Spiroketones and a Biphenyl Analog from Stems and Leaves of Larrea nitida and Their Inhibitory Activity against IL-6 Production

Bioactivity-guided fractionation for the stems of leaves of Larrea nitida Cav., using interleukin-6 (IL-6) inhibitory assay in human mast cells (HMC-1), led to the isolation of three new compounds with an unprecedented skeleton in nature (1–3) and three known compounds (4–6). Their structures were elucidated through extensive spectroscopic analysis. The three new compounds were elucidated as two new spiroketones, nitidaones A (1), and B (2) and one new biphenyl analog, nitidaol (3). The known compounds were identified as nordihydroguaiaretic acid (4), 7,3′,4′-tri-O-methylquercetin (5) and ayanin (6). All the isolates were tested for their inhibitory activity against IL-6 production in HMC-1 cells. Of them, compounds 1, 3–6 showed potent anti-inflammatory activity, with IC50 values of 12.8, 17.5, 14.9, 22.9, and 17.8 µM, respectively.


Introduction
The genus Larrea (Zygophyllaceae) has been used for ethnobotanical purposes by the native peoples of northwest, central, and southeast Argentina. This genus comprises five species seen in North and South America: L. ameghinoi Speg., L. nitida Cav., L. divaricata Cav., L. mexicana Moric., and L. tridentata (DC.) Coville [1,2]. Phytochemical studies on the genus Larrea reported the occurrence of flavonoids, lignans, naphthoquinones, saponins, and tannins [3,4]. These plants have been used for the treatment of cancer, inflammation, and menstrual pains [5][6][7]. Previous phytochemical investigations on L. nitida have reported the presence of flavonoids and lignans [8,9]. The biological activities of its extracts or individual ingredients included antifungal activity associated with Argentinean Andean propolis, antioxidant activity, and estrogenic activity [8][9][10]. As a part of our search for IL-6 production inhibitors from higher plants [11][12][13], L. nitida was selected for further isolation work due to IL-6 production inhibitory activity in the initial screening. Herein, the structural elucidation of three new compounds along with three known compounds and their IL-6 production inhibitory activity are described.
together via a spiral carbon at C-4, as depicted in Figure 1. The acetyl group was affixed to C-10 from the observed correlations between δC 173.1 and both H-10 and methyl protons (δH 2.00). Therefore, the structure of compound 1 was determined to be 4-(1-(4-methoxyphenyl)-4-oxocyclohexa-2,5dienyl)-2,3-dimethylbutyl acetate, named nitidaone A.  (Table 1) were similar to those of 1 except for the absence of two olefinic protons. Instead of olefinic protons, methylene signals at δH 2.40-2.29 (2H) and δH 2.24-2.14 (2H) were observed in the spectrum of 2, assignable to H-5 and H-6. Furthermore, the HMBC correlations of H-7 with C-4 (δC 44.7) and C-1′ (δC 137.0) and H-2′,6′ with C-4 confirmed its spiral skeleton ( Figure 1). Taken together with all data, this compound was elucidated It has been reported that 2-cyclohexenones empirically give a negative Cotton effect (n→π * ) in the 340 nm region if the ring has a half-chair conformation with a pseudo-axial disposition of an aryl group ( Figure 2a) [17]. From a negative Cotton effect at 339 nm in the CD spectrum of 2, it was affirmed that the cyclohexenone ring of this compound prefers the half-chair conformation with a pseudo-axial aromatic group at room temperature ( Figure 2b). Therefore, the absolute configuration of C-4 was established to be R.
The exciton splitting theory supported its R configuration. From the UV and CD spectrum, the negative exciton-split Cotton effect at 220 nm was observed due to the π→π * intramolecular charge-transfer transition of a 4-methoxyphenyl chromophore and a 2-cyclohexenone chromophore [18,19]. The negative exciton-split Cotton effect indicated that the two electric transition moments of these chromophores were rotated in a counterclockwise direction, as shown in Figure 2c. . Also, a methoxy group at δH 3.82 (3H, s, OCH3) and an acetyl group at δH 1.95 (3H, s, OCOCH3) was observed ( Table 2).
It has been reported that 2-cyclohexenones empirically give a negative Cotton effect (n→π * ) in the 340 nm region if the ring has a half-chair conformation with a pseudo-axial disposition of an aryl group (Figure 2a) [17]. From a negative Cotton effect at 339 nm in the CD spectrum of 2, it was affirmed that the cyclohexenone ring of this compound prefers the half-chair conformation with a pseudo-axial aromatic group at room temperature ( Figure 2b). Therefore, the absolute configuration of C-4 was established to be R.
The exciton splitting theory supported its R configuration. From the UV and CD spectrum, the negative exciton-split Cotton effect at 220 nm was observed due to the π→π * intramolecular chargetransfer transition of a 4-methoxyphenyl chromophore and a 2-cyclohexenone chromophore [18,19]. The negative exciton-split Cotton effect indicated that the two electric transition moments of these chromophores were rotated in a counterclockwise direction, as shown in Figure 2c.  (Table 2).

Plant Material
The stems and leaves of L. nitida Cav. were collected at Jarilla, Chile in 2007 and identified by Dr. Joongku Lee, Korea Research Institute of Bioscience and Biotechnology. A voucher specimen (access number FBM026-052) was deposited in the herbarium of the Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.

Extraction and Isolation
Air-dried stems and leaves of L. nitida (52 g) were pulverized and extracted with MeOH to yield the crude extract (14 g). A portion of this extract (12 g) was subjected to a reverse-phase silica gel column chromatography eluting with a gradient of H 2 O-MeOH (90:10-0:100) to give 17 sub-fractions (LN01-17). Of these sub-fractions, LN 8 and 11 were found to inhibit IL-6 production (over 50% inhibition at 20 µg/mL) in HMC-1 cells stimulated by PMA + A23187 ( Figure S6). LC8 (1 g) was fractionated into 14 sub-fractions using a reversed phase silica gel column chromatography with a gradient mixture of MeOH-H 2 O (25:75 to 100:0).

Interleukin-6 Determination
Cells were seeded at 1 × 10 6 /mL per well in 24-well tissue culture plates and pretreated with the indicated concentration of compounds for 30 min before stimulation by PMA (50 nM) + A23187 (1 µM). After 24 h, the supernatant was decanted into a new micro-centrifuge tube, and the amount of interleukin-6 (IL-6) was determined using an enzyme-linked immunosorbent assay (ELISA) kit according to the procedures described by the manufacturer (BD Biosciences, San Jose, CA, USA). All subsequent steps took place at room temperature, and all standards and samples were assayed in duplicate [13].