Analysis of phytochemical in a Malaysian medicinal plant and the bioavailability of dietary hydroxycinnamates

The first study was looking into the phytochemical analysis of herbal medicinal plant from Malaysia. Phenolic compounds in an aqueous infusion from leaves of Ficus deltoidea (Moraceae), a well-known herbal tea in Malaysia, were analysed by HPLC coupled to photodiode-array and fluorescence detectors and an electrospray ionization tandem mass spectrometer. Following chromatography of extracts on a reverse phase C12 column 25 flavonoids were characterized and/or tentatively identified, with the main constituents being flavan-3-ol monomers, proanthocyanidins and C-linked flavone glycosides. The proanthocyanidins were dimers and trimers comprising (epi)catechin and (epi)afzelechin units. No higher molecular weight proanthocyanidin polymers were detected. The antioxidant activity of F. deltoidea extract was analysed using HPLC with on-line antioxidant detection. This revealed that 85% of the total antioxidant activity of the aqueous F. deltoidea infusion was attributable to the flavan-3-ol monomers and the proanthocyanidins In terms of the bioavailability of hydroxycinnamates with the high occurrence of p-coumaroylquinic acid in F. deltoidea extract, further study was carried out using radiolabeled caffeic acid. In the study, male Sprague-Dawley rats ingested 140 x 106 dpm of [3-14C]trans-caffeic acid and over the ensuing 72 h period body tissues, plasma, urine and faeces were collected and the overall levels of radioactivity determined. Where sufficient radioactivity had accumulated samples were analysed by HPLC with on-line radioactivity and tandem mass spectrometric detection. Nine labeled compounds were identified, the substrate and its cis isomer, 3-O- and 4-O-sulphates and glucuronides of caffeic acid, 4-O-sulphates and glucuronides of ferulic acid and isoferulic acid-4-O-sulphate. Four unidentified metabolites were also detected. After passing down the gastrointestinal tract the majority of the radiolabeled metabolites were excreted in urine with minimal accumulation in plasma. Only relatively small amounts of unidentified 14C-labeled metabolites were expelled in faeces. There was little or no accumulation of radioactivity in body tissues, including the brain. The overall recovery of radioactivity 72 h after ingestion of [3-14C]caffeic acid was ~80% of intake. The role of colonic microflora in the metabolism of caffeic acid was carried using an in vitro model of the human colonic microflora. Caffeic acid (55 µmoles) was incubated with human faecal materials obtained from five Asian donors, and caffeic acid degradation was monitored from 0-8 h. Faecal samples were analysed by GC-MS, where major phenolic acids identified were dihydrocaffeic acid, 3-(3-hydroxyphenyl)propionic acid and phenylacetic acid. Caffeic acid was quickly degraded by the colonic microflora, as it disappeared after 4 h of incubation in two of the faecal samples. The degree of degradation of caffeic acid was significantly influenced by the addition of glucose as well as individual variations in the density and the composition of microflora. These findings support extensive metabolism of caffeic acid in the colon, depending on the substrate concentration and the supplement of glucose which resulting the formation of simple phenolics

Endogenous antioxidant and LOX-mediated systems contribute to the hepatoprotective activity of aqueous partition of methanol extract of Muntingia calabura L. leaves against paracetamol intoxication =

Methanol extract of Muntingia calabura L. (family Muntingiaceae) leaf has been reported to exert various pharmacological activities including hepatoprotection. The present study was carried out to identify the most effective hepatoprotective partition derived from the extract and to determine the mechanisms of action involved. The extract was partitioned using solvents with different polarity to yield petroleum ether (PEMC), ethyl acetate (EAMC), and aqueous (AQMC) extracts. Each extract, at 250 mg/kg, was subjected to the paracetamol (PCM)-induced hepatotoxic assay and several parameters such as liver weight, liver/body weight ratio, serum liver enzymes' level, and histopathological examinations were determined. Each partition was also tested for their antioxidant and anti-inflammatory potentials. The most effective extract (AQMC) was prepared in additional dose of 50 and 500 mg/kg, and then subjected to the same liver toxicity test in addition to the endogenous antioxidant enzymes assay. Moreover, AQMC was also subjected to the phytochemical screening and HPLC analysis. Overall, from the results obtained: AQMC exerted significant (p < 0.05): (i) antioxidant activity when assessed using the DPPH, SOD and ORAC assays with high TPC detected; (ii) anti-inflammatory activity via LOX, but not XO pathway; (iii) hepatoprotective activity indicated by its ability to reverse the effect of PCM on the liver weight and liver/body weight ratio, the level of serum liver enzymes (ALT, AST, and ALP), and activity of several endogenous antioxidant enzymes (SOD and CAT). Phytochemicals analyses demonstrated the presence of several flavonoid-based bioactive compounds such as gallic acid and quercetin, which were reported to possess hepatoprotective activity. In conclusion, AQMC exerts hepatoprotective activity against the PCM-induced toxicity possibly by having a remarkable antioxidant potential and ability to activate the endogenous antioxidant system possibly via the synergistic action of its phytoconstituents

Antinociceptive Activity of Methanolic Extract of Clinacanthus nutans

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 (β-funaltrexamine, naltrindole, and nor-binaltorphimine); (iii) prechallenged against antagonist of nonopioid systems, namely, α2-noradrenergic (yohimbine), β-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, β-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, β-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

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 β–funaltrexamine (β–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) affect the locomotor activity of treated mice. The antinociceptive activity of PECN was significantly (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

Antinoceptive 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 (β-funaltrexamine, naltrindole, and nor-binaltorphimine); (iii) prechallenged against antagonist of nonopioid systems, namely, α2-noradrenergic (yohimbine), β-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 () reversed the nociceptive effect of all models in a dose-dependent manner. Moreover, the antinociceptive activity of 500 mg/kg MECN was significantly () inhibited by (i) antagonists of μ-, δ-, and κ-opioid receptors; (ii) antagonists of α2-noradrenergic, β-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, β-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

Methanol extract of Muntingia calabura leaves attenuates CCl4-induced liver injury: possible synergistic action of flavonoids and volatile bioactive compounds on endogenous defence system

Context: Muntingia calabura L. (Muntingiaceae) exerts antioxidant and anti-inflammatory activities, thus, it might be a good hepatoprotective agent. Objective: This study investigates the effect of methanol extract of M. calabura leaves (MMCL) on hepatic antioxidant and anti-inflammatory activities in CCl4-induced hepatotoxic rat. Materials and methods: Sprague Dawley rats (n = 6) were treated (p.o.) with 10% DMSO (Groups 1 and 2), 50 mg/kg N-acetylcysteine (Group 3) or, 50, 250, or 500 mg/kg MMCL (Groups 4–6) for 7 consecutive days followed by pretreatment (i.p.) with vehicle (Group 1) or 50% CCl4 in olive oil (v/v) (Groups 2–6) on day 7th. Plasma liver enzymes and hepatic antioxidant enzymes and pro-inflammatory cytokines concentrations were measured while liver histopathology was examined. Results: MMCL, at 500 mg/kg, significantly (p < 0.05) ameliorated CCl4-induced hepatotoxicity by decreasing the plasma level of alanine transaminase (429.1 versus 168.7 U/L) and aspartate transaminase (513.8 versus 438.1 U/L) as well as the tissue level of nitric oxide (62.7 versus 24.1 nmol/g tissue). At 50, 250, or 500 mg/kg, MMCL significantly (p < 0.05) reduced the tumour necrosis factor α (87.8 versus 32.7 pg/mg tissue), interleukin-1β (1474.4 versus 618.3 pg/mg tissue), and interleukin-6 (136.7 versus 30.8 pg/mg tissue) while increased the liver catalase (92.1 versus 114.4 U/g tissue) and superoxide dismutase (3.4 versus 5.5 U/g tissue). Additionally, qualitative phytochemicals analysis showed that MMCL contained gallic acid, ferulic acid, quercetin, and genistein. Discussion and conclusions: MMCL ability to attenuate CCl4-induced hepatotoxicity could be helpful in the development of hepatoprotective agents with fewer side effects

Methanol extract of Dicranopteris linearis leaves attenuate pain via the modulation of opioid/NO-mediated pathway

Dicranopteris linearis leaf has been reported to exert antinociceptive activity. The present study elucidates the possible mechanisms of antinociception modulated by the methanol extract of D. linearis leaves (MEDL) using various mouse models. The extract (25, 150, and 300 mg/kg) was administered orally to mice for 30 min priot to subjection to the acetic acid-induced writhing-, hot plate- or formalin-test to establish the antinociceptive profile of MEDL. The most effective dose was then used in the elucidation of possible mechanisms of action stage. The extract was also subjected to the phytochemical analyses. The results confirmed that MEDL exerted significant (p < 0.05) antinociceptive activity in those pain models as well as the capsaicin-, glutamate-, bradykinin- and phorbol 12-myristate 13-acetate (PMA)-induced paw licking model. Pretreatment with naloxone (a non-selective opioid antagonist) significantly (p < 0.05) reversed MEDL effect on thermal nociception. Only l-arginine (a nitric oxide (NO) donor) but not N(ω)-nitro-l-arginine methyl ester (l-NAME; a NO inhibitor) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a specific soluble guanylyl cyclase inhibitor) significantly (p < 0.05) modified MEDL effect on the writhing test. Several polyphenolics and volatile antinociceptive compounds were detected in MEDL. In conclusion, MEDL exerted the opioid/NO-mediated antinociceptive activity, thus, justify D. linearis as a potential source for new analgesic agents development

Analysis of Flavone C

Clinacanthus nutans (family Acanthaceae) has been used for the treatment of inflammation and herpes viral infection. Currently, there has not been any report on the qualitative and quantitative determination of the chemical markers in the leaves of C. nutans. The C-glycosidic flavones such as shaftoside, isoorientin, orientin, isovitexin, and vitexin have been found to be major flavonoids in the leaves of this plant. Therefore, we had developed a two-step method using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC) for the rapid identification and quantification of the flavones C-glycosides in C. nutans leaves. The TLC separation of the chemical markers was achieved on silica gel 60 plate using ethyl acetate : formic acid : acetic acid : water (100 : 11 : 11 : 27 v/v/v/v) as the mobile phase. HPLC method was optimized and validated for the quantification of shaftoside, orientin, isovitexin, and vitexin and was shown to be linear in concentration range tested (0.4–200 μg/mL, r2 ≥ 0.996), precise (RSD ≤ 4.54%), and accurate (95–105%). The concentration of shaftoside, orientin, vitexin, and isovitexin in C. nutans leave samples was 2.55–17.43, 0.00–0.86, 0.00–2.01, and 0.00–0.91 mmol/g, respectively