Journal of Chromatographic Science, 2016, Vol. 54, No. 3, 291–294 doi: 10.1093/chromsci/bmv158 Advance Access Publication Date: 3 November 2015 Article Article Volatile Profile of Herniaria fontanesii Growing Spontaneously in Tunisia Saoussen Hammami1,*, Ridha El Mokni2, Khaled Faidi1, Mohamed Hédi El Aouni2, Zine Mighri1, and Rajesh K. Joshi3 1 Research Unit 13ES63, Applied Chemistry and Environment, Faculty of Sciences, Monastir University, Monastir 5000, Tunisia, 2Faculty of Sciences of Bizerte, Laboratory of Botany and Plant Ecology, Zarzouna, Bizerte 7021, Tunisia, and 3 Department of Phytochemistry, Regional Medical Research Centre, Nehru Nagar, Belgaum, Karnataka 590010, India *Author to whom correspondence should be addressed. Email: saoussenhammami@voila.fr Received 20 June 2015; Revised 5 September 2015 Abstract The essential oil extracted from Desfontaine’s rupturewort, Herniaria fontanesii J. Gay subsp. fontanesii growing wildly in Tunisia, was analyzed using GC and GC–MS techniques. The free radical scavenging capacity and total phenol contents of three crude extracts having different polarities (n-hexane, ethyl acetate and methanol) were examined. Thus, a total of 35 constituents were identified in the Desfontaine’s rupturewort essential oil representing 89.8% of the whole constituents. The oil was dominated by hexadecanoic acid, caryophyllene oxide, terpin-4-ol, khusimone and trans-sabinene hydrate. The total phenolic contents ranged from 16.91 to 92.27 mg of gallic acid/g of dry weight and they were found to be significantly higher in methanol than in polar ethyl acetate and hexane extracts. Correlations were observed between the phenolic contents and the antioxidant properties. Thus, the antioxidant activity of the methanol extract was superior to that of all samples tested (IC50 = 0.21 ± 0.04 mg/mL). Introduction Aromatic plants constitute a rich source of essential oils widely used in folk and modern medicine in the treatment of infectious diseases. Actually, a revival interest has been attributed to the extraction of natural essential oils formed especially by terpenoid hydrocarbons, oxygenated monoterpenes and sesquiterpenes which are widely studied for their great usage in cosmetic, food and pharmaceutical industries as ingredients for fragrances, aroma, flavors and safe medicines without side-effects (1). Caryophyllaceae is one of the large dicotyledonous families, comprising about 80 genera and 2100 species, known for its ornamental plants, generally rich in triterpenoids and saponins which are very often responsible for a wide range of medicinal uses such as anti-inflammatory, antispasmodic, antidiabetic and anticarcinogenic properties (2). The rupturewort (genus Herniaria L., Caryophyllaceae) belonging to the subfamily Paronychioideae included 45 species distributed in Africa, Europe and Asia (3). With the aim of our contribution to the search of plant-derived bioactive molecules from the crude and volatile extracts (4), we have been interested in the chemical and biological investigation of the Desfontaine’s rupturewort, Herniaria fontanesii J. Gay subsp. fontanesii, one of the five species within the Herniaria L. genus, widely distributed in Tunisia (5–7). The subspecies fontanesii is a spontaneous perennial plant in Tunisia; it has several woody stems at the base with fragile branches with discarded internodes. The more or less hairy leaves are linear to lanceolate with small stipules. Flowers are mostly green in axillary few-flowered glomeruli; triangular haired bracts. Its calyx tube is pubescent with hooked hairs. Outer sepals are slightly fleshy and bristly. Forth Stamens have large anthers (up to 5 mm) (5, 8, 9). The effectiveness of Herniaria fontanesii Gray from Moroccan origin in the treatment of lithiasis or diuresis has been described (10). Previous chemical investigation conducted on the crude extracts from the same species growing in Morocco led to the isolation and structure elucidation of flavonol derivatives, triterpenoid saponins and triterpene glycosides (10). As far as we know, there is no report on volatile constituents of H. fontanesii, and this is the first study on the chemical composition of essential oils, phenolic contents and antioxidant effects of crude extracts derived from the subspecies harvested in Tunisia. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com Downloaded from https://academic.oup.com/chromsci/article-abstract/54/3/291/2754803 by guest on 08 April 2018 291 Hammami et al. 292 Experimental Plant material and isolation of essential oil Aerial parts of H. fontanesii were collected in the southern-east of Tunisia (Region of Chenini-Gabes; coordinates 33°52′39, 26″N, 10°03′ 54, 17″E, altitude 24 m above sea level) in May 2014. Harvested samples were identified by one of the authors (R.E.M), botanist in the Laboratory of Botany and Plant Ecology (SNA-214), Faculty of Sciences of Bizerte, Zarzouna, Bizerte, Tunisia, where voucher specimens have been deposited. Two-hundred grams of air-dried material were subjected to hydrodistillation for 3 h in a Clevenger-type apparatus. The volatile oils were dried over anhydrous sodium sulfate and stored in sealed vials at −4°C for further analysis. Preparation of extracts Gas chromatography The GC analysis of the oil was carried out using Varian 450 gas chromatograph equipped with FID, using stationary phase CP Sil-8-CB (30 m × 0.25 mm i.d., 0.25 µm film thickness) column under the experimental conditions reported (11, 12). Nitrogen was the carrier gas at a flow rate of 1.0 mL min−1. Temperature was programmed from 60 to 220°C at 3°C/min. and for injector and detector temperatures were 230 and 250°C, respectively. The injection volume was 1.0 µL of 1% solution diluted in n-hexane; the split ratio was 1 : 50. Gas chromatography–mass spectrometry The GC–MS analysis of the oil was carried out using Thermo Scientific Trace Ultra GC interfaced with a Thermo Scientific ITQ 1100 Mass Spectrometer fitted with TG-5 (30 m × 0.25 mm i.d., 0.25 µm film thicknesses) column. The oven temperature was programmed from 60 to 220°C at 3°C/min using helium as the carrier gas at a flow rate of 1.0 mL min−1. The injector temperature was 230°C; the injection volume was 0.1 µL of 1% solution diluted in n-hexane; the split ratio 1 : 50. MS was applied at 70 eV with a mass scan range of 40–450 amu. All the experimental parameters were taken from those reported earlier (12, 13). Identification of the components Identification of the components was done on the basis of linear retention index (RI, determined with reference to homologous series of Figure 1. GC–TIC chromatogram of the essential oil of Herniaria fontanesii . Downloaded from https://academic.oup.com/chromsci/article-abstract/54/3/291/2754803 by guest on 08 April 2018 n-alkanes C8–C25, under identical experimental conditions), MS library search (NIST 08 MS Library (Version 2.0 f ) and WILEY MS 9th Edition) and in comparison with MS literature data (14). The relative amounts of individual components were calculated based on the GC peak area (FID response) without using the correction factor. Determination of total phenolic contents Aliquots of 0.2 mL of ethyl acetate extracts (1 mg/mL) were mixed with 1 mL of Folin–Ciocalteu reagent. A reagent blank using ethyl acetate was prepared. After 5 min incubation at room temperature, 0.8 mL of sodium carbonate solution (7.5%) was added. Samples were incubated at room temperature for 1 h and the absorbance was measured at 765 nm versus the prepared blank. A calibration curve was performed in parallel under the same operating conditions using gallic acid as the positive control. The results are expressed as milligrams gallic acid equivalent per gram of dry extract (mg GAE/g) (15). Antioxidant activity DPPH radical scavenging assay. 2,2′-diphenyl-1-picrylhydrazyl (DPPH) free radical assay was carried out to measure the free radical scavenging activity as reported previously (16). A volume of 1.0 mL of each ethanol solution of H. fontanesii prepared at different concentrations was mixed with an equal volume of ethanolic solution of DPPH (0.1 mM). The disappearance of the DPPH was measured after 30 min of incubation at room temperature. The inhibition percentage of the DPPH radical by the extract was calculated according to the formula of Yen and Duh (17): %RSA ¼  Acontrol Asample Acontrol  × 100 where Acontrol is the absorbance of a control sample (t = 0 h), and Asample is the absorbance of a tested sample at the end of the reaction (t = 1 h). The concentration providing 50% inhibition (IC50) was calculated from the graph plotting percentage of free radical scavenging activity (% RSA) against the Tunisian H. fontanesii extract concentration. Volatile Profile of Herniaria fontanesii Results Chemical composition Volatile oil of H. fontanesii was obtained by hydrodistillation with a yield of 2 × 10−2%. Thirty-five components that were identified using GC and GC–MS accounted for 89.8% of the whole constituents (Figure 1). The qualitative and quantitative chemical compositions are represented in Table I. As can be seen from the table, the essential oil was composed of 30.1% of oxygenated monoterpene fraction, 16.3% of oxygenated sesquiterpene fraction, 7.9% of sesquiterpene hydrocarbons, 0.5% of monoterpene hydrocarbon fraction and 35% of other compounds. Hexadecanoic acid (18.6%), caryophyllene oxide (9.3%), terpin-4-ol (7.5%), khusimone (6.7%) and trans-sabinene hydrate (6.1%) are the major constituents. Phenolic content and DPPH radical scavenging activity The total phenolic content of various extracts from the Desfontaine’s rupturewort harvested from a natural population in the southerneast of Tunisia was measured. The results given in Table II showed that the amounts of phenolic derivatives ranged from 16.9 to 92.3 mg GAE g−1. These contents increased significantly from the less polar to the most polar extract. Thus the methanol gave the highest yield for total phenolic extraction (92.3 mg GAE g−1). Furthermore, a correlation was observed between the total phenolic contents and the antioxidant power. The determined EC50 values denoted the concentration of the extract required to scavenge 50% of DPPH radical. Thus, the highest ability to scavenge free radicals was observed for methanol extract (EC50 = 0.21 mg mL−1), while the weakest scavengers were in hexane apolar extract (EC50 = 1.3 mg mL−1) (Figure 2). Discussion Within the aim of the contribution to the screening of medicinal plants growing in Tunisia, searching for natural crude and volatile extracts having considerable antioxidant potentials and which may reduce the body’s oxidative damage, we have been interested in the chemical and biological investigation of H. fontanesii essential oils. The antioxidant activities and the total phenolic contents of three crude extracts of Herniaria dry material were investigated in the study. Palmitic acid has been detected as the major component (18.6%). Furthermore, the essential oil was rich in terpene derivatives (54.8%) dominated by caryophyllene oxide (9.3%), terpin-4-ol (7.5%) and khusimone (6.7%) as the major odorants. Despite the fact that some authors ascribed fatty acids as artery-clogging molecules have detrimental role on human health (18), hexadecanoic acid (synonym: palmitic acid) produced in a large amount in many plants has been used in many cosmetics, shampoos and various commercialized cosmetic products. Recently, its contribution to the apoptotic effect and cell cycle arrest of human neuroblastoma cells has been identified (19). It is also important to point out that caryophyllene oxide possesses antimicrobial properties against a wide range of bacteria and fungi; terpin-4-ol is known for its broad spectrum activity against microorganisms (20–22). Moreover, the evaluation of free radical scavenging activity of three crude extracts from Herniaria species has been extensively performed during the study, via the most commonly used and the highly sensitive DPPH method. Since the reductive potentials of the tested extracts are strictly related to the polarities of the corresponding phytochemicals, the methanol extract is ranked as the most antioxidant extract exhibiting the strongest activity on scavenging DPPH Downloaded from https://academic.oup.com/chromsci/article-abstract/54/3/291/2754803 by guest on 08 April 2018 293 Table I. Chemical Composition of the Desfontaine’s Rupturewort, H. fontanesii Harvested in the Southern-east of Tunisia Compound RI % Identification Sabinene α-Terpinene o-Cymene β-Phellandrene γ-Terpinene cis-Sabinene hydrate Terpinolene trans-Sabinene hydrate cis-p-Menth-2-en-1-ol trans-p-Menth-2-en-1-ol Borneol Terpin-4-ol α-Terpineol cis-Piperitol trans-Piperitol Thymol Carvacrol Eugenol β-Caryophyllene β-Ionone-5,6-epoxide E-β-Ionone Viridiflorene n-Pentadecane γ-Cadinene Isopentyl salicylate Pentyl salicylate n-Tridecanol Caryophyllene oxide Khusimone trans-isolongifolanone Myristic acid Pentadecanol Hexadecanoic acid Octadecanol n-Tricosane Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others Total identified 944 982 984 992 1,022 1,026 1,053 1,058 1,084 1,102 1,131 1,148 1,161 1,169 1,182 1,282 1,292 1,349 1,450 1,506 1,510 1,519 1,537 1,555 1,564 1,609 1,623 1,636 1,661 1,670 1,833 1,873 2,030 2,132 2,331 T 0.3 T T 0.2 2.1 1.6 6.1 4.7 2.2 1.5 7.5 2.9 0.9 0.6 0.4 0.5 1.1 3.9 4.1 1.2 3.1 1.4 0.9 0.8 0.3 0.7 9.3 6.7 0.3 0.8 1.4 18.6 3.3 0.4 0.5 30.1 7.9 16.3 35 89.8 RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, Retention index relative to C8–C25 n-alkanes on TG-5 column; MS, NIST and Wiley library and the literature; t, trace (<0.1%). Table II. Total Phenolic Content (TPC) of Various Extracts From H. fontanesii Harvested in the Southern-east of Tunisia Sample Total phenolics (mg GAE g−1, DW) Hexanic extract Ethyl acetate extract Methanol extract 16.91 ± 3.08 31.52 ± 2.31 92.27 ± 6.15 radicals with an EC50 value of 210 µg mL−1. This may be related to the high content of the methanol extract, compared with others less polar extracts, in flavonoids and also in phenolic derivatives for which the hydroxyl groups can act as protons donating and can be considered as the first responsible chemicals of the scavenging activity. 294 Figure 2. DPPH radical scavenging activity of the various solvent extracts from the Desfontaine’s rupturewort, H. fontanesii compared with Quercetin. Conclusion According to the literature, this the first study on the chemical composition and antioxidant activities of the Desfontaine’s rupturewort growing spontaneously in Tunisia. 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