Phytochemical profile of Rosmarinus officinalis and Salvia officinalis extracts and correlation to their antioxidant and anti-proliferative activity
Highlights
► The anti-proliferative activity of rosemary and sage extracts was monitored. ► Cytotoxic and antioxidant activity were correlated to phytochemical composition. ► Both extracts exerted direct cytocydal effect via up-regulation of NO and TNF-α. ► Rosemary extract developed more pronounced cytotoxic and immunomodyfying activity. ► The primary cytotoxic components were determined.
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.
Section snippets
Plant material, reagents and standards
R. officinalis and S. officinalis were commercial samples. All solvents were of appropriate purity and were purchased from various suppliers. Acetic acid (glacial) was of analytical grade from Merck (Darmstadt, Germany). Folin–Ciocalteu, phenol reagent and aluminium chloride were obtained from Fluka (Switzerland), DPPH (∼90%) was obtained from Sigma–Aldrich (Steinheim, Germany). Standard compounds were: ursolic acid (90%), caffeic acid (98%) and betulinic acid (90%) from Aldrich (Steinheim,
The phytochemical composition of extracts – LC–MS analysis
The LC/DAD/ESI-MSn analysis of the rosemary extract led to the separation and identification of the majority of the constituents; overall, 17 compounds were identified belonging to three representative classes of constituents: diterpenes, flavonoids and triterpenic acids. Rosmarinic acid was the only hydroxycinnamic derivative that was identified. Because polyphenols contain one or more hydroxyl and/or carboxylic acid groups, MS data were acquired in negative ionization mode. Identification of
Conclusions
The phytochemical analysis of R. officinalis and S. officinalis extracts revealed the presence of several constituents, classified in the diterpene, triterpenoid and flavonoid natural product classes, most of them being common for both extracts. The quantitatively dominant compounds in these extracts, quantified by HPLC and NMR methods, were ursolic acid followed by carnosic acid in the rosemary extract, while in the sage extract, they were ursolic acid followed by oleanolic acid. Also, another
Acknowledgements
This work was co-funded by Esthir Gkani Foundation, (Ioannina, Greece) and the Project of Ministry of Education and Science, Republic of Serbia (No.: 173013). Special thanks are given to the Mass Spectrometry Unit and NMR center of University of Ioannina for providing access to LC-MS/MS, HPLC and NMR facilities. The authors have declared no conflict of interest.
References (30)
- et al.
Topical anti-inflammatory activity of Salvia officinalis L. leaves: the relevance of ursolic acid
Journal of Ethnopharmacology
(2001) - et al.
Comparison of different extraction procedures for the comprehensive characterization of bioactive phenolic compounds in Rosmarinus officinalis by reversed-phase high-performance liquid chromatography with diode array detection coupled to electrospray time-of-flight mass spectrometry
Journal of Chromatography A
(2011) - et al.
Antioxidant activity and free radical scavenging capacity between Korean medicinal plants and flavonoids by assay-guided comparison
Plant Science
(2002) - et al.
Evaluation of apparent formation constants of pentacyclic triterpene acids complexes with derivatized β- and γ-cyclodextrins by reversed phase liquid chromatography
Journal of Chromatography A
(2004) - et al.
Green processes for the extraction of bioactives from rosemary: chemical and functional characterization via ultra-performance liquid chromatography-tandem mass spectrometry and in-vitro assays
Journal of Chromatography A
(2010) - et al.
Rosmarinic acid inhibits angiogenesis and its mechanism of action in vitro
Cancer Letters
(2006) - et al.
A potential role of macrophage activation in the treatment of cancer
Critical Reviews in Oncology/Hematology
(2002) - et al.
Rapid and novel discrimination and quantification of oleanolic and ursolic acids in complex plant extracts using two-dimensional nuclear magnetic resonance spectroscopy-comparison with HPLC methods
Analytica Chimica Acta
(2009) - et al.
Screening of radical scavenging activity of some medicinal and aromatic plant extracts
Food Chemistry
(2004) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays
Journal of Immunological Methods
(1983)