Chemical composition, antioxidant, antibacterial and cholinesterase inhibitory activities of three Juniperus species

: The chemical composition, antioxidant, antibacterial and cholinesterase inhibitory activities of three Juniperus species were studied. The contents of total phenolic and 10 phenolic compounds were highest in Juniperus rigida Sieb.et Zucc., of which catechin and cumaric acid were the predominant phenolic compounds, but were lowest in Juniperus sibirica Burgsd. GC-MS analysis showed the highest contents of essential oils were in J. rigida (92.61%), followed by Juniperus formosana Hayata (87.30%) and J. sibirica (84.89%). The a -pinene was the most dominant compound in J. rigida (23.99%) and J. formosana (9.71%), however, it has not been detected in J. sibirica . Ethanol extracts showed the higher radical scavenging capacity in ABTS, FRAP and DPPH assays than essential oils. The essential oils and ethanol extracts of J.sibirica showed the strong antibacterial activity against Salmonella typhimurium and Escherichia coli . Three Juniperus species showed certain acetylcholinesterase and butyrylcholinesterase inhibitions and J. formosana showed better cholinesterase inhibitory.


Determination of total phenolic content (TPC) and flavonoid content (TFC).
The total phenolic contents (TPC) of the ethanol extracts were determined by the previous studies using the Folin-Ciocalteu colorimetric method (Basgedik et al.2010;Mocan et al.2014). Samples solutions were diluted to the concentration of 2 mg/mL. The phenolic contents of samples was estimated from the gallic acid equivalent based on a calibration curve of gallic acid standard solutions. All values were expressed as millimole gallic acid equivalent per g of dry weight (mmol equiv. GAE /g). Data were reported as means ± SD for three replicates.
Determination of the total flavonoid contents (TFC) were carried out according previously published procedure using the NaBH 4 /chloranil-based (SBC) assay (He et al.2008). Sample solutions were diluted to a concentration of 2 mg/mL.
A calibration curve was generated using different concentrations of quercetin (0.1-10.0 mM). The total flavonoid contents of ethanol extracts of needles of Juniperus was expressed as millimoles of quercetin equivalents per g dry weight (mmol equiv. QUE/g). Data were expressed as means ± SD for three replicates.

Validation of the HPLC Procedure.
The quantitative determination of each compound was performed using the external standard method by using pure standards with concentrations ranging from 1 to 1200 μg/mL. In previous study, our team validated the HPLC procedure via analysis of precision, reproducibility and repeatability. All relative standard deviation (RSD) of the relative peak areas were less than 3%, suggesting that the method of the analysis was deemed repeatable and accurate.

GC-MS analysis of essential oils.
The GC-MS analysis of the essential oils were carried out using a TRACE1310-ISQLT Ultra System. TG-5MS capillary column was used. Helium was the carrier gas and the flow rate was 1mL/min. One microliter diluted sample were injected into the GC by an autosampler and split ratio was 1/40. Temperature program was at 50 °C for 1 min and then increase to 130℃ at 3°C /min held for 1 min. After that, it was raised to 150 ° C at a rate of 2 ° C / min for 1 min; finally, it was raised to 200 ° C at a rate of 10 ° C / min for 1 min. An electron impact ionization system with ionization energy of 70 eV. The electron ionization spectra with a mass scan range of 40-460 m/z were used and solvent delay time was 2.5 min.
The DPPH radical scavenging capacity was analyzed by the method described by Yen and Chen (1995) with some modifications (Yen et al. 1995). Trolox was the positive control, and all experiments were tested in triplicate. IC 50 (mg/mL) were known as the standard of antioxidant capacity and were obtained from linear regression analysis. A lower IC 50 value indicates a higher DPPH radical scavenging ability.

ABTS• + radical cation scavenging assay.
The ABTS radical scavenging assay was determined by Yang et al. (2013) andMocan et al. (2015) with some modification (Yang et al. 2013;Mocan et al. 2015). In this work, Trolox was used as the standard antioxidant and phosphate-buffered saline (PBS) solutions were used as blank samples. The results were expressed in terms of micromoles of trolox equivalents per g dry extract weight (µmol eq. trolox/g). All determinations were performed in triplicate.

Ferric reducing power (FRAP) assay.
The FRAP assay was used to test the ferric reducing ability of the extracts according to Benzie and Strain (Benzie et al. 1996), with some modification. Trolox was employed as the standard solution. The results were expressed micromoles of trolox equivalent per g. The absorbances of samples were measureed using the spectrophotomet at 593nm. Each analysis group was performed in triplicate.