Phytosterols and diterpenoids isolated from Euphorbia graminea Jacq. (Asteraceae) leaves exhibit antiproliferative effects against human MCF-7 breast and NCI-H460 lung cancer cell lines

Emmanuel O. Ikpefan et al., ISSN: 2814-1423 11 Nigerian Journal of Pharmaceutical and Biomedical Research Vol. 8 Issue.1 April, 2024. p-ISSN: 2579-1419 e-ISSN: 2814-1423 Phytosterols and diterpenoids isolated from Euphorbia graminea Jacq. (Asteraceae) leaves exhibit antiproliferative effects against human MCF-7 breast and NCI-H460 lung cancer cell lines Emmanuel O. Ikpefan 1, Cletus A. Ukwubile 2, *, Edwin O. Omeje 3, Samuel O. Ovbiagele 4, Azhar Mudassar 5 , Zadar Ali Shah 6. 1. Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmacy, Delta State University Abraka, Nigeria. 2* Department of Pharmacognosy, Faculty of Pharmacy, University of Maiduguri, Maiduguri, Nigeria. 3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Nigeria Nsukka, 41001, Nsukka, Nigeria. 4 Ovbioise Hospital, 2 Isibor Agbontaen Street, Aduwawa, Benin City, Edo State, Nigeria. 5 University of Karachi, International Centre for Chemical and Biological Sciences, Dr Panjwani Center for Molecular Medicine and Drug Research, Karachi, Pakistan. 6 H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan. *Corresponding author: Email: [email protected], doi.org.10.55639/njpbr.1021.002 Abstract The plant Euphorbia graminea is a tree that has been used in Nigeria for the treatment of various diseases such as cancer, inflammations, diabetes, and ulcers in folk medicine. The present work was aimed at isolating phytosterols and diterpenoids from the leaf extract with potential anticancer and antiproliferative effects against selected cancer cell lines. The chloroform fraction from the 90 % aqueous methanol leaves extract of E. graminea was subjected to vacuum liquid chromatographic fractionation and the fractions were tested at the concentration of 1-100 µg/mL on MCF-7 and NCI-H460 cell lines. Data obtained were statistically analysed using one-way ANOVA followed by Dunnett’s post hoc test (p < 0.05; p = 0.001). The results showed that the chloroform fraction of Euphorbia graminea has demonstrated significant anti-cancer activities against MCF-7 breast and NCI-H460 lung cancer cell lines. Repeated bioassay-chromatographic fractionation of the active fractions yielded four known compounds as triacontan-1-ol, dotriacontan-1-ol, stigmasterol, stigmasta-5-en-3βol and a new compound abietane-11, 23 diene-16-oic-14-one whose identities were unequivocally confirmed through detailed NMR and Mass spectroscopic analyses. Against MCF-7 cell lines, dotriacontan-1-ol and abietane-11, 23 diene-16-oic-14-one produced LC50 of ~40 and ~91 µM respectively, compared to the ~10 µM produced by standard drug doxorubicin (p < 0.05; p = 0.001). The LC50 against NCI-H460 was observed to be >100 µM for all compounds while doxorubicin gave 15 µM. The isolated compounds from this plant are reported here for the first time. Among the compounds, Dotriacontan-1-ol and Abietane-11, 23 diene-16-oic-14one were observed to elicit a selective antiproliferative activity on MCF-7 and NCI-H460 respectively hence could be considered as selective natural anticancer agents. However, the mechanism with which these compounds exhibit this activity was not studied and should be considered for future research. Keywords: Anticancer, Euphorbia graminea, Antiproliferation, SRB assay, Abietane-11, 23 diene-16-oic14-one. Emmanuel O. Ikpefan et al., ISSN: 2814-1423 12 Introduction Plants have a continued history of application in the treatment of cancer (Tavares-Carreón et al., 2020). Although conventional cancer treatments such as radiotherapy and chemotherapy already exist, they have a variety of side effects such as neurological, cardiac, renal, and pulmonary toxicity, which can severely harm a person's health. The growing number have side effects and the high cost of medication has shifted the focus of research toward herbal medicines. As a result, an alternative method for developing anticancer drugs that are less toxic and more potent than those currently available on the market is needed(Onyancha et al., 2018). The prevalence of cancer globally is on the increase ranging from cervical, prostate, lung aetiology, breast, blood, and colon. In terms of etiology, breast cancer is the most common cancer after skin cancer is diagnosed in women worldwide. Men and women can be diagnosed with breast cancer, but it is most common in women. On the other hand, lung cancer affects the lungs, especially in people who smoke. The non-small cell lung cancer is the most prevalent of all lung cancers with about 85 % of cases globally (Abuzaid, et al,2020). Cancers remain one of the diseases that have little or no cure globally because, various treatment measures such as chemotherapy, radiotherapy, and surgery have yielded little or no solution at all. In Nigeria, various medicinal plants have been used and are continuously used to manage and treat various types of cancers. These ethnobotanical prescriptions in Nigeria’s traditional medicine have produced great success in cancer therapy, and one such plant used is Euphorbia graminea Jacq. which was confirmed from the world flora online (Ikpefan et al., 2020). The plant Euphorbia graminea is commonly called the grass leaf spurge and was previously known as Agaloma graminea (Ikpefan et al., 2023) belongs to the class of untapped plant species. It has been reported as an invasive species which is native to northern Mexico and Peru (Dilleniaceae et al., 2022). Apart from its native origin, it has been reported to have spread to other parts of the world such as Taiwan, Galapagos Islands and Nigeria (Tchimene et al., 2016). The plant is morphologically made up of tiny, glanduliform stipules, petaloid gland appendages and leaves with entire margin (Aigbokhan & Ekutu, 2015). It has been reported to have leaves that are alternate below and opposite above and have apex which ranges from acuminate to acute, bases which are acute to obtuse surfaces which are pubescent and shapes which are ovaterounded to oblong. In Nigeria and other parts of the world, there is little or no information documented on the phytochemistry and biological activities of E. graminea. However, traditional healers in some parts of the world such as Columbia have reported the use of the plant in the treatment of numerous diseases such as skin infections, ulcers, warts, and cancer (Tchimene et al., 2016). It has also been reported to be poisonous to ruminants (Dilleniaceae et al., 2022). In Nigeria, there are no existing reports on the phytochemical contents of this plant, despite the uses of this plant for treating various disease conditions. Previous research on this plant showed that the chloroform fraction outperformed the aqueous fraction and the methanol extract in terms of antiproliferative activity (Antiproliferative et al., 2021). This current research was aimed at isolating, characterize and elucidate structurally antiproliferative phytosterols and diterpenoids from chloroform fraction of E. graminea and Emmanuel O. Ikpefan et al., ISSN: 2814-1423 13 study their effects on MCF-7 breast and NCIH460 lung cancer cell lines. Materials and Methods Collection of plant materials The fresh leaves of E. graminea was collected in the evening hours at a location within the University of Benin campus, Nigeria. Authentication of the plant was made by a taxonomist Mr. Adewale of the International Institute for Forest Research, Ibadan, Nigeria. A voucher specimen number of FHI 109024 was deposited for the plant. The leaves were then dried under-shade for two weeks, reduced into fine powder using milling machine and stored for further use. Extraction and purification of bioactive fraction of extract A total of 2.5 kg of the powdered leaves of E. graminea were exhaustively extracted by applying a Soxhlet apparatus using 95 % aqueous methanol (5.5 L) as the extracting solvent. The extract was dried in vacuo at 40 0C and was subjected to exhaustive solventsolvent partitioning using chloroform and 70 mL distilled water. A portion of the active chloroform fraction (98.50 g) previously obtained from the extract (200 g) as earlier reported (State et al., 2023), was subjected to standard vacuum liquid chromatography on normal-phase silica gel using C6H14 (100 %), C6H14-CH2Cl2(1:1; 1:3), CH2Cl2 (100 %),CHCl2CH3COOC2H5 (3:1,1:1,1:3), CH3COOC2H5(100%), CH3COOC2H5– CH3OH(1:1) and CH3OH (100 %) (1.5 L) in increasing polarity, to afford ten (10) fractions which were concentrated in vacuo at 45 0C .Analytical thin layer chromatographic analyses of the Vacuum Liquid Chromatographic fractions of the chloroform fraction was carried out on a pre-coated aluminium plate of Silica gel GF254 using DCM: CH3OH (9.4: 0.6). After development, the plates were sprayed with ceric sulphate spray and subsequently heated for 5 min at 110C (Ukwubile et al., 2019). The coloured spots were noted and their Rf values were recorded. Based on observations made on the results obtained, coupled with the TLC profile, the chloroform VLC sub-fraction of E. graminea were bulked into CA (1-4), 5-9(CB) and 10(CC) accompanied by biological test at concentration between 1-100 µg/mL. Silica gel column chromatography purification of sub-fraction Column chromatographic purification of subfraction CB (5-9/74.56g) using silica gel (60120 mesh) and mobile phases using 800 mL of n-hexane (100%), n-hexane-ethyl acetate (99: 1, 97:3, 95:5,93:7,90:10,80:20, 70:30, 50:50), CH3COOC2H5 (100%), CH3COOC2H5-CH3OH (90:10, 80:20, 50:50) and CH3OH (100%) further resulted in one hundred and twelve (112) fractions which were bulked into seven sub-fractions (1-2, 3-6, 7-12, 13-14, 15-18, 1924, 25-112) after thin layer chromatographic analysis using n-hexane-ethyl acetate (3:2) solvent system. All fractions were screened at a single concentration of 75 µg/mL. Fractions1314, 15-18 and 19-24 precipitated out and were purified by washing with DCM and Hexane (HPLC grade solvents) and the resulting pure compounds were coded as EGC13, EGC15 and EGC19. Sub-fractions 25-112 [116.202mg] were re-chromatographed using reverse phase chromatographic analysis on C18-reversed phase silica gel (40-63 μm) and was eluted with 300 mL of 100 % H2O, H2O:CH3OH (70:30, 60:40, 50:50, 40: 60, 30:70, 20:80, and 10:90) and 100 % CH3OH to give 13 fractions (RC1RC13) were subjected to biological activities using MCF-7 and NCI-H460 cell lines at a Emmanuel O. Ikpefan et al., ISSN: 2814-1423 14 single concentration of 50 µg/mL. Sub-fractions RC-13 (68.55mg) re-chromatographed by preparative TLC analyses on 0.5 mm Silica gel GF254 using n-hexane-ethyl acetate (3:2) and developed three times. Using UV at 254 and 356nm, four bands were scrapped, eluted, concentrated and coded as S-1, S-11, S-2 and S21. Isolate compounds S-1, S11 and S2, S21 were combined and coded as S1and S2 respectively whose purity were confirmed by TLC analyses using chloroform-methanol (3:2) and n-hexane-ethyl acetate (3:2) as solvent systems (Ukwubile et al., 2019) Characterization and structure elucidation of isolated compounds The structures of the isolated compounds were determined using MS, 1D and 2D NMR (1H/ 1H COSY, NOESY; 1H/ 13C HSQC and HMBC) spectroscopic analysis carried out on a Bruker Avance DRX spectrometer at 500 MHz (1H) or 125 MHz (13C), with CDCl3 as solvent. Twodimensional experiments (1H/ 1H COSY, NOESY; 1H/ 13C HSQC and HMBC) were set up and processed with standard Bruker software. The signals of the deuterated solvent were taken as reference (Hurlimann, n.d.). SRB antiproliferative assay The cells (MCF-7 and NCI-H460) were cultured with 10% FBS, 1% l-glutamine and antibiotic solutions (penicillin-streptomycinamphotericin) supplemented with DMEM (Dulbecco's Adapted Eagle's Medium). The SRB assay method by Mbaoji et al. (Akah et al., 2012) and Nguyen et al. (Bessède et al., 2014), were adopted with slight modifications. Briefly, cells (10000 cells/well/100 µL were seeded for confluence formation and incubated in CO2 incubator at 37 ºC for 24 h. The antiproliferative activities of fractions (1-100 μg/mL) and isolated compounds (1-100 µM) were carried out on breast (MCF-7) and lung (NCI-H460) cancer cell lines using the sulforhodamine-B assay. The various concentrations of the fractions (1-100 µg/mL) and isolated compound (1-100 µM) were added (100 µL/well) in appropriate wells and incubated for 48 h. Appropriate controls and blanks (drug and extract) were prepared. At the end of 48 h, time zero-1 (Tz1 plate) and time zero-2 (Tz2 plate) plates were fixed with gentle addition of 50 % w/v cold TCA (50 µL/well) before and after the addition of the test samples in experimental plates. These were left at room temperature for 30 min, washed (3x) and dried overnight. After 48 h, experimental plates were also fixed. The dried fixed plates were stained with 100 µL of sulforhodamine solution (0.4 % wt/vol prepared in 1 % acetic acid) for 10 min followed by washing (5x) with 1 % acetic acid to remove excess stain and air-dried. Finally, 100 µL of tris base solution (pH 10.2, 10 mM) was added and absorbance was recorded at 545 nm using a micro-plate reader. The results of the extracts, fractions and compounds were presented as GI50 (growth inhibition of 50 % of cell population), TGI (total growth inhibition) and LC50 (lethal/killing concentration for 50 % of cell population) and expressed in μg/mL (Pillai et al., 2022). Statistical analysis All data were expressed as mean ± SD (n=3). The values of p < 0.05 were considered statistically significance using one-way analysis of variance (one-way ANOVA) followed by Dunnett’s post hoc test as was analyzed using GraphPad Prism version 9 (UK). Results The results in Table 1 showed that the bulked VLC sub-fractions (CA-CC) of E. graminea on MCF-7 and NCI-H460 cell lines at concentrations between 1-100 µg/mL showed fraction CB (5-9) was the most active as it Emmanuel O. Ikpefan et al., ISSN: 2814-1423 15 produced cytotoxicity of 1.26 and 3.89 % at 75 and 100 µg/mL respectively. At similar concentrations, CA (1-4) and CC (10) produced growth inhibitions of 84.53, 94.40 and 41.55 and 58.60 % respectively. LC50 for the three fractions were observed to be greater than 100 µg/mL while fraction CB (5-9) had TGI and GI50 of 76 and 23 µg/mL (Table 1). Table 1. Effects of the VLC fractions of E. graminea on MCF-7 cell lines Bulked VLC Fractions Conc. (µg/mL) (%) Growth Inhibition/cytotoxicity GI50 LC50 TGI (µg/mL) CA(1-4) 1.0 25 50 75 100 0.00 ± 0.00 +30.53 ± 2.94 +54.08 ± 2.94 +84.53 ± 1.53 +94.40 ± 1.0 97.82± 2.0* ˃100 ˃100 CB (5-9) 1.0 25 50 75 100 +12.50 ± 0.04 +66.08 ± 1.31 +80.10± 1.01 –1.26 ± 0.07 –3.89 ± 0.29 22.72± 0.0* ˃100 76±0* CC (10) 1.0 25 50 75 100 0.00 ± 0.00 0.00 ± 0.00 +12.22± 0.14 +41.55 ± 0.19 +58.60 ± 0.25 ˃100 ˃100 ˃100 - sign represents cytotoxicity, + sign represents growth inhibition. GI50 = growth inhibition of 50% of cell population, TGI = total growth inhibition, and LC50 = lethal/killing concentration for 50% of cell population. Column chromatographic fractionation of CB gave 112 fractions bulked into C1-C6. While fractions C4 (13-14) and C5 (15-24) precipitated and were cleaned up and coded as EGC13 and EGC15 respectively, fraction C6 (25-112) was subjected to reverse phase chromatographic analysis yielding 94 sub-fractions bulked into RCI-RC10 bulked fractions with RC10 showing activity against the cell lines (RCI-RC9 were inactive). *Statistically significant at p < 0.05 (one-way ANOVA followed by Dunnett’s post hoc; P = 0.001). In addition, the trend of activity on NCI-H460 was also like that of MCF-7 but there was no cytotoxic effect even at the maximum concentration of 100 µg/mL. At 75 and 100 µg/mL, CA produced 29.65 and 61.67 % growth inhibition while CB gave 81.26 and 89.85 % respectively against NCI-H460. However, CC was observed to be inactive at 100 µg/mL. Both the TGI and LC50 were greater than 100 µg/mL for all the fractions. Hence, fractions CB (5-9) was the most active and was selected for further purification (Table 2). Emmanuel O. Ikpefan et al., ISSN: 2814-1423 16 Table 2. Effects of the bulked VLC fractions of E. graminea on NCI-H460 Cell VLC fractions Conc. (µg/mL) (%) Growth Inhibition GI50 LC50 TGI (µg/mL) CA(1-4) 1.0 25 50 75 100 0.00± 0.00 0.00 ± 0.00 +14.20 ± 0.42 +29.65 ± 1.46 +61.67 ± 8.07 92 ± 4* ˃100 ˃100 CB(5-9) 1.0 25 50 75 100 +19.73 ± 0.06 +53.57 ± 2.55 +67.54± 1.22 +81.26 ± 4.84 +89.85 ± 1.21 54 ± 0.05* ˃100 ˃100 CC(10) 100 ˂50 >100 >100 >100 GI50 = growth inhibition of 50% of cell population, TGI = total growth inhibition, and LC50 = lethal/killing concentration for 50% of cell population. *Statistically significant at p < 0.05 (one-way ANOVA followed by Dunnett’s post hoc; P = 0.001). The antiproliferative studies of the subsequent chromatographic sub-fractions (C1-C6) from CB (5-6), at single concentration of 75 µg/mL showed higher antiproliferative activities on both cell lines as they recorded cytotoxicity of - 73. 00 and -61.0 % as well as -13.52 and -11.18 % on MCF-7 an NCI-H460 cell lines, respectively (Plate 1). Antiproliferative activities of the bulked reversed phase column chromatographic fractions at a single concentration of 50 µg/mL against MCF-7 breast and NCI-H460 lung cell lines showed RC10 produced cytotoxicity of 22.95 and 9.30 %, respectively which resulted in cytotoxic effects of the compounds against the cell lines and subsequent prevention of metastasis (Plate 1). However, sub-fractions RC1-RC9 were recorded to be inactive on both cell lines with their GI50, LC50 and TGI ˃50. Fractions RC10 were subjected to preparative thin layer chromatographic analysis and two compounds S1 and S2 were isolated. At 75 and 100 µM, compound EGC 19 (Plate 1; Table 3) and S2 were observed to produce growth inhibitory effects of 54, 78, 80 and 91 %, respectively against MCF-7 cell lines with GI50 of 62 and 54 µM. Emmanuel O. Ikpefan et al., ISSN: 2814-1423 17 Plate 1. Photomicrographs of cancer cells exposed to various concentrations of Abietane-11, 23 diene16-oic-14-one. The red arrows indicate live cancer cells and black arrows indicate apoptotic cells, 40x. 1 EGC 13 2 EGC 15 1 2 Untreated Treated MCF-7 NCI-H460 Emmanuel O. Ikpefan et al., ISSN: 2814-1423 18 12 3 1 2 3 Plates Solvent system R f 1 C6H14 :CH3COOC2H5 (9.2 : 0.8) 0.88 Plates Solvent system R f 1 C6H14 :CH3COOC2H5 (9.2 : 0.8) 0.52 S1 S2 Plate 2. Chromatograms showing compounds EGC13, EGC15, EGC19, S1 and S2 from the column chromatographic analysis of the bioactive fraction of E. graminea. AdsorbentSilica gel (C6H14:CH3COOC2H5 (9.8:0.2; 9:1). Spray reagentCeric acid sulphate, heated at 110C. At the same concentrations, while compound EGC 19 recorded GI50, LC50 and TGI’s of 19.4,40.5 and 32.6 µM, S1 was observed to produce GI50, LC50, TGI of 34, 91.4 and 56.27±8.23µM respectively (Table 3). The higher activity of EGC15 (Plate 2) and S1 was similarly observed against NCI-H460 cell lines with the former and latter having GI50 and TGI of 50 and 89 µM and 63 and 94 µM respectively. However, they were observed to produce LC50 higher than 100 µM (Plate 2; Tables 3 and 4). These cytotoxic effects displayed by these compounds were statistically significant (p<0.05) when compared to doxorubicin standard drug. 1 2 EGC 19 Emmanuel O. Ikpefan et al., ISSN: 2814-1423 19 Table 3. Growth Inhibitory and Cytotoxic Effect of the isolated compounds on MCF-7 Cell Lines Compound Conc. (µM) % Growth Inhibition/ cytotoxicity GI50 LC50 TGI (µM) EGC 13 100 ˂50 ˃100 ˃100 ˃100 EGC15 1.0 25 50 75 100 +14 ± 2.51 +54 ± 3.80 –65 ± 5.33 –73 ± 3.70 –93 ± 3.14 19.4 ± 0.33* 40.5 ± 0.21* 32.60±5.01 EGC19 1.0 25 50 75 100 0.00 ± 0.00 0.00 ± 0.00 +18.00 ± 1.81 +54.98 ± 7.80 +78.00± 3.17 62.40± 0.12* ˃100 ˃100 S1 1.0 25 50 75 100 +3.50 ± 1.00 +14.90 ± 2.00 +63.00 ± 18.00 +84.60 ± 8.00 – 4.30 ± 1.40 33.65 ± 0.74* 91.40±6.95* ˃100 S2 1.0 25 50 75 100 +13 ± 1.55 +28 ± 2.17 +46 ± 0.75 +80 ± 1.45 +91 ± 1.61 53.90 ± 5.85* ˃100 ˃100 Doxorubicin 0.01 0.1 0.5 5.0 10.0 +4 .00± 1.0.00 +37.00 ± 5.00 +81.00 ± 2.00 –18.00 ± 3.00 –70.00 ± 2.00 0.55 ± 0.08* 10±0.2* 11 ± 1.0* - sign represents cytotoxicity, + sign represent growth inhibition, GI50 = growth inhibition of 50% of cell population, TGI = total growth inhibition, and LC50 = lethal/killing concentration for 50% of cell population. *Statistically significant at p < 0.05 (one-way ANOVA followed by Dunnett’s post hoc; P = 0.001). Emmanuel O. Ikpefan et al., ISSN: 2814-1423 26 Fig. 4. Mass spectra sheet (GCMS) of sample S1 (of Abietane-11, 23 diene-16-oic-14-one) a. b. Fig. 5. Proposed structure (a) and configuration (b) of S1 (Abietane-11, 23 diene-16-oic-14-one). Emmanuel O. Ikpefan et al., ISSN: 2814-1423 27 EGC 13 EGC 15 Fig. 6. Proposed structures of other compounds (1-4) isolated from E. graminea. Discussion From the current study, unlike compound S1, compounds EGC 13 (Triacontan-1-ol), EGC 15 (Dotriacontan-1-ol), EGC 19 (β-sitosterol) and S2 (β-stigmasterol) had spectra data similar to already existing compounds isolated from plants (Lalthanpuii & Lalchhandama, 2020). The TLC was used to check the purity of isolated compounds by obtaining a single spot of the plate (Plate 2). Unlike the breast cancer cell lines (MCF-7) (Plate 1), the effects of the fractions against lung cancer cells (NCI-H460) were observed to be growth inhibitory rather than cytotoxic effects. Lung cancers have been reported to be the most difficult form of cancer to treat (Tavares-Carreón et al., 2020). This may explain why the fractions and compounds only retarded the growth and not cytotoxic. Relating the activities of compound EGC 19 (βSitosterol), S1 (Abietane-11, 23 diene-16-oic14-one) (Tables 5 and 6) and S2 (βStigmasterol) revealed some variation in their biological activities. These variations could be due to the extra α, β-unsaturated γ-lactone ring in S1 and double bonds in S2. The aim of the present study at isolating the anti-proliferative bioactive constituents from the chloroform fraction of E. graminea was achieved. This was due to the fact that a total four known compounds comprising of two fatty alcohols (Triacontan-1-ol and Dotriacontan-1-ol) and two stigmastane (β-sitosterol and βstigmasterol) (Lalthanpuii & Lalchhandama, 2020), as well as a new diterpene (Abietane-11, EGC 19 S2 Emmanuel O. Ikpefan et al., ISSN: 2814-1423 28 23 diene-16-oic-14-one l) were isolated and their structures were elucidated via spectroscopic methods. The most important data were gained from NMR measurements. 1D and 2D NMR (1H NMR and 13C NMR) spectra were recorded for all the compounds. To the best of our knowledge, these compounds were isolated for the first time from this plant. The anticancer mechanism of these diterpenoids and phytosterols are not fully understood but is believed that they induce apoptosis in cancer cells by controlling the signal pathway of PI3K/Akt as well as production of mitochondrial-derived reactive oxygen species (mROS), while their growth inhibitory effects are majorly dependent on the modulatory effect against cyclin proteins and cyclin-dependent kinase (Porter & Jänicke, 1999) . Our study showed the presence of five bioactive compounds from chloroform fraction of E. graminea leave extract. The isolated compounds including the new Abietane-11, 23 diene-16-oic-14-one was isolated from this plant for the first time, and they were observed to exhibit a selective antiproliferative effects against the cell lines as they were more active on MC-7 compared to NCI-H460 cell lines. The results of this study show that E. graminea can significantly inhibit the growth of human MCF7 and NCI-H460 cells and contains a contains a new compound, Abietane-11, 23 diene-16-oic14-one, whose novelty requires further investigation The results suggest that Abietane11, 23 diene-16-oic-14-one is viable and this compound offers a new avenue for chemotherapeutic intervention after it has been clinically investigated. The study further affirms the use of E. graminea extract for the treatment and management of cancers in Nigeria. Acknowledgements The authors are grateful to Cancer Research Laboratories especially those in Pakistan for technical assistance helps. 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