* (\*denotes MRSA strains) and *Klebsiella pneumoniae*. ND, not determined.

The traditional use of medicinal plants is useful as a guideline in the quest for new drugs. Furthermore plants are a source of novel lead compounds which would generally not have been synthesised. Extraction of these biologically active lead compounds may be expensive and slow and the activity of lead compounds may also not be sufficient to encourage commercialisation. These compounds may also have undesirable side effects, it is therefore important to periodically review the results of research conducted, ruling out unnecessary variation of parameters, to determine the most promising structures. The process of drug

We thank the University of KwaZulu-Natal Competitor Fund for financial support.

*pylori* growth. *Journal of Medicinal Food,* Vol. 13, pp. 189-195.

Basile, A.; Conte, B.; Rigano, D.; Senatore, F. & Sorbo, S. (2010). Antibacterial and antifungal

Cook, N.C. & Samman, S. (1996). Flavonoids – chemistry, metabolism, cardioprotective effects, and dietary sources. *Nutritional Biochemistry,* Vol. 7, pp. 66-76.

properties of acetonic extract of *Feijoa sellowiana* fruits and its effect on *Helicobacter* 

R2,R4,R5,R7,R8=OH >25\* ND

R2,R4,R5,R8=OH R7=OCH3

<sup>37</sup>R1=lavandulyl

<sup>38</sup>R2,R4,R5,R7,R8=OH

<sup>39</sup>R1=geranyl

<sup>40</sup>R2,R4,R7,R8= OH

<sup>41</sup>R2,R4,R5,R7,R8=OH

<sup>42</sup>R1=lavandulyl

<sup>43</sup>R2,R4,R7,R8=OH

<sup>44</sup>R1=lavandulyl

design and development could then be accelerated.

**6. Conclusion** 

**7. Acknowledgment** 

**8. References** 

*S. Aureus K. Pneumoniae* 


Table 6. Biflavonoids of the following structure isolated from plants and their activities against *Staphylococcus aureus* (\*denotes MRSA strains) and *Klebsiella pneumoniae*. ND, not determined.



Table 7. Flavones of the following structure isolated from plants and their activities against *Staphylococcus aureus* (\*denotes MRSA strains) and *Klebsiella pneumoniae*. ND, not determined.

#### **6. Conclusion**

82 Antimicrobial Agents

**Compound Structure Susceptibility (MIC,** μ**g/ml)** 

17 R1,R2=OCH3 (2*S*, 2''*S*-enantiomer) 17.5\* ND

19 R1,R2=OH (2*S*-enantiomer) 37\* ND

Table 6. Biflavonoids of the following structure isolated from plants and their activities against *Staphylococcus aureus* (\*denotes MRSA strains) and *Klebsiella pneumoniae*. ND, not

O

R5

R1

R4

R1=lavandulyl R2,R4,R5,R8=OH R3=prenyl R7=OCH3

R2

R3

<sup>26</sup>R2,R4,R6,R7=OH

<sup>27</sup>R2,R4,R7=OH

<sup>34</sup>R1=lavandulyl

35

O

**Compound Structure Susceptibility (MIC,** μ**g/ml)** 

20 R5=OH 7.8 7.8

36 R1=prenyl 6.25\* ND

R2=OH (2*S*, 2''*S*-enantiomer) 35.9\* ND

R6

R8 R9

R9=*O*-glc-rha 4 16

R9=*O*-glc-rha 4 16

R2,R4,R7,R8=OH 3.13-6.25\* ND

R7

O

R2

O

*S. Aureus K. Pneumoniae* 

*S. Aureus K. Pneumoniae* 

3.13-6.25\* ND

HO OH

R1

O

OH O

HO

<sup>18</sup>R1=OCH3

determined.

The traditional use of medicinal plants is useful as a guideline in the quest for new drugs. Furthermore plants are a source of novel lead compounds which would generally not have been synthesised. Extraction of these biologically active lead compounds may be expensive and slow and the activity of lead compounds may also not be sufficient to encourage commercialisation. These compounds may also have undesirable side effects, it is therefore important to periodically review the results of research conducted, ruling out unnecessary variation of parameters, to determine the most promising structures. The process of drug design and development could then be accelerated.

#### **7. Acknowledgment**

We thank the University of KwaZulu-Natal Competitor Fund for financial support.

#### **8. References**


**5** 

*Croatia* 

**Antibacterial Activity** 

*2Galapagos Research Center Ltd., Zagreb,* 

*Dedicated to all our colleagues engaged* 

**of Novel Sulfonylureas, Ureas** 

Mirjana Bukvić Krajačić1,2 and Miljenko Dumić<sup>3</sup> *1GlaxoSmithKline Research Centre Zagreb, Zagreb,* 

*3Department of Biotechnology, University of Rijeka, Rijeka,* 

*worldwide in discovery and development of azithromycin,* 

*on the occasion of the 30th anniversary of its invention (1981-2011)* 

One of the 20th century's significant achievements is a discovery of azithromycin **(1)** and its development to commercial product for effective treatment of various infective diseases. Owing to its exceptional therapeutic and biopharmaceutical properties, it has come to be one of the most successful antibiotics worldwide. For the discovery of azithromycin, in addition to receiving numerous awards, in the year 2000, PLIVA's scientists Slobodan Djokic and Gabrijela Kobrehel together with the representatives from the US-based Pfizer, Gene Michael Bright and Arthur E. Girard, (Anonymous, 2000) were granted the honourable titles of "Heroes of Chemistry" by the American Chemical Society (ACS), a non-profit association of American chemists and chemical engineers, and the largest association of scientists in the world. This prestigious award is taken to be also recognition of the achievement of PLIVA's entire team working on azithromycin. The success of azithromycin has positioned PLIVA among the few pharmaceutical companies in the world that have developed their own blockbuster drug, and has entitled Croatia to join a small group of nations that have

Nowadays, on the occasion of the 30th anniversary of azithromycin's invention (1981-2011) an increasing prevalence of antibiotic-resistant pathogens suggests that we deeply entered into a "Post-Antimicrobial Era" (Cohen 1992; Travis 1994; Kirst 1996b). Investment in newer anti-infective platforms is essential and urgent in order to achieve a significant progress in

our understanding of bacterial resistance and new approaches how to control it.

**1. Introduction** 

developed a new antibiotic.

**and Thioureas of 15-Membered Azalides** 

