Chemical constituents from Epimedium koreanum Nakai and their chemotaxonomic significance

Abstract In this study, 21 compounds were isolated from the aerial parts of Epimedium koreanum Nakai, including 6 phenols (1–5 and 10), 4 lignans (6–9) and 11 flavonoids (11–21). The chemical structures of those isolates were established after extensive one-and two-dimensional nuclear magnetic resonance and mass spectroscopy analyses. This is the first report of compounds 2, 3, 4 and 9 in E. koreanum and of compounds 5 and 7 in the family Berberidaceae. The chemotaxonomic significance of the isolated compounds is discussed.


Introduction
Epimedium koreanum Nakai (Berberidaceae) is a perennial, low-growing medicinal plant, which is widely distributed in eastern Asia: China, Korea and Japan (Ma et al. 2011). The aerial parts of E. koreanum have been traditionally used as a therapeutic supplement for treatment of forgetfulness, impotence and spermatorrhoea in China, Korea and Japan. Previous phytochemical research of the aerial parts of this medicinal plant resulted in the isolation of various constituents, including flavonoids, lignans and alkaloids. Studies of pharmacological applications and clinical practice have illustrated that various extracts and some of individual chemical components from E. koreanum possessed diverse bioactive effects, such as antibacterial, anti-oxidative, anti-osteoporosis activities (Meng et al. 2005).

Chemotaxonomic significance
Flavonoids and their derivatives are major constituents of the genus Epimedium. The present phytochemical research of the aerial parts of E. koreanum led to isolation of 11 flavonoids, which could be subdivided into three groups: anhydroicaritin (11-13, 15, 19), desmethylanhydroicaritin (14, 16-18) and kaempferol (20, 21). Anhydroicaritin and desmethylanhydroicaritin, which possess a pentenyl group at C-8 position, are the most distinctive compounds of the genus Epimedium (Ma et al. 2011). All of these, epimedin B (13), icariine (15) and icariside II (19) have been used as reference compounds to evaluate the quality and pharmaceutical applications of several species in genus Epimedium, such as E. koreanum, E. brevicornum, E. pubescens (Xu et al. 2017). Epimedoside E (14), epimedoside A (16), hexandraside E (17) and ikarisoside B (18) are desmethylanhydroicaritin flavonoids, which are widely found in the genus Epimedium. Epimedoside E has been reported from E. wushanense and E. sagittatum (Li et al. 2012). Epimedoside A was obtained from E. diphyllum, E. wushanense and E. acuminatum before (Mizuno et al. 1988). Hexandraside E previously was isolated from E. brevicornum, while ikarisoside B was isolated from E. wushanense (Li et al. 1995). Korepimedoside C (11) and epimedokoreanoside I (12), which were identified as Anhydroicaritin with acetylated sugar groups, are characteristic compounds of E. koreanum (Sun et al. 1998;Liu et al. 2005). Nevertheless, both types are common in the genus Epimedium and other genus in the family Berberidaceae. Vancouveria hexandra (family Berberidaceae) is native to the Pacific coast of North America (Kim et al. 2004). Epimedin B (13), epimedoside E (14), icariine (15), epimedoside A (16) and hexandraside E (17) have also been isolated from V. hexandra (Yamamoto et al. 1993). The occurrence of these 8-isoprenylated flavonol glycosides in both E. koreanum and V. hexandra supports the allied nature of the genus Epimedium and Vancouveria and the use of V. hexandra as a substitute material in Epimedii Herba (Kim et al. 2004). The presence of various prenylflavonoids has chemotaxonomic significance for E. koreanum.
The present phytochemical investigation of constituents in the aerial parts of E. koreanum led to the isolation of four lignans, of which (-)-olivil (6), lariresinol (7) and (+)-cycloolivil (8) consist of two phenylpropanoid units connected by a 3β-(8-8′) bond (Ma et al. 2011). Compounds 6 and 7 are further classified as furan types, while (+)-cycloolivil (8) has a carbocyclic ring. Compound 9 is a neolignan glycoside that contains two phenylpropanoids linked by an 8-O-4′ bond. (-)-Olivil (6) and compound 9 were first time isolated from E. koreanum, which were obtained only from E. sagittatum and E. grandiflorum previously. (+)-Cycloolivil (8) is a common lignin that has been isolated from many species in the genus, including E. brevicornum and E. diphyllum. Lariresinol (7) was isolated from the family Berberidaceae for the first time, although its glycoside has already been isolated from E. leptorrhizum.
Five phenolic acids and one phenolic glycoside were identified in this study, of which protocatechuic acid (2), naringeninic acid (3) and caffeic acid (4) have not been previously isolated from E. koreanum. Protocatechuic acid (2) and naringeninic acid (3) have been isolated from E. sagittatum, while caffeic acid (4) has been isolated from E. brevicornum (Hsieh et al. 2003). Methyl chlorogenate (5) was isolated from the family Berberidaceae for the first time, although compounds with similar structures have been reported from E. sagittatum. Epimedoicarisoside A (10) is a 9, 10-dihydrophenanthrene derivative, which was isolated only from E. koreanum (Li et al. 1995). The finding suggests that this non-flavonoid compound has chemotaxonomic significance for E. koreanum.

Conclusion
In this study, 21 compounds were isolated from MeOH extract of the aerial parts of E. koreanum. To our knowledge, this is the first report of the compounds protocatechuic acid (2), naringeninic acid (3), caffeic acid (4), and compound 9 from E. koreanum and methyl chlorogenate (5) and lariresinol (7) from the Berberidaceae. Prenyl flavonoids (11-19) are considered as specific markers of Epimedium species. Of them, epimedoicarisoside A (10), korepimedoside C (11) and epimedokoreanoside I (12) can be considered as chemotaxonomic markers of E. koreanum. Overall, results of the present study highlight the taxonomic importance of a wide range of compounds in E. koreanum, that complement the current chemotaxonomic profile of the family Berberidaceae.

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

Funding
This work was supported by the researcher fund of Chungnam National University, and also supported by a grant (K17281) awarded to the Korean Institute of Oriental Medicine by the Ministry of Education, Science and Technology (MEST).