Nanomedicine

Ranjita Misra , Sanjeeb K. Sahoo , in Methods in Enzymology, 2012

7.3 Determination of minimum inhibitory concentration and minimum bactericidal concentration of doxycycline

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are determined by the microdilution method ( Wagenlehner et al., 2006). Briefly, DH5α bacterial culture containing 0.5 McFarland (1.5   ×   108 colony forming units/ml) of organisms in Luria broth is added to various concentrations of doxycycline and doxycycline-loaded nanoparticles ranging from 0.1 to 8   μg/ml. The MIC concentration of doxycycline is defined as the lowest concentration inhibiting visible growth of bacteria after overnight incubation of above cultures at 37   °C. The MBC is measured by subculturing the broths used for MIC determination onto fresh agar plates. MBC is the lowest concentration of a drug that results in killing 99.9% of the bacteria being tested. The MIC or the lowest concentration inhibiting visible growth of bacteria is found to be 6   μg/ml in case of native doxycycline and 4   μg/ml in case of doxycycline-loaded nanoparticles in our study. MBC is found to be 8 μg/ml for native doxycycline and 6 μg/ml for doxycycline loaded nanoparticles for DH5α bacterial culture. This suggests that a higher concentration of drug is required to kill bacteria completely. However, it is noteworthy that even though 8   μg/ml concentrations of native doxycycline killed 99.9% of bacteria in our study, the microbes multiplied when transferred to fresh medium, indicating that even at a higher dose the antibiotics are not able to inhibit growth of bacteria completely. The native drug gradually loses its effect after 24   h, and bacteria which escaped drug action can multiply at a faster rate when given suitable condition. Therefore, a sustained release of a formulation is required which can control the growth of bacteria for a longer period of time. However, it is worth mentioning that MIC and MBC values are less in doxycycline-loaded nanoparticles than that of native doxycycline in our study. The reason may be the better penetration of smaller nanoparticles into the bacterial cells and better delivery of doxycycline to its site of action.

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Designing and testing single tablet for tuberculosis treatment through electrospinning

Ibrahim A. Hassounah , ... Roop L. Mahajan , in Fabrication and Self-Assembly of Nanobiomaterials, 2016

11.2.3.3 MIC and MBC tests

Minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) tests are used often to determine the activity of the drug on certain species of bacteria. MIC is defined as the lowest concentration of antimicrobial or drug that will inhibit the visible growth of bacteria after overnight incubation ( Levison, 2004), while MBC is the lowest concentration of antibacterial agent required to kill a particular bacterium (Wiegand et al., 2008).

The MIC test is done by the use of both broth dilution assay as demonstrated by Wiegand et al. (2008). For each drug, a broth plate with 96 wells (8 rows×12 columns) was inoculated with different concentrations of the drugs, bacteria, and media (Figure 11.10). The antituberculosis drugs used were diluted by a two-fold method; starting from 50 to 2.44×10−2  µg/ml. The drugs were dissolved in water (from rows B–D) and THF (rows E–G) and mixed with bacterial suspension of M. avium (McFarland standard of 0.5, approximated cell density of r 1×108  CFU/ml) and the media Middlebrook 7H9 Broth. The first row was kept as control and it contained only media without M. avium or drug. The last row (row H) contained only media and bacterial suspension without any drugs. The plate was then incubated at 35   °C for 1 week. The MIC values are determined in broth dilution assay by seeing the first well which contains clear solution without observable turbidity resulting from bacterial growth.

Figure 11.10. Schematic illustration showing the setup of the broth dilution assay.

 Republished from Hassounah et al. (2014).

For the MBC test, the agar plate assay was used. The diluted drugs and the bacterial suspension in each well of the broth plate were inoculated using a cathra replicator with a 3-mm pin. First the cathra replicators were sterilized on flame for 1   min and then they were cooled down to room temperature before use. Thereafter, the cathra replicators were immersed in the bacterial suspension in each well and 5   µg of the sample were inoculated onto the agar plate containing the media Middlebrook 7H10 Agar. The agar plate was then incubated at 35   °C for 1 week, which allowed the M. avium to grow on the agar media. The MBC values are determined by observing the first clear area on the agar plate that has no observable bacterial growth.

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Alkaloids From Apocynaceae

Abhijit Dey , ... Madhubrata Chaudhury , in Studies in Natural Products Chemistry, 2017

Cryptolepis Alkaloids

Neocryptolepine from the Cr. sanguinolenta root bark inhibited the yeast Candida albicans with MIC and MBC values of 62.5 and 250  μg/mL, respectively. Cryptolepine HCI inhibited Epidermophyton floccosum, Trichophyton rubrum, and Microsporum canis with MIC values of 30   μg/mL (against E. floccosum and T. rubrum) and 4   μg/mL (against M. canis). Moreover, the total alkaloid fraction inhibited M. canis with IC50 value of 0.2   μg/mL [141]. Cryptolepine from C. sanguinolenta roots inhibited C. albicans and Saccharomyces cerevisiae with MIC values ranging over 5–20   μg/mL against S. cerevisiae strains and MIC   =   40–160   μg/mL for Candida and other stains[142].

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Recent Progress in the Chemistry of Pandanus Alkaloids

Mario A. Tan , Hiromitsu Takayama , in The Alkaloids: Chemistry and Biology, 2019

4 Pharmacology of the Pandanus Species

Investigation of antibacterial activity using the microwell assay for known alkaloids pandamarilactone-1, pandamarilactone-32, pandamarilactonine-A and pandamarilactonine-B revealed that pandamarilactonine-A exhibited a minimum inhibitory concentration of 15.6   μg/mL and minimum bactericidal concentration of 31.25   μg/mL against Pseudomonas aeruginosa ATCC 27853. 7 All alkaloids showed weak to no activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Although there is a limited data on the biological activities of the Pandanus alkaloids, several pharmacological studies were conducted on the extracts of various Pandanus species. The methanolic extract of P. odoratissimus showed significant antiinflammatory activity at a dose of 100   mg/kg in carrageenan-induced acute (68%) and formalin-induced chronic (64.2%) paw edema in rats. 6 In vitro investigation of the ethanolic extract of P. amaryllifolius using cell cycle progression analysis, the Annexin V assay, and TUNEL assay showed that it mediated mitochondrial activated apoptosis pathways in MDA-MB-231 breast cancer cells. The proposed mechanism in the induction of apoptosis involved the up regulation of the tumor suppressor protein p53 and the downregulation of the inhibitor of apoptosis protein XIAP. 4 P. foetidus leaf extracts, including the methanol, petroleum ether, chloroform, and aqueous fractions, were investigated for their antidiarrheal activity and cytotoxicity. 8 The extract showed antidiarrheal activity, reducing the number of defecations and maintaining stool consistency. The methanol and chloroform fractions reduced the castor oil induced enteropooling and fluid accumulation, whereas the aqueous fraction reduced gastrointestinal motility. The brine shrimp assay for cytotoxicity showed that the chloroform extract gave an LC50 of 106.97   μg/mL.

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Emerging New Therapeutics Against Key Gram-Negative Pathogens

D. Obrecht , ... K. Dembowsky , in Annual Reports in Medicinal Chemistry, 2011

3.3 Ceragenins (CSA-13)

Ceragenins are cholic acid-derived antimicrobial agents that mimic the activity of endogenous AMPs and act via membrane depolarization [5,65]. CSA-13 (23 ) is the most potent of the ceragenin class with broad-spectrum antimicrobial activity. MBCs for 23 against E. coli (minimal bactericidal concentration (MBC)=1.5 μg/ml), K. pneumoniae (MBC   =   2.5   μg/ml), and P. aeruginosa (MBC   =   3.5   μg/ml) were described [65]. In particular, 23 shows also some activity against a MDR P. aeruginosa strain [66]. The in vitro activities of CSA-13, alone or in combination with colistin, tobramycin, and ciprofloxacin, were investigated using 50 P. aeruginosa isolates from CF patients. Synergistic interactions were mostly seen for CSA-13 in combination with colistin. Therefore, CSA-13 seems to be an interesting candidate for the treatment of P. aeruginosa strains in CF patients, alone or in combination. However, additional studies on safety, efficacy, and PK are needed [67].

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Antibacterial Potential of Diterpenoids

R.C.S. Veneziani , ... L.C. Oliveira , in Studies in Natural Products Chemistry, 2017

Diterpenes Against Periodontitis and Endodontic Infections

Periodontitis can be defined as a local infection and inflammation in tooth-supporting tissues that can result in tooth loss if untreated. It is suggested that the same bacteria involved in periodontal infections are also related to more severe systemic conditions like cardiovascular and respiratory infections, brain abscesses, diabetes, and low birth weight complications [53,95,96]. As well as caries disease, periodontitis also has a polymicrobial nature. On the tooth surfaces, for example, the main early or primary colonizers are streptococci and actinomyces. Over time, the proportions of these Gram-positive facultative anaerobic bacteria decrease, and Gram-negative anaerobes eventually become more established, especially at the interface between the teeth and gums [53].

The diterpenes and diterpene-containing natural products have also been considered very promising against bacteria associated with periodontitis. In this sense, Sangwan et al. [95] show that 4-epi-pimaric acid (Fig. 4.20; 5.1) isolated from aerial parts of Aralia cachemirica displayed antibacterial activity against different strains that are often associated with caries disease and periodontal infection. Their study included the determination of minimal inhibitory concentration (MIC) values of Gram-positive noncariogenic bacteria, such as Actinomyces viscosus (ATCC 15987; 4.00   μg/mL/13.2   μM) and Gram-negative anaerobic periodontal and supragingival pathogens like Porphyromonas gingivalis (ATCC 33277; 8.00   μg/mL/26.5   μM) and Prevotella intermedia (ATCC 25611; 8.00   μg/mL/26.5 μM). The minimal bactericidal concentration (MBC) values of the 4- epi-pimaric acid for these bacteria were two to four times higher than their respective MIC values, indicating its bactericidal effect. In a similar manner, Hernandez et al. [97] have found that ent-trachyloban-19-oic acid (Fig. 4.6; 2.27) isolated from Iostephane heterophylla was able to kill the P. gingivalis inoculum at 70.80   μg/mL/234.3   μM.

Fig. 4.20. Chemical structure of 4-epi-pimaric acid (5.1).

Oleoresin, sold in Brazil as "Copaíba oils," is extracted from plants of the genus Copaifera (Fabaceae). Chemically, the nonvolatile fractions of Copaifera oleoresins are characterized by the presence of diterpenes [98]. Our research group has concentrated efforts on the isolation of these diterpenes and on the investigation of their antibacterial potential against bacteria related to periodontal diseases. In Souza et al. [96], we described the isolation of four labdan-type diterpenes from the oleoresin of C. langsdorffii, and we have shown that (−)-copalic acid (Fig. 4.16, 4.34) displayed low minimal inhibitory and bactericidal concentration values (MIC   =   MBC   =   3.1   μg/mL/10.3   μM) against the key pathogen (P. gingivalis) involved in this infectious disease.

The interesting results obtained for (−)-copalic acid against the main pathogen related to periodontitis motivated us to conduct further investigations to better understand additional features of its activity. Time-kill curve assays performed with this metabolite against P. gingivalis revealed that this compound inhibited the growth of the inoculums in the first 12   h (bacteriostatic effect) and exerted its bactericidal effect thereafter (between 12 and 24   h). Also, (−)-copalic acid showed an additive effect when evaluated in association with chlorhexidine dihydrochloride, which is the active ingredient of several commercially available oral care products used to treat periodontal diseases. The time curve profile resulting from this combination showed that this association needed only six hours for the bactericidal effect to be noted (Fig. 4.21).

Fig. 4.21. Time-kill curve showing the reduction of time to completely kill the P. gingivalis inoculums using the association of (−)-copalic acid and clorexidine dihydrochloride.

Our research group also proposed that the use of standardized extracts based on Copaifera oleoresins can be an important strategy in the development of novel oral care products to control diseases such as periodontitis [96]. We also evaluated the oleoresin of C. reticulata (CRO) against 19 bacterial strains that can be considered the causative agents of tooth decay and periodontitis and assessed the CRO cytotoxic potential. In this study, the presence of the diterpenes ent-agathic acid 15-methyl ester (5.2), ent-polyalthic acid (5.3)—Fig. 4.22, and ent-copalic acid (Fig. 4.16; 4.34) were detected through HPLC-MS/MS [53].

Fig. 4.22. Chemical structures of ent-agathic acid 15-methyl ester (5.2) and ent-polyalthic acid (5.3).

In this work, antimicrobial assays also included determination of MIC and MBC, determination of the minimum inhibitory concentration of biofilm (MICB50), time-kill assay, and checkerboard dilution. Through these assays, it was possible to determine that CRO MIC and MBC values ranged from 6.25 to 200.00   mg/mL and that CRO took 4   h to show its bactericidal activity against Fusobacterium nucleatum, S. mitis; 6   h against Prevotella nigrescens; 12   h against Porphyromonas gingivalis, L. casei; and finally 18   h against S. salivarius and S. mutans [53].

The endodontic infection can be defined as the infection of the canal root system, and the chemomechanical preparation of this canal using antimicrobial agents constitutes the initial step in endodontic treatment [99]. The genera Peptostreptococcus, Prevotella, Porphyromonas, Fusobacterium, Bacterioides, and Actinomyces are often related to this infection and our research group has also investigated the potential of diterpenes against such microorganisms [100,101]. We have tested pimarane-type diterpenes (Fig. 4.16, 4.25–4.30) originated from V. arenaria as well as the dehydroabietic acid (Fig. 4.13, DA) isolated from oleoresin of Pinus elliottii against a panel of representative microorganisms responsible for dental root canal infections [100,101].

Among all tested compounds obtained from V. arenaria, pimaradienoic acid (Fig. 4.16, 4.26) was shown to be the most active by displaying MBC values between 1.0 and 10.0   μg/mL against P. gingivalis, P. nigrescens, P. intermedia, P. buccae, B. fragilis, A. naeslundii, P. micros, and A. actinomycetemcomitans. Moreover, this study of such diterpenes also allowed us to propose some structure-activity relationship (SAR) considerations about the antibacterial activity of the diterpenes, which are discussed in the next section.

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Medicinal Chemistry Approaches to Tuberculosis and Trypanosomiasis

Steven J. Berthel , ... Nader Fotouhi , in Annual Reports in Medicinal Chemistry, 2019

5.1 Inhibitors of ClpP1P2 protease complex

Recently, a series of boronate inhibitors of ClpP1P2 have been discovered based on the successful proteasome inhibitor bortezomib. A systematic search for the substrate specificity of the Mtb ClpP1P2 led to the discovery of dipeptide and tripeptide boronates with submicromolar ClpP1P2 peptidase activity, 47 as exemplified by compounds in Table 7; Ac-Pro-Lys-boroMet (34), Ac-His-Lys-boroMet (35), and Ac-Ala-Lys-boroMet (36). N-terminal capping with a picolinoyl or 3,5-difluorophenyl acetyl groups further enhanced activity leading to compound 38 with a Ki of 65   nM. The on-target activity of the peptide boronates was demonstrated by the inhibition of degradation of GFP-ssrA in Mtb. The boronates, however, exhibited modest antibacterial activity with MIC50s in the low micromolar range. It is likely that peptidyl boronates may have limited cell permeability or suffer from partial instability inside Mtb.

Table 7. SAR of the boronate inhibitors of the ClpP2P2 protease.

Compound Inhibitor IC50 (μM) MIC50 (μM)
34 Ac-Pro-Lys-boroMet 0.46 12
35 Ac-His-Lys-boroMet 0.78 6
36 Ac-Ala-Lys-boroMet 0.8 3
37 N-(Picolinoyl)-Trp-Lys-boroMet 0.18 3
38 N-(2-(3,5-Difluorophenyl)acetyl)-Lys-boroMet 0.065 6

More recently, another series of boronate derivatives were prepared by modifying the P1 and P2 sites of the bortezomib core structure, and led to the identification of compounds 40 and 41 (Table 8) with good protease activity, MIC50, as well as encouraging selectivity over human proteasome. 48 The MBC99.9 (minimum bactericidal concentration required to kill 99.9% of the bacterial population, i.e., to induce a 1000-fold kill) was 50   μM against M. tuberculosis. Compound 41 given i.v. at a dose of 10   mg/kg exhibited a mean elimination half-life of 3.7   h, a moderate plasma clearance of 0.5   l/h/kg, and volumes of distribution at steady state (Vss) and during the terminal phase (Vz) of 2.1 and 2.5   L/kg, respectively. Given orally, the bioavailability was 14%, and at 100   mg/kg Cmax of 6.9   μg/mL and AUC0–t(last) of 22.8   μg h/mL were achieved suggesting that this series of boronates may be ultimately optimized to achieve plasma and tissue level required to see in vivo efficacy.

Table 8. SAR of boronates at P1 and P2 subsites.

Compound P1 P2 IC50 for ClpP1P2 (μM) MIC50 (μM) MIC90 (μM) IC50 for HepG2 proteasome Selectivity ↑ over 39
39
H 6   ±   0.5 4.3   ±   1.3 12   ±   0.5 0.005 1
40
7.5   ±   4.5 0.8 3 0.53   ±   0.1 107
41
1.5   ±   0.5 2.5   ±   1.3 6   ±   2.8 0.37   ±   0.1 74

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Microbiological Quality in Cosmetics

Gabriel A. March , ... Raúl Ortiz de Lejarazu , in Analysis of Cosmetic Products (Second Edition), 2018

Bacterial Resistance in Cosmetics

The modes of action of preservatives are much broader than those of antibiotics used in clinical settings. The mode of action of antibiotics is known at the molecular level because they act via specific biochemical reactions. However, preservatives act in a more general way by denaturing cellular proteins or by affecting membrane permeability; in this way, transport of nutrients and energy generation are blocked (Brannan, 1995).

There are some methods for determining the effectiveness of antimicrobial preservatives in cosmetics. One of them is the challenge test. However, to select adequate preservatives to add into cosmetic products, the susceptibility of bacteria commonly isolated from cosmetic products and manufacturing plants to preservatives should be previously determined. Despite the importance of these studies, as of this writing no standard method for evaluating the susceptibility of bacteria to preservatives can be found. Nevertheless, the minimum inhibitory concentration (MIC) method, described for antimicrobial drugs in clinical microbiology, can be considered as a useful approach for studying the phenotypic behaviour of bacteria when incubated in the presence of preservatives (Mokhtari, 2008 ). In this case, MIC is defined as the lowest concentration of preservative at which the growth of a tested microorganism is completely inhibited. The minimum bactericidal concentration (MBC) method, rarely used in clinical microbiology, can also be used to evaluate the susceptibility of bacteria to antibiotics. The MBC is defined as the lowest concentration of antibacterial agent that reduces the viability of the initial bacterial inoculum by ≥99.9% (French, 2006). The application of these methods would enable one to select the most suitable preservatives for a specific cosmetic product and, moreover, it could be possible to approximate the preservative concentration suitable for avoiding microbial growth.

The problem of bacterial resistance to preservatives in cosmetic products should be focused on the group non-fermenting gram-negative rods, which include the species P. aeruginosa and B. cepacia complex. P. aeruginosa is the most frequently isolated bacterium from recalled contaminated cosmetic products in the EU and the United States (Lundov and Zachariae, 2008; Wong et al., 2000) because of its resistance to some of the preservatives commonly used in cosmetics. Ferrarese et al. (2003) noted that, against the preservatives imidazolidinil urea, dimethylol dimethyl hydantoin, methylisothiazolinone and parabens mix in phenoxyethanol, the strains of P. aeruginosa isolated from contaminated cosmetic products and industrial plants had enhanced resistance compared to collection bacterial strains, which have not been in contact with preservatives. Many publications have demonstrated that Pseudomonas spp., via progressive subculture in the presence of sublethal concentrations of the preservatives benzalkonium chloride (Mc Cay et al., 2010), phenoxyethanol (Abdel Malek and Badran, 2010) and isothiazolinone biocides (Brozel and Cloete, 1994; Winder et al., 2000), are able to develop resistance to them. Moreover, Mokhtari (2008) calculated the MICs of parabens and isothiazolinones in S. aureus, E. coli, P. aeruginosa, C. albicans and Aspergillus niger and noted that among the microorganisms tested, P. aeruginosa provided the highest MICs. Regarding B. cepacia complex, Rushton et al. (2013) calculated the MIC and MBC of eight commercially available preservatives (benzisothiazolinone, benzethonium chloride, dimethylol dimethyl hydantoin, methylisothiazolinone, methylisothiazolinone/chloromethylisothiazolinone at the proportion 3:1, methylparaben, phenoxyethanol and sodium benzoate) in 83 strains. They found that, for six of the eight preservatives evaluated, MIC and/or MBC values were above the maximum level permitted for use in personal care products. They also observed that maximum permitted levels of dimethylol dimethyl hydantoin (0.3%) and phenoxyethanol (1%) had the greatest activity against B. cepacia complex, inhibiting and killing all strains tested. Rose et al. (2009) identified two strains belonging to B. cepacia complex with chlorhexidine MBCs of 1000   mg/L and 31 B. cepacia complex strains with cetylpyridinium chloride MBCs in excess of 1000   mg/L. It is important to note that chlorhexidine and cetylpyridinium chloride are used in a variety of commercial products in concentrations ranging from 0.1% to 4% (1000–40,000   mg/L). Hence, the lowest concentration of chlorhexidine and cetylpyridinium used in cosmetic products may not be enough to kill strains of B. cepacia complex.

Antibacterial agents and antibiotics share the same resistance problem: resistance will certainly increase as the drug persists, especially at low levels for long periods of time. In clinical settings, owing to misuse and overuse of antibiotics, bacteria resistant to all standard antimicrobial agents have emerged (Sopirala et al., 2010). Preservatives should be used at optimal concentrations, not only to kill bacteria and avoid the emergence of bacterial strains resistant to preservatives, but also to exclude the occurrence of adverse reactions such as skin irritation. Moreover, preservatives should be compatible with all ingredients of a cosmetic product and its packaging and should be active in the complete formulation and stable over the range of pH values (Mokhtari, 2008). Thus, a major challenge for cosmetics microbiology is to ensure that preservatives are able to kill all bacteria that might be present in the cosmetic and do not lead to adaptive antimicrobial resistance. For a better understanding of bacterial resistance mechanisms to preservatives, some papers about phenotypic changes in Enterobacter gergoviae and B. cepacia in the adaptive response to preservatives have appeared in the literature (Periame et al., 2015a,b; Rushton et al., 2013). It is important to make progress in this field and discover the molecular mechanism of how bacteria become resistant to preservatives.

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Biologically Active Compounds from the Genus Centaurium s.l. (Gentianaceae)

Branislav Šiler , Danijela Mišić , in Studies in Natural Products Chemistry, 2016

Antimicrobial Actions

The most intensively studied biological activities of various extracts of centauries are, naturally, their antimicrobial activities against a vast number of both Gram-positive and Gram-negative bacteria species/strains and fungi as well. Thus, Šiler et al. [83] have reported that C. pulchellum methanol extracts have shown antibacterial activity with MICs (minimum inhibitory concentrations) and MBCs (minimum bactericidal concentrations) in the range of 0.05–0.2 mg/mL, depending on the bacterial species and have possessed similar effects as the commercial antibiotics streptomycin and ampicillin. Antifungal activities of the same extracts have ranged in MIC of 0.1–1.0 mg/mL and MFCs (minimum fungicidal concentrations) of 1.0–2.0 mg/mL possessing antifungal potential of the same level as the commercial fungicides bifonazole and ketoconazole against Aspergillus fumigatus, yet up to 10 times lower against Aspergillus versicolor, Aspergillus niger, and Penicillium funiculosum. In the assay against Trichoderma viride, plant extracts have shown 10 times higher MICs and three times higher MFCs than ketoconazole.

Similarly, Šiler et al. [59] have published that the methanol extracts of five centaury species: C. erythraea, C. littorale, C. tenuiflorum, C. pulchellum, and S. spicata have had powerful antibacterial effects with MICs range between 0.05 and 0.25 mg/mL and MBCs for 0.1–0.5 mg/mL in more or less the same range as the commercial antibiotic. High antimicrobial activity of the methanol extract of various centaury species is therewith ascribed to secoiridoid glycosides (monoterpenes) and phenolics, including flavonoids and xanthones. Plant extracts have also shown great antifungal activity with inhibitory (MIC) activity at 0.1–0.4 mg/mL and fungicidal (MFC) at 0.2–0.6 mg/mL. Moreover, the activity of C. erythraea essential oil to Escherichia coli has been reported to be almost identical to the inhibition zones of ampicillin with Gram-positive bacteria demonstrating increased resistance to the C. erythraea oil, compared to Gram-negative ones [42].

As Šiler et al. [59] have noticed, above-ground plant parts of each centaury species studied therewith could be recommended for the human usage, as a rich source of natural antioxidants but also as a strong antimicrobial treatment. The authors also presume that these plants, taken in food and beverages, may support endogenous defense mechanisms and protection of human body against oxidative stress and/or pathogenic bacteria and fungi. In words of food safety and food preservation, as additives in food and beverages, these species could possibly prevent/control growth of pathogens and thus improve their preservation. Furthermore, results published in this study [59] indicate that C. erythraea, which presents the official drug Centaurii herba and natural source of food flavoring, could be safely substituted by other centaury species characterized in the study.

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Bioactive Natural Products

Remziye Aysun Kepekçi Burcu Yener İlçe Sibel Demir Kanmazalp , in Studies in Natural Products Chemistry, 2021

Tests to determine the antimicrobial effect of biomaterials

After the development of a newly synthesized wound-dressing material, it should be shown that the biomaterial is safe for human usage, and various methods are used to assess the antimicrobial activity of the newly synthesized medicinal biomaterial compounds manufactured for use in health care. Here we provide a summary of these methods.

Determination of minimum inhibitory concentration (MIC)

The lowest concentration of the tested antimicrobial agent (in this case, biomaterials) that leads to a visible growth inhibition of a microorganism (in this case, wound-associated bacterial strains) after overnight incubation is known as the MIC [109]. The mentioned concentration is bacteriostatic, which means it's not necessary to kill them, but enough just to stop their reproduction. The dilution methods used to determine MICs—either agar dilutions on agar plates or broth dilutions in broth—are considered the optimum approach in antimicrobial susceptibility testing. The most common wound-associated bacterial strains are gram-negative Escherichia coli, Pseudomonas aeruginosa, and Morganella morganii, and gram-positive Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, Streptococcus agalactiae, and Corynebacterium striatum [49,109].

Determination of minimum bactericidal concentration (MBC)

This refers to the lowest concentration of the tested antimicrobial agent (in this case, biomaterials) that is needed to kill a particular bacterium (in this case, wound-associated bacterial strains) under specific conditions and over longer time periods, such as 24 h. MBC is determined after the broth dilution phase of a MIC test has been completed. Antimicrobial agents are usually regarded as bactericidal if the MBC is no more than four times the MIC [109,110].

Kirby-Bauer test

This test, known also as the disk-diffusion method or agar diffusion test, is still the most widely used approach to the assessment of susceptibility of bacteria to antimicrobials. Adjusted concentrations of the bacterial suspensions are spread over the surface of an appropriate agar, such as Mueller-Hinton (MH) or Nutrient agar, using a spreader. The most commonly used test strains are gram-positive S. aureus and B. subtilis, and gram-negative E. coli and P. aeruginosa. Disks of filter paper are soaked in solutions containing different known concentrations of an antimicrobial compound (plant extracts) and placed on the surface of a Mueller-Hinton (MH) agar plate. The antibacterial activity is the "zone of inhibition" measured around the disk in millimeters [39].

Agar well-diffusion method

The agar well-diffusion method is similar to the disk-diffusion method, which is preferred when the antimicrobial agent is more condense and nonoil. In this assay, instead of placing a disk, small wells are punched inside the agar using a sterilized borer (6–8   mm), and the antimicrobial agent solution (wound-dressing material suspension) at the desired concentration is poured into the wells on each plate. The growth inhibition of the microbial strain, as an indicator of antimicrobial activity, is measured using a caliper and is assessed from the diameter of the inhibition zone surrounding the well [111].

Zone of inhibition

The aim of this method is the same as the two methods above, with the only difference being that the dressing material is placed gently directly onto the agar surface using sterile forceps [24].

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