Oleracea Extract Research Articles

Targeting Cannabinoid Receptors and Fatty Acid Amide Hydrolase: An Innovative Food-Grade Delivery System of Zingiber officinale and Acmella oleracea Extracts as Natural Adjuvant in Pain Management

Background: Anti-inflammatory and analgesic properties are displayed by several natural products, in particular Acmella oleracea and Zingiber officinale. Literature data suggests that both botanical extracts can be useful as inflammation/pain modulators, so technological efforts have been made to associate them together in a unique delivery new food-grade formulation, Mitidol, with the purpose of improving their beneficial effects. Objective: The aim of the present work was to assess if Mitidol and its active ingredients are able to modulate the endocannabinoid 2 receptor system, one of the major systems responsible for positive effects of Cannabis treatment. Cannabinoid 2 receptor represents an attractive pharmacological target in obtaining an anti-inflammatory/analgesic effect with low central nervous system side-effects. Methods: The new food-grade Phytosome, together with all the botanical ingredients and active compounds, were tested in a cell-based assay in human recombinant Cannabinoid 2 Receptor cells, in order to evaluate a possible agonist effect on those receptors, and in a Fatty Acid Amide Hydrolase Inhibition Activity assay, to evaluate potential inhibition of that hydrolase, which is responsible for degradation of the endogenous cannabinoid anandamide. Results: Zingiber officinale showed potent activity in Fatty Acid Amide Hydrolase Inhibition Activity assay, being about six fold more effective than the flavonoid Kaempferol. Acmella oleracea proved to be highly active in Fatty Acid Amide Hydrolase inhibition and Cannabinoid 2 activation. Conclusion: Our data suggest a strong rationale for the use of Mitidol as a natural adjuvant in pain management.

Design and Evaluation of Vaginal Douche Formulation for the Ethanolic Extract of Cabbage (Brassica oleracea var. Capitata alba) as Anti-Flour Albus

The purpose of this present study was to determine the most suitable vaginal douche formulations of the Brassica oleracea var. capitata alba ethanolic extract as anti-flour albus. Determination of the basic vaginal douche formula was carried out using different thickener substances, as follows: carbopol 980, esaflor HM22 and SMC-20 viscolam. The concentration of acnibio Ac as a preservative with no antifungal activity against Candida albicans, was determined. The B. oleracea extract with different concentration (7 & 8.75%w/v) were formulated in the suitable basic formula. All formulas were stored at room temperature for 56 d and periodically evaluated for the stability of organoleptic characteristics, pH, and density. The effect of formulation on the active substance, existence was analyzed using thin layer chromatography (TLC). The antifungal effectivity of the formulas were evaluated using the agar diffusion method and its antifungal strength was compared to Povidone iodine solution. Among basis formula, the formula with SMC-20 viscolam provided the best base solubility. As a result of preservative concentration test, it was found that acnibio Ac at a concentration of 0.0125% w/v did not provide antifungal activity against C. albicans. The evaluation results showed that all vaginal douche formulas with varying extract concentrations were stable. Based on TLC results, the formulation did not showed the loss of active substance in the extract. The formula with cabbage extract concentration of 8.75% w/v revealed the best results and provided a good strength as an herbal preparation in compared to povidone iodine, because it has a small comparative value of 1: 0.2576. Thus, the cabbage leaf ethanolic extract concentration of 8.75% w/v can be recommended as reliable and effective active agents to be formulated as a vaginal douche using the studied formula.

Effect of pH and added sugar on stability of color, anthocyanin content and phenolic content of Clitoria ternatea, Ipomoea tricolor and Brassica oleracea extracts

Effect of pH and added sugar on stability of color, anthocyanin content and phenolic content of Clitoria ternatea, Ipomoea tricolor and Brassica oleracea extracts

Effect of pH and added sugar on stability of color, anthocyanin content and phenolic content of Clitoria ternatea, Ipomoea tricolor and Brassica oleracea extracts

Effect of pH and added sugar on stability of color, anthocyanin content and phenolic content of Clitoria ternatea, Ipomoea tricolor and Brassica oleracea extracts

Primena D-optimalnog dizajna eksperimenata i sveobuhvatne reološke analize u razvoju prirodnih anti-aging kremova

This work is focused on the development and evaluation of completely natural anti-aging creams, with the aid of the experimental design. Model formulations, differing in the content of emollients (10%/15%/20%) and emulsifier (1%/2%/3%) and containing the Acmella oleracea extract as a model anti-aging active were prepared by the cold process and evaluated regarding rheological behavior (con-tinuous rotational and oscillatory tests) and physical stability (dynamic-mechanical thermoanalysis-DMTA test). To study the influence of varied formulation parameters and their interaction on the critical rheological characteristics of the developed creams, a D-optimal design within the response surface method was applied. The data acquired from rheological characterization revealed favorized pseudoplastic (shear-thinning) flow behavior with the yield point, and dominating elastic behavior (storage modulus G' > loss modulus G") in both, amplitude and frequency sweeps, which together with a rather small and constant structural change obtained in the DMTA test indicated satisfying overall physical stability of formulated creams. The results of D-optimal design showed a significant individual, as well as a mutual effect of the tested formulation factors (emollient concentration, emulsifier concentration) affecting critical quality attributes (apparent viscosity, yield point, flow point, G', G", structural change) of developed anti-aging creams. Based on optimization results, the model formulation containing 20% of the emollient mixture and 2% of the emulsifier was selected as preferred in terms of required rheological properties and, thus, desired stability and quality, and it could be considered as a promising candidate worth exploring further for efficacy and in vivo skin performances.

Oxidative stabilization of corn oil with spinach extract

The present study was aimed to analyze the antioxidant activity of spinach (Spinacia oleracea) extract in oxidative stabilization of refined, bleached and de-odorized (RBD) corn oil (CO). Corn oil was supplemented with three different concentrations 600, 1200 and 1800 ppm of methanolic spinach extract and then stored at 25oC and 60oC. Different oxidation measuring parameters such as peroxide value (PV), free fatty acid (FFA) value, iodine value (IV), conjugated dienes (CD), conjugated trienes (CT) and para-anisidine value (PAV) were examined. The potency of spinach extract as natural antioxidant was found to be as effective as the synthetic antioxidants BHA and BHT used as standards and highest stabilization was observed in the order CO-1800>CO-1200>CO-BHT> CO-BHA>CO-600>CO-control. The results showed a significant effect of spinach extract in enhancing the oxidative stability of corn oil.
 Bangladesh J. Sci. Ind. Res.54(4), 375-382, 2019

Phytochemical Screening and Toxicological Study of Neptunia oleracea Lour. (Mimosaceae) Extracts, Plant Used in Traditional Medicine

Neptunia oleracea (Lourd.) Mimosaceae is a plant commonly used in traditional medicine for the treatment of several pathologies such as dysentery, jaundice, leucorrhoea, troubles of earache, among others.
 Aim: The purpose of this study was to carry out preliminary phytochemical screening, acute toxicity studies and to evaluate the effect of the aqueous and hydro-ethanolic extracts of N. oleracea on intestinal motility in vivo.
 Methodology: Preliminary qualitative phytochemical screening was conducted using standard procedures while acute toxicity studies was performed using OECD method. The effect of N. oleracea extracts on intestinal motility was evaluated using on normal and acetylcholine-induced transits.
 Results: Preliminary qualitative phytochemical screening of aqueous and hydro-ethanolic extracts of N. oleracea revealed the presence of similar constituents including steroids, triterpenoids, saponins, tannins, flavonoids, anthocyanidins, coumarins and carbohydrates. Alkaloids was absent in both the extracts. The oral median lethal dose (LD50) for both extracts was estimated to be 5000 mg/kg.
 The effect of extracts on intestinal peristalsis in mice showed that the aqueous and hydro-ethanolic extracts of N. oleracea stimulate normal intestinal transit by 1.29 and 8.54% respectively at the dose of 50 mg/kg body weight, thus there was inhibition at higher doses. These extracts potentiate acetylcholine-induced intestinal transit by 23.9 and 14.39% respectively at 500 mg/kg body weight.
 Conclusion: The findings of this study showed that the aqueous and hydro-ethanolic extracts of Neptunia oleracea contain bioactive constituents that have practically no toxic effect. This could justify the many forms of use of this plant in traditional medicine.

CosIng database analysis and experimental studies to promote Latin American plant biodiversity for cosmetic use

Although Latin America is one of the regions with the greatest plant diversity on the planet and, its commercial exploitation has not impacted the economic growth of Latin American countries, with the exception of Brazil. The great plant biodiversity of Latin American countries is an opportunity for the development of sustainable and innovative cosmetic ingredients and products. Some studies have focused on identifying the potential cosmetic use of Latin American plants. However, their commercial use is not possible due to their limited availability as raw materials, their safety has not been proven, and their lack of inclusion in international lists. This study analyzed plant species in the CosIng database for UV-filtering, UV-absorbing, bleaching, skin-protecting, and antioxidant functions and assayed the properties of potential plants by in vitro assays to promote the cosmetic use of biodiverse Latin American plants. The results showed that the majority of cosmetic ingredients listed in CosIng are from Asiatic plants, while 9 % are from Latin American plants. Approximately half of the registered plants in CosIng are commercialized as cosmetic ingredients. In addition, 28 % of the plants registered in CosIng occur in Colombia, but of these, only 30 % are native. The experimental analysis showed that the Bixa orellana, Theobroma cacao, and Eugenia stipitata extracts had the highest skin protective activity due to their capacity to inhibit collagenase, elastase, and hyaluronidase enzymes. B. orellana and Euterpe oleracea extracts showed the highest antioxidant capacity, phenol contents, and spectrophotometric sun protection factor (SPFspectrophotometric) values. B. orellana, E. oleracea, and E. stipitata extracts showed photoprotective properties in human dermal fibroblasts due to the reduction of cell death, matrix metalloproteinase-1 (MMP-1) production, and reactive oxygen spices (ROS) production and an increase in pro-collagen production after ultraviolet (UV) exposure. Finally, a list of Latin American plants with possible cosmetic applications is presented proving the great potential of Latin American plant biodiversity and its low commercial exploitation in the cosmetic industry. This study evidences the antioxidant and skin protecting actions of Latin American plants and it is presented as a tool for the identification of vegetable species of Latin American biodiversity with potential cosmetic use to sustainable and short-term commercial exploitation.

1-Carbomethoxy-β-Carboline, Derived from Portulaca oleracea L., Ameliorates LPS-Mediated Inflammatory Response Associated with MAPK Signaling and Nuclear Translocation of NF-κB.

Portulaca oleracea is as a medicinal plant known for its neuroprotective, hepatoprotective, antidiabetic, antioxidant, anticancer, antimicrobial, antiulcerogenic, and anti-inflammatory activities. However, the specific active compounds responsible for the individual pharmacological effects of P. oleracea extract (95% EtOH) remain unknown. Here, we hypothesized that alkaloids, the most abundant constituents in P. oleracea extract, are responsible for its anti-inflammatory activity. We investigated the phytochemical substituents (compounds 1–22) using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS) and screened their effects on NO production in lipopolysaccharide (LPS)-induced macrophages. Compound 20, 1-carbomethoxy-β-carboline, as an alkaloid structure, ameliorated nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), and proinflammatory cytokines associated with the mitogen-activated protein kinase (MAPK) pathways, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Subsequently, we observed that compound 20 suppressed nuclear translocation of nuclear factor κB (NF-κB) using immunocytochemistry. Moreover, we recently reported that compound 8, trans-N-feruloyl-3’, 7’-dimethoxytyramine, was originally purified from P. oleracea extracts. Our results suggest that 1-carbomethoxy-β-carboline, the most effective anti-inflammatory agent among alkaloids in the 95% EtOH extract of P. oleracea, was suppressing the MAPK pathway and nuclear translocation of NF-κB. Therefore, P. oleracea extracts and specifically 1-carbomethoxy-β-carboline may be novel therapeutic candidates for the treatment of inflammatory diseases associated with the activation of MAPKs and NF-κB.

Analgesic properties of a food grade lecithin delivery system of Zingiber officinale and Acmella oleracea standardized extracts in rats

This study investigated whether (i) the 5:1 combination of standardized extracts of Zingiber officinale and Acmella oleracea is endowed with analgesic effects and (ii) the phospholipid-based formulation of Zingiber officinale and Acmella oleracea extracts (ZAP) potentiated the analgesic effects of the plain extract combination (PEC). To this end, rats were exposed to acute pain (Tail Flick test) and chronic, inflammatory pain [Von Frey monofilament test and Randall–Selitto paw pressure test in rats treated intraplantarily with complete Freund's adjuvant (CFA)]. The plain combination of per se ineffective doses of the two extracts produced analgesic effects in healthy rats. ZAP was more potent and effective than the corresponding doses of PEC. ZAP also produced analgesic effects in CFA-treated rats. Studies are now warranted to assess whether the analgesic properties of ZAP may generalize to humans.

Anti-Asthmatic Effects of Portulaca Oleracea and its Constituents, a Review.

ObjectivesThe medicinal plants are believed to enhance the natural resistance of the body to infections. Some of the main constituents of the plant and derived materials such as, proteins, lectins and polysaccharides have anti-inflammatory effects. Portulaca oleracea (P. oleracea) were used traditionally for dietary, food additive, spice and various medicinal purposes. This review article is focus on the anti-asthmatic effects of P. oleracea and its constituents.MethodsVarious databases, such as the PubMed, Scopus, and Google Scholar, were searched the keywords including “Portulaca oleracea”, “Quercetin”, “Anti-inflammatory”, “Antioxidant”, “Cytokines”, “Smooth muscle “, and “ Relaxant effects “ until the end of Jul 2018.ResultsP. oleracea extracts and its constituents increased IFN-γ, IL-2, IFNγ/IL-4 and IL- 10/IL-4 ratio, but decreased secretion of TNF-α, IL-4 and chemokines in both in vitro and in vivo studies. P. oleracea extracts and quercetin also significantly decreased production of NO, stimulated β-adrenoceptor and/or blocking muscarinic receptors in tracheal smooth muscles. Conclusion: P. oleracea extracts and quercetin showed relatively potent anti-asthmatic effects due to decreased production of NO, inflammatory cytokines and chemokines, reduced oxidant while enhanced antioxidant markers, and also showed potent relaxant effects on tracheal smooth muscles via stimulatory on β-adrenoceptor or/and blocking muscarinic receptors.

Comparing the effects of Portulaca oleracea seed hydro-alcoholic extract, valsartan, and vitamin E on hemodynamic changes, oxidative stress parameters and cardiac hypertrophy in thyrotoxic rats

The present study compared the effects of Portulaca oleracea (P. oleracea) seed hydro-alcoholic extract, valsartan, and vitamin E on hemodynamic changes, oxidative stress markers and cardiac hypertrophy in a model of thyrotoxicosis. The hyperthyroid state was induced by intraperitoneal injection of levothyroxine (100 µg/kg) for 4 weeks in male adult rats. After 2 weeks, vitamin E (20 mg/kg), valsartan (8 mg/kg), and P. oleracea seed extract (400 mg/kg) were administered in three groups of thyrotoxic rats. The control group was given a daily injection of normal saline. Systolic blood pressure and heart rate were measured on three occasions with tail cuff. At the end of the fourth week, the animals were scarified and serum samples and heart tissue were collected for biochemical and histological studies. The levothyroxine increased heart rate and systolic blood pressure. A lower heart rate and reduced systolic blood pressure were observed in groups receiving valsartan and P. oleracea extract. The heart weight/body weight ratio increased in groups treated with levothyroxine, but in a microscopic study, cardiomyocyte width was not different between the groups. Levothyroxine increased the level of malondyaldehide and NO metabolite but reduced the thiol concentration, superoxide dismutase, and catalase activities. However, treatment with vitamin E and P. oleracea extract increased the thiol concentration, superoxide dismutase and catalase activities while decreasing malondyaldehide level. In addition, treatment with P. oleracea extract and valsartan decreased NO metabolite level. Treatment with P. oleracea extract improved levothyroxine induced oxidative stress and hemodynamic changes. These effects may be for antioxidant components.

Effects of Portulaca Oleracea Extract on Acute Alcoholic Liver Injury of Rats.

The present study was envisaged to investigate the chemical constituents and the intervention effects of Portulaca oleracea extract (POE) on acute alcoholic liver injury of rats. The chemical composition of POE was detected by high performance liquid chromatography (HPLC). Sixty male Wistar rats were divided into 6 groups: Normal control (NC) group, acute alcoholic liver injury model group (ALI), low, medium and high dose of POE (25, 50, 100 mg/kg) groups and bifendate (BF, 3.75 mg/kg) group. Each group was given by intragastrical administration for 7 days. Alcoholic liver injury was induced in the experimental model by administering 50% ethanol at 8 mL/kg and repeated administration after 6 h, for a period of 7 days. The results showed that pretreatment with POE significantly reduced the ethanol-elevated serum level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and triglyceride (TG). The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in liver were enhanced followed by administration of POE, while the content of nitric oxide (NO) and malondialdehyde (MDA) was found to decrease. Hepatic content of tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) was also reduced by POE treatment. These results indicated that POE could increase the antioxidant capacity and relieve the inflammatory injury of the liver cells induced by ethanol. Meanwhile, in our study, POE reduced the expression of miR-122, acetyl coenzyme A carboxylase (ACC) 1 mRNA and protein and increased the expression of lipoprotein lipase (LPL) mRNA and protein in liver, which indicated that POE could improve the lipid metabolism disorder induced by ethanol. Our findings suggested that POE had protective effects on acute alcoholic liver injury of rats.

Attenuating Effect of Portulaca oleracea Extract on Chronic Constriction Injury Induced Neuropathic Pain in Rats: An Evidence of Anti-oxidative and Anti-inflammatory Effects.

Neuropathic pain responds poorly to drug treatments. The present study investigated the therapeutic effect of Portulaca oleracea, in chronic constriction injury (CCI)-induced neuropathic pain in rats. Neuropathic pain was performed by putting four loose ligatures around the sciatic nerve. CCI resulted in the development of heat hyperalgesia, mechanical allodynia and cold allodynia accompanied by an increase in the contents of TNF-α, IL1β, malondialdehyde, with a reduction in total thiol content. Administration of Portulaca oleracea (100 and 200 mg/kg intraperitoneal) for 14 days in CCI rats significantly alleviated pain-related behaviors, oxidative damage and inflammatory cytokines in a dose-dependent manner. In conclusion, it is suggested that the antinociceptive effects of Portulaca oleracea might be due to antioxidant and anti-inflammatory properties.

Hypnotic Effect of Portulaca oleracea on Pentobarbital-Induced Sleep in Mice

In Iranian Traditional Medicine, the herbs with cold and wet temperament can help to improve insomnia. Portulaca oleracea has a cold and wet temperament, so the present study was carried out to investigate the sleep-prolonging effect of Portulaca oleracea. This work was an experimental study on mice which were randomly divided into these groups: saline (control); Diazepam: positive control); hydro-alcoholic extract of Portulaca oleracea (12.5, 25, 50, 75 and 100 mg/kg) by Soxhlet apparatus and maceration; in the effective (dose25 mg/kg), different fractions of extract were tested. Ethyl acetate fraction (EAF:); N hexane fraction (n-HF); water fraction (WF). All the test compounds were injected intraperitoneally (IP) 30 minutes before pentobarbital administration (30 mg/kg). Duration and latency of pentobarbital-induced sleep were recorded. Also, LD50 of Portulaca oleracea extract was determined and the possible neurotoxicity of the extract was tested on neural PC12 cells. Besides, 30 min after administration of hydroalcoholic extract (HAE) motor coordination (rota-rod test) was assessed. HAE increased the duration of pentobarbital-induced sleep at doses of 25, 50, 75 and 100 mg/kg. The hypnotic effect of HAE was comparable to that induced by diazepam. Similarly, WF, EAF, and NHF at 25 mg/kg could increase sleep duration. The sleep latency was decreased by HAE and NHF but not by WF and EAF. The LD50 value for HAE was found to be 4.8 g/Kg. HAE and its fractions did not show neurotoxic effect in cultured PC12-cell line, also HAE did not affect the animals performance on the rotarod test. The present data demonstrated that Portulaca oleracea potentiates sleeping behaviors. The main components responsible for the hypnotic effects of this plant is most likely a non-polar agents which is found in NHF. Isolation of the active constituents may yield a novel sedative drug.

Plant Growth Inhibitory Activity of the Extracts of Acmella oleracea and its Growth Inhibitory Substances

Aqueous methanol extracts of Acmella oleracea inhibited the growth of roots and shoots of cress ( Lepidum sativum), lettuce ( Lactuca sativa), Italian ryegrass ( Lolium multiflorum), and barnyard grass ( Echinochloa crus-galli) with the extract concentration-dependent manner. The extracts were then purified and 2 growth inhibitory substances were isolated and identified by spectral data as ( E,E)-2,4-undecadien-8,10-diynoic acid isobutylamide (compound 1) and nona-(2 Z)-en-6,8-diynoic acid 2-phenylethylamide (compound 2). Compound 1 inhibited the growth of cress roots and shoots at concentrations greater than 0.3 mM and that of barnyard grass root at concentrations greater than 0.03 mM. Compound 2 inhibited the growth of cress shoots and barnyard grass roots at concentrations greater than 1 mM. The inhibition on the seedling growth indicates that both compounds had growth inhibitory activity and may contribute to the inhibitory effect of A. oleracea extracts. This is the first report of having growth inhibitory activity of compounds 1 and 2 on plants.

Innovative pH sensors developed from ultrafine fibers containing açaí (Euterpe oleracea) extract

This work describes the development of ultrafine fibers with açaí (Euterpe oleracea Mart.) extract (AE) for use as pH sensors with potential applications in the food industry. The fibers were produced by electrospinning with polymeric solutions composed of 7% (w v−1) polycaprolactone, 2% (w v−1) polyethylene oxide and 3% (w v−1) AE solubilized in chloroform and methanol (3:1). The mean diameter of the fibers was 1635 ± 277 nm, with hydrophilic characteristics (contact angle < 90°), a melting point of 58 °C and a maximum degradation temperature of 408 °C. The total color difference (ΔΕ) of the colorimetric response was greater than 5, corresponding to the human ability for color differentiation. This new material can be used as a pH sensor for foods such as pork and fish to ensure quality and safety for the consumer, who can visually check the condition of the products.

Red cabbage and broccoli (Brassica oleracea) extracts inhibits cell proliferation by inducing apoptosis in colorectal cell lines

Red cabbage and broccoli (Brassica oleracea) extracts inhibits cell proliferation by inducing apoptosis in colorectal cell lines

Effects of the Portulaca oleracea Extract on Gentamicin-Induced Nephrotoxicity in Male Rats

Background: Some data suggest the protective effect of Portulaca oleracea against renal failure and its association with the preservation of the renal antioxidant status and the regulation of apoptosis. Objectives: The current study aimed to examine the renoprotective effects of treatment with Portulaca oleracea (PO), a prominent hydrogen sulfide donor, in a 5/6 nephrectomy animal model. Methods: In this experimental study, 32 adult male Wistar rats, initially weighting 200 - 250 g, were housed under standard conditions since the beginning of April 2017 until the end of January 2018. The effect of PO extract on renal dysfunction induced by gentamicin was studied. The male rats were treated with two selected doses of PO (i.e., 200 and 600 mg/kg p.o.) and normal saline (5 mL/kg p.o.) for 28 consecutive days. Gentamicin (80 mg/kg/day intraperitoneally [i.p.]) was administered to two groups for seven days, and they were considered as the PO200 and PO600 gentamicin groups, respectively, while the group administered normal saline (5 mL/kg p.o.) for 35 consecutive days was considered as the control group. The rats were anesthetized on day 36. Then, they were sacrificed under deep anesthesia, and plasma and tissue samples were obtained. Results: Treatment with PO decreased the renal histological damages and apoptosis induced by gentamicin and enhanced renal function parameters compared to the gentamicin group. Conclusions: The present findings provide strong evidence to support the traditional medicinal use of this herb by the tribal people in the treatment of renal impairment. Finally, these results support the therapeutic effect of PO in preventing the development of renal dysfunction.

Ameliorating effect of Brassica oleracea (broccoli) extract on the myocardial damage induced by polycystic ovary syndrome in the adult female albino rat

Abstract Polycystic ovary syndrome (PCOS) is one of the most common and complex hormonal disorders affecting the females in the childbearing period. It is associated with several comorbidities recognized as cardiovascular risk factors.