Foegraecumoside O and P, a Pair of Triterpenoid Saponins with a 4/5/6 Fused Tricyclic Oleanane Carbon Skeleton from Lysimachia foenum-graecum Hance

Lysimachia foenum-graecum Hance (Primulaceae) is a medicinal plant used for cold, pain, ascariasis, etc., in China. Triterpenoid saponins have been found to be the main components of this genus. In this work, a pair of oleanane-type triterpenoid saponins with an unprecedented 4/5/6 fused tricyclic skeleton, foegraecumoside O (1) and foegraecumoside P (2) were isolated from the butanol fraction of the aerial parts of L. foenum-graecum. Their structures were determined using chemical methods and extensive spectroscopic analyses, along with quantum chemical calculations. Compound 2 displayed moderate cytotoxicity against HepG2, MGC-803, T24, NCI-H460, A549, and A549/CDDP (drug-resistant lung-cancer cell line) with IC50 at 12.4–19.2 μM in an MTT assay, comparing with the positive control doxorubicin, which had IC50 at 0.53–4.92 μM, but was inactive for A549/CDDP. Furthermore, a possible biosynthetic pathway for forming compounds 1 and 2 was proposed.


Introduction
There is a long history of human beings fighting with cancer [1]. After the development and application of multiple generations of anticancer drugs, drug resistance was gradually produced, due to which the effects of anticancer drugs declined progressively. Therefore, there is always need to develop new potential therapeutic molecules, especially those molecules that are active against drug-resistant cancers [2,3].
The Lysimachia genus (Primulaceae) comprises approximately 180 species that are widespread in temperate and subtropical regions, 138 of which can be found in China [4]. Lysimachia foenum-graecum Hance is a species that is distributed mainly in the Guangxi Zhuang Autonomous Region and the Guangdong, Hunan, and Yunnan Provinces of China [4]. It is famous due to its pleasant smell after drying; therefore, it is commonly used as perfumery material. The aerial parts of this plant have been used for the treatment of colds, headaches, sore throats, toothaches, abdominal pain, ascariasis, and other diseases in traditional Chinese medicine [5]. Previous studies indicated that triterpenoid saponins are the main chemical constituents of this genus, and many of them were found to be cytotoxic [6][7][8][9][10][11][12]. Among them, our group has contributed to the discoveries of new components from L. clethroides, L. fortune, and L. foenum-graecum [8,[10][11][12]. In previous phytochemistry investigation, many triterpenoid saponins and a few flavonoids have been isolated from L. foenum-graecum [10,[12][13][14][15][16][17]. Some reported saponins were found to possess a cytotoxic property against cancer cell lines, including NCI-H460, MGC-803, HepG2, T24, A549, and A549/CDDP. [10,12,13].
In terms of exploring the more active natural molecules from this genus, a pair of novel compounds, foegraecumoside O (1) and foegraecumoside P (2), featuring unique 4/5/6 fused tricyclic skeletons linked between C-18 and C-30, were purified and identified from L. foenum-graecu ( Figure 1). Based on the presence of the chiral center at C-30, which makes the two compounds different, quantum chemical calculations were performed to simulate the theoretical NMR data of the optical isomers. Thereafter, the absolute configurations of the isomers were able to be determined. Herein, the isolation, structural characterization, inhibitory effect on the growth of tumor cells, and proposed biosynthesis of these two molecules were discussed.
Molecules 2023, 28, x FOR PEER REVIEW 2 of 13 been isolated from L. foenum-graecum [10,[12][13][14][15][16][17]. Some reported saponins were found to possess a cytotoxic property against cancer cell lines, including NCI-H460, MGC-803, HepG2, T24, A549, and A549/CDDP. [10,12,13]. In terms of exploring the more active natural molecules from this genus, a pair of novel compounds, foegraecumoside O (1) and foegraecumoside P (2), featuring unique 4/5/6 fused tricyclic skeletons linked between C-18 and C-30, were purified and identified from L. foenum-graecu ( Figure 1). Based on the presence of the chiral center at C-30, which makes the two compounds different, quantum chemical calculations were performed to simulate the theoretical NMR data of the optical isomers. Thereafter, the absolute configurations of the isomers were able to be determined. Herein, the isolation, structural characterization, inhibitory effect on the growth of tumor cells, and proposed biosynthesis of these two molecules were discussed.

Structure Elucidation
The aerial parts of L. foenum-graecum were extracted by 95% EtOH. Then, the obtained crude was suspended in water and extracted with EtOAc and n-BuOH, successively. Two compounds, 1 and 2 ( Figure 1), were isolated through multiple chromatography columns from the BuOH fraction and then purified using reverse-phase HPLC.

Structure Elucidation
The aerial parts of L. foenum-graecum were extracted by 95% EtOH. Then, the obtained crude was suspended in water and extracted with EtOAc and n-BuOH, successively. Two compounds, 1 and 2 ( Figure 1), were isolated through multiple chromatography columns from the BuOH fraction and then purified using reverse-phase HPLC.
Compound 2, with a negative optical rotation in MeOH, [α] 20 D − 11.3 (c 0.1, MeOH), was purified as a white amorphous powder. It was deduced to have an identical molecular formula to that of compound 1 according to its HR-ESI-MS data ( Figure S19). The IR spectrum displayed absorptions at 3426 (OH), 2940 (CH 3 , CH 2 , and CH), at 1075 (C-O-C) cm −1 . The 1 H and 13 C NMR data of compound 2 closely resembled those of compound 1 (  Figure 3). Thus, compound 2 was proved as shown in Figure 1. NMR data of compounds 1 and 2 are assigned in Table 1. cules 2023, 28, x FOR PEER REVIEW the structure of compound 2 is practically same to that of compound configuration of H-30, which was confirmed as β-orientated through H-30 (δH 3.96) with H2-12 (δH 1.73, 1.85) and H-29 (δH 1.18) observed in trum of compound 2 ( Figure 3). Thus, compound 2 was proved as s NMR data of compounds 1 and 2 are assigned in Table 1.

NMR Calculation
To further verify the configuration of the hydroxy at C-30, a quantum chemical calculation of the 1 H NMR and 13 C NMR chemical shifts for the aglycones of compounds 1 and 2 was conducted using the gauge-including atomic orbitals (GIAO) method at the mPW1PW91/6-311+G (2d, p) in pyridine with the IEFPCM model [19,20]. As shown in Figure 4, as well as Tables S1 and S3, the experimental 13 C NMR values of the aglycone of compound 1 has an R 2 (coefficient of determination) of 0.9917 with theoretical values of the aglycone of compound 1 ( Figure 4A) but had one of 0.9845 with the aglycone of compound 2 ( Figure 4B). Similarly, the experimental 13 C NMR values of the aglycone of compound 2 had an R 2 of 0.9914 with theoretical values of the aglycone of compound 2 ( Figure 4D) but had one of 0.9818 with the aglycone of 1 ( Figure 4C). The linear regression fitting of 1 H NMR ( Figure 5, Tables S2 and S3) displayed that the experimental 1 H NMR data of the aglycone of compound 1 owns an R 2 of 0.9451 ( Figure 5A) but had one of 0.8604 with the aglycone of compound 2 ( Figure 5B). Likewise, the experimental 1 H NMR values of the aglycone of compound 2 had an R 2 of 0.9588, with theoretical values of the aglycone of compound 2 ( Figure 5D) but had one of 0.8719 with the aglycone of compound 1 ( Figure 5C). It is therefore concluded that the calculated 1 H NMR and 13 C NMR chemical shifts for the aglycones of compounds 1 and 2 showed a better agreement with the experimental values with a higher correlation coefficient. Consequently, structures of compounds 1 and 2 were further confirmed.
Molecules 2023, 28, x FOR PEER REVIEW 6 of 13 NMR data of the aglycone of compound 1 owns an R 2 of 0.9451 ( Figure 5A) but had one of 0.8604 with the aglycone of compound 2 ( Figure 5B). Likewise, the experimental 1 H NMR values of the aglycone of compound 2 had an R 2 of 0.9588, with theoretical values of the aglycone of compound 2 ( Figure 5D) but had one of 0.8719 with the aglycone of compound 1 ( Figure 5C). It is therefore concluded that the calculated 1 H NMR and 13 C NMR chemical shifts for the aglycones of compounds 1 and 2 showed a better agreement with the experimental values with a higher correlation coefficient. Consequently, structures of compounds 1 and 2 were further confirmed.

Cytotoxicity Assay
Compounds 1 and 2 were evaluated for their cytotoxic activities against fiv cancer cell lines in vitro using the MTT method with doxorubicin as the positiv ( Table 2). Compound 2 showed moderate cytotoxicities against NCI-H460, M HepG2, and T24, with IC50 values of 18.4, 12.4, 19.2, and 15.0 μM, respectively. more, compound 2 was tested on drug-sensitive and drug-resistant lung-cancer (A549 and A549/CDDP, respectively), and it displayed moderate cytotoxicity A549/CDDP, with an IC50 value of 16.0 μM and a resistance factor (RF) of 0.94 (T a Results are expressed as means ± SD (n = 3); b positive control.

Cytotoxicity Assay
Compounds 1 and 2 were evaluated for their cytotoxic activities against five human cancer cell lines in vitro using the MTT method with doxorubicin as the positive control ( Table 2). Compound 2 showed moderate cytotoxicities against NCI-H460, MGC-803, HepG2, and T24, with IC 50 values of 18.4, 12.4, 19.2, and 15.0 µM, respectively. Furthermore, compound 2 was tested on drug-sensitive and drug-resistant lung-cancer cell lines (A549 and A549/CDDP, respectively), and it displayed moderate cytotoxicity against A549/CDDP, with an IC 50 value of 16.0 µM and a resistance factor (RF) of 0.94 (Table 3).

Discussion
Triterpenoids and their saponins play an important role in natural product chemistry. Among them, the oleanane-type skeleton is a classical pentacyclic skeleton and commonly exists in many families, such as Araliaceae [24], Fabaceae [25], Campanulaceae [26], Polygalaceae [27], etc. In the previous phytochemical investigation of the Primulaceae family, it was found that oleanane-type skeletons, especially 13,28-epoxyoleanane skeletons, were the general feature of the main components of this family [28]. As a genus from the Primulaceae family, many oleanane-type triterpenoid saponins, more than half of which having beard 13,28-epoxyoleanane skeletons, have been isolated from the Lysimachia genus [6][7][8][9][10][11][12][13][14][15]. In this work, two epimers featuring a unique 4/5/6 fused tricyclic skeleton linked between C-18 and C-30 were isolated from L. foenum-graecu. This is the first example of this novel skeleton within the large family of oleanane-type triterpenoids. Therefore, it is an important discovery for the chemical diversity of triterpenoids.
As a pair of epimers with different configurations of 30-OH, compounds 1 and 2 displayed high stereoselectivity against cancer cell lines. Compound 2, with 30α-OH, was active against several cancer cell lines, including the drug-resistant lung-cancer cell line A549/CDDP. Whereas compound 1 with 30α-OH was proven to be inactive against

Discussion
Triterpenoids and their saponins play an important role in natural product chemistry. Among them, the oleanane-type skeleton is a classical pentacyclic skeleton and commonly exists in many families, such as Araliaceae [24], Fabaceae [25], Campanulaceae [26], Polygalaceae [27], etc. In the previous phytochemical investigation of the Primulaceae family, it was found that oleanane-type skeletons, especially 13,28-epoxyoleanane skeletons, were the general feature of the main components of this family [28]. As a genus from the Primulaceae family, many oleanane-type triterpenoid saponins, more than half of which having beard 13,28-epoxyoleanane skeletons, have been isolated from the Lysimachia genus [6][7][8][9][10][11][12][13][14][15]. In this work, two epimers featuring a unique 4/5/6 fused tricyclic skeleton linked between C-18 and C-30 were isolated from L. foenum-graecu. This is the first example of this novel skeleton within the large family of oleanane-type triterpenoids. Therefore, it is an important discovery for the chemical diversity of triterpenoids.
As a pair of epimers with different configurations of 30-OH, compounds 1 and 2 displayed high stereoselectivity against cancer cell lines. Compound 2, with 30α-OH, was active against several cancer cell lines, including the drug-resistant lung-cancer cell line A549/CDDP. Whereas compound 1 with 30α-OH was proven to be inactive against all those tested cell lines. Meanwhile, with an RF of 0.94, compound 2 displayed a similar inhibitory effect against normal lung-cancer cells and drug-resistant lung-cancer cells, showing its potential to be a new approach for drug resistance. The results provide a potential lead compound for further investigation in treating diseases related to cancer and drug resistance to cancer.

General Experimental Procedures
A PerkinElmer model 341 polarimeter was used to record optical rotations. A Thermo-Scientific Exactive mass spectrometer was used to carry out HR-ESI-MS spectrum. A PerkinElmer Spectrum Two FT-IR spectrometer was used to obtain IR spectra. The 1D and 2D NMR spectra were performed on Bruker AV-500 or AV-600 MHz spectrometers in deuterated pyridine. Chemical shifts (δ) were reported in ppm relative to the solvent signals, and coupling constants (J) were calculated and reported in Hz. A Shimadzu LC-6AD HPLC with an RID-10A detector and an YMC-Pack ODS-A column (250 mm × 20 mm, 5 µm) was used to perform purification of two compounds. A Jasco LC-4000 Analytical HPLC was used to analyze extracts, fractions, and semipure samples. Different materials, including silica gel (200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China), MCI gel (CHP20, 75-150 µm, Mitsubishi Chemical Corporation, Tokyo, Japan), and ODS (50 µm, YMC, Kyoto, Japan), were used for column chromatography (CC) for isolation of pure compounds. Precoated silica gel GF254 plates (Qingdao Marine Chemical Factory) were used for TLC analysis. Spots were detected on TLC by observing them under UV light and then heating after spraying with 10% H 2 SO 4 in EtOH. Solvents used for extraction and chromatography column were all analytical grade and manufactured by Xilong Scientific, Shantou, China. HPLC-grade methanol and acetonitrile were manufactured by Tedia, Plzen, Czech Republic.

Plant Material
The aerial parts of L. foenum-graecum were collected in Jinxiu, Guangxi Zhuang Autonomous Region, People's Republic of China. Shaoqing Tang from Guangxi Normal University authenticated the species. A voucher specimen (NO. LF-2014022) was deposited in the archive of the State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin, China.

Acid Hydrolysis
Each compound (2 mg) was dissolved in 1 M HCl (1,4-dioxane-H 2 O, 1:1, 5 mL; Xilong Scientific, Shantou, China) and then stirred at 80 • C for 8 h. The reaction mixture was extracted with CH 2 Cl 2 (Xilong Scientific, Shantou, China) after cooling. Afterward, each aqueous layer was evaporated under a vacuum (50 • C in water bath), and then diluted with H 2 O for multiple times to provide a neutral residue. Each residue was subjected to analytical HPLC (Jasco LC-4000, Tokyo, Japan) under the following conditions: Shodex Asahipak NH2P-50 4E column (250 mm × 4.6 mm, 5 mm); Jasco OR-4090 optical rotation detector; mobile phase, CH 3 CN:H 2 O (78:22, v/v); flow rate 1 mL/min. The absolute configurations of sugar units in compounds 1 and 2, composed of glucose, arabinose, and rhamnose, were confirmed by comparing their retention times and optical rotations with those of authentic samples (National Institute for Food and Drug Control, Beijing, China) [18]. Authentic sugars had retention times of t R : 6.5 min (L-rhamnose, negative optical rotation), 7.7 min (L-arabinose, positive optical rotation), and 11.3 min (D-glucose, positive optical rotation).

Quantum Chemical Calculations
NMR calculations were carried out via Gaussian 09, following the protocol adapted from Michael et al. [29]. At first, structures were optimized at B3LYP/6-31+G(d,p) theory level in gas phase. Then, the NMR calculations were conducted using the gauge-including atomic orbitals (GIAO) method at mPW1PW91/6-311+G (2d, p) in pyridine using the IEFPCM model. Finally, the TMS-corrected NMR chemical-shift values were fitted to the experimental values using the ordinary least-squares linear-regression (OLSLR) method. The calculated 13 C NMR and 1 H NMR chemical-shift values of TMS in pyridine were 187.32 ppm and 31.73 ppm, respectively.

Statistical Analysis
The data were processed with Student's t-test using SPSS software (17.0; IBM ® , USA), with a significance level of p < 0.05. IC 50 values were determined through a Probit test in SPSS. All the tests were repeated in three independent experiments.

Conclusions
In conclusion, two oleanane-type triterpenoid saponins, foegraecumoside O (1) and foegraecumoside P (2), with unique 4/5/6 fused tricyclic skeletons, were isolated from L. foenum-graecum. They represent the very first example of oleanane-type triterpenoid saponins bearing a ring linked between C18 and C30. This discovery expands the structural diversity of the oleanane-type triterpenoid saponins. Furthermore, these two compounds are epimers that have opposite configurations of 30-OH. Interestingly, only two with 30α-OH were found to be active to cancer cell lines NCI-H460, MGC-803, HepG2, T24, A549, and A549/CDDP. It provides a potential lead molecule in drug development for treating diseases related to cancer and drug resistance to cancer.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.