Potential anticancer properties of the water extract of Inontus obliquus by induction of apoptosis in melanoma B16-F10 cells

Ethnopharmacological relevance

Inonotus obliquus (Chaga mushroom), one of the widely known medicinal mushrooms, has been used to treat various cancers in Russia and most of Baltic countries for many centuries.

Aim of the study

To examine the anti-proliferative effects of Inonotus obliquus extract on melanoma B16-F10 cells. Furthermore, to assess the anti-tumor effect of Inonotus obliquus extract in vivo in Balb/c mice.

Materials and methods

The water extract of Inonotus obliquus was studied for anti-proliferative effects on the growth and morphology of B16-F10 melanoma cells and for anti-tumor effect using in vivo in Balb/c mice.

Results

Inonotus obliquus extract not only inhibited the growth of B16-F10 cells by causing cell cycle arrest at G₀/G₁ phase and apoptosis, but also induced cell differentiation. These effects were associated with the down-regulation of pRb, p53 and p27 expression levels, and further showed that Inonotus obliquus extract resulted in a G₀/G₁ cell cycle arrest with reduction of cyclin E/D1 and Cdk 2/4 expression levels. Furthermore, the anti-tumor effect of Inonotus obliquus extract was assessed in vivo in Balb/c mice. Intraperitoneal administration of Inonotus obliquus extract significantly inhibited the growth of tumor mass in B16-F10 cells implanted mice, resulting in a 3-fold (relative to the positive control, ^*p < 0.05) inhibit at dose of 20 mg/kg/day for 10 days.

Conclusion

This study showed that the water extract of Inonotus obliquus mushroom exhibited a potential anticancer activity against B16-F10 melanoma cells in vitro and in vivo through the inhibition of proliferation and induction of differentiation and apoptosis of cancer cells.     

Anti-inflammatory activity of ethyl acetate fraction of the seeds of Brucea Javanica

Ethnopharmacological relevance

The seeds of Brucea javanica (L.) Merr. (Yadanzi in Chinese) have been used for the treatment of inflammation, dysentery, malaria, and cancer in Chinese traditional medicine. However, the anti-inflammatory mechanism of Brucea javanica has not been fully elucidated. This study examined the anti-inflammatory activity of ethyl acetate fraction of the seeds of Brucea javanica (EA-BJ) in vitro and in vivo.

Materials and methods

The anti-inflammatory activity of EA-BJ and its ability to modulate the production of NO, PGE2, TNF-α, IL-1β, IL-6 and IL-10 inflammatory mediators in lipopolysaccharide-activated RAW 264.7 macrophage were evaluated. Moreover, the anti-inflammatory activity of EA-BJ was also in vivo assayed by carrageenan induced paw edema in mice.

Results

In vitro assays showed remarkable anti-inflammatory activity of EA-BJ, through the inhibition of production of NO, PGE2, TNF-α, IL-1β and IL-6 inflammatory mediators and induction of production of IL-10 anti-inflammatory cytokine. In vivo assays showed anti-inflammatory activity for decrement of the paw edema in carrageenan induced paw edema test.

Conclusion

The results obtained in vitro and in vivo showed that possible anti-inflammatory effects of EA-BJ may be attributed to inhibition pro-inflammatory mediators production, NO, PGE2, TNF-α, IL-1β and IL-6 and to increase production of IL-10 anti-inflammatory cytokine. The seeds of Brucea javanica may thus prove beneficial in the treatment of inflammatory diseases.     

Echinacea increases arginase activity and has anti-inflammatory properties in RAW 264.7 macrophage cells,indicative of alternative macrophage activation

Ethnopharmacological relevance

The genus Echinacea is a popular herbal immunomodulator. Recent reports indicate that Echinacea products inhibit nitric oxide (NO) production in activated macrophages.

Aim of the study

In the present study we determined the inhibitory effects of alcohol extracts and individual fractions of alcohol extracts of Echinacea on NO production, and explored the mechanism underlying the pharmacological anti-inflammatory activity.

Materials and methods

Alcohol extracts of three medicinal Echinacea species, Echinacea angustifolia, Echinacea pallida and Echinacea purpurea, were prepared using Soxhlet apparatus and fractionated using HPLC. NO production by LPS activated RAW 264.7 macrophage cells was measured using a Griess reagent and iNOS detected using immunoblotting. In addition, effects on arginase activity were measured in RAW 264.7 cells stimulated with 8-bromo-cAMP +/− LPS.

Results

Alcohol extracts of all three Echinacea species significantly inhibited NO production by lipopolysaccharide (LPS)-activated the RAW 264.7 macrophage cell line; among them Echinacea pallida was the most active. The Echinacea-mediated decrease in NO production was unlikely due to a direct scavenging of NO because the extracts did not directly inhibit NO released from an NO donor, sodium nitroprusside. An immunoblotting assay demonstrated that the extract of Echinacea pallida inhibited inducible nitric oxide synthase (iNOS) protein expression in LPS-treated macrophages. The enzymes iNOS and arginase metabolize a common substrate, l-arginine, but produce distinct biological effects. While iNOS is involved in inflammatory response and host defense, arginase participates actively in anti-inflammatory activation. Arginase activity of RAW 264.7 cells stimulated with 8-bromo-cAMP was significantly increased by alcohol extracts of all three Echinacea species. The polar fraction containing caffeic acid derivatives enhanced arginase activity, while the lipophilic fraction containing alkamides exhibited a potential of inhibiting NO production and iNOS expression.

Conclusions

These results suggest that the anti-inflammatory activity of Echinacea might be due to multiple active metabolites, which work together to switch macrophage activation from classical activation towards alternative activation.     

Davilla elliptica and Davilla nitida: Gastroprotective,anti-inflammatory immunomodulatory and anti-Helicobacter pylori action

Ethnopharmacological relevance

Davilla elliptica and Davilla nitida are species commonly found in the Brazilian Cerrado biome.

Aim of the study

Based on ethnopharmacological and phytochemical analyses, methanolic extracts from leaves of Davilla elliptica (EDE) and Davilla nitida (EDN) were evaluated for their anti-ulcer, anti-inflammatory, immunological and anti-Helicobacter pylori activities.

Materials and methods

The gastroprotective action of both extracts was evaluated in rodent experimental models (HCl/ethanol, ethanol or NSAID). We also evaluated anti-inflammatory (carrageenin-induced rat hind paw edema), immunomodulatory (murine peritoneal macrophages) and antibacterial action of both extracts against a standard strain of Helicobacter pylori.

Results

EDE and EDN (500 mg/kg) were able to protect gastric mucosa against HCl/ethanol solution (EDE 63%; EDN 59%), absolute ethanol (EDE 95%; EDN 88%), and also against injurious effect of NSAID (EDE 77%; EDN 67%). When EDE and EDN were challenged with sulfhydryl depleter compound, the gastroprotective action of both extracts was completely abolished. EDE had gastroprotective effect related to increase of glutathione bioavailability and stimulated higher levels of NO, H₂O₂ and TNF-α production. Otherwise EDN showed better anti-Helicobacter pylori action than EDE. Neither extracts presented anti-inflammatory activity by oral route.

Conclusion

The phytochemical investigation showed that both extracts possess phenolic acid derivatives, acylglycoflavonoids and condensed tannins with evident quantitative variations that probably influenced the pharmacological differences between extracts.     

Anti-inflammatory action of hydroalcoholic extract,dichloromethane fraction and steroid α-spinasterol from Polygala sabulosa in LPS-induced peritonitis in mice

Ethnopharmacological relevance

Polygala sabulosa A. W. Bennett is a small herb popularly known as “timutu-pinheirinho” that is widely distributed in southern Brazil and that is used to treat disorders of the bowel and kidney and as a topical anesthetic and expectorant in folk medicine. This study was designed to investigate the anti-inflammatory properties of the hydroalcoholic extract (HEPs), CH₂Cl₂ fraction and the steroid α-spinasterol obtained from the aerial parts of Polygala sabulosa in a model of acute inflammation induced by intraperitoneal injection of bacterial lipopolysaccharide in mice.

Materials and methods

The anti-inflammatory effect of HEPs (3–300 mg/kg, i.g.), CH₂Cl₂ fraction (0.003–30 mg/kg, i.g.) and steroid α-spinasterol (0.001–1 mg/kg, i.p. or 1–10 mg/kg, i.g.), were evaluated in mice subjected to the acute inflammation caused by intraperitoneal (i.p.) injection of lipopolysaccharide (LPS, 0.02 µg/kg). The anti-inflammatory activity of the HEPs, CH₂Cl₂ fraction and steroid were assessed by determining the total numbers of leukocytes and differential cell counts (neutrophils and mononuclear cells) and levels of pro-inflammatory (IL-1β, TNF-α, IL-6) or anti-inflammatory (IL-10) cytokines in peritoneal fluid.

Results

The administration of HEPs (3–300 mg/kg, i.g.) completely inhibited inflammatory cell infiltration (300 mg/kg, i.g.) and it reduced TNF-α (100–300 mg/kg) and IL-1β (100 mg/kg) levels in LPS-injected mice. Furthermore, the administration of CH₂Cl₂ fraction (0.003–30 mg/kg, i.g.) or α-spinasterol (0.001–10 mg/kg, by i.p. or i.g.) significantly reduces inflammatory cell infiltration in LPS-injected mice. Moreover, dexamethasone (0.5 mg/kg, i.p., used as a positive control) inhibited inflammatory cell infiltration and reduced the levels of TNF-α, IL-1β and IL-6 in LPS-injected mice.

Conclusions

Taken together, these results provide the first experimental evidence demonstrating that HEPs have significant anti-inflammatory effects on LPS-induced inflammation. These effects appear to be, at least in part, due to the presence of α-spinasterol. These findings support the widespread use of Polygala sabulosa in popular medicine and demonstrate that this plant has therapeutic potential for the development of phytomedicines with anti-inflammatory properties.     

In vivo anti-inflammatory and wound healing activities of Centaurea iberica Trev. ex Spreng

Aim of the study

Several Centaurea species (Asteraceae) are used in Turkish folk medicine to alleviate pain and inflammatory symptoms in rheumatoid arthritis, high fever, head ache and for wound healing. Particularly, the aerial part of Centaurea iberica Trev. ex Spreng. has been practiced on wounds for healing. In order to evaluate the anti-inflammatory and wound healing activities of the plant, extracts were prepared with variety of solvents: hexane, chloroform, ethyl acetate and aqueous methanol (85%) from the aerial parts of Centaurea iberica.

Materials and methods

The incision by using tensiometer and excision models were used in order to assess the effect of the plant extracts on wound healing in mice and rats. Results were also evaluated histopathologically. In vivo inhibitory effect of the extracts on acetic acid-induced increase in capillary permeability was studied for the assessment of anti-inflammatory activity.

Results

The wound healing effect was comparatively evaluated with a reference ointment Madecassol^®. Noteworthy wound healing activity was observed for the ointment formulation prepared with 1% methanol extract. The results of histopathological evaluation supported the outcome of both incision and excision wound models. Moreover, the methanol extract exerted remarkable wound healing activity and also demonstrated a significant and dose-dependent anti-inflammatory activity.

Conclusion

The experimental study revealed that Centaurea iberica displays remarkable wound healing and anti-inflammatory activity.     

Substantiation of the ethnopharmacological use of Conyza sumatrensis (Retz.) E.H.Walker in the treatment of malaria through in-vivo evaluation in Plasmodium berghei infected mice

Ethnopharmacological relevance

Scientific validation of ethnopharmacologically used plants and their utilization for therapeutic interventions can be a source of affordable treatment especially for neglected diseases in endemic areas. Conyza sumatrensis is a plant which finds its use in treating malaria like fevers but lacks proper scientific validation. Our study has tried to address this gap by authenticating its traditional use for the treatment of malaria.

Aim of the study

Evaluate the antimalarial activity of extracts derived from Conyza sumatrensis for its ethnopharmacological validation.

Materials and methods

Shade dried leaves were extracted with varying concentrations of ethanol and concentrated for bio-evaluation. Swiss albino mice infected with 1×10⁶ parasitized red blood cells, were orally administered with test extracts for 7 days in two sets of experiments. The first set was used to evaluate alcoholic, hydroalcoholic and aqueous extracts while the second set was used to evaluate the dose response of alcoholic extract ranging from 500–1600 mg/kg. Mean survival time, parasitaemia and haemoglobin levels were considered to interpret the antimalarial potential. Phytochemical analysis for the presence of flavonoids, alkaloids tannins, total phenolics, riboflavin and thiamine was also carried out.

Results

Among the three extracts administered at 1000 mg/kg, chemo suppression was significantly (p<0.001) observed in the alcoholic extract (62.59±12.52%) followed by hydroalcoholic (41.81±19.04%, p<0.01) and aqueous (32.04±19.04%, P<0.05) indicating that the active constituents leach out in ethanol. The dose response study involving the ethanol extract concluded the optimum dose to be 1000 mg/kg, as also evidenced by the haemoglobin levels.

Conclusion

The plant exhibits moderate antimalarial activity which can be further prospected for active fractions or pure molecules for adjunctive therapy.     

Orally administered aqueous extract of Inonotus obliquus ameliorates acute inflammation in dextran sulfate sodium (DSS)-induced colitis in mice

Ethnopharmacological relevance

Chaga mushroom (Inonotus obliquus) has been used in folk medicine to treat several disorders through its various biological functions. I. obliquus is claimed to produce general immune-potentiating and strengthening, antiinflammatory, and antitumor properties, but its effects on intestinal inflammation (ulcerative colitis) are clearly not understood.

Aim of the study

To determine the effects and mode of action of an aqueous extract of I. obliquus (IOAE) on experimental colitis in mice induced by dextran sulfate sodium (DSS).

Materials and methods

Female 5-week-C57BL/6 mice were randomized into groups differing in treatment conditions (prevention and treatment) and doses of IOAE (50 and 100 mg/kg body weight). Mice were exposed to DSS (2%) in their drinking water over 7 day to induce acute intestinal inflammation. In colon tissues, we evaluated histological changes by hematoxylin and eosin staining, levels of iNOS by immuno-histochemical staining, and neutrophil influx by myeloperoxidase assay. mRNA expression of pro-inflammatory mediators TNF-α, IL-1β, IL-6, and IFN-γ was determined by RT-PCR.

Results

Histological examinations indicated that IOAE suppressed edema, mucosal damage, and the loss of crypts induced by DSS. IOAE markedly attenuated DSS-induced iNOS levels and myeloperoxidase accumulation in colon tissues, demonstrating its suppressive effect on infiltration of immune cells. In addition, IOAE significantly inhibited mRNA expression of pro-inflammatory cytokines induced by DSS in colon tissues.

Conclusion

Our results suggest anti-inflammatory effect of IOAE at colorectal sites due to down-regulation of the expression of inflammatory mediators. Suppression of TNF-α and iNOS together with IL-1β by IOAE denotes that it might be a useful supplement in the setting of inflammatory bowel disease.     

In vivo anti-inflammatory activities of Ocimum suave in mice

Ethnopharmacological relevance

Ocimum suave has been used in the Ethiopian traditional medicine to relieve pain, fever, inflammation and other disease conditions.

Aim of the study

The aim of the present study was to investigate the anti-inflammatory activities of the aqueous and ethanol leaf extracts and some fractions of Ocimum suave in mice.

Materials and methods

The crude extracts were screened for their anti-inflammatory activities on carrageenan-induced mouse paw edema at three dose levels. The butanol and aqueous fractions of the aqueous extract were also evaluated for their anti-inflammatory activities using carrageenan, histamine and serotonin-induced mouse paw edema at three dose levels. Normal saline and aspirin were employed as negative and positive control groups, respectively.

Results

Both ethanol and aqueous extracts significantly decreased carrageenan-induced inflammation at all the three doses used. However, greater paw edema inhibition was observed with the aqueous extract. The two fractions also showed significant reduction of inflammation against inflammatory models in which the aqueous residue exhibited the highest inhibition.

Conclusions

From the present findings, it can be concluded that the ethanol and aqueous leaf extracts as well as butanol and aqueous fractions of Ocimum suave have shown anti-inflammatory properties.