Anti-Human Coronavirus (anti-HCoV) Triterpenoids from the Leaves of Euphorbia neriifolia Fang-Rong Changa, Chiao-Ting Yena, Mohamed EI-Shazlya,b, Wen-Hsun Lina, Ming-Hong Yenc, Kuei-Hsiang Lind and Yang-Chang Wue,f,g,* aGraduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 80708, Taiwan b Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, Ain-Shams University, Cairo 11566, Egypt c College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan d School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan e School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan f Natural Medicinal Products Research Center, China Medical University Hospital, Taichung 40402, Taiwan g Center for Molecular Medicine, China Medical University Hospital, Taichung 40402, Taiwan yachwucmu.edu.tw Received: August 27th, 2012; Accepted: October 2nd, 2012 Euphorbia neriifolia L. is a spiny herb native to Southeast Asia and currently cultivated in southern Taiwan. From the ethanolic extract of E. neriifolia leaves, 23 compounds were isolated, including 22 triterpenoids and one flavonoid glycoside. The anti-human coronavirus (HCoV) activity of the separated triterpenoids was studied revealing the structure-activity relationship (SAR) of these isolates. 3-Friedelanol exhibited more potent anti-viral activity than the positive control, actinomycin D, which implies the importance of the friedelane skeleton as a potential scaffold for developing new anti-HCoV-229E drugs. Keywords: Euphorbia neriifolia, Euphorbiaceae, Human coronaviruses (HCoVs), Triterpenoids, Structure-activity relationship (SAR). Coronavirus (CoV), a genus of the Coronaviridae family, is a positive-strand RNA virus with the largest viral genome of all RNA viruses (2732 kb) 1. The first studies on human CoVs (HCoV) appeared in the mid-1960s 2. Almost 40 years later, a CoV was identified as the causative agent of the severe acute respiratory syndrome (SARS), which caused a global health threat 3. A highly effective global public health response prevented further spread of this virus resulting in the eradication of SARS-CoV from its endemic areas. However, recent research pointed out the emergence of new HCoV species calling for immediate action to prevent another health disaster. So far only few treatments have been developed against SARS, such as interferon and glycyrrhizin 4. The potent anti-viral activity of glycyrrhizin as a triterpenoidal saponin and the wide distribution of this class of secondary metabolites in the plant kingdom imply an intriguing opportunity in finding new drug leads with a triterpenoidal nucleus targeting HCoVs. Triterpenoids are abundant in terrestrial plants with over 90 families containing different types of this class of secondary metabolites. Among plant families rich in triterpenoids is Euphorbiaceae with 300 genera and around 7,500 species. The largest and most diverse genera of Euphorbiaceae is Euphorbia with 1600 species distributed worldwide ranging from annual weeds to trees 5. E. neriifolia L. is a spiny herb, popularly known as sehund or thohar. It is of South-Asian origin, including India and Sri Lanka and cultivated nowadays in the southern warm region of Taiwan 6. In traditional medicine, the leaves are used as an aphrodisiac, diuretic, and as a treatment for common cold, bleeding piles and anal fistula. Phytochemical investigation of the hydroalcoholic extract of E. neriifolia leaves revealed the presence of sugars, tannins, flavonoids, alkaloids, and triterpenoidal saponins 5. The use of E. neriifolia as a remedy for common cold and its richness in triterpenoids were the driving force to isolate and purify the major triterpenoids of E. neriifolia aiming to deduce their anti-HCoV activity. The fresh leaves of E. neriifolia, collected from Pingtung, Taiwan, were extracted with ethanol. The EtOH extract was concentrated and subjected to silica gel column chromatography (CC) successively eluted with a gradient mixture of n-hexane, CHCl3, EtOAc, and MeOH. The obtained fractions were purified using normal- and reversed-phase HPLC to yield 22 triterpenoids and one flavonoid glycoside. The structures of the isolated compounds were identified by comparing their spectral data with the published values and are summarized as: 3-friedelanol (1) 7, 3-friedelanol (2) 7, 3-acetoxy friedelane (3) 8, friedelin (4) 9,10, glutinone (5) 9, glutin-5-en-3-ol (6) 10, glutinol acetate (7) 10, lupenone (8) 11, epitaraxerol (9) 11, epitaraxeryl acetate (10) 12, taraxeryl acetate (11) 13, -amyrin (12) 11,13, -amyrin acetate (13) 13, dammarenediol II acetate (14) 14, cabraleadiol monoacetate (15) 15, 3-simiarenol (16) 16, simiarenone (17) 17, cycloartanol (18) 18, 24-oxocycloart-25-en-3-ol (19) 19, (23Z)-cycloart-23-ene-3,25-diol (20) 20, cycloeucalenol (21) 21, 29-norcycloartanol (2