Centella asiatica (L.) Urb. and Hydrocotyle umbellata L. identification and quality assessment: A methodology comparison.

INTRODUCTION: Centella is an important genus in the Apiaceae family. It includes Centella asiatica, which has significant edible and medicinal values. However, this species is easily confused due to its similar morphological traits to Hydrocotyle umbellata, hindering its utilization in the consumer and pharmacological industries. OBJECTIVE: The study aims to differentiate these two closely related plant species using reliable methods of confirming the authenticity of natural herbal medicines. METHODS: Our work mainly focuses on the basic morphological characteristics, chemical markers, genetic fingerprints, and their biological responses. RESULTS: The plants can be clearly differentiated using their leaf shapes, stipules, petioles, inflorescences, and fruit structures. Although the phytochemical compositions of the C. asiatica extract were similar to that of H. umbellata which included flavonoids, tannins, and saponins important to the plant's ability to reduce inflammation and promote healing of wounds, the H. umbellata extract showed significantly higher toxicity than that of C. asiatica. High-performance liquid chromatography analysis was used to identify chemical fingerprints. The result revealed that C. asiatica had major triterpene glycoside constituents including asiaticoside, asiatic acid, madecassoside, and madecassic acid, which have a wide range of medicinal values. In contrast, triterpenoid saponins were not identified in H. umbellata. Furthermore, using SCoT1-6 primers was possible to effectively and sufficiently created a dendrogram which successfully identified the closeness of the plants and confirmed the differences between the two plant species. CONCLUSION: Therefore, differentiation can be achieved through the combination of morphometrics, molecular bioactivity, and chemical analysis.

Pantoea ananatis, a plant growth stimulating bacterium, and its metabolites isolated from Hydrocotyle umbellata (dollarweed).

A bacterium growing on infected leaves of Hydrocotyle umbellata, commonly known as dollarweed, was isolated and identified as Pantoea ananatis. An ethyl acetate extract of tryptic soy broth (TSB) liquid culture filtrate of the bacterium was subjected to silica gel chromatography to isolate bioactive molecules. Indole was isolated as the major compound that gave a distinct, foul odor to the extract, together with phenethyl alcohol, phenol, tryptophol, N-acyl-homoserine lactone, 3-(methylthio)-1-propanol, cyclo(L-pro-L-tyr), and cyclo(dehydroAla-L-Leu). This is the first report of the isolation of cyclo(dehydroAla-L-Leu) from a Pantoea species. Even though tryptophol is an intermediate in the indoleacetic acid (IAA) pathway, we were unable to detect or isolate IAA. We investigated the effect of P. ananatis inoculum on the growth of plants. Treatment of Lemna paucicostata Hegelm plants with 4 × 109 colony forming units of P. ananatis stimulated their growth by ca. five-fold after 13 days. After 13 days of treatment, some control plants were browning, but treated plants were greener and no plants were browning. The growth of both Cucumis sativus (cucumber) and Sorghum bicolor (sorghum) plants was increased by ca. 20 to 40%, depending on the growth parameter and species, when the rhizosphere was treated with the bacterium after germination at the same concentration. Plant growth promotion by Pantoea ananatis could be due to the provision of the IAA precursor indole.

Hydrocotyle umbellata L.; a natural source of bioactives to mitigate diabetes mellitus and its complications.

Metabolomics study of Hydrocotyle umbellata L. revealed the richness of its aerial parts in phenolics primarily; quercetin and its glycoside derivatives, which are well-reported to exert antidiabetic activity owing to their powerful antioxidant capacity. Hence, the antioxidant and antidiabetic potentials of the quercetin standardized ethanolic extract of H. umbellata aerial parts were investigated. The antioxidant activity was examined by using in-vitro 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay, while the antidiabetic activity was examined by using in-vitro α-glucosidase inhibitory assay and further confirmed by in-vivo experiments using streptozotocin-induced diabetes in rat model. Interestingly, the standardized ethanolic extract showed significant in-vitro antioxidant activity, and effectively inhibited Saccharomyces cerevisiae α-glucosidase enzyme activity. Moreover, it significantly reduced fasting blood glucose, triglycerides, and cholesterol levels. Thus, H. umbellata is a potential natural candidate to attenuate diabetes mellitus and its altered lipid profile complications, which could be attributed to its quercetin and quercetin glycosides content.

Vascular relaxing effect of Hydrocotyle umbellata L. is mediated by blocking of l-type Ca2+ channels.

ETHNOPHARMACOLOGICAL RELEVANCE: Hydrocotyle umbellata L. is a medicinal herb for the treatment of some health problems including hypertension, according to traditional medicine. Even so, its vascular effects and the pharmacological action mechanisms have not been analyzed. AIM OF THE STUDY: This experiment aimed to analyze the effects of hydroalcoholic extract of Hydrocotyle umbellata L. (HEHU) on isolated vessels and verify the interaction of hibalactone (chemical marker) against Cav1.2 channels using molecular docking. MATERIALS AND METHODS: Vascular reactivity experiments were performed using rat aortas with (E+) or without endothelium (E-) in an isolated organ bath. Computational molecular docking approaches were used to show the direct effect on L-type Ca2+ Channels. RESULTS: HEHU (0-560 µg/mL) induced relaxation of the pre-contracted arteries in a concentration-dependent manner. The maximum effect was higher in E+ (76.8 ± 4.1%) as compared to E- (47.3 ± 5.5%). Pre-treatment of E+ arteries with L-NAME or ODQ reduced the relaxation to similar level of E- arteries. The treatment of arteries with MDL-12,330 A, diclofenac, propranolol and atropine did not change the relaxation induced by HEHU. The contraction caused by internal Ca2+ release induced by caffeine was reduced after HEHU treatment. Moreover, the HEHU also impaired the contraction induced by Ca2+ influx stimulated with phenylephrine or high KCl. The docking study demonstrated the effectiveness of hibalactone in blocking the Cav1.2 channel. CONCLUSIONS: These findings show that HEHU induces vascular relaxation which is potentiated (but not dependent) by endothelial cells. Blocking of Ca2+ influx seems to be the main mechanism for the vascular effects of HEHU.

In-vivo Anti-inflammatory Activity of Hydrocotyle umbellata L. aerial parts and Isolation of the Main Phytochemicals.

Hydrocotyle umbellata L. (Family Araliaceae) populary known as Acaricoba, is indicated in folk medicine for treatment of several inflammatory disorders. The goal of the present study is to evaluate the anti-inflammatory activity of the defatted ethanolic extract (DEE) of the aerial parts using carrageenan-induced rat paw oedema method. The levels of the pro-inflammatory cytokine interleukin-6 (IL-6) and the inflammatory mediator prostaglandin E2 (PGE2) were assessed using ELISA. The DEE at dose level 100 mg/kg showed significant decrease in oedema volume after 2 and 3 h, equivalent to 70.75% and 95.92% of the activity of the standard anti-inflammatory indomethacin, respectively. DEE significantly decreased the concentrations of the excessively produced IL-6 and PGE2 (24 ± 2.1 and 2374 ± 87 pg/mL compared to 16 ± 2 and 2419 ± 95 pg/mL induced by indomethacin). Chemical investigation was carried out to isolate and identify the bioactive compounds that might be responsible for this activity. The total phenolic (79.28 ± 0.1 mg) and total flavonoid (57.99 ± 0.1 mg) contents of the DEE were quantified spectrophotometricaly and expressed as gallic acid and rutin equivalents/g dry weight, respectively. The DEE was subjected to further fractionation using solvents of increasing polarities. Purification of the ethyl acetate fraction using different chromatographic techniques led to the isolation of five compounds, which were identified through 1D and 2D and UV/Vis spectral data. The five compounds were: quercetin, avicularin, quercitrin, hyperoside, and neochlorogenic acid. Several biological studies confirmed the important role of caffeoyl quinic acid and quercetin derivatives as anti-inflammatory bioactive compounds.

Antinociceptive, anti-inflammatory and anxiolytic-like effects of the ethanolic extract, fractions and Hibalactone isolated from Hydrocotyle umbellata L. (Acariçoba) - Araliaceae.

Hydrocotyle umbellata Linn. (Araliaceae) is specie used in the treatment of inflammatory diseases. Crude extract (E-HU) was prepared from H. umbellata subterraneous parts and fractionated by liquid-liquid partition, resulting hexane fraction (HF-HU), dichloromethane fraction (DF-HU), ethyl acetate fraction (EAF-HU) and aqueous fraction (AF-HU). The hibalactone (HU-1) was isolated from the DF-HU and its structure was elucidated by 1H NMR and 13C NMR Spectroscopy, mass spectrometry and crystallographic x-ray analysis. The formalin-induced nociception was used to evaluate antinociceptive activity; carrageenan-induced edema and pleurisy tests to evaluate anti-inflammatory activity and light-dark box to evaluate anxiolytic-like activity in mice. The acute oral treatments with E-HU (1000mg/kg), DF-HU (150mg/kg), EAF-HU (400mg/kg) and HU-1 (33mg/kg) decreased the licking time in both phases of the formalin test. In the carrageenan-induced inflammation models, the treatment with the same doses of E-HU, DF-HU, EAF-HU and HU-1 reduced the paw edema formation and leukocytes account into pleural cavity. In silico findings suggest that hibalactone present anti-inflammatory activity by interacting with the enzymes 5-lipoxygenase and cyclooxygenase-2. In the light dark box, the treatments with DF-HU, EAF-HU and HU-1 revealed an anxiolytic like effect. Thus, the E-HU and fractions of H. umbellata showed antinociceptive, anti-inflammatory and anxiolytic like activities, as also hibalactone, a possible phytoconstituent responsible for the biological effects of this specie.

Anxiolytic-like and sedative effects of Hydrocotyle umbellata L., Araliaceae, extract in mice

The plant Hydrocotyle umbellata L., Araliaceae (water pennywort), is widely used in Brazilian folk medicine to reduce anxiety. This work investigates the anxiolytic-like effects of the ethanol extract from H. umbellata subterraneous parts as well as the extract's other putative central nervous system effects that could justify its common use. Oral dosing of the extract (0.3 and 1 g/kg) clearly showed an anxiolytic-like profile in the elevated plus maze test where it increased the percentage of entries into and the time spent in the open arms of the maze. In the marble-burying test, the extract induced anxiolytic-like effects only at a dose of 1 g/kg, which also causes mild sedative properties in other models. The sedated state was characterized by a slight reduction in spontaneous exploratory activity during the open field test and a potentiating of pentobarbital-induced hypnosis. No signs of motor impairment were detected in the rota rod or chimney tests. The extract did not show antidepressant properties in mice as assessed by the forced swimming test. These results support the use of H. umbellata in Brazilian folk medicine as an anxiolytic and contribute to the scientific knowledge of this possible phytotherapeutic resource.

Caracterização anatômica e química da folha e do sistema radicular de Hydrocotyle umbellata (Apiaceae) / Anatomical and chemical characterization of the leaf and root system of Hydrocotyle umbellata (Apiaceae)

A família Apiaceae (Umbelliferae), também denominada de pioneira das praias, é capaz de habitar locais de alto teor de salinidade, além de suportar a ação dos ventos e das ondas. O presente trabalho teve como objetivo o estudo da anatomia foliar e radicular e analisar constituintes químicos das folhas e do sistema radicular, caracterizando grupos químicos biologicamente ativos presentes nesses órgãos que permitam aplicações farmacológicas. O material foi coletado na região de Proteção Ambiental Iguape, Cananéia, Peruíbe. As folhas são dorsiventrais, com epiderme unisseriada, bordo regular, parênquima paliçádico com duas a três camadas e parênquima lacunoso com oito e nove camadas de células. É anfiestomática, apresentando grandes câmaras subestomáticas com maior incidência de estômatos na epiderme abaxial. O feixe vascular é colateral apresentando células de esclerênquima em forma de meia lua ao redor do xilema e do floema. O pecíolo apresenta contorno irregular, está envolvido por colênquima em toda sua extensão e apresenta grande quantidade de canais secretores entre os feixes vasculares. O rizoma apresenta contorno irregular com variação de 10 a 15 camadas de células de parênquima constituindo o córtex. O cilindro central é constituído por feixes colaterais delimitados pela endoderme. O periciclo é sinuoso e envolve totalmente os feixes vasculares. Sob a epiderme há uma faixa contínua de colênquima. A medula é constituída de células de parênquima de parede fina. H. umbellata apresentou triterpenos, saponinas, flavonóides, compostos poliacetilênicos e leucoceramidas. Folhas e rizomas apresentaram constituintes químicos semelhantes, com diferenças apenas na intensidade dos picos, o que denota diferença quantitativa entre as substâncias presentes. O rendimento do extrato do rizoma é menor que o rendimento das folhas.

The Apiaceae family (Umbelliferae), also called pioneer of beaches, is capable to inhabit areas of high salinity levels. It supports the action of the winds as well as the waves. The objectives of the present work are the study of the foliar and root anatomy and its chemical constituents, in order to find biological active agents that allow pharmacological applications. The plants were collected in an environmental protected region (Área de Proteção Ambiental de Iguape, Cananéia, Peruíbe, SP). The leaves are dorsiventral, with one cellular layer epidermis, palisade parenchyma having two or three layers and lacunary parenchyma presenting eight or nine layers of cells. The leaves are amphistomatical, presenting great substomatal chambers with bigger incidence of stomata in abaxial epidermis. The vascular bundles are collateral, presenting half moon sclerenchyma form around the xylem and the phloem. Petiole shows irregular contour, involved by collenchyma in all extension and presents great amount of secretory ducts between vascular bundles. Rhizome presents irregular contour with ten to fifteen layers of cortical parenchyma cells. The central cylinder has collateral bundles delimited by the endodermis. Pericycle is sinuate and totally involves vascular bundles. Under the epidermis there is a continuous band of collenchyma. The medulla has thin wall parenchyma cells. H. umbellata presented triterpenoids, saponins, flavonoids, poliacetylenes compounds and leucoceramides. Leaves and rhizomes presented similar chemical components, with slightly differences on spectral peaks. The yield of rhizome extract was smaller than the leaves extract.