Polyphenols of Frangula alnus and Peganum harmala Leaves and Associated Biological Activities

Frangula alnus and Peganum harmala populations growing in Saudi Arabia might be rich sources of natural compounds with important biological activities. A high performance liquid chromatography diode array revealed several polyphenols in the leaf extracts for the first time, including p-coumaric acid, rosmarinic acid, chlorogenic acid, ferulic acid, quercitrin, rutoside, quercetin and trifolin in F. alnus; and hydrocaffeic acid, protocatechuic acid, rosmarinic acid, caffeic acid and cynaroside in P. harmala. F. alnus and P. harmala showed strong antioxidant effects attributed to the polyphenolic composition of leaves and reduction of reactive oxygen species (ROS) accumulation. F. alnus and P. harmala leaf extracts showed cytotoxic effects against Jurkat, MCF-7, HeLa, and HT-29 cancer cells using MTT and flow cytometry assays. These activities were attributed to the polyphenolic composition of leaves including quercitrin, trifolin and cymaroside, as well as the activation of caspase family enzymes 2, 6, 8 and 9 in treated cancer cells compared to control. The current findings of this study include a novel comprehensive investigation on the polyphenol composition and anticancer effects of leaf extracts of F. alnus and P. harmala from natural populations in Saudi Arabia.


Introduction
Natural populations of medicinal plants have been a valuable source of therapeutic compounds and drug discovery [1]. These compounds include polyphenols which may reduce certain age-associated illnesses including cancer and Alzheimer's disease by confronting escalated cellular damage through significant reduction of reactive oxygen species (ROS) and inflammatory status [2,3]. The antiproliferative and cytotoxic activities of polyphenols against human cancer cells are also attributed to cell cycle arrest and intermolecular regulation of known genes [4][5][6][7][8][9].

Antiproliferative Effects
Antiproliferative effects of F. alnus and P. harmala extracts were tested against an array of cancer cells including Jurkat, HeLa, MCF-7, and HT-29. [6,27,28]. HEK-293 normal human cells were also used. To measure the changes in cell viability (antiproliferative effect), we used MTT. The leaf extracts obtained before were solubilized in DMSO (1%). Leaf extract solutions (serial concentrations) were added to prepared MEM medium containing 0.1 mM nonessential amino acids, 10% FBS, 1 mM sodium pyruvate, and 17.8 mM NaHCO 3 in 75 cm 2 flasks. The medium contained cancer/normal cells 4 × 10 −4 cells µL -1 . A washing step was performed using PBS. The MTT solution (12 mM) was mixed the medium. Isopropanol (0.04 N HCl) was mixed as well and left for 40 min. A positive control (vinblastine sulfate and taxol) and negative control (untreated) were used. Absorbance was measured at 570 nm wavelength using the following equation: The inhibition activity percentage = (AB 570 nm ) C − (AB 570 nm ) s (AB 570 nm ) C × 100 where: AB is absorbance (AB 570nm ) C and (AB 570 nm ) s are the absorbances of the control and sample, respectively.

Cytotoxic Effects
The IC 50 values of each prepared extract were calculated by plotting percentage of viable cells against the concentration of the extract in µg mL −1 . These IC 50 values were employed in the flow cytometry experiment. The apoptotic cell populations were determined for selected cancer cells using an FAC Scan, USA [6,27,29].

Antioxidant Activity
The antioxidant effects of F. alnus and P. harmala extracts were explored in three different experiments: ferric reducing antioxidant power (FRAP), β-carotene bleaching and 2,2-diphenyl-1-picrylhydrazyl (DPPH) [28,[30][31][32][33][34]. The IC 50 (µg/mL) values were defined as the amount of extract scavenging 50% of β-carotene bleaching/DPPH solution/FRAP reagent. These values were calculated by plotting the inhibition percent against extract concentration. In the DPPH experiment, serial concentrations of the extracts were incubated in methanolic DPPH solution previously prepared (5 mL of 0.004%) for 30 min in the dark at room temperature. The absorbance was measured at Plants 2020, 9, 1086 4 of 15 517 nm. A positive control was used in the experiment (butylated hydroxytoluene, BHT). In the β-carotene-bleaching experiment, the absorbance was measured at 470 nm. In the FRAP experiment, Trolox (positive control) was used and the absorbance was measured at 593 nm. All experiments were repeated thrice.

ROS Intercellular Accumulation
This assay determined the ability of the obtained leaf extracts to reduce the intracellular levels of ROS in HeLa, Jurkat, T24, and MCF-7 cancer cells. The assay employs the fluorogenic dye (H 2 DCF-DA) [35]. The cancer cells were exposed to leaf extracts or polyphenols IC 50 values (determined by DPPH). DCF fluorescence was determined after 90 min of treatment at 485 nm. Hydrogen peroxide (H 2 O 2 ) was used as a positive control.

Caspase Activity by Colorimetric Assay
The effect of F. alnus and P. harmala extracts on caspase activity in cancer cells was determined using the Protease Sampler Kit (Invitrogen, Carlsbad, CA). The cells were cultured for 1 d in RPMI growth medium containing the IC 50 of the extracts/polyphenols, then harvested and tested for caspase activity according to the protocol of the manufacturer. Briefly, control and treated cells were resuspended in chilled cell lysis buffer (50 mL) then incubated on ice for 10 min. The lysates were centrifuged for 1 min (10,000 g). The concentration of the protein was calculated using Bradford's method. The reaction buffer was added to the protein and incubated 2 h at 37 • C. The reaction buffer contained 200 mM substrate VEID (caspase-6), IETD-pNA (caspase-8), LEHD-pNA (caspase-9), and VDVAD-pNA (caspase-2). Absorbance was measured at 405 nm. The relative caspase activity (expressed as % of untreated control) was determined by the comparison of the absorbance of pNA from an apoptotic sample with the control.

Statistical Analyses
Least significant difference (LSD) was calculated by using SAS software. The values are means ± SDs of three series of experiments.

Antioxidant Effects
F. alnus and P harmala extracts showed strong antioxidant effects comparable to selected polyphenols as shown in Table 2. F. alnus showed significantly higher antioxidant effects than P. harmala as detected by different assays including β-carotene bleaching DPPH and FRAP. F. alnus polyphenols such as trifolin, p-coumaric acid, and rosmarinic acid showed strong antioxidant effects (low IC 50 ). P. harmala polyphenols including hydrocaffeic acid, cynaroside and protocatechuic acid showed strong antioxidant effects. Rosmarinic acid antioxidant activities were comparable to antioxidant standards. Table 2. Antioxidant activities of F. alnus and P. harmala and identified polyphenols (quercitrin, trifolin, p-coumaric acid, cynaroside, rutoside, rosmarinic acid, quercetin, protocatechuic acid, chlorogenic acid, hydrocaffeic acid and ferulic acid) using different assays (expressed as IC 50 in µg/mL).

MTT Assay
The antiproliferative effects of F. alnus and P. harmala extracts against selected cancer cells were evaluated using the MTT test (Table 3). There were antiproliferative effects of F. alnus and P. harmala extracts, as well as selected polyphenols, against all cancer cells. The normal cells of HEK-293 were not affected by the extracts. F. alnus showed higher antiproliferative activities than P. harmala. Noticeable antiproliferative effects were found when applying polyphenols such as quercitrin, cynaroside, trifolin, p-coumaric acid and rutoside against cancer cells.

Flow Cytometry
The cytotoxic activities of F alnus and P. harmala extracts, as well as quercitrin, trifolin, and cymaroside, were studied using the flow cytometry technique (Figure 3). The experiments showed obvious apoptotic cell accumulation following 2 d of exposure in the upper and lower right quadrant.

ROS Accumulation Assay
F. alnus and P. harmala extracts, as well as quercitrin, trifolin, and cynaroside, reduced the accumulation of ROS in treated cells compared to control using H 2 DCFDA fluorescence ( Figure 4). The highest reduction of ROS was found when using trifolin, and cynaroside in all cells after 90 min of incubation. H 2 O 2 showed the highest accumulation of ROS cancer cells.

Detection of Caspase Activity
The effects of F. alnus and P. harmala leaf extracts on caspase 2, 6, 8 and 9 activities were studied in selected cancer cells ( Figure 5). The results showed that increased caspase activity occurred after F. alnus and P. harmala treatment in all cancer cell lines compared to the control cells. In caspase 2, the treatment with F. alnus and P. harmala leaf extracts showed the highest activities in Jurkat cells. In caspase 6 and 9, the treatment with F. alnus and P. harmala leaf extracts showed the highest activities in HeLa cells. In caspase 8, the treatment with F. alnus and P. harmala leaf extracts showed the highest activities in T24 cells.

ROS Accumulation Assay
F. alnus and P. harmala extracts, as well as quercitrin, trifolin, and cynaroside, reduced the accumulation of ROS in treated cells compared to control using H2DCFDA fluorescence ( Figure 4). The highest reduction of ROS was found when using trifolin,and cynaroside in all cells after 90 min of incubation. H2O2 showed the highest accumulation of ROS cancer cells.

Detection of Caspase Activity
The effects of F. alnus and P. harmala leaf extracts on caspase 2, 6, 8 and 9 activities were studied in selected cancer cells ( Figure 5). The results showed that increased caspase activity occurred after F. alnus and P. harmala treatment in all cancer cell lines compared to the control cells. In caspase 2, the treatment with F. alnus and P. harmala leaf extracts showed the highest activities in Jurkat cells. In caspase 6 and 9, the treatment with F. alnus and P. harmala leaf extracts showed the highest activities in HeLa cells. In caspase

Discussion
This is the first study exploring the polyphenolic compositions of F. alnus and P. harmala. The common raw material of F. alnus is the bark, which contain frangulins, anthraquinone glycoside derivatives, anthtaquinone monoglycosides (emodin) and glucofrangulins components [11,12]. In the current study, we revealed five phenolic acids and four flavonoids. Rosmarinic and chlorogenic acids were the main phenolic acids, while quercitrin and trifolin were the major flavonoids (Table 1). Recently, Nejabatdoust et al. [36] studied hydroalcoholic, ethanolic and methanolic bark extracts of F. alnus collected from Iran. They determined the total phenolic composition in the extracts and only identified 2,4-di-tert-butylphenol and butylated hydroxytoluene with the GC/MS analysis. They indicated no presence of phenolic acids and flavonoids. Maleš et al. [11] quantified the glucofrangulins and the phenolic compounds in Croatian Rhamnus and Frangula species. In the methanolic bark extracts of the Croatian F. alnus they estimated, using a spectrophotometric method, the total flavonoids, phenolic acids and total polyphenols components and their contents ranged from 0.05 to 0.08%, 1.21-1.44% and 5.57-8.30%, respectively. The variation of the polyphenolic composition of this species might be affected by an environmental factor such as light, which may influence the tannins in this species [15]. Indeed, high temperature and sunny conditions in Saudi Arabia influences the chemical composition of this species.
P. harmala is a species known by the presence of specific harmala alkaloids, which are at least 5.9% of the dry weight [37]. In the current study, the extracts of P. harmala contained four phenolic acids, with hydrocaffeic and protocatechuic acids as the major compounds. In addition, high amount of one flavonoid (cynaroside) were detected (Table 1). Sodaeizadeh et al. [38] studied the Iranian P. harmala using HPLC and determined seven phenolic acids in leaves and four phenolic acids in roots. 4-Hydroxybenzoic acid was the dominant compound some plant parts (leaves and roots), whereas caffeic acid was the highest in other parts such as the stems. In the current study, we investigated Arabian origin leaf extracts and characterized different phenolic acid components (only caffeic acid was a common compound between the two studies). We, additionally, confirmed hydrocaffeic, rosmarinic and protocatechuic acids. From flavonoids, Sharaf et al. [39] studied the leaf extracts of the Egyptian P. harmala and confirmed other compounds including peganetin, acacetin 7-0-rhamnoside, 7-0-[6"-O-glucosyl-2"-O-(3"'-acetylrhamnosyl)glucoside, 7-O-(2"'-0-rhamnosyl-2"-O-glucosylglucoside) and glycoflavone 2"'-O-rhamnosyl-2"-O-glucosylcytisoside using TLC, and NMR methods. In the current study, and from our collection of widely distributed flavonoids, we tentatively determined only cynaroside (luteolin 7-glucoside). Previous investigation showed that environmental factors may influence the chemical composition of this species [40]. They reported elevated composition of vasicine, choline and sucrose in May as well as increases in betaine, lysine, 4-hydroxyisoleucine and proline in August. Furthermore, increases in phosphorylcholine, glucose, acetic acid and vasicinone were reported in December.
The antioxidant effects of F. alnus are mainly attributed to some leaves' polyphenols including trifolin, p-coumaric acid, rosmarinic acid, quercetin, chlorogenic acid and ferulic acid. Trifolin is a kaempferol 3-galactoside flavonoid, and few studies detected this polyphenol in plants. Leaf extracts of Zanthoxylum bungeanum showed strong antioxidant effects in a previous study and were attributed to specific polyphenols such as trifolin (31.24 mg/g) [41]. In our study, p-coumaric acid showed noticeable antioxidant effects, which is in agreement with pervious investigations on other plants [42,43]. Rosmarinic acid antioxidant activities were comparable to antioxidant standards. In a previous investigation on the Saudi-origin Artemisia abrotanum, a strong antioxidant activity was detected and it was attributed to several polyphenols including rosmarinic acid [44]. The antioxidant activity of P. harmala is mainly attributed to polyphenols such as hydrocaffeic acid, cynaroside and protocatechuic acid. Elsholtzia bodinieri showed antioxidant effects attributed to specific flavonoids including cynaroside [45]. Protocatechuic acid is a phenolic acid commonly found in plants and has a strong antioxidant activity [45]. This activity is attributed to chelating metal ions and scavenging free radicals. Hydrocaffeic acid is not commonly found in plants as the caffeic acid. In the current study, we found high concentrations in the leaves of P. harmala. These high concentrations are associated with the high antioxidant effects.
Most of the previous investigations on F. alnus focused on the bark, which is commonly used as laxative in herbal and alternative medicines. For example, the bark extract of F. alnus (Croatian origin) showed antioxidant effects attributed to emodin content [12]. From our knowledge, this is the first report confirming the antioxidant activities and polyphenolic composition of leaf methanolic extracts of F. alnus. The seed extracts of Indian-origin P. harmala showed high antioxidant properties in a previous investigation [17]. Previous investigation on Tunisian-origin P. harmala leaf extracts showed strong antioxidant and antibacterial activities [18]. However, no polyphenols were identified. Algerian-origin P. harmala leaf extracts showed bactericidal activities against S. aureus but no polyphenols were associated with this activity [19].
There were antiproliferative activities of F. alnus and P. harmala extracts against cancer cells (Table 3). F. alnus showed higher antiproliferative activities than P. harmala. In addition, there were antiproliferative activities when applying polyphenols such as quercitrin, cynaroside, trifolin, p-coumaric acid and rutoside against [22] cancer cells. Flow cytometry showed obvious apoptotic cell accumulation when applying F. alnus and P. harmala methanolic leaf extracts, as well as quercitrin, trifolin and cynaroside. In a previous investigation on Bosnian-origin F. alnus, a mild cytotoxic effect was found in the plant extracts against HeLa cancer cells but no activity was found when using the extracts of Serbian-origin plants [46]. No studies revealed a complete polyphenolic picture of this species. The antiproliferative and cytotoxic activities of F. alnus found here are related to the main polyphenols detected, such as quercitrin and trifolin. A previous investigation revealed that the quercetin flavonoid has antiproliferative activities against RAW264.7 cancer cell lines [47]. Other studies revealed that quercetin has cytotoxic activity against lung cancer cells [48]. Trifolin is a galactosideconjugated kaempferol that is formed by the kaempferol 3-ogalactosyltransferase and has apoptotic activity against lung cancer cells attributed to intrinsic and extrinsic pathways [49].
Previous investigations showed that P. harmala seeds and isolated compounds such as vasicinone, harmine, peganine and harmalacidine have antiproliferative and cytotoxic activities against Med-mek carcinoma, UCP-med sarcoma and Jurkat [20,21]. Harmine inhibited cell growth and vasicinone showed strong antiproliferating activity. The Chinese-origin P. harmala showed high amounts of 4-hydroxyisoleucine, asparagine, proline, lysine, vasicine and sucrose in the methanolic extracts of leaves [22]. However, no studies associated the leaf polyphenols with the cytotoxic activities of leaf extracts because larger interest was given for P. harmala seeds. In the current study, the antiproliferative and cytotoxic activities of P. harmala leaf extracts were mainly attributed to major polyphenols including cynaroside, hydrocaffeic acid and protocatechuic acid. Only a single previous investigation showed that cynaroside isolated from Turkish-origin Teucrium chamaedrys has antiproliferative activities against HeLa cells [50]. However, the current investigation is the first report studying the cytotoxic activities of cynaroside against HeLa, as well as other, cancer cells. Protocatechuic acid (3,4-dihydroxybenzoic acid) has anticancer effects against known cancer cells [51][52][53].
The caspase family proteases play an essential role in the apoptosis mechanism by controlling the pathway of apoptosis [54]. We found strong evidence that these caspase enzymes are activated in cells when treated with F. alnus and P. harmala leaf extracts. Caspases-8 and 9 are considered as large prodomains and initiator caspases, while caspase 6 is small prodomain [55]. In caspase-3 deficient cancer cells such as MCF-7, caspase 6, 8 and 9 could be involved in apoptosis [56]. Caspase 2 is not classified yet as an initiator or as an effector. However, it is involved in the cell death by activation within the p-53 protein with death domain PIDDosome [54]. From our knowledge, the effects of F. alnus and P. harmala leaf extracts on caspases activities have not been studied before.

Conclusions
The novel findings of this study can be summarized in the exploration of the polyphenol composition, and associated anticancer activities, of methanolic leaf extracts of F. alnus and P. harmala from natural populations in Saudi Arabia. Several polyphenols were identified by HPLC-DAD in the leaf extracts, including p-coumaric acid, rosmarinic acid, chlorogenic acid, ferulic acid, quercitrin, rutoside, quercetin and trifolin, in F. alnus, and hydrocaffeic acid, protocatechuic acid, caffeic acid, rosmarinic acid and cynaroside, in P. harmala. F. alnus and P. harmala leaf extracts showed antiproliferative and cytotoxic activities against cancer cells using MTT and flow cytometry assays. These anticancer activities were attributed to the polyphenolic composition of leaves including quercitrin, trifolin and cynaroside, which resulted in necrotic cell accumulation during apoptotic phases. F. alnus and P. harmala showed strong antioxidant effects attributed to the polyphenolic composition of leaves including quercitrin, trifolin, p-coumaric acid, rutoside, rosmarinic acid, quercetin, chlorogenic acid and ferulic acid in F. alnus and cynaroside, hydrocaffeic acid and protocatechuic acid in P. harmala. These antioxidant effects were associated with reduced ROS production in treated cells compared to controls. This is the first study investigating the antioxidant activities of hydrocaffeic acid and showing strong antioxidant activities. Finally, this is the first study revealing the activation of caspase family proteases in cancer cells by F. alnus and P. harmala leaf extracts.