**3.4. Evaluation of antibacterial activity of host-guest complex**

**3.3. Evaluation of antioxidant activity of host-guest complex**

tions ranged from 5 to 50 μg/mL.

276 Cyclodextrin - A Versatile Ingredient

radicals [110].

Antioxidant activity was evaluated in terms of DPPH scavenging capacity (%) of free and encapsulated guava leaf oil compared to a synthetic chemical antioxidant (BHT) at concentra-

It was established that the components responsible for the antioxidant activity of guava leaf oil are limonene, α-pinene and β-caryophyllene [146]. While limonene has a moderate antioxidant activity [147], β-caryophyllene and α-pinene show weak and moderate DPPH scavenging activity, respectively [146, 147]. Unfortunately, the encapsulated guava leaf oil gave slightly lower DPPH scavenging activity than that of the free guava leaf oil. This could be because HPβCD blocks the functional groups of the active compounds that react with DPPH

In the case of yarrow oil carvacrol as a major component shows strong antioxidant activity (72% DPPH scavenging at 50 μg/mL). The most effective antioxidants usually contain aromatic or phenolic rings, which interrupt the free radical chain reaction by donating H• to the free radicals [148]. The encapsulated yarrow oil gave slightly lower antioxidant activity than the activity of the free yarrow oil. It was a result of the HPβCD was blocking the functional groups of active compounds during reacting with DPPH radicals [110]. However, the encap-

Black pepper oil shows antioxidant activity with 54% DPPH scavenging (50 μg/mL black pepper oil) (**Figure 5**). It was established that the components responsible for the antioxidant activity are β-caryophyllene, limonene and α-pinene [146]. β-caryophyllene, a major component of black pepper oil, was found to give a weak DPPH scavenging activity [146]. Limonene, a minor composition, has been reported to give a moderate antioxidant activity and another component, α-pinene, also possesses a moderate antioxidant property [147]. It should be

However, the inclusion complexes have been reported to increase the stability of the essential oils [13, 14]. After exposure to sunlight, the DPPH scavenging of free guava leaf oil drastically decreased around 43–54% at all tested concentrations (5–50 μg/mL), which was likely due to limonene and pinene sensitive to sunlight [149]. Then, the inclusion complexation of guava leaf oil with HPβCD could protect the active components against the effect of light. In effect, after sunlight exposure, the DPPH radical scavenging capacity of the encapsulated guava leaf

Similar results were found for yarrow essential oil, where DPPH radical scavenging (with concentration range from 5 to 35 μg/mL of essential oil) decreased around 41–51% after exposure to sunlight for 12 h. The yarrow oil with higher concentration range (40–50 μg/mL) exhibited lower loss of DPPH radical scavenging (36–37%). Obviously, as in the previous case, the encapsulation of yarrow oil in HPβCD could protect the active components against the effect of sunlight. The complexation with HPβCD improved the stability of yarrow oil by 27–30% in

The DPPH radical scavenging capacity of black pepper oil drastically decreased after 12 h exposure to sunlight (**Figure 4**). At the sample concentration range of 5–25 μg/mL, the DPPH

sulation has been reported to increase the stability of the essential oils [13, 14].

noted that free HPβCD did not show antioxidant activity.

oil was more stable than the free guava leaf oil by 26–38%.

a similar range that guava leaf oil (26–38% -*vide supra*-).

**Table 4** shows minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of essential oil for *Staphylococcus aureus* and *Escherichia coli*.

Guava leaf oil displayed antibacterial activity against both bacteria with MIC value of 500 μg/ mL, that could be attributed to guava leaf oil monoterpenes (such as limonene) which have been found to play efficient role in antimicrobial activity via membrane structures increasing membrane fluidity and permeability [150]. Pure limonene was reported to give antimicrobial activity against *S. aureus* and *E. coli* with MIC values of 8.0 and 10.0 μg/mL, respectively [151].

The antibacterial activity of guava leaf oil was improved after encapsulation in HPβCD by 4 and 2 times against *S. aureus* and *E. coli*, respectively. It has been reported that inclusion complexes with HPβCD could increase aqueous solubility of the encapsulated guests, thus improving the antimicrobial efficiency of essential oils at lower concentrations [99] due to a better accessibility of the active compounds to cells [111].

Yarrow oil exhibited antibacterial activity against *S. aureus* and *E. coli* with the MIC values of 250 μg/mL and 500 μg/mL, respectively. The antimicrobial activity of yarrow essential oil might be because its oxygenated phenolic compounds, such as carvacrol and thymol, have been reported to give strong antimicrobial activity. These compounds were found to increase membrane permeability and membrane disruption of microbial cells (*Pseudomonas aeruginosa* and *S. aureus*) [152]. Antimicrobial potential of oxygenated phenolic compounds, were also reported in the literature [153–157]. In addition, *S. aureus*, a representative for Gram-positive bacteria, was more sensitive to tested samples than *E. coli*. This was because the external surface of outer membrane of *E. coli* that composes of lipopolysaccharides and proteins is more


\* Values were based on the actual concentrations of essential oil encapsulated in the HPβCD (calculated from encapsulation efficiency).

**Table 4.** Minimum inhibitory and bactericidal concentration (MIC, MBC) against *Staphylococcus aureus* and *Escherichia coli* for both free and encapsulated essential oil.

tolerate to the tested samples, and the O-side chains of the lipopolysaccharides of *E. coli* has a hydrophilic surface protecting the hydrophobic molecules to enter the bilayer [146].

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The antibacterial efficacy of yarrow oil was much improved after encapsulated in HPβCD by 4 and 8 times against *S. aureus* and *E. coli*, respectively, while antibacterial activity of black pepper oil was improved by 4 times against both *S. aureus* and *E. coli*. As quote above, inclusion complex formation with HPβCD could increase aqueous solubility and improve antimicrobial efficacy at lower concentrations of encapsulated compounds [99]. As the primitive sites for antimicrobial action were found at the cell membrane and inside the cytoplasm, HPβCD may enhance the accession of essential oils to these regions by improving water solubility of essential oils [111].
