Toxicological and pharmacological prospection in vitro and in vivo oil resins, leaf extracts and isolated compounds from Copaifera oblongifolia Mart. ex Hayne and C. duckei Dwyer

Author(s)

Marivane Lemos

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2016-08-26

Examining board members

Jairo Kenupp Bastos; Sérgio Ricardo Ambrosio; Thiago Mattar Cunha; Luiz Alberto Beraldo de Moraes

• Advisor: Jairo Kenupp Bastos; Helen Sheridan

Abstract

Medicinal plants are a rich source of potentially bioactive molecules. In Brazil, about 40% of the population use medicinal plants as an alternative therapy. However, most Brazilian native plants have no scientific studies proving their effectiveness and safety. Additionally, due to the enormous Brazilian biodiversity, the number of studied species is still small, and it represents a vast field to be explored. In this context, oleoresins from plants belonging to the genus Copaifera have few studies regarding its chemical, pharmacological and toxicological properties. For its aerial parts such as leaves, the studies are also scarce. Within this context, this project sought to assess the toxicology of oleoresins and leaf extracts of C. oblongifolia Mart. ex Hayne and C. duckei Dwyer, and understand preliminarily the mechanisms involved in wound healing processes and cytoprotection of gastric ulcers, as well as the anti-inflammatory and antinociceptive processes, commonly assigned and previously described in the literature for the copaiba oleoresin. Furthermore, there are few studies of C. oblongifolia Mart. ex Hayne and C. duckei Dwyer. Based on the fragmentation patterns obtained in the GC-MS and UHPLC-(ESI)-HRMS analyses it was concluded that oleoresins possess compounds of the sesquiterpene and diterpene class, as described in the literature. In leaf extracts, it was observed the presence of flavonoids and galoilquinic acid derivatives, as previously described for the leaves of this species, and epicatechins, described in the Fabaceae family, but not reported thus far in species of Copaifera spp. The results of the cytotoxic tests on neoplastic (AGS and THP-1) and non-neoplastic lines (CHO-k1 and L929) by cell viability assay for acid phosphatase, and cell death assay determined by propidium iodide in flow cytometry for AGS and THP-1 cells demonstrate that oleoresins, extracts and compounds isolated from C. oblongifolia and C. duckei present preferred cytotoxic activity on neoplastic cells, thus confirming the antitumor activity already described in literature. In the evaluation of acute toxicity by a fixed dose, leaf extracts from C. oblongifolia, and C. duckei and polyalthic acid are safe at doses up to 2000 mg / kg. The administration of oleoresin promotes hematological, biochemical and histological changes, mainly linked to liver, kidney and pancreatic metabolism. C. duckei oleoresin is toxic at a dose of 2000 mg / kg, promoting death of animals after 48 hours of its administration. In the experiment with repeated doses in 90 days there was no death of animals treated with 1, 10 and 100 mg / kg of C. duckei oleoresin, but there were hematological, biochemical and histological changes, although less obvious, in hepatic, renal and pancreatic metabolism. Regarding gastroprotection, the oleoresins and extracts from C. oblongifolia and C. duckei leaves promote cytoprotective effects on gastric mucus in EtOH/HCl and NSAIDs models. The dichloromethane and ethyl acetate fractions from crude leaves extract of both species Copaifera spp. contain compounds which may be responsible for gastric cytoprotection. Sesquiterpenes, diterpenes, flavonoids and galoilquinic acid derivatives were also evaluated, demonstrating gastroprotective activities in the model of ulcers induced by EtOH/HCl. Oleoresin and leaf extracts from C. oblongifolia and C. duckei present gastric healing activity in the model of ulcers induced by acetic acid, and increase cell proliferation in the L929 wound scratch assay. C. oblongifolia and C. duckei oleoresin do not increase the stomach pH, but decrease the amount of H + ions secreted. C. oblongifolia and C. duckei leaf extracts significantly increased the quantity of mucus, favoring gastric defense. Oleoresin and leaf extract of C. oblongifolia are active against the bacteria H. pylori, both in vitro and in vivo. C. oblongifolia and C. duckei leaf extracts have higher antioxidant activity - in vitro and in vivo - compared to their oleoresins. In part, the metabolites present in oleoresins and in leaf extracts of C. oblongifolia and C. duckei promote healing and cytoprotective effects, interfering in the secretion of acid, as demonstrated in enzyme test against H +, K + -ATPase, increasing the secretion of mucus and favoring the antioxidant defense mechanisms. In order to verify whether the antinociceptive activity was related to ab effect in the CNS of animals, we evaluated oleoresins and leaf extracts in the function and motor coordination of animals, through the Open field and Rota rod test. No treatment interfered in the function and motor coordination of animals. C. duckei oleoresin and its major substance, polyalthic acid were tested for anti-inflammatory and antinociceptive activity, both in the acute and chronic models. Different methods were used such as writhing by acetic acid, acute nociception formalin, thermal nociception by tail-flick, mechanical hyperalgesy, paw edema and air pouch (exudate assay in air pouch) induced by various agents, such as formalin, carrageenan, dextran, zymosan and LPS. Additionally, there were preliminarily investigated activities against chronic pain induced by brachial plexus avulsion (APB) and hyperalgesy induced by CFA for 15 days. In all experiments there was a reduction of in nociceptive response and inflammatory activity, suggesting that there is antinociceptive activity in the peripheral level, by inhibition of the nociceptive signal or by a decrease of mediators that stimulate nociceptors, which decrease cell migration and concentration of myeloperoxidase (MPO) at the site of injury. In THP-1 cells stimulated by LPS, treatments with oleoresins, extracts and Major compounds from C. oblongifolia and C. duckei decreased levels of proinflammatory cytokines INF-?, IL-1?, IL-6 and TNF-?. IL-10 levels were partially reduced with these treatments. The action of proinflammatory cytokines suggests both acute and chronic anti-inflammatory activity, considering that the experiment has the times of 24 h and 72 h and this decrease is dose-dependent. Furthermore, C. duckei oleoresin and polyalthic acid and interfere with NF-?B levels determined in the luciferase assay. These results indicate that there may be an interaction with the TLR4 and TLR2 receptors that affect the NF-?B pathway and decrease the inflammatory and apoptotic events from this pathway, confirming the studied activities. The antinociceptive and anti-inflammatory activity observed in the treatment with Copaifera spp oleoresin. or its isolated substances may be due to a decrease in the release or synthesis of mediators related to the inflammatory response, which results in a decreased nociceptive stimulus. The described cytotoxic activities indicate that there is potential antitumor effect that justifies the study for the development of substances having antitumor activity, especially regarding to gastric cancers and those related to the immune system, considering the cytotoxic selectivity in AGS and THP-1 tumor cell lines. The results presented herein are new, considering the scarce studies regarding C. oblongifolia, and long-term use of oleoresins and extracts from Copaifera. Moreover, phytochemicals findings suggest that there is potential in the chemical research, as many substances have not yet been isolated from these species. Furthermore, these results contribute to the confirmation of the pharmacological activities assigned commonly to the species of Copaifera spp., and this is the first time that oleoresins are found to be effective for the treatment of chronic inflammatory and painful disorders. Thus, this study makes the studied plants an alternative target for further studies concerning the pharmacologic mechanisms of action involved, providing support for further studies that will serve for the development of a new phytotherapy medicine in Brazil. (AU)

• FAPESP's process: 12/09727-8 - Toxicological and pharmacological in vitro and in vivo prospection of oil resins, leaf extracts and isolated compounds from Copaifera oblongifolia Mart. ex Hayne and C. duckei Dwyer
• Grantee: Marivane Lemos
• Support Opportunities: Scholarships in Brazil - Doctorate