Hydrolysis and diffusion of raffinose oligosaccharides family products in chickpeas, lentils, and beans under different pH and temperature steeping conditions.

Soaking pulses in water is a traditional practice widely used both by many households and by the food industry, and depending on the specific conditions used, can effectively reduce α-galactosides. Monitoring changes in α-galactoside content in pulses under different steeping conditions can provide insights into the degradation mechanisms and help overcome the barrier to consumption caused by digestive problems. In this study, we analyzed the impact of steeping at different temperatures (30, 45, 60, 75, and 90 °C) and at different pH (4.0, 5.0, and 6.0) on α-galactosides content in chickpeas, lentils, and beans. Our results showed that the lower the pH, the faster the α-galactosides were reduced. Moreover, steeping at lower temperatures (30 °C and 45 °C) favored hydrolysis of α-galactosides, whereas steeping at higher temperatures (60, 75, and 90 °C) favored diffusion. Soaking at 45 °C at a pH of 4.0 for 3 h resulted in acceptable levels of α-galactosides (less than 1 g/100 g), i.e. a reduction of up to 65 % in chickpeas, 85 % in lentils, and 52 % in beans.

NADES-based extraction optimization and enrichment of Cyanidin-3-O-galactoside from Rhododendron arboreum Sm.: Kinetics and thermodynamics insights.

Cyanidin-3-O-galactoside (Cy3-gal) is the most widespread anthocyanin that has been found to be applicable to nutraceutical and pharmaceutical ingredients. Nevertheless, the process of separation and purification, susceptibilities to heat, and pH inactivation present some limitations. In the present study, natural deep eutectic solvents (NADES) with an ultrasonic-assisted extraction method were briefly studied, and the recovery of Cy3-gal from Rhododendron arboreum was highlighted. The NADES, consisting of choline chloride and oxalic acid (1:1), was screened out as an extractant, and single-factor experiments combined with a two-site kinetic model were employed to describe the extraction process. Further, the work investigated ultrasound-assisted adsorption/desorption to efficiently purify Cy3-gal using macroporous resins. The optimal extraction conditions to attain maximum Cy3-gal yield was 30% water in a 50:1 (mL/g) solvent-to-sample ratio, 11.25 W/cm3 acoustic density, and 50% duty cycle for 16 min of extraction time. Under these conditions, the results revealed 23.07 ± 0.14 mg/g of Cy3-gal, two-fold higher than the traditional solvents. Furthermore, of the different resins used, Amberlite XAD-7HP showed significantly (p < 0.05) higher adsorption/desorption capacities (12.82 ± 0.18 mg/g and 10.97 ± 0.173 mg/g) and recovery (48.41 ± 0.76%) percent over other adsorbents. Experiments on the degrading behavior (40-80 °C) of the recovered Cy3-gal were performed over time, and the first-order kinetic model better explained the obtained data. In conclusion, the study asserts the use of ultrasonication with NADES and XAD-7HP resin for the improved purification of Cy3-gal from the crude extract.

Syntheses of α(2,8) Sialosides Containing NeuAc and NeuGc by Using Double Carbonyl-Protected N-Acyl Sialyl Donors.

We report on the syntheses of NeuAc and NeuGc-containing glycosides via the use of double carbonyl-protected N-acetyl sialyl donors. The 7-O,9-O-carbonyl protection of an N-acyl-5-N,4-O-carbonyl-protected sialyl donor markedly increased the α-selectivity during glycosylation, particularly when glycosylating the C-8 hydroxyl group of sialic acids. The N-acyl carbamates were selectively opened with ethanethiol under basic conditions to provide N-acyl amines. It is noteworthy that N-glycolyl carbamate was more reactive to nucleophiles by comparison with the N-acetyl carbamate due to the electron-withdrawing oxygen in the N-acyl group and however, allowed selective opening of the carbamates without the loss of N-glycolyl groups. To demonstrate the utility of the approach, we began by synthesizing α(2,3) and α(2,6) sialyl galactosides. Glycosylation of the hydroxy groups of galactosides at the C-6 position with the NeuAc and NeuGc donors provided the corresponding sialyl galactoses in good yields with excellent α-selectivity. However, glycosylation of the 2,3-diol galactosyl acceptor selectively provided Siaα(2,2)Gal. Next, we prepared a series of α(2,8) disialosides composed of NeuAc and NeuGc. Glycosylation of NeuGc and NeuAc acceptors at the C-8 hydroxyl group with NeuGc and NeuAc sialyl donors provided the corresponding α(2,8) disialosides, and no significant differences were detected in the reactivities of these acceptors.

Placental galectins: a subfamily of galectins lose the ability to bind ß-galactosides with new structural features†.

Galectins are a phylogenetically conserved family of soluble ß-galactoside binding proteins. There are 16 different of galectins, each with a specific function determined by its distinct distribution and spatial structure. Galectin-13, galectin-14, and galectin-16 are distinct from other galectin members in that they are primarily found in placental tissue. These galectins, also referred to as placental galectins, play critical roles in regulating pregnancy-associated processes, such as placenta formation and maternal immune tolerance to the embedded embryo. The unique structural characteristics and the inability to bind lactose of placental galectins have recently received significant attention. This review primarily examines the novel structural features of placental galectins, which distinguish them from the classic galectins. Furthermore, it explores the correlation between these structural features and the loss of ß-galactoside binding ability. In addition, the newly discovered functions of placental galectins in recent years are also summarized in our review. A detailed understanding of the roles of placental galectins may contribute to the discovery of new mechanisms causing numerous pregnancy diseases and enable the development of new diagnostic and therapeutic strategies for the treatment of these diseases, ultimately benefiting the health of mothers and offspring.

Unexpected Nucleophile Masking in Acyl Transfer to Sterically Crowded and Conformationally Restricted Galactosides.

Design and synthesis of orthogonally protected monosaccharide building blocks are crucial for the preparation of well-defined oligosaccharides in a stereo- and regiocontrolled manner. Selective introduction of protecting groups to partially protected monosaccharides is nontrivial due to the often unpredictable electronic, steric, and conformational effects of the substituents. Abolished reactivity toward a commonly used Lewis base-catalyzed acylation of O-2 was observed in conformationally restricted 4,6-O-benzylidene-3-O-Nap galactoside. Investigation of analogous systems, crystallographic characterization, and quantum chemical calculations highlighted the overlooked conformational and steric considerations, the combination of which produces a unique passivity of the 2-OH nucleophile. Evaluating the role of electrophile counterion and auxiliary base in the acylation of the sterically crowded and conformationally restricted galactoside system revealed an alternative Brønsted base-driven reaction pathway via nucleophilic activation. Insights gained from this model system were utilized to access the target galactoside intermediate within the envisioned synthetic route. The acylation strategy described herein can be implemented in future syntheses of key monomeric building blocks with unique protecting group hierarchies.

Neutralizing the Impact of the Virulence Factor LecA from Pseudomonas aeruginosa on Human Cells with New Glycomimetic Inhibitors.

Bacterial adhesion, biofilm formation and host cell invasion of the ESKAPE pathogen Pseudomonas aeruginosa require the tetravalent lectins LecA and LecB, which are therefore drug targets to fight these infections. Recently, we have reported highly potent divalent galactosides as specific LecA inhibitors. However, they suffered from very low solubility and an intrinsic chemical instability due to two acylhydrazone motifs, which precluded further biological evaluation. Here, we isosterically substituted the acylhydrazones and systematically varied linker identity and length between the two galactosides necessary for LecA binding. The optimized divalent LecA ligands showed improved stability and were up to 1000-fold more soluble. Importantly, these properties now enabled their biological characterization. The lead compound L2 potently inhibited LecA binding to lung epithelial cells, restored wound closure in a scratch assay and reduced the invasiveness of P. aeruginosa into host cells.

Discovery of potent 1,1-diarylthiogalactoside glycomimetic inhibitors of Pseudomonas aeruginosa LecA with antibiofilm properties.

In this work, ß-thiogalactoside mimetics bearing 1,1-diarylmethylene or benzophenone aglycons have been prepared and assayed for their affinity towards LecA, a lectin and virulence factor from Pseudomonas aeruginosa involved in bacterial adhesion and biofilm formation. The hit compound presents higher efficiency than previously described monovalent inhibitors and the crystal structure confirmed the occurrence of additional contacts between the aglycone and the protein surface. The highest affinity (160 nM) was obtained for a divalent ligand containing two galactosides. The monovalent high affinity compound (Kd = 1 µM) obtained through structure-activity relationship (SAR) showed efficient antibiofilm activity with no associated bactericidal activity.

Structural and evolutionary insights into the multidomain galectin from the red abalone Haliotis rufescens with specificity for sulfated glycans.

Galectins are an evolutionarily ancient family of lectins characterized by their affinity for ß-galactosides and a conserved binding site in the carbohydrate recognition domain (CRD). These lectins are involved in multiple physiological functions, including the recognition of glycans on the surface of viruses and bacteria. This feature supports their role in innate immune responses in marine mollusks. Here, we identified and characterized a galectin, from the mollusk Haliotis rufescens (named HrGal), with four CRDs that belong to the tandem-repeat type. HrGal was purified by affinity chromatography in a galactose-agarose resin and exhibited a molecular mass of 64.11 kDa determined by MALDI-TOF mass spectrometry. The identity of HrGal was verified by sequencing, confirming that it is a 555 amino acid protein with a mass of 63.86 kDa. This protein corresponds to a galectin reported in GenBank with accession number AHX26603. HrGal is stable in the presence of urea, reducing agents, and ions such as Cu2+ and Zn2+. The recombinant galectin (rHrGal) was purified from inclusion bodies in the presence of these ions. A theoretical model obtained with the AlphaFold server exhibits four non-identical CRDs, with a ß sandwich folding and the representative motifs for binding ß-galactosides. This allows us to classify HrGal within the tandem repeat galectin family. On the basis of a phylogenetic analysis, we found that the mollusk sequences form a monophyletic group of tetradomain galectins unrelated to vertebrate galectins. HrGal showed specificity for galactosides and glucosides but only the sulfated sugars heparin and ι-carrageenan inhibited its hemagglutinating activity with a minimum inhibitory concentration of 4 mM and 6.25 X 10-5% respectively. The position of the sulfate groups seemed crucial for binding, both by carrageenans and heparin.

Protective effects and mechanism of amino acids as chokeberry cyanidin and its glycoside protectant under the condition of vitamin C coexistence.

In the presence of vitamin C, cyanidin and cyanidin glycosides are degraded during the processing and storage of food products. To solve this issue, we investigated the protective effects and mechanism of action of five amino acids on the stability of cyanidin and its glycosides from chokeberry. The results showed that 0.3% tryptophan most effectively inhibited the degradation of cyanidin and its glycosides in the presence of vitamin C, under ultraviolet, dark, and sucrose-rich conditions. Fluorescence spectrum analysis showed that tryptophan could form noncovalent binding complexes with cyanidin-3-O-galactoside and cyanidin through hydrophobic and electrostatic forces and hydrogen bonds. Molecular docking results showed that the indole structure of tryptophan could form hydrophobic interactions with cyanidin-3-O-galactoside and cyanidin via hydrogen bonding, resulting in greater protection. Therefore, tryptophan could effectively protect cyanidin and its glycosides in cyanidin- and cyanidin glycoside-rich food products.

Multiplexed assessment of engineered bacterial constructs for intracellular ß-galactosidase expression by redox amplification on catechol-chitosan modified nanoporous gold.

Synthetic biology approaches for rewiring of bacterial constructs to express particular intracellular factors upon induction with the target analyte are emerging as sensing paradigms for applications in environmental and in vivo monitoring. To aid in the design and optimization of bacterial constructs for sensing analytes, there is a need for lysis-free intracellular detection modalities that monitor the signal level and kinetics of expressed factors within different modified bacteria in a multiplexed manner, without requiring cumbersome surface immobilization. Herein, an electrochemical detection system on nanoporous gold that is electrofabricated with a biomaterial redox capacitor is presented for quantifying ß-galactosidase expressed inside modified Escherichia coli constructs upon induction with dopamine. This nanostructure-mediated redox amplification approach on a microfluidic platform allows for multiplexed assessment of the expressed intracellular factors from different bacterial constructs suspended in distinct microchannels, with no need for cell lysis or immobilization. Since redox mediators present over the entire depth of the microchannel can interact with the electrode and with the E. coli construct in each channel, the platform exhibits high sensitivity and enables multiplexing. We envision its application in assessing synthetic biology-based approaches for comparing specificity, sensitivity, and signal response time upon induction with target analytes of interest.

Structure investigation on a novel 2-halo-2,3-unsaturated-N-galactoside, NMR and X-ray diffraction of a monoclinic multidomain crystal.

Single crystal X-ray and NMR investigations on multidomain structured N-(4,6-di-O-acetyl-2,3-dideoxy-α-D-threo-hex-2-en-2-iodo-pyranosyl)-methylsulfonamide are reported. This is the first crystallographic diffraction data report related to a 2-halo-2,3-unsaturated galactoside derivative. A complete structural study, including conformations and crystal packing, was performed by analyzing the spectroscopic data in solid state (XRD) and in solution (NMR).

Compression-induced Phase Transition in Adsorbed Monolayers of Alkylgalactosides at the Air/Water Interface.

Compression-induced formation of condensed-phase domains in adsorbed monolayers of alkylgalactosides (AGs) at the air/water interface was observed. When an aqueous solution of AGs was poured into a Langmuir trough, the AG molecules were spontaneously adsorbed from the solution at the air/water interface to form the adsorbed or Gibbs monolayer in an expanded, liquid-like phase at equilibrium. The monolayer was subsequently laterally compressed by the barriers of the trough, while simultaneously observing the system using a Brewster angle microscope (BAM). The surface pressure-film area isotherm upon compression showed a kink at a surface pressure (πkink) comparable to or several mN・m-1 higher than the surface pressure at the critical micelle concentration (πCMC), followed by a plateau region. BAM observations revealed that condensed-phase domains were formed in the homogeneous expanded phase at the plateau. Hence, the plateau corresponds to a first-order phase transition from the expanded phase to the condensed phase. As expected, the compressed adsorbed monolayer was in a metastable state because the surface pressure decreased with time, and the condensed-phase domains disappeared when compression was discontinued. The transient formation of a quasi-stable condensed phase may originate from the combined effect of the lower solubility of AG molecules in water, moderately strong attractive intermolecular interactions between AG molecules at the air/water interface, and high-rate compression.

Sulfated glycans containing NeuAcα2-3Gal facilitate the propagation of human H1N1 influenza A viruses in eggs.

When human influenza viruses are isolated and passaged in chicken embryos, variants with amino acid substitutions around the receptor binding site of hemagglutinin (HA) are selected; however, the mechanisms that underlie this phenomenon have yet to be elucidated. Here, we analyzed the receptor structures that contributed to propagation of egg-passaged human H1N1 viruses. The analysis included seasonal and 2009 pandemic strains, both of which have amino acid substitutions of HA found in strains isolated or passaged in eggs. These viruses exhibited high binding to sulfated glycans containing NeuAcα2-3Gal. In MDCK cells overexpressing the sulfotransferase that synthesize Galß1-4(SO3--6)GlcNAc, production of human H1N1 viruses was increased up to 90-fold. Furthermore, these sulfated glycans were expressed on the allantoic and amniotic membranes of chicken embryos. These results suggest that 6-sulfo sialyl Lewis X and/or NeuAcα2-3Galß1-4(SO3--6)GlcNAc are involved in efficient propagation of human H1N1 viruses in chicken embryos.

The assessment of Pseudomonas aeruginosa lectin LecA binding characteristics of divalent galactosides using multiple techniques.

Pseudomonas aeruginosa is a widespread opportunistic pathogen that is capable of colonizing various human tissues and is resistant to many antibiotics. LecA is a galactose binding tetrameric lectin involved in adhesion, infection and biofilm formation. This study reports on the binding characteristics of mono- and divalent (chelating) ligands to LecA using different techniques. These techniques include affinity capillary electrophoresis, bio-layer interferometry, native mass spectrometry and a thermal shift assay. Aspects of focus include: affinity, selectivity, binding kinetics and residence time. The affinity of a divalent ligand was determined to be in the low-nanomolar range for all of the used techniques and with a ligand residence time of approximately 7 h, while no strong binding was seen to related lectin tetramers. Each of the used techniques provides a unique and complementary insight into the chelation based binding mode of the divalent ligand to the LecA tetramer.

Structural insights in galectin-1-glycan recognition: Relevance of the glycosidic linkage and the N-acetylation pattern of sugar moieties.

Galectins, soluble lectins widely expressed intra- and extracellularly in different cell types, play major roles in deciphering the cellular glycocode. Galectin-1 (Gal-1), a prototype member of this family, presents a carbohydrate recognition domain (CRD) with specific affinity for ß-galactosides such as N-acetyllactosamine (ß-d-Galp-(1 â†’ 4)-d-GlcpNAc), and mediate numerous physiological and pathological processes. In this work, Gal-1 binding affinity for ß-(1 â†’ 6) galactosides, including ß-d-Galp-(1 â†’ 6)-ß-d-GlcpNAc-(1 â†’ 4)-d-GlcpNAc was evaluated, and their performance was compared to that of ß-(1 â†’ 4) and ß-(1 â†’ 3) galactosides. To this end, the trisaccharide ß-d-Galp-(1 â†’ 6)-ß-d-GlcpNAc-(1 â†’ 4)-d-GlcpNAc was enzymatically synthesized, purified and structurally characterized. To evaluate the affinity of Gal-1 for the galactosides, competitive solid phase assays (SPA) and isothermal titration calorimetry (ITC) studies were carried out. The experimental dissociation constants and binding energies obtained were compared to those calculated by molecular docking. These analyses evidenced the critical role of the glycosidic linkage between the terminal galactopyranoside residue and the adjacent monosaccharide, as galactosides bearing ß-(1 â†’ 6) glycosidic linkages showed dissociation constants six- and seven-fold higher than those involving ß-(1 â†’ 4) and ß-(1 â†’ 3) linkages, respectively. Moreover, docking experiments revealed the presence of hydrogen bond interactions between the N-acetyl group of the glucosaminopyranose moiety of the evaluated galactosides and specific amino acid residues of Gal-1, relevant for galectin-glycan affinity. Noticeably, the binding free energies (ΔGbindcalc) derived from the molecular docking were in good agreement with experimental values determined by ITC measurements (ΔGbindexp), evidencing a good correlation between theoretical and experimental approaches, which validates the in silico simulations and constitutes an important tool for the rational design of future optimized ligands.

Benzimidazole-galactosides bind selectively to the Galectin-8 N-Terminal domain: Structure-based design and optimisation.

We have obtained the X-ray crystal structure of the galectin-8 N-terminal domain (galectin-8N) with a previously reported quinoline-galactoside ligand at a resolution of 1.6 Å. Based on this X-ray structure, a collection of galactosides derivatised at O3 with triazole, benzimidazole, benzothiazole, and benzoxazole moieties were designed and synthesised. This led to the discovery of a 3-O-(N-methylbenzimidazolylmethyl)-galactoside with a Kd of 1.8 µM for galectin-8N, the most potent selective synthetic galectin-8N ligand to date. Molecular dynamics simulations showed that benzimidazole-galactoside derivatives bind the non-conserved amino acid Gln47, accounting for the higher selectivity for galectin-8N. Galectin-8 is a carbohydrate-binding protein that plays a key role in pathological lymphangiogenesis, modulation of the immune system, and autophagy. Thus, the benzimidazole-derivatised galactosides represent promising compounds for studies of the pathological implications of galectin-8, as well as a starting point for the development of anti-tumour and anti-inflammatory therapeutics targeting galectin-8.

Indolyl Septanoside Synthesis for In Vivo Screening of Bacterial Septanoside Hydrolases.

Building-up and breaking-down of carbohydrates are processes common to all forms of life. Glycoside hydrolases are a broad class of enzymes that play a central role in the cleavage of glycosidic bonds, which is fundamental to carbohydrate degradation. The large majority of substrates are five- and six-membered ring glycosides. Our interest in seven-membered ring septanose sugars has inspired the development of a way to search for septanoside hydrolase activity. Described here is a strategy for the discovery of septanoside hydrolases that uses synthetic indolyl septanosides as chromogenic substrates. Access to these tool compounds was enabled by a route where septanosyl halides act as glycosyl donors for the synthesis of the indolyl septanosides. The screening strategy leverages the known dimerization of 3-hydroxy-indoles to make colored dyes, as occurs when the ß-galactosidase substrate X-Gal is hydrolyzed. Because screens in bacterial cells would enable searches in organisms that utilize heptoses or from metagenomics libraries, we also demonstrate that septanosides are capable of entering E. coli cells through the use of a BODIPY-labeled septanoside. The modularity of the indolyl septanoside synthesis should allow the screening of a variety of substrates that mimic natural structures via this general approach.

Chromo-fluorogenic probes for ß-galactosidase detection.

ß-Galactosidase (ß-Gal) is a widely used enzyme as a reporter gene in the field of molecular biology which hydrolyzes the ß-galactosides into monosaccharides. ß-Gal is an essential enzyme in humans and its deficiency or its overexpression results in several rare diseases. Cellular senescence is probably one of the most relevant physiological disorders that involve ß-Gal enzyme. In this review, we assess the progress made to date in the design of molecular-based probes for the detection of ß-Gal both in vitro and in vivo. Most of the reported molecular probes for the detection of ß-Gal consist of a galactopyranoside residue attached to a signalling unit through glycosidic bonds. The ß-Gal-induced hydrolysis of the glycosidic bonds released the signalling unit with remarkable changes in color and/or emission. Additional examples based on other approaches are also described. The wide applicability of these probes for the rapid and in situ detection of de-regulation ß-Gal-related diseases has boosted the research in this fertile field.

The Protective Effect of Anthocyanins Extracted from Aronia Melanocarpa Berry in Renal Ischemia-Reperfusion Injury in Mice.

BACKGROUND: Our previous research showed the antioxidant activity of anthocyanins extracted from Aronia melanocarpa of black chokeberry in vitro. Ischemia acute kidney injury is a significant risk in developing progressive and deterioration of renal function leading to clinic chronic kidney disease. There were many attempts to protect the kidney against this progression of renal damage. Current study was designed to examine the effect of pretreatment with three anthocyanins named cyanidin-3-arabinoside, cyanidin-3-glucodise, and cyaniding-3-galactoside against acute ischemia-reperfusion injury in mouse kidney. METHODS: Acute renal injury model was initiated by 30 min clamping bilateral renal pedicle and followed by 24-hour reperfusion in C57Bl/6J mice. Four groups of mice were orally pretreated in 50 mg/g/12 h for two weeks with cyanidin-3-arabinoside, cyanidin-3-glucodise, and cyaniding-3-galactoside and anthocyanins (three-cyanidin mixture), respectively, sham-control group and the renal injury-untreated groups only with saline. RESULTS: The model resulted in renal dysfunction with high serum creatinine, blood urea nitrogen, and changes in proinflammatory cytokines (TNF-ɑ, IL-1ß, IL-6, and MCP-1), renal oxidative stress (SOD, GSH, and CAT), lipid peroxidation (TBARS and MDA), and apoptosis (caspase-9). Pretreatment of two weeks resulted in different extent amelioration of renal dysfunction and tubular damage and suppression of proinflammatory cytokines, oxidative stress, lipid peroxidation, and apoptosis, thus suggesting that cyanidins are potentially effective in acute renal ischemia by the decrease of inflammation, oxidative stress, and lipid peroxidation, as well as apoptosis. CONCLUSION: the current study provided the first attempt to investigate the role of anthocyanins purified from Aronia melanocarpa berry in amelioration of acute renal failure via antioxidant and cytoprotective effects.

Screening and identification of bioactive components resistant to metallo-beta-lactamase from Schisandra chinensis (Turcz.) Baill. by metalloenzyme-immobilized affinity chromatography.

The screening and identification of bioactive components, which are effectively resistant to metallo-beta-lactamase (MßL), were studied in the alcohol extract of Schisandra chinensis (Turcz.) Baill. by metalloenzyme-immobilized affinity chromatography. Taking bizinc metalloenzyme beta-lactamase II from Bacillus cereus (Bc II) and monozinc metalloenzyme CphA from aeromonas hydrophila (CphA) as examples, we studied the feasibility of this scheme based on the construction of metalloenzyme-immobilized chromatographic model. It was found that the Bc II- and CphA-immobilized chromatographic column could be used not only to explore the interaction between the MßL and their specific ligands, but also to screen the bioactive components from traditional Chinese medicine. The Bc II-and CphA-immobilized columns were used to screen the bioactive components from the alcohol extract of Schisandra chinensis (Turcz.) Baill. Time-of-flight tandem mass spectrometry analysis and molecular docking revealed that isobutyl 3-O-sulfo-ß-D-galactopyranoside is the effective bioactive components that could bind with metalloenzyme Bc II. It is believed that our current work may provide a methodological reference for screening MßL inhibitors from traditional Chinese medicine.