Antinociceptive activity of petroleum ether fraction obtained from methanolic extract of Clinacanthus nutans leaves involves the activation of opioid receptors and NO-mediated/cGMP-independent pathway.

BACKGROUND: Methanol extract (MECN) of Clinacanthus nutans Lindau leaves (family Acanthaceae) demonstrated peripherally and centrally mediated antinociceptive activity via the modulation of opioid/NO-mediated, but cGMP-independent pathway. In the present study, MECN was sequentially partitioned to obtain petroleum ether extract of C. nutans (PECN), which was subjected to antinociceptive study with aims of establishing its antinociceptive potential and determining the role of opioid receptors and L-arginine/nitric oxide/cyclic-guanosine monophosphate (L-arg/NO/cGMP) pathway in the observed antinociceptive activity. METHODS: The antinociceptive potential of orally administered PECN (100, 250, 500 mg/kg) was studied using the abdominal constriction-, hot plate- and formalin-induced paw licking-test in mice (n = 6). The effect of PECN on locomotor activity was also evaluated using the rota rod assay. The role of opioid receptors was determined by pre-challenging 500 mg/kg PECN (p.o.) with antagonist of opioid receptor subtypes, namely ß-funaltrexamine (ß-FNA; 10 mg/kg; a µ-opioid antagonist), naltrindole (NALT; 1 mg/kg; a δ-opioid antagonist) or nor-binaltorphimine (nor-BNI; 1 mg/kg; a κ-opioid antagonist) followed by subjection to the abdominal constriction test. In addition, the role of L-arg/NO/cGMP pathway was determined by prechallenging 500 mg/kg PECN (p.o.) with L-arg (20 mg/kg; a NO precursor), 1H-[1, 2, 4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 2 mg/kg; a specific soluble guanylyl cyclase inhibitor), or the combinations thereof (L-arg + ODQ) for 5 mins before subjection to the abdominal constriction test. PECN was also subjected to phytoconstituents analyses. RESULTS: PECN significantly (p < 0.05) inhibited nociceptive effect in all models in a dose-dependent manner. The highest dose of PECN (500 mg/kg) also did not significantly (p > 0.05) affect the locomotor activity of treated mice. The antinociceptive activity of PECN was significantly (p < 0.05) inhibited by all antagonists of µ-, δ-, and κ-opioid receptors. In addition, the antinociceptive activity of PECN was significantly (p < 0.05) reversed by L-arg, but insignificantly (p > 0.05) affected by ODQ. HPLC analysis revealed the presence of at least cinnamic acid in PECN. CONCLUSION: PECN exerted antinocicpetive activity at peripheral and central levels possibly via the activation of non-selective opioid receptors and modulation of the NO-mediated/cGMP-independent pathway partly via the synergistic action of phenolic compounds.

Antinociceptive activity of methanolic extract of Muntingia calabura leaves: further elucidation of the possible mechanisms.

BACKGROUND: Muntingia calabura (Elaecoparceae) is a medicinal plant traditionally used, particularly, by the Peruvian people to alleviate headache and cold, pain associated with gastric ulcers or to reduce the prostate gland swelling. Following the recent establishment of antinociceptive activity of M. calabura leaf, the present study was performed to further elucidate on the possible mechanisms of antinociception involved. METHODS: The methanol extract of M. calabura (MEMC) was prepared in the doses of 100, 250 and 500 mg/kg. The role of bradykinin, protein kinase C, pottasium channels, and various opioid and non-opioid receptors in modulating the extract's antinociceptive activity was determined using several antinociceptive assays. Results are presented as Mean ± standard error of mean (SEM). The one-way ANOVA test with Dunnett's multiple comparison was used to analyze and compare the data, with P < 0.05 as the limit of significance. RESULTS: The MEMC, at all doses, demonstrated a significant (p < 0.05) dose-dependent antinociceptive activity in both the bradykinin- and phorbol 12-myristate 13-acetate (PMA)-induced nociception. Pretreatment of the 500 mg/kg MEMC with 10 mg/kg glibenclamide (an ATP-sensitive K+ channel inhibitor), the antagonist of µ-, δ- and κ-opioid receptors (namely 10 mg/kg ß-funaltrexamine, 1 mg/kg naltrindole and 1 mg/kg nor-binaltorphimine), and the non-opioid receptor antagonists (namely 3 mg/kg caffeine (a non-selective adenosinergic receptor antagonist), 0.15 mg/kg yohimbine (an α2-noradrenergic antagonist), and 1 mg/kg pindolol (a ß-adrenoceptor antagonist)) significantly (p < 0.05) reversed the MEMC antinociception. However, 10 mg/kg atropine (a non-selective cholinergic receptor antagonist), 0.15 mg/kg prazosin (an α1-noradrenergic antagonist) and 20 mg/kg haloperidol (a non-selective dopaminergic antagonist) did not affect the extract's antinociception. The phytochemicals screening revealed the presence of saponins, flavonoids, tannins and triterpenes while the HPLC analysis showed the presence of flavonoid-based compounds. CONCLUSIONS: The antinociceptive activity of MEMC involved activation of the non-selective opioid (particularly the µ-, δ- and κ-opioid) and non-opioid (particularly adenosinergic, α2-noradrenergic, and ß-adrenergic) receptors, modulation of the ATP-sensitive K+ channel, and inhibition of bradikinin and protein kinase C actions. The discrepancies in MEMC antinociception could be due to the presence of various phytochemicals.

Antiulcer activity of Muntingia calabura leaves involves the modulation of endogenous nitric oxide and nonprotein sulfhydryl compounds.

Abstract Context: Muntingia calabura L. (Muntingiaceae) is a native plant species of the American continent and is widely cultivated in warm areas in Asia, including Malaysia. The plant is traditionally used to relieve pain from gastric ulcers. Objective: This study was designed to determine the antiulcer activity of a methanol extract of M. calabura leaves (MEMC) and the possible mechanisms of action involved. Materials and methods: An acute toxicity study was conducted using a single oral dose of 2000 mg/kg MEMC. The antiulcer activity of MEMC was evaluated in absolute ethanol- and indomethacin-induced gastric ulcer rat models. MEMC was administered orally (dose range 25-500 mg/kg) to rats fasted for 24 h. The animals were pretreated with NG-nitro-l-arginine methyl esters (l-NAME) or N-ethylmaleimide (NEM) prior to MEMC treatment to assess the possible involvement of endogenous nitric oxide (NO) and nonprotein sulfhydryl (NP-SH) compounds in the gastroprotective effect of MEMC. Results: As the administered dose did not cause toxicity in the rats, the oral median lethal dose (LD50) of MEMC was >2000 mg/kg in rats. MEMC exerted significant (p < 0.001) gastroprotective activity in the ethanol- and indomethacin-induced ulcer models dose-dependently. Histological evaluation supported the observed antiulcer activity of MEMC. l-NAME and NEM pretreatment significantly (p < 0.05) reversed and abolished the gastroprotective effect of MEMC, respectively. Discussion and conclusion: The results obtained indicate that MEMC has significant antiulcer activity that might involve the participation of endogenous NO and NP-SH compounds. These findings provide new pharmacological information regarding the potential use of M. calabura.

Modulation of POPDC1 Expression by Phenothiazine and Trifluoperazine Suppress Colon Cancer Growth and Migration.

OBJECTIVE: The aim of this study was to investigate the effects of CaM antagonist, PTZ, and TFP on cell proliferation and migration of colon cancer cells and its impact on POPDC protein expression. METHODS: The 50% inhibitory concentration (IC50) of PTZ and TFP in SW1116, SW480, HCT-15, and COLO205 colon cancer cell lines are measured using MTT. Western blot and immunocytochemistry were used to determine the expression of PCNA, cyclin D1 (CD1), and POPDC proteins. Cell migration was observed using a scratch wound-healing assay. RESULTS: Treatment with PTZ and TFP inhibited colon cancer cells growth in a dose-dependent manner. PTZ and TFP significantly inhibited the activation of proliferation markers, PCNA and CD1, and the migration of colon cancer cells. Furthermore, POPDC protein was significantly suppressed in all cell types of colon cancer, particularly in SW480. Finally, the CaM antagonist upregulates the POPDC1 expression in colon cancer cells. CONCLUSION: These findings suggest that CaM antagonists suppress colon cancer cells proliferation via downregulation of CD1 and PCNA. In addition, POPDC protein could be used as a biomarker in colon cancer, and CaM antagonist could be used to regulate POPDC1 expression. This study suggests that targeting POPDC1 with CaM inhibition could be a potential therapeutic strategy for colon cancer treatment. 
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Antinociceptive Activity of Petroleum Ether Fraction of Clinacanthus nutans Leaves Methanolic Extract: Roles of Nonopioid Pain Modulatory Systems and Potassium Channels.

Methanolic extract of Clinacanthus nutans Lindau leaves (MECN) has been reported to exert antinociceptive activity. The present study aimed to elucidate the possible antinociceptive mechanisms of a lipid-soluble fraction of MECN, which was obtained after sequential extraction in petroleum ether. The petroleum ether fraction of C. nutans (PECN), administered orally to mice, was (i) subjected to capsaicin-, glutamate-, phorbol 12-myristate 13-acetate-, bradykinin-induced nociception model; (ii) prechallenged (intraperitoneal (i.p.)) with 0.15 mg/kg yohimbine, 1 mg/kg pindolol, 3 mg/kg caffeine, 0.2 mg/kg haloperidol, or 10 mg/kg atropine, which were the respective antagonist of α 2-adrenergic, ß-adrenergic, adenosinergic, dopaminergic, or muscarinic receptors; and (iii) prechallenged (i.p.) with 10 mg/kg glibenclamide, 0.04 mg/kg apamin, 0.02 mg/kg charybdotoxin, or 4 mg/kg tetraethylammonium chloride, which were the respective inhibitor of ATP sensitive-, small conductance Ca2+-activated-, large conductance Ca2+-activated-, or nonselective voltage-activated-K+ channel. Results obtained demonstrated that PECN (100, 250, and 500 mg/kg) significantly (P<0.05) inhibited all models of nociception described earlier. The antinociceptive activity of 500 mg/kg PECN was significantly (P<0.05) attenuated when prechallenged with all antagonists or K+ channel blockers. However, only pretreatment with apamin and charybdotoxin caused full inhibition of PECN-induced antinociception. The rest of the K+ channel blockers and all antagonists caused only partial inhibition of PECN antinociception, respectively. Analyses on PECN's phytoconstituents revealed the presence of antinociceptive-bearing bioactive compounds of volatile (i.e., derivatives of γ-tocopherol, α-tocopherol, and lupeol) and nonvolatile (i.e., cinnamic acid) nature. In conclusion, PECN exerts a non-opioid-mediated antinociceptive activity involving mainly activation of adenosinergic and cholinergic receptors or small- and large-conductance Ca2+-activated-K+ channels.

Antinociceptive Activity of Methanolic Extract of Clinacanthus nutans Leaves: Possible Mechanisms of Action Involved.

Methanolic extract of Clinacanthus nutans Lindau leaves (MECN) has been proven to possess antinociceptive activity that works via the opioid and NO-dependent/cGMP-independent pathways. In the present study, we aimed to further determine the possible mechanisms of antinociception of MECN using various nociceptive assays. The antinociceptive activity of MECN was (i) tested against capsaicin-, glutamate-, phorbol 12-myristate 13-acetate-, bradykinin-induced nociception model; (ii) prechallenged against selective antagonist of opioid receptor subtypes (ß-funaltrexamine, naltrindole, and nor-binaltorphimine); (iii) prechallenged against antagonist of nonopioid systems, namely, α2-noradrenergic (yohimbine), ß-adrenergic (pindolol), adenosinergic (caffeine), dopaminergic (haloperidol), and cholinergic (atropine) receptors; (iv) prechallenged with inhibitors of various potassium channels (glibenclamide, apamin, charybdotoxin, and tetraethylammonium chloride). The results demonstrated that the orally administered MECN (100, 250, and 500 mg/kg) significantly (p < 0.05) reversed the nociceptive effect of all models in a dose-dependent manner. Moreover, the antinociceptive activity of 500 mg/kg MECN was significantly (p < 0.05) inhibited by (i) antagonists of µ-, δ-, and κ-opioid receptors; (ii) antagonists of α2-noradrenergic, ß-adrenergic, adenosinergic, dopaminergic, and cholinergic receptors; and (iii) blockers of different K+ channels (voltage-activated-, Ca2+-activated, and ATP-sensitive-K+ channels, resp.). In conclusion, MECN-induced antinociception involves modulation of protein kinase C-, bradykinin-, TRVP1 receptors-, and glutamatergic-signaling pathways; opioidergic, α2-noradrenergic, ß-adrenergic, adenosinergic, dopaminergic, and cholinergic receptors; and nonopioidergic receptors as well as the opening of various K+ channels. The antinociceptive activity could be associated with the presence of several flavonoid-based bioactive compounds and their synergistic action with nonvolatile bioactive compounds.

Methanolic Extract of Clinacanthus nutans Exerts Antinociceptive Activity via the Opioid/Nitric Oxide-Mediated, but cGMP-Independent, Pathways.

The objectives of the present study were to determine the mechanisms of antinociceptive effect of methanol extract of Clinacanthus nutans (Acanthaceae) leaves (MECN) using various animal nociceptive models. The antinociceptive activity of orally administered 10% DMSO, 100 mg/kg acetylsalicylic acid (ASA), 5 mg/kg morphine, or MECN (100, 250, and 500 mg/kg) was determined using the acetic acid-induced abdominal constriction (ACT), formalin-induced paw licking (FT), and hot plate tests (HPT). The role of opioid and nitric oxide/cyclic guanosine monophosphate (NO/cGMP) systems was also investigated. The results showed that MECN produced a significant (p < 0.05) antinociceptive response in all nociceptive models with the recorded ED50 value of 279.3 mg/kg for the ACT, while, for the early and late phases of the FT, the value was >500 mg/kg or 227.7 mg/kg, respectively. This antinociceptive activity was fully antagonized by naloxone (a nonselective opioid antagonist) but was partially reversed by l-arginine (l-arg; a nitric oxide [NO] precursor), Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME; an NO synthase inhibitor), or their combinations thereof. In contrast, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ; a soluble guanylyl cyclase inhibitor) enhanced the extract's antinociception. UHPLC analysis revealed the presence of several flavonoid-based compounds with antinociceptive action. In conclusion, MECN exerted the peripherally and centrally mediated antinociceptive activity via the modulation of the opioid/NO-mediated, but cGMP-independent, systems.

Antinociceptive effect of semi-purified petroleum ether partition of Muntingia calabura leaves

ABSTRACT Muntingia calabura L., Muntingiaceae, is a medicinal plant for various pain-related diseases. The aims of the present study were to determine the antinociceptive profile and to elucidate the possible mechanisms of antinociception of petroleum ether partition obtained from crude methanol extract of M. calabura leaves using various animal models. The antinociceptive profile of petroleum ether fraction (given oral; 100, 250 and 500 mg/kg) was established using the in vivo chemicals (acetic acid-induced abdominal constriction and formalin-induced paw licking test) and thermal (hot plate test) models of nociception. The role of glutamate, TRPV1 receptor, bradykinin, protein kinase C, potassium channels, and various opioid and non-opioid receptors in modulating the partition's antinociceptive activity was also determined. The results obtained demonstrated that petroleum ether partition exerted significant (p < 0.05) antinociception in all the chemicals-, thermal-, capsaicin-, glutamate-, bradykinin, and phorbol 12-myristate 13-acetate (PMA)-induced nociception models. The antinociceptive activity was reversed following pretreatment with opioid antagonists (i.e. naloxone, β-funaltrexamine, naltrindole and nor-binaltorphimine), and the non-opioid receptor antagonists (i.e. pindolol (a β-adrenoceptor), haloperidol (a non-selective dopaminergic), atropine (a non-selective cholinergic receptor), caffeine (a non-selective adenosinergic receptor), and yohimbine (an α2-noradrenergic)). In addition, pretreatment with L-arginine (a nitric oxide (NO) donor), NG-nitro-L-arginine methyl esters (L-NAME; an inhibitor of NO synthase (NOS)), methylene blue (MB; an inhibitor of cyclic-guanosine monophosphate (cGMP) pathway), or their combination failed to inhibit petroleum ether partition's antinociception. In conclusion, petroleum ether partition exerts antinociceptive activity at the peripheral and central levels via the modulation of, partly, the opioid (i.e. µ, κ and δ) and several non-opioids (i.e. β-adrenergic, dopaminergic, cholinergic, adenosinergic, and α2-noradrenergic) receptors, glutamatergic, TRPV1 receptors, PKC and K+ channels systems, but not L-arg/NO/cGMP pathway.