Inhibition of the NLRP3 Inflammasome by a Quercus Serrata Extract and Isolation of the Component Compounds for the Treatment of Arthritis.

Quercus serrata belongs to the Fagaceae family. There are 600 known species of Quercus worldwide. Q. serrata is distributed nationally in Korea, Japan, and China and grows to a height of 10-15 m. It exhibits a light grey bark with longitudinal furrows; the leaves are 6-12 cm long and 2.5-5 cm wide. The Quercus genus reportedly exhibits several types of bioactivity, including antioxidant, anti-inflammatory, antifungal, antimicrobial, and anticancer activity. Additionally, it has been reported that Quercus produces diverse phytochemicals, including tannins, flavonoids, and triterpenoids. Herein, we describe the column chromatographic isolation of five compounds from a Q. serrata extract. The compounds included caffeic acid (1), myricetin-3-O-cellobioside (2), phloroglucinol (3), (S)-2,3-HHDP-D-glucopyranoside (4), and pedunculagin (5). We assessed the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity, antioxidant activity, NLR family pyrin domain-containing 3 (NLRP3) inflammasome (including NLRP3, ASC, and caspase-1) inhibitory effects, and collagenase inhibition activity of the Q. serrata extract and its constituent compounds. Our results indicated that the Q. serrata extract and the isolated constituent compounds showed inhibitory activity with reference to nitric oxide production, inflammasome component expression, and collagenase activity. Our findings imply that the Q. serrata extract and the isolated constituent compounds are potential candidates for the treatment of inflammatory diseases such as arthritis.

Antiacne and Anti-Inflammatory Effects of Phenolic Compounds from Quercus acutissima Carruth. Leaves.

Quercus plants are widely distributed in Korea and have been used for their antiallergic and anti-inflammatory properties to treat dermatitis. The phenolic compounds of Quercus acutissima Carruth (QA) are estimated to have antioxidant and anti-inflammatory activities, based on the results of previous studies with Quercus mongilica, Quercus stenophylla, Quercus gilva Blame., and Quercus acuta Thunb. We yield QA extract and the isolated phenolic compounds (hyperoside (1), astragalin (2), kaempferol 3-O-(6″- galloyl)-ß-D-glucopyranoside (KGG) (3), quercetin 3-O-(6″-O-galloyl)-ß-D-glucopyranoside (QGG) (4), pedunculagin (5), and casuarinin (6)) and were identified using NMR. Among them, KGG (3) and QGG (4) were isolated for the first time from QA. QA extract and the isolated phenolic compounds demonstrated antioxidative, anti-inflammatory, and antiacne activities in RAW 264.7 mouse macrophage cells in vitro. 3-6 demonstrated strong inhibitory activities in the DPPH scavenging and NO production assay and anti-inflammatory and antiacne activities through western blotting (NLRP3, IL-1ß, and 5α-reductase). The most outstanding activity in all experiments was casuarinin (6). The study findings suggest potential therapeutic candidates for acne.

Anti-Inflammatory Effects of Fermented Bark of Acanthopanax sessiliflorus and Its Isolated Compounds on Lipopolysaccharide-Treated RAW 264.7 Macrophage Cells.

The fermentation was carried out on the bark of Acanthopanax sessiliflorus (AS). Acanthopanax species have been used in traditional medicine as tonics, sedatives, and antispasmodics. An activity-guided isolation of the fermented bark of A. sessiliflorus (FAS) yielded several phytochemicals: acanthoside D (1), acanthoside B (2), daucosterol (3), protocatechuic acid (4), chlorogenic acid methyl ester (5), ciwujiatone (6), syringaresinol (7), farnesol (8), 3,4-dicaffeoylquinic acid (9), and falcarindiol (10). HPLC analysis showed that content of lignan glycoside (1) was decreased and 4 and 7 were increased after fermentation. Anti-inflammatory activities on FAS showed the decrease of nitric oxide (NO) production, and inhibitory activities of iNOS and COX-2, proinflammatory cytokines (IL-6 and tumor necrosis factor-α), and collagenase. The aglycone, syringaresinol (7), which was increased through fermentation showed enhanced activity than 1. Thus, FAS may have the potential to treat inflammatory disorders, such as arthritis.

Anti-Acne Vulgaris Effects of Pedunculagin from the Leaves of Quercus mongolica by Anti-Inflammatory Activity and 5α-Reductase Inhibition.

Quercus mongolica (QM)-a member of the Fagaceae family-has been used as traditional medicine in Korea, China and Mongolia as a treatment for inflammation of oral, genital or anal mucosa and for external inflammation of skin. To treat acne vulgaris (AV), we evaluated the inhibition of inflammatory cytokines (IL-6 and IL-8) of QM leaf extract (QML) and its main compound, pedunculagin (PD) in vitro and 5α-reductase inhibitory activity by western blotting. As results, QML and PD showed potent NO production inhibitory activity compared with the positive control (PC), NG-monomethyl-L-arginine (L-NMMA). QML and PD was also showed the decreases of IL-6 and IL-8 compared with the PC, EGCG and exhibited potent 5α-reductase type 1 inhibitory activities compared with the PC, dutasteride.

Anti-Inflammatory Effects of Alnus Sibirica Extract on In Vitro and In Vivo Models.

Alnus sibirica extracts (ASex) have long been used in Oriental medicine to treat various conditions. To provide a scientific basis for this application and the underlying mechanism, we investigated the anti-inflammatory effects of ASex in vitro and in vivo. The in vitro model was established using human dermal fibroblasts (HDFs) treated with inflammatory stimulants (lipopolysaccharide, tumor necrosis factor-alpha, interferon-gamma). Lactate dehydrogenase and reverse transcription-polymerase chain reaction showed that ASex inhibited the increased expression of acute-phase inflammatory cytokines. The in vivo model was established by inducing skin inflammation in NC/Nga mice via the repeated application of house dust mite (HDM) ointment to the ears and back of the mice for eight weeks. HDM application increased the severity of skin lesions, eosinophil/mast cell infiltration, and serum immunoglobulin E levels, which were all significantly decreased by ASex treatment, demonstrating the same degree of protection as hydrocortisone. Overall, ASex showed excellent anti-inflammatory effects both in vitro and in vivo, suggesting its potential as an excellent candidate drug to reduce skin inflammation.

Anti-acne vulgaris effect including skin barrier improvement and 5α-reductase inhibition by tellimagrandin I from Carpinus tschonoskii.

BACKGROUND: Carpinus tschonoskii (CT) has been previously studied for various activities in the improvement of skin diseases. In the present study, we examined the in vitro anti-acne vulgaris (AV) effect of CT leaves (CTL) and tellimagrandin I (TI), one of the main ellagitannins from CT, including skin barrier improvement and 5α-reductase inhibitory activity. METHODS: To test the anti-AV activities of CTL and TI, firstly, anti-oxidative and anti-inflammatory activities including DPPH radical scavenging activity, nitric oxide (NO) inhibitory activity, and cytokines [interleukin (IL)-6 and IL-8] were tested. Skin barrier improvement experiments were tested using developing cornified envelope (CE) formation, and filaggrin mRNA expression level was determined by RT-PCR. The 5α-reductase inhibitory activity was determined by measuring the testosterone levels in rat liver microsomes. RESULTS: CTL and TI showed potent anti-oxidative activity and anti-inflammatory activities. Especially, the cytokine production inhibitory activities of TI were found to be similar to the positive control, epigallocatechin gallate (EGCG). CTL and TI enhanced the CE formation and filaggrin mRNA expression levels and showed potent activities compared to that in the positive control, 1.5 mM Ca2+. In additionally, CTL and TI showed 5α-reductase inhibitory activities in a dose-dependent manner. CONCLUSION: The results showed that CTL and TI inhibit AV endogenous factors such as 5α-reductase and inflammatory cytokines and affect exogenous factors such as developing skin barrier function (CE and filaggrin levels). Therefore, CTL and TI may be plant-derived agent, promising in the treatment of acne vulgaris.

Alnus Sibirica Extracts Suppress the Expression of Inflammatory Cytokines Induced by Lipopolysaccharides, Tumor Necrosis Factor-α, and Interferon-γ in Human Dermal Fibroblasts.

The effects of Alnus sibirica (AS) extracts on cytokine expression induced by inflammatory stimulants were examined in human dermal fibroblasts (HDFs) and RAW264.7 cells. The anti-oxidative effect and effect on cell viability of AS extracts were evaluated, and four extracts with the highest anti-oxidative effects were selected. HDFs and RAW264.7 cells were treated with inflammatory stimulants, and the expression of cytokines involved in acute (IL-6 and IL-10) and chronic (IL-18) inflammation, the initiation of the immune response (IL-33), and non-specific immune responses (IL-1ß, IL-8, and TNF-α) were determined using a reverse-transcription polymerase chain reaction. LPS increased the expression of all the cytokines, except for IL-18; however, AS extracts, particularly AS2 and AS4, reduced this increase, and TNF-α treatment markedly increased the expression of cytokines related to non-specific immune responses. IFN-γ treatment induced no significant changes, except for increased IL-33 expression in HDFs. AS extracts inhibited the increase in the expression of IL-33 and other cytokines in HDFs. Thus, the exposure of HDFs and RAW264.7 cells to inflammatory stimulants increased the expression of cytokines related to all the inflammatory processes. HDFs are involved not only in simple tissue regeneration but also in inflammatory reactions in the skin. AS2 and AS4 may offer effective therapy for related conditions.

Anti-Inflammatory Effects of Phenolic Compounds Isolated from Quercus Mongolica Fisch. ex Ledeb. on UVB-Irradiated Human Skin Cells.

Quercus mongolica Fisch. ex Ledeb. (QM) has been used as an oriental traditional medicine to relieve hemorrhoids, fever, and enteritis. We screened the inhibitory activities of the extracts and compounds (1-6) isolated from QM on the production of inflammatory cytokines and chemokines to evaluate their anti-inflammatory activities. Further, we evaluated the expression levels of cytokines, chemokines, and immune factors on pedunculagin (PC, 1), which was selected from isolated compounds (1-6) because of its potential anti-inflammation effect. Additionally, we evaluated whether the inflammation mitigation effects of PC (1) following UVB exposure in keratinocytes occurred because of nuclear factor (NF)-κB and signal transducer and activator of transcription (STAT)/Janus kinase (JAK) activation. PC (1) remarkably suppressed interleukin (IL)-6, IL-10, IL-13, and monocyte chemoattractant protein-1 (MCP-1) mRNA expression and reduced the mRNA expression level of Cyclooxygenase-2 (COX-2) and also reduced the phosphorylation of p38 mitogen-activated protein kinases (p38), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) in a concentration-dependent manner.

Inhibitory Effects on NO Production and DPPH Radicals and NBT Superoxide Activities of Diarylheptanoid Isolated from Enzymatically Hydrolyzed Ehthanolic Extract of Alnus sibirica.

Alnus sibirica (AS) is geographically distributed in Korea, Japan, Northeast China, and Russia. Various anti-oxidant, anti-inflammation, anti-atopic dermatitis and anti-cancer biological effects of AS have been reported. Enzymatic hydrolysis decomposes the sugar bond attached to glycoside into aglycone which, generally, has a superior biological activity, compared to glycoside. Enzymatic hydrolysis of the extract (EAS) from AS was processed and the isolated compounds were investigated-hirsutanonol (1), hirsutenone (2), rubranol (3), and muricarpon B (4). The structures of these compounds were elucidated, and the biological activities were assessed. The ability of EAS and the compounds (1-4) to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and Nitroblue tetrazolium (NBT) superoxide, and to inhibit NO production was evaluated in vitro. EAS showed more potent antioxidant and anti-inflammatory activity than AS. All investigated compounds showed excellent antioxidant and anti-inflammatory activities.

Anti-Obesity Activities of Chikusetsusaponin IVa and Dolichos lablab L. Seeds.

Obesity, a condition where excess body fat accumulates to the extent, causes a negative effect on health. Previously, we reported the extract of Dolichos lablab L. (DLL-Ex) inhibited high-fat diet (HFD)-induced increases in body weight and body fat mass and ameliorated increases in body weight. In the present work, we studyed the molecular mechanism for the inhibitory effect of DLL-Ex or Chikusetsusaponin IVa (CS-IVa), as isolated from Dolichos lablab L. (DLL) seeds extract, on adipocyte differentiation. We evaluated the effect of DLL-Ex, an anti-obesity agent, and CS-IVa, an active component of DLL-Ex, on 3T3-L1 cell differentiation via Oil red O assay and Q-PCR, along with their effects on CCAAT element binding protein alpha (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS), and fatty acid-binding protein 4 (FABP4) mRNA transcriptions. FAS and FABP4 protein expression levels after exposure to CS-IVa were also tested. The results showed that DLL-Ex and CS-IVa have potent inhibitory activity on adipocyte differentiation. Therefore, DLL and CS-IVa may be developed as a functional food material to treat obesity.

Inhibitory Activity of Allergic Contact Dermatitis and Atopic Dermatitis-Like Skin in BALB/c Mouse through Oral Administration of Fermented Barks of Alnus sibirica.

Phytochemical isolation of fermented Alnus sibirica (FAS) which was produced by using Lactobacillus plantarum subsp. argentoratensis, exhibited multiple and different composition compared with the original plant. Anti-allergic contact dermatitis (anti-ACD)/anti-atopic dermatitis (anti-AD) activities (visual observation and regulation of Th1/Th2 cytokines and IgE in blood) of FAS and the barks of Alnus sibirica extract (AS) and the two diarylheptanoids, hirsutenone (1) and muricarpon B (2), which are major components of FAS, were measured in vitro and in vivo. FAS, AS and the two compounds showed potent anti-oxidative, anti-inflammatory, anti-ACD and anti-AD activity. In particular, FAS showed more potent biological activity than AS. Thus, fermentation might be a prominent way to enhance the biological activity compared with the original plant. In addition, compounds (1) and (2) might be developed as functional materials or herbal medicines for ACD and AD.

Anti-Inflammatory and Anti-Urolithiasis Effects of Polyphenolic Compounds from Quercus gilva Blume.

Quercus gilva Bume (QGB, family Fagaceae) is a tall evergreen oak species tree that grows in warm temperate regions in Korea, Japan, China and Taiwan. Quercus plants have long been the basis of traditional medicines. Their clinical benefits according to traditional medicine include relief of urolithiasis, tremors and inflammation. In the present study, the anti-urolithiasis activity including anti-inflammatory and anti-oxidative activities, of some phenolic compounds isolated from QGB were described. Seven compounds were isolated and identified as picraquassioside D (1), quercussioside (2), (+)-lyoniresinol-9'α-O-ß-d-xylopyranoside (3), (+)-catechin (4), (-)-epicatechin (5), procyanidin B-3 (6), and procyanidin B-4 (7). Compounds 5-7 showed potent anti-oxidative and anti-inflammatory activities. These compounds were further tested for their inhibition of the gene expression of the inflammatory cytokines. The three compounds 5-7 showed dose-dependent inhibitory activities on gene expression of COX-2 and IL-1ß. In vivo, urolithiasis was induced more effectively in an animal model of acute urolithiasis by the administration of QGB extract. These results indicate the potential of compounds from QGB in the treatment of urolithiasis.

Antioxidative and Anti-Inflammatory Activities of Galloyl Derivatives and Antidiabetic Activities of Acer ginnala.

Chromatographic isolation of the 80% MeOH extract of Acer ginnala (AG) yielded seven galloyl derivatives: gallic acid (1), ginnalin B (2), acertannin (3), maplexin D (4), maplexin E (5), quercetin-3-O-(2''-galloyl)-α-L-rhamnopyranoside (6), and kaempferol-3-O-(2''-galloyl)-α-L-rhamnopyranoside (7). This is the first study to report the isolation of compounds 4 and 5 from AG. Galloyl derivatives 3-7 exhibited potent radical scavenging activities, with 5 and 7 showing particularly strong inhibitory activities against nitric oxide production in lipopolysaccharides- (LPS-) stimulated RAW264.7 cells. In addition, oral administration of AG extract (500 mg/kg b.w.) improved symptoms of hyperglycemia and blunted the increases in serum GOT/GPT levels in a rat model of streptozotocin-induced diabetes. These results suggest that galloyl derivatives (1-7) are antioxidant and anti-inflammatory agents and that AG extract has potential as a functional material or novel herbal medicine for treating diabetes mellitus.

Efficient methods for isolating five phytochemicals from Gentiana macrophylla using high-performance countercurrent chromatography.

Efficient high-performance countercurrent chromatography methods were developed to isolate five typical compounds from the extracts of Gentiana macrophylla. n-Butanol-soluble extract of G. macrophylla contained three hydrophilic iridoids, loganic acid (1), swertiamarin (2) and gentiopicroside (3), and a chromene derivative, macrophylloside D (4) which were successfully isolated by flow rate gradient (1.5 mL/min in 0-60 min, 5.0 mL/min in 60-120 min), and consecutive flow rate gradient HPCCC using n-butanol/0.1% aqueous trifluoroacetic acid (1:1, v/v, normal phase mode) system. The yields of 1-4 were 22, 16, 122, and 6 mg, respectively, with purities over 97% in a flow rate gradient high-performance countercurrent chromatography, and consecutive flow rate gradient high-performance countercurrent chromatography gave 1, 2, 3 (54, 41, 348 mg, respectively, purities over 97%) and 4 (13 mg, purity at 95%) from 750 mg of sample. The main compound in methylene chloride soluble extract, 2-methoxyanofinic acid, was successfully separated by n-hexane/ethyl acetate/methanol/water (4:6:4:6, v/v/v/v, flow-rate: 4 mL/min, reversed phase mode) condition. The structures of five isolates were elucidated by 1 H, 13 C NMR and ESI-Q-TOF-MS spectroscopic data which were compared with previously reported values.

Inhibitory Activities of Phenolic Compounds Isolated from Adina rubella Leaves Against 5α-Reductase Associated with Benign Prostatic Hypertrophy.

Adina rubella Hance (AR), a plant native to Korea, has been used as traditional medicine for dysentery, eczema, intoxication, and external hemorrhages. Previous phytochemical studies of AR have reported several components, including terpenoids, phenolics, and alkaloids. The current study evaluated the anti-oxidative and anti-inflammatory activities and 5α-reductase inhibition of isolated compounds of AR leaves to find a potential therapeutic agent for benign prostatic hypertrophy (BPH). Repeated chromatographic isolation of an 80% acetone extract of AR leaves yielded seven phenolic compounds: caffeic acid (1), chlorogenic acid (2), methyl chlorogenate (3), quercetin-3-rutinoside (4), kaempferol-3-O-α-l-rhamnopyranosyl-(1→6)-ß-d-glucopyranoside (5), hyperoside (6), and grandifloroside (7). Compound 7 is a novel compound in AR. Caffeoyl derivatives 1-3 and 7 showed good anti-oxidative activities. In particular, caffeic acid (1) and grandifloroside (7) showed potent anti-inflammatory activities, and 7 also exhibited potent inhibitory activity against TNF-α and 5α-reductase. Our results show that the extract and grandifloroside (7) from leaves of AR might be developed as a source of potent anti-oxidative and anti-inflammatory agents and therapeutic agent for BPH.

The Hot-Water Extract of Smilacis Chinae Rhizome Suppresses 2,4-Dinitrochlorobenzene and House Dust Mite-Induced Atopic Dermatitis-Like Skin Lesions in Mice.

Smilacis Chinae Rhizome (SCR) has been used as an oriental folk medicine for various biological activities. However, its effect on atopic dermatitis (AD) remains undetermined to date. We assessed the effect of orally administered hot-water extract of SCR on AD-like skin lesions in mice and its underlying mechanisms. AD-like murine model was prepared by repeated alternate application of house dust mite (Dermatophagoides farinae) extract (DFE) and 2,4-dinitrochlorobenzene (DNCB) for 4 weeks, topically to the ears. Daily oral administration of SCR for 3 and 4 weeks significantly reduced inflammatory ear thickening, with the effect being enhanced at the earlier start and longer period of administration. This effect was accompanied by a significant decrease in both Th2 and Th1 serum antibodies (total IgE, DFE-specific IgE, and IgG2a). Histological analysis showed that SCR markedly decreased the epidermal/dermal ear thickening and the dermal infiltration of inflammatory cells. Furthermore, SCR suppressed DFE/DNCB-induced expression of IL-4, IL-13, IL-17, IL-18, TSLP, and IFN-γ genes in the ear tissue. Taken together, our observations demonstrate that chronic oral administration of SCR exerts beneficial effect in mouse AD model, suggesting that SCR has the therapeutic potential as an orally active treatment of AD by modulating both Th1 and Th2 responses.

Two new phenolic compounds from the leaves of Alnus sibirica Fisch. ex Turcz.

Two new phenolic compounds, 4-O-glucopyranosyl-5-O-caffeoylshikimic acid (1) and 2,3-digalloyl oregonin (2), were isolated along with eight known phenolic compounds (3-10) from an 80% acetone extract of Alnus sibirica leaves. The chemical structures of these compounds were elucidated using 1D/2D nuclear magnetic resonance and high resolution-MS. The anti-oxidative activities of these compounds were determined by assaying their 1,1-diphenyl-2-picrylhydrazyl radical and nitroblue tetrazolium superoxide anion scavenging activity. All of the isolated phenolic compounds (1-10) exhibited potent anti-oxidative activities. In particular, 2 and 4, which are diarylheptanoids, and 10 which is ellagitannin exhibited excellent anti-oxidative activities with almost the same potency as that of the positive controls L-ascorbic acid and allopurinol.

The comparison of DPPH-scavenging capacity and anti-inflammatory effects of phenolic compounds isolated from the stems of Stewartia koreana Nakai.

The Stewartia koreana Nakai (SK) had been used in oriental traditional medicine as a remedy for acute gastroenteritis, liver diseases, quadriplegia and pain. The antioxidant activity guided isolation 80% methyl extract from stems of SK yielded eight phenolic compounds. We evaluated the anti-oxidative and anti-inflammatory effects of these compounds via assays of 1,1-diphenyl-2-picrylhydazyl (DPPH) radicals and inhibition of nitric oxide (NO) production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells. The results demonstrated that syringaresinol (6) exhibited significant DPPH radical-scavenging activity and inhibitory effects on NO production compared with its positive controls, ascorbic acid and L-NMMA, respectively.

Topical application of Rosa multiflora root extract improves atopic dermatitis-like skin lesions induced by mite antigen in NC/Nga mice.

The roots of Rosa multiflora THUNB. (RM) has been used in oriental traditional medicines as remedies for scabies, rheumatic arthralgia and stomatitis which were practicably related with today's inflammatory and allergic diseases. In the present study, we evaluated whether RM root extract (RME) and its major constituent, 2-(3,4-dihydroxyphenyl)-6-(4-hydroxyphenyl)-8-(2,4-dihydroxyphenyl)-2,3-trans-6,7-cis-7,8-trans-3,4,7,8-tetrahydro-2H,6H-pyrano[2,3-f] chromene-3,7,9-triol (RM-3) belongs to condensed tannins, improve atopic dermatitis (AD)-like skin lesions in NC/Nga mice induced by mite antigen. Topical application of RME as well as RM-3 improved skin severity and suppressed mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) on skin tissues, in addition, significantly reduced T helper 2 (Th2) immune responses via interleukin 10 (IL-10) up-regulation. Thus, RME, contains lots of condensed tannins such as RM-3 which possesses potent anti-inflammtory and immune-modulatory effects, may be useful for treatment of skin allergies and can be developed as new alternative herbal therapy against AD.

Anti-inflammatory activity of sulfate-containing phenolic compounds isolated from the leaves of Myrica rubra.

Three sulfated phenolic compounds, juglanin B (11R)-O-sulfate (1), myricetin 3´-O-sulfate (2), and ampelopsin 3´-O-sulfate (3), were isolated from the leaves of Myrica rubra. Compound 1 was a new sulfated lignan, 2 was a new sulfated flavone, and 3 was a known sulfated flavone. The structures of the new compounds (1 and 2) were determined by acid hydrolysis and spectroscopic methods, including IR, FAB-MS, 1D and 2D NMR. The inhibitory activities of compounds 1-3 and their hydrolysates (1a-3a) against LPS-induced cytokine (TNF-α, IL-1ß, and IL-6) production in macrophage RAW 264.7 cells were evaluated. The 2 new compounds (1 and 2) and their aglycones (1a and 2a) significantly reduced LPS-induced expression of iNOS and COX-2 proteins.