AIM OF THE STUDY: This study aimed to compare the differential toxicity and teratogenicity (if any) of the hot water (HW) and cold water (CW) extracts of both wild and cultivated sclerotium on zebrafish (Danio rerio) embryos.

MATERIALS AND METHODS: Zebrafish embryos were treated with varying concentrations of the sclerotial HW and CW extracts (0.3-500 μg/mL) for 72 h until hatching. The hatching, mortality and heartbeat rate of the embryos as well as the potential teratogenic effect of the extracts were assessed in embryos post-treatment with the extracts.

RESULTS: While the sclerotial HW extracts were nontoxic (LC50 > 500 μg/mL), the sclerotial CW extracts delayed the hatching of the embryos up to 48 h and showed slight toxicity with LC50 values of 398.4 μg/mL and 428.3 μg/mL for the cultivated and wild sclerotium, respectively. The sclerotial CW extracts also induced minor tachycardia in zebrafish larvae. Phenotypic assessment revealed that, while yolk sac edema was observed at high concentrations (300 and 500 μg/mL) of all extracts, curved trunk and bent tail were only observed in the embryos treated with CW extracts of wild sclerotium (300 and 500 μg/mL) but not for CW extracts of cultivated sclerotium at similar concentrations.

AIM OF THE STUDY: To determine the inhibitory properties of FD aqueous extract on pro-inflammatory mediators involved in lipopolysaccharide (LPS)-induced microglial cells.

METHODS: Vitexin and isovitexin in the extract were quantified via high performance liquid chromatography (HPLC). The extract was evaluated for its cytotoxicity activity via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Pre-treatment with the extract on LPS-induced microglial cells was done to determine its antioxidant and anti-neuroinflammatory properties by measuring the level of reactive oxygen species (ROS), nitric oxide (NO), tumour necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) via 2'-7'-dichlorofluorescin diacetate (DCFDA) assay, Griess assay and Western blot respectively.

RESULTS: The extract at all tested concentrations (0.1 μg/mL, 1 μg/mL, 10 μg/mL, 100 μg/mL) were not cytotoxic as the percentage viability of microglial cells were all above ~80%. At the highest concentration (100 μg/mL), the extract significantly reduced the formation of ROS, NO, TNF-α, IL-1β and IL-6 in microglial cells induced by LPS.

AIM OF THE STUDY: This study aimed to use a computational target fishing approach to predict the possible therapeutic effect of Marantodes pumilum and evaluated their effectivity.

MATERIALS AND METHODS: This study involves a computational approach to identify the potential targets by using target fishing. Several databases were used: PubChem database to obtain the chemical structure of interested compounds; Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) server and the SWISSADME web tool to identify and select the compounds having drug-likeness properties; PharmMapper was used to identify top ten target protein of the selected compounds and Online Mendelian Inheritance in Man (OMIM) was used to predict human genetic problems; the gene id of top-10 proteins was obtained from UniProtKB to be analyzed by using GeneMANIA server to check the genes' function and their co-expression; Gene Pathway established by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) of the selected targets were analyzed by using EnrichR server and confirmed by using DAVID (The Database for Annotation, Visualization and Integrated Discovery) version 6.8 and STRING database. All the interaction data was analyzed by Cytoscape version 3.7.2 software. The protein structure of most putative proteins was obtained from the RCSB protein data bank. Thedocking analysis was conducted using PyRx biological software v0.8 and illustrated by BIOVIA Discovery Studio Visualizer version 20.1.0. As a preliminary evaluation, a cell viability assay using Sulforhodamine B was conducted to evaluate the potential of the predicted therapeutic effect.

RESULTS: It was found that four studied compounds are highly correlated with three proteins: EFGR, CDK2, and ESR1. These proteins are highly associated with cancer pathways, especially breast cancer and prostate cancer. Qualitatively, cell proliferation assay conducted shown that the extract has IC50 of 88.69 μg/ml against MCF-7 and 66.51 μg/ml against MDA-MB-231.

AIM OF THE REVIEW: This review aims to compile the preclinical and clinical studies that had been done on EGCG to investigate its protective effect on cardiovascular and metabolic diseases in order to provide a systematic guidance for future research.

MATERIALS AND METHODS: Research papers related to EGCG were obtained from the major scientific databases, for example, Science direct, PubMed, NCBI, Springer and Google scholar, from 1995 to 2017.

RESULTS: EGCG was found to exhibit a wide range of therapeutic properties including anti-atherosclerosis, anti-cardiac hypertrophy, anti-myocardial infarction, anti-diabetes, anti-inflammatory and antioxidant. These therapeutic effects are mainly associated with the inhibition of LDL cholesterol (anti-atherosclerosis), inhibition of NF-κB (anti-cardiac hypertrophy), inhibition of MPO activity (anti-myocardial infarction), reduction in plasma glucose and glycated haemoglobin level (anti-diabetes), reduction of inflammatory markers (anti-inflammatory) and the inhibition of ROS generation (antioxidant).

AIM OF THE STUDY: To evaluate the immune stimulatory effects of F3 from S. crispus in NMU-induced rat mammary tumor model.

MATERIALS AND METHODS: Immunohistochemistry analysis of cellular immune parameters (CD4+ or CD8+ T cells, CIITA, MHC-II and CD68) was performed on NMU-induced rat mammary tumor nodules, followed by evaluation of the serum level of 34 cytokines using the cytokine antibody array.

RESULTS: Significant increase in MHC-II, CD4+ and CD8+ T cell and CIITA expression by tumor cells was observed in F3-treated rats compared to the tumor control group. F3-treated rats also displayed a significant decrease in the serum level of CCL2 and CD68+ infiltrating macrophages. Serum IFN-γ level in this group was increased by 1.7-fold suggesting enhanced infiltration of T cells, and upregulation of CIITA and MHC-II expression in the tumor cells might be triggered by F3-induced production of IFN-γ.

MATERIALS AND METHODS: Firstly, M. oleifera leaf were extracted in various solvents (aqueous, 50%, 70% and 100% ethanolic extracts) and standardized by reference standards using UHPLC technique. The extracts were then tested for cell migration and proliferation using HDF and HEK cell lines. M. oleifera leaf aqueous extract was then incorporated into alginate-pectin (SA-PC) based film dressing. The film dressings were characterized for the physicochemical properties and the bioactives release from the M. oleifera leaf extract loaded film dressing was also investigated using Franz diffusion cells.

RESULTS: All extracts were found to contain vicenin-2, chlorogenic acid, gallic acid, quercetin, kaempferol, rosmarinic acid and rutin. Among all M. oleifera extracts, aqueous standardized leaf extracts showed the highest human dermal fibroblast and human keratinocytes cells proliferation and migration properties. Among the film formulations, SA-PC (3% w/v) composite film dressing containing M. oleifera aqueous leaf extract was found to possess optimal physicochemical properties as wound dressing.

AIM OF THE STUDY: To evaluate kratom's effects towards hematological and clinical-chemistry parameters among regular kratom users in Malaysia.

METHODS: A total of 77 subjects (n=58 regular kratom users, and n=19 healthy controls) participated in this cross-sectional study. All the surveys were conducted through face-to-face interview to elicit subject's socio-demographic characteristics and kratom use history. A full-blood test was also administered. Laboratory analysis was conducted using GC-MS to determine mitragynine content in the acquired kratom samples in order to relate mitragynine consumption with possible alterations in the blood parameters of kratom users.

RESULTS: Findings showed that there were no significant differences in the hematological and clinical-chemistry parameters of traditional kratom users and healthy controls, except for HDL and LDL cholesterol values; these were found to be above the normal reference range for the former. Similarly, long-term kratom consumption (>5 years), and quantity of daily kratom use (≥3 ½ glasses; mitragynine content 76.3-114.8mg) did not appear to alter the hematological and biochemical parameters of kratom users.

AIM OF THE STUDY: Recent studies have demonstrated a potent anticancer potential of P. macrocarpa, especially against HeLa cell. The objective of this study was to investigate the regulation of miRNAs on MDA-MB-231 treated with P. macrocarpa ethyl acetate fraction (PMEAF).

MATERIALS AND METHODS: The regulation of miRNAs on MDA-MB-231 cells treated with PMEAF was studied through IIlumina, Hi-Seq. 2000 platform of Next Generation Sequencing (NGS) and various in silico bioinformatics tools.

RESULTS: The PMEAF treatment against MDA-MB-231 cells identified 10 upregulated and 10 downregulated miRNAs. A set of 606 target genes of 10 upregulated miRNAs and 517 target genes of 10 downregulated miRNAs were predicted based on computational and validated databases by using miRGate DB Query. Meanwhile, results from DAVID Bioinformatics Resources 6.8 specified the functional annotation of the upregulated miRNAs involvement in cancer pathway by suppressing the oncogenes and downregulating miRNAs by expressing the tumour suppressor genes in the regulation of apoptosis pathway.

MATERIALS AND METHODS: The inhibitory effects of hexane (LHXN), dichloromethane (LDCM), ethyl acetate (LEA) and methanol (LMEOH) extracts from leaves of PS on Aβ-induced production and mRNA expression of pro-inflammatory mediators in BV-2 microglial cells were assessed using colorimetric assay with Griess reagent, ELISA kit and real-time RT-PCR respectively. Subsequently, MTT reduction assay was used to evaluate the neuroprotective effects of PS leaf extracts against Aβ-induced microglia-mediated neurotoxicity in SH-SY5Y neuroblastoma cells. The levels of tau proteins phosphorylated at threonine 231 (pT231) and total tau proteins (T-tau) were determined using ELISA kits.

RESULTS: Polar extracts of PS leaves (LEA and LMEOH) reduced the Aβ-induced secretion of pro-inflammatory cytokines (IL-1β and TNF-α) in BV-2 cells by downregulating the mRNA expressions of pro-inflammatory cytokines. The inhibition of nitric oxide (NO) production could be due to the free radical scavenging activity of the extracts. In addition, conditioned media from Aβ-induced BV-2 cells pre-treated with LEA and LMEOH protected SH-SY5Y cells against microglia-mediated neurotoxicity. Further mechanistic study suggested that the neuroprotective effects were associated with the downregulation of phosphorylated tau proteins.

MATERIALS AND METHODS: International and Thai databases were searched from inception to February 2017. Clinical trials investigating effects of PM menopausal or postmenopausal women were included. Outcomes were self-reported menopausal symptoms, serum reproductive hormones, urino-genital tract function, and bone surrogates. Methodological quality was assessed by Cochrane risk-of-bias v2.0, and a 22-parameter quality score based on the CONSORT checklist for herbal medicines.

RESULTS: Eight studies (9 articles) used data from 309 menopausal patients. Five-studies demonstrated that PM was associated with climacteric scores reduced by ~50% compared to baseline. Other PM studies using limited numbers of placebo participants suggested improved vaginal and other urogenital tract symptoms. Bone alkaline phosphatase halved (suggesting lowered bone turnover). Variable serum reproductive hormone levels suggested menopausal status differed between studies. PM active ingredients and sources were not defined. Adverse event rates (mastodynia, vaginal spotting, dizziness) were similar in all groups (PM, conjugated equine estrogen, and placebos) but serum C-reactive protein doubled. These studies had design and reporting deficiencies, high risks of biases, and low quality scores.

AIM OF THE REVIEW: This review aims to provide a comprehensive report on the ethnomedicinal use, phytochemistry, pharmacological activities, molecular mechanisms, and nutritional values of C. nutans. The present review will open new avenues for further in-depth pharmacological studies of C. nutans for it to be developed as a potential nutraceutical and to improve the available products in the market.

MATERIAL AND METHODS: All the available information on C. nutans was collected using the key words "Clinacanthus nutans" and/or "ethnomedicine" and/or "phytochemicals" and/or "anticancer" and/or "anti-inflammatory" and/or "antiviral" through an electronic search of the following databases: PubMed, Web of Science, EMBASE, Cochrane Library, Clinical Trials.org, SciFinder Scholar, Scopus, and Google Scholar. In addition, unpublished materials, Ph.D. and M.Sc. dissertations, conference papers, and ethnobotanical textbooks were used. The Plant List (www.theplantlist.org) and International Plant Name Index databases were used to validate the scientific name of the plant.

RESULTS: The literature supported the ethnomedicinal uses of C. nutans as recorded in Thailand, Indonesia, and Malaysia for various purposes. Bioactivities experimentally proven for C. nutans include cytotoxic, anticancer, antiviral, anti-inflammatory, immunomodulatory, antidiabetic, antioxidant, antihyperlipidemic, antimicrobial, and chemotherapeutic (in aquaculture) activities. Most of these activities have so far only been investigated in chemical, cell-based, and animal assays. Various groups of phytochemicals including five sulfur-containing glycosides, eight chlorophyll derivatives, nine cerebrosides, and a monoacylmonogalactosyl glycerol are present in C. nutans. The presence of two glycerolipids, four sulfur-containing compounds, six known flavones, a flavanol, four flavonols, two phytosterols, one polypeptide, and various phenolics and fatty acids largely influences its diverse bioactivities. Numerous reports justify the ethnomedicinal use of C. nutans as an antiviral agent in treating herpes simplex virus and varicella-zoster virus infections and as part of a traditional anticancer anti-inflammatory concoction agent for various inflammatory diseases. C. nutans tea was reported to have a good percentage of carbohydrate, crude protein, minerals, essential amino acids, nonessential amino acids, and essential fatty acids. Acute, subacute, and subchronic toxicity studies demonstrated that oral administration of ethanol and methanol extracts of C. nutans to male Swiss albino mice and male Sprague-Dawley (SD) rats, respectively, did not lead to any toxicity or adverse effects on the animal behavior and organs when used in amounts as high as 2g/kg.

THE AIM OF THE REVIEW: The aim of this review is to summarize the current studies on ethnomedicinal practices, phytochemistry, pharmacological studies and a potential study of Acalypha indica in different locations around the world. This review updates related information regarding the potential therapeutic treatments and also discusses the toxicity issue of Acalypha indica.

MATERIALS AND METHODS: This review was performed through a systematic search related to Acalypha indica including the ethnomedicinal practices, phytochemistry and pharmacological studies around the world. The data was collected from online journals, magazines, and books, all of which were published in English, Malay and Indonesian. Search engine websites such as Google, Google Scholar, PubMed, Science Direct, Researchgate and other online collections were utilized in this review to obtain information.

RESULTS: The links between ethnomedicinal practices and scientific studies have been discussed with a fair justification. Several pharmacological properties exhibited certain potentials based on the obtained results that came from different related studies. Based on literature studies, Acalypha indica has the capability to serve as anthelmintic, anti-inflammation, anti-bacterial, anti-cancer, anti-diabetes, anti-hyperlipidemic, anti-obesity, anti-venom, hepatoprotective, hypoxia, and wound healing medicine. For the traditional practices, the authors also mentioned several benefits of consuming the raw plant and decoction.

AIMS OF STUDY: The aim of the present study is to evaluate the repeated dose toxicity of the standardized aqueous extract administered daily for 30 days through oral administration at its effective hypoglycemia doses.

MATERIALS AND METHODS: The seeds were dried, ground and extracted in deionized water. A HPLC-photodiode array method was developed and validated for the standardization of both the hypoglycemia agents, namely bruceine D and E in aqueous extract. Both normoglycemia and streptozotocin (STZ)-induced diabetic rats were fed orally with 15, 30 and 60mg/kg body weight of standardized aqueous extract. The blood glucose was measured at 0-8h. In repeated dose toxicity, similar doses were administered orally to rats for 30 days. At the end of 30 days, the blood was withdrawn and subjected to biochemical and haematology analysis while organs were harvested for histology analysis.

RESULTS: Oral administration of standardized aqueous extract exhibited a dose-response relationship in both the normoglycemia and STZ-induced diabetic rats. Daily oral administration of 15, 30 and 60mg/kg standardized aqueous extract for 30 days to rats did not show signs to toxicity in its biochemical, haematology and histology analysis.

AIM OF STUDY: Although anticancer activity has been reported for the plant, the goal of the study was designed to isolate and characterize the active metabolites from G. mangostana and measure their cytotoxic properties. In this research, the mechanism of antiproliferative/cytotoxic effects of the tested compounds was investigated.

MATERIALS AND METHODS: The CHCl3 fraction of the air-dried fruit hulls was repeatedly chromatographed on SiO2, RP18, Diaion HP-20, and polyamide columns to furnish fourteen compounds. The structures of these metabolites were proven by UV, IR, 1D, and 2D NMR measurements and HRESIMS. Additionally, the cytotoxic potential of all compounds was assessed against MCF-7, HCT-116, and HepG2 cell lines using SRB-U assay. Antiproliferative and cell cycle interference effects of potentially potent compounds were tested using DNA content flow cytometry. The mechanism of cell death induction was also studied using annexin-V/PI differential staining coupled with flow cytometry.

RESULTS: The CHCl3 soluble fraction afforded two new xanthones: mangostanaxanthones V (1) and VI (2), along with twelve known compounds: mangostanaxanthone IV (3), β-mangostin (4), garcinone E (5), α-mangostin (6), nor-mangostin (7), garcimangosone D (8), aromadendrin-8-C-β-D-glucopyranoside (9), 1,2,4,5-tetrahydroxybenzene (10), 2,4,3`-trihydroxybenzophenone-6-O-β-glucopyranoside (11), maclurin-6-O-β-D-glucopyranoside (rhodanthenone) (12), epicatechin (13), and 2,4,6,3`,5`-pentahydroxybenzophenone (14). Only compound 5 showed considerable antiproliferative/cytotoxic effects with IC50's ranging from 15.8 to 16.7µM. Compounds 3, 4, and 6 showed moderate to weak cytotoxic effects (IC50's ranged from 45.7 to 116.4µM). Using DNA content flow cytometry, it was found that only 5 induced significant cell cycle arrest at G0/G1-phase which is indicative of its antiproliferative properties. Additionally, by using annexin V-FITC/PI differential staining, 5 induced cells killing effect via the induction of apoptosis and necrosis in both HepG2 and HCT116 cells. Compound 3 produce necrosis and apoptosis only in HCT116 cells. On contrary, 6 induced apoptosis and necrosis in HepG2 cells and moderate necrosis in HCT116 cells.

METHODS: Using the PRISMA 2020 Protocol, a systematic search of the publications was undertaken from the MEDLINE, CENTRAL, Science Direct, PubMed, and Google Scholars for randomized control trials published through 31st January 2022 to determine the effectiveness of Salvadora persica-extract mouthwash relative to chlorhexidine gluconate as anti-plaque and anti-gingivitis properties.

AIM OF THE STUDY: To assess the effectiveness of miswak in maintaining periodontal health among adults.

MATERIALS AND METHODS: We searched for randomised controlled trials (RCTs) investigating the effect of miswak published in PubMed, EBSCOHOST (Dentistry & Oral Sciences), SCOPUS, and Cochrane Database for Systematic Review (CDSR) from inception to May 08, 2022. The primary outcomes of interest were changes in the periodontal health measured with plaque and gingivitis scores as well as subgingival bacteria load. The quality of evidence was assessed using the Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) approach while the estimates of effect were pooled using a random-effects model.

RESULTS: Ten eligible articles were identified, of which 9 could be analysed quantitatively. The remaining report was included as part of the qualitative analysis. The meta-analysis showed that miswak was comparable with the toothbrush in reducing the mean plaque score (p= 0.08, SMD: 0.39, and 95% CI: -0.05 to 0.83) and mean gingivitis score (p= 0.37, SMD: 0.13, and 95% CI: -0.16 to 0.43). Even higher certainty of evidence for the effect of miswak on mean plaque reduction on labial surface of anterior teeth. However, the adjunctive effect of miswak was significantly more superior for reducing plaque (p= 0.01, SMD: 0.68, and 95% CI: 0.14 to 1.22) and gingivitis score (p= 0.04, SMD: 0.66, and 95% CI: 0.03 to 1.29).

AIM OF THE STUDY: As allergy could be mediated by both IgE and IgG, we further evaluated the anti-allergy potential of CNAE in both in vitro model of IgG-induced macrophage activation and in vivo anaphylaxis models to further dissect the mechanism of action underlying the anti-allergic properties of CNAE.

MATERIAL & METHODS: The anti-allergy potential of CNAE was evaluated in in vivo anaphylaxis models of ovalbumin-challenged active systemic anaphylaxis (OVA-ASA) and IgE-challenged passive systemic anaphylaxis (PSA) using Sprague Dawley rats as well as IgG-challenged passive systemic anaphylaxis (IgG-PSA) using C57BL/6 mice. Meanwhile, in vitro model of IgG-induced macrophage activation model was performed using IC-21 macrophages. The release of soluble mediators from both IgE and IgG-mediated pathways were measured using enzyme-linked immunosorbent assay (ELISA). The signaling molecules targeted by CNAE were identified by performing Western blot.

RESULTS: IgG, platelet-activating factor (PAF) and IL-6 was suppressed by CNAE in OVA-ASA, but not IgE. In addition, CNAE significantly suppressed PAF and IL-6 in IgG-PSA but did not suppress histamine, IL-4 and leukotrienes C4 (LTC4) in IgE-PSA. CNAE also inhibited IL-6 and TNF-α by inhibiting the phosphorylation of ERK1/2 in the IgG-induced macrophage activation model.

AIM OF THE REVIEW: The present review aimed to comprehensively summarise the current researches on the traditional and scientific applications of the genus Pterocarpus with regard to the phytochemical content, in vivo and in vitro bioactivities, as well as clinical evidence that may be useful for future drug development.

MATERIALS AND METHODS: Information about the Pterocarpus genus were obtained from local classic herbal literature and electronic databases, such as PubMed, Scopus, and Google Scholar. The scientific name of the species and its synonyms were checked with the information of The Plant List. Additionally, clinical trial results were obtained from the Cochrane library.

RESULTS: Several phytochemical constituents of the plants, e.g., flavonoids, isoflavonoids, terpenoids, phenolic acids, and fatty acids have been reported. There are about 11 species of Pterocarpus that have been scientifically studied for their biological activities, including anti-inflammatory, anti-microbial, analgesic, and anti-hyperglycemic. Of which, the anti-hyperglycemic activity of the extracts and phytochemicals of P. indicus and P. marsupium is particularly remarkable, allowing them to be further studied under clinical trial.

AIM OF STUDY: This study aimed to examine the anti-tumor activities of L. rhinocerus TM02®, using two different sample preparations [cold water extract (CWE) and fraction] via various routes of administration (oral and intraperitoneal) on an MCF7-xenograft nude mouse model. This study also investigated the inhibitory effect of TM02® CWE and its fractions against COX-2 in vitro using LPS-induced RAW264.7 macrophages, on the basis of the relationship between COX-2 and metastasis, apoptosis resistance, as well as the proliferation of cancer cells.

MATERIALS AND METHODS: The first preparation, L. rhinocerus TM02® sclerotium powder (TSP) was dissolved in cold water to obtain the cold water extract (CWE). It was further fractionated based on its molecular weight to obtain the high (HMW), medium (MMW) and low (LMW) molecular weight fractions. The second preparation, known as the TM02® rhinoprolycan fraction (TRF), was obtained by combining the HMW and MMW fractions. TSP was given orally to mimic the daily consumption of a supplement; TRF was administered intraperitoneally to mimic typical tumorous cancer treatment with a rapid and more thorough absorption through the peritoneal cavity. Another experiment was conducted to examine changes in COX-2 activity in LPS-induced RAW264.7 macrophages after a 1-h pre-treatment with CWE, HMW, and MMW.

RESULTS: Our results revealed that intraperitoneal TRF-injection (90 μg/g BW) for 20 days reduced initial tumor volume by ∼64.3% (n = 5). The percentage of apoptotic cells was marginally higher in TRF-treated mice vs. control, suggesting that induction of apoptosis as one of the factors that led to tumor shrinkage. TSP (500 μg/g BW) oral treatment (n = 5) for 63 days (inclusive of pre-treatment prior to tumor inoculation) effectively inhibited tumor growth. Four of the five tumors totally regressed, demonstrating the effectiveness of TSP ingestion in suppressing tumor growth. Although no significant changes were found in mouse serum cytokines (TNF-α, IL-5, IL-6 and CCL2), some increasing and decreasing trends were observed. This may suggest the immunomodulatory potential of these treatments that can directly or indirectly affect tumor growth. Pre-treatment with CWE, HMW and MMW significantly reduced COX-2 activity in RAW264.7 macrophages upon 24 h LPS-stimulation, suggesting the potential of L. rhinocerus TM02® extract and fractions in regulating M1/M2 polarization.