Protective Effects of Galium Verum L
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Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii Vol. 25 issue issue 3, 2015, pp.207-214 © 2015 Vasile Goldis University Press (www.studiauniversitatis.ro) PROTECTIVE EFFECTS OF GALIUM VERUM L. EXTRACT ON THE HYPOTHALAMIC – PITUITARY – ADRENAL AXIS UNDER ANAKINETIC STRESS CONDITIONS, IN RATS. HISTOLOGICAL ASPECTS Ioana Roman1,*, Vlad Al. TOMA1,2, Anca D. Farcaș1,2 1Department of Experimental Biology and Biochemistry, Institute of Biological Research Cluj-Napoca, Branch of National Institute of Research and Development for Biological Sciences, Bucharest, Romania 2National Institute for Research and Development of Isotopic and Molecular Technologies, 67-103 Donat Str., 400293, Cluj-Napoca, Romania ABSTRACT: 24 female Wistar rats (120 ± 10 g) were divided into 4 groups: - control group (C); - Galium verum treated group (E), (25 mg extract/100 g bw); - anakinetic stressed group (S); - stress + Galium verum extract (25 mg extract/100 g bw) treated group (SE). Results revealed different reactivity of hypothalamo- hypophyseal-adrenal axis in the three experimental groups. The extract administration, in conditions of exposure to stress, resulted in an enhancement of NPV neurosecretory activity – associated with a possible stimulation of CRH release, a possible activation of adenohipophyseal hormones, as well as stimulation of adrenal steroid hormones. In S group, the effects of stress were opposite to those of E and SE groups. Histological results of the study proved that the administration of Galium verum vegetal extract in condition of anakinetic stress exposure induced important morphological changes at the all constitutive assembly of hypothalamo-hypophyseal-adrenal axis. These results justify the stimulation of secretory activity of the axis. Keywords: Galium verum extract; rats; hypothalamo-hypophyseal-adrenal axis; anakinetic stress. INTRODUCTION: Chronic psychological stress is one of the major blocking toxins and free radicals by competitive factors that contribute to several pathological disorders inhibition for some receptor in the cell membrane. (Brown, 1993; Cohen et al., 2007; Gouir and Medicinal plants and other plants, which contain Matuszewich, 2005). Immobilization/restraint stress is more polyphenols, are a category of plants sought due an easy and convenient method to induce both to their possible use in phytotherapy. The most popular psychological, an escape reaction and physical stress, a Galium species of our country with medicinal uses muscle work which leads to restricted mobility and include: Galium verum L. and Galium mollugo L aggression (Lee et al., 2015; Ramanova et al., 1994; Genus Galium, Fam. Rubiaceae, comprises about Singh et al., 1999). 300 species spread across the globe. It occupies large However, it is well known that intensive stress areas in Europe and Asia, from the plains to the response results in the production of reactive oxygen mountain stage at an altitude of 2,500 m. Romanian species (ROS) e.g. hydrogen peroxide, hydroxyl flora is among 28 species (Flora RPR, 1961) and 35 radical and superoxide anion radical that cause lipid species of Galium (Ciocârlan, 2000). These include: 16 peroxidation, especially in membranes and can play an species with white flowers, 6 species with yellow important role in tissue injury. It has been suggested flowers, two species with green flowers, two species that chronic stress and high level of glucocorticoids, with purple flowers and two species with undefined the adrenal steroids secreted during stress, affect flowers. The most known officinal species of various processes involving ROS and increase ROS by spontaneous Romanian flora are the species: Galium approximately 10% basally (Cohen et al., 2012; verum and Galium mollugo. Galium species (Lady's Kovacs et al., 1996). Presence and harmful effects of Bedstraw or Yellow Bedstraw) are known from the free radicals were imposed to find remedies. Last most ancient times as officinal plants with a variety of decades of research have identified substances with therapeutic properties: diuretic, depurative, slightly antioxidant effects, which may neutralize or limit the sedative and antispasmodic weak. harmful effects of free radicals. In this category belong Although in Romania have made a series of studies polyphenols, too. on Galium species (Gîrd and Florea, 2006; Tămaş et Currently there is an interest for identification of al., 2006; Hemcinschi et al., 2008; Hemcinschi et al., some species of plants with a rich content in 2009; Milića et al., 2013), generally Galium species polyphenols. The mechanisms of polyphenols action have not been fully studied in terms of chemical, are complex and little known. The most remarkable pharmaceutical (Bruneton, 1993), nor in terms of aspects relate to antioxidant activity which means biological effects which that extracts may have on the reducing the free radicals production and lipid animal body. peroxidation processes, especially in the cell Due to its chemical compounds: iridioides membranes, which provides protection of the entire (asperuloside) with anti-inflammatory, analgesic, cell. Another mechanism of polyphenols action is sedative effects; flavonosides with diuretic and anti- inflammatory action, phenyl-propane compounds, Correspondence*: Ioana Roman, Institute of Biological Research Cluj-Napoca, Department of Experimental Biology and Biochemistry, 48 Republicii Str, 400015, Cluj-Napoca, Romania, Tel.+40-(0264)-591238, Fax. +40-(0264)-591238, email: [email protected] Article published: September 2015 Roman I., Toma V.Al., Farcaș A.D. tannins with healing action, citric acid, minerals, MATERIALS AND METHODS: enzymes, plant has many therapeutic properties: Extract preparation depurative (blood cleansing), diuretics; cholagogue The hydroalcoholic extract of Galium verum was (improve bile secretion); astringent, healing, anti- obtained at the Faculty of Pharmacy, Cluj-Napoca. The inflammatory and vulnerary (healing of vegetal material (Galii veri herba) was harvested on wounds); diaphoretic (increases sweat gland secretion); July 2012 from the Becaş Colony meadows, Cluj- appetizers, flavoring. These therapeutic properties Napoca. The vegetal material was dried at room gives the plant a lot of phytotherapeutic uses temperature and then crushed to a fine powder (VI FR (Demirezer et al, 2006; Orhan et al., 2012). X sieve). The fluid extract (1 : 1) was obtained with Thus, we proposed to study the protective effects of alcohol 70˚ using the percolation technique of Squib Galium verum extract on hypothalamic – hypophyseal (Stoian and Savopol, 1977) using 1 p alcohol for 1 p - adrenal axis activity in rats under anakinetic stress Galii verii herba (pulvis). Qualitative analysis of the condition in order to obtain phytopharmaceutical polyphenolic compounds (flavonoids, phenyl-propane preparations used in phytotherapy to protect this compounds) of the Galium verum extract was done by structures which may be potentially affected by stress. thin-layer chromatography (TLC), (Wagner et al., 1996). (Table 1.) Table 1. Quantitative determination of total polyphenolic, flavonoid, and phenyl - propane compounds from Galium verum extract. Extractive Total polyphenols Flavonoids Phenyl - propane solution concentration concentration compounds concentration Fluid extract 1040.8 mg gallic 567 mg 341.75 mg caffeic acid rutoside/100 g acid/100 g extract /100g extract extract Histopathological investigations Animals The brain, pituitary and adrenals were removed and The experiments were performed on mature white weighed. Fragments of tissues were fixed in Bouin Wistar female rats, weighing 120 ± 10 g. Animals were fluid for 24 hours, the parts being processed for obtained from the „Iuliu Hatieganu” Medicine and inclusion in paraffin (Mureşan et al., 1974). The Pharmacy University, Cluj-Napoca biobase and kept fragment was sectioned at the Reichert - Austria type under standardized zoohigienical conditions, microtome with a thickness of 7 μm. The sections were respectively with free access to pet food (standaridsed deparaffinized using two changes of 50 ml of xylene pellet for rodent, Cantacuzino Institute) and water ad for ten minutes at room temperature. The sections were libitum, standard environmental conditions and 12 : 12 rehydrated in a sequence of 50 ml of 100%, 96%, 70% circadian cycle, in accordance to the European ethanol, for ten minutes. The tissues were washed once Communities Council Directive 2010/63/UE and in distilled water, and stained with specific dyes. The according to the approval of the Ethics Committee and general morphology of the brain and to highlight the Animal Protection for Experiments from the Institute neurosecretory activity of periventricular regions of the of Biological Research, NIRDBS branch, Cluj-Napoca, hypothalamus (Nissl granulations), was determined Romania. using Kluver-Barrera method (Kluver-Barrera, 1953). Experimental design The staining was performed using 5% Luxol fast blue Animals were divided into 4 experimental groups 1% cresyl violet (Mureșan et al., 1974), which stains of 6 animals each, as follows: 1 - control group, C; 2 - both the nucleoli and the rough endoplasmic reticulum extract group (E), treated for 15 days with a in (neurosecretion) in perikaryons. The histological hydroalcoholic extract of Galium verum in amount of examination of the slides was performed on an 25 mg extract/100 g body weight (bw), by intragastric Olympus BX-51 microscope, and the images were gavage, á jeun; 3 - anakinetic stressed group,