Extraction of Teucrium manghuaense and Evaluation of the Bioactivity of Its Extract

The ethanol extract of Teucrium manghuaense grown in Hainan province (China) was analysed by GC and GC/MS. Of the constituents 84–96% were identified on the basis of their GC retention times and their mass spectra in regard to authentic compounds. The results revealed that it contained 9-methyl-9-azabicyclo[4.2.1]nonane (7.43%), 2-methylpyrrolidine (19.42%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (10.84%), and squalene (28.55%), as major components, constituting 66.24% of the extract. The chemical characterization of the ethanol extract by GC/MS also allowed identification of 1-octen-3-one (3.41%), 2-pentyl-piperidine (2.25%), 1-(2-methyl-1-propenyl)-piperidine (4.63%), 2,2′-diethoxy-5,5′-bi-1-pyrroline (0.41%), (Z,Z,Z)-9,12,15-octadecatrieniic acid, 2,3-dihydroxypropyl ester (1.56%), vitamin E (2.95%) and stigmasterol (1.39%). Finally, the antioxidant and anti-tumor activities of the ethanol extract have been evaluated. Results show that the extract of Teucrium manghuaense leaf possesses strong DPPH−, hydroxyl radical scavenging activity and anti-tumor activity.


Introduction
In the human body superoxide, hydroxyl radicals and hydrogen peroxide are produced by the reduction of oxygen [1]. It is well established that oxygen radicals are involved in various pathological states such as cancer, cardiovascular disorders, arthritis, inflammation, and liver diseases [2,3]. Antioxidants can protect the human body from free radical damage, and prevent cancer and coronary heart disease and aging process [4].
Teucrium is a genus of perennial plants, of the family Labiatae. These species are herbs, shrubs or subshrubs, most common in Mediterranean climates. Humans value Teucrium as ornamental plants and a source of aromatics, and some species have culinary and/or medicinal value. Several are used as food plants [5]. A common examples for this genus is Teucrium manghuaense Sun ex S. Ghow. Teucrium manghuaense are rich in essential oils (0.7-4%), and have been proven to show a wide range of biological activities important to human health, for example, antiviral, antitumoral, antiinflammatory and anticoagulant activity [6]. In this study, we extracted bioactive compounds from Teucrium manghuaense and characterized the ethanol extract by GC-MS. In addition, we examined the bioactivity of the extract, including antioxidant and anti-tumor activities.

Materials
Teucrium manghuaense was obtained from the Spice and Beverage Research Institute at the Xing Long Tropical Botanical Garden of Chinese Academy of Tropical Agricultural Sciences (Hainan province, China). Leaves of Teucrium manghuaense was dried (a relative humidity between 2.8% and 3.4%) and ground into fine powder (70 mesh) using a mechanical grinder. Mouse leukemic L1210 cells was obtained from the Shanghai Cellular Institute of China Scientific Academy, Human Colon Carcinoma LoVo Cell Line kindly offered from the Pharmacological Laboratory in Shanghai Jiao Tong University. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Methotrexate (MTX) was purchased from Shanghai Hualian Pharmacy Co Ltd. All other solvents and chemicals were of analytical grade.

Extraction of bioactive compounds
The bioactive compounds of ground Teucrium manghuaense leaf were extracted using ethanol as solvent. Extraction was carried out using a shaking incubator at room temperature for 2 h followed by filtration through Whatman No.1 filter paper. The residue was re-extracted twice in the same manner and the three filtrates were combined. The ethanolic extract was concentrated using a rotary evaporator at 55 °C to near dryness. The extraction rate was 4.2%.

GC-MS analyses
The ethanolic extracts compositions were analysed by GC-MS performed using a Trace GC/MS gas chromatograph coupled to an ion trap detector [7]. The fused-silica column was a SLB-5MS silica column (Supelco, Bellafonte, PA, USA) (30 m × 0.25 mm i.e., film thickness 0.25 cm). GC-MS data were obtained using the following conditions: carrier gas helium (He 99.999%); flow rate 1.0 mL·min -1 ; the split ratio 1/70 (v/v). An aliquot of 100 mg of distilled oils were diluted with 1 mL acetone, as were the extracts, and 1.0 μL was injected into the GC-MS system. The oven temperature program was: 70 °C for 13 min, from 70 to 280 °C at 6 °C min -1 , and holding 280 °C for 16 min. The injector, transfer line and ion trap temperatures were 250, 280 and 200 °C, respectively. The electron impact (70 eV) spectra were recorded at 1 scan/s with a filament emission current of 10 μA. The identification of volatile compounds was based both on comparison of the linear retention indexes (RI) calculated using the Van der Dool and Kratz's equation with those reported in the literature [8] and by the matching of mass spectra of the compounds with the reference mass spectra of two libraries (Wiley5 and Nist05) coupled with the software of GC-MS and Adams' library [9]. For the major chromatographic peaks, identification was also confirmed using authentic standards.

Nuclear magnetic resonance
The 400 MHz 1 H-NMR spectra of all sample batches were analyzed on an AMX-II 600 MHz spectrometer (Bruker Instruments, Inc.), 32 scans were collected into 64 K data points over a spectral width of 4789 Hz (12 ppm) with the transmitter offset at 5.00 ppm, yielding a digital resolution of 0.15 Hz per point. 1 H-NMR high-temperature spectra at 353 K were measured at 300.13 MHz using a QMP probe. Thirty two scans were collected into 64 K data points over a spectral width of 6172.84 Hz (20.56 ppm). A flip angle of 30° was used. The acquisition time was 5.31 s, followed by a relaxation delay of 1 s. An exponential line broadening window function of 0.3 Hz was used in the data processing. 13 C-NMR spectra were recorded at 25 °C using a Bruker Avance 600 NH 3 spectrometer operating at 14 T and equipped with a 5 mm broad-band probe. For the quantitative 13 C-NMR study the following experimental parameters were used: 90 pulse width (14 s, -3.0 dB), pulse delay of 38 s, spectral width of 21186.44 Hz (120 ppm), 64 K sampled points with 1.5 s to acquire them.

The DPPHradicals scavenging activity
Experiments were carried out according to Kordali et al. [10] using a slight modification. Briefly, the extract was diluted with 0.1 mL of solvent. Then 0.5 mM DPPH solution in methanol was prepared, and 0.5 mL of this DPPH solution was mixed with 0.1 mL of various amounts of the extract and vortexed thoroughly. Then, 4 mL of methanol was added to the solution and allowed to stand for 60 min in a dark room. The absorbance was measured at 516 nm using a Shimadzu UV-1601 spectrophotometer (Shimadzu, Kyoto, Japan). Decreasing the absorbance of the DPPH solution indicates an increase in DPPH radical scavenging activity. This activity is given as percent DPPH radical scavenging, which is calculated with the equation: %DPPH radical scavenging = [(control absorbance -sample absorbance)/control absorbance] × 100%.
Control contained 0.5 mL of DPPH solution and 0.1 mL of methanol. Vitamin C at varying concentrations was used as positive control. Data were reported as means ± SD for three replicates.

The hydroxyl radical scavenging activity
The hydroxyl free radical scavenging activity (FRSA) was assayed by using the 1,10-phenanthroline-Fe 2+ oxidative method [11]. After incubation at 37 °C for 1 h, the absorbance was measured at 536 nm. Hydroxyl FRSA was evaluated as the inhibition rate of 1,10-phenanthroline-Fe 2+ oxidation by hydroxyl radical. The FRSA was calculated using the following equation: where As is the absorbance in the presence of sample and H 2 O 2 ; Ac is the absorbance in the presence of H 2 O 2 without sample; Ab is the absorbance without sample and H 2 O 2 . Each assay was performed in triplicate.

NMR
The 1 H-NMR spectral data (Figure 2) of the extract sample showed that a chemical shift between δ H 4.96 and 5.05 confirm the presence of six vinyl methyls, between δ H 1.83 and 1.99, ten methylenes, and between δ H 1.47 and 1.53 eight methyl groups. The 13 C-NMR spectral data (Figure 3) of the extract sample showed that a chemical shift between δ C 16.2 and 25.9 confirmed the presence of eight methyls, between δ C 27.4 and 40.6 ten methylenes, and between δ C 125.1 and 135.5 six vinyl methyls. The modified Mosher's method was carried out based on the chemical shift of the major compound only. Based on the result of the above spectral data, examination of the ∆δ values [δ(−) − δ(+)] showed a negative and positive chemical shift distribution to various protons, which can confirm the presence of squalene.

Determination of DPPH radical scavenging capacity
DPPH has been widely used to test the ability of compounds or plant extracts to act as free radical scavengers, for example Teucrium [13]. Figure 4b shows the percent of DPPH radical scavenging capacity with vitamin C as reference. The experimental data revealed that at various levels the extract was likely scavenge free radicals. From Figure 4a we observed that a dose-response relationship was found in the DPPH radical scavenging capacity; the activity increased as the extract concentration increased. Within concentrations used in the experiment, 0.63 mg/mL ethanolic extract was the strongest scavenger with 93.6%, then 0.5 mg/mL vitamin C as standard with 96.5% (Figure 4b). The 50% inhibition concentration (IC 50 ) of the ethanolic extract was determined to be 0.22 mg/mL. From the results, DPPH radical scavenging activity of the extract was weaker than the reference vitamin C at lower concentration. However, it displayed the strong scavenging capacity at higher concentration. Therefore, the ethanolic extract of Teucrium manghuaense could act as a natural antioxidant

Determination of hydroxyl radical scavenging activity
The antioxidant activity of the ethanolic extract of Teucrium manghuaense was evaluated by the hydroxyl radical scavenging capacity method. Figure 5b shows the percent of hydroxyl radical scavenging capacity with vitamin C as reference. The activity increased as the concentration increased for the extract. At 0.73 mg/mL, scavenging abilities of ethanol extract of Teucrium manghuaense on hydroxyl radicals were 39.3% (Figure 5a). However, the scavenging activity decreased when the concentration of the extract exceeded 2.5 mg/mL. At 0.5 mg/mL, scavenging abilities of vitamin C on hydroxyl radicals were 87.3% (Figure 5b). It indicated that the enthanol extract was less effective in its scavenging ability than vitamin C, which scavenged 87.3% of hydroxyl radicals at 0.5 mg/mL (Figure 5b). However, at 2.5 mg/mL, the extract of Teucrium manghuaense showed a scavenging ability of 85.2%. Cheng, Cui and Chen [14] reported that ethanolic extracts of Teucrium manghuaense exhibited good hydroxyl radical scavenging ability and attributed to this the alleged anticarcinogenic properties of Teucrium manghuaense extracts. These results indicate that some compounds contained in the ethanolic extracts of Teucrium manghuaense are indeed effective scavengers for hydroxyl free radicals. Accordingly, it was anticipated that the high scavenging ability of Teucrium manghuaense extracts might confer some antimutagenic properties.

Determination of anti-tumor activity
Anti-tumor activity of the samples were evaluated using mouse leukemic L1210 cells and human Colon Carcinoma LoVo Cell by enzymatic reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide). As shown in Figure 6, the ethanolic extract inhibited the mouse leukemic L1210 cells and human Colon Carcinoma LoVo Cells growth obviously in a dose-dependent manner. The IC 50 of the ethanolic extract was 122.8 mg/L on mouse leukemic L1210 cells and 106.0 mg/L on human Colon Carcinoma LoVo Cell, respectively. In contrast to those in the control (methotrexate), the inhibition rate in the ethanolic extract group was significantly lower at all concentrations (p < 0.05). The IC 50 of methotrexate was 0.41 mg/L on mouse leukemic L1210 cells and 0.26 mg/L on human Colon Carcinoma LoVo Cell, respectively.