The ingredient of G. tenax were extracted by CO₂-SFE and analyzed by GC-MS. Chemical composition was separated by programmed temperature gas chromatography, and then individual MS fragments were analyzed. The percentage composition of the extract was computed by normalization to the GC peak areas without using correction factors. Identification of constituents was based on comparison of their Kovats Index (KI) and the MS fragmentation pattern with reference compositions in the database of the NIST Mass Spectral Search Program. The GC-MS total ion chromatogram (TIC) of the G. tenax extraction is shown in Figure 1.

Thirty compounds were identified in all of the oils analyzed, twenty-three of which are listed in Table 1. The compounds that are not listed in Table 1 are alkanes. 78% were identified by GC-MS combined with KI. The others were not identified in the database of the NIST Mass Spectral Search Program, possibly because the comparison of the obtained mass spectra with those of reference compounds was too low to enable identification. The identified components include six sesquiterpenes (14.39%), three ketones (5.02%), seven fatty acids and their esters (29.1%), two phenols (1.71%), and three sterols (12.81%) (Table 1).

Extraction of volatile components using conventional processes, such as solvent extraction, is conducted at high pressure and temperature and is time-consuming. Supercritical fluid extraction (SFE) is a rapid, selective, effective and convenient technique for sample preparation before the analysis of compounds. Supercritical fluids have been used as solvents for a wide variety of applications, such as essential oil extraction, and more than 90% of all analytical SFE is performed with CO₂. Under conditions of low pressure and low temperature, and no influence of solvent, use of CO₂-SFE is a good way to extract the volatile constituents of G. tenax. Some compounds were identified by GC-MS (Table 1). These compounds have physiological activity, including antioxidant, anti-inflammatory, and antimicrobial activities. Sesquiterpenes are formed from a 15-carbon skeleton of terpenoids. Widely distributed in nature, the structure of the skeleton is different, and the diverse biological activity of sesquiterpenes has been shown to include antitumor, antibacterial, antiviral, and antimalarial activities [18,19]. Sesquiterpenes can also protect against alcohol-induced gastric mucosal lesions and oxidative damage [20]. (−)-Thujopsene, cedrol, (+)-cuparene, α-curcumene, (−)-β-bisabolene, α-zingiberene are sesquiterpenes were identified. Thujopsene has shown potent antibacterial activity, for example against Phytophthora ramorum at 2.0–3.0 ppm [21]. Inhaling vaporized Cedrol ((64.0 ± 7.7) × 10⁻⁹ M) can affect autonomic nervous functions in humans, such as induce sedative effects, decrease heart rate and blood pressure [22], and (+)-cuparene, α-curcumene and (−)-β-bisabolene mostly exhibit a strong physiological activity [23,24]. Vanillylacetone and fitone both have some physical activity, especially vanillylacetone. For instance, zingerone acts mainly by increasing systemic superoxide dismutase activity to protect the effect of zingerone against 6-hydroxydopamine-induced dopamine reduction, (65 nmol/kg, ip) [25]. Zingerone (10 μg/mL) has also been shown to mitigate radiation-induced mortality and cytogenetic damage, which may be attributed to inhibition of the radiation-induced decline in endogenous antioxidant levels and scavenging of radiation-induced free radicals [26]. Zingerone exerts its potent anti-inflammatory action by increasing HNF-4 and PPAR activities, while suppressing NF-kappaB activity at a dose of either 2 or 8 mg/kg/day for 10 days. [27].

Due to the complex nature of phytochemicals and determination of antioxidant activity according to the reaction mechanism, we used three methods to measure the antioxidant properties of G. tenax extracts: a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, a β-carotene/linoleic acid-coupled oxidation reaction, and a deoxyribose degradation assay. All three methods detected increasing antioxidant activity with increasing dose. Butylated hydroxytoluene (BHT), a strong antioxidant, was used as a positive control and exhibited the highest antioxidant activity. The G. tenax extract showed dose-dependent scavenging activity (Figure 2).

Reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), superoxide anion (O₂^•−), and hydroxyl radical (OH^•), are linked to cell damage [28]. Previous literature has demonstrated that the interaction of a potential antioxidant with DPPH depends on its structural conformation. The number of DPPH molecules that are reduced seems to be correlated with the number of electron-donating hydroxyl groups in the antioxidant molecule [29]. This structural requirement could be linked to the presence of phenolic compounds, which are known to be widely distributed in natural herb and spice extracts. Phenolic compounds have a high reducing ability to eliminate free radicals because of both their alcoholic hydroxyl group and conjugated π electrons of the benzene ring [30]. The percentage of inhibition of DPPH by G. tenax extracts was evaluated at different extract volumes (Table 2 and Figure 2). In the β-carotene/linoleic acid-coupled oxidation assay, antioxidants are capable of reducing the rate of chain reaction initiated during lipid peroxidation mainly by scavenging the intermediate peroxyl free radicals formed when linoleic acid is oxidized. This also depends on the hydrogen-donating ability of antioxidants [12]. Although hydrogen peroxide itself is not the most reactive, its in vivo toxicity can be partly attributed to hydroxyl radical formation in the cells. Addition of hydrogen peroxide to cells in culture can lead to transition metal ion-dependent OH^• mediated oxidative DNA damage [31], as well as other detrimental effects on nucleic acids [32], protein [33], and lipids [34]. Deoxyribose on exposure to hydroxyl radicals, generated by Fenton reaction, degrades into fragments and generates a pink chromophore on heating with thiobarbituric acid (TBA) at low pH [35]. We measured the ability of G. tenax extracts to scavenge hydroxyl radicals by studying the competition between deoxyribose and G. tenax extract for hydroxyl radicals (Table 2 and Figure 2). Phenolic compounds we identified in G. tenax extracts, such as p-hydroxybenzaldehyde and 2,2′-methylenebis(6-tert-btyl-4-methylphenol), acted as effective antioxidants and free radical terminators in the deoxyribose degradation assay. Vanillylacetone and fatty acids, the active compounds we identified, may also be related to antioxidant activity of G. tenax. Accumulated evidence demonstrates the antioxidant activity of essential fatty acid components extracted from various plants. Therefore, tetradecanoic acid, n-hexadecanoic acid, linoleic acid and oleic acid present in G. tenax may contribute to the antioxidant activity [36].

The antimicrobial activity of G. tenax was assessed both qualitatively and quantitatively by disc diffusion. G. tenax extracts exhibited moderate broad-spectrum antimicrobial action. Zones of inhibition exhibited a dose-dependent relationship with increasing concentrations of extract. Minimum inhibitory concentration (MIC) values for inhibition of Staphyloccocus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli were 3.9, 7.8, 15.6, and 3.9 mg/mL, respectively (Table 3).

Many diseases are caused by microbial infection, indicating the need for more antimicrobial agents [37]. Gloiopeltis is often used to treat intestinal diarrhea. Although much is known about the biological activities of Gloiopeltis extracts, little is known about the anti-diarrheal mechanisms. Gloiopeltis extracts display broad biological effects, such as the ability to regulate the immune system, inhibit bacterial growth, and prevent adhesion of bacteria to target surfaces. We demonstrate that G. tenax extracts inhibit bacterial growth, suggesting that Gloiopeltis extracts exert their antibacterial activity directly, rather than indirectly by enhancing the immune system.

GC-MS analysis identified compounds, within G. tenax extracts, with known antibacterial activity. Sesquiterpenes, such as thujopsene, (+)-cuparene and cedrol, all have antibacterial activity [21,38,39]. Zingerone (vanillylacetone) and its derivatives can inhibit enterotoxigenic Escherichia coli heat-labile enterotoxin-induced diarrhea in mice [40]. Studies have shown that zingerone might exert beneficial therapeutic effects on hypermotility-induced diarrhea by abrogating excessive gastrointestinal motility [41]. Therefore, vanillylacetone is the likely active constituent responsible for the antidiarrheal efficacy of G. tenax. A number of free fatty acids are known to possess antibacterial activity against Gram-positive bacteria, as well as their esters [42,43]. Some synthetic fatty acid analogs of cholesterol show excellent antibacterial activity in vitro [44]. The antimicrobial activity and therapeutic efficacy of oleic acid in a liposomal formulation against methicillin-resistant S. aureus is known [45]. The presence of a hydroxyl group in compounds, especially phenolic components, such as 4-hydroxybenzaldehde found in G. tenax extract, can destabilize the bacterial membrane to increase the activity of antimicrobials. Such components at low concentrations might be involved in some type of synergism with the other active compounds.

G. tenax was provided and authenticated by the Nan Ao Marine Biological Research Station of Shantou University in Guangdong Province. It was collected at Nan Ao Island. The study area is on the Tropic of Cancer, located 116°56′–117°09′E and 23°23′–23°29′N off the city of Shantou, eastern Guangdong Province, neighboring the Fujian Province, between Hong Kong and Taiwan. This region is at the northern edge of algae distribution in East Asia.

Alkane standard solutions of C8–C20 (mixture No. 04070) and C21–C40 (mixture No.04071) were from Fluka Chemika (Buchs, Switzerland). 2,2-Diphenyl-1-picrylhydazyl (DPPH), β-carotene, butylated hydroxytoluene (BHT), ascorbic acid, and linoleic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 2-Deoxy-D-ribose was purchased from Amresco. 2-Thiobarbituric acid (TBA) and EDTA were obtained from Aladdin (Shanghai, China). All other chemicals and solvents (ethanol, H₂O₂, tween-40) were of analytical grade. Ultrapure water was used for the experiments.

CO₂-SFE involved use of an HA221-50-06 extractor (Huaan Supercritical Equipment Co., Nantong, Jiangsu, China). The instrument was run with CO₂ for both the extraction and cooling gases. The extraction pressure and temperature were 300 bars and 45 °C, respectively. G. tenax powder was placed into 5 L extraction thimbles. The samples were extracted with pure CO₂, with ethanol used as an entrainer. The amount of ethanol, 1 L, was approximately equal to the sample volume. Fractions were collected in both separators into two separate containers. The addition of a polar co-solvent (ethanol 95%) led to increased efficiency of extraction.

GC-MS analysis involved an Agilent 7890 GC equipped with a quadrupole 5975 mass spectrometer (Agilent Technologies, CA, USA) and an HP-5MS column (i.d., 0.25 mm; length, 30 m; film thickness, 0.25 μm). Oven temperature was maintained at 100 °C for 2 min initially and then sequentially raised to 140 °C at a rate of 10 °C/min, to 170 °C at a rate of 1 °C/min, and to 280 °C/min at a rate of 8 °C/min where the temperature was finally held for 10 min. The MS conditions were: ionization voltage, 70 eV; emission current, 10 mAmp; scan rate, 1 scan/s; mass range, 45–450 M/Z; trap temp., 150 °C; transfer line temp., 280 °C. Operating conditions were: injector temp., 280 °C; FID temp., 280 °C.; carrier(He) flow rate, 0.8 mL/min. Samples were injected splitless. Two microliters of sample, dissolved in ethyl acetate was injected.

Identification of the constituents was based on comparison of their Kovats Index (KI) and the MS fragmentation pattern with reference compositions in the database of the NIST Mass Spectral Search Program (NIST 08 mass spectral database, National Institute of Standards and Technology, Washington, DC, USA). To determine the KI value of the components, a commercial aliphatic hydrocarbon mixture (Sigma-Aldrich) was added to the essential oil before injecting it into the GC/MS equipment and analyzed under the same conditions as above. The following quasi-linear equation for the temperature-programmed Kovats Index was used: {KI(x) = 100 × Z + 100 × [t(x) − t(z)]/[t(z + 1) − t(z)]} (1) where KI(x) is the temperature-programmed Kovats Index of interest and t(z), t(z + 1), and t(x) were the retention times in minutes of the two standard n-alkanes containing z and z + 1 carbons and index of interest, respectively: t(z) < t(x) < t(z + 1).

Antimicrobial susceptibility testing for G. tenax involved the Kirby-Bauer (KB) disk diffusion method against Gram-positive and -negative bacteria. Microorganisms included Staphylococcus aureus (American Type Culture Collection (ATCC) No. 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Briefly, a suspension of the tested microorganism (0.1 mL of 10⁸ cells/mL) was spread on solid media plates. Filter paper discs (6 mm in diameter) were impregnated with 10 µL of different concentrations of G. tenax extract and placed on the inoculated plates, then incubated at 37 °C for 24 h.

A broth microdilution method was used to determine the MIC against the susceptible microorganisms using the KB method according to the National Committee for Clinical Laboratory Standards. A serial doubling dilution of G. tenax extract from 0.24 to 500 μg/μL was prepared in 50 μL Mueller-Hinton broth (MHB) in each well of a 96-well microtiter plate. Freshly grown microbial suspensions in MHB were standardized to a cell density of 1.5 × 10⁸ (McFarland No. 0.5) and added to the wells (50 μL). After incubation at 37 °C for 24 h, the MIC was defined as the lowest concentration of G. tenax extract at which the microorganism did not demonstrate visible growth.

Assays were carried out in triplicate, and each analysis of all samples was performed in duplicate; results displayed are averages. Absorbance values are expressed as means ± SD. Differences between variables were tested by one-way ANOVA with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Values of P < 0.05 were considered statistically significant.