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Article

Assessment of Phenolic Content, Antioxidant and Anti-Aging Activities of Honey from Pittosporum undulatum Vent. Naturalized in the Azores Archipelago (Portugal)

by
Soraia Santos
1,
Miguel Maia
2,
Ana Barros
1 and
Irene Gouvinhas
1,*
1
Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB)/Institute for Innovation, Capacity Building and Sustainability of Agri-Food Production (Inov4Agro), University of Trás-os-Montes and Alto Douro (UTAD), Quinta de Prados, 5000-801 Vila Real, Portugal
2
APISMAIA, Produtos & Serviços, Rua Almirante Reis, 91-A-2, 4490-463 Póvoa de Varzim, Portugal
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(3), 1788; https://doi.org/10.3390/app13031788
Submission received: 6 January 2023 / Revised: 25 January 2023 / Accepted: 28 January 2023 / Published: 30 January 2023
(This article belongs to the Special Issue Food-Related Bioactive Compounds: Extraction, Bioavailability, and Applications)
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Versions Notes

Abstract

Pittosporum undulatum Vent. is an invasive species scattered across all of the Azores’s Islands. Identifying processes to obtain economic returns from the plant is of great interest. This work aims to evaluate honey from P. undulatum from the Azores by determining its phenolic content and biological activities, to enhance its value and equate its potential applications in the food, cosmetic, and/or pharmaceutical industries. Herein, the phenolic content and antioxidant capacity were evaluated by spectrophotometric methods. Furthermore, and for the first time, anti-aging capacity was determined in honey samples. The total phenols content revealed values from 20.82 to 112.13 mg GA/100 g, from 10.25 to 103.26 mg GA/100 g for ortho-diphenols, and from 2.94 to 40.96 mg CAT/100 g for flavonoids content. Regarding the antioxidant capacity, the values ranged from 0.05 to 2.27 mmol Trolox/100g. Concerning the anti-aging capacity, promising results were obtained, namely for tyrosinase inhibitory capacity, with values ranging between 4.36% and 9.37%, while the values of elastase inhibitory capacity ranged from 37.52% to 45.88%. This study allowed us to understand the phytochemicals and biological activities of honey from P. undulatum, enhancing the possible health benefits, namely for potential anti-aging treatment, valorizing, at the same time, a national food product.

1. Introduction

Pittosporum undulatum Vent. belongs to the genus Pittosporum and the family of Pittosporaceae. This genus covers 200 species scattered worldwide, is native to Australia, and has been introduced in several countries, including Portugal [1]. Indeed, this non-indigenous plant is present in all Azores archipelago, firstly to protect orange tree plantations but also due to the geographical and climatic conditions that tend to spread it uncontrollably [2,3].
Pittosporum undulatum is an evergreen tree, called Victoria box tree (Figure 1), which grows in temperate/sub-tropical climatic conditions and can achieve 4–13 m of high with wavy-edged leaves, and flowers are white-creamy and aromatic, being grouped in loose umbellate cymes [4,5].
This plant is a source of wood and compost for pineapple and honey; however, it does not significantly impact public economic activities [5]. In 2000, a research group analyzed the extracted oils of P. undulatum for potential medical properties and biological activities, namely a preliminary evaluation of the antithrombin and some antibacterial activity [7].
The management strategy and costs associated with the control and removal of invasive plants take much work to implement, requiring alternatives that lead to economic returns, such as taking advantage of the metabolites produced by invasive species while eradicating them. In this sense, there is no intention of valuing these species to delay or discourage their eradication, but rather to conduct studies on chemical composition and pharmacological application at the same time as control actions are being maintained. The honey from this species can be characterized by the particularity of having a pollen percentage of P. undulatum higher than 30% in its composition and could be used as a product for several industries, such as pharmaceutical and cosmetics, adding value to this tree [3]. Furthermore, according to the Azores government, this type of honey has a Protected Designation of Origin (PDO) with its well-established organoleptic characteristics. Its specifications were published in the Official Journal of the European Union (OJEU) Reg. no 1107/96 L 148 on 21/6/199 and modified on 12/11/2019 (2019/C 384/09) [8].
Since ancient times, honey has been used as a carbohydrate source and a natural drug which is still the case until the present time. Its recognition in light of its bioactive characteristics and flavor increases its demand as a nutraceutical food and an essential product for several industries, such as cosmetics, pharmaceuticals, and food [9,10,11].
This substance is a concentrated aqueous solution composed of carbohydrates, amino acids, minerals, organic acids, and phenolic compounds, among others [10]. However, the botanical source is a vital factor influencing honey’s composition and, consequently, its medical properties [12]. Notably, several authors reported that honey could act as a vehicle for the plant’s medical properties, making monofloral honey a highly valued food product [13,14]. Non-volatile compounds, including glycosides and phenolic compounds, can be responsible for these health benefits [15].
Phenolic compounds are secondary metabolites synthesized by plants as a response to biotic and abiotic stresses. These phytochemicals have different biological effects, such as antioxidants, antimicrobial, anti-inflammatory, and anti-aging, preventing and/or inhibiting the reactive oxygen species (ROS) that cause oxidative stress [16,17,18]. Indeed, oxidative stress and these radical species can affect the biosynthetic activity of skin cells, improving skin aging by the modulation of numerous aging-related enzymes, including tyrosinase and elastase [19]. Nowadays, one of the leading skin problems is skin aging. In this sense, its treatment and care are vital to reverse these effects [20]. Skin aging is a natural and biological process that can arise from intrinsic and extrinsic factors, including genetic changes, vitamin deficiencies, hormonal disorders, environmental toxins, UV radiation, and inadequate care [21]. Oxidative stress is also a notable factor, forming free radicals and influencing intrinsic and extrinsic aging [20,22].
Foods with enzyme inhibitory activity can be used as a clinically helpful nutraceutical and a disease-prevention functional food element [22]. Honey is increasingly used as an ingredient in cosmetics due to its moisturizing, emollient, nourishing, and antibacterial properties. It is also reported to have enzyme inhibitory capacity, such as tyrosinase, that could be explained by the presence of polyphenols and flavonoids [22,23,24,25].
Enzyme inhibitors are mainly bioactive secondary metabolites that bind to enzymes and reduce their biological and catalytic activities. Besides, the inactivation of an enzyme can decrease or eliminate pathogens or correct metabolic disorders [24].
The inhibition of human degradative skin enzymes has been used as a tool in drug discovery to correct pathological conditions, becoming fundamental in pharmacology as substitutes for the synthetic and chemical drugs currently used [21,23,24].
Tyrosinase is an enzyme involved in melanogenesis and responsible for skin pigmentation and, consequently, for the protection of damage caused by radiation, including ultraviolet (UV) radiation. However, if the enzyme is overproduced, it can result in an excessive accumulation of epidermal pigmentation, leading to several disorders, such as age spots. Elastase is also an enzyme associated with skin aging since it is the main one responsible for elastin hydrolysis, an extracellular protein (which provides elasticity and resilience) causing loss of skin firmness [19,20,23,26,27].
According to Codex Alimentarius [28], due to the rapidly expanding honey trade industry, it is touted that “rules of the road” are required to be established that consider the requirements to guarantee equal conditions, standardization, security, and transparency from production to commercialization. In this sense, to ensure honey quality, Codex Alimentarius referred to many parameters, including a maximum 5-hydroxymethylfurfural (HMF) content (40 mg/kg) which is related to honey freshness, being affected by overheating and/or storage time, since it is a chemical compound that results from the decomposition of monosaccharides or the Maillard reaction [10,29,30].
In this sense, this study aims to determine for the first time the phenolic content and some biological properties, namely the antioxidant and anti-aging capacity of monofloral honey from P. undulatum, allowing to prove if this food product can add value to an invasive species, and in addition, it can be considered as an alternative to generating an economic return to an invasive species.

2. Materials and Methods

2.1. Chemicals

The methanol (MeOH), 5-hydroxymethylfurfural (HMF), gallic acid, sodium carbonate, sodium acetate, catechin, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS•+), 6-hydroxy-2,5,7,8-tetra-methylchromone-2-carboxylic acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl (DPPH•), tyrosinase from mushroom, kojic acid, potassium phosphate, elastase from porcine pancreas, and N-succinyl-Ala-Ala-Ala-p-nitroanilide were purchased from Sigma-Aldrich (Steinheim, Germany). Folin-Ciocalteu’s reagent, sodium molybdate 2-hydrate (Na2MoO4.2H2O), and sodium hydroxide (NaOH) were acquired from Panreac (Panreac Química S.L.U., Barcelona, Spain). Aluminum chloride, sodium nitrite, and sodium hydroxide were purchased from Merck (Merck, Darmstadt, Germany).

2.2. Sampling

The sampling was constituted by six monofloral honey from P. undulatum from Ilha Terceira, Azores Archipelago (Portugal) in 2021 to accomplish this work. All kinds of honey were obtained by centrifugation and were not pasteurized.

2.3. Sample Preparation

Four grams of each honey sample was weighed, and 50 mL of MeOH/H2O (20:80, v/v) mixture was added and homogenized according to the modified method described by Kuçuk et al. [31] to determine the phenolic content and antioxidant capacity. Then, 4 mL of samples were stored for one week at 4 °C, and the remaining were stored at −80 °C.
One gram of each honey sample was weighed, with 5 mL of distilled water added and homogenized according to the modified method previously reported by Petrillo et al. [23] to determine the anti-aging inhibition. The samples were used immediately and discarded after analysis.
Four grams of each honey sample was weighed and dissolved in 10% methanol before being filtered to 0.45 μm [diameter 13 mm, polytetrafluoroethylene (PTFE)] to determine the HMF profile.
Each sample was analyzed in triplicate (n = 3) for each protocol.

2.4. Pollen Analysis

The pollen analysis was performed through the melissopalynology method [32], aiming to confirm if the analyzed honey samples are monofloral honey and which other pollen types are present that could further influence their biological properties.
The pollen grain types were counted and identified using a VWR (model BL224T1-630-2672, UK) light microscope in 1000× magnification. The botanical origin was determined based on the relative frequencies of nectariferous species. At least 300 nectariferous pollen grains per sample were counted. Pollen types from nectarless species were also recorded and counted separately.

2.5. Determination of HMF of Honey by High-Performance Liquid Chromatography—Ultraviolet Detector (HPLC-UV)

For the determination of HMF, the samples were injected into a Thermo Scientific Dionex UltiMate 3000 Series system (Thermo Fisher Scientific, Inc., Waltham, USA), composed of an RS quaternary pump, WPS-3000RS autosampler (maintained at 4 °C), TCC-3000RS column compartment (maintained at 35 °C), and a UV detector. The chromatographic separations were carried out using a C18-type column maintained at 35 °C (dimensions 4.5 × 15 mm).
The HPLC-UV technique was based on an isocratic mode with one mobile phase, solvent A (10% methanol solution), flowing at a rate of 1.0 mL/min and between 100 and 200 bar pressures. The HMF content of the samples was calculated by comparing the corresponding peak areas of the sample and those of the standard solutions, considering the dilution. The results showed a linear connection between the concentration and HMF peak area and were expressed in ppm. At the detection wavelength of 284 nm, chromatograms were acquired during the data-collecting process. A stock solution of HMF (standard) was prepared at a concentration of 1.0 mg/mL in distilled water.

2.6. Phenolic Content of Honey

The total phenols, ortho-diphenols, and flavonoids were quantified through spectrophotometric methods and adapted to 96-well microplates (Frilabo, Milheirós, Portugal), according to the method previously reported by Gouvinhas et al. and Breda et al. [33,34] to determine the phenolic content.
The content of total phenols in honey samples was determined by the Folin-Ciocalteu method. Firstly, 20 µL of each honey sample was added directly to each well, followed by 100 µL of the Folin-Ciocalteu reagent previously diluted with water (1:10 H2O) and then 80 µL of a 7.5% sodium carbonate (Na2CO3) were added. The mixtures were protected from light, for 30 min in the oven at 40–45 °C. The absorbance was read at a wavelength of 750 nm in the microplate reader (Thermo Fisher Scientific, Lisbon, Portugal).
Sodium molybdate (40 µL) was added to evaluate ortho-diphenols in the honey sample (160 µL). Afterward, the mixture was incubated at room temperature for 15 min protected from the light. The absorbance was measured at 375 nm.
For both assays, gallic acid was used as standard. The results are expressed in mg of gallic acid (GA)/100 g (g) of the sample.
The aluminum complex method was employed to determine the flavonoid content in the honey samples. Sodium nitrite (28 µL) was added to honey samples (24 µL) and incubated at room temperature for 5 min. Then, 10% aluminum chloride (28 µL) was added to the mixture. After 6 min, 1 M sodium hydroxide (120 µL) was added, and the absorbance of the mixture was measured at 510 nm after agitation for 30 s in a microplate reader. Catechin was used as standard. The results are expressed in mg of catechin (CAT)/100 g (g) of the sample.

2.7. Antioxidant Capacity of Honey

The antioxidant capacity of the honey samples was determined by DPPH (8.87 mM), ABTS (7 mM), and FRAP spectrophotometric methods, according to the methodology described by Yu et al. [33] with some modifications and adapted to microscale using 96-well microplates (Frilabo, Milheirós, Portugal). For the DPPH assay, a DPPH working solution (190 µL) was prepared and added to the honey samples (10 µL) and reacted for 15 min at room temperature. The absorbance was read at 520 nm with hydro-methanol used blank. In ABTS methodology, an ABTS working solution (188 µL) and sample dilutions (12 µL) were mixed and incubated for 30 min at room temperature. For the FRAP assay, the honey sample (20 µL) reacted with a FRAP solution (180 µL) composed of 10-volume acetate buffer (300 mM, pH = 3.6), 1-volume TPTZ (10 mM dissolved in hydrochloric acid), and 1-volume ferric chloride (20 mM in water). The mixture was maintained at 37 °C for 10 min before use. After 10 min of incubation at 37 °C, the absorbance of the mixture was read at 593 nm. Trolox was used as the standard solution. All the results are expressed in mmol Trolox (T)/100 g.

2.8. Anti-Aging Capacity of Honey

The inhibition of two enzymes, elastase, and tyrosinase, was determined for anti-aging evaluation. The elastase inhibition was determined by a colorimetric method previously described and adapted by Apraj and Pandita [20]. It was added, in a microplate, Tris-HCl buffer (0.2 mM, pH 8) (160 µL), followed by substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide (0.8 mM, in buffer) (20 µL) to the honey samples (50 µL) and the negative control (50 µL) (buffer) and reacted at room temperature protected for the light for 10 min. Then, the enzyme (1 U/mL) (20 µL) was added and incubated under the same conditions for 20 min. Afterward, the absorbance was measured at 410 nm. The percentage of inhibition was calculated using Equation (1).
Inhibition   % = A b s 410 c o n t r o l − A b s 410 s a m p l e A b s 410 c o n t r o l × 100
All control and samples were analyzed in triplicate and the results of the elastase inhibition are expressed in percentage (%).
A spectrophotometric-adapted method previously reported by Shim [35] was implemented to evaluate tyrosinase inhibition. Firstly, it was added, in a microplate, 10 µL of honey samples and negative control (buffer), followed by 20 µL of the enzyme (1000 U/mL) and 170 µL of a solution composed of L-tyrosine solution (1 mM), phosphate buffer (50 mM, pH 6.5) and distilled water (10:10:9). The reaction was incubated for 10 min in the oven at 37 °C, and then the absorbance was measured at 590 nm. The percentage of inhibition (%) was calculated according to Equation (2):
Inhibition   % = A b s 590 c o n t r o l − A b s 590 s a m p l e A b s 590 c o n t r o l × 100
The results of the tyrosinase inhibition are expressed in percentage (%).

2.9. Statistical Analysis

All data were analyzed using IBM SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA), using analysis of variance (ANOVA) and a multiple range test (Tukey’s test), for a p-value < 0.05. Correlation analysis (Pearson’s coefficient, r-value) was performed to understand the effect of the chemical composition on the bioactivity of the honey extracts, by Graphpad Prism (version 9.0.1, GraphPad Software, USA).
The results of the samples are presented as mean values ± standard deviation (n = 3).

3. Results and Discussion

3.1. Pollen Analysis

Monofloral honey originated predominantly from a single floral source to at least 45%, however, as previously referred, for this specific honey the pollen composition from P. undulatum is over 30% [36]. The six honey samples under study were submitted to pollen analysis to guarantee they could be considered monofloral honey from P. undulatum. In Table 1, the values registered are the predominant, secondary, and minority pollen percentages per sample.
The samples showed several pollen types, highlighting the P. undulatum, C. sativa, Eucalyptus spp., and Acacia spp. among them. All honey samples are monofloral and had origin in P. undulatum. The sample with a higher percentage of Victoria box tree was sample 6, with a presence of 69%, followed by sample 4, with 65%, with a secondary predominance pollen type, Eucalyptus spp. On the other hand, samples 2, 3, and 5, besides their higher percentage in Victoria box tree pollen, had as a secondary pollen predominance pollen from C. sativa. In contrast, sample 1 revealed being the sample with the most variety of pollen type percentages.

3.2. HMF Content

The HMF is a component that naturally occurs in honey and results from the caramelization and Maillard reactions. The ratio of this compound increases with time, which makes it an important quality parameter for the evaluation of honey freshness, storage conditions, and overheating [36,37]. Furthermore, high concentrations of this component can adversely affect human health, including respiratory tract, eyes, skin, mucous membranes irritation, and potential carcinogenicity [37,38].
According to Codex Alimentarius [21], HMF content should be at a maximum of 40 mg/kg in general honey, except for honey used for industrial use. From Table 2, it was possible to verify that all samples are suitable for marketing or human consumption, since these values were much lower than the recommended limit value, ranging between 5.20 ± 0.04 mg/kg and 15.37 ± 0.02 mg/kg.

3.3. Phenolic Content of Honey

Féas et al. [36] state that the results in this food matrix should be interpreted as a quantitative estimation because sugar reactions can also react with the Folin-Ciocalteu reagent. However, since the sugar component provides an equal contribution, it was possible to conclude that the differences between the values result from the phenol content variations. This methodology is applied for its sensibility, precision, reproductivity, and fast response, which makes it the most attractive method. In this sense, the honey samples showed a total phenols content ranging from 21.37 ± 1.35 mg GA/100 g (sample 6) to 30.31 ± 0.42 mg GA/100 g (sample 1) (Table 2). The highest value was from sample 1 with significant differences (p < 0.001) from the remaining samples, except for sample 2.
Regarding the ortho-diphenols content, as shown in Table 2, for the honey samples from P. undulatum the results varied from 21.25 ± 0.12 mg GA/100 g (sample 4) to 28.47 ± 0.59 mg GA/100 g (sample 1), with significant differences from the remaining honey samples. As the previously evaluated parameter, sample 1 presented the highest value, with significant differences (p < 0.001) from the others five honey samples.
Concerning the flavonoid content, the honey samples showed values ranging between 4.41 ± 0.35 CAT/100 g (sample 6) and 7.67 ± 0.84 CAT/100 g (sample 1). Sample 1, once again, was the sample with the highest content with significant differences (p < 0.01) from the remaining ones, which the presence of different pollen types could justify. Indeed, the differences between samples could be explained by the nectar and pollen composition since a small percentage of other pollen types was observed besides being monofloral, Furthermore, they are influenced by climatic conditions, soil composition, the methodology used in honey extraction from the wax, the packing material, and storage time [39,40,41].
To the best of our knowledge, no studies in the literature determined the phenolic content of honey from P. undulatum. However, several authors studied these parameters in other kinds of honey from different floral sources.
Ferreira et al. [42] evaluated three monofloral honeys from different botanical sources: Rosmarinus officinalis L., Echium vulgare L., and Erica australis L. for their total phenols content. The results obtained in this work ranged from 22.62 ± 0.02 mg/100 g (rosemary honey) to 72.78 ± 0.02 mg/100 g (heather honey) [42]. Alves et al. [43] also studied several honey samples from different botanical sources. Similarly, they verified that rosemary honey samples had the lowest total phenols content (approximately 40.0 mg GA/100 g) when compared with heather honey, which also showed the highest value (approximately 130.0 mg GA/100 g). In 2020, it was also studied the same parameter for Romanian honey from different botanical sources (thyme (Thymus spp.), rape (Brassica spp.), mint (Mentha piperita L.), raspberry (Rubus idaeus L.), and sunflower (Helianthus spp.) honey) and registered values ranging from 18.91 mg GA/100 g (thyme honey) to 23.71 mg GA/100 g (mint honey) [44]. It is possible to verify that our values are similar when comparing the referred works since the same range as those of the mentioned authors was observed.
Concerning flavonoid content, there are also similar data for different types of honey in the literature. A research group from Peru analyzed eucalyptus honey and acquired a flavonoid content from 1.41 mg CAT/100 g to 1.63 mg CAT/100 g for this honey sample [45]. For the same type of honey, other authors verified a mean value of 5.63 ± 2.3 CAT/100 g [23]. For heather honey, an Algeria research group also reported the flavonoid content and registered 7.0 mg CAT/100 g [46], a similar result for an Estonia research group (6.4 mg CAT/100 g) [47]. By comparing the cited publications, it is feasible to confirm that our values are comparable and fall within the same range as those of the authors mentioned above.
Concerning the ortho-diphenols, to the best of our knowledge there are no studies in the literature that determined this content on honey samples, being the present study the first one to present these results.

3.4. Antioxidant Capacity of Honey

The antioxidant capacity was evaluated through three assays (DPPH, ABTS, and FRAP). Significant differences between samples were verified after analyzing the data in Table 3 (p < 0.001).
For the DPPH assay, the honey samples exhibited significant differences (p < 0.001), and the values ranged between 0.039 ± 0.007 mmol T/100 g (sample 3) and 0.447 ± 0.022 mmol T/100 g (sample 2). In ABTS methodology, it was possible to verify that sample 4 showed the lowest value (0.079 ± 0.002 mmol T/100 g), while sample 1 showed the highest antioxidant capacity for this assay (0.346 ± 0.020 mmol T/100 g). In both assays, samples 3, 4, 5, and 6 did not present significant differences from each other.
Concerning the FRAP assay, sample 6 showed that the lowest antioxidant capacity (0.178 ± 0.003 mmol T/100 g) not being significantly different from sample 5. In contrast, sample 1 exhibited the highest scavenging activity (0.566 ± 0.010 mmol T/100 g) with significant differences from the remaining honey samples.
In summary, the highest antioxidant capacity was verified in sample 1 for ABTS and FRAP assays (0.346 ± 0.020 mmol T/100 g and 0.566 ± 0.010 mmol T/100 g, respectively) with significant differences from the remaining samples. On the other hand, for DPPH, sample 2 showed the highest scavenging capacity (0.447 ± 0.022 mmol T/100 g) with significant differences from the other samples. For sample 1, as previously referred, the differences observed from the remaining samples could be justified by the differences in pollen types present in its composition.
As far as we know, no studies in the literature evaluated the scavenging capacity and reducing the power of honey from P. undulatum. For other types of honey, some authors studied these parameters (DPPH, ABTS, and FRAP); however, for the DPPH assay, it was impossible to compare the available data in the literature since different reaction conditions were employed.
For example, Gonçalves et al. [48] analyzed commercial Portuguese honey, including eucalyptus and heather honey samples, and reported DPPH radical scavenging activity values of 17.3% for eucalyptus honey and 32.7% for heather honey. Another study determined the radical scavenging capacity of honey from Trifolium spp., and obtained an inhibition percentage of 23.3% using the DPPH method. In contrast, the same honey samples exhibited a FRAP value of only 0.089 mmol TE/100 g, which is a lower value compared to our study [49].
A Spanish research group applied a similar methodology to the ABTS assay developed in the present work, analyzing three heather honey samples and obtaining values ranging from 0.123 ± 0.002 mmol T/100 g to 0.449 ± 0.021 mmol T/100 g [50]. When compared with our results, it is possible to verify that they are within the range of the values obtained in our study.
According to Barros et al. [51] the phenolic compounds are correlated with antioxidant capacity, which could justify the present results since sample 1 was the sample with higher phenolic content and generally showed the highest antioxidant capacity. However, the samples that demonstrated the lowest phenolic content did not present the lowest antioxidant capacity, which could indicate that honey may contain other components or/and factors that could affect the antioxidant capacity.

3.5. Anti-Aging Capacity of Honey

All six honey samples were evaluated for their tyrosinase and elastase inhibitory activity, aiming to understand if honey is suitable to be applied in anti-aging products. From the data in Table 3, it was possible to verify that the tyrosinase inhibition percentage ranged from 4.36% (sample 2) to 9.37% (sample 6) with significant differences (p < 0.05). The elastase inhibition percentage ranged from 37.52% (sample 2) to 45.88% (sample 1) with also significant differences (p < 0.001). In both analyses, sample 1 was the one that presented the highest inhibitory capacity, with no significant differences from samples 5 and 6. These results could also be justified by its pollen composition and the previously mentioned factors associated with the environmental conditions and honey’s manipulation.
As far as we know, currently, there is no data about the capacity of honey from P. undulatum to inhibit these two enzymes related to the skin’s aging process. Thus, the present study is the first to document this type of honey’s anti-elastase and anti-tyrosinase properties.
For both enzymes, only a few studies registered the inhibition capacity of honey for tyrosinase inhibition, but from different botanical sources.
In the literature, it is registered that some varieties of Thai honey presented inhibition percentages between 22.71% and 87.73% [52]. At the same time, other studies reported anti-tyrosinase activity in different honey samples, including monofloral and multifloral kinds of honey, and obtained values ranging from 51.25% to 90.06% [53]. In both works, the activity was higher than the one determined in the current work.
For anti-elastase activity, to the best of our knowledge, only a Mauritius Island team research tested the inhibition capacity of honey on elastase, observing no inhibitory activity [27]. In this sense, our research group was the first to register elastase inhibition by honey samples.
However, other authors investigated the anti-aging capacity of other materials. Currently, the search for natural compounds has gained an increasing demand due to their fewer side effects and become more prevalent in cosmetic formulations. Several authors already tried to investigate the potential use of bioactive compounds from a diverse matrix with potential skin applications. Our research group has already found anti-elastase and anti-tyrosinase activity in agro-food by-products, namely, grape stems from the winery industry, obtaining inhibition percentages between 41.47% and 53.83% for tyrosinase and between 67.98% and 98.02% for elastase [19]. Here, a higher percentage of elastase inhibition was found than tyrosinase inhibition as obtained in the present work.
Concerning elastase inhibition, in Actinidia arguta (kiwiberry) leaves, excellent results were also described regarding elastase inhibition, achieving values of 65.62% [54]. A plant from the Asteraceae family (Podospermum canum) also revealed enzyme inhibitory effects with a value of 51.7% [55]. Other authors that investigate the essential oil composition of sour orange (Citrus aurantium) leaves from Algeria found an interesting level of elastase inhibition of about 70% [56]. All these values revealed a present higher inhibitory capacity for these enzymes than those found in the current work.

3.6. Correlation Analysis

A Pearson’s correlation was performed to establish the correlation between all parameters analyzed (Figure 2).
Concerning the correlation between phenolic and HMF contents and antioxidant and anti-aging capacities, it was possible to verify some significant positive correlations (p ≤ 0.05).
Regarding the phenolic content, there was a significant and positive correlation between total phenols and antioxidant assays (rDPPH = 0.790, rABTS = 0.0.670, rFRAP = 0.645), that corroborate the influence of phenolic compounds on the antioxidant capacity as mentioned previously [51]. Ortho-diphenols also showed significant and positive correlations for anti-aging assays (rtyrosinase = 0.880, relastase = 0.606) and flavonoids contents were significantly and positively correlated with ABTS, FRAP, and HMF (rABTS = 0.600, rFRAP = 0.714, rHMF = 0.498). In fact, concerning the HMF content, some studies reported a relationship between the flavonoids and HMF components, associated with its production, i.e., compounds such as catechins can inhibit or reduce the production of HMF, probably due to less reactivity of the sugars [56]. In some cases, negative correlations were found, which can be explained by synergistic/antagonistic effects between compounds.

4. Conclusions

Currently, consumers are more interested in natural products with health benefits. In this regard, honey has established itself as a natural food source of phenolic compounds with biological properties frequently associated with its chemical composition.
In the current work, it was possible to conclude that honey from P. undulatum showed significant results for phenolic content and biological activities (antioxidant and anti-aging capacities), demonstrating that they can be used in pharmaceutical and cosmetic industries, including skin care products against skin aging and offer value to the cosmeceutical business. Additionally, this study demonstrated the honey’s anti-elastase capacity for the first time.
However, to ensure consumer safety, which is essential to the success of this application, it is crucial to identify the compounds responsible for these activities.
Besides, the honey analyzed in this work was from P. undulatum an invasive plant in all Azores Islands with few economic benefits, constant expansion, and hard to control. As such, this study contributed with added value and economical returns from a species that has long been considered a problem, drawing benefits from it and adding an economic value to this part of the archipelago.

Author Contributions

Conceptualization, A.B. and I.G.; methodology, S.S., M.M., and I.G.; software, S.S.; validation, A.B. and I.G.; investigation, S.S. and M.M.; data curation, S.S., M.M., and I.G.; writing—original draft preparation, S.S.; writing—review and editing, M.M., A.B. and I.G.; supervision, A.B. and I.G.; funding acquisition, A.B. and I.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by National Funds by FCT—Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 13 01788 g001
Figure 1. Victoria box tree from Azores (adapted from SIARAM—Governo dos Açores) [6].
Figure 1. Victoria box tree from Azores (adapted from SIARAM—Governo dos Açores) [6].
Applsci 13 01788 g001
Applsci 13 01788 g002
Figure 2. Pearson correlation between phenolic content, antioxidant capacity, anti-aging capacity, and HMF content. Significance: * significant at p < 0.05; ** significant at p < 0.01.
Figure 2. Pearson correlation between phenolic content, antioxidant capacity, anti-aging capacity, and HMF content. Significance: * significant at p < 0.05; ** significant at p < 0.01.
Applsci 13 01788 g002
Table 1. Pollen spectrum of types of honey studied. Data presented are about predominant, secondary, and minority pollen percentages per sample.
Table 1. Pollen spectrum of types of honey studied. Data presented are about predominant, secondary, and minority pollen percentages per sample.
SamplesPredominant Pollen
(>30%)		Secondary Pollen
(16–30%)		Minority Pollen
(3–15%)
SpeciesPercentage (%)SpeciesPercentage (%)SpeciesPercentage (%)
1Pittosporum undulatum Vent.51--Eucalyptus spp.12
Trifolium spp.8
Castanea sativa Mill.7
Acacia spp.5
Scrophularia spp.5
Lathyrus spp.3
2Pittosporum undulatum Vent.51Castanea sativa Mill.19Acacia spp.9
Eucalyptus globulus Labill.7
Ranunculus spp.3
3Pittosporum undulatum Vent.48Castanea sativa Mill.21Acacia spp.14
Eucalyptus globulus Labill.9
Ranunculus spp.3
4Pittosporum undulatum Vent.65Eucalyptus spp.17Raphanus raphanistrum L.5
Acacia spp.3
5Pittosporum undulatum Vent.50Castanea sativa Mill.24Trifolium spp.8
Acacia spp.7
Eucalyptus globulus Labill.5
6Pittosporum undulatum Vent.69--Acacia spp.11
Eucalyptus globulus Labill.10
Castanea sativa Mill.3
Table 2. Quantification of HMF by HPLC-UV (mg/kg) and phenolic content (total phenols (mg GA/100 g), ortho-diphenols (mg GA/100 g), and flavonoids (mg CAT/100 g) methods.
Table 2. Quantification of HMF by HPLC-UV (mg/kg) and phenolic content (total phenols (mg GA/100 g), ortho-diphenols (mg GA/100 g), and flavonoids (mg CAT/100 g) methods.
Samples123456p-Value
Quality parameterHMF (mg/kg)13.94 ± 0.11 e10.67 ± 0.04 c9.78 ± 0.02 b15.37 ± 0.02 f12.72 ± 0.04 d5.20 ± 0.041***
Phenolic contentTotal phenols
(mg GA/100 g)		30.31 ± 0.42 c26.01 ± 0.87 bc23.28 ± 1.37 ab21.37 ± 1.35 a25.87 ± 0.00 b20.83 ± 1.42 a***
Ortho-diphenols
(mg GA/100 g)		28.47 ± 0.59 e22.82 ± 0.24 b24.91 ± 0.35 c21.25 ± 0.12 a26.96 ± 0.00 d27.29 ± 0.47 d***
Flavonoids
(mg CAT/100 g)		7.67 ± 0.84 d6.78 ± 0.00 bc7.67 ± 0.84 bc7.23 ± 0.42 bc5.88 ± 0.35 ab4.41 ± 0.35 a**
The values are presented as mean ± standard deviation (n = 3). Different letters in each row indicate significantly different results (ANOVA, p < 0.05). Significance: non-significant, N.S. (p > 0.05); ** significant at p < 0.01; *** significant at p < 0.001.
Table 3. Determination of antioxidant capacity [DPPH, ABTS, and FRAP methods (mmol T/100 g)] and anti-aging capacity (%) of honey samples.
Table 3. Determination of antioxidant capacity [DPPH, ABTS, and FRAP methods (mmol T/100 g)] and anti-aging capacity (%) of honey samples.
Samples123456p-Value
Antioxidant capacityDPPH
(mmol T/100 g)		0.362 ± 0.007 b0.447 ± 0.022 c0.039 ± 0.007 a0.045 ± 0.004 a0.050 ± 0.010 a0.098 ± 0.003 a***
ABTS
(mmol T/100 g)		0.346 ± 0.020 c0.203 ± 0.016 b0.092 ± 0.001 a0.079 ± 0.002 a0.102 ± 0.002 a0.129 ± 0.005 a***
FRAP
(mmol T/100 g)		0.566 ± 0.010 d0.612 ± 0.010 c0.335 ± 0.013 b0.321 ± 0.003 b0.184 ± 0.005 a0.178 ± 0.003 a***
Anti-aging capacityTyrosinase inhibition
(%)		9.37 ± 0.20 bc4.36 ± 0.28 a6.34 ± 0.59 a4.65 ± 0.95 a6.89 ± 0.06 ab9.87 ± 0.70 b***
Elastase inhibition
(%)		45.88 ± 0.65 b37.59 ± 2.16 a41.28 ± 0.33 ab38.70 ± 2.85 ab42.72 ± 2.84 ab39.85 ± 1.20 ab*
The values are presented as mean ± standard deviation (n = 3). Different letters in each row indicate significantly different results (ANOVA, p < 0.05). Significance: non-significant, N.S. (p > 0.05); * significant at p < 0.05; *** significant at p < 0.001.
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Santos, S.; Maia, M.; Barros, A.; Gouvinhas, I. Assessment of Phenolic Content, Antioxidant and Anti-Aging Activities of Honey from Pittosporum undulatum Vent. Naturalized in the Azores Archipelago (Portugal). Appl. Sci. 2023, 13, 1788. https://doi.org/10.3390/app13031788

AMA Style

Santos S, Maia M, Barros A, Gouvinhas I. Assessment of Phenolic Content, Antioxidant and Anti-Aging Activities of Honey from Pittosporum undulatum Vent. Naturalized in the Azores Archipelago (Portugal). Applied Sciences. 2023; 13(3):1788. https://doi.org/10.3390/app13031788

Chicago/Turabian Style

Santos, Soraia, Miguel Maia, Ana Barros, and Irene Gouvinhas. 2023. "Assessment of Phenolic Content, Antioxidant and Anti-Aging Activities of Honey from Pittosporum undulatum Vent. Naturalized in the Azores Archipelago (Portugal)" Applied Sciences 13, no. 3: 1788. https://doi.org/10.3390/app13031788

APA Style

Santos, S., Maia, M., Barros, A., & Gouvinhas, I. (2023). Assessment of Phenolic Content, Antioxidant and Anti-Aging Activities of Honey from Pittosporum undulatum Vent. Naturalized in the Azores Archipelago (Portugal). Applied Sciences, 13(3), 1788. https://doi.org/10.3390/app13031788

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