Cyclodextrin inclusion complex of a multi-component natural product by hot-melt extrusion

This study aimed to investigate whether hot-melt extrusion (HME) processing can promote molecular encapsulation of a multi-component natural product composed of volatile and pungent hydrophobic substances (ginger oleoresin (OR)) with cyclodextrins. 6-Gingerol and 6-shogaol, the biomarkers of ginger OR, were quantified by HPLC. Phase-solubility studies were performed using β-cyclodextrin (βCD) and hydroxypropyl-β-cyclodextrin (HPβCD) for ginger OR complexation. Solid complexes were then prepared by thermal (HME)– and solvent (slurry (SL))–based methods. Morphology, thermal behavior, solubility, in vitro dissolution, and in vivo anti-inflammatory activity were evaluated. HPβCD gave rise to AL-type complexes with ginger OR, whereas βCD led to materials with limited solubility. Ginger OR was complexed with HPβCD by HME without significant change in gingerol and shogaol content. Additionally, thermogravimetric analysis (TGA) suggested higher volatile retention in HME complexes than in SL ones. Shogaol and gingerol solubility and dissolution significantly increased from SL and HME complexes compared with ginger OR. In turn, 1:2 OR/HPβCD HME complex showed higher 6-shogaol solubility than SL, associated with a gradual release. The carrageenan-induced pleurisy test showed that the anti-inflammatory activity of ginger OR was maintained after complexation with HPβCD. The complexes significantly decrease the levels of IL-1β and inhibit cell migration. HME complex showed performance equivalent to the positive control and superior to the SL material. Taken together, these results indicate that HME can be useful for promoting the molecular encapsulation of complex natural products that contain volatile and thermolabile substances. HME complexes showed better in vivo and in vitro performance than complexes prepared using the solvent-based method.


Introduction
Ginger (Zingiber officinale, Roscoe) rhizome has been extensively used in food products due to its pungent properties. Additionally, the use of ginger as a phytomedicine is being increasingly investigated, and several pharmacological activities have been described [1]. 6-Gingerol is the major metabolite in ginger rhizomes, presenting numerous biological activities, including anticancer [2] and antiinflammatory [1] that make it a potential therapeutic agent [3]. Other compounds, particularly the 6-shogaol, also have important biological activities, including the distinguished anti-inflammatory action reported for this natural product [4]. Both compounds are present at high concentrations in ginger oleoresin (OR), a thick liquid extracted from ginger rhizomes. The use of ginger OR is preferable as a raw material for pharmaceutical purposes, not only because it has high concentrations of secondary metabolites but also because it is more resistant to microbiological contamination and its composition is less affected by seasonal variation [5]. Yet, this material is susceptible to chemical degradation and shows a sticky behavior that complicates its handling [5].
It has been reported that 6-gingerol could be converted into 6-shogaol through a dehydration-hydration reversible reaction, which is pH-and temperature-dependent [6,7]. Additionally, the poor water solubility of gingerols and shogaols hampers their therapeutic application [8,9]. Therefore, the therapeutic use of the ginger OR could be enhanced by stabilizing its main constituents while transforming it into a non-pungent water-soluble free-flowing powder.
Cyclodextrins are cyclic oligosaccharides with a high capacity to form inclusion complexes with hydrophobic compounds, modifying their properties. The utility of such labile encapsulation includes increasing water solubility, modulating dissolution, masking unpleasant tastes, and enhancing stability [2,10]. The inclusion complexation of some isolated ginger constituents has already been reported [9,11]. For instance, intestinal absorption of 6-shogaol was enhanced due to its inclusion into the β-cyclodextrin (βCD) cavity [9]. Additionally, Silva et al. [11] reported the solubility and dissolution improvement of 6-gingerol from the βCD/6-gingerol complex.
Despite the great number of studies on the formation of inclusion complexes of apolar compounds with cyclodextrins, the use of this approach with multi-component guests is less frequent [8]. In the case of natural products, the therapeutic effects are typically the result of a synergistic action of several compounds. Hence, it is essential to ensure that the cyclodextrins could interact with the various secondary metabolites of the vegetal substrate to improve its pharmacokinetics. At the same time, there is a great technological challenge, considering that multiple compounds will compete for the hydrophobic cavity of the cyclodextrin.
In this sense, a gingerol-enriched extract from ginger rhizome was complexed with γ-cyclodextrin and incorporated into a food matrix with a minimum color difference, resulting in a product with good antioxidant activity for nutraceutical purposes [8]. To the best of our knowledge, there is no study reporting the effects of the ginger OR complexation with cyclodextrins on the dissolution of 6-gingerol and 6-shogaol. Additionally, there are no studies on OR complexation with hydroxypropyl-β-cyclodextrin (HPβCD), a more soluble and safer βCD derivative.
In general, cyclodextrin complexation in solid-state has been performed using solvent-based methods [12]. Melting methods such as hot-melt extrusion (HME) present several advantages, including avoiding drying steps and organic solvents, and allowing fast production with low costs [13]. Few studies have reported inclusion complexation with cyclodextrins using HME [14][15][16], and none of them described the complexation of multi-component natural products.
Based on this, this work aimed to explore the complexation of ginger OR using a more water-soluble cyclodextrin derivative and to evaluate the possible complexation of this natural product in solid-state using a melting method. For this, phase-solubility studies were performed to understand the nature of the 6-shogaol and 6-gingerol complexes with βCD or HPβCD. Next, ginger OR was complexed with HPβCD using solvent and melting methods. The solid complexes were characterized by thermogravimetry (TGA), solubility, and in vitro dissolution. Lastly, the antiinflammatory activity of the ginger OR and its complexes with HPβCD was determined using a carrageenan-induced pleurisy model.

6-Gingerol and 6-shogaol quantitation
The quantitation of 6-gingerol and 6-shogaol, the ginger OR markers, was performed by high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity II HPLC system equipped with a UV detector (G7114A), quaternary pump (G7111B), and auto-injector system 1 3 (G7129A) (Agilent Technologies, Santa Clara, CA, USA). An RP-18 column (250 × 4.6 mm, 5 μm) was used at 38 °C. The mobile phase was comprised of a 30:30:40 (v/v/v) mixture of acetonitrile, methanol, and water (in the first 10 min) and acetonitrile:water (53.5:46.5, v/v) mixture for the next 14 min. The flow rate was 1.2 mL/min, the injection volume was 20 μL, and the detection wavelength was set at 280 nm. The method was validated in accordance with International Conference on Harmonization guidelines to be used in the makers' content determination of the ginger OR and the inclusion complexes produced as well as in the dissolution studies. Particularly, selectivity was investigated against the formulation compounds used, i.e., βCD, HPβCD, HPMC, PEG400, and talc.
Methanolic solutions of the 6-gingerol and 6-shogaol analytical standards were prepared in the range from 3 to 18 µg/mL to obtain analytical curves. Ginger OR and OR/ HPβCD complexes were dispersed in methanol solution (0.3 mg/mL of OR), filtered (PVDF membrane, 0.45 µm), and injected into the HPLC system. The markers' contents of the OR/HPβCD solid inclusion complexes produced here were obtained by the recovery percentage (%R) defined as the average 6-shogaol plus 6-gingerol in each sample calculated as a function of the theoretical amount of such markers in the complex.

Phase-solubility studies
The solubility studies were carried out according to the method described by Higuchi and Connors [17]. First, an excess amount of ginger OR (20 mg) was added to screwcapped vials with 5 mL of ethanol:water (25:75 v/v) containing βCD or HPβCD (0 to 16 mM). Next, the vials (n=5) were shaken at 25 °C for 48 h in an orbital shaker SL-233 (Solab, São Paulo, Brazil). Then, the samples were filtered using a 0.45-µm PVDF filter (Millex ® , Millipore Corporation, Darmstadt, Germany), diluted in methanol, and quantified using the HPLC method described in the "6-Gingerol and 6-shogaol quantitation" section. The apparent stability constants (Kc) of 6-gingerol and 6-shogaol were calculated from the linear regression of the phase-solubility curves, according to Eq. 1. The experiments were performed in triplicate.
where S o is the intrinsic solubility of 6-gingerol and 6-shogaol measured in the ginger OR without cyclodextrin.

Inclusion complexation using a solvent-based method
Slurry complexation was carried out by adding absolute ethanol to a beaker containing HPβCD at 10:1 (v/m) solvent:cyclodextrin ratio. Next, different amounts of the ginger OR were added to achieve a 1:1 and 1:2 molar guest:host ratios ( Table 1). The molar weight of the OR was calculated considering the molar weight of the 6-gingerol (294 g/mol) and 6-shogaol (276 g/mol), and their ratio in the OR. Then, the samples were stirred for 12 h by a magnetic stirring device (RT ® 10 power magnetic stirrer, IKA, Staufen, Germany) operating at 440 rpm. Finally, the mixtures were dried at room temperature for 7 days in a desiccator. For comparison purposes, physical mixtures were prepared by adding the ginger OR in an agate mortar containing powdered HPβCD. All samples (SL complexes) were kept in airtight amber glass containers stored at 4 °C.

Inclusion complexation by hot-melt extrusion
Ginger OR:HPβCD complexes were prepared at 1:1 and 1:2 molar guest:host ratios ( Table 1). The OR/HPβCD binary mixture corresponded to 50% of the total mass of the formulation. The other 50% was comprised of HPMC (41%, m/m), PEG400 (5%, m/m), and talc (4%, m/m). All formulation compounds were mixed in a mortar and manually fed into a lab-scale vertical twin-screw hot-melt extruder equipped with a backflow channel for recirculation (EHM 5, LabMaq do Brasil Ltd., São Paulo, Brazil). A 16-mm twin-screw with a helix angle of 1.97° was used. The HME of the samples was performed using a temperature gradient (120/130/140 °C) applied to the 3 heating zones of the equipment. The screw speed was 75 rpm, and the molten material was recirculated inside the extruder barrel for 3 min. The extruded material was collected, cooled to room temperature, and milled using a mortar and pestle. The powder fraction from 180 to 125 μm was selected for further analysis.

Solubility study
Ginger OR and its complexes in solid-state with HPβCD were added (in excess) in test tubes containing 10 mL HCl 0.1N solution. First, the tubes were kept under magnetic stirring (440 rpm) for 24h at 37 °C. Next, the suspensions were filtered (0.45 µm), diluted in methanol, and quantified by HPLC (n = 5).

Thermogravimetry
Thermogravimetric analyses (TGA) were carried out in a thermobalance TGA/DTA-60 (Shimadzu ® , Kyoto, Japan). Ginger OR, neat HPβCD, OR/HPβCD complexes (SL and HME), and their corresponding physical mixtures were placed in platinum crucibles (5 mg) and heated at 10 °C min −1 from 25 to 650 °C. Analyses were performed under a dynamic nitrogen atmosphere (50 mL min −1 ). Mass loss of each complex was compared to that determined from their corresponding physical mixture with a view of shedding light on OR/HPβCD interaction.

Morphological characterization
The macrostructure of the OR/HPβCD complexes was evaluated by recording scanning electron microscopy (SEM) images using a JEOL JSM 6610 (Tokyo, Japan) apparatus equipped with an energy-dispersive spectrometry (EDS) X-ray detector (Thermo Scientific, Waltham, MA, USA). Samples were deposited on glass slides and coated with gold using a Denton Vacuum sputter coater (Desk V, Moorestown, USA) for 2 min. SEM images were captured with 50 and 300× magnification using 5 kV.

In vitro dissolution
6-Gingerol and 6-shogaol in vitro dissolution were evaluated, under non-sink conditions, using a DT 80 dissolution system (Erweka® GmbH, Langen, Germany) equipped with a USP type II apparatus. OR/HPβCD complexes obtained by SL and HME methods, as well as the ginger OR mixed with microcrystalline cellulose (30:70, m/m), were placed in 500 mL of dissolution media (HCl 0.1N, pH 1.2), which was kept at 37 °C, and stirred at 50 rpm. Each dissolution cube had an equivalent of 140.8 mg ginger OR in all cases. Samples were withdrawn at predetermined intervals (5,15,30,45, 60, 90, and 120 min) and filtered through a 0.45-µm PVDF membrane. After each sampling, an equal volume of fresh dissolution medium was replaced to the cube. The filtrate was diluted in methanol and analyzed by the HPLC method previously described.
The experiments were performed in triplicate.

Animals
Albino Swiss mice (3-month-old, 28-32g) were randomly housed in appropriate cages ( ). An additional control group of healthy animals that did not receive any treatment was used (Sham). Samples were always administered 60 min before carrageenan pleural injection. Pleurisy was induced by intrapleural administration of 100 μL of 1% (w/v) carrageenan suspension in sterile saline solution [18]. An adapted 13 × 5 needle was inserted into the right side of the thoracic cavity to inject the carrageenan suspension. Four hours after the carrageenan administration, the animals were euthanized, and the pleural inflammatory exudate was collected through pleural lavage with 1 mL of PBS containing ethylenediaminetetraacetic acid (EDTA; 10 mM). The exudate volume was centrifuged (1500 rpm, 10 min), and the supernatant was collected for the determination of cytokine levels in the pleural fluid. The cells were resuspended in 500 µL PBS, and an aliquot of 10 μL was diluted with Turk's solution (1:20). The total leukocytes were counted in a Neubauer chamber using a light microscope, examining four external quadrants [19].

Determination of TNF-α and IL-1β levels in the pleural fluid
Tumor necrosis factor-alpha (TNF-α) and interleukin 1 beta (IL-1β) levels in the pleural cavity were assessed 4 h after carrageenan injection. TNF-α and IL-1β levels in the pleural exudate were determined following the manufacturer's protocol (BD-Bioscience Pharmingen, San Diego, CA, USA). All analyses were done in duplicate. The plates were read in an ELISA microplate reader (ASYS ® ) at a wavelength of 450 nm. Finally, the concentration of cytokines was obtained by interpolating the standard curve and the results are expressed as pg/mL. The kit's sensitivity is 8 pg/mL (IL-1β and TNF-α).

Statistical analysis
Data were expressed as mean ± SEM. Differences of groups were analyzed using one-way analyses of variance (ANOVA) followed by Tukey's test. The statistical analyses were performed using the GraphPad Prism 5.0 software (GraphPad Prism Software Inc., San Diego, CA, USA).

Results and discussion
Quantification of 6-gingerol and 6-shogaol 6-Gingerol and 6-shogaol content in ginger OR was determined using a validated reverse-phase chromatographic method. Figure 1 shows the chromatograms of the ginger OR (Fig. 1a) and analytical standards (Fig. 1b). Retention times for gingerol and shogaol in OR were 9.03 ± 0.08 min and 21.90 ± 0.29 min, respectively. The 24-min running time is shorter than the previous methods described, which required between 30 and 60 min [20][21][22][23][24]. In addition, high resolutions among 6-gingerol and 6-shogaol and the other OR constituents were achieved (peak resolution of 3.04 and 3.11, respectively). The method was linear with correlation coefficients 0.9999 and 0.9998 for 6-gingerol and 6-shogaol, respectively, meeting the validation requirements and proving the ability of the method to provide proportionality between the area and the concentration values in the range tested (3 to 18 μg mL −1 ). The high values of the angular coefficients of the curves (246200 and 316703, for 6-gingerol and 6-shogaol, respectively) indicate the appropriate response of the method to the changes in the concentration imposed. ANOVA shows non-significant linearity deviation (0.03 < 3.26 for 6-gingerol and 0.10 < 3.26 for 6-shogaol) and significant linear regression (432.22 > 4.75 for 6-gingerol and 504.64 > 4.75 for 6-shogaol). The residual graph showed non-biased data collection (Fig. S1, Supplemental Material). The method was also accurate (97.67-99.19% for gingerol and 99.69-99.92% for shogaol) and precise (coefficient of variation of 1.45% for gingerol, and 1.83% for shogaol) with a quantification limit of 159 and 1027 ng mL −1 for gingerol and shogaol, respectively. Selectivity was investigated (βCD, HPβCD, HPMC, PEG400, and talc), and no interference was observed in drug retention time (Fig. S2, Supplemental  Material). Furthermore, the area of the peaks did not change in the presence of the formulation constituents. 6-Gingerol and 6-shogaol content in OR was 5.15 g. 100 g −1 and 3.98 g. 100 g −1 , respectively.

Phase-solubility studies
The phase-solubility diagram of 6-gingerol in HPβCD is shown in Fig. 2a. The curve showed that the 6-gingerol solubility linearly increased with HPβCD concentration, featuring a type A L curve, as proposed by Higuchi and Connors [17]. The Kc of this complex was 9.89 M −1 which is a value below the usual range found for drug inclusion complexes reported in the literature (50 to 2000 M −1 ) [25]. In its turn, the phase-solubility diagram of 6-shogaol (A L -type; Fig. 2b) showed higher affinity to HPβCD cavity with a Kc of 80.12 M −1 , which is expected since 6-shogaol is rather more apolar than 6-gingerol.
Despite the differences in 6-shogaol and 6-gingerol affinity to the HPβCD cavity, both showed low Kc values, which can be attributed to the competition for the cyclodextrin cavity with several other guests presented in a natural product such as OR ginger [8]. As far as we know, this is the first time that HPβCD complexes with 6-gingerol and 6-shogaol were reported. Moreover, the inclusion complexation of oleoresins with cyclodextrins is poorly documented in the literature [12,[26][27][28][29]. Importantly, there is no previous report on ginger OR/HPβCD inclusion complex.
On the other hand, the complexation of 6-gingerol in ginger OR with βCD resulted in the formation of a Bs-type complex. Figure 2a shows an initial increase in 6-gingerol solubility (at low βCD concentration) followed by a decrease. In this profile, the inclusion complex formed is poorly soluble. Indeed, increasing the βCD concentration produced more inclusion complexes that rapidly reached saturation in the medium and precipitate. From that point onwards, the progressive increase in the concentration of βCD shifts the balance toward the formation of more inclusion complexes that precipitate. Thus, in practice, a reduction in the amount of compound dissolved in the medium is observed. Silva et al.
(2021) also reported the formation of Bs-type complex with βCD using high-purity 6-gingerol. Unlike the observed for gingerol, shogaol/ βCD complex gave rise to A L -type curve (Kc; 15.91 M −1 ) (Fig. 2b). There is only one report on the inclusion complexation of 6-shogaol with βCD that shows a Kc value of 1017.24 M −1 , about 64-fold higher than that found here. This can be attributed to the multiple guests present in ginger OR and is evidence that the cyclodextrins interact with the other compounds of the natural extract.
In sum, phase-solubility studies of 6-gingerol and 6-shogaol in ginger OR clearly showed the advantages of using HPβCD as host since it gave rise to A L curves. Accordingly, only HPβCD complexes were prepared by slurry and hot-melt extrusion methods.

Ginger OR complexation by slurry and hot-melt extrusion methods
The A L -type phase-solubility curves for HPβCD discussed in the "Phase-solubility studies" section indicated the formation of inclusion complexes with 1:1 stoichiometry (OR/HPβCD) in solution. To produce solid-state complexes, which are limited molecular mobility systems, in addition to the equimolar ratio, complexes in the molar ratio of 1:2 (OR/HPβCD) were also produced. SL complexes prepared at 1:1 and 1:2 OR/ HPβCD molar ratio resulted in yellowish granulated material (Fig. 3). SEM images showed a large particle size of the 1:1 SL complex, likely related to the greater amounts of OR. The macroscopic aspect of the 1:1 SL complex showed a darker and more cohesive material than the 1:2 OR/HPβCD complex (Fig. 3). 6-Gingerol content in 1:2 and 1:1 SL complexes was This data suggested the uniform distribution of the 6-gingerol in the samples. On the other hand, 6-shogaol content in 1:2 and 1:1 SL complexes was 84.3 ± 3.3% and 83.0 ± 1.0%, respectively, suggesting some loss of this hydrophobic marker during complex preparation by slurry complexation. 1:1 and 1:2 OR/HPβCD complexes were prepared by HME. Preliminary tests were performed to determine the range of the extruder operating conditions in preparing these complexes. The temperature range used (120/130/140 °C) allowed for high yield production, without OR losses, and enabled processing under moderate torque. Indeed, the inclusion complexation by HME did not result in any decrease of %R compared with pure OR (p > 0.05) (Fig. S3, Supplemental Material). The 6-gingerol content in 1:2 and 1:1 HME complexes was, respectively, 92.5 ± 1.3% and 99.0 ± 2.6% of the theoretical value expected, suggesting the uniform distribution of this marker in HME samples. 6-Shogaol content, in its turn, was 110.2 ± 7.8% and 109.6 ± 8.3%, suggesting some non-uniformity of this marker in HME samples. It is important to note that ANOVA showed no individual difference in the 6-shogaol and 6-gingerol content of the HME complexes compared with pure OR (p > 0.05). The reversible conversion of 6-gingerol into 6-shogaol was reported as insignificant within 1 h in solution at 37 °C [7]; however, the reaction is favorable at high temperatures [6]. In the present study, the recirculation time during HME trials (3 min) was insufficient to generate an effective conversion between the markers. These findings showed the feasibility of using a thermal process to obtain stable inclusion complexes containing ginger OR. HME complexes showed a denser and homogeneous aspect than SL ones (Fig. 3), explained by their lower OR concentration (Table 1). SEM images of the milled OR/CD HME complexes show the presence of small and agglomerated particles (Fig. 3).

Solubility study
The solubility of ginger OR markers was evaluated in a simulated gastric medium to assess the feasibility of the oral administration of the inclusion complexes produced. From the free ginger OR, the solubility of 6-gingerol (16.09 µg/ mL) was 8.6-fold higher than that of 6-shogaol (1.87 µg/ mL). Indeed, the higher polarity of 6-gingerol is due to the presence of a hydroxyl moiety in the alkyl chain of such compound [24].
Gingerol and shogaol solubility in OR/HPβCD complexes prepared by SL or HME are presented in Fig. 4. 6-Gingerol solubility was significantly improved in all OR/HPβCD complexes prepared by both methods at 1:1 and 1:2 OR/ HPβCD ratio, when compared to ginger OR (p < 0.05) (Fig. 4a). 6-Gingerol solubility was not influenced by the preparation method (p > 0.05).
In its turn, 6-shogaol solubility in SL complexes showed a 147-fold improvement compared to ginger OR (p < 0.05) (Fig 4b). The comparison among SL complexes prepared at 1:1 and 1:2 molar ratios showed that the shogaol solubility was significantly higher when HPβCD amount was greater (OR/HPβCD 1:2) in the formulation (p < 0.05). Inclusion complexation using HME technology resulted in an even greater improvement of 6-shogaol solubility compared to pure OR (162-fold) (p < 0.05). Additionally, shogaol solubility in HME complexes was significantly higher than that observed for the corresponding SL complexes (p < 0.05). This finding suggests the higher complexation efficiency of HME complexes prepared under higher temperatures and shear forces. Malaquias et al. [15] had shown that HME was 3-fold more effective than traditional complexation methods when the poorly watersoluble fluconazole was extruded with hydroxypropyl cellulose and βCD or HPβCD. Fig. 3 Scanning electron micrographs and visual aspect of ginger oleoresin/cyclodextrin complexes (OR/HPβCD) prepared by slurry complexation (SL) or hot-melt extrusion (HME) Moreover, possibly, the conditions used in the SL complexation may not have guaranteed the necessary dispersion of more apolar compounds, like shogaol, whereas the heat and shear forces applied during HME processing may have promoted a closer contact between this compound and the cyclodextrin. Until now, no studies have compared solventbased and HME methods in the preparation of inclusion complexes with cyclodextrins.

Thermogravimetry
Solubility improvement of the pungent compounds suggests their complexation with HPβCD. Furthermore, the ginger OR is also composed of several volatile compounds, like the α-zingiberene, α-farnesene, β-bisabolene, β-sesquiphellandrene, cineol, geraniol, camphene, and citral among others which contribute to its biological activities [1]. TG analysis is a very suitable tool for inferring the complexation of volatile compounds in cyclodextrins. Comparing the complexes and their corresponding physical mixtures allows for qualitative suggestions of host and guest interactions in different samples [30]. To investigate the complexation of volatile components in ginger OR, the mass loss differences between solid complexes and their corresponding physical mixtures at different temperature intervals were used. The TG curve of pure ginger OR is shown in Fig. S4 (Supplemental Material). This curve shows three mass loss steps from 25 to 400 °C. Figure 5 shows the mass loss in the interval from 25 to 130 °C (Fig. 5a) and from 130 to 290 °C (Fig. 5b). Mass loss from OR/HPβCD complexes (SL and HME) prepared at 1:1 and 1:2 molar ratios in the first temperature range was lower than that calculated from their PM counterparts, which suggests a successful inclusion of the OR volatile constituents. Conversely, it may indicate a loss of these volatile compounds during the complexation procedures. To shed light on this issue, mass loss from SL and HME complexes was compared with the mass loss from the corresponding PM at a higher temperature interval (130 to 290 °C). A higher mass loss from the complexes could be observed, supporting the suggestion of a successful complexation of volatile materials by using both methods.
Particularly, it is important to highlight the mass loss behavior of the 1:2 OR/HPβCD (HME) complex. This material showed the biggest mass loss difference in the 25-130 °C interval compared with its corresponding physical mixture. This finding strongly suggested that HME was more efficient in promoting the complexation of the volatile ginger constituents. Therefore, it could be used to produce cyclodextrin complexes containing volatile compounds, similarly to what has already been described for other volatile natural products [31]. Consequently, a proper choice of temperature and residence time is needed to guarantee the quality of the final product. Figure 6a shows the shogaol dissolution from ginger OR and OR/HPβCD complexes. Shogaol dissolution from all complexes was significantly higher than from ginger OR (p Fig. 4 6-Gingerol (a) and 6-shogaol (b) solubility in acid medium from ginger OR, and OR/HPβCD complexes prepared by slurry complexation (SL) or hot-melt extrusion (HME). *Significantly lower than all OR/HPβCD complexes; # lower than 1:2 SL and 1:1 and 1:2 HME complexes; ** higher than 1:2 SL and 1:1 HME complexes (one-way ANOVA, followed by Tukey's test) < 0.05), denoting the impact of the inclusion complexation of ginger OR in accelerating the biomarkers' dissolution. It can be noted that a lower % shogaol was dissolved at the end of the experiment (120 min) from 1:1 OR/HPβCD complexes than that from 1:2 samples prepared using the same method, in agreement to solubility data in acid medium. Similarly, 1:2 OR/HPβCD complex prepared by HME showed higher shogaol dissolution (at 120 min) than that observed from the corresponding SL complex (p < 0.05). This finding is, once again, in agreement with the solubility data previously discussed. Notably, the shogaol dissolution from HME complexes was more gradual than from SL samples (Fig. 6a), which can be explained by the swelling of the HPMC extrudate at the beginning of the dissolution assay. Low molecular weight HPMC such as Benecel E3 is used to fine-tune drug release in immediate release systems.

In vitro shogaol and gingerol dissolution from OR/ HPβCD complexes
Additionally, one can hypothesize that shogaol is interacting with the polymeric matrix retarding its release. Indeed, Benecel E3 has a high average percent of methoxy groups [32], increasing its affinity for hydrophobic drugs. Compared to high-molecular-weight HPMC with a similar percent of methoxy groups (E5, E15, and E50), Benecel E3 showed a higher ability to interact with poorly soluble drugs [33].
Dissolution tests of SL complexes prepared with the exact composition of HME ones were not performed because it was impossible to prepare homogeneous samples containing 41% (m/m) of HPMC by the slurry procedure. Figure 6b shows the % gingerol dissolved from ginger OR and OR/HPβCD complexes. At 120 min, all complexes showed higher gingerol dissolution than ginger OR (p < 0.05). In fact, at the end of the assay, more than 90% of 6-gingerol was dissolved from the complexes, against only 75% from the uncomplexed ginger OR. The OR/HPβCD complexes presented similar dissolution regardless of the production method or the drug:cyclodextrin molar ratio. Similar to what was observed for shogaol, gingerol was gradually dissolved from the HME samples.
In general, the benefits of inclusion complexation of ginger OR in solid-state were remarkable. The highest effects were observed for shogaol due to its higher affinity for the HPβCD cavity. The performance of the 1:2 OR/HPβCD complex produced by HME is noteworthy. Solubility and dissolution data, as well as the TGA findings, pointed to its superiority. Additionally, this complex was produced using no solvent in a fast and potentially continuous process. Altogether, these results show the great potential of the HME method for cyclodextrin complexation of multi-component guests composed of botanical extracts. The next step was to investigate the in vivo anti-inflammatory effects of the selected OR/HPβCD complexes (1:2 OR/CD prepared by SL or HME).

In vivo study
The anti-inflammatory profile of HME, SL, and ginger OR (200 and 500 mg/kg v.o.) was evaluated by the carrageenaninduced pleurisy model, and the data are summarized in Fig. 7. This model allows studying acute inflammation by following different mediators and determining the effects of drugs in the inflammatory response. Carrageenan increases local vascular permeability, causing fluid extravasation and leukocyte infiltration [34][35][36][37], and activates the immune cells, inducing an exacerbated production of proinflammatory cytokines TNF-α and IL-1β [34,36,38].
The results show that the administration of carrageenan into the pleural space of mice induced a significant increase in total leukocyte count (p < 0.001), and upregulated TNF-α (p < 0.001) and IL-1β (p < 0.001) production compared with the control group of healthy animals (SHAM group). Treatment with OR/HPβCD 1:2 (HME) complex and ginger OR (500 mg/kg, v.o.) significantly decreased the total leukocyte count (p < 0.001, p < 0.05) compared to the control group. The positive control indomethacin (10 mg/kg, v.o.) had the same effect (p < 0.001) (Fig. 7a). Additionally, HME, SL, and ginger OR (500 mg/kg v.o.) caused a significant reduction in IL-1β levels (p < 0.01) in pleural fluid when compared to the control group (Fig. 7b). Notably, this response was similar to that obtained using the positive control of indomethacin. Finally, no significant difference was observed in TNF-α production in HME-, SL-, and ORtreated mice (p > 0.05) (Fig. 7c).
Our findings showed that the HME complex mitigated the cell migration induced by carrageenan. This effect was equivalent to that presented by the positive control, indomethacin. In all cases, the inhibition of cell migration may be associated with the inhibition of proinflammatory cytokine IL-1β release (Fig. 7b), since IL-1β plays a key role in respect of cell accumulation in the pleural cavity. Moreover, this cytokine exerts a critical modulatory action in the inflammatory reaction, leading to the increase in prostanoid levels and activating the inflammatory pathway [34]. Then, it is feasible that the anti-inflammatory activity of ginger constituents is associated with the inhibition The analyses were performed 4 h after carrageenan injection to evaluate the recruitment of total leukocytes (a) and levels of IL-1β (b) and TNF-α (c). Data were expressed as mean ± SEM. *p < 0.05, **p < 0.01, and *** p <0.001 vs control groups; ## p < 0.01 and ### p < 0.001 vs sham groups (one-way ANOVA, followed by Tukey's test) (n=8, per group). SHAM, control group of healthy animals; Control, vehicle (0.1% polysorbate 80 aqueous dispersions, 0.1 mL/10g, v.o.) 1 3 of the production of inflammatory final mediators. In this context, ALmohaimeed et al. [39] reported that oral ginger administration downregulated IL-1β expression in the hippocampus of diabetic animals, confirming that ginger acts in the IL-1β signaling pathway. In addition, literature studies have also shown that 6-shogaol, a component of ginger oleoresin, mitigates leukocyte infiltration into inflamed tissue accompanied by a reduction in inflammatory mediators, including COX-2, MAPK, and IL-1β [40,41], corroborating in part our findings.
It is interesting to note that the SL complex was not able to significantly reduce the cell migration even in the highest dose studied (Fig. 7a). 6-Shogaol solubility from the SL complex was lower than that of HME (Fig. 4a). Additionally, TG data suggested that the HME complex has higher volatile content than the SL one; therefore, the in vivo superiority of the HME complex can be attributed to the higher inclusion of the 6-shogaol and volatile constituents in this material. Indeed, the anti-inflammatory activity of ginger rhizome has been attributed to volatile components and pungent compounds [1]. Lantz et al. [4] reported that the in vitro anti-inflammatory activity of ginger can be related to inhibition of PGE2-production and LPS-induced COX-2 expression. These authors also showed that ginger constituents are less effective in inhibiting TNF-α, which agrees with the results presented here.

Conclusion
The HPβCD showed a greater affinity for the biomarkers of ginger OR (6-shogaol and 6-gingerol) compared to βCD. In fact, the more efficient complexation of the cyclodextrin derivative promoted the formation of inclusion complexes in solid-state with increases in solubility of these biomolecules in several orders of magnitude (more than 160 times for some samples). Moreover, the solid complexation promoted a notorious stabilization of the volatile compounds present in ginger OR, which are co-responsible for their pharmacological properties.
Regarding the production of solid-state complexes, a novel production method of cyclodextrin complexes with a multicomponent natural product by using hot-melt extrusion was proposed. Mainly, inclusion complexes containing ginger OR and HPβCD prepared by HME (1:2 molar ratio) showed a higher 6-shogaol solubility and a gradual dissolution compared to the 1:2 slurry complex. Additionally, the HME complex presented a superior in vivo anti-inflammatory activity than ginger OR or SL complex. Taken together, these results show that HME can be used to promote molecular encapsulation of natural materials that contain volatile and thermolabile substances, allowing for better in vivo and in vitro performance compared to complexes prepared by solvent-based traditional methods.