Pharmacokinetics-based elucidation on disparity in clinical effectiveness between varieties of Zhi Zhu Wan,a Traditional Chinese Medical formula

Aim

Zhi Zhu Wan (ZZW) is a classical Chinese medical formulation used for the treatment of functional dyspepsia that attributed to Spleen-deficiency Syndrome. ZZW contains Atractylodes Rhizome and Fructus Citrus Immaturus, the later originates from both Citrus aurantium L. (BZZW) and Citrus sinensis Osbeck (RZZW). The present study is designed to elucidate disparities in the clinical efficacy of two ZZW varieties based on the pharmacokinetics of naringenin and hesperetin.

Mehtod

After oral administration of ZZWs, blood sample was collected from healthy volunteers at designed time points. Naringenin and hesperetin were detected in plasma by RP-HPLC, pharmacokinetic parameters were processed using mode-independent methods with WINNONLIN.

Results

After oral administration of BZZW, both naringenin and hesperetin were detected in plasma, and demonstrated similar pharmacokinetic parameters. Ka was 0.384 ± 0.165 and 0.401 ± 0.159, T_1/2(ke)(h) was 5.491 ± 3.926 and 5.824 ± 3.067, the AUC (mg/L h) was 34.886 ± 22.199 and 39.407 ± 19.535 for naringenin and hesperetin, respectively. However, in the case of RZZW, only hesperetin was found in plasma, but the pharmacokinetic properties for hesperetin in RZZW was different from that in BZZW. T_max for hesperetin in RZZW is about 8.515 h, and its C_max is much larger than that of BZZW. Moreover, it was eliminated slowly as it possessed a much larger AUC value.

Conclusion

The distinct therapeutic orientations of the Chinese medical formula ZZWs with different Fructus Citrus Immaturus could be elucidated based on the pharmacokinetic parameters of constituents after oral administration.     

A simple high-performance liquid chromatographic method for the determination of brazilin and its application to a pharmacokinetic study in rats

Ethnopharmacological relevance

Caesalpinia sappan is a medicinal plant native to China popularly used to treat chronic pelvic inflammation, dysmenorrhea and hysteromyoma. Its main bioactive component is brazilin which had presented antibacterial, anti-inflammatory and anti-platelet aggregation activities. To establish a sensitive, selective, reproducible, and accurate high performance liquid chromatographic (HPLC) method for the quantitative determination of brazilin in plasma, and study the pharmacokinetics of brazilin in rats after intravenous administration of brazilin.

Materials and methods

Rats received intravenous injection of 25, 50 and 100 mg/kg of brazilin. Concentrations of brazilin in plasma were determined by HPLC method at different time points and all pharmacokinetic parameters were estimated by non-compartmental analysis with WinNonLin 6.2 software.

Results

After single intravenous doses of 25, 50 and 100 mg/kg brazilin in rats, the main PK parameters were as follows: C_max were 18.1±4.1, 46.7±8.7 and 82.2±9.6 µg/mL; AUC_0–24 were 20.4±4.3, 48.7±6.8 and 90.4±10.3 µg h/mL; and t_1/2 were 5.4±1.5, 5.8±0.9 and 6.2±1.2 h, respectively.

Conclusion

It showed that the brazilin was eliminated moderately in rat by intravenous injection route with t_1/2 of 6 h and showed a dose-dependence profile of C_max and AUC_0–24 at the doses of 25~100 mg/kg of brazilin for injection in rats.     

Bioguided isolation of myricetin-3-O-β-galactopyranoside with antinociceptive activity from the aerial part of Davilla elliptica St.-Hil

Ethnopharmacological relevance

Davilla elliptica St.-Hil. (Dilleniaceae) is a medicinal plant traditionally used in Brazil to treat inflammatory processes, to relieve pain, as diuretic, gastro- and hepatoprotective agents.

Aim of the study

To undertake the fractionation of the ethanolic extract from Davilla elliptica leaves guided by an antinociceptive assay.

Materials and methods

The antinociceptive activity was evaluated through the formalin test in mice. Extract fractionation was performed by percolation through silica gel and partition between immiscible solvents, followed by successive column chromatography over Sephadex LH-20 and preparative RP-HPLC. Structure elucidation of the isolated compound was accomplished by spectroscopic data.

Results

The EtOAc and MeOH fractions derived from the crude extract reduced significantly the licking time in the late phase of the formalin test. The bioguided fractionation of the MeOH fraction resulted in the isolation of myricetin-3-O-β-galactopyranoside, which produced significant inhibition on nociception induced by formalin (ID₅₀=0.26 mg/kg; p.o.).

Conclusions

These results point out that myricetin-3-O-β-galactopyranoside contributes for the antinociceptive effect of Davilla elliptica extract, a constituent considerably more potent than diclofenac, employed as reference drug.     

Antinociceptive activity of Amaranthus spinosus in experimental animals

Aim of the study

50% ethanol extract (ASE) of Amaranthus spinosus (whole plant) has been evaluated for antinociceptive and antiinflammatory activities.

Materials and methods

Analgesic and antiinflammatory activities were studied by measuring nociception by formalin, acetic acid, hot plate, tail immersion method while inflammation was induced by carrageenan.

Results

ASE had significant dose dependent percentage protection against acetic acid (0.6% of 10 ml) induced pain and the effects were also compared to aspirin, morphine and naloxone while formalin induced pain (0.05 ml of 2.5%) was significantly blocked only at higher dose (400 mg/kg) in first phase. ASE significantly blocked pain emanating from inflammation at all the doses in second phase. The reaction time in hot plate was increased significantly and dose dependently where as pretreatment with naloxone rigorously reduced the analgesic potentials of ASE. Further in tail immersion test the same dose dependent and significant activity was observed. Aspirin had no effect on thermal induced pain i.e. hot plate and tail immersion tests but showed an effect on writhing test.

Conclusions

Our investigation show that Amaranthus spinosus possess significant and dose dependant antiinflammatory activity, it has also central and peripheral analgesic activity.     

Artemisia copa aqueous extract as vasorelaxant and hypotensive agent

Ethnopharmacological relevance

Artemisia copa Phil. (Asteraceae) is a medicinal plant commonly used in traditional medicine in Argentina.

Aim of the study

The vasorelaxant and hypotensive activities of the aqueous extract of Artemisia copa have been investigated.

Materials and methods

The in vitro effect of the extract and isolated compounds from Artemisia copa was investigated using isolated rat aortic rings. The acute effect caused by the intravenous (i.v.) infusion (0.1–300 mg/kg) on blood pressure and heart rate was evaluated in spontaneous hypertensive rats. In addition, a phytochemical analysis of the extract was performed by HPLC.

Results

Artemisia copa had a relaxant effect in endothelium-intact aortic rings that had been pre-contracted with 10⁻⁷ M phenylephrine (E_max=96.7±1.3%, EC₅₀=1.1 mg/ml), 10⁻⁵ M 5-hydroxytriptamine (E_max=96.7±3.5%, EC₅₀=1.5 mg/ml) and 80 mM KCl (E_max=97.9± 4.4%, EC₅₀=1.6 mg/ml). In denuded aortic rings contracted by phenylephrine, a similar pattern was observed (E_max=92.7±6.5%, EC₅₀=1.8 mg/ml). l-NAME, indomethacin, tetraethylammonium and glibenclamide were not able to block the relaxation induced by the extract. Nevertheless, the pre-treatment with Artemisia copa attenuated the CaCl₂-induced contraction in a concentration-dependent manner (E_max: 86% of inhibition for 3 mg/ml and 52% de-inhibition for 1 mg/ml). This pre-treatment also induced a significant attenuation of the norepinephrine-induced contraction in a concentration-dependent manner (E_max: 72.7% of inhibition for 3 mg/ml and 27% de inhibition for 1 mg/ml) in a Ca²⁺ free medium. Upon analyzing the composition of the extract, the presence of p-coumaric acid, isovitexin, luteolin and chrysoeriol were found. Luteolin (CE₅₀: 1.5 μg/ml), chrysoeriol (CE₅₀: 13.2 μg/ml) and p-coumaric acid (CE₅₀: 95.2 μg/ml), isolated from the aqueous extract, caused dilatation of thoracic aortic rings pre-contracted with phenylephrine. Artemisia copa administered i.v. also induced a decrease in the mean arterial pressure but did not affect the heart rate in hypertensive rats.

Conclusions

The aqueous extract of Artemisia copa proved to have vasorelaxing and hypotensive effects through the inhibition of Ca²⁺ influx via membranous calcium channels and intracellular stores. The presence of luteolin, chrysoeriol and p-coumaric acid found in this plant could be involved in this effect.     

α-Glucosidase inhibitory activity of selected Philippine plants

Ethnopharmacological relevance

Antidesma bunius Spreng. (Phyllantaceae), Averrhoa bilimbi L. (Oxalidaceae), Biophytum sensitivum (L.) DC. (Oxalidaceae), Ceriops tagal (Perr.) C.B. Rob. (Rhizophoraceae), Kyllinga monocephala Rottb. (Cyperaceae), and Rhizophora mucronata Lam. (Rhizophoraceae) are used as remedies to control diabetes. In the present study, these plants were screened for their potential α-glucosidase inhibitory activity.

Materials and methods

The 80% aqueous ethanolic extracts were screened for their α-glucosidase enzyme inhibitory activity using yeast alpha glucosidase enzyme.

Results

Except for A. bilimbi with IC₅₀ at 519.86±3.07, all manifested a significant enzyme inhibitory activity. R. mucronata manifested the highest activity with IC₅₀ at 0.08±1.82 μg mL⁻¹, followed by C. tagal with IC₅₀ at 0.85±1.46 μg mL⁻¹ and B. sensitivum with IC₅₀ at 2.24±1.58 μg mL⁻¹.

Conclusion

This is the first report on the α-glucosidase inhibitory effect of the six Philippine plants; thus, partly defining the mechanism on why these medicinal plants possess antidiabetic properties.     

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 100 mg/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.     

Antiinflammatory and neurological activity of pyrithione and related sulfur-containing pyridine N-oxides from Persian shallot (Allium stipitatum)

Ethnopharmacological relevance

Persian shallot (Allium stipitatum) is a bulbous plant native to Turkey, Iran and Central Asia. It is frequently used in folk medicine for the treatment of a variety of disorders, including inflammation and stress. Antiinflammatory and neurological activities of pyrithione and four related sulfur-containing pyridine N-oxides which are prominent constituents of Allium stipitatum were tested.

Methods

The antiinflammatory activity was tested by the ability of the compounds to inhibit cyclooxygenase (COX-1 and COX-2), whereas the neurological activities were evaluated by assessing the compounds ability to inhibit monoamine oxidase-A (MAO-A) and acetylcholinesterase (AChE). The compounds? affinity for the serotonin transport protein (SERT) and the GABA_A–benzodiazepine receptor were also investigated.

Results

2-[(Methylthio)methyldithio]pyridine N-oxide showed very high antiinflammatory effects which are comparable with those of common pharmaceuticals (IC₅₀ of 7.8 and 15.4 µM for COX-1 and COX-2, respectively). On the other hand, neurological activities of the compounds were rather modest. Some compounds moderately inhibited AChE (IC₅₀ of 104–1041 µM) and MAO-A (IC₅₀ of 98–241 µM) and exhibited an affinity for the SERT and GABA_A–benzodiazepine receptor.

Conclusions

Our findings may help to rationalize the wide use of Persian shallot for the treatment of inflammatory disorders.     

Antiinflammatory and antinociceptive activities of Blechnum occidentale L. extract

Aim of study

Blechnum occidentale L. is a terrestrial fern that ranges from the United States to South America, and is employed in Brazilian folk medicine. In the present study we investigated the antinociceptive and antiinflammatory activities of the methanolic extract of Blechnum occidentale L. (MEB) in animal models of pain and inflammation to support its medicinal use in treatment of inflammatory and pulmonary diseases, urinary infections and liver diseases.

Materials and methods

The antinociceptive activity of MEB was evaluated using the writhing, formalin, and tail flick tests. The antiinflammatory activity of MEB was evaluated in carrageenan-induced paw oedema and neutrophil migration. In order to discard possible non-specific muscle relaxant or sedative effects of MEB, mice motor performance was evaluated in the rota rod test and its toxicity evaluated over 14 days.

Results

Intraperitoneal (IP) administration of MEB (0.01–100 mg/kg) produced a dose-related antinociception on acetic acid-induced writhing in mice. Oral administration of MEB, at a different range of doses (100–400 mg/kg), also produced significant antinociceptive effect on the writhing test. Furthermore, treatment with MEB (100 and 200 mg/kg IP) inhibited significantly both the early and late phases of formalin-induced hypernociception in rats. In contrast, treatment with MEB (100 and 200 mg/kg IP) did not prevent the thermal nociception in the tail flick test. The IP administration of MEB (100 and 300 mg/kg) significantly reduced the paw oedema induced by carrageenan. Moreover, systemic treatment with MEB (11–300 mg/kg) reduced the neutrophil migration in the carrageenan-induced migration to the peritoneal cavity. In the rota rod test, MEB-treated mice did not show any significant motor performance alterations with the dose of 300 mg/kg. In addition, over the study duration of 14 days, there were no deaths or toxic signs recorded in the mice given 100 or 1000 mg/kg of MEB.

Conclusion

The results described here are the first report of pharmacological studies of Blechnum occidentale L. and indicate that this plant has antinociceptive and antiinflammatory activities which support its folk medicine use.     

Sesquiterpene lactones with antinociceptive and antipyretic activity from two Centaurea species

Ethnopharmacological relevance

Several Centaurea species are used to alleviate pain and inflammatory symptoms in rheumatoid arthritis, high fever, and head ache in Turkish folk medicine.

Aim of the study

The effectiveness of extracts, fractions and subfractions from dried Centaurea solstitialis L. subsp. solstitialis (CSS) (Asteraceae) roots and aerial parts were studied on mice.

Materials and methods

The antinociceptive and antipyretic effects of Centaurea solstitialis L. subsp. solstitialis have been investigated by using p-benzoquinone-induced writhing reflex for antinociceptive activity and Freund's Complete Adjuvant-induced pyrexia model for antipyretic activity assessment in mice.

Results

The ethanolic extract from the aerial parts of the plant was shown to possess significant antinociceptive (p < 0.01) and antipyretic activities (p < 0.01). The extract was then submitted to subsequent solvent extractions and chromatographic processes. Through bioassay-guided fractionation and isolation procedures two sesquiterpene lactones, solstitialin A and acetyl solstitialin, were isolated and defined as the active components of CSS. On the other hand, a comparative study was conducted on another species, Centaurea depressa Bieb., which has no similar folkloric utilization. Following the same fractionation chart same compounds were defined as the active ingredients.

Conclusion

Results of the present study proved that aerial part of CSS possesses antinociceptive and antipyretic activities supporting the folkloric assertion in Turkish folk medicine. However, these effects seem not limited to CSS, some other Centaurea species, in fact, having no folkloric use might be equally active.     

Investigation on modulation of human P-gp by multiple doses of Radix Astragali extract granules using fexofenadine as a phenotyping probe

Ethnopharmacology

Herb–drug interactions may potentially affect drug efficacy and/or the likelihood of adverse drug reactions. Radix Astragali (RA) extract formulation is usually prescribed for long-term use for patients with immunodeficiency, diabetes, nephropathy or cardiovascular diseases. Its use in combination with P-glycoprotein (P-gp) substrates is possible in clinical practice. Currently there is little knowledge about whether concomitant use of RA extract has an influence on disposition of P-gp substrate.

Aim of the study

This study was to investigate whether continuous and multiple doses of RA extract granules had modulatory effects on human P-gp.

Material and methods

A randomised, placebo-controlled, two-period crossover pharmacokinetic drug interaction study was conducted in healthy Chinese volunteers. Fexofenadine was used as a P-gp phenotyping probe. Fourteen volunteers received RA extract granules or placebo (4 g bid) for 7 days and then received a single oral dose of 120 mg fexofenadine. Fexofenadine plasma concentrations were determined by HPLC. Pharmacokinetic parameters were calculated by non-compartmental method and bioequivalence evaluation was performed.

Results

Pharamcokinetic parameters in the placebo phase were as follows: T_1/2 (3.75±1.47 h), C_max (745.11±137.41 μg/L), T_max (2.25±0.47 h), AUC_(0−t) (3894.27±923.45 μg h/L), AUC_(0−∞) (3993.84±912.97 μg h/L). Pharamcokinetic parameters in the RA extract phase were as follows: T_1/2 (4.00±1.24 h), C_max (709.44±170.03 μg/L), T_max (2.21±0.51 h), AUC_(0−t) (3832.72±1077.60 μg h/L), AUC_(0−∞) (3983.53±1019.83 μg h/L). The influence of RA extract on fexofenadine C_max and AUC lacks statistical significance. Fexofenadine in the two phases were bioequivalent. In the placebo phase, T_1/2 of fexofenadine in ABCB1 3435T mutation allele carriers was longer compared to ABCB1 3435CC carriers (4.43±1.44 h vs. 2.54±0.21 h, p<0.05). However, RA extract pretreatment abolished such genotype-related difference due to the lengthened T_1/2 in ABCB1 3435CC carriers. There was no association of the C3435T polymorphism with C_max and AUC_(0−t) in subjects with two pretreatments.

Conclusion

One-week administration of RA extract granules did not have a statistically significant impact on systematic exposure to fexofenadine, suggesting that RA extract is not a potent modulator of P-gp in vivo. RA extract appears to have ABCB1 C3435T genotype-dependent inhibitory effect on elimination rather than absorption of a P-gp substrate. Further investigations are necessary in patients who receive long-term use of RA extract formulation and combined P-gp substrates, especially in those ABCB1 3435CC carriers.     

Pharmacokinetic study of four flavones of Glycyrrhiza in rat plasma using HPLC–MS

Aim of the study

This study aimed to develop a specific HPLC–MS method for simultaneous quantification of four flavones of Glycyrrhiza in rat plasma after oral administration and to describe the pharmacokinetics of four flavones in rat plasma.

Materials and methods

A simple, sensitive and selective method for simultaneous determination of four flavones of Glycyrrhiza in rat plasma, i.e., liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin, by high performance liquid chromatography–tandem mass spectrometry (HPLC–MS) with negative electrospray ionization mode, was developed and validated. The method was applied to investigate the pharmacokinetics of four flavones in rat plasma after oral administration of Glycyrrhiza flavones. Chromatographic separation was accomplished on an Agilent TC-C18 column (4.6 mm×250 mm, and 5 μm), with gradient elution by using a mixture of methanoic acid (A) and acetonitrile (B) as the mobile phase at a flow rate of 0.8 mL/min.

Results

The calibration curves for four flavones had good linearity higher than 0.997 in the measured range. Relative standard deviations (RSDs) of the intra- and inter-day precision at different levels were all less than 4.8%. The pharmacokinetic profile of four flavones in rat plasma was fitted with a two-compartment model detected by a simple, rapid and accurate HPLC–MS method. Time (h) to reach peak concentration (μg/mL) of liquiritin (2.69±0.04), isoliquiritin (10.16±0.02), liquiritigenin (2.83±0.02), and isoliquiritigenin (0.28±0.01) was 2.02±0.23, 1.97±0.20, 0.48±0.02, and 1.93±0.36, respectively. The distribution and elimination half-life (h) and area under the concentration–time curve (μg/mL–h) from t=0 to last time of liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin were 1.02±0.48/2.27±0.53/16.97±0.43, 2.04±1.01/2.38±0.80/69.20±5.24, 0.35±0.10/4.26±0.16/14.83±0.11, and 1.18±0.32/3.04±0.22/2.10±0.09, respectively. Isoliquiritin presented the phenomenon of double peaks and the others appeared together in a single and plateau absorption phase. Isoliquiritigenin had the lowest oral bioavailability because of C_max and AUC_0−∞. Liquiritigenin had the fastest absorption and distribution rate and the lowest elimination rate according to T_max, t_1/2α, and t_1/2β.

Conclusions

This paper first reported on identification and determination of four flavones of Glycyrrhiza in rat plasma and their respective pharmacokinetic characteristics. The results provided a meaningful basis for better understanding the absorption mechanism of Glycyrrhiza and evaluating the clinical application of this medicine.     

Antinociceptive effect of ethanol extract of leaves of Lannea coromandelica

Ethnopharmacological relevance

Lannea coromandelica (Houtt.) Merr. is a plant locally called “Jiga”, found all over Bangladesh. Leaf of the plant is traditionally used in the treatment of local swellings, pains of body, toothache etc. This study evaluated the antinociceptive effect of the ethanol extract of L. coromandelica leaves (EELC).

Materials and methods

The antinociceptive activity of the extract (at the doses of 50, 100, and 200 mg/kg) was evaluated by using chemical- and heat-induced pain models such as acetic acid-induced writhing, hot plate, tail immersion, formalin, and glutamate test. To verify the possible involvement of opioid receptor in the central antinociceptive effect of EELC, naloxone was used to antagonize the effect. Besides, the involvements of ATP-sensitive K⁺ channel and cGMP pathway were also justified by using glibenclemide and methylene blue.

Results

EELC demonstrated significant dose-dependent antinociceptive activity in the chemical- and heat-induced nociception in mice models (p<0.05). These findings imply the involvement of both peripheral and central antinociceptive mechanisms. The use of naloxone confirmed the association of opioid receptors in the central antinociceptive effect. EELC also showed the involvements of ATP-sensitive K⁺ channel and cGMP pathway for antinociceptive activity.

Conclusions

This study reported the antinociceptive activity of the leaf of L. coromandelica and rationalized the traditional use of the leaf in the treatment of different painful conditions.     

Ethnopharmacology guided screening of traditional Indian herbs for selective inhibition of Plasmodium specific lactate dehydrogenase

Ethnopharmacological relevance

Medicinal plants traditionally used to treat malaria can provide quality leads towards identifying novel anti-malarial drugs. Here we combined this approach with target based drug discovery and explored Plasmodium specific lactate dehydrogenase (LDH) inhibitory activity of 8 Indian plants which are ethnically used to treat malaria.

Methods

LDH from Indian Plasmodium falciparum and Plasmodium vivax strains, were cloned and expressed in Escherichia coli, followed by purification of recombinant enzymes (rPfLDH and rPvLDH respectively). Extracts of 8 plants in different organic and aqueous solvents, were screened for their inhibitory activity on rPfLDH, rPvLDH and mammalian LDHs. Phyllanthus amarus aqueous extract was further tested for in vitro parasiticidal activity.

Results

Aqueous extract of Phyllanthus amarus Schum. and Thonn. and chloroform extract of Murraya koenigii (L.) Spreng. exhibited profound and exclusive inhibitory effect on Plasmodium falciparum LDH (IC₅₀=11.2 μg/ml±0.4) and Plasmodium vivax LDH (IC₅₀=6.0 μg/ml±0.6) respectively. Moreover, Phyllanthus amarus aqueous extract also demonstrated antiplasmodial activity in vitro, on Chloroquine sensitive and resistant strains of Plasmodium falciparum (IC₅₀=7.1 μg/ml±0.5 and 6.9 μg/ml±0.7 respectively).

Conclusion

Target specific screening of traditional herbs used in malaria treatment has proffered Phyllanthus amarus and Murraya koenigii extracts as hits which can optimistically provide novel antimalarial drugs.     

Involvement of glutamate and cytokine pathways on antinociceptive effect of Pfaffia glomerata in mice

Ethnopharmacological relevance

Pfaffia glomerata (Spreng) Pedersen (Amaranthaceae) is a medicinal plant known in Brazil as “Paratudo” and “Brazilian ginseng” and is commonly used as tonic, antidiabetic and to treat gastric disorders.

Aim of the study

This study evaluates the possible mechanism by which hydroalcoholic extract (HE) of Pfaffia glomerata exerts its antinociceptive effect.

Materials and methods

The HE was evaluated in acetic acid and glutamate models of pain or by biting behavior following intrathecal (i.t.) administration of agonists of excitatory aminoacids (EAA) receptors glutamate and pro-inflammatory cytokines, IL-1β and TNF-α in mice.

Results

Oral administration of HE produced dose-dependent inhibition of acetic acid-induced visceral pain and glutamate-induced pain, with ID₅₀ of 64.6 (47.7–87.5) mg/kg and ID₅₀ of 370.8 (253.4–542.7) mg/kg, respectively. The HE (300 mg/kg, p.o.) antinociception, in the acetic acid test, was not affected by i.p. treatment of animals with naloxone. In addition, HE (300 mg/kg, p.o.) inhibited the pain-related behaviors induced by i.t. injection of trans-ACPD and TNF-α, but not by NMDA, AMPA, kainate or IL-1β.

Conclusions

Our results suggest that inhibition of glutamatergic metabotropic receptors and TNF-α may account for the antinociceptive action reported for the HE in models of chemical pain used in this study.     

Evidence for a role of 5-HT1A receptor on antinociceptive action from Geissospermum vellosii

Ethnopharmacological relevance

Geissospermum vellosii is a tree widely found throughout the Amazonic forest and frequently used by the native population for painful disorders.

Aim of the study

The present study examined the antinociceptive effects of Geissospermum vellosii in behavioral models of nociception.

Materials, methods and results

Oral administration of crude extract of Geissospermum vellosii or its dichloromethane fraction (1–100 mg/kg) inhibited formalin-induced inflammatory nociception and acetic acid-induced visceral nociception. The antinociceptive effect of Geissospermum vellosii was unrelated with motor dysfunctions. Furthermore, the alkaloid 12-metoxy-1-methyl-aspidospermidine (0.001–1 mg/kg), isolated from the dichloromethane fraction, also produced antinociception. The antinociception caused by the dichloromethane fraction was significantly attenuated by pre-treatment of mice with p-chlorophenylalanine methyl ester (PCPA, an inhibitor of serotonin synthesis, 100 mg/kg once a day for 4 consecutive days) and WAY-100635 (a 5-HT_1A receptor antagonist, 0.3 mg/kg). In contrast, dichloromethane fraction antinociception was not affected by pre-treatment of animals with ketanserin (a 5-HT₂ receptor antagonist, 0.3 mg/kg) or ondansetron (a 5-HT₃ receptor antagonist, 0.5 mg/kg).

Conclusions

Together, these results indicate that Geissospermum vellosii produces antinociception through an interaction with 5-HT_1A receptors. Furthermore, the alkaloid 12-metoxy-1-methyl-aspidospermidine contributes to the antinociceptive properties reported for Geissospermum vellosii.     

Venom's antinociceptive property in the primitive ant Dinoponera quadriceps

Ethnopharmacological relevance

In northeastern Brazil, Dinoponera (Ponerinae) ants macerate are used to treat ear ache and its sting, rheumatism, and back pain. Such a popular use is a relevant fact that called for experimental evaluation of the antinociceptive activity of Dinoponera venom.

Materials and methods

Dinoponera quadriceps venom (DqV; 5–500 μg/kg; i.v.) or morphine (3.4 mg/kg; s.c.) were evaluated in mice models of nociception (n=8 animals/group). Negative controls received sterile saline (0.9% NaCl; i.v.).

Results

DqV showed 64% protein content and exhibited antinociceptive activity, without affecting motor function, in the tests: formalin (72%), writhing (52%), von Frey (71%) and hot plate (45%). The antinociceptive activity was abolished under protein denaturant conditions.

Conclusions

This study provided the first demonstration of the antinociceptive property of Dinoponera quadriceps venom in mice models of chemical, mechanical and thermal nociception, corroborating the popular use and suggesting its potential therapeutic utilization in painful conditions.     

Isolation of seselin from Clausena anisata (Rutaceae) leaves and its effects on the feeding and development of Lucilia cuprina larvae may explain its use in ethnoveterinary medicine

Ethnopharmacological relevance

The leaves of Clausena anisata are used traditionally to expel maggots from wounds of animals in Zimbabwe. We have previously proved in the laboratory that the plant certainly affects the behaviour and growth of blowfly larvae. The objective of this study was to isolate and identify the active compounds responsible for this activity.

Materials and methods

The acetone extract of Clausena anisata leaf powder was separated by solvent–solvent partition into five fractions. The n-hexane fraction was the most active in the larvicidal assay and therefore subjected to open column chromatography on silica gel.

Results

The isolated compound was identified by nuclear magnetic resonance (NMR) and mass spectroscopy (MS) as the pyranocoumarin, seselin, chemically known as 2′,2′-dimethylpyranocoumarin. It inhibited feed intake in the first and second instars of blowfly larvae at the minimum concentration tested of 1 ppm resulting in significant lower mass pupae (13.5±0.5 mg and 22.4±0.4 mg for the first and second instar larvae respectively) compared to the solvent control group (26.19±0.8 mg) (p<0.05).

Conclusions

This is the first report of the isolation of seselin from the leaves of Clausena anisata and the first report of the compound having an effect against blowfly larvae.     

Characterization of the antinociceptive and anti-inflammatory activities of fractions obtained from Copaifera multijuga Hayne

Ethnopharmacological relevance

Copaifera multijuga Hayne (Leguminosae) is a tree that produces an oleoresin, which is extensively commercialized in Brazil as capsules or crude oil for the treatment of several disorders. Ethnopharmacological studies show a diversity of indications such as anti-inflammatory and epidermal wound cicatrization.

Aim of the study

In the present work three fractions obtained from Copaifera multijuga oleoresin (hexane (HF), chloroform (CF), and methanol (MF) from a KOH impregnated silica gel column chromatography, representing the three main classes of compounds in the Copaifera genus (hydrocarbon sesquiterpenes, oxygenated sesquiterpenes and acidic diterpenes), were evaluated using antinociceptive and anti-inflammatory models.

Materials and methods

HF, CF, and MF (doses ranging between 1 and 150 mg/kg, depending on the model used), Copaifera multijuga oleoresin (CMO, 100 mg/kg, p.o.) and the reference drug morphine (5 mg/kg, p.o.) were evaluated using models for analgesia (acetic acid-induced contortions and tail flick) or inflammation (rat paw oedema and increase in vascular permeability). To elucidate the mechanism of action from the fractions, animals were pre-treated with naloxone (opioid receptor antagonist, 5 mg/kg, i.p.).

Results

Fractions significantly inhibited (in a concentration-dependant way) the number of contortions induced by acetic acid and the second phase of formalin-induced licking response. Similar results were observed in the tail flick model. The central antinociceptive effect for HF and CF at the doses of 50 and 100 mg/kg was higher than the one observed for morphine (1 mg/kg). Administration of naloxone inhibited the antinociceptive effect of fractions indicating that HF, CF, and MF may be acting on opioid receptors. All three fractions also inhibited rat paw oedema and the increase in vascular permeability induced by several phlogistic agents (carrageenan, histamine, and serotonin).

Conclusions

Our results indicate that fractions obtained from Copaifera multijuga Hayne demonstrate an antinociceptive effect probably mediated by opioid receptors, and anti-inflammatory activity through inhibition of histaminergic and serotoninergic pathways.     

In vitro xanthine oxidase inhibitory activity of the fractions of Erythrina stricta Roxb.

Aim of the study

To assay the in vitro xanthine oxidase inhibitory activity of the various fractions of the hydromethanolic extract of the leaves of Erythrina stricta and to determine its enzyme inhibition mechanism.

Materials and methods

Xanthine oxidase inhibitory activity was assayed spectrophotometrically under aerobic conditions and the degree of enzyme inhibition was determined by measuring the increase in absorbance at 295 nm associated with uric acid formation. Enzyme kinetics was carried out using Lineweaver-Burk plots using xanthine as the substrate.

Results

Among the fractions tested, the chloroform fraction exhibited highest potency (IC₅₀ 21.2 ± 1.6 μg/ml) followed by the pet–ether (IC₅₀ 30.2 ± 2.2 μg/ml), ethyl acetate (IC₅₀ 44.9 ± 1.4 μg/ml) and residual (IC₅₀ 100 ± 3.3 μg/ml) fractions. The IC₅₀ value of allopurinol used, as the standard was 6.1 ± 0.3 μg/ml. Enzyme inhibition mechanism indicated that the mode of inhibition was of a mixed type.

Conclusion

These results suggest that the use of Erythrina stricta for the treatment of gout could be attributed to its xanthine oxidase inhibitory activity.