Preparation of a Highly Water-dispersible Powder Containing Hydrophobic Polyphenols Derived from Chrysanthemum Flower with Xanthine Oxidase-inhibitory Activity

Abstract

We aimed to develop a highly water-dispersible dry powder of hydrophobic polyphenols derived from chrysanthemum flower. A dry powder resulting from the mixture of an ethanolic extract of chrysanthemum flower (CF) with transglycosylated rutin (rutin-G) and sucrose fatty acid esters (SEs) was successfully formulated. This powder dispersed easily in water, and the mean particle size in water remained at ∼150 nm without apparent changes even after one-week storage. Furthermore, the particle size of the dispersed powder prepared by heat-and-acid treatment shifted below 100 nm after one-week storage. The xanthine oxidase-inhibitory activity of the resulting CF–rutin-G–SEs powder was almost equivalent to that of chrysanthemum flower oil according to in vitro analysis. This technique for preparing a highly water-dispersible dry powder from natural products containing hydrophobic polyphenols should be applicable to the development of functional food products.

Introduction

Self-medication with over-the-counter drugs and complementary medications has recently been promoted to suppress the escalation of healthcare costs on a global scale. Many studies on functional foods containing natural compounds have been conducted worldwide (González-Castejón and Rodriguez-Casado, 2011; Shakeri, et al., 2016; Suvarna et al., 2017). Natural compounds have been attracting attention as functional foods, pharmaceuticals, and nutraceuticals owing to their potential therapeutic properties for human healthcare. Specifically, the number of pharmaceutical products and dietary supplements derived from phytochemicals is rising in proportion to the worldwide economic growth (Chang et al., 2016). Although natural products derived from phytochemicals contain many hydrophobic bioactive materials that may serve as pharmaceuticals or functional foods, the low absorption via oral administration is cause for concern regarding the application of hydrophobic bioactive materials as supplements, due to their poor solubility or dispersibility in water (Si and Liu, 2014). For instance, hydrophobic components extracted from glycyrrhiza with an organic solvent have various biological activities such as antioxidant, antibacterial, and antiallergic effects (Chakotiya et al., 2016). Because it is difficult to disperse these hydrophobic compounds in water, the application of these substances to the development of nutritional supplements such as functional foods and drinks has been limited (Panza et al., 2015). The flower portion of chrysanthemum (Chrysanthemum morifolium Ramat) has also been eliciting interest as a dietary and medicinal substance. The flowers contain essential oils, flavonoids, and their glycosides (Mladenova et al., 1988). Because chrysanthemum flower has various biological activities such as antioxidant, antimutagenic, and anti-HIV effects (Kim and Lee, 2005; Lee et al., 2003; Miyazaki and Hisama, 2003), CFO (Kaneka Chrysflavone™) was developed as an oily fraction containing polyphenols derived from chrysanthemum flowers (Honda et al., 2014). The serum uric-acid-lowering effect of CFO and its mode of action have been reported. These data have shown that CFO attenuates the increase in serum uric-acid levels by the following two mechanisms: suppression of uric-acid production via inhibition of xanthine oxidase and enhancement of uric-acid excretion by upregulation of excretion-related genes. In spite of these attractive functions, the use of CFO in supplements and clinical applications is challenging because of its poor dispersibility in water. To overcome these problems, an effective approach to increasing dispersibility and wettability is required to improve the physicochemical and nutraceutical potential of CFO.

Solubilization has attracted considerable interest as an effective way to improve the dispersibility of various hydrophobic compounds (Cheng et al., 2016). Cyclodextrins have been used to prepare powders of oil components. Some researchers succeeded in encapsulating tea tree oil by direct complexation with solid amorphous β-cyclodextrin (Shrestha et al., 2017). Sasako et al. (2016) prepared a capsule-like substance of a flavor component using cyclodextrin and phytosterol ester. Our recent studies revealed that the specific properties of transglycosylated compounds, including transglycosylated hesperidin (hesperidin-G), transglycosylated stevia (stevia-G), and transglycosylated rutin (rutin-G), can be employed as effective excipients to prepare solubilized formulations of poorly water-soluble materials (Sato et al., 2015; Tozuka et al., 2010; Uchiyama et al., 2010). Indeed, we have successfully enhanced the nutraceutical functions of food materials with low water solubility such as quercetin, ipriflavone, and sesamin (Fujimori et al., 2015, 2016; Sato et al., 2017). To implement the practical use of this technology in the food industry, a further reduction in the amount of the excipient is desired. Further supplementation (with an additive agent) of the binary system of a bioactive hydrophobic compound and a transglycosylated-compound solution may be a promising way to significantly enhance dissolution and stabilize the supersaturated state (Fujimori et al., 2017; Kadota et al., 2016a, 2016b; Uchiyama et al., 2012). Nonetheless, these extra additives were not approved as food additives.

In this study, our purpose was to develop a highly dispersible and water-soluble powder derived from CFO using transglycosylated materials and approved surfactants by a spray-drying method. Four surfactants, which have been widely used as emulsifying agents of food additives, were screened as additional excipients for enhancing the water dispersibility of hydrophobic compounds. To select the optimal powder formulation, we tested whether the resulting powder prepared by spray-drying can be dispersed easily in water, and whether the mean particle size in water can be maintained before and after heat-and-acid treatment. We compared the inhibitory effects of the optimized CF powder formulation and CFO on xanthine oxidase in an in vitro experiment, as previously reported for CFO (Honda et al., 2014).

Materials and Methods

Materials    Stevia-G, rutin-G, and hesperidin-G were kindly gifted by Toyo Sugar Refining Co., Ltd. (Tokyo, Japan). The ethanolic extract (CF) was prepared from chrysanthemum flowers harvested in China in accordance with the Japanese Pharmacopoeia, 16th edition. CFO (Kaneka Chrysflavone™) is an oily food fraction comprising an ethanolic extract of chrysanthemum flowers, medium-chain triglycerides (MCT, Riken Vitamin Co., Ltd., Tokyo, Japan), and diglycerol monooleate (Riken Vitamin Co., Ltd.) at ∼20% (w/w), ∼60% (w/w), and ∼20% (w/w), respectively. Four nonionic surfactants were selected. Two sucrose fatty acid esters (SEs; DK ester SS^®, hydrophile-lipophile balance [HLB] value = 19.0; DK ester F160^®, HLB value = 15.0) were supplied by DKS Co., Ltd. (Kyoto, Japan), and two polyglycerol esters of fatty acids (SY-glyster MS-5S^®, HLB = 11.6; SY-glyster MS-3S^®, HLB = 8.4) were supplied by Sakamoto Yakuhin Kogyo Co., Ltd. (Osaka, Japan). Ascorbic acid, xanthine, and xanthine oxidase were purchased from Sigma-Aldrich (Missouri, USA). All other chemicals and solvents were of reagent or HPLC grade.

Preparation of CF formulations by evaporation    The powder of CF with transglycosylated materials was prepared via solvent evaporation to select an optimized excipient. For the preparation of the CF–transglycosylated-material powder, the ethanolic extract of chrysanthemum (200 mg, including 40 mg of solid content) was dissolved in 60 mL of ethanol, and the transglycosylated additives (2000 mg) and/or surfactants (200 mg) were dissolved in 140 mL of distilled water. The powder of a bicomponent or tricomponent preparation in CF and transglycosylated rutin and/or surfactants was generated via solvent evaporation. The solutions were mixed in an ethanol–water solution (3:7 v/v) using a magnetic stirrer for 1 min at ambient temperature. The obtained emulsified samples were evaporated to dryness in an R-3 rotary evaporator (Büchi, Tokyo, Japan) under pressure at ca. 4 kPa in a water bath maintained at 50°C. After evaporation of the liquids, the solidified samples containing CF and transglycosylated rutin and/or surfactants were micronized using a mortar and pestle. All the evaporated samples were dried in a desiccator with blue silica gel under reduced pressure for 1 day before testing of their physicochemical properties.

Preparation of formulations of CF-transglycosylated additive by spray-drying    After selection of optimized formulations of CF–rutin-G–surfactant, composite particles of CF–rutin-G–surfactant were prepared by the spray-drying method. CF (400 mg) and transglycosylated food additive (2000 mg) or nonionic surfactants (200 mg) were dissolved in an ethanol-water solution (3:7 v/v). This solution was fed into a spray dryer (B-290, Büchi) at a rate of 5.5 mL/min. The drying air flow rate was expected to be around 35 m³/h, and the compressed air supplied separately for the spray nozzle was 473 L/h at 41 kPa. The inlet and outlet temperatures of the drying chamber were maintained at 120 and 70°C, respectively. All spray-dried particles were dried in a desiccator with blue silica gel under reduced pressure for 1 day before testing of their physicochemical properties.

Dynamic light scattering analysis    Particle size distributions of the CF samples were measured by dynamic light scattering (Nanotrac UPA-UT151, MicrotracBEL, Corp., Osaka, Japan). The resulting powdered CF formulations were dispersed in water. The UPA detection range was 0.003–6.000 µm. Particle sizes in the undiluted suspension were evaluated within an appropriate high-concentration range, and the evaluation was performed in triplicate.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity    The DPPH activity of stevia-G, rutin-G, hesperidin-G, and ascorbic acid was measured to evaluate antioxidant activity as reported elsewhere (Chen et al., 2013; Kadota et al., 2016c). The prepared 2 mL of a 0.1 mM solution of DPPH in ethanol was mixed with each sample solution of 2 mL at different concentrations as follows: stevia-G, rutin-G, and hesperidin-G ranged from 0.65 to 65 mM, and ascorbic acid as a reference ranged from 2.8 to 280 mM. The mixtures were incubated for 30 min in the dark. Absorbance of the sample solutions was measured using a UV spectrometer (UV-2900, Hitachi, Tokyo, Japan) at 517 nm. The DPPH-scavenging effect was calculated by means of the following formula (Brand-Williams et al., 1995; Wu et al., 2008):   

Analysis of powder particle size    Particle size distributions of the samples were determined by the laser scattering method on an MT3000II instrument (MicrotracBEL, Corp., Osaka, Japan). The measurement was conducted at 0.4 MPa. The particle size is expressed as the volume median diameter.

Scanning electron microscopy (SEM)    The particle morphology of each sample was evaluated using a scanning electron microscope (Miniscope^® TM3030, Hitachi High-Technologies Corporation, Tokyo, Japan). SEM images were captured randomly at magnification levels at 1000×. An acceleration voltage of 15 kV during each observation was performed. This SEM system does not require metal stubs because it has a highly sensitive rough vacuum secondary electron detector.

Powder X-ray diffraction (PXRD)    PXRD patterns were recorded by means of a powder X-ray diffractometer (Miniflex, Rigaku Corporation, Tokyo, Japan) with Cu Kα radiation generated at 15 mA and 40 kV. The scanning rate was 4°/min over a 2θ range of 4–35° at a step size of 0.02°.

Heat-and-acid treatment    The experiments with CF formulations dispersed in water were conducted next. The concentration of each formulation was set to 1 mg/mL in water. The addition of citric acid into dispersed liquids was terminated at pH 2.9. The liquids were heated to 95 ± 5°C under acidic conditions and then cooled to 20 ± 5°C.

In vitro xanthine oxidase-inhibitory activity    A 0.02 U/mL xanthine oxidase solution (50 µL) was prepared in a microplate, and then CFO solution or the optimized CF formulation at various concentrations, ranging from 50 to 300 µg/mL (50 µL), was added to the microplate. These solutions were incubated for 15 min at 25°C. The assay was initiated by addition of a 400 µg/mL substrate xanthine solution (100 µL) with incubation for 15 min at 25°C. After completion of the enzymatic reaction, absorbance of each formulation (A_formulation) at a wavelength of 295 nm was measured on a microplate reader (Multi-Detection Microplate Reader, Synergy HT, Bio-Tek, Instruments Inc., Winooski, VT, USA). The contrast solution was prepared by the same procedure, and the absorbance of the contrast (A_contrast) was measured. All assays were performed in triplicate. The inhibition rate of xanthine oxidase activity was calculated using the following equation (Wan et al., 2016):   

Results and Discussion

Selection of the optimal transglycosylated compound    Three transglycosylated compounds have been applied as additive agents for improving the dispersibility of hydrophobic compounds in water (Fujimori et al., 2015; Sato et al., 2015; Uchiyama et al., 2010). Nevertheless, the dispersing effects of these transglycosylated compounds in water differ depending on the combination of hydrophobic compounds and the excipient, indicating the relevance of the molecular interaction between hydrophobic compounds and the transglycosylated compounds (Tozuka et al., 2012; Zhang et al., 2011, 2014). Thus, selection of an optimal transglycosylated compound suitable for the target compounds was required. Powdered CF–transglycosylated compound mixture was prepared in a rotary evaporator to select the transglycosylated compound suitable for CF, and the water dispersibility of CF–transglycosylated compound mixtures (containing rutin-G, hesperidin-G, or stevia-G) was studied. The images in Figure 1 show aqueous suspensions of redispersed evaporated CF with rutin-G, hesperidin-G, or stevia-G at a specific weight ratio (1/10) before and after heat-and-acid treatment. The upper images show the state of formulations shortly after dispersing the evaporated powder of CF–transglycosylated compound mixtures in water. On the other hand, the lower images show the appearance of CF–transglycosylated compound mixtures treated under a heat-and-acid condition after dispersing their powder in water. CF is a lipophilic component and is almost undispersible in water. The suspended state was observed for any of the formulations shortly after the powdered CF-transglycosylated compound mixtures were dispersed in water. There were hardly any differences in appearance among these formulations before and after heat-and-acid treatment.

Fig. 1.

Samples of redispersed evaporated CF wit. (A) rutin-G, (B) hesperidin-G, or (C) stevia-G. (I) Soon after dispersal in water, (II) after heat-and-acid treatment.

To validate CF as a supplement, the degradation of CF by UV light should be prevented by the addition of an antioxidant. To address these problems, the antioxidant activity was measured in each transglycosylated compound. Figure 2 shows the common logarithm of half-inhibitory concentrations (IC₅₀ values) of rutin-G, hesperidin-G, stevia-G, and ascorbic acid toward DPPH (50 mM). The antioxidant effect of rutin-G was more pronounced than that of stevia-G, hesperidin-G, or ascorbic acid. This means that among these compounds, rutin-G could be used at the lowest concentration to obtain a 50% antioxidant effect. Furthermore, the interfacial activity of the solutions containing these transglycosylated compounds was compared, and rutin-G showed little surface tension, comparable to water (72 mN/m at 37°C), even when we increased the concentration of rutin-G (Fujimori et al., 2016). In addition to the improvement in water dispersibility of hydrophobic compounds, the toxicity of rutin-G to Caco-2 cells was found to be negligible even at high concentrations (100 mg/mL), indicating its potential as a safe pharmaceutical additive compared to other conventional surfactants used to enhance solubility (Tozuka et al., 2012). Based on these observations, rutin-G was selected as an optimal excipient for the preparation of a CF powder formulation.

Fig. 2.

The antioxidant activities of ascorbic acid, rutin-G, hesperidin-G, and stevia-G. All analyses were performed in triplicate, and values were expressed as mean ± SE, n = 3.

Selection of the optimal sucrose fatty acid esters    To develop functional powdered CF–transglycosylated compound as a nutritional supplement that can be added to a drink, an easily dispersible powder without precipitation is required. As shown in Figure 1, the dispersed powder of CF–rutin-G in water was in a suspended form. We previously demonstrated that further supplementation (with an additive agent) of the binary system compound–transglycosylated compound solution may be a promising method to significantly enhance dissolution and stabilize the supersaturated state (Fujimori et al., 2017; Kadota et al., 2016b). Nonetheless, these extra additives are not approved in the food industry. Four surfactants, which have been widely used as emulsifying agents, were screened to further improve CF dispersibility in water. The images in Figure 3 show the aqueous suspensions of a redispersed powder of CF-rutin-G with DK ester SS^®, DK ester F160^®, SY-glyster MS-5S^®, or SY-glyster MS-3S^® prepared by evaporation. The aqueous suspension of redispersed powder of CF–rutin-G with DK ester SS^®, which yielded the highest HLB values among the four surfactants, produced an apparently clear solution as compared to the others. In addition, that formulation maintained a clear-solution state after the heat-and-acid treatment. The addition of DK ester SS^® to the binary formulation of CF–rutin-G resulted in a preparation with a smaller particle size. The dispersing effects of the four surfactants differed depending on the HLB, indicating that a surfactant with a higher HLB value could better disperse CFO into water. Surfactants with a higher HLB value could enhance the emulsification of hydrophobic compounds like CFO, because surfactants with a high HLB value (above 8) exhibited hydrophilic properties in water. A further reduction in the amount of the excipient and scale-up are desired for practical use of this technology.

Fig. 3.

Appearance of the samples and a change in particle size distribution of redispersed evaporated CF-rutin-G with (A) DK ester SS^®, (B) DK ester F160^®, (C) SY-glyster MS-5S^®, or (D) SY-glyster MS-3S^®. (I) Soon after dispersal in water, (II) after heat-and-acid treatment.

Spray-dried powder of CF-rutin-G with DK ester SS^®    The powderization of an oily component such as CF is a fairly demanding process (Takashige et al., 2017). Microencapsulation of the oil components of flavors in carrier matrices containing sugars by spray drying is of great importance in the flavor and food industries (Paramita et al., 2010). Powdered CF–rutin-G with DK ester SS^® was prepared by the spray-drying method to obtain monodispersed fine particles. SEM images of the CF samples were taken to assess the surface morphology (Figure 4). Powdered CF–rutin-G with DK ester SS^® prepared by spray-drying was compared with the same powder prepared by evaporation. Powdered CF- rutin-G with DK ester SS^® prepared by evaporation looked like typical flaky evaporated materials, whereas CF–rutin-G with DK ester SS^® prepared by spray-drying consisted of uniform spherical fine particles, and the particle size of the spray-dried powder was smaller than the particles in the evaporation group (Table 1). Particle size is a critical factor in improving the dispersibility of a powder in water because of the increased surface area obtained by the micronization of particles (Bucton and Beezer, 1992). The wettability of CF within the formulation could be enhanced because rutin-G and DK ester SS^® are highly hydrophilic materials.

Fig. 4.

SEM images of (a) untreated rutin-G, (b) untreated DK ester SS^®, (c) evaporated CF-rutin-G with DK ester SS^®, and (d) spray-dried CF–rutin-G with DK ester SS^®.

Table 1. Particle size distribution (µm) of an evaporated powder of CF–rutin-G with DK ester SS^®, and a spray-dried powder of CF–rutin-G with DK ester SS^®. These particle sizes were measured using a MT3300EXII laser diffraction-based apparatus (Data represents the mean ± SE of 3 experiments.).

Sample	D10	D50	D90
CF–rutin-G–DK ester SS® 1:5:0.5 evaporated powder	10.78±0.21	40.17±0.25	193.54±2.17
CF–rutin-G–DK ester SS® 1:5:0.5 spray-dried powder	1.37±0.25	4.03±0.45	9.27±0.51

PXRD analysis was carried out for crystallinity evaluation (Figure 5). The sharp peaks associated with DK ester SS^® are characteristic of its crystalline form, whereas rutin-G yielded a typical broad amorphous band. None of the spray-dried and evaporated CF-rutin-G with DK ester SS^® preparations showed an intense peak associated with DK ester SS^® in the PXRD analysis. These results suggest that CF could be encapsulated into each additive; this phenomenon may be attributable to the preferable miscibility of the materials during the spray-drying and evaporation processes. These observations are suggestive of the encapsulation of CF within rutin-G and DK ester SS^®. Amorphous forms following encapsulation could have higher wettability and dispersibility than the crystalline state after the higher-energy state (Puri et al., 2010).

Fig. 5.

PXRD patterns of (a) untreated DS ester SS^®, (b) untreated rutin-G, (c) spray-dried CF–rutin-G with DK ester SS^®, and (d) evaporated CF–rutin-G with DK ester SS^®.

Figure 6 shows the particle size distributions and the appearance of the CF–rutin-G formulation with DK ester SS^® under each condition. The particle size distribution of the powder shortly after dispersal in water represented an almost unimodal pattern, with a mean volume particle diameter of 150 nm, and that of the powder after heat-and-acid treatment revealed a similarly unimodal pattern, with a mean volume particle diameter of 100 nm. Here, the mean particle size of the powder decreased after heat-and-acid treatment. This effect was due to the promotion of microemulsion by addition of heat to the formulation in water (Liang et al., 2017). According to the appearance after storage in water for one week and after heat-and-acid treatment for one week, few changes were observed. Nevertheless, the particle size distribution of both suspensions had a bimodal pattern because of slight precipitation. Because the observable sedimentation did not form a caking powder, the precipitate was easily redispersible. The dispersible powder of CF-rutin-G with DK ester SS^® has the potential to enhance the nutraceutical performance of CF.

Fig. 6.

Appearances of the samples, and a change in particle size distributions of CF–rutin-G with DK ester SS^® (a) shortly after dispersal in water, (b) after heat-and-acid treatment, (c) after storage in water for a week, (d) after heat-and-acid treatment for a week.

In vitro xanthine oxidase-inhibitory activity    The serum uric-acid-lowering effect of CFO has been demonstrated by Honda et al. (2014). CFO attenuates the increase in serum uric-acid levels by inhibiting xanthine oxidase and enhancing uric-acid excretion via upregulation of its excretion-related genes. The inhibition of xanthine oxidase (a key enzyme in purine metabolism that produces uric acid) by the newly developed powder formulation of CF–rutin-G with DK ester SS^® was studied in comparison with CFO. Figure 7 shows the comparison of the xanthine oxidase-inhibitory activity between CFO and the optimized CF formulation at five concentrations. The inhibition rate of CF formulations was almost equivalent to that of CFO, indicating that the rutin-G and DK ester SS^®-containing CF powder formulation had no adverse consequences for xanthine oxidase-inhibitory activity. Given that both CFO and CF formulations inhibited xanthine oxidase in a dose-dependent manner, the CF formulations are expected to attenuate increases in serum uric-acid levels as effectively as with CFO.

Fig. 7.

A comparison of xanthine oxidase-inhibitory activity between CFO (○) and the CF-rutin-G/DK ester SS^® powder (Δ).

Conclusion

We succeeded in developing a highly dispersible and water-soluble powder derived from CFO using transglycosylated materials and officially approved surfactants as food additives by a spray-drying method. The particle size distribution of the resulting powdered CF–rutin-G–SEs shortly after dispersal in water had an almost unimodal pattern, with a mean volume particle diameter of 150 nm, and that of the powder after heat-and-acid treatment showed a similar unimodal pattern, with a mean volume particle diameter of 100 nm. The wettability of CF within the formulation was enhanced because rutin-G and DK ester SS^® are highly hydrophilic materials. According to in vitro analysis of xanthine oxidase inhibition, the xanthine oxidase-inhibitory activity of the resulting CF–rutin-G–SEs powder is almost equivalent to that of CFO. The CF powder formulation showed better water-dispersibility as compared to CFO. According to these findings, the proposed method for preparing a highly water-dispersible dry powder from natural products containing hydrophobic polyphenols should be an effective and versatile approach to the development of nutritional supplements and functional foods whose oil formulation has previously not been applicable. Nevertheless, details of the bioavailability of the CF powder are still unknown and warrant further research via oral absorption. Further studies on the bioavailability of the CF powder formulation will most likely show a markedly improved xanthine oxidase-inhibitory activity.

Acknowledgements    The authors are grateful to Toyo Sugar Refining Co., Ltd. (Tokyo, Japan) for gifting the transglycosylated compounds. We also thank MicrotracBEL Corp., Ltd. for measurement of particle size using a laser diffraction-based apparatus.

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