Alkaloid Constituents of Ficus hispida and Their Antiinflammatory Activity

Four new alkaloids, ficuhismines A–D (1–4), together with three known ones, were isolated from Ficus hispida. Their structures were elucidated by spectroscopic analysis and chemical method. The new compounds represent the first amine alkaloids with a rhamnosyl moiety (1–2) or with a N-oxide motif (2–4) from the genus Ficus. Compound 2 showed potent inhibitory effect in nuclear factor-κB (NF-κB) pathway luciferase assay with IC50 value of 0.52 ± 0.11 μM. Electronic supplementary material The online version of this article (10.1007/s13659-020-00233-5) contains supplementary material, which is available to authorized users.


Introduction
The genus Ficus (Moraceae) comprises approximately 1000 species all over the world. They mainly distribute in tropical and subtropical areas and show diversity particularly in Southeast Asia [1]. In addition to the well-known fig ("Wu Hua Guo") and banyan trees, many Ficus plants possess important medicinal values. The plant Ficus hispida L.f. is a herbal medicine that has been used in China and India as a remedy for bronchitis, dysentery, rheumatism, and skin disorders [2,3]. The extracts from its different parts have been reported to show antiinflammatory, antidiabetic, antitumor, and hepatoprotective activities [4][5][6][7]. Primarily known for the rich flavonoids and triterpenoids [8], alkaloids with antitumor and vasorelaxant activities have also been discovered from F. hispida [9,10]. Some alkaloids and α-glucosidase inhibitory flavonoids from this plant have been reported previously by our group [11].
Nuclear factor-κB (NF-κB) is a key regulator of inflammation. Activation of NF-κB initiates inflammation-associated metabolic disease, such as obesity, type 2 diabetes, and atherosclerosis [12]. In order to discover antiinflammatory natural products against metabolic diseases, alkaloid constituents of F. hispida were reinvestigated, and their inhibitory effects in NF-κB pathway luciferase assay were evaluated. Seven alkaloids including four new ones (ficuhismines A-D, 1-4) were isolated from the twigs and leaves of F. hispida (Fig. 1). The new compounds represent the first amine alkaloids with a rhamnosyl moiety (1-2) or with a N-oxide motif (2-4) from the genus Ficus. Compound 2 showed potent NF-κB inhibitory activity. In this paper, the structural identification and bioactivity evaluation of the alkaloids are discussed.
Compound 3 was assigned the molecular formula C 14 H 21 NO 3 by the (+)-HRESIMS ion at m/z 252.1595 [M+H] + (calcd for C 14 H 22 NO 3 , 252.1594). Its 1D (Table 1) and 2D NMR data (Fig. 2) indicated that 3 was an aglycone of 2. The structure of 3 was thus elucidated, and the compound was named ficuhismine C.
All the isolated alkaloids were tested in vitro for their antiinflammatory effects. Ficuhismine B (2) exhibited significant inhibitory activity in NF-κB pathway luciferase assay with IC 50 value of 0.52 ± 0.11 μM. Bortezomib (PS-341) was used as the positive control (IC 50 = 0.12 ± 0.04 μM) in this test. All the other compounds were inactive. It seemed that both l-rhamnosyl and N-oxide moieties are necessary for the NF-κB inhibition.
Alkaloids are one of the main bioactive ingredients of F. hispida. Amphetamine [9,11], piperidine [9], pyrrolidine [10,11], and isoquinoline [15] alkaloids have been reported from this plant previously. In this research, amine alkaloids with a rhamnosyl group (1-2) or with a N-oxide moiety (2)(3)(4) were discovered for the first time from the genus Ficus. Compound 2 with both rhamnosyl and N-oxide moieties showed potent NF-κB inhibitory activity, which may provide useful information for mining or designing drug leads against inflammation-related metabolic diseases.

Plant Material
The

Extraction and Isolation
The plant material (10.0 kg) was ground and percolated with 95% EtOH (30 L) at room temperature. The filtrate was evaporated under reduced pressure to produce a crude extract (1.5 kg). It was dissolved in tartaric acid solution (pH 2.0), basified using saturated sodium carbonate solution to pH 13, and then partitioned with CHCl 3 to give a total alkaloid extract (1.

Luciferase Assay
HEK293/NF-κB cells were prepared as reported [16]. The luciferase assay procedure was performed according to the reported methods [17]. The IC 50 values were calculated using Graphpad prism 8.