A proanthocyanidin-rich extract from Cassia abbreviata exhibits antioxidant and hepatoprotective activities in vivo

Author’s Accepted Manuscript A proanthocyanidin-rich extract from Cassia abbreviata exhibits antioxidant and hepatoprotective activities in vivo Mansour Sobeh, Mona F. Mahmoud, Mohamed A.O. Abdelfattah, Haroan Cheng, Assem M. ElShazly, Michael Wink www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(16)31245-4 https://doi.org/10.1016/j.jep.2017.11.007 JEP11098

To appear in: Journal of Ethnopharmacology Received date: 9 October 2016 Revised date: 10 May 2017 Accepted date: 6 November 2017 Cite this article as: Mansour Sobeh, Mona F. Mahmoud, Mohamed A.O. Abdelfattah, Haroan Cheng, Assem M. El-Shazly and Michael Wink, A proanthocyanidin-rich extract from Cassia abbreviata exhibits antioxidant and hepatoprotective activities in vivo, Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2017.11.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A proanthocyanidin-rich extract from Cassia abbreviata exhibits antioxidant and hepatoprotective activities in vivo

Mansour Sobeha*1, Mona F. Mahmoudb, Mohamed A.O. Abdelfattahc, Haroan Chenga, Assem M. El-Shazlyd, Michael Winka*

a

Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer

Feld 364, 69120-Heidelberg, Germany b

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Zagazig University,

Zagazig 44519, Egypt c

Department of Chemistry, American University of the Middle East, 15453, Kuwait

d

Department of Pharmacognosy, Faculty of Pharmacy, Zagazig University, Zagazig 44519,

Egypt

*

Corresponding authors: Professor Dr. Michael Wink Institut für Pharmazie und Molekulare

Biotechnologie, Universität Heidelberg, Im Neuenheimer Feld 364, 69120- Heidelberg, Germany, [email protected]; Tel.: +49 6221 54 4880; fax: +49 6221 54 4884

Abstract: Ethnopharmacological relevance: Cassia abbreviata is a small to medium sized branched umbrella-shaped deciduous tree. It is widely spread in the tropics, especially in Africa, having a long history in traditional medicine

1

[email protected] 1

for the treatment of numerous conditions such as headaches, diarrhea, constipation, some skin diseases, malaria, syphilis, pneumonia, stomach troubles, uterine pains, and against gonorrhea.

Aim of the study: We investigated the phytochemical constituents of a root extract from Cassia abbreviata using HPLC-PDA-ESI-MS/MS. We also determined the antioxidant activities in vitro and in vivo using the nematode Caenorhabditis elegans as a model organism. The hepatoprotective activities in case of D-galactosamine (D-GaIN)-induced hepatotoxicity were studied in a rat model.

Materials and methods: HPLC-PDA-ESI-MS/MS analysis allowed the identification of the secondary metabolites of the methanol extract. DPPH and FRAP assays were used to determine the antioxidant activities in vitro. Using the C. elegans model, survival rates under juglone induced oxidative stress, intracellular ROS content, quantification of Phsp-16.2: GFP expression and subcellular DAF-16: GFP localization were investigated to determine the antioxidant activities in vivo. The in vivo hepatoprotective potential of the root extract was evaluated for D-galactosamine (D-GaIN)induced hepatotoxicity in rats. The activity of the liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT), in addition to liver peroxidation product malondialdehyde (MDA) and glutathione content (GSH) as well as albumin and total bilirubin concentration were determined. A histopathological study was also performed.

Results and conclusion:

2

C. abbreviata root extract is rich in polyphenolics, particularly proanthocyanidins. HPLC-PDAESI-MS/MS analysis resulted in the identification of 57 compounds on the bases of their mass spectra. (epi)-Catechin, (epi)-afzelechin, (epi)-guibourtinidol, and (ent)-cassiaflavan monomers as well as their dimers, trimers, and their diastereomers are the main components of the extract. The total phenolic content amounted for 474 mg/g root extract expressed as gallic acid equivalent using the Folin-Ciocalteu method. The extract exhibited powerful antioxidant activity with EC50 of 6.3 μg/mL in DPPH and 19.15 mM FeSO4 equivalent/mg sample in FRAP assay. In C. elegans model the extract (200 μg/mL) was able to increase the survival rate by 44.56% and reduced the ROS level to 61.73 %, compared to control group. Pretreatment of rats with 100 mg extract/kg (b. wt.) reduced MDA by 47.36% and elevated GSH by 59.1%. The extract caused a significant reduction of ALT, AST and GGT activities by 11, 35.7 and 65%, respectively. The findings of this study suggest that the proanthocyanidin-rich extract from C. abbreviata may be an interesting candidate for hepatoprotective activity in case of hepatocellular injury.

Graphical abstract:

3

Abbreviations: ANOVA: analysis of variance; ALT: alanine aminotransferase; AST: aspartate aminotransferase; C. elegans: Caenorhabditis elegans; D-GaIN: D-galactosamine; DPPH: 2,2-diphenyl-1picrylhydrazyl; FRAP: ferric reducing antioxidant power; GGT: gamma-glutamyltransferase; GFP: green fluorescent protein; GSH: reduced glutathione content; HSP: heat shock protein; HPLC-PDA-ESI-MS/MS: high performance liquid chromatography-photodiode array detectorelectrospray ionization mass spectrometry; MDA: malondialdehyde; ROS: reactive oxygen species Key words: Cassia abbreviata, proanthocyanidins, HPLC-PDA-ESI-MS/MS, antioxidant, hepatoprotective, in vivo, Caenorhabditis elegans

4

1. Introduction Medicinal plants have been used in folk medicine for ages against a wide range of ailments and health disorders in the form of herbal infusions and decoctions. Several health benefits of medicinal plants are attributed to certain chemical entities known as secondary metabolites among them tannins as a biologically highly active class (van Wyk and Wink 2004; 2015). Tannins have shown powerful potential as antioxidant, antimicrobial, antiviral, antitumor, antidiarrheal, and anti-inflammatory agents beside their ability to treat some cardiovascular diseases (Gali et al. 1992; Kashiwada et al. 1992; Souza et al. 2007; Diouf et al. 2009; Lee et al. 2010). Moreover, several epidemiological correlations have been established between the regular intake of tannins and the decreased incidence of chronic diseases. These broad biological properties have promoted the application of tannin-rich plants in various pharmacological and nutritional studies (Sakagami et al. 2000; Beninger and Hosfield 2003; Koleckar et al. 2008; Abbas and Wink 2009). Cassia abbreviata is a small to medium sized branched umbrella-shaped deciduous tree with very distinctive cylindrically shaped fruits. It is widely spread in the tropics, especially in Africa in the whole region spanning from Somalia to South Africa (Mojeremane et al. 2005). Phytochemical investigations of this plant have revealed many secondary metabolites belonging to different chemical classes such as anthraquinones, phenolics, triterpenoids, organic acids, and proanthocyanidins (Dehmlow et al. 1998; Erasto and Majinda 2003; Mongalo and Mafoko 2013). Several guibourtinidins (flavan-3-ol derivatives) e.g. (2R, 3S)-guibourtinidol and its diastereomers have been isolated from the bark and heartwood (Nel et al. 1999). Additionally, a 2,4-trans-7,4′-dihydroxymethoxyflavan was isolated and characterized from polar extracts of shredded leaves and twigs of C. abbreviata (Dehmlow et al. 1998). Moreover, two novel trimeric

5

proanthocyanidins, cassinidin A (3,7,4'-trihydroxyflavan-(4β→8)-3,5,7,4'-tetrahydroxyflavan(3'→6)-3,5,7,2',4'-pentahydroxyflavan) and cassinidin B (3,7,2',4'-tetrahydroxyflavan-(4α→8)3,5,7,4'-tetrahydroxyflavan-(4α→6)-3,5,7,2',4'-pentahydroxyflavan) (Erasto and Majinda 2003), in addition to 2,3-dihydro-5-hydroxy-8-methoxy-2-(4-methoxyphenyl)chromen-4-one and 3,4dihydro-2-(4-hydroxyphenyl)-4-methoxy-2H-chromen-7-ol) (Kiplagat et al. 2012), were isolated from the root bark. Traditionally, the fruits, leaves, bark and roots of C. abbreviata are used to treat numerous conditions: The bark was employed to treat headaches, diarrhea, constipation, some skin diseases, and malaria. The roots are taken orally for the treatment of syphilis and their decoction is used for the treatment of pneumonia, stomach troubles, and uterine pains, and against gonorrhea. Extracts from the roots and bark exhibited anti-schistosomiasis (Bruschi et al., 2011; Leteane et al., 2012) and anti-malaria activities (Kiplagat et al. 2012). Furthermore, antimicrobial (Mulubwa and Prakash 2015), anthelmintic, antiviral (Molgaard et al. 2001; Leteane et al. 2012), anti-diabetic and antioxidant activities were reported (Shai et al. 2011). Recently, evidence was provided that a leaf extract can inhibit CYP450 in a concentration dependent manner (Thomford et al. 2016). In this study, we comprehensively investigated the polyphenolic constituents of a methanol extract from roots of C. abbreviata using HPLC-MS/MS coupled with a PDA detector, the method of choice to analyse complex mixtures of non-volatile secondary metabolites (Clifford et al. 2003; Theodoridis et al. 2008; Link et al. 2015). Moreover, the antioxidant activities as well as the hepatoprotective activities of the extract were investigated in vitro and in in vivo models (Caenorhabditis elegans and D-galactosamine-induced hepatotoxicity in rats).

6

2. Material and methods 2.1 Plant material Roots of Cassia abbreviata Oliv. (Fabaceae - Caesalpinioideae) were collected from Lupaga Site in Shinyanga, Tanzania by Dr. C. D. Rubanza (Tanzania Forestry Research Institute, Shinyanga). The trypanocidal activities have been investigated before for this plant in our lab (Nibret et al. 2010). The identity of the plant was confirmed by DNA barcoding carried out in our laboratory using rbcL as a marker gene. The voucher specimens (leaves and root) were deposited at the Department of Biology, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University under the accession number P7291 and P7294 at IPMB Heidelberg (Germany), respectively. The dried root (100 g) was grinded and exhaustively extracted with 100% methanol at room temperature for an overall extraction period of 3 d. The combined extracts were reduced under vacuum at 40 °C until 500 mL, centrifuged, and then the soluble fraction was further evaporated till dryness. The residue was frozen at -70 °C, and then lyophilized yielding fine dried powder (27 g). 2.2 Drugs and chemicals Chemicals were purchased from AppliChem (Darmstadt, Germany), Fluka (Buchs, Switzerland) and Sigma Aldrich GmbH (Sternheim, Germany). D-galactosamine was supplied by Sigma Chemicals (ST. Louis. Mo, USA). All solvents for extraction and separation were of analytical grade. 2.3 High performance liquid chromatography (HPLC-PDA-MS/MS) The phytochemical analysis of polyphenolic compounds was carried out using high performance liquid chromatography–Mass spectrometry (HPLC-PDA-MS/MS). The LC system

7

was Thermofinigan (Thermo electron Corporation, USA) coupled with an LCQ-Duo ion trap mass spectrometer with an ESI source (ThermoQuest). The separation was achieved using a C18 reversed-phase column (Zorbax Eclipse XDB-C18, rapid resolution, 4.6 × 150 mm, 3.5 µm, Agilent, USA). A gradient of water and acetonitrile (ACN) (0.1 % formic acid each) was applied from 5% to 30% ACN over 60 min with flow rate 1 mL/min with a 1:1 split before the ESI source. The samples were injected automatically using autosampler surveyor ThermoQuest. The instrument was controlled by Xcalibur software (XcaliburTM 2.0.7, Thermo Scientific). The MS operated in the negative mode with a capillary voltage of − 10 V, a source temperature of 200 °C, and high purity nitrogen as a sheath and auxiliary gas at a flow rate of 80 and 40 (arbitrary units), respectively. Collision energy of 35% was used in MS/MS fragmentation. The ions were detected in a full scan mode and mass range of 50-2000 m/z. 2.4 Biological activity experiments 2.4.1 Antioxidant activities in vitro Determination of total phenolic content as well as antioxidant activities in in vitro assays (DPPH and FRAP) were performed as previously described by us (Sobeh et al. 2016) 2.4.2 Antioxidant activity in vivo 2.4.2.1 Caenorhabditis elegans strains and maintenance Nematodes were maintained under standard conditions [20 °C, on nematode growth medium (NGM), fed with living E. coli OP50]. Age synchronized cultures were obtained by sodium hypochlorite treatment of gravid adults; the eggs were kept in M9 buffer for hatching and larvae obtained were subsequently transferred to S-media seeded with living E. coli OP50 (D.O600 = 1.0) (Stiernagle 2006). In the current work, the following strains of C. elegans were used: Wild

8

type (N2), TJ375 [Phsp-16.2: GFP(gpls1)] and TJ356. All of them obtained from the Caenorhabditis Genetic Center (CGC), University of Minnesota, U.S.A. 2.4.2.2 Survival Assay under juglone induced oxidative stress Early larval stage (L1) wild type worms were treated with different concentrations of the extract (100 and 200 µg extract/mL), except the control group, and maintained at 20 °C for 48 h in S-medium; then a single dose with a final concentration of 80 µM juglone was added to the media. The survivors were counted after 24 h after the addition of the pro-oxidant naphthoquinone juglone (Abbas and Wink 2014). The results are presented as percentage of live worms. 2.4.2.3 Intracellular ROS content Early larval stage (L1) wild type worms were treated with two different concentrations of the extract (100 and 200 µg extract/mL), except the control group, and maintained at 20 °C for 48 h in S-medium. Then the worms were incubated in M9 buffer contained 20 μM H2DCF-DA at 20 °C for 30 min. Fluorescence intensity was measured with a fluorescence microscope (Keyence, BZ-9000, Osaka, Japan) and quantified by Image J 1.48 software (National Institutes of Health, Bethesda, MD) (Sobeh et al. 2016). 2.4.2.4 Quantification of Phsp-16.2: GFP expression Early larval stage (L1) wild type worms were treated with two different concentrations of the extract (100 and 200 µg extract/mL), except the control group, and maintained at 20 °C for 48 h in S-medium. Then the worms were exposed to 20 μM juglone for 24 h to induce oxidative stress. Fluorescence intensity was measured with a fluorescence microscope (as before) and quantified by Image J 1.48. software as described before (Peixoto, et al. 2016a) 2.4.2.5 Subcellular DAF-16: GFP localization

9

Early larval stage (L1) transgenic stain TJ356 worms were treated with two different concentrations of the extract (100 and 200 µg extract/mL) at 20 °C for 24 h in S-medium. Images were taken by a fluorescence microscope (Keyence, BZ-9000, Osaka, Japan). According to the localization of the fusion DAF-16::GFP construct, worms were sorted as showing cytosolic, intermediate and nuclear Daf-16 localisation (Link et al. 2016). 2.4.3. In vivo hepatoprotective activities 2.4.3.1 Animals Adult male Wistar rats (180–200 g; Zagazig University, Zagazig, Egypt) were used in the present study. All the animals were maintained under standard husbandry condition with food and water ad libitum. The experimental procedures were approved by the Institutional Animal Ethics Committee of the Faculty of Pharmacy, Zagazig University (approval number P1-6-2016) and animals were handled following the International Animal Ethics Guidelines, ensuring minimum animal suffering. 2.4.3.2 Study protocol Animals were randomly divided into 3 experimental groups (six animals each): control, dgalactosamine (D-GalN), C. abbreviata extract (100 mg/kg). Control group received only the vehicle. D-GalN group received D-GalN, 800 mg/kg dissolved in normal saline and given via i.p route as a single dose. Extract (suspended in gum acacia solution, 10 mg/mL saline w/v) was given separately to rats by orogastric gavage. One hour later, rats of this group were injected with D-GalN in the same previous dose via i.p. route. Twenty four hours following D-GalN injection, animals were anaesthetized with subcutaneous injection of 1.2 g/kg urethane (Sigma, St. Louis, MO, USA), blood was collected from the retro-orbital plexus and centrifuged (3000 x g, 4 °C, 20 min) for separation of plasma. The obtained plasma was used to analyze liver

10

enzymes, albumin and total bilirubin. Thereafter, animals were sacrificed and livers were separately dissected and blood was washed off with cold saline; livers were divided into two parts; one part was immediately flash-frozen in liquid nitrogen and kept at -80 °C for measurement of tissue parameters and the other part was kept in 10% formalin for histopathological examination. 2.4.3.3 Biochemical analysis 2.4.3.3.1 Liver enzymes activities Quantitative determination of ALT and AST according to (Murray 1984; Young 1995). Kinetic colorimetric method according to (Szasz G. et al. 1974) was used to determine GGT. 2.4.3.3.2 Liver function tests Albumin was determined using modified bromocresol green colorimetric method (Doumas et al. 1997). The total bilirubin concentration was determined in the presence of caffeine by the reaction with diazotized sulphanilic acid to produced an intensely colored diazo dye (Balistreri and Shaw 1987). 2.4.3.3.3 Determination of oxidative stress The production of reactive oxygen species (ROS) in response to D-GalN-induced liver injury was evaluated in liver tissues by measuring the elevated lipid peroxidation product malondialdehyde (MDA) and the reduced glutathione (GSH) contents. These markers were determined photometrically in homogenized liver tissue according to (Ohkawa et al. 1979) and (Beutler et al. 1963), respectively. 2.4.3.3.4 Biochemical kits The plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using commercially available analytical kits (Diamond Co, Egypt), while gamma-

11

glutamyltransferase (GGT) activities, albumin and total bilirubin levels were measured using kits of Spectrum Co, Egypt. Biodiagnostic Co. kits, Egypt were used for glutathione reduced (GSH) and lipid peroxidation product (malondialdehyde) quantification. 2.4.3.3.5 Histopathological study Liver tissues were fixed in 10% formalin and embedded in paraffin. Sections of 5 - 6 μm thickness were stained with hematoxylin and eosin (H & E) then examined under light microscope for determination of histopathological changes. The histological analysis was performed by a person blinded for treatments. 2.5 Statistical analysis All data are expressed as mean ± SEM. Statistical analysis was performed by ANOVA followed by Tukey post hoc-test using a computer-based curve fitting program (prism 5, Graphpad, CA, USA). A p- value < 0.05 was considered statistically significant.

3. Results and discussion 3.1 Chemical composition of C. abbreviata root The HPLC technique coupled to electrospray ionization mass spectrometry and diode array detection was employed to identify the polyphenols in the root extract. Identification of compounds was based on MS spectra in comparison to literature data. Because of complex stereochemistry of these compounds, the prefix- (epi) or (ent) was given to relate various stereochemical relationships. Altogether, 57 secondary metabolites were identified and their assignment and fragmentation are summarized in Table 1. The LC-MS base peaks in the negative ionization mode ESI (-) are shown in Fig. 1 and structures of some selected compounds from Table 1 are presented in Fig. 2.

12

Free (epi)-catechin (7, 12) and (epi)-afzelechin (18) were identified at the retention times 7.16, 11.3 and 15.57 min, respectively. (epi)-Afzelechin showed a [M-H]- at m/z 273 and daughter ions at m/z 229, 189 and 123 while (epi)-catechin gave a [M-H]- at m/z 289 and daughter ions at m/z 245, 205, 179 (Gu et al. 2003; Buendia et al. 2009; Kuhnert et al. 2010; Tala et al. 2013). An interesting and a rare (epi)-catechin glucoside (6) was identified at m/z 451 and daughter ion at m/z 289. The loss of 162 amu is in agreement with glucosyl moiety (Fig. 3a) (Gu et al. 2003; Buendia et al. 2009; Kuhnert et al. 2010; Tala et al. 2013). Fig. 3 Two peaks (33, 39) showed a molecular ion peak at [M-H]- at m/z 529 with daughter ions at m/z 273 [M-H-256], corresponding to the loss of a guibourtinidol moiety. They were identified as (epi)-guibourtinidol-(epi)-afzelechin (Fig. 3b) as previously described (Messanga et al. 1998). Seven additional peaks (40- 43, 46, 47, 51) showed a molecular ion peak [M-H]- at m/z 529 with daughter ions at m/z 289 [M-H-240], indicating the loss of cassiaflavan moiety. These compounds were identified as (ent)-cassiaflavan-(epi)-catechin, their retention times are documented in Table 1, and a representative mass spectrum is illustrated in Fig. 3c (Coetzee et al. 2000). Several (epi)-guibourtinidol-(epi)-catechin dimers (28, 30-32, 34, 35, 38) were identified via molecular ion peak at m/z 545 and fragment at 289 [M-H- 256] (Fig. 3 d), which was consistent with the reported data (Malan et al. 1996). Altogether, 12 peaks (13, 15, 16, 19, 21-25, 27, 29, 37) showed molecular ion peak [M-H]- at m/z 561 and daughter ion at 289 [M-H-272] which indicated that the corresponding compounds are mixed dimers consisting of (epi)-catechin-(epi)afzelechin, based on fragments and data of literature (Fig. 3 e) (de Souza et al. 2008; Omar et al. 2011).

13

Three peaks (3, 9, 11) showed a molecular ion peak [M-H] at m/z 577 and MS2 experiments with the precursor ion at m/z 289 have led to the identification of these compounds as catechin dimers ((epi)-catechin-(epi)-catechin) that matches previously described data (de Souza et al. 2008; Tala et al. 2013). The retention times of the dimers are documented in Table 1 and representative mass spectrum is illustrated in Fig. 3f. Procyanidin dimer monogallate (14, 17) was detected with a [M-H]- at m/z 729 and daughter ions at m/z 577 [M-H- 152], 559 [M-H- 170], 289 [M-H- 440]; the compound was identified as procyanidin dimer consisting of (epi)-catechin and (epi)-catechin gallate as previously described (Tala et al. 2013). Other tannins with higher polymerization degree (45, 49) were evidenced by a molecular ion at m/z 801 and fragments at m/z 545 [M-H- 256], 289 [M-H- 256 - 256] were tentatively identified as (epi)-guibourtinidol-(epi)-guibourtinidol-(epi)-catechin. A trimeric proanthocyanidin (8) with a molecular ion peak [M-H]- at m/z 865 and daughter ions at m/z 739 [M-H- 126], 577 [M-H-288] was identified as a trimer of (epi)-catechin as previously reported (Tala et al. 2013), representative spectra are shown in Fig. 4a and 3b, respectively. Fig. 4 Furthermore, theaflavin (20), cassanidin A (44) (previously described in C. abbreviata (Erasto and Majinda 2003)) as well as quercetin rhamnoside (36) (Sousa de Brito 2007) and kaempferol rhamnoside (26) (Barros et al. 2012) were identified based on their molecular weights and fragmentation patterns. The ion at m/z 769 is consistent with three condensed flavan-3-ol units (55 - 57). The fragments at m/z 529 [M-H-240], and 289 [M-H-240-240] indicate the presence of (ent)-cassiaflavan-(ent)-cassiaflavan-(epi)-catechin isomers, the proposed fragmentation and a representative spectrum are shown in Fig. 5. The retention times are documented in Table 1. Fig. 5

14

The MS2 spectra of m/z 785 (52-54) showed product ions at m/z 545 [M-H- 240] assigned to loss of (ent)-cassiaflavan and at m/z 289 [M-H-240-256] assigned to loss of (ent)-cassiaflavan and (epi)-guibourtinidol, hence this trimer was identified as an (epi)-catechin-(epi)guibourtinidol-(ent)-cassiaflavan; the proposed fragmentation and a representative spectrum are shown in Fig. 6, their retention times are documented in Table 1. Fig. 6 3.2 Biological activities 3.2.1 Antioxidant activity in vitro The total phenolic content (determined by the Folin-Ciocalteu method) of the root extract amounted to 474 mg as gallic acid equivalents (GAE)/g extract. Plants containing high concentrations of flavonoids, particularly proanthocyanidins, have been promoted for their health effects which are in part due to their antioxidant properties (Wu et al. 2004). The antioxidant activity of the proanthocyanidin-rich extract was assessed by measuring its potential to scavenge the DPPH as well as to reduce Fe(III) in FRAP assay. We could show that the root extract exhibited promising antioxidant activities in both assays (Table 2). The high antioxidant activities are most probably due to the high tannin content. The previously reported antioxidant potential of tannins (Luximon-Ramma et al. 2002; Beninger and Hosfield 2003; Koleckar et al. 2008; Abbas and Wink 2009; Diouf et al. 2009) very well agrees with our data. Table 2 3.2.2 Antioxidant activity in vivo in C. elegans: 3.2.2.1 Survival assay and intracellular ROS content under juglone induced oxidative stress: To evaluate the antioxidant effect of the root extract in vivo, the C. elegans model was employed, which is useful in this context (Su and Wink 2015; Wang and Wink 2016). The pro-

15

oxidant naphthoquinone juglone from Juglans regia was used to induce ROS-mediated toxicity in the nematodes. Juglone is an allelopathic substance, found mainly in the leaves, roots, and barks of plants belonging to the family Juglandaceae. It interferes with some vital metabolic enzymes leading to stunning the growth of some other plants and organisms such as parasitic worms. Being a naphthoquinone in nature, toxicity of juglone involves also formation of naphthosemiquinone and reactive oxygen species leading to cytotoxicity (Doherty et al. 1987). When the wild type nematodes were exposed to 80 μM juglone only 40% survived. An improved survival rate was observed in nematodes pretreated with 200 μg extract/mL (Fig. 7 a). We assume that the polyphenols from the root extract were absorbed (at least partially) and able to scavenge ROS in vivo. The extract was thus able to protect the worms against the deleterious effects of the juglone-induced free radicals. As shown in Fig. 7 b, the intracellular ROS level was significantly reduced in wild-type nematodes to 61.73% (compared to control group; 100%) which were treated with 200 μg extract/mL. Similar results have been observed for other polyphenols and anthocyanins (Chen et al. 2013; Abbas and Wink 2014; Su and Wink 2015; Peixoto et al. 2016a; Sobeh et al. 2016). Fig. 7 3.2.2.2 Quantification of Phsp-16.2::GFP expression and subcellular DAF-16:: GFP localization To address the possible mechanism of the in vivo antioxidant activities of the root extract, a transgenic strain was used which can express the green fluorescent protein (GFP) coupled to heat shock protein HSP-16.2. Oxidative stress was induced by incubating the worms with 20 μM juglone for 24 h, and the expression of Phsp-16.2::GFP was measured by the fluorescence intensity in the pharynx. As shown in Fig. 8 a, the root extract (200 μg/mL) reduced HSP-16.2

16

expression significantly. Similar effects have been described for other phenolic antioxidants (Chen et al. 2013a; Su and Wink 2015). Fig. 8 To further investigate potential upstream mediating factors of the regulation of heat shock protein expression, the transcription factor Daf-16/FOXO was investigated. The transcription factor is inactive when in the cytosol and active in the nucleus (Chen et al. 2013b). Fig. 8b illustrates that both concentrations of 100 μg/mL and 200 μg/mL of the root extract induced a nuclear translocation of DAF-16::GFP, indicating that the in vivo antioxidant of C. abbreviata extract may involve DAF-16/FOXO regulated signalling pathway. Similar mechanisms have been observed for other polyphenols (Chen et al. 2013a; Abbas and Wink 2014; Su and Wink 2015; Peixoto et al. 2016b; Sobeh et al. 2016). 3.2.3 Hepatoprotective activities Acute liver failure (ALF) is known as a severe life threatening disease with a high mortality rate characterized by rapid loss of liver function as well as necrosis of a large number of hepatocytes. The only treatment option shown to improve the outcome of ALF is liver transplantation and it is considered the most commonly used therapy. However, it is limited due to organ rejection, lack of donors, in addition to high costs. Although several studies were performed to find out treatments for various liver diseases using several oral hepatoprotective agents, few beneficial liver drugs are currently available in practice. Herbal medicines have been used in the treatment of liver disorders for a long time (such as silymarin from Silybum marianum) and multi-ingredient herbal preparation are now available in the market for this purpose (Madrigal-Santillan et al. 2014; Namdeo and Syed 2014).

17

ALF may be caused by exposure to different agents such as viruses, alcohol, and chemicals, or by autoimmune diseases. D-Galactosamine (D-GalN) is considered a specific hepatotoxic agent. It depletes the uridine triphosphate pool and inhibits synthesis of macromolecules such as RNA and in consequence proteins, resulting in hepatotoxicity that is similar to that of human viral and drug-induced hepatitis (Decker and Keppler 1974). Overproduction of reactive oxygen species from hepatocytes, infiltrated leukocytes, and activated Kupffer cells, accompanied by increased activity of the pro-inflammatory cytokines and their signaling pathway, contribute to D-GalN induced acute liver damage (Ghosh et al. 2012). Furthermore, previous studies showed that D-GalN increased the expression of toll like receptors and their signaling pathway (Mahmoud et al. 2014). In the current study, D-GalN induced a significant liver injury manifested by dramatic increase in liver enzyme activities (ALT, AST and GGT) in the blood as compared to controls (p< 0.05, Table 3). The increased activities of liver enzymes may be attributed to the ability of D-GalN to suppress the formation of essential uridine nucleotides. This causes inhibition of the synthesis of mRNA and corresponding proteins (Yoo et al. 2008) which leads to changes in the membrane permeability of hepatic cells resulting in leakage of liver enzymes into the blood and elevation of bilirubin level. Another important indication of increased membrane permeability observed in the present study was increased lipid peroxidation manifested by increased MDA hepatic content. 3.2.3.1 Effect of the root extract in D- GalN-induced hepatic injury To assess expected hepatoprotective effect of the extract, the activities of necrosis markers such as transferases (AST, ALT and GGT) were measured in serum. Pretreatment of the rats

18

with the root extract in a dose of 100 mg/kg induced a significant reduction of ALT, AST and GGT activities -11, -35.7 and -65%, respectively as shown in Table 3. Table 3 3.2.3.2 Extract effect on liver function markers Hepatic dysfunction induced by D-GalN injection was monitored by serum albumin and total bilirubin measurements. Following 24 h of D-GalN treatment, a dramatic increase in total bilirubin level was observed in treated rats as compared to control (p< 0.05, Table 3). Pretreatment with root extract attenuated total bilirubin elevation by 23.8% when compared to DGalN treated group (p< 0.05, Table 3). On the other hand, a significant effect on serum albumin was not observed (p˃ 0.05, Table 3). 3.2.3.3 Extract effect on oxidative stress Intraperitoneal D-GaIN administration induced a significant oxidative stress manifested by an increase in the lipid peroxidation product MDA and depletion of GSH levels. Oral pretreatment with the root extract reduced MDA by 47.36% and elevated GSH by 59.1% (Fig. 9a and 9b, respectively) when compared to D-GaIN group. Fig. 9 3.2.3.4 Extract effect in rats with D-GalN-induced histopathological changes Following D-GalN administration, hepatic parenchyma showed intense microscopic changes represented as focal hepatic necrosis infiltrated by mononuclear cells (Fig. 10b). Various reversible and irreversible changes as acute cell swelling and intense microsteatosis to hepatic cells were also noticed. Programmed cell death (apoptosis) involving single or clusters of the hepatic cells was detected and some apoptotic bodies engulfed by the adjacent cells could be noted as well. Some portal areas showed vasculitis and endotheliosis of their vascular intimae.

19

The majority of portal areas revealed intense portal fibrosis infiltrated by mononuclear cells and contain proliferative bile ductules. The aforementioned lesions usually invade the interlobular tissue resulted in its mass thickening. The hepatic capsule and sub capsular tissues appeared also thickened by mature fibrous tissue. Fig. 10 In extract treated rats, the hepatic parenchyma revealed minimal changes represented as focal vacuolar and hydropic degenerations together with normal hepatic features in the majority of hepatic lobules. Few portal areas showed mild eosinophils infiltration (Fig. 10c), hyperplasia of Kupffer cells and minute fibrous strands in portal area and interlobular tissue could be barely observed. 4. Conclusions To find new plant based pharmaceuticals, to increase confidence among users and to contribute to the development of traditional medicine sector, phytochemical and pharmacological studies on Cassia abbreviata root were performed on its methanol extract utilizing the rapid and sensitive HPLC-PDA-ESI-MS/MS technique. The analysis resulted in identifying different types of phenolics present mainly as proanthocyanidins in different isomeric forms. A methanol root extract exerted promising biological activities such as in vivo hepatoprotective effect against DGaIN-induced liver toxicity in rats, and antioxidant potential both in vivo in C. elegans organism and in vitro in DPPH and FRAP assays. Such diversity of biological properties shown by the studied extract may be attributed to the presence of high content of phenolics (proanthocyanidins) that was determined by Folin-Ciocalteu method. This type of secondary metabolites interferes with almost all proteins. In view of these findings, the reported utilization of C. abbreviata in traditional medicine is plausible and rational.

20

Acknowledgments The authors would like to thank Dr. C. D. Rubanza (Tanzania Forestry Research Institute, Shinyanga, Tanzania) for plant collection and Dr. B. Wetterauer (IPMB) for his help in collecting HPLC-MS/MS data.

Conflict of interest The authors declare no conflict of interest References: Abbas, S., Wink, M., 2009. Epigallocatechin gallate from green tea (Camellia sinensis) increases lifespan and stress resistance in Caenorhabditis elegans. Planta Med. 75, 216-221. Abbas, S., Wink, M., 2014. Green tea extract induces the resistance of Caenorhabditis elegans against oxidative stress. Antioxidants. 3, 129-143. Balistreri, W., Shaw, L., 1987. "Liver function." Fundamentals of clinical chemistry. 3rd ed. Philadelphia: WB Saunders p. 729-761. Barros, L., Duenas, M., Carvalho, A. M., Ferreira, I. C., Santos-Buelga, C., 2012. Characterization of phenolic compounds in flowers of wild medicinal plants from Northeastern Portugal. Food Chem. Toxic. 50, 1576-1582. Barros, L., DueNas, M., Ferreira, I. C. F. R., Carvalho, A. M., Santos-Buelga, C., 2011. Use of HPLC–DAD–ESI/MS to profile phenolic compounds in edible wild greens from Portugal. Food Chem. 127, 169-173.

21

Beninger, C. W., Hosfield, G. L., 2003. Antioxidant activity of extracts, condensed tannin fractions, and pure flavonoids from Phaseolus vulgaris L. seed coat color genotypes. J. Agri. Food Chem. 51, 7879-2883. Beutler, E., Duron, O., Kelly, B. M., 1963. Improved method for the determination of blood glutathione. J. Lab. Clin. Med. 61, 882-888. Bruschi, P., Morganti, M., Mancini, M., Signorini, M. A., 2011. Traditional healers and laypeople: a qualitative and quantitative approach to local knowledge on medicinal plants in Muda (Mozambique). J. Ethnopharmacol. 138, 543-563. uendia, .,

il, M. ., Tudela, . ., ady, . L., Medina, . ., oria, C., Lope , . M., Tom s-

Barberan, F. A., 2009. HPLC-MS analysis of proanthocyanidin oligomers and other phenolics in 15 strawberry cultivars. J. Agri. Food Chem. 58, 3916-3926. Chen,

., Mu ller, D., Richling, E., Wink, M., 2013. Anthocyanin-rich purple wheat prolongs

the life span of Caenorhabditis elegans probably by activating the DAF-16/FOXO transcription factor. J. Agr. Food Chem. 61, 3047-3053. Chen, W., Rezaizadehnajafi, L., Wink, M., 2013a. Influence of resveratrol on oxidative stress resistance and life span in Caenorhabditis elegans. J. Pharm. Pharmacol. 65, 682-688. Chen, W., Sudji, I. R., Wang, E., Joubert, E., van Wyk, B.-E., Wink, M., 2013b. Ameliorative effect of aspalathin from rooibos (Aspalathus linearis) on acute oxidative stress in Caenorhabditis elegans. Phytomedicine. 20, 380-386. Clifford, M. N., Johnston, K. L., Knight, S., Kuhnert, N., 2003. Hierarchical scheme for LC-MS identification of chlorogenic acids. J. Agr. Food Chem. 51, 2900-2911.

22

Coetzee, J., Mciteka, L., Malan E., Ferreira, D., 2000. Structure and synthesis of the first procassinidin

dimers

based

on

epicatechin,

and

gallo-

and

epigallo-catechin.

Phytochemistry. 53, 795-804. de Brito, E.S., de Araújo, M. C., Lin, L. Z., Harnly J., 2007. Determination of the flavonoid components of cashew apple (Anacardium occidentale) by LC-DAD–ESI/MS. Food Chem. 105, 1112-1118. de Souza, L. M., Cipriani, T. R., Iacomini, M., Gorin, P. A., Sassaki, G. L., 2008. HPLC/ESI-MS and NMR analysis of flavonoids and tannins in bioactive extract from leaves of Maytenus ilicifolia. J. Pharmaceutic. Biomed. 47, 59-67. Decker, K., Keppler, D., 1974. Galactosamine hepatitis: key role of the nucleotide deficiency period in the pathogenesis of cell injury and cell death. Rev. Physiol. Biochem. Pharmacol. 71, 77-106. Dehmlow, E. V., van Ree, T., Guntenhaner, M., 1998. 2, 4-Trans-, 7 4’-dihydroxy-4methoxyflavan from Cassia abbreviata. Phytochemistry. 49, 1805-1806. Diouf, P. N., Stevanovic, T., Cloutier, A., 2009. Study on chemical composition, antioxidant and anti-inflammatory activities of hot water extract from Picea mariana bark and its proanthocyanidin-rich fractions. Food Chem. 113, 897-902. Doherty, M. D. A., Rodgers, A., Cohen, G. M., 1987. Mechanisms of toxicity of 2‐and 5‐hydroxy‐1, 4‐naphthoquinone; absence of a role for redox cycling in the toxicity of 2‐hydroxy‐1, 4‐naphthoquinone to isolated hepatocytes. J. Appl. Toxicol. 7, 123-129. Doumas, B. T., Watson, W. A., Biggs, H. G., 1997. Albumin standards and the measurement of serum albumin with bromcresol green. Clin. Chim. Acta. 258, 21-30.

23

Erasto, P., Majinda, R., 2003. Bioactive proanthocyanidins from the root bark of Cassia abbreviata. Int. J. Biol. Chem. Sci. 5, 2170-2179. Gali, H. U., Perchellet, E. M., Klish, D. S., Johnson, J. M., Perchellet, J.-P., 1992. Antitumorpromoting activities of hydrolyzable tannins in mouse skin. Carcinogenesis, 13, 715-718. Ghosh, M., Das, J., Sil, P. C., 2012. D (+) galactosamine induced oxidative and nitrosative stress-mediated renal damage in rats via NF-κ and inducible nitric oxide synthase (iNO ) pathways is ameliorated by a polyphenol xanthone, mangiferin. Free Radical. Res. 46, 116132. Gu, L., Kelm, M. A., Hammerstone, J. F., Beecher, G., Holden, J., Haytowitz, D., Prior, R. L., 2003. Screening of foods containing proanthocyanidins and their structural characterization using LC-MS/MS and thiolytic degradation. J. Agr. Food. Chem. 51, 7513-7521. Kashiwada, Y., Nonaka, G. i., Nishioka, I., Chang, J.-J., Lee, K.-H., 1992. Antitumor agents, 129. Tannins and related compounds as selective cytotoxic agents. J. Nat. Prod. 55, 10331043. Kiplagat, D. M., Akala, H. M., Liyala, P. O., Wangui, J. M., Odhiambo, R. A., Omolo, J. O., 2012. Antiplasmodial activity of flavan derivatives from rootbark of Cassia abbreviata Oliv. J. Saudi. Chem. Soc. DOI 10.1016/j.jscs.2012.10.002 Koleckar, V., Kubikova, K., Rehakova, Z., Kuca, K., Jun, D., Jahodar, L., Opletal, L., 2008. Condensed and hydrolysable tannins as antioxidants influencing the health. Mini-Rev. Med. Chem. 8, 436-447. Kuhnert, N., Drynan, J. W., Obuchowicz, J., Clifford, M. N., Witt, M., 2010. Mass spectrometric characterization of black tea thearubigins leading to an oxidative cascade hypothesis for thearubigin formation. Rapid Commun. Mass. Spectrom. 24, 3387-3404.

24

Lee, C.-J., Chen, L.-G., Liang, W.-L., Wang, C.-C., 2010. Anti-inflammatory effects of Punica granatum Linné in vitro and in vivo. Food Chem. 118, 315-322. Leteane, M. M., Ngwenya, B. N., Muzila, M., Namushe, A., Mwinga, J., Musonda, R., Moyo, S., Mengestu, Y. B., Abegaz, B. M., Andrae-Marobela, K., 2012. Old plants newly discovered: Cassia sieberiana DC and Cassia abbreviata Oliv. Oliv. root extracts inhibit in vitro HIV-1c replication in peripheral blood mononuclear cells (PBMCs) by different modes of action. J. Ethnopharmacol. 141, 48-56. Link, P., Roth, K., Sporer, F., Wink, M., 2016. Carlina acaulis exhibits antioxidant activity and counteracts

β

toxicity

in

Caenorhabditis

elegans."

Molecules

21,

871.

doi:10.3390/molecules21070871 Link, P., Wetterauer, B., Fu, Y., Wink, M., 2015. Extracts of Glycyrrhiza uralensis and isoliquiritigenin counteract amyloid-β toxicity in caenorhabditis elegans. Planta. Med. 81, 357-362. Luximon-Ramma, A., Bahorun, T., Soobrattee, M. A., Aruoma, O. I., 2002. Antioxidant activities of phenolic, proanthocyanidin, and flavonoid components in extracts of Cassia fistula. J. Agr. Food Chem. 50, 5042-5047. Madrigal-Santillan, E., Madrigal-Bujaidar, E., Alvarez-Gonzalez, I., Sumaya-Martínez, M. T., Gutiérrez-Salinas, J., Bautista,

M., Morales-González, A., García-Luna, Y., González-

Rubio, M., Aguilar-Faisal, J., 2014. Review of natural products with hepatoprotective effects. World J. Gastroenterol. 20, 14787-14804. Mahmoud, M. F., Hamdan, D. I., Wink, M., El-Shazly, A. M., 2014. Hepatoprotective effect of limonin, a natural limonoid from the seed of Citrus aurantium var. bigaradia, on d-

25

galactosamine-induced liver injury in rats. Naunyn Schmiedebergs Arch. Pharmacol. 387: 251-261. Malan, E., Swinny, E., Ferreira, D., Steynberg, P., 1996. The structure and synthesis of proguibourtinidins from Cassia abbreviata. Phytochemistry 41, 1209-1213. Messanga, B. B., Ghogomu, R., Sondengam, B. L., Martin, M.-T., Blond, A., Brouard J.-P., Bodo, B., 1998. Calodenin C: A new guibourtinidol-(4α→8)-afzelechin from Ochna calodendron. Planta Med. 64, 760-761. Mojeremane, W., Legwaila, G. M., Mogotsi, K. K., Tshwenyane, S. O., 2005. Monepenepe (Cassia abbreviata): a medicinal plant in Botswana. Int. J. Bot. 1, 108-110. Molgaard, P., Nielsen, S. B., Rasmussen, D. E., Drummond, R. B., Makaza, N., Andreassen, J., 2001. Anthelmintic screening of Zimbabwean plants traditionally used against schistosomiasis. J. Ethnopharmacol. 74, 257-264. Mongalo, N., Mafoko, B., 2013. Cassia abbreviata Oliv. A review of its ethnomedicinal uses, toxicology, phytochemistry, possible propagation techniques and pharmacology. Afr. J. Pharm. Pharmacol. 7, 2901-2906. Morimoto, S., Nonaka, G.-I., Chen, R.-F., Nishioka, I., 1988. Tannins and related compounds. LXI: Isolation and structures of novel bi-and triflavanoids from the leaves of Cassia fistula L. Chem. Pharm. Bull. 36, 39-47. Mulubwa, M., Prakash, S., 2015. Antimicrobial activity and potency of Cassia abbreviata Oliv. stem bark extracts. Int. J. Pharm. Pharmaceut. Sci. 7, 426-428. Murray, R. 1984. Alanine aminotransferase. Kaplan A et al. Clin Chem. The C.V. Mosby Co. St Louis. Toronto. Princeton: 1088.

26

Namdeo, A. G., Syed, S. H., 2014. Current status of natural products for the treatment of liver disease-A review. Int. J. Phyto. Pharm. 4, 37-43. Nel, R. J., Mthembu, M., Coetzee, J., Van Rensburg, H., Malan, E., Ferreira, D., 1999. The novel flavan-3-ol, (2R, 3S)-guibourtinidol and its diastereomers. Phytochemistry. 52, 1153-1158. Nibret, E., Ashour, M. L., Rubanza C. D., Wink, M., 2010. Screening of some Tanzanian medicinal plants for their trypanocidal and cytotoxic activities. Phytother. Res. 24, 945-947. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351-358. Omar, M. H., Mullen, W., Crozier, A., 2011. Identification of proanthocyanidin dimers and trimers, flavone C-glycosides, and antioxidants in Ficus deltoidea, a Malaysian herbal tea. J. Agr. Food. Chem. 59, 1363-1369. Peixoto, H., oxo, M.,

rstin, ., o hrig, T., Richling, E., Wink, M., 2016a. An anthocyanin-

rich extract of Acai (Euterpe precatoria Mart.) increases stress resistance and retards agingrelated markers in Caenorhabditis elegans. J. Agr. Food Chem. 64, 1283-1290. Peixoto, H., Roxo, M., Krstin, S., Wang, X., Wink, M., 2016b. Anthocyanin-rich extract of Acai (Euterpe precatoria Mart.) mediates neuroprotective activities in Caenorhabditis elegans. J. Funct. Food. 26, 385-393. Sakagami, H., Jiang, Y., Kusama, K., Atsumi, T., Ueha, T., Toguchi, M., Iwakura, I., Satoh, K., Ito, H., Hatano, T., Yoshida, T., 2000. Cytotoxic activity of hydrolyzable tannins against human oral tumor cell lines - A possible mechanism. Phytomedicine. 7, 39-47. Shai, L., Magano, S., Lebelo, S., Mogale, A., 2011. Inhibitory effects of five medicinal plants on rat alpha-glucosidase: Comparison with their effects on yeast alpha-glucosidase. J. Med. Plants Res. 5, 2863-2867.

27

Sobeh, M., ElHawary, E. A., Peixoto, H., Labib, R. M., Handoussa, H., Swilam, N., El-Khatib, A. M., Sharapov, F., Mahmoud, T., Krstin, S., Linscheid, M. W., Singab, A. N. B., Wink, M, Ayoub, N., 2016. Identification of phenolic secondary metabolites from Schotia brachypetala Sond. (Fabaceae) and demonstration of their antioxidant activities in Caenorhabditis elegans. PeerJ DOI 10.7717/peerj.2404 Souza, S., Aquino, L., Jr Ac, M., Bandeira, M., Nobre, M., Viana, G., 2007. Antiinflammatory and antiulcer properties of tannins from Myracrodruon urundeuva Allemao (Anacardiaceae) in rodents. Phyt. Res. 21, 220-225. Stiernagle, T., 2006. Maintenance of C. elegans, WormBook, ed. The C. elegans Research Community." WormBook.[February 11, 2006]. http://www. wormbook. org. Su, S., Wink, M., 2015. Natural lignans from Arctium lappa as antiaging agents in Caenorhabditis elegans. Phytochemistry 117, 340-350. Szasz G., Persijn J. P., and C. E. 1974. Kinetic method for quantitative determination of gammaglutamyl transpeptidase. Z Klin. Chem. Klin. Biochem. 12, 228. Tala, V. R. S., Candida da Silva, V., Rodrigues, C. M., Nkengfack, A. E., Campaner dos Santos, L., Vilegas, W., 2013. Characterization of proanthocyanidins from Parkia biglobosa (Jacq.) G. Don. (Fabaceae) by flow injection analysis-electrospray ionization ion trap tandem mass spectrometry and liquid chromatography/electrospray ionization mass spectrometry. Molecules 18, 2803-2820. Theodoridis, G., Gika, H. G., Wilson, I. D., 2008. LC-MS-based methodology for global metabolite profiling in metabonomics/metabolomics. TrAC. 27: 251-260. Thomford, N. E., Dzobo, K., Chopera, D., Wonkam, A., Maroyi, A., Blackhurst, D., Dandara, C., 2016. In Vitro reversible and time-dependent CYP450 inhibition profiles of medicinal

28

herbal plant extracts Newbouldia laevis and Cassia abbreviata: implications for herb-drug interactions. Molecules 21: 891. doi: 10.3390/molecules21070891. van Wyk, B.-E., Wink, M., 2004. Medicinal plants of the world: an illustrated scientific guide to important medicinal plants and their uses, Timber Press. van Wyk, B.-E., Wink, M., 2015. Phytomedicines, herbal drugs, and poisons, University of Chicago Press. Wang, E., Wink, M., 2016. Chlorophyll enhances oxidative stress tolerance in Caenorhabditis elegans and extends its lifespan. PeerJ 4: e1879. Wu, X., Gu, L., Prior, R. L., McKay, S., 2004. Characterization of anthocyanins and proanthocyanidins in some cultivars of Ribes, Aronia, and Sambucus and their antioxidant capacity. J. Agr. Food Chem. 52, 7846-7856. Yoo, Y.-M., Nam, J.-H., Kim, M.-Y., Choi, J., Park, H.-J., 2008. Pectolinarin and pectolinarigenin of Cirsium setidens prevent the hepatic injury in rats caused by Dgalactosamine via an antioxidant mechanism. Biol. Pharm. Bull. 31, 760-764. Young, D. S., 1995. Effects of drugs on clinical laboratory tests. AACC.

Fig. 1 Total ion chromatogram of the methanol root extract of C. abbreviata (LC-MS base peak in the negative ionization mode ESI) Fig. 2 Structures of selected compounds from Table 1 Fig. 3 Negative ion ESI-MS spectra of selected compounds from Table 1 Fig. 4 Product ion spectra of different compounds in negative mode ESI-MS from Table 1 Fig. 5 ESI-MS Schematic representation of fragmentation of MS2 of m/z 769 [M-H]-

29

Fig. 6 ESI-MS Schematic representation of fragmentation of MS2 of m/z 785 [M-H]Fig. 7 (a) Stress resistance of N2 worms under juglone treatment (80 μM). Survival rates were significantly increased after pre-treatment of the nematodes with the C. abbreviata extract (200 μg/ml). Data are presented as percentage of survival (mean ± SEM, n=3; untreated worms had a survival of 100%). (b): Intracellular ROS content in N2 worms containing the indicator H2DCFDA. Data are presented as the percentage of fluorescent pixel related to control. ROS content were significantly decreased after pre-treatment of the nematodes with the root extract (200 μg/mL, n=3). * p < 0.05 related to control was analysed by one-way ANOVA. Fig. 8 (a): Phsp-16.2::GFP expression in mutant TJ375 worms. Data are presented as the percentage of fluorescence pixel related to control. The level of Phsp-16.2::GFP was significantly reduced after pre-treatment of the nematodes with the root extract (200 μg/mL). (b): Translocation of DAF-16::GFP in mutant TJ356 worms. The percentage of worms exhibiting a specific DAF-16 subcellular localization pattern is documented, namely, cytosolic, intermediate, and nuclear translocation of DAF-16::GFP in mutant TJ356 worms. ** p < 0.01 related to control was analysed by one-way ANOVA. Fig. 9 Effect of oral administration of the root extract (100 mg/kg) in rats with D-galactosamineinduced liver injury (D-GalN, 800 mg/kg, single IP dose): (a) malondialdehyde content (MDA, nmol/g tissue) in liver, (b) reduced glutathione content (GSH, mg/g tissue) in liver. Results are expressed as mean ± SE. (*) significant difference compared to normal control group (@) significant difference compared to D-GalN treated group at p< 0.05. n = 6; by One Way ANOVA and Tukey post hoc test.

30

Fig. 10 Photomicrographs are representative of cross sections from 6 rats (staining with hematoxylin and eosin, 200×) ; A: Liver of healthy rats with normal hepatocytes and vesicular nuclei arranged in branching cords around the central vein with normal portal area, central vein and normal ducts; B: liver of D-galactosamine treated rats showing focal hepatic necrosis infiltrated by mononuclear cells (arrow head) with microsteatosis of the adjacent hepatocytes (arrows); C: liver of C. abbreviata root extract (100 mg/kg) treated rats with minute fibrous strands in portal area and interlobular tissue with reversible degenerative changes mainly vacuolar degeneration in the hepatic cells.

31

32

33

34

35

Table 1: Chemical composition of the methanolic root extract of C. abbreviata by LC-ESIMS/MS

Peak

tR

[M

(mi

-

n)

H]-

Compound Identification

MS/MS

References

fragment

(m/

No.

z) 2.9 3

(Thomford et al.

1

Sibricose A3

461 167, 329

2016)

2

Quinic acid derivative

3.9

491 191, 275, 371

3

(epi)-Catechin-(epi)-catechin*

5.3

577 289,

407, (de Souza et al.

36

2

425, 559

2008)

6.1 4

(epi)-Catechin glucoside * (epi)-Catechin gallate*

5 6

(epi)-Catechin glycoside*

3

451 179, 245, 289 (Gu et al. 2003)

6.4

(Kuhnert et al.

5

441 289

2010)

6.6

451 289

(Gu et al. 2003)

7.1 7

(epi)-Catechin* (epi)-Catechin-(epi)-catechin-(epi)-

8

catechin*

6 7.3 1 8.1

9

(epi)-Catechin-(epi)-catechin*

4

289 179, 205, 245 407,

577,

865 695, 739 289,

(Tala et al. 2013) 407, (de Souza et al.

577 425, 559

2008)

8.1 10

Theaflavin derivative

4 9.4

11

(epi)-Catechin-(epi)-catechin*

2

771 563, 677, 724 289,

407,

577 425, 559

(Tala et al. 2013)

11. 12

(epi)-Catechin*

3 11.

13

(epi)-Catechin-(epi)-afzelechin*

63 12.

289 179, 205, 245 (Tala et al. 2013) 271,

289,

561 409, 543 289,

14

Procyanidin dimer mono gallate*

17

729 559, 577

15

(epi)-Catechin-(epi)-afzelechin*

13.

561 271,

(Omar et al. 2011) 425, (Tala et al. 2013) 289, (de Souza et al.

37

16

(epi)-Catechin-(epi)-afzelechin*

52

409, 543

14.

271,

96 14.

17

Procyanidin dimer mono gallate

96

2008) 289,

561 409, 543 289,

(Omar et al. 2011) 425,

729 559, 577

(Tala et al. 2013)

15. 18

(epi)Afzelechin

57 15.

19

(epi)-Catechin-(epi)-afzelechin*

69

(Buendia et al. 273 123, 189, 229 2009) 271,

289,

561 409, 543

(Omar et al. 2011)

15. 20

Theaflavin

87 16.

21

(epi)-Catechin-(epi)-afzelechin*

76 17.

22

(epi)-Catechin-(epi)-afzelechin*

09 17.

23

(epi)-Catechin-(epi)-afzelechin*

99 18.

24

(epi)-Catechin-(epi)-afzelechin*

6 19.

25

(epi)-Catechin-(epi)-afzelechin*

16

(Kuhnert et al. 563 289, 443 271,

2010) 289,

561 409, 543 271,

(Omar et al. 2011) 289, (de Souza et al.

561 409, 543 271,

2008) 289,

561 409, 543 271,

(Omar et al. 2011) 289, (de Souza et al.

561 409, 543 271,

2008) 289,

561 409, 543

(Omar et al. 2011)

431 285

(Barros et al. 2011)

19. 26

Kaempferol rhamnoside

3

38

19. 27

(epi)-Catechin-(epi)-afzelechin*

64 19.

28

(epi)-Guibourtinidol-(epi)-catechin*

89 20.

29

(epi)-Catechin-(epi)-afzelechin*

2

271,

289,

561 409, 543 205, 545 391, 409 271, 561 409, 543

(Omar et al. 2011) 289, (Malan et al. 1996) 289, (de Souza et al. 2008)

21. 30

(epi)-Guibourtinidol-(epi)-catechin*

03

545 289, 409, 527 (Malan et al. 1996)

21. 31

(epi)-Guibourtinidol-(epi)-catechin*

23

545 289, 409, 527 (Malan et al. 1996)

22. 32

(epi)-Guibourtinidol-(epi)-catechin*

42

(Thomford et al. 545 289, 409, 527 2016)

23. 33

(epi)-Guibourtinidol-(epi)-afzelechin*

68

(Messanga et al. 529 273, 419, 511 1998)

24. 34

(epi)-Guibourtinidol-(epi)-catechin*

76

(Thomford et al. 545 289, 409, 527 2016)

24. 35

(epi)-Guibourtinidol-(epi)-catechin*

95

Quercetin rhamnoside

25.

36

88 26.

545 289, 409, 527 (Malan et al. 1996) (Sousa de Brito 447 151, 179, 301 2007) 271,

289, (de Souza et al.

37

(epi)-Catechin-(epi)-afzelechin*

41

561 409, 543

2008)

38

(epi)-Guibourtinidol-(epi)-catechin*

26.

545 289, 409, 527 (Thomford et al.

39

39

(epi)-Guibourtinidol-(epi)-afzelechin*

84

2016)

27.

(Messanga et al.

59 31.

40

(ent)-Cassiaflavan-(epi)-catechin*

93 32.

41

(ent)-Cassiaflavan-(epi)-catechin*

8 33.

42

(ent)-Cassiaflavan-(epi)-catechin*

44 33.

43

(ent)-Cassiaflavan-(epi)-catechin*

57

529 273, 419, 511 1998) 245,

289, (Coetzee et al.

529 419, 511 245,

2000) 289, (Coetzee et al.

529 419, 511 245,

2000) 289, (Coetzee et al.

529 419, 511 245,

2000) 289, (Coetzee et al.

529 419, 511

2000)

34. 44

45

Cassanidin A

34

(epi)-Guibourtinidol-(epi)-guibourtinidol-

35.

(epi)-catechin*

24 36.

46

(ent)-Cassiaflavan-(epi)-catechin*

23 37.

47

(ent)-Cassiaflavan-(epi)-catechin*

58 39.

48

49

(ent)-Cassiaflavan-(epi)-afzelechin*

34

(epi)-Guibourtinidol-(epi)-guibourtinidol-

39.

(epi)-catechin*

71

(Erasto and 817 289, 409, 665 Majinda 2003) 289,

409,

801 545, 765 245,

289, (Coetzee et al.

529 419, 511 245,

2000) 289, (Coetzee et al.

529 419, 511 239,

2000) 273, (Morimoto et al.

513 403, 495 289,

1988) 409,

801 545, 765

40

42. 50

(epi)-Fisetinidol–A–(epi)-catechin*

9 44.

51

52

53

54

55

56

57

(ent)-Cassiaflavan-(epi)-catechin*

08

(epi)-Catechin-(epi)-guibourtinidol-(ent)-

46.

cassiaflavan*

8

(epi)-Catechin-(epi)-guibourtinidol-(ent)-

48.

cassiaflavan*

49

(epi)-Catechin-(epi)-guibourtinidol-(ent)-

49.

cassiaflavan*

94

(ent)-Cassiaflavan-(ent)-cassiaflavan-

52.

(epi)-catechin*

11

(ent)-Cassiaflavan-(ent)-cassiaflavan-

52.

(epi)-catechin*

41

(ent)-Cassiaflavan-(ent)-cassiaflavan-

56.

(epi)-catechin*

03

267,

289,

559 425, 513 245,

289, (Coetzee et al.

529 419, 511 289,

2000) 529,

785 545, 767 289,

529,

785 545, 767 289,

529,

785 545, 767

769 289, 529, 751

769 289, 529, 751

769 289, 529, 751

*prefix (epi or ent) represents both possibilities (e.g. (epi)Catehin represents either catechin or epicatechin)

41

Table 2 Antioxidant activities of the methanol root extract of C. abbreviata

Sample Root extract Ascorbic acid

DPPH

FRAP

(EC50 µg/mL)

(mM FeSO4 equivalent/mg sample)

6.2 ± 0.5

19.15 ± 0.13

2.92 ± 0.29

-

-

24.04 ± 1.23

Quercetin

Table 3 Effect of the root extract (100 mg/kg) in rats with D-galactosamine- induced liver injury (DGalN, 800 mg/kg, single i.p. dose) on liver enzymes (serum ALT, AST and GGT activities), total bilirubin and serum albumin levels. Parameters

Control

D-GalN

root extract

% Reduction or elevation

ALT (U/L)

83.36 ± 2.75

98.26* ± 0.97

87.43± 2.64

- 11.0%

AST (U/L)

17.00 ± 1.30

41.40* ± 2.02

30.50± 3.18

- 35.7%

GGT (U/L)

2.44 ± 0.56

6.69* ± 1.42

2.32@± 0.27

- 65.3%

T. bilirubin (mg/dL)

0.41 ± 0.03

1.09* ± 0.16

0.83± 0.13

-23.8%

Albumin(g/dL)

4.38 ± 0.22

3.84 ± 0.07

4.19 ± 0.16

+ 9.1%

Results are expressed as mean ± SE. (*) significant difference compared to normal control group (@) significant difference compared to D-GalN treated group at p< 0.05. n = 6; by One Way ANOVA and Tukey post hoc test

42