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Anti-proliferative and immunomodulatory activities of fractions from methanol root extract of Abrus precatorius L

Anti-proliferative and immunomodulatory activities of fractions from methanol root extract of... Background: Abrus precatorius possesses various therapeutic properties including anticancer potentials. This study evaluated the anti-proliferative activities of fractions of methanol root extract of A. precatorius on breast and cervical cancer cells and their immunomodulatory effect. Phytochemical screening was done by FTIR and GCMS. In vitro anti-proliferative effect was evaluated on human breast cancer (AU565) and cervical cancer (HeLa) cells and on murine fibroblast (NIH 3 T3) cells. Antioxidant activity was performed via DPPH radical scavenging assay. The immunomodulatory potential of fractions was evaluated by inhibition of phagocytes oxidative burst (ROS), Nitric oxide (NO) and proinflammatory cytokine TNF-α. Results: A. precatorius fractions showed different chemical groups and were somewhat selective in antiproliferative activity against studied cancer cells. Ethyl acetate fraction showed the most significant antiproliferative activity with IC values of 18.10 μg/mL and 11.89 μg/mL against AU565 and HeLa cells respectively. Hexane fraction significantly (p < 0.05) inhibited HeLa cells (IC 18.24 ± 0.16 μg/mL), whereas aqueous fraction showed mild inhibition (IC 50 50 46.46 ± 0.14 μg/mL) on AU565 cell proliferation. All fractions showed no cytotoxicity against NIH-3 T3 murine fibroblast normal cells. All fractions showed potent and significant (p < 0.001) DPPH radical scavenging activity as well as suppressed phagocytic oxidative burst. Hexane (< 1 μg/mL), ethyl acetate (< 1 μg/mL), and butanol (5.74 μg/ mL) fractions potently inhibited the cytokine TNF- α, hexane (< 1 μg/mL) and ethyl acetate (< 1 μg/mL) fractions also potently inhibited NO. Conclusions: The antiproliferative activities and suppressive effect on the phagocytic oxidative burst, NO and proinflammatory cytokine might be due to the synergistic actions of bioactive compounds especially flavonoids present in the assayed fractions and therefore, suggest chemotherapeutic use of A. precatorius in cancer treatment. Keywords: Abrus precatorius, Breast cancer, Cervical cancer, Cytokines, Cytotoxicity, Tumor necrosis factor, oxidative burst Introduction from natural product sources. Cancer is broadly de- Cancer is one of the leading causes of death in both de- scribed as a group of diseases characterized by uncon- veloped and underdeveloped countries. It has generated trolled growth and spread of abnormal cells, associated considerable scientific and commercial interest especially with resistance to normal growth-inhibitory signals, un- in the progressive discovery of new anticancer agents controlled activation of growth signals, impairment of apoptosis, promotion of angiogenesis, invasion of sur- rounding tissues and metastasis [1, 2]. * Correspondence: okoroemeka2003@gmail.com Breast cancer is the most commonly diagnosed cancer Biochemistry Department, Babcock University Ilishan-Remo, PMB 21244, Ikeja, Nigeria in several sub-Saharan African countries, a shift from a Dr. Panjwani Center for Molecular Medicine and Drug Research (PCMD), trend in which cervical cancer was the leading cause of International Center for Chemical and Biological Sciences, University of cancer-related deaths among women in Africa over the Karachi, Karachi 75270, Pakistan Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Okoro et al. Clinical Phytoscience (2019) 5:45 Page 2 of 9 past decade [1]. The reasons for this shift still remain but not limited to antidiabetic [19, 20], anti- unclear but have a strong correlation with prevalent risk inflammatory [21] activities. factors such as obesity, early menarche, late childbearing, Based on the above mentioned considerations, this and lifestyle associated with urbanization and economic study evaluated the antiproliferative and immunosup- development [3]. pressive activities of methanolic crude extract and frac- The generation of reactive oxygen species (ROS) dur- tions of A. precatorius roots which will be able to ing oxidative burst is considered as one of the major simultaneously modulate human neutrophils’ oxidative mechanisms by which phagocytes exert their tumoricidal burst, restraining the inflammatory process, and inhibit functions [4]. Phagocytes such as macrophages undergo the growth of breast (AU565) and cervical (HeLa) cancer oxidative burst in response to antigenic stimuli with gen- cell lines. To underscore the broad-spectrum activity of eration and release of different reactive oxygen metabo- the plant, two cancer cell lines of different origin were lites thereby making oxidative burst as defense function adopted for the study primarily due to their invasive [5]. Oxidative stress is closely linked with carcinogenesis metastatic nature. due to the interplay of ROS in relation to other epigen- etic factors in the induction, promotion, and modulation Materials and methods of breast and cervical cancer [6]. Plant material Nitric oxide (NO) is a short-lived pleiotropic regulator, Young roots of A. precatorius were collected from Imota that plays critical roles in numerous physiological as well Ikorodu, Nigeria. A voucher specimen (IFE-17655) was as pathological processes [7]. Its role in tumor develop- deposited at the Herbarium of Obafemi Awolowo Uni- ment is somewhat complex [8]. However, reported roles versity, Ile-Ife, Nigeria. of NO such as genotoxic mechanisms, antiapoptotic ef- fects, induction, and promotion of angiogenesis, limita- tion of host immune response against tumor, and Extract preparation promotion of metastasis has been implicated in various Young fresh A. precatorius roots were thoroughly types of cancer [9] and NO tumor-promoting effect ap- washed, oven-dried (Uniscope SM9053) at 40 °C for 96 h pears to be both time and concentration-dependent [10]. to a constant weight and pulverized. Methanol (70%) (1: Overproduction of reactive oxygen (ROS) and nitrogen 10) crude extraction of pulverized A. precatorius roots (RNS) species by phagocytes, namely neutrophils, may was done for 48 h using a shaker water bath (Uniscope result in chronic inflammation and initiation of the SM101) at 40 °C and filtered (Whatman No.1110 mm). multistage process of various cancer development in- The filtrate was concentrated using a Rotary evaporator cluding breast and cervical cancer [11]. (Stuart RE 300) to obtain the crude methanol extract, Mitogen-activated protein (MAP) kinases in different reconstituted in distilled water (1:5) and subjected to cell types are involved in the production of extracellular liquid-liquid partitioning using solvents of increasing po- polypeptides or glycoproteins called cytokines. Cytokine larity to obtain partially purified – n-hexane, ethyl acet- activity is influenced by the microenvironment in which ate, n-butanol, and aqueous fractions respectively. The they are produced and as such may have pro- (Th1) or fractions were further concentrated to a constant weight anti-inflammatory (Th2) actions [12]. Tumor necrosis under reduced pressure using a Rotary evaporator at factor (TNF-α), an inflammatory cytokine that is highly 40 °C and freeze-drying for the aqueous fraction. expressed in breast and cervical carcinomas play an im- portant role in the regulation of both induction and pro- tection in breast and cervical cancer [13, 14], and Phytochemical screening apoptosis [15]. To identify the chemical constituents and possible Abrus precatorius (family: Fabaceae) is a perennial, functional groups, A. precatorius fractions were sub- well-branched, twinning and climbing herb that bears a jected to Fourier Transform Infra-red (FTIR) spec- characteristic bright red colored seeds with a black troscopy measured on VECTOR22, Resolution 2 cm blotch at the hilum [16]. It is endemic in the tropics and (10 scans) and Gas chromatography-mass spectrom- commonly known as Rosary bean. It is known as Otuo- etry (GCMS) analysis using Agilent 5975C gas chro- biribiri (Igbo-Ohafia), Idon zakara (Hausa), Oju ologbo matograph combined with inert XL EI/CI MSD and (Yoruba) in Nigeria. The roots of A. precatorius contain Triple-Axis Detector source at 270 °C at 70 eV. The proteins, glycosides, phenolic compounds, fatty acid, injector was set at 270 °C with splitting ratio 1:30. A fatty acid esters, anthocyanins, and minerals [17]. A. pre- mass spectral survey was performed using the NIST catorius has been reported from folklore to have poten- mass spectral program. The concentrations of the tial antitumor properties [18]. Other reported identified compounds were calculated using area ethnopharmacological and therapeutic activities include normalization over the FID response method. Okoro et al. Clinical Phytoscience (2019) 5:45 Page 3 of 9 Cytotoxicity screening USA) and data reported as mean ± standard error of Cytotoxic activity of extract and fractions of A. precator- mean. N-acetylcysteine and Gallic acid were used as ref- ius roots, was evaluated in 96-well flat-bottomed micro- erence compounds. plates by using the standard MTT (3-[4, 5- dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium brom- Oxidative burst assay ide) colorimetric assay. For this purpose, cell lines were The studies on human blood cells were performed after cultured in Minimum Essential Medium Eagle, supple- an approval from independent ethics committee, ICCBS, mented with 5% of fetal bovine serum (FBS), 100 IU/ml UoK, No: ICCBS/IEC-008-BC-2015/Protocol/1.0. of penicillin and 100 μg/ml of streptomycin in 75cm Luminol-enhanced chemiluminescence assay was per- flasks, and kept in 5% CO incubator at 37 °C. 200 μLof formed, as described by Helfand et al. [24]. Briefly, 25 μL cell suspension were seeded in round bottom 96 well of diluted human whole blood / (1 × 10 ) isolated PMNs plate at the density of 10,000 cell/well and incubated at ++ in HBSS (Hanks Balanced Salt Solution, containing 37 °C in 5% CO incubator for 24 h. Exponentially grow- calcium chloride and magnesium chloride) [Sigma, St. ing cells were harvested, counted with a hemocytometer Louis, USA] was incubated with 25 μL of various con- and diluted with the medium. Cell culture with the con- centrations (0.1–250 μg/mL) of A. precatorius fractions. centration of 6 × 10 cells/ml was prepared and intro- ++ Control wells received HBSS and cells, but no com- duced (100 μL/well) into 96-well plates. After overnight pounds. The test was performed in white half area 96 incubation, the medium was removed and 200 μLof well plates [Costar, NY, USA], which was incubated at fresh medium was added with different concentrations 37 °C for 15 min in the thermostat chamber of lumin- of 10–100 μg/mL of fractions and 0.23–30 μg/mL for 3 ometer [Labsystems, Helsinki, Finland]. After incubation, T3 cells. 25 μL of serum-opsonized zymosan (SOZ) [Fluka, Buchs, After 48 h, 200 μL MTT (0.5 mg/ml) was added to Switzerland] and 25 μL of intracellular reactive oxygen each well and incubated further for 4 h. Subsequently, species detecting probe, luminol [Research Organics, 100 μL of DMSO was added to each well. The extent of Cleveland, OH, USA] were added into each well, except MTT reduction to formazan within cells was calculated ++ blank wells (containing only HBSS ). The level of the by measuring the absorbance at 570 nm, using a micro- ROS was recorded in the luminometer in terms of rela- plate reader (Spectra Max Plus, Molecular Devices, CA, tive light units (RLU) for 50 mins in the repeated scan USA). The cytotoxicity was recorded as concentration mode. Ibuprofen was used as a standard drug. causing 50% growth inhibition (IC ) for cell lines [22]. Doxorubicin and Cyclohexamide served as standard drugs. The percent inhibition was calculated by using the fol- Cell lines lowing formula: The cell lines used in this study including AU565 (hu- man breast adenocarcinoma, CRL-2351) and NIH-3 T3 ðÞ mean OD of test compound−mean OD of negative control %Inhibition ¼ 100−  100 (mouse embryonic fibroblast, CRL-1658) were purchased ðÞ mean OD of positive control−mean OD of negative control from ATCC, Manassas, USA, THP-1 (Human monocytic leukemia) and J774.2 (mouse macrophages) were pur- DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging chased from ECACC, Salisbury, UK and HeLa cells (hu- assay man cervical adenocarcinoma) were purchased from DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging CLS, Germany by Dr. Panjwani Centre for Molecular activities of extract and fractions of A. precatorius roots, Medicine and Drug Research (PCMD), International was done using the method described by Sagar & Singh Center for Chemical and Biological Sciences) (ICCBS), [23]. DPPH solution (95 μl, 300 μM) in Ethanol was University of Karachi, Karachi-75270, Pakistan. NIH-3 mixed with test solution (5 μl, 500 μM). The reaction T3, HeLa and J774.2 cells were grown in DMEM sup- was allowed to progress for 30 min at 37 °C and absorb- plemented with 10% Fetal bovine serum (FBS) and 1% ance monitored by the multiplate reader, SpectraMax340 penicillin/streptomycin and AU565 in ATCC modified at 517 nm. Upon reduction, the color of the solution Rosewell Park Memorial Institute (RPMI) medium faded (Violet to pale yellow). Percent Radical Scavenging supplemented with 90% FBS, 1% penicillin, 1% Activity (%RSA) was determined by comparison with a streptomycin. The cells were grown in 75 cc culture DMSO containing control. The concentration that flask and upon reaching 75% confluency were har- causes a decrease in the initial DPPH concentration by vested and used for experimental purpose. For cyto- 50% is defined as IC value. The IC values of the frac- toxicity on NIH-3 T3 and HeLa (6 × 10 cells/mL), 50 50 tions were calculated using EZ-Fit Enzyme kinetics soft- AU565 (6 × 10 cells/mL) and for NO assay from ware program (Perrella Scientific Inc. Amherst, MA, J774.2 (1 × 10 cells/mL) were used. Okoro et al. Clinical Phytoscience (2019) 5:45 Page 4 of 9 Nitric oxide assay dihydroxyflavanone, 4H-1-Benzopyran-4-one, 5,7-dihy- The mouse macrophage cell line J774.2 was cultured in droxy-2-(3-hydroxy-4,5-dimethoxyphenyl)-6,8- 75 cc flasks IWAKI (Asahi Techno Glass, Japan) in dimethoxy, 3-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)- DMEM Sigma-Aldrich (Steinheim, Germany) supple- 4H-chromen-4-one), triterpenes (lupeol, α-amyrin), aro- mented with 10% fetal bovine serum GIBCO (N.Y U. S) matic carboxylic acids (Benzenepropanoic acid), aro- 1% streptomycin/penicillin. Flasks were kept at 37 °C in matic alcohols (1H-Indole-3-(ethanol), as shown in an atmosphere of humidified air containing 5% CO , Fig. 1 and Table 1. cells were seeded in 96-well plate (10 cells/mL) and were induced by 30 μg/mL Escherichia coli lipopolysac- Cytotoxicity screening charide (LPS) (DIFCO Laboratories Michigan, USA). The antiproliferative activities of the crude extract and Three different concentrations of fractions (1, 10 and fractions of A. precatorius are presented in Table 2. 100 μg/mL) were added simultaneously with LPS and The results of the cytotoxicity of A. precatorius frac- the plate was incubated for 48 h at 37 °C in 5% CO . The tions against breast adenocarcinoma (AU565) cell line FBS concentration was 5%. Nitrite accumulation in the showed that ethyl acetate and aqueous fraction signifi- culture supernatant was measured using the Griess re- cantly (p < 0.05) inhibited breast adenocarcinoma prolif- agent [25]. eration (IC 18.10 ± 2.68 μg/mL, IC 46.46 ± 0.14 μg/ 50 50 mL respectively). Crude extract, hexane, and butanol Cytokine production and quantification fractions were found to be inactive against breast adeno- Human monocytic leukemia cells THP-1 cells were carcinoma. The ethyl acetate and hexane fractions also maintained in RPMI-1640 containing 5.5 mmol/L glu- showed significant (p < 0.05) inhibition of cervical cancer cose (BioM Laboratories, Chemical Division, Malaysia), (HeLa) cell proliferation (IC 11.89 ± 0.63, 18.24 ± 50 μmol/L mercaptoethanol (Merck Darmstadt, 0.16 μg/mL respectively). Crude extract, butanol, and Germany), 10% FBS (fetal bovine serum), 2 mmol/L; L- aqueous fractions were found to be inactive against cer- glutamine (PAA Laboratories, GmbH, Pasching, vical cancer cells. All the fractions showed no cytotox- Austria). Cells were grown in 75 cc flasks, harvested and icity against 3 T3 murine fibroblast normal cells thus 2.5 × 10 cells/mL was then plated in 24-well tissue cul- indicating their safety (Table 2). ture plates. 20 ng/mL of phorbol myristate acetate (PMA), (SERVA, Heidelberg, Germany) was added In vitro antioxidant activity of A. precatorius fractions followed by incubation for 24 h at 37 °C in 5% CO to The results of the DPPH Radical Scavenging Activity are convert them into a macrophage-like cell. Cells were presented in Table 3. then stimulated with E. coli Lipopolysaccharide B, All assayed fractions of A. precatorius root methanol (DIFCO Laboratories, Michigan, USA) at a final concen- extract showed significant (p < 0.001) DPPH scavenging tration of 50 ng/mL and treated with fractions using activity compared with standards Gallic acid and N- three concentrations (1, 10 and 100 μg/mL) and then in- acetyl cysteine. Hexane fraction inhibited DPPH radical cubated for 4 h at 37 °C in 5% CO . The supernatants formation with significant (p < 0.001) IC 0.010 ± 0.002 2 50 collected were analyzed for the level of TNF -α using the mg/mL compared with the positive controls, making it human TNF-α Duo Set ELISA (R&D Systems, Minneap- the most active fraction in DPPH radical scavenging ac- olis, USA), and according to manufacturer’s instructions tivity Methanolic crude extract inhibited DPPH forma- [25]. tion with an IC of 0.087 ± 0.002 mg/mL; whereas the ethyl acetate, butanol, and aqueous fractions inhibited Statistical analysis the formation of DPPH radicals with IC 0.079 ± 0.005 Data from all experiments were statistically evaluated mg/mL, 0.098 ± 0.002 mg/mL, and IC 0.086 ± 0.002 using one-way analysis of variance (ANOVA) on Soft- mg/mL respectively compared with the standards Gallic Max Pro software and results expressed as mean ± acid (0.0032 ± 0.0001 mg/mL), and N-acetyl cysteine standard deviation (SD) and mean ± standard error of (0.0141 ± 0.0001 mg/mL) respectively (Table 3). mean (SEM). P < 0.05 at 95% and p < 0.001 at 99.9% con- fidence level was considered statistically significant for Immunomodulatory activities differences in mean and were obtained by Student’st- The results of immunomodulatory activities are pre- test analysis. sented in Table 4. Methanolic crude extract and ethyl acetate fraction of Results A. precatorius showed potent inhibition of whole blood Phytochemical analysis ROS with IC of < 10 μg/mL respectively, whereas hex- Phytochemical screening revealed the presence of several ane (30.5 ± 0.3 μg/mL) and butanol (21.0 ± 0.5 μg/mL) chemical groups: flavonoids/isoflavones (5-Methoxy-3,7- fractions mildly inhibited whole blood ROS. Hexane, Okoro et al. Clinical Phytoscience (2019) 5:45 Page 5 of 9 −1 − 1 Fig. 1 Chromatogram of FTIR of A. precatorius. Resolution 2 cm (10 scans). Intense peaks around 1500-3450 cm indicating the presence of flavonoids, isoflavonoids, carbonyl groups, and hydroxyl flavones ethyl acetate and butanol fractions of A. precatorius invasion, metastasis, and angiogenesis, and induce pro- showed significant (p < 0.001) suppression of oxidative apoptotic pathways [30, 31]. The observed cytotoxic ac- burst generated from zymosan activated polymorpho- tivity may be attributed to the phytoconstituents such as nuclear cells (PMNs) (IC 0.6 ± 0.003 μg/mL, 0.6 ± flavonoids, and terpenes present in these fractions, 0.002 μg/mL, 6.1 ± 0.8 μg/mL respectively) and also which may have worked synergistically to exert these ef- found to significantly (p < 0.001) inhibit proinflammatory fects [32]. cytokine TNF-α with IC values of < 1 μg/mL for hex- A handful of ‘red line’ events which propel tumor cells ane and ethyl acetate and 5.74 ± 0.05 μg/mL for butanol and their derivatized progenies into full-blown uncon- fraction respectively. The hexane and ethyl acetate frac- trolled metastasis have made cancer a complex idio- tion also inhibited the NO production with IC values pathic disorder. Deregulation of cell proliferation of < 1 μg/mL respectively. The aqueous fraction was alongside suppressed apoptosis, provide leverage for all found to be inactive (Table 4). cancer evolution and progression. Uncontrolled cell div- ision is a primary determinant and underlying factor in Discussion the progression of cancer tumors. To evaluate fractions Phytochemical screening showed that they are abundant from root methanol extract of A. precatorius as a poten- in flavonoids, terpenes, alkaloids, and glycosides. Flavo- tial therapy for cancer, different fractions were assayed noids, as well as terpenes, possess anticancer properties against human breast cancer cells (AU565) and cervical [26] through their effects on signal transduction in cell cancer (HeLa) cell lines. The antiproliferative effects proliferation and angiogenesis [27]. Flavonoids have also were quantified in terms of cytotoxicity and IC values been implicated in playing a major role in attenuating determined. One of the reliable criteria in the assess- the development of tumors via their antioxidant effects ment of any anticancer drug is a decrease in tumor vol- [28]. Flavonoids may interfere with the activation of the ume and viable tumor cell count and an increase in proinflammatory nuclear factor-kB (NF-kB) and tumor non-viable tumor cell count. The results of this study activator protein-1 (AP-1) while inducing cell cycle ar- show an anticancer effect of A. precatorius fractions rest and apoptosis [29]. Flavonoids repress molecular against breast adenocarcinoma and cervical cancer. The targets that stimulate proliferation, inflammation, results reveal that the fractions were somewhat selective Okoro et al. Clinical Phytoscience (2019) 5:45 Page 6 of 9 Table 1 Identified compounds from methanolic crude extract of A. precatorius roots Peak Compound Formula t Area Remarks # (min) % 1 Benzoic acid C H O 7.88 0.07 antimicrobial, antimutagenic, antiestrogenic, 7 6 2 hypoglycemic, anti-inflammatory 2 1-Dodecanol C H O 15.23 0.38 An insect attractant, pesticide 12 26 3 Benzaldehyde, 4-hydroxy-3,5-dimethoxy- C H O 19.65 0.08 Antioxidant, antimicrobial and hypoglycemic 9 10 4 4 1-Hexadecanol C H O 19.88 0.21 Emulsifier 16 34 5 Ethanol, 2-(dodecyloxy)- C H O 20.76 0.13 14 30 2 6 1H-Indole-3-ethanol C H NO 21.83 0.46 Estradiol metabolism 10 11 7 Hexadecanoic acid, methyl ester C H O 26.83 5.31 Antioxidant, anti-inflammatory 17 34 2 8 Dibutyl phthalate C H O 28.32 0.54 Antibacterial 16 22 4 9 n-Hexadecanoic acid C H O 28.58 0.75 Antibacterial, antifungal 16 32 2 10 Heptadecanoic acid, methyl ester C H O 31.51 0.36 Antimicrobial 18 36 2 11 9,12-Octadecadienoic acid (Z,Z)-, methyl ester C H O 35.99 7.28 Antibacterial 19 34 2 12 11-Octadecenoic acid, methyl ester C H O 36.44 5.08 Antimicrobial 19 36 2 14 Octadecanoic acid, methyl ester C H O 37.89 1.27 Antimicrobial, anti-inflammatory, anticancer 19 38 2 15 9,12-Octadecadienoic acid (Z,Z)- C H O 38.22 0.46 Antibacterial 18 32 2 16 cis-Vaccenic acid C H O 38.54 0.63 Antibacterial, hypolipidemic 18 34 2 17 Benzene, 1,1′-tetradecylidenebis- C H 43.96 1.11 Antimicrobial 26 38 18 5-Methoxy-3,7-dihydroxyflavanone C H O 47.19 2.66 Antioxidant, anti-inflammatory, anticancer 16 14 5 19 1,2-Benzenedicarboxylic acid, diisooctyl ester C H O 47.29 1.71 Antimicrobial, antioxidant, anticancer 24 38 4 20 3-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-chromen-4-one C H O 49.87 7.48 Anticancer, antihypertensive, antioxidant 16 12 5 21 13-Docosenamide, (Z)- C H NO 50.15 0.94 Antimicrobial 22 43 22 2,2,4-Trimethyl-3-(3,8,12,16-tetramethyl-heptadeca-3,7,11,15- C H O 51.09 0.51 30 52 tetraenyl)-cyclohexanol 23 Benzaldehyde, 4-hydroxy-3,5-dimethoxy-, [(4-hydroxy-3,5- C H N O 51.88 1.65 Indicator for laccase and peroxidase activity 18 20 2 6 dimethoxyphenyl)methylene]hydrazone 24 4H-1-Benzopyran-4-one, 5,7-dihydroxy-2-(3-hydroxy-4,5- C H O 52.69 2.93 antibacterial 19 18 9 dimethoxyphenyl)-6,8-dimethoxy- 25 6,6,7-Trimethyl-9-oxo-3-oxabicyclo(3.3.1) nonane 2,4- C H N O 53.59 9.16 17 22 4 5 dinitrophenylhydrazone 26 1,1′-Biphenyl-5-carboxylic acid, 2,3,4,4′-tetramethoxy-6′- C H O 53.76 1.13 Antibacterial 19 20 8 methoxycarbon 27 Silane, dimethyl(3-ethylphenoxy)tetradecyloxy- C H O Si 54.71 41.09 Antimicrobial 24 44 2 28 Anthiaergosta-1,5,7,9-tetraene C H 55.77 2.47 28 42 29 Cyclohexan-1-one-3α,5β-diacetic acid, 2α-(5-hydroxy-2,4- C H O 56.14 0.83 Antibacterial 22 30 8 dimethoxyphenyl)-, diethyl ester 30 α-Amyrin C H O 56.64 0.47 Antimicrobial, antifungal, anti-inflammatory, 30 50 anticancer 31 Lupeol C H O 57.31 0.89 Antiprotozoal, anti-inflammatory, antitumor 30 50 32 Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, C H O 60.73 1.07 Antifungal. antimicrobial 35 62 3 octadecyl ester in their activity against cancer cells with ethyl acetate some anticancer bioactive compounds which will be iso- fraction showing significant anticancer activity against lated and assayed in further study. Mild cytotoxicity of HeLa cell lines and AU565 cell lines whereas hexane the crude extract was observed against 3 T3 murine fraction showed significant inhibition against HeLa cells fibroblast normal cells implying safety and selectivity. only. The crude extract did not show any observable in- These findings are in agreement with a similar report in hibition in the assayed cancer cell lines and therefore in- which dietary flavonoid luteolin inhibited the invasion of dicate that by partial purification, the fractions contain cervical cancer [29]. Okoro et al. Clinical Phytoscience (2019) 5:45 Page 7 of 9 Table 2 Antiproliferative activities of methanolic crude extract Table 4 Immunomodulatory activities of methanolic crude and fractions of A. precatorius roots extract and fractions A. precatorius roots Fraction/Drug WB ROS PMNs ROS NO TNF-α AU565 HeLa 3 T3 IC ±SD IC ±SD IC ±SD IC ±SD 50 50 50 50 Fraction/Drug IC ±SD IC ±SD IC ±SD 50 50 50 Crude < 10.0 NT NT NT Crude NA NA 25.80 ± 0.91 Hexane 30.5 ± 0.3 0.6 ± 0.003 < 1 < 1 Hexane NA 18.24 ± 0.16 NA Ethyl acetate < 10 0.6 ± 0.002 < 1 < 1 Ethyl acetate 18.10 ± 2.68 11.89 ± 0.63 24.80 ± 2.19 Butanol 21.0 ± 0.5 6.1 ± 0.8 NT 5.74 ± 0.05 Butanol NA NA NA Aqueous 12.0 ± 1.0 > 250 NT NT Aqueous 46.46 ± 0.14 NA NA Standard (Ibuprofen) 11.2 ± 1.9 Doxorubicin 0.54 ± 0.04 0.80 ± 0.10 NT: Not tested Values are presented as mean ± SD of triplicates at p < 0.001 by Student’st Cyclohexamide 0.80 ± 0.10 test analysis. WB Whole Blood, NO Nitric Oxide, TNF Tumor necrosis factor, ROS NA: Inactive Reactive Oxygen Species, PMNs Polymorphonuclear cells Values are means of three replicates ± standard deviation (SD). Significant p value (p < 0.05) were obtained by Student’s t test analysis. Composite Macrophages have been implicated in neoplasm de- treatments were compared using one-way analysis of variances (ANOVA) struction via infiltration into the tumor site and partici- pation in inflammatory reaction. They generate reactive One of the basic and routine assays which provide oxygen species through the oxidative burst process front line information on the antiradical activity of which has been fingered as a major mechanism for their plant extracts is DPPH radical scavenging assay. The antimicrobial and tumoricidal functions [34]. Agents results of the DPPH scavenging activity from our such as zymosan used in this study can induce the se- work suggest that fractions of A. precatorius are good quence of oxidative reactions and are known as trigger- sources of antioxidants compounds. However, ethyl ing agents; other substances which can modify the acetate and butanol fractions appear to be excellent magnitude of the response are known as modulating sources of potent antioxidant secondary metabolites. agents. Therefore, the result of this study reveals the This maybedue to the abundance of variousflavo- modulatory role of fractions of A. precatorius thus indi- noids - 4H-1-Benzopyran-4-one, 5,7-dihydroxy-2-(3- cating their anti-inflammatory potentials. hydroxy-4,5-dimethoxyphenyl)-6,8-dimethoxy, 5- Cytokines are critical for tumor immunosurveillance and Methoxy-3,7-dihydroxyflavanone, 3-Hydroxy-2-(4-hy- have demonstrated therapeutic anti-tumor activity in mur- droxy-3-methoxyphenyl)-4H-chromen-4-one, triter- ine models and in the clinical treatment of several human penes and other non-phenolic constituents with cancers where they play complex and often opposing antioxidant effects such as α-Amyrin and Lupeol as roles in the development of the immune system, host shown by the phytochemical screening results. The defense, and tumor immunobiology [35, 36]. Tumor observed IC values of the fractions which were a lit- necrosis factor (TNF-α) is produced by macrophages tle higher compared with the standard drugs may be and is involved in cell activation, co-stimulation, and duetothe crudenatureofthe fractions unlike the inflammation processes. Hexane, ethyl acetate, and pure forms of the drugs. The findings of this study butanol fractions potently suppressed TNF-α with are congruent with a previous report by Mir et al. IC of < 1 μg/mL respectively for hexane and ethyl [33] in which leaf extracts of A. precatorius demon- acetate and 5.74 μg/mL for butanol fraction. The re- strated antioxidant and antiproliferative activities. sult from this study showed that fractions of A. pre- catorius suppressed the expression of TNF-α, hence inhibiting signaling and communication among cancer Table 3 DPPH Radical Scavenging Activities of methanolic cells and is congruent with a similar report by Kang- crude extract and fractions of A. precatorius roots samaksin et al. [37]. Fraction/Standard IC ± SEM Nitric oxide has been reported to have tumor-promoting Crude 0.087 ± 0.002 roles via formation of toxic and mutagenic species, direct modification of DNA– strand breaks, oxidation and de- Hexane 0.010 ± 0.002 amination of nucleic acids, inhibition of systems required Ethyl Acetate 0.079 ± 0.005 to repair DNA lesions, and inhibition of cytochrome C re- Butanol 0.098 ± 0.002 lease [9, 38]. The hexane and ethyl acetate fraction of A. Aqueous 0.086 ± 0.002 precatorius inhibited NO production with IC values of < Gallic Acid 0.003 ± 0.0001 1 μg/mL respectively, thus suggesting a reversal of antia- N-acetylcysteine 0.014 ± 0.0001 poptotic effect and genotoxic mechanisms of NO and im- Values are expressed as mean ± SEM, p < 0.001 munomodulatory potentials of A. precatorius. Okoro et al. Clinical Phytoscience (2019) 5:45 Page 8 of 9 Conclusion 4. Benedetti S, Nuvoli B, Catalani S, Ga R. Reactive oxygen species a double- edged sword for mesothelioma. Oncotarget. 2015;6(19):16848–65. This work provides experimental evidence that methanol 5. Alpay M, Backman L, Cheng X, Dukel M, Kim W. Oxidative stress shapes root extract of A. precatorius contains bioactive com- breast cancer phenotype through chronic activation of ATM-dependent pounds that exhibit anticancer, antioxidant and immu- signaling. Breast Cancer Res Treat. 2015;151(1):75–87. 6. Calaf GM, Urzua U, Termini L, Aguayo F. Oxidative stress in female cancers. nomodulatory potential without toxic effects on normal Oncotarget. 2018;9(34):23824–42. cells. The modulatory effect on cytokines and antiprolif- 7. Switzer CH, Cheng RY-S, Ridnour LA, Glynn SA, Ambs S, Wink DA. Ets-1 is a erative activities might be due to the synergistic actions transcriptional mediator of oncogenic nitric oxide signalling in estrogen receptor negative breast cancer. Breast Cancer Res. 2012;14:R125. of bioactive compounds present in them especially the 8. Habib S, Ali A. Biochemistry of nitric oxide. Indian J Clin Biochem. 2011; ethyl acetate and butanol fractions and therefore suggest 26(1):3–17. https://doi.org/10.1007/s12291-011-0108-4. chemotherapeutic use of A. precatorius in cancer treat- 9. Choudhari SK, Choudhary M, Badge S, Gadbail AR, Joshi V. Nitric oxide and cancer: a review. World J Surg Oncol. 2013;11:118. https://doi.org/10.1186/ ment. However, extensive studies to characterize and 1477-7819-11-118. elucidate the mechanism of action of the active princi- 10. Muntané J, De la Mata M. Nitric oxide and cancer. World J Hepatol. 2010; ples present in these fractions on antiproliferative activ- 2(9):337–44. https://doi.org/10.4254/wjh.v2.i9.337. 11. Tavares-da-Silva EJ, Varela CL, Pires AS, Encarnação JC, Abrantes AM, Botelho ities are currently in progress. MF, et al. Combined dual effect of modulation of human neutrophils’ oxidative burst and inhibition of colon cancer cells proliferation by Acknowledgments hydroxycinnamic acid derivatives. Bioorg Med Chem. 2016. https://doi.org/ E.E. Okoro acknowledges the World Academy of Sciences for the 10.1016/j.bmc.2016.05.065. advancement of Science in Developing Countries (TWAS), Trieste, Italy, for 12. de Oliveira C, Sakata R, Issy A, Gerola L, Salomao R. Cytokines and pain. Rev ICCBS-TWAS Fellowship at the H.E.J. Research Institute of Chemistry, ICCBS, Bras Anestesiol. 2011;61:255–65. University of Karachi, Karachi, Pakistan. 13. Esquivel-Vela’zquez M, Ostoa-Saloma P, Palacios-Arreola MI, Nava-Castro KE, Castro JI, Morales-Montor J. The role of cytokines in breast cancer Authors’ contributions development and progression. J Interferon Cytokine Res. 2015;35(1):1–16. EEO, ORO and AJ conceptualized and designed the experiment. EEO, AJ and https://doi.org/10.1089/jir.2014.0026. SS carried out the experiments. EEO wrote the manuscript. ORO, AJ, CMI and 14. Paradkar PH, Joshi JV, Mertia PN, Agashe SV, Vaidya RA. Role of cytokines in FDO read and approved the final manuscript for submission. genesis, progression and prognosis of cervical cancer. Asian Pac J Cancer Prev. 2014;15(9):3851–64. https://doi.org/10.7314/APJCP.2014.15.9.3851. Funding 15. Aliyu M, Odunola OA, Farooq AD, Mesaik AM, Choudhary MI, Fatima B, et al. This research work was fully funded by the International Centre for Chemical Acacia honey modulates cell cycle progression, pro-inflammatory cytokines and Biological Sciences, ICCBS, University of Karachi, Karachi, Pakistan and and calcium ions secretion in PC-3 cell lines. J Cancer Sci Therapy. 2012;4: The World Academy of Science (TWAS) Trieste, Italy, for the advancement of 401–7. https://doi.org/10.4172/1948-5956.1000174. Science in Developing Countries. 16. Summet G, Bhatia M, Siddiqui N. Abrus Precatoirus (L.): an evaluation of traditional herb. Indo Am J Pharmaceutical Res. 2013;3(4):3295–315. Availability of data and materials 17. Lebri M, Tilaoui M, Bahi C, Achibar H, Akhramez S, Fofie Y, et al. Not applicable. Phytochemical analysis and in vitro anticancer effect of aqueous extract of Abrus precatorius Linn. Der Pharma Chemica. 2015;7(8):112–7. Ethics approval and consent to participate 18. Narendra G, Atul B. Ethnobotanical and Phytopharmacological potential of The studies on human blood cells were performed after approval from Abrus precatorius L.: A review. Asian Pac J Trop Biomed. 2014;4(1):S27–34. independent ethics committee, ICCBS, UoK, No: ICCBS/IEC-008-BC-2015/ 19. Umamahesh B, Veeresham C. Antihyperglycemic and insulin secretagogue Protocol/1.0. activities of Abrus precatorius leaf extract. Pharm Res. 2016;8:303–8. 20. Abo K, Fred-Jaiyesimi A, Jaiyesimi A. Ethnobotanical studies of medicinal Consent for publication plants used in the management of diabetes mellitus in South Western Not applicable. Nigeria. J Ethnopharmacol. 2008;115:67–71. 21. Khadse C, Kakde R, Chandewar A. Anti-inflammatory activity of methanol Competing interests extract fractions of Abrus precatorius leaves. Int J PharmTech Res. 2013;5(3): The authors declare that they have no competing interests. 1426–33. 22. Mosmann T. J Immunol Methods. 1983;65:55–63. Author details 23. Sagar BK, Singh RP. 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Anti-proliferative and immunomodulatory activities of fractions from methanol root extract of Abrus precatorius L

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Medicine & Public Health; Medicine/Public Health, general; Gynecology; Pneumology/Respiratory System; Gastroenterology; Pediatrics; Diabetes
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Abstract

Background: Abrus precatorius possesses various therapeutic properties including anticancer potentials. This study evaluated the anti-proliferative activities of fractions of methanol root extract of A. precatorius on breast and cervical cancer cells and their immunomodulatory effect. Phytochemical screening was done by FTIR and GCMS. In vitro anti-proliferative effect was evaluated on human breast cancer (AU565) and cervical cancer (HeLa) cells and on murine fibroblast (NIH 3 T3) cells. Antioxidant activity was performed via DPPH radical scavenging assay. The immunomodulatory potential of fractions was evaluated by inhibition of phagocytes oxidative burst (ROS), Nitric oxide (NO) and proinflammatory cytokine TNF-α. Results: A. precatorius fractions showed different chemical groups and were somewhat selective in antiproliferative activity against studied cancer cells. Ethyl acetate fraction showed the most significant antiproliferative activity with IC values of 18.10 μg/mL and 11.89 μg/mL against AU565 and HeLa cells respectively. Hexane fraction significantly (p < 0.05) inhibited HeLa cells (IC 18.24 ± 0.16 μg/mL), whereas aqueous fraction showed mild inhibition (IC 50 50 46.46 ± 0.14 μg/mL) on AU565 cell proliferation. All fractions showed no cytotoxicity against NIH-3 T3 murine fibroblast normal cells. All fractions showed potent and significant (p < 0.001) DPPH radical scavenging activity as well as suppressed phagocytic oxidative burst. Hexane (< 1 μg/mL), ethyl acetate (< 1 μg/mL), and butanol (5.74 μg/ mL) fractions potently inhibited the cytokine TNF- α, hexane (< 1 μg/mL) and ethyl acetate (< 1 μg/mL) fractions also potently inhibited NO. Conclusions: The antiproliferative activities and suppressive effect on the phagocytic oxidative burst, NO and proinflammatory cytokine might be due to the synergistic actions of bioactive compounds especially flavonoids present in the assayed fractions and therefore, suggest chemotherapeutic use of A. precatorius in cancer treatment. Keywords: Abrus precatorius, Breast cancer, Cervical cancer, Cytokines, Cytotoxicity, Tumor necrosis factor, oxidative burst Introduction from natural product sources. Cancer is broadly de- Cancer is one of the leading causes of death in both de- scribed as a group of diseases characterized by uncon- veloped and underdeveloped countries. It has generated trolled growth and spread of abnormal cells, associated considerable scientific and commercial interest especially with resistance to normal growth-inhibitory signals, un- in the progressive discovery of new anticancer agents controlled activation of growth signals, impairment of apoptosis, promotion of angiogenesis, invasion of sur- rounding tissues and metastasis [1, 2]. * Correspondence: okoroemeka2003@gmail.com Breast cancer is the most commonly diagnosed cancer Biochemistry Department, Babcock University Ilishan-Remo, PMB 21244, Ikeja, Nigeria in several sub-Saharan African countries, a shift from a Dr. Panjwani Center for Molecular Medicine and Drug Research (PCMD), trend in which cervical cancer was the leading cause of International Center for Chemical and Biological Sciences, University of cancer-related deaths among women in Africa over the Karachi, Karachi 75270, Pakistan Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Okoro et al. Clinical Phytoscience (2019) 5:45 Page 2 of 9 past decade [1]. The reasons for this shift still remain but not limited to antidiabetic [19, 20], anti- unclear but have a strong correlation with prevalent risk inflammatory [21] activities. factors such as obesity, early menarche, late childbearing, Based on the above mentioned considerations, this and lifestyle associated with urbanization and economic study evaluated the antiproliferative and immunosup- development [3]. pressive activities of methanolic crude extract and frac- The generation of reactive oxygen species (ROS) dur- tions of A. precatorius roots which will be able to ing oxidative burst is considered as one of the major simultaneously modulate human neutrophils’ oxidative mechanisms by which phagocytes exert their tumoricidal burst, restraining the inflammatory process, and inhibit functions [4]. Phagocytes such as macrophages undergo the growth of breast (AU565) and cervical (HeLa) cancer oxidative burst in response to antigenic stimuli with gen- cell lines. To underscore the broad-spectrum activity of eration and release of different reactive oxygen metabo- the plant, two cancer cell lines of different origin were lites thereby making oxidative burst as defense function adopted for the study primarily due to their invasive [5]. Oxidative stress is closely linked with carcinogenesis metastatic nature. due to the interplay of ROS in relation to other epigen- etic factors in the induction, promotion, and modulation Materials and methods of breast and cervical cancer [6]. Plant material Nitric oxide (NO) is a short-lived pleiotropic regulator, Young roots of A. precatorius were collected from Imota that plays critical roles in numerous physiological as well Ikorodu, Nigeria. A voucher specimen (IFE-17655) was as pathological processes [7]. Its role in tumor develop- deposited at the Herbarium of Obafemi Awolowo Uni- ment is somewhat complex [8]. However, reported roles versity, Ile-Ife, Nigeria. of NO such as genotoxic mechanisms, antiapoptotic ef- fects, induction, and promotion of angiogenesis, limita- tion of host immune response against tumor, and Extract preparation promotion of metastasis has been implicated in various Young fresh A. precatorius roots were thoroughly types of cancer [9] and NO tumor-promoting effect ap- washed, oven-dried (Uniscope SM9053) at 40 °C for 96 h pears to be both time and concentration-dependent [10]. to a constant weight and pulverized. Methanol (70%) (1: Overproduction of reactive oxygen (ROS) and nitrogen 10) crude extraction of pulverized A. precatorius roots (RNS) species by phagocytes, namely neutrophils, may was done for 48 h using a shaker water bath (Uniscope result in chronic inflammation and initiation of the SM101) at 40 °C and filtered (Whatman No.1110 mm). multistage process of various cancer development in- The filtrate was concentrated using a Rotary evaporator cluding breast and cervical cancer [11]. (Stuart RE 300) to obtain the crude methanol extract, Mitogen-activated protein (MAP) kinases in different reconstituted in distilled water (1:5) and subjected to cell types are involved in the production of extracellular liquid-liquid partitioning using solvents of increasing po- polypeptides or glycoproteins called cytokines. Cytokine larity to obtain partially purified – n-hexane, ethyl acet- activity is influenced by the microenvironment in which ate, n-butanol, and aqueous fractions respectively. The they are produced and as such may have pro- (Th1) or fractions were further concentrated to a constant weight anti-inflammatory (Th2) actions [12]. Tumor necrosis under reduced pressure using a Rotary evaporator at factor (TNF-α), an inflammatory cytokine that is highly 40 °C and freeze-drying for the aqueous fraction. expressed in breast and cervical carcinomas play an im- portant role in the regulation of both induction and pro- tection in breast and cervical cancer [13, 14], and Phytochemical screening apoptosis [15]. To identify the chemical constituents and possible Abrus precatorius (family: Fabaceae) is a perennial, functional groups, A. precatorius fractions were sub- well-branched, twinning and climbing herb that bears a jected to Fourier Transform Infra-red (FTIR) spec- characteristic bright red colored seeds with a black troscopy measured on VECTOR22, Resolution 2 cm blotch at the hilum [16]. It is endemic in the tropics and (10 scans) and Gas chromatography-mass spectrom- commonly known as Rosary bean. It is known as Otuo- etry (GCMS) analysis using Agilent 5975C gas chro- biribiri (Igbo-Ohafia), Idon zakara (Hausa), Oju ologbo matograph combined with inert XL EI/CI MSD and (Yoruba) in Nigeria. The roots of A. precatorius contain Triple-Axis Detector source at 270 °C at 70 eV. The proteins, glycosides, phenolic compounds, fatty acid, injector was set at 270 °C with splitting ratio 1:30. A fatty acid esters, anthocyanins, and minerals [17]. A. pre- mass spectral survey was performed using the NIST catorius has been reported from folklore to have poten- mass spectral program. The concentrations of the tial antitumor properties [18]. Other reported identified compounds were calculated using area ethnopharmacological and therapeutic activities include normalization over the FID response method. Okoro et al. Clinical Phytoscience (2019) 5:45 Page 3 of 9 Cytotoxicity screening USA) and data reported as mean ± standard error of Cytotoxic activity of extract and fractions of A. precator- mean. N-acetylcysteine and Gallic acid were used as ref- ius roots, was evaluated in 96-well flat-bottomed micro- erence compounds. plates by using the standard MTT (3-[4, 5- dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium brom- Oxidative burst assay ide) colorimetric assay. For this purpose, cell lines were The studies on human blood cells were performed after cultured in Minimum Essential Medium Eagle, supple- an approval from independent ethics committee, ICCBS, mented with 5% of fetal bovine serum (FBS), 100 IU/ml UoK, No: ICCBS/IEC-008-BC-2015/Protocol/1.0. of penicillin and 100 μg/ml of streptomycin in 75cm Luminol-enhanced chemiluminescence assay was per- flasks, and kept in 5% CO incubator at 37 °C. 200 μLof formed, as described by Helfand et al. [24]. Briefly, 25 μL cell suspension were seeded in round bottom 96 well of diluted human whole blood / (1 × 10 ) isolated PMNs plate at the density of 10,000 cell/well and incubated at ++ in HBSS (Hanks Balanced Salt Solution, containing 37 °C in 5% CO incubator for 24 h. Exponentially grow- calcium chloride and magnesium chloride) [Sigma, St. ing cells were harvested, counted with a hemocytometer Louis, USA] was incubated with 25 μL of various con- and diluted with the medium. Cell culture with the con- centrations (0.1–250 μg/mL) of A. precatorius fractions. centration of 6 × 10 cells/ml was prepared and intro- ++ Control wells received HBSS and cells, but no com- duced (100 μL/well) into 96-well plates. After overnight pounds. The test was performed in white half area 96 incubation, the medium was removed and 200 μLof well plates [Costar, NY, USA], which was incubated at fresh medium was added with different concentrations 37 °C for 15 min in the thermostat chamber of lumin- of 10–100 μg/mL of fractions and 0.23–30 μg/mL for 3 ometer [Labsystems, Helsinki, Finland]. After incubation, T3 cells. 25 μL of serum-opsonized zymosan (SOZ) [Fluka, Buchs, After 48 h, 200 μL MTT (0.5 mg/ml) was added to Switzerland] and 25 μL of intracellular reactive oxygen each well and incubated further for 4 h. Subsequently, species detecting probe, luminol [Research Organics, 100 μL of DMSO was added to each well. The extent of Cleveland, OH, USA] were added into each well, except MTT reduction to formazan within cells was calculated ++ blank wells (containing only HBSS ). The level of the by measuring the absorbance at 570 nm, using a micro- ROS was recorded in the luminometer in terms of rela- plate reader (Spectra Max Plus, Molecular Devices, CA, tive light units (RLU) for 50 mins in the repeated scan USA). The cytotoxicity was recorded as concentration mode. Ibuprofen was used as a standard drug. causing 50% growth inhibition (IC ) for cell lines [22]. Doxorubicin and Cyclohexamide served as standard drugs. The percent inhibition was calculated by using the fol- Cell lines lowing formula: The cell lines used in this study including AU565 (hu- man breast adenocarcinoma, CRL-2351) and NIH-3 T3 ðÞ mean OD of test compound−mean OD of negative control %Inhibition ¼ 100−  100 (mouse embryonic fibroblast, CRL-1658) were purchased ðÞ mean OD of positive control−mean OD of negative control from ATCC, Manassas, USA, THP-1 (Human monocytic leukemia) and J774.2 (mouse macrophages) were pur- DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging chased from ECACC, Salisbury, UK and HeLa cells (hu- assay man cervical adenocarcinoma) were purchased from DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging CLS, Germany by Dr. Panjwani Centre for Molecular activities of extract and fractions of A. precatorius roots, Medicine and Drug Research (PCMD), International was done using the method described by Sagar & Singh Center for Chemical and Biological Sciences) (ICCBS), [23]. DPPH solution (95 μl, 300 μM) in Ethanol was University of Karachi, Karachi-75270, Pakistan. NIH-3 mixed with test solution (5 μl, 500 μM). The reaction T3, HeLa and J774.2 cells were grown in DMEM sup- was allowed to progress for 30 min at 37 °C and absorb- plemented with 10% Fetal bovine serum (FBS) and 1% ance monitored by the multiplate reader, SpectraMax340 penicillin/streptomycin and AU565 in ATCC modified at 517 nm. Upon reduction, the color of the solution Rosewell Park Memorial Institute (RPMI) medium faded (Violet to pale yellow). Percent Radical Scavenging supplemented with 90% FBS, 1% penicillin, 1% Activity (%RSA) was determined by comparison with a streptomycin. The cells were grown in 75 cc culture DMSO containing control. The concentration that flask and upon reaching 75% confluency were har- causes a decrease in the initial DPPH concentration by vested and used for experimental purpose. For cyto- 50% is defined as IC value. The IC values of the frac- toxicity on NIH-3 T3 and HeLa (6 × 10 cells/mL), 50 50 tions were calculated using EZ-Fit Enzyme kinetics soft- AU565 (6 × 10 cells/mL) and for NO assay from ware program (Perrella Scientific Inc. Amherst, MA, J774.2 (1 × 10 cells/mL) were used. Okoro et al. Clinical Phytoscience (2019) 5:45 Page 4 of 9 Nitric oxide assay dihydroxyflavanone, 4H-1-Benzopyran-4-one, 5,7-dihy- The mouse macrophage cell line J774.2 was cultured in droxy-2-(3-hydroxy-4,5-dimethoxyphenyl)-6,8- 75 cc flasks IWAKI (Asahi Techno Glass, Japan) in dimethoxy, 3-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)- DMEM Sigma-Aldrich (Steinheim, Germany) supple- 4H-chromen-4-one), triterpenes (lupeol, α-amyrin), aro- mented with 10% fetal bovine serum GIBCO (N.Y U. S) matic carboxylic acids (Benzenepropanoic acid), aro- 1% streptomycin/penicillin. Flasks were kept at 37 °C in matic alcohols (1H-Indole-3-(ethanol), as shown in an atmosphere of humidified air containing 5% CO , Fig. 1 and Table 1. cells were seeded in 96-well plate (10 cells/mL) and were induced by 30 μg/mL Escherichia coli lipopolysac- Cytotoxicity screening charide (LPS) (DIFCO Laboratories Michigan, USA). The antiproliferative activities of the crude extract and Three different concentrations of fractions (1, 10 and fractions of A. precatorius are presented in Table 2. 100 μg/mL) were added simultaneously with LPS and The results of the cytotoxicity of A. precatorius frac- the plate was incubated for 48 h at 37 °C in 5% CO . The tions against breast adenocarcinoma (AU565) cell line FBS concentration was 5%. Nitrite accumulation in the showed that ethyl acetate and aqueous fraction signifi- culture supernatant was measured using the Griess re- cantly (p < 0.05) inhibited breast adenocarcinoma prolif- agent [25]. eration (IC 18.10 ± 2.68 μg/mL, IC 46.46 ± 0.14 μg/ 50 50 mL respectively). Crude extract, hexane, and butanol Cytokine production and quantification fractions were found to be inactive against breast adeno- Human monocytic leukemia cells THP-1 cells were carcinoma. The ethyl acetate and hexane fractions also maintained in RPMI-1640 containing 5.5 mmol/L glu- showed significant (p < 0.05) inhibition of cervical cancer cose (BioM Laboratories, Chemical Division, Malaysia), (HeLa) cell proliferation (IC 11.89 ± 0.63, 18.24 ± 50 μmol/L mercaptoethanol (Merck Darmstadt, 0.16 μg/mL respectively). Crude extract, butanol, and Germany), 10% FBS (fetal bovine serum), 2 mmol/L; L- aqueous fractions were found to be inactive against cer- glutamine (PAA Laboratories, GmbH, Pasching, vical cancer cells. All the fractions showed no cytotox- Austria). Cells were grown in 75 cc flasks, harvested and icity against 3 T3 murine fibroblast normal cells thus 2.5 × 10 cells/mL was then plated in 24-well tissue cul- indicating their safety (Table 2). ture plates. 20 ng/mL of phorbol myristate acetate (PMA), (SERVA, Heidelberg, Germany) was added In vitro antioxidant activity of A. precatorius fractions followed by incubation for 24 h at 37 °C in 5% CO to The results of the DPPH Radical Scavenging Activity are convert them into a macrophage-like cell. Cells were presented in Table 3. then stimulated with E. coli Lipopolysaccharide B, All assayed fractions of A. precatorius root methanol (DIFCO Laboratories, Michigan, USA) at a final concen- extract showed significant (p < 0.001) DPPH scavenging tration of 50 ng/mL and treated with fractions using activity compared with standards Gallic acid and N- three concentrations (1, 10 and 100 μg/mL) and then in- acetyl cysteine. Hexane fraction inhibited DPPH radical cubated for 4 h at 37 °C in 5% CO . The supernatants formation with significant (p < 0.001) IC 0.010 ± 0.002 2 50 collected were analyzed for the level of TNF -α using the mg/mL compared with the positive controls, making it human TNF-α Duo Set ELISA (R&D Systems, Minneap- the most active fraction in DPPH radical scavenging ac- olis, USA), and according to manufacturer’s instructions tivity Methanolic crude extract inhibited DPPH forma- [25]. tion with an IC of 0.087 ± 0.002 mg/mL; whereas the ethyl acetate, butanol, and aqueous fractions inhibited Statistical analysis the formation of DPPH radicals with IC 0.079 ± 0.005 Data from all experiments were statistically evaluated mg/mL, 0.098 ± 0.002 mg/mL, and IC 0.086 ± 0.002 using one-way analysis of variance (ANOVA) on Soft- mg/mL respectively compared with the standards Gallic Max Pro software and results expressed as mean ± acid (0.0032 ± 0.0001 mg/mL), and N-acetyl cysteine standard deviation (SD) and mean ± standard error of (0.0141 ± 0.0001 mg/mL) respectively (Table 3). mean (SEM). P < 0.05 at 95% and p < 0.001 at 99.9% con- fidence level was considered statistically significant for Immunomodulatory activities differences in mean and were obtained by Student’st- The results of immunomodulatory activities are pre- test analysis. sented in Table 4. Methanolic crude extract and ethyl acetate fraction of Results A. precatorius showed potent inhibition of whole blood Phytochemical analysis ROS with IC of < 10 μg/mL respectively, whereas hex- Phytochemical screening revealed the presence of several ane (30.5 ± 0.3 μg/mL) and butanol (21.0 ± 0.5 μg/mL) chemical groups: flavonoids/isoflavones (5-Methoxy-3,7- fractions mildly inhibited whole blood ROS. Hexane, Okoro et al. Clinical Phytoscience (2019) 5:45 Page 5 of 9 −1 − 1 Fig. 1 Chromatogram of FTIR of A. precatorius. Resolution 2 cm (10 scans). Intense peaks around 1500-3450 cm indicating the presence of flavonoids, isoflavonoids, carbonyl groups, and hydroxyl flavones ethyl acetate and butanol fractions of A. precatorius invasion, metastasis, and angiogenesis, and induce pro- showed significant (p < 0.001) suppression of oxidative apoptotic pathways [30, 31]. The observed cytotoxic ac- burst generated from zymosan activated polymorpho- tivity may be attributed to the phytoconstituents such as nuclear cells (PMNs) (IC 0.6 ± 0.003 μg/mL, 0.6 ± flavonoids, and terpenes present in these fractions, 0.002 μg/mL, 6.1 ± 0.8 μg/mL respectively) and also which may have worked synergistically to exert these ef- found to significantly (p < 0.001) inhibit proinflammatory fects [32]. cytokine TNF-α with IC values of < 1 μg/mL for hex- A handful of ‘red line’ events which propel tumor cells ane and ethyl acetate and 5.74 ± 0.05 μg/mL for butanol and their derivatized progenies into full-blown uncon- fraction respectively. The hexane and ethyl acetate frac- trolled metastasis have made cancer a complex idio- tion also inhibited the NO production with IC values pathic disorder. Deregulation of cell proliferation of < 1 μg/mL respectively. The aqueous fraction was alongside suppressed apoptosis, provide leverage for all found to be inactive (Table 4). cancer evolution and progression. Uncontrolled cell div- ision is a primary determinant and underlying factor in Discussion the progression of cancer tumors. To evaluate fractions Phytochemical screening showed that they are abundant from root methanol extract of A. precatorius as a poten- in flavonoids, terpenes, alkaloids, and glycosides. Flavo- tial therapy for cancer, different fractions were assayed noids, as well as terpenes, possess anticancer properties against human breast cancer cells (AU565) and cervical [26] through their effects on signal transduction in cell cancer (HeLa) cell lines. The antiproliferative effects proliferation and angiogenesis [27]. Flavonoids have also were quantified in terms of cytotoxicity and IC values been implicated in playing a major role in attenuating determined. One of the reliable criteria in the assess- the development of tumors via their antioxidant effects ment of any anticancer drug is a decrease in tumor vol- [28]. Flavonoids may interfere with the activation of the ume and viable tumor cell count and an increase in proinflammatory nuclear factor-kB (NF-kB) and tumor non-viable tumor cell count. The results of this study activator protein-1 (AP-1) while inducing cell cycle ar- show an anticancer effect of A. precatorius fractions rest and apoptosis [29]. Flavonoids repress molecular against breast adenocarcinoma and cervical cancer. The targets that stimulate proliferation, inflammation, results reveal that the fractions were somewhat selective Okoro et al. Clinical Phytoscience (2019) 5:45 Page 6 of 9 Table 1 Identified compounds from methanolic crude extract of A. precatorius roots Peak Compound Formula t Area Remarks # (min) % 1 Benzoic acid C H O 7.88 0.07 antimicrobial, antimutagenic, antiestrogenic, 7 6 2 hypoglycemic, anti-inflammatory 2 1-Dodecanol C H O 15.23 0.38 An insect attractant, pesticide 12 26 3 Benzaldehyde, 4-hydroxy-3,5-dimethoxy- C H O 19.65 0.08 Antioxidant, antimicrobial and hypoglycemic 9 10 4 4 1-Hexadecanol C H O 19.88 0.21 Emulsifier 16 34 5 Ethanol, 2-(dodecyloxy)- C H O 20.76 0.13 14 30 2 6 1H-Indole-3-ethanol C H NO 21.83 0.46 Estradiol metabolism 10 11 7 Hexadecanoic acid, methyl ester C H O 26.83 5.31 Antioxidant, anti-inflammatory 17 34 2 8 Dibutyl phthalate C H O 28.32 0.54 Antibacterial 16 22 4 9 n-Hexadecanoic acid C H O 28.58 0.75 Antibacterial, antifungal 16 32 2 10 Heptadecanoic acid, methyl ester C H O 31.51 0.36 Antimicrobial 18 36 2 11 9,12-Octadecadienoic acid (Z,Z)-, methyl ester C H O 35.99 7.28 Antibacterial 19 34 2 12 11-Octadecenoic acid, methyl ester C H O 36.44 5.08 Antimicrobial 19 36 2 14 Octadecanoic acid, methyl ester C H O 37.89 1.27 Antimicrobial, anti-inflammatory, anticancer 19 38 2 15 9,12-Octadecadienoic acid (Z,Z)- C H O 38.22 0.46 Antibacterial 18 32 2 16 cis-Vaccenic acid C H O 38.54 0.63 Antibacterial, hypolipidemic 18 34 2 17 Benzene, 1,1′-tetradecylidenebis- C H 43.96 1.11 Antimicrobial 26 38 18 5-Methoxy-3,7-dihydroxyflavanone C H O 47.19 2.66 Antioxidant, anti-inflammatory, anticancer 16 14 5 19 1,2-Benzenedicarboxylic acid, diisooctyl ester C H O 47.29 1.71 Antimicrobial, antioxidant, anticancer 24 38 4 20 3-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-chromen-4-one C H O 49.87 7.48 Anticancer, antihypertensive, antioxidant 16 12 5 21 13-Docosenamide, (Z)- C H NO 50.15 0.94 Antimicrobial 22 43 22 2,2,4-Trimethyl-3-(3,8,12,16-tetramethyl-heptadeca-3,7,11,15- C H O 51.09 0.51 30 52 tetraenyl)-cyclohexanol 23 Benzaldehyde, 4-hydroxy-3,5-dimethoxy-, [(4-hydroxy-3,5- C H N O 51.88 1.65 Indicator for laccase and peroxidase activity 18 20 2 6 dimethoxyphenyl)methylene]hydrazone 24 4H-1-Benzopyran-4-one, 5,7-dihydroxy-2-(3-hydroxy-4,5- C H O 52.69 2.93 antibacterial 19 18 9 dimethoxyphenyl)-6,8-dimethoxy- 25 6,6,7-Trimethyl-9-oxo-3-oxabicyclo(3.3.1) nonane 2,4- C H N O 53.59 9.16 17 22 4 5 dinitrophenylhydrazone 26 1,1′-Biphenyl-5-carboxylic acid, 2,3,4,4′-tetramethoxy-6′- C H O 53.76 1.13 Antibacterial 19 20 8 methoxycarbon 27 Silane, dimethyl(3-ethylphenoxy)tetradecyloxy- C H O Si 54.71 41.09 Antimicrobial 24 44 2 28 Anthiaergosta-1,5,7,9-tetraene C H 55.77 2.47 28 42 29 Cyclohexan-1-one-3α,5β-diacetic acid, 2α-(5-hydroxy-2,4- C H O 56.14 0.83 Antibacterial 22 30 8 dimethoxyphenyl)-, diethyl ester 30 α-Amyrin C H O 56.64 0.47 Antimicrobial, antifungal, anti-inflammatory, 30 50 anticancer 31 Lupeol C H O 57.31 0.89 Antiprotozoal, anti-inflammatory, antitumor 30 50 32 Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, C H O 60.73 1.07 Antifungal. antimicrobial 35 62 3 octadecyl ester in their activity against cancer cells with ethyl acetate some anticancer bioactive compounds which will be iso- fraction showing significant anticancer activity against lated and assayed in further study. Mild cytotoxicity of HeLa cell lines and AU565 cell lines whereas hexane the crude extract was observed against 3 T3 murine fraction showed significant inhibition against HeLa cells fibroblast normal cells implying safety and selectivity. only. The crude extract did not show any observable in- These findings are in agreement with a similar report in hibition in the assayed cancer cell lines and therefore in- which dietary flavonoid luteolin inhibited the invasion of dicate that by partial purification, the fractions contain cervical cancer [29]. Okoro et al. Clinical Phytoscience (2019) 5:45 Page 7 of 9 Table 2 Antiproliferative activities of methanolic crude extract Table 4 Immunomodulatory activities of methanolic crude and fractions of A. precatorius roots extract and fractions A. precatorius roots Fraction/Drug WB ROS PMNs ROS NO TNF-α AU565 HeLa 3 T3 IC ±SD IC ±SD IC ±SD IC ±SD 50 50 50 50 Fraction/Drug IC ±SD IC ±SD IC ±SD 50 50 50 Crude < 10.0 NT NT NT Crude NA NA 25.80 ± 0.91 Hexane 30.5 ± 0.3 0.6 ± 0.003 < 1 < 1 Hexane NA 18.24 ± 0.16 NA Ethyl acetate < 10 0.6 ± 0.002 < 1 < 1 Ethyl acetate 18.10 ± 2.68 11.89 ± 0.63 24.80 ± 2.19 Butanol 21.0 ± 0.5 6.1 ± 0.8 NT 5.74 ± 0.05 Butanol NA NA NA Aqueous 12.0 ± 1.0 > 250 NT NT Aqueous 46.46 ± 0.14 NA NA Standard (Ibuprofen) 11.2 ± 1.9 Doxorubicin 0.54 ± 0.04 0.80 ± 0.10 NT: Not tested Values are presented as mean ± SD of triplicates at p < 0.001 by Student’st Cyclohexamide 0.80 ± 0.10 test analysis. WB Whole Blood, NO Nitric Oxide, TNF Tumor necrosis factor, ROS NA: Inactive Reactive Oxygen Species, PMNs Polymorphonuclear cells Values are means of three replicates ± standard deviation (SD). Significant p value (p < 0.05) were obtained by Student’s t test analysis. Composite Macrophages have been implicated in neoplasm de- treatments were compared using one-way analysis of variances (ANOVA) struction via infiltration into the tumor site and partici- pation in inflammatory reaction. They generate reactive One of the basic and routine assays which provide oxygen species through the oxidative burst process front line information on the antiradical activity of which has been fingered as a major mechanism for their plant extracts is DPPH radical scavenging assay. The antimicrobial and tumoricidal functions [34]. Agents results of the DPPH scavenging activity from our such as zymosan used in this study can induce the se- work suggest that fractions of A. precatorius are good quence of oxidative reactions and are known as trigger- sources of antioxidants compounds. However, ethyl ing agents; other substances which can modify the acetate and butanol fractions appear to be excellent magnitude of the response are known as modulating sources of potent antioxidant secondary metabolites. agents. Therefore, the result of this study reveals the This maybedue to the abundance of variousflavo- modulatory role of fractions of A. precatorius thus indi- noids - 4H-1-Benzopyran-4-one, 5,7-dihydroxy-2-(3- cating their anti-inflammatory potentials. hydroxy-4,5-dimethoxyphenyl)-6,8-dimethoxy, 5- Cytokines are critical for tumor immunosurveillance and Methoxy-3,7-dihydroxyflavanone, 3-Hydroxy-2-(4-hy- have demonstrated therapeutic anti-tumor activity in mur- droxy-3-methoxyphenyl)-4H-chromen-4-one, triter- ine models and in the clinical treatment of several human penes and other non-phenolic constituents with cancers where they play complex and often opposing antioxidant effects such as α-Amyrin and Lupeol as roles in the development of the immune system, host shown by the phytochemical screening results. The defense, and tumor immunobiology [35, 36]. Tumor observed IC values of the fractions which were a lit- necrosis factor (TNF-α) is produced by macrophages tle higher compared with the standard drugs may be and is involved in cell activation, co-stimulation, and duetothe crudenatureofthe fractions unlike the inflammation processes. Hexane, ethyl acetate, and pure forms of the drugs. The findings of this study butanol fractions potently suppressed TNF-α with are congruent with a previous report by Mir et al. IC of < 1 μg/mL respectively for hexane and ethyl [33] in which leaf extracts of A. precatorius demon- acetate and 5.74 μg/mL for butanol fraction. The re- strated antioxidant and antiproliferative activities. sult from this study showed that fractions of A. pre- catorius suppressed the expression of TNF-α, hence inhibiting signaling and communication among cancer Table 3 DPPH Radical Scavenging Activities of methanolic cells and is congruent with a similar report by Kang- crude extract and fractions of A. precatorius roots samaksin et al. [37]. Fraction/Standard IC ± SEM Nitric oxide has been reported to have tumor-promoting Crude 0.087 ± 0.002 roles via formation of toxic and mutagenic species, direct modification of DNA– strand breaks, oxidation and de- Hexane 0.010 ± 0.002 amination of nucleic acids, inhibition of systems required Ethyl Acetate 0.079 ± 0.005 to repair DNA lesions, and inhibition of cytochrome C re- Butanol 0.098 ± 0.002 lease [9, 38]. The hexane and ethyl acetate fraction of A. Aqueous 0.086 ± 0.002 precatorius inhibited NO production with IC values of < Gallic Acid 0.003 ± 0.0001 1 μg/mL respectively, thus suggesting a reversal of antia- N-acetylcysteine 0.014 ± 0.0001 poptotic effect and genotoxic mechanisms of NO and im- Values are expressed as mean ± SEM, p < 0.001 munomodulatory potentials of A. precatorius. Okoro et al. Clinical Phytoscience (2019) 5:45 Page 8 of 9 Conclusion 4. Benedetti S, Nuvoli B, Catalani S, Ga R. Reactive oxygen species a double- edged sword for mesothelioma. Oncotarget. 2015;6(19):16848–65. This work provides experimental evidence that methanol 5. Alpay M, Backman L, Cheng X, Dukel M, Kim W. Oxidative stress shapes root extract of A. precatorius contains bioactive com- breast cancer phenotype through chronic activation of ATM-dependent pounds that exhibit anticancer, antioxidant and immu- signaling. Breast Cancer Res Treat. 2015;151(1):75–87. 6. Calaf GM, Urzua U, Termini L, Aguayo F. 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