The natural triterpene 3ß,6ß,16ß-trihydroxy-lup-20(29)-ene obtained from the flowers of Combretum leprosum induces apoptosis in MCF-7 breast cancer cells.

BACKGROUND: The 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene (TTHL) is a pentacyclic triterpene obtained from the medicinal plant Combretum leprosum Mart. In folk medicine, this plant is popularly known as mofumbo, cipoaba or mufumbo, and is used to treat several diseases associated with inflammation and pain. METHODS: We investigated the antitumor efficacy of TTHL isolated from C. leprosum. The TTHL cytotoxic effect was investigated in MRC5, MCF-7, HepG2, T24, HCT116, HT29, and CACO-2 cells after 24, 48, 72 and 120 h of treatment. The mechanisms of cell death and DNA damage induction were investigated by flow cytometry and comet assay, respectively. RESULTS: The results indicated that TTHL induced a time- and concentration-dependent growth inhibition in all human cancer cell lines. The cytotoxicity was more pronounced in MCF-7 breast cancer cells, with an IC50 of 0.30 µg/mL at 120 h. We therefore evaluated the cell death mechanism induced by TTHL (IC20, IC50, and IC80) in MCF-7 cells at 24 h. We found that the treatment with IC50 and IC80 TTHL for 24 h induced apoptosis in 14% (IC50) and 52% (IC80) of MCF-7 cells. The apoptosis induced by TTHL was accompanied by increased levels of both cleaved caspase-9 and intracellular ROS. In order to further understand the biological mechanism of TTHL-induced cytotoxicity, we have also investigated its effect on different Saccharomyces cerevisiae yeast strains. The mutant strains sod1Δ, sod2Δ, and sod1Δsod2Δ, which are deficient in superoxide dismutase antioxidant defenses, were hypersensitive to TTHL, suggesting that its capacity to disturb cellular redox balance plays a role in drug toxicity. Moreover, TTHL induced mutagenicity in the yeast strain XV185-14c. CONCLUSIONS: Taken together, the results suggest that TTHL forms covalent adducts with cellular macromolecules, potentially disrupting cellular function and triggering apoptosis.

Combretum leprosum Mart. (Combretaceae): potential as an antiproliferative and anti-inflammatory agent.

ETHNOPHARMACOLOGICAL RELEVANCE: Combretum leprosum is a species that is popularly used in Brazil as a healing agent to treat skin problems and lesions. In this study we investigated the possible potential of this extract to treat inflammatory and hyperproliferative skin conditions. MATERIALS AND METHODS: Classical models of skin inflammation such as TPA- and croton oil-induced mouse ear oedema were applied in order to verify the potential topical anti-inflammatory activity of the ethanolic extract from flowers of Combretum leprosum. RESULTS: Topical application of ethanolic extract promoted a dose-dependent inhibition of phorbol ester-induced ear oedema, reduced myeloperoxidase activity and IL-6 tissue levels with inhibition comparable to dexamethasone (positive control). Histological and immunohistochemical analysis revealed that ethanolic extract also suppressed cell infiltration. Ethanolic extract altered inflammatory parameters on a chronic skin inflammation model induced by repeated applications of croton oil, decreasing ear oedema, epidermal hyperproliferation and cell infiltration. In addition, immunohistochemical analysis showed that the extract decreased PCNA expression on the epidermis. CONCLUSION: Taken together, these results suggest that the extract from flowers of Combretum leprosum could be considered as a new potential tool for the treatment of several skin inflammatory diseases since it reversed the skin inflammatory and hyperproliferative process in a very significant manner. Further investigations are needed in order to verify the cellular mechanism and safety of Combretum leprosum extract.

In vitro and in vivo immunostimulatory activity of Woodfordia fruticosa flowers on non-specific immunity.

CONTEXT: Woodfordia fruticosa Kurz. (Lythraceae), a non-rasayana immunomodulatory Indian medicinal plant, used traditionally as an anthelmintic, in dysentery, leprosy, blood diseases, leucorrhea, and menorrhagia. OBJECTIVE: To investigate the effect of ethanol extract of W. fruticosa flowers on non-specific immune responses in mice. MATERIALS AND METHODS: In vitro immunomodulatory activity of the extract was examined on murine peritoneal macrophage phagocytosis (nitroblue tetrazolium (NBT) dye reduction, lysosomal enzyme activity, nitric oxide and myeloperoxidase) and on proliferation of bone marrow cells by sulforhodamine B (SRB) assay, while the in vivo potential on macrophages and bone marrow cells was evaluated by using carbon clearance test and cyclophosphamide-induced myelosuppression, respectively. RESULTS: Significant increase in the release of myeloperoxidase, nitric oxide lysosomal enzyme and superoxide from macrophages along with significant increase in phagocytic index in carbon clearance test indicate stimulatory activity of the extract on macrophages. The extract also demonstrated 60% increase in bone marrow cell proliferation and offer protection towards cyclophosphamide-induced myelosuppression which represents the stimulation of bone marrow activity. DISCUSSION: Significant increase in mediators released from macrophages and phagocytic index in carbon clearance test suggests the release of cytokines from macrophages and stimulation of reticulo-endothelial system. Proliferation of bone marrow cells indicates the plausible release of colony stimulating factors, which further stimulates the immune system through generation of immune cells. CONCLUSION: The result described here indicates the immunostimulatory activity of ethanol extract of W. fruticosa flowers by stimulating non-specific immune responses, macrophages and bone marrow cells.

Antinociceptive properties of the ethanolic extract and of the triterpene 3beta,6beta,16beta-trihidroxilup-20(29)-ene obtained from the flowers of Combretum leprosum in mice.

The present study examined the antinociceptive effects of the ethanolic extract (EE) and of the triterpene 3beta,6beta,16beta-trihidroxilup-20(29)-ene obtained from the flowers of Combretum leprosum in chemical and thermal behavioural models of pain in mice. The EE (10-1000 mg/kg) given orally (p.o.), 1 h prior to testing, produced dose-dependent inhibition of acetic acid-induced visceral pain, with mean ID50 value of 131.9 mg/kg. In the formalin test, the EE (10-300 mg/kg, p.o.) also caused significant inhibition of both the early (neurogenic pain) and the late (inflammatory pain) phases of formalin-induced licking, however, it was more potent and efficacious in relation to the late phase of the formalin test, with mean ID50 values for the neurogenic and the inflammatory phases of approximately 300 and 88.8 mg/kg, respectively. The EE (10-1000 mg/kg, p.o.) also caused significant and dose-dependent inhibition of capsaicin- and glutamate-induced pain, with mean ID50 values of 160.5 and 38.3 mg/kg, respectively. Furthermore, the triterpene 3beta,6beta,16beta-trihidroxilup-20(29)-ene (1-30 mg/kg), given p.o., 1 h prior to testing, also produced dose-related inhibition of glutamate-induced pain, with a mean ID50 value of 5.6 mg/kg. When assessed in a thermal model of pain, the EE (10-300 mg/kg, p.o.) and fentanyl (100 microg/kg, s.c.) caused a significant and marked increase in the latency response on the hot-plate test (50 degrees C). The antinociception caused by EE (100 mg/kg, p.o.) in the glutamate test was significantly attenuated by intraperitoneal (i.p.) treatment of mice with naloxone (opioid receptor antagonist, 1 mg/kg), pindolol (a 5-HT 1A/1B receptor/beta adrenoceptor antagonist, 1 mg/kg), WAY100635 (a 5-HT 1A receptor antagonist, 0.7 mg/kg) or ketanserin (a 5-HT 2A receptor antagonist, 0.3 mg/kg). In contrast, EE (100 mg/kg, p.o.) antinociception was affected neither by L-arginine (precursor of nitric oxide, 600 mg/kg) nor by ondansetron (a 5-HT3 receptor antagonist, 0.5 mg/kg) i.p. treatment. It was not associated with non-specific effects such as muscle relaxation or sedation. Together, these results indicate that EE produces dose-related antinociception in several models of chemical and thermal pain through mechanisms that involve an interaction with opioid and serotonergic (i.e., through 5-HT 1A/1B and 5-HT 2A receptors) systems.