| Viola yedoensis Makino (Violaceae) is a small perennial herb with violet flowers distributed in China, Korea, Russia and Japan. The dried whole plant (including the roots) is known as’Violae Herba’and is used as a heat-clearing and detoxifying traditional Chinese medicine under the name of’ZiHuaDiDing’. It has been used to treat inflammatory diseases such as sores, boils, jaundice, acute nephritis, diarrhea, and snake bites. Modern pharmacological studies showed that it has anti-inflammatory, immunoregulation, antioxidantive and anti-HIV activities. This herb has a variety of chemicals, such as flavonoids, coumarins, organic acids and microelements, among which flavonoids and coumarins are regarded as the most important markers.In this study, we investigated the antiviral, anti-bacterial, and antioxidant effects of V. yedoensis, which are closely related to its detoxifying function. The results showed that the alcoholic extract and aqueous extract of this herb have no antiviral activity. The n-butanol fraction of the alcoholic extract was found to possess the inhibitory effect against Escherichia coli. The the ethyl acetate and n-butanol fractions of the alcoholic extract had DPPH radical scavenging capacity. Taking together, we can conclude that the anti-bacterial and antioxidant effects are related to the detoxifying function of V. yedoensis, and the ethyl acetate and n-butanol fractions of the alcoholic extract were responsible for its detoxifying function.Since the chemical components of the petroleum ether and ethyl acetate fractions of this herb were previously investigated systematically in our group, this study only focused on the chemical constituents of the n-butanol fraction of the alcoholic extract of this herb. As a result,11compounds were isolated by various column chromatography, and identified as apigenin-6,8-di-C-β-D-glucopyranoside (1), apigenin-6-C-β-D-glucopyranosyl-8-C-a-L-arabinopyranoside (2), apigenin-6-C-β-D-glucopyranosyl-8-C-β-L-arabinopyranoside (3), apigenin-6-C-P-D-glucopyranosyl-8-C-β-D-xylopyranoside (4), apigenin-6-C-a-L-arabinopyranosyl-8-C-β-D-xylopyrano-side (5), apigenin-6,8-di-C-a-L-arabinopyranoside (6), kaempferol-3-O-β-D-sophoro-syl-7-O-α-L-rhamnopyranoside (7), kaempferol-3,7-di-α-a-L-rhamnopyranoside (8), kaempferol-3-O-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside (9), adenosine (10) and esculetin (11). Compound8was isolated from this herb for the first time, and compounds4and7were isolated from Viola for the first time.All the compounds are evaluated for the antioxidant capacity against DPPH radicals, and esculetin (11) had remarkable antioxidant activity, while flavone C-glycosides and flavone O-glycosides showed weak antioxidant activity. The antioxidant activity of flavone C-glycosides are lower than those of flavone O-glycosides. Compounds1and2are the main constituents of the n-butanol fraction, which had no anti-bacterial activity against Staphylococcus aureus, Escherichia coli, Pneumonic diplococcus, methicillin-resistant S. aureus (MRSA) and Pseudomonas aeruginosa. These results indicated that the flavone C-glycosides (the main constituents of the n-butanol fraction), had no anti-bacterial activity but showed antioxidant activities.The flavone C-glycosides of V. yedoensis possesses high polarity, similar structures, and large amounts and high temperature were required for their NMR determination. As a consequence, it is difficult to isolate the flavone C-glycosides from this herb, and only the main flavone C-glycosides can be obtained and evaluated by traditional separation and structural elucidation methods. In this study, the HPLC-PDA-ESI-IT-MS" method was firstly used to identify or characterize the flavone C-glycosides of V. yedoensis, The fragmentation patterns of5isolated flavones C-glycosides were studied and summarized:1) The MS1-5produced useful structural information for C-glycosides, and MS fragmentation beyond MS6is irregular;2) the cross-ring ions0,2X-of the sugar parts in MS2and MS3is the characteristic ions of flavone C-glycosides;3) the0,2X0,2X-ions (base peaks) in MS3can be used to identify the aglycone; and the ions [M-60-H]-and [M-120-H]-can be used as the characteristic ions to identify the existence of pentose and hexose, respectively;4) once the C-8glycosyl of flavone C-glycosides is not its preferential conformation, the cross-ring reaction of the C-8-glycosyl would take place first, instead of C-6reported in the references. Depending on these fragmentation patterns, combined with the PDA behaviors and retention times,35flavone glycosides were identified and characterized, including3flavone O-glycosides and32flavone C-glycosides (including1flavone mono-C-glycoside,26flavone di-C-glycosides, and5acetyl-flavone C-glycosides). The acetyl-flavone C-glycosides were reported for the first time from V. yedoensis. As a result, the LC-MS" method is simple and rapid for the identification/characterization of flavone C-glycosides present in V. yedoensis.According to the characteristics of fragmentation patterns of the flavone C-glycosides, an HPLC-ESI-IT-MS/MS method coupled to microwave assisted extraction (MAE) and solid phase extraction (SPE) was developed and validated to quantify three main flavone C-glycosides (apigenin-6,8-di-C-β-D-glucopyranoside, apigenin-6-C-β-D-glucopyranosyl-8-C-α-L-arabinopyranoside, and apigenin-6,8-di-C-a-L-arabinopyranoside) in V. yedoensis for the first time. The chromatographic and mass spectrometric conditions:Waters C18column (2.1×100mm,1.7μm), mobile phase:acetonitrile and water (0min, A=5%;0-30min, A=5%-12%;30-40min, A=12%-13%;40-45min, A=13%-100%), flow rate:180μL/min, selected reaction monitoring (SRM) mode. The linear ranges of the3standards were0.034-34.333μg/mL,0.068-68.000μg/mL, and0.051-50.667μg/mL, respectively. The contents of the three flavone C-glycosides in fourteen batches of Violae Herba were determined. The results showed that the newly developed method was sensitive (the limits of detection were0.017-0.034μg/mL and the limits of quantitation were0.034-0.051μg/mL), rapid and accurate. |
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