Therapeutic Potential of Hydroxypropyl-β-Cyclodextrin-Based Extract of Medicago sativa in the Treatment of Mucopolysaccharidoses

• Abstract
• Introduction
• Results
• Discussion
• Materials and Methods
• Plant materials and chemicals
• Preparation of extracts
• Total phenol and total flavonoid content
• Free radical scavenging and chelating activity
• Experimental design
• HPLC analysis of isoflavones
• Determination of sulphated glycosaminoglycans in skin fibroblasts
• Supporting information
• Acknowledgements
• References

Abstract

Mucopolysaccharidoses are inherited metabolic disorders resulting in the dysfunction of enzymes involved in the degradation of glycosaminoglycans, leading to severe clinical symptoms and a significantly shortened life span of patients. Flavonoids are recognized as glycosaminoglycan metabolism modulators, able to correct glycosaminoglycan cell storage. Therefore, the aim of this work was the development of an efficient and eco-friendly extraction process of phytochemicals from Medicago sativa by simultaneous use of ultrasound extraction and hydroxypropyl-β-cyclodextrin complexation, and investigation of the potential of such an extract as a glycosaminoglycan metabolism modulator. The Box-Behnken design and response surface methodology were used in order to optimize the extraction process, considering hydroxypropyl-β-cyclodextrin concentration, ultrasonic power, and extraction time as the key parameters. The dependent variables included total phenolicand total flavonoid content, DPPH radical scavenging activity, and Fe²⁺ chelating activity, due to the importance of oxidative stress in the pathology of mucopolysaccharidoses. The developed technology using hydroxypropyl-β-cyclodextrin led to more selective flavonoid extraction from M. sativa than obtained either by the use of water or ethanol. The lyophilization of extracts resulted in products with high radical scavenging activity, suitable for further use. The application of 20 mM hydroxypropyl-β-cyclodextrin solution, 432 W ultrasonic power, and an extraction time of 45 min resulted in an extract with both the highest total flavonoid content and the lowest radical scavenging activity IC₅₀. This extract reduced the levels of glycosaminoglycans in skin fibroblasts of mucopolysaccharidose III patient in a dose-dependent manner. At concentrations of 3 and 6 µg/mL, the observed levels of glycosaminoglycans were reduced by 41.2 and 51.1 %, respectively, clearly demonstrating the validity of the selected approach.

Introduction

Medicago sativa L. (Fabaceae), commonly known as alfalfa or lucerne, is a perennial herbaceous plant that has long been used as a traditional herbal remedy in Europe, Asia, and America. It is used to improve memory, to cure kidney pain, cough, and sore muscles, as a rejuvenator, diuretic, and galactagogue as well as an antidiabetic, antioxidant, anti-inflammatory, antifungal, antiasthmatic, and antimicrobial agent. In addition, its sprouts are used as food. Alfalfa is a rich source of phenolic antioxidants, which greatly contribute to its observed medicinal properties [1], [2].

MPS are inherited metabolic disorders caused by a deficiency of enzymes required for the stepwise breakdown of GAGs [3], [4]. Fragments of partially degraded GAGs accumulate in the lysosomes, resulting in cellular dysfunction and clinical abnormalities. Depending on the nature of the lacking or defective enzyme and the kind(s) of stored GAG(s), 11 types and subtypes of MPS have been distinguished. These are rare conditions, with an estimated total incidence of all MPS types of approximately 1 in 20 000 live births. Clinical manifestations can vary in different MPS, as well as in distinct patients within the same disorder, and include hepatosplenomegaly, multiple dysostosis, reduced growth, recurrent infections, and a chronic degenerative progression of the disease [3].

Despite extensive studies focused on the development of novel therapies for MPS, only several therapeutic options are currently available to patients as approved procedures. Therapies used for a relatively large number of MPS patients, such as bone marrow transplantation and enzyme replacement therapy (ERT), are notably ineffective for neurological symptoms due to the poor distribution of enzymes in the central nervous system. Therefore, there is a need for continuous studies in order to enhance therapeutic strategies of MPS. One such strategy is the implementation of the nonenzymatic substrate reduction therapy (SRT) using GAG metabolism modulators. It was believed that the primary storage of GAGs and their deposition in tissues was solely responsible for the signs and symptoms associated with the MPS [5], but recent research suggests a more complex picture [6], implying that oxidative stress is one of the main factors implicated in MPS pathogenesis [7], [8]. Therefore, implementation of the nonenzymatic substrate reduction therapy using various flavonoids, such as antioxidants and GAG metabolism modulators, could be a possible strategy to enhance the therapeutic efficiency of MPS [9].

Ultrasound has great potential in the extraction of bioactive compounds from the plant material. It shows high extraction efficiency as well as low energy and solvent consumption, resulting in more pure products. A main mechanism behind highly efficient ultrasound-assisted extraction has been attributed to mechanical, cavitation, and thermal effects, which can result in the disruption of cell walls, particle size reduction, and enhanced mass transfer across cell membranes. All of this enhances solvent contact with the extractable cell material, resulting in higher extraction yields in a relatively short extraction time [10], [11]. Besides the reactor configuration, it is important to optimize several operating parameters, including extraction time, temperature and, most importantly, the solvent type. The type, quantity, and concentration of the solvent have a prominent effect on extraction efficiency. The effectiveness of the ultrasound-assisted extraction depends on the solventʼs capacity to adsorb and transmit the energy of the ultrasound. Besides viscosity, vapor pressure and surface tension of the solvent are the two key factors that impact the cavitation intensity and, generally, cavitation intensity decreases as vapor pressure and surface tension increases. The polarity of the solvent is another important characteristic controlling extraction efficiency. Water is the most favored solvent due to its eco-friendly nature and low cost, but many times water may not be able to extract the contents completely and it is necessary to use organic solvents, such as ethanol, methanol, and hexane [11].

In order to avoid the use of organic solvents and to develop an eco-friendly extraction procedure, we have opted to use aqueous CD solutions as the extraction medium. Such an approach was successfully applied to extract phenolic compounds from grapes and their pomace, as well as to prepare mistletoe extracts with antioxidant and cytostatic activity [12], [13]. Also, one-pot ultrasound-assisted water extraction of resveratrol from Polygonum cuspidatum Siebold & Zucc. (Polygonaceae) was successfully accomplished by CD application [14]. All this prompted us to investigate in-depth the applicability of this technology in the extraction of bioactive compounds from M. sativa. CDs are a group of structurally related oligosaccharides, able to improve solubility, chemical stability, and bioavailability of different compounds through an inclusion complex formation. Chemical modification of parent CDs consisting of 6, 7, or 8 glucopyranose units (αCD, βCD, and γCD) resulted in numerous derivatives with improved solubility and complexation efficiency. In general, CDs are biocompatible and nontoxic when applied orally, while some derivatives, such as HPβCD, sulphobuthyl-β-cyclodextrin (SBEβCD), and γCD, are also suitable for parenteral application [15], [16].

A wide range of reports has been published regarding the encapsulation of natural polyphenolic compounds by CDs for food and drug delivery purposes. The majority of these publications demonstrated improved the water solubility, stability, and bioavailability of different polyphenols due to inclusion complex formation. In general, CD derivatives such as HPβCD were more efficient as complexation agents compared to parent βCD and, in many cases, an increased antioxidant capacity of CD encapsulated polyphenols was observed [16], [17]. Taking all of this into account, it seems that the use of CD aqueous solutions as an extraction medium could be a valid approach to efficiently extract bioactive compounds from the plant material, especially taking into account the results of Yatsu et al. [18], who demonstrated the possibility of the simultaneous complexation of daidzein, genistein, and glycitein from an enriched soy fraction with the HPβCD. An additional benefit of the CD-based extraction could be in their ability to chemically stabilize sensitive molecules by inclusion complex formation, considering that ultrasound may trigger or accelerate some chemical reaction in the sample, such as degradation of phenolic acids as well as degradation and oxidation of flavonoids [19]. Finally, the addition of CDs would allow the preparation of free-flowing, highly soluble powdered extracts, which are more convenient for further use [20]. In order to produce such products, a lyophilization procedure could be employed. This is a well-established drying process used to convert aqueous solution of unstable materials, such as polyphenols, into solid form. In the lyophilized solid products, chemical or physical degradation processes are inhibited or sufficiently decelerated, resulting in improved long-term stability [21].

Taking all of this into consideration, the aim of this work was to efficiently extract bioactive polyphenols from M. sativa by the combined use of CDs and ultrasound. Optimization of the ultrasound-assisted extraction procedure will be achieved by the application of the RSM. Such an approach would allow the determination of critical technological parameters of the extraction procedure, thus facilitating a rational process optimization in order to produce extracts rich in phenolic compounds with a maximum antiradical and ChA. An additional goal would include the comparison of the developed technology with conventional extraction procedures, performed by the use of water and ethanol as the extraction medium. The effect of the drying technology on the key properties of the extracts prepared will also be examined. Finally, the therapeutic potential of the selected dry extract will be evaluated by measuring the GAG reduction level in the skin fibroblast, obtained from MS III patients.

Results

The process variables and experimental data of 17 runs performed are shown in [Table 1]. The amount of TP and TF in the extracts was in the range of 1.98–2.47 mg/mL and 0.41–0.75 mg/mL, respectively. For the extraction of M. sativa we used 0.08 g of herbal material per mL of the solvent. This means that, according to the extraction conditions, it was possible to extract 24.8–30.9 mg of TP and 5.1–9.4 mg of TP from 1 g of herbal material, respectively. All extracts prepared presented notable RSA. The RSA IC₅₀ values of the non-dried extracts were in the range of 1.10–10.53 HME mg/mL. The observed activity in this assay for the lyophilized extract was significantly higher (paired t-test, p < 0.05), ranging from 1.40 HME mg/mL to 3.51 HME mg/mL. BHA, used as a positive control in this assay, presented an RSA IC₅₀ of 0.043 ± 0.004 mg/mL. ChA IC₅₀ values were in the range of 0.17–0.77 HME mg/mL (non-dried extracts) and 0.22–0.65 HME mg/mL (lyophilized extracts), while the ChA activity of EDTA (positive control) tested under the same conditions was (3.04 ± 0.64) × 10^− 3mg/mL. The chelating activities of the non-dried and lyophilized extracts did not differ significantly (paired t-test, p > 0.05). To estimate the efficiency of the CD-based extraction, a comparison of TP and TF levels in the extracts prepared by the use of water and ethanol was performed. The use of either water or ethanol, as the extraction medium, results in a more efficient extraction of TP ([Table 1]). On the contrary, the use of HPβCD resulted in a more efficient TF extraction, whose levels were up to 3.75 times higher. Furthermore, the extracts prepared without HPβCD displayed a notably higher RSA IC₅₀ and ChA IC₅₀ ([Table 1]).

By applying multiple regression analysis on the experimental data and by analysis of variance (ANOVA) for the selected models ([Table 2]), it was found that the relationship between the response variables and independent variables could be expressed either by the two-factor interaction (TP) or by the quadratic polynomial equations (TF, RSA, ChA). Polynomial equations obtained are presented in [Table 3], while the corresponding response surface plots are presented in [Fig. 1]. The F value of the models was higher than 3.36 and the p values of the models were lower than 0.05, which indicates that the models are significant and can be used to optimize the extraction variables. Lack of fit test in all the models was statistically insignificant and indicates that the fitting model is adequate to describe the experimental data. The lowest coefficient of determination belonged to the model for TP (r ² = 0.6687). The determination coefficients for the other responses were much higher (r ² = 0.9175, 0.8897, and 0.9357 for TF, RSA and ChA, respectively).

The selected variables (HPβCD concentration, time of extraction, and ultrasonication power) significantly affected at least one of the responses as linear (X_i), quadratic (X_ii) variables, and/or their interaction (X_ij). Among them, the HPβCD concentration displayed the greatest influence over the investigated responses and affected the TF and RSA as linear (X₁), and RSA and ChA as a quadratic term (X₁₁). In addition, the interaction of the HPβCD concentration and ultrasonication power (X₁₃) significantly influenced TP, TF, and ChA. The significant quadratic terms were also time of the extraction (X₂₂; ChA) and ultrasonication power (X₃₃; RSA and ChA; [Table 3]).

The suitability of the model equations for predicting the optimum response values was tested using the selected optimum conditions. The aims were to maximize TP and TF, as well as to minimize the RSA IC₅₀ and ChA IC₅₀ of the extracts. The optimum conditions were rounded to the nearest whole numbers. The predicted and experimental values of the response variables are shown in [Table 4]. The conditions for the maximal TF and minimal RSA IC₅₀ were the same. The results of most of the determinations agreed closely with the predicted value (the difference was between 4.4 and 10.2 %), indicating that the RSM model is satisfactory and relatively accurate, taking into account the complexity of sample matrices and the coexistence of multiple forms of bioactive phytochemicals and their interaction with other cellular components, which all pose a significant challenge for their accurate estimation and optimization.

Due to the observed health-related effects of flavonoids, as well as their role in oxidative stress in MPS, the extract with the maximized TP and minimized RSA IC₅₀ (MS2; [Table 4]) was subjected to the in vitro biological studies using skin fibroblasts taken from MPS III patients as a model. In this experiment, genistein was used as a positive control, taking into account its well-documented efficiency [9], [22]. Indeed, genistein in the concentration of 6 mg/mL reduced the GAG levels in the treated cells 61.6 % compared to the untreated ones (p < 0.05; [Fig. 2]). Knowing that, the amount of genistein, and also daidzein, glycitein, and formononetin, in MS2 was quantified. The corresponding chromatogram is presented in Supporting Information (Fig. 1 S, Supporting Information). The amount of dry matter in the extract was determined to be 84.8 mg/mL, while the content of daidzein, formononetin, genistein, and glycitein was 0.95, 1.34, 0.4, and 2.24 mg/g of dry matter, respectively. The extract tested was also capable of significantly reducing the amount of GAG in the cells of MPS III patients in a dose-dependent manner, clearly demonstrating its therapeutic potential ([Fig. 2]). The activity of MS2 was approximately 25 % lower than the activity of the pure genistein, taken as a positive control.

Discussion

Polyphenols are a large group of phytochemicals that are recognized as being responsible for the health benefits associated with the consumption of food, herbs, and spices. M. sativa is a rich source of phenolic antioxidants that have numerous beneficial properties. Luteolin and kaempferol, for example, can act as anti-inflammatory agents [23], [24], while luteolin can display neuroprotective properties [24]. In addition, quercetin has shown antiproliferative effects on several cell lines [25]. These and other dietary polyphenols, as well as their metabolites, can also reduce the risk of the development of type 2 diabetes complications, cardiovascular diseases, and even cancer [26]. Therefore, instead of determining the amount of individual secondary metabolites, we aimed at assessing the amount of total phenolic compounds and flavonoids. The prepared extracts were rich in TP and TF. Owing to their structure, flavonoids and other polyphenolic compounds are quite polar, but poorly soluble in water, which is limiting their extraction by the aqueous medium. While comparing the TP and TF content in the extracts prepared by the use of water and ethanol as an extraction medium ([Table 1], Runs 18 and 19, respectively), it seems that the application of HPβCD leads to a more selective extraction of flavonoids from the plant material, while the extraction of other phenols is less efficient. This could be primarily related to the higher affinity of flavonoids for the inclusion complex formation with HPβCD, while the affinity of simple phenolic components for the interaction with the lipophilic CD cavity would be less pronounced. Zhang et al. [27] have demonstrated that the stability constant of the inclusion complex of caffeic acid with HPβCD is 112 M⁻¹, while for flavonols such as myricetin, quercetin, and kaempferol, the stability constants are in the range of 5494–12 456 M⁻¹ [28], clearly demonstrating their high efficiency for inclusion complexation. On the other hand, the complex structure of tannins would impair their interaction with HPβCD by steric reasons [17]. The most important outcome of the inclusion complex formation is in the solubility increase of the included molecule [15]. Shulman and coworkers have proven that naringerin solubility increased over 400 times when complexed with HPβCD [29]. Similarly, a pronounced increase of solubility upon complexation with HPβCD was also demonstrated for other flavonoids [16]. Therefore, it could be reasonably presumed that during the extraction procedure, cavitation phenomena caused by applied ultrasound would result in the liberation of different phytochemicals from the plant material and in the formation of their supersaturated solution. The dissolved molecules would be able to interact with HPβCD present in the extraction medium according to their corresponding affinity. The simultaneous CD complexation of several different molecules was already demonstrated for isoflavones present in an enriched soy fraction [18] as well as in the case of ferulic and galic acid coencapsulation in HPβCD [30]. After equilibration of the system, the components that form the most stabile complexes with HPβCD would remain in the solution, thus being more efficiently extracted. The other compounds with a lower affinity for complexation with HPβCD would not be so efficiently extracted. Such an assumption would explain the significant influence of HPβCD concentration on TF levels in the extracts. The other reason that could explain the higher levels of flavonoids in extracts prepared with HPβCD would be the fact that those compounds are prone to chemical degradation by the applied ultrasound [19]. Inclusion complexation of such compounds by HPβCD could efficiently prevent such degradation [16], leading to higher concentrations of TF in CD-based extracts. The efficient extraction of the phytochemicals present in the plant material depends on the concentration of CD molecules in the extraction media ([Table 1]). When the higher concentrations of CDs are present in the extraction fluid, the higher quantity of bioactive compounds could interact with them, thus being efficiently extracted from the plant material. At some point, when all bioactive compounds are extracted, an additional increase of CD concentration would not result in higher concentrations of the phenolic compounds in the extract. In preliminary experiments, we have seen that the use of HPBCD concentrations higher than 25 mM does not give a further increase of phenolic contents in the extracts prepared, but only leads to the bulkiness of the extract prepared.

A significant (p < 0.05) but negative correlation was observed between the TF and RSA IC₅₀ (r ² = 0.4825), indicating that flavonoids play an important role in the observed RSA. Furthermore, extracts obtained by CD extraction showed a notably higher RSA and ChA compared to ones prepared by the use of water or ethanol as an extraction medium ([Table 1]). Although HPβCD alone does not possess antiradical activity [30], it has been shown to modulate the antioxidant activity of the included molecule. Literature data showed that the complexation of resveratrol with HPβCD leads to almost double antioxidant activity compared to that determined in the absence of CD [31]. Also, the antioxidant activity of kaempferol, quercetin, and myricetin increased upon complexation with HPβCD [32]. In the case of caffeic acid, a slight decrease in antioxidant activity has been observed upon complexation with HPβCD [33]. The opposite effect of HPβCD on the activity could be explained by the structure and the strength of the complexes formed [30], [33], [34]. Hydrogen atom transfer and electron transfer from the phenolic moiety to the radical are the most frequent oxidation mechanisms included in the antioxidant action of phenolic compounds [35]. Therefore, if a polyphenol is oriented in such way that the hydroxyl groups responsible for the quenching of the peroxide radicals are located inside the central cavity of the CD molecule, its antioxidant activity will be reduced in proportion to the strength of this interaction. If the hydroxyl groups remain outside the CD cavity, their antioxidant activity will remain unchanged, or it could be increased, depending on the hydrogen bond reorganization upon inclusion complex formation. In fact, the inclusion complexation might break intermolecular hydrogen bonds present in the free polyphenolic molecule, thereby facilitating the transfer of the proton and increasing the antioxidative activity of the included molecule [30], [33]. Also, it is possible that inclusion complex formation would result in the formation of a new hydrogen bond between the hydroxyl groups of the polyphenols with oxygen atoms of CD. Such bonds would weaken the covalent bonds between hydrogen and oxygen in the hydroxyl group of polyphenols, which in turn would make hydrogen donation becoming easier [34]. In this regard, the structure of the CD derivative used as complexation agent plays a critical role. The derivatives that are able to make hydrogen bonds with the included molecule, such as HPβCD, would, in general, contribute to the increase of antioxidant activity, as observed in the case of luteolin complexes with HPβCD [36]. On the other hand, flavonoid complexation with methylated βCD derivatives (RAMEB) decreases their antioxidant activity due to the reduced ability of RAMEB to form hydrogen bonds [37]. The above-listed mechanisms could therefore explain the increased RSA of the extracts prepared by the use of HPβCD when compared to that of CD free extracts prepared by the use of water and ethanol as the extraction medium ([Table 1]).

Surprisingly, the extracts prepared by the use of HPβCD also presented a several times higher chelating ability compared to that prepared by the use of water and ethanol as the extraction medium ([Table 1]). The interaction of aliphatic polyalcohols with metal ions is generally weak and usually not significant in acidic and neutral solutions. However, their deprotonation at high pH values turns them into strong and efficient metal ion binding agents. Therefore, the acidity of terminal OH- groups plays a key role in the CD complexation of metal ions. A polynuclear complex of multideprotonaded CDs with iron (II) was prepared in alkaline solution [38], but such a complex does not exist at physiological pH values and therefore cannot explain the pronounced chelating ability of the extracts prepared. As discussed previously, CD complexation could enhance deprotonation of the hydroxyl groups of flavonoids. This leads to their ionization and consequent free electron pair formation available for complexation with Fe²⁺ ions [39], probably contributing to the observed increase of ChA.

The preparation of the extracts in a solid state is an important step in product development, resulting in more technologically favorable properties of the prepared product. This relates, in first line, to the improved long-term storage stability, but also to the easier handling during shipping and storage. Lyophilization is an important and well-established drying process used to improve the long-term stability of sensitive drugs, especially therapeutical proteins [21]. The lyophilized product obtained is generally amorphous and porous, which contributes to its rapid dissolution in contact with biological fluid [40]. Furthermore, lyophilization is a standard procedure used to prepare highly soluble CD inclusion complexes in the solid state [41]. Taking all of this into account, we have applied freeze-drying technology in order to obtain extracts in the solid state. This technology has also been used to stabilize different vegetables and fruits, such as avocados, mangos, and bananas [42], [43], [44]. It has been demonstrated that lyophilization has little or no effect on the polyphenol content in the plant material, which makes it a technology of choice in the drying of polyphenol-rich products. Therefore, considering the favorable effects of lyophilization on the content of polyphenolic compounds, we have chosen radical scavenging and ChA of the dry extracts prepared as quality control parameters ([Table 1]). When comparing the ChA and RSA activity of extracts prior to and after lyophilization, it seems that the selected drying technology has no significant influence on the ChA of the products prepared, but at the same time, it significantly increased the RSA the extract prepared, additionally contributing to the quality of the products prepared. A similar effect was also observed in the case of lyophilized lipophilic extracts of avocado [42].

RSM is an effective statistical procedure that uses a minimum set of experiments for determination of the coefficients of a mathematical model and optimization of the conditions [45]. This pproach also allows for a rapid detection of the factors whose variations gave rise to significant changes of the considered responses. BBD is one of the most widely used designs in RSM. It is a three-level design suitable for fitting second-order response surfaces. It is a spherical and revolving design that has been successfully applied in the optimization of chemical and physical processes because of its reasonable design and excellent outcomes [46]. However, regardless of the used design, it is important to test the appropriateness of the RSM mathematical model for predicting the optimal variances and adequately representing the real relationship between the selected parameters [45]. In the presented work, the BBD was successfully applied for developing suitable mathematical models for finding a relationship between extraction conditions (HPβCD concentration, time, and ultrasonication power) and selected response variables (TP, RSA IC₅₀, and ChA IC₅₀). The suitability of the model was confirmed by the appropriate F values, which were significant for the selected models and not significant for their respective lack of fit. However, even though the models were significant, the coefficients of determination have shown that they cannot fully explain the relationship between independent and dependent variables. The lowest coefficient of determination belonged to the model for TP, where more than 30 % of the data variation could not be explained by the selected two-factor interaction model. However, the attempts to fit the measured data to other models (linear and quadratic) resulted in the non-significant model. The selected models for the considered responses were verified by preparing the extracts with the most desirable properties, including maximal TP and TF, as well as minimal RSA IC₅₀ and ChA IC₅₀. The optimization results have shown that the conditions for preparing the extract with the highest TF coincided with the conditions for preparing the extract with a minimal RSA IC₅₀. Such a result is not surprising, taking into account the observed negative correlation between TF and RSA, further confirming that mainly flavonoids are responsible for the antiradical effect of M. sativa extracts. Therefore, only three extracts were prepared: MS1 (maximal TP), MS2 (maximal TF and minimal RSA IC₅₀), and MS3 (minimal ChA IC₅₀; [Table 4]). A comparison with [Table 1] reveals that the conditions for preparing MS2 closely resembled the ones already used in the design (Run 17). Close agreement of predicted and experimental values reported in [Table 4] confirms the validity of the model. The results of the optimization of the model have shown that the most phenols are extracted during a longer extraction time and using a higher ultrasonication power, while the desirable HPβCD concentration was 5 mM (the lowest one used in the experiment). The unfavorable influence of HPβCD on TP was confirmed by the greater efficiency of water as the solvent for the phenols of M. sativa ([Table 1]). TF, on the other hand, was extracted best by using a low ultrasonication power (the lowest used in the experiment) and a relatively high HPβCD concentration (20 mM). Such a result could be expected taking into account that HPβCD is acting as the carrier of the extracted compounds, solubilizing the flavonoids present in M. sativa during the extraction, as discussed previously. The ultrasonication power is also an important parameter for the extraction of flavonoids, primarily causing the liberation of the present phytochemicals from the plant material, which then become available for the interaction with HPβCD. Although CD complexation could slow down the oxidative degradation of flavonoids caused by ultrasound, its intensity should be carefully regulated in order to avoid their degradation. Therefore, the optimal TF extraction was obtained at the lowest levels of the ultrasound applied ([Table 4]). Also, a longer extraction time was needed to successfully isolate the high levels of TF, which could be related to the reactor configuration. An ultrasonic bath was used in all extraction procedures, resulting in indirect ultrasound irradiation, which is less potent and thus requires a longer extraction time. But at the same time, such an experimental setup allowed more precise control of the temperature in the extraction medium, which was kept at 20 °C. Even though the extraction efficiency was not optimized for isoflavone content, it is interesting to note that the ethanolic extract of M. sativa showed a similar isoflavone profile (Fig. 1 S, Supporting Information). It is known that flavonoids are more prone to decomposition when treated with ultrasound at temperatures higher that 40 °C [11]. The same conditions were the most appropriate for the preparation of the extracts with the strongest antiradical capacity. For maximal ChA, a moderate concentration of HPβCD and ultrasound strength was the best conditions. All of the responses were optimal at the longest extraction time applied.

Having in mind the importance of oxidative stress in MPS pathogenesis, as well as the observed influence of flavonoids on GAG synthesis, the obvious choice of the extract for the in vitro biological testing was MS2, which contained the most TF among the prepared extracts and showed remarkable RSA ([Table 4]). Flavonoids were previously reported to partially inhibit GAG synthesis and reduce GAG storage in cells derived from MPS patients. Among them, genistein (an isoflavone) has been studied intensively, and it was proposed that this compound can downregulate GAG production by blocking phosphorylation of the EGF receptor, thus impairing a signal transduction pathway necessary for activation of genes coding for enzymes involved in this anabolic process [9]. Because of this, genistein was selected as the positive control for this study. Compared to pure genistein, the GAG-reducing ability of MS2 was somewhat weaker. However, it is important to note that the extract tested contains only 0.04 % of genistein and 0.84 % of TF. This implicates that the activity of the MS2 could not be related only to its genistein content but most probably also to the other phytochemicals extracted. At the same time, the synergistic action of isoflavonoids, such as daidzein, formononetin, genistein, and glycitein as well as other components present in the extract should also be considered. This is in agreement with results of other studies, which indicated that different isoflavonoids and other flavonoids, either natural or synthetic, can also significantly modulate GAG synthesis and storage while acting through an EGF-independent mode. In addition to that, enhanced effects of combinations of various flavonoids on GAG synthesis and accumulation, relative to single compounds from this group, have also been reported [9]. Combinations of various flavonoids, such as apigenin, daidzein, kaempferol, and naringenin, resulted in significantly more effective inhibition of GAG synthesis than the use of any of these compounds alone [22]. Our results are suggesting that the combination of phytochemicals extracted from M. sativa by the simultaneous use of HPβCD and ultrasound can be considered an effective aid capable of reducing GAG synthesis. Further studies are necessary to investigate their safety and potential therapeutic efficiency in the treatment of MPS.

Materials and Methods

Plant materials and chemicals

Aerial parts of M. sativa were collected in the surroundings of Zagreb (lake Jarun, 45°46′43.3″N 15°54′46.8″E), dried, and stored in a well-ventilated room, protected from light. The plant material was identified by Vedran Šegota, expert associate of the Herbarium Croaticum at the Division of Botany, Department of Biology, Faculty of Science, University of Zagreb. Voucher specimens are deposited in the Herbarium of the Department of Pharmaceutical Botany with Pharmaceutical Botanical Garden “Fran Kušan”, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia (Reg. No. HFK-HR-5–2013). DMEM, FBS, and AAS (antibiotic/antymicotic solution) were purchased from Sigma. L-Glutamine and DPBS-CMF (Dulbeccoʼs phosphate buffer saline calcium and magnesium free) were purchased from EuroClone. Trypsin/EDTA solution was purchased from Life Technologies. Blyscan™ Sulphated Glycosaminoglycan Assay Kit was purchased from Biocolor Ltd. Butylated hydroxyanisole (BHA) (≥ 99 %), daidzein (≥ 97 %), ethylenediaminetetraacetic acid (EDTA, ≥ 99 %), Folin-Ciocalteu phenol reagent, DPPH, formononetin (≥ 98 %), gallic acid monohydrate (≥ 99 %), genistein (≥ 97 %), glycinetin (≥ 97 %), Hoechst 33 258 dye, and quercetin dehydrate (≥ 98 %) were purchased from Sigma-Aldrich. Methanol was HPLC grade. Other reagents were of analytical grade.

Preparation of extracts

Prior to the extraction, the dried aerial parts of M. sativa were milled and passed through a sieve of 850 µm mesh size. Powdered plant material (2 g) was combined with a selected amount of HPβCD and 25 mL of water in a 50-mL Erlenmeyer flask. The extraction was performed in a temperature-controlled ultrasonic bath (Bandelin SONOREX Digital 10 P DK 156 BP) set to 20 °C using a selected ultrasonication power. Upon extraction, the mixture was filtered using folded filter papers S&S 589/1 1/2 and diluted with water to a volume of 25.0 mL. A portion (15 mL) of each extract was lyophilized (Christ Alpha 1–4 LD Freeze Dryer). All of the extracts were stored at + 4 °C in the dark until use. Before use, aliquots of the lyophilized extracts were dissolved in water. Prior to each determination, all of the extracts (non-dried and lyophilized) were centrifuged at 3000 rpm for 10 min.

Total phenol and total flavonoid content

TP in extracts was determined by the Folin–Ciocalteau colorimetric method according to Singleton et al. [47], while the TF was assessed by the method of Kumazawa et al. [48]. For these determinations, the modifications were used as described in Jug et al. [49]. The amounts of the analyzed substances in the extracts are expressed as mg/mL from calibration curves recorded for the standards and expressed as standard equivalents. Namely, TP and TF were expressed as gallic acid and quercetin equivalents, respectively. Measurements were performed using a Stat Fax 3200 (Awareness Technologies) microplate reader.

Free radical scavenging and chelating activity

Free RSA was evaluated using the DPPH free radical method [37], while the ChA toward ferrous ions was studied according to the method described by Decker and Welch [50], using the modifications as described by Zovko Končić et al. [51]. The RSA for the DPPH free radical was calculated according to equation (1).

RSA = (A_control – A_sample)/A_control × 100  (1)

Where A_control is the absorbance of the negative control (e.g., blank DPPH solution without test compound) and A_sample is the absorbance of the DPPH solution containing the extract. RSA was calculated as the concentration of the extract which scavenges 50 % of DPPH free radicals present in the solution and has an RSA = 50 % (RSA IC₅₀) and is expressed as mg of the herbal material equivalents per mL of the extract (HME mg/mL). ChA was calculated according to equation (2).

ChA = (A_control – A_sample)/A_control × 100  (2)

Chelating activity was expressed as ChA IC₅₀, the concentration that chelates 50 % of Fe²⁺ ions and is expressed as mg of the herbal material equivalents per mL of the extract (HME mg/mL). BHA and EDTA were used as positive controls in the RSA and ChA assays, respectively.

Experimental design

Design Expert software version 8.0.6 (Stat-Ease) was employed for the regression analysis and the optimization of the results. A three-level three-factor BBD was employed to determine the best combination of independent extraction variables for the selected dependent variables (responses). HPβCD concentration (X₁), time (X₂), and ultrasonication power (X₃) were chosen as the independent variables, and their coded and uncoded levels are presented in [Table 5]. TP, TF, RSA IC₅₀, and ChA IC₅₀ were selected as the responses ([Table 1]). Experimental data were fitted to a quadratic polynomial model as described by the following two-factor interaction (3) or quadratic equation (4):

Where Y is the dependent variable; A₀, A_i, A_ij, and A_ii are the regression coefficients for intercept, linearity, interaction, and square (where applicable), respectively; X_i and X_j are the independent variables.

HPLC analysis of isoflavones

Prior to HPLC analysis, glycosides were hydrolyzed by the addition of 400 µl of 5 M HCl to 1 mL of extract in a hermetically sealed tube. The solution was heated for 2 h in a boiling water bath [52]. After hydrolysis, the mixture was allowed to cool down to room temperature. The extracts were centrifuged and the supernatant was filtered through a PTFE membrane (0.45 µm) before HPLC analysis. The genistein was quantified using an HPLC instrument (Agilent 1200 series, Agilent Technologies) equipped with a variable wavelength detector and an Ultropac-C18 column (5 µm, 250 mm × 4.6 mm, LKB). The solutions of the standards and the extracts were filtered through a 0.45-µm syringe filter. The injection volume was 20 µL. Solutions of formic acid (0.1 %) in methanol and ultrapure water were used as solvents A and B, respectively. Separation was performed at 40 °C using the following protocol: 0 min 33 % B, 7 min 45 % B, 15 min 50 % B, 25 min 60 % B, 30 min 70 % B, 35 min 0 % B, 37 min 33 % B. These conditions were maintained for 10 min and then returned to the initial ones within 3 min. The flow rate was 1.0 mL/min. The peaks were observed and quantified at 254 nm. The peak assignment and identification of the isoflavones was based on the comparison of the retention times of the peaks in the sample chromatogram with those of the standards. Additionally, the identity of individual peaks was confirmed by peak overlapping when running a simultaneous injection of the sample and the standard. Isoflavones were quantified according to respective standard calibration curves. Limit of detection (LOD) and limit of quantification (LOQ) were determined using equations (5) and (6), respectively:

LOD = 3 × δ/S(5)

LOQ = 10 × δ/S(6)

Where δ is the standard error of the linear regression and S is the slope of the calibration curve regression line. The method was proven suitable for the determination of genistein (y = 6394.5 x + 70; r ² = 0.999 986; LOD = 0.0063 µg; LOQ = 0.019 µg), daidzein (5531.7 x + 23.7, r ² = 0.999 999, LOD = 0.0018 µg, LOQ = 0.0056 µg), formononetin (4215.4 x + 5.8, r ² = 0.999 997, LOD = 0.0031 µg, LOQ = 0.0093 µg), and glycitein (5700.6 x – 34.8, r ² = 0.999 992, LOD = 0.0055 µg, LOQ = 0.0168 µg) in the extracts prepared.

Determination of sulphated glycosaminoglycans in skin fibroblasts

Skin fibroblast lines were initiated from forearm skin biopsies obtained from MPS III patients, diagnosed on the basis of standard biochemical and enzymatic assays for levels of urinary GAGs and activity of lysosomal hydrolases, respectively. The research was performed in accordance with ethical committee approval (Class: 643–03/l4-QL/04; No. 251–62-03-L4–33; Zagreb, 16th of May 2014). Cells were cultured to early confluence in 25 cm² flasks in DMEM supplemented with 10 % heat inactivated FBS and AAS at 37 °C in a humidified atmosphere with 5 % (v/v) CO₂. Cells were treated for 72 h either with genistein, as a positive control, or the selected extract, both dissolved in 2 % DMSO at concentrations of 3 and 6 µg/mL, respectively. Preliminary experiments showed that exposure of cells to 2 % DMSO did not affect their viability and GAG production.

Sulphated glycosaminoglycans (s-GAG) were measured using the Blyscan proteoglycan and s-GAG assay, which is based on the specific binding of the cationic dye, 1,9-dimethylmethylene blue [53]. Results were normalized to the DNA content and measured flourometrically using the Hoechst 33 258 method for double-stranded DNA [54]. All of the measurements were performed in triplicate.

Supporting information

A chromatogram of the MS2 extract recorded at 254 nm, subjected to in vitro biological testing, and a chromatogram of the ethanolic extract (Run 19) are available as Supporting Information.

Acknowledgements

The authors are thankful to Vedran Šegota, an expert associate of the Herbarium Croaticum at the Division of Botany, Department of Biology, Faculty of Science, University of Zagreb, for help in the identification of the plant material. The financial support of the University of Zagreb in the frame of the project Isolation and modulation of antioxidative properties of plant polyphenols by the use of CDs is kindly acknowledged.

Conflict of Interest

The authors declare no conflict of interest.

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Correspondence

• References

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  CrossrefPubMedSearch in Google Scholar
• 34 Nguyen TA, Liu B, Zhao J, Thomas DS, Hook JM. An investigation into the supramolecular structure, solubility, stability and antioxidant activity of rutin/cyclodextrin inclusion complex. Food Chem 2013; 136: 186-192
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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