Phytochemical profile of Rosmarinus officinalis and Salvia officinalis extracts and correlation to their antioxidant and anti-proliferative activity

Abstract

The goal of this study was to monitor the anti-proliferative activity of Rosmarinus officinalis and Salvia officinalis extracts against cancer cells and to correlate this activity with their phytochemical profiles using liquid chromatography/diode array detection/electrospray ion trap tandem mass spectrometry (LC/DAD/ESI-MSⁿ). For the quantitative estimation of triterpenic acids in the crude extracts an NMR based methodology was used and compared with the HPLC measurements, both applied for the first time, for the case of betulinic acid. Both extracts exerted cytotoxic activity through dose-dependent impairment of viability and mitochondrial activity of rat insulinoma m5F (RINm5F) cells. Decrease of RINm5F viability was mediated by nitric oxide (NO)-induced apoptosis. Importantly, these extracts potentiated NO and TNF-α release from macrophages therefore enhancing their cytocidal action. The rosemary extract developed more pronounced antioxidant, cytotoxic and immunomodifying activities, probably due to the presence of betulinic acid and a higher concentration of carnosic acid in its phytochemical profile.

Introduction

Medicinal plants have served as rich sources of pharmacologically active substances. Herbs have been used in a diverse array of purposes including medicine, nutrition, flavorings, beverages, dyeing, repellents, fragrances, cosmetics, charms, smoking and industrial uses. Today, herbs are still found in 40% of prescription drugs (Newman & Cragg, 2007). Lamiaceae plants are now cultivated worldwide, mainly for use as culinary and medicinal herbs, and are widely studied as natural antioxidant sources since they are enriched in polyphenols. Their potent bioactivity and relatively low toxicity have rendered them useful ingredients in complementary alternative medicine and as nutritional supplements. Rosemary (Rosmarinus officinalis L.) and sage (Salvia officinalis) leaf are popular herbal teas and essential-oil containing drugs. Rosemary and sage are rich sources of di- and triterpenoids, phenolic acids, and flavonoids. Carnosic acid, carnosol and rosmarinic acid are the main antioxidant compounds present in them (Cuvelier, Berset, & Richard, 1994).

Rosemary extract has shown anti-proliferative effects on various tumor cell lines (Cheung & Tai, 2007). Carnosol and carnosic acid isolated from rosemary leaves have also presented anticancer properties, as determined in HL-60 cells (Bai et al., 2010). The cytotoxic activity of sage has not been studied in detail, but it has been shown that rosmarinic acid and sage extracts induce apoptosis in colorectal cancer cell lines (Xavier, Lima, Fernandes-Ferreira, & Pereira-Wilson, 2009). These findings suggest that Lamiaceae herbs contain several compounds with anti-proliferative activity against different cancers. As for their immunomodifying properties, the rosemary extract was found to be mainly anti-inflammatory (Cheung & Tai, 2007), while sage extract exhibited pro-inflammatory effects during its anti-leishmanial activity (Radtke, Yeap Foo, Lu, Kiderlen, & Kolodziej, 2003). There are still no studies looking into the anti-proliferative activity of sage and rosemary extracts and also at the effects of their major constituents on rat insullinoma RINm5F cells. There is also no general investigation of their phytochemical composition and there has been no comparative evaluation of their antioxidant and cytotoxic activities.

The aim of the present study was to characterize the composition of plant extracts rich in biophenols belonging to the Lamiacea family (ethyl acetate extracts of R. officinalis and S. officinalis) using LC/DAD/ESI-MSⁿ. The main compounds of the extracts were quantified using HPLC and NMR methods. Furthermore, extracts were studied colorimetrically for their total phenolic and flavonoid contents. Their antioxidant activities were evaluated using the DPPH (1,1-diphenyl-2-picrylhydrazyl) method. In addition, the effect of these plant extracts on viability, apoptosis and NO production was investigated in rat insulinoma RINm5F cells and their potential immunomodifying properties were monitored.