Cytotoxic and antiviral activities of Jatropha variegata and Jatropha spinosa in relation to their metabolite profile

Jatropha variegata and Jatropha spinosa (family: Euphorbiaceae) are utilized in Yemeni traditional medicine to treat respiratory tract infection and in different skin conditions such as wound healing, as antibacterial and hemostatic. In this study, we evaluated the cytotoxicity and the antiviral activities of the methanolic J. variegata (leaves: Ext-1, stems: Ext-2, and roots: Ext-3), and J. spinosa extracts (aerial parts: Ext-4 and roots: Ext-5), in addition to their methylene chloride fractions of roots extracts (F-6 and F-7, respectively). All samples were tested against three human cancer cell lines in vitro (MCF-7, HepG2, and A549) and two viruses (HSV-2 and H1N1). Both plants showed significant cytotoxicity, among them, the methylene chloride fractions of roots of J. variegata (F-6) and J. spinosa roots (F-7) showed the highest activity on MCF-7 (IC50 = 1.4 and 1 μg/mL), HepG2 (IC50 = 0.64 and 0.24 μg/mL), and A549 (IC50 = 0.7 and 0.5 μg/mL), respectively, whereas the IC50 values of the standard doxorubicin were (3.83, 4.73, and 4.57 μg/mL) against MCF-7, HepG2, and A549, respectively. These results revealed that the roots of both plants are potential targets for cytotoxic activities. The in vitro results revealed potential antiviral activity for each of Ext-3, Ext-5, F-6, and F-7 against HVS-2 with IC50 of 101.23, 68.83, 4.88, 3.24 μg/mL and against H1N1 with IC50 of 51.29, 27.92, 4.24, and 3.06 μg/mL respectively, whereas the IC50 value of the standard acyclovir against HVS-2 was 83.19 μg/mL and IC50 value of the standard ribavirin against H1N1 was 52.40 μg/mL .The methanol extracts of the roots (Ext-3 and Ext-5) of both plants were characterized using UPLC/MS. A total of 73 metabolites were annotated, including fourteen diterpenoids, eleven flavonoids, ten phenolic acid conjugates, twelve fatty acids and their conjugates, five triterpenes and steroids, two sesquiterpenes, and six coumarins. The cytotoxicity and antiviral activities determined in the present work are explained by the existence of flavonoids, coumarins and diterpenes with commonly known cytotoxicity and antiviral activities.


Biological activity
Cell culture Nawah Scientific Inc. (Mokatam, Cairo, Egypt) provided the hepatocellular carcinoma (HepG2), lung cancer (A-549) , breast adenocarcinoma (MCF-7) cells and human skin fibroblast (HSF) cells for this study.The cells were kept in DMEM medium supplemented with 100 mg/mL streptomycin, 100 units/mL penicillin, and 10% heat-inactivated fetal bovine serum at 37 °C in a humidified, 5% (v/v) CO 2 atmosphere.Additionally, cells for HSV type 2, H1N1, and Vero were purchased from Nawah Scientific Inc. in Egypt.In DMEM medium supplemented with 10% fetal bovine serum and 0.1% antibiotic/antimycotic solution, Vero cells were cultured.We tested the cytotoxicity prior to this assay by seeding cells at a density of 2 × 10 4 cells/well in a 96-well culture plate.The following day, we cultured the cells in culture media containing serially diluted samples for 48 h before removing the medium and washing the cells with PBS.

Cytotoxicity assay
The SRB assay was used to determine cell viability as described by Skehan 29 .In 96-well plates, aliquots of 100 L of cell suspension (5 × 103 cells) were seeded and incubated in complete media for 24 h.The cells were subsequently treated with 100 L medium containing various doses of medicines.Cells were fixed 72 h after drug exposure by replacing medium with 150 L of 10% TCA and incubating at 4 °C for 1 h.After removing the TCA solution, the cells were washed 5 times with distilled water.Aliquots of 70 L SRB solution (0.4% w/v) were added and incubated at room temperature for 10 min in the dark.The plates were cleaned three times with 1% acetic acid and air-dried overnight.

Antiviral assay
Cell viability was determined by inhibiting the Cytopathic Effect (CPE) with crystal violet.Vero E6 cells and Vero cells were cultured in DMEM medium with 10% fetal bovine serum and 0.1% antibiotic/antimycotic solution.Gibco BR (Grand Island, NY, USA) provided the antibiotic and antimycotic solution, trypsine-EDTA, fetal bovine serum, and DMEM medium.Schmidtke et al. described the crystal violet method for evaluating antiviral and cytotoxicity activity 30 .In brief, one day before infection, Vero cells were planted at a density of 2 × 104 cells/well in a 96-well culture plate.The culture media was withdrawn the next day, and the cells were rinsed with PBS.The crystal violet method was used to measure H1N1 infectivity, which monitored CPE and allowed the percentage of cell viability to be estimated.The antiviral potency of each test sample was assessed using a concentration range of 0.1-100 g/mL that had been twice diluted.The assay was conducted with viral controls (virus-infected, non-drug treated cells) and cell controls (non-infected, non-drug treated cells).The culture plates were cultured for three days at 37 °C in 5% CO2, and the progression of the cytopathic effect was seen under a light microscope.The cell monolayers were fixed and stained using a 0.03% crystal violet solution in 2% ethanol and 3% formalin after being cleaned with PBS.After the plates had dried and been washed, a spectrophotometric measurement at 540/630 nm was used to determine the optical density of each individual well.This allowed for the calculation of the percentage of antiviral activities of the tests compounds according to Pauwels, et al. 31 using the following equation: Antiviral activity = [(mean optical density of cell controls − mean optical density of virus controls)/ (optical density of test − mean optical density of virus controls)] × 100% using the DIAS.Based on these results, the 50% CPE inhibitory dose (ID 50 ) was calculated.

UHPLC-DAD-QTOF-MS/MS profiling of methanol extracts
The metabolite profiling of the methanol extracts of roots of J. variegata and J. spinosa (Ext-3 and Ext-5, respectively) with potential cytotoxicity and antiviral activities were analyzed using UHPLC-DAD-QTOF-MS/MS analysis.Some classes of compounds, such as sesquiterpenes and diterpenes, were well-identified in the positive ionization mode, whereas flavonoids, phenolic acids, and quinic acid derivatives could be detected in the negative ionization mode 32 .

Phenolic compounds
The UHPLC-MS analysis of tested samples revealed the presence of 11 flavonoids, 10 phenolic acid conjugates, 6 coumarins, and 1 lignan were identified.Flavonoids were found primarily as C-linked hexoses (5 flavonoids), www.nature.com/scientificreports/which were easily distinguished by a neutral loss of 120 Da., as observed in compounds as homoorientin (3) and vitexin (10), vitexin was differentiated from its structural isomer isovitexin by the intensity of the fragment ion at 283 m/z, also homoorientin was differentiated from orientin by the intensity of the fragments 298 and 327 m/z.Flavonoids O-glycosides were characterized by the neutral loss of 162 Da for hexoses as observed in eriodictyol-hexoside (1) quercetin-O-hexoside (4) and naringenin O-hexoside (9).Most of these flavonoids were detected in Jatropha species 33,35,36 .1.Among the phenolic acid esters that have been annotated, only tetradecyl ferulate has been documented in the genus and has been isolated from both J. curcas 45 , and J. multifida 46 , and was previously detected in J. integerrima 33 .

Nitrogenous compounds
In J. variegata and J. spinosa extracts, seven simple nitrogenous compounds were identified, including choline and its glycoside.Other simple nitrogenous compounds including arginine and N-gucosylarginine, as well as indol carboxyaldehyde, betaien, and adenine, were also detected in the extract.

Terpenoids
The most frequent secondary metabolite group found in J. variegata and J. spinosa roots was composed of terpenoids, including two sesquiterpenes, five triterpenes and steroids, and 14 metabolites of diterpenes.Free diterpenoids esters, predominantly phorbol esters, were the first to be eluted in this investigation at retention time (2.8-3.5 min), which could easily be distinguished because to the neutral loss of acetic acid (60 Da), as seen in 12-deoxyphorbol, 4,9,12-trideoxyphorbol, and 12-deoxyphorbol.Additionally, at retention time 4.3-6.2min, free diterpenoids were eluted, and were tentatively identified as jatrophone, curcusone, spruceanol, mutifidone, jatropholone B, jatrocurcasenone E, and ferruginol, containing one to three oxygen atoms.Most of these diterpenes were isolated and previously identified in the genus Jatropha 36,37 47 .
Two sesquiterpenes were identified in both species, namely cuparene (66) and dihydro-pulicaric acid (67).Dihydro-pulicaric acid (67), a dihydroderivative of pulicaric acid, was identified in the genus Jatropha and in the family Euphorbiaceae for the first time, but was previously identified in Pulicaria crispa and P. incise 48

Fatty acids and their conjugates
Fatty acids were found in the form of free fatty acids and fatty acid glycerides, which accounted for 12 metabolites.The most prevalent fatty acids were unsaturated (C-18), including oxo-phytodienoic (C

Miscellaneous compounds
Six miscellaneous compounds were annotated in J. variegata and J. spinoa extracts including a sugar, a lignan, an anthraquinone, a dicarboxylic acid and two tricarboxylic acid derivatives, namely sucrose, eudesmin/ epieudesmin, emodine, azalic acid, ethyl aconitate hexoside, and ethyl aconitate dihexoside, respectively.Most of these compounds were identified in the genus Jatropha 33,35 .Only emodin anthraquinone was not detected in the genus Jatropha but was identified in Euphorbiaceae family 50 .The elimination of CO to create m/z 243 was followed by the loss of one hydroxyl group to produce m/z 225 in the fragmentation of emodin (C 15 H 10 O 5 ).

In vitro cytotoxic activity
This is the first report evaluating the in vitro cytotoxic activities of J. variegata and J. spinosa extracts against three human cancer cell lines, namely breast adenocarcinoma (MCF-7), hepatocellular carcinoma (HepG2), and lung cancer (A-549).The curves of the concentration response for the doxorubicin control drug, methanol extract and methylene chloride fractions against the three human cells lines are shown in Figs. 2, 3, 4, 5, 6.The results of the in vitro antiproliferative assay with human cancer cell lines are displayed in the Table 2.
The cytotoxic activity of the most effective anticancer extracts (Ext-3 and Ext-5) and fractions (F-6 and F-7) was evaluated against non-cancerous cells, human skin fibroblast (HSF) cell line to test their selectivity for cancer cells and results are shown in Table 2.The tested extracts and fractions showed considerable cytotoxic activity against HSF cells with IC 50 values of 1.54 ± 0.07, 1.08 ± 0.02 and for Ext-3 and Ext-5 and 0.69 ± 0.02 and 0.34 ± 0.01 for F-6 and F-7, respectively.Therefore, the selectivity index (SI) was calculated for the active extracts (Ext-3 and Ext-5) and fractions (F-6 and F-7) and shown in Table 2.The potent antiproliferative activity of F-6 was accompanied with high selectivity in case of HepG2 and A-549 (SI 1.078 and 0.986, respectively) and a moderate selectivity for MCF-7 (SI 0.49).Also, F-7 displayed strong SI in case of HepG2 (SI 1.417).
The diverse bioactive metabolites in Jatropha species may be responsible for the variations in the efficiency of the extracts against cancer cells.So, using UHPLC-DAD-QToF, untargeted metabolite profiling of the MeOH extracts was carried out.The LC-MS profile of the extracts showed the presence of six coumarins, including propacin, fraxidin, fraxetin, 3,4-dihydrocoumarine, coumarin, and 7-methyl coumarin, as well as major components including diterpenes (seen in positive mode) and flavonoids (indicated in negative ion mode).These coumarins, flavonoid and terpenoids enhance the active MeOH extracts as revealed in LC-MS and are well known for their anticancer properties.

In vitro antiviral activity
The antiviral activities of CH 2 Cl 2 fractions F-6 and F-7 of J. variegata and J. spinosa roots are presented in Table 4.These fractions showed the most significant activities against Human Herpes Simplex Type 2 (HSV-2) and Human Influenza (H1N1) cell lines (IC 50 4.88, 4.24, 3.4, and 3.06 μg/mL, respectively), whereas methanol extracts Ext-3 and Ext-5 of J. vriegata and J. spinosa roots were less active (IC 50 > 50 μg/mL) as shown in Figs. 7, 8, 9, 10, whereas acyclovir showed IC 50 of 83.19 µg/ml against HSV-2 cell line and ribavirin showed IC 50 of 52.40 µg/ml against H1N1 cell line.F-6 and F-7 factions showed greater activities than other extracts when compared to standard drugs, with in vitro antiviral activities ranging from 3.06 to 4.88 μg/ mL.The determination of selectivity indices for the extracts and fractions revealed significant differences.The extracts Ext-3 and Ext-5, with IC 50 values greater than 50 μg/mL, exhibited weak selectivity, ranging from SI 0.2 to 1.71.On the other hand, F-6 demonstrated high selectivity in terms of antiviral activity against HSV-2 (SI 5.9) and H1N1 (SI 3.4).Similarly, F-7 displayed good selectivity against H1N1 (SI 5.2).Both Fractions (F-6 and F-7) showed higher SI than each of acyclovir and ribavirin (Table 4).

Biological findings
Cancer is a disease caused by the unregulated division of aberrant cells in a single location of the body, and it has the potential to infiltrate surrounding tissues 51 .There are about 200 different forms of cancer, with the most prevalent being breast, colon, lung, and prostate cancer.Brain tumors, carcinomas, leukaemias, lymphomas, and sarcomas are the five forms of cancer.Cancer is predicted to impact one in every two persons in the UK over their lifetime, and it is currently the world's second biggest cause of death 52 .
In both the treatment and prevention of cancer, natural therapies have been extremely important.Vincristine in 1963 and vinblastine in 1965, the vinca alkaloids isolated from Catharanthus spp.(Madagascar periwinkle plants) growing in the Philippines and Jamaica, were the first naturally derived anticancer drugs approved by the Food and Drug Administration (FDA) in the United States 53 , the most prevalent genital cancer is cervical cancer, which is also one of the main reasons why women die.It accounts for about 12% of all female malignancies worldwide, particularly in underdeveloped nations 54 .
The increasing number of cancer-related fatalities has prompted researchers to search for novel anticancer medications.Synthetic drug-based chemotherapeutic treatments have been used to treat cancer, but their usage has been limited by their high cost and potential for toxicity.The study of plant-based anticancer drugs is now advancing rapidly.According to a recent assessment, the majority of secondary metabolites isolated from numerous plant families have shown potential for development as anticancer medicines 55,56 .According to the WHO, natural drugs have the potential to treat between 65 and 80% of human diseases.Natural products are less expensive than synthetic drugs and have fewer side effects, which has led to an increase in the use of medicines derived from natural products 57 .www.nature.com/scientificreports/

HEPG2-Ext
The possible cytotoxic properties of J. variegata and J. spinosa, two well-known bioactive medicinal herbs, were therefore examined in this work, as well as their potential impact on the cytotoxic profile of MCF-7, HEPG2, and A549 cell lines.To the best of our knowledge, no in vivo or in vitro studies of J. variegata and J. spinosa's anticancer and antiviral activities in various types of malignancies have been published before.This is the initial study of J. variegata and J. spinosa's effects on cancerous and viral cells.

Conclusions
In conclusion, our study demonstrated the promising cytotoxicity and antiviral activities of various extracts and fractions derived from J. variegata and J. spinosa.These beneficial effects can be attributed to the high content of flavonoids and terpenoids present in these extracts and fractions.
Metabolomics profiling of J. variegata and J. spinosa revealed the presence of numerous compounds with well-established cytotoxic and antiviral activities.Among them, 14 diterpene compounds such as jatrophone, ferrugenol, curcusone, multifidone and 11 Flavonoids such as taxifolin, vitexin, and quercetin.These findings highlight the potential of J. variegata and J. spinosa extracts and fractions as a source of bioactive compounds for the development of cytotoxic and antiviral agents.
Overall, our results underscore the importance of exploring phytoconstituents derived from J. variegata and J. spinosa, as they hold promise for the development of novel therapeutic interventions against cancer and viral infections.Further research and investigations are warranted to elucidate the mechanisms of action and evaluate the potential clinical applications of these bioactive compounds.

Figure 1 .
Figure 1.Base peak chromatograms (BPC) of the MeOH extracts of J. variegata and J. spinosa roots in negative (A, B) and positive (C, D) ionization modes, respectively.