Study On Extraction And Separation Of Essential Oil And Phloroglucinols From Dryopteris Frangrans And Antibacterial Activity

Dryopleris fragrans (L.) schott, a member of the Dryopteris genus, is a deciduous perennial herb characterized by distinctive fragrance and persistent old fronds. This aromatic medicinal plant normally occurs on talus slopes and cliff crevices, and mainly distributed in Asia-temperate, Europe and North America. Especially in Wu Da Lian Chi (Heilongjiang province, China), a place of interest with volcanic geological landforms, it possessed a strong affinity for lava and molten rocks formed after volcanic eruption. The indigenous plant has been used in traditional folk medicine for decades in treating various dermatosis (such as psoriasis, acne, erythra and dermatitis) and using as an effective agent of antibacterial, analgesic, antioxidant, anti-tumour as well as antirheumatic arthritis. In the present study, extraction and separation of essential oil and phloroglucinols from D. frangrans were investigated. The antibacterial activities of purified constituents were studied.The results were as follows:1. The analysis method of essential oil and four phloroglucinols from D. fragrans were developed.(1) GC-MS combined with retain index were used to identify the constituents of the essential oil of D. fragrans. Eighteen compounds, representing89.65%of the oil, were identified. The essential oil was found to be rich in sesquiterpenes.(2) A RP-HPLC method was developed for the simultaneous separation and determination of four phloroglucinols from D. fragrans.Chromatography column:ODS C18reversed phase column (100mm×4.6mm. i.d.,2.6μm); mobile phase:acetonitrile-water-Formic acid; column temperature:25℃. This method was repeatable, accurate and stable, which was suitable for the determination of phloroglucinols in D. fragrans as well as in other plants.2The solvent-free microwave extraction was developed for extraction of the essential oil from D. fragrans. A central composite design combined with response surface methodology was applied to optimize the technique. The influence parameters of extraction time, irradiation power and humidity were investigated. The optimal operating condition was as follows: extraction time34min, irradiation power500W, humidity50%.Under the opimized condition, the extraction yield of essential oil was0.33%, and R.S.D. was5.19%. In the results of response surface analysis, extraction time is the main factor which affects the microwave extraction process followed by irradiation power and humidity. The relationship between the extraction yield and the influence factor is not simple linearity. There is strong interaction between extraction time and irradiation power. The solvent-free microwave extraction was an efficient technique for extraction of the essential oil from D. fragrans. After extracted by solvent-free microwave extraction, the residua can be used as raw material to extract other target components.3. The negative-pressure cavitation extraction (NPCE) was developed for extraction of four main phloroglucinols from D. fragrans. The effects of liquid/solid ratio, extraction time, air flow, pressure and number of extration cycles on the recovery of flavonoids were optimized. The optimum parameters were as follows:liquid/solid ratio10:1(mL/g), extraction time20min, air flow30mL/min, pressure-0.04～-0.05Mpa,3extraction cycles.Under the above opimized conditions, the relative recoveries of dryofragin, aspidin BB, aspidin PB and aspidinol were all higher than94%. Negative pressure is the impetus of NPCE; and the extraction process was performed under the obturation condition and normal temperature. So the method was energy saving, solvent free and avoiding the thermal decomposition, which was suitable for the extraction of active components in D. fragrans as well as in other plants.4. The method of separation and enrichment of aspidinol, aspidin PB, dryofragin and aspidin BB from D. fragrans was studied.(1) Resin column adsorption was applied for separation and enrichment of aspidinol and aspidin PB. Considering both contents and recoveries, the optimal enrichment and separation conditions were confirmed as follows:30%ethanol5BV was used for removing impurities,50%ethanol10BV was selected to wash aspidinol and70%ethanol15BV to elute aspidin PB, the flow rate was3BV/h.(2) Resin column chromatography was applied for separation and enrichment of dryofragin and aspidin BB. Considering both contents and recoveries, the optimal enrichment and separation conditions were confirmed as follows:70%ethanol10BV was used for removing impurities,80%ethanol30BV was selected to wash dryofragin and95%ethanol20BV to elute aspidin BB, the flow rate was6BV/h.(3)After AB-8resin column adsorption, the content of aspidinol increased from1.40mg/g to10.4mg/g, increased7.4times; the content of aspidinol increased from4.9mg/g to41.8mg/g, increased8.52times.The recoveries of aspidinol and aspidin PB were85.73%and78.41%, separately. After AB-8resin column chromatography, the content of dryofragin and aspidin BB increased to34.4mg/g and56.9mg/g, separately, increased8.39and5.99times. The recoveries of dryofragin and aspidin BB were91.22and75.64%, respectively.5. Medium-pressure silica gel column chromatography and recrystallization were applied for purification of aspidinol, aspidin PB, dryofragin and aspidin BB from D. fragrans were studied. A series of schemes were employed for identification the structure of the purified compounds. (1)1he condition of medium-pressure silica gel column chromatography and recrystallization were determined. After determined by HPLC, the purities of aspidinol, aspidin PB, dryofragin and aspidin BB were96.2%,95.1%,97.7%and95.3%, separately, with the recoveries of78.26%,63.27%,73.24%and65.63%.(2)The products were identified as the target compounds by the reaction of physical and chemical property combined with UV spectrum, mass spectrum (ESI-MS-MS) and nuclear magnetic resonance (1H-NMR和13C-NMR).6. Antibacterial activities of different fractions, four main phloroglucinols and essential oil from D. fragrans were studied. The results were as follows:(1)The antibacterial activities of different fractions were tested, and the bacterium include Propionibacterium Acnes, Staphylococcus aureus, Staphylococcus epidermidis, Bacilus subtillis, Escherichia coli, Proteus vuLgaris, Pseudomonas aeruginos and Candida albicans. All fractions showed antimicrobial activity towards both gram-positive bacteria and gram-negative bacteria. The most sensitive stain towards petroleum ether, ethyl acetate, n-butanol and water fractions was Escherichia coli with the MIC values of1.95,1.95,3.9and3.9mg/mL(2)The MIC and MBC values of D. fragrans essential oil towards P. Acnes were0.0391%and0.0782%(v/v). The antibacterial activity of D. fragrans essential oil towards P. Acnes was better than that of reported Rosmarinus officinalis essential oil and nearly to that of Syzygium aromaticum essential oil.(3)The most sensitive stains towards four phloroglucinols were Staphylococcus epidermidis, Staphylococcus aureus, Bacilus subtillis, Proteus vμLgaris, Candida albicans and Propionibacterium Acnes.The MIC values of dryofragin towards Candida albicans were125μg/mL. The bactericidal effect of dryofragin approach to that of positive control penicillium and better than that of chloromycetin and erythromycin. The bactericidal effect of aspidinol, aspidin PB and aspidin BB towards Staphylococcus epidermidis, Staphylococcus aureus, Bacilus subtillis, Proteus vμLgaris and Candida albicans are nearly to or better than that of positive control (penicillium, chloromycetin and erythromycin). The MIC values of dryofragin, aspidinol, aspidin BB and aspidin PB towards P. Acnes were15.63,15.63,31.25and125μg/mL.(4) According to time-kill curves of aspidin BB (MBC), all the P. Acnes were killed in5hours. The results of SDS-PAGE showed the molecular mechanisms of aspidin BB against P. acnes, aspidin BB could inhibit the two special proteins (26kDa and33kDa). The DNA (?)leavage activity of aspidin BB towards P. Acnes showed that aspidin BB inhibits the growth of P. Acnes by DNA cleaving.In conclusion, the method of solvent-free microwave extraction (SFME) and negative- pressure cavitation extraction (NPCE) was suit for extraction of essential oil and four main phloroglucinols from D.fragrans. Resin column adsorption and Resin column chromatography could enrich the four main phloroglucinols effectively. After extraction, separation and purification, the purities of aspidinol, aspidin PB, dryofragin and aspidin BB were96.2%,95.1%,97.7%and95.3%, separately, with the recoveries of46.07%,36.63%,45.80%and38.66%. Different fractions, purified constituents and essential oil from D.fragrans possessed antibacterial activities. Aspidin BB showed great antimicrobial activity. The results benefit of develop and make full use of plant medicinal.