New natural furfural derivatives from the leaves and stems of Pogostemon cablin

Abstract A new furfural derivative, pogoscafuran A (1), a new natural product, HMF levulinate (2), together with four known compounds (3–6) were isolated from an extract of the leaves and stems of Pogostemon cablin (Blanco) Benth. Their structures were elucidated on the basis of extensive spectroscopic analyses and single-crystal X-ray crystallography. Compound 1 was the first example of natural furfural derivative with a unique C5–C1′ linkage between a molecule of furfural and 3-methyl-2-cyclopentenone moiety. The plausible biogenetic pathway for the new compound 1 was proposed. All these isolated compounds were tested for their inhibitory effects on the nitric oxide (NO) production induced by lipopolysaccharide in RAW264.7 cells, and only compound 1 exhibited weak inhibitory activity. Graphical Abstract


Introduction
Furfural derivatives, consisting of an aromatic furan ring and a formyl group, are a class of heterocyclic small molecules. Most of the natural furfural derivatives are existent in various plants, and some of them possess remarkable biological activities (Li et al. 2012;Huang et al. 2019;Zhang et al. 2020). Recently, five new structurally diverse furfurals, hostines A-E, showing significant nitric oxide (NO) inhibitory activities, were isolated from the flowers of Hosta plantaginea (Zhang et al. 2020). (5-formylfuran-2yl)methyl-4-hydroxy-2-methylenebutanoate with pronounced anti-neuroinflammatory activity, was obtain from the roots of Polygala tricornis (Li et al. 2012).
Pogostemon cablin (Blanco) Benth. (Labiatae), known as 'Guang-Huo-Xiang' in traditional Chinese medicine (TCM), is a fragrant herbaceous plant widely cultivated in Guangdong, Guangxi, and Fujian provinces of China and several other countries of Southeast Asia (Editor Committee for Flora of China 1997). Its leaves and stems have long been used in TCM for the treatment of various diseases such as diarrhea, gastroenteritis, and intestinal infections (Zhu et al. 2017). Previous studies on chemical composition of this plant have led to the identification of numerous sesquiterpenoids (Li et al. 2011;Tong et al. 2013;Dai et al. 2018;Zhou et al. 2018), flavonoids (Park et al. 1998), and phenylpropanoid glycosides (Kim et al. 2015). Some of these constituents exhibited diverse pharmacological properties such as anti-inflammatory (Li et al. 2011), vasorelaxant (Zhou et al. 2018, and cytotoxic (Park et al. 1998) activities. In this study, a new furfural derivative (1), a new natural product (2), and four known compounds (3-6) were isolated from the leaves and stems of P. cablin. Their structures were characterized on the basis of extensive spectroscopic data analyses and singlecrystal X-ray diffraction experiments. Compound 1 showed weak NO inhibitory effect (IC 50 ¼ 44.15 ± 4.37 lM). Herein, we described the isolation and structural elucidation of these compounds, as well as their NO inhibitory effects.

Results and discussion
The dried leaves and stems of P. cablin were powdered and extracted with 75% EtOH. The crude extract was suspended in H 2 O and then partitioned sequentially with petroleum ether and EtOAc. The EtOAc fraction was separated by repeated column chromatography over silica gel, LH-20 gel, and HPLC to give compounds 1-6 ( Figure 1).
Compound 1 was isolated as colourless crystals. The molecular formula of 1 was established as C 11 H 10 O 3 , with seven degrees of unsaturation, by the HRESIMS data at m/z 213.0516 [M þ Na] þ (calcd for C 11 H 10 O 3 Na þ , 213.0522). In the IR spectrum, the absorption bands of conjugated carbonyl groups (1699 and 1652 cm -1 ) were observed. The 1 H NMR spectroscopic data displayed characteristic signals for a a-substituted furfural moiety [d H 9.64 (1H, s, H-6), 7.29 (1H, d, J  double bond (d C 153.2, 151.4, 123.0 and 111.9), a tetrasubstituted double bond (d C 175.2 and 128.4), two sp 3 methylenes (d C 35.1 and 33.1) and a methyl (d C 19.6). The aforementioned functionalities accounted for five degrees of unsaturation, and the remaining two degrees of unsaturation suggested a bicyclic ring system for 1.
Compound 2 was previously reported as a synthetic product, HMF levulinate (Qin et al. 2016). This is the first time to report compound 2 as a natural product, and its UV and IR data were also reported in this study.
A plausible biosynthetic pathway for the new compound 1 was proposed in Scheme S1. Compound 1 should be originated from 5-hydroxymethylfurfural (3). The reduction of formyl group in compound 3 would produce intermediate i, which then underwent dehydration to give intermediate ii. The hydration of ii followed by hydrogenation could afford intermediate iv.
Aldol condensation between C-5 and C-6 in iv would afford v, which underwent reduction of hydroxymethyl would lead to the intermediate 3-methyl-2-cyclopentenone. Finally, this intermediate was coupled with a molecule of furfural to produce compound 1.
Since several furfural derivatives have been reported to possess anti-inflammatory activity (Li et al. 2012;Zhang et al. 2020), compounds 1-6 were tested for their inhibitory effects on the nitric oxide (NO) production induced by lipopolysaccharide in RAW264.7 cells. However, the results revealed that only compound 1 showed weak inhibitory activity with an IC 50 value of 44.15 ± 4.37 lM. Quercetin was used as a positive control (IC 50 ¼ 15.98 ± 1.64 lM).

General experimental procedures
X-ray data were collected using an Agilent Xcalibur Nova X-ray diffractometer. UV data were obtained using Shimadzu UV-2450 spectrophotometry, and IR spectra were obtained using a Bruker Tensor 27 spectrometer. Melting points were measured on an X-4 melting instrument. 1D and 2D NMR spectra were measured on a Bruker AM-400 spectrometer. HRESIMS were performed on a Waters Micromass Q-TOF spectrometer. Semipreparative HPLC separations were carried out by photodiode array (PDA) analysis using a Wufeng LC-100 apparatus with a Kromasil 100-5 C 18 column (250 Â 10 mm, 5 lm). TLC analysis was carried out on silica gel plates (Marine Chemical Ltd., Qingdao, China). Silica gel (300-400 mesh, Qingdao Haiyang Chemical Co., Ltd.) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used for column chromatography (CC).

Plant material
The leaves and stems of P. cablin were collected from Guangdong Province, China, in July 2020. The identification of the plant was done by one of the author (Y. Wang). A voucher specimen (201007-PC) was deposited at School of Environmental Science and Engineering, Southern University of Science and Technology.

Measurement of NO production
The NO concentration was measured by the Griess reagent. Briefly, RAW264.7 cells were treated with LPS (1.0 lg/mL) and compounds for 24 h. After that, the 50 lL of culture supernatant was collected to react with the same volume of Griess reagent for 10 min at room temperature in the dark. Then, the absorbance was measured at 540 nm using a microplate reader. Inhibition (%) ¼ (1 -(A LPS þ sample -A untreated )/(A LPS À A untreated ) Â 100. The experiments were performed in triplicates. Quercetin was used as a positive control.

Conclusion
In summary, a new furfural derivative (1), a new natural product (2), and four known compounds (3-6) were identified from the leaves and stems of P. cablin. The structures of these isolated compounds were elucidated by extensive analysis of NMR spectroscopic data and single-crystal X-ray diffraction. To the best of our knowledge, pogoscafuran A (1) was the first natural furfural derivative with a C5-C1 0 linkage between a molecule of furfural and cyclopentanone moiety. Compound 1 showed weak NO inhibitory activity, and its plausible biogenetic pathway was also proposed. Altogether, these isolated furfural derivatives increased the structural diversity of this type compounds.