| Salvia bowleyana Dunn, a perennial herb of genus Salvia, is widely distributed in the southern provinces and Fujian is one of the main distribution area. Its taproot thickened, can be used to replace S. miltiorrhiza in folk medicines, and the efficacy has been proved. At present, there are few studies on the research of effective ingredients, activities of S. bowleyana. Therefore, this paper uses S. bowleyana distributed in Fujian as the study object. On the basis of determination of salvianolic acid B contents in the root, stem and leaf of S. bowleyana, our research mainly focuses on the separation, purification and identification of antioxidant activity of root phenolic acid compounds, aiming to explore the potential value of S. bowleyana. The main results are as follows:1. high performance liquid chromatography was adopted to measure salvianolic acid B contents in the root, stem and leaf of S. bowleyana produced in Fujian, and S. milliorrhiza produced in Anhui was used to be the comparison. The results showed that the salvianolic acid contents in the root, stem and leaf of S. bowleyana are separately 7.42%,2.12% and 2.20%, among which the root salvianolic acid B content is significantly higher than S. miltiorrhiza (6.79%) (p< 0.05). Therefore, the salvianolic acid B contents in different organs are in the following order (descending order):Root> leaf ≈stem. Therefore, we selected the root as the material to carry out the follow-up study.2. phenolic acid extract in root of S. bowleyana was exerted as the raw material, Sephadex LH-20 glucan gel was selected as the medium for column chromatography, and the ethanol liquids with different volume fraction was used successively as the eluent. Five compounds through fractionation purification were obtained, which were named as compounds A-E. As the results of further calculation, the yields of 5 compounds in the S. bowleyana were 4.26%,0.33%,0.72%,1.23% and 0.54%, respectively.3. Purity test of five compounds were carried out through HPLC. The results showed that the purities of compounds A-E were 93.26%,94.26%,93.56%,85.51% and 93.56%, respectively. Of which, the compounds A and C had almost the same retention time,4.57 min and 4.58 min respectively. In addition, they showed the same ultraviolet spectrum characteristics. Therefore, these two materials were most likely of the same kind.4. Furthermore, HPLC-MS first-order mass spectrum measurement were carried out. The results showed the following:the molecular weights of compound A and B were 718 and 360 respectively. Comparing with the database, we speculated that the formulas are C36H30O16 and C18H16O8; Molecular weights of compound C and D were 718 and 538 respectively. Comparing with the database, we speculated that the formulas are C36H30O16 and C27H22O12 respectively; Molecular weight of compound E was 718. Comparing with the database, we speculated that its molecular formula is C36H30O16. Combining with the HPLC peak time and UV spectra characteristics, compound A and C were conduded to be the same material. Because compound A and E have different peak times, we speculate that they are the different materials.5. The structure analysis on 5 compounds were carried out through HPLC-MS2, and the results showed the following:Ion fragments produced through cracking of Compounds B and D respectively conformed to the behaviors of rosmarinic acid and lithospermic acid, compound B was identified to be rosmarinic acid and compound D to be lithospermic acid. Ion fragments produced through cracking of Compounds A, C and E basically conform to behaviors of salvianolic acid B and E, respectively. Combining with other spectral data and related literatures, we have concluded that compound A and C are all salvianolic acid B, while compounds E may be salvianolic acid E, waiting for further validation. The lithospermic acid was isolated from S. bowleyana for the first time.6. five different systems were adopted to examine iron reducing & antioxidant capacity, removal ability of DPPH·,· OH and O2-, and the protection for DNA oxidative damage etc. of the four phenolic acid compounds. The results showed that in FRAP method system, total iron reduction & antioxidant capacity of the four kinds of compounds were successively as follows (in descending order):Salvianolic acid B> salvianolic acid E>lithospermic acid> rosmarinic acid; In DPPH· method system, the four compounds had strong removal ability, and the effect was significantly better than Trolox (p<0.05), the effects were successively the following (strength in descending order):salvianolic acid E> salvianolic acid B> lithospermic acid> rosmarinic acid, the PEC50 values were respectively 0.13 mmol·L-1,0.14 mmol·L-1,0.25 mmol·L-1,0.32 mmol·L-1; in the rhodamine B-Fenton fluorescence system (OH), the removal abilities of the four compounds for OH were successively as follows (strength in descending order):salvianolic acid B>salvianolic acid E> rosmarinic acid> lithospermic acid; in the PMS-NADH-NBT system (O2-·), the removal abilities of the four compounds for O2-· were successively as follows (strength in descending order):salvianolic acid E> rosmarinic acid> lithospermic acid> salvianolic acid B, and the removal abilities were all stronger than Vc and Trolox. As for the protection effect for DNA lipid overoxidation damage caused by AAPH, we have found that the four compounds have remarkable protective effect on DNA damage, which is ranked as follows (strength in descending order):salvianolic acid E> salvianolic acid B> lithospermic acid> rosmarinic acid. As we can see from above, the 4 compounds in the extract of root of S. bowleyana have strong anti-oxidant ability, strong removal ability for artificial free radicals and oxygen free radicals, and they have good protection effect on the anti-oxidant damage effect of DNA.In a word, the organ of S. bowleyana root is rich in salvianolic acid series compounds, especially salvianolic acid B; the extraction rate is high, and the antioxidant activity is strong, therefore, the subject is worthy of in-depth research and development. |
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