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Objective: To perform testing for cytokines involved in dermal inflammatory reactions and to document and compare the effects of an oleander extract (OE), oleandrin, and oclacitinib on biomarkers relevant to allergic reactions. The effects of these compounds under inflamed culture conditions are of direct importance to the treatment of canine atopic dermatitis., Methods: Testing involved primary canine dermal fibroblasts and the canine DH82 macrophage cell line; both cell types are important for initiating, regulating, and resolving dermal allergic reactions via cytokine communication., Results: Under inflamed conditions, OE and oleandrin downregulated key cytokines secreted by canine dermal fibroblasts and the DH82 macrophage cell line; all of which are treatment targets in dermatitis. In the DH82 macrophage cultures, the most noteworthy reductions involved IL-6, IL-12/IL-23p40, interferon-γ, tumor necrosis factor-α, VEGF, and nerve growth factor-β. Oclacitinib triggered reductions of some cytokines involved in allergic reactions, including TGF-β1, IL-12/IL-23p40, and tumor necrosis factor-α; however, these reductions were less robust than the reductions triggered by OE and oleandrin and accompanied by increases in other cytokines involved in dermal inflammation, including IL-6, interferon-γ, and nerve growth factor-β. In cultures of primary dermal fibroblasts, OE and oleandrin reduced the levels of IL-8 and monocyte chemoattractant protein-1, whereas oclacitinib had little or no effect., Conclusions: Oleander extract and oleandrin directly modulate immune responses under inflamed conditions. Moreover, OE and oleandrin appear to provide a more beneficial overall cytokine regulation than oclacitinib under inflamed culture conditions., Clinical Relevance: These results suggest that OE and oleandrin are efficacious agents to treat canine atopic dermatitis. Future studies should evaluate the efficacy of these compounds in dogs affected by atopic dermatitis.

Mosquitoes, one of the deadliest animals on the planet, cause millions of fatalities each year by transmitting several human illnesses. Synthetic pesticides were previously used to prevent the spread of diseases by mosquitoes, which was effective in protecting humans but caused serious human health problems, environmental damage, and developed mosquito pesticide resistance. This research focuses on exploring new, more effective, safer, and environmentally friendly compounds to improve mosquito vector management. Phytochemicals are possible biological agents for controlling pests and many are target-specific, rapidly biodegradable, and eco-friendly. The potential of extracts of Lantana camara, Melia azedarach, Nerium oleander, Ricinus communis, and Withania somnifera against 3rd instar Culex pipiens (Common house mosquito) larvae was evaluated. Methanol extracts had more toxic effects against Cx. pipiens larvae (95-100%, 24 h post-treatment) than aqueous extracts (63-91%, 24 h post-treatment). The methanol extracts of Nerium oleander (LC 50  = 158.92 ppm) and Ricinus communis (LC 50  = 175.04 ppm) were very effective at killing mosquito larvae, 24 h after treatment. N. oleander (LC 50  = 373.29 ppm) showed high efficacy in aqueous plant extracts. Among the different extracts of the five plants screened, the methanol extract of R. communis recorded the highest ovicidal activity of 5% at 800 ppm concentration. Total developmental duration and growth index were highly affected by R. communis and M. azedarach methanol extracts. In field tests it was clear that plant extracts decreased mosquito larval density, especially when mixed with mosquito Bti briquette, with stability up to seven days for N. oleander. GC-MS results showed that the methanol extract had a higher number of chemical compounds, particularly with more terpene compounds. A high-performance liquid chromatography (HPLC) technique was used to detect the existence of non-volatile polyphenols and flavonoids. All five methanol extracts showed high concentrations of active ingredients such as gallic acid, chlorogenic acid (more than 100 μg/ml) and the rosmarinic acid was also found in all the five extracts in addition to 17 active polyphenols and flavonoids presented at moderate to low concentrations. Molecular modeling of 18 active ingredients detected by the HPLC were performed to the vicinity of one of the fatty acid binding proteins of lm-FABP (PDB code: 2FLJ). Rutin, Caffeic acid, coumaric acid and rosmarinic acid which presented densely in R. communis and N. oleander showed multiple and stable intermolecular hydrogen bonding and π-π stacking interactions. The inhibition ability of the fatty acid binding protein, FABP4, was evaluated with remarkable receptor inhibition evident, especially with R. communis and N. oleander having inhibitory concentrations of IC 50  = 0.425 and 0.599 µg/mL, respectively. The active phytochemical compounds in the plants suggest promising larvicidal and ovicidal activity, and have potential as a safe and effective alternative to synthetic insecticides., (© 2024. The Author(s).)

Cancer drug resistance remains a major obstacle in clinical oncology. As most anticancer drugs are of natural origin, we investigated the anticancer potential of a standardized cold-water leaf extract from Nerium oleander L., termed Breastin. The phytochemical characterization by nuclear magnetic resonance spectroscopy (NMR) and low- and high-resolution mass spectrometry revealed several monoglycosidic cardenolides as major constituents (adynerin, neritaloside, odoroside A, odoroside H, oleandrin, and vanderoside). Breastin inhibited the growth of 14 cell lines from hematopoietic tumors and 5 of 6 carcinomas. Remarkably, the cellular responsiveness of odoroside H and neritaloside was not correlated with all other classical drug resistance mechanisms, i.e., ATP-binding cassette transporters (ABCB1, ABCB5, ABCC1, ABCG2), oncogenes (EGFR, RAS), tumor suppressors (TP53, WT1), and others (GSTP1, HSP90, proliferation rate), in 59 tumor cell lines of the National Cancer Institute (NCI, USA), indicating that Breastin may indeed bypass drug resistance. COMPARE analyses with 153 anticancer agents in 74 tumor cell lines of the Oncotest panel revealed frequent correlations of Breastin with mitosis-inhibiting drugs. Using tubulin-GFP-transfected U2OS cells and confocal microscopy, it was found that the microtubule-disturbing effect of Breastin was comparable to that of the tubulin-depolymerizing drug paclitaxel. This result was verified by a tubulin polymerization assay in vitro and molecular docking in silico. Proteome profiling of 3171 proteins in the NCI panel revealed protein subsets whose expression significantly correlated with cellular responsiveness to odoroside H and neritaloside, indicating that protein expression profiles can be identified to predict the sensitivity or resistance of tumor cells to Breastin constituents. Breastin moderately inhibited breast cancer xenograft tumors in vivo. Remarkably, in contrast to what was observed with paclitaxel monotherapy, the combination of paclitaxel and Breastin prevented tumor relapse, indicating Breastin's potential for drug combination regimens.

This study investigates the antioxidant activities of lipid, protein, and carbohydrate extracts from the marine mollusk Perna canaliculus . Lipids were extracted using acetone, which was followed by protein extraction using the broad-spectrum enzyme Alcalase and then carbohydrate extraction using cetylpyridinium chloride. Eighty white BALB/c mice were divided into eight groups according to the administered extracts. Groups 1 and 5 were the control and toxin control groups, respectively. Groups 2, 3, and 4 were administered lipid, protein, and carbohydrate extracts, respectively. The other groups were administered P. canaliculus extracts as well as gentamicin and acetaminophen, known as ethanolic extracts, derived from Nerium oleander to induce oxidation stress. All groups showed significant improvements in body weight ( p < 0.05). The lipid extract group showed a significant decrease in low-density lipoprotein cholesterol ( p < 0.05) and a significant increase in high-density lipoprotein cholesterol ( p < 0.05). After the toxin injection, all groups treated with P. canaliculus extracts showed increased antioxidant effects on hepatocytes ( p < 0.05). The lipid extracts induced antioxidant effects to protect the kidney by increasing lipid peroxidation ( p < 0.05) and catalase activities ( p < 0.05). Also, protein extracts showed antioxidant effects by increasing glutathione and catalase levels significantly ( p < 0.005). In conclusion, P. canaliculus extracts, especially lipids and proteins, have potent antioxidant activities that protect vital organs from oxidation stress., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Ahmad A. Alghamdi et al.)

Introduction: The Nerium oleander plant contains cardenolides that may cause human poisoning when ingested. A long-standing belief holds that it is possible to be poisoned by eating hot dogs or other foods cooked on Nerium oleander branch skewers. Oleandrin levels in frankfurters cooked on fresh and dry Nerium oleander skewers were measured., Methods: Hot dogs were cooked separately on either dried or fresh oleander branch skewers using a disposable charcoal grill. The hot dogs were then frozen and transported to an analytical laboratory where oleandrin content was measured via liquid chromatography/mass spectroscopy (LC/MS)., Results: The oleandrin content of hot dogs cooked on dried and fresh skewers did not exceed 343 ng and 701 ng, respectively., Conclusion: Hot dogs cooked on Nerium oleander skewers contain a negligible amount of oleandrin with respect to that sufficient to cause human poisoning. Reports of poisonings occurring in this manner are most likely the result of an urban myth.

Nerium oleander L., commonly known as oleander, is a toxic shrub and also a medicinal plant. All parts of oleander are rich in cardiac glycosides that inhibits Na + /K + -ATPase and induce inotropic effect on the cardiomyocytes. Several pre-clinical and clinical reports indicate acute toxicity due to intentional, accidental and suicidal oleander consumption. Contrarily, oleander is used for the treatment of diverse ailments in traditional medicinal practices around the globe and several evidence-based pre-clinical studies indicated metabolic and immunological health benefits of polyphenol-rich oleander extracts. Thus, the current review aims to address this pharmaco-toxicological conundrum of oleander by addressing the possible role of gut microflora in the differential oleander toxicity. Additionally, a comprehensive account of ethnopharmacological usage, metabolic and immunological health benefits has been documented that supplement the conflicting arguments of pharmaco-toxicological properties of oleander. Finally, by addressing the gap of knowledge of ethnomedicinal, pharmacological and toxicological reports of oleander, the current review is expected to pave the way to address the differential pharmaco-toxicological effects of oleander., (Copyright © 2020. Published by Elsevier Masson SAS.)

Background: Along with swift economic evolution and continuous amelioration of lifestyle, people at present are paying more attention to health issues. Synthetic drugs will be compensated with other natural ones that belong to natural origin. Plants have always been considered as sources of several compounds that are used in many fields, especially human and animal health, starting from boosting immunity to the treatment of infectious diseases caused by some pathogenic microbes such as bacteria, fungi as well as viruses. This study aimed to incorporate some types of plants within the antimicrobial portfolio through the examination of different six plants which were Cichorium intybus, Cinnamomum camphora, Commiphora myrrha, Foeniculum vulgare, Nerium oleander, and Spartium junceum. As well, attempting to identify the active constituents of their extracts using GC-MS., Materials and Methods: All selected plants were analyzed to determine their phytochemical composition such as phenolics, alkaloids, flavonoids, terpenoids, and so on. The extraction step was done by sophisticated equipment called supercritical fluid extractor SFE through adjustment of specific conditions include temperature, time, flow rate and pressure to change the behavior of CO 2 . Testing the antimicrobial activity of each plant extract via agar well diffusion method through the formation of clear zones against a wide range of test microorganisms including both Gram-positive and Gram-negative bacteria as well as yeasts. Finally, attempting to primarily identify the constituents of each plant extract using GC-MS., Results and Discussion: The crude extract of F. vulgare showed the highest potency against C. albicans, E. faecalis and S. typhimurium, it contains some unique compounds such as squalene, eugenol and isoeugenol while, Extract of C. intybus showed a moderate activity especially against C. lipolytica and MRSA and it includes Vitamin A like compound which indicates antioxidant property., Conclusion: Conclusively, fennel gave a promising result as a good wide spectrum antimicrobial agent because it contains some compounds act as antimicrobial agents such as eugenol which was used as food preservatives in addition to squalene which acts as an antioxidant and antimycotic agent so, it will be useful especially while it was used in highly purified form excluding all undesirable subcomponents.

Shrubs represent the most affordable and accessible form of feed that livestock can rely on to acquire both essential and non-essential elements of life. In addition to their inherent toxins, they contain endogenous substances commonly referred to as 'antinutritive factors' (ANFs) that often interfere with the utilisation of nutrients. Their abundance may lead to severe clinical trauma. Hence, the objective of the study was to investigate the effects of different extraction techniques on Nerium oleander L. and animal feeds as well as to quantify oxalates. Organic (hexane, acetone and methanol) sequential and aqueous (infusion and decoction) extractions were explored. Qualitative and quantitative analyses were conducted to determine the presence of various phytochemicals and oxalate contents as putative ANFs, respectively. The results showed higher extraction yields of 22.6% and 43.1% in the decoction and infusion of N. oleander, respectively. The quantification methods were validated for linearity, accuracy and precision. Oxalate contents of 6.76 ± 0.245 (0.65%) mg/g and 5.74 ± 0.236 mg/g dry weight (0.55%) were obtained in N. oleander and feeds, respectively. This difference was statistically significant with p < 0.05. Percentage recoveries of 98.5 (percent relative standard deviation [% RSD] = 2.3), 85.7 (% RSD = 1.03) and 80.3 (% RSD = 1.22) at 76%, 95% and 112% fortifications were obtained, respectively. Relative standard deviation for precision was 0.99% and 1.13% at 0.33 mg and 0.39 mg fortifications, respectively, while reproducibility showed 2.21% RSD. Therefore, these methods can be used to provide a valuable basis for qualitative determination of ANFs, particularly in shrub foliage.

Background: Nerigoside (NG), a cardenolide isolated from a commonfolk medicine, Nerium oleander Linn. (Apocynaceae), has not been explored for its biological effects. To date, cardenolides have received considerable attention in pharmacology studies due to their direct effects of apoptosis-induction or growth-inhibitory against tumor in vitro and in vivo. Whether and how NG exerts anticancer effects against colorectal cancer remains to be elucidated., Purpose: The aim of this study was to investigate the anticancer effect of NG in human colorectal cancer cells., Methods: To test anticancer effect, we compared potency of NG in two colorectal cancer cell lines, HT29 and SW620 by WST-1 and colony proliferation assays. And we investigated mechanism of anticancer activities by analyzing players in apoptotic and ERK/GSK3β/β-catenin signaling pathways in HT29 and SW620 cells treated with NG., Results: In this study, we showed that NG markedly suppressed the cell viability and colony formation of colorectal cancer cells HT29 and SW620, with no significant toxic effect on non-cancer cells NCM460. Annexin V-FITC/PI and CFSE labeling results revealed that NG suppressed cell proliferation in low concentration, along with reducing expression of PCNA, while NG induced apoptosis in high concentration,. Meanwhile, NG significantly arrested cell migration by reversal of EMT and cell cycle on G2/M. Then, we found that the ERK and GSK3β/β-catenin signaling pathway were noticeably blocked in CRC cells after treatment with NG. According to western blot, NG upregulated the expression of p-GSK3β/GSK3β and decreased especially the expression of β-catenin in nuclear. In addition, Wnt signaling and its target genes were suppressed in response to NG. Then, the Ser9 phosphorylation of GSK3β can be reduced / raised by GÖ 6983 / LiCl, respectively. Thus, we further confirmed that the GSK3β/β-catenin axis is involved in NG-prevented cell proliferation., Conclusion: NG inhibited the growth of colorectal cancer cells by suppressing ERK/GSK3β/β-catenin signaling pathway. And the GSK3β/β-catenin axis is involved in preventing cell proliferation and migration by NG-treatment. These results suggest that NG may be used to treat colorectal cancer, with better outcome by combining with GSK3β inhibitor to block Wnt pathway., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Introduction: Weeds are an important factor in determining production in the agricultural sector. Currently, weeds have been managed by employing synthetic weedicides chemicals. These chemicals have a negative impact on the environment through accumulation in soil and water, in addition to their effect on biological diversity., Material and Methods: A study was undertaken to evaluate the activity of the aqueous extract of leaves from; Nerium (Nerium oleander L), olives (Olea europaea L) and castor (Ricinus communis L) on the control of Cyperus rotundus L. The study comprised of two components: three plant species, and the uses of three planta extracts concentration (5%, 7.5% and 10%) and their impact on germination and inhibition of seedling growth., Results: The results from using Nerium extract in comparison with Tibenuron and the two other plants extracts gave 90% inhibition at concentrations of 5% and 10%, while the concentration of 2.5% gave the lowest inhibition rates for germination of all plant extracts under study. The second experiment carried out using 10% concentration of the Nerium extract gave complete inhibition (100%), compared with the same concentrations of other plants extract and weedicides., Conclusions: The current study indicates the possibility of using natural products of plant origin as alternatives to chemical herbicides as one of the means of biological control to reducing environment pollution.

Many studies have been carried out in order to determine the toxicity of medicinal plants. The objective of this study was to compare and analyze the hepatic response against two doses of Nerium oleander, (N. oleander) “kaner” leaf decoction. Aqueous leaf decoction was injected intramuscularly into both hind limbs of male rats (200&#x2213;10g), assigned into three categories (n=4): control group with no treatment; group I, injected with 5 ml/ kg; and group II injected with 10 ml/ kg of leaf decoction, respectively. Animals were sacrificed 6 h after administration and hepato-histological changes were then observed. The decoction induced an acute phase reaction reflected by a more significant recruitment of inflammatory cells in group II than in group I and controls, as observed by histological studies. These results indicated that both doses can induce an acute-phase condition. Hence, traditional practice of medicinal plants without preliminary dose assessment must not be administered.

Background: Nerium oleander L. (syn. Nerium indicum Mill, Nerium odorum Aiton) belongs to the family Apocynaceae. It is used for its anti-inflammatory, anti-diabetic, anti-cancer and hepatoprotective activities in traditional medicine. Previous pharmacognostic studies suggested that 70 % hydro-methanolic extracts of oleander possess potent free radical scavenging and anti-inflammatory activities, both of which are helpful against hepatotoxicity., Methods: Hydro-methanolic extracts of oleander stem and root were evaluated for their hepatoprotective activities in acute CCl4 intoxicated mouse through in vitro and in vivo studies. Silymarin was used as positive reference. Antioxidant enzymes, pro-inflammatory markers and liver enzymatic and biochemical parameters were studied. The extracts were further chemically characterized using Fourier Transform Infrared (FTIR) spectroscopy and Gas chromatography-mass spectrometry (GC-MS)., Results: CCl4 toxicity caused fatty liver formation by increase of relative liver weight (32.53 g) compared to control group (16.08 g). The elevated liver enzymatic and biochemical parameters due to CCl4 toxicity were considerably normalized by the extracts treatment under both in vivo and in vitro models. Oleander stem (NOSE) and root (NORE) extracts increased the reduced hepatic catalase activity 27.37 and 25.25 %, whereas peroxidase activity was increased 18.19 and 22.78 %, respectively. The extent of lipid peroxidation was significantly (p < 0.01) lowered 20.76 % (NOSE) and 21.12 % (NORE) compared to CCl4 group. The levels of pro-inflammatory tumor necrosis factor-α (TNF-α) was lowered 71.33 % (NOSE) and 61.60 % (NORE). Histopathological study demonstrated substantial reduction of hepatocellular necrosis, fatty infiltration, sinusoidal dilation, bile duct proliferation, vascular congestion, leukocyte infiltration in the silymarin and extract treated groups. Furthermore, various bioactive compounds were identified in the extracts such as apocynin, tocopherol, squalene, vanillin, isoeugenol, amyrin, lupeol etc., Conclusion: The present study provided convincing evidence that oleander extracts possess potent hepatoprotective capacity which was primarily governed by its antioxidant and anti-inflammatory activities. The collegial bioactivities of the phytochemicals may be accredited behind the hepatoprotective activity of oleander.

Aim: The aim of this study was to analyze the ethyl acetate extract of Nerium indicum (NIE) flower for its antioxidant effect in anxious Sprague-Dawley rats., Materials and Methods: Animals were divided into six groups ( n = 6) and treated with 200 mg/kg and 400 mg/kg p.o. of NIE for 21 days to assess its preventive and curative effects. Anxiety was induced by isolating animals socially for 21 days. Elevated plus maze (EPM) and light and dark model were used for measuring anxiety in animals. Oxidative stress parameters such as lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) in blood and brain tissue homogenate were monitored after 21 days of social isolation in animals., Results: Rats were treated with NIE 200 mg/kg and 400 mg/kg p.o. Both the treatments showed a significant ( P < 0.001) increase in the number of open arm entries and time spent in open arm in EPM when compared with the negative control. Results also demonstrated that there was a significant ( P < 0.001) increase in the number of lightbox entries and time spent in light box in light and dark model when compared with negative control. There was a significant ( P < 0.001) improvement in endogenous anti-oxidants such as SOD, CAT, reduced GSH, and decreased levels of LPO in blood and brain tissue when compared with the negative control., Conclusion: The present study suggests the role of NIE in the treatment of anxiety, possibly by modulating the oxidative stress.

We previously reported neuroprotective activity of the botanical anti-cancer drug candidate PBI-05204, a supercritical CO2 extract of Nerium oleander, in brain slice and in vivo models of ischemic stroke. We showed that one component of this neuroprotective activity is mediated through its principal cardiac glycoside constituent, oleandrin, via induction of the potent neurotrophic factor brain-derived neurotrophic factor (BDNF). However, we also noted that the concentration-relation for PBI-05204 in the brain slice oxygen-glucose deprivation (OGD) model is considerably broader than that for oleandrin as a single agent. We thus surmised that PBI-05204 contains an additional neuroprotective component(s), distinct from oleandrin. We report here that neuroprotective activity is also provided by the triterpenoid constituents of PBI-05204, notably oleanolic acid. We demonstrate that a sub-fraction of PBI-05204 (Fraction 0-4) containing oleanolic and other triterpenoids, but without cardiac glycosides, induces the expression of cellular antioxidant gene transcription programs regulated through antioxidant transcriptional response elements (AREs). Finally, we show that Fraction 0-4 provides broad neuroprotection in organotypic brain slice models for neurodegeneration driven by amyloid precursor protein (APP) and tau implicated in Alzheimer's disease and frontotemporal dementias, respectively, in addition to ischemic injury modeled by OGD.

Objective: Nerium indicum Mill (syn. N. oleander L. and N. odorum Aiton; family: Apocynaceae) is a medicinal plant, used in the treatment of diverse ailments including various chronic inflammatory diseases in traditional medicine. We have previously demonstrated the immunomodulatory activity of a bioactive fraction of Nerium indicum leaf (NILE) by studying up-regulation of interleukin-2 (IL-2), IL-10, interferon-gamma and down regulation of IL-4, tumor necrosis factor-alpha (TNF-α), nitric oxide, cyclooxygenase-1 (COX-1) and COX-2 activities. Therefore, this study aimed to confirm the anti-inflammatory activity of NILE by inhibition of prostaglandin E₂ (PGE₂) activity in murine splenic lymphocytes in vitro., Materials and Methods: Murine lymphocytes were isolated from spleen and stimulated with 5 ΅g/mL concanavalin A in RPMI-1640, supplemented with 50 U/mL penicillin, 50 U/mL streptomycin, 50 U/mL nystatin and 10% fetal bovine serum. Different concentrations (0-80 μg/mL) of NILE were added and the cells were cultured for 48 h. The culture supernatants were thereafter collected by centrifugation and assayed for expression of PGE₂ level. The data were analyzed statistically., Results: The results demonstrated a 2.26-fold inhibition of PGE2 level at 80 μg/mL of NILE. Half maximum inhibitory concentration (IC₅₀) was calculated to be 44.95 ± 0.45 ΅g/mL. Linear correlation analysis of the dose-dependent PGE₂ inhibition with other pro- and anti-inflammatory mediators demonstrated high inter-correlation between the parameters., Conclusions: Thus, the present study remains in accordance with our previous report and confirms the anti-inflammatory claim of N. indicum, mentioned in the traditional medicine.

Objectives: Nerium oleander is traditionally used in various diseases because of its medicinal properties. One of its uses is in musculoskeletal disorder. The aim of the study was to evaluate the skeletal muscle relaxant activity of the aqueous extract of Nerium oleander flowers (AENOF) in albino rats in comparison with diazepam., Materials and Methods: A total of 20 Swiss albino rats aged 6-7 weeks, of either sex, weighing about 100-150 g, were taken, and after acute toxicity studies two different doses were selected. The animals were divided into four different groups. The first group was kept as the control (normal saline), second as the standard (diazepam) and the remaining two groups as Test I and Test II, and given different doses of the AENOF. Skeletal muscle relaxant activity (motor coordination) on Rotarod and locomotor activity on photoactometer was performed. Statistical analysis was carried out by using analysis of variance, followed by Dunnett's multiple comparison tests., Results: The result from the Actophotometer test and Rotarod test showed that the extract of AENOF significantly reduced (P < 0.05) the motor coordination of the tested animals., Conclusions: Our data indicates that AENOF possesses skeletal muscle relaxant activities.

CCl4 is a potent environmental toxin which cause liver damage through free radical mediated inflammatory processes. In this study, hepatoprotective capacity of Nerium indicum leaf extract (NILE) was evaluated on CCl4 induced acute hepatotoxicity in murine model. Animals were divided into 5 groups and treated as following: control group (received only normal saline), CCl4 group (received only CCl4), silymarin group (received CCl4 and 100mg/kg silymarin), NILE low group (received CCl4 and 50mg/kg NILE) and NILE high group (received CCl4 and 200mg/kg NILE). After 10 consecutive days of treatment, the levels of hepatic biochemical markers, malondialdehyde (MDA) content, peroxidase and catalase activities were measured as well as histopathological study was performed. Furthermore, liver explant cultures were set up as following: control (no treatment), CCl4 group (contained 25 μl/ml CCl4), silymarin group (contained 25 μl/ml CCl4 and 100 μg/ml silymarin), NILE low group (contained 25 μl/ml CCl4 and 25 μg/ml NILE) and NILE high group (contained 25 μl/ml CCl4 and 100 μg/ml NILE). Hepatic transaminases and phosphatases, tumor necrosis factor-α (TNF-α) expression, nitric oxide (NO) release and cell viability were studied on the explant cultures. Phytochemical fingerprinting of NILE was performed by gas chromatography-mass spectrometry (GC-MS). The results showed that the biochemical parameters were overexpressed due to CCl4 administration, which were significantly normalized by NILE treatment. The findings were further supported by histopathological evidences showing less hepatocellular necrosis, inflammation and fibrosis in NILE and silymarin treated groups, compared to CCl4 group. GC-MS analysis revealed presence of different bioactive phytochemicals with hepatoprotective and antioxidant properties. Therefore, the present study indicate that NILE possesses potent hepatoprotective capacity to ameliorate haloalkane xenobiotic induced injured liver in murine model., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Objectives: The current study aims to investigate the possible role of NO distillate either for therapeutic or for protective potential in diabetic cardiomyopathy., Background: Protective and restorative effects of distillated Nerium oleander (NO) on the diabetes-induced electrophysiological and structural alterations were investigated., Methods: Type 2 diabetes was induced by combination of single dose streptozotocin injection and high fat diet for four weeks. Experimental groups were designed as follows: control, diabetic, restorative-NO treated diabetic and protective-NO treated diabetic. Intracellular action potentials (AP) and contractile activities were measured form left ventricular papillary muscle strips as well as histopathological examination of heart tissue and biochemical examinations of serum were performed., Results: Type 2 diabetes induced AP prolongation was prevented with both ways of NO treatments. Moreover, treatments produced nearly complete restorations of diabetes-induced depressed amplitude and altered kinetics of contractile activities. In parallel to electrophysiological parameters, both histopathological and biochemical results indicates the NO induced beneficial effects on the diabetes related alterations., Conclusion: Distillated Nerium oleander (NO) can be a highly potential therapeutic or preventive agent on the diabetes induced excitation-contraction coupling alterations (Tab. 3, Fig. 3, Ref. 23).

Background: Nerium oleander has been widely studied for medicinal purposes for variety of maladies. N. oleander has also been reported having noxious effects because of its number of components that may show signs of toxicity by inhibiting plasma lemma Na+, K+-ATPase. The present study was performed to scrutinize the toxic effect of N. oleander leaves extract and its clinical and pathological features in wistar rats., Results: Hematological analysis showed significant variations in RBCs count (P = 0.01), Hb (P = 0.001), Hct (P = 0.0003), MCV (P = 0.013), lymphocyte count (P = 0.015), neutrophil count (P = 0.003), monocyte count (P = 0.012) and eosinophil count (P = 0.006). Histopathological studies have shown that in T1 group noticeable infiltration of inflammatory cells was found with low level of vascular damage. In T2 group, increased proportion of binucleated and inflammatory cells, hepatic necrosis, widening of sinusoidal spaces and mild level of vascular damage was observed., Conclusion: Taken together these findings we can conclude that N. oleander leaves extract significantly affects on experimental animals due to its toxicity. Efforts must be exerted to purify different chemical components from extract with no inflammation as this plant is utilized in folk medicine with narrow therapeutic indices.

Background: Oleander interferes with serum digoxin measurements using various immunoassays. The potential interference of oleander and its active ingredient, oleandrin, with a relatively new homogenous sequential chemiluminescent digoxin assay based on luminescent oxygen channeling technology (LOCI digoxin assay, Siemens Diagnostics) has not been previously reported., Methods: Aliquots of a digoxin-free serum pool were supplemented with increasing concentrations of oleandrin, or with oleander extract, followed by measuring the apparent digoxin concentrations using the LOCI digoxin assay using Vista 1500 analyzer. Mice were fed oleandrin or oleander extract, and their blood digoxin levels at 1 and 2 h were measured with the LOCI digoxin assay. In addition, two digoxin serum pools were prepared by combining sera of patients receiving digoxin; aliquots of both pools were supplemented with oleandrin or oleander extract and digoxin concentrations were again measured. Attempts to overcome this interference were made by measuring free digoxin concentration using a third digoxin pool., Results: Significant apparent digoxin concentrations were observed after supplementing aliquots of the drug-free serum pool with oleandrin or oleander extract. Mice fed with oleandrin or oleander extract also showed apparent digoxin levels 1 and 2 h after feeding. Digoxin values were also falsely lower or elevated (bidirectional interference) when aliquots of digoxin serum pools were further supplemented with oleandrin or oleander extract depending on concentration; this interference was not eliminated by free digoxin monitoring., Conclusions: Oleandrin interferes with LOCI digoxin assay., (© 2013 Wiley Periodicals, Inc.)

Objective: To investigate the molecular effects of Nerium oleander leaf distillate on paclitaxel and vincristine resistant (MCF-7/Pac and MCF-7/Vinc) cells and sensitive (MCF-7/S) cell lines., Methods: Nerium oleander (N. oleander) leaf extract was obtained by hydrodistillation method. The toxicological effects of N. oleander distillate, previously suggested as medicinal food supplement, on drug resistant cells were evaluated by XTT tests. MDR modulation potential of the plant material was evaluated by flow cytometry and fluorescent microscopy. Paclitaxel and vincristine were applied to the sublines in combination with N. oleander distillate., Results: Fractional inhibitory indices show that N. oleander distillate did not increase the antiproliferative effects of anticancer drugs. N. oleander treatment in to MCF-7/Pac and MCF-7/Vinc did not inhibit P-gp activity and MDR1 gene expression level., Conclusions: As a result it may be suggested that although N. oleander distillate has some medicinal effects as food supplement it may not be suitable as an MDR modulator for drug resistant breast cancer cells.

Because cardenolides specifically inhibit the Na(+)K(+)-ATPase, insects feeding on cardenolide-containing plants need to circumvent this toxic effect. Some insects such as the monarch butterfly rely on target site insensitivity, yet other cardenolide-adapted lepidopterans such as the oleander hawk-moth, Daphnis nerii, possess highly sensitive Na(+)K(+)-ATPases. Nevertheless, larvae of this species and the related Manduca sexta are insensitive to injected cardenolides. By radioactive-binding assays with nerve cords of both species, we demonstrate that the perineurium surrounding the nervous tissue functions as a diffusion barrier for a polar cardenolide (ouabain). By contrast, for non-polar cardenolides such as digoxin an active efflux carrier limits the access to the nerve cord. This barrier can be abolished by metabolic inhibitors and by verapamil, a specific inhibitor of P-glycoproteins (PGPs). This supports that a PGP-like transporter is involved in the active cardenolide-barrier of the perineurium. Tissue specific RT-PCR demonstrated expression of three PGP-like genes in hornworm nerve cords, and immunohistochemistry further corroborated PGP expression in the perineurium. Our results thus suggest that the lepidopteran perineurium serves as a diffusion barrier for polar cardenolides and provides an active barrier for non-polar cardenolides. This may explain the high in vivo resistance to cardenolides observed in some lepidopteran larvae, despite their highly sensitive Na(+)K(+)-ATPases.

To gain insight into the hepatohistological alterations in noninjured rat liver, Nerium oleander (N.O.) leaves extract was injected intramuscularly to induce an acute phase reaction (APR). Histopathological changes were studied after 3, 12, and 24 h time course of sterile muscle abscess. Tissue integrity and any infiltration of inflammatory cells in the liver were investigated by Hematoxylin and Eosin and ED1 peroxidase stainings. The administration of N.O. leaves extract (10 mL/kg) in H & E stained sections showed a general vacuolization of cytoplasm resulting loss of polarity with prominent nucleoli after 3 h of induction. At 12 h, eccentric nuclei were also observed in the sections. Marked infiltration of leucocytes with predominate macrophages was also found after 24 h as seen by ED1 positive staining. In the present study, a possible relationship between serum hepcidin and total iron level was also investigated in vivo. An early increase of hepcidin and total iron level (3 h) with a maximum at 12 h (P < 0.01; P < 0.001) was observed. These changes indicate that sterile muscle abscess may induce APR resulting in hepatic damage which is evident with the recruitment of inflammatory cells into the organ.

Indian system of medicine describes the usage of certain very toxic plant based drugs after performing a detoxification process (Shodhana samskara). Nerium indicum is traditionally used as a medicine though known to cause severe allergic symptoms, tachycardia and gastrointestinal effects leading to fatalities. In this study, the detoxification (shodhana) for Nerium indicum was scientifically validated based on phytochemical and toxicity profiles. Shodhana was performed according to traditional literature. HPTLC densitometric studies were performed for the pre- and post-shodhana powders followed by sub-acute toxicity evaluation in rats. Preparative TLC and LC-MS showed the reduction of oleandrin peak in the post-shodhana sample. Prominent features of cardiotoxicity including tachycardia were noted in the pre-shodhana Nerium treated animals along with mortality. However, no such toxicity was encountered in the post-shodhana Nerium treated animals. Hence, using the recommended detoxification (shodhana), the toxicity of an important medicinal plant was significantly nullified. Such studies provide a scientific support towards our traditional medicinal practices using modem analytical and experimental methodologies and may prove to be very useful in establishing standard scientific procedures for routine and safe use of traditional medicines.

Cardiac glycosides from fresh leaves of Nerium indicum were evaluated for its molluscicidal activity against Pomacea canaliculata (golden apple snail: GAS) under laboratory conditions. The results showed that LC(50) value of cardiac glycosides against GAS was time dependent and the LC(50) value at 96 h was as low as 3.71 mg/L, which was comparable with that of metaldehyde at 72 h (3.88 mg/L). These results indicate that cardiac glycosides could be an effective molluscicide against GAS. The toxicological mechanism of cardiac glucosides on GAS was also evaluated through changes of selected biochemical parameters, including cholinesterase (ChE) and esterase (EST) activities, glycogen and protein contents in hepatopancreas tissues of GAS. Exposure to sublethal concentrations of cardiac glycosides, GAS showed lower activities of EST isozyme in the later stages of the exposure period as well as drastically decreased glycogen content, although total protein content was not affected at the end of 24 and 48 h followed by a significant depletion at the end of 72 and 96 h. The initial increase followed by a decline of ChE activity was also observed during the experiment. These results suggest that cardiac glycosides seriously impair normal physiological metabolism, resulting in fatal alterations in major biochemical constituents of hepatopancreas tissues of P. canaliculata., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A new cardenolide diglycoside (1) was isolated from Nerium oleander together with ten known cardenolide diglycosides 2-11. The structure of compound 1 was established on the basis of their spectroscopic data. The in vitro anti-inflammatory activity of compounds 1-11 was examined on the basis of inhibitory activity against the induction of the intercellular adhesion molecule-1 (ICAM-1). Compounds 2-5 were active at an IC(50) value of less than 0.8 µM. The cytotoxicity of compounds 1-11 was evaluated against three human cell lines normal human fibroblast cells (WI-38), malignant tumor cells induced from WI-38 (VA-13), and human liver tumor cells (HepG2). Compound 3 was active toward VA-13 cells, and compounds 2-5 were active toward HepG2 cells at IC(50) values of less than 1.3 µM. The multidrug resistance (MDR)-reversal activity of compounds 1-11 was evaluated on the basis of the amount of calcein in MDR human ovarian cancer 2780AD cells in the presence of each compound. Compounds 1 and 8 showed moderate effects on calcein accumulation.

Oleander poisoning can be detected by digoxin immunoassays and for last two decades the fluorescence polarization immunoassay (FPIA) has been used for rapid detection of oleander poisoning in clinical laboratories. Recently, Abbott Laboratories (Abbott Park, IL) discontinued this assay. Therefore, we explored the possibility of using another digoxin assay (Dimension Vista Flex Reagent Cartridge, Tina Quant, EMIT 2000 and old FPIA assay for comparison) for rapid detection of oleander poisoning. When aliquots of drug-free serum pools were supplemented with pure oleandrin or oleander extract, we observed the highest apparent digoxin values using Dimension Vista digoxin assay (Flex Reagent Cartridge). We also observed significant apparent digoxin values in vivo in sera of mice both 1 and 2  hr after feeding with oleander extract. When a serum pool prepared from patients taking digoxin was further supplemented with various amounts of oleander extract, the highest falsely elevated digoxin values were observed with Dimension Vista digoxin assay. Monitoring free digoxin using Dimension Vista digoxin assay (Flex Reagent Cartridge) did not eliminate this interference. Digibind neutralized digoxin-like factors of oleander extract and such effect can be monitored by observing significant reduction in apparent free digoxin levels in the presence of Digibind as measured in the protein-free ultrafiltrate using Dimension Vista digoxin assay (Flex Reagent Cartridge)., (© 2011 Wiley-Liss, Inc.)

Two new cardenolide monoglycosides, cardenolides B-1 (1) and B-2 (2) were isolated from Nerium oleander, together with oleagenin (3) which is the first isolated compound from natural sources. The structure of compounds 1-3 were established on the basis of their spectroscopic data.

Ursolic acid (1) and kaempferol (3) are two major constituents of the Mediterranean plant Nerium oleander L. Microbial metabolism of (1) with Aspergillus flavus (ATCC 9170) resulted in the formation of 3-oxo-ursolic acid derivative, ursonic acid (2). On the other hand, Cunninghamella blakesleeana (ATCC 8688A) was able to convert (3) into kaempferol 3-O-beta-D-glucopyranoside (4) as well as the new natural product kaempferol 4'-sulfate (5). Incubation of kaempferol with Mucor ramannianus (ATCC 9628) led to the isolation of one metabolite identified as kaempferol 4'-O-alpha-L-rhamnopyranoside (6). Transformation of kaempferol to the new compound kaempferol 7-O-beta-D-4-O-methylglucopyranoside (7) and herbacetin 8-O-beta-D-glucopyranoside (8) was observed after fermentation with Beauveria bassiana (ATCC 13144). Cytotoxic as well as antioxidant activities of the isolated metabolites were determined.

This work proposes a viable remediation method based on the use of natural organic substances (NOSs) that characterize the Mediterranean region to improve the ecological system. A series of experiments, including variable conditions, such as temperature, pH, contact time, and pesticide concentration, were performed to demonstrate the efficiency of endosulfan sulfate removal from water by NOSs. Experimental results showed that the pH and temperature of pesticide solutions negatively affect the adsorption process. The maximum adsorption capacity for a specific initial concentration of endosulfan sulfate (0.5 microg/L) was achieved with Origanum compactum (75%), followed by Cistus ladaniferus and Raphanus raphanistrum (72 and 68%, respectively). The adsorption tests gave very satisfying results and point to the possible application of these supports as a remediation technique to prevent pesticide contamination of aquatic ecosystems.

Nerium indicum is an India-Pakistan-originated shrub belonging to the oleander family. The ingestion of leaves of N. indicum before a meal is known to effect the lowering of postprandial glucose levels in Type II diabetic patients and this plant is now used as a folk remedy for Type II diabetes in some regions of Pakistan. In the present study, the hot-water extract of N. indicum leaves was found to reduce the postprandial rise in the blood glucose when maltose or sucrose was loaded in rats. It was also found that the extract strongly inhibited alpha-glucosidase, suggesting that the suppression of the postprandial rise in the blood glucose is due to the occurrence of some inhibitors of alpha-glucosidase in the leaves. We, therefore, tried to isolate the active principles from the leaf extract, using alpha-glucosidase-inhibitory activity as the index. Employing Sephadex G-15, silica gel and reversed-phase HPLC, we isolated two active compounds. The UV, mass and NMR spectrometric analyses established that the chemical structures of these compounds are 3-O-caffeoylquinic acid (chlorogenic acid) and its structural isomer, 5-O-caffeoylquinic acid. Both compounds were shown to inhibit alpha-glucosidases in a non-competitive manner. The authentic chlorogenic acid was found to suppress the postprandial rise in the blood glucose in rats and also inhibited the absorption of the glucose moiety from maltose and glucose in the everted gut sac system prepared from rat intestine. These results demonstrate that chlorogenic acid is one of the major anti-hyperglycemic principles present in the leaves of N. indicum. Furthermore, among polyphenol compounds tested, quercetin and catechins were shown to have strong inhibitory activity against alpha-glucosidase.

A female, aged 43 and a male, aged 66, experienced gastrointestinal and cardiovascular symptoms after a meal including snail stew. Twelve hours after the ingestion, they presented with nausea, vomiting, diarrhea, and cardiovascular symptoms typical of acute toxic digoxin ingestion and were hospitalized. The man's electrocardiogram was altered, and the woman's was normal. Serum digoxin levels, measured on a Roche COBAS Integra 800 with the Roche On-Line Digoxin reagent, were 1.14 and 1.00 nmol/L, respectively. Potassium levels were normal in both patients. The serum digoxin concentration decreased on the second day, and symptoms resolved on the third day with patients fully recovered (i.e., reversion to a normal sinus rhythm). Cardiac-glycoside-like intoxication symptoms follow the ingestion of leaves or flowers of Nerium oleander. The consumed snails were suspected to be responsible for the intoxication. In the homogenized snail tissue, the concentration expressed in digoxin equivalents was 0.282 nmol/g. The presence of oleandrin and oleandrigenin in the snails was confirmed by liquid chromatography-tandem mass spectrometry analysis, which was performed on a ionic-trap Finnigan LXQ instrument using an electrospray ionization interface. High-pressure liquid chromatographic separation was performed on a C18 column with a gradient of methanol/water. An extract of oleander leaves was used as reference.

Italian stone pine (Pinus pinea L.) and oleander (Nerium oleander L.) leaves, bark and wood samples were collected at different sites around an industrial area (Huelva, SW Spain) and compared with samples of the same species from a background site. Samples were analysed with respect to the following pollutants: Al, Ba, Cr, Cu, Fe and Pb by ICP-AES. The suitability of different plant parts as biomonitors of pollution was investigated. In pine samples from the polluted sites the ratio of concentrations between bark and wood was high for Al, Ba, Cu and Fe, whereas no differences were found in samples from the unpolluted area. No differences were detected in oleander for the same ratio. In the oleander species, the ratio between leaves and wood concentration allowed to distinguish between control and polluted sites. The ratio of the concentration between leaves and wood was elevated for Al, Ba and Fe in pine samples from the polluted sites. The ratio of the concentration in bark or leaves to their concentration in wood might be useful to detect inorganic atmospheric pollutants.

Objective: To evaluate the molluscicidal activities of three Chinese plants N. indicum Mill, R stenoptera DC, and R. japonicum Houtt, and to clarify the molluscicidal mechanism., Methods: N-butanol extracts and water extracts of the three plants were obtained. The reactions of EST isozyme, glycogen and total protein of snails to the plant extracts were studied., Results: EST electrophoresis showed that EST was an important antidotal enzyme system and reacted strongly to environment. EST changed greatly during the whole exposure period so that it could be viewed as a pathological index of toxicity. Extracts decreased the glycogen content of the snails' soft tissues greatly, and also the protein content., Conclusion: All extracts show strong molluscicidal activity. The LD50 value of the water extract of N. indicum Mill is as low as 13.2 mg/L. EST can be viewed as a pathological index of toxicity. The energy metabolism abnormity is the key reason for the molluscicidal activities. The biochemical mechanism needs further research.

Nerium oleander L. (Oleander) leaves grown in Palermo city (Sicily, Italy) were collected from six sampling sites representing either areas of high traffic and urbanisation density or areas far away from traffic (e.g. city gardens). Concentration of Al, Ba, Cr, Cu, Fe, Pb, Mg, Mn, and Zn were determined in leaf samples during two years. Multivariate analysis classified the sampling sites in four groups based on the metal content in vegetal leaves in agreement with traffic and human activity site. Many elements studied (Al, Ba, Fe, Mn Mg) arise from the soil composition and others such as Cr, Cu, Pb and Zn as pollutant of the soil. On the other hand, about 30% of Al, Fe Cr, Cu and Pb originate from aerial deposition on leaves. Although the results presented should be handled with caution N. oleander can be considered as a means of assessing dust contamination in the urban environment.

Degeneration of neurons is a key problem in Alzheimer's disease (AD) and neuroprotection is a possible way to safeguard neurons from neurodegeneration. Polysaccharides isolated from Chinese medicinal herbs have been investigated extensively for their anti-tumor and immune stimulating effects. Yet, little is known about the effects of polysaccharides in neurons. Recently, two pure polysaccharides isolated from the flowers of Nerium indicum were shown to stimulate proliferation and differentiation of PC12 pheochromocytoma cells, an effect similar to that observed from nerve growth factor. In this notion, it is hypothesized that polysaccharides isolated from the flowers of N. indicum could exhibit beneficial effects in neurons. In this study, we isolated, characterized and investigated two new polysaccharides from the flowers of N. indicum for their neuroprotective effects on neurons against serum-deprivation and beta-amyloid (Abeta) peptide toxicity in primary rat cortical neuronal cultures. Pretreatment of the polysaccharides significantly reduced the number of apoptotic neurons revealed by DAPI staining when neurons were exposed to serum-free medium. Besides, the polysaccharides could also decrease the activity of caspase-3 triggered by Abeta peptides. Western blot analysis indicated that polysaccharides stimulated the phosphorylation of PDK-1 (Serine 241) and Akt (Threonine 308). In conclusion, the polysaccharides J2, J3 and J4 isolated from N. indicum provide a lead for future development of neuroprotective agent against neuronal death in neurodegenerative diseases and the neuroprotective mechanism may primarily rely on activation of Akt survival signaling pathway.

The diethyl ether, chloroform, acetone and methanol extract of Nerium indicum leaf were evaluated for their piscicidal activity against common freshwater air breathing predatory fish Channa punctatus. The rank of order of toxicity (LC50) of the leaf extract was, diethyl ether extract (17.34 mg/l)>acetone (40.01 mg/l)>chloroform (40.61 mg/l)>and methanol (106.37 mg/l). There was a significant negative correlation between LC50 values and exposure periods. Thus increase in exposure period, LC50 decreases from 17.34 mg/l (24 h) to >13.58 mg/l (96 h) in the diethyl ether extract. Similar trends were also observed in acetone, chloroform and methanol extracts. Exposure of sub-lethal doses (40% and 80% of LC50) of the diethyl ether extract of N. indicum leaf (which has maximum piscicidal activity) for 24 or 96 h caused significant alteration in the level of total protein, total free amino acid, nucleic acid, glycogen, pyruvate, lactate and activity of enzyme protease, phosphatases, alanine aminotransferase, aspartate aminotransferase and acetylcholinesterase in liver and muscle tissue. The alterations in all the above biochemical parameters were significantly (P<0.05) time and dose dependent. There was a significant recovery in all the above biochemical parameters, in both liver and muscle tissues of fish after the seventh day of the withdrawal of treatment. Thus, the leaf extracts of N. indicum have potent piscicidal activity against fish C. punctatus and also significantly affect both aerobic and anaerobic pathway of respiration in fish.

To clarify the pathway for biosynthesis of sinapyl alcohol in angiosperms, tracer experiments using stable isotopes were performed on robinia ( Robinia pseudoacacia L.), oleander ( Nerium indicum Mill.), magnolia ( Magnolia kobus DC.) and Arabidopsis thaliana (L.) Heynh. Precursors used in the experiment were (13)C- and (2)H ( D)-labeled [8-(13)C, 3-OCD(3)]ferulic acid and [8-(13)C, 3,5-OCD(3)]sinapic acid. The incorporation of labeled precursor into lignin was confirmed by gas chromatography-mass spectrometry of the products of derivatization followed by reductive cleavage. Crude extracts of differentiating xylem or stems from these plants were also assayed for 4-coumarate-CoA ligase (4CL; EC 6.2.1.12) activity using sinapic acid and ferulic acid as substrates. In robinia and oleander, 4CL activity toward sinapic acid was detected, and labeled sinapic acids were incorporated into syringyl lignin. These results indicate that robinia and oleander have a pathway that produces sinapyl alcohol from sinapic acid via sinapoyl-CoA. By contrast, in magnolia and Arabidopsis, 4CL activity toward sinapic acid could not be detected, and labeled sinapic acid was not incorporated into lignin. These results suggest that syringyl lignin biosynthesis in angiosperms operates via multiple pathways that depend on the species.