3.1 Antimicrobial activity

The compounds were screened for their antibacterial and antifungal activity according to standard protocols [36].

The antimicrobial activity was studied using Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Sarcina lutea ATCC 9341, Bacillus cereus ATCC 14579), Gram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853), and pathogenic yeasts (Candida albicans ATCC 10231, Candida glabrata ATCC MYA 2950, Candida parapsilosis ATCC 22019). All these strains were obtained from the Culture Collection of the Department of Microbiology, Faculty of Pharmacy, "Grigore T. Popa" University of Medicine and Pharmacy, Iasi, Romania.

Compd. R υC-H arom cm<sup>1</sup> υC=O side chain cm<sup>1</sup> υC=O lactone cm<sup>1</sup> IIa H3C▬ 3070 1750 1680 IIb H3C▬CH2▬CH2▬ 3080 1740 1690

Compd. R υNH2 cm<sup>1</sup> υCO-NH cm<sup>1</sup> υC-N cm<sup>1</sup> IIIa H3C▬ 3423, 3331 1612 1271 IIIb H3C▬CH2▬CH2▬ 3411, 3340 1610 1260

Compd. R υN-H cm<sup>1</sup> υC=S cm<sup>1</sup> IVa H3C▬ 3210 1240 IVb H3C▬CH2▬CH2▬ 3150 1210

Compd. R υS-H cm<sup>1</sup> υC=N cm<sup>1</sup> υS-C cm<sup>1</sup> Va H3C▬ 2380 1610 621 Vb H3C▬CH2▬CH2▬ 2350 1620 638

Table 7.

Table 8.

Table 9.

Table 10.

146

IR spectral data of compounds IIa–IIb.

Phytochemicals in Human Health

IR spectral data of compounds IIIa–IIIb.

IR spectral data of compounds IVa–IVb.

IR spectral data of compounds Va–Vb.

Antimicrobial activity was evaluated by agar disc diffusion method (CLSI, 2014). A small amount of each microbial culture was diluted in sterile 0.9% NaCl until the turbidity was equivalent to McFarland standard no. 0.5 (106 CFU/ml). The suspensions were further diluted 1:10 in Mueller-Hinton agar for bacteria and Sabouraud agar for yeasts and then spread on sterile Petri plates (25 ml/Petri plate). Sterile stainless steel cylinders (5 mm internal diameter; 10 mm height) were applied on the agar surface in Petri plates. Then, 0.1 ml of each compound (10 mg/ml in DMSO) was added into the cylinders. The DMSO solvent was also tested in order to assess its intrinsic antimicrobial activity. Commercial available discs containing ampicillin (25 μg/disc), chloramphenicol (30 μg/disc), and nystatin (100 μg/disc) were also placed on the agar surface. The plates were incubated at 37°C for 24 h (bacteria) and at 24°C for 48 h (yeasts). After incubation the diameters of inhibition zones were read in triplicate. Statistical analysis of the results included the calculation of standard deviation (Tables 12 and 13).

The qualitative screening of the antimicrobial activity was performed in order to identify the antimicrobial spectrum of the tested compounds. The inhibitory effects of the synthetic compounds against Gram-positive and Gram-negative bacteria and fungi are given in Tables 12 and 13 [35].

According to the results of the antibacterial studies, the efficacy of the tested compounds against Gram-positive bacteria was higher than that exhibited for Gram-negative bacteria. All the synthesized compounds were very active against S. aureus ATCC 25923, the most active compounds being Ib, IIb, IIIb, and IVb. The replacement of the methyl radical in the fourth position with the propyl group was correlated with an increased activity against S. aureus ATCC 25923.

We found a moderate action against B. cereus ATCC 14579, the most active being the umbelliferone derivatives with a methyl group attached to C4: IIa, Ia, and IVa. Against Escherichia coli ATCC 25922, the investigated compounds had a weaker action than the controls ampicillin and chloramphenicol. The most active was the

Compd./reference Diameters of the growth inhibition zone (mm)

Ia 24 1.83 21 0.52 34 1.83 Ib 10 0.91 10 0.79 10 0.54 IIa 25 0.52 27 0.54 35 1.83 IIb 9 1.83 NA NA IIIa 19 1.79 24 0.52 24 1.79 IIIb 12 1.83 11 0.54 11 0.54 IVa 23 0.91 16 0.52 25 1.08 IVb 10 0.54 12 1.08 NA Va 16 1.79 21 1.83 21 0.54 Vb 9 0.54 9 0.52 NA Nystatin (100 μg/disc) 25 0.52 25 0.52 24 0.00

C. glabrata ATCC MYA 2950 C. parapsilosis ATCC 22019

C. albicans ATCC 10231

Coumarin Derivatives with Antimicrobial and Antioxidant Activities

DOI: http://dx.doi.org/10.5772/intechopen.88096

The presence of the methyl group attached to the coumarin ring in the fourth position had a positive influence on the anti-Pseudomonas ATCC 27853 potential of the compounds, all the tested 4-propyl-coumarin derivatives being inactive. We have noticed a very important action against the investigated Candida strains; all tested compounds were found to be very active against fungi. The compounds IIa and Ia had a greater inhibitory potential against C. parapsilosis ATCC 22019 than nystatin. The introduction of the sulfur atom appeared to be

In order to evaluate the antioxidant activity of synthesized compounds, we use three antioxidant assays: DPPH radical inhibition, total reducing power, and nitric

The DPPH assay is based on assessing the substances' ability to reduce the stable radical (diphenylpicrylhydrazyl) to diphenylpicrylhydrazine. The DPPH free radical, bearing an odd electron, gives a strong absorption maximum at λ = 517 nm (purple color). When the odd electron of the DPPH radical pairs with a hydrogen atom from an antioxidant, the reduced form DPPH-H is created, and the color turns

A possible mechanism that can explain the antioxidant effect of the coumarin hydrazide derivatives is related to the keto-enol forms of the substances, the enol

The experimental procedure for the DPPH assay was adapted from literature [27, 28, 37], only slight modifications being made. Briefly, 2.5 ml solution of DPPH (2,2-diphenyl-1-picrylhydrazyl) radical 0.1 mM in methanol was added over 0.5 ml

group being capable to easily donate the hydrogen (Figure 8) [38].

compound that contains a thiadiazole ring, Vb.

Data are mean SD (n = 3); NA, no activity.

Antifungal activity of compounds I–V.

Table 13.

correlated with a good anti-Candida activity.

3.2 Antioxidant activity

oxide (NO) inhibition.

149

from purple to yellow [36, 37].

The tested compounds exhibited excellent antibacterial activity against S. lutea, the most active derivatives being IIb, IVb, Ib, and IIIb.


#### Table 12.

Antibacterial activity of compounds I–V.


Coumarin Derivatives with Antimicrobial and Antioxidant Activities DOI: http://dx.doi.org/10.5772/intechopen.88096

#### Table 13.

Antimicrobial activity was evaluated by agar disc diffusion method (CLSI, 2014). A small amount of each microbial culture was diluted in sterile 0.9% NaCl until the turbidity was equivalent to McFarland standard no. 0.5 (106 CFU/ml). The suspensions were further diluted 1:10 in Mueller-Hinton agar for bacteria and Sabouraud agar for yeasts and then spread on sterile Petri plates (25 ml/Petri plate). Sterile stainless steel cylinders (5 mm internal diameter; 10 mm height) were applied on the agar surface in Petri plates. Then, 0.1 ml of each compound (10 mg/ml in DMSO) was added into the cylinders. The DMSO solvent was also tested in order to assess its intrinsic antimicrobial activity. Commercial available discs containing ampicillin (25 μg/disc), chloramphenicol (30 μg/disc), and nystatin (100 μg/disc) were also placed on the agar surface. The plates were incubated at 37°C for 24 h (bacteria) and at 24°C for 48 h (yeasts). After incubation the diameters of inhibition zones were read in triplicate. Statistical analysis of the results

The qualitative screening of the antimicrobial activity was performed in order to identify the antimicrobial spectrum of the tested compounds. The inhibitory effects of the synthetic compounds against Gram-positive and Gram-negative bacteria and

According to the results of the antibacterial studies, the efficacy of the tested compounds against Gram-positive bacteria was higher than that exhibited for Gram-negative bacteria. All the synthesized compounds were very active against S. aureus ATCC 25923, the most active compounds being Ib, IIb, IIIb, and IVb. The replacement of the methyl radical in the fourth position with the propyl group was

The tested compounds exhibited excellent antibacterial activity against S. lutea,

B. cereus ATCC 14579

26 0.04 36 0.00 NA 21 0.79 NA

22 0.00 38 0.00 24 0.00 21 0.52 NA

E. coli ATCC 25922

Pseudomonas aeruginosa ATCC 27853

included the calculation of standard deviation (Tables 12 and 13).

correlated with an increased activity against S. aureus ATCC 25923.

Compd./reference Diameters of the growth inhibition zone (mm)

S. lutea ATCC 9341

Ia 14 0.52 25 0.79 25 1.52 12 0.79 8 0.93 Ib 27 1.29 29 0.83 NA NA NA IIa 14 0.91 22 0.79 26 0.79 11 0.52 8 1.43 IIb 27 0.52 30 NA NA NA IIIa 15 0.54 20 0.79 22 0.79 10 0.79 9 0.79 IIIb 25 0.52 28 NA NA NA IVa 17 1.08 25 0.91 24 1.52 12 0.93 9 1.43 IVb 25 1.08 30 0.83 NA NA NA Va 14 0.52 25 0.52 20 1.43 10 1.52 8 0.52 Vb 21 1.43 25 0.79 NA 13 0.83 NA

the most active derivatives being IIb, IVb, Ib, and IIIb.

S. aureus ATCC 25923

Ampicillin (25 μg/disc)

Table 12.

148

Chloramphenicol (30 μg/disc)

Data are mean SD (n = 3); NA, no activity.

Antibacterial activity of compounds I–V.

fungi are given in Tables 12 and 13 [35].

Phytochemicals in Human Health

Antifungal activity of compounds I–V.

We found a moderate action against B. cereus ATCC 14579, the most active being the umbelliferone derivatives with a methyl group attached to C4: IIa, Ia, and IVa.

Against Escherichia coli ATCC 25922, the investigated compounds had a weaker action than the controls ampicillin and chloramphenicol. The most active was the compound that contains a thiadiazole ring, Vb.

The presence of the methyl group attached to the coumarin ring in the fourth position had a positive influence on the anti-Pseudomonas ATCC 27853 potential of the compounds, all the tested 4-propyl-coumarin derivatives being inactive.

We have noticed a very important action against the investigated Candida strains; all tested compounds were found to be very active against fungi. The compounds IIa and Ia had a greater inhibitory potential against C. parapsilosis ATCC 22019 than nystatin. The introduction of the sulfur atom appeared to be correlated with a good anti-Candida activity.

#### 3.2 Antioxidant activity

In order to evaluate the antioxidant activity of synthesized compounds, we use three antioxidant assays: DPPH radical inhibition, total reducing power, and nitric oxide (NO) inhibition.

The DPPH assay is based on assessing the substances' ability to reduce the stable radical (diphenylpicrylhydrazyl) to diphenylpicrylhydrazine. The DPPH free radical, bearing an odd electron, gives a strong absorption maximum at λ = 517 nm (purple color). When the odd electron of the DPPH radical pairs with a hydrogen atom from an antioxidant, the reduced form DPPH-H is created, and the color turns from purple to yellow [36, 37].

A possible mechanism that can explain the antioxidant effect of the coumarin hydrazide derivatives is related to the keto-enol forms of the substances, the enol group being capable to easily donate the hydrogen (Figure 8) [38].

The experimental procedure for the DPPH assay was adapted from literature [27, 28, 37], only slight modifications being made. Briefly, 2.5 ml solution of DPPH (2,2-diphenyl-1-picrylhydrazyl) radical 0.1 mM in methanol was added over 0.5 ml

Figure 8. Proposed mechanism for antioxidant activity of coumarin hydrazides.

of methanolic solution of the tested compound (1 mg/ml). The absorbance of the DPPH solution at 517 nm was determined spectrophotometrically before (Acontrol) and 15 minutes after adding the solutions of the compounds (Atest), and the percentage of activity was calculated. Ascorbic acid was used as a reference compound:

% radical scavenging activity ¼ ðAcontrol � AtestÞ � 100=Acontrol

where Acontrol is the absorbance of the control sample (DPPH solution without test sample) and Atest is the absorbance of the test sample (DPPH solution + test compound).

Out of the tested compounds, the most active DPPH free radical scavengers were the coumarin hydrazide derivatives (IIIa–IIIb, IVa–IVb). The activities of IVa were similar to that of the standard, the inhibition percentage being over 90%, the introduction of sulfur atoms in the molecule having a positive influence on the scavenging potential. Compounds IIIa and IVa, containing a methyl group, were slightly more active than their analogues with propyl radical (Table 14) [19].

Fe(III) reduction is often used as an indicator of electron-donating activity. In the reducing power assay, antioxidants with electron-donating abilities reduce ferricyanide to ferrocyanide by donating an electron. The amount of ferrocyanide is monitored by measuring the formation of Perl's Prussian blue at 700 nm. Increasing absorbance at 700 nm indicates an increase in reducing ability [39]. Within this assay, EC50 values are the effective concentrations at which the absorbance is 0.5.

The solution of the test compound (0.5 ml) at different concentrations in methanol was mixed with phosphate buffer (1.25 ml, 0.2 mol/l, pH 6.6) and potassium ferricyanide 1% (1.25 ml), and the mixture was incubated at 50°C for 20 min. At the end of the incubation period, trichloroacetic acid 10% (1.25 ml) was added to the mixture and centrifuged at 3000 rpm for 10 min. The upper layer solution was collected, and 2.5 ml were mixed with distilled water (2.5 ml) and ferric chloride 0.1% (0.5 ml). The absorbance was measured after 15 min at 700 nm against a blank. The EC50 values were calculated by linear interpolation between values above and below 50% activity. Ascorbic acid was used as reference [28, 30, 36, 37].

The reducing power of the tested compounds was modest, and the results are presented in Table 15. The only substances that were moderately active were the hydrazide derivatives IIIa and IVa, but their activity was inferior to that exhibited by the reference substance (ascorbic acid) [19].

The calculated values for EC50 are shown in Table 16. This method could not be applied to compound IIIb due to the formation of an abundant precipitate in the

Compd. Extinction Ia 0.0599 Ib 0.0105 IIa 0.0234 IIb 0.0010 IIIa (1 mg/ml) 1.3432

Compd. Inhibition percentage (%)

Ia 21.4 Ib 21.2 IIa 19 IIb 19.83 IIIa 73 IIIb 53.56 IVa 95 IVb 87.67 Va 31.51 Vb 25.13

Coumarin Derivatives with Antimicrobial and Antioxidant Activities

DOI: http://dx.doi.org/10.5772/intechopen.88096

IVa (1 mg/ml) 0.6592

IVb 0.4887 Va 0.0472 Vb 0.3063

(0.8 mg/ml) 1.1237 (0.6 mg/ml) 1.0821 (0.4 mg/ml) 0.9674 (0.2 mg/ml) 0.5499

96.8

(0.8 mg/ml) 0.5900 (0.6 mg/ml) 0.4854 (0.4 mg/ml) 0.4258 (0.2 mg/ml) 0.2785

2.8261

Nitric oxide is involved in a variety of biological functions (neurotransmission, vascular homeostasis, antimicrobial and antitumor activities). NO was primarily

process.

151

Table 15.

Ascorbic acid 1 mg/ml

Ascorbic acid 1 mg/ml

DPPH inhibition percentages of compounds I–V (1 mg/ml).

Table 14.

The reducing power of compounds I–V.

#### Coumarin Derivatives with Antimicrobial and Antioxidant Activities DOI: http://dx.doi.org/10.5772/intechopen.88096


#### Table 14.

of methanolic solution of the tested compound (1 mg/ml). The absorbance of the DPPH solution at 517 nm was determined spectrophotometrically before (Acontrol) and 15 minutes after adding the solutions of the compounds (Atest), and the percentage of activity was calculated. Ascorbic acid was used as a reference compound:

Proposed mechanism for antioxidant activity of coumarin hydrazides.

Figure 8.

Phytochemicals in Human Health

150

% radical scavenging activity ¼ ðAcontrol � AtestÞ � 100=Acontrol

where Acontrol is the absorbance of the control sample (DPPH solution without test sample) and Atest is the absorbance of the test sample (DPPH solution + test compound). Out of the tested compounds, the most active DPPH free radical scavengers were the coumarin hydrazide derivatives (IIIa–IIIb, IVa–IVb). The activities of IVa were similar to that of the standard, the inhibition percentage being over 90%, the introduction of sulfur atoms in the molecule having a positive influence on the scavenging potential. Compounds IIIa and IVa, containing a methyl group, were slightly more active than their analogues with propyl radical (Table 14) [19]. Fe(III) reduction is often used as an indicator of electron-donating activity. In the reducing power assay, antioxidants with electron-donating abilities reduce ferricyanide to ferrocyanide by donating an electron. The amount of ferrocyanide is monitored by measuring the formation of Perl's Prussian blue at 700 nm. Increasing absorbance at 700 nm indicates an increase in reducing ability [39]. Within this assay, EC50 values are the effective concentrations at which the absorbance is 0.5. The solution of the test compound (0.5 ml) at different concentrations in methanol was mixed with phosphate buffer (1.25 ml, 0.2 mol/l, pH 6.6) and potassium ferricyanide 1% (1.25 ml), and the mixture was incubated at 50°C for 20 min. At the end of the incubation period, trichloroacetic acid 10% (1.25 ml) was added to the mixture and centrifuged at 3000 rpm for 10 min. The upper layer solution was collected, and 2.5 ml were mixed with distilled water (2.5 ml) and ferric chloride 0.1% (0.5 ml). The absorbance was measured after 15 min at 700 nm against a blank. The EC50 values were calculated by linear interpolation between values above and below 50% activity. Ascorbic acid was used as reference [28, 30, 36, 37]. The reducing power of the tested compounds was modest, and the results are presented in Table 15. The only substances that were moderately active were the hydrazide derivatives IIIa and IVa, but their activity was inferior to that exhibited

by the reference substance (ascorbic acid) [19].

DPPH inhibition percentages of compounds I–V (1 mg/ml).


#### Table 15.

The reducing power of compounds I–V.

The calculated values for EC50 are shown in Table 16. This method could not be applied to compound IIIb due to the formation of an abundant precipitate in the process.

Nitric oxide is involved in a variety of biological functions (neurotransmission, vascular homeostasis, antimicrobial and antitumor activities). NO was primarily


(10 mM) and 0.5 ml phosphate buffer saline (pH = 7.4) were added to each tube. The solutions were incubated at 25°C for 150 minutes. After the incubation, over 0.5 ml of the incubated solution 1 ml of sulfanilic acid 0.33% was added, and the mixture was left for 5 min at room temperature; after this period of time, 1 ml naphthylethylene diamine (NED) HCl reagent 0.1% was added, and the solutions were incubated for another 30 min. The absorbance was measured at 546 nm [38]. Most of the investigated compounds were moderate NO inhibitors (Table 17) [19].

Coumarin Derivatives with Antimicrobial and Antioxidant Activities

DOI: http://dx.doi.org/10.5772/intechopen.88096

We have synthesized some coumarin derivatives starting from 4-methyl-7 hydroxycoumarin with antimicrobial and antioxidant activities to different reaction steps. The IR and NMR spectra of the synthesized compounds were in accordance with the assigned structures. All the synthesized compounds were very active against S. aureus ATCC 25923, and they exhibited excellent antibacterial activity against S. lutea. The presence of the methyl group attached to the coumarin ring in the fourth position had a positive influence on the anti-Pseudomonas ATCC 27853 potential of the compounds, all the tested 4-propyl-coumarin derivatives being inactive. Against the investigated Candida strains, all tested compounds were found to be very active. The introduction of the sulfur atom appeared to be correlated with a good anti-Candida activity. The most active DPPH free radical scavengers were the coumarin hydrazide derivatives, the activities of these being similar to that of the standard. The reducing power of the tested compounds was modest, and only the hydrazide derivatives were moderately active. Most of the investigated

The interest in the synthesis of coumarin derivatives has been gaining importance over the last decades, reflecting the importance of such compounds in both medical and chemical research. Future goals for this field of research include the discovery, synthesis, and development of compounds which display increased potency, as well as fueling structure–activity relationship studies aimed at understanding the modes of action of the most biologically active members of these

Although coumarin is a simple molecule and many of its derivatives have been known for more than a century, it continues to maintain the interest of researchers being a plentiful source of potential drug candidate because of their significant

4. Concluding remarks

compounds were moderate NO inhibitors.

The authors declare no conflict of interest.

classes of products.

therapeutic potential.

Conflict of interest

153

#### Table 16.

The calculated values of EC50.

described as a regulator of vascular tones in the cardiovascular system. Beyond this function it can prevent platelet activation, limit leukocyte adhesion to the endothelium, and regulate myocardial contractility, and it is involved in immune system reactions.

Despite the possible beneficial effects of NO, it also contributes to oxidative damage. In general, the overwhelming production of NO contributes to the pathogenesis of both acute and chronic inflammatory processes, and NO has been recognized as one of the main signaling molecules involved in these processes [23, 40]. Therefore, compounds that act like nitric oxide inhibitors have beneficial effects.

The NO inhibition assay is based on the diazotization of sulfanilic acid at acid pH by nitric oxide. The reaction product is subsequently coupled stoichiometrically with N-(1-naphthyl)ethylenediamine, forming a colored azo compound which is measured spectrophotometrically at a peak absorbance of 548 nm [36].

0.5 ml of the tested coumarin derivative solution, as well as ascorbic acid (standard compound), was taken in separate tubes, and 2.0 ml of sodium nitroprusside


Table 17. NO inhibition activity of compounds I–V. Coumarin Derivatives with Antimicrobial and Antioxidant Activities DOI: http://dx.doi.org/10.5772/intechopen.88096

(10 mM) and 0.5 ml phosphate buffer saline (pH = 7.4) were added to each tube. The solutions were incubated at 25°C for 150 minutes. After the incubation, over 0.5 ml of the incubated solution 1 ml of sulfanilic acid 0.33% was added, and the mixture was left for 5 min at room temperature; after this period of time, 1 ml naphthylethylene diamine (NED) HCl reagent 0.1% was added, and the solutions were incubated for another 30 min. The absorbance was measured at 546 nm [38].

Most of the investigated compounds were moderate NO inhibitors (Table 17) [19].
