Cytotoxic activity of 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol from Eulophia nuda against human cancer cells

Ethnopharmacological relevance

Eulophia nuda L. (Orchidaceae) is a medicinally important terrestrial orchid used for the treatment of tumours and various health problems by the local healers throughout the Western Ghats region in Maharashtra (India).

Aim of the study

To isolate the active molecule from Eulophia nuda and to study its cytotoxic potential against human cancer cells.

Materials and methods

The crude methanolic extract of Eulophia nuda tubers was fractionated by stepwise gradient of the solvents—chloroform–methanol to isolate the pure compound. Isolated pure compound was assessed for its cytotoxic potential against human breast cancer cell lines, MCF-7 and MDA-MB-231 using MTT assay. Structure elucidation of the isolated active compound was carried out by extensive spectroscopic analysis including ¹H NMR, ¹³C NMR, NOESY, COSY, LC–MS and IR.

Results

The isolated active molecule was identified as phenanthrene derivative 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol. This compound showed good antiproliferative activity against human breast cancer cell lines MCF-7 (91%) and MDA-MB-231 (85%) at 1000 μg/ml concentration.

Conclusion

9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol from Eulophia nuda tubers showed good growth suppressive effect against human cancer cell lines MCF-7 and MDA-MB-231 making it a potential biomolecule against human cancer.     

Cytotoxic and antibacterial triterpenoids derivatives from Clematis ganpiniana

Ethnopharmacological relevance

The roots and rhizomes of Clematis are commonly used as an analgesic, abirritative, antibacterial, antiphlogistic, anticancer and diuretic agent. The Naxi people traditionally used Clematis ganpiniana's (Lévl. Et Vant.) as a diuretic agent, an anti-inflammatory and anticancer remedy.

Aim of study

To investigate the cytotoxic and antibacterial components from Clematis ganpiniana.

Materials and methods

The aboveground part of Clematis ganpiniana was isolated by chromatographic techniques. Structures of isolated compounds were identified by spectroscopic methods and comparison with published data. Their cytotoxic, apoptosis and antibacterial activities of purified components were also performed.

Results

By bioassay-guided fractionation techniques and chemical characterization, four triterpene glycosides were isolated and their cytotoxicity against cancer cells and antibacterial activity were tested. They showed significant inhibitory activities against MCF-7, MDA-MB-231 with IC50 value of 0.7–16.5 μg/ml, and significant apoptosis for MCF-7 and MDA-MB-231. Moreover, compound 4 showed weak wide-spectrum antibacterial activity.

Conclusion

These results provide promising baseline information for the potential use of Clematis ganpiniana as well as some of the isolated compounds in the treatment of cancer and infectious disease.     

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.     

Anti-cancer activity of compounds from Bauhinia strychnifolia stem

Ethnopharmacological relevance

The stem and root of Bauhinia strychnifolia Craib (Fabaceae family) have been traditionally used in Thailand to treat fever, alcoholic toxication, allergy and cancer. An EtOH extract of Bauhinia strychnifolia showed good inhibitory activity against several cancer cell lines including HT-29, HeLa, MCF-7 and KB. As there has been no previous reports on chemical constituents of Bauhinia strychnifolia, this study is aimed to isolate the pure compounds with anti-cancer activity.

Materials and methods

Five pure compounds were isolated from EtOH extract of Bauhinia strychnifolia stem using silica gel, dianion HP-20 and sephadex LH-20 column chromatography and were tested for their cytotoxic effects against HT-29, HeLa, MCF-7 and KB cell lines using the Sulforhodamine B (SRB) assay.

Results

Among five compounds, 3,5,7,3′,5′-pentahydroxyflavanonol-3-O-α-l-rhamnopyranoside (2) possessed very potent activity against KB (IC₅₀=0.00054 μg/mL), HT-29 (IC₅₀=0.00217 μg/mL), MCF-7 (IC₅₀=0.0585 μg/mL) and HeLa cells (IC₅₀=0.0692 μg/mL). 3,5,7-Trihydroxychromone-3-O-α-l-rhamnopyranoside (3) also showed good activity against HT-29 (IC₅₀=0.02366 μg/mL), KB (IC₅₀=0.0412 μg/mL) and MCF-7 (IC₅₀=0.297 μg/mL), respectively. The activity of 2 (IC₅₀=0.00054 μg/mL) against KB cell was ten times higher than that of the positive control, Camptothecin (anti-cancer drug, IC₅₀=0.0057 μg/mL). All compounds did not show any cytotoxicity with normal cells at the concentration of 1 μg/mL.

Conclusion

This is the first report of compounds 2 and 3 on anti-cancer activity and based on the anti-cancer activity of extracts and pure compounds isolated from Bauhinia strychnifolia stem, it might be suggested that this plant could be useful for treatment of cancer.     

An in vitro study on antiproliferative and antiestrogenic effects of Boerhaavia diffusa L. extracts

Ethnopharmacological relevance

Boerhaavia diffusa L. (Nyctinaceae) is a plant of tropical region used in Indian traditional medicine for the treatment of human ailments including abdominal tumor, jaundice, dyspepsia, menstrual disorders, etc. This plant also has antilymphoproliferative, antimetastatic and immunomodulatory effects.

Aim of the study

This study aimed to assess the antiproliferative and antiestrogenic properties of methanol extract of Boerhaavia diffusa (BME) in MCF-7 breast cancer cell lines.

Materials and methods

The effective concentration range of BME on cell viability was analyzed using MTT assay. Hydroxylapatite assay (HAP) was carried out to confirm the competitive binding of BME to the estrogen receptor (ER). The effect of BME on the expression of a selected estrogen responsive gene pS2 was analyzed by RT-PCR. The ability of BME to alter the cell cycle phases and distributions were studied using FACS analysis.

Results

Treatment with varying concentrations of BME (20–320 μg/mL) resulted in moderate to very strong growth inhibition in MCF-7 cell lines. BME competed with [³H]-estradiol for binding to ER with IC₅₀ value of 320 ± 25 μg/mL. RT-PCR analysis revealed that BME reduced the mRNA expression of pS2 indicating the antiestrogenic action of BME. BME treatment for 48 h resulted in a remarkable increase in the number of MCF-7 cells in the G0-G1 fraction from 69.1% to 75.8%, with a reciprocal decrease of cells in all other phases indicating cell cycle arrest at G0-G1 phase.

Conclusions

The results demonstrate that Boerhaavia diffusa possess antiproliferative and antiestrogenic properties and suggest that it may have therapeutic potential in estrogen dependent breast cancers.     

Antiproliferative and apoptosis inducing activity of Markhamia tomentosa leaf extract on HeLa cells

Ethnopharmacological relevance

Markhamia tomentosa (Benth) K. Schum ex. Engl. (Bignoniaceae), a tree widely dispersed in West Tropical Africa, is used traditionally to treat various diseases as it possesses antimicrobial, antioxidant, analgesic, anticancer and anti-inflammatory activities.

Materials and methods

This study evaluates the cytotoxic effect and underlying mechanisms of the ethanolic extract of Markhamia tomentosa on HeLa and MCF-7 cancer cell lines and non-cancerous Vero cell line. Brine shrimp lethality test was used for preliminary screening. Cytotoxicity was determined using the MTT assay and IC₅₀ was calculated. Effect of Markhamia tomentosa on the cell cycle was monitored by flow cytometry and the apoptosis-induction capability confirmed by exposure of phosphatidylserine to the outer leaflet of the plasma membrane. Loss of mitochondrial membrane potential was analysed by flow cytometry using JC-1.

Results

Markhamia tomentosa was toxic to brine shrimps with LD₅₀ of 31.62 µg/ml. Cell viability and growth of HeLa cells was inhibited by the extract with an IC₅₀ of 189.1±1.76 µg/ml at 24 h post treatment. However, no cytotoxic effect was observed in MCF-7 and Vero cell lines. The extract induced cell cycle arrest in HeLa cells in the G₀/G₁ phase resulting in cell death after 24 h exposure. Induction of apoptosis in HeLa cells was substantiated by Annexin V-FITC/PI double staining showing phosphatidylserine translocation and depolarisation of the mitochondrial membrane potential by flow cytometry of JC-1 stained cells.

Conclusion

The ethanolic extract of Markhamia tomentosa induces G₀/G₁ in HeLa cells followed by induction of the intrinsic pathway of apoptosis.     

Anti-inflammatory activity of Mitraphylline isolated from Uncaria tomentosa bark

Ethnopharmacological relevance

Uncaria tomentosa (Willd. ex Roem. & Schult.) DC. (Rubiaceae) is widely used by populations living in South America to treat many ailments associated with inflammatory disorders. Mitraphylline was shown to be the major pentacyclic oxindolic alkaloid present in the bark chloroformic extract of this plant. Its activity against cytokines involved in inflammation process was tested in a murine model in vivo.

Materials and methods

Mice received mitraphylline once a day for 3 days at 30 mg/kg/day by oral route. Then, they were subjected to bacterial lipopolysaccharide (LPS) endotoxin (15 mg/kg) and the LPS-induced production of 16 different cytokines was determined by Elisa multiplex. Control group received dexamethasone orally at 2 mg/kg/day. Toxicity on K565 cells and murine peritoneal macrophages, in vitro, at doses up to 100 μM was monitored by XTT-colorimetric assay.

Results and conclusions

For the first time mitraphylline was tested in vivo against a large range of cytokines that play a crucial role in inflammation. Mitraphylline inhibited around 50% of the release of interleukins 1α, 1β, 17, and TNF-α. This activity was similar to dexamethasone. It also reduced almost 40% of the production of interleukin 4 (IL-4) while the corticoid did not. Lastly it did not show any toxicity on K565 cells nor murine macrophages at doses up to 100 μM.     

Evaluation of the wound healing property of Boesenbergia longiflora rhizomes

Ethnopharmacological relevance

The rhizomes of Boesenbergia longiflora (Wall.) Kuntze (Zingiberaceae) have been traditionally used for treatment of inflammatory bowel disease, ulcerative colitis, aphthous ulcer and abscess by decoction with alcohol.

Aim of the study

The rhizomes of Boesenbergia longiflora were carried out to investigate for anti-inflammatory and wound healing activities in order to support the traditional use.

Material and methods

The ethanolic extract of Boesenbergia longiflora and its fractions were tested using relevant in vitro anti-inflammatory and wound healing assays. For the in vitro studies, murine macrophage RAW264.7 cells and mouse fibroblast L929 cells were assessed for anti-inflammatory and fibroblast stimulatory activities, respectively. In vivo anti-inflammatory activity was determined by carrageenan-induced rat paw edema model as well as acute toxicity estimated by the up-and-down method in mice.

Results

The present study has demonstrated that the ethanolic extract of Boesenbergia longiflora rhizomes possesses a potent anti-inflammatory and wound healing activities. Among the isolated fractions, the CHCl₃ fraction showed potent anti-inflammatory effect through nitric oxide inhibitory activity (IC₅₀=5.5 μg/ml) and reduction of carrageenan-induced rat paw edema (ED₅₀=222.7 mg/kg), whereas this fraction exhibited wound healing property via fibroblast migration on both day 1 (77.3%) and day 2 (100%) as well as enhanced collagen production (187.5 μg/ml) at concentration of 3 μg/ml, compared to that of the controls, 39.4% for fibroblast and 60.8 μg/ml for collagen, respectively. The anti-inflammatory mechanism of the CHCl₃ fraction is found to suppress the iNOS and COX-2 mRNA expression.

Conclusion

The scientific investigation of wound healing activity of Boesenbergia longiflora rhizomes support the Thai traditional uses for treatment of inflammatory bowel disease, ulcerative colitis, aphthous ulcer and abscess. The EtOH extract and CHCl₃ fraction exert potential wound healing property through NO inhibition, anti-oxidant effect and stimulation of fibroblast migration and collagen production. The phytochemical screening revealed that the CHCl₃ fraction of Boesenbergia longiflora rhizomes contains diarylheptanoids, flavonoids and terpenes. The isolation of the compounds responsible for the wound healing effect is now in progress.     

In vitro antiplasmodial activity of plants used in Benin in traditional medicine to treat malaria

Aim of the study

The aim of the study was to evaluate the in vitro antiplasmodial activity of crude extracts of 12 plant species traditionally used in Benin for the treatment of malaria in order to validate their use.

Materials and methods

For each species, dichloromethane, methanol and total aqueous extracts were tested. The antiplasmodial activity of extracts was evaluated using the measurement of the plasmodial lactate dehydrogenase activity on chloroquine-sensitive (3D7) and resistant (W2) strains of Plasmodium falciparum. The selectivity of the different extracts was evaluated using the MTT test on J774 macrophage-like murine cells and WI38 human normal fibroblasts.

Results

The best growth inhibition of both strains of Plasmodium falciparum was observed with the dichloromethane extracts of Acanthospermum hispidum DC. (Asteraceae) (IC₅₀ = 7.5 μg/ml on 3D7 and 4.8 μg/ml on W2), Keetia leucantha (K. Krause) Bridson (syn. Plectronia leucantha Krause) (Rubiaceae) leaves and twigs (IC₅₀ = 13.8 and 11.3 μg/ml on 3D7 and IC₅₀ = 26.5 and 15.8 μg/ml on W2, respectively), Carpolobia lutea G.Don. (Polygalaceae) (IC₅₀ = 19.4 μg/ml on 3D7 and 8.1 μg/ml on W2) and Strychnos spinosa Lam. (Loganiaceae) leaves (IC₅₀ = 15.6 μg/ml on 3D7 and 8.9 μg/ml on W2). All these extracts had a low cytotoxicity.

Conclusion

Our study gives some justifications for the traditional uses of some investigated plants.     

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.     

Pharmacological mechanisms underlying the vascular activities of Loranthus ferrugineus Roxb. in rat thoracic aorta

Aim of the study

The present study was aimed to investigate the pharmacological basis for the use of Loranthus ferrugineus in hypertension.

Materials and methods

Loranthus ferrugineus methanol extract (LFME) was obtained using Soxhelt extractor and then successively fractionated using chloroform, ethyl acetate and n-butanol. The n-butanol fraction of LFME (NBF-LFME) was studied using isolated rat thoracic aorta.

Results

NBF-LFME (1.0 × 10⁻⁵ to 3.0 mg/ml) was found to be the most potent to concentration-dependently relax the endothelium-intact phenyephrine (PE, 1 μM)- and high K⁺ (80 mM)-precontracted rat aortic rings. Removal of the endothelium completely abolished the vascular relaxing properties of NBF-LFME. Pretreatment with atropine (1 μM), l-NAME (10 μM), indomethacin (10 μM) and methylene blue (10 μM) significantly blocked NBF-LFME-mediated relaxation. Endothelium-dependent and -independent relaxations induced by acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, were significantly enhanced in aortic rings pretreated with NBF-LFME when compared to those observed in control aortic rings. On the contrary, glibenclamide (10 μM), propranolol (1 μM) and prazosin (0.01 μM) did not alter NBF-LFME-induced relaxation.

Conclusions

The results suggest that NBF-LFME induced vascular relaxation by stimulating muscarinic receptors, activating the endothelium-derived nitric oxide-cGMP-relaxant pathway, promoting prostacyclin release and/or possibly through its ability to lengthen the released nitric oxide half-life. The present data further supports previous in vivo findings and explain the traditional use of Loranthus ferrugineus as an anti-hypertensive agent.     

Chebulagic acid,a COX–LOX dual inhibitor isolated from the fruits of Terminalia chebula Retz., induces apoptosis in COLO-205 cell line

Ethnopharmacological relevance

Terminalia chebula has an esteemed origin in Indian mythology; its fruits are used to treat many diseases such as digestive, diabetes, colic pain, chronic cough, sore throat, asthma, etc.

Aim of the study

The water or ethanolic extracts of the fruits were reported to have anti-oxidant, anti-inflammatory, anti-cancer and radio-protector properties. The present study is to isolate and identify the compounds that inhibit COX and 5-LOX, the key enzymes involved in inflammation and carcinogenesis.

Materials and methods

The ethanolic extract of the fruits was fractionated by RP-HPLC and fractions were tested for enzyme inhibition activity against COX and 5-LOX. One of the fractionated compounds showed potent dual inhibition against COX and 5-LOX. It was identified as chebulagic acid by LC–MS, NMR and IR analyses. The chebulagic acid was also tested for anti-proliferative activity.

Results

Chebulagic acid showed potent COX–LOX dual inhibition activity with IC₅₀ values of 15 ± 0.288, 0.92 ± 0.011 and 2.1 ± 0.057 μM for COX-1, COX-2 and 5-LOX respectively. It also showed anti-proliferative activity against HCT-15, COLO-205, MDA-MB-231, DU-145 and K562 cell lines. Further mechanistic studies on COLO-205 cells revealed induction of apoptosis by chebulagic acid.

Conclusions

Chebulagic acid, a COX-2 and 5-LOX dual inhibitor isolated from the fruits of Terminalia chebula, induces apoptosis in COLO-205 cells.     

Cytotoxic polyphenols against breast tumor cell in Smilax china L.

Ethnopharmacological relevance

Smilax china L., referred to ‘Ba Qia’ (or ‘Jin Gang Teng’) in China, is a small vine that grows in the southern parts of China. The roots and tubers of S. china L. have been applied not only as traditional Chinese medicine (TCM) for treatment of diuretic, rheumatic arthritic, detoxication, lumbago, gout, tumor, and inflammatory diseases, but also as food in some area of China.

Aim of study

To investigate the breast tumor cell toxic components in S. china L. continuously and systematically.

Materials and methods

Three fractions and six polyphenols were isolated from roots and tubers of S. china L. under bioassay-guided screenings. The structures of six compounds were elucidated by spectroscopic methods and comparison with published data. Their breast tumor cytotoxicity and apoptosis of purified components were performed.

Results

Six polyphenols were obtained on the basis of a bioassay-guided separation of the ethyl acetate extract, and their breast tumor cytotoxic activities were tested. They showed anti-tumor activities against MCF-7 and MDA-MB-231 with IC50 value of 2.1–38.9 μg/mL, and can induce apoptosis for MCF-7 and MDA-MB-231.

Conclusions

Among these six polyphenols, five (1, 3–6) were reported for the 1st time with in vitro activities on anti-breast tumor cell. It is likely that these polyphenols are the active components of S. china L. responsible for the anti-breast tumor cell activities.     

Anti-inflammatory activity of Markhamia tomentosa (Benth.) K. Schum. Ex Engl. ethanolic leaf extract

Ethnopharmacological relevance

The leaves of Markhamia tomentosa (Benth.) K. Schum (Bignoniaceae) are used traditionally for the treatment of oedema and rheumatoid arthritis in Nigeria.

Aim of the study

The aim of the work was to investigate the anti-inflammatory activity of the ethanolic leaf extract of Markhamia tomentosa.

Materials and methods

The extract was screened using the carrageenan-induced paw oedema in rats, xylene-induced oedema in mice and the formalin-induced oedema in mice at 50, 100, 200 mg/kg doses p.o respectively. The mechanism by which the extract mediated the anti-inflammatory activity was assessed using the histamine-induced rat paw oedema and serotonin-induced rat paw oedema at the highest dose (200 mg/kg).

Results

The results showed that the extract produced a significant dose-dependent inhibition in carrageenan-induced, xylene-induced and the formalin tests. The extract exerted a significant inhibition of 54.55% (P<0.0001) and 42.11% (P<0.01) at 90 min in the histamine-induced and serotonin-induced rat paw oedema models respectively.

Conclusions

These findings suggest that the ethanolic leaf extract of Markhamia tomentosa possesses anti-inflammatory activity possibly mediated by histamine. The results justify the use of the plant in the preparation of ethno medicines used in the treatment of ailments associated with inflammation.     

Effects of ethanolic dried leaf extract of Lecaniodiscus cupanioides on antioxidant enzymes and biochemical parameters in rats

Ethnopharmacological relevance

Lecaniodiscus cupanioides is widely used in West African folk medicine for the treatment of inflammatory conditions, fevers and bacterial infections.

Aim of the study

To evaluate the potential toxic effects of the ethanolic dried leaf extract of Lecaniodiscus cupanioides (LC) on antioxidant enzymes in selected organs and biochemical parameters.

Materials and Methods

Crude ethanolic extract of Lecaniodiscus cupanioides dried leaves was prepared. A 90-day sub-chronic toxicity study was conducted using albino rats. Reconstituted Lecaniodiscus cupanioides was administered at a dosage of 400, 800 and 1600 mg/kg (high dose) with a control group receiving 10 ml/kg orally. Histopathological studies of major organs and blood chemistry analysis were performed on blood obtained via cardiac puncture after euthanization. Selected organs (liver, kidney and brain) were harvested for antioxidant and histopathological assessments.

Results

The extract produced significant (p<0.05) increases in the weights of liver, kidney and brain at 800 mg/kg and 1600 mg/kg compared to the control. Biochemical analysis showed significant increase in Alanine transferase (ALT) at 800 mg/kg and 1600 mg/kg. Assay for antioxidant enzymes showed a reversible decrease in the activity of Catalase (CAT), Superoxide dismutase (SOD) and Glutathione (GSH) with an increase in Malondialdehyde (MDA) at 800 mg/kg and 1600 mg/kg Lecaniodiscus cupanioides. Histopathological study showed reversible congestion in the brain, liver, and kidney at 800 mg/kg and 1600 mg/kg.

Conclusion

Findings in this study reveal that the ethanolic dried leaf extract of Lecaniodiscus cupanioides has the potential for inhibiting in vivo antioxidant enzymes activity and causing hepatotoxicity after prolonged exposure.     

Traditionally used Thai medicinal plants: In vitro anti-inflammatory,anticancer and antioxidant activities

Aims of the study

In order to assess traditional Thai claims about the therapeutic potential of medicinal plants and to select plants for future phytochemical research, nine plant species with anti-inflammatory uses were selected from Thai textbooks and assessed for their in vitro anti-inflammatory, antiproliferative and antioxidant activities.

Methods

Nuclear factor-kappaB (NF-κB) inhibitory effects in stably transfected HeLa cells were determined by luciferase assay, and effects on LPS-induced pro-inflammatory mediators prostaglandin E2 (PGE2), interleukin (IL)-6, IL-1β, and tumour necrosis factor (TNF)α in primary monocytes were assessed by ELISA. Cytotoxic activities were examined against HeLa cells, human leukaemia CCRF-CEM cells and the multidrug-resistant CEM/ADR5000 subline using the MTT and XTT tests. However, a redox status has been linked with both inflammation and cancer, antioxidant effects were also assessed using the DPPH, lipid-peroxidation, and Folin-Ciocalteau methods.

Results

Among all the nine species, Gynura pseudochina var. hispida and Oroxylum indicum showed the most promising NF-κB inhibitory effects with the lowest IC₅₀ values (41.96 and 47.45 μg/ml, respectively). Muehlenbeckia platyclada did not inhibit the NF-κB activation but effectively inhibited the release of IL-6, IL-1β and TNF-α with IC₅₀ values ranging between 0.28 and 8.67 μg/ml. Pouzolzia indica was the most cytotoxic against CCRF-CEM cells and the multidrug-resistant CEM/ADR5000 cells (9.75% and 10.48% viability, at 10 μg/ml, respectively). Rhinacanthus nasutus was the most potent cytotoxicity against HeLa cells (IC₅₀ 3.63 μg/ml) and showed specific cytotoxicity against the multidrug-resistant CEM/ADR5000 cells (18.72% viability at 10 μg/ml, p < 0.0001 when compared to its cytotoxicity against CCRF-CEM cells). Moreover, Oroxylum indicum showed a high level of antioxidant activity by inhibiting lipid-peroxidation (IC₅₀ 0.08 μg/ml).

Conclusions

This study provides in vitro evidence for the use of the Thai plants, most importantly Gynura pseudochina var. hispida, Oroxylum indicum and Muehlenbeckia platyclada as Thai anti-inflammatory remedies and these plants are now a priority for further phytochemical research.     

Anti-inflammatory activity of flavonoids from Cayaponia tayuya roots

Ethnopharmacological relevance

Taiuiá or tayuya (Cayaponia tayuya, Cucurbitaceae) is a climbing, lignified plant with a large swollen root that has traditionally been used as an anti-inflammatory and anti-rheumatic agent in the folk medicine of Brazil, Peru, and Colombia.

The aim of the study

We have assayed the pharmacological properties of a flavonoid fraction obtained from the butanol extract of Cayaponia tayuya roots using two models of topical mouse ear oedema, paying special attention to its influence on the induction on pro-inflammatory enzymes and peptidic mediators.

Material and methods

The in vivo experiments involved both the acute oedema induced by a single application of TPA and the subchronic inflammation brought on by repeated applications of TPA. The effects on the induction of pro-inflammatory enzymes and peptidic mediators in RAW 264.7 macrophages were analyzed with the aid of Western blot analysis.

Results

The extract was identified as a mixture of flavonoids in which vicenin-2, spinosin, isovitexin, and a mixture of swertisin and isoswertisin were found. In acute TPA-induced oedema in mouse ears, the flavonoid-enriched fraction (at a dose of 0.5 mg/ear) inhibited the oedema by 66% (4.2 ± 0.6 mg vs. 12.3 ± 1.4 mg, P < 0.01) while in the subchronic model, the inhibition reached 37% at a dose of 0.5 mg/ear × 7 applications (7.5 ± 0.6 mg vs. 11.9 ± 1.3 mg, P < 0.05). When assayed in vitro, the flavonoid showed no toxicity at 33.45 μg/mL on RAW 264.7 macrophages. Although the nitric oxide production in these cells was moderately reduced (42%) at 33.45 μg/mL, the flavonoid-enriched fraction had no effect on TNF-α production. In addition, at 22.30 μg/mL, the test sample inhibited both iNOS and COX-2 expression by 98% and 49%, respectively.

Conclusion

These results indicate that the anti-inflammatory activity of flavonoids from tayuya roots most likely stems from their inhibition of the induction of the enzymes COX-2 and iNOS.     

Biological effects of aqueous extract from Turkey vulture Cathartes aura (Cathartidae) meat

Ethnopharmacological relevance

Cathartes aura is a bird used in the Mexican traditional medicine for the empirical treatment of cancer, injuries, infections and burns.

Materials and methods

The in vitro immunomodulatory effects of Cathartes aura extract (CAE) were evaluated estimating its effects on proliferation of human peripheral blood mononuclear cells and murine splenocytes. The effects of CAE (1–200 μg/ml) on NO production, pinocytosis and lysosomal enzyme activity were assayed in murine macrophages RAW 264.7. The cytotoxic effects of CAE (1–500 μg/ml) on tumorigenic and non tumorigenic cells were evaluated using the MTT assay.

Results

In the absence of LPS, CAE induced the proliferation of murine splenocytes (119%), enhanced the pinocytosis (113%) and lysosomal enzyme activity (141%) in murine macrophages with a similar potency than lypopolisaccharides 1 μg/ml. In addition, CAE exerted cytotoxic effects mainly on human cervical cancer cells (IC₅₀=117 μg/ml) but lacked toxic effects on non tumorigenic cells (IC₅₀>500 μg/ml).

Conclusion

Cathartes aura exerts immunostimulatory and cytotoxic activities     

Water extract of Rheum officinale Baill. induces apoptosis in human lung adenocarcinoma A549 and human breast cancer MCF-7 cell lines

Ethnopharmacological relevance

Rheum officinale Baill. (Da Huang) is one of the herbs commonly used in traditional Chinese medicine formulae against cancer. The traditional decoction is similar to the water extract used in the present study.

Aim of the study

The water extract of Da Huang was investigated to see if it possesses anticancer effects through apoptotic pathways.

Materials and methods

Human lung adenocarcinoma A549 and human breast cancer MCF-7 cell lines were treated with different concentrations of Da Huang water extract at different time intervals. Growth inhibition was detected by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and colony formation assays; apoptosis was detected by cell morphologic analysis, DNA fragmentation analysis and COMET assay.

Results

Da Huang water extract was found to have significant growth inhibitory effects on both A549 and MCF-7 cell lines with IC₅₀ values 620 ± 12.7 and 515 ± 10.1 μg/ml, respectively. Growth inhibitory effects were dose- and time-dependent. A significant decrease in cell number, DNA fragmentation and single DNA strand breakages were observed in the Da Huang water extract treated A549 and MCF-7 cells.

Conclusions

This suggests that the water extract of Da Huang exerts potential anticancer activity through growth inhibition and apoptosis on MCF-7 and A549 cells lines.     

Insulin releasing and alpha-glucosidase inhibitory activity of ethyl acetate fraction of Acorus calamus in vitro and in vivo

Ethnopharmacological relevance

The radix of Acorus calamus L. (AC) is widely used in the therapy of diabetes in traditional folk medicine of America and Indonesia, and we previously reported the insulin sensitizing activity of the ethyl acetate fraction of AC (ACE).

Aim of the study

To investigate the insulin releasing and alpha-glucosidase inhibitory activity of ACE in vitro and in vivo.

Materials and methods

Insulin releasing and alpha-glucosidase inhibitory effects of different fractions from AC were detected in vitro using HIT-T15 cell line and alpha-glucosidase enzyme. Furthermore, effects of ACE orally on serum glucose were detected in fasted and glucose/amylum challenged normal mice.

Results

AC and ACE increased insulin secretion in HIT-T15 cells as gliclazide did. As in vivo results, ACE (400 and 800 mg/kg) significantly decreased fasting serum glucose, and suppressed the increase of blood glucose levels after 2 g/kg glucose loading in normal mice. In addition, ACE as a mixed-type inhibitor inhibited alpha-glucosidase activity in vitro with an IC₅₀ of 0.41 μg/ml, and 100 mg/kg of it clearly reduced the increase of blood glucose levels after 5 g/kg amylum loading in normal mice.

Conclusions

Apart from its insulin sensitizing effect, ACE may have hypoglycemic effects via mechanisms of insulin releasing and alpha-glucosidase inhibition, and thus improves postprandial hyperglycemia and cardiovascular complications.