Unveiling the anti-cancer potential of Euphorbia greenwayi : cytotoxicity, cell migration, and identification of its chemical constituents

Background Different herbal phytochemicals have potential in cancer treatment, Euphorbia genus has valuable secondary metabolites and is used in traditional medicine to treat various ailments. However, the specific constituents and biological activity of Euphorbia greenwayi remain largely unexplored. Results Euphorbia greenwayi aerial parts were extracted using methanol. Consequently, the methanol extract was then fractionated with solvents of different polarities viz., n ‑hexane, chloroform, and ethyl acetate. All were screened for their cytotoxic activity against different cell lines; MCF‑7, HepG‑2, and SW620. The n ‑hexane (HF) and chloroform (CF) fractions showed considerable activity against all tested cell lines especially MCF‑7 with IC 50 values at 18.6 ± 0.2 and 17.5 ± 0.6 μg/ml respectively. Therefore, a cell migration assay on the MCF‑7 cell line was applied to both fractions as well as investigation and isolation of the main active constituents. Lupeol, β-sitosterol, and cycloartenol were isolated from the nonpolar fractions of E. greenwayi for the first time. Conclusions Euphorbia greenwayi aerial parts exhibit considerable anti‑cancer effects via cytotoxicity. Three chemical constituents with promising cytotoxic activity are identified.


Background
Worldwide, cancer ranks as the second leading cause of death.The most prevalent cancers are colon, liver, and breast cancers.Cancer is characterized by its widespread occurrence globally.It exhibits notably high mortality rates according to statistical data.Lifestyle and genetic predisposition are commonly acknowledged as the primary factors contributing to its development.
Phytochemicals derived from herbs and traditional medicine are becoming more widely recognized as effective cancer treatments.Recent clinical trials have demonstrated the beneficial effects of herbal medications on cancer patients' quality of life, survival rates, and immune system control when used in conjunction with traditional treatments.Numerous phytochemicals, including phenolic compounds, terpenoids, lignans, tannins, alkaloids, and others, have been studied from herbal sources and show potent antioxidant qualities that can suppress cell division and boost the immune system, improving prevention [1][2][3][4].
The ornamental medicinal plant species, Euphorbia greenwayi P.R.O.Bally & S. Carter is a member of the Euphorbiaceae (Spurge) family [5].Euphorbia is considered the third biggest genus of flowering plants having milky poisonous latex.It consists of many species that are used in traditional medicine to cure a wide range of illnesses.This might be related to the wealth of their secondary metabolites [6,7].Different studies reported that Euphorbiaceae members consist mainly of terpenes, flavonoids, and tannins, which are known for their antioxidant, hepatoprotective, and anti-tumour properties [8][9][10].As a member of the Euphorbia genus, E. greenwayi possesses succulent quality produces milky latex and may grow up to 1.2 m tall [11].It was introduced to Egypt a short time ago, but it is native to Tanzania and East Africa.Upon reviewing the available literature, little information was reported on E. greenwayi; one study compared the immune-boosting capabilities of fifteen plant extracts from the Euphorbiaceae family, demonstrating E. greenwayi's mild antiviral activity.[10].Moreover, another report proved that the hydroalcoholic extract of E. greenwayi has significant antimicrobial potential [12].Our recently published work demonstrated its antiinflammatory and antioxidant potential [13].
The current study intends to assess the cytotoxic activity of the E. greenwayi methanol extract and its fractions.Additionally, an in vitro migration assay (wound healing activity) for the most active fractions is conducted to determine the tumour cell migration capacity of cell lines and, consequently, their invasiveness and potential to generate metastases.The chemical components of the most active fractions are determined using spectroscopic and chromatographic techniques.

Phytochemical screening
A phytochemical screening is essential to determine the active substances responsible for the biological activity that plants are known to display and to evaluate a plant's potential medicinal usefulness.It also provides the foundation for more precise compound identification and investigation.Tannins, flavonoids, unsaturated sterols, and/or terpenes were found, together with carbohydrates and/or glycosides.As shown in Table 1, there were no volatile oils, alkaloids, nitrogenous bases, anthraquinones, or saponins.

Compound EA 1
Compound (EA 1 ) 20 mg was isolated as white amorphous powder.It is soluble in n-hexane, chloroform and insoluble in methanol, m.p. 215-216ºC.; the R f values were 0.59 on silica TLC using n-hexane:ethyl acetate (95:5 v/v) as developer.It gave a pink color when sprayed with 10% H 2 SO 4 ..It also gave positive Salkowski and Liebermann-Burchard tests [14].The 1 HNMR spectrum   [16,17].Compound (EA1) was identified as Lupeol based on the data presented above, comparison to published data [13,18] and co-chromatography with a standard sample (Fig. 1).It was separated for the first time from E. greenwayi.

Antitumor activity (IC 50 )
According to the cell viability assay (Table 3) HF and CF were the most cytotoxic fractions of the 3 tested cancerous cell lines.Accordingly, those 2 fractions were further tested to find their (IC 50 ) using Sulforhodamine B (SRB) analysis in comparison with doxorubicin as a reference antitumor drug.As shown in Table 4 both HF and CF have a moderate to low activity against all tested cell lines.In specific the breast adenocarcinoma (MCF-7) cells were the most susceptible cell line against both fractions with IC 50 values at 18.6±0.2µg/ml against HF and 17.5±0.6µg/ml against CF.
It can be deduced that the aerial parts of E. greenwayi have a moderate antitumor activity especially against MCF-7 cell line.The United States National Cancer Institute (NCI) stated that any crude extract with IC 50 value ≤ 20 µg/ml is considered an active cytotoxic agent [29].The  This was confirmed after tracing the anticancer potential of the 3 isolated compounds (lupeol, β-sitosterol, and cycloartenol) that was already proven in previous studies [30][31][32][33][34].This reveals a good correlation between antitumor potential and nonpolar constituents of E. greenwayi like sterols and terpenes.

Anti-Migration Activity of MCF-7 Cell Line
A wound healing assay, conducted in vitro, aims to assess the migratory potential of cell lines treated with the most potent fractions.This assay helps evaluate the cells' ability to migrate, thereby indicating their invasiveness and the likelihood of generating metastases.Based on the antitumor activity results, only HF and CF were continued in this study, because those fractions showed cytotoxic activity superior to ME and EF against MCF-7 which was the most susceptible cell line among the 3 cell lines tested.
HF and CF antimigration assay was performed using 2 doses (subtoxic and a lethal dose (IC 50 )), 1.9 and 19 μg/ ml respectively for HF and 1.7 and 17 μg/ml respectively for CF.Figures 2, 3 and 4 demonstrated the MCF-7 monolayer which was scratched and treated with selected fractions.The wound area was monitored and imaged every 24 hours for 72 hours.Finally, the migration rate was calculated and compared with the negative control Figs. 5 and 6.
As shown in Figs. 5 and 6 both the subtoxic and lethal doses of both HF and CF don't exhibit an anti-migratory effect.

Discussion
The exploration of medicinal plants has garnered increased attention as a means to discover more effective treatments for various cancer types.Presently, a substantial portion of pharmaceutical agents, especially in cancer therapy, comprises natural products.Taxol, vinblastine, and camptothecin are illustrative examples, distinguished by their unique structures and mechanisms of action, with their discovery primarily attributed to isolation from natural sources.In this sense, the Euphorbia genus is distinguished by its richness in biologically active phytoconstituents with promising cytotoxic activity [35][36][37].In this regard, E. greenwayi was chosen to be the subject of our study because of the little-known information regarding its primary components and its biological activity.
E. greenwayi showed positive presence of sterols, triterpenes, and phenolic compounds; hence its methanol extract (ME) was fractionated using n-hexane (HF), chloroform (CF), and ethyl acetate (EF) to test these fractions for cytotoxic activity against MCF-7, HepG-2, and SW-620 cell lines.(HF) and (CF) showed significant cytotoxic activity against MCF-7 with IC 50 values at 18.6 ± 0.2 and 17.5 ± 0.6 µg/ml respectively.However, they showed significant cytotoxic effect on HepG-2 and SW-620 at higher doses.This confirms the susceptibility of MCF-7 against (HF) and (CF).On the other hand, ME and EF didn't exhibit any cytotoxic activity against the 3 cell lines at all tested concentrations.Wound healing assay for cancer metastasis is highly reproducible method to study cancer cell in vitro.By this method we can develop an additive treatment combined with the main drugs in order to decrease migration of cancer cell to another organs.Based on the previous findings (HF) and (EF) were tested against MCF-7 cell migration yet they didn't display a significant antimigratory effect.These findings don't contradict the results of antitumor assay, but rather suggest that both (HF) and (CF) are cytotoxic to MCF-7 cells in a non-apoptotic cell death mechanisms other than decreasing cell migration or inhibiting the cell motility [38].Also, it was already established that some of the most effective anticancer drugs such as carboplatin and paclitaxel were observed to induce the migration of cancer cells in different kinds of cancers [39].
Driven by the antitumor assay findings we decided to explore the constituents of the nonpolar fractions (HF) and (CF) of E. greenwayi.Phytochemical analysis reveals the separation of three compounds from (HF) and (CF): lupeol, β-sitosterol, and cycloartenol.The 3 compounds are isolated from E. greenwayi for the first time.Based on previous data and the pharmacological effects of the three compounds, we realised that they all exhibited cytotoxic activity against various cancer cell lines [30, 32-34, 40, 41].This supports the antitumor effects of E. greenwayi's nonpolar fractions.In addition, in our recently published work through LC-MS we identified several nonpolar compounds in E. greenwayi [13] with reported cytotoxic activity such as taraxasterol [42], ingenol dibenzoate [43], and ingenol mebutate [44].

Plant material
Collection, handling, and authentication of plant material was previously discussed in our recently published work [13].

Extraction and fractionation
Twelve Kg of E. greenwayi fresh plant was macerated with absolute methanol till exhaustion (12 L × 3).The methanol extract was evaporated under a vacuum at 40°C.The crude extract (40 g) was suspended in 200 ml distilled water.The aqueous suspension was successively fractionated by partition with n-hexane, chloroform, and ethyl acetate.The results of the fractionation are summarized in (Fig. 7).

Phytochemical screening
Dried aerial parts of E. greenwayi (40 g) underwent a phytochemical screening to identify the different phytochemical components that were found in it.These components included volatiles, carbohydrates and/or glycosides, alkaloids and/or nitrogenous bases, saponins, anthraquinones, unsaturated sterols and/or triterpenes, tannins, and flavonoids [45,46].The Pio-chem corporation in Cairo, Egypt provided all the chemicals, which were of high purity.Among the substances utilized following instructions were glacial acetic acid, concentrated ammonia, alcoholic KOH, FeCl 3 , HCl, Dragendorff 's reagent, methanol, chloroform, and H 2 SO 4 [47,48].

Compound isolation
N-hexane (3.6 g) and chloroform (1.3 g) fractions showed similar spots, so both fractions were added together.Fractionation was done through column chromatography using a silica gel (Merck) (200 g, 100 cm X 5 cm).Gradient elution started with 100% n-hexane then 5% increments of ethyl acetate, till the elution reaches 100% ethyl acetate.Fractions, each of 15 ml, were collected, concentrated under reduced pressure, and monitored using thin liquid chromatography (TLC).A system consisting of n-hexane: ethyl acetate with a different ratio was used as a developer.10% H 2 SO 4 was used as a spraying reagent for spot visualization.Similar fractions were pooled together.Fraction (I) was fractionated through column silica gel using n-hexane/methylene chloride gradient elution resulting in compound (EA 1 ) separation.

Structure elucidation of the purified compound
NMR spectroscopic analysis used a Bruker spectrometer at 400 MHz for ( 1 H NMR) and 100 MHz ( 13 C NMR) according to [49].The UPLC-ESI-MS/MS negative and positive ion modes were executed on a Waters Corporation, Milford, MA01757, USA, XEVO TQD triple quadrupole mass spectrometer.
In this experimental procedure, 100 μL aliquots of a cell suspension containing 5 × 10 3 cells were dispensed into individual wells of 96-well plates and incubated in complete media for a duration of 24 h.Subsequently, the cells were subjected to treatment with another 100 μL of media containing various concentrations of drugs.Following 72 h of exposure to the drugs, the cells were fixed by replacing the media with 150 μL of a 10% trichloroacetic acid (TCA) solution and incubated at 4 °C for 1 h.After removal of the TCA solution, the cells underwent five washes with distilled water.Subsequently, 70 μL aliquots of a sulforhodamine B (SRB) solution at a concentration of 0.4% w/v were added to each well, and the plates were incubated in darkness at room temperature for 10 min.Following this incubation period, the plates underwent three washes with 1% acetic acid and were then allowed to air-dry overnight.To dissolve the protein-bound SRB stain, 150 μL of a tris(hydroxymethyl) aminomethane (TRIS) solution was added at a concentration of 10 mM.The absorbance of the resulting solution was measured at 540 nm using a BMG LABTECH ® -FLUOstar Omega microplate reader (Ortenberg, Germany).

Cell migration (wound healing) assay
N-hexane and chloroform fractions were evaluated for their potential to inhibit wound healing in cancerous cell lines according to [51,52].Since MCF-7 (Breast Adenocarcinoma) was the most susceptible cell line, it is chosen to be used in this assay.Both fractions were evaluated in 2 concentrations, lethal dose (IC 50 ) and Subtoxic dose.
Cells were seeded at a density of 2 × 10 5 cells per well on a 12-well plate that had been pre-coated for scratch wound assay.They were cultured overnight in a medium consisting of 5% fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) at 37°C and 5% CO 2 .The following day, horizontal scratches were carefully introduced into the confluent cell monolayer.Subsequently, the plate underwent thorough washing with phosphate-buffered saline (PBS).Control wells were replenished with fresh medium, while wells designated for drug treatment were supplied with fresh medium containing the specified drug.Images were captured at designated time intervals using an inverted microscope.The plate was maintained at 37°C and 5% CO 2 between  Wound width is the distance between the edges of the scratches in average; as cell migration is induced the wound width decreases.
Migration rate is determined according to the formula below: MR = IW − FW / t where MR is the rate of cell migration, IW is the average wound width at 0 h, FW is the average final wound width, and t is duration of migration (in hours).

Conclusion
In conclusion, the investigation into the anti-cancer properties of E. greenwayi has revealed promising findings.The study encompassed cytotoxicity assays, evaluations of cell migration, and identification of its chemical constituents.
The cytotoxicity assessments demonstrated considerable potency within specific fractions (n-hexane and chloroform fractions) of E. greenwayi, notably highlighting considerable toxicity against cancerous cell lines.
Furthermore, the identification of chemical constituents within E. greenwayi provides valuable insights into potential bioactive compounds responsible for its anticancer effects.These constituents may serve as a foundation for further research and development of novel anti-cancer agents.
The collective findings underscore the significance of E. greenwayi as a potential source of compounds with anticancer properties.Continued exploration and elucidation of its mechanisms and active compounds could pave the way for the development of new therapeutic strategies in combating cancer.

Fig. 1
Fig. 1 Chemical structure of the identified compounds

Table 2
demonstrated the characteristic deshielded proton at δH 3.3

Table 1
Preliminary phytochemical screening

Table 2 )
. Mass spectrum of isolated compound showed molecular ion m/z 427 [M + H] corresponding to the molecular formula C 30 H 51 O.It gave MS spectra with a base peak at m/z 409 which resulted from loss of 1 water molecule [M + H − H 2 O] + .MS 2 also showed characteristic peaks at m/z 257, 271 and 285, 191, 203 and 217 and compared with published data