New Terpenoids from Potentilla freyniana Bornm. and Their Cytotoxic Activities

Two new A-ring contracted triterpenoids, madengaisu A and madengaisu B, and one undescribed ent-kaurane diterpenoid, madengaisu C, along with 20 known compounds were isolated from the roots of Potentilla freyniana Bornm. The structures were elucidated using extensive spectroscopic techniques, including 1D and 2D-NMR, HR-ESI-MS, ECD spectra, IR, and UV analysis. Moreover, all isolated constituents were evaluated for their anti-proliferative activity against RA-FLS cells and cytotoxic activities against the human cancer cell lines Hep-G2, HCT-116, BGC-823, and MCF-7. Ursolic acid and pomolic acid displayed moderate inhibitory activity in RA-FLS cells with IC50 values of 24.63 ± 1.96 and 25.12 ± 1.97 μM, respectively. Hyptadienic acid and 2α,3β-dihydroxyolean-12-en-28-oic acid 28-O-β-d-glucopyranoside exhibited good cytotoxicity against Hep-G2 cells with IC50 values of 25.16 ± 2.55 and 17.66 ± 1.82 μM, respectively. In addition, 2α,3β-dihydroxyolean-13(18)-en-28-oic acid and alphitolic acid were observed to inhibit HCT-116 cells (13.25 ± 1.65 and 21.62 ± 0.33 μM, respectively), while madengaisu B and 2α,3β-dihydroxyolean-13(18)-en-28-oic acid showed cytotoxic activities against BGC-823 cells with IC50 values of 24.76 ± 0.94 and 26.83 ± 2.52 μM, respectively, which demonstrated that triterpenes from P. freyniana may serve as therapeutic agents for RA and cancer treatment.


Introduction
Potentilla L. is a major genus belonging to the family Rosaceae with more than 200 species in the world, and many of them are widely used as ethnomedicine. Previous phytochemical research has shown that the genus Potentilla L. contains amounts of bioactive compounds, such as triterpenoids, flavonoids, and tannins, etc. Pharmacological studies also reported that Potentilla species possess significant hypoglycemic, anti-inflammatory, anti-tumor, antioxidative, and antibacterial activities [1].
Potentilla freyniana Bornm. is a perennial herb and is widely distributed in China, Russia, Japan, and Korea. It is also called "Ma Deng Ai" or "San Zhang ye" in the Dong ethnomedicine of China. The commonly used medicinal parts of the herb include the roots and whole grass, which have the effect of clearing heat and detoxifying, dispersing blood stasis, and stopping bleeding for the treatment of trauma bleeding, enteritis dysentery, stomatitis, and other diseases [2]. Previous phytochemical studies on P. freyniana afforded 2 of 12 29 compounds, mainly including flavonoids and triterpenoids, but there have been few investigations on biological activities [3].
To further investigate the pharmacologically active constituents of P. freyniana, a phytochemical study on the roots of P. freyniana was conducted, which led to the isolation of two undescribed A-ring contracted triterpenoids (1 and 2) and one new ent-kaurane diterpenoid (22) along with 20 known compounds (Figure 1) from the CH 2 Cl 2 -soluble layer. All isolates  were evaluated for their cytotoxic activities against RA-FLS cells and human cancer cell lines, including HepG2 (human liver carcinoma), HCT-116 (human colorectal carcinoma), BGC-823 (human gastric carcinoma), and MCF-7 (human breast carcinoma), to explore biologically significant metabolites. Herein we deal with the isolation and structure elucidation of the new compounds (1)(2)22) as well as their anti-proliferative activity against RA-FLS cells and cytotoxic activities against the human cancer cell lines Hep-G2, HCT-116, BGC-823, and MCF-7.
Molecules 2021, 26, x FOR PEER REVIEW 2 of 12 29 compounds, mainly including flavonoids and triterpenoids, but there have been few investigations on biological activities [3].
To further investigate the pharmacologically active constituents of P. freyniana, a phytochemical study on the roots of P. freyniana was conducted, which led to the isolation of two undescribed A-ring contracted triterpenoids (1 and 2) and one new ent-kaurane diterpenoid (22) along with 20 known compounds (Figure 1) from the CH2Cl2-soluble layer. All isolates  were evaluated for their cytotoxic activities against RA-FLS cells and human cancer cell lines, including HepG2 (human liver carcinoma), HCT-116 (human colorectal carcinoma), BGC-823 (human gastric carcinoma), and MCF-7 (human breast carcinoma), to explore biologically significant metabolites. Herein we deal with the isolation and structure elucidation of the new compounds (1-2, 22) as well as their antiproliferative activity against RA-FLS cells and cytotoxic activities against the human cancer cell lines Hep-G2, HCT-116, BGC-823, and MCF-7.

Structural Elucidation
Compound 1 was obtained as a white and amorphous powder, and the molecular formula was determined to be C30H46O3 by HR-ESI-MS (m/z 477.3342 [M + Na] + , calculated for C30H46NaO3, 477.3345), indicating eight degrees of unsaturation ( Figure S3). The IR spectrum showed characteristic absorptions ( Figure S2) of hydroxyl (3325 cm −1 ), methyl
Previous reports have shown that an A-ring contracted skeleton was found in ursane-, oleanane-, and dammarane-type triterpenes. In general, the O-containing groups at the C-2 or C-3 position of the six-membered A-ring go through oxidation reactions, which leads to selective C(2)-C(3) bond cleavage and further recyclization to form an Apentacycle triterpene [24]. In our study, compounds 2-4 belong to A-ring contracted type triterpenes, which may derive from ursolic acid (6) and corresponding derivatives, and their intermediate products include compounds 7-12. The plausible biosynthetic pathway of compounds 2-4 and 6-12 is shown in Figure 8, and the oleanane-type triterpenes (1, 5, and 16) also have a similar pathway. Meanwhile, aglycones are easily glycosylated at C-28 to form relevant triterpenoid saponins (13,14,19, and 20) [25].

Biological Activities
The cytotoxic activities of all compounds (1-23) against RA-FLS cells and human cancer cell lines (Hep-G2, HCT-116, BGC-823, and MCF-7) were evaluated in vitro using the MTT method. As shown in Table 3, 6 and 7 showed good bioactivities against RA-FLS

Cytotoxicity Assessment
The cytotoxicity activities were evaluated by the MTT assay [26]. Firstly, the tested compounds (1-23) were diluted in corresponding media from freshly made solutions in DMSO (50 mM) to working concentrations (0-40 µM). The cells were seeded in 96-well plates. After 24 h incubation, the cells were exposed to the test compounds at the indicated concentrations for 24 h under 5% CO 2 at 37 • C. Then, 100 µL of MTT (0.5 mg/mL) was added to each well and incubated for 4 h. Subsequently, the supernatant was removed from the formazan crystals, and 100 µL DMSO was added to each well. Finally, the absorbance was measured by a microplate reader using a wavelength of 492 nm to determine cell viability rate.

Conclusions
As a common Dong ethnomedicine in China, the roots of P. freyniana have been widely used for heat clearing and detoxifying. Moreover, ursane-type triterpenes are some of the most common pentacyclic triterpenes in ethnic medicine, with various pharmacological activities and low toxicity [27]. In this work, twenty-three compounds, comprising two unusual A-ring contracted triterpenoids named madengaisu A and B (1 and 2) and one new ent-kaurane diterpenoid named madengaisu C (22) along with 20 known compounds, were isolated and identified, thirteen of which were classified as ursane-type triterpenoids. In addition, this article is the first to report the diterpenoid constituents from the genus Potentilla L. Compounds 5, 11, 15, 16, 19, 22, and 23 were isolated from the genus Potentilla L. for the first time, and compounds 4, 6, 7, 10, and 20 were isolated from this plant for the first time, which enriched the chemical metabolite diversity of diterpenoids and triterpenoids in this plant. Additionally, compounds 2, 4, 6, 7, 16, 19, and 21 showed good anti-RA and/or anti-tumor activities, which demonstrated that triterpenes from P. freyniana may serve as therapeutic agents for RA and cancer treatment.