Two New Iridoid Glucosides from the Whole Plant of Patrinia scabiosifolia Link

As a traditional Chinese medicine, Patrinia scabiosifolia Link has been used to treat various inflammatory-related diseases, and recent studies have shown that it possesses potent anti-inflammatory activity. Therefore, phytochemical investigation on whole plants of P. scabiosifolia were carried out, which led to the isolation of two new iridoid glucosides, patriniscabiosides A (1) and B (2), together with six known compounds (3–8). The structural elucidation of all compounds was performed by HRESIMS and extensive spectroscopic analyses including IR, 1D, 2D NMR, and electronic circular dichroism (ECD). All the isolated compounds were tested for their anti-inflammatory activity using the NF-κB-Dependent Reporter Gene Expression Assay, and compound 3 displayed anti-inflammatory activity through the inhibition of the NF-κB pathway, with an inhibitory rate of 73.44% at a concentration of 10 μM.


Introduction
Growing evidence supports the idea that inflammation plays an important role in various human diseases including cerebrovascular, cardiovascular, pulmonary, blood, liver, and intestinal diseases [1]. Nuclear transcription factor κB (NF-κB) is an important transcription factor which regulates the expression of a variety of genes involved in immune and inflammatory responses, and the activation of the NF-κB pathway can lead to the induction of many inflammatory cytokines [2]. Therefore, compounds targeting the NF-κB signaling pathway are considered promising anti-inflammatory agents.
Iridoids, a large and still expanding class of monoterpenoids, are a type of secondary metabolites that can be found in many folk medicinal plants. Recent studies revealed that iridoids exhibit a wide range of bioactivities, including anti-inflammatory [3][4][5], antitumor [6], neuroprotective [7][8][9], and hepatoprotective effects [10,11], and can serve as promising lead compounds in drug discovery.
As part of an ongoing project to search for novel bioactive compounds, the whole plants of P. scabiosifolia was further investigated, leading to the isolation of two new iridoid glucosides (1, 2) ( Figure 1) and six known iridoids and iridoid glucosides (3~8). Compound 3 was shown to possess anti-inflammatory activity by the inhibition of the NF-κB pathway.

Results and Discussions
The connection between the three fragments could be further determined by the HMBC spectrum. The correlation from H-1 to C-7 and from H-7 to C-1 indicated that the glucopyranosyl unit was attached to 7-OH, which was also supported by the downfield chemical shift of C-7 (δc 83.7). The correlation from H-6 to C-1" determined that the isovaleryl ester moiety was attached to 6 -OH. Thus, the planar structure of 1 was established.
The relative configuration of 1 was assigned on the basis of the ROESY spectrum. The correlations H-5/Me-11 and H-5/H-9 indicated that H-5, H-9, and Me-11 were on the same face of the ring, while the correlation H-9/H10 determined H-8 to be on the other face. Meanwhile, the correlation H-7/H-8 in combination with the absence of correlation fpr H-7/H-5, H-7/H-9, H-7/H-10b suggested that H-7 was on the same face as H-8. The absolute configuration of 1 was further confirmed by electronic circular dichroism (ECD) ( Figure S10, See Supplementary Materials), which showed a negative Cotton effect at 217.5 nm [27], suggesting the absolute configuration to be 4R,5R,7S,8S,9S. Thus, the structure of compound 1 was confirmed, as shown in Figure 1, and the compound was named patriniscabioside A.

Chemical Structure of Compound 2
Compound 2 was obtained as a yellow oil and was assigned the molecular formu C21H34O10, as deduced from the [M + HCOO] − ion at m/z 491.21338 (calcd for C22H35O 491.21340). The IR spectrum suggested the presence of hydroxyl (3377 cm −1 ) and carbon (1747 cm −1 ) groups. The 1 H and 13 C NMR data of 2 (Table 1) resembled those reported f patrinoside [28], except for the sugar moiety. The analysis of the molecular formula, combination with the chemical shift δc 36.

Chemical Structure of Compound 2
Compound 2 was obtained as a yellow oil and was assigned the molecular formula C 21 [28], except for the sugar moiety. The analysis of the molecular formula, in combination with the chemical shift δc 36.4 of 2, suggested the presence of a deoxysaccharide unit. The 1 H-1 H COSY correlation (Figure 3) between H-4" (δ H 1.92, 1.36) and H-3" (δ H 3.60), H-5" (δ H 3.53) revealed that deoxygenation occurred at the C-4" position, which was further supported by the HMBC correlation from H-2" (δ H 3.10), H-6" (δ H 3.57) to C-4" (δc 36.4). Thus, a planar structure was established.

Chemical Structures of Compounds 3-8
The known compounds were identified as (4R,5R

Chemical Structures of Compounds 3-8
The known compounds were identified as (4R,5R

Biological Activities of Compounds 1-8
All the isolated compounds were assayed for their anti-inflammatory, hepatoprotective, and cytotoxic activities. Among these, compound 3 displayed antiinflammatory activity by inhibiting the NF-κB pathway, with an inhibitory rate of 73.44% at the concentration of 10 μM, while the other compounds showed weak activity, with an inhibitory rate lower than 50% at 10 μM ( Table 2). None of these compounds showed hepatoprotective or cytotoxic activities.

Biological Activities of Compounds 1-8
All the isolated compounds were assayed for their anti-inflammatory, hepatoprotective, and cytotoxic activities. Among these, compound 3 displayed anti-inflammatory activity by inhibiting the NF-κB pathway, with an inhibitory rate of 73.44% at the concentration of 10 µM, while the other compounds showed weak activity, with an inhibitory rate lower than 50% at 10 µM ( Table 2). None of these compounds showed hepatoprotective or cytotoxic activities.
Iridoids are an important type of chemical constituents of the genus Patrinia. Previous investigations showed that some iridoids from P. heterophylla and P. scabra possessed significant anti-inflammatory activity [35][36][37]. However, studies on the iridoids in P. scabiosifolia were mainly focused on their cytotoxic and AChE inhibitory activities, and studies on the anti-inflammatory activity of the iridoids from this plant are still insufficient [22,25]. In our work, eight compounds were isolated from P. scabiosifolia. Structurally, compounds 1, 3, and 4 possess the same skeleton, and compound 3 is the aglucone of compound 1, while compounds 2, 6, 7, and 8 are of the same type, and compound 6 is the aglucone of compound 2. By comparing the chemical structures of compounds 1, 3, and 4 and their anti-inflammatory activities, we found that compound 3, regarded as the aglucone of compound 1, had enhanced the anti-inflammatory activity. In addition, when replaced the 4-CH 3 with a double bond, as in compound 4, the anti-inflammatory activity dramatically decreased. In contrast, structures such as those of compounds 2, 6, 7, and 8 exhibited no NF-κB inhibition activity. These findings may be helpful for further investigation of structure-activity relationships on this type of compounds.

Extraction and Isolation
The air-dried plant material (20 kg) of P. scabiosifolia was extracted three times with 95% EtOH under reflux for three hours each time and concentrated under reduced pressure to afford a crude residue (3650 g). Part of the residue (2100 g) was extracted by the Soxhlet method sequentially with petroleum ether, dichloromethane, ethyl acetate, and methanol to afford petroleum ether (80 g), CH 2 Cl 2 (210 g), EtOAc (80 g), and MeOH (1500 g) soluble extracts.

Anti-Inflammatory Assay for Compounds 1-8
The anti-inflammatory activity of all the isolated compounds was assayed in 293T cells transiently transfected with the pNF-κB-Luc expression plasmid. The pRL-CMV-Renilla plasmid was co-transfected as a control. After transfection, the cells were pretreated with the test compounds for 1 h and then stimulated with lipopolysaccharide (LPS, 1 µg/mL) for 24 h. A luciferase assay was performed with the Dual-Luciferase Reporter Assay System following the manufacturer's instructions (Promega, Madison, WI, USA). The assay was conducted according to a previous published paper [38]. Compounds 1-8 were dissolved in DMSO at a concentration of 10 µM. JSH23 [4-methyl-N 1 -(3-phenylpropyl)-benzene-1,2diamine] was used as positive control.

Conclusions
As a result, two new iridoid glucosides, patriniscabiosides A (1) and B (2), together with six known iridoids and iridoid glucosides (3~8), were isolated from the whole plants of Patrinia scabiosifolia, and their structures were elucidated by various spectroscopic analyses including 1D and 2D NMR, IR, UV, and HRESIMS. The absolute configuration of the new compounds was further confirmed by ECD. Among these, compound 1 is an iridoid glucoside with an isovaleryl moiety attached to the 6 -position of the glucose, and compound 2 possesses a rare 4-deoxyglucose moiety. In addition, compounds 4, 7, 8 are reported from P. scabiosifolia for the first time. Compound 3 displayed potent anti-inflammatory activity through the inhibition of the NF-κB pathway, with an inhibitory rate of 73.44% at a concentration of 10 µM. The cytotoxic and hepatoprotective activities of compounds 1-8 were also tested, but were not relevant. According to our bioassay results, we propose that the iridoid lactone skeleton, as well as the methyl group at C-4 can contribute to the anti-inflammatory activity, while its glucoside would, somehow, weaken its bioactivity. We believe our findings will provide important information for researchers in future work; further studies on the anti-inflammatory mechanism of the isolated compound are still needed.