Stilbenes: Characterization, bioactivity, encapsulation and structural modifications. A review of their current limitations and promising approaches.

Stilbenes are phenolic compounds naturally synthesized as secondary metabolites by the shikimate pathway in plants. Research on them has increased in recent years due to their therapeutic potential as antioxidant, antimicrobial, anti-inflammatory, anticancer, cardioprotective and anti-obesity agents. Amongst them, resveratrol has attracted the most attention, although there are other natural and synthesized stilbenes with enhanced properties. However, stilbenes have some physicochemical and pharmacokinetic problems that need to be overcome before considering their applications. Human clinical evidence of their bioactivity is still controversial due to this fact and hence, exhaustive basis science on stilbenes is needed before applied science. This review gathers the main physicochemical and biological properties of natural stilbenes, establishes structure-activity relationships among them, emphasizing the current problems that limit their applications and presenting some promising approaches to overcome these issues: the encapsulation in different agents and the structural modification to obtain novel stilbenes with better features. The bioactivity of stilbenes should move from promising to evident.

Molecular encapsulation and bioactivity of gnetol, a resveratrol analogue, for use in foods.

BACKGROUND: Gnetol is a stilbene whose characterization and bioactivity have been poorly studied. It shares some bioactivities with its analogue resveratrol, such as anti-inflammatory, anti-thrombotic, cardioprotective and anti-cancer activities. However, the low solubility of stilbenes may limit their potential applications in functional foods. Encapsulation in cyclodextrins could be a solution. RESULTS: The antioxidant activity of gnetol was evaluated by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation and ferric reducing antioxidant power methods (Trolox equivalents 13.48 µmol L-1 and 37.08 µmol L-1 respectively at the highest concentration) and it was higher than that of resveratrol, and depending on the method, similar or higher to that of oxyresveratrol. Spectrophotometric and spectrofluorimetric characterization of gnetol is published for the first time. Moreover, its water solubility was determined and improved almost threefold after its molecular encapsulation in cyclodextrins, as well as its stability after storage for a week. A physicochemical and computational study revealed that cyclodextrins complex gnetol in a 1:1 stoichiometry, with better affinity for like 2-hydroxypropyl-ß-cyclodextrin (KF  = 4542.90 ± 227.15 mol-1  L). Temperature and pH affected the encapsulation constants. CONCLUSION: These results could increase interest of gnetol as an alternative to the most studied stilbene, resveratrol, as well as aid in the development of more stable inclusion complexes that improve its aqueous solubility and stability so that it can be incorporated into functional foods. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Enhanced production of 2-phenylethanol by salicylic acid and cyclodextrins in cell suspension cultures of the unexplored filamentous fungus Monochaetinula geoffroeana.

BACKGROUND: 2-Phenylethanol (PEA) is a higher aromatic alcohol with a rose-like odor, which is used in several industries. Although PEA can be synthesized, consumers are increasingly concerned about the toxicity of chemically synthesized products, and prefer natural aroma compound. PEA occurs naturally in the environment but concentrations are too low to justify extraction. RESULTS: The present study offers a novel biological source of PEA: the filamentous fungi Monochaetinula geoffroeana. We report the highest recorded yield of PEA of fungal origin to date: 6.52 g L-1 . The volatility and low water solubility of PEA can affect its use in many industries, for which reason complexation studies of PEA and cyclodextrins were carried out using the phase solubility technique. PEA formed 1:1 stoichiometric inclusion complexes with natural and modified CDs, the highest encapsulation constant being obtained with MßCD (K1:1  = 299.88 L mol-1 ). The complexation process significantly increased the water solubility of PEA. A computational study showed a high degree of correlation between computed scores and experimental values. Furthermore, this study reports the role of salicylic acid as an effective elicitor for improved PEA production by the studied fungi. Supplementation with 10 µmol L-1 salicylic acid increased PEA production from 6.52 to 10.54 g L-1 . CONCLUSION: The best treatment to enhance PEA production by M. geoffroeana under laboratory conditions was to use salicylic acid 10 µmol L-1 . Due to the commercial importance of PEA, further investigation is needed to improve PEA production by M. geoffroeana and to optimize culture conditions in order to standardize yields. © 2021 Society of Chemical Industry.

Identification of distinctive physiological and molecular responses to salt stress among tolerant and sensitive cultivars of broccoli (Brassica oleracea var. Italica).

BACKGROUND: Salt stress is one of the main constraints determining crop productivity, and therefore one of the main limitations for food production. The aim of this study was to characterize the salt stress response at the physiological and molecular level of different Broccoli (Brassica oleracea L. var. Italica Plenck) cultivars that were previously characterized in field and greenhouse trials as salt sensitive or salt tolerant. This study aimed to identify functional and molecular traits capable of predicting the ability of uncharacterized lines to cope with salt stress. For this purpose, this study measured different physiological parameters, hormones and metabolites under control and salt stress conditions. RESULTS: This study found significant differences among cultivars for stomatal conductance, transpiration, methionine, proline, threonine, abscisic acid, jasmonic acid and indolacetic acid. Salt tolerant cultivars were shown to accumulate less sodium and potassium in leaves and have a lower sodium to potassium ratio under salt stress. Analysis of primary metabolites indicated that salt tolerant cultivars have higher concentrations of several intermediates of the Krebs cycle and the substrates of some anaplerotic reactions. CONCLUSIONS: This study has found that the energetic status of the plant, the sodium extrusion and the proline content are the limiting factors for broccoli tolerance to salt stress. Our results establish physiological and molecular traits useful as distinctive markers to predict salt tolerance in Broccoli or to design novel biotechnological or breeding strategies for improving broccoli tolerance to salt stress.

Separating and Identifying the Four Stereoisomers of Methyl Jasmonate by RP-HPLC and using Cyclodextrins in a Novel Way.

INTRODUCTION: Several authors have reported on the different bioactivities of methyl jasmonate (MeJA) stereoisomers. However, no simple, precise and cheap method for separating and identifying them using reversed-phase high performance liquid chromatography (RP-HPLC) has been developed. OBJECTIVE: (1) To create a simple, precise and cheap method for separating and identifying the four stereoisomers present in commercial racemic mixtures of MeJA and (2) to identify the four stereoisomers using molecular docking techniques and coinjection. Materials and Methods - RP-HPLC using a 250 mm C18 column and different proportions of cyclodextrins (CDs) and organic solvents was applied to a commercial sample of racemic MeJA. RESULTS: The results show that the best conditions for separating the MeJA stereoisomers are: 20% methanol in the mobile phase, a temperature of 45 °C and a 16 mM concentration of methyl-ß-cyclodextrin (M-ß-CD). A simple C18 250 mm column and a flow rate of 1.25 mL/min were used. The reduction in the retention time of MeJA observed when M-ß-CD is added to the mobile phases was used to determine the complexation constants of the guest/CD complex and compared with the obtained when other CDs were used. The KF for M-ß-CD (117.49 ± 5.9 1/M) was obtained with a 1:1 stoichiometry. The four stereoisomers were identified by molecular docking techniques and coinjection of a commercially available rosemary essential oil. CONCLUSION: The new method identified and classified the four stereoisomers of MeJA in the following ordination: (-)epiMeJA, (-)MeJA; (+)MeJA and (+)epiMeJA. These results could be used to improve the elicitation of cell cultures with only the best isomer. Copyright © 2016 John Wiley & Sons, Ltd.

Cyclodextrins and antioxidants.

In recent years, the growth of the functional foods industry has increased research into new compounds with high added value for use in the fortification of traditional products. One of the most promising functional food groups is those enriched in antioxidant compounds of a lipophilic nature. In spite of the numerous advantages reported for such antioxidant molecules, they may also have disadvantages that impede their use in functional foods, although these problems may well avoided by the use of encapsulant agents such as cyclodextrins. This explains the recent increase in the number of research papers dealing with the complexation of different guest molecules possesing important antioxidant properties using natural and modified cyclodextrins. This paper presents a review of the most recent studies on the complexes formed between several important types of antioxidant compounds and cyclodextrins, focusing on the contradictory data reported in the literature concerning to the antioxidant activity of the host/guest molecule complexes, the different complexation constants reported for identical complexes, the bioavailability of the antioxidant compound in the presence of cyclodextrins and recommendation concerning the use of natural or modified cyclodextrins. Moreover, the use of cyclodextrins as antibrowning agents to prevent enzymatic browning in different foods is revised. Finally, we look at studies which suggest that cyclodextrins act as ''secondary antioxidants," enhancing the ability of traditional antioxidants to prevent enzymatic browning.

Improvement of the Physicochemical Limitations of Rhapontigenin, a Cytotoxic Analogue of Resveratrol against Colon Cancer.

It has been argued that methoxylated stilbenes are better candidates for oral administration than hydroxylated stilbenes, including resveratrol, as they share many biological activities but have better bioavailability. By contrast, they have a disadvantage to consider, i.e., their lower hydrophilic character that leads to precipitation issues in the final product. In this work, we analysed and compared the growth inhibition of colorectal cancer cells of the methoxylated stilbene rhapontigenin and some analogues and overcame potential problems in the development of fortified products by designing inclusion complexes. Among several cyclodextrins, we found the one that best fit the molecule by physicochemical and bioinformatics assays. The stoichiometry and the encapsulation constants with natural and modified cyclodextrins were determined by fluorescence spectroscopy. The most promising complexes were analysed at different temperature and pH conditions, determining the thermodynamic parameters, to discover the optimal conditions for the preparation and storage of the products. The results showed that rhapontigenin solubility and stability were significantly improved, achieving a sevenfold increase in water solubility and maintaining more than 73% of the stilbene after three months. These findings could be of great interest for industries that aim to deliver novel bioactive compounds with higher solubility and lower degradation.

Antiproliferative Effects in Colorectal Cancer and Stabilisation in Cyclodextrins of the Phytoalexin Isorhapontigenin.

Isorhapontigenin has been proposed as a better alternative for oral administration than the famous resveratrol, as it shares many biological activities, but with a structure that could make its delivery easier. Although this hydrophobic structure could enhance bioavailability, it could also be a disadvantage in the development of products. In this research, we study the antiproliferative activity of this stilbene against colorectal cancer and overcome its limitations through molecular encapsulation in cyclodextrins. The cytotoxic activity against human colorectal cancer cells of isorhapontigenin was similar to that of resveratrol or piceatannol, supporting its use as a bioactive alternative. The study of the encapsulation through fluorescence spectroscopy and molecular docking revealed that the complexation satisfies a 1:1 stoichiometry and that HP-ß-CD is the most suitable CD to encapsulate this stilbene. Through a spectrophotometric assay, it was observed that this CD could double the basal water solubility, exceeding the solubility of other hydroxylated stilbenes. The stability of these inclusion complexes was higher at a pH below 9 and refrigeration temperatures. Moreover, the use of CDs retained more than 78% of isorhapontigenin after storage for 12 weeks, compared to 15% in free form. Overall, these findings could help design novel formulations to better deliver isorhapontigenin.

The use of cyclodextrins as solubility enhancers in the ORAC method may cause interference in the measurement of antioxidant activity.

The ability of cyclodextrins to enhance the water solubility of lipophilic compounds is used to modify the water-based Oxygen Radical Absorbance Capacity (ORAC) method to measure antioxidant activity in vitro. However, the use of these solubility enhancers may alter fluorescent readings, which has led to contradictory results being described in the literature. The low specificity of these oligosaccharides and their controlled release effect can result in cyclodextrins forming inclusion complexes with other reagents in the assay, changing the kinetics. In this study, the cause of cyclodextrins' interference in the ORAC method is evaluated through a physicochemical and computational approach. Cyclodextrins showed a clear increase in the fluorescent signal both in the presence and absence of the antioxidant oxyresveratrol, the precise effect being dependent on the type and concentration of cyclodextrin. Although the glucidic nature of cyclodextrins could play a minimal role in this effect, it seems that the main cause was the encapsulation of other substrates in the reaction, fluorescein and AAPH.

Development if healthy milk and yogurt products for reducing metabolic diseases using cyclodextrin and omega-3 fatty acids from fish oil.

The food industry is constantly attempting to develop better products that will have a positive effect on health. Feiraco® and Clesa®, expressed their intention to create novel products using UNICLA® milk as a matrix to develop functional foods. In this respect, ß-cyclodextrin (ß-CD) at 1% was able to reduce the cholesterol concentration in Feiraco-UNICLA® milk products by around 87-85%. Products were fortified with omega-3 from fish oil with α- and ß-CD acting as carriers. It was possible to add around 50% of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) Recommended Dietary Allowances (RDA), with a high diet proportion of fibre and similar organoleptic properties to commercial omega-3 products. 80% of a sensory panel found our formulations satisfactory. The final product was stable, and the bioaccessibilty of the fatty acids added to the milk was around 74%. These results as a whole satisfy the aid of Feiraco® and Clesa® to develop improved products.

Cyclodextrin-Based Nanosponges as Perse Antimicrobial Agents Increase the Activity of Natural Antimicrobial Peptide Nisin.

At present, antibiotic resistance is considered a real problem. Therefore, for decades scientists have been looking for novel strategies to treat bacterial infections. Nisin Z, an antimicrobial peptide (AMP), can be considered an option, but its usage is mainly limited by the poor stability and short duration of its antimicrobial activity. In this context, cyclodextrin (CD)-based nanosponges (NSs), synthesized using carbonyldiimidazole (CDI) and pyromellitic dianhydride (PMDA), were chosen for nisin Z loading. To determine the minimum inhibitory of nisin Z loaded on CD-NS formulations, agar well diffusion plates were used. Then, the bactericide concentrations of nisin Z loaded on CD-NS formulations were determined against Gram-positive (Staphylococcus aureus) and -negative (Escherichia coli) bacteria, using microdilution brain heart infusion (BHI) and tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The minimum and bactericide inhibitory values of the nisin complex with NSs were potentially decreased against both bacteria, compared with the nisin-free sample, while the nisin complex with ß-CD showed lower antibacterial activity. The antimicrobial effect was also demonstrated by free NSs. Furthermore, the total viable counts (TVCs) antibacterial experiment indicated that the combination of nisin Z in both PMDA and CDI ß-CD-based NSs, especially CDI, can provide a better conservative effect on cooked chicken meat. Generally, the present study outcomes suggest that the cross-linked ß-CD-based NSs can present their own antimicrobial potency or serve as promising carriers to deliver and enhance the antibacterial action of nisin Z.

Study of the fluorescence and interaction between cyclodextrins and neochlorogenic acid, in comparison with chlorogenic acid.

Neochlorogenic acid, a less-studied isomer of chlorogenic acid, has been seen to posses antioxidant, antifungal, anti-inflammatory and anticarcinogenic effects, which makes it an interesting candidate for incorporation in functional foods. However, its poor solubility in water and susceptibility to oxidation make such a task difficult. To overcome that, its encapsulation in cyclodextrins (CDs) is proposed. The fluorescence of neochlorogenic acid in different pH conditions was analyzed, and caffeic acid was proved to be the fluorescent moiety in the molecule. An encapsulation model whereby the ligand poses two potential complexation sites (caffeic and D-(-)-quinic moieties), showed that α-CD and HP-ß-CD formed the best inclusion complexes with neochlorogenic acid, followed by M-ß-CD, ß-CD and γ-CD. Molecular docking with the two best CDs gave better scores for α-CD, despite HP-ß-CD providing stabilization through H-bonds. The encapsulation of chlorogenic acid led to a similar CD order and scores, although constants were higher for α-CD, ß-CD and M-ß-CD, lower for HP-ß-CD, and negligible for γ-CD. The protonation state affected these results leading to a different order of CD preference. The solubility and the susceptibility to oxidation of neochlorogenic acid improved after complexation with α-CD and HP-ß-CD, while the antioxidant activity of both isomers was maintained.

Cyclodextrins as Anti-inflammatory Agents: Basis, Drugs and Perspectives.

Inflammation is a biological response of the immune system to harmful stimuli. Importantly, inflammation is also a hallmark of several human diseases such as cancer or diabetes. Novel drugs to treat this response are constantly researched, but the formulation is usually forgotten. Cyclodextrins (CDs) are a well-known excipient for complexing and drug delivery. Anti-inflammatory drugs and bioactive compounds with similar activities have been favored from these CD processes. CDs also illustrate anti-inflammatory activity per se. This review tried to describe the capacities of CDs in this field, and is divided into two parts: Firstly, a short description of the inflammation disease (causes, symptoms, treatment) is explained; secondly, the effects of different CDs alone or forming inclusion complexes with drugs or bioactive compounds are discussed.

Advances and Classification of Cyclodextrin-Based Polymers for Food-Related Issues.

Cyclodextrins (CDs) are a good alternative to reduce or enhance different biomolecule characteristics and have demonstrated great results in food science. However, CDs present intrinsic limitations that can be solved by derivative synthesis. This review represents a survey of the state of the art of CD-based materials and their uses in food science. A deep review of the structure is carried out and different groups for ordination are suggested. After that, different applications such as cholesterol complexation or its use as sensors are reviewed. The derivatives show novel and promising activities for the industry. A critical perspective of the materials suggests that they might not present toxicity, although more studies are required. These points suggest that the research in this field will be increased in the following years.

Characterization of Resveratrol, Oxyresveratrol, Piceatannol and Roflumilast as Modulators of Phosphodiesterase Activity. Study of Yeast Lifespan.

Our desire to live longer has led to an ever-increasing number of novel antiaging products. However, few molecules have any real effect and new ones need to be studied before they can be used commercially. In this contribution, activation of the caloric restriction (CR) pathway was studied using different three (resveratrol, oxyresveratrol and piceatannol)-a family with demonstrated bioactivity on phosphodiesterase activity. The high-affinity phosphodiesterase type 2 (PDE2) of Saccharomyces cerevisiae was expressed in Escherichia coli, purified and characterized. The activity and the inhibitory activity of each stilbene was studied, and the findings were compared in vitro and in silico with those obtained with roflumilast-a human PDE4 inhibitor widely used in chronic obstructive pulmonary diseases. Finally, an in vivo chronological lifespan assay using WT S. cerevisiae and ΔPDE2 S. cerevisiae strains was carried out. It was demonstrated that stilbenes can modulate yPDE2 activity, increasing the lifespan of the yeast by 18% over a control (in combination with other pathways). In addition, roflumilast increased the lifespan in the WT strain. The findings as a whole would increase the range of lifespan products available and suggest novel uses for approved drugs.

Cyclic Oligosaccharides as Active Drugs, an Updated Review.

There have been many reviews of the cyclic oligosaccharide cyclodextrin (CD) and CD-based materials used for drug delivery, but the capacity of CDs to complex different agents and their own intrinsic properties suggest they might also be considered for use as active drugs, not only as carriers. The aim of this review is to summarize the direct use of CDs as drugs, without using its complexing potential with other substances. The direct application of another oligosaccharide called cyclic nigerosyl-1,6-nigerose (CNN) is also described. The review is divided into lipid-related diseases, aggregation diseases, antiviral and antiparasitic activities, anti-anesthetic agent, function in diet, removal of organic toxins, CDs and collagen, cell differentiation, and finally, their use in contact lenses in which no drug other than CDs are involved. In the case of CNN, its application as a dietary supplement and immunological modulator is explained. Finally, a critical structure-activity explanation is provided.

Lifespan extension in Caenorhabditis elegans by oxyresveratrol supplementation in hyper-branched cyclodextrin-based nanosponges.

In this work, the increase of the Caenorhabditis elegans (C. elegans) lifespan extension using hyper-branched cyclodextrin-based nanosponges (CD-NS) complexing oxyresveratrol (OXY), and the possible inhibition of C. elegans phosphodiesterase type 4 (PDE4) were evaluated. The titration displacement of fluorescein was used to calculate the apparent complexation constant (KF) between CD-NS and OXY. Moreover, PDE4 was expressed in E. coli, purified and refolded in presence of cyclodextrins (CDs) to study its possible inhibition as pharmacological target of OXY. The apparent activity was characterized and the inhibitory effect of OXY on PDE4 displayed a competitive in vitro inhibition corroborated in silico. A maximum increase of the in vivo life expectancy of about 9.6% of using OXY/CD-NS complexes in comparison with the control was obtained, in contrast to the 6.5% obtained with free OXY. No effect on lifespan or toxicity with CD-NS alone was found. These results as a whole represent new opportunities to use OXY and CD-NS in lifespan products.

Recent advances in the treatment of Niemann pick disease type C: A mini-review.

Niemann Pick disease Type C (NPC) is a recessive rare disease caused by the mutation on NPC1 and/or NPC2 genes changing the processing of the Low-density proteins (LDL) resulting in an accumulation of lipids in the cells. Until today there is not a cure, the current treatment is based on palliative affairs to reduce the symptoms and prevent its appearance. Among all the treatments proposed the use of cyclodextrins (CDs), nanocarriers which can complex cholesterol, is one of the most useful alternatives. Indeed, for several years 2-hydroxypropyl-ß-CD (HPß-CD) is approved as orphan drug for FDA and EMA to the treatment. However, different CDs based materials are created each year to improve the cholesterol uptake. This review is focused on the novelty of CD based materials for NPC treatment.

Study of oxyresveratrol complexes with insoluble cyclodextrin based nanosponges: Developing a novel way to obtain their complexation constants and application in an anticancer study.

The complexation of the bioactive compound oxyresveratrol (OXY) with a polymer called cyclodextrin-based nanosponge (CD-NS) and its application was studied.A new methodology is used to calculate, an apparent inclusion complex constant (KFapp) between a ligand and CD-NSs. Moreover, the KFapp of resveratrol was also evaluated and compared. The complex of OXY with the nanosponge ß-CDI 1:4, was studied in vitro using DSC, TGA and FTIR techniques and its drug loading and release behavior were studied. An in vitro digestion showed higher protection of OXY complexed than free OXY. The bioactivity enhancing capacity of OXY was also studied against prostate (PC-3) and colon (HT-29 and HCT-116) cancer cell lines, where it showed stronger cell viability inhibition than the free drug. The findings as a whole represent a new opportunity for studying the complexation of drugs in CD-NSs and the use of oxyresveratrol as an ingredient in nutraceutical products.

An improved "ion pairing agent free" HPLC-RP method for testing cAMP Phosphodiesterase activity.

Current HPLC methods for analyzing cAMP Phosphodiesterase activity (PDE) use salts, limiting the life of the columns. For this reason, we have developed an improved "ion pairing agent free" method, using a simple 150 mm C18-hydro column at 30 °C and two phases: (a) water with 0.1% acetic acid and (b) 85/15 w/w MeOH/tetrahydrofuran with 0.1% acetic acid. Using this method the peaks for cAMP and AMP were obtained with good resolution (R ≈ 1.35) and sensitivity (5·10-9 mols) in only 15 min. Moreover, the method was applied to the GMP/cGMP pair obtaining the same sensitivity and resolution (R ≈ 1.38). The precision and accuracy were tested and the method was verified with a Type IV Phosphodiesterase reaction, which produced AMP from cAMP. The method is cleaner and less aggressive, and represents an interesting alternative to currently used methods.