Phytomedicine 53 (2019) 9–17 Contents lists available at ScienceDirect Phytomedicine journal homepage: www.elsevier.com/locate/phymed Hepatoprotective activity of Erythrina × neillii leaf extract and characterization of its phytoconstituents T Riham O. Bakra, , Marwa A.A. Fayedb, Ahmed M. Fayezc, Salma K. Gabra, Ahlam M. El-Fishawyd, Taha S.El-Alfyd ⁎ a Department of Pharmacognosy, Faculty of Pharmacy, October University for Modern Sciences and Arts,, Giza 11787, Egypt Department of Pharmacognosy, Faculty of Pharmacy, University of Sadat City, Egypt c Department of Pharmacology, Faculty of Pharmacy, October University for Modern Sciences and Arts, Giza 11787, Egypt d Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt b A R T I C LE I N FO A B S T R A C T Keywords: Erythrina × neillii Alkaloids Flavonoids Antihepatotoxic Background: Natural antioxidants and anti-inflammatory agents have the ability to restore normal balance to destructed liver cells. The genus Erythrina has attracted attention for its broad spectrum of physiological activities and its rich polyphenolic and alkaloid contents. Hypothesis/Purpose: The major phytoconstituents of Erythrina × neillii, an ornamental coral tree and a hybrid between E. herbacea and E. humeana that was not previously studied, were investigated. The hepatoprotective effect and underlying mechanisms were also assessed. Study design and methods: The main phytoconstituents in the different fractions of the alcoholic leaf extract (dichloromethane and ethyl acetate) were identified using high resolution high-performance liquid chromatography coupled with mass spectrometry (HR-HPLC-MS-MS) based on the fragmentation pattern and molecular formula of the identified compounds and on previous literature. In addition, the hepatoprotective, anti-inflammatory and antioxidant activities of three doses of E. × neillii alcoholic leaf extract (100, 250, 500 mg/kg) were investigated in methotrexate (MTX)-intoxicated rats and were compared with those of silymarin-treated rats. Liver function parameters were obtained, and a histopathological study was performed. In addition, the anti-inflammatory mediators and the antioxidant system in the liver tissues were assessed. Results: The dichloromethane extract revealed an abundance of alkaloids (25), in addition to tentatively identifying flavone (1), flavanone (1) and three fatty acids. Additionally, thirty-six compounds belonging to different classes of phytoconstituents with a predominance of flavonoids (21), O/C-flavone and flavonol glycosides, followed by alkaloids (9), fatty acids (4) and (2), and phenolic glycoside were identified in the ethyl acetate extract. Compared with MTX, alcoholic leaf extract (500 mg/kg) ameliorated the MTX-induced alterations by improving several biochemical marker levels, fighting oxidative stress in serum and liver tissues, and decreasing inflammatory mediators; this finding was further confirmed by the histopathological study. Conclusion: This study reveals E. × neillii, a rich source of flavonoids and alkaloids, which could be further exploited to provide a promising and safe antihepatotoxic agent source. Introduction plants and their formulations, as they increase the rate of the natural healing process of the liver. Hence, the search for an effective liver protective drug persists (Stickel and Schuppan, 2007). Experimental models are used to validate the hepatoprotective activity of those systems used in traditional medicine. Methotrexate Liver diseases represent extremely serious health problems with large economic losses in modern society. In the traditional system of medicine, liver diseases have been successfully treated using medicinal Abbreviations: MTX, methotrexate; HR-HPLC/MS/MS, high-performance liquid chromatography coupled with mass spectrometry; ROS, reactive oxygen species; TNF, tumour necrosis factor; SEM, standard error of the means; ANOVA, analysis of variance; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; MDA, malondialdehyde; GSH, blood reduced glutathione; SOD, superoxide dismutase; MPO, myeloperoxidase; DM, dichloromethane; EA, ethyl acetate; LC, liquid chromatography; LD50, lethal dose ⁎ Corresponding author. E-mail address: romar@msa.eun.eg (R.O. Bakr). https://doi.org/10.1016/j.phymed.2018.09.231 Received 10 February 2018; Received in revised form 26 August 2018; Accepted 28 September 2018 0944-7113/ © 2018 Elsevier GmbH. All rights reserved. Phytomedicine 53 (2019) 9–17 R.O. Bakr et al. obtained from J.T. Baker (Deventer, Netherlands), MilliQ water was used for LC analysis. ALT, AST, ALP, MDA, GSH and SOD were obtained from Biodiagnostic, Egypt. TNF-α ELISA kits were obtained from Uscn Life Science Inc., USA. Silymarin and methotrexate (MTX) were obtained from Loba-chemie Co. (Mumbai, Maharashtra, India). Petroleum ether, dichloromethane, ethyl acetate, n-butanol, and ethanol were obtained from El-Nasr Pharm. Chem. Co. (Cairo, Egypt). (MTX) is one of the drugs used to induce hepatotoxicity. The pathogenesis of MTX-induced hepatotoxicity has been attributed to a redox imbalance, either due to depletion of the antioxidant system and/or generation of reactive oxygen species (ROS), suggesting that antioxidants may have a therapeutic benefit (Pravenec et al., 2013). The genus Erythrina is a member of flowering plants in the pea family (Fabaceae); it consists of more than 200 species and contributes greatly to folk medicine. Various species are utilized as tranquilizers against insomnia, inflammation, colic, syphilis, rheumatic pain, jaundice and liver ailments (García-Mateos et al., 2001). Therefore, the species of Erythrina represent an interesting field of study. Phytochemical studies have demonstrated a variety of phytoconstituents, including terpenes and alkaloids, as well as flavonoids, especially isoflavones, pterocarpans, flavanones and isoflavanones (Chawla and Kapoor, 1995; Hussain et al., 2016; Majinda et al., 2005). Due to this wide variation in active constituents, the documented bioactivities of isolated metabolites showed a wide range of medicinal uses, including antimicrobial activity with high efficacy against resistant organisms and anti-inflammatory, antidiabetic, antidepressant, cytotoxic and muscle relaxant activities (Anupama et al., 2012; Nyandoro et al., 2017; Setti-Perdigão et al., 2013). In a continuation of our investigation of Erythrina × neillii Mabberley & Lorence, the hybrid derived from the cross between E. herbacea and E. humeana (Gabr et al., 2017), where no reports dealing with its phytoconstituents or biological activities were found. Herein, we report an investigation of dichloromethane and ethyl acetate extracts using HR-HPLC-MS-MS, and we highlight some of its biological activities. Plant extraction Two hundred grams of powdered leaves were extracted with 70% ethanol and then fractionated beginning with petroleum ether (b.r.40–60 °C) followed by dichloromethane, ethyl acetate and butanol. The extraction with each solvent continued until exhaustion. Each extract was distilled off under reduced pressure and dried to a constant weight. Dichloromethane (DM) and the ethyl acetate (EA) extracts were subjected to HR-HPLC-MS-MS. HR-HPLC-MS\MS analysis The identification of the major metabolites in the DM and EA fractions was performed using HR-HPLC-MS-MS. The mobile phase consisted of 2% acetic acid (pH 2.6) (A) and 80% methanol (B) using gradient elution; from 5 to 50% B at 30 °C at a flow rate of 100 μl/min (Hassaan et al., 2014). Eluted compounds were detected from 120 to 1500 m/z using a Bruker micro-TOF-Q Daltonics (API) time-of-flight mass spectrometer (Bremen, Germany) equipped with an ion spray (pneumatically assisted electrospray) in the negative and positive ion modes using the following instrument settings: nebulizer gas, nitrogen, 1.6 bar; dry gas, argon, 200 °C; capillary, 4000 V. The data were processed using X-Calibur 2.2 SP1 software from Thermo Scientific (Berlin, Germany), and the major compounds were tentatively identified by comparing their mass spectra with the reference literature and searching the phytochemical dictionary of natural products database. Materials and methods Plant material Leaves of E. × neillii Mabberley & Lorence were collected during the flowering period in October 2012 from El-Zohria garden, Cairo. The leaves were kindly identified by Dr. Gwilym P. Lewis, Legume Research Leader, Comparative Plant & Fungal Biology. Royal Botanic Gardens, Kew, UK. The voucher specimen was deposited at the Herbarium of the Faculty of Pharmacy, MSA University, under registration number (RS020) and at the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University (no.1-11-2012), and duplicates were housed in the Kew Herbarium (K). Biological assays Acute toxicity study The lethality test (LD50) was estimated orally in rats, according to the OECD guideline no. 420. Overnight fasted rats were divided into sixteen groups of six rats each. The rats were treated with increased doses (1000, 2000, 3000, 4000 and 5000 mg/kg body weight) of the alcoholic extract of the leaves of E. × neillii suspended in the vehicle. The control group was given vehicle alone (water) and was kept under the same conditions. Animals were observed for 24 h for signs of toxicity and number of deaths. They were observed continuously for one hour for any gross behavioural changes such as drowsiness, restlessness, writhing, convulsions, piloerection and symptoms of toxicity and mortality, if any, and then periodically for the next 6 h, and again at 24 h for signs of acute toxicity. Furthermore, the incidence of mortality for each group was recorded. Food and water were provided ad libitum (no. 2001). Animals Adult male Wistar albino rats, weighing 200–250 g, were purchased from the National Institute of Ophthalmology, Giza, Egypt. The animals were kept at the animal centre, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), Egypt. The animals were housed under appropriate temperature and humidity conditions (25 ± 2 °C and 60–70% humidity). Animals were fed standard pellet chow (El-Nasr Chemical Co., Abou-Zaabal, Cairo, Egypt) and were supplied with water ad libitum. All the procedures involving animals and their care were in conformity with the institutional guidelines (ethics committee for animal experimentation, Faculty of Pharmacy, Cairo University) and complied with national and international laws on the care and use of laboratory animals. Experimental design for hepatotoxicity A total of forty-eight rats were divided into six groups of eight animals each. Three different doses of E. × neillii alcoholic extract (100, 250 and 500 mg/kg) were tested, and the schedule of treatment was as follows: Group (1): Rats received saline for five days and served as a normal control group. Group (2): Rats received MTX (20 mg/kg, ip) at a single dose and then received saline for four days and served as a negative control group. Groups (3, 4 and 5): Rats received MTX (20 mg/ kg, ip) at a single dose and then received oral E. × neillii alcoholic extract (100, 250, 500 mg/kg, respectively) for four days. Group (6): Rats received MTX (20 mg/kg, ip) at a single dose and then received oral silymarin (100 mg/kg) for four days (De et al., 2015). Chemicals, biochemicals and instrumentation For HR-HPLC-MS-MS, chromatographic separation was performed using an HPLC system consisting of a liquid chromatograph (Agilent 1200 Waldbronn, Germany), equipped with a high-performance auto sampler, binary pump, and PDA detector G 1314 C (SL), and a Superspher 100 RP-18 (75 × 4 mm i.d.; 4 μm) column (Merck, Darmstadt, Germany). Acetonitrile and formic acid (LCMS grade) were 10 Phytomedicine 53 (2019) 9–17 R.O. Bakr et al. Nitrogenous compounds in the DM fraction of E. × neillii detected in the positive mode showed essentially similar fragmentation patterns. Most of the identified alkaloids showed methyl losses from the methoxy group (15 Da). Pk 1, identified as erythratine/11-methoxyerysopine/ erysotinone/ erysosalvine with a molecular ion peak at m/z 316.15427 [M+H]+, and fragments at m/z 301 [M+H−CH3]+, 285 [M+H−OCH3]+, 284 [M+H−OCH4]+, 283 [M+H−OCH5]+, 258 [M+H−C3H6O]+, 245 [M+H−C4H7O]+ showed a typical fragmentation pattern as previously described (Chawla and Kapoor, 1995). Pk 2 was tentatively identified as erysotine (m/z 318.16975, M+H) and showed fragments at m/z 287 [M+H−OCH3]+ and 260 [M +H−C3H6O]+. Pk 4 was tentatively identified as erysopine (m/z 286.14342, M+H), showing fragments at m/z 271 [M+H−15]+, 255 [M+H−31]+, while the fragment at m/z 228 [M+H−58]+ corresponds to an RDA fragmentation (Amer et al., 1991). All other alkaloids typically have the same fragmentation pattern and similar molecular ion peaks, as shown in Table 1. Serum and tissue preparation Blood samples were collected from the retro-orbital plexus twentyfour hours after the last dose of treatments under light ether anaesthesia in non-heparinized tubes, and the serum was separated after centrifugation and stored at −20 °C. Animals were later sacrificed; the livers were excised and divided into two portions: the first portion was used for the histopathological study, and the second portion was homogenized for the determination of MDA, GSH and SOD levels. Biochemical analysis Biochemical parameters assessing liver functions (ALT, AST and ALP) were determined spectrophotometrically using commercial kits (Beutler et al., 1963). The liver homogenate was centrifuged, and the MDA, GSH and SOD tissue contents were determined for an estimation of antioxidant activity (Nishikimi et al., 1972). To determine the antiinflammatory activity, MPO and TNF-α were evaluated using an ELISA kit (Hycult Biotechnology, USA) (Grisham et al., 1990). Histopathological study Autopsy samples from the liver were fixed in 10% formalin and embedded in paraffin. Tissue sections (4 μm) were stained with haematoxylin and eosin (H&E) and then examined microscopically (Bott, 2014). Flavonoids and fatty acids A minor appearance of flavonoids was observed in the DM (Table 1), where one flavanone (Pk 26, prenyl naringenin) and one flavone aglycone (Pk 28, apigenin) have been tentatively identified in addition to three fatty acids (Table 1, Pk 27, 29, 30) identified as 7oxotetradecanedioic acid, 12 oxododecanoic acid and 10,16 dihydroxyhexadecanoic acid, respectively, in comparison with previous literature (Farag et al., 2016a,b; Stevens et al., 1997). Statistical analysis The results are expressed as the means ± standard error of the mean (SEM). Comparisons between different groups were carried out by one-way analysis of variance (ANOVA) followed by the TukeyKramer test. The level of significance was set at p < 0.05. GraphPad software InStat (version 2) was used to carry out statistical analysis. Ethyl acetate fraction Alkaloids. The elution order of secondary metabolites in the EA fraction (Fig. 2) followed a sequence of decreasing polarity, whereby cinnamates and alkaloids eluted first, followed by flavonoid glycosides, and finally triterpenes and fatty acids (Farag et al., 2016a). A glycosylated form of Erythrina alkaloids was identified in the E. × neillii EA fraction analysed by HR-HPLC-MS-MS (Table 2). The fragmentation of the glycoside is carried out by a neutral loss of the glycosyl unit to yield a protonated aglycone ion that fragments in the same pattern as the free protonated alkaloid. Pk 1 was tentatively identified as 15-β-D-glucoerysopine or 16-β-D-glucoerysopine with a pseudomolecular ion peak at m/z 448.19587[M+H]+, and fragments at m/z 433 denoting the loss of a methoxyl group followed by a fragment at m/z 269 [M+H−OCH3-hex]+, showing the loss of a hexose moiety (Wanjala and Majinda, 2000)..Pk 2 (m/z 344.14913,M +H) with fragments at m/z 329 [M+H−CH3]+ and 311[M+H−33]+ helped in its tentative identification as 11-β-hydroxyerysotramidine (Juma and Majinda, 2004). Pk 3 (m/z 344.18560, M+H) showed characteristic fragments at m/z 329 [M+H−15]+ corresponding to the loss of a methyl group, in addition to a peak at 286 [M+H−58]+, which corresponds to an RDA fragmentation, beside fragments at m/z 273 [M+H−71]+ and 272 [M+H−72]+, which helped in its tentative identification as erythristemine (Amer et al., 1991). Similarly, Pk 4 (m/z 316.15395, M+H) presented a similar MS fragmentation spectrum and fragments at m/z 314, 298 [M +H−18]+, which represents the loss of a molecule of water, and 284 [M+H−31]+ corresponded to the loss of a methoxy group and was therefore tentatively identified as erysovine-N-oxide (Cui et al., 2009). Pk 6 (m/z 298.14310, M+H) showed a characteristic base peak at m/z 267 denoting the loss of OCH3 in addition to the loss of the allylic substituent at C-3, which helped in its tentative identification as erythraline (Mohammed et al., 2012), while Pk 10 was tentatively identified as erythrartine-N-oxide (m/z 346.16382, M+H) with a characteristic fragment at m/z 330 [M+H−O]+ (Juma and Majinda, 2004). Erythraline, erythrartine and erystrotrine were previously identified in E. herbacea (Garin-Aguilar et al., 2005). Results and discussion Chemical analysis HR-HPLC/MS/MS analysis Mass spectroscopy can be an extremely useful tool when dealing with small quantities of material. The combination of ESI positive and negative modes permitted the tentative identification of thirty compounds in the DM fraction and a total of thirty-six compounds in the EA fraction by the interpretation of their fragmentation patterns combined with information from the available literature. The identities, retention times, and observed molecular and fragmentation information for the tentatively identified peaks are presented in Tables 1 and 2. Notably, this is the first comprehensive metabolite profile of E. × neillii plants. Dichloromethane fraction The DM fraction (Fig. 1) showed the prevalence of alkaloids. Nitrogenous compounds are better detected in the positive mode, warranting the importance of acquiring data in both positive and negative ionization modes. The Erythrina alkaloids are of the spiran system and are divided into two groups: those that contain a conjugated 1,6-diene system [e.g., erythraline] and those that contain an isolated l (6)-double bond [e.g., erythratine]. The mass spectra of all the aromatic diene alkaloids showed essentially the same fragmentation pattern, which showed major peaks at [M+H] +, [M+H−15 / M+H−CH3]+, [M +H−31 / M+H−OCH3]+, [M+H−33 / M+H−OCH5]+, [M +H−58 / M+H−C3H6O]+, [M+H−71 / M+H−C4H7O]+, [M and [M +H−72, [M+H−84 / M+H−C5H8O]+ +H−85]+(Dyke and Quessy, 1981). The fragmentations of the second group of Erythrina alkaloids, those having a l (6)-double bond, were more complex and varied than those of the above group. The ions [M+H−15]+ and [M+H−31]+ were of relatively minor importance. One ion that was of general importance in the spectra of this second group was that corresponding to a retro-DielsAlder reaction (RDA) in ring A [M+H−58]+ (Wu and Huang, 2006). Flavonoids. Analysis of the EA fraction of E. × neillii in the negative mode revealed the tentative identification of fifteen flavones with C11 12 Peak no. Rt (min) Molecular formula Error ppm Molecular ion m/z ± MS/MS Proposed compound 1 3.87 C18H21NO4 −0.173 316.15427 [M+H]+ 301 [M+H−15]+, 285 [M+H−31]+, 284 [M+H−32]+, 283 [M+H−33]+, 258 [M+H−58]+, 245 [M+H−71]+ 2 3 4.66 4.79 C18H23NO4 C17H19NO3 −0.738 0.175 318.16975 [M+H]+ 286.14382 [M+H]+ 300, 287 [M+H−31]+,260 [M+H−58]+, 259 [M+H−59]+, 239, 228 255, 254 [M+H−32]+, 237 4 5 6 7 4.83 5.19 5.55 8.73 C17H19NO3 C18H19NO4 C24H31NO8 C19H23NO4 −1.223 −0.115 −1.716 −0.650 286.14342 314.13816 462.21145 330.16977 8 9.66 C17H17NO3 −0.844 284.12788 [M+H]+ Erythratine 11-methoxyerysopine Erysotinone Erysosalvine Erysotine Erysoline Erysonine Erysopine Erythrinine Gluco-erysodine 11-β-hydroxyerysotrine or erysotrine-Noxide erythrocarine 9 10.70 C18H19NO4 −0.524 314.13852 [M+H]+ 10 11 10.78 11.80 C18H19NO4 C18H21NO3 −0.285 −0.600 314.13840 [M+H]+ 300.15924 [M+H]+ 12 13 12.18 13.81 C19H25NO4 C19H23NO3 −3.296 −0.255 332.18454 [M+H]+ 314.17489 [M+H]+ 14 15.83 C18H21NO4 −0.285 316.15405 [M+H]+ + [M+H]+ [M+H]+ [M+H]+ [M+H]+ 271 299 431 314 [M+H−15], 255 [M+H−31]+, 254 [M+H−32]+, 253 [M+H−33]+, 237, 228. [M+H−15]+,296, 286, 270, 254, 240, 237, 226 [M+H−31]+, 430, 269, 268, 242 [M+H−15]+, 312, 299 [M+H−31]+, 298, 280, 270, 256, 229,153 269 [M+H−15]+, 266, 253 [M+H−31]+, 252 [M+H−32]+, 251 [M+H−33]+, 234, 225 [M+H−58]+, 217, 207 299 [M+H−15]+, 296, 283 [M+H−31]+, 282 [M+H−32]+, 264, 256 [M+H−58]+, 243 [M+H−71]+, 229 299 [M+H−15]+, 298, 296, 283 [M+H−31]+, 282, 264, 256, 229, 211 269 [M+H−31]+, 268 [M+H−32]+, 251 314, 301 [M+H−31]+, 300 [M+H−32]+, 298, 282, 271, 253, 242, 229 299 [M+H−15]+, 283 [M+H−31]+, 270, 256 [M+H−58]+, 254, 242 [M+H−72]+, 230 [M+H−84]+, 229 [M+H−85]+ 298, 284, 266, 257, 229 15 16 17 17.65 17.89 18.00 C18H19O4N C16H17NO4 C19H21NO4 −0.588 0.817 −2.574 314.13850 [M+H] 288.12327 [M+H]+ 328.15349 [M+H]+ 282, 264, 223, 176 271, 253, 235, 217, 180, 161 310, 296 [M+H−32]+, 266, 252, 178 18 18.25 C18H19NO3 −0.560 298.14321 [M+H]+ 19 18.43 C19H21NO4 −2.178 328.15362 [M+H]+ 283 [M+H−15]+, 280, 267 [M+H−31]+, 266 [M+H−32]+, 265 [M+H−33]+, 249, 240 [M+H−58]+, 227 [M+H−71]+, 226 [M+H−72]+, 214 [M+H−84]+, 175, 143, 137 313 [M+H−15]+, 298 [M+H−30]+, 297 [M+H−31]+, 296 [M+H−32]+, 286, 271, 237, 191, 178 + 11-oxoerysodine Erysodienone Erysodine Erysovine Erythratidine Erysotrine Erythravine or erythramine Erythratinone 8-oxo-α/β-erythroidine Erysotramidine/ coreximine/ scoulerine Erythraline 271.15282 [M–H]– 253, 240, 227, 191, 173, 155, 135, 121, 111 7-oxotetradecanedioic acid 269.04527 [M–H]− 213.14862 [M+H]+ 289.23760 [M+H]+ 251, 225, 209, 201, 181, 161 195 Apigenin 12-oxododecanoic acid 271 [M+H−18]+, 253, 229 (10S)-10,16-dihydroxyhexadecanoic acid 20 21 22 23 24 25 26 19.62 20.38 23.56 27.57 33.58 44.65 46.00 C19H19NO4 C21H25NO5 C20H25NO4 C25H33NO9 C19H23NO5 C18H15NO4 C20H20O5 −3.050 −1.503 −0.575 −1.018 −0.518 −1.820 −2.375 326.13769 372.17999 344.18506 492.22179 346.16472 310.10682 341.13754 27 46.65 C14H24O5 3.023 28 29 64.72 68.88 C15H10O5 C12H22O3 3.048 0.464 30 70.87 C16H33O4 0.913 Boldface digits reflects the base peak (100% abundance). Fragment ions are listed in order of relative abundances. [M+H] [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ Phytomedicine 53 (2019) 9–17 308, 294 [M+H−32] , 280, 267 341 [M+H−31]+, 340 [M+H−32]+ 329 [M+H−15]+, 313 [M+H−31]+, 312 [M+H−32]+, 298, 286 [M+H−58]+, 281, 269, 250, 230 474, 330 330 [M+H−15]+, 328, 314, 296, 268, 258, 238, 192, 183, 165 292, 279 [M+H−31]+, 278 [M+H−32]+ 323, 311, 281, 187, 175, 161, 137 11-methoxyerythraline or 8-oxoerythrinine Erythrabine Erythrascine Erythristemine 11-β-methoxyglucoerysodine Erythratine-N-oxide Crystamidine Prenyl naringenin + R.O. Bakr et al. Table 1 The tentatively identified metabolites in the E. × neillii dichloromethane fraction using HPLC/MS-MS. Phytomedicine 53 (2019) 9–17 R.O. Bakr et al. Table 2 The tentatively identified metabolites in E. × neillii ethyl acetate extract using the HR-HPLC/MS-MS technique. Peak no. Rt min. Molecular formula ∆ mass ppm Molecular ion m/z ± MS/MS Proposed compound 1 2.91 C23H29NO8 −1.614 448.19587 [M+H]+ 433 [M+H−OCH3]+, 269 [M+H−OCH3-hex]+ 2 5.24 C19H21NO5 −0.347 344.14913 [M+H]+ 3 8.12 C20H25NO4 −0.03 344.18560 [M+H]+ 329 [M+H−CH3] +, 311 [M+H−33] +, 299, 277, 271,267, 255, 241, 223 329 [M+H−15]+, 313 [M+H−31]+, 312 [M+H−32]+, 286 [M+H−58]+, 273 [M+H−71]+, 272 [M+H−72]+ 314, 298 [M+H−H2O]+, 284 [M+H−32]+, 266, 255, 237, 176, 154 315, 312, 299, 283, 267, 251, 235, 223,214 15-β-D-glucoerysopine or 16-β-D-glucoerysopine 11-β-hydroxyerysotramidine + 4 10.45 C18H21NO4 −1.217 316.15395 [M+H] 5 10.50 C19H23NO4 −2.286 330.16923 [M+H]+ 6 7 11.29 12.62 C18H19NO3 C20H23NO5 -0.670 −0.277 298.14310 [M+H]+ 358.16480 [M+H]+ 8 9 10 18.91 22.66 22.71 C15H18O8 C19H23NO5 C19H23NO5 2.541 −1.587 −3.118 325.09262[M–H]− 346.16435 [M+H]+ 346.16382 [M+H]+ 285, 283, 267, 239, 213, 175 343 [M+H−CH3]+,327 [M+H−OCH3]+, 312, 297, 288, 281, 270, 255, 226, 209, 182 163 [M–H−162] 330 [M+H−O]+, 329, 315 [M+H−OCH3]+, 297, 283 330 [M+H−O]+, 329, 315 [M+H−OCH3]+, 283, 255. 11 12 13 24.02 28.37 39.15 C18H24O13 C21H22O11 C27H30O15 14 40.74 C26H28O14 2.087 3.211 1.692 0.880 0.448 447.11425 449.10928 593.15108 [M–H]− 595.16627 [M+H]+ 563.13998 [M–H]− 385, 324, 310, 285 [M–H−162] 179, 153 [M–H−162−132] 287 [M–H−162], 269, 259 575 [M–H–H2O], 473 [M–H−120], 503 [M–H−90], 383, 353 577, 559, 541, 529, 457, 433 545, 503, 473 [M–H−90], 443 [M–H−120], 425, 383, 353 15 42.08 C26H28O15 1.198 579.13514 [M–H]− 16 43.35 C21H20O11 2.085 447.09312 489 [M–H−90], 459 [M–H−120], 399 [459−60], 369 [M–H−90] 429 [M–H−H2O], 357 [M–H−90], 327 [M–H−120] 17 18 19 20 44.57 49.08 49.85 51.41 C25H26O13 C21H20O11 C22H22O11 C27H30O14 1.769 2.0107 1.891 1.747 533.12991 447.09313 [M–H]− 461.10871 [M–H]− 577.15619 [M–H]− 473 [M–H−60], 443 [M–H−90], 383, 353 387, 327 [M–H−120], 285 [M–H−162] 341 [M–H−120], 371 [M–H−90], 443.23 503 [M–H−74], 457 [M–H−120], 487 [M–H−90], 471 [M–H−106], 383, 353 21 22 23 24 53.22 54.40 54.41 54.78 C20H18O9 C15H10O5 C21H19O12 C27H30O14 2.497 0.775 2.176 1.885 401.09 [M–H]− 271.06031 [M+H]+ 463.08811 [M–H]− 577.15627 25 55.15 C26H28O13 1.559 547.14547 [M–H]- 311 [M–H−120], 341 [M–H−90], 383 253, 239, 226, 211, 198, 152 341, 301, 443 559 [M–H−18], 487 [M–H−90], 473 [M–H−104], 457 [M–H−120], 383, 353 529, 473, 457, 443, 425, 383, 353 − 26 27 55.46 55.94 C20H18O11 C28H30O15 1.853 1.410 433.07734 [M–H] 605.15095 28 29 30 31 32 33 34 56.10 57.34 60.74 62.73 65.92 71.93 72.93 C21H20O11 C21H18O11 C20H32O10 C21H20O10 C21H20O10 C18H30O3 C18H32O3 2.264 0.833 2.079 2.335 2.011 2.588 2.468 447.0932 [M–H]− 447.09256 [M+H]+ 431.19207 [M–H]− 431.09828 [M–H]− 431.10 [M–H]− 293.21188 [M–H]− 295.22750 [M–H]− 371, 301, 269, 179, 161 545 [M–H−60], 515 [M–H−90], 473 [M–H−104], 443, 383, 353 327, 301, 285, 269 427, 333, 271 371, 341, 311, 269, [M–H−162] 371, 341, 311, 285, 269, 179, 161 369, 341, 311, 269 275, 265, 249, 231, 223, 211, 183 295, 277, 251, 235, 185, 183, 171, 155 35 36 75.97 78.77 C18H30O2 C18H32O2 3.042 2.877 277.2175[M–H]− 279.23[M–H]− 259, 233, 231, 221, 195, 179 279, 266, 261, 259, 250, 235, 204, 96 Erythristemine Erysovine-N-oxide 11-β-hydroxyerysotrine Erythraline 11-β-methoxyerysotramidine Coumaroyl glucoside 11-α-hydroxyerysotrine N-oxide 11-β-hydroxyerysotrine Noxide [(erythrartine-N-oxide)] Gentisic acid pentosyl hexoside Eriodyctiol hexoside Apigenin 6,8C-dihexoside Apigenin 6-C-pentoside 8-Chexoside Arabinopyranosylorientin Luteolin 6-C-hexoside (isoorientin) Apigenin 6-C-dipentoside Kaempferol hexoside Diosmetin glucoside Apigenin -6-C-rhamnoside 8-Chexoside Apigenin pentoside Apigenin Quercetin hexoside Apigenin 6-C-hexoside-rhamnoside Apigenin -6-C-di-pentoside-methyl ether Quercetin pentoside Apigenin-8-C-hexoside6-C-pentoside acetate Quercetin rhamnoside Apigenin glucuronide Apigenin O-hexoside Kaempferol rhamnoside Apigenin 6-C-hexoside (isovitexin) Hydroxy-octadecatrienoic acid 15-hydroxy-9,11-octadecadienoic acid Linolenic acid Linoleic acid Boldface digits reflects the base peak (100% abundance). Fragment ions are listed in order of relative abundances. 18, 31), based on their MS/MS fragmentation. Pk 13 (593.15108, M–H) showed a base peak at m/z 473 [M–H−120]− in addition to fragments at m/z 503 [M–H−90]− and 575 [M–H−H2O]− denoting a C-hexosyl flavone. Additional hexose was revealed by fragments at m/z 383 and 353. The very low intensity of a fragment at m/z 341 [M–H−sugar-90]− and [M–H−sugar-H2O]− suggested a symmetric Di-C-glucoside (Becchi and Fraisse, 1989). Therefore, Pk 13 was tentatively identified as apigenin 6,8C-dihexoside. Pk 14 (m/z 563.13998, M–H) showed a characteristic fragment at m/z 545 [MH−18]−, denoting the loss of one molecule of water. The fragment at m/z 473 [M–H−90]−, appearing as a base peak in addition to a fragment at m/z 443 [M–H−120]−, denotes a hexosyl moiety at C-8, while the fragment at m/z 503 [M–H−60]− >50% denotes the and O-glycosylation, five flavonols, and one flavanone (Table 2), whereby the nature of the sugars could be revealed by elimination of the sugar residue, that is, 176 amu (hexuronic acid), 162 amu (hexose: glucose or galactose), and 132 amu (pentose) (Farag et al., 2016a). Apigenin showed twelve conjugates with the predominance of Cglycosylation detected by the neutral loss of 120 amu (0,2 cross-ring cleavage), 90 amu (0,3 cross-ring cleavage), and 18 amu (loss of H2O). Apigenin conjugates showed C-6 derivatives (Pk 24, 32), C-8 (Pk 13) and a combination of both (Pk 14, 20, 27) in addition to Oglycosylation (Pk 30). Luteolin conjugate appeared at Pk 15 and 16 and was identified as luteolin pentosyl hexoside and luteolin C-6 hexoside, in addition to diosmetin (Pk 19). Flavonols were also identified, including quercetin (Pk 23, 26, 28) and kaempferol (Pk 13 Phytomedicine 53 (2019) 9–17 R.O. Bakr et al. Fig. 1. Total ion chromatograms of E. × neillii dichloromethane fraction. A: positive mode. B: negative mode. Peak numbers follow those used for compound identification as listed in Table 1. acid (Pk 36, 279.23, M–H) were identified in the negative mode in the EA (Farag et al., 2016a,b). attachment of an extra C-linked pentose at C-6. The fragment at m/z 353 [M–H−120−90]− and 383 [M–H−120–60]− confirmed the tentative identification of Pk 14 as apigenin 6-C-pentosyl-8-Chexoside. Pk17 (533.12991, M–H) was tentatively identified based on fragments at m/z 443 [M–H−90]−, appearing as the base peak and m/z 473 [M–H−60]−, revealing a pentoside loss. Pk 17 was tentatively identified as apigenin 6-C-dipentoside. Pk 25 (547.14547, M–H) showed the same fragmentation pattern with an extra mass difference of 14 amu in molecular ion and was identified as apigenin-6-C-dipentoside-methyl ether. Pk 20 (577.15619, M–H) showed a base peak at m/z 503 [M–H−74]−, followed by fragments at m/z 457 [M–H−120]− and 473 [M–H−104]−, assigning a typical rhamnoside fragmentation pattern. The appearance of fragments at m/z 487, 383 and 353 denoted an extra hexosyl moiety. The high intensity of rhamnosyl fragments confirmed its attachment at 6-C and the hexosyl at 8-C. Therefore, Pk 20 was tentatively identified as apigenin-6-C-rhamnoside 8-C-hexoside (Ferreres et al., 2003). The O-glycosides were also identified with apigenin O-hexoside (Pk 30) in addition to the tentatively identified flavonols as kaempferol hexoside (Pk 18). Biological assays Preliminary acute toxicity study To assess the E. × neillii extract safety margin, acute toxicity was monitored followed by any behavioural or apparent changes. In the acute toxicity study, compared to vehicle-treated rats, the treated rats with different doses of E. × neillii extract (up to 4000 mg/ kg b. wt.) were alive and had no adverse effects related to their general behaviour, appearance, body weight and food consumption, while 5000 mg/ kg b.wt led to 20% mortality. This finding indicates a wide safety margin of up to 4 g/kg according to (OECD, 2001). For further studies, the concentration was fixed as 1/20 LD50 (250 mg/kg b.wt). Hepatoprotective activity Effect of E. × neillii on liver function parameters. The ability of the total alcoholic extract to protect against MTX-induced hepatotoxicity was assessed in a rat animal model in which silymarin was chosen as a positive control. Treatment of the rats with MTX resulted in a significant (P ≤ 0.05) increase in liver aminotransferases (AST and ALT) and ALP levels, indicative of hepatocyte damage in accordance with previously reported data (Tunali-Akbay et al., 2010). In addition, MTX led to a significant increase in MDA in accordance with (Kose et al., 2012) and a reduction of GSH activity; GSH is an Oxygenated fatty acids With a delayed appearance, hydroxy-octadecatrienoic acid (Pk 33, 293.21188, M–H), 15-hydroxy-9,11-octadecadienoic acid (PK 34, 295.2275, M–H), linolenic acid (Pk 35, 277.2175, M–H), and linoleic 14 Phytomedicine 53 (2019) 9–17 R.O. Bakr et al. Fig. 2. Total ion chromatograms of E. × neillii ethyl acetate fraction. A: positive mode. B: negative mode. Peak numbers follow those used for compound identification as listed in Table 2. Table 3 Effects of the alcoholic extract of E. × neillii on various biochemical parameters in MTX-intoxicated rats. Parameter ASTc (U/ml) ALTd(U/ml) ALPe(U/ml) Control 10.85 ± 1.09 24.16 ± 1.92 59.89 ± 4.33 MTX 117.50a ± 9.75 137.33a ± 11.86 156.44a ± 12.58 E. × neillii extract 100 mg/kg 250 mg/kg 500 mg/kg Silymarin (100 mg/kg) 55.89b ± 4.33 46.37b ± 3.20 78.23b ± 5.45 46.78b ± 3.62 39.25b ± 3.12 71.36b ± 6.61 28.67b ± 2.11 31.78b ± 2.65 67.22b ± 4.17 24.21b ± 1.83 26.33b ± 1.67 62.33 ± 5.88 Data are presented as the mean ± SEM, n = 6. a Significantly different from the normal control group at P ≤ 0.05. b Significantly different from the MTX control group at P ≤ 0.05. c Aspartate aminotransferase. d Alanine aminotransferase. e Alkaline phosphatase. and ALT levels, in addition to a 58% decrease in ALP levels; these effects were comparable to those of silymarin (80% and 60%, respectively). At the oxidant status level, compared with silymarin, the administration of E. × neillii at 100, 250 and 500 mg/kg in the MTX-intoxicated rats significantly mitigated lipid peroxidation activity, with a significant decrease in the hepatic tissue content of MDA and a significant increase in the hepatic tissue content of GSH and hepatic tissue activity of SOD in a dose-dependent manner (Table 4). endogenous protective antioxidant known to protect from free oxygen radicals in hepatic tissue. The silymarin control group, along with the E. × neillii-administered groups, ameliorated the MTX-induced alterations, and a well-characterized antihepatotoxic activity was observed (Table 3). Treatment of rats with different doses (100, 250 and 500 mg/kg) of the alcoholic extract of the leaves of E. × neillii for 4 days resulted in significant decreases in serum AST, ALT and ALP levels in a dose-dependent manner compared with those in the MTX and silymarin control groups (P < 0.05; Table 3). The best effect was observed at a dose of 500 mg/kg of E. × neillii, which contributed to a 76% decrease in AST Anti-inflammatory activity of E. × neillii extract. The exposure of rats to 15 Phytomedicine 53 (2019) 9–17 R.O. Bakr et al. Table 4 Effects of the alcoholic extract of E. × neillii on oxidative stress in MTX-intoxicated rats. Parameter MDAc nmol/g.tissue GSHd mg/g.tissue SODe U/mg.tissue Control 96.27 ± 9.11 422.78 ± 32.65 96.25 ± 6.88 MTX 271.18a ± 17.02 128.93a ± 10.12 33.29a ± 3.12 E. × neillii extract 100 mg/kg 250 mg/kg 500 mg/kg Silymarin (100 mg/kg) 149.33b ± 13.50 171.67 ± 14.17 59.38b ± 4.56 139.78b ± 10.23 322.33b ± 23.83 75.34b ± 7.27 125.67 b ± 8.89 345.67 b ± 29.62 81.17b ± 7.67 110.75b ± 9.77 351.27b ± 27.89 87.66b ± 6.97 Data are presented as the mean ± SEM, n = 8. a Significantly different from the normal control group at P ≤ 0.05. b Significantly different from the MTX control group at P ≤ 0.05. c Malondialdehyde. d Reduced glutathione. e Superoxide dismutase. Table 5 Effects of the alcoholic extract of E. × neillii on inflammatory mediators in MTX-intoxicated rats. Parameter TNF-α1 pg/mg.tissue MPO2 U/mg.tissue Control 85.28 ± 7.02 26.87 ± 1.12 MTX 297.30a ± 28.89 93.25a ± 8.95 E. × neillii extract 100 mg/kg 250 mg/kg 500 mg/kg Silymarin (100 mg/kg) 139.67b ± 11.11 58.48b ± 5.23 124.56b ± 10.18 44.50b ± 4.11 103.67b ± 9.27 37.75b ± 3.12 109.82b ± 8.76 40.33b ± 3.70 Data are presented as the mean ± SEM, n = 8. 1 Tumour necrosis factor-alpha. 2 Myeloperoxidase. a Significantly different from the normal control group at P ≤ 0.05. b Significantly different from the MTX control group at P ≤ 0.05. Fig. 3. Histological structure of the liver cells showing changes in the normal liver cells after the application of MTX and different doses of E. × neillii alcoholic extract compared with silymarin. A: Normal control group showing a normal histological structure of the central vein and surrounding hepatocytes, B: Methotrexate control group showing severely degenerated surrounding hepatocytes and fatty changes with a dilated and congested central vein, C: E. × neillii 100 mg/kg identifying inflammatory cell infiltration in the portal area and the few micro fat vacuoles in the cytoplasm of hepatocytes, D: E. × neillii 250 mg/kg showing few haemorrhages and mild dilatation in the central vein with fat vacuoles in the hepatocytes, E: E. × neillii 500 mg/kg almost normal hepatocytes, a normal central vein with mild congestion in hepatic sinusoids. F: Silymarin 100 mg/kg showing almost normal hepatocytes with mild vascular degeneration. (Table 5). Histopathological examination confirmed the improvement in biochemical parameters. The liver excised from the normal control group rats showed a normal histological structure of the hepatocytes and central vein with no histopathological alteration (Fig. 3A). In contrast, the liver of the MTX control rats showed severe dilatation and congestion in the central vein with degeneration in the surrounding MTX resulted in a significant increase in the hepatic tissue content of the inflammatory mediators TNF-α and MPO compared with that of the control group, while treatment of rats with 100, 250 and 500 mg/kg alcoholic extract resulted in a significant decrease in the hepatic tissue content of TNF-α and MPO in a dose-dependent manner. Compared with silymarin, the dose of 500 mg/kg of the alcoholic extract decreased the hepatic tissue content of TNF-α and MPO significantly 16 Phytomedicine 53 (2019) 9–17 R.O. Bakr et al. hepatocytes in addition to the fatty changes (Fig. 3B). Treatment of the intoxicated rats with 100 mg extract of E. × neillii resulted in a liver showing congestion in the central vein, few inflammatory cells infiltrating in the portal area with few micro fat vacuoles in the cytoplasm of hepatocytes (Fig. 3C), while rats given 250 mg extract of E. × neillii showed mild dilatation in the central vein and very few micro fat vacuoles in hepatocytes (Fig. 3D). The best effect was observed in the liver of rats given the 500 mg extract of E. × neillii, showing almost normal hepatocytes and a normal central vein with mild congestion in hepatic sinusoids; a similar pattern was seen with silymarin (Fig. 3E and F, respectively). This study showed that E. × neillii extract improved liver function, possibly due to the free radical scavenging and antioxidant activities of its constituents, especially flavonoids, which can impair the activation of MTX into the reactive form. 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