Toxoplasmocidal and Cytotoxic Activities Guided Isolation and Characterization of an Undescribed Bioflavonoid-di-C-glucoside from Cycas rumphii Miq. Cultivated in Egypt

Toxoplasmosis and cancer are serious worldwide diseases, and the available drugs cause serious side effects. Investigation for new alternative therapies from natural sources is now an increasing concern. Herein, we carried out, for the first time, an in vitro screening of Cycas rumphii Miq. leaves for toxoplasmocidal effect, using Viruluent RH Toxoplasma gondii, and cytotoxic activity against HEPG-2, HCT-116 and HELA cancer cell lines using MTT assay. Among the tested extracts, the ethyl acetate fraction was the most effective against T. gondii, with an EC50 of 3.51 ± 0.2 µg/mL compared to cotrimoxazole (4.18 ± 0.01 µg/mL) and was the most potent against the tested cell lines, especially HEPG-2, with an IC50 of 6.98 ± 0.5 µg/mL compared to doxorubicin (4.50 ± 0.2 µg/mL). Seven compounds were isolated from the ethyl acetate fraction by extensive chromatographic techniques and fully elucidated using different spectroscopies. Compound (7) is an undescribed 4′, 4′′′ biapigenin di-C-glucoside, which showed a strong cytotoxic activity. Four known biflavonoids (1, 2, 4 and 5) in addition to a phenolic acid ester (3) and a flavonoid glycoside (6) were also isolated. Compounds (1, 3 and 6) were reported for the first time from C. rumphii.


Introduction
Toxoplasmosis is caused by Toxoplasma gondii, which is an obligate parasite [1]. The life cycle of this parasite occurs only in definitive hosts, such as domestic cats and wild felids, which pass the infection to humans [2]. The seroprevalence of this parasite in humans ranges from 30% in America and Europe to 60% in Africa [3]. This infection is a serious danger to immunocompromised people, leading to death, and to pregnant women, causing congenital toxoplasmosis and abortion [4].
The first treatment line of toxoplasmosis includes the combination of pyrimethamine, sulfadiazine and folic acid. However, pyrimethamine causes teratogenicity and other serious side effects [5,6].
Cancer is one of the prevalent diseases worldwide causing death, therefore attracting the interest of scientists to deliver new pipelines for treatment and to lower or avoid the known side effects of the currently used drugs.
The genus Cycas is a rich source of flavonoids, biflavonoids, phenolic acids, tannins, lignans, fatty acids and sterols, which exhibit a plethora of biological activities [7][8][9]. Cycas rumphii Miq. is a member of the cycadaceae family native to Indonesia [10]. It is traditionally used in Southeast Asian countries, such as India, Bangladesh and Indonesia for the treatment of wounds, ulcers, boils, itchy skin lesions and sore throat. In addition, it is rumphii Miq. is a member of the cycadaceae family native to Indonesia [10]. It is traditionally used in Southeast Asian countries, such as India, Bangladesh and Indonesia for the treatment of wounds, ulcers, boils, itchy skin lesions and sore throat. In addition, it is used for nephrotic pain, headache, bloody vomiting and flatulence [11,12]. The plant extract showed a potential antimicrobial activity against a range of human pathogenic bacteria [8]. There are no reports on the phytochemicals of Cycas rumphii Miq. having antitoxoplasma and cytotoxic properties. Thus, we aimed to evaluate the toxoplasmocidal and cytotoxic potentials of Cycas rumphii Miq. and to isolate and characterize the phytochemicals which could be responsible for the resulting activity.

Toxoplasmocidal Activity
This study reports, for the first time, an in vitro evaluation for the potential toxoplasmocidal activity of C. rumphii leaves against T. gondii RH strain tachyzoites. The total methanol extract of the C. rumphii leaves exhibited an EC50 of 5.15 ± 0.3 µg/mL, while the positive control drug (cotrimoxazole) showed an EC50 value of 4.18 ± 0.3 µg/mL. These results motivated us to investigate the toxplasmocidal activity of the different prepared fractions from the methanol extract. Amongst the tested extracts, the ethyl acetate fraction was the most potent, with an EC50 of 3.51 ± 0.2 µg/mL, which is lower than that of cotrimoxazol ( Figure 1, Supplementary material Table S1). The results indicated a promising toxoplasmocidal activity of C. rumphii leaves against T. gondii RH strain.

Cytotoxic Activity
The present study is the first report for assessing the cytotoxic activity of the C. rumphii leaves against six different cell lines (Supplementary Material Table S2). The results showed that the total methanol extract had a strong cytotoxic activity against HEPG-2, HELA and HCT-116 with IC50 values of 10.09 ± 0.9, 11.79 ± 1.0 and 12.58 ± 1.1 µg/mL, respectively, according to the classification of Hossan and Abu Melha, 2014 [13] ( Figure  2). Interestingly, the total methanol extract showed an IC50 value of 53.72 ± 3.7 µg/mL against normal cell line (WISH), while the positive control (doxorubicin) exhibited an IC50 value of 7.79 ± 0.5 µg/mL. Thus, the methanol extract of C. rumphii leaves was considered safer on normal cells and more specific to cancer cells than doxorubicin. The different

Cytotoxic Activity
The present study is the first report for assessing the cytotoxic activity of the C. rumphii leaves against six different cell lines (Supplementary Material Table S2). The results showed that the total methanol extract had a strong cytotoxic activity against HEPG-2, HELA and HCT-116 with IC 50 values of 10.09 ± 0.9, 11.79 ± 1.0 and 12.58 ± 1.1 µg/mL, respectively, according to the classification of Hossan and Abu Melha, 2014 [13] (Figure 2). Interestingly, the total methanol extract showed an IC 50 value of 53.72 ± 3.7 µg/mL against normal cell line (WISH), while the positive control (doxorubicin) exhibited an IC 50 value of 7.79 ± 0.5 µg/mL. Thus, the methanol extract of C. rumphii leaves was considered safer on normal cells and more specific to cancer cells than doxorubicin. The different fractions of the methanol extract were tested against the most affected cell lines (HEPG-2, HELA and HCT-116). The results revealed that the ethyl acetate fraction was the most potent amongst the tested fractions with very strong cytotoxic activity. This fraction exhibited IC 50 values of 6.98 ± 0.5, 7.94 ± 0.8 and 8.70 ± 0.9 µg/mL against the HEPG-2, HELA and  Table S3). According to the biological evaluation, the ethyl acetate fraction showed the highest activity. Thus, phytochemical investigation of this fraction was carried out to isolate and characterize the compounds mediating the exhibited biological potential.
fractions of the methanol extract were tested against the most affected cell lines (HEP HELA and HCT-116). The results revealed that the ethyl acetate fraction was the m potent amongst the tested fractions with very strong cytotoxic activity. This fraction hibited IC50 values of 6.98 ± 0.5, 7.94 ± 0.8 and 8.70 ± 0.9 µg/mL against the HEPG-2, HE and HCT-116 cell lines, respectively ( Figure 2, Supplementary material Table S3). Acc ing to the biological evaluation, the ethyl acetate fraction showed the highest activ Thus, phytochemical investigation of this fraction was carried out to isolate and cha terize the compounds mediating the exhibited biological potential.

Biological Activities of Pure Compounds
Cytotoxic Activity Compounds (1-7) were evaluated for the cytotoxic activity against the HEPG-2, HELA and HCT-116 cell lines. The results showed that compound (3) was the most potent against HEPG-2, HELA and HCT-116, with IC50 values of 8.67 ± 0.6, 10.08 ± 0.9 and 6.24 ± 0.5 µg/mL, respectively, followed by compound (6), which exhibited a strong cytotoxic activity, with IC50 values of 14.49 ± 1.1, 23.32 ± 2.0 and 11.63 ± 0.9 µg/mL, respectively. Finally, compound (7) showed a strong cytotoxic activity, with IC50 values of 21.47 ± 1.8, 15.66 ± 1.3 and 18.17 ± 1.4 µg/mL, respectively ( Figure 5). Additionally, the selectivity indices for the ethyl acetate fraction and its constituents against these cancer cell lines (HEPG-2, HELA and HCT-116) were calculated as reported in the literature data [30] (Supplementary Material Table S4). The higher the magnitude of the selectivity index of a test material, the greater its selectivity. In this study, the ethyl acetate fraction and its isolated compound (3) showed higher selectivity for the cancer cells than the normal cells compared to doxorubicin.

Biological Activities of Pure Compounds Cytotoxic Activity
Compounds (1-7) were evaluated for the cytotoxic activity against the HEPG-2, HELA and HCT-116 cell lines. The results showed that compound (3) was the most potent against HEPG-2, HELA and HCT-116, with IC 50 values of 8.67 ± 0.6, 10.08 ± 0.9 and 6.24 ± 0.5 µg/mL, respectively, followed by compound (6), which exhibited a strong cytotoxic activity, with IC 50 values of 14.49 ± 1.1, 23.32 ± 2.0 and 11.63 ± 0.9 µg/mL, respectively. Finally, compound (7) showed a strong cytotoxic activity, with IC 50 values of 21.47 ± 1.8, 15.66 ± 1.3 and 18.17 ± 1.4 µg/mL, respectively ( Figure 5). Additionally, the selectivity indices for the ethyl acetate fraction and its constituents against these cancer cell lines (HEPG-2, HELA and HCT-116) were calculated as reported in the literature data [30] (Supplementary Material Table S4). The higher the magnitude of the selectivity index of a test material, the greater its selectivity. In this study, the ethyl acetate fraction and its isolated compound (3) showed higher selectivity for the cancer cells than the normal cells compared to doxorubicin.

General Experimental Procedures
Solvents used were of HPLC analytical grade ≥99.9% and were purchased from Sigma Co. NMR experiments were performed using a Bruker Avance III spectrometer (Rheinstetten, Germany), with 400 MHz for 1 H and 100 MHz for 13 C and DEPT-Q NMR. ESI-MS spectra were recorded by Advion compact mass spectrometer (CMS) (New York, NY, USA). High-resolution mass spectra were measured using the Q-TOF-LC/HRMS system (6530) from Agilent Technologies Co. (Waldbronn, Germany) equipped with an autosampler (G7129A) with an ESI ionization source. Melting point determination was carried out using a Gallenkamp melting point apparatus from Hanon Co. (Jinan, China). Optical rotation was measured using a Polax-2L Polarimeter (Atago Co., Tokyo, Japan). UV spectra were recorded using a UV/Vis spectrophotometer (UV-1800 from Shimadzu Co., Tokyo, Japan). IR spectra were measured as KBr discs using an FT/IR-6100 spectophotometer from Jasco Co. (Tokyo, Japan). An ELISA Processor II Microplate Reader EXL800 from Biotek Co. (Winooski, VT, USA) was used for the cytotoxic assessment.

Plant Material
The leaves of C. rumphii Miq. were collected from El-Abd Garden at 68 kilos from the desert Cairo-Alexandria Road in July 2018. It was kindly provided and identified by the researcher Rabea Sharawy (Agronomist and palm researcher). A voucher sample (No. PGG-012) was deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Tanta University, Egypt.

Extraction and Isolation
The plant material was dried in shade, reduced to powder and stored in tightly closed containers. The plant powder (5 Kg) was extracted with methanol by cold maceration till exhaustion. The total methanol extract was evaporated under reduced pressure at 40 • C to yield a green residue (294 g). The methanol extract residue (274 g) was suspended in 50% aqueous methanol (1.5 L) and was successively fractionated with petroleum ether (40-60 • C), methylene chloride, ethyl acetate and n-butanol to yield 38.03 g, 8.10 g, 20.1 g and 59.20 g, respectively.
Acid hydrolysis of compound (7): The procedure in reference [28] was followed with some modifications. Compound 7 (1 mg) was dissolved in 2 N HCl/methanol mixture (1:1, 2 mL) and heated at 100 • C for 1 h. Then, the solution was evaporated to remove the residual methanol, and the left solution was neutralized with NaHCO 3 . The sugar in the hydrolysis product was detected by paper chromatography alongside authentic sample of β-D-glucose using the solvent system n-butanol:acetic acid:water (4:1:5).  Figure S28).

Toxoplasmocidal Activity
The assay was carried out using virulent RH T. gondii strain, which was obtained from the Medical Parasitology Department, Faculty of Medicine, Alexandria University, Egypt. Different concentrations of the total methanol extract of the C. rumphii leaves and its different fractions were tested for toxplasmocidal activity according to the method reported by [31]. The mean effective concentration (EC 50 ) in µg/mL was calculated and compared to that of cotrimoxazole as a reference drug.
The cytotoxicity assay was carried out according to the reported procedures using the MTT assay method [32,33]. The assay was carried out using seven different concentrations (1.56, 3.125, 6.25, 12.5, 25, 50 and 100 µg/mL) of the total methanol extract of C. rumphii in DMSO against the tested cancer cell lines and one normal cell line (WISH) to ensure the safety of the plant extract on normal cells. The most affected cancer cell lines were tested against the extracts of petroleum ether, methylene chloride, ethyl acetate and n-butanol fractions and the constituents of the most active fraction were also tested using the same previous concentrations. Doxorubicin was used as a positive control. IC 50 was calculated and the cytotoxic effect was assessed according to the classification of Hossan and Abu Melha, 2014 [13].

Statistical Analysis
All experiments were carried out at least three times, and the data were expressed as the mean ± standard error of the mean (SEM).

Conclusions
In conclusion, the biological screening for C. rumphii different fractions indicated that the ethyl acetate fraction has potent toxoplasmocidal and cytotoxic activities against various cell lines. Therefore, the phytochemical investigation of the most active fraction of the C. rumphii leaves was carried out and resulted in the isolation of a previously undescribed biflavonoid C-glycoside in addition to six known compounds, three of them were isolated for the first time from C. rumphii leaves. These compounds were identified using different spectroscopic techniques and compared to the data reported in the literature. In addition, the isolated compounds were evaluated for cytotoxic activity using the MTT assay method. The results showed that compounds (3, 6 and 7) have significant cytotoxic activity. Future studies for in vivo assessment of these activities are necessary.