Two new triterpene glycosides with antiproliferative activities on HepG2 from Phytolacca acinosa fruit fermentation broth

Abstract Two new oleanane-type triterpene glycosides, phytolasides A (1) and B (2), and six known ones (3–8), were isolated from Phytolacca acinosa fruit fermentation broth. Their structures were elucidated by HR-ESI-MS and 1 D- and 2 D-NMR spectroscopic methods. Antiproliferation of compounds 1 and 2 against HepG2 cells was examined by using CCK8 assays. Graphical Abstract


Introduction
Phytolacca acinosa Roxb. commonly known as Yeluobo belongs to Phytolaccaceae family. A variety of constituents including triterpene glycosides, flavonoids, and polysaccharides, sterol, phenolic acids, fatty oil, organic acids and alkaloids were detected in this plant species (Wang et al. 2008;Daoud and Huma 2014). Phytolacca acinosa Roxb. and Phytolacca americana L., recorded in the Chinese Pharmacopoeia (2020 edition), are commonly used traditional Chinese medicine. It is reported that this plant has a variety of functions including anti-inflammatory, anti-tumor, anti-fungal and antibacterial activities (Naito et al. 2002;Ma et al. 2019).
Previous chemical studies of this species have mainly focused on the roots, but few on the fruits. In order to make full use of Phytolacca acinosa fruit resources, our research group took the lead in using fermentation method to Phytolacca acinosa fruit. It is often used as an anti hepatoma drug in Hunan province, China. The anticarcinogenic activity of the fermentation broth has not yet been fully explored Singh et al. 2020).
In light of this, the present study discloses the isolation and structure elucidation of two new triterpene glycosides from the Phytolacca acinosa fruit fermentation broth: phytolasides A (1) and B (2), together with six known compounds (3-8). After the antiproliferative activity screening, 1 and 2 displayed certain inhibitory effect on HepG2 cells.

Results and discussion
Repeated column chromatography from n-butanol extracts of the Phytolacca acinosa fruit fermentation broth afforded 8 compounds that were classified as triterpene glycosides, carbohydrate, amino acid and dipeptides compounds ( Figure 1). Among them, 1 and 2 are new compounds. 3-8 as known compounds were identified. Except for 3 and 4, the remaining constituents were separated from Phytolacca for the first time.
The relative configuration of 1 was further determined by analysis of the NOESY spectrum (Supplementary material, Figure S2). The NOE correlations of H-2 and H-3 with H-24, and H 3 -25 with H 3 -23, indicated the assignment of a-configuration for H-2, H-3, and H-24. The NOE correlations of H-5 with H-9, H-9 with H 3 -27, H-18 with H-1 0 , H 3 -25 with H 3 -26, and H 3 -27 with H 3 -29 indicated that H-18, H 3 -25, H 3 -26 were b-configuration, and H-5, H-9, H 3 -27 and H 3 -29 were a-configuration. After acid hydrolysis of saponin (Compound 1), the presence of D-glucose was confirmed by TLC and optical rotation analyses [a] þ 42.0 (c 0.1, H 2 O) (Luo et al. 2013). The configuration of glycosidic linkage of the glucopyranoside moiety in 1 was determined to be b on the basis of the J value of the anomeric proton signal at d H 5.34 (1H, d, J ¼ 7.9 Hz, H-1 0 ). Thus, the structure of 1 was deduced to be 2b,3b-dihydroxy-30-carbonyl-28-O-b-D-glucopyranosyl phytolaccagenin, which is a new triterpene glycoside named as phytolaside A.
Phytolaside B (2) was found to have the molecular formula C 37 H 58 O 12 by using HRESIMS (m/z 693.3873 [M -H] -, calcd for 693.3850), which indicates the presence of 9 degrees of unsaturation. The 1 H NMR spectrum of 2 reveals the presence of five singlet methyl at d H 0.80 (3H, s), 0.90 (3H, s), 1.16 (3H, s), 1.17 (3H, s) and 1.28 (3H, s), two methenyl with hydroxyl at d H 3.57 (1H, d, J ¼ 3.9 Hz) and 4.07 (1H, m); a glucose terminal hydrogen proton d H 5.34 (1H, d, J ¼ 8.2 Hz), and a methoxyl: d H (3.69, s, -OCH 3 ). The 13 C-NMR and DEPT spectra contain resonances for 37 carbons, including five methyl, 11 methylene, 11 methine (ten sp 3 , one sp 2 ), and nine quaternary carbons (maintain two carbonyls: d C 177.6, 178.8 and one olefinic: d C 124.5, 144.6, These NMR data showed similarity to those of phytolaside A (1). Their only difference is that carbonyl C-30 has been methylated based on the chemical shift C-30 (d C 178.8) and HMBC correlations (Supplementary material, Figure S1) from OCH 3 (d H 3.69)/C-30. Next, the relative configuration of 2 was also identified by analysis of the NOESY spectrum (Supplementary material, Figure S2). After acid hydrolysis of saponin (Compound 2), the presence of D-glucose was confirmed by TLC and optical rotation analyses [a] þ 46 (c 0.1, H 2 O). The configuration of glycosidic linkage of the glucopyranoside moiety in 2 was determined to be b on the basis of the J value of the anomeric proton signal at d H 5.34 (1H, d, J ¼ 7.9 Hz, H-1 0 ). Thus, the structure of 2 was deduced to be 2b,3b-dihydroxy-30-methoxycarbonyl-28-O-b-D-glucopyranosyl phytolaccagenin, which is a new triterpene glycoside named as phytolaside B.
Among two new compounds tested for in vitro inhibition on HepG2 cells (Ali et al. 2014) (Supplementary material, Table S1). The result showed that the IC 50 values of the positive control doxorubicin was 1.463 ± 0.059 lM. Compounds 1 and 2 showed positive effects with IC 50 of 12.524 ± 0.659 lM and 14.738 ± 0.725 lM, respectively. The results suggested phytolaside A and phytolaside B both inhibited the proliferation of HepG2 cells.

Preparation of Phytolacca acinosa fruit fermentation broth
The fruit was harvested in September 2018 and identified by Professor Qiuhong Wang of Guangdong Pharmaceutical University as the mature fruit of Phytolaccaceae plant Phytolacca acinosa Roxb. (number as par-201809). The Phytolacca acinosa fruit fermentation broth (number as parf-201809) and fruit specimens are stored in Department of Traditional Chinese Medicine, Guangdong Pharmaceutical University.
The Phytolacca acinosa fruit fermentation broth was made by Mr. Wenlong Liu. The production process is as follows: (1) Preparation of fermentation mother broth: take mature and fresh Phytolacca acinosa fruit, crush, ferment, stir continuously, optionally, add an appropriate amount of inorganic salts and white granulated sugar, adjust the culture conditions and fully ferment, that is, the fermentation mother broth is prepared; (2) Preparation of the first fermentation broth: take fresh and mature Phytolacca acinosa fruit, wash it, drain the water, air it in a cool place until it is 60 $ 70% dry, put it in a non-ferrous fermentation container, add 2 $ 3% fermentation mother broth, mix it evenly with Phytolacca acinosa fruit, ferment it aerobically at 20 $ 30 C for 30 $ 60 days, add an appropriate amount of white granulated sugar and organic acid to adjust the sugar content and acidity, and separate the fermentation broth, that is, the first fermentation broth is prepared; (3) Preparation of the second fermentation broth: put the first fermentation broth in a non-ferrous container, place it in a cold place of 5 $ 25 C, seal it away from light and stand still. Under anaerobic conditions, fully ferment it again for more than 30 days to obtain the second fermentation broth, namely Phytolacca acinosa fruit fermentation broth.

Extraction and isolation
The Phytolacca acinosa fruit fermentation broth (10 L) were freezed drying and extracted with 70% methanol (3 Â 3 L) under ultrasound (40 KHz, 25 C, 30 min) three times. After vacuum filtration, the filtrates were condensed using a rotary evaporator under reduced pressure. The residue was then suspended in water and extracted three times by cyclohexane, ethyl acetate, and n-butanol. The n-butanol (105 g) was chromatographed on a column of silica gel and eluted successively with a gradient of CH 2 Cl 2 -MeOH (100:0 to 0:100) to yield eleven fractions (Fr.1 À Fr.11).

Acid hydrolysis of 1 and 2
A solution of 1 (5 mg) and 2 (5 mg) in 2 N HCl was heated at 90 C on a water bath for 2 h. After cooling, the reaction mixture was extracted by EtOAc. The aqueous layer was neutralized with 2 N NaOH and dried by freeze-drying. The residue was purified by semipreparative HPLC with gradient aqueous MeOH to afford pure sugars. They were identified as D

Antiproliferation assay
Antiproliferation assay included the HepG2 cells inhibition evaluation. The exponential growth phase cells (1 Â 10 5 cells/well) were seeded into a 96well mipcrotiter plate in the absence or presence of test compounds of seven different concentrations in triplicate. After incubation in a 5% CO 2 incubator at 37 C for 48 h, cell viability was measured by the CCk8 method, as described previously (Shi et al. 2019). The concentration causing the reduction of viable cells by 50% (IC 50 ) was determined. The results showed that compounds 1 and 2 displayed inhibitory effects on HepG2 cells. The absorbance was documented with a microplate reader at a wavelength of 570 nm. The experiments were conducted a minimum of three times.

Conclusion
In summary, this report describes the isolation and structural elucidation of eight compounds from the Phytolacca fruit fermentation broth. Among them were six known compounds, two new compounds, phytolasides A (1) and B (2) that was isolated for the first time as new compounds. Compounds 1 and 2 exert antiproliferative effects on HepG2 cells.