Flavonoids from Selaginella doederleinii Hieron and Their Antioxidant and Antiproliferative Activities

Selaginella doederleinii Hieron. (S. doederleinii) is a traditional herb that is widely used in China to treat several ailments, but mainly cancer. Studies have been carried out to determine the phytochemicals ascribed to its pharmacological activity. However, both phytochemical and pharmacological profiles have not been fully explored as few compounds have been reported. This study evaluated the flavonoid content of the ethanol extract and its four fractions (petroleum ether, dichloromethane, ethyl acetate, and n-butanol) together with their antioxidant activity (DPPH and FRAP assays). Further, the antiproliferative activity was evaluated. Two new secondary metabolites (1 and 3) were isolated from S. doederleinii, which comprised of an apigenin skeleton with a phenyl attached at C-8 of ring A and an acetyl group. Additionally, other known metabolites 2 and 4–16 were isolated, whereby compounds 2, 4, 5, 8, 12, 15, and 16 were reported for the first time in this species. These compounds were evaluated for their antioxidative potentials by both DPPH and FRAP assays, and for their antiproliferative activities by the MTT assay on three human cancer cell lines: colon cancer (HT-29), cervical cancer (HeLa), and lung cancer (A549). Compound 7 exhibited the best activity on the three cancer cell lines (HT-29, HeLa, A549) by inhibiting the rate of growth of the cancer cells in a dose-dependent manner with IC50 values of 27.97, 35.47, and 20.71 µM, respectively. The structure–activity relationship of the pure compounds was highlighted in this study. Hence, the study enriched both the phytochemical and pharmacological profiles of S. doederleinii.


Introduction
Cancer has persistently remained a global health concern by claiming many human lives [1]. In some developed countries, cancer incidences and the rate of mortality for many cancers have been reported to be decreasing. However, in developing countries, both morbidity and mortality rates are escalating at an alarming rate [2]. Screening and developing new anticancer chemotherapeutic drugs have remained an urgent approach in cancer management and mitigation [3]. Additionally, since a number of these phytochemicals' solubility in water is poor, studies on the administration of plant extracts and pure isolated compounds to the delivery system are imperative. This would amplify their oral bioavailability and control the release of the drug payloads [4].

Extraction and Separation
The dried plant materials (8.0 kg) were extracted by maceration with 75% ethanol (4 times, 3 days/time) at room temperature. The ethanol extract was evaporated under reduced pressure to obtain a residue (638.2 g). The obtained ethanol extract was then suspended in water for liquid-liquid extraction and successively extracted with petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EA), and n-butanol (n-BuOH) to obtain their corresponding fractions.

Determination of the Total Flavonoid Content (TFC)
The TFC analysis was evaluated using the colorimetric method as described [33,34], with some modifications. Briefly, 80 µL of a diluted sample solution was mixed with NaNO 2 (80 µL 5% w/v) solution and then shaken for 6 min. AlCl 3 (80 µL 10% w/v) was added and allowed to stand for 6 min. Then, NaOH (400 µL 4% w/v) solution was added and allowed to react for 15 min. Afterward, the absorbance of the reaction mixture was read at 510 nm with a UV/VIS spectrophotometer (UV-11000, MAPADA, Shanghai, China) with methanol used as the blank. The TFC of each sample was evaluated in triplicate and expressed as rutin equivalents, which were determined from a rutin calibration curve (100-600 µg/mL), and the results were expressed as mg RE/g sample.
2.5. In Vitro Antioxidant Assays 2.5.1. DPPH (2,2-diphenyl-1-picrylhydrazyl) Assay The DPPH assay of S. doederleinii ethanol extract and four fractions was assessed as described in [35,36], with some minor modifications. Firstly, the DPPH solution was prepared with methanol at a concentration of 0.1 mM. Then, 10 µL of prepared samples and standards (vitamin C and BHT) of 9.375-250 µg/mL were added to 190 µL of the DPPH solution in each well of a 96-well plate. The mixture was shaken and incubated in darkness for 30 min. The absorbance of the reaction mixture was then taken at 517 nm using a multifunctional microplate reader (Tecan, Infinite M20PRO, Switzerland) with methanol being used as the blank. The analysis was done in triplicates and the results were expressed as the inhibition rate (%) and IC 50 values. The DPPH radical scavenging activity was then calculated and expressed as follows: where A 0 is the control absorbance and A 1 is the sample/standard control absorbance.

Ferric Reducing Antioxidant Power (FRAP) Assay
This assay was evaluated on the ethanol extract and its fractions (PE, DCM, EA, and n-BuOH) of S. doederleinii according to the reported method, with some slight modifications [37]. Firstly, a working solution, FRAP reagent comprised of 300 mM acetate buffer of pH 3.6, 20 mM FeCl 3 ·6H 2 O solution, and 10 mM TPTZ (2,4,6-tri(2-pyridyl)-S-triazine) solution in a ratio of 10:1:1 (v/v/v), was used. The working solution was then heated to 37 • C before use and 190 µL of FRAP working solution was mixed with 10 µL of sample in a 96-well plate. The mixture was then incubated at 37 • C for 10 min. The absorbance of the mixture was recorded by a microplate reader at a wavelength of 593 nm. The tests were done in triplicates and a standard curve was established. Eventually, the antioxidant activities were calculated and expressed as mmol Fe 2+ /g of the sample.

Antiproliferative Activity
The antiproliferative activity was performed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method [38], with some modifications. Three human cancer cell lines, colon cancer (HT-29), cervical cancer (HeLa), and lung cancer (A549), were tested. The three cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), which was supplemented with 10% fetal bovine serum (FBS). The 90 µL cell suspension was added to each well and then the 96-well cell culture plates were maintained at 37 • C in a 5% CO 2 atmosphere for 24 h to culture. Afterwards, 10 µL of samples at different concentrations (final concentration 6.25, 12.5, 25, 50, and 100 µM) were added to the wells in triplicates and the positive control was also set up. After incubating for 48 h, 15 µL of MTT (5 mg/mL) was added to each well and incubated at 37 • C for 4 h. Afterwards, 100 mL of DMSO was then added to each well and shaken for 15 min to dissolve the precipitates formed. The OD value of each well was measured at 590 nm with a microplate spectrophotometer reader (Tecan Infinite M200 PRO, TECAN, Männedorf, Switzerland). Then, the IC 50 values were calculated by GraphPad Prism 8.0.1 Software (GraphPad Software Inc., San Diego, CA, USA).

Statistical Analysis
All the experiments were performed and data were expressed as mean ± standard deviation (SD) of triplicate values. Data analysis was performed by SPSS statistics 22 software (IBM Corporation, New York, NY, USA) using one-way ANOVA Duncan's multiple range test and the significance difference was considered at p < 0.05. The IC 50

Total Flavonoid Content
With S. doederleinii being used traditionally to treat cancer for decades and flavonoids having been shown as its main active constituents [25], it was necessary to evaluate its total flavonoid content (TFC). The flavonoid content was calculated using the equation, (y = 0.0013x + 0.0146, R 2 = 0.9967), which was obtained by the calibration curve and ranged from 340.8 ± 1.0 to 72.2 ± 8.7 mg RE/g-with the dichloromethane fraction expressing the highest content and n-butanol the least, as shown in Figure 1; the order of the other constituents were: (2) ethyl acetate extract, (3) petroleum ether, and (4) crude extract with values of 310.3 ± 3.1, 104.2 ± 2.0, and 84.0 ± 3.6 mg RE/g, respectively. As seen from Figure 1, it could be noted that the TFC values of the DCM and EA fractions were close in range, with DCM being higher by 1.1 times. On the other hand, ethanolic extracts of Selaginella tenera and Selaginella inaequalifolia exhibited slightly higher TFC values of 125.6 ± 4.3 and 138.4 ± 2.1 mg RE/g, respectively [39], compared to our ethanol extract TFC content. The difference in values could be attributed to the extraction methodology and the geographical locations of the two species [40].

Total Flavonoid Content
With S. doederleinii being used traditionally to treat cancer for decades and flavonoids having been shown as its main active constituents [25], it was necessary to evaluate its total flavonoid content (TFC). The flavonoid content was calculated using the equation, (y = 0.0013x + 0.0146, R 2 = 0.9967), which was obtained by the calibration curve and ranged from 340.8 ± 1.0 to 72.2 ± 8.7 mg RE/g-with the dichloromethane fraction expressing the highest content and n-butanol the least, as shown in Figure 1; the order of the other constituents were: (2) ethyl acetate extract, (3) petroleum ether, and (4) crude extract with values of 310.3 ± 3.1, 104.2 ± 2.0, and 84.0 ± 3.6 mg RE/g, respectively. As seen from Figure  1, it could be noted that the TFC values of the DCM and EA fractions were close in range, with DCM being higher by 1.1 times. On the other hand, ethanolic extracts of Selaginella tenera and Selaginella inaequalifolia exhibited slightly higher TFC values of 125.6 ± 4.3 and 138.4 ± 2.1 mg RE/g, respectively [39], compared to our ethanol extract TFC content. The difference in values could be attributed to the extraction methodology and the geographical locations of the two species [40].

In-Vitro Antioxidant Potential of S. doederleinii Extracts
The ethanol extract and its fractions were evaluated for their scavenging potential and expressed different inhibition percentages. From Figure 2, the EA fraction expressed the highest inhibition percentage (DPPH) at a sample concentration of 250 µg/mL, followed by n-BuOH, DCM, ethanol extract, and PE with 80.9, 79.7, 69.5, 56.6, and 55.4%, respectively. The IC50 values for ethanol extract, its fractions, and positive controls were shown in Table 1. The EA fraction exhibited the best antioxidant activity, followed by DCM according to their IC50 values, while ethanol extract exhibited the lowest activity.

In-Vitro Antioxidant Potential of S. doederleinii Extracts
The ethanol extract and its fractions were evaluated for their scavenging potential and expressed different inhibition percentages. From Figure 2, the EA fraction expressed the highest inhibition percentage (DPPH) at a sample concentration of 250 µg/mL, followed by n-BuOH, DCM, ethanol extract, and PE with 80.9, 79.7, 69.5, 56.6, and 55.4%, respectively. The IC 50 values for ethanol extract, its fractions, and positive controls were shown in Table 1. The EA fraction exhibited the best antioxidant activity, followed by DCM according to their IC 50 values, while ethanol extract exhibited the lowest activity. However, the FRAP assay indicated that the DCM fraction had the highest reducing ability, followed by the EA fraction with 2.6 ± 0.1 and 1.7 ± 0.0 mmol Fe 2+/ g, respectively. Our fraction exhibited a slightly lower antioxidant activity compared to the DPPH assay results reported by Wang et al. [32]. In both assays, the ethanol extract and PE fraction exhibited the lowest scavenging activity, while both the DCM and EA fractions depicted strong activities, which were closely attributed with their TFC yields.
However, the FRAP assay indicated that the DCM fraction had the highest reducing ability, followed by the EA fraction with 2.6 ± 0.1 and 1.7 ± 0.0 mmol Fe 2+/ g, respectively. Our fraction exhibited a slightly lower antioxidant activity compared to the DPPH assay results reported by Wang et al. [32]. In both assays, the ethanol extract and PE fraction exhibited the lowest scavenging activity, while both the DCM and EA fractions depicted strong activities, which were closely attributed with their TFC yields.

Antiproliferative Activity of S. doederleinii Extracts
The antioxidant assays and TFC values revealed that both DCM and EA were the most active fractions of S. doederleinii when compared to the others. In this regard, both extracts were evaluated for their antiproliferative activity on three cancer cell lines: HT-29, HeLa, and A549 at different concentrations ranging from 12.5 to 200 µg/mL. The inhibition rates are shown in Figure 3, while the IC50 values are shown in Table 2. The inhibition rate of the solvent was almost zero, which confirmed that the solvent used did not influence the cytotoxicity of the samples. Additionally, the toxicity investigation revealed that the solvents did not influence cell viability. The EA fraction exhibited the best antiproliferative activity against the HT-29 and HeLa cell lines by inhibiting the cell growth rate in a dose-dependent manner with IC50 values of 55.6 ± 1.3 and 69.2 ± 1.3 µg/mL, respectively. The DCM fraction exhibited the best activity against the A549 cell line with an IC50 value of 55.9 ± 12.6 µg/mL. Song et al. [41] evaluated the anticancer activities of the extracts of S. doederleinii collected from different provinces in China against the A549 cancer cell line. Comparing the activities of the extracts with those of ours, our DCM extract Data were expressed as means ± standard deviation (n = 3). The mean values denoted by letters (a-f) are significantly different at level p < 0.05 by one-way ANOVA DMRT. Nt denote, not tested.

Antiproliferative Activity of S. doederleinii Extracts
The antioxidant assays and TFC values revealed that both DCM and EA were the most active fractions of S. doederleinii when compared to the others. In this regard, both extracts were evaluated for their antiproliferative activity on three cancer cell lines: HT-29, HeLa, and A549 at different concentrations ranging from 12.5 to 200 µg/mL. The inhibition rates are shown in Figure 3, while the IC 50 values are shown in Table 2. The inhibition rate of the solvent was almost zero, which confirmed that the solvent used did not influence the cytotoxicity of the samples. Additionally, the toxicity investigation revealed that the solvents did not influence cell viability. The EA fraction exhibited the best antiproliferative activity against the HT-29 and HeLa cell lines by inhibiting the cell growth rate in a dosedependent manner with IC 50 values of 55.6 ± 1.3 and 69.2 ± 1.3 µg/mL, respectively. The DCM fraction exhibited the best activity against the A549 cell line with an IC 50 value of 55.9 ± 12.6 µg/mL. Song et al. [41] evaluated the anticancer activities of the extracts of S. doederleinii collected from different provinces in China against the A549 cancer cell line. Comparing the activities of the extracts with those of ours, our DCM extract exhibited better activity than most of the fractions. Our EA fraction exhibited better antiproliferative activity on the HeLa cancer cell line compared with that reported by Wang et al. [42], which had an IC 50 value of 76.1 ± 1.9 µg/mL. These results explained the traditional use of S. doederleinii to cure and manage cancers. To this end, flavonoids expressed in the TFC results could be presumed to play a role in the antiproliferative activity of this species (both DCM and EA fractions) by suppressing the formation of cancers that emerge from oxidative stress. Accordingly, for a better understanding and exploration of this species towards cancer, the DCM fraction was selected for isolation work to identify the responsible bioactive phytochemicals. tional use of S. doederleinii to cure and manage cancers. To this end, flavonoids expressed in the TFC results could be presumed to play a role in the antiproliferative activity of this species (both DCM and EA fractions) by suppressing the formation of cancers that emerge from oxidative stress. Accordingly, for a better understanding and exploration of this species towards cancer, the DCM fraction was selected for isolation work to identify the responsible bioactive phytochemicals.

Isolation and Structure Elucidation
A phytochemical examination of the DCM fraction of the whole plant of S. doederleinii using different column chromatography yielded two new compounds (1 and 3). Besides the new compounds, 14 other known compounds ( Figure 4) were isolated and their chemical structures were determined by comparison of their NMR data (both 1 H and 13 C), as per existing literature.
Compound 1 was isolated as a yellow amorphous powder. Its molecular formula was deduced as C25H20O8, owing to a molecular ion peak observed at m/z 449.1227 [M + H] + (calculated for 449.1231) in the HR-ESI-MS, as shown in Figure S2, which was per the 1 H NMR and 13 C NMR spectroscopic data (Table 3) Hz, H-3′, 5′) indicated the parasubstitution of ring B. Two aromatic singlets were allocated to H-3 and H-6. The aromatic singlet at δH 6.68 was assigned to H-3 as it showed an HMBC correlation ( Figure S8) with C-10 (δC 103.8) and C-2 (δC 164.6), and δH 6.60 was assigned to H-6 since H-8 was involved

Isolation and Structure Elucidation
A phytochemical examination of the DCM fraction of the whole plant of S. doederleinii using different column chromatography yielded two new compounds (1 and 3). Besides the new compounds, 14 other known compounds ( Figure 4) were isolated and their chemical structures were determined by comparison of their NMR data (both 1 H and 13 C), as per existing literature.
Compound 1 was isolated as a yellow amorphous powder. Its molecular formula was deduced as C 25 H 20 O 8, owing to a molecular ion peak observed at m/z 449.1227 [M + H] + (calculated for 449.1231) in the HR-ESI-MS, as shown in Figure S2, which was per the 1 H NMR and 13 C NMR spectroscopic data (  , H-3 ). An AA XX coupling system signal at δ H 7.56 (2H, d, J = 8.9 Hz, H-2 , 6 ) and δ H 6.93 (2H, d, J = 8.9 Hz, H-3 , 5 ) indicated the para-substitution of ring B. Two aromatic singlets were allocated to H-3 and H-6. The aromatic singlet at δ H 6.68 was assigned to H-3 as it showed an HMBC correlation ( Figure S8) with C-10 (δ C 103.8) and C-2 (δ C 164.6), and δ H 6.60 was assigned to H-6 since H-8 was involved in the linkage between the flavonoid unit and the benzene ring (ring D). This was confirmed by the HMBC correlations from H-6 (δ H 7.93) to C-8 (δ C 105.6), as shown in structure 1 in Figure 5. All 25 carbon resonances were resolved in the 13 C NMR spectrum (Table 3 and Figure S4) and were further classified by a DEPT spectrum ( Figure S5). They were categorized as 3 methyl (oxygenated), 9 methines (unsaturated), and 13 quaternary carbons (2 carbonyl).   The three methoxy groups were assigned to be attached to C-7, C-4 , and C-2 , which were determined by HMBC signals from δ H 3.82 to δ C 163.3 (C-7), δ H 3.86 to δ C 164.4 (C-4 ), and δ H 3.77 to δ C 163.1 (C-2 ). Besides the tri-substituted benzene ring (ring D), the remaining signals disclosed that Compound 1 had a flavonoid skeleton. The singlet proton at δ H 6.60 (H-6) suggested that ring A could be substituted either at C-6 or C-8, and the HMBC studies have shown that the 1,2,5-trisubstituted benzene ring (ring D) was linked to C-8 by the correlation from δ H 7.93 to δ C 105.6 (C-8). It was concluded that compound 1 was an apigenin derivative and the chemical structure was determined as 3-(5-hydroxy-7methoxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen-8-yl)-4-methoxybenzoic acid.  (Table 3 and Figure S12). The singlet methyl group at δ H 2.57 (3H, s, H-8 ), along with the carbonyl carbon at δ C 199.6, revealed an acetyl group that, based on the HMBC correlation of H-4 and H-8 to C-7 ( Figure 5), was attached to C-5 of ring D. Additionally, an AA XX coupling system signal at δ H 7.64 (2H, d, J = 8.9 Hz, H-2 /6 ) and 6.94 (2H, d, J = 8.9 Hz, H-3 /5 ) indicated the para-substitution of ring B, and the two aromatic singlets at δ H 6.68 and 6.40 were assigned to H-3 and H-6, respectively. All 24 carbons were displayed in the 13 C NMR spectrum ( Figure S12), which included 15 carbons for the apigenin skeleton, 6 for the phenyl (ring D), 2 for the acetyl group at δ C 199.6 and 26.9, and 1 for the methoxyl at δ C 56.0. The HMBC spectrum ( Figure S16

Antioxidant Activities of Isolated Compounds from S. doederleinii
The isolated compounds from S. doederleinii were evaluated for their antioxidant activity by DPPH and FRAP assays. All the examined compounds exhibited radical scavenging abilities at different concentrations with the lowest and highest concentrations of 6.25 and 100 µM, respectively, as shown in Figure 6. Compound 14 expressed the best antioxidant activity among the tested compounds with an IC50 value of 89.3 ± 4.0 µM, while the positive control (Vitamin C) had an IC50 value of 20.3 ± 0.2 µM. The radical scavenging ability of the isolated compounds from S. doederleinii is attributed to the hydroxy groups in their structures, which donate a hydrogen atom to neutralize the free radicals, hence suppressing their oxidation potentials. The tested compounds expressed close free radical scavenging abilities even at the highest concentration of 100 µM, except compound 14 which had a higher value as compared to the rest. The FRAP assay results (Figure 7) also revealed that compound 14 exhibited the highest ferric reducing ability with a value of 1.4 ± 0.03 mM Fe 2+ /g, followed by compound 4 with a value of 1.1 ± 0.02 mmol Fe 2+/ g, which also exhibited the second highest DPPH radical scavenging rate at concentration of 100 µM. Vitamin C was used as the positive control on the FRAP assay and it exhibited an ion-reducing capacity with a value of 7.8 ± 1.2 mM Fe 2+ /g. The antioxidant activity of the isolated compounds from S. doederleinii has not been reported before, hence our work reports this for the first time. Flavonoids derived from plants have been reported to be strong antioxidants [56]. Bedir et al. [44] evaluated the antioxidant activity of flavonoids and four biflavonoids (Amentoflavone, Bilobetin, Ginkgetin, and Sciadopitysin). The flavonoids exhibited noble antioxidant activity. However, none of the four biflavonoids evaluated exhibited strong antioxidant activity. Another study by Orčić et al. [57] revealed low antioxidant activity of biflavonoids isolated from Hypericum perforatum species, whereas the monomer flavonoids exhibited strong antioxidant activities. Previous studies in the same species had reported low antioxidant activities of isolated biflavonoids. This is in support of our findings, whereby flavonoid compound 14 exhibited the strongest   (Table 3). By comparing the 1 H NMR and 13 C NMR data of compound 3 to that of 4, it was observed that there was an additional methoxyl that was attached at C-2 of ring D, according to the HMBC correlations from δ H 3.82 to C-2 (δ C 163.3). The structure of compound 4 was thus determined as 8-(5-acetyl-2-methoxyphenyl)-5,7-dihydroxy-2-(4methoxyphenyl)-4H-chromen-4-one [43]. This is the first time its spectroscopic data and its isolation from natural resources have been reported.
Compound 5 was isolated as a yellow powder. It shared the same skeleton structure with compounds 3 and 4 but with three methoxy groups. 1 H NMR and 13 C NMR data gave the molecular formula as C 26 H 22 O 7 . The 1 H NMR and 13 C NMR spectroscopic data (Table 3) of 5 closely resembled that of 4, except that the hydroxyl at C-7 of ring A was substituted by a methoxy group. The HMBC studies of this compound indicated that the three methoxy groups are attached at C-7, C-4 , and C-2 , as indicated in Figure 5. Hence, compound 5 was determined as 8-(5-acetyl-2-methoxyphenyl)-5-hydroxy-7-methoxy-2-(4methoxyphenyl)-4H-chromen-4-one [43]. The spectroscopic data of 5 is also being reported for the first time in this study, as well as its isolation from natural resources.

Antioxidant Activities of Isolated Compounds from S. doederleinii
The isolated compounds from S. doederleinii were evaluated for their antioxidant activity by DPPH and FRAP assays. All the examined compounds exhibited radical scavenging abilities at different concentrations with the lowest and highest concentrations of 6.25 and 100 µM, respectively, as shown in Figure 6. Compound 14 expressed the best antioxidant activity among the tested compounds with an IC 50 value of 89.3 ± 4.0 µM, while the positive control (Vitamin C) had an IC 50 value of 20.3 ± 0.2 µM. The radical scavenging ability of the isolated compounds from S. doederleinii is attributed to the hydroxy groups in their structures, which donate a hydrogen atom to neutralize the free radicals, hence suppressing their oxidation potentials. The tested compounds expressed close free radical scavenging abilities even at the highest concentration of 100 µM, except compound 14 which had a higher value as compared to the rest. The FRAP assay results (Figure 7) also revealed that compound 14 exhibited the highest ferric reducing ability with a value of 1.4 ± 0.03 mM Fe 2+ /g, followed by compound 4 with a value of 1.1 ± 0.02 mmol Fe 2+ /g, which also exhibited the second highest DPPH radical scavenging rate at concentration of 100 µM. Vitamin C was used as the positive control on the FRAP assay and it exhibited an ion-reducing capacity with a value of 7.8 ± 1.2 mM Fe 2+ /g. The antioxidant activity of the isolated compounds from S. doederleinii has not been reported before, hence our work reports this for the first time. Flavonoids derived from plants have been reported to be strong antioxidants [56]. Bedir et al. [44] evaluated the antioxidant activity of flavonoids and four biflavonoids (Amentoflavone, Bilobetin, Ginkgetin, and Sciadopitysin). The flavonoids exhibited noble antioxidant activity. However, none of the four biflavonoids evaluated exhibited strong antioxidant activity. Another study by Orčić et al. [57] revealed low antioxidant activity of biflavonoids isolated from Hypericum perforatum species, whereas the monomer flavonoids exhibited strong antioxidant activities. Previous studies in the same species had reported low antioxidant activities of isolated biflavonoids. This is in support of our findings, whereby flavonoid compound 14 exhibited the strongest antioxidant activity compared to the rest of the tested compounds, which were mainly biflavonoids. antioxidant activity compared to the rest of the tested compounds, which were mainly biflavonoids.

Antiproliferation Activity of Compounds Isolated from S. doederleinii
All the isolated compounds were evaluated for their antiproliferation activity against three human cancer cell lines: HT-29, HeLa, and A549 by the MTT method. All the compounds showed antiproliferation activity on the three tested cancer cell lines to different degrees. Interestingly, these compounds expressed some level of antiproliferation on cancer cell line A549, which could give an insight into its major traditional use for lung cancer treatment and management. Among the 16 compounds, three (8,9,16) expressed the best activity by inhibiting the rate of cell growth in a dose-dependent manner on the three cancer cell lines, and their IC50 values were shown in Table 4. Interestingly, the three were biflavonoids, which have continued to be of interest in the search for cancer drugs [28,58].   antioxidant activity compared to the rest of the tested compounds, which were mainly biflavonoids.

Antiproliferation Activity of Compounds Isolated from S. doederleinii
All the isolated compounds were evaluated for their antiproliferation activity against three human cancer cell lines: HT-29, HeLa, and A549 by the MTT method. All the compounds showed antiproliferation activity on the three tested cancer cell lines to different degrees. Interestingly, these compounds expressed some level of antiproliferation on cancer cell line A549, which could give an insight into its major traditional use for lung cancer treatment and management. Among the 16 compounds, three (8,9,16) expressed the best activity by inhibiting the rate of cell growth in a dose-dependent manner on the three cancer cell lines, and their IC50 values were shown in Table 4. Interestingly, the three were biflavonoids, which have continued to be of interest in the search for cancer drugs [28,58].

Antiproliferation Activity of Compounds Isolated from S. doederleinii
All the isolated compounds were evaluated for their antiproliferation activity against three human cancer cell lines: HT-29, HeLa, and A549 by the MTT method. All the compounds showed antiproliferation activity on the three tested cancer cell lines to different degrees. Interestingly, these compounds expressed some level of antiproliferation on cancer cell line A549, which could give an insight into its major traditional use for lung cancer treatment and management. Among the 16 compounds, three (8,9,16) expressed the best activity by inhibiting the rate of cell growth in a dose-dependent manner on the three cancer cell lines, and their IC 50 values were shown in Table 4. Interestingly, the three were biflavonoids, which have continued to be of interest in the search for cancer drugs [28,58]. Compounds 8 and 16 exhibited noble activities on cancer cell line A549 as compared to their activities on the other cell lines. This affirmed the DCM fraction antiproliferative activity on cancer cell line A549, which exhibited the best activity compared to the other cell lines. Additionally, it supports the main use of this species, which is traditionally in the treatment and management of lung cancer.

Structure-Activity Relationship of S. doederleinii Phytochemicals
The structure-activity relationship study of our results was interesting, with all compounds exhibiting obvious cytotoxicity on the three cancer cell lines. The two amentoflavone derivatives (8 and 9) exhibited antiproliferation activity, with 8 having the best activity on the HeLa and A549 cancer cell lines and 9 having the best activity on the HT-29 cell line, as shown in Table 4. Compound 8 exhibited better activity than 9, this could be attributed to the OH at C-5,7 of ring A and C-4 of ring B of the first flavonoid unit as compared to 9, which had OCH 3 at C-7,4 . This confirms the importance of OH at C-5,7 of ring A and at C-3 ,4 of ring B [59][60][61]. When comparing the antiproliferation of the hinokiflavone derivatives, compound 16 exhibited more interesting activity than 15 with the best activity on the three human cancer cell lines among the tested compounds. The noble activity of 16 was enhanced by the methoxy group at C-7 of ring A of the second flavonoid unit. This was in accordance with Du et al. [62], who established that methylation at ring A enhances the antiproliferative activity of flavones.

Conclusions
In this study, the TFC, antioxidant (DPPH and FRAP assays), and antiproliferative potentials of the ethanol extract and its fractions were evaluated. The DCM and EA fractions depicted good potency on the three bioassays. The phytochemical investigation was carried out to identify the phytochemicals responsible for its antioxidant and antiproliferative activities. This resulted in the isolation of 16 compounds, including two new compounds (1 and 3). The isolated compounds were then evaluated for their antioxidative and antiproliferative potentials. All the evaluated compounds exhibited some free radical scavenging ability. Compound 14 expressed the best antioxidant activity on the DPPH assay and the highest ferric reducing antioxidant ability on the FRAP assay. The antiproliferative activity was tested by MTT assay on three human cancer cell lines: HT-29, HeLa, and A549. Compound 16 (7 -methyl ether Tetrahydrohinokiflavone) exhibited the strongest activity by inhibiting the rate of cell growth in a dose-dependent manner on the three cancer cell lines. Compounds 8 and 16 exhibited noble antiproliferative activities on the A549 cancer cell line, hence they could be promising lung cancer drug candidates. The study has therefore supported the traditional use of S. doederleinii in cancer treatment and identified the bioactive chemical constituents responsible for its pharmacological properties. Additionally, the study has enriched the phytochemical constitution of S. doederleinii as well as its pharmacological profile. However, we strongly suggest more isolation work to expand the phytochemical profile of this species with new compounds of different classes as it has been reported in other species of the genus Selaginella.
Author Contributions: M.G. conceived of, designed, and supervised the study. F.W.M., Y.L., G.C. and Y.Z. performed the experiments, analyzed the data. F.W.M. wrote the original manuscript. Y.L. revised the manuscript and co-supervised the study. All authors have read and agreed to the published version of the manuscript.
Funding: This research was partly supported by the Natural Science Foundation of Hubei Province, grant number 2020CFB486, to Ye Liu.