rip t Fractionation and bioassay-guided isolation of antihypertensive components of senecio serratuloides Charlotte Mungho Tata, Deprek Ndinteh, Benedicta Ngwenchi Nkeh-Chungag, Opeopluwa Oyehan Oyedeji and Constance Rufaro Sewani-Rusike us c Accepted Manuscript Version an This is the unedited version of the article as it appeared upon acceptance by the journal. A final edited version of the article in the journal format will be made available soon. M As a service to authors and researchers we publish this version of the accepted manuscript (AM) as soon as possible after acceptance. Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). Please note that during production and pre-press, errors may be discovered which could affect the content. pt ed © 2020 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. ce Publisher: Cogent OA Journal: Cogent Medicine Ac DOI: http://dx.doi.org/10.1080/2331205X.2020.1716447 1 t Fractionation and bioassay-guided isolation of antihypertensive somponents of senecio serratuloides us c rip Charlotte Mungho Tata1,2, Derek Ndinteh2, Benedicta Ngwenchi Nkeh-Chungag3, Opeopluwa Oyehan Oyedeji4, and Constance Rufaro Sewani-Rusike1* 1 Ac ce pt ed Corresponding author: Constance Rufaro Sewani-Rusike consewa@hotmail.com M an Department of Human Biology, Faculty of Health Sciences, Walter Sisulu University, Mthatha 5117, South Africa 2 Department of Chemical Sciences, Faculty of Science, University of Johannesburg, South Africa 3 Department of Biological Sciences, Faculty of Natural Sciences, Walter Sisulu University, Mthatha 5117, South Africa 4 Department of Chemistry, Faculty of Science and Agriculture, University of Fort Hare, PBX1314 Alice, 5700 Eastern Cape Province, South Africa 2 Abstract Senecio serratuloides commonly referred to as “two day cure” is used in folk medicine for rip t treating hypertension and wounds in South Africa. This study was aimed at isolating and testing the antihypertensive effects of bioactive compounds from S. serratuloides. Senecio serratuloides us c was serially extracted using solvents of increasing polarity. Phytochemical analysis, antioxidant capacity and antihypertensive properties of fractions were investigated. Bioactive compounds were isolated from ethyl acetate and methanol fractions, their antihypertensive effects and effect an on urine norepinephrine concentration was determined. Ethyl acetate and methanol fractions had all eight phytochemicals tested, better antioxidant capacity and significantly (p<0.001) prevented M the increase in blood pressure induced by Nω-Nitro-L-arginine methyl ester hydrochloride. The isolated bioactive compounds were phytosteroids and Estran-3-one, 17-(acetyloxy)-2-methyl-, ed (2à,5à,17á)- which was isolated from methanol fraction had significantly (p<0.001) better antihypertensive effects through the 4 hour period of the study. Senecio serratuloides may be a ce pt potential source of antihypertensive lead compounds. Keywords: Senecio serratuloides; Serial extraction; Norepinephrine; Oxidative stress; Ac Hypertension; Antioxidants 3 Introduction Hypertension (HTN) is the central pathophysiologic contributor to cardiovascular morbidity and rip t mortality (1). Increased sympathetic nervous system (SNS) activity and reactive oxygen species (ROS) are implicated in the pathogenesis of HTN (2). The role of the SNS in HTN is confirmed us c by increase in circulating plasma levels of catecholamines like norepinephrine in normotensive individuals with a family history of HTN or people with borderline HTN (3). Several factors are potentially capable of activating the SNS, some of which includes; baroreflex dysfunction, an chemoreceptor activation, renin-angiotensin system and other humoral systems (4,3). M Activation of the SNS and other systems like the renin angiotensin system results in increased formation of reactive oxygen species (ROS) which in turn activate the SNS even further (2). ed Increased production of ROS decreases nitric oxide (NO) bioavailability by direct inactivation through formation of peroxynitrite (5) and also by inhibition of eNOS activity through oxidation pt of 4-tetrahydrobiopterin leading eNOS uncoupling (6). NO is known to mediate vasodilation, inhibit platelet aggregation, and prevent leukocyte adhesion to endothelial cells (7). Therefore ce inhibition of NO has deleterious effects on the cardiovascular system. For instances inhibition of Ac NOS using Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) results in NADPH activation and subsequent production of ROS (8,9). In experimental models and human subjects, administration of antioxidant compounds such as vitamin C, Vitamin E, Polyphenols, Allopurinol and Selenium have been shown to have antihypertensive effects through decreasing ROS formation or increasing levels of NO (10,11,12). 4 There are major advances in the development of therapeutic treatments of HTN (13). However despite these advances, the global prevalence of HTN is on the increase due to multiple factors one of which is directly associated with antihypertensive therapy, mainly involving compliance problems (14,15). Non-compliance is a major problem attributed to associated side effects of rip t current antihypertensive drugs. Most of these drugs are not accessible and/or affordable and in many cases, none of them can control HTN singly (16,17). In addition to lack of compliance it is us c estimated that up to 30% of patients with HTN are unresponsive to available drug regimens (18). Therefore there is need for novel agents with better efficacy and little or no side effects. an The plant kingdom may be an alternative for novel agents because it includes a large number of species which produce diverse bioactive compounds with different biological activities (19). M These bioactive compounds include flavonoids, polyphenols, saponins, alkaloids, tanins, triterpenoids, phytosteroids and glycosides. Flavonoids are scavengers of free radicals (20) and ed they prevent oxidation of low density lipoproteins (21). They are associated with improvement of sympatho-vagal balance, decrease systolic blood pressure (SBP) and heart rates thus reducing pt cardiovascular risk and mortality (22). Polyphenols have vasorelaxant effects, decreasing BP by ce increasing endothelial nitric oxide bioavailability via their antioxidant action and their capacity to activate vascular endothelial nitric oxide synthase (23). Saponins block the renin-angiotensin- Ac aldosterone system resulting in decrease total peripheral resistance and consequently decrease systemic HTN (24). Some alkaloids bind strongly to protein receptors on the membrane of secretary vesicles found in the intracellular cytosol of presynaptic neurons and prevent neurotransmitters from being incorporated into the presynaptic vesicle. This prevents and dampens the promulgation of nervous signals in the primary sympathetic neurons of the brain 5 and peripheral nervous system (25). Triterpenoids and phytosteroids lower serum lipid levels thus reducing the risk of atherosclerosis and hence HTN (26,27). An example of a plant which is used in folk medicine for treating HTN in Eastern Cape, South t Africa and thus maybe a source of antihypertensive agents is Senecio serratuloides that is used rip singly or in combination with other herbs (Personal communication, Mahlakata). Senecio serratuloides is also used singly or in combination with other plants to treat wounds such as cuts, us c internal and external sores (including those resulting from sexually transmitted infections), burns, swollen gums and chest pain (28)(29)(30)(31). A study in our laboratory reported the antihypertensive effect of the hydoethanolic extract of S. serratuloides (32). The plant has also an been reported to have phenols, tannins, flavonoids and gallotannins and to possess anti- M inflammatory, anticholinesterase, antioxidant and wound healing properties (28)(29). This study was aimed at serially fractionating S. serratuloides using solvents of increasing polarities in ed order to simplify fractions and enhance isolation of bioactive compounds from the fractions since each solvent extracts different phytochemical groups. The antihypertensive properties of the pt fractions and bioactive compounds were investigated. ce Materials and Methods Chemicals and Drugs Ac Nω-Nitro-L-arginine methyl ester, 2,2’- azinobis (3-ethylbenzothiazoline-6-sulfonic acid), 1,1diphenyl-2-picryl-hydrazil, gallic acid, ascorbic acid, 6-Hydroxy-2,5,7,8-tetramethyl-chroman-2carboxylic acid (trolox) and quercetin were purchased from Sigma-Aldrich Chemical Co. (St Lois, Mo, USA), Captopril was purchased from Pharmacare Ltd. (South Africa) and Norepinephrine ELISA kit from Cloud-Clone Corp. (Texas, USA). All solvents (hexane, dichloromethane, ethyl acetate, methanol) were of analytical grade. 6 Plant material Senecio serratuloides whole plant (stems, leaves and roots) was supplied by Mr Fikile Mahlakata of Lusikisiki, Eastern Cape, South Africa. It was authenticated by Dr Immelman of the Kei t Herbarium, Walter Sisulu University where a voucher specimen (Tata 1/13967) was deposited. rip Whole plant material was air-dried in the laboratory and crushed using a mortar and pestle. Serial Exhaustive Extraction us c Serial extraction of 1221 g of the crushed plant was done using non polar and polar solvents in the order n-hexane, dichloromethane, ethyl acetate and methanol. The dry material was extracted an (three times) with 5 L of n-hexane for 7 days at room temperature. The filtrate was collected by passing the mixture through Whatman No.1 filter paper using a Bϋchner funnel. The filtrate was M concentrated under reduced pressure using a rotatory evaporator (Heidolph Laboroto 4000, Germany) at temperatures not exceeding 40oC (33). The marc was further extracted three times ed with dichloromethane. The procedure was repeated with ethyl acetate and methanol. Once concentrated to small volumes, the fractions were placed in pre-weighed labelled beakers and pt allowed to dry completely; hexane, dichloromethane and ethyl acetate fractions were dried at room temperature while the methanol fraction was dried at 35oC. The total mass of fraction ce extracted by each solvent was calculated as percentage yield using the formula: Ac %Yield = mass of fraction/mass of plant material*100 Phytochemical characterisation Phytochemical screening of fractions for the presence of phytoconstituents was done following the procedures as described by Mir et al. (34). Phenolic compounds were quantified employing Folin's reagent using gallic acid as standard (35). Flavonoid content was quantified following procedures as described by Irshad et al. (36) using quercetin as standard. 7 Antioxidant Capacity of extract fractions Radical Scavenging Activity Radical scavenging activity was evaluated by 2 methods; DPPH (1,1-diphenyl-2-picryl-hydrazil) and ABTS (2,2’- azinobis (3-ethylbenzothiazoline-6-sulfonic acid). The DPPH assay was done rip t following the method described by Yadav et al. (35) using ascorbic acid as standard and ABTS was done following method described by Thaipong et al. (37) using trolox as standard. us c Total Antioxidant Capacity FRAP (Ferric Reducing Antioxidant Power) was done following the method described by Irshad an et al. (36) using ascorbic acid as standard. Chromatography of ethyl acetate and methanol fractions M Thin layer and column chromatography were done following protocols described by Bajpai et al. (38). Aluminium-backed TLC plates (Merck Silica F254 plates) were used. Plates were ed developed under ultraviolet (UV) light at 254 nm and 356 nm (CAMAG universal UV lamp). For visualization of non-fluorescing spots plates were dipped in concentrated sulphuric acid, pt incubated at 600C for 5 mins. The column for column chromatography was packed by slurry ce packing and solvents of different polarities were passed through the column at uniform rate under gravity to further fractionate the fractions. Each fraction was collected separately in a Ac beaker (250 ml) and numbered consecutively for further analysis on TLC. The fractions were concentrated to approximately 1/100 of original volume using a rotatory evaporator (BUCHI, Germany) at 800C. TLC was done on concentrated fractions and those that had the same bands on chromatoplates were mixed and all the fractions were allowed to dry in vials. Crystals were formed in some of the vials and were referred to as bioactive compounds while the fractions that dried up into pastes were referred to as sub-fractions. Two bioactive compounds (CSSA and 8 CSSB) and two sub-fractions (CSSX1 and CSSX2) were isolated from ethyl acetate fraction (SSEA) and one bioactive compound (CSSD) and two sub-fractions (CSSY1 and CSSY2) from methanol fraction (SSMOH). t Identification of bioactive compounds and sub-fractions rip Characterization of bioactive compounds and GC/MS of sub-fractions was done in Department of Applied Chemistry, University of Johannesburg, Doornforntein Campus, South Africa. us c Animals Swiss albino mice weighing 20-25 g were used for acute toxicity and female Wistar rats an weighing 200-240 g were used for HTN prevention study. Animals were housed six per cage in animal holding facilities of Walter Sisulu University which were maintained at 23-240C. The M rooms were lit by day light and dark at night. The animals had free access to rat chow (Epol, grade-BR 1, SA) and water. All animal procedures were in accordance with South African ed National Standards (NSPCA) and EU committee guidelines and were approved by the Research and Ethics Committee of the Faculty of Health Sciences, Walter Sisulu University (Protocol # pt 051/15). ce Hypertension Study design for fractions, sub-fractions and bioactive compounds Table 1 Ac Animals in group 1 were treated with normal saline, those in group 2 were treated with L-NAME and normal saline while those in groups 3 to 7 were co-treated with L-NAME (20 mg/kg) and fraction (150 mg/kg) or sub-fraction (5 mg/kg) or bioactive compound (5 mg/kg) orally once daily for two days (Table 1) (39). Measurement of blood pressure 9 Blood pressure was measured in conscious rats, using non-invasive tail-cuff plethysmography (CODATM 8 Non-Invasive Blood Pressure System, Kent Scientific Corporation, USA) as per manufacturer’s instructions. Baseline BP was measured for all groups. 9-16 hrs after the last treatment with fractions, BP was measured. Meanwhile on day 2, BP was measured at 1, 2 and 4 rip t hrs after treatment (40) for the groups treated with sub-fractions or bioactive compounds. us c Urine collection 24 hrs urine was collected in acidified (300 µl of 3 M HCl) graduated cylinders by placing rats individually in metabolic cages. Collected urine was stored at −20°C for later analysis. The an quantity of water consumed was also monitored. M Determination of norepinephrine concentration in urine Norepinephrine (NE) concentration in urine was determined using an ELISA kit (CEA907Ge; ed Cloud-Clone Corp., USA) which employed a competitive inhibition enzyme immunoassay technique, as per manufacturer’s instructions. All samples were run in one assay. Intra-assay pt coefficient of variability was <10%. There was no significant cross reactivity or interference ce between NE and analogues. Detection range of assay was between 61.7 and 5000 pg/ml. Statistical analysis Ac Results were expressed as mean ± standard error (SEM). Statistical analyses were carried out using Graphpad Prism version 5.03 for Windows (GraphPad Software, San Diego, CA, USA). One-way analysis of variance (ANOVA) followed by Tukey’s posthoc test for multiple comparisons were performed to determine differences between treatment groups. A p-value less than 0.05 were considered statistically significant. 10 Results Percentage yield of fractions Serial extraction using hexane, dichloromethane, ethyl acetate and methanol yielded 4 fractions SSHex, SSDCM, SSEA and SSMOH respectively. The highest yield was obtained with rip t methanol with a percentage of 12.74%, followed by dichloromethane (1.15%), hexane (0.92%) and ethyl acetate (0.81%). us c Phytochemical constituents Qualitative phytochemical screening showed that SSEA and SSMOH had the highest number of an phytochemicals followed by SSHex and SSDCM (Table 2). Results from quantitative analysis of phenols and flavonoids showed that SSMOH and SSEA had the highest phenol contents while M SSDCM had higher flavonoid content (Table 2). Antioxidant capacity ed Table 2 pt Results from ABTS and DPPH assays showed that SSEA and SSMOH had lower IC50 values and hence better scavenging properties. Results from FRAP assay showed that SSMOH and ce SSEA equally had better reducing power than SSDCM and SSHex (Table 3). Table 3 Ac Sub-fractions and compounds isolated from ethyl acetate and methanol fractions Two bioactive compounds (CSSA and CSSB) were isolated from SSEA and one (CSSD) from SSMOH. Sub-fractions CSSX1 and CSSX2 were isolated from SSEA while CSSY1 and CSSY2 were isolated from SSMOH. GC-MS analysis revealed that compounds with relative abundance of 1% and above were 17 in CSSX1, 6 in CSSY1 and 3 in CSSY2. The most abundant 11 compounds are shown in Table 4. The three bioactive compounds identified by NMR are shown in Table 5. Table 4 Effects of Fractions on Systolic and Diastolic Blood Pressure rip t Table 5 us c Administration of L-NAME (20 mg/kg) to female Wistar rats for 2 days significantly increased SBP and DBP in LN group by 16 and 27 % respectively compared to NT group that only observed 0.4 and 3 % increase in SBP and DBP respectively. SSEA and SSMOH significantly an prevented this increase in BP compared to SSDCM and SSHex. It was observed that SBP increased by 7 and 8 % in rats treated with L-NAME and SSEA or SSMOH respectively M compared to 16 % increase in LN group. DBP increased by 4 % in SSEA rats and decreased by 5 % in SSMOH rats compared to 27 % increase in LN group (Figure 1). ed Figure 1 Effects of sub-fractions on blood pressure, heart rate and norepinephrine concentration pt Sub-fractions CSSX1 and CSSX2 isolated from SSEA and CSSY1 and CSSY2 from SSMOH ce were investigated for acute antihypertensive activity over a period of two days. Results from cotreatment of rats with L-NAME and sub-fractions showed that L-NAME significantly increased Ac SBP and DBP by 23 and 37 % respectively compared to NT group that observed 2 and -1 % change in SBP and DBP respectively. CSSX1 and CSSX2 from SSEA significantly (p<0.001) prevented this increase in BP in the first hour after treatment while CSSY1 and CSSY2 from SSMOH had no significant effect on BP (Table 6). 12 L-NAME caused progressive decrease in heart rates in all treatment groups. CSSX1, CSSY1 and CSSY2 significantly (p<0.001) lowered HR even further from the 1hr, 2hrs to 4hr compared to LN control (figure 2). L-NAME also significantly (p<0.001) decreased norepinephrine concentration in the LN group compared to the NT group and all the groups that were co-treated norepinephrine levels compared to NT control group (Figure 2). us c Table 6 rip t with L-NAME and sub-fractions or captopril equally had significantly (p<0.001) lower Figure 2 an Effect of sub-fractions on water intake and urine output LN treatment group consumed significantly (p<0.05) lower volume of water compared to NT M control group. Water intake in CSSX2 group was significantly (p<0.05) higher compared to LN control group and this was reflected in significantly higher urine output in this group compared ed to NT and LN control groups (Table 7). pt Table 7 ce Effects of bioactive compounds on blood pressure, heart rates and norepinephrine concentration Ac Bioactive compounds CSSA and CSSB isolated from SSEA and CSSD from SSMOH were investigated for acute antihypertensive activity over a period of two days. Results showed that LNAME significantly increased BP 1, 2 and 4 hours after treatment by 23, 20 and 17 % for SBP and 37, 29 and 17 % for DBP compared to NT group with 2, 0.4 and -4 % for SBP and -1, 3 and -1 for DBP respectively. CSSD significantly prevented L-NAME-induced increase in SBP at 1, 2 and 4 hrs after treatment but its effect on DBP was not significant at the 4th hour after treatment. 13 CSSB was significantly active in preventing increase in SBP from 2 hrs to 4 hrs (p<0.01) after treatment and its effect on DBP was noticed at 1 and 2 hrs after treatment. CSSA only had significant effect on SBP and DBP 1 hr after treatment (Table 8). t Comparing HR after treatment with HR at baseline, L-NAME caused progressive decrease in rip heart rates in all treatment groups with a significantly (p<0.05) lower HR observed 4 hrs after treatment. CSSA showed the same trend found in LN group whereas CSSD significantly us c (p<0.001) decreased HR from the 1st to the 4th hr. L-NAME significantly (p<0.001) decreased norepinephrine concentration in the LN group (52.76±10 pg/ml) compared to the NT group an (175.04±25 pg/ml). All the groups that were co-treated with L-NAME and bioactive compounds or captopril equally had significantly (p<0.001) lower norepinephrine levels compared to NT M control group (Figure 3). ed Table 8 Figure 3 pt Effect of bioactive compounds on water intake and urine output ce Rats treated with CSSD had significantly (p<0.05) higher water intake than rats treated with LNAME. This was reflected in significantly higher urine output in this group compared to NT and Ac LN control groups. There was however no significant difference in water intake and urine output in CSSA and CSSB compared to NT and LN controls (Table 9). Table 9 Discussion 14 Results from this study revealed that the highest percentage yield was gotten with methanol and the least with ethyl acetate. Ethyl acetate and methanol fractions (SSEA and SSMOH) had more phytochemicals, better antioxidant and antihypertensive properties than dichloromethane and hexane fractions (SSDCM and SSHex). L-NAME increased BP and decreased urinary t Among the three phytosteroid compounds rip norepinephrine concentration and heart rates. isolated, estran-(CSSD) from SSMOH had better antihypertensive properties compared to the us c other compounds and CSSX1 and CSSX2 sub-fractions from SSEA had better antihypertensive properties compared to the other sub-fractions. The polarity of solvents used in extraction determines the difference in type, composition, and an bioactivity of phytochemicals extracted (41). Ethyl acetate is a semipolar solvent that can dissolve sterols, alkaloids, glycosides, terpenoids, and flavonoids. Methanol is polar and can M dissolve polar compounds such as sugar, amino acid, glycosides, phenolic compounds, flavonoids, terpenoid, saponin, tannin, flavone, phenone, and polyphenol (42). Although the two ed solvents had great disparity in yield, they extracted similar phytochemicals some of which were pt not found in SSDCM and SSHex. Hexane is non-polar and can dissolve non polar compounds, such as lignin, wax, lipid, aglycon, sterol and terpenoid (42). This suggests that S. serratuloides ce had fewer phytochemicals with non-polar properties. The high phytochemical content of SSEA and SSMOH was reflected in their antioxidant and Ac antihypertensive capacities. Phytochemicals such as sterol, flavonoid, saponin, tannin, phenol, alkaloid and cardiac glycoside have been proven to have antioxidant activity (39,38). The mechanisms of action of these antioxidants include suppressing reactive oxygen species formation either by inhibition of enzymes or chelating trace elements involved in free radical production; scavenging reactive oxygen species; up-regulating or protecting antioxidant defences 15 (44). The high antioxidant capacity of SSEA and SSMOH was reflected in their better antihypertensive properties. Their efficacy against acute L-NAME induced HTN suggested that they may have vasoactive properties. Previous studies have indicated the possibility of plant extracts in acting as vasorelaxants, for instance; extracts of saffron have been shown to decrease rip t contractility and heart rate of guinea-pig isolated perfuse hearts by blocking Ca2+ channels, opening potassium channels and antagonizing β-adrenoreceptors (45). Extracts and constituents us c of celery have also been reported to lower arterial pressure in humans, possibly by lowering levels of circulating catecholamines and decreasing vascular resistance (46). The mechanism of action of extract components with vasoactive properties may be similar to that of an neurotransmitters which modulate the activities of receptors directly by binding to the relevant receptor proteins or indirectly by diffusing into postsynaptic membranes and altering the M membrane physicochemical properties (43,44). Besides interacting with functional proteins (enzymes, receptors, and ion channels) as the primary targets, bioactive phytochemicals like ed flavonoids, terpenoids, alkaloids have been presumed to act on lipid bilayers and modify pt membrane physicochemical properties (48). All the bioactive compounds isolated were phytosteroids. Since CSSD (estran-) was active from ce the first to fourth hour after treatment, it is possible that the compound and its metabolites had antihypertensive properties. On the other hand, it may have a long half-life, long clearance time Ac and hence high bioavailability. Bioavailability is considered predictive of clinical outcomes (49). The activity of CSSB (pregnan-) only began two hours after treatment suggesting that the activity may have been as a result of its metabolites. CSSA (stigmastan-) was only active in the first hour after treatment suggesting that its metabolites may not have antihypertensive properties or it may have a short half-life, fast rate of clearance and thus decreased bioavailability. The 16 amphipathic nature of etran-(CSSD) and pregnan- (CSSB) may have been responsible for partitioning of the molecules into hydrophobic and hydrophilic media thus affecting their duration and bioavailability. Pharmacologically, the parent drug and its metabolites may act by similar mechanisms, different mechanisms, or even by antagonism (50). CSSD (estran-) equally rip t provoked excretion of higher amount of 24 hr urine compared to the other compounds. This suggested that CSSD may have diuretic properties. Diuretics act by diminishing sodium us c reabsorption at different sites in the nephron, thereby increasing urinary sodium/water losses, decreasing blood volume and hence BP (51). Studies have shown that phytosterols may act as adjuvants in the prevention and treatment of cardiovascular diseases by reducing blood an cholesterol levels (52). This is achieved through competition between phytosterols and cholesterol in the intestinal lumen since they have similar chemical structures. The more M hydrophobic plant sterols are retained, causing a decrease in cholesterol absorption and its consequent elimination in the faeces (53). In addition to the hypocholesterolemic and ed antiatherosclerotic effects of phytosterols, some studies have shown that they exert other pt biological activities such as anti-inflammatory properties (54) and antioxidant potential (55) all of which are important in preventing cardiovascular diseases. Considering Lipinki's rule of 5 on ce drug and drug candidates as stated by (56): (molecular weight < 500 Da; lipophilicity, logP (the logarithm of the partition coefficient between water and 1-octanol) <5; H bond acceptors <10 Ac and H bond donors < 5), these bioactive compounds may be considered as good drug candidates because their molecular weights are less than 500, they are not too polar and not too hyprophobic. The presence of 1,2 benzenedicarboxylic acid-diisooctyl ester, 6-methyl-3-pyrinol, hexadecane, pentadecane and cis-9-[6.1.0]non-2yne in sub-fraction CSSX1 may be responsible for its better 17 antihypertensive properties. Some of these compounds may be adrenergic antagonists while others like cis-9-oxabicyclo[6.1.0]non-2-yne, 2-butoxy-ethanol, 6-methyl-3-pyridinol and 1,2benzenedicarboxylic acid-diisooctyl ester which are capable of donating and/or accepting hydrogen bonds may have antioxidant properties. rip t The decreased NE and heart rates observed in this study may suggest that the SNS had no role in the initiation of L-NAME induced HTN or maybe L-NAME augmented the release, reuptake and us c metabolism of NE. In line with the first suggestion, Fellet et al. (57) showed that a bolus injection of L-NAME increased mean arterial pressure similarly in intact rats and in rats submitted to complete autonomic blockade. They proposed that the effect of L-NAME an administration on BP may probably be due to a direct vasoconstrictor effect caused by the decreased vascular NO synthesis. From this proposal it is possible that decreased HR induced by M L-NAME maybe as a result of activation of baroreceptor afferents by increased BP which resulted in activation of the parasympathetic vagal innervations of the heart. Considering the ed second suggestion, a study done by Kvetnansky et al. (58) revealed significant elevation of NE pt metabolites in L-NAME treated animals in spite of unchanged levels of plasma NE thus suggesting that L-NAME increases release, turnover, reuptake and metabolism of NE in the ce sympathoneural system. In line with this suggestion, Saeed et al. (59) found that the magnitude of interstitial NE can increase far greater than that in plasma and thus they suggested that NE Ac movement into the circulation decreases with baroreceptor unloading. Studies have also shown that spillover of NE from the interstitium may be attenuated by decreased blood flow due to increased peripheral resistance leading to accumulation of interstitial NE (55,56). Therefore the decreased concentrations of NE in urine witnessed in this study may have resulted from interstitial accumulation and/or metabolism which resulted in increased BP. The increased 18 vascular resistance may have triggered a compensatory reflex that overcame its direct stimulatory effects on the heart and resulted in decreased heart rates. In line with the fact that L-NAME upregulates NE release and metabolism, the bioactive compounds and some sub-fractions that decreased BP may have acted as adrenergic antagonists rip t or they had the ability to decrease interstitial NE concentration. Decreased heart rates induced by L-NAME was consistent with previous studies (57,58). Nevertheless, studies have also shown no us c change (39) or increased (64) heart rates in this model. These discrepancies could be due to the dose of L-NAME or route of administration or duration of the study. Zatz and Baylis (65) proposed that the relationship between NO and SNS is highly complex with direct interactions at an various adrenergic receptor subtypes and indirect interactions through baroreceptor control of BP, providing numerous and sometimes opposing influences. This complexity may be M responsible the controversies in literature. ed Conclusion Overall, our results provide evidence indicating that Senecio serratuloides may be a potential pt source of novel agents for treating HTN due to its wide range of phytochemicals and antioxidant properties. Its phytosteroids may serve as vital lead compounds for treating HTN and other ce cardiovascular diseases. Ac Acknowledgements The plant was supplied by Mr Fikile M. Mahlakata and identified by Dr Immelman. Funding This work was supported the National Research Foundation (NRF), South Africa under grant number NRFUID93177 and National Institute of Minority Health Disparities/National Institutes of Health under grant number 5T37MD001810. 19 and Health Disclosure of Interest The authors report no conflict of interest About the Author rip t Our group is interested in probing for novel treatments for non-communicable diseases like hypertension and Diabetes from indigenous medicinal plants. In a bid to determine the us c mechanism of action of these plant extracts and isolates, we investigate their in vitro and ex vivo antioxidant capacities and equally carry out several Biochemical, immunological and histopathological assays on samples obtained from experimental animals after treatment with the an extracts or isolates. Public interest statement M Senecio serratuloides is prescribed by traditional healers in Eastern Cape, South Africa for treating hypertension. Since these traditional healers claim they have been having positive results ed over the years, it is likely that this plant has phytoconstituents that can be exploited by pharmaceutical industries. In this study, Senecio serratuloides was extracted by sequential pt fractionation using four solvents (hexane, dichloromethane, ethyl acetate and methanol). The ce ethyl acetate and methanol fractions had better antioxidant capacity and antihypertensive properties and thus were subjected to thin layer and column chromatography for isolation of Ac bioactive compounds. Three phytosteroids were isolated; two from ethyl acetate fraction and one from the methanol fraction. The phytosteroid isolated from the methanol fraction had better antihypertensive properties. References 20 Dharmashankar K, Widlansky ME. Vascular endothelial function and hypertension: insights and directions. Curr Hypertens Rep. 2010 Dec;12(6):448–55. 2. Campese VM. Oxidative Stress and Sympathetic Activity in Hypertension. Am J Hypertens [Internet]. 2010 May 1;23(5):456. Available from: http://dx.doi.org/10.1038/ajh.2010.19 3. Mancia G, Grassi G. The autonomic nervous system and hypertension. Circ Res. 2014;114(11):1804–14. 4. Saino A, Pomidossi G, Perondi R, Morganti A, Turolo L, Mancia G. Modulation of sympathetic coronary vasoconstriction by cardiac renin-angiotensin system in human coronary heart disease. Circulation. 2000 May;101(19):2277–83. 5. Gonzalez J, Valls N, Brito R, Rodrigo R. Essential hypertension and oxidative stress: New insights. World J Cardiol. 2014 Jun;6(6):353–66. 6. Dinh QN, Drummond GR, Sobey CG, Chrissobolis S. Roles of inflammation, oxidative stress, and vascular dysfunction in hypertension. Biomed Res Int. 2014;2014:406960. 7. Adaramoye OA, Nwosu IO, Farombi EO. Sub-acute effect of N(G)-nitro-l-arginine methyl-ester (L-NAME) on biochemical indices in rats: Protective effects of Kolaviron and extract of Curcuma longa L. Pharmacognosy Res [Internet]. 2012 Sep 28;4(3):127– 33. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424838/ 8. Bunbupha S, Pakdeechote P, Kukongviriyapan U, Prachaney P, Kukongviriyapan V. Asiatic acid reduces blood pressure by enhancing nitric oxide bioavailability with modulation of eNOS and p47phox expression in L-NAME-induced hypertensive rats. Phytother Res. 2014 Oct;28(10):1506–12. 9. Berkban T, Boonprom P, Bunbupha S, Welbat JU, Kukongviriyapan U, Kukongviriyapan V, et al. Ellagic Acid Prevents L-NAME-Induced Hypertension via Restoration of eNOS and p47phox Expression in Rats. Nutrients. 2015 Jun;7(7):5265–80. 10. Kizhakekuttu TJ, Widlansky ME. Natural antioxidants and hypertension: promise and challenges. Cardiovasc Ther. 2010 Aug;28(4):e20-32. 11. Kadkhodaee M, Sedaghat Z. Novel renoprotection methods by local and remote conditioning. J Ren Inj Prev [Internet]. 2014 Nov 3;3(2):37–8. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4206047/ 12. Baradaran A, Nasri H, Rafieian-Kopaei M. Oxidative stress and hypertension: Possibility of hypertension therapy with antioxidants. J Res Med Sci. 2014 Apr;19(4):358–67. 13. Cushman WC, Whelton PK, Fine LJ, Wright JT, Reboussin DM, Johnson KC, et al. SPRINT Trial Results: Latest News in Hypertension Management. Hypertension [Internet]. 2016 Feb 9;67(2):263–5. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4768735/ Ac ce pt ed M an us c rip t 1. 21 Armario P, Waeber B. Therapeutic strategies to improve control of hypertension. J Hypertens. 2013 Mar;31 Suppl 1:S9-12. 15. Jarari N, Rao N, Peela JR, Ellafi KA, Shakila S, Said AR, et al. A review on prescribing patterns of antihypertensive drugs. Clin Hypertens [Internet]. 2016 Mar 27;22:7. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4808570/ 16. Marshall IJ, Wolfe CDA, McKevitt C. Lay perspectives on hypertension and drug adherence: systematic review of qualitative research. BMJ Br Med J [Internet]. 2012 Jul 9;345. Available from: http://www.bmj.com/content/345/bmj.e3953.abstract 17. Mugabo P, Raji IA. Effects of aqueous leaf extract of Asystasia gangetica on the blood pressure and heart rate in male spontaneously hypertensive Wistar rats. BMC Complement Altern Med [Internet]. 2013;13(1):283. Available from: https://doi.org/10.1186/14726882-13-283 18. Calhoun DA, Booth JN 3rd, Oparil S, Irvin MR, Shimbo D, Lackland DT, et al. Refractory hypertension: determination of prevalence, risk factors, and comorbidities in a large, population-based cohort. Hypertens (Dallas, Tex 1979). 2014 Mar;63(3):451–8. 19. Atanasov AG, Waltenberger B, Pferschy-Wenzig E-M, Linder T, Wawrosch C, Uhrin P, et al. Discovery and resupply of pharmacologically active plant-derived natural products: A review. Biotechnol Adv. 2015 Dec;33(8):1582–614. 20. Kooshki A, Hoseini BL. Phytochemicals and Hypertension. Shiraz E-Medical J [Internet]. 2014;15(1):e19738. Available from: http://emedicalj.com/en/articles/20410.html 21. de Paula TP, Steemburgo T, de Almeida JC, Dall’Alba V, Gross JL, de Azevedo MJ. The role of Dietary Approaches to Stop Hypertension (DASH) diet food groups in blood pressure in type 2 diabetes. Br J Nutr. 2012 Jul;108(1):155–62. 22. Duarte A, Mostarda C, Irigoyen MC, Rigatto K. A single dose of dark chocolate increases parasympathetic modulation and heart rate variability in healthy subjects. Rev Nutr [Internet]. 2016;29:765–73. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S141552732016000600765&nrm=iso 23. Zhao Y, Wang J, Ballevre O, Luo H, Zhang W. Antihypertensive effects and mechanisms of chlorogenic acids. Hypertens Res. 2012 Apr;35(4):370–4. 24. Chen M, Long Z, Wang Y, Liu J, Pian H, Wang L, et al. Protective effects of saponin on a hypertension target organ in spontaneously hypertensive rats. Exp Ther Med. 2013 Feb;5(2):429–32. 25. Lobay D. Rauwolfia in the Treatment of Hypertension. Integr Med A Clin J [Internet]. 2015 Jun;14(3):40–6. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4566472/ Ac ce pt ed M an us c rip t 14. 22 Abumweis SS, Barake R, Jones PJH. Plant sterols/stanols as cholesterol lowering agents: A meta-analysis of randomized controlled trials. Food Nutr Res. 2008;52. 27. Machaba KE, Cobongela SZZ, Mosa RA, Oladipupo LA, Djarova TG, Opoku AR. In vivo anti-hyperlipidemic activity of the triterpene from the stem bark of Protorhus longifolia (Benrh) Engl. Lipids Health Dis [Internet]. 2014 Aug 15;13:131. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246574/ 28. Fawole OA, Amoo SO, Ndhlala AR, Light ME, Finnie JF, Van Staden J. Antiinflammatory, anticholinesterase, antioxidant and phytochemical properties of medicinal plants used for pain-related ailments in South Africa. J Ethnopharmacol. 2010;127(2):235–41. 29. Gould AN, Penny CB, Patel CC, Candy GP. Enhanced cutaneous wound healing by Senecio serratuloides (Asteraceae/Compositae) in a pig model. South African J Bot [Internet]. 2015;100:63–8. Available from: http://www.sciencedirect.com/science/article/pii/S0254629915002896 30. De Wet H, Nciki S, van Vuuren SF. Medicinal plants used for the treatment of various skin disorders by a rural community in northern Maputaland, South Africa. J Ethnobiol Ethnomed. 2013 Jul;9:51. 31. De Wet H, Nzama VN, Van Vuuren SF. Medicinal plants used for the treatment of sexually transmitted infections by lay people in northern Maputaland, KwaZulu-Natal Province, South Africa. South African J Bot [Internet]. 2012;78:12–20. Available from: http://dx.doi.org/10.1016/j.sajb.2011.04.002 32. Tata CM, Sewani-Rusike CR, Oyedeji OO, Gwebu ET, Mahlakata F, Nkeh-Chungag BN. Antihypertensive effects of the hydro-ethanol extract of Senecio serratuloides DC in rats. BMC Complement Altern Med. 2019;19(1):1–10. 33. Ruttoh EK, Tarus PK, Bii CC, Machocho AK, Karimie LK, Okemo PO. Antibacterial activity of Tabernaemontana stapfiana britten (Apocynaceae) extracts. African J Tradit Complement Altern Med AJTCAM. 2009 Mar;6(2):186–94. 34. Mir M amin, Sawhney SS, Jassal MMS. Qualitative and quantitative analysis of phytochemicals of Taraxacum officinale. Vol. 2, Journal of Pharmacy and Pharmocology. 2013. 1–5 p. Ac ce pt ed M an us c rip t 26. 35. Yadav M, Yadav A, Yadav JP. In vitro antioxidant activity and total phenolic content of endophytic fungi isolated from Eugenia jambolana Lam. Asian Pac J Trop Med. 2014;7(S1):S256–61. 36. Irshad M, Zafaryab M, Singh M, Rizvi MMA. Comparative Analysis of the Antioxidant Activity of Cassia fistula Extracts. Int J Med Chem [Internet]. 2012;2012:1–6. Available from: http://www.hindawi.com/journals/ijmc/2012/157125/ 37. Thaipong K, Boonprakob U, Crosby K, Cisneros-Zevallos L, Hawkins Byrne D. 23 Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. J Food Compos Anal [Internet]. 2006;19(6):669–75. Available from: http://www.sciencedirect.com/science/article/pii/S0889157506000081 Bajpai VK, Majumder R, Park JG. Isolation and purification of plant secondary metabolites using column-chromatographic technique. Bangladesh J Pharmacol. 2016;11(4):844–8. 39. Biancardi VC, Bergamaschi CT, Lopes OU, Campos RR. Sympathetic activation in rats with L-NAME-induced hypertension. Brazilian J Med Biol Res = Rev Bras Pesqui medicas e Biol. 2007 Mar;40(3):401–8. 40. Tavares T, Sevilla M-A, Montero M-J, Carron R, Malcata FX. Acute effect of whey peptides upon blood pressure of hypertensive rats, and relationship with their angiotensinconverting enzyme inhibitory activity. Mol Nutr Food Res. 2012 Feb;56(2):316–24. 41. Dehkharghanian M, Adenier H, A. Vijayalakshmi M. Study of flavonoids in aqueous spinach extract using positive electrospray ionisation tandem quadrupole mass spectrometry. Vol. 121, Food Chemistry. 2010. 863–870 p. 42. Sri Widyawati P, Budianta TDW, Kusuma FA, Wijaya EL. Difference of solvent polarity to phytochemical content and antioxidant activity of Pluchea indicia less leaves extracts. Int J Pharmacogn Phytochem Res. 2014;6(4):850–5. 43. Tapas AR, Sakarkar DM, Kakde RB. Flavonoids as Nutraceuticals : A Review. 2008;7(September):1089–99. 44. Kumar S, Pandey AK. Chemistry and Biological Activities of Flavonoids : An Overview. 2013;2013. 45. Boskabady MH, Shafei MN, Shakiba A, Sefidi HS. Effect of aqueous-ethanol extract from Crocus sativus (saffron) on guinea-pig isolated heart. Phytother Res. 2008 Mar;22(3):330– 4. 46. Houston MC. Nutraceuticals, vitamins, antioxidants, and minerals in the prevention and treatment of hypertension. Prog Cardiovasc Dis. 2005;47(6):396–449. 47. Cantor RS. Receptor desensitization by neurotransmitters in membranes: are neurotransmitters the endogenous anesthetics? Biochemistry. 2003 Oct;42(41):11891–7. 48. Tsuchiya H. Membrane Interactions of Phytochemicals as Their Molecular Mechanism Applicable to the Discovery of Drug Leads from Plants. Molecules. 2015 Oct;20(10):18923–66. 49. Chow S-C. Bioavailability and Bioequivalence in Drug Development. Wiley Interdiscip Rev Comput Stat. 2014;6(4):304–12. 50. Lin JH, Lu AY. Role of pharmacokinetics and metabolism in drug discovery and Ac ce pt ed M an us c rip t 38. 24 development. Pharmacol Rev. 1997 Dec;49(4):403–49. Ribeiro EF, de Fatima Reis C, de Carvalho FS, Abreu JPS, Arruda AF, Garrote CFD, et al. Diuretic effects and urinary electrolyte excretion induced by Aspidosperma subincanum Mart. and the involvement of prostaglandins in such effects. J Ethnopharmacol. 2015 Apr;163:142–8. 52. Racette SB, Lin X, Lefevre M, Spearie CA, Most MM, Ma L, et al. Dose effects of dietary phytosterols on cholesterol metabolism: a controlled feeding study. Am J Clin Nutr. 2010 Jan;91(1):32–8. 53. Sanclemente T, Marques-Lopes I, Puzo J, Garcia-Otin AL. Role of naturally-occurring plant sterols on intestinal cholesterol absorption and plasmatic levels. J Physiol Biochem. 2009 Mar;65(1):87–98. 54. Navarro A, De las Heras B, Villar A. Anti-inflammatory and immunomodulating properties of a sterol fraction from Sideritis foetens Clem. Biol Pharm Bull. 2001 May;24(5):470–3. 55. Ferretti G, Bacchetti T, Masciangelo S, Bicchiega V. Effect of phytosterols on copper lipid peroxidation of human low-density lipoproteins. Nutrition. 2010 Mar;26(3):296–304. 56. Leeson P. Drug discovery: Chemical beauty contest. Vol. 481, Nature. England; 2012. p. 455–6. 57. Fellet AL, Di Verniero C, Arza P, Tomat A, Varela A, Arranz C, et al. Effect of acute nitric oxide synthase inhibition in the modulation of heart rate in rats. Brazilian J Med Biol Res = Rev Bras Pesqui medicas e Biol. 2003 May;36(5):669–76. 58. Kvetnansky R, Pacak K, Tokarev D, Jelokova J, Jezova D, Rusnak M. Chronic blockade of nitric oxide synthesis elevates plasma levels of catecholamines and their metabolites at rest and during stress in rats. Neurochem Res. 1997 Aug;22(8):995–1001. 59. Saeed N, Khan MR, Shabbir M. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complement Altern Med [Internet]. 2012;12(1):221. Available from: https://doi.org/10.1186/1472-6882-12-221 60. Chang PC, Kriek E, van der Krogt JA, van Brummelen P. Does regional norepinephrine spillover represent local sympathetic activity? Hypertens (Dallas, Tex 1979). 1991 Jul;18(1):56–66. 61. Xing J, Koba S, Kehoe V, Gao Z, Rice K, King N, et al. Interstitial norepinephrine concentrations in skeletal muscle of ischemic heart failure. Am J Physiol Heart Circ Physiol. 2007 Aug;293(2):H1190-5. 62. Sozer V, Uzun H, Gelisgen R, Kaya M, Kalayci R, Tabak O, et al. The effects of atorvastatin on oxidative stress in L-NAME-treated rats. Scand J Clin Lab Invest. 2013 Oct;73(7):591–7. Ac ce pt ed M an us c rip t 51. 25 Sung JH, Jo YS, Kim SJ, Ryu JS, Kim MC, Ko HJ, et al. Effect of Lutein on L-NAMEInduced Hypertensive Rats. Korean J Physiol Pharmacol Off J Korean Physiol Soc Korean Soc Pharmacol. 2013 Aug;17(4):339–45. 64. Cunha RS, Cabral AM, Vasquez EC. Evidence that the autonomic nervous system plays a major role in the L-NAME-induced hypertension in conscious rats. Am J Hypertens. 1993 Sep;6(9):806–9. 65. Zatz R, Baylis C. Chronic nitric oxide inhibition model six years on. Hypertens (Dallas, Tex 1979). 1998 Dec;32(6):958–64. Ac ce pt ed M an us c rip t 63. 26 t ### ### ### 30 ### ### 20 ** ### *** ** ### ## 10 20 *** 10 0 -10 0 *** *** an *** us c Change in DBP *** *** H SS M O SS EA SS D C M SS H ex C A P Treatment groups M Treatment groups N T H SS M O SS EA ex C M SS D P SS H C A LN N T -20 LN Change in SBP rip 40 30 Ac ce pt ed Figure 1. Changes in systolic and diastolic blood pressure in rats treated with fractions. Values are expressed as mean ±SEM. n = 6; SSHex-hexane fraction; SSDCM-dichloromethane fraction; SSEA-ethyl acetate fraction; SSMOH-methanol fraction. * indicates comparison between the treatment groups and L-NAME (LN) control group; # indicates comparison between the treatment groups and normotensive control group (NT). ** p< 0.01, *** p ˂ 0.001; ##p<0.01, ### p< 0.001. 27 t an Ac ce pt ed M Figure 2. Effect of sub-fractions on heart rates and norepinephrine concentration in urine. Values are expressed as mean±SEM. n = 6; NT = normotensive control; LN = L-NAME control; CPT = captopril; CSSX1 & CSSX2- sub-frations from SSEA; CSSY1 & CSSY2- sub-frations from SSMOH. * p< 0.05, ** p ˂ 0.01, *** p ˂ 0.001 compared to L-NAME (LN) control group; #p<0.05, ## p< 0.01, ###p<0.001 compared to normotensive control group. 28 Y2 SS Y1 Treatment groups C 2 SS C SX N T us c 2 SY CS SS Y1 X2 C SS X1 C SS PT C C Treatment groups 1 50 0 LN 0 ### CS 100 100 rip 200 150 SX * 200 PT *** CS 300 ** ** **** C * LN * *** 400 NE Conc(pg/ml) 250 NT Heart Rate(Beats/min) 500 * *** 300 200 100 0 200 t * *** 150 100 rip * ### us c 400 NE Conc(pg/ml) 250 50 an Treatment groups Ac ce pt ed Figure 3. Effect of bioactive compounds on heart rates and norepinephrine concentration in urine.Values are expressed as mean±SEM. n = 6; NT - normotensive control; LN - L-NAME control; CPT = captopril; CSSA - stigmastan-, CSSB - pregnan-, CSSD - estran-. * p< 0.05, ** p ˂ 0.01, *** p ˂ 0.001 compared to L-NAME (LN) control group; #p<0.05, ## p< 0.01, ###p<0.001 compared to normotensive control group. 29 SD CS SB CS SA CS T CP LN NT SD M Treatment groups CS SB CS SA CS CP LN T 0 NT Heart Rate(Beats/min) 500 Table 1. Animal treatment groups Ac ce pt ed M an us c rip t Group (n=6) Fractions Sub-fractions Bioactive compounds 1 (NT) Normal saline Normal saline Normal saline 2 (LN) Normal saline+L-NAME Normal saline+L-NAME Normal saline+L-NAME 3 Captopril+L-NAME Captopril+L-NAME Captopril+L-NAME 4 SSHex+L-NAME CSSX1+L-NAME CSSA+L-NAME 5 SSDCM+L-NAME CSSX2+L-NAME CSSB+L-NAME 6 SSEA+L-NAME CSSY1+L-NAME CSSD+L-NAME 7 SSMOH+L-NAME CSSY2+L-NAME NT – normotensive group, LN– L-NAME group, SSHex - hexane fraction, SSDCM dichloromethane fraction, SSEA - ethyl acetate fraction, SSMOH - methanol fraction CSSX and CSSY - fraction codes; CSSA, CSSB and CSSD – compound codes. 30 Table 2. Phytochemical constituents of fractions from S. serratuloides Ac ce pt ed M an us c rip t Phytochemical SSHex SSDCM SSDCM SSMOH Alkaloids _ + + + Phenols _ _ + + Steroids + + + + Tannins _ _ + + Saponins + + + + Flavonoids _ _ + + Terpenes + _ + + Glycosides + _ + + Polyphenols (µgGAE/mg extract) 64.3±1 47.3±3 114.5±2 185.9±1 Flavonoid (µgQE/mg extract) ) 20.5±0.1 61±0 26.8±0.3 34.8±0.8 + Phytochemical present; - Phytochemical absent; GAE - gallic acid equivalent; QE - quecertin equivalent 31 Table 3. Radical Scavenging (IC50) and Total Antioxidant Capacity of Fractions Ac ce pt ed M an us c rip t SSHex SSDCM SSEA SSMOH ABTS (IC50 mg/ml) 11.79 2.38 1.09 0.41 DPPH (IC50 mg/ml) # # 0.61 0.18 FRAP (µgAAE/mg extract) 37.8±2 52.4±0.4 61.1±1 157.6±1 AAE - ascorbic acid equivalent, # - very weak scavenging properties as percentage inhibition at the concentrations examined were far lower than 50. SSHex - hexane fraction, SSDCM dichloromethane fraction, SSEA - ethyl acetate fraction, SSMOH - methanol fraction. 32 Table 44. Prominen nt compoun nds in sub-ffractions frrom ethyl accetate and methanol fractions f % and RT 111.33% 221.64 m mins 6-methyl-3-pyridinol 88.56% 111.75 m mins Hexadecanee 77.42 % 111.41 m mins Pentadecanne 66.71 % 110.14 m mins 2-butoxy-etthanol CSSY2 Cis-9oxabicyclo[[6.1.0] non-2-yne ce Propperties H bond acceptoor; W-390 g/moll MW MF(C8H17COO)2 C6H4 rip us c H bond donor and acceptor; W-109 g/moll MW MF-C6H7NO M an MW W-226 g/moll; MF-C16H34 33.7 % 33.65 mins MW W-212 g/moll; MF-C15H32 H bond donor and acceptor; W-118g/mol MW MF-C6H14O2 pt CSSY1 Structure t N Name 1,2benzenedicaarboxyli c acid, diisoooctyl ester ed Code CSSX1 33.02 % 22.61 mins H-bbond acceptoor; MW W-122 g/moll MF-C8H10O Ac CSSX aand CSSY - fraction coddes; MW-m molecular weeight; MF-m molecular foormula; RT-rretention time; strructures andd properties from Chem mSpider and PubChem ddatabase. 33 Table 55. Bioactive compound ds: structurres and Prooperties CSSD Estran-3one e, 17(acetyloxy)2-m methyl-, (2à à,5à,17á)- MW - 332.48 g/mol; M M - C21H32O3 MF t Pre egnan-20one e, 3,17bis[[oxy]-, Omethyloxime, (3á á,5à)- Prroperties MW - 396 g//mol; M M C29H48 MF- rip CSSB structure us c me Nam Stiggmastan- 3, 5-diiene MW - 332.52; MF M C22H36O2 an code CSSA Ac ce pt ed M CSSA, CSSB C and CSSDC comppound codees; MW-mollecular weigght, MF-moolecular form mula 34 Table 6. Effect of sub-fractions on systolic and diastolic blood pressure Ac ce pt ed M an us c rip t Time/hrs NT LN CPT CSSX1 CSSX2 CSSY1 CSSY2 SBP 0 146±3 146±1 147±1 149±2 146±1 146±0.4 149±2 1 149±4 180±3 168±4a 141±1c 161±3c 169±2 171±1 2 147±3 175±5 170±3 166±5 166±3 174±4 170±2 4 140±1 171±2 160±3 151±5b 160±4 158±2 165±2 DBP 0 113±5 110±2 117±3 114±1 119±4 120±3 117±5 1 112±2 152±4 141±5 108±7c 130±3a 132±4a 142±1 2 117±2 142±6 140±4 130±8 136±5 142±7 138±3 4 112±1 129±2 128±5 122±3 127±3 167±3 135±3 Values are expressed as mean±SEM. n = 6; NT = normotensive control; LN = L-NAME control; CPT = captopril; CSSX1 & CSSX2-sub-fractions from SSEA, CSSY1 & CSSY2 - sub-fractions from SSMOH. a=* p< 0.05, b=** p ˂ 0.01, c=*** p ˂ 0.001 compared to L-NAME (LN) control group. 35 Table 7. Effect of sub-fractions on water intake and urine output Ac ce pt ed M an us c rip t NT LN CPT CSSX1 CSSX2 CSSY1 CSSY2 Water intake/ml 30±2 22±2# 28±2 27±2 31±2* 21±0.2# 24 Urine output/ml 8±1 8±0.8 13±0.7 13±0.9 14±2#* 12±0.9 11±1.6 Values are expressed as mean±SEM. n = 6; NT = normotensive control; LN = L-NAME control; CPT = captopril; CSSX1, CSSX2, CSSY1 and CSSY2 = sub-fractions consisting of several phytochemicals. * p< 0.05, compared to L-NAME (LN) control group; #p<0.05 compared to normotensive control group. 36 Table 8. Effect of bioactive compounds on systolic and diastolic blood pressure Ac ce pt ed M an us c rip t Time/hrs NT LN CPT CSSA CSSB CSSD SBP 0 146±3 146±1 147±1 147±2 147±6 147±2 1 149±4 180±3 168±4a 163±2b 171±2 153±1c 2 147±3 175±5 170±3 170±2 160±4a 153±2c 4 140±1 171±2 160±3 163±1 151±3c 157±2b DBP 0 113±5 110±2 117±3 121±3 117±7 113±4 1 112±2 152±4 141±5 130±4b 135±3a 108±3c 2 117±2 142±6 140±4 140±3 123±4a 115±3c 4 112±1 129±2 128±5 131±2 123±3 115±6 Values are expressed as mean±SEM. n = 6; NT = normotensive group; LN = L-NAME group; CPT = captopril; CSSA = stigmastan-, CSSB = pregnan-, CSSD = estran-. a-* p< 0.05, b-** p ˂ 0.01, c-*** p˂ 0.001 compared to L-NAME (LN) control group. 37 Table 9. Effect of bioactive compounds on water intake and urine output Ac ce pt ed M an us c rip t NT LN CPT CSSA CSSB CSSD Water intake/ml 30±2 22±2 28±2 25±2 24±4 31±2* Urine output/ml 8±1 8±0.8 13±0.7#* 10±1 10±1 15±1##*** Values are expressed as mean±SEM. n = 6; NT = normotensive groug; LN = L-NAME group; CPT = captopril; CSSA = stigmastan-, CSSB = pregnan-, CSSD = estran-.* p< 0.05, *** p ˂ 0.001 compared to L-NAME (LN) control group; #p<0.05, ## p< 0.01, compared to normotensive control group. 39