Cymbopogon citratus and its polyphenols as potential phytotherapeutic products: an in vivo approach

Tese de doutoramento em Ciências Farmacêuticas, na especialidade de Farmacognosia e Fitoquímica, apresentada à Faculdade de Farmácia da Universidade de CoimbraCymbopogon citratus (DC). Stapf (Poaceae), commonly known as lemongrass, is a tropical perennial shrub originated from the Southeast Asia. This plant is reported to possess antifungal, insecticidal, anti-diabetic, anti-septic, anti-mutagenic, anti-carcinogenic activities as well as anti-inflammatory. In fact, aqueous extracts of dried leaves are used all over the year in folk medicine for the treatment of peptic ulcers and inflammatory conditions. Recently, some phenolic compounds, such as luteolin and apigenin glycosides and condensed tannins, were identified and related to both antioxidant and anti-inflammatory properties. The purposes of this work were to i) validate an analytical method for quantification of phenolic compounds of C. citratus; ii) study the influence of harvest time and plant quality on the phenolic composition and antioxidant activity; iii) characterize the tannins; iv) validate the traditional uses of lemongrass infusion as anti-inflammatory in vivo; v) obtain a topical formulation to evaluate the phenolic compounds permeation and their anti-inflammatory activity; vi) trace the pharmacokinetic profile of the main phenolic compounds in rats. Three different extracts: infusion (CcI), 50% aqueous ethanol (CcM50%) and ethanol (CcM100%) extracts were prepared and a simple and efficient RP-HPLC-PDA method was successfully validated for simultaneous identification and quantification of phenolic acids and flavonoids. Infusions were also obtained from different harvest dates (April, June, July, August and September) and quality grades (High, Medium and Low). It was verified that the content on polyphenols and the antioxidant capacity of CcI is strongly related with the quality of the plant. The total phenols assay showed a substantial loss from August to September. It was possible to find out the best month to harvest the plant to get the most of each phenolic group: April and June for hydroxycinnamic acids; June and September for flavonoids; June, July and August for tannins. Regardless the group of phenolic compound addressed, its content was always inversely proportional to the degree of leaves ageing. For all tested oxidant species, the high-quality samples exhibited the best antioxidant results. CcI was fractionated by column chromatography and polyphenol-rich fractions, namely phenolic acids (CcPA), flavonoids (CcF) and tannins (CcT) were obtained. CcT was characterized by HPLC-PDA-ESI/MSn, revealing the presence of proanthocyanidin hetero-dimers, along with some common procyanidin dimers. These hetero-dimeric flavan structures have been described for the first time in lemongrass and consist of apigeniflavan or luteoliflavan units linked to a flavanone, either naringenin or eriodictyol, and may occur as aglycone or glycosylated forms. For the in vivo assays, CcI, CcF and CcT were tested. CcI administered before and after ethanol stimulus, significantly reduced the incidence and severity of gastric lesions and, consequently, the ulcer index, corroborating the traditional medicinal use of this plant to ameliorate gastritis and/or peptic ulcers symptoms. On the other hand, CcI, CcF and CcT were orally administered to rats, in order to evaluate the anti-inflammatory effect at the carrageenan-induced paw edema assay. The observed effect by CcI (68.24 mg/kg), 82.30% of edema inhibition, was very similar to that obtained by the reference NSAID used (diclofenac, 10 mg/kg), 84.00%. On the other hand, flavonoid (7.42 mg/kg) and the tannin-rich (5.96 mg/kg) fractions significantly contributed for the anti-inflammatory activity on the edema volume (59 and 61%, respectively). The topical anti-inflammatory activity of CcI was also addressed. The results suggest that flavonoids, mainly, luteolin 7-O-neohesperidoside, cassiaoccidentalin B, carlinoside and cynaroside, may contribute to the topical anti-inflammatory effect. CcF (0.6%), CcT (0.3%) and CcF+CcT (0.66%+0.34%) topical formulations were also tested, and the results obtained suggest that tannins and flavonoids also possess a significant activity and that a synergistic mechanism of action may occur. In fact, edema inhibitions of 43%, 47% and 59% were respectively verified, being CcF+CcT effect very close to that of 1% diclofenac (65.9%). Pharmacokinetic analysis was performed in plasma, liver and kidney and showed that the compounds present in CcI are not detected in vivo after a single-dose oral administration. In contrary, the metabolites, luteolin 7-O-glucuronide and luteolin 3’-O-sulfate, present at the highest bioavailability, are probably the main responsible for the anti-inflammatory activity previously reported. In conclusion, this work has developed a method to quantify the phenolic compounds contained in C. citratus; pointed the importance of harvesting and storing the plant material, in order to take the maximum advantages from the phenolic compounds use; and demonstrated, in safe doses, its anti-inflammatory activity, using an in vivo approach, which supports the traditional use of lemongrass infusion. Furthermore, C. citratus leaves flavonoids and tannins were highlighted as bioactive compounds, encouraging the development of new anti-inflammatory drugs or nutraceuticals