Juniperus sibirica Burgsdorf. as a novel source of antioxidant and anti-inflammatory agents

Abstract

In order to examine antioxidant properties of methanol extracts of cones and needles of the unexplored Juniperus sibirica Burgsdorf. (Cupressaceae) species, various assays which measure free radical scavenging ability were carried out: DPPH, hydroxyl, superoxide anion and nitric oxide radical scavenger capacity test and reducing power (FRAP) assay. In all of the tests the extracts showed a potent antioxidant effect compared with BHT, a well-known synthetic antioxidant. In addition, the anti-inflammatory activity considering inhibitory potency toward COX-1 and 12-LOX was observed. Both extracts showed markedly anti-inflammatory activity, with needles having somewhat higher potency concerning both assays, reaching IC₅₀ at 1.29 mg/mL towards COX-1 and IC₅₀ at 1.34 mg/mL towards 12-LOX. Besides, in the extracts examined the total phenolic and flavonoid amounts were also determined, together with presence and content of the selected flavonoids: luteolin-7-O-glucoside, apigenin-7-O-glucoside, luteolin, apigenin, rutin and quercetin, which were studied using LC–MS/MS technique. LC–MS/MS analysis showed a noticeable content of natural products according to which the examined J. sibirica Burgsdorf. species could well be regarded as a promising new source of bioactive natural compounds, which can be used both as a food supplement and a remedy.

Introduction

The genus Juniperus L. belongs to the family of Cupressaceae and comprises about 70 species which are widely distributed in the Northern Hemisphere. In Serbia, five autochthonous species from Juniperus genus are distributed (Juniperus communis L., Juniperus oxycedrus L., Juniperus sibirica Burgsdorf., Juniperus sabina L. and Juniperus excelsa M.B.). Furthermore, besides the species mentioned, Juniperus chinensis L., Juniperus foetidissima Willd., Juniperus horizontalis Mnch., Juniperus squamata Buchana-Hamilton in Lamberts and Juniperus virginiana L. are cultivated as horticultural plants and used for decoration (Jovanovic, 1992). Nevertheless, only a few of them had previously been investigated. All over the world plants from this genus have always been regarded as a well-known traditional remedy due to their numerous therapeutic properties, such as: anti-inflammatory, diuretic, antiseptic (bacterial and fungal), anthelmintic, hypoglycaemic, hypotensive, analgesic and abortifacient. In medicinal application all parts of plant are used together with the essential oil. Furthermore, these plants are extensively used worldwide in the folk medicine for healing various disorders: common cold, urinary and kidney infections, dermatological disorders, bronchitis, pneumonia, dysentery, haemorrhage, rheumatic arthritis, stomachache, diarrhoea, haemorrhoids and for regulation of the menstruation and in relieving menstrual pains (Akkol et al., 2009, Kozan et al., 2006, Miceli et al., 2009, Thomas et al., 2007). Moreover, plants from the Juniperus L. genus, mostly their cones, have found great application in different European cuisines as a spice which adds “sharp and clear flavour”, preferably for pickling meat, particularly the wild bird and game meat (Montagne, 1999). In some traditional recipes for preparing the meat specialties, the juniper cones are used as distinctive ingredients giving the unique taste (Loizzo et al., 2007). Nowadays, juniper cones are used for flavouring different alcoholic drinks. In Serbia, the “Klekovača”, a type of juniper brandy, is a very popular alcoholic drink, well-known for its unique aroma with a nice juniper aftertaste, which is very good for appetite whetting. Furthermore, in the production process of gin (Vichi, Riu-Aumatell, Mora-Pons, Buxaderas, & Lopez-Tamames, 2005) and the authentic “kozicowe” beer from Poland (Łuczaj & Szymański, 2007) juniper cones are also used for flavouring.

Amongst the Juniperus L. genus, the most renowned species used in traditional medicine are J. communis L. (Agelet and Vallès, 2003, Gautam et al., 2007, González-Tejero et al., 2008) and J. oxycedrus L. (Kozan et al., 2006, Moreno et al., 1998). In addition, medicinal application of some other species like: Juniperus phoenicea L. (Agelet and Vallès, 2003, Al-Qura’n, 2009, González-Tejero et al., 2008), Juniperus drupacea Labill. (Kozan et al., 2006), J. excelsa M. Bieb. (Gautam et al., 2007), J. oxycedrus L. subsp. macrocarpa (Sm.) Ball. (González-Tejero et al., 2008), Juniperus thurifera L. (González-Tejero et al., 2008) are also reported. Despite their great healing potency, there are very few reports on biological activity and chemical composition of Juniperus L. genus. Although the data about chemical composition of the essential oil from cones and needles of Juniperus plants had already been published (Angioni et al., 2003), only few of the studies investigated chemical constituents other than volatile oils. It was reported that the extracts from Juniperus L. genus contain phenolic compounds, primary flavonoids, neolignans, phenylpropanoids (Abul’saeva and Pashinina, 1981, Iida et al., 2007, Nakanishi et al., 2002), and terpenoids (Topçu et al., 1999).

J. sibirica Burgsdorf. (Syn. J. communis L. 1753 var. saxatilis Pall. 1789 and J. sibirica L. 1753) is one of the five species from the Juniperus genus which can be found in Serbia. At the same time, it grows widely in Russia, where it was determined for the first time. Most probably this species expanded to the region of the Balkan Peninsula, including Serbia. To the best of our knowledge, there are no literature data concerning either its chemical constituents or its biological activities and medicinal properties.

Having in mind the significance of oxidative stress and inflammatory process in the development of many severe chronic diseases, we found it worthwhile to investigate the antioxidant and anti-inflammatory activity of this plant species, unexplored in this respect. Besides potential use of J. sibirica Burgsdorf. in medical care, its usage in production of food and beverage could also be proposed, especially concerning the modern concept of so called functional food, food which possesses both the nutritional and healthy properties. Taking this into account, the study of the antioxidant activity of both the cones and the needles of J. sibirica was performed evaluating their ability to reduce and neutralise free radicals (DPPH, HO, ${\text{O}}_2^{-}$ and NO), as well as measuring total reducing potential of the samples examined.

At the same time, the anti-inflammatory activity of the obtained extracts was evaluated. Anti-inflammatory potential was determined by an in vitro assay based on the inhibitory effect on the biosynthesis of 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid (12-HHT), and 12(S)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12-HETE). 12-HHT and 12-HETE are inflammation mediators derived from arachidonic acid metabolism, which is catalysed by enzymes of inflammatory response, cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX), respectively, in human platelets. Furthermore, the advantage of this type of experiment is avoidance of the undesirable in vivo tests on experimental animals, since the tests commonly used to detect the anti-inflammatory activity is carrageenan induced paw edema in rats. Knowing that phenolic compounds are the most responsible for the antioxidant activity (Havsteen, 2002), and also stand as well-known anti-inflammatory agents, the content and composition of several flavonoid compounds have been determined.