Secondary metabolites from Gentiana cruciata L. and their anti-inflammatory and analgesic activities

Abstract A previously unreported secoiridoid glycoside, cruciatoside (1) was isolated from the aerial parts of Gentiana cruciata L. along with ten known compounds eustomoside (2), eustomorusside (3), gentiopicroside (4), 6'-O-β-D-glucopyranosyl gentiopicroside (5), loganic acid (6), isoorientin (7), isovitexin (8), isovitexin 2''-(E)-ferulate (9), mangiferin (10), and 2-methyl-inositol (11). The chemical structures of the isolates were elucidated based on extensive 1 D and 2 D NMR experiments as well as HRMS analysis. All isolates were evaluated for their in vitro anti-inflammatory and analgesic activities. Compounds 9, 4, and 7 (200 µM) showed moderate anti-inflammatory activity by inhibiting nitrite production from LPS-induced RAW 264.7 macrophage cells, with the inhibition rates of 39.5%, 25.8% and 22.9% respectively without exhibiting substantial cytotoxicity. Besides, 1, 2, 4, and 7 exerted the highest decrease in IL-6 levels. Moreover, compound 4 showed in vitro analgesic activity by decreasing the PGE2 level comparable to the reference drugs. Graphical Abstract


Introduction
The genus Gentiana (Gentianaceae) is a large genus containing around 400 species distributed worldwide (Pan et al. 2016). Some of these species have long been used as a folk medicine for the treatment of various disorders. G. kurroo Royle, also known as 'Indian Gentian', is traditionally used against fever, syphilis, bronchitis, and urinary complaints (Alphonse et al. 2022), while G. scabra Bunge roots are utilized as an antidiabetic remedy in traditional Korean medicine (Suh et al. 2015). The genus Gentiana is represented by 12 species in the flora of T€ urkiye (Pritchard 1978). Among these species, G. cruciata is consumed as a folk remedy in the Black Sea region of Anatolia. Its flowering aerial part infusion is prepared to relieve kidney pain whereas whole plant decoction is used as tonic (Sezik et al. 1992). In Serbia, the same species has been used against a broad range of medical conditions, such as gastrointestinal complaints, anaemia, and diabetes (Jari c et al. 2015). The extracts of this species were reported to possess in vivo hepatoprotective (Mihailovi c et al. 2014) and antioxidant activities (Mihailovi c et al. 2015). Few previous phytochemical studies have concluded the presence of iridoids, secoiridoids, flavonoids, and xanthones in G. cruciata (Szucs et al. 2002;Hayta et al. 2011;Xu et al. 2017;Olennikov et al. 2019). However, there is no detailed study on the isolation of its secondary metabolites and their in vitro antiinflammatory and analgesic activities. In our continuing effort to search for new bioactive constituents from Turkish Gentiana species (Kırmızıbekmez et al. 2022), G. cruciata was investigated and 11 secondary metabolites ( Figure 1) were isolated including a new secoiridoid glycoside. Furthermore, all the isolated compounds were tested in vitro for their potential anti-inflammatory and analgesic activities. Herein, the isolation, structural elucidation, and biological activities of isolated compounds were described.
Compound 1 was isolated as a colorless amorphous powder. The molecular formula, C 23 H 28 O 13 , was determined based on the sodiated ion peak [M þ Na] þ at m/z 535.1425 (C 23 H 28 O 13 Na þ , calcd for 535.1422) in its HRESIMS, indicating 10 degrees of unsaturation. It showed UV absorption maxima at 231 and 282 nm, while its IR spectrum showed bands at 3349 (OH), 1711 (C ¼ O), and 1617 (C ¼ C), 1451, and 1413 cm À1 (aromatic ring), respectively. The 1 H NMR spectrum of (Spectrum S5) 1 displayed the signals due to presence of an olefinic ( secoiridoid glycoside containing a lactone moiety. Moreover, an anomeric proton signal was observed at d H ¼ 4.65 with a coupling constant of 7.9 Hz. The spectrum also contained five aromatic signals at d H ¼ 8.06 (dd, J ¼ 7.8, 1.3 Hz, 2H), 7.61 (m) and 7.48 (t, J ¼ 7.8 Hz). The 13 C-JMOD NMR spectrum ( Figure S6) of 1 contained 23 resonances, six of which were arising from a b-glucopyranose unit while seven for a benzoyl moiety. The remaining ones were characteristic of a C 10 -secoiridoid skeleton (Bock et al. 1976). All of these data showed a signal pattern similar to eustomorusside (3) (Uesato et al. 1979), except for the presence of a benzoyl unit in 1. Furthermore, H 2 -10 signals (d H 4.47 and 4.23) of 1 were found to be shifted downfield around 1 ppm compared to those of eustomorusside, implying the site of acylation. 2 D NMR experiments (COSY, HSQC, and HMBC) supported the proposed structure and the attachment points of b-glucopyranose and benzoyl units to the aglycone of 1. Thus, the longrange correlation from C-1 (d C 96.0) to H-1 0 (d H 4.65) in its HMBC spectrum (Figures S9 and S11) confirmed the glycosidation site to be C-1. The cross-peak between benzoyl carbonyl signal at d C 167.9 and H 2 -10 (d H 4.47 and 4.23) positioned benzoyl moiety at C-10. The relative configuration of 1 was established by the NOESY spectrum ( Figure  S10) and coupling constant values (Uesato et al. 1979). The NOE correlation between H-8 and H-1 and between H-8 and Ha-10 (d H 4.23) positioned them on the same side of the molecule (a). Moreover, J 9,1 (1.2 Hz) and J 9,8 (5.7 Hz) values of 1 were found to be superimposable with those of eustomorusside. According to these results, the structure of 1 was determined as a 10-O-benzoyleustomorusside and named cruciatoside based on the species name of G. cruciata.
Cruciatoside showed structural similarity to eustomorusside, which can be classified as sweroside-morroniside type secoiridoid glycoside (Inouye and Uesato 1986). Eustomorusside and eustomoside were first isolated from Eustoma russellianum, which also belongs to the Gentianaceae family (Uesato et al. 1979). These two secoiridoid glycosides were also obtained from different Gentiana species previously (Mpondo et al. 1990;Çalış et al. 1990) but were isolated from G. cruciata for the first time. Thus, the occurrence of such sweroside-morroniside type secoiridoid glycosides implies the chemotaxonomic relationship between the genera Gentiana and Eustoma within the Gentianaceae family. This study also represents the first record of the occurrence of isovitexin 2 0 '-(E)-ferulate, 6 0 -O-b-D-glucopyranosyl gentiopicroside, and 2-methyl inositol in G. cruciata.
All isolates were screened for their cytotoxic activities on RAW264.7 cells at three different concentrations for 24 h of exposure prior to evaluating their in vitro antiinflammatory and analgesic activities. The results showed that none of the compounds exerted remarkable toxicity at the highest tested concentration (200 mM), with relative cell viabilities above 70% compared to the control group (Table S1, Figure S2). Hence, 200 mM was selected for further biological studies. Among the tested compounds, only 1, 6, and 11 showed dose-dependent cytotoxicity with increasing concentration, which were statistically insignificant ( Figure S2).
The anti-inflammatory activities of the isolates were evaluated by measuring the stable metabolite of nitric oxide (NO), nitrite levels and secretion of pro-inflammatory cytokine IL-6 level in RAW264.7 cells activated by LPS (Table S1, Figure S3). Among the tested compounds, 9 (200 mM) showed the most potent anti-inflammatory activity with a 39.46 ± 7.68% relative nitrite inhibition rate higher than both positive controls, IND and L-NAME (Table S1) which is followed by 4 and 7 with the inhibition rates of 25.84, and 22.90%, respectively (Table S1). Compounds 1, 2, 4, and 7 exerted the highest decrease in IL-6 levels. (Figure S3). Thus, it can be concluded that 1, 4, 7, and 9 showed a potent anti-inflammatory activity through both NO and IL-6 pathways. The isolated compounds were also evaluated for their in vitro analgesic activities through evaluating of PGE 2 levels in LPS-activated RAW264.7 cells (Table S1). However, only 4 led to a decrease in PGE 2 level comparable to reference drug indomethacin.

Plant material
Gentiana cruciata was collected from Devrekani, Kastamonu, T€ urkiye, in August 2019. The plant material was identified by one of us, Prof. Dr. Hasan Kırmızıbekmez and a voucher specimen (YEF 19041) have been deposited at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Yeditepe University, _ Istanbul, T€ urkiye.

Conclusion
A new secoiridoid glycoside, cruciatoside (1), along with ten known compounds were isolated from the aerial parts of G. cruciata. The chemical structures of the isolates were determined based on extensive 1 D and 2 D NMR experiments as well as HRMS analysis. All isolated compounds were evaluated for anti-inflammatory and analgesic activities. Among the tested compounds, 1, 4, 7, and 9 showed a potent anti-inflammatory activity through nitrite and IL-6 pathways, while compound 4 also exerted an analgesic activity by decreasing the PGE 2 level. Therefore, compounds 1, 4, 7, and 9 might be considered potential candidates for the in vivo anti-inflammatory and analgesic activity studies.