Encapsulation of bougainvillea (Bougainvillea spectabilis) flower extract in Urtica dioica L. seed gum: Characterization, antioxidant/antimicrobial properties, and in vitro digestion

Abstract Bougainvillea spectabilis extract (BSE), a rich source of bioactive compounds like phenolic, flavonoid, and anthocyanin, was used for encapsulation with Urtica dioica L. seed gum. The extract was obtained using shaking, bath, and probe ultrasound. The results showed that probe ultrasound extract was more efficient, as reflected by the higher value of total phenolic (4354.15 mg GAE/100 g FW), flavonoid (2431.25 mg CE/100 g FW), and anthocyanin content (106.57 mg CGE/100 g FW). BSE was encapsulated in U. dioica L. seed gum at 1:1 and 1:2 core to coating ratio. In both DPPH radical scavenging and FRAP assay, higher antioxidant activity was observed in the encapsulated extract than in the free extract. Encapsulated extracts exhibited 87.9 nm average diameter (polydispersity index below 0.23) and negative zeta potential. The average minimum inhibitory concentration (MIC) of nanoparticles against Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Salmonella enterica was 112.5, 87.5, 112.5, and 87.5 μg/ml, respectively, while MIC of the free extract against S. aureus and E. coli was 150 and 125 μg/ml, respectively. The phenolic compounds are gradually released from the nanoparticles in the gastric and small intestine phase, whereas free extract released phenolic compounds quickly after entering the gastric phase. Considering antioxidant/antimicrobial activity and release properties, nanoparticles with a 1:1 ratio of core to coating had the potential to use as an effective natural preservative agent in food products besides the delivery of bioactive compounds to the human body.

The extraction process is substantially the most vital step in obtaining valuable phytochemical compounds. The method of extraction interferes with the process of obtaining bioactive compounds (Abarca-Vargas et al., 2016;Debiasi et al., 2021). Nowadays, the conventional methods of extraction have been replaced by green procedures like ultrasound-assisted extraction due to its long extraction time, low extraction yield, degradation of bioactive compounds, and high solvent consumption (Debiasi et al., 2021;Razavi & Kenari, 2021).
Despite all the health benefits, anthocyanin and phenolics have been shown to have limited bioavailability after intake of a meal rich in them (Bao et al., 2019;Rahnemoon et al., 2021;Zhao et al., 2017).
Encapsulating of bioactive compounds in diverse wall materials is a promising technology that can protect them in such conditions and improve their activity and bioavailability (Rahnemoon et al., 2021).
Natural proteins, polysaccharides, and lipids are promising coating materials for the encapsulation of bioactive compounds which affects the stability and characteristics of final particles (Razavi & Kenari, 2021). Hydrocolloids are high-molecular-weight biopolymers with an extensive range of functions. Seeds are generally traditional source of different gums (Zamani & Razavi, 2021). Nettle (Urtica dioica L.) is a herb belonging to the Urticaceae family (Kutlu et al., 2020), which produces the most immense amount of mucilage (Zamani & Razavi, 2021). The rheological, functional, and physicochemical properties of nettle seed gum were studied before (Kutlu et al., 2020;Zamani & Razavi, 2021), but its usage possibilities in encapsulation have not been investigated before.
Bougainvillea spectabilis extract (BSE) can be encapsulated in colloidal dispersions with droplet sizes between 20 and 200 nm, referred to as a nanoemulsion. These systems efficiently support the use of bioactive compounds in food products by reducing the impact on taste, as well as by enhancing their antimicrobial/antioxidant activities and bioavailability (Seibert et al., 2019). Although the antimicrobial and antioxidant properties of BSE have been studied previously, the bioavailability and preservation potential of encapsulated BSE has not been previously investigated. Therefore, the objective of this study was to evaluate: (1) the effect of extraction method on bioactive compounds of BSE, (2) the antimicrobial and antioxidant activities of free and nanoencapsulated BSE, and (3) the efficiency of U. dioica L. seed gum in increasing the bioavailability of bioactive compounds in the simulated gastrointestinal tract.

| Materials
Air parts (flower) of B. spectabilis were collected from Sari in May 2021. Nettle seed was purchased from a local grocery. All reagents and chemicals were purchased from Sigma-Aldrich.

| Extract production and extraction yield
The extracts of B. spectabilis flower were obtained using three different methods following the guidelines reported by Han et al. (2018) with slight modification. Five grams of the fine powder was added to 100 ml ethanol:water (50:50 V/V). The mixture was shaken in a shaker incubator (Unimax 1010, Heidolph) at 35°C for 1 h. For bath ultrasound extraction, the mixture was placed in an ultrasound water bath (Elma) at 35°C for 1 h (37 kHz-250 W). For probe ultrasound mLabs) extraction, the mixture was sonicated with a probe ultrasound set at 20 kHz (280 W) and 50% intensity at 35°C for 1 h. Then, all treatments were immediately centrifuged at 3000 rpm for 15 min, and supernatants were oven dried overnight at 45°C (Han et al., 2018). The extraction yield of extracts was calculated using the following equation: W 1 = the total weight of dried extracts obtained after drying and W 2 = the total weight of ground flower taken for each extraction method (Abarca-Vargas et al., 2016).

| Determination of the content of bioactive compounds and antioxidant activity
Total phenolic, flavonoid, and anthocyanin content of BSEs were determined using the method described by Kuspradini et al. (2016).
Antioxidant activity of the extract was measured according to the DPPH radical scavenging activity and ferric reducing antioxidant activity (Razavi & Kenari, 2021).

| MIC and MBC determination
The assays were performed using two gram-negative bacteria strains  Different dilutions of BSE in both free and nanoencapsulated forms were obtained by serial dilution and added to tubes at the same concentration. All tubes were incubated for 24 h at 37°C, and after that, each sample was cultured on Mueller-Hinton agar again and incubated at 37°C for 24 h. Finally, the growth or lack of development of bacteria in different concentrations for free and nanoencapsulated extract of B. spectabilis was observed, and MIC and MBC were determined.

| Extraction of nettle seed gum
Seed gum was provided under the condition described by Kutlu et al. (2020) with slight modification. Briefly, 100 g seeds were soaked in 1 L distilled water at 60°C for 1 h under a magnetic stirrer.
Then, the mucilage separated from swollen seeds, passed through a vacuum filter, and oven dried at 45°C. The dried gum was milled and stored in a dry and cool place until use (Kutlu et al., 2020).

| Nanoemulsion preparation
Nanoemulsions were prepared by the method of Kenari et al. (2020) with slight modification. First, seed gum was dispersed in deionized water to achieve total solid content of 30%. The extract (25 mg) at 1000 ppm of concentration was added to 20 ml sunflower oil and 5 ml emulsifier Tween 80 and stirred for 2 h at 25°C until primary emulsion formed. Then, gum solution at two different ratios (1:1 and 1:2) of emulsion to gum solution was added to the emulsion gradually and homogenized using an ultraturrax (T25D, IKA) at 15,000 rpm and 10°C for 10 min .

| Characterization of nanoemulsion
Particle size, polydispersity index (PDI), and zeta potential of nanoemulsions were evaluated by dynamic light scattering technology using Zetasizer (S90) instrument at 25°C. The nanoemulsion was diluted 20-fold in deionized water to escape multiple scattering effects (Razavi et al., 2020). The encapsulation efficiency (EE) of BSE was calculated by measuring the amount of total phenolic compounds (TPC) of the extract before and after encapsulation. The ratio of unencapsulated TPC on TPC before encapsulation has been considered as EE (Rahnemoon et al., 2021). Nanoemulsions were dried using a freeze dryer (VaCO5, Zirbus) at 0.017 mPa and −57°C for 48 h. Then, the morphology of nanoemulsions was examined using a scanning electron microscope (Mira 3 LMU, Tescan, Czech Republic). For this purpose, samples were attached to aluminum stubs and covered with a thin layer of gold. The voltage device was 15 kV, and images were taken at 1000× magnification (Razavi et al., 2020).

| Simulated gastrointestinal condition
Simulated fluid for gastric (SGF) and small intestine (SIF) were prepared as described by Razavi et al. (2020). For the stomach digestion, 1.4 ml of SFG was mixed with 100 ml of nanoemulsion and put in a shaker incubator at 37°C for 2 h with continuous agitating at 100 rpm. For the intestinal digestion, 2.4 ml of SIF was mixed with 100 ml of digesta from the stomach stage and shook in a shaker incubator at 37°C for 2 h. In all experiments, the digesta was collected at 0, 30, 60, 90, and 120 min of digestion. After digestion, the samples were immediately centrifuged at 2000 rpm for 15 min, and after filtration, the filtrate was used to determine the content of bioactive compounds released from the nanoemulsion (Razavi et al., 2020).

| Statistical analysis
The results were statistically analyzed by one-way analysis of variance (ANOVA) with Duncan test (p < .05) and T test using SPSS version 20.0 statistical software (SPSS, Inc.). All experiments were carried out in triplicate.

| Extraction yield and content of bioactive compounds
Extraction is the first step in analyzing bioactive compounds from plant materials (Abarca-Vargas et al., 2016). The results of extraction yield (EY) obtained by different methods are illustrated in Table 1 The antioxidant and antimicrobial activity of BSE is related to its high phenolic, flavonoid, and anthocyanin content (Ydjedd et al., 2017). Therefore, evaluating the content of bioactive compounds in the extract is very important.  (Kuspradini et al., 2016). In this study, a higher TAC (106.57 mg CGE/100 g FW) was observed in the extract obtained from probe ultrasound extraction. Anthocyanins have great potential in the pharmaceutical and food industries because they act as antioxidant agents by donating hydrogen to highly reactive species (Kuspradini et al., 2016).

| Antioxidant activity
The reactive oxygen species were involved in the expansion of cancer and cardiovascular diseases (Compaore et al., 2013). DPPH and  Table 2. Free extract showed lower antioxidant activity than encapsulated extract in both evaluating methods. The nanoemulsion prepared at a 1:1 ratio of core to coating exhibited higher antioxidant activity than extract at a 1:2 ratio of core to coating, which is related to the higher amount of BSE. Also, the partial antioxidant activity of the nanoemulsion may be related to nettle seed gum. Zamani and Razavi (2021) evaluated the antioxidant activity between vitamin C and nettle seed gum.
Their result revealed that the antioxidant activity of the nettle seed gum at a concentration of 1000 ppm was 38.1% compared to the vitamin C which was 67.9%. They also reported the presence of hydroxyl groups in the nettle seed gum which is responsible for the appearance of radical scavenging (Zamani & Razavi, 2021

| Characterization of nanoemulsion
Nanoemulsions are important vehicles for delivering bioactive compounds into various food products, drugs, and nutraceuticals (Almadiy et al., 2016). The particle size of nanoemulsion is a critical parameter closely associated with the aqueous dispersibility, aggregation, and fusion of nanoparticles (Zhao et al., 2017). The results of particle size of different nanoemulsions are shown in Table 4. The particle diameter of samples ranged from 76.5 to 99.3 nm, and significantly increased by increasing the ratio of gum solution. When the amount of gum solution is more than emulsion, a sufficient amount of gum molecules is available to interact with the emulsion droplet, which may result in decreasing the aggregation and reducing the particle size. Rahnemoon et al. (2021) formulated sodium alginate nanocapsules containing pomegranate peel extract at the 2:1 and 4:1 ratio of alginate to extract. They stated a decrease in particle size from 218.9 to 177.8 nm when the alginate ratio increased from 2 to 4. In another study conducted by Kenari et al. (2020), the chitosan:sage seed gum nanoparticles containing Iranian golpar extract were prepared at different extract to wall material ratios (0.10, 0.25, and 0.40) and the particle size decreased when wall material increased. Another reason for bigger particle size and wide size distribution may be due to Bacteria Free extract Nanoencapsulated extract (core to coating ratio)   (Table 4). The zeta potential of more than +30 mV and lower than −30 mV has good stability. According to the result, the stability of 1:2 nanoemulsions is higher than the 1:1 nanoemulsion. It is attributed to lower zeta potential. Therefore, the properties of nanoemulsions depend on the ingredients that participate in forming the nanoemulsion, encapsulated compounds, dispersing medium, and preparation conditions (Rahnemoon et al., 2021). The negative zeta potential for encapsulated extract in native gums also was reported for Iranian golpar in sage seed gum , phenolic compounds of sesame seed in native seed gums (Esmaeilzadeh , and Fumaria parviflora extract in Arabic gum (Razavi & Kenari, 2021).
EE is an important property to describe the effectiveness of the encapsulation process. It points to the possibility of the coating material holding the core substance inside of nanocapsules. The EE ranged from 76.6% to 83.4%. The ratio of core to coating affects EE, and by increasing ratio from 1:2 to 1:1, the EE was increased. It is related to the higher viscosity of emulsion and higher extract concentration. Zhao et al. (2017) reported that higher anthocyanin addition resulted in a significant increase in the EE. Also, Kenari et al. (2020) reported higher EE occurs due to using a larger amount of extract.

| Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) images give information about the morphology of nanoparticles and their appropriate surfaces (Ydjedd et al., 2017). Figure 1 presents SEM images of nanoemulsions prepared in different core to coating ratios. The morphology of nanoemulsion with both core to coating ratios was partly spherical in shape. Spherical nanoparticles indicate that coating materials are stable and effectively surround core materials (Razavi & Kenari, 2021).
Although the surface of nanoparticles was not uniform, no porose was observed on the surfaces. Minimum agglomeration was observed in nanoparticles, which indicates strong encapsulating properties of nettle seed gum as wall materials. Similar morphology was observed in nanoencapsulated bioactive compounds in gum coatings Razavi & Kenari, 2021;Robert et al., 2010).

| In vitro release
The digestion process is the first step, before absorption and metabolism, in modulating the bioavailability of bioactive compounds by affecting their bioaccessibility (Ydjedd et al., 2017). Figure 2 shows the release of phenolic compounds during the in vitro gastric and intestinal digestion phase. Generally, the release of phenolic compounds from both nanoemulsion was increased gradually with an extension of the digestion time from 0 to 120 min after digestion respectively. In addition, the release of phenolic from nanoemulsion by the intestinal phase was higher than the release by the gastric phase. This may be because of the strong solubility of coating substances at higher pH of the intestinal phase. This result means that both nanoencapsulated extracts are protected against the condition changes of digestion like enzymes and pH variation. These results revealed that the encapsulated BSE could be effectively absorbed in the small intestine. These results are in accordance with the results of Mohamed et al. (2018), who reported a higher release of betalain from microcapsules in the small intestine phase than gastric phase. However, for free extract, the phenolic compounds are more released in the gastric phase as compared to the nanoencapsulated BSE. These results are in line with the report by Ydjedd et al. (2017), who reported that the release of TPC increased during digestion and TPC in the gastric phase was higher than small intestine phase.

| CON CLUS ION
The obtained results demonstrate that BSE in both free and nanoencapsulated forms can scavenge free radicals due to a higher amount of phenolic, flavonoid, and anthocyanin compounds.
Additionally, it exhibits antimicrobial activity against S. aureus, E. coli, L. monocytogenes, and Sa. enterica. Nettle seed gum could improve the antioxidant and antimicrobial activity of extract. The core to coating ratio 1:1 showed gradual release of bioactive compounds in comparison to ratio 1:2. In conclusion, it could be said that BSE at a concentration of 1000 mg/L, which is nanoencapsulated in U. dioica L. seed gum, is a natural functional compound with antioxidant/ antimicrobial properties and higher bioavailability. Future research is needed for evaluating the characteristics of fabricated nanoemulsion as natural preservative agents in food products.

ACK N OWLED G M ENTS
The authors are grateful to the Research Vice-Chancellery of Sari Agricultural Sciences and Natural Resources University Foundation for their financial support (Grant number: 02-1400-04) to carry out this research.

CO N FLI C T O F I NTE R E S T
Authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.

E TH I C A L A PPROVA L
The study does not involve any human or animal testing.