Phenolics from Lagotis brevituba Maxim

Abstract A phytochemical investigation on Lagotis brevituba led to the isolation and characterisation of 11 phenolic compounds: p-hydroxy-benzoic acid 1, methyl 3,4-dihydroxybenzoate 2, vanillic acid 3, protocatechuic acid 4, caffeic acid 5, glucose ester of (E)-ferulic acid 6, p-coumaric acid 7, vanillin 8, diosmetin-7-O-β-d-glucoside 9, chrysoeriol 10 and luteolin 11. Their structures were elucidated using spectroscopic methods and by comparison with data in the literature. Compounds 1–6 were first obtained from the genus Lagotis, and compounds 1–9 were isolated from L. brevituba for the first time. Compound 4 and 11 displayed remarkable antioxidant activities against DPPH radical (IC50 = 5.60 ± 0.09, 27.5 ± 0.06 mg/L, respectively), which were superior to positive control rutin. And compound 11 was also superior to rutin in ABTS assay (IC50 = 2.04 ± 0.13 mg/L).


Introduction
The genus Lagotis (Scrophulariaceae), whose members are mainly distributed in the Himalayas, Pamirs, Kalakoram, Qilian Range and Hengduan Range, comprises about 30 species (Editorial Committee of Flora of China 1979). Lagotis brevituba is a perennial species that usually grows on mountain slopes at an altitude of 3800-4800 m. It is a Tibetan medicinal plant that is distributed in the Qinghai, Gansu and Xizang provinces of China. The whole plant is traditionally used as a Tibetan medicine to treat fever, nephritis, lung disease, hypertension and atherosclerosis (Xi et al. 2010). Pharmacological experiments have shown that ethanol extracts of L. brevituba have antibacterial activities, and the total flavonoids in the herb are responsible for the anti-inflammatory activities (Liu et al. 2010;Zhang et al. 2012). On account of the flavonoids also belonging to phenolics, we decided to study phenolic components in L. brevituba. Furthermore, L. brevituba is a plant that is characteristic of the high mountain environment. Because of the inaccessibility of its habitat, there have been few phytochemical investigations on this species to date. The present study was conducted to investigate the total phenolic content (TPC) and total flavonoid content (TFC) and isolate 11 phenolic compounds from ethanolic extracts of L. brevituba. They were identified as p-hydroxy-benzoic acid 1 (Meng et al. 2014), methyl 34-dihydroxybenzoate 2 (Li et al. 2009), vanillic acid 3 (Choi et al. 2007), protocatechuic acid 4 , caffeic acid 5 (Damtoft & Jensen 1994), glucose ester of (E)-ferulic acid 6 (Beate & Peter 2001), p-coumaric acid 7 (Yang et al. 2005), vanillin 8 (Challice et al. 1980), diosmetin-7-O-β-d-glucoside 9 (Abduwaki et al. 2014), chrysoeriol 10 (Chen et al. 1989) and luteolin 11 (Abduwaki et al. 2014). All the isolates were tested for their antioxidant activities using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and 2,2-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical scavenging activity (Li et al. 2016;Pui & Mirko 2016).

Results and discussion
Eleven naturally occurring phenolic compounds were isolated from ethanolic extracts of L. brevituba and their structures were elucidated. Compounds 1-6 were first obtained from the genus Lagotis, and compounds 1-9 were isolated from L. brevituba for the first time ( Figure 1).
Compounds 7 and 9 have been reported from Lagotis yunnanensis; compounds 8 and 11 have been reported from L. yunnanensis, Lagotis alutacea and Lagotis brachystachya; and compound 10 has been reported from L. yunnanensis and L. brachystachya (Chen et al. 1989;Yang et al. 2003Yang et al. , 2004Yang et al. , 2005Liu et al. 2015). Interestingly, compounds 10 and 11 were also previously identified in L. brevituba (Shi et al. 2006;Xi et al. 2010). The presence of compounds 7-11 reveals a close chemotaxonomic relationship among the four species of Lagotis. Compound 4 has been reported from the genera Rehmannia and Veronica, and compound 5 has been reported from the genus Scrophularia (Jong et al. 2009;Li et al. 2012;. The presence of these compounds suggests that there might be a close chemotaxonomic relationship among these genera. Compound 6, a rare compound, has not been isolated from other species of Lagotis. It has previously been reported only from Riesling wine (Beate & Peter 2001). Hence, 6 may be a chemotaxonomic marker for L. brevituba, although further phytochemical investigations of other Lagotis species should be conducted to confirm the chemotaxonomic significance of 6. Compound 1 has been isolated from family of the Solanaceae (Meng et al. 2014). Compound 2 has been isolated from family of the Polygonaceae (Li et al. 2009), and compound 3 has been isolated from family of the Solanaceae (Choi et al. 2007). Further studies on the distribution of 1-3 in the genus Lagotis should be conducted to evaluate their chemotaxonomic significance.
TPC and TFC were determined using colorimetric method (Zhou et al. 2011) (Table 1). The antioxidant activities of the obtained compounds were evaluated by DPPH radical scavenging assay and ABTS radical scavenging assay (Table 2).  Table 1. tPcs and tFcs of ethanolic extract. a each value is expressed as mean ± sd (n = 3).

Conclusion
The present study provides data on the isolation of 11 phenolic compounds (1-11), as well as the DPPH radical scavenging activities of the isolated compounds from L. brevituba. Compounds 1-6 were firstly obtained from the genus Lagotis, and compounds 1-9 were isolated from L. brevituba for the first time. Based on the evidences above, it is believable that L. brevituba has close relationship with some species of Lagotis genus such as L. yunnanensis, L. alutacea and L. brachystachya. The antioxidant activities results showed that compounds 4 and 11 exhibited strong activities against DPPH radical, which were superior to positive control rutin. And compound 11 (IC 50 = 2.04 ± 0.13 mg/L) was also superior to rutin in ABTS assay.

Disclosure statement
No potential conflict of interest was reported by the authors.