Evaluation of the toxicology, anti-lipase, and antioxidant effects of Callistemon citrinus in rats fed with a high fat-fructose diet.

CONTEXT: Callistemon citrinus Skeels (Myrtaceae) exhibits many biological activities. OBJECTIVE: This study analyzes for the first time, the toxicity, obesogenic, and antioxidant effects of C. citrinus in rats fed with a high fat-fructose diet (HFFD). MATERIALS AND METHODS: Four studies using male Wistar rats were conducted: (a) 7 groups (n = 3): control (corn oil) and ethanol extract of C. citrinus leaf (single oral dose at 100-4000 mg/kg) for acute toxicity; (b) 2 groups (n = 8): control (corn oil) and C. citrinus (1000 mg/kg/day) for 28 days for subacute toxicity; (c) 3 groups (n = 4) with single oral dose of lipid emulsion: control (lipid emulsion), C. citrinus and orlistat (250 and 50 mg/kg, respectively) for lipid absorption; (d) 4 groups (n = 6): control (normal diet) and 3 groups fed with HFFD: HFFD only, C. citrinus and simvastatin (oral dose 250 and 3 mg/kg, respectively) for 13 weeks. Antioxidant enzymes and biomarkers were evaluated and inhibition of pancreatic lipase was determined in vitro. RESULTS: Toxicological studies of C. citrinus showed no differences in biochemical parameters and lethal dose (LD50) was higher than 4000 mg/kg. C. citrinus inhibited pancreatic lipase activity, with IC50 of 392.00 µg/mL, and decreased lipid absorption by 70%. Additionally, it reduced the body weight 22%, restored the activities of antioxidant enzymes, and reduced the biomarkers of oxidative stress. CONCLUSIONS: Callistemon citrinus showed an effect against oxidative stress by reducing biomarkers and induced antioxidant system, without toxic effects.

Antibacterial and cytotoxicity activities and phytochemical analysis of three ornamental plants grown in Mexico.

This study evaluates the antibacterial, cytotoxic activities, and phytochemical composition, of Callistemon citrinus, Hibiscus rosa-sinensis and Plumbago auriculata leaves and flowers, three ornamental plants in Mexico. However, in other countries offers a range of other uses. Ethanol extracts of C. citrinus leaf and flower presented stronger antibacterial activity than the extracts obtained from the other two plants. C. citrinus leaf showed low cytotoxicity (LC50 <600 µg/mL) on the brine shrimp test, whereas the ethanol extracts of H. rosa-sinensis and P. auriculata leaves showed no cytotoxic activity. Flower extracts obtained from the three plants did no exhibit cytotoxicity. GC-MS analysis revealed that the ethanol extract of P. auriculata leaf contained lupeol triterpene and lupeol acetate, neither of them have been previously reported in this genus. Gamma sitosterol was present in the leaf and flower extracts of P. auriculata. Higher contents of linoleic and linolenic acids were found in extracts of H. rosa-sinensis leaves and flowers. The ability of the ethanol extracts of C. citrinus leaves and flowers to inhibit the growth of Gram-positive and Gram-negative bacteria indicates a potentially broad antimicrobial spectrum. Moreover, the absence of cytotoxicity suggests the potential use of this plant to treat microbial infections.

Use of Callistemon citrinus as a gastroprotective and anti-inflammatory agent on indomethacin-induced gastric ulcers in obese rats.

Background: Obesity leads to an elevated risk of developing gastrointestinal disease such as gastric ulcers. Callistemon citrinus leaf extract has shown antioxidant, antimicrobial, hepatoprotective, and chemoprotective effects against colon cancer. The aim of this study is to evaluate the gastroprotective effect of C. citrinus leaf extract on indomethacin-induced gastric ulcers in obese rats. Methods: Gastric ulcers were induced in female obese Wistar rats using a single oral dose of indomethacin (IND). In the first stage, the rats were fed with a high fat sugar diet (HFSD) for 15 weeks to induce obesity and, at the same time, the diet of the other group of animals included daily administration of ethanolic C. citrinus leaf extract (250 mg/kg) in addition to HFSD. In the second stage, gastric ulcers were induced with IND (30 mg/kg). The gastroprotective activity of C. citrinus, the inflammatory enzyme activities, and cytokines in the stomach were determined. Results: C. citrinus produced a reduction of gastric lesions caused by IND. Myeloperoxidase (MPO), cyclooxygenase-2 (COX-2), and 5-lipoxygenase (5-LOX) activities also decreased. Although inflammatory biomarkers such as TNFα, IL-6, AOPP, and leptin were significantly decreased by C. citrinus, adiponectin levels increased. Moreover, C. citrinus decreased weight gain and morphological and biochemical parameters. Conclusion: The use of indomethacin in rats fed with a high fat-sugar diet increased gastric ulcers. Gastroprotective effect of C. citrinus in obese rats is attributed to the reduction of pro-inflammatory cytokines and the inflammatory enzymes.

Development and Evaluation of Phytosomes Containing Callistemon citrinus Leaf Extract: A Preclinical Approach for the Treatment of Obesity in a Rodent Model.

Callistemon citrinus has several biological effects; it is anti-inflammatory, anti-obesogenic, antioxidant, hepatoprotection, and chemoprotective. Its bioactive compounds include terpenoids, phenolic acids, and flavonoids which have low oral bioavailability and absorption. This study aimed at developing phytosomes of C. citrinus to improve oral bioavailability and absorption. Phytosomes were formulated with soybean phosphatidylcholine and C. citrinus leaf extract using the thin layer sonication method. Phytosomes were evaluated by scanning electron microscopy (SEM), entrapment efficiency, solubility, and particle size determination. Antioxidant capacity and total phenolic, flavonoid, and terpenoid contents were also measured. The in vivo anti-obesogenic activity was evaluated. Phytosomes loaded with C. citrinus (P C.c) extract had small spherical shapes. The average particle size was 129.98 ± 18.30 nm, encapsulation efficiency 80.49 ± 0.07%, and solubility 90.00%; the stability study presented no significant changes in the average particle size at 20 °C. P C.c presented high antioxidant capacity. For the first time, ellagic acid is reported in this plant. The in vivo obesity study showed a strong anti-obesogenic activity of phytosomes with C. citrinus to reduce 40% body weight as well as morphometric and biochemical parameters.

Role of the major terpenes of Callistemon citrinus against the oxidative stress during a hypercaloric diet in rats.

1,8-Cineole, limonene and α-terpineol are the major terpenes present in Callistemon citrinus. This study reports for the first time that terpenes attenuate the oxidative stress in rats fed with high-fat-sucrose diet (HFSD) via antioxidant and anti-inflammatory mechanisms. Thirty-six male Wistar rats were divided into six groups (n = 6). Control (fed standard food, HFSD (fed with 41.7% fat and 16.6% sucrose), HFSD + 1,8-cineole (0.88 mg/kg body weight), limonene (0.43 mg/kg body weight), α-terpineol (0.32 mg/kg body weight) and a mixture of the three terpenes, given daily by gavage for 15 weeks. Morphometric and biochemical parameters were taken. Paraoxonase (PON1), reduced glutathione (GSH), lipid peroxidation products malondialdehyde (MDA) and hydroxyalkenals (HNE), advanced oxidation protein products (AOPP) and pro-inflammatory cytokines were measured in liver homogenates. All terpenes showed a remarkable reduction in weight gain, fat deposition, serum glucose and, triacylglycerol levels. However, terpenes presented different effects on the hepatic cell and the oxidative biomarkers. Conversely, the three terpenes and the mixture showed the same positive effect on the tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), leptin and adiponectin levels. Finally, 1,8-cineole, limonene and α-terpineol demonstrate significant anti-inflammatory effects and differential effects on the oxidative stress, suggesting the importance of these terpenes in Callistemon citrinus activities.

Protective effect of Callistemon citrinus on oxidative stress in rats with 1,2-dimethylhydrazine-induced colon cancer.

Callistemon citrinus has terpenes effective in inducing antioxidant enzymes, an important mechanism involved in cancer chemoprevention. This study investigated the chemopreventive efficacy of herbal preparation of C. citrinus leaves against the oxidative stress produced during the colorectal cancer (CRC) in male Wistar rats. The amelioration of toxicity in a model of CRC induced with 1,2-dimethylhydrazine (DMH) was determined by assessing antioxidant enzymes, phase II enzymes activities and lipid peroxidation (LPO) products after 22 weeks of treatment. C. citrinus was administered at a daily oral dose of 250 mg/kg. The activities in proximal, middle and distal colon, liver, kidney and heart were determined. C. citrinus showed a strong antioxidant activity that correlated with the high content of phenolics and terpenoids. DMH treated animals showed a decrease of the enzymes activity in most tissues and the level of reduced glutathione (GSH). Conversely, the levels of lipid peroxidation products were increased. Macroscopic examination revealed the protective effect of C. citrinus in damaged organs caused by DMH. Moreover, histopathological examination of the liver displayed normal structure in the C. citrinus-treated group, unlike the DMH-treated group. C. citrinus supplementation significantly maintained or increased the antioxidant enzyme activities, whereas lipid peroxidation products levels were reduced to values similar to the level of control group. The ability of C. citrinus to induce the antioxidant system reduced the damage of oxidative stress, which makes this plant a good candidate to be used as a prevention agent in treatment of diseases such as colorectal cancer.

Global gene expression analyses of the alkamide-producing plant Heliopsis longipes supports a polyketide synthase-mediated biosynthesis pathway.

BACKGROUND: Alkamides are plant-specific bioactive molecules. They are low molecular weight N-substituted α-unsaturated acyl amides that display biological explicit activities in different organisms from bacteria, fungi, insects to mammals and plants. The acyl chain has been proposed to be biosynthesized from a fatty acid; however, this has not been demonstrated yet. Heliopsis longipes (Asteraceae) accumulates in root a C10 alkamide called affinin in its roots, but not in leaves. The closely related species Heliopsis annua does not produce alkamides. To elucidate the biosynthetic pathway of the alkamides acyl chain, a comparative global gene expression analysis contrasting roots and leaves of both species was performed. METHODS: Transcriptomics analysis allowed to identify genes highly expressed in H. longipes roots, but not in tissues and species that do not accumulate alkamides. The first domain searched was the Ketosynthase (KS) domain. The phylogenetic analysis using sequences of the KS domain of FAS and PKS from different organisms, revealed that KS domains of the differentially expressed transcripts in H. longipes roots and the KS domain found in transcripts of Echinacea purpurea, another alkamides producer species, were grouped together with a high bootstrap value of 100%, sharing great similarity. Among the annotated transcripts, we found some coding for the enzymatic domains KS, AT, ACP, DH, OR and TE, which presented higher expression in H. longipes roots than in leaves. The expression level of these genes was further evaluated by qRT-PCR. All unigenes tested showed higher expression in H. longipes roots than in any the other samples. Based on this and considering that the acyl chain of affinin presents unsaturated bonds at even C numbers, we propose a new putative biosynthesis pathway mediated by a four modules polyketide synthase (PKS). RESULTS: The global gene expression analysis led to the selection of a set of candidate genes involved in the biosynthesis of the acyl chain of affinin, suggesting that it may be performed by a non-iterative, partially reductive, four module type I PKS complex (PKS alk) previously thought to be absent from the plant kingdom.

Chemopreventive effect of Callistemon citrinus (Curtis) Skeels against colon cancer induced by 1,2-dimethylhydrazine in rats.

PURPOSE: Callistemon citrinus (Curtis) Skeels is a shrub native of Australia. In spite of containing an important number of bioactive compounds (1,8-cineole, limonene and α-terpineol) recognized as a potential chemotherapeutic agents, it is only used as an ornamental plant in Mexico. This study investigated the chemopreventive effect of C. citrinus leaves extract on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats. METHODS: Twenty-four rats were divided into 3 groups of eight rats. Group 1 served as negative control, groups 2 and 3 were given subcutaneous injections of DMH (65 mg/kg b.w.) twice a week the first 2 weeks, and then one the third week. In addition, group 3 was administrated with leaves extracts (250 mg/kg b.w., orally daily) during the 22 weeks of the experiment. Animals were killed and the presence of colon tumors and aberrant crypt foci (ACF) were scored for number and distribution pattern along the colon. The activity of two-phase II enzymes quinone reductase (QR) and glutathione S-transferase (GST) was determined in the liver and three segments of the colon: proximal, middle and distal. RESULTS: The results show that rats feed with C. citrinus leaves extract significantly reduced the size of tumors, the number of ACF and the crypt multiplicity. Additionally, C. citrinus leaves extract increased or maintained the activity of QR and GST in the different tissues as compared with DHM-treated group (p > 0.05). CONCLUSION: This study demonstrates that Callistemon citrinus extract could have a chemopreventive effect against colon carcinogenesis.

Antioxidant Activity of the Essential Oil and its Major Terpenes of Satureja macrostema (Moc. and Sessé ex Benth.) Briq.

BACKGROUND: The aim of this study was to investigate the in vitro antioxidant activity of Satureja macrostema (Moc. and Sessé ex Benth.) Briq. (Lamiaceae) essential oil, a Mexican medicinal plant known as nurite. MATERIALS AND METHODS: Fresh aerial parts of S. macrostema plants cultivated in greenhouse for 3 months were subjected to hydrodistillation in a Clevenger apparatus to obtain essential oil. Volatile compounds were identified by gas chromatography (GC) and GC/mass spectrometry. Antioxidant effectiveness of essential oil and its major terpenes of S. macrostema was examined by three different radical scavenging methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and total antioxidant capacity (TAC). The concentrations tested were 0.001, 0.01, 0.1, and 1 mg/mL. RESULTS: The major volatile compounds were caryophyllene, limonene, linalool, pulegone, menthone, and thymol. S. macrostema essential oil showed the highest free radical scavenging activity with DPPH and ABTS methods (53.10% and 92.12%, respectively) at 1 mg/mL and 98% with TAC method at 0.1 mg/mL. Thymol exerted the highest antioxidant capacity with 0.1 mg/mL, reaching 83.38%, 96.96%, and 98.57% by DPPH, ABTS, and TAC methods. Caryophyllene, limonene, linalool, pulegone, and menthone exhibited an antioxidant capacity <25% with the DPPH and ABTS methods; however, limonene showed a TAC of 85.41% with 0.01 mg/mL. CONCLUSION: The essential oil of S. macrostema and thymol showed a free radical scavenging activity close to that of the synthetic butylated hydroxytoluene. SUMMARY: The major volatile compounds of essential oil of Satureja macrostema were caryophyllene, limonene, linalool, pulegone, menthone and thymolThe essential oil of S. macrostema showed a high free radical scavengingThymol exerted the highest antioxidant capacity by DPPH, ABTS and TAC methods. Abbreviations used: GC: Gas Chromatography; DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid; TAC: Total antioxidant capacity.

Valine and phenylalanine as precursors in the biosynthesis of alkamides in Acmella radicans.

Acmella radicans (Asteraceae) produces at least seven alkamides, most with either an isobutyl- or phenylethyl group as the amine moiety. These moieties suggest that the amino acids valine and phenylalanine are the biosynthetic precursors of these alkamides. On the basis of labeled feeding experiments using either L-[2H8]valine or L-[2H8]phenylalanine we present evidence for the involvement of these two amino acids in the biosynthesis of (2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide (affinin) (1), (2Z,4E)-N-(2-phenylethyl)-2,4-octadienamide (2), (2E)-N-(2-phenylethyl)-nona-2-en-6,8-diynamide (3), and 3-phenyl-N-(2-phenylethyl)-2-propenamide (4). Alkamides were isolated from young A. radicans plants and analyzed by gas chromatography-mass spectrometry (GC-MS). Additionally, in cell free in vitro experiments based on isobutyl and phenylethylamide biosynthesis, using a colorimetric assay and GC-MS, valine and phenylalanine decarboxylase activities were assayed in the soluble extract of A. radicans leaves.

Arbuscular mycorrhizal symbiosis increases the content of volatile terpenes and plant performance in Satureja macrostema (Benth. ) Briq / La simbiosis micorrícica arbuscular aumenta el contenido de terpenos volátiles y el rendimiento vegetal en Satureja macrostema (Benth.) Briq

We studied the effect of Rhizophagus irregularis on plant performance and volatile terpenes content of the Mexican native medicinal plant Satureja macrostema (Benth.) Briq. (Lamiaceae) in greenhouse conditions. The growth parameters considered in this research and the composition of volatile components were quantified monthly in mycorrhizal and non-mycorrhizal plants. The essential oil was collected from aerial parts and analyzed by gas chromatography-mass spectrometry. Colonization by R. irregularis significantly increased biomass, shoot and root length, and the amount of volatile terpenes. The more concentrated volatile terpenes were limonene, β- linalool, menthone, pulegone, and verbenol acetate. It is concluded that the use of R. irregularis allows optimal growth of S. macrostema plants in low fertility soils and increased production of the main components of the essential oil.

El efecto de Rhizophagus irregularis sobre el rendimiento vegetal y la producción de los terpenos volátiles de Satureja macrostema (Benth.) Briq. (Lamiaceae), una planta medicinal nativa mexicana, fue estudiado en condiciones de invernadero. Los parámetros de crecimiento considerados en esta investigación y los componentes volátiles, fueron cuantificados mensualmente en plantas con y sin micorrizas. El aceite esencial fue colectado de la parte aérea y fue analizado por técnicas de cromatografía de gases-espectrometría de masas. La colonización de R. irregularis aumentó significativamente la biomasa, longitud de tallo y raíz, y la cantidad de terpenos volátiles. Los terpenos volátiles mayoritarios fueron limoneno, β-linalol, mentona, pulegona y acetato de verbenol. Se concluye que el uso de R. irregularis permitió un óptimo crecimiento de las plantas de S. macrostema en suelos de baja fertilidad, con un aumento de los componentes principales del aceite esencial.