Polyphenols Isolated from Xanthoceras sorbifolia Husks and Their Anti-Tumor and Radical-Scavenging Activities

Xanthoceras sorbifolia Bunge. is used in traditional medicine in North China. To evaluate the anti-tumor and radical-scavenging activities of X. sorbifolia husks polyphenols and determine their structure-activity relationships, 37 polyphenols 1–37 were obtained by bioassay-guided fractionation. Two new compounds 1–2, and compounds 5, 6, 8, 9, 11, 14–17, 21–25, 27–29, 31, 33, 34, 36, and 37 were isolated from the genus Xanthoceras for the first time. Compounds 1–37 did not show strong cytotoxicity against the four tested tumor cell lines (A549, HepG2, MGC-803, and MFC) compared to paclitaxel and under the conditions tested in the anti-tumor assay, but compounds 3, 4, 7, 8, 10, 18–20, 25, 26, 29, 30, 32, and 35 exhibited stronger radical-scavenging activity than ascorbic acid in a 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt assay. This was the first report on the anti-tumor and radical-scavenging activities of the polyphenols isolated from X. sorbifolia husks. Overall, the present study contributed valuable information concerning X. sorbifolia husks use in medicine and pharmacology.


Introduction
Xanthoceras sorbifolia Bunge. is the only species in the genus Xanthoceras (family Sapindaceae) and is distributed in North China [1]. This species is a kind of woody oil-bearing crop, traditionally used in herbal medicine for curing atherosclerosis, rheumatism, hyperpiesia, chronic hepatitis, and child enuresis [2], and it has been included in the 1977 edition of the China Pharmacopoeia [3]. Chemical studies of X. sorbifolia husks, which are considered byproducts, showed that the husks contained a variety of compounds, including polyphenols [4], triterpenoids [5], and sterols [6]. Medical research showed that X. sorbifolia husk components improved learning ability and memory [7], had anti-cancer effects [8], inhibited tyrosinase [9], cured cardiovascular diseases [10], had anti-oxidant properties [11], and inhibited pancreatic lipase activity [12]. However, anti-tumor and radical-scavenging activities have not yet been reported for the polyphenols isolated from X. sorbifolia husks.

Chemotaxonomic Significance
The polyphenols identified from X. sorbifolia husks provided an image concerning the chemotaxonomic situation of the genus Xanthoceras within the family Sapindaceae. The main polyphenols isolated from X. sorbifolia husks were protocatechuic acid (6.01 mg/100 g husks), epicatechin (5.24 mg/ 100 g husks), catechin (3.34 mg/100 g husks), rutin (2.81 mg/100 g husks), myricetin-3-O-rutinoside (1.37 mg/100 g husks), quercetin (1.19 mg/100 g husks), and quercitrin (1.12 mg/100 g husks); quercetin and myricetin were the major aglycons in X. sorbifolia husks. Previous phytochemical studies of the genus showed that their polyphenolic pool comprised mostly of flavonoids and phenolic acids [4], but these compounds were not quantified. The present study is the first to isolate and quantify the polyphenolic compounds from X. sorbifolia husks, which might be important for the chemotaxonomy of the genus and family.

Anti-Tumor Effects of X. sorbifolia Polyphenols
In general, the anti-tumor activities of natural products are evaluated by testing their ability to directly inhibit the proliferation of tumor cells, or their capacity to induce immune cells to secrete cytokines that could act on tumor cells [15]. Tests for the anti-tumor activity of the polyphenols isolated from the husks (1-37) at a concentration of 50 µg/mL, revealed that all compounds had null or weak cytotoxicity, when compared to that of paclitaxel and under the conditions described in the present study. The percentage inhibition of compounds 1-37 at 50 µg/mL against four tumor cell lines is summarized in Figures 3 and 4. Protocatechuic acid (7) showed no effect on the lung adenocarcinoma (A549) cell line, similarly to that described in a previous study [50]; however, in that study, protocatechuic acid (7) exhibited anti-tumor activity against other cancer cell lines, at high concentrations. Previous studies [34,51] also reported that epicatechin (19) had no effect or a weak effect on the A549, liver cancer (HepG2), and gastric carcinoma (MGC-803) cell lines, supporting the results found in the present study. It has also been previously reported [52] that catechin (18) [13,34,53]. Although myricetin 3-O-rutinoside (37) showed no or weak anti-tumor activity against A549, HepG2, MGC-803, and murine foregastric carcinoma (MFC) cell lines (as shown in Figures 3 and 4), it was firstly investigated in the present study. Similar to that found in a previous study [33], quercetin (20) had no effect on the MGC-803 cell line. As there were few reports on the in vitro anti-tumor activity of polyphenols on the MFC cell line, the present study represents a major addition to the knowledge on this subject. Overall, polyphenols isolated from X. sorbifolia husks did not strongly inhibit the proliferation of some cancer cell lines, compared to paclitaxel, under the conditions examined in this research. Interestingly, we found that almost half of the compounds exhibited negative inhibition effects, the mechanism was worthy of further study and could be studied by other tumor cell lines or even animal models. showed that their polyphenolic pool comprised mostly of flavonoids and phenolic acids [4], but these compounds were not quantified. The present study is the first to isolate and quantify the polyphenolic compounds from X. sorbifolia husks, which might be important for the chemotaxonomy of the genus and family.

Anti-Tumor Effects of X. sorbifolia Polyphenols
In general, the anti-tumor activities of natural products are evaluated by testing their ability to directly inhibit the proliferation of tumor cells, or their capacity to induce immune cells to secrete cytokines that could act on tumor cells [15]. Tests for the anti-tumor activity of the polyphenols isolated from the husks (1-37) at a concentration of 50 µg/mL, revealed that all compounds had null or weak cytotoxicity, when compared to that of paclitaxel and under the conditions described in the present study. The percentage inhibition of compounds 1-37 at 50 µg/mL against four tumor cell lines is summarized in Figures 3 and 4. Protocatechuic acid (7) showed no effect on the lung adenocarcinoma (A549) cell line, similarly to that described in a previous study [50]; however, in that study, protocatechuic acid (7) exhibited anti-tumor activity against other cancer cell lines, at high concentrations. Previous studies [34,51] also reported that epicatechin (19) had no effect or a weak effect on the A549, liver cancer (HepG2), and gastric carcinoma (MGC-803) cell lines, supporting the results found in the present study. It has also been previously reported [52] that catechin (18) had a weak effect on HepG2 and MGC-803 cell lines. Figures 3 and 4 evidence that rutin (35) exhibited no or weak effects on A549, HepG2, and MGC-803 cell lines, in agreement with previous reports [13,34,53]. Although myricetin 3-O-rutinoside (37) showed no or weak anti-tumor activity against A549, HepG2, MGC-803, and murine foregastric carcinoma (MFC) cell lines (as shown in Figures 3 and 4), it was firstly investigated in the present study. Similar to that found in a previous study [33], quercetin (20) had no effect on the MGC-803 cell line. As there were few reports on the in vitro anti-tumor activity of polyphenols on the MFC cell line, the present study represents a major addition to the knowledge on this subject. Overall, polyphenols isolated from X. sorbifolia husks did not strongly inhibit the proliferation of some cancer cell lines, compared to paclitaxel, under the conditions examined in this research. Interestingly, we found that almost half of the compounds exhibited negative inhibition effects, the mechanism was worthy of further study and could be studied by other tumor cell lines or even animal models.

Radical-Scavenging Activity
There is considerable evidence that free radicals induce oxidative damage to biomolecules and play an important role in cancer and cardiovascular diseases [54]. The 2,2′-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay has been a popular radical-scavenging test for natural components [55] and, therefore, it was used in the present study. The XSB fraction presented the highest radical-scavenging activity among X. sorbifolia husk extracts ( Figure 5). Then the bioassay-guided fractionation of X. sorbifolia husks led to the isolation of 37 polyphenols. The evaluation on the radicalscavenging activity of the polyphenols at a concentration of 50 µg/mL showed that compounds 1-37 had strong radical-scavenging activity ( Figure 6), and, therefore, all compounds were subject to ABTS assay to determine their scavenging capability. As shown in Figure 6 and Table 3, the main polyphenols (protocatechuic acid, epicatechin, (+)-catechin, rutin, myricetin-3-O-rutinoside, quercetin, and quercitrin) exhibited strong radical-scavenging activities, compared to ascorbic acid. Thus, the husks' main compounds strongly contributed to the striking radical-scavenging activity of the XSB fraction. As free radical damage is indicated to be the main cause of cancer [56], although X. sorbifolia polyphenols did not strongly inhibited the proliferation of cancer cell lines under the conditions described in the present research (Figures 3 and 4), they might indirectly present anti-tumor activity by reducing oxidative damage [57].

Radical-Scavenging Activity
There is considerable evidence that free radicals induce oxidative damage to biomolecules and play an important role in cancer and cardiovascular diseases [54]. The 2,2 -azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay has been a popular radical-scavenging test for natural components [55] and, therefore, it was used in the present study. The XSB fraction presented the highest radical-scavenging activity among X. sorbifolia husk extracts ( Figure 5). Then the bioassay-guided fractionation of X. sorbifolia husks led to the isolation of 37 polyphenols. The evaluation on the radical-scavenging activity of the polyphenols at a concentration of 50 µg/mL showed that compounds 1-37 had strong radical-scavenging activity ( Figure 6), and, therefore, all compounds were subject to ABTS assay to determine their scavenging capability. As shown in Figure 6 and Table 3, the main polyphenols (protocatechuic acid, epicatechin, (+)-catechin, rutin, myricetin-3-O-rutinoside, quercetin, and quercitrin) exhibited strong radical-scavenging activities, compared to ascorbic acid. Thus, the husks' main compounds strongly contributed to the striking radical-scavenging activity of the XSB fraction. As free radical damage is indicated to be the main cause of cancer [56], although X. sorbifolia polyphenols did not strongly inhibited the proliferation of cancer cell lines under the conditions described in the present research (Figures 3 and 4), they might indirectly present anti-tumor activity by reducing oxidative damage [57].

Discussion on the SAR of Polyphenols
The polyphenols isolated from X. sorbifolia husks can be divided into three categories ( Figure 1): flavonoids, phenolic acids, and coumarins. The twenty-eight flavonoids can be further classified into flavonols, flavones, flavanones, flavan-3-ols, and flavanonols. Flavonoids with vicinal phenolic hydroxyls presented strong radical-scavenging activity, in agreement with that previously reported [58]. In comparison with the flavonoids bearing saccharide groups, different characteristics of the sugar side chain also play important roles in their radical-scavenging effect. The flavonoids 36, 37 possess the same aglycone, but flavonoid glycoside 37 with a disaccharide chain exhibited weaker radical-scavenging effect than 36 with a monosaccharide chain, which suggested that the presence of a disaccharide chain might reduce the radical-scavenging effect. Thus, the antioxidant activities of these flavonoids would depend on not only the substituent groups on the aglycone, but also the sugar moieties. Phenolic acids with both carboxyl and vicinal phenolic hydroxyls also showed strong radical-scavenging activity. However, coumarins with a single phenolic hydroxyl exhibited stronger radical-scavenging activity than coumarins with vicinal phenolic hydroxyls.

Acidic Hydrolysis of the New Compound and Sugar Analysis
The new compound 2 (2 mg) was added to a solution of concentrated HCl (0.5 mL), and refluxed for 3 h, by adding H 2 O (1.5 mL)/dioxane (3 mL). After dilution with H 2 O, the reaction mixture was subjected to extraction twice with ethyl acetate (EtOAc). The H 2 O layers of compound 2 were then neutralized with NaHCO 3 and concentrated to dryness under reduced pressure. The residue was re-dissolved in H 2 O for TLC analysis.

Anti-Tumor Assay
Compounds 1-37 were evaluated for their anti-tumor activity against three human cancer cell lines, the lung adenocarcinoma (A549), liver cancer (HepG2), and gastric carcinoma (MGC-803) cell lines, and one murine foregastric carcinoma (MFC) cell line, which were obtained from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum (both from Hyclone, South Logan, UT, USA), in 5% CO 2 at 37 • C. The anti-tumor assay was performed according to the MTT method [58], with some modifications. In brief, A549, HepG2, MGC-803, and MFC cells were seeded into 96-well plates (5 × 10 3 cells/well) for 20-24 h under the above conditions, treated with 50 µg/mL of each tested compound, and further incubated for 24 h under the same conditions. After this period, 20 µL of MTT stock solution (5 mg/mL in PBS) were added to each well and samples were incubated for another 4 h, under the same conditions. Supernatants were then removed, and 150 µL of dimethyl sulfoxide were added to each well. After 10 min, absorbance was determined on a Multiskan MK 3 Automated Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA) at 490 nm, using paclitaxel as the positive control. The percentage inhibition was calculated according to: where AA is the inhibition percentage, Ao is the absorbance of the blank sample, and Ai is the absorbance of the test sample.

ABTS Radical-Scavenging Assay
Samples (extracts and polyphenols from X. sorbifolia husks) ability to scavenge the ABTS radical cation was measured following a previously reported method [59], with some modifications, and using L-ascorbic acid as the positive control. Assays were performed in 96-well plates and absorbance was measured at 734 nm. The radical-scavenging activity of each sample was expressed as the percentage inhibition of the ABTS radical and determined according to Equation (1).

Statistical Analysis
All experiments were carried out in three replicates to ensure reproducibility. Sample concentrations providing 50% scavenging capability (SC 50 ) were obtained by fitting dose-response data to a four-parametric logistic nonlinear regression model, using GraphPad Prism 5.0 software (GraphPad, La Jolla, CA, USA).

Conclusions
In conclusion, quercetin and myricetin polyphenols were the main aglycons in X. sorbifolia husks, and this might be of great chemotaxonomic importance within the genus Xanthoceras and the family Sapindaceae. Pharmacological studies showed that, although compounds 1-37 did not show strong cytotoxicity against the four tumor cell lines (A549, HepG2, MGC-803, and MFC), compared to paclitaxel and under the conditions described in the present research, compounds 3, 4, 7, 8, 10, 18-20,  25, 26, 29, 30, 32, and 35 showed stronger radical-scavenging activity than ascorbic acid in the ABTS assay. This was the first report on the anti-tumor and radical-scavenging activities of the polyphenols isolated from X. sorbifolia husks. The results obtained in the present study contribute important baseline information on the biological activity of X. sorbifolia husks, which might contribute for their pharmacological application.