Quality Studies on Cynometra iripa Leaf and Bark as Herbal Medicines.

Cynometra iripa Kostel. is a Fabaceae species of mangrove used in traditional Ayurvedic medicine for treating inflammatory conditions. The present study aims to establish monographic botanical and chemical quality criteria for C. iripa leaf and bark as herbal substances and to evaluate their in vitro antioxidant potential. Macroscopic and microscopic qualitative and quantitative analyses, chemical LC-UV/DAD-ESI/MS profiling, and the quantification of key chemical classes were performed. Antioxidant activity was evaluated by DPPH and FRAP assays. Macroscopically, the leaf is asymmetrical with an emarginated apex and cuneate base. Microscopically, it shows features such as two-layered adaxial palisade parenchyma, vascular bundles surrounded by 3-6 layers of sclerenchyma, prismatic calcium oxalate crystals (5.89 ± 1.32 µm) along the fibers, paracytic stomata only on the abaxial epidermis (stomatal index-20.15), and non-glandular trichomes only on petiolules. The microscopic features of the bark include a broad cortex with large lignified sclereids, prismatic calcium oxalate crystals (8.24 ± 1.57 µm), and secondary phloem with distinct 2-5 seriated medullary rays without crystals. Chemical profile analysis revealed that phenolic derivatives, mainly condensed tannins and flavonoids, are the main classes identified. A total of 22 marker compounds were tentatively identified in both plant parts. The major compounds identified in the leaf were quercetin-3-O-glucoside and taxifolin pentoside and in the bark were B-type dimeric proanthocyanidins and taxifolin 3-O-rhamnoside. The total phenolics content was higher in the leaf (1521 ± 4.71 mg GAE/g dry weight), while the total flavonoids and condensed tannins content were higher in the bark (82 ± 0.58 mg CE/g and 1021 ± 5.51 mg CCE/g dry weight, respectively). A total of 70% of the hydroethanolic extracts of leaf and bark showed higher antioxidant activity than the ascorbic acid and concentration-dependent scavenging activity in the DPPH assay (IC50 23.95 ± 0.93 and 23.63 ± 1.37 µg/mL, respectively). A positive and statistically significant (p < 0.05) correlation between the phenol content and antioxidant activity was found. The results obtained will provide important clues for the quality control criteria of C. iripa leaf and bark, as well as for the knowledge of their pharmacological potential as possible anti-inflammatory agents with antioxidant activity.

Quality, Safety and Biological Studies on Campylanthus glaber Aerial Parts.

In Cabo Verde, several endemic species are used in traditional medicine. However, no scientific studies have been conducted on the quality, efficacy, and safety of most of these plants. This study focused on establishing the botanical and chemical identification parameters required for a quality monograph of Campylanthus glaber Benth. aerial parts, a medicinal plant of Cabo Verde traditionally used to treat fever and muscular pain. In addition, in vitro antioxidant and antihyperglycemic activity, cytotoxicity, and genotoxicity were assessed for this medicinal plant. Optical microscopy, LC/UV-DAD-ESI/MS, and colorimetric assays were used for botanical, chemical, and biological studies, respectively. Cytotoxicity was assessed by the MTT assay with HepG2 cells, and genotoxicity by the Ames test. Microscopically, the xeromorphic leaf of C. glaber presents a thick cuticle (13.6-25.5 µm), thick-walled epidermal cells, anomocytic-type stomata, glandular trichomes (stalk length = 49.4-120.8 µm), and idioblasts containing calcium oxalate microcrystals. The chemical screening of aqueous and hydroethanolic extracts of this medicinal plant revealed the presence of organic acids, iridoids, phenylethanoids, and flavonoids as the main classes of marker compounds, with malic acid, citric acid, and verbascoside being the main marker compounds identified. Both extracts showed similar LC/UV-DAD/ESI-MS qualitative profiles and DPPH radical scavenger activity (IC50 = 130.9 ± 1.4; 134.3 ± 3.1 µg/mL). The hydroethanolic extract inhibited both α-amylase and α-glucosidase enzymes in a dose-dependent manner. Both extracts showed no cytotoxicity (up to 1000 µg/mL) by the MTT assay and no genotoxic potential with or without metabolic activation up to 5 mg /plate. The results obtained are an important contribution to the monographic quality assessment of C. glaber aerial parts and suggest that this medicinal plant may be safe and potentially used as an herbal drug raw material for pharmaceutical purposes.

Vernonia britteniana Root Phytochemical Studies, In Vitro Cercaricidal Activity on the Larval Stage of Schistosoma mansoni and Antioxidant Activities.

Vernonia britteniana Hiern. (Asteraceae) is a medicinal plant used in traditional Angolan medicine against schistosomiasis. Our study aimed to investigate the phytochemical composition and the cercaricidal and antioxidant activities in vitro of a traditional herbal preparation (Water-Vbr) and a 70% hydroethanolic extract (EtOH70%-Vbr) prepared with this medicinal plant. The activity of the extracts against Schistosoma mansoni cercariae was assessed at different extract concentrations (500, 438, and 125 µg/mL) and at different time intervals, and the phytochemical profiles were obtained by LC-UV-ESI/MS-MS. In addition, the major chemical classes of the identified metabolites were quantified by colorimetry, and the antioxidant potential was assessed using the DPPH and FRAP methods. After 30 min, 100% cercarial mortality was observed at a concentration of 500 µg/mL after exposure, and after 120 min, an LC50 of 438 µg/mL was observed for both extracts. Phenolic acid derivatives (chlorogenic acid, caffeic acid; 3,4-di-O-caffeoylquinic acid; 3,5-di-O-caffeoylquinic acid; and 4,5-di-O-caffeoylquinic acid) and triterpenoids (stigmastane-type steroidal saponins; vernoamyoside D and vernonioside D1; vernoamyoside B; and vernoniamyoside A and C) were identified as the main secondary metabolites. The Water-Vbr extract showed the highest antioxidant activity-DPPH: IC50 = 1.769 ± 0.049 µg/mL; FRAP: mean = 320.80 ± 5.1325 µgAAE/g.

Identification of Marker Compounds and In Vitro Toxicity Evaluation of Two Portuguese Asphodelus Leaf Extracts.

The leaves of Asphodelus bento-rainhae subsp. bento-rainhae, an endemic Portuguese species, and Asphodelus macrocarpus subsp. macrocarpus have been used as food, and traditionally as medicine, for treating ulcers, urinary tract, and inflammatory disorders. The present study aims to establish the phytochemical profile of the main secondary metabolites, together with the antimicrobial, antioxidant and toxicity assessments of both Asphodelus leaf 70% ethanol extracts. Phytochemical screenings were conducted by the TLC and LC-UV/DAD-ESI/MS chromatographic technique, and quantification of the leading chemical classes was performed by spectrophotometric methods. Liquid-liquid partitions of crude extracts were obtained using ethyl ether, ethyl acetate, and water. For in vitro evaluations of antimicrobial activity, the broth microdilution method, and for the antioxidant activity, the FRAP and DPPH methods were used. Genotoxicity and cytotoxicity were assessed by Ames and MTT tests, respectively. Twelve known compounds including neochlorogenic acid, chlorogenic acid, caffeic acid, isoorientin, p-coumaric acid, isovitexin, ferulic acid, luteolin, aloe-emodin, diosmetin, chrysophanol, and ß-sitosterol were identified as the main marker compounds, and terpenoids and condensed tannins were found to be the major class of secondary metabolites of both medicinal plants. The ethyl ether fractions demonstrated the highest antibacterial activity against all the Gram-positive microorganisms, (MIC value of 62 to 1000 µg/mL), with aloe-emodin as one of the main marker compounds highly active against Staphylococcus epidermidis (MIC value of 0.8 to 1.6 µg/mL). Ethyl acetate fractions exhibited the highest antioxidant activity (IC50 of 800 to 1200 µg/mL, respectively). No cytotoxicity (up to 1000 µg/mL) or genotoxicity/mutagenicity (up to 5 mg/plate, with/without metabolic activation) were detected. The obtained results contribute to the knowledge of the value and safety of the studied species as herbal medicines.

The Genus Cynometra: A Review of Ethnomedicine, Chemical, and Biological Data.

Cynometra L. is a Fabaceae genus that is widely distributed throughout the tropics, consisting of tropical forest trees with ecological and economic importance since they are used as food and herbal medicines by the populations of their natural habitats. Our goal is to provide a review of the research data concerning the potential of this botanical genus as a source of herbal medicines and secondary metabolites that are useful for human health. To that end, scientific databases, including PubMed, Science Direct, ISI Web of Science, Scopus, and Google Scholar, were searched using the following terms: Cynometra, medicine, chemical, biological activity, toxicity, and "AND" as the Boolean connector. Eleven Cynometra species (9.7%) were reported to be used in traditional medicine to treat different ailments. A total of 185 secondary metabolites of various chemical classes, mainly flavonoids and terpenoids, were identified in eight Cynometra species (7.1%). Vitexin was the only flavonoid identified as bioactive in the sequence of bioguided studies on this botanical genus. Ten species (8.8%) were submitted to in vitro and in vivo biological activity assays. The main evaluated activities were in vitro antioxidant, antimicrobial, cytotoxic, and in vivo anti-inflammatory activities, but no human clinical trials or safety data about this genus were found. Cynometra cauliflora and Cynometra ramiflora were the most studied species. The present work confirms the use of Cynometra species as a source of medicinal plants. However, more experimental studies must be conducted to better understand this botanical genus's usefulness as a source of raw materials for pharmaceutical use.

Monographic Quality Parameters and Genotoxicity Assessment of Asphodelus bento-rainhae and Asphodelus macrocarpus Root Tubers as Herbal Medicines.

Root tubers of Asphodelus bento-rainhae subsp. bento-rainhae (AbR), an endemic species with relevant interest due to conservation concerns, and Asphodelus macrocarpus subsp. macrocarpus (AmR) have been traditionally used for culinary and medicinal purposes, mainly associated with skin infection and inflammation. The present study aims to establish the quality control criteria for the proper characterization of dried root tubers of both species as herbal substances, together with their preclinical safety assessments. Botanical identification using macroscopic and microscopic techniques and phytochemical evaluation/quantification of the main classes of marker secondary metabolites, including phenolic compounds (flavonoid, anthraquinone, condensed and hydrolysable tannin) and terpenoids were performed. Additionally, in vitro genotoxicity/mutagenicity was evaluated by Ames test. Evident morphological differences in the development of tubercles (3.5 × 1 cm in AbR and 8.7 × 1.4 cm in AmR) and microscopicly in the arrangements and characteristics of the vascular cylinder (metaxylem and protoxylems) were found. Anatomical similarities such as multiple-layered epidermis (velamen) and the cortex area with thin-walled idioblasts (134 ± 2.9 µm and 150 ± 27.6 µm) containing raphide crystals (37.2 ± 14.2 µm and 87.7 ± 15.3 µm) were observed between AbR and AmR, respectively. Terpenoids (173.88 ± 29.82 and 180.55 ± 10.57 mg OAE/g dried weight) and condensed tannins (128.64 ± 14.05 and 108.35 ± 20.37 mg CAE/g dried weight) were found to be the main class of marker secondary metabolites of AbR and AmR extracts, respectively. No genotoxicity (up to 5 mg/plate, without metabolic activation) was detected in these medicinal plants' tested extracts. The obtained results will contribute to the knowledge of the value of the Portuguese flora and their future commercial cultivation utilization as raw materials for industrial and pharmaceutical use.

Acute and Chronic Toxicity of Neem Oil to the Endoparasitoid Palmistichus elaeisis (Hymenoptera: Eulophidae).

Palmistichus elaeisis Delvare and LaSalle 1993 (Hymenoptera: Eulophidae) and neem oil are two control alternatives for the integrated management of defoliating lepidopterans. The aim of this study was to evaluate the acute and chronic toxicity of neem oil compared to the synthetic insecticide deltamethrin, on the endoparasitoid P. elaeisis, in generations F0, F1, F2, and F3. Females of P. elaeisis were exposed to neem solutions at concentrations of 0, 1.87, 3.75, 7.50, 15.00, 30.00, and 60.00 mg ml-1, to determine the dose-response relationship and estimate the neem LC50. The sublethal effects on the parasitoid P. elaeisis in generations F0 to F3 were evaluated with these same concentrations of neem and 0.033 mg ml-1 of deltamethrin. The neem LC50 was estimated at 3.92 mg ml-1. The LC50 for P. elaeisis is 3.83 times lower than that recommended by the neem manufacturer for pest control, demonstrating high acute toxicity to this natural enemy. The chronic toxicity of both the commercial dose and those below it to P. elaeisis caused low sublethal effects. The correct concentration of neem oil in pest control is important, and its use should be performed with caution in integrated pest management programs using the endoparasitoid P. elaeisis to avoid causing interference between the two pest control techniques.

Toxicity of Essential Oils to Diaphania hyalinata (Lepidoptera: Crambidae) and Selectivity to Its Parasitoid Trichospilus pupivorus (Hymenoptera: Eulophidae).

Diaphania hyalinata (Linnaeus, 1767) is one of the main pests of the cucurbit crops. Biological control and botanicals are used in integrated pest management (IPM), especially in agro-ecological agricultures where the use of synthetic pesticides is restricted. Toxicological effects of plant essential oils on target and nontarget organisms should be evaluated to determine its use in IPM. The toxicity of ginger, peppermint, oregano, and thyme essential oils to D. hyalinata eggs, larvae, and pupae and their selectivity to the parasitoid Trichospilus pupivorus and the LC50, LC99 and the relative selectivity index (RSI) of these botanicals were determined. The eggs of D. hyalinata were more sensitive to the botanicals applied than its larvae and pupae, with higher toxicity of oregano and thyme essential oils, followed by those of peppermint and ginger. Topical application was the least toxic method to D. hyalinata larvae. Ginger, peppermint, and thyme essential oils were more toxic by ingestion and oregano by contact to D. hyalinata larvae. The essential oil concentrations applied to D. hyalinata pupae prevent the emergence of adults of this insect with the oregano essential oil showing the greatest toxicity. Peppermint, ginger, thyme, and oregano essential oils were selective to T. pupivorus with RSI50 of 5.40, 1.38, 8.15, and 6.98 and RSI99 of 1.54, 2.53, 3.90, and 4.16 respectively. The ginger, peppermint, oregano, and thyme essential oils were toxic to immature D. hyalinata and selective to T. pupivorus females presenting potential as an alternative control in the IPM of this pest in Cucurbitaceae crops.

Colorectal cancer: can nutrients modulate NF-kappaB and apoptosis?

BACKGROUND & AIMS: NF-kappaB may promote carcinogenesis by altering cell cycle, inflammatory responses and apoptosis-related gene expression, though cell mechanisms relating diet and colorectal cancer (CRC) remain unveiled in humans. This study in patients with CRC aimed to explore potential interactions between the dietary pattern, nutrient intake, expression of NF-kappaB, apoptosis and tumour histological aggressiveness. METHODS: Usual diet was assessed by diet history; nutrient composition was determined by DIETPLAN software. Histologically classified patient tissue samples (adenoma, adenocarcinoma and normal surrounding mucosa) were obtained via biopsies during colonoscopy (n=16) or surgery (n=8). NF-kappaB expression was determined by immunohistochemistry and apoptosis by TUNEL assay. RESULTS: NF-kappaB expression and apoptosis were higher in tumours (p<0.01), greater along with histological aggressiveness (p<0.01). Highest intake terciles of animal protein, refined carbohydrates, saturated fat, n-6 fatty acids and alcohol were associated with higher NF-kappaB, apoptosis and histological aggressiveness (p<0.01); the opposite tissue characteristics were associated with highest intake terciles of n-3 fatty acids, fibre, vitamin E, flavonoids, isoflavones, beta-carotene and selenium (p<0.002). Additionally, higher n-6:n-3 fatty acids ratio (median 26:1) was associated with higher NF-kappaB (p<0.006) and apoptosis (p<0.01), and more aggressive histology (p<0.01). Conversely, lower n-6:n-3 fatty acids ratio (median 6:1) was associated with lower NF-kappaB (p<0.002) and apoptosis (p<0.002), and less aggressive histology (p<0.002). CONCLUSIONS: NF-kappaB expression and apoptosis increased from adenoma to poorly differentiated adenocarcinoma. This degenerative transition, recognized as key in carcinogenesis, appear to have been influenced by a diet promoting a pro-inflammatory milieu that can trigger NF-kappaB.

Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53.

Drug resistance to 5-fluorouracil (5-FU) is still a major limitation to its clinical use. In addition, the clinical value of p53 as a predictive marker for 5-FU-based chemotherapy remains a matter of debate. Here, we used HCT116 human colorectal cancer cells expressing wild-type p53 and investigated whether inhibition of Fas expression by interference RNA modulates 5-FU-induced apoptosis. Cells were treated with 5-FU (1, 4 or 8 microM) for 8-48 h. Cell viability was evaluated by trypan blue dye exclusion. Apoptosis was assessed by changes in nuclear morphology and caspase activity. The interference RNA technology was used to silence Fas expression. Caspase activation, p53, Fas, cytochrome c, and Bcl-2 family protein expression was evaluated by immunoblotting. 5-FU was cytotoxic in HCT116 cells (p<0.001). Nuclear fragmentation and caspase-3, -8 and -9 activities were also markedly increased in HCT116 cells after 5-FU (p<0.001). In addition, wild-type p53 and Fas expression were 25- and 4-fold increased (p<0.05). Notably, when interference RNA was used to inhibit Fas, 5-FU-mediated nuclear fragmentation and caspase activity were markedly reduced in HCT116 cells. Finally, western blot analysis of mitochondrial extracts from HCT116 cells exposed to 5-FU showed a 6-fold increase in Bax, together with a 3-fold decrease in cytochrome c (p<0.001). In conclusion, 5-FU exerts its cytotoxic effects, in part, through a p53/Fas-dependent apoptotic pathway that involves Bax translocation and mitochondrial permeabilization.