Relaxant effects of Artemisia ludoviciana on isolated rat smooth muscle tissues

Ethnopharmacological relevance

Artemisia ludoviciana spp. mexicana (Willd. Ex.) Spring D.D. Keck (Asteraceae), known as “estafiate” is employed for the treatment of diarrhea, dysentery, parasites, abdominal pain, vomiting, stomach ache, and also as antispasmodic agent. The aim of the present study was to evaluate the relaxant effect of hexanic (HEAl), dichloromethanic (DEAl) and methanolic (MEAl) extracts on isolated trachea, ileum and aorta rat rings, and to establish the tracheo-relaxant mode of action of DEAl.

Materials and methods

All extracts were investigated based on their capacity of to inhibit the rat ileum spontaneous contraction, to relax contraction induced by noradrenaline (0.1 μM) on endothelium-intact and endothelium-denuded thoracic aorta rat rings, and also to inhibit contraction provoked by carbachol (1 μM) on rat trachea.

Results

Organic extracts had no spasmolytic action on ileum strips compared to positive control (papaverine, p < 0.05). On the other hand, all extracts induced a significant concentration- and partial endothelium-dependent vasorelaxant activity. Extracts also showed significant relaxant effect on pre-contracted tracheal tissue in a concentration-dependent manner. In last two experiments, DEAl was the most potent and efficient extract; however, it was less potent than papaverine and theophylline, used as positive controls (p < 0.05). In tracheal preparation, DEAl shifted to the right, in a parallel manner, the concentration-response curves induced by carbachol (p < 0.05). Also, DEAl induced a significant relaxant effect on the contraction produced by potassium chloride (KCl, 80 mM). Pre-incubation with 1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ, 10 μM), indomethacin (10 μM), N_ω-nitro-l-arginine methyl ester (l-NAME, 10 μM), glibenclamide (10 μM) and 2-aminopyridine (2-AP, 100 μM) did not modify the DEAl-relaxant curves.

Conclusions

Functional experiments suggest that the most active extract, DEAl, induced its relaxant effect by possible muscarinic receptors antagonism and calcium channel blockade in tracheal rings. On the other hand, significant vasorelaxant activity showed by DEAl is partially endothelium-dependent. Finally, spasmolytic activity induced by the extracts in the rat ileum was not significant, which suggests that the antidiarrheic effect of the plant is related to antimicrobial and antiparasitic properties previously described.     

Bacopa monnieri and its constituents is hypotensive in anaesthetized rats and vasodilator in various artery types

Ethnopharmacological relevance

Bacopa monnieri (Brahmi) provides traditional cognitive treatments possibly reflecting improved cerebral hemodynamics. Little is known about the cardiovascular actions of Brahmi. We sought to assess its effects on blood pressure and on isolated arteries, thus providing insights to clinical applications.

Materials and methods

Intravenous Brahmi (20-60 mg/kg) was tested on arterial blood pressure and heart rate of anaesthetized rats. In vitro vasorelaxation was assessed in arteries, with and without blockers of nitric oxide synthase (L-NAME), cyclooxygenase (indomethacin), and mechanical de-endothelialisation. The effects of Brahmi on Ca²⁺ influx and release from stores were investigated.

Results

Intravenous Brahmi extract (20-60 mg/kg) decreased systolic and diastolic pressures without affecting heart rate. Brahmi evoked relaxation in isolated arteries in order of potency: basilar (IC50 = 102 ± 16 μg/ml) > mesenteric (171 ± 31) > aortae (213 ± 68) > renal (IC50 = 375 ± 51) > tail artery (494 ± 93) > femoral arteries (>1000 μg/ml). Two saponins, bacoside A3 and bacopaside II, had similar vasodilator actions (IC50 = 8.3 ± 1.7 and 19.5 ± 6.3 μM). In aortae, without endothelium or in L-NAME (10-4 M), Brahmi was less potent (IC50 = 213 ± 68 to 2170 ± 664 and 1192 ± 167 μg/ml, respectively); indomethacin (10-5 M) was ineffective. In tail artery, Brahmi inhibited K⁺-depolarization induced Ca²⁺ influx and Ca²⁺ release from the sarcoplasmic reticulum by phenylephrine (10-5 M) or caffeine (20 mM).

Conclusions

Brahmi reduces blood pressure partly via releasing nitric oxide from the endothelium, with additional actions on vascular smooth muscle Ca²⁺ homeostasis. Some Brahmi ingredients could be efficacious antihypertensives and the vasodilation could account for some medicinal actions.     

Anti-inflammatory, antioxidant, anti-tyrosinase and phenolic contents of four Podocarpus species used in traditional medicine in South Africa

Ethnopharmacological relevance

Species of Podocarpus are used traditionally in their native areas for the treatment of fevers, asthma, coughs, cholera, chest complaints, arthritis, rheumatism, venereal diseases and distemper in dogs.

Aims of the study

To investigate the antioxidant, anti-inflammatory and anti-tyrosinase activities of four Podocarpus species, Podocarpus elongatus, Podocarpus falcatus, Podocarpus henkelii and Podocarpus latifolius, used in traditional medicine in South Africa. Phytochemical analysis to determine the phenolic contents was also carried out.

Materials and methods

DPPH, FRAP and β-carotene-linoleic acid assays were used to determine the antioxidant/radical scavenging activities of these species. Anti-inflammatory activity of these species was assayed against two cyclooxygenase enzymes (COX-1 and COX-2). Tyrosinase inhibition activity was analysed using the modified dopachrome method with l-DOPA as the substrate. Phenolics were quantitatively determined using spectrophotometric methods.

Results

Stems of Podocarpus latifolius exhibited the lowest EC₅₀ (0.84 μg/ml) inhibition against DPPH. The percentage antioxidant activity based on the bleaching rate of β-carotene ranged from 96% to 99%. High ferric reducing power was observed in all the extracts. For COX-1, the lowest EC₅₀ value was exhibited by stem extracts of Podocarpus elongatus (5.02 μg/ml) and leaf extract of Podocarpus latifolius showed the lowest EC₅₀ against COX-2 (5.13 μg/ml). All extracts inhibited tyrosinase activity in a dose-dependent manner with stem extract of Podocarpus elongatus being the most potent with an EC₅₀ value of 0.14 mg/ml. The total phenolic content ranged from 2.38 to 6.94 mg of GAE/g dry sample.

Conclusion

The significant pharmacological activities observed support the use of these species in traditional medicine and may also be candidates in the search for modern pharmaceuticals in medicine, food and cosmetic industries.     

Evaluation of the use of Cocos nucifera as antimalarial remedy in Malaysian folk medicine

Ethnopharmacological relevance

White flesh extract of Cocos nucifera (coconut) was studied to ascertain the ethnopharmacological standing of its antimalarial usage in Malaysian folk medicine.

Materials and methods

The crude methanol extract was investigated for phytochemical constituents and acute oral toxicity. Antimalarial activity of different extract doses of 50, 100, 200 and 400 mg/kg were investigated in vivo against Plasmodium berghei (NK65) infections in mice during early, established and residual infections. Chloroquine (20 mg/kg) and pyrimethamine (1.2 mg/kg) were used as reference drugs.

Results

The results revealed that the extract contained some phytochemical constituents and is toxicologically safe by oral administration. The extract significantly reduced the parasitaemia by the 200 and 400 mg/kg doses in the all three in vivo assessment assays. However, the extract did not significantly increase the survival time of the infected mice.

Conclusions

The observed pharmacological activities suggest that the Malaysian folkloric medicinal application of Cocos nucifera has a pharmacological basis.     

Procyanidin-rich fractions from Parkia biglobosa (Mimosaceae) leaves cause redox-sensitive endothelium-dependent relaxation involving NO and EDHF in porcine coronary artery

Aim of the study

Parkia biglobosa leaves are traditionally used as an antihypertensive agent in Benin. The present study assessed the vasorelaxant activity of different Parkia biglobosa leaf extracts using isolated porcine coronary artery rings.

Materials and methods

A hydroalcoholic leaf extract was submitted to a multi-step liquid-liquid fractionation with solvents of increasing polarity and the polyphenolic content of the different fractions was analyzed. Vascular reactivity of the different extracts was assessed using porcine coronary artery rings, in the presence or absence of specific pharmacological inhibitors.

Results

The hydroalcoholic, ethyl acetate and butanolic extracts contained mainly procyanidins and monomeric flavonoids. Parkia biglobosa leaf crude extract induced a redox-sensitive endothelium-dependent relaxation mediated by both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). The fractionation of the butanolic extract generated 6 fractions, two of which induced stronger vasorelaxation than the original extract and they had a higher phenolic content.

Conclusions

Parkia biglobosa leaf extract is able to induce endothelium-dependent NO- and EDHF-mediated relaxation in porcine coronary artery rings. The vasorelaxant activity is dependent on their phenolic content and appears to involve mainly procyanidins.     

Pentacyclic triterpenoids and ceramide mediate the vasorelaxant activity of Vitex cienkowskii via involvement of NO/cGMP pathway in isolated rat aortic rings

Ethnopharmacological relevance

Vitex cienkowskii Kotschy & Peyritsch is a deciduous tree, prescribed by Cameroonian traditional healers as one of the most popular plant widely used in many disorders including cardiovascular diseases. The preliminary pharmacological studies carried out on Vitex cienkowskii showed its vasorelaxant activities on guinea-pig aortic rings.

Aim of the study

The present work evaluated the vasorelaxant activity of extract and isolated compounds from Vitex cienkowskii.

Materials and methods

Rat aortic rings were used to evaluate the in vitro vascular effect of the extract. The antioxidant activity was determined by measuring the reduction of the free radical 1,1-diphenyl-1-picryl-hydrazyl (DPPH).

Results

Vitex cienkowskii induced significant relaxation in a concentration- and endothelium-dependent manner (EC₅₀ = 12.12 μg/ml, CH₂Cl₂-MeOH, 1:1) and did not produce a vasorelaxant effect on contraction evoked by KCl (60 mM). In order to determine its mode of action, Vitex cienkowskii-induced relaxant effect was evaluated in the presence of indomethacin (10 μM), L-NAME (100 μM), ODQ (1 μM) and SQ22356 (100 μM). Relaxation was significantly blocked by L-NAME and ODQ. These results indicate that Vitex cienkowskii-mediated relaxation is endothelium dependent, probably due to NO release, and the consequent activation of vascular smooth muscle soluble guanylate cyclase (sGC), a signal transduction enzyme that forms the second messenger cGMP. Bio-guided study of Vitex cienkowskii allowed the isolation of the known pentacyclic triterpenoids and a ceramide. It is the first report of salvin A, maslinic acid and a ceramide from Vitex cienkowskii. The activity induced by these compounds indicated that they may be partly responsible for the vasorelaxant effect of the plant extract.A dose of 40 mg/kg of CH₂Cl₂-MeOH (1:1) extract administered intravenously induced a decrease of mean arterial pressure but did not affect the heart rate. Moreover the plant extracts were found to be highly active in the DPPH radical scavenging assay.

Conclusion

Vitex cienkowskii extract possesses antioxidant property, vasorelaxing, and hypotensive effect linked to the endothelium related factors, where nitric oxide is involved.     

South African plants used in traditional medicine to treat epilepsy have an antagonistic effect on NMDA receptor currents

Ethnopharmacological relevance

Several Searsia species (Anacardiaceae), including Searsia dentata and Searsia pyroides, are used in South Africa traditional medicine to treat epilepsy. Ethanol leaf extracts of these plants have been shown to act as possible antagonists of N-methyl-d-aspartate (NMDA)-type glutamate receptors.

Materials and methods

Leaf material of three Searsia species were collected from the Botanical Garden at the University of KwaZulu-Natal, Pietermaritzburg; dried and extracted with ethanol in an ultrasound bath. Filtered and dried extracts were resuspended in DMSO (100 mg/ml) and diluted in the recording solution. The effect of Searsia dentata, Searsia pyroides and Searsia glauca extracts was investigated in dissociated cerebellar granule cells (CGCs) from 8-day-old rats and in transiently transfected HEK (human embryonic kidney) 293 cells (HEK), expressing either NR1a/NR2A or NR1a/NR2B receptors. In both systems we measured whole-cell currents elicited by 0.5 mM NMDA (CGCs) or 50 μM glutamic acid (HEK) at −60 mV in 0 Mg and 30 μM glycine and NMDA driven Ca influx in Fura2-loaded CGC.

Results

Searsia dentata and Searsia pyroides ethanol extracts caused a dose-dependent decrease of NR current with ED₅₀ close to 0.03 mg/ml in CGC and a similar inhibition (80% with 1 mg/ml) in HEK cells, while Searsia glauca was much less effective. The inhibition was dependent on time of incubation and slightly favored by opening of the NR channel. It was hardly reversible during the recording time, but was not caused by accelerated run-down or by interaction with the modulatory redox site. Searsia pyroides ethanol extract also depressed the NMDA stimulated increase in intracellular Ca.

Conclusions

The data confirm the specificity of Searsia dentata and Searsia pyroides and justify their use in traditional medicine. These plants may combine one or more γ-aminobutyric acid (GABA)_A agonists with one or more NMDA antagonists, thus representing an efficient treatment for epilepsy.     

The anti-hypertensive effect of Danshen (Salvia miltiorrhiza) and Gegen (Pueraria lobata) formula in rats and its underlying mechanisms of vasorelaxation

Ethnopharmacological relevance

Radix Salviae miltiorrhizae (Danshen) and Radix Puerariae lobatae (Gegen) have long been used in traditional Chinese Medicine and serve as the principal herbs in treating cardiovascular disease.

Aims of the study

In the present study, an aqueous extract comprising Danshen and Gegen in the ratio of 7:3 (DG) was investigated for its anti-hypertension in vivo and vasodilative activities ex vivo.

Materials and methods

The anti-hypertensive effect of DG extract was investigated in spontaneously hypertensive rat (SHR) by measuring systolic blood pressure (SBP). Oral administration of DG extract was started at age of 6 weeks and 14 weeks for the preventive and therapeutic studies, respectively. Blood pressure was measured by tail-cuff method biweekly for 12 weeks. The ex vivo vasodilative activities of DG extract, its dependency on endothelium and the involvement of nitric oxide, prostacyclin and potassium channels were investigated using isolated rat aorta ring in organ bath.

Results

For in vivo study, systolic blood pressure was significantly reduced in DG extract-treated groups (90.2 and 300 mg/kg) as compared with the SHR control in both preventive and therapeutic studies. However, DG extract was unable to suppress or delay the onset of hypertension in the preventive study. For ex vivo study, the results showed that DG extract induced a concentration-dependent relaxation in aorta and persisted response was observed with the removal of endothelium. Besides, pretreatment with a non-selective potassium channel inhibitor tetraethylammonium (TEA) also significantly inhibited DG extract-induced vasodilation. Further investigations on specific potassium channel blockers revealed that ATP-sensitive potassium (K_ATP) channel inhibitor glibenclamide, inward rectifier potassium (Kir) inhibitor barium chloride and voltage-dependent potassium (K_v) channel inhibitor 4-aminopyridine, but not BK_Ca channel inhibitor iberiotoxin, exerted significant inhibition on DG extract-induced vasodilation.

Conclusions

The results of in vivo SHR animal model suggested that DG aqueous extract possessed blood pressure lowering effect on both pre- and post-hypertensive rats, which could be explained by its endothelium-independent vasodilation via the opening of K_ATP, Kir and K_v channels.     

Polysaccharide fractions of Caesalpinia ferrea pods: potential anti-inflammatory usage

Ethnopharmacological relevance

Caesalpinia ferrea (Caesalpinioideae), known as pau-ferro or juca, has been used in the traditional medicine in North and Northeast of Brazil in inflammatory disorder, among others. Thus, experimental evaluation of the anti-inflammatory activity of extracts and fraction polysaccharides of Caesalpinia ferrea pods, and correlation with its anti-inflammatory activity and popular use is important.

Materials and methods

Total polysaccharides (TPL) were applied to ion exchange chromatography and eluted stepwise. Paw edema was induced s.c. by λ-carrageenan, dextran, histamine, serotonin, compound 48/80, bradykinin, prostaglandin E₂ (PGE₂) or l-arginine and analyzed by plethysmometry and protein leakage by spectrophotometry. Peritonitis was induced i.p. by carrageenan or N-formyl-methionyl-leucyl-phenylalanine (fMLP) and analyzed 4 h later for leukocyte migration and protein leakage. Animals were treated i.v. with TPL or polysaccharide fractions (0.01, 0.1, 1 mg/kg) 30 min before stimuli in both models. Toxicity (variation of body/organ mass and hematological/biochemical parameters) was evaluated after the seven-day treatment with the most active polysaccharide fraction (1 mg/kg; i.v.).

Results

Chromatography of TPL (2.8% yield) provided three major polysaccharide fractions (FI, FII, FIII). At 1 mg/kg, TPL inhibited the paw edema induced by carrageenan (60%) and FIII (fraction presenting high carbohydrate and low protein content) inhibited the inflammatory parameters in the paw edema induced by the following stimuli: carrageenan (70%), dextran (53%), histamine (65%), serotonin (62%), bradykinin (60%), PGE₂ (63%), nitric oxide (61%) and compound 48/80 (36%). Additionally, FIII at 1 mg/kg inhibited the carrageenan-induced edema in animals with intact mast cells, but only the late phase of those with degranulated mast cells elicited by compound 48/80. Moreover, FIII inhibited cell migration and protein leakage in the model of peritonitis elicited by carrageenan (88%) and fMLP (64%), being well tolerated by animals.

Conclusions

Extracts and polysaccharide fractions of Caesalpinia ferrea pods exhibit potent anti-inflammatory activity via negative modulation of histamine, serotonin, bradykinin, PGE₂ and NO released in the carrageenan-induced edema, showing involvement of mast cells. FIII could be interfering not only in the vascular, but also in cellular inflammatory events, revealing to be an important active component of traditionally prepared remedies used to treat inflammatory states.     

Exploring Tagetes erecta Linn flower for the elastase, hyaluronidase and MMP-1 inhibitory activity

Ethnopharmacological relevance

Traditionally Tagetes erecta Linn flower is claimed to treat skin diseases like sores, burns, wounds, ulcers, eczema and several other skin ailments. The aim of the present experiment was to evaluate the anti-wrinkle potential of standardized flower extract of Tagetes erecta.

Materials and methods

The Tagetes erecta extract and fractions were screened for hyaluronidase, elastase and matrix metalloproteinase (MMP-1) inhibitory activity compared with the activity of standard oleanolic acid. Syringic acid and β-amyrin were obtained from the extract and quantified through RP-HPLC. Also the compounds were evaluated for anti-wrinkle activity.

Results

The methanol extract showed significant (^aP < 0.05) hyaluronidase and elastase inhibition with IC50 of 11.70 ± 1.79 μg mL⁻¹ and 4.13 ± 0.93 μg mL⁻¹ respectively and better MMP-1 inhibition compared to standard oleanolic acid. The isolated compounds syringic acid and β-amyrin found to inhibit enzymes comparable to oleanolic acid. The RP-HPLC analysis revealed that good amounts of syringic acid and β-amyrin (2.30%, w/w and 0.06%) are present in Tagetes erecta.

Conclusion

Tagetes erecta flower showed effective inhibition of hyaluronidase, elastase and MMP-1. Therefore, this experiment further rationalizes the traditional uses of this plant, which may be useful as an anti-wrinkle agent.     

Characterization of the antinociceptive and anti-inflammatory activities from Cocos nucifera L. (Palmae)

Aim of the study

Cocos nucifera cultivated in Brazil is known as “coco-da-Bahia” or “coqueiro-da-Índia”. The tea from the husk fiber is widely used to several inflammatory disorders. Crude extract and fractions obtained from Cocos nucifera “common variety” were evaluated to test the anti-inflammatory and antinociceptive activities.

Materials and methods

Crude extract (CE, 50, 100, and 150 mg/kg), fraction 1 (F1, molecular weight lesser than 1 kDa, 1, 10, and 50 mg/kg), fraction 2 (F2, molecular weight higher than 1 kDa, 1, 10, and 50 mg/kg), and the references drugs morphine (5 mg/kg), acetilsalicilic acid (200 mg/kg), prometazine (30 mg/kg), and metisergide (5 mg/kg) were evaluated on models of analgesia and inflammation.

Results

CE, F1, and F2 significantly develop peripheral and central antinociceptive activity but with less effect on supra-spinal regions of the brain. Administration of the opioid antagonist, naloxone (5 mg/kg) inhibited the antinociceptive effect indicating that Cocos nucifera crude extract and fractions may be acting in opioid receptors. CE and F1 also inhibited rat paw edema induced by histamine, and serotonin.

Conclusions

results demonstrated that Cocos nucifera and its fractions have antinociceptive and anti-inflammatory activities which confirm the popular use of this plant in several inflammatory disorders.     

Normal and delayed wound healing is improved by sesamol, an active constituent of Sesamum indicum (L.) in albino rats

Ethno-pharmacological relevance

The seeds of Sesamum indicum Linn. (Pedaliaceae) has been used traditionally for the treatment of wounds in Buldhana district of Maharashtra state. Sesamol is the main anti-oxidative constituent contained mainly in the processed sesame seed oil which has not been explored scientifically for its wound healing activity.

Aim of the study

To investigate the influence of sesamol (SM) on wound repair, both in normal and dexamethasone (DM) delayed healing processes in albino rats.

Materials and methods

Incision, excision and dead space wounds were inflicted on albino rats (180-220 g) of either sex, under ketamine anaesthesia. Group I served as control, group II received SM 50 mg/kg i.p., group III was treated with dexamethasone (DM) i.m. (0.17 mg/kg) and SM + DM was given to group IV. The tensile strength, wound contraction, hydroxyproline, lysyl oxidase and total RNA and DNA levels (in granulation tissue) were measured.

Results

The tensile strength significantly (p < 0.05) increased with SM at 471.40 ± 14.66 g when compared to control at 300.60 ± 9.16 g in normal and DM suppressed healing. No significant change was observed in duration of wound contraction and lysyl oxidase when compared to control at 2.98 ± 0.10 mg. SM treated rats showed a significant (p < 0.05) rise in hydroxyproline levels at 6.45 ± 0.45 mg when compared to control at 1.75 ± 0.20 mg.

Conclusion

These results indicate that sesamol could be a promising drug in normal as well as delayed wound healing processes.     

Hepatoxicity of major constituents and extractions of Radix Polygoni Multiflori and Radix Polygoni Multiflori Praeparata

Ethnopharmacological relevance

Radix Polygoni Multiflori (RPM) and Radix Polygoni Multiflori Praeparata (RPMP) were traditionally widely used as Chinese herbal medicine. However, liver adverse reactions caused by RPM or RPMP were frequently reported all around the world recent years. The aim of this study was to study the cytotoxicities of RPM, RPMP and their major constituents on human liver cell L-02 simultaneously.

Materials and methods

Multi-assays, including MTT assay, neutral red uptake (NRU) assay, LDH leakage percentage and liver enzyme secretion (AST, ALT and ALP) were used. Cytotoxicities of major chemical constituents of RPM, 2, 3, 5, 4′-tetrahydroxy-stilbene-2-O-β-D-glucoside (TSG), physcion and emodin, were tested. The cytotoxicities of water, 50% ethanol and 95% ethanol extractions of RPM and RPMP were tested. HPLC-DAD analysis was carried to reveal the content change of TSG, physcion and emodin after the processing procedure.

Results

The TD₅₀ of TSG, physcion and emodin in MTT assay were >10,000 μM, 2853.61 μM and 520.37 μM. In the NRU assay, the TD₅₀ of TSG, physcion and emodin were much smaller (1401.53 μM, 1140.00 μM, and 3.80 μM). Emodin induced much severe liver enzyme secretion than TSG and physcion. Cell proliferation and LDH leakage rate showed no difference between RPM and RPMP extractions, but ALP, AST and ALT secretions in RPMP extractions were significant lower than that of PMR groups. Water extractions of RPM and RPMP were less toxic than any other solvent in most of the assays. Positive correlation was found between the TSG/emodin ratio and MTT survival rate. The emodin/physcion ratio also showed positive correlation with the LDH leakage percentage.

Conclusions

In conclusion, Radix Polygonum multiflorum and Radix Polygonum multiflorum Praeparata were not liver injure inducing in our in vitro assays. However, the processing produce of RPM could reduce its effect on both cell proliferation and enzyme secretion of liver cell. Judging from cell proliferation, integrity of cell membrane and enzyme secretion, three major chemical constituents of RPM: TSG, physcion and emodin showed no, moderate and severe cytotoxicity against human liver cell line L-02 respectively. Chemical constituents-cytotoxicity relationship investigation revealed that TSG and physcion probably had attenuating effect to emodin. The attenuating mechanisms were still under investigation.     

Apocynum venetum leaf aqueous extract inhibits voltage-gated sodium channels of mouse neuroblastoma N2A cells

Ethnopharmacological relevance

Apocynum venetum Linn. (Apocynaceae family), also called Luobuma, is a shrub which grows widely in the Xinjiang Autonomous Region of China. Its leaves are used in herbal tea for the treatment of hypertension, anxiety and depression. Animal studies have also shown that Apocynum venetum leaf extract (AVLE) also exerts anti-depressant and anti-anxiety activities. The effects of AVLE on neuronal tissues in vitro are not fully understood.

Materials and methods

Using the whole-cell voltage-clamp method, we studied the effects of AVLE on ion channels in cultured mouse neuroblastoma N2A cells.

Results

AVLE inhibited voltage-gated inward Na⁺ current in a reversible and concentration-dependent manner (half-inhibitory concentration was 18 μg/ml and maximum inhibition at 100 μg/ml). AVLE specifically promoted steady-state inactivation of Na⁺ channels but did not affect voltage-dependence of activation. The inhibitory effect was not use-dependent and was not affected by 300 μM L-NAME, suggesting that NO was not involved in the action of AVLE in neuronal cells. AVLE also had a mild inhibitory effect on voltage-gated K⁺ channels, but did not affect ATP-sensitive K⁺ channels.

Conclusions

Since voltage-gated Na⁺ and K⁺ channels are associated with neuronal excitability and therefore affect neurotransmission, the modulation of neuronal ion channels by AVLE may exert neuropharmacological effects. In particular, the inhibition of voltage-gated Na⁺ currents by AVLE may in part account for the psychopharmacological effects of this herbal remedy.     

Antisecretory actions of Baccharis trimera (Less.) DC aqueous extract and isolated compounds: analysis of underlying mechanisms

Ethnopharmacological relevance

Baccharis trimera (Less.) DC. (Asteraceae) is a species native to South America used in Brazilian folk medicine to treat gastrointestinal and liver diseases, kidney disorders and diabetes. Previous studies from this laboratory confirmed the antacid and antiulcer activities of the plant aqueous extract (AE) in rat and mouse models.

Aim of the study

To investigate the mechanisms involved in the antacid action of AE and isolated compounds from Baccharis trimera.

Materials and methods

AE was assayed in vivo in cold-restraint stress gastric ulcers and in pylorus-ligated mice. Nine fractions (F2-F10) previously isolated from AE were assayed in vitro on acid secretion measured as [¹⁴C]-aminopyrine ([¹⁴C]-AP) accumulation in rabbit gastric glands, and on gastric microsomal H⁺, K⁺-ATPase preparations. Chlorogenic acids (F2, F3, F6, F7), flavonoids (F9), an ent-clerodane diterpene (F8) and a dilactonic neo-clerodane diterpene (F10) have been identified in these fractions.

Results

Intraduodenal injection of AE (1.0 and 2.0 g/kg) in 4 h pylorus-ligated mice decreased the volume (20 and 50%) and total acidity (34 and 50%) of acid secretion compared to control values. Administered orally at the same doses AE protected against gastric mucosal lesions induced in mice by restraint at 4 °C. Exposure of isolated rabbit gastric glands to fractions F8 (10-100 μM) and F9 (10-300 μg/ml) decreased the basal [¹⁴C]-AP uptake by 50 and 60% of control (Ratio = 6.2 ± 1.1), whereas the remaining fractions were inactive. In the presence of the secretagogues F2 and F4 (30-300 μg/ml) decreased the [¹⁴C]-AP uptake induced by histamine (His) with a 100-fold lower potency than that of ranitidine. F5 and F6 reduced the [¹⁴C]-AP uptake stimulated by carbachol (CCh), but they were 10 to 20-fold less potent than atropine. F8 (diterpene 2) and F9 (flavonoids) decreased both the His- and CCh-induced [¹⁴C]-AP uptake, whereas F10 (diterpene 1) was inactive against the [¹⁴C]-AP uptake stimulated by secretagogues. Diterpene 2 was the most active of all tested compounds being 7-fold less potent than ranitidine and equipotent to atropine in reducing acid secretion in vitro. This compound also reduced the gastric H⁺, K⁺-ATPase activity by 20% of control, while the remaining fractions were inactive on the proton pump in vitro.

Conclusions

The results indicate that Baccharis trimera presents constituents that inhibit gastric acid secretion by acting mainly on the cholinergic regulatory pathway. The plant extract also contains compounds that exert moderate inhibition of the histaminergic regulatory pathway of acid secretion and the gastric proton pump. Altogether these active constituents appear to provide effective inhibition of acid secretion in vivo, which may explain the reputed antiulcer activity of the plant extract.     

Pharmacological actions of the South African medicinal and functional food plant Sceletium tortuosum and its principal alkaloids

Ethnopharmacological relevance

The South African plant Sceletium tortuosum has been known for centuries for a variety of traditional uses, and, more recently, as a possible source of anti-anxiety or anti-depressant effects. A standardised extract Zembrin^® was used to test for pharmacological activities that might be relevant to the ethnopharmacological uses, and three of the main alkaloids were also tested.

Materials and methods

A standardised ethanolic extract was prepared from dried plant material, along with the purified alkaloids mesembrine, mesembrenone and mesembrenol. These were tested on a panel of receptors, enzymes and other drug targets, and for cytotoxic effects on mammalian cells.

Results

The extract was a potent blocker in 5-HT transporter binding assays (IC₅₀ 4.3 μg/ml) and had powerful inhibitory effects on phosphodiesterase 4 (PDE4) (IC₅₀ 8.5 μg/ml), but not other phosphodiesterases. There were no cytotoxic effects. Mesembrine was the most active alkaloid against the 5-HT transporter (K_i 1.4 nM), while mesembrenone was active against the 5-HT transporter and PDE4 (IC₅₀'s < 1 μM).

Conclusions

The activity of the Sceletium tortuosum extract on the 5-HT transporter and PDE4 may explain the clinical effects of preparations made from this plant. The activities relate to the presence of alkaloids, particularly mesembrine and mesembrenone.     

Standardized extract of Dicksonia sellowiana Presl. Hook (Dicksoniaceae) decreases oxidative damage in cultured endothelial cells and in rats

Aims

Aging and a variety of pathologies, including cancer, diabetes, cardiovascular and inflammatory diseases have been associated with reactive oxygen species (ROS), such as superoxide anion (O₂⁻), hydroxyl radical (OH) and hydrogen peroxide (H₂O₂) generation. Plant polyphenols bear radical scavenging/antioxidant activity. A phytomedicinal preparation obtained from aerial parts of Dicksonia sellowiana (Dicksoniaceae), a native plant from Central and South America, has been widely used in Brazil against asthma and presents beneficial effects in several other diseases, including cardiovascular disturbance. In this work, we investigated whether Dicksonia sellowiana, which is also known to contain high levels of polyphenols, presents antioxidant activity.

Methods

The antioxidant activity of the hydroalcoholic extract obtained from Dicksonia sellowiana leaves (HEDS) was investigated by in vitro and in vivo tests.

Results

HEDS (0.1-100 μg/mL) exhibited a strong scavenging activity against all reactive species tested (DPPH, O₂⁻,OH and H₂O₂; IC₅₀ = 6.83 ± 2.05, 11.6 ± 5.4, 2.03 ± 0.4, and 4.8 ± 0.4 μg/mL, respectively). HEDS strongly protected endothelial cells against H₂O₂-induced oxidative stress by mechanisms other than increasing catalase activity. In addition, HEDS protected cell membrane from oxidative damage. HEDS, (20 and 40 mg/kg) inhibited lipid peroxidation in vivo (29.8% and 24.5%, respectively).

Conclusions

According to our results, we can speculate that the traditional uses of Dicksonia sellowiana for cardiovascular diseases, asthma and skin diseases could be, at least in part, related to the potent antioxidant and endothelial protective activities of the plant.     

Phaleria macrocarpa (Scheff.) Boerl fruit aqueous extract enhances LDL receptor and PCSK9 expression in vivo and in vitro

Ethnopharmacological relevance

Phaleria macrocarpa (Scheff.) Boerl (Pm) has been shown to reduce cholesterol level in vitro and in vivo experiment.

Aim of the study

This study investigated the effects of Pm fruit on weight control and mechanistic basis of its anti-hypercholesterolemic effect in both in vivo and in vitro.

Materials and method

In the in vivo study, thirty six male Sprague Dawley were randomized to six groups. Five groups were induced into hypercholesterolemia by giving 3% cholesterol enriched-diet for 52 days while one group acted as control. The rats were then treated with Pm extract (0, 20, 30 and 40 mg/ml) or simvastatin for 84 days. The following parameters were determined: (1) body weight, (2) blood lipid profile (total cholesterol, triglyceride, HDL and LDL) and (3) hepatic LDL receptor (160 kDa and 120 kDa) and PCSK9 proteins. In the in vitro study, HepG2 cells were cultured in serum-free RPMI supplemented with 0.2% BSA with or without LDL and in the presence of Pm extract (0, 0.1, 2, 40 and 1000 μg/ml) or simvastatin (4.60 μg/ml) for 24 h. The abundance of both LDL receptor and PCSK9 proteins and mRNA were investigated.

Results

Pm extract significantly (P < 0.05) reduced body weight gain, total cholesterol, triglycerides, HDL LDL levels and upregulated hepatic LDL receptor as well as PCSK9 proteins of hypercholesterolemic rats. These results were supported by studies in HepG2 cells whereby Pm extract also significantly upregulated both LDL receptor and PCSK9 at protein and mRNA levels.

Conclusion

This study enhances the potential usage of Pm fruit for controlling the body weight of obese people and for treating hypercholesterolemia.     

Acute and subchronic oral toxicity studies of Nelumbo nucifera stamens extract in rats

Ethnopharmacological relevance

Since the use of Nelumbo nucifera stamens in herbal medicines as well as in cosmetic products are highly prevalent in Thailand and increasing worldwide, acute and subchronic toxicity studies to confirm the safe use of Nelumbo nucifera stamens are warranted.

Aim of the study

Acute and subchronic oral toxicity studies of Nelumbo nucifera stamens extract in rats were performed in the present study in order to evaluate its safety.

Materials and methods

In acute toxicity study, Nelumbo nucifera stamens extract was administered by oral gavage to Sprague-Dawley rats (5 males and 5 females) at a dose of 5000 mg/kg. In subchronic toxicity study, the extract at doses of 50, 100, and 200 mg/kg/day were given orally to groups of rats (6 rats/dose/sex) for 90 consecutive days.

Results

The extract at a dose of 5000 mg/kg produced no treatment-related signs of toxicity or mortality in any of the animals tested during 14 days of the study. In the repeated dose 90-day oral toxicity study, there was no significant difference in body weight between the control and all treatment groups with the exception of the body weight of the female group treated with 200 mg/kg/day of the extract which was statistically significantly less than that of its control counterpart on day 90 but the percent weight changes of both groups were almost similar. Some statistically significant differences in hematological and biochemical parameters as well as in some internal organ weights of both male and female rats treated with the extract at the highest dose were observed. However, no abnormality of internal organs was observed in both gross and histopathological examinations.

Conclusions

These results suggest that the oral lethal dose of Nelumbo nucifera stamens extract for male and female rats is in excess of 5000 mg/kg and the no-observed-adverse-effect level (NOAEL) of the extract for both male and female rats is considered to be 200 mg/kg/day.