Involvement of the L-arginine-nitric oxide pathway in the antinociception caused by fruits of Prosopis strombulifera (Lam.) Benth

Ethnopharmacological relevance

Prosopis strombulifera (Lam.) Benth. is a rhizomatous shrub that grows in the north and central zone of Argentina. In folk medicine, the fruits of this plant have been used as an astringent, anti-inflammatory and odontalgic agent and anti-diarrheic.

Aim of the study

To investigate the antinociceptive effect of ethanol (EE), chloroform (CE) and ethyl acetate (EtOAcE) extracts of Prosopis strombulifera fruits and the involvement of the l-arginine-nitric oxide pathway in this effect.

Materials and methods

The antinociceptive effects of the EE, CE and EtOAcE of Prosopis strombulifera fruits were evaluated in vivo using the formalin-induced pain test in mice with aspirin and morphine as reference antinociceptive compounds. The participation of the l-arginine-nitric oxide pathway in the antinociceptive effect was investigated in the same animal model using l-arginine as a nitric oxide (NO) precursor. The in vitro inhibitory effect of the extracts on LPS-induced nitric oxide production and iNOS expression was investigated in a J774A.1 macrophage-derived cell line.

Results

CE (300 mg/kg), in contrast to EE and EtOAcE, caused significant inhibition (p < 0.05) of the in vivo nociceptive response. Moreover, CE (100–1000 mg/kg, p.o.) produced a dose-dependent inhibition of the neurogenic and the inflammatory phases of the formalin test with inhibition values (at 600 mg/kg) of 42 ± 7% and 62 ± 7%, respectively. CE inhibition was more potent in the inflammatory phase, with an ID₅₀ of 400.1 (252.2–634.8) mg/kg. The antinociception caused by CE (600 mg/kg, p.o.) was significantly attenuated (p < 0.05) by i.p. treatment of mice with l-arginine (600 mg/kg). In addition, CE (100 μg/mL) produced significant in vitro inhibition (p < 0.001) of LPS-induced NO production, which was not observed with EE and EtOAcE at the same concentration. The inhibition of NO production by CE (10–100 μg/mL) was dose-dependent, with an IC₅₀ of 39.8 (34.4–46.1) μg/mL, and CE significantly inhibited LPS-induced iNOS expression in J774A.1 cells.

Conclusions

This study supports, in part, the ethnomedical use of Prosopis strombulifera fruits by showing that its CE produces moderate antinociception in vivo. The findings also provide scientific information for understanding the molecular mechanism involved in the analgesic effect of this plant.     

Evaluation of the wound healing potential of Wedelia trilobata (L.) leaves

Ethnopharmacological relevance

Wedelia trilobata (L.) Hitchc (Asteraceae) leaves are used in the treatment of wounds by traditional healers. Despite the use of this plant in wound healing, there is a scarcity of scientific data to support its therapeutic application.

Aim of the study

To investigate the wound healing potential of Wedelia trilobata (L.) leaves commonly employed by traditional healers and to clarify its traditional use in a scientific investigation.

Materials and methods

An ethanolic extract of Wedelia trilobata leaves was subjected to column chromatography. Hexane, ethyl acetate (WEA) and chloroform:methanol (50:50) (WCM) fractions were obtained. The fractions were tested using relevant in vitro wound healing assays. Antioxidant activity was measured by the DPPH assay. The fibroblast proliferation, oxidative stress using hydrogen peroxide, an in vitro scratch assay, and increasing collagen content was determined using fibroblast L929. Minimum inhibitory concentrations (MICs) were determined against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa.

Results

WEA (3 μg/mL) promoted fibroblast L929 survivability up to more than 90% before and more than 85% after hydrogen peroxide induced oxidative stress. WEA (3 μg/mL) induced a 70% migration rate in the in vitro scratch assay and the collagen content was increased to 261 μg/mL compared to the control (57.5 μg/mL). WCM exhibited a scavenging activity for DPPH with an IC₅₀ value of 179.5 μg/mL comparable to BHT (139.3 μg/mL). WEA was active against Gram positive bacteria Staphylococcus aureus, Staphylococcus epidermidis with MIC values of 62.5 and 31.25 μg/mL, respectively.

Conclusion

These scientific findings of wound healing activity supports the traditional claims for Wedelia trilobata (L.) leaves. The WEA displayed antibacterial and fibroblast stimulatory activities while WCM exhibited antioxidant to indicate its potential wound healing properties. However further studies to isolate the antibacterial, antioxidant and fibroblast stimulatory compounds that contribute to the wound healing properties of this plant are needed.     

Aegiceras corniculatum extract suppresses initial and late phases of inflammation in rat paw and attenuates the production of eicosanoids in rat neutrophils and human platelets

Aim of the study

The present study is designed to explore the anti-inflammatory potential of Aegiceras corniculatum Linn. Blanco stems extracts and their mechanism of action against various pro-inflammatory mediators and to validate its traditional use against inflammatory diseases.

Materials and methods

Rat paw edema and peritonitis models were employed for in vivo studies. For in vitro studies human platelets and rat neutrophils were stimulated with Ca²⁺-ionophore A23187 leading to the production of various pro-inflammatory metabolites, i.e., 12-HTT, 12-HETE and LTB₄ and 5-HETE which were quantified by HPLC.

Results

The highly polar methanol extract (100 mg/kg) caused ∼90% reduction in the carrageenan- and prostaglandin E2-induced paw edema in rats. It also caused the inhibition of cycloxygenase-1 metabolite, 12-HHT (IC₅₀ 41.1 ± 1.5 μg/ml) with a concomitant rise in 12-lipoxygenase metabolite, 12-HETE in A23187 stimulated human platelets. Conversely, the non-polar hexane extract attenuated (IC₅₀ 0.36 ± 0.12 μg/ml) 12-HETE formation with a parallel rise in 12-HHT, thereby displaying a selectivity towards 12-lipoxygenase. Non-polar hexane extract also antagonized the production of 5-lipoxygenase metabolites, i.e., leukotriene B₄ and 5-HETE in the rat neutrophils. Furthermore, ethyl acetate extract inhibited both COX and 5-LOX with a marked decline in the production of 12-HHT (IC₅₀ 0.08 ± 0.002 μg/ml) and LTB₄ (IC₅₀ 0.86 ± 0.03 μg/ml), respectively. The anti-inflammatory effect of hexane and ethyl acetate extracts was also reflected by the diminution of carrageenan-induced cell infiltration in rat peritoneum. Additionally, plant extracts caused ∼60% suppression in dextran-induced paw edema implying that they also ameliorate histamine and serotonin release.

Conclusion

Hexane, ethyl acetate and methanol extracts derived from Aegiceras corniculatum possess significant anti-inflammatory activity via multiple mechanisms and validate their traditional use against inflammation-related diseases.     

Beneficial effects of the ethanol extract of Caesalpinia pyramidalis on the inflammatory response and abdominal hyperalgesia in rats with acute pancreatitis

Ethnopharmacological relevance

Caesalpinia pyramidalis Tul. (Fabaceae) is a plant found in the Northeast of Brazil that is popularly used to treat inflammation. Acute pancreatitis (AP) is an inflammatory disease for which abdominal pain is a relevant symptom. As there is no specific therapy for AP, we investigated the effect of the ethanol extract from the inner bark of C. pyramidalis (EECp) on the AP induced by common bile duct obstruction (CBDO) in rats.

Material and methods

AP was induced in male Wistar rats (200–250g, n=6–8) through laparotomy and subsequent CBDO. Animals were euthanized after 6 (G6h) or 24 h (G24h) of induction. In the G6h protocol, animals were pretreated with EECp (100–400 mg/kg, p.o.) or vehicle (Tween 80; 0.2%) 1 h before CBDO or sham surgery. For the G24h protocol, rats were pretreated with EECp (400 mg/kg, 1 h before CBDO or 1 h before and 12 h after CBDO) or vehicle. The following parameters were measured: inflammatory/oxidative (myeloperoxidase activity and malondialdehyde formation in the pancreas and lung, leukocyte counts in the blood and serum nitrate/nitrite), enzymatic (serum amylase and lipase levels) and nociceptive (abdominal hyperalgesia).

Results

Induction of AP by CBDO significantly increased all the parameters evaluated in both G6h and G24h protocols when compared with the respective sham group. In the G6h protocol, the EECp pretreatment (400 mg/kg) significantly reduced all these parameters, besides completely inhibiting abdominal hyperalgesia. The same profile of reduction was observed from two administrations of EECp in the G24h protocol, while one single dose of EECp was able to significantly reduce pancreatic MDA, serum lipase levels, leukocyte counts in the blood and abdominal hyperalgesia without affecting the other parameters in the G24h protocol. Furthermore, rutin was found in the EECp.

Conclusions

Our results demonstrated that EECp decreases inflammation, lipoperoxidation and hyperalgesia in CBDO-induced AP, making it of interest in future approaches to treat this condition.     

Evaluation of the antinociceptive, anti-inflammatory and gastric antiulcer activities of the essential oil from Piper aleyreanum C.DC in rodents

Ethnopharmacological relevance

Piper aleyreanum is a small tree that is widely distributed in tropical and subtropical regions, mostly in North and South America, and is used as an immunomodulator, analgesic and antidepressant in folk medicine.

Aim of the study

This study was designed to investigate the antinociceptive, anti-inflammatory and gastric antiulcer activities of the essential oils from the aerial parts of Piper aleyreanum (EOPa) in rodents.

Materials and methods

The antinociceptive and anti-inflammatory effects of orally administered EOPa were evaluated in mice subjected to the formalin and pleurisy models, respectively. We also pretreated the rats with EOPa before acute ethanol-induced gastric lesions and measured gastric lesion extension and mucus and glutathione (GSH) levels in the gastric mucosa. Finally, we performed a phytochemical analysis of EOPa.

Results

The chemical composition of EOPa was analyzed by gas chromatography and mass spectrometry (GC/MS), which identified 35 compounds, representing 81.7% of total oil compounds. Caryophyllene oxide (11.5%), β-pinene (9%), spathulenol (6.7%), camphene (5.2%), β-elemene (4.7%), myrtenal (4.2%), verbenone (3.3%) and pinocarvone (3.1%) were the major oil constituents. The oral administration of EOPa (10–1000 mg/kg) significantly inhibited the neurogenic and inflammatory phases of formalin-induced licking, with ID50 values of 281.2 and 70.5 mg/kg, respectively. The antinociception caused by EOPa (100 mg/kg, p.o.) was not reversed by naloxone (1 or 5 mg/kg, i.p.) in the formalin test. EOPa (100–300 mg/kg, p.o.) did not affect animal motor coordination in an open-field model. In carrageenan-induced pleurisy, EOPa (1–100 mg/kg, p.o.) significantly decreased the total cell count, neutrophils and mononuclear cells with mean ID50 values of 53.6, 21.7 and 43.5 mg/kg, respectively. In addition, EOPa (1–30 mg/kg, p.o.) protected the rats against ethanol-induced gastric lesions with an ID50 value of 1.7 mg/kg and increased the mucus and GSH levels of the gastric mucosa to levels similar to those of the non-lesioned group.

Conclusions

These data show for the first time that EOPa has significant antinociceptive and anti-inflammatory actions, which do not appear to be related to the opioid system. EOPa also has interesting gastroprotective effects related to the maintenance of protective factors, such as mucus production and GSH. These results support the widespread use of Piper aleyreanum in popular medicine and demonstrate that this plant has therapeutic potential for the development of phytomedicines with antinociceptive, anti-inflammatory and gastroprotective properties.     

Effect on blood pressure of a dietary supplement containing traditional medicinal plants of Côte d'Ivoire

Ethnopharmacological relevance

A medicinal composition containing salt (sodium chloride) is given as a traditional dietary supplement to hypertensive patients (TDSHP) in Côte d’Ivoire. It consists of whole plant of Bidens pilosa (Asteraceae) and fresh leaves of Moringa oleifera (Moringaceae).

Aim of the study

The aim of this study was to establish the scientific basis for the use of this traditional recipe rich in sodium chloride in hypertension settings.

Materials and methods

We used a total aqueous extract of this traditional dietary supplement containing medicinal plants (Bidens pilosa, Moringa oleifera) and salt (sodium chloride). Experiment was carried out to evaluate its effect on arterial blood pressure of rabbits. The experimental device used for recording blood pressure in rabbits is based on the principle of Ludwig mercury manometer.

Results

TDSHP between 5 × 10⁻⁸ and 5 × 10⁻² mg/kg caused a dose-dependent hypotension. TDSHP elicited drops in blood pressure ranging between 7.14 ± 4 and 100 ± 7.5%, compared to normal blood pressure of rabbits. Fifty percent effective dose of TDSHP was 3.95 × 10⁻⁴ mg/kg. Similarly as the hypotension induced by acetylcholine, the one caused by TDSHP at dose of 3.95 × 10⁻⁴ mg/kg in rabbit was progressively inhibited by atropine, dosed between 5 × 10⁻⁴ to 5 × 10⁻² mg/kg. The percentage drop of recorded blood pressure ranged from 50.3 ± 1.87 to 3.71 ± 1.09% compared to the normal value of blood pressure. In the presence of atropine, TDSHP effect was partially inhibited. The same increasing doses of TDSHP reduced significantly the increase of blood pressure induced by adrenaline dosed at 4.76 × 10⁻⁴ mg/kg from 89.3 ± 2.19 to 1.19 ± 0.59%.

Conclusion

The consumption of this traditional dietary supplement is justified in hypertensive patients according to its composition and its ability to reduce blood pressure has been demonstrated experimentally. TDSHP should not be considered as an antihypertensive drug, it remains to us a salt substitute to be taken with moderation with strict adherence to the traditional dose.     

Antifibrotic effects of Artemisia capillaris and Artemisia iwayomogi in a carbon tetrachloride-induced chronic hepatic fibrosis animal model

Ethnopharmacological relevance

Artemisia capillaris and Artemisia iwayomogi, both members of the Compositae family, have been indiscriminately used for various liver disorders as traditional hepatotherapeutic medicines in Korea for many years.

Aim of the study

In this study, the anti-hepatofibrotic effects of Artemisia capillaris and Artemisia iwayomogi were comparatively analyzed using a carbon tetrachloride (CCl₄)-induced liver fibrosis rat model.

Materials and methods

Hepatic fibrosis was induced via a 10-week course of intraperitoneal CCl₄ injections (50% dissolved in olive oil, 2 mL/kg, twice per week). Water extract of Artemisia capillaris (AC) or Artemisia iwayomogi (AI) was orally administered six times per week from the 5th to the 10th week.

Results

AI (50 mg/kg) significantly attenuated the CCl₄-induced excessive release of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in serum (p < 0.05), and hydroxyproline and malondialdehyde (MDA) contents in liver tissue (p < 0.05). Further, AI markedly ameliorated the depletion of total antioxidant capacity (TAC), glutathione (GSH), and superoxide dismutase (SOD) in liver tissue (p < 0.01). Unexpectedly, AC did not exert any effects on the above parameters. Histopathological and immunohistochemical analyses revealed that AI drastically reduced inflammation, necrosis, fatty infiltration, collagen accumulation, and activation of hepatic satellite cells in liver tissue. These changes were not observed with AC treatment. Several critical genes of fibrosis-related cytokines including transforming growth factor beta (TGF-β), platelet-derived growth factor beta (PDGF-β), and alpha smooth muscle actin (α-SMA) were more prominently downregulated by AI compared to AC treatment.

Conclusion

Our results show that AI exerts greater hepatoprotective and anti-fibrotic effects as compared with AC via enhancing antioxidant capacity and downregulating fibrogentic cytokines.     

Antinociceptive effectiveness of triterpenes from rosemary in visceral nociception

Ethnopharmacological relevance

Rosemary is a species used worldwide as a common spice, but also in folk medicine for their therapeutic properties against abdominal pain. The rationale of this study was to examine the involvement of triterpenes and to compare their effectiveness in the antinociceptive effect of an ethanol extract of Rosmarinus officinalis L. (Lamiaceae).

Material and methods

Fractionation and HPLC analyses allowed the identification of a mixture of micromeric (121 mg/g), oleanolic (64 mg/g) and ursolic (83 mg/g) acids as partial antinociceptive responsible in an ethyl acetate fraction of R. officinalis by using the acetic acid-induced abdominal constrictions model in mice.

Results

These triterpenes individually evaluated produced a significant and dose-dependent antinociceptive response with similar potency as follows: ED₅₀=1.1 mg/kg (0.9–1.3 mg/kg), 2.1 mg/kg (1.6–2.6 mg/kg) and 1.6 mg/kg (1.1–2.1 mg/kg), respectively, by using the intraperitoneal (i.p.) route of administration in mice. Their maximal antinociceptive efficacy resembled that produced by ketorolac (10 mg/kg, i.p.), a common clinic analgesic.

Conclusion

Our results provide evidence that these triterpenes participate in the antinociceptive activity of R. officinalis. In addition, each individual triterpene showed a similar potency to that observed with ketorolac, a non-steroidal anti-inflammatory drug, in this experimental model.     

Inhibitory effects of adlay bran (Coix lachryma-jobi L. var. ma-yuen Stapf) on chemical mediator release and cytokine production in rat basophilic leukemia cells

Ethnopharmacological relevance

Adlay (Job's tears, Coix lachryma-jobi L. var. ma-yuen Stapf) has long been used in China to treat rheumatism.

Aim of the study

We investigated the anti-allergic effects of adlay bran on rat basophilic leukemia (RBL)-2H3 cells.

Materials and methods

To evaluate the anti-allergic effects of adlay bran, the release of histamines and cytokines were measured using ELISA. To explore the mechanism of these effects, the protein expression levels were determined using western blotting.

Results

A 40.8 μg/mL concentration of the ethyl acetate fraction of the ethanolic extracts of adlay bran (ABE-EtOAc) effectively inhibited mast cell degranulation. The 40–100% EtOAc/Hex subfractions of ABE-EtOAc inhibited histamine release with an IC₅₀ of 71–87 μg/mL. Moreover, the ABE-EtOAc subfractions suppressed the secretion of interleukin (IL)-4, IL-6 and tumor necrosis factor-α in the RBL-2H3 cells, indicating that adlay bran can inhibit cytokine secretion in the late phase of the allergic reaction. In addition, adlay bran reduced the intracellular production of reactive oxygen species, inhibited the phosphorylation of Akt and decreased the expression of protein kinase C. Furthermore, six phenolic acids and one flavone were isolated. Of these compounds, luteolin showed the most potent inhibitory activity (IC₅₀ = 1.5 μg/mL).

Conclusion

Adlay bran extract reduced the release of histamines and cytokines and suppressed the production of Akt. These combined effects influenced the signal transduction in RBL-2H3 cells, thereby revealing the mechanisms of the anti-allergic effects of adlay.     

Potentiality of standardized extract and isolated flavonoids from Zuccagnia punctata for the treatment of respiratory infections by Streptococcus pneumoniae: in vitro and in vivo studies

Ethnopharmacological relevance

Zuccagnia punctata Cav. (Fabaceae) is a monotypic species distributed in western Argentina and is traditionally used for the treatment of bacterial and fungal infections. The aim of this study was to demonstrated the antibacterial activity of the Zuccagnia punctata standardized extract and the structurally related non-methoxylated flavonoids with similar pattern of substitution and differences in ring C present in this plant species: 7-hydroxyflavanone (HF), 2′,4′-dihydroxychalcone (DHC) and 3,7-dihydroxyflavone (DHF), against Streptococcus pneumoniae clinical isolates using in vitro and in vivo models.

Materials and Methods

MIC values of natural products were determined by agar macrodilution method. In vivo activities were investigated in a Streptococcus pneumoniae infection model in mice. Lung and blood samples were obtained for bacterial cell counts. The serum was used by biochemical analysis (alanine transaminase, aspartate transaminase, urea and creatinine) in order to evaluate the toxicity of natural products.

Results

All samples showed antimicrobial activity in vitro with MIC values between 50 and 500 μg/ml. Zuccagnia punctata extract (1 mg/mice) and HF (1 mg/mice) significantly reduced the number of viable Streptococcus pneumoniae in lung (p < 0.01) while lower quantities has not effect. Therefore, the present study has shown that intake once or twice a day of 1 mg of Zuccagnia punctata extract or HF for seven days did not result in toxicity.

Conclusions

Our results showed that Zuccagnia punctata extract as well as one of its isolated flavonoids, 7-hydroxyflavanone, could be useful for the development of a novel respiratory infections treatment.     

Central depressant activity of butanol fraction of Securinega virosa root bark in mice

Ethnopharmacological relevance

Securinega virosa is a commonly used medicinal plant in African traditional medicine in the management of epilepsy and mental illness. Previous studies in our laboratory showed that the crude methanol root bark extract of the plant possesses significant behavioral effect in laboratory animals. In an attempt to isolate and characterize the biological principles responsible for the observed activity, this study is aimed at evaluating the central depressant activity of the butanol fraction of the methanol root bark extract of Securinega virosa.

Materials and methods

The medial lethal dose of the butanol fraction was estimated using the method of Lorke. Preliminary phytochemical screening was conducted on the butanol fraction using standard protocol. The behavioral effect of the butanol fraction (75, 150 and 300 mg/kg) was evaluated using diazepam induced sleep test, hole-board test, beam walking assay, staircase test, open field test and elevated plus maze assay, all in mice.

Results

The median lethal dose of the butanol fraction was estimated to be 1256.9 mg/kg. The preliminary phytochemical screening revealed the presence of tannins, saponins, alkaloids, flavonoids, cardiac glycosides, similar to those found in the crude methanol extract. The butanol fraction significantly (P < 0.001) reduced the mean onset of sleep in mice and doubled the mean duration of sleep in mice at the dose of 75 mg/kg. The butanol fraction and diazepam (0.5 mg/kg) significantly (P < 0.01–0.001) reduced the number of head dips in the hole-board test suggesting sedative effect. The sedative effect of the butanol fraction was further corroborated by its significant (P < 0.01–0.001) reduction of the number of step climbed and rearing in the staircase test. The butanol fraction did not significantly increase the time taken to complete the task and number of foot slips in the beam walking assay, suggesting that it does not induce significant motor coordination deficit. Diazepam (2 mg/kg), the standard agent used significantly (P < 0.01) increased the number of foot slips. In the open field test, the butanol fraction significantly reduced the number of square crossed as well as the number of rearing. However, the butanol fraction did not significantly alter the behavior of mice in the elevated plus maze assay, while diazepam (0.5 mg/kg) significantly (P < 0.05) increased the time spent in the open arm and reduced the number of closed arm entry.

Conclusion

The findings of this study suggest that the butanol fraction of Securinega virosa root bark contains some bioactive principles that are sedative in nature.     

Antiulcerogenic activity of hydroalcoholic fruit extract of Pithecellobium dulce in different experimental ulcer models in rats

Ethnopharmacological relevance

The ethnopharmacological importance of Pithecellobium dulce is evidenced by its traditional use for gastric complications. The aim of the study is to evaluate the gastroprotective activity and the mechanism of action of hydroalcoholic fruit extract of P. dulce (HAEPD) in rats by using chemical and stress induced ulcer models.

Materials and methods

Gastric ulcer was induced by administering alcohol (or) acetylsalicylic acid (or) hypothermic restraint stress to rats pretreated with HAEPD (200 mg/kg b wt for 30 day). Volume of gastric fluid, pH, acidity, activities of pepsin, H⁺, K⁺-ATPase, myeloperoxidase, mucin content, nucleic acids, glycoproteins and prostaglandin E₂ (PGE₂) levels were assessed in gastric tissues.

Results

Ulcer score was significantly minimized in HAEPD administered animals. pH and acidity of gastric fluid were significantly minimized and the mucin, PGE₂ levels were significantly maintained in drug pre administered animals. The activities of H⁺, K⁺- ATPase and myeloperoxidase were found to be significantly elevated in ulcer control animals and found to be decreased in drug pretreated animals. The cell proliferation was found to be enhanced in drug received animals. The total protein bound carbohydrate to total protein ratio was found to be significantly maintained by HAEPD. The effects were found to be comparable with that of standard drug omeprazole.

Conclusion

It is concluded that HAEPD possess a potent antiulcer activity probably by acting as cytoprotective and antiacid secretory agent.     

Hepatoprotective and antioxidant activity of Amaranthus spinosus against CCl4 induced toxicity

Aim

50% ethanolic extract (ASE) of Amaranthus spinosus (whole plant) was evaluated for in vitro antioxidant and hepatoprotective activity.

Methods

The total phenolics and reducing capacity of ASE was determined using standard curve of gallic acid (0–1.0 mg/ml) and butylated hydroxy anisole. In vitro antioxidant activity was determined by DPPH, superoxide, hydroxyl radicals, hydrogen peroxide and nitric oxide scavenging methods. The hepatoprotective activity of ASE was evaluated at 6, 7, 8, 9 and 10 μg/ml concentration against CCl₄ (1%) induced toxicity in freshly isolated rat hepatocytes and HepG2 cells.

Results

ASE was found to contain 336 ± 14.3 mg/g total polyphenolics expressed as gallic acid equivalent while the reducing capacity was 2.26 times of BHA. ASE showed significant antioxidant activity in DPPH assay (IC₅₀ 29 μg/ml), scavenges superoxide (IC₅₀ ∼ 66–70 μg/ml), hydrogen peroxide (IC₅₀ ∼120–125 μg/ml), hydroxyl radicals (IC₅₀ ∼140–145 μg/ml) and nitric oxide (IC₅₀ ∼ 135–140 μg/ml). ASE (6, 7, 8, 9 and 10 μg/ml) was able to normalise the levels of biochemical parameters in isolated rat hepatocytes intoxicated with CCl₄. A dose dependent increase in percentage viability was observed in CCl₄ intoxicated HepG2 cells.

Conclusions

ASE possesses significant hepatoprotective activity which might be due to antioxidant defence factors and phenolics might be the main constituents responsible for activity.     

Cytotoxicity and modes of action of four Cameroonian dietary spices ethno-medically used to treat Cancers: Echinops giganteus, Xylopia aethiopica, Imperata cylindrica and Piper capense

Ethnopharmacological relevance.

Echinops giganteus, Imperata cylindrica, Piper capense and Xylopia aethiopica are four medicinal spices used in Cameroon to treat cancers.

Aim of the study

The above plants previously displayed cytotoxicty against leukemia CCRF-CEM and CEM/ADR5000 cell lines as well as human pancreatic MiaPaCa-2 cells. The present study aims at emphasizing the study of the cytotoxicity and the modes of action of the above plants on a panel of ten cancer cell lines including various sensitive and drug-resistant phenotypes. The study has been extended to the isolation of the bioactive constituents from Echinops giganteus.

Materials and methods

The cytotoxicity of the extracts was determined using a resazurin reduction assay, whereas the caspase-Glo assay was used to detect the activation of caspases 3/7, caspase 8 and caspase 9 in cells treated with the four extracts. Flow cytometry was used for cell cycle analysis and detection of apoptotic cells, analysis of mitochondrial membrane potential (MMP) as well as measurement of reactive oxygen species (ROS).

Results

The four tested extracts inhibited the proliferation of all tested cancer cell lines including sensitive and drug-resistant phenotypes. Collateral sensitivity of cancer cells to the extract of Echinops giganteus was generally better than to doxorubicin. The recorded IC₅₀ ranges were 3.29 µg/mL [against human knockout clones HCT116 (p53^−/−) colon cancer cells] to 14.32 µg/mL (against human liver hepatocellular carcinoma HepG2 cells) for the crude extract from Echinops giganteus, 4.17 µg/mL (against breast cancer cells transduced with control vector MDA-MB231 cells) to 19.45 µg/mL (against MDA-MB-231 BCRP cells) for that of Piper capense, 4.11 µg/mL (against leukemia CCRF-CEM cells) to 30.60 µg/mL (against leukemia HL60AR cells) for Xylopia aethiopica, 3.28 µg/mL [against HCT116 (p53^−/−) cells] to 33.43 µg/mL (against HepG2 cells) for Imperata cylindica and 0.11 µg/mL (against CCRF-CEM cells) to 132.47 µg/mL (against HL60AR cells) for doxorubicin. The four tested extracts induced apoptosis in CCRF-CEM cells via the alteration loss of MMP whilst that of Piper capense also enhanced the production of ROS.

Conclusion

The studied plants are potential cytotoxic drugs that deserve more detailed exploration in the future, to develop novel anticancer drugs against sensitive and otherwise drug-resistant phenotypes.     

Immunomodulatory potential of Enicostema axillare (Lam.) A. Raynal, a traditional medicinal plant

Ethnopharmacological relevance

Enicostema axillare (Lam.) A. Raynal., (Gentianaceae) has been used in traditional Indian system of medicine as depurative and for the treatment of psoriasis, intermittent fever and cancer. Ethnobotanical survey conducted in Theni District indicated a high consensus for this plant as blood purifier and to treat dermatopathy and venereal infections. The present study evaluated the immunomodulatory activity of the methanol extract of Enicostema axillare.

Materials and methods

In vivo immunomodulatory activity of Enicostema axillare methanol extract (100 and 200 mg/kg b.w) was evaluated by assessing its effect on the total and differential leukocyte count, organ weight, hemaggultinating antibody titer, plaque forming cells, quantitative hemolysis of SRBC and delayed type hypersensitivity. Sheep red blood cells (SRBC, 5 × 10⁸ cells/0.1 mL) were used to immunize the animals. In vitro immunomodulatory potential of the extract was studied using peritoneal macrophages by evaluating its effects on NBT reduction, NO production and cytokine release.

Results

The animals treated with Enicostema axillare methanol extract showed a significant (P ≤ 0.05) increase in weight of the thymus and spleen. The total leukocyte and lymphocyte count was increased significantly (P < 0.005) by the treatment. There was no significant alteration in neutrophil count. A dose dependent increase in antibody titer value was observed. A decreased response to DTH reaction induced by SRBC was recorded. A potential phagocytic response was seen on treatment with the extracts at 10 and 25 μg/mL. The extract inhibited the release of pro-inflammatory cytokines and production of NO significantly in a dose dependent manner.

Conclusion

These findings suggested that the methanol extract of Enicostema axillare acted on both humoral and cell mediated immune functions and decreased the release of pro-inflammatory cytokines in the peritoneal macrophages.     

Evaluation of diuretic activity of Amaranthus spinosus Linn. aqueous extract in Wistar rats

Ethnopharmacological relevance

Traditional Siddha medicine literature claims that the Amaranthus spinosus Linn. (family: Amaranthaceae) whole plant possesses diuretic property.

Aim of the study

To evaluate the diuretic potential of Amaranthus spinosus aqueous extract (ASAE) in rats.

Material and methods

Different concentrations of ASAE (200, 500, 1000, 1500 mg/kg), thiazide (10 mg/kg) and vehicle were orally administered to rats (n = 6 animals per group) and their urine output was collected after 24 h. Volume, pH, Na⁺, K⁺ and Cl⁻ concentrations of urine were estimated.

Results

ASAE produced increase in Na⁺, K⁺, Cl⁻ excretion, caused alkalinization of urine, showed strong saluretic activity and carbonic anhydrase inhibition activity. These effects were observed predominantly at 500 mg/kg dose and there was no dose–response relationship.

Conclusion

Our study strongly suggests that the Amaranthus spinosus is acting as a thiazide like diuretic with carbonic anhydrase inhibitory activity which restates the claim as diuretic herb in Siddha medicine.     

Effect of Tulbaghia violacea on the blood pressure and heart rate in male spontaneously hypertensive Wistar rats

Ethnopharmacological relevance

Tulbaghia violacea Harv. (Alliaceae) is a small bulbous herb which belongs to the family Alliaceae, most commonly associated with onions and garlic. In South Africa, this herb has been traditionally used in the treatment of various ailments, including fever, colds, asthma, paralysis, hypertension and stomach problems. The aim of this study was to evaluate the effect of methanol leaf extracts (MLE) of Tulbaghia violacea on the blood pressure (BP) and heart rate (HR) in anaesthetized male spontaneously hypertensive rats; and to find out the mechanism(s) by which it acts.

Materials and methods

The MLE of Tulbaghia violacea (5–150 mg/kg), angiotensin I human acetate salt hydrate (ang I, 3.1–100 μg/kg), angiotensin II human (ang II, 3.1–50 μg/kg), phenylephrine hydrochloride (phenylephrine, 0.01–0.16 mg/kg) and dobutamine hydrochloride (dobutamine, 0.2–10.0 μg/kg) were infused intravenously, while the BP and HR were measured via a pressure transducer connecting the femoral artery and the Powerlab.

Results

Tulbaghia violacea significantly (p < 0.01) reduced the systolic, diastolic, and mean arterial BP; and HR dose-dependently. Ang I, ang II, phenylephrine and dobutamine all increased the BP dose-dependently. The hypertensive effect of ang I and the HR-increasing effect of dobutamine were significantly (p < 0.01) decreased by their co-infusion with Tulbaghia violacea (60 mg/kg). However, the co-infusion of ang II or phenylephrine with Tulbaghia violacea (60 mg/kg) did not produce any significant change in BP or HR when compared to the infusion of either agent alone in the same animal.

Conclusions

Tulbaghia violacea reduced BP and HR in the SHR. The reduction in BP may be due to actions of the MLE on the ang I converting enzyme (ACE) and β₁ adrenoceptors.     

Hyperpigmentant activity of leaves and flowers extracts of Pyrostegia venusta on murine B16F10 melanoma

Ethnopharmacological relevance

Pyrostegia venusta is a native Brazilian plant which has a variety of uses in traditional folk medicine including the treatment of vitiligo. However, its effectiveness on melanogenesis is not yet elucidated.

Aim of the study

This study aimed to investigate the melanogenic activity of hydroalcoholic extracts from the leaves and flowers of P. venusta on murine B16F10 melanoma cells.

Materials and methods

Different concentrations of the hydroalcoholic extracts of flowers and leaves of P. venusta were evaluated in trials of spontaneous melanin content (4 days), and cell viability by the MTT assay in murine B16F10 cells, and in the mushroom tyrosinase activity in vitro.

Results

Both extracts, leaves (0.1; 0.3; 1 and 3 μg/mL) and flowers (0.03 and 0.1 μg/mL) increased the melanin content in a concentration dependent manner after 4 days of incubation on melanoma cells. Leaves extract promoted enhancement of melanogenesis with maximum effect of 33.3 ± 3% (3 μg/mL), and the flower extract increased in 23.4 ± 3% (0.1 μg/mL). The cell viability test using MTT showed that in the same tested concentrations of both extracts no cell death was detected. Actually, either extract was not able to cause any change in the tyrosinase activity. HPLC analysis of P. venusta extracts found 0.09% and 1.08% of allantoin on leaves and flowers extracts, respectively.

Conclusions

The leaves and flowers extracts of P. venusta stimulates B16F10 melanogenesis at very low concentrations. These findings support the folk medicinal use of P. venusta on the treatment of hypopigmentation diseases, such as vitiligo.     

Bioactivity guided isolation of analgesic and anti-inflammatory constituents of Cnestis ferruginea Vahl ex DC (Connaraceae) root

Ethnopharmacological relevance

Cnestis ferruginea (CF) Vahl ex DC (Connaraceae) is a shrub widely used in Traditional African Medicine (TAM) for the treatment of various painful and inflammatory conditions.

Aim of the study

To isolate the active pharmacological constituents responsible for the anti-inflammatory and antinociceptive properties of the methanolic root extract of C. ferruginea.

Materials and methods

The crude methanolic root extract of CF was sequentially fractionated into four sub extracts (chloroform, ethylacetate, n-butanol and the remaining aqueous fraction). The aqueous–butanol fractions, having showed significant inhibition of inflammation and pain, were subjected to fractionation through successive column chromatography on silica gel 60–120 mesh, eluted with a gradient of CHCl₃–MeOH.Sixty five fractions were collected; fractions with similar TLC profiles were grouped into seven major fractions (1–7). Fraction 4 being the most active in bioassay was rechromatographed to obtain CF-2. Analgesic activity was evaluated using the acetic acid-induced writhing and hot plate tests in mice while carrageenan induced paw oedema test was used to investigate the anti-inflammatory actions of the fractions obtained.

Result

Amentoflavone (CF-2) was isolated from the aqueous/n-butanol fraction. CF-2 (12.5, 25 and 100 mg/kg; p.o) produced significant (P<0.05) dose dependent inhibition of pain response elicited by acetic acid and increased nociceptive reaction latency in hot plate test. In addition it produced significant (P<0.05) dose-dependent inhibition of oedema in the carrageenan-induced inflammation.

Conclusion

This study showed that amentoflavone is responsible for the analgesic and anti-inflammatory activity of Cnestis ferruginea.     

Antispasmodic and bronchodilator activities of Artemisia vulgaris are mediated through dual blockade of muscarinic receptors and calcium influx

Aim of the study

The present study describes antispasmodic, antidiarrheal, bronchodilatory and tracheo-relaxant activities of Artemisia vulgaris to rationalize some of its traditional uses.

Materials and methods

Crude extract of Artemisia vulgaris (Av.Cr) was studied in the isolated tissue preparations of rabbit jejunum and guinea-pig trachea, as well as in the in vivo castor oil-induced diarrhea and bronchodilatory techniques.

Results

Av.Cr which tested positive for alkaloids, coumarins, flavonoids, saponins, sterols, tannins and terpenes caused concentration-dependent (0.03–10 mg/mL) relaxation of jejunum spontaneous contractions. Av.Cr inhibited the carbachol (CCh, 1 μM) and K⁺ (80 mM)-induced contractions in a pattern, similar to that of dicyclomine. Av.Cr shifted the Ca²⁺ concentration–response curves to right, like that caused by verapamil and dicyclomine. Av.Cr produced rightward parallel shift in CCh-curves, followed by non-parallel shift at higher concentration with the suppression of the maximum response, similar to that caused by dicyclomine. It exhibited protective effect against castor oil-induced diarrhea and CCh-mediated bronchoconstriction in rodents. In trachea, Av.Cr relaxed the CCh (1 μM) and K⁺ (80 mM)-induced contractions and shifted the CCh-curves to right.

Conclusion

These results indicate that Artemisia vulgaris exhibits combination of anticholinergic and Ca²⁺ antagonist mechanisms, which provides pharmacological basis for its folkloric use in the hyperactive gut and airways disorders, such as abdominal colic, diarrhea and asthma.