| Rosmarinus officinalis L.is a perennial aromatic plant of the genus Rosemary L in the family Rosmarix in the family Lipaceae.R.officinalis L.has a variety of natural active ingredients,because of its antibacterial,anti-inflammatory,antioxidant,anti-tumor and other biological activities,can be widely used in biomedicine,cosmetics,food and other fields.The efficient extraction of rosemary ingredients and the exploration of the efficacy of different varieties of extracts are an important part of the research and utilization of their natural active ingredients.Therefore,this article uses three varieties of rosemary with different leaf types(leaf types: short-leaf,broad-leaved,long-leaved)(respectively: R.officinalis ‘Severn Sea’,R.officinalis ‘Lockwood de Forest’,R.officinalis ‘ Albus’)as the research object,first using water distillation method to prepare essential oils,and volatile components for gas combination analysis,and then the solid residue for component extraction,optimize solvent(ethanol)reflux extraction,ultrasonic auxiliary extraction,subcritical extraction,supercritical extraction and other four extraction methods,non-volatile component analysis of 12 extracts of R.officinalis L.of different leaf types,explore bacteriostatic activity,antioxidant activity and antitumor activity,to provide a scientific basis for the high-value utilization of R.officinalis L.active ingredients.The main findings are as follows:(1)Analysis of volatile components of R.officinalis L.three varieties such as R.officinalis ’Severn Sea’,R.officinalis ’Lockwood de Forest’,R.officinalis ’Albus’ were extracted,and the extracts were isolated and identified by GC-MS technology.51 kinds of volatile components,compared with the map,the composition of different varieties of R.officinalis L.is basically the same,but the relative content is different,of which the main common components are eucalyptol(11.16 ~ 20.72%),verbena ketone(1.69 ~ 13.51%),dextran(3.95 ~ 9.43%),β-pinene(6.00 ~ 10.92%),pinenen(5.38 ~ 8.21%)and other substances.These chemical components are rich in physiological activity,so they can be used in chemicals,food,biomedicine,cosmetics and other fields.(2)Optimization of R.officinalis L.ingredient extraction conditions.Through the one-factor combined orthogonal experiment,the extraction conditions of ethanol reflux extraction,ultrasonic assisted extraction,subcritical extraction and supercritical extraction of rosemary components were optimized,and the results showed that the optimal extraction conditions for solvent reflux were: 1:25 feed-liquid ratio,extraction time 1.5 h,temperature 80°C,and the extraction rates of the three varieties were 23.90%,23.77% and 22.67%,respectively,and the optimal extraction conditions for ultrasound assistance were: extraction time 50 min,feed-to-liquid ratio 1:25,and power 600 W The extraction temperature is 80 °C,and the extraction rate of the three varieties is 23.11%,21.67%,and 21.48%,respectively;the optimal extraction conditions for subcritical are: the material-liquid ratio is 1:20,the extraction temperature is 60 °C,the extraction time is 30 min,and the extraction rate of the three varieties is 7.34%,7.14%,and 7.01%,respectively;the optimal extraction conditions of the supercritical are: extraction pressure 400 bar,extraction temperature 30℃,extraction time 3 h,and the extraction rate of the three varieties is 6.00%,5.28%、5.79%。 The order of the extraction rate is: ethanol reflux extraction> ultrasonic auxiliary extraction> subcritical extraction> supercritical extraction.(3)Analysis of group changes before and after R.officinalis L.extraction.Infrared spectroscopy analysis of 12 kinds of R.officinalis L.extracts using rosemary powder as a contrast material found that the substances in rosemary are mainly polysaccharides,proteins,lipids,flavonoids,etc.The composition of the group was the same before and after extraction,indicating that 12 rosemary extracts were effectively extracted.(4)Analysis of non-volatile components of rosemary extract.12 kinds of R.officinalis L.extracts are rich in active ingredients,of which terpenes,flavonoids,sugars and other compounds are relatively high content,and the extracted substances are mainly sage phenol,luteolin,oleanolic acid,sage acid and so on.The results showed that there were differences in the active ingredients in the three leaf types of R.officinalis L.extracted by four methods.(5)Analysis of bacteriostatic activity of rosemary extract.The bacteriostatic properties of 12 kinds of R.officinalis L.extracts were evaluated and analyzed by filter paper method,and the results showed that R.officinalis L.extract had obvious inhibitory effects on Staphylococcus aureus,Bacillus cereus and Bacillus subtilis,but no inhibitory effect on E.coli and Salmonella.Using the bacteriostatic APC index and the minimum bactericidal concentration to comprehensively evaluate the bacteriostatic activity,in terms of varieties,R.officinalis ’Severn Sea’ extract had the strongest bacteriostatic activity;in terms of extraction method,supercritical extraction had the strongest inhibitory effect,while ethanol reflux extraction had the weakest inhibitory effect.(6)Antioxidant activity analysis of rosemary extract.By reducing force,removal of DPPH,OH,ABTS free radical ability to determine,it shows that R.officinalis L.extract has a strong antioxidant capacity,and is positively correlated with the concentration,and the scavenging ability at higher concentrations is comparable to VC.When the concentration of R.officinalis L.extract is 512 μg/m L,the scavenging ability of DPPH and ABTS has reached more than 90%;for OH,short-leaf and broadleaved R.officinalis L.extract has a strong antioxidant capacity,and its scavenging ability has reached more than 90% at 18 mg/m L,while the antioxidant results of longleaf rosemary extract are not ideal,especially ethanol reflux extract,the scavenging ability is only 80% at 20 mg/m L;12 extracts have shown a certain reducing ability.At concentrations of 512 μg/m L,the absorbance values of R.officinalis L.extract were all below 0.5.(7)Analysis of antitumor activity of rosemary extract.By using CCK-8 method,the inhibition effect of 12 R.officinalis L.extracts on the proliferation of Hela(cervical cancer cells),Hep G2(liver cancer cells),MCF-7(human breast cancer cells),and SGC-7901 cells(human gastric cancer cells)was studied in in vitro anti-tumor experiments,and the results showed that R.officinalis L.extract had good anti-tumor activity.The 12 extracts have a strong inhibitory effect on SCG-7901 and MCF-7cells,with a cell fatality rate of more than 93%;for Hep G2 cells,11 extracts have extremely strong anti-tumor activity,and their cell fatality rate is above 94%,while the subcritical extraction of mid-leaf R.officinalis L.extract(R.officinalis‘Lockwood de Forest’)has slightly poor antitumor activity,and its cell fatality rate is80.7%.For Hela cells,the three methods of solvent(ethanol)reflux extraction,ultrasonic assisted extraction,and supercritical extraction have strong anti-tumor activity,and their cell fatality rate is more than 93%,followed by subcritical extraction.(8)Comprehensive evaluation of the TOPSIS method.Sampling TOPSIS method,the yield yield,bacteriostatic activity,antioxidant capacity and anti-tumor activity of 12 extracts of 3 rosemary cultivars were comprehensively evaluated,and the results showed that the extract of R.officinalis ‘Severn Sea’ extracted from supercritical extraction had good development potential.This paper provides a theoretical basis for the analysis,extraction method and biological activity analysis of R.officinalis ingredients,and provides a scientific basis for further in-depth development and comprehensive utilization of R.officinalis L resources. |
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