Acmella oleracea (L.) R.K. Jansen: Alkylamides and phenolic compounds in aerial parts and roots of in vitro seedlings.

Acmella oleracea L. is an edible and medicinal plant commonly known for its local anaesthetic effect induced by the alkylamide spilanthol. It is also rich of secondary metabolites of biological interest, mainly phenolic acids and glycosylated flavonoids. This study evaluated for the first time alkylamides and phenolic compounds in aerial parts and roots of seedlings grown in vitro and produced from single seed and from regenerating lines. The extracts obtained showed similar chemical profiles and the caffeic acid derivatives were the most abundant phenolic compounds. Spilanthol was prevalent in the aerial parts, especially in samples of seedlings obtained from regenerating lines, in which reached maximum value of 1.72 mg/g dried matter (DM). The roots contained a lower content of alkylamides, while showing twice as much total phenols (11.19 mg/g DM) as the aerial parts. A hexane fractionation step allowed to recover spilanthol and its derivatives in a concentrated extract, which reached a value of 63.4 mg/g dried extract in the aerial parts from seedlings from regenerating lines. Hydroalcoholic dried extracts showed high yields (30-45 % on dried matter) and those obtained from aerial parts contained up to 5.69 mg/g of spilanthol.

Local effects of natural alkylamides from Acmella oleracea and synthetic isobutylalkyl amide on neuropathic and postoperative pain models in mice.

Neuropathic and postoperative pain are clinical conditions that impair the patient's quality of life. The current pharmacotherapy of both painful states is ineffective and accompanied by several side effects. In order to develop new therapeutics targets, the secondary metabolites of plants have been extensively studied. Acmella oleracea ("jambu") is a native plant from the Amazon region and rich in alkylamides, bioactive compounds responsible for inducing anesthetic and chemesthetic sensations. We previously demonstrated that the intraplantar administration of an hexanic fraction (HF) rich in alkylamides from jambu and the synthetic isobutylalkyl amide (IBA) at 0.1 µg/20 µL can promote antinociceptive and anti-inflammatory effects. Thus, this study aimed to evaluate the local effect of HF and IBA (0.1 µg/20 µL) on neuropathic (partial sciatic nerve ligation, PSNL) and postoperative pain (plantar incision surgery, PIS) models in mice. Seven days after the PSNL, the mechanical (von Frey test) and cold (acetone-evoked evaporative cooling) allodynia, and digital gait parameters were analyzed. The intraplantar HF and IBA treatments attenuated the mechanical and cold allodynia as well as the static (max. Contact and print area) and dynamic (stand duration) parameters of digital gait analyses. On the day after PIS, the mechanical allodynia, heat hyperalgesia (hot plate, 52 ± 0.1°C), and spontaneous nociception scores were evaluated. Topical treatment with HF reduced the mechanical allodynia, heat hyperalgesia, and spontaneous nociception scores. In contrast, IBA treatment only partially reduced the mechanical allodynia. In summary, the local treatment with HF was effective on both neuropathic and postoperative pain, as opposed to IBA, which only had an effect on neuropathic pain.

Acaricidal activity of Acmella oleracea (Asteraceae) extract against Rhipicephalus microplus: What is the influence of spilanthol?

The present study was carried out to evaluate and compare the acaricidal activity of different fractions of Acmella oleracea methanolic extract, containing 0.0 % (F1), 24.5 % (F2), 48.0 % (F3) and 100 % (F4) of spilanthol, on unfed larvae and engorged females from the same Rhipicephalus microplus population. To obtain these fractions, the crude extract was subjected to different extraction procedures using increasingly polarized solvents to isolate the spilanthol compound. The Larval Packet Test was used to evaluate acaricidal activity in unfed larvae at concentrations ranging from 0.2 to 25.0 mg/mL, while for engorged females, the Adult Immersion Test was performed at concentrations from 3.1 to 25.0 mg/mL. The F1 fraction showed no activity on unfed larvae, while a control percentage of 44.6 % was observed at a concentration of 25.0 mg/mL for engorged females. For unfed larvae, the F2 fraction resulted in 95.7 % mortality at a concentration of 1.6 mg/mL, with a control percentage of 92.7 % for engorged females at a concentration of 12.5 mg/mL. Fractions F3 and F4 had similar activity against unfed larvae, with mortality >84.0 % from the concentration of 0.8 mg/mL. This similarity between the fractions was also observed for engorged females from a concentration of 12.5 mg/mL, resulting a control percentage >94.0 %. These results demonstrate that the presence of spilanthol is an important factor for the acaricidal activity of A. oleracea extract. Fraction extracts with 24.5, 48 and 100 % of spilanthol have similar acaricidal activity on R. microplus.

Pharmacological potential of alkylamides from Acmella oleracea flowers and synthetic isobutylalkyl amide to treat inflammatory pain.

Acmella oleracea ("jambu") is an Amazonian plant rich in alkylamides. Its flowers are widely used in folk medicine to treat toothache due to tingling, numbness, and local anaesthesia caused in the mouth. Our group previously demonstrated that the intraplantar (i.pl.) injection of an alkylamide-rich hexane fraction (HF) obtained from jambu flowers and a synthetic isobutylalkyl amide (IBA) displayed antinociceptive and anesthetic effects in acute pain models. Thus, here we evaluated the effects of HF and IBA on carrageenan-induced acute inflammation. Mice were pretreated with HF or IBA (0.01, 0.1, and 1 µg/20 µL, i.pl.) 15 min before carrageenan injection (300 µg/20 µL, i.pl.). Mechanical allodynia and paw oedema were evaluated previously (basal) and at 0.5 until 6 h following carrageenan. Both HF and IBA at 0.1 µg promoted effective and long-lasting antiallodynic and anti-oedematogenic activities until 3 and 5 h, respectively, in comparison to the different doses evaluated. At the inflammatory peak, the plantar surfaces were excised for measurement of inflammatory and oxidative stress parameters. HF and IBA (0.1 µg) reduced the myeloperoxidase activity, TNF-α and IL-1ß levels, prevented the production of lipid hydroperoxides, and the decrease of antioxidant agents, namely superoxide dismutase and catalase activities, and glutathione contents. Furthermore, only HF maintained IL-10 levels and decreased PGE2 synthesis. On the basis of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, HF and IBA are devoid of antioxidant activity in vitro. Collectively, our results demonstrated the promising anti-inflammatory effect of local pretreatment with alkylamides, supporting the potential of these molecules to treat acute inflammatory pain conditions.

Distinct mechanisms underlying local antinociceptive and pronociceptive effects of natural alkylamides from Acmella oleracea compared to synthetic isobutylalkyl amide.

Acmella oleracea (jambu), is used as ingredient for food and in folk medicine to relief toothache. Jambu edible flowers are rich in alkylamides, mainly spilanthol, which are responsible to evoke chemesthetic sensations. This study aimed to investigate the local effects promoted by the intraplantar injection of the hexanic fraction (HF) rich in alkylamides from jambu flowers and compare to synthetic isobutylalkyl amide (IBA). Swiss male mice were intraplantarly administrated with HF and IBA (0.1-30 µg/20 µL), and the underlying mechanisms associated to the antinociceptive (0.1 µg) and pronociceptive (30 µg) effects were evaluated in chemical and sensorial tests. HF and IBA at 0.1 µg promoted analgesia in neurogenic and inflammatory phases of formalin test, against glutamate-induced nociception and independent of the activation of endogenous opioidergic system and dependent of TRPV1 modulation, whereas only HF reduced both nociception and mast cell degranulation in hindpaw induced by compound 48/80. However, both potentiated the TRPA1-mediated nociception. In contrast, HF and IBA (30 µg)-evoked nociceptive behaviors were reduced by the activation of opioidergic system, by TRPA1 antagonist and TRP nociceptive fibers desensitization. In addition, 30 µg IBA-evoked nociception by activation of TRPV1, and 30 µg HF by mast cell degranulation. Furthermore, on the contrary of IBA, HF elevated both mechanical and thermal paw threshold. Altogether, these results indicate that alkylamides could elicited dual effects, adding new evidences and mechanisms for these opposite actions in different doses. Although further research is needed, we confirmed that alkylamides displays local analgesic and/or anesthetic effects.

Morphological alterations in the ovaries of Amblyomma cajennense semi-engorged ticks exposed to ethanolic extract of Acmella oleracea.

The present study analyzed the effects of different concentrations of Acmella oleracea crude ethanolic extract (EEAO) on the development of germ cells from semi-engorged Amblyomma cajennense females in order to evaluate the potential of this natural chemical as a strategy to control these important ectoparasites. A hundred semi-engorged females were divided into five groups (duplicates) (10 animals/group): Control 1 (distilled water); Control 2 (solvent ethanol 50% and DMSO 1%); and Treatment I to III (3.1, 6.2, and 12.5 mg/mL of EEAO, respectively). For the exposure of the ticks to the extract was used the Adult Immersion Test. After the exposition, the ovaries were removed and submitted to histological analysis using Harris hematoxylin and aqueous eosin. The histochemical tests were performed using PAS and Bromophenol blue staining techniques, for the detection of total polysaccharides and total protein, respectively. The extract caused significant alterations in the oocytes, including changes in the shape of the cells, disorganization, and cytoplasmic vacuolation, decrease in the number of yolk granules and germ vesicle fragmentation. These alterations were more intense in the oocytes in initial developmental stages (I and II). The results obtained in this study confirm the cytotoxic potential of the ethanolic extract of A. oleracea on the germ cells of A. cajennense females, opening up the possibility to use this extract as an alternative to control these ectoparasites.

Activity of the extract of Acmella oleracea on immature stages of Amblyomma sculptum (Acari: Ixodidae).

This study evaluated the acaricidal activity of the methanol extract of Acmella oleracea with 0.187% of spilanthol against immature stages of Amblyomma sculptum. The packet test was used to evaluate the extract's activity on unengorged larvae and nymphs, testing concentrations of 0.4 to 50 mg/mL for larvae and 12.5 to 200.0 mg/mL for nymphs. For the engorged stages, the immersion test was used, at concentrations of 0.4 to 50 mg/mL for larvae and 12.5 to 200.0 mg/mL for nymphs. The methanol extract caused 100% mortality of the unengorged larvae and nymphs starting at concentrations of 12.5 and 200.0 mg/mL, respectively. For engorged larvae and nymphs, the mortality was 100% starting from concentrations of 12.5 and 150.0 mg/mL, respectively. The LC50 for unengorged larvae was 3.2 mg/mL, while for engorged larvae it was 6.6 mg/mL. For unengorged nymphs, the LC50 was 38.5 mg/mL, but it was not possible to calculate the corresponding value for engorged nymphs because the data did not fit the probit model. These results demonstrate that the methanol extract of A. oleracea has acaricidal activity against different immature stages of A. sculptum.

Potential action of extract of Acmella oleracea (L.) R.K. Jansen to control Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) ticks.

The use of synthetic acaricides is currently the main method to control ticks. However, the indiscriminate use of these chemicals can lead to the selection of resistant individuals and in the accumulation of chemical residues in the environment, contaminating the soil and water streams, consequently affecting the flora, fauna, and the human beings as well. In this sense, the objective of this study was to investigate the acaricidal effect of crude ethanolic extract of Acmella oleracea (L.) R.K. Jansen aerials parts at different concentrations on fed males and semi-engorged females of A. cajennense s.s. An in vitro bioassay (Adult Immersion Test) was carried out to determine the lethal concentration 50 (LC50) of ethanolic extract, calculated by Probit analysis. The results showed that the fed males were sensitive to all the concentrations of A. oleracea ethanolic extract, and mortality rate progressively increased (15-65%) in higher ethanolic extract concentrations. However, semi-engorged females were not sensitive to all the concentrations used here. In the highest concentration (100mg/mL), a mortality rate of 100% was observed after 72h of exposure, indicating that the acaricidal effect would probably be dose-dependent. The LC50 values obtained for the fed A. cajennense s.s males and semi-engorged females were 29.4534mg/mL (limits: 24.4467-41.3847mg/mL) and LC50=17.6335mg/mL (limits: 5.2506-23.5335mg/mL), respectively.

Antinociceptive effects of ethanolic extract from the flowers of Acmella oleracea (L.) R.K. Jansen in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil, Acmella oleracea (L.) R.K. Jansen, popularly known as "jambu", has been used by some communities from Amazon region to treat toothache. In this study we examined the antinociceptive effect of the ethanolic extract obtained from the flowers of Acmella oleracea (EEAO) in animal models of nociceptive (chemical and thermal) and neuropathic (partial sciatic nerve ligation) pain. MATERIALS AND METHODS: Adult male mice were treated by intraperitoneal route (i.p.) with EEAO before the induction of nociceptive response by formalin, capsaicin and cinnamaldehyde, thermal heat hyperalgesia (hot plate test) and mechanical allodynia (traumatic sciatic nerve injury). Acute toxicity and non-specific sedative effects were evaluated. RESULTS: EEAO (10, 30 and 100 mg/kg) reduced both neurogenic and inflammatory phases of the formalin- and also capsaicin- and cinnamaldehyde-induced orofacial nociception. Interestingly, EEAO at 100mg/kg (i.p.) also reversed capsaicin-induced heat hyperalgesia assessed as the latency to paw withdrawal in the hot plate test. Also in the hot plate test, paw withdrawal latency was increased by EEAO (100 mg/kg) and this response was only partially reversed by naloxone. Furthermore, EEAO (100 mg/kg) also reduced mechanical allodynia caused by partial sciatic nerve ligation for 3 h. The estimated LD50 value was 889.14 mg/kg and EEAO did not alter the locomotion of animals in the open-field test. CONCLUSION: Taken together, our data show that EEAO produces prevalent antinociceptive effects and does not cause adverse effects. The presence of N-alkylamides, including spilanthol, suggests that the therapeutic effect of EEAO is related to its highest anesthetic activity.