Extraction and formulation of plant substances

The aim of this thesis was to investigate the extraction of potential valuable neutraceuticals from different rhizomes: Iris germanica L. and Curcuma longa. On the one hand, three different curcuminoids (curcumin, dimethoxy-, and bisdemethoxycurcumin) from Curcuma longa were extracted. On the other hand, extraction and isolation of isoflavones and benzophenones of Iris germanica L. were performed. Most of the isolated compounds from Iris germanica L. were tested for their potential anti-inflammatory and antibacterial abilities. First, curcuminoids were successively extracted from Curcuma longa using a green, sustainable, bio-degradable and food-approved surfactant-free microemulsion (SFME) consisting of water, ethanol (EtOH) and triacetin (TriA) exhibiting high extraction yields (15.28 mg curcuminoids per g Curcuma longa). The best yield was achieved by the usage of a SFME consisting of 40/24/36 H2O/EtOH/TriA (wt%). The maximum of solubility of curcumin was investigated in the binary mixture EtOH/TriA via UV measurements. The binary mixture EtOH/TriA (40/60 in wt%) turned out to be the best one for the solubilisation of curcumin. The structuring of the SFME was previously investigated with DLS and conductivity measurements. It was also found and demonstrated that the addition of water to the binary mixture of EtOH/TriA was responsible for the structuring of the SFME and for the high extraction yield. Indeed, bisdemethoxycurcumin, one of the curcuminoids, is the most “polar” curcuminoid and therefore, the most sensitive to water. Compared to the binary mixture EtOH/TriA (40/60 in wt%), the extraction yield of demethoxycurcumin and especially bisdemethoxycurcumin could be increased by 14 % and 32 % respectively. Recycling and up-concentration of the SFME with curcuminoids was attempted. Therefore, the SFME was re-used to perform several extraction cycles and to concentrate the curcuminoids in the SFME. One of the goals of this study was to solubilise the curcuminoids in an aqueous solution. Therefore, different purification methods (hydro distillation, vacuum distillation and freeze-drying) were used to remove the essential oils of Curcuma longa and to enhance the relative purity of the extract. Purification of the extract was achieved by freeze-drying the rhizome of Curcuma longa, as it leads to high relative purity of the extract (about 94%) through repetitive lyophilisation cycles and did not destroy the curcuminoids before extraction. Using an appropriate composition of the SFME (50/32.5/17.5 H2O/EtOH/TriA in wt%), dilution of the curcuminoids extract solution with water and stabilisation against day light and precipitation were achieved. The extraction efficiencies of the curcuminoids were further enhanced using different additives, which were solubilised in the water phase of the SFME. Meglumine has been found to be the best additive while using pyroglutamic acid (PCA) as pH regulator of the SFME and as hydrotrope for curcumin. Using meglumine with and without PCA, high extraction efficiencies of the curcuminoids were achieved: 17.3 mg curcuminoids per g Curcuma longa using a SFME (15/34/51 H2O/EtOH/TriA in wt%) at pH 9 containing 5 wt% meglumine neutralised with PCA in pure water and 18.3 mg curcuminoids per g Curcuma longa using a SFME (5/38/57 H2O/EtOH/TriA in weight percent) at pH 11.5 containing 15 wt% meglumine without PCA in pure water. A simple water extraction (water containing 15 wt% of meglumine) achieved the best extraction efficiency for bisdemethoxycurcumin (3.46 ± 0.62 mg per g Curcuma longa). Further, another SFME consisting of water, sodium salicylate (NaSal) and ethyl acetate (EtOAc) was investigated concerning its capacity to solubilize, stabilize and separate the three curcuminoids. The extraction efficiency of one curcuminoid could be enhanced using different SFME compositions: H2O/NaSal/EtOAc 17/12/71 and 7/13/80 (in wt%) for respectively bisdemethoxy- and demethoxycurcumin and the pure EtOAc for curcumin. The presence of NaSal in the SFME enhanced the stability of curcumin and the other two curcuminoids in solution, because of its antioxidant and UV absorbing properties. In the second part of this thesis, isoflavones and benzophenones were successively extracted and isolated from the rhizomes and the roots of Iris germanica L. by chromatographic methods (silica gel column followed by high-performance liquid chromatography (HLPC) and semi preparative HPLC). Using NMR and LC-MS data, structures of eleven isolated compounds were revealed. Some of the isolated compounds were tested as potential anti-inflammatory agents but had unexpected pro-inflammatory properties, of which the rhizome extract showed the highest pro-inflammatory activity. The isolates and the extracts were also tested as potential antibacterial agents. None of the extracts nor isolates were active against the bacterium S. aureus. Only the iris butter (the essential oil of orris) showed a potent antibacterial activity, certainly due to the presence of irones. The iris butter showed also an antibacterial activity against the bacterium E. coli

Improvement of the Solubilization and Extraction of Curcumin in an Edible Ternary Solvent Mixture

A water-free, ternary solvent mixture consisting of a natural deep eutectic solvent (NADES), ethanol, and triacetin was investigated concerning its ability to dissolve and extract curcumin from Curcuma longa L. To this purpose, 11 NADES based on choline chloride, acetylcholine, and proline were screened using UV–vis measurements. A ternary phase diagram with a particularly promising NADES, based on choline chloride and levulinic acid was recorded and the solubility domains of the monophasic region were examined and correlated with the system’s structuring via light scattering experiments. At the optimum composition, close to the critical point, the solubility of curcumin could be enhanced by a factor of >1.5 with respect to acetone. In extraction experiments, conducted at the points of highest solubility and evaluated via HPLC, a total yield of ~84% curcuminoids per rhizome could be reached. Through multiple extraction cycles, reusing the extraction solvent, an enrichment of curcuminoids could be achieved while altering the solution. When counteracting the solvent change, even higher concentrated extracts can be obtained

TRAF4 is a novel phosphoinositide-binding protein modulating tight junctions and favoring cell migration

Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is frequently overexpressed in carcinomas, suggesting a specific role in cancer. Although TRAF4 protein is predominantly found at tight junctions (TJs) in normal mammary epithelial cells (MECs), it accumulates in the cytoplasm of malignant MECs. How TRAF4 is recruited and functions at TJs is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)-binding domain crucial for its recruitment to TJs. Of interest, this property is shared by the other members of the TRAF protein family. Indeed, the TRAF domain of all TRAF proteins (TRAF1 to TRAF6) is a bona fide PIP-binding domain. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer that binds up to three lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts as a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJs and favoring cell migration

Curcumin extracts from Curcuma Longa – Improvement of concentration, purity, and stability in food-approved and water-soluble surfactant-free microemulsions

Curcumin was extracted from Curcuma Longa employing a green, bio-based, and food-agreed surfactant-free microemulsion (SFME) consisting of water, ethanol, and triacetin. Concerning the high solubility of curcumin in the examined ternary mixtures, it was attempted to produce highly concentrated tinctures of up to a total of similar to 130 mg/mL curcuminoids in the solvent by repeatedly extracting fresh rhizomes in the same extraction mixture. The amount of water had a significant influence on the number of cycles that could be performed as well as on the extraction of the different curcuminoids. In addition, the purity of single extracts was enhanced to 94% by investigating several purification steps, e.g. vacuum distillation and lyophilization. Through purification before extraction, the water insoluble curcumin extract could be solubilized indefinitely in an aqueous environment. Additional stability tests showed that solutions of curcumin can be stable up to five months when concealed from natural light

Enhancement of water solubilization of quercetin by meglumine and application of the solubilization concept to a similar system

International audienceIn the present work, meglumine, an aminocarbohydrate derived from glucose with a secondary amine group, was found to be able to increase the water-solubility of the well-known and beneficial flavonoid quercetin. pH proves to be a fundamental factor for this purpose. The solubility enhancement as a function of the pH as well as the mechanism behind this phenomenon were investigated. The state of charge of quercetin depending on pH has been estimated from the calculation of the pKa by COSMO-RS. A compromise between a satisfying solubility enhancement (6–7-fold) and reasonable stability against oxidation (70–75 % of quercetin still intact after 2 h) was found around pH 8. The solubilization mechanism is believed to be based on proton exchange between quercetin and the amine function of meglumine, and on the ability of meglumine to form numerous hydrogen bonds with water molecules through its five hydroxyl groups. Finally, it is shown that the underlying solubilization mechanism can be extended both to other polyphenols and to other hydrotropes

Novel green production of natural-like vanilla extract from curcuminoids

The demand for natural vanilla extract, and vanillin in particular, by far exceeds the current production, as both the cultivation of vanilla beans and the extraction of vanillin are laborious. For this purpose, most vanillin used today is produced synthetically, contrary to the general trend toward bio-based products. The present study deals with the synthesis of nature-based vanillin, starting with the more accessible rhizomes of the plant Curcuma longa. Besides vanillin, vanillic acid and p-hydroxybenzaldehyde are synthesized that way, which are also found in the natural vanilla bean. The extraction of the curcuminoids and, finally, their conversion to the flavors are performed using visible light and food-grade chemicals only. A binary mixture of ethanol and triacetin, as well as a surfactant-free microemulsion consisting of water, ethanol, and triacetin, are investigated in this context. The results exceed the literature values for Soxhlet extraction of vanilla beans by a factor > 7

NADES-based surfactant-free microemulsions for solubilization and extraction of curcumin from Curcuma Longa

A choline chloride + lactic acid (1:1) natural deep eutectic solvent (NADES) is used as an adjuvant to ethanol/ triacetin mixtures to solubilize and extract curcumin from Curcuma Longa. The obtained NADES/ethanol/triacetin mixtures are homogeneous, transparent and of low viscosity even in the absence of water. Dynamic light scattering revealed significant nanostructures, typical of surfactant-free microemulsions. A twofold increase of curcumin solubility and remarkable extraction power (yield of similar to 90%) can be achieved in the ternary system including the NADES, although curcumin is hydrophobic and the used NADES are very polar. Due to the elevated solubility of curcumin, more extraction cycles can be made than in the previously published aqueous systems with the same amount of solution. As a result, less solvent is required to achieve the same extraction yield

Solubilization and extraction of curcumin from Curcuma Longa using green, sustainable, and food-approved surfactant-free microemulsions

Curcumin is a powerful coloring agent widely used in the food industry. Its extraction from the plant Curcuma longa is commonly done with aqueous solvent solutions. In contrast to the conventional extraction methods, the present study aimed to compare two different green and bio-based surfactant-free microemulsion (SFME) extraction systems, which are approved for food and yield a higher extracting power of curcuminoids. Two SFMEs, water/ethanol/triacetin and water/diacetin/triacetin, were investigated via dynamic light scattering. Curcumin solubility in binary mixtures consisting of ethanol/triacetin or diacetin/triacetin was studied both experimentally and theoretically using UV-Vis measurements and COSMO-RS. The SFMEs were further examined and compared to a common ethanol/water (80/20) extraction mixture with respect to their extracting ability using high performance liquid chromatography. The SFMEs containing ethanol were found to extract similar to 18% more curcuminoids than the SFMEs containing diacetin and similar to 53% more than the ordinary ethanol/water mixture

Extraction of curcumin from Curcuma longa using meglumine and pyroglutamic acid, respectively, as solubilizer and hydrotrope

In preceding papers, it could be shown that mixtures of water, ethanol, and triacetin can significantly boost the extraction of curcuminoids out of Curcuma longa, compared to the usually applied water-ethanol mixtures. To further improve it, additives can be beneficial. We show that meglumine and pyroglutamic acid are among the most promising of these additives. First, the influence of meglumine and pyroglutamic acid on the phase diagram of ternary mixtures consisting of water, ethanol and triacetin has been investigated: the addition of meglumine to the water phase only slightly increases the miscibility gap, whereas pyroglutamic acid increases it significantly in the oil-rich region. The solubility of curcumin could be significantly enhanced through the addition of meglumine. Extractions were successfully conducted extracting similar to 18 mg of curcuminoids per g Curcuma longa by adding 15 wt% of meglumine to the amount of water in the extraction mixtures consisting of water, ethanol and triacetin, which is an increase of similar to 20% compared to the best extraction mixture water/ethanol/triacetin from the previous study. A simple water/meglumine extraction was also investigated. Around 13 mg curcuminoids per g Curcuma longa could be extracted. Bisdemethoxycurcumin contributes 3.46 mg to the total amount, which is a very good extraction yield (increase of 19% compared to the best extraction mixture from the previous study). The purity of the extract reached 87% just by extracting with water and meglumine. For comparison, the same purity was achieved using the ternary water/ethanol/triacetin system, but only after freeze drying of the rhizomes before extraction. (C) 2021 Elsevier B.V. All rights reserved

Metastatic Lymph Node 51, a novel nucleo-cytoplasmic protein overexpressed in breast cancer

International audienceMetastatic Lymph Node 51 (MLN51) cDNA was isolated by differential screening of a human breast cancer metastasis cDNA library. MLN51 cDNA encodes a novel human protein of 703 residues that shares no significant homology to any known protein. However MLN51 is well conserved between vertebrate and invertebrate species suggesting an important biological function. The amino terminal half of the protein contains a coiled-coil domain and two potential nuclear localization signals (NLS). The carboxy terminal half contains one SH2 and four SH3 binding motifs. The coiled-coil domain promotes MLN51 oligomerization in transfected cells. When transiently expressed, the MLN51 protein is mainly found in the cytoplasm with a weak nuclear staining. However, deletion of the carboxy terminal half of the protein allows the targeting of the protein to the nucleus, demonstrating that the NLSs are functional. MLN51 is ubiquitously expressed in normal tissues. Human breast carcinomas show MLN51 overexpression in malignant epithelial cells. The uncommon association of protein-protein interaction domains often found either in nuclear or in cytoplasmic signaling proteins raises a possible nucleo-cytoplasmic function for MLN51