Anti-inflammatory Ursane- and Oleanane-Type Triterpenoids from Vitex negundo var. cannabifolia Man-Man Li, †,‡ Xiao-Qin Su, †,‡ Jing Sun, †,‡ Yu-Fan Gu, †,‡ Zheng Huang, †,‡ Ke-Wu Zeng, ⊥ Qian Zhang, † Yun-Fang Zhao, † Daneel Ferreira, § Jordan K. Zjawiony, § Jun Li,* ,† and Peng-Fei Tu* ,† † Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China ‡ School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, People’s Republic of China ⊥ State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, People’s Republic of China § Department of BioMolecular Sciences, Division of Pharmacognosy, and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677-1848, United States *S Supporting Information ABSTRACT: Six new polyoxygenated triterpenoids, cannabi- folins A−F(1−6), and eight known triterpenoids, 7−14, were isolated from the leaves of Vitex negundo var. cannabifolia. The absolute configuration of cannabifolin A (1) was determined by single-crystal X-ray crystallographic analysis. Compounds 1 and 2 represent a class of rare natural pentacyclic triterpenoids bearing cis-fused C/D rings and are the first examples of 12,19- epoxy ursane- and oleanane-type triterpenoids. Compounds 3, 7, 8, and 14 exhibited inhibition of nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophages with IC 50 values in the range 24.9−40.5 μM. T he genus Vitex (Verbenaceae) consists of small trees and shrubs, with about 250 species distributed mainly in tropical and subtropical regions. About 14 species, seven varieties, and three forms are found in mainland China. 1 Many Vitex plants have been used in traditional medicine worldwide to treat a wide range of ailments, such as menstrual disorders, premenstrual dysphoric disorders, corpus luteum insufficiency, colds, cough, asthma, rheumatism, inflammatory joint con- ditions, allergy, venereal diseases, malaria, wounds, skin diseases, snake bite, and body pain. 2,3 More than 30 species of Vitex have been investigated biologically and phytochemically in the past decades. Crude extracts and pure compounds from Vitex species have been reported to exhibit a wide array of bioactiv- ities including antibacterial, antimalarial, antifeedant, antioxidant, antiviral, antiproliferative, anti-inflammatory, hepatoprotective, antiaging, skin-whitening, antipyretic, analgesic, and potential effects on menopausal symptoms. Phytochemical investigations have indicated the presence of flavonoids, diterpenoids, phytoecdysteroids, iridoid glycosides, triterpenoids, phenyl- propanoids, phenolic glycosides, and essential oils in Vitex plants. 2−4 Vitex negundo L. var. cannabifolia (Sieb. et Zucc.) Hand.-Mazz. (syn.: Vitex cannabifolia Sieb. et Zucc.), a shrub or small tree, is mainly distributed in the Yangzi River basin of the People’s Republic of China. Different parts of V. negundo var. cannabifolia are used in traditional Chinese medicine for the treatment of various diseases. Its fruits, leaves, and stems have been used to treat colds, cough, asthma, stomachache, diarrhea, and beriberi. In turn, the roots are utilized for the treatment of colds, headache, toothache, malaria, and rheumatic arthralgia, 5 while the essential oil from the leaves is used to treat chronic bronchitis. 6 Previous phytochemical studies on the different parts of V. negundo var. cannabifolia have resulted in the isola- tion of diterpenoids, 7,8 flavonoids, 7−10 iridoid glycosides, 7,9 lignans, 9 and phenolic glycosides. 9,10 In the course of a search for anti-inflammatory agents from medicinal plants of the genus Vitex, the EtOAc-soluble fraction from a 95% EtOH extract of the leaves of V. negundo var. cannabifolia was found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages (89% inhibition at 40 μg/mL). Subsequent isolation of the bioactive fraction afforded six new polyoxygenated triterpenoids, cannabifolins A−F(1−6), together with eight known triterpenoids (7−14). Compounds 1 and 2 are rare natural pentacyclic triterpenoids with cis-fused C/D rings and represent the first examples of 12,19-epoxy ursane- and oleanane-type triterpenoids. Herein, the isolation and structural elucidation of the new compounds as well as an evaluation of their in vitro anti-inflammatory activities are described. Received: June 22, 2014 Article pubs.acs.org/jnp © XXXX American Chemical Society and American Society of Pharmacognosy A dx.doi.org/10.1021/np500509q | J. Nat. Prod. XXXX, XXX, XXX−XXX