Phytochemical composition and antioxidant, anti-inflammatory and antimicrobial activities of Juniperus macrocarpa Sibth. et Sm.

Highlights

• Extracts and essential oils of cones and leaves of J. macrocarpa were analyzed.

• 44 phenolics were identified using LC–MS/MS, with rutin as the most dominant.

• 27 terpenes were identified using GC–MS, with monoterpenes as the most dominant.

• Cones and leaves showed moderate antioxidant and noticeable antimicrobial activity.

• Significant anti-inflammatory activity of cones was evaluated.

Abstract

The phenolic and terpene profiles, as well as antioxidant, anti-inflammatory and antimicrobial activities of extracts and essential oils of Juniperus macrocarpa species were studied. The content of 45 phenolics was determined using LC–MS/MS. Accordingly, significantly different compositions were found between leaves and seed cones extracts, while the dominant compounds were rutin, catechin, quercitrin, epicatechin and amentoflavone. GC–MS showed simple terpene composition, with monoterpenes being dominant and α-pinene as the most abundant compound. Extracts and essential oils of J. macrocarpa showed moderate antioxidant activity comparable to that of butylated hydroxyanisole (BHA). Seed cones showed significant anti-inflammatory activity, with high pro-inflammatory activity of extract towards production of PGE₂. Essential oils demonstrated considerable antimicrobial activity against Gram positive bacteria, especially leaves against Clostridium perfringens. J. macrocarpa showed noteworthy biopotential and its usage in food and beverage manufacturing and as a source of potent herbal drugs should be considered.

Introduction

Juniperus macrocarpa Sibth. et Sm. (Syn. Juniperus oxycedrus L. subsp. macrocarpa) belongs to the genus Juniperus L., of which there are 67 species and 34 varieties, evergreen shrubs or trees, mostly distributed throughout the Northern Hemisphere. Plants of this genus, mostly Juniperus communis L. and J. oxycedrus L., are widely used in cooking as a spice, preferably for pickling game birds and meat. In some traditional recipes, juniper seed cones are an important ingredient, such as in the cabbage dish “bigos” and the Polish traditional sausage “kiełbasa jałowcowa” (Loizzo et al., 2007). Additionally, the seed cones of Juniperus drupacea Labill. are used in Turkey for making traditional jam (Lesjak et al., 2011). The species Juniperus are also used in food production as a preservative (Mrabet et al., 2008). Furthermore, the unique taste and aroma of the seed cones of common juniper (J. communis) are an indispensable additive in the manufacture of gin, the Italian liquor “Gineprino”, Serbian brandy “Klekovača” and “piwo kozicowe” beer from Poland (Lesjak et al., 2011), among others. Moreover, species from genus Juniperus L., mainly the extracts and essential oils of needles or leaves (flake leaves), seed cones and wood, are well-known in the traditional medicine of ancient civilizations of Europe and Asia for their numerous pharmacological properties. These include diuretic, antirheumatic, antiseptic, antimicrobial, antifungal, antihelmintic, anti-inflammatory, immunomodulatory, antioxidant, analgesic and antituberculotic activities, as well as being potent abortion inducer. (Akkol et al., 2009, Lesjak et al., 2011, Orhan et al., 2011).

In spite of the widespread use in beverage manufacture and food preparation and the great healing power of the Juniperus species, there are very few reports concerning their chemical compositions and biological activities. However, although the data on the chemical composition of essential oil of Juniperus plants were published, only a few studies investigated in details chemical constituents other than volatile oils, such as phenolics (Lesjak et al., 2011). There is one preliminary study on the phenolic profile of leaves (Stassi, Verykokidou, Loukis, & Harvala, 1998), one cursory study about phenolics in seed cones (Taviano et al., 2013), only a few studies about the volatile compounds of this species (Adams et al., 1999, Amri et al., 2011, Hanène et al., 2012, Medini et al., 2009, Sezik et al., 2005, Stassi et al., 1995, Valentini et al., 2003, Velasco-Negueruela et al., 2005), and one concerning its fatty acid composition (Güvenç, Küçükboyaci, & Gören, 2012). Regarding the biological activities of J. macrocarpa, there is a limited number of reports on the antioxidant activity (Hanène et al., 2012, Massei et al., 2006, Taviano et al., 2013) and anti-inflammatory and antinociceptive potency (Akkol et al., 2009) of extracts, while two reports have discussed the antimicrobial efficacy of the essential oil (Medini et al., 2009, Stassi et al., 1996).

Phenolics and volatile compounds render a significant biological potential, especially in preventing oxidative stress, inflammation and bacterial infection, where all of them have a promoter role in the development of many severe chronic diseases. Therefore, we investigated in depth the phenolics and terpenoid profile, as well as the antioxidant, anti-inflammatory and antimicrobial potential of the extracts and essential oils of J. macrocarpa. Accordingly, a LC–MS/MS technique was employed to evaluate the content of 45 phenolics (Orčić et al., 2014), and the results for the first time report details of phenolic profile of leaves and seed cones extracts of J. macrocarpa. Additionally, GC–MS was applied for determination of essential oil composition (Lesjak et al., 2013). The antioxidant potential was determined using various assays related to free radical (2,2-diphenyl-1-picrylhydrazyl radical (DPPH); Lesjak et al., 2013)., reactive oxygen (HO, ${\text{O}}_2^{-}$; Lesjak et al., 2011, Nishikimi et al., 1972) and reactive nitrogen species (NO; Green et al., 1982) scavenging ability. In addition, the inhibitory potential of lipid peroxidation (LP; Lesjak et al., 2013). and reducing power (ferric reducing antioxidant power (FRAP) assay; Benzie & Strain, 1996) were determined. Results considering antioxidant activity were compared with the synthetic antioxidant butylated hydroxyanisole (BHA). In addition, the total phenolic and flavonoid contents were determined (Lesjak et al., 2011). Anti-inflammatory activity was determined with an assay based on the inhibitory potential of samples on the biosynthesis of 12(S)-hydroxy(5Z,8E,10E)-heptadecatrienoic acid (12-HHT), thromboxane B₂ (TXB₂), prostaglandin E₂ (PGE₂) and 12(S)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12-HETE), in human platelets (Lesjak et al., 2013). 12-HHT, TXB₂, PGE₂ and 12-HETE are inflammation mediators derived from arachidonic acid metabolism, which is catalyzed by enzymes of inflammatory response, cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX). Furthermore, the antimicrobial activity of essential oils was evaluated against six reference bacterial strains using the disk diffusion (Prabuseenivasan, Jayakumar, & Ignacimuthu, 2006) and agar dilution method (NCCLS, 2002).