Growth arrest and induction of apoptotic and non-apoptotic programmed cell death by, Physalis minima L. chloroform extract in human ovarian carcinoma Caov-3 cells

Ethnopharmacological relevance

The decoction of the whole plant of Physalis minima L. is traditionally consumed to treat cancer. Its anticancer property has been previously verified (using in vitro cytotoxicity assays) against NCI-H23 lung, CORL23 lung and MCF7 breast cancer cell lines but the mechanism underlying the anticancer potency towards ovarian carcinoma cells remain unclear.

Aim of the study

The present study is aimed to systematically determine the cytotoxicity and possible cell death mechanism elicited by the chloroform extract of Physalis minima in human ovarian Caov-3 carcinoma.

Materials and methods

Cytotoxicity of the extract was measured using the methylene blue assay. The mechanism of cell death was determined using four independent methods, namely DeadEnd™ assay to label the DNA fragmentation nuclei cells, RT-PCR analysis to determine the mRNA expression level of three apoptotic genes (c-myc, p53 and caspase-3 genes), Transmission Electron Microscope (TEM) analysis to describe the ultra structural characteristics and annexin V and propidium iodide staining to confirm the types and stages of cell deaths.

Results

Cytotoxicity screening of the extract on Caov-3 cells exhibited concentration- and time-dependent inhibitory effects. A combination of apoptotic and autophagic programmed cell death was detected. The apoptotic characteristic was initially determined by DNA fragmentation followed by the expression of c-myc and p53 genes that was much earlier than caspase-3. Apoptotic ultra structural changes (including clumping and magination of chromatin, blebbing and convolution of nucleus membrane and formation of apoptotic bodies) and autophagy (Type II non-apoptotic programmed cell death) with distinct vacuolated morphology were detected in TEM analysis. The existence of these programmed cell deaths was then corroborated using annexin V and propidium iodide staining.

Conclusions

The chloroform extract of Physalis minima exerted anticancer effect due to a combination of apoptotic and autophagic cell death mechanisms on Caov-3 cells. The induction of these programmed cell deaths was mediated via c-myc, p53 and caspase-3 dependent pathway. The results could provide a valuable insight in cancer therapy.     

Physalin A induces apoptosis via p53-Noxa-mediated ROS generation,and autophagy plays a protective role against apoptosis through p38-NF-κB survival pathway in A375-S2 cells

Ethnopharmacological relevance

Physalin A is a bioactive withanolide isolated from natural plant Physalis alkekengi L. var. franchetii (Mast.) Makino, a traditional Chinese herbal medicine named Jindenglong which has long been used for the treatment of cough, sore throat, hepatitis, eczema, dysuria and tumors in China.

Aim of the study

Based on the previous study that physalin A induced cytotoxic effect in human melanoma A375-S2 cells, this study was designed to further illustrate the molecular mechanisms underlying.

Materials and methods

Cell viability was evaluated in A375-S2 cells by MTT assay, and the mechanisms involved in physalin A-induced A375-S2 cell death were investigated by phase contrast microscopy and fluorescence microscopy, siRNA transfection, flow cytometry and western blot analysis.

Results

We demonstrated that physalin A decreased the proportion of viable A375-S2 cells in a time- and dose-dependent manner, and exposure of A375-S2 cells to physalin A led to both apoptosis and autophagy. Moreover, physalin A-induced apoptosis was triggered by activation of p53-Noxa pathway and intracellular reactive oxygen species (ROS) generation. The administration of ROS scavengers NAC and GSH resulted in the complete inhibition of physalin A-induced ROS generation and apoptosis. Application of p53 inhibitor PFT-α or transfection with Noxa-siRNA could also lead to the same results. Autophagy, demonstrated by the punctuate distribution of monodansylcadaverine staining, as well as the change of LC3-II/LC3-I proportion and Beclin 1 activation, played a protective role against apoptosis via up-regulation of the p38-NF-κB survival pathway in A375-S2 cells. Additionally, inhibition of autophagy by the specific autophagic inhibitor 3MA or blocking the p38-NF-κB pathway with p38 inhibitor SB203580 or NF-κB inhibitor PDTC obviously promoted physalin A-induced apoptosis.

Conclusions

Physalin A induced apoptotic cell death via p53-Noxa-mediated ROS generation, and autophagy played a protective role against apoptosis through up-regulating the p38-NF-κB survival pathway in A375-S2 cells. These results stated the possibility that physalin A would be a potential agent for the treatment of melanoma in the future.     

Evaluation of anti-tussive activity of Chamaedorea tepejilote

Aim of the study

Evaluate the potential inhibition of cough in a mouse model induced by sulfur dioxide gas of methanol and chloroform extracts of aerial parts of Chamaedorea tepejilote.

Material and methods

The antitussive effect was examined using the Ogyhara method. The extracts and theobromine were orally administered to mice. Each extract was administered to one group (400 mg/Kg) and theobromine (2 mg), and negative control received PVP. The frequency of cough was measured at 60, 90 min after the administration of chloroform extract.

Results

The water extract of C. tepejilote had no effect on the pharmacological model used in this study. The effects of the methanol and chloroform extracts of this plant on sulfur-dioxide-induced cough in experimental animals show activity dose-dependently in the range between 100 to 500 mg/Kg. At doses of 400 mg/kg, the methanol and chloroform extracts inhibited cough by 41.72% and 49.1%, respectively; these results are comparable to the effect produced by the antitussive agent theobromine (53.4% inhibition).

Conclusions

Both extracts exhibited significant antitussive activity, and the effect of the chloroform extract was dose dependent. The antitussive activity of this extract was comparable to that of theobromine. The effect of chloroform extract on the trachea was also tested; it did not relax pre-contractions in guinea pig trachealis induced KCl, carbachol or histamine.     

Anticancer potential of aqueous extract of alocasia macrorrhiza against hepatic cancer in vitro and in vivo

Ethnopharmacological relevance

Alocasia macrorrhiza has been used as a folk medicine for cancer treatment in the Southwest of China.

Aim of the study

The purpose of this study is to confirm the anticancer activity of aqueous extract of alocasia macrorrhiza against hepatic cancer and to elucidate its mechanism of action.

Materials and methods

Human normal liver cells and hepatocellular carcinoma cells were tested in vitro for cytotoxicity, colony formation inhibition, EdU incorporation, AO/EB staining apoptotic cells, apoptotic DNA fragmentation, and cell cycle distribution in response to alocasia macrorrhiza extract. The mRNA and protein expressions of PPARγ, Cyclin D1, Rb, P21, Bax, Bcl-2 and caspase-3 were detected through RT-PCR and Western blotting; the tumor growth inhibition in vivo was tested by oral administration of the extract.

Results

Alocasia macrorrhiza aqueous extract exhibited proliferation inhibition and apoptosis effects on human hepatocellular carcinoma cells in vitro, inhibited hepatoma growth in vivo.

Conclusion

Alocasia macrorrhiza extract has potential cytotoxic and apoptotic effect on human hepatocellular carcinoma cells and inhibits hepatoma growth in vivo, its mechanism of action might be associated with the inhibition of DNA synthesis, cell cycle (G₀/G₁) arrest, apoptosis induction through up-regulation the mRNA and protein expressions of PPARγ, Rb, Bax and capase-3genes and down-regulation of the expressions of Cyclin D1 and Bcl-2 genes.     

A petrol ether extract of the roots of Onosma paniculatum induces cell death in a caspase dependent manner

Aim of the study

Traditional Chinese medicine (TCM) has become very popular in Western countries during the last years. Zicao, a remedy of TCM, has been traditionally used to treat cancer, and, its main constituents, naphthoquinones, have been reported to possess antitumor activity (4 and 20). Here, we prepared extracts of different polarities of Onosma paniculatum Bur. & Franch., a plant which is amongst others used as Zicao, but, much less investigated. The extracts were analyzed concerning their growth inhibitory and apoptosis-inducing activity in various tumor cells.

Materials and methods

Cell viability was measured by XTT viability and a growth inhibition assay. Effects on the cell cycle and caspase-3 were determined by flow cytometry.

Results

From three different extracts, a petrol ether extract showed significant growth inhibitory effect, cell cycle influence and caspase-3 dependent induction of apoptosis which was time and dose dependent.

Conclusion

To further determine the activity and mechanism of action of the petrol ether extract, we would like to isolate and identify the active principle and investigate the effects in more detail.     

Antiplasmodial activity of root extract and fractions of Croton zambesicus

Aim of the study

Antiplasmodial activity of root extract and fractions of Croton zambesicus were evaluated to ascertain the folkloric claim of its antimalarial activity and elucidate its antiplasmodial mechanism of action.

Material and method

The crude ethanolic root extract (27–81 mg/kg) and gradient fractions ( n- hexane, chloroform, ethyl acetate and methanol; 54 mg/kg) of Croton zambesicus were investigated for antiplasmodial activity against chloroquine - sensitive Plasmodium berghei infections in mice. The antiplasmodial activity during early and established infections as well as the prophylactic activity were investigated. Chloroquine (5 mg/kg) and pyrimethamine (1.2 mg/kg) were used as positive controls. Thin films made from tail blood of each mouse were used to assess the level of parasitaemia of the mice. Oxidant generation potentials of the crude extract and fractions was also evaluated to elucidate their mechanism of action.

Results

The crude root extract (27 – 81 mg/kg) demonstrated significant (P < 0.01–0.001) schizonticidal activity during early and established infections and also had prophylactic activity. The activity was comparable to that of the standard drug used (chloroquine 5 mg/kg, pyrimethamine 1.2 mg/kg). Methanol, ethyl acetate and chloroform fractions had comparative in vivo antiplasmodial activity and oxidant generation potentials.

Conclusion

The antiplasmodial activity of this root extract and fractions which is likely to be through peroxidation confirms the folkloric use of this plant.     

In vitro and in vivo aphrodisiac properties of Corchorus depressus Linn. on rabbit corpus cavernosum smooth muscle relaxation and sexual behavior of normal male rats

Ethnopharmacological relevance

Corchorus depressus Linn. has been used as an aphrodisiac in traditional Indian medicine to treat male sexual dysfunction and impotency.

Aim of the study

The petroleum ether, chloroform, ethyl acetate, n-butanol and aqueous fractions of 95% methanol extract of Corchorus depressus were screened initially for their in vitro aphrodisiac activity on rabbit corpus cavernosum smooth muscle. The chloroform fraction (CDC) was found to be the most active and therefore investigated further on general mating behavior, libido and potency of normal male Wistar albino rats in comparison with the standard drug, Sildenafil citrate.

Materials and methods

Animals were divided into the following groups: Control, SC CDC 100, CDC 200, and CDC 400, who received saline, Sildenafil citrate or the chloroform fraction of Corchorus depressus at doses of 100, 200 or 400 mg/kg b.wt., respectively. The route of administration for all the groups was oral dosing, which was once in a day for 45 days. To analyze the mating behavior, female rats with estrus phase were used.

Results

The chloroform fraction of methanolic extract of Corchorus depressus significantly reduced ML, IL, PEI and III. There was a significant increase in the MF, IF and EL and serum testosterone levels throughout the study period. The potency test significantly increased erections, quick flips, long flips and total reflex. In vitro aphrodisiac activity was significantly higher in chloroform fraction at a concentration of 25.0 mg/ml, which induced 71.4% relaxation. The combined results of the above mentioned models indicate that the chloroform fraction of Corchorus depressus produces a significant increase in sexual activity as exhibited by 25 mg/ml in vitro and 400 mg/kg in vivo. In comparison with the control, all the drug-treated groups have shown drug-induced effects for a few parameters.

Conclusions

In vitro and in vivo studies provide valuable experimental evidence that the chloroform fraction of methanolic extract of Corchorus depressus possesses aphrodisiac property. This study further substantiates the ethnopharmacological claims of Corchorus depressus as a sexual stimulating agent and offers a significant potential for studying the effect on male sexual response and its dysfunctions.     

Anti-tumor activity and relative mechanism of ethanolic extract of Marsdenia tenacissima (Asclepiadaceae) against human hematologic neoplasm in vitro and in vivo

Ethnopharmacological relevance

Marsdenia tenacissima, a traditional Chinese medicinal herb, endemic to Yunnan Province is widely used to treat cough, asthma, expectorant, esophageal cancer, gastric cancer, lung cancer, and hepatocellular carcinoma. The aim of this study was to evaluate in vitro and in vivo anti-hematologic neoplasm potential of the ethanolic extract of this herb (crude ethanolic extract of Marsdenia tenacissima, CME) and by using different assays to elucidate its possible mechanism of action.

Materials and methods

The cytotoxicity of CME on tumor cells and peripheral blood mononuclear cells (PBMCs) was evaluated using MTT and apoptosis assays. We also tested the effect of CME on colony formation inhibition and cell cycle distribution of tumor cells. The protein expressions of Cyclin D1, Bax, Bcl-2, caspase-3 and caspase-9 were detected through Western blotting. In vivo anti-tumor effect was evaluated by measuring tumor volume changes, measuring tumor weight, evaluation of tumor microvessel density (MVD) and TUNEL staining by using immunohistochemistry staining in tumor models of nude mice.

Results

Marsdenia tenacissima ethanolic extract exhibited effects of proliferation inhibition and induction of apoptosis on human hematologic neoplasm tumor cells in vitro, as well as hematologic neoplasm growth in vivo.

Conclusion

This study clearly indicated that the ethanolic extract of Marsdenia tenacissima displayed strong anti-tumor effects against hematologic neoplasm cells and could induce tumor cells apoptosis in vitro and in vivo, and also had a significant anti-angiogenic effect in vivo against tumor cell apoptosis. Its multi-mechanism of action might be associated with the cell cycle (G0/G1) arrest, induction of apoptosis through up-regulation protein expressions of Bax, caspase-9 and caspase-3 genes and down-regulation of the expressions of Cyclin D1 and Bcl-2 genes, a decrease in tumor microvessel density and an increase of TUNEL-positive cells in vivo. These findings provided the molecular theoretical basis of clinical application.     

Hypericum grandifolium Choisy: A species native to Macaronesian Region with antidepressant effect

Aim of the study

Various species of Hypericum genus have been used in the Canary Islands as sedative, diuretic, vermifuge, wound healing, antihysteric and antidepressant agent. Studies have shown that methanol extract of Hypericum grandifolium Choisy is active in tetrabenazine-induced ptosis and forced swimming tests. In the current study, the aqueous, butanol and chloroform fractions obtained from the methanol extract as well as three sub-fractions derived from the chloroform fraction were evaluated for their central nervous effects in mice, particularly their antidepressant activity.

Materials and methods

The central nervous effect of different fractions and sub-fractions of Hypericum grandifolium was evaluated in mice using various behavioural models including locomotor and muscle relaxant activity, forced swimming test, effect on normal body temperature, barbiturate-induced sleep, tetrabenazine-induced syndrome and 5-hydroxytryptohan-induced head twitches and syndrome.

Results

We found that the butanol and chloroform fractions and all sub-fractions showed an antidepressant effect in the forced swimming test, the chloroform fraction being the most active. They produced no effects or only a slight depression of locomotor activity. Chloroform fraction significantly increased the pentobarbital-induced sleeping time, produced a slight but significant hypothermia and antagonized tetrabenazine-induced ptosis, whereas the butanol fraction produced a slight potentiation of 5-HTP-induced head twitches and syndrome.

Conclusions

The present results, together with previous pharmacological and phytochemical data, indicated that Hypericum grandifolium possess antidepressant-like effects in mice and that different constituents, such as the flavonoids and the benzophenone derivatives, could be responsible at least in part for the antidepressant effects observed for this species.     

Chrysanthemum morifolium Ramat (CM) extract protects human neuroblastoma SH-SY5Y cells against MPP-induced cytotoxicity

Ethnopharmacological relevance

Chrysanthemum morifolium Ramat (Asteraceae) has (CM) long been used in Korean and Chinese traditional herbal medicines with numerous therapeutic applications.

Aim of the study

To evaluate the neuroprotective activities of Chrysanthemum morifolium (CM) extract against 1-methyl-4-phenylpridinium ions (MPP⁺), Parkinsonian toxin through oxidative stress and impaired energy metabolism, in human SH-SY5Y neuroblastoma cells and the underlying mechanisms.

Materials and methods

The effects of CM against MPP⁺-induced cytotoxicity and neuronal cell viability, oxidative damage, the expression of Bcl-2 and Bax, caspase-3 and poly(ADP-ribose) polymerase (PARP) proteolysis were evaluated by using SH-SY5Y neuroblastoma cells.

Results

CM effectively inhibited the cytotoxicity and improved cell viability. CM also attenuated the elevation of reactive oxygen species (ROS) level, increase in Bax/Bcl-2 ratio, cleavage of caspase-3 and PARP proteolysis.

Conclusion

These results demonstrate that CM possesses potent neuroprotective activity and therefore, might be a potential candidate in neurodegenerative diseases such as Parkinson's disease.     

Anti-inflammatory mechanism of Kaempferia parviflora in murine macrophage cells (RAW 264.7) and in experimental animals

Ethnopharmacological relevance

The rhizomes of Kaempferia parviflora Wall. ex Baker have been used in Thailand for treatment of gout, apthous ulcer, peptic ulcer and abscesses.

Aim of the study

In our previous study, the crude ethanol extract of Kaempferia parviflora and its compound (5, 5-hydroxy-3,7,3′,4′-tetramethoxyflavone), was reported to show nitric oxide (NO) inhibition in RAW 264.7 cells. The present study is thus investigated the anti-inflammatory mechanism of Kaempferia parviflora extract and compound 5 against inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expressions.

Materials and methods

The extract of Kaempferia parviflora and its compound were tested against NO and prostaglandin E₂ (PGE₂) releases using RAW264.7 cells as well as studied on anti-inflammatory activity in carrageenan-induced rat paw edema and acute toxicity in mice.

Results

The results revealed that the ethanol extract of Kaempferia parviflora markedly inhibited PGE₂ release with an IC₅₀ value of 9.2 μg/ml. This plant extract and compound 5 also suppressed mRNA expression of iNOS in dose-dependent manners, whereas COX-2 mRNA expression was partly affected. According to the in vivo study, chloroform and hexane fractions greater decreased rat paw edema than ethanol, ethyl acetate and water fractions.

Conclusion

The mechanisms for anti-inflammatory activity of Kaempferia parviflora and compound 5 are mainly due to the inhibition of iNOS mRNA expression but partly through that of COX-2 mRNA.     

Exploring the wound healing activity of Arnebia densiflora (Nordm.) Ledeb. by in vivo models

Ethnopharmacological relevance

The roots of Arnebia densiflora (Nordm.) Ledeb. (Boraginaceae), which is an endemic plant for Turkey, have been used for healing of wounds in folk medicine. The extract of the roots in olive oil and mostly the root barks was applied onto open wounds for rapid healing.

Aim of the study

To evaluate the wound healing activity of the roots, extracts were prepared with different solvents; hexane, chloroform, ethyl acetate and methanol, respectively from the roots of Arnebia densiflora.

Materials and methods

Incision by using tensiometer and excision models were employed on mice and rats.

Results

Significant wound healing activity was observed with the ointment formulation prepared by using hexane extract at 1% concentration on the mentioned models. The results of histopathological examination supported the outcome of both incision and excision wound models. The wound healing effect was comparatively evaluated with a reference ointment Madecassol^®.

Conclusion

The experimental data demonstrated that Arnebia densiflora displayed remarkable wound healing activity.     

Hexane extract from Polygonum multiflorum attenuates glutamate-induced apoptosis in primary cultured cortical neurons

Ethnopharmacological relevance

Polygonum multiflorum has traditionally had wide use as an anti-aging treatment in East Asian countries. We investigated the neuroprotective effects of Polygonum multiflorum against glutamate-induced neurotoxicity with a focus on the anti-apoptotic mechanism in primary cultured cortical neurons.

Material and methods

Cell viability, cytotoxicity, morphological, flow cytometry, Western blot, and caspase activity assays were performed for examination of the neuroprotective effects of active hexane extract from Polygonum multiflorum (HEPM).

Results

Pretreatment with HEPM resulted in significantly decreased glutamate-induced neurotoxicity in a concentration-dependent manner and also resulted in drastically inhibited glutamate-induced apoptosis. Treatment with HEPM resulted in decreased expression of glutamate-induced death receptor (DR)4, and enhanced expression of glutamate-attenuated anti-apoptotic proteins, including Bcl-2, XIAP, and cIAP-1, and slightly reduced glutamate-induced cleavage of Bid. In addition, treatment with HEPM resulted in suppressed glutamate-induced activation of caspase-8, caspase-9, and caspase-3, and, subsequently, decreased degradation of poly(ADP-ribose) polymerase, β-catenin, and phospholipase Cγ1 protein, which are downstream targets of activated caspase-3.

Conclusions

The results of this study demonstrated that HEPM exerts a neuroprotective effect against glutamate-induced neurotoxicity via inhibition of apoptosis. This protection may be mediated through suppression of DR4 and up-regulation of Bcl-2, XIAP, and cIAP-1, as well as inhibition of caspase activation, resulting in prevention of apoptosis of cortical neurons.     

Cytotoxic effects of Echinacea purpurea flower extracts and cichoric acid on human colon cancer cells through induction of apoptosis

Ethnopharmacological relevance

Echinacea is a top-selling herbal supplement that acts as immunostimulant. It has been used to treat common cold, coughs, bronchitis and upper respiratory infections. It is also a popular product used in anticancer therapy. The cytotoxic effects of Echinacea on cancer cells are still not clear. The aims of this study were to provide a preliminary validation of the effects of 50% aqueous ethanol extract of Echinacea purpurea flowers and its major compound, cichoric acid, on human colon cancer cells Caco-2 and HCT-116.

Materials and methods

The cytotoxic effects of Echinace flower extracts and cichoric acid on cell viability, telomerase activity, DNA fragmentation, β-catenin, caspase-9, and cleavage of poly-ADP-ribose polymerase (PARP) of human colon cancer cell were examined.

Results

The results showed a significant inhibition of proliferation in E. purpurea flower extract and cichoric acid in a dose- and time-dependent manner. Cichoric acid treatment decreased telomerase activity in HCT-116 cells. Moreover, cichoric acid effectively induced apoptosis in colon cancer cells, which were characterized by DNA fragmentation, activation of caspase-9, cleavage of PARP and downregulation of β-catenin.

Conclusions

Our data indicate that cichoric acid has a strong growth-inhibitory effect against colon cancer cells, presumably resulting from the reduced telomerase activity and the induction of apoptosis. The exact mechanism of action should still be determined in future studies. Overall, the effects of 50% aqueous ethanol extract of E. purpurea flowers and cichoric acid may have provided in vitro evidence for the use as chemotherapeutic agents.     

On the potential of Tephrosia purpurea as anti-Helicobacter pylori agent

Aim of the study

Since Tephrosia purpurea (Linn.) Pers. (Fabaceae) has traditional use in curing different types of wounds including gastroduodenal ulcers, it was of interest to evaluate the in vitro anti-Helicobacter pylori activity profile of the plant extract and its fractions with a view to examining its therapeutic potential, if any.

Materials and methods

Employing clinical isolates and standard strains of Helicobacter pylori, the extract and fractions were bioevaluated in terms of MIC and MBC values, acid stability, time-kill kinetics, drug resistance, and synergistic potential.

Results

The methanolic extract showed promising activity against clinical isolates and standard strains of Helicobacter pylori, including metronidazole-resistant strains. Fractionation of the extract revealed the n-hexane and chloroform fractions to possess marked activity. The extract and the less polar fractions remained functionally active in acidic condition similar to stomach environment, exhibited consistent bacteriostatic activity during repeated exposure, and demonstrated synergism, complete or partial, even with antibiotic-resistant strains.

Conclusion

Apolar fractions of Tephrosia purpurea may have therapeutic potential in combating Helicobacter pylori mediated gastroduodenal disorders.     

Investigation on the morphological protective effect of 5-hydroxymethylfurfural extracted from wine-processed Fructus corni on human L02 hepatocytes

Aim

To determine the mode of action of 5-hydroxymethylfurfural (5-HMF) extracted from wine-processed Fructus corni on hepatoprotective activities, the effects of 5-HMF on H₂O₂-induced human L02 hepatocytes injury was examined.

Mthods

Hepatocytes L02 injured by H₂O₂ was treated by 5-HMF. The morphological changes of the cells were observed under inverted phase-contrast, fluorescence, and transmission electron microscopy and the activities of caspase-9 and caspase-3 were tested by enzyme-linked immunosorbent detector.

Results

It revealed that 5-HMF improved the morphology of H₂O₂-treated human L02 hepatocytes, and also inhibited the level of caspase-9 and caspase-3 of them.

Conclusions

These results suggested a morphological hepatocyte protective effect and the anti-apoptosis mechanism by 5-HMF.     

Anti-tumor and pro-apoptotic activity of ethanolic extract and its various fractions from Polytrichum commune L.ex Hedw in L1210 cells

Ethnopharmacological relevance

Polytrichum commune L.ex Hedw is a traditional Chinese herb for treatment of fever, hemostatic, uterine prolapse and especially for lymphocytic leukemia, but the antitumor effect and its potential mechanism remains unclear.

Aims of the study

The present study was to investigate the possible anti-proliferative activity of ethanolic extract and the organic fractions from P. commune on murine leukemia L1210 cells.

Materials and methods

The content of ethanolic extract and its fractions was performed on HPLC analysis with gradient elution. L1210 cells were treated with different concentrations of ethanolic extract and its fractions at different time intervals. Cell viability was evaluated using MTT assay. Apoptotic cell death was monitored by nuclear condensation and confirmed by exposure of phosphatidylserine to outer leaflet of plasma membrane. Loss of mitochondrial membrane potential was analyzed by flow cytometry using rhodamine 123 staining.

Results

The obtained results showed that the cell viability of L1210 cells was reduced by ethanolic extract of P. commune in a concentration-dependent manner, and the IC₅₀ value was about 77.22 μg/ml at 24 h post treatment. The ethylacetate fraction displayed higher anti-tumor effect than that of chloroform and butanol fractions with 32.29 μg/ml (IC₅₀ value, 48 h). Microscopy studies revealed that ethanolic extract and ethylacetate fraction treated cells showed morphological characteristics of apoptosis such as chromatin condensation and DNA aggregation. Further, Annexin V-PE/ 7-AAD double staining showing the out leaflet of phosphatidylserine and the decline of mitochondrial membrane potential by flow cytometry confirmed that the extracts do, in fact, induce apoptosis in L1210 cells.

Conclusion

This is the first report on anti-tumor and pro-apoptotic effect of P. commune in cultured leukemia cells, which provides scientific basis for its usefulness as traditional medicine. Further studies are needed to confirm the precise mechanism not only the crude extract but as well the monomeric chemical substances of Polytrichum commune L.ex Hedw.     

Antioxidant and anti-inflammatory activities of aqueous extract of Centipeda minima

Ethnopharmacological relevance

Centipeda minima (L.) is traditionally used in Chinese folk medicine for the treatments of rhinitis, sinusitis, relieving pain, reducing swelling, and treating cancer for a long history in Taiwan. However, there is no scientific evidence which supports the use in the literature.

Aim of the study

The aim of this study was to evaluate the antioxidant and anti-inflammatory activities of the aqueous extract of Centipeda minima (ACM).

Materials and methods

The following activities were investigated: antioxidant activities [2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), DPPH (1,1-diphenyl-2-picrylhydrazyl)], and anti-inflammatory [lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW264.7 macrophages and paw-edema induced by λ-carrageenan (Carr)]. We also investigated the anti-inflammatory mechanism of ACM via studies of the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) and the levels of malondialdehyde (MDA) in the edema paw. Serum NO, tumor necrosis factor α (TNF-α), and interleukin-1β (IL-1β) were also measured in vivo. In HPLC analysis, the fingerprint chromatogram of ACM was established.

Results

ACM showed the highest TEAC and DPPH radical scavenging activities, respectively. ACM also had highest contents of polyphenol and flavonoid contents. We evaluated that ACM and the reference compound of protocatechualdehyde and caffeic acid decreased the LPS-induced NO production in RAW264.7 cells. Administration of ACM showed a concentration dependent inhibition on paw edema development after Carr treatment in mice. The anti-inflammatory effects of ACM could be via NO, TNF-α, and IL-1β suppressions and associated with the increase in the activities of antioxidant enzymes. Western blotting revealed that ACM decreased Carr-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions.

Conclusions

Anti-inflammatory mechanisms of ACM might be correlated to the decrease in the level of Malondialdehyde (MDA), iNOS, and COX-2 via increasing the activities of CAT, SOD, and GPx in the edema paw. Overall, the results showed that ACM demonstrated antioxidant and anti-inflammatory activity, which supports previous claims of the traditional use for inflammation and pain.     

Anti-inflammatory and anti-nociceptive activities of Platanus orientalis Linn. and its ulcerogenic risk evaluation

Ethnopharmacological relevance

Leaves of Platanus orientalis Linn. are used in folk medicine as a wound-healer and ophthalmologic agent. Phytol derivatives from the leaves of plane-tree show anti-ulcer activity. Its analgesic and anti-inflammatory effects for knee pain were known to Persian scientists and hakims.

Materials and methods

The ethanolic extract of Platanus orientalis Linn. and its various fractions were given at a dose of 100 mg/kg po and 200 mg/kg po for testing their anti-inflammatory activity by carrageenan induced hind paw edema. The analgesic activity of the ethanolic extract and its fractions has been carried out by tail-flick method and writhing test at a dosage of 200 mg/kg po. Gastric ulceration studies have been further carried out to study the ulcerogenic risk evaluation of the ethanolic extract and its various fractions at a dose of 600 mg/kg body weight.

Results

Among the tested fractions, chloroform fraction exhibited better inhibition (68.33%) at 200 mg/kg po dosage when compared to the standard drug Ibuprofen (66.66%) after 3 h in the carrageenan induced hind paw edema. The ethanolic extract and all its fractions especially the chloroform (p<0.01) showed significant analgesic activity with insignificant ulceration as compared to the standard drug i.e. Ibuprofen. The histopathological study of ethanolic extract and its fractions revealed that none of them cause ulcer.

Conclusion

The present study indicates that Platanus orientalis Linn. has significant anti-inflammatory and analgesic effect.