Phytomedicine Plus 2 (2022) 100182 Contents lists available at ScienceDirect Phytomedicine Plus journal homepage: www.sciencedirect.com/journal/phytomedicine-plus Phytochemical characterization, antihyperglycaemic and antihyperlipidemic activities of Setaria megaphylla in alloxan-induced diabetic rats Jude E. Okokon a, 1, *, Koofreh Davies b, 3, Lekara John b, 2, Klinton Iwara b, 2, Wen-Wu Li c, 5, Paul S. Thomas d, 4 a Department of Pharmacology and Toxicology Faculty of Pharmacy, University of Uyo, Uyo, Nigeria Department of Physiology, Faculty of Basic Medical Sciences, University of Uyo, Uyo, Nigeria c School of Pharmacy and Bioengineering, Faculty of Medicine and Health Science, Keele University, Stoke-on-Trent, ST4 7QB, United Kingdom d Department of Pharmacognosy and Natural Medicine, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria b A R T I C L E I N F O A B S T R A C T Keywords: Diabetes mellitus Medicinal plants Hypoglycaemic agents Insulin 1-Triacontanol Stigmasterol Background: Setaria megaphylla (Steud) Dur & Schinz (Poaceae), a grass used ethnomedically by herbalists in Nigeria for the treatment of diabetes was evaluated for antidiabetic, hypolipidemic and pancreas protective potentials in rats. Methods: Solvents fractions (hexane, dichloromethane (DCM), ethyl acetate (EA) and methanol ) were investigated for antidiabetic, hypolipidemic and pancreas protective potentials in alloxan-induced diabetic rats. Glibenclamide was used as positive control. The fasting blood glucose (FBG) level, serum insulin, glycosylated hemoglobin (Hb1Ac), oral glucose tolerance test (OGTT) and lipids levels were determined. Histopathological study of the pancreas was done. Isolation of phytochemicals and their subsequent identification using Fourier transform infrared spectroscopy, gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were carried out. Results: Treatment of alloxan-induced diabetic rats with the leaf fractions caused significant (p < 0.05–0.001) reduction in FBG of treated diabetic rats in acute and prolonged studies as well as OGTT with DCM, EA and hexane fractions having pronounced activities. The leaf fractions also caused significant (p < 0.01) decreases in Hb1Ac levels and increases in serum insulin levels. The leaf fractions further caused lowering of serum total cholesterol, triglycerides, low density lipoprotein (LDL), very low density lipoprotein (VLDL) with increased high density lipoprotein (HDL) level in the treated diabetic rats. Histopathological study of pancreas revealed protective effect by the leaf fractions. 1-Triacontanal, 1-triacontanol, 1-dotriacontanol, 1-triacontyl cerotate, and stigmasterol were isolated and identified from the active DCM and EA fractions of this plant for the first time . Conclusion: The leaf fractions of S.megaphylla possess hypoglycemic, insulin secretion stimulatory, hypolipidemic, and pancreas protective potentials which may be due to the activities of the phytochemical constituents. List of abbreviations DCMDichloromethane EAFBGOGTT- Ethyl acetate Fasting blood glucose Oral glucose tolerance test ; ATR, Attenuated total reflection; FT-IR, Fourier transform infrared spectroscopy; ROS, Reactive oxygen species. * Corresponding author. E-mail address: judeokokon@uniuyo.edu.ng (J.E. Okokon). 1 Jude E. Okokon designed and supervised the work 2 Lekara John and Klinton Iwara carried the animal studies 3 Koofreh Davies did the statistical analysis 4 Paul S. Thomas carried out the isolation and purification of the compounds 5 Li Wen Wu did the GC–MS and NMR analysis and interpretation of the spectra and also edited the work. https://doi.org/10.1016/j.phyplu.2021.100182 Received 19 October 2021; Received in revised form 15 November 2021; Accepted 27 November 2021 Available online 30 November 2021 2667-0313/© 2021 The Authors. Published by Elsevier B.V. This is an open access (http://creativecommons.org/licenses/by-nc-nd/4.0/). article under the CC BY-NC-ND license J.E. Okokon et al. Hb1AcGC–MSNMRLDLVLDLHDLWHOH & ET C- Phytomedicine Plus 2 (2022) 100182 Materials and methods Glycosylated hemoglobin Gas chromatography-mass spectrometry Nuclear magnetic resonance spectroscopy Low density lipoprotein Very low density lipoprotein High density lipoprotein World Health Organisation Heamatoxylin and eosin Total cholesterol Plant material and extraction Fresh leaves of the plant were collected from Akwa Anwa forest, Uruan in Akwa Ibom State, Nigeria in August 2020 and identified by a taxonomist in the Department of Botany, University of Uyo, Uyo. A voucher specimen (UUPH. 221 d) of the plant was deposited at herbarium of Department of Pharmacognosy and Natural Medicine, University of Uyo, Uyo. The leaves were washed, chopped into pieces, dried and powdered. The powder (1.5 kg) was successively and gradiently macerated in 7.5 L of each of these solvents at room temperature (28 ± 2 ◦ C) for 72 h; n-hexane, dichloromethane (DCM), ethyl acetate (EA) and methanol to give corresponding their fractions. The liquid filtrates obtained were concentrated to dryness at 40 ◦ C using rotary evaporator and these were preserved in a desicator until used for the proposed experiments. Introduction Diabetes mellitus (DM) is a chronic metabolic disease affecting economic and social activities of all countries of the world with estimated millions of deaths worldwide due to high blood glucose and risks of associated complications, which often result in premature death (WHO, 2017). There are three major types of DM: type 1 diabetes (T1DM, insulin dependent), type 2 diabetes mellitus (T2DM), and gestational diabetes mellitus. T2DM contributes to more than 90% of all DM cases. According to International Diabetes Federation in 2019, the total adult population stands at 463 million with diabetes, which may increase to 578 million and 700 million by 2030 and 2045, respectively (Saeedi et al., 2019). The increasing prevalence of diabetes is worrisome and calls for an urgent remedial steps as the orthodox medicines used in the management of DM seem to be inadequate and challenging (Saeedi et al., 2019). The current use of orthodox drugs and insulin in the management of diabetes does not offer satisfactory treatment outcome as the drugs are associated with unpleasant side effects such as hypoglycemia, hypersensitivity, gastrointestinal disturbances, hepatotoxicity, renotoxicity and heart failure ((Basu et al., 2015); Sudasinghe and Peiris,2018). Herbal preparations are highly patronized for the management of diabetes due to their low cost, accessibility, relative safety and many biological activities compared to conventional anti-diabetic drugs (Badri et al., 2006; Khan et al., 2012; Peiris et al., 2015). Medicinal plants have been extensively investigated for the potential treatment of DM (Marles and Farnsworth, 1995; Ezuruike and Prieto, 2014; Tran et al., 2020). Various phytochemicals such as ginsenosides in Panax ginseng, cucurbitane type terpene glycosides in Momordica charantia, glycyrrhizin in Glycyrrhiza glabra and berberines in Coptis chinensis have been found to possess antidiabetic activity (Park and Jang, 2017). In particular, metformin, one of the most common drugs for DM, is derived from guanidine compounds in Galega officinalis (Bailey, 2004). Hence there is the need to search for new antidiabetic compounds from medicinal plants with potentials to circumvent the present challenges faced in DM management. Setaria megaphylla (Steud) Dur & Schinz (Poaceae) is a perennial pasture grass distributed widely in tropical and subtropical countries of the world (Van Oudtshoorn, 1999). It is called ‘nkwongo’ in ibibio or broad leafed brittle grass. Although no detailed ethnopharmacological survey has been carried out on medicinal plants used by the Ibibios in Akwa Ibom State, reports of ethnomedicinal use of the plant (leaves and roots) by herbalists from Ibibio tribe in Niger Delta region of Nigeria for the treatment of diabetes have been published (Okokon et al., 2006; Okokon and Antia, 2007, 2007a). Preliminary reports indicated antidiabetic and hypoglycaemic activities of the leaves and roots (Okokon and Antia, 2007; Okokon et al., 2007a). Other activities of the leaves include; in vitro and in vivo antimalarial (Clarkson et al., 2004; Okokon et al., 2007b, 2017), anti-inflammatory, analgesic (Okokon et al., 2006), cytotoxic, immunomodulatory and antileishmanial (Okokon et al., 2013) and antidepressant (Okokon et al., 2016). Phytochemical constituents of the leaf extract identified through various screening and analytical methods have been reported (Okokon et al., 2006; Okokon et al., 2013). Here, we report the antihyperglycaemic and hypolipidemic potentials of the solvent leaf fractions of Setaria megaphylla in alloxan-induced diabetic rats, and further isolation and identification of phytochemicals in these fractions. Purification of compounds from dichloromethane and ethyl acetate fractions of Setaria megaphylla Dichloromethane and ethyl acetate fractions (15 g) found to be active during the experiment were bulked and subjected to silica gel column chromatography (Merck, 60–120 mesh) and gradient-eluted with n-hexane containing increasing quantity of dichloromethane, followed by increasing quantity of ethyl acetate and methanol. Eluates (20 mL each) were collected, monitored on silica thin layer chromatography (TLC) plates (Merck, Germany) in hexane: DCM: EA (2:1:1) using vanillin-sulphuric acid as spray reagent. Fifty-one fractions were obtained and bulked together based on their similar TLC characteristics (Rf values, color reaction with spray reagents) to give four semi-pure residues coded F1–F4. F2 was further purified using preparative TLC; carefully dissolved in dichloromethane and applied across the coated silica gel plate (20 × 20 cm, 0.25 mm) using a micro-Pasteur pipette (Simax, India) 1 cm above the bottom edge of the plate. The plates were developed using n-hexane: dichloromethane (4:1) solvent system in a Chromatank (USA). The chromatogram obtained showed two distinctly resolved layers which were carefully scrapped, separated, filtered and concentrated in vacuo. Further TLC evaluations indicated a single spot in each layer which were denoted X1 (10 mg) and X16/17 (8 mg). F3 on further purification with preparative TLC using hexane and dichloromethane (1:4) yielded one pure compound N15 (10 mg). Purification of F4 with preparative TLC using hexane and DCM (4:1) produced a pure compound PJ3 (8 mg). This was filtered and concentrated to give a white crystalline compound. The chemical structures of isolated pure compounds were elucidated using spectroscopic analyses. Animals Wistar rats (males and females) used for these experiments were obtained from Animal house in University of Uyo. The animals were accommodated in standard plastic cages and maintained on pelleted Feed (Guinea Feed) and water ad libitum. Permission and approval for animal studies were obtained from College of Health Sciences Animal Ethics committee, University of Uyo (UU/CHS/AE/21/024). Induction of experimental diabetes using alloxan monohydrate Intraperitoneal injection of freshly prepared alloxan monohydrate solution (150 mg/kg i.p) in ice cold 0.9% saline (NaCl solution), was used to induce diabetes in overnight fasted forty (40) Wistar rats (males and females) weighing (140–160 g). Immediately after the induction, 2 mL of 5% dextrose solution was orally administered to the animals to reduce the effect of initial hypoglycaemia (Okokon and Nyong, 2018). Rats with moderate diabetes, (i.e. with blood glucose levels of 200 mg/dL and above), after a post-induction rest period of 72 h, which was 2 J.E. Okokon et al. Phytomedicine Plus 2 (2022) 100182 allowed for the diabetes to be fully developed (Lenzen, 2008) were considered diabetic and selected for the experiments. The sample size for the study was calculated using resource equation method of Festing (2006) and the diabetic animals were divided into 6 (six) treatment groups of 6 rats each. Based on determined median lethal dose (LD50) of 2.4 ± 0.5 g/kg, (Okokon et al., al.,2006), the rats were treated with 200 mg/kg/day of respective solvent fractions of S. megaphylla leaf orally for 14 days as follows; group 1 was given 10 mL/kg/day of normal saline, group 2, 5 mg/kg/day of glibenclamide, group 3 – 6 were respectively administered 200 mg/kg/day of n-hexane, dichloromethane, ethyl acetate and methanol fractions of S. megaphylla leaf. (1984). Determination of the effect of the solvent fractions on the lipid profile (Serum TG, TC, HDL, LDL, VLDL levels) of the treated diabetic rats Serum total cholesterol (TC), triglyceride and high density lipoprotein (HDL) levels of the diabetic rats were measured using Randox diagnostic kits, low and very low-density lipoprotein (LDL and VLDL) were calculated from the formula of Friedwald et al. (1972). Gas chromatography-Mass spectrometry (GC–MS) analysis Assessment of oral glucose tolerance in treated diabetic rats GC–MS was carried out on an Agilent 7890A gas chromatograph, coupled with an Agilent MS model 5975C MSD with triple axis detector (Agilent Technologies, USA). The system was equipped with a HP5-MS column 5% phenyl-methylpolysiloxane, 30 m × 0.25 mm × 0.25 µm (Agilent Technologies, USA). The carrier gas was helium with a gas flow under a constant pressure of 10 psi. The injector temperature was set at 280 ◦ C. The initial oven temperature was 160 ◦ C and increased to 320 ◦ C at 10 ◦ C/min, and the final temperature was held for 6 min at 320 ◦ C. The mass spectrometer was operated in the electron ionization mode at 70 eV. The 0.2–0.4 mg of each compounds were dissolved in CHCl3 (5 mg/mL) (X16/17 and PJ3) or treated with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (N15) for 20 min at room temperature before injection of 1 µL to GC–MS system (Aldulaimi et al., 2017). The compounds were identified by comparison of spectral data and fragmentation patter with reference compounds in the NIST 2011 database. Oral glucose tolerance test (OGTT) was evaluated in a group of 36 diabetic rats induced with diabetes using alloxan as stated above. After confirmation and selection of the diabetic rats on the 3rd day, the rats were fasted for 24 h but allowed free access to water. After which the animals were weighed and randomised into 6 groups of 6 rats each. The basal Blood glucose levels (BGL) of the animals were determined by tailtipping method using digital Glucometer. Thereafter, the diabetic rats were orally administered with 200 mg/kg of the respective solvent fractions (n-hexane, dichloromethane, ethyl acetate and methanol). Thirty (30) min post-administration of fractions the animals were given oral glucose load (2 g/kg). BGL were determined at 30, 60, 90, 120 and 180 min post glucose administration (Rabintossaporn et al., 2009; Okokon and Mandu, 2018). Determination of hypoglycemic potential of the solvent fractions of S. megaphylla in alloxan-induced diabetic rats Nuclear magnetic resonance (NMR) spectroscopy FBG of diabetic rats were determined at hourly intervals during the periods of acute study, i.e. 1–3, 5 and 7 h interval, after a single dose of the fractions and at intervals of 1–3, 5, 7 and 14 days during prolonged study by “the tail-tipping method” using microprocessor digital blood glucometer (WHO, 1980). Administration of fractions and drugs were carried out at a scheduled time daily and the animals were fasted every evening prior to days for measurement of the FBG concentrations. 1 H and 13C NMR spectra for the isolated compounds were obtained on a Bruker 400 and 100 MHz instrument, respectively. Chemical shifts were reported in δ (ppm) using the solvent CDCl3 or CDCl3 + CD3OD (9:1) as standard and coupling constants (J) were measured in Hertz. Fourier transform infrared (FT-IR) spectroscopy Fourier transform infrared spectrometer (Nicolet AVATAR360, Thermo Fisher Scientific) fitted with the attenuated total reflection (ATR) accessory was used to record FTIR spectra with a resolution of 4.0 cm-1 from 4000 to 800 cm-1. Determination of the body weights changes of the treated diabetic rats The body weights of the experimental animals were monitored before and after induction of diabetes and at the end of the study (Day15). Statistical analysis Data obtained from this work are expressed as MEAN ± SEM and were analysed statistically using one way ANOVA followed by TukeyKramer multiple comparison test using Instat Graphpad software, San Diego, USA. Differences between means were considered significant at 5% level of significance ie p ≤ 0.05. Collection of blood samples and organs Twenty-four (24 h) after 14 days of treatment with fractions/drugs, the rats were weighed and sacrificed under light diethyl ether vapor. Blood samples were collected by cardiac puncture into plain centrifuge tubes and centrifuged immediately at 1500 rpm for 15 min to separate the serum at room temperature to avoid haemolysis. These blood samples were used for biochemical assays. The pancreas of the diabetic rats used in the study were surgically removed, weighed, fixed in 10% formaldehyde, processed, stained using Heamatoxylin and eosin (H & E) method and microscopically examined under the microscope at magnification (X400) Results Effect of solvent fractions on body and pancreas weights of rats There were considerable changes in the body weights of the treated and untreated alloxan-induced diabetic rats (Table 1). Treatment of the diabetic rats with the leaf fractions caused significant (p < 0.05–0.001) increases in the body weight of the diabetic rats when compared to control. The highest increases were recorded in EA fraction-treated group (9.64%), followed by hexane group (8.30%) (Table 1). Significant (p < 0.001) decreases in the pancreas weights of the diabetic rats compared to control was observed following treatment of alloxan –induced diabetic rats with leaf fractions of S. megaphylla with the hexane fraction exerting the highest reduction (Table 1). Determination of insulin and glycosylated hemoglobin levels in the diabetic rats Serum insulin levels were measured with an ultra-sensitive rat insulin ELISA kit (Alpco Diagnostics) (Finlay and Dillard, 2007), while glycosylated hemoglobin was measured by method of Nathan et al. 3 J.E. Okokon et al. Phytomedicine Plus 2 (2022) 100182 and hexane fraction treated groups. This was followed by DCM and EA groups (Table 2). The serum insulin level of hexane group was comparable to that of the standard drug, glibenclamide (Fig. 2). Table 1 Effect of leaf fractions of Setaria megaphylla on body and pancreas weights of diabetic rats. Treatment Control normal saline Glibenclamide Dose mg/ kg – 10 n-hexane fraction 200 Dichloromethane fraction Ethyl acetate fraction Methanol fraction 200 200 200 Weight (g) Day 0 Day 15 154.6 ± 11.18 147.3 ± 0.85 144.96 ± 1.81 145.26 ± 4.67 144.4 ± 8.50 152.46 ± 12.10 148.3 ± 14.60 157.68 ± 11.20 157.0 ± 24.61 151.25 ± 10.84 158.33 ± 16.02 159.33 ± 15.16 % Increase Weights of pancreas (g) −4.07 0.71 ± 0.03 7.04 8.30 0.44 ± 0.01a 0.50 ± 0.02 4.12 0.53 ± 0.01 9.64 0.54 ± 0.04 4.50 0.58 ± 0.03 Effect of solvents fractions on oral glucose tolerance test (OGTT) Table 2 shows the effect of different solvent fractions on oral glucose tolerance test of alloxan-induced diabetic rats. The leaf fractions caused lowering of BGL of the diabetic rats. These reductions were significant (p < 0.01–0.05) when compared with control and peaked effects were recorded at 180 min with the various fractions producing (34.07–50.29%) inhibition of elevation of BGL, while the methanol fraction had the highest percentage inhibition of 50.98% followed by hexane (50.29%) and dichloromethane (43.84%) (Table 4). a a a Hypolipidemic effect of leaf fractions a a Data are expressed as MEAN ± SEM, Significant at p < 0.001, when compared to control. (n = 6). The leaf fractions of S. megaphylla did not cause any significant effect (p > 0.05) on the levels of total cholesterol, triglyceride, HDL, LDL and VLDL. Although there were prominent decreases in LDL and VLDL levels, as well as increases in HDL levels of the fractions-treated groups, these changes were not significant (p > 0.05) when compared to control group. The standard drug, glibenclamide, caused significant (p < 0.05) reductions in LDL and VLDL levels (Table 5). Effects on the FBG levels of alloxan-induced diabetic rats during acute treatment The leaf fractions, 2 h post treatment, exerted considerable reductions in FBG of the diabetic rats which were significant at the 7 h (p < 0.05–0.001) with the dichloromethane and EA fractions-treated groups having the most significant (p < 0.01) reductions of FBG. The effect of the dichloromethane fraction was stronger than that of standard drug, glibenclamide (7 h) (Table 2). Histological studies of the pancreas Histopathological study of pancreas of untreated diabetic rats treated with normal saline only (10 mL/kg) at X400 revealed distorted pancreas with areas of islets cells degeneration with very few cells, degranulated islet cells and degranulated β-cells and α-cells as well as areas of reduced islet cells. Pancreas of diabetic rats treated with glibenclamide and leaf fractions revealed some normal areas though with few areas of reduced islet cells of Langerhans than normal, degranulated or degenerated cell, thereby showing a significant protective effect (Fig. 3). Antidiabetic activities of the leaf fractions during prolonged treatment The leaf fractions caused significant (p < 0.05–0.001) and sustained lowering of FBG levels of the diabetic rats throughout the duration of the study. The effect of the standard drug, glibenclamide was higher than that of the solvents fractions. On the last day of the study (day 14), the FBG levels of all the groups treated with the fractions were reduced significantly (p < 0.001) with EA fraction exerting the highest effect followed by n-hexane and dichloromethane fractions which exert comparable effects (Table 3). Identification of compounds Five compounds were isolated and identified using FT-IR, 1H and 13C NMR (Supplementary materials) and GC-MS along with comparison with literature data as 1-triacontanal, 1-triacontanol, 1-dotriacontanol, 1-triacontyl cerotate, and stigmasterol (Fig. 4). These compounds are reported for the first time from S. megaphylla. 1-Triacontanal (X16/17), white powder. IR (ATR, νmax, cm−1): 2921, 2854, 1726, 1479, 1461. 1H NMR (400 MHz, CDCl3, δ, ppm), δH 9.70 (1H, t, J = 1.83 Hz, H-1), 2.41 (2H, td, J = 7.34, 1.83 Hz, H-2), 1.63 (2H, m, H-3), 1.25 (16H, brs), 0.88 (3H, t, J = 7.0 Hz, H-30); 13C NMR (100 MHz, CDCl3, δ, ppm): 14.11 (C-30), 22.10 (C-29), 22.69 (C-28), 29.18 (C-27), 29.36 (C-26), 29.43 (C-25), 29.59 (C-4), 29.70 (C-(5–24)), 31.93 (C-3), 43.92 (C-2), 202.95 (C-1). GC–MS (Rt, 17.918 min). m/z (relative%): 418.4 [M - H2O]+ (10), 268.1 (5), 147.0 (5), 96 (50), 82 (60), 71.1 (95), 57.1 (100). GC–MS was consistent with data reported by Tulloch. (1987). The NMR data of 1-triacontanal were first reported. 1-Triacontanol and 1-Dotriacontanol (ratio 1:3 by GC–MS, N15), Effect of leaf fractions on glycosylated hemoglobin level There was significant (p < 0.001) reduction of Hb1Ac levels in fractions-treated groups with the hexane and dichloromethane groups exerting the highest reductions. The effect of the hexane group was more than that of the standard drug, glibenclamide. The hexane group was followed by DCM and EA groups (Fig. 1). Effect of leaf fractions on insulin level The solvent fractions treatment significantly (p < 0.05–0.01) increased the serum insulin levels of the respective treated groups relative to control. These increases were highest in the glibenclamide Table 2 Antidiabetic effect of leaf fractions of Setaria megaphylla on BGL of alloxan- induced diabetic rats during acute study. Treatment Dose mg/kg Blood glucose level mg/dl in hours 0 hr 1 hr 2 hr 3 hr 5 hr Control normal saline Glibenclamide n-hexane fraction Dichloromethane fraction Ethyl acetate fraction Methanol fraction 10 mL/kg 10 200 200 200 200 342.0 344.0 334.6 314.3 301.0 319.6 243.0 ± 2.94 203.3 ± 75.76 198.6 ± 40.20 182.0 ± 58.34 187.6 ± 59.53 233.33 ± 77.28 248.66 ± 57.56 192.6 ± 55.36 234.6 ± 57.81 154.33 ± 42.55 170.33 ± 57.62 214.33 ± 92.96 312.6 ± 171.3 ± 273.0 ± 156.6 ± 188.0 ± 230.6 ± ± 47.60 ± 87.55 ± 58.85 ± 77.62 ± 72.38 ± 87.18 243.0 ± 49.56 272.6 ± 87.41 242.0 ± 42.93 202.33±68.00 217.3 ± 70.33 276.0 ± 77.39 Data are expressed as MEAN ± SEM, Significant at ap < 0.05, bp < 0.01, cp < 0.001, when compared to control. (n = 6). 4 7 hr 65.59 64.86 71.93 64.87a 76.70 84.76 326.22 ± 45.28 184.3 ± 61.18 c 275.5 ± 44.23c 113.33 ± 13.77c 186.2 ± 45.23c 226.7 ± 65.33c J.E. Okokon et al. Phytomedicine Plus 2 (2022) 100182 Table 3 Antidiabetic effect of solvent leaf fractions of Setaria megaphylla on blood glucose level of alloxan- induced diabetic rats during prolonged study. Treatment Dose mg/kg Blood glucose level mg/dl in hours 0 hr 24 hr 3rd day 5th day 7th day 10th day 14th day Control normal saline Glibenclamide n-hexane fraction Dichloromethane fraction Ethyl acetate fraction Methanol fraction 10 mg/ml 10 200 200 200 200 342.0 ± 344.0 ± 334.6 ± 314.3 ± 301.0 ± 319.6 ± 312.6 159.3 148.5 123.0 134.0 143.0 206.33±27.84 85.6 ± 3.52b 133.3 ± 4.91c 118.0 ± 4.04c 123.6 ± 2.60c 190.3 ± 4.57 196.66±34.78 76.0 ± 6.92c 73.0 ± 2.30c 94.66 ± 6.00c 90.33 ± 2.02c 83.66±7.42 176.3 ± 10.13 74.0 ± 6.65c 79.66 ± 7.21c 80.00 ± 4.93c 92.33 ± 21.75c 77.0 ± 10.97c 145.3 ± 22.78 61.3 ± 6.36c 78.0 ± 8.08c 77.66 ± 4.33c 90.66 ± 2.99c 70.66 ± 8.19c 47.60 87.55 58.85 77.62 72.38 87.18 320.0 ± 325.6 ± 346.0 ± 184.3 ± 248.3 ± 354.3 ± 52.15 52.44 86.52 40.53 90.03 74.46 ± 65.59 ± 5.34 c ± 9.20c ± 5.94c ± 4.00c ± 9.57 Data is expressed as MEAN ± SEM, Significant at ap < 0.05, bp < 0.01, cp < 0.001, when compared to control. (n = 6). Fig. 1. Effect of solvent leaf fractions of Setaria megaphylla on glycosylated hemoglobin concentration of alloxan-induced diabetic rats. Data is expressed as MEAN ± SEM. Significant at *p < 0.05, **p < 0.01, ***p < 0.001, when compared to control. (n = 6). Stigmasterol ((24S)−5,22-Stigmastadien-3β-ol, PJ3); white powder. FT-IR (ATR, νmax, cm−1): 2918, 2851, 1732, 1479, 1290. C29H48O, MW= 412.37. 1H NMR (400 MHz, CDCl3) δ: 0.69 (3H, d, J = 7.3 Hz, CH3–21), 0.81 (3H, t, J = 7.0, CH3–29), 0.90 (3H, d, J = 6.4 Hz, CH3–26), 1.01 (3H, d, J = 7.5 Hz, CH3–27), 1.25 (3H, s, CH3–18), 3.53 (1H, m, H-3), 5.02 (dd, 1H, J = 8.6, 15.0 Hz, H-22), 5.16 (1H, dd, J = 6.6, 15.2 Hz, H-23) and 5.34 (1H, d, J = 4.9 Hz, H-6). 13C NMR (100 MHz, CDCl3) δ: 140.77 (C-5), 138.32 (C-22), 129.30 (C-23), 121.72 (C6), 71.84 (C-3), 56.89, 55.99, 50.19, 42.33, 40.49, 39.70, 37.28, 36.53, 31.92, 31.69,29.71, 28.92, 25.41, 24.38, 21.22 (C-21), 21.09 (C27), 19.41 (C-19), 18.99 (C-26), 12.25 (C-29), 12.06 (C-18). GC–MS (Rt, 17.813 min), m/z (%): 412.2 [M]+(25), 351.3 (5), 300.1 ((8), 255.2 (20), 213.1 (8), 145.0 (25), 105.0 (40), 55. 0 (100). NMR data are consistent with those reported (Chen et al., 2021). white powder, FT-IR (ATR, νmax, cm−1): 3275, 2918, 2854, 1467, 1064. H NMR (400 MHz, CDCl3+CH3OD (9:1)), δH 0.80 (t, J = 7.0 Hz, 3H), 1.22 (brs, 54–58 H), 1.50 (2H, m), 3.60 (t, J = 5.0 Hz, 2H); 13C NMR (100 MHz, CDCl3+CH3OD (9:1)), δ: 62.62 (C-1), 32.56 (C-2), 31.86 (C3), 29.62 (C-4), 29.56 (20C and 22C, C-5–25 and 5–27), 29.40, 29.28, 25.70, 22.61, 13.98. GC–MS, TMSi-Triacontanol: m/z (Relative%): 595.5 [M-15]+ (100), 75 (35), 57.0 (40), 43.0 (35). TMSiDotriacontanol: m/z (relative %): 523.6 [M-15]+ (100), 75 (35), 57.0 (40), 43.0 (35). These NMR data are in agreement with those reported for 1-triacontanol (Mahmuod et al., 2021), and 1-dotriacontanol (Parmer et al., 1998). 1-Triacontyl cerotate (X1). White solid, IR (ATR, νmax, cm−1): 2925, 2853, 1743, 1471, 1175; 1H NMR (400 MHz, CDCl3) δ: 4.05 (t, J = 6.8 Hz, 2H, -CH2O), 2.30 (t, J = 7.0 Hz, 2H, -CH2CO), 1.61 (quin, J = 7.0 Hz, 4H, -CH2CH2O and –CH2CH2CO), 1.33–1.24 (m, 98 H, 49 x CH2), 0.88 (t, J = 7.0 Hz, 6H, 2 x CH3); 13C NMR (100 MHz, CDCl3) δ 14.12, 22.70, 25.06, 25.96, 28.68, 29.18, 29.27, 29.37, 29.49, 29.54, 29.62, 29.67, 29.71, 31.94, 34.44 (CH2CO), 64.41 (O–CH2), 174.62 (COO). FT-IR and NMR data are consistent with reported data (Snehunsu et al., al.,2015). 1 Discussion Diabetes mellitus, a chronic metabolic disorder, is reportedly associated with increased generation of free radicals especially reactive 5 J.E. Okokon et al. Phytomedicine Plus 2 (2022) 100182 Fig. 2. Effect of solvent leaf fractions of Setaria megaphylla on insulin levels of alloxan-induced diabetic rats. Data is expressed as MEAN ± SEM. Significant at *p < 0.05, **p < 0.01, ***p < 0.001, when compared to control. (n = 6). Table 4 Effect of solvent leaf fractions of Setaria megaphylla on Oral glucose tolerance of diabetic rats. Treatment Control normal saline Glibenclamide n-hexane fraction Dichloromethane fraction Ethyl acetate fraction methanol fraction Dose mg/kg Blood glucose level (mg/dL) in min 0 min 30 min 60 min 120 min 180 min – 10 200 200 200 200 154.18 ± 44.68 170.28 ± 15.48 176.0 ± 51.22 151.7 ± 2.16 174.74 ± 48.78 183.6 ± 44.10 254.54 ± 33.68(65.09) 97.2 ± 11.80(42.91) 91.8 ± 13.60(47.84) 151.2 ± 8.46(0.32) 137.88 ± 15.22(21.09) 117.54 ± 11.70(35.98) 215.94 ± 39.36(40.05) 67.14 ± 9.00(60.57) 97.2 ± 12.80(44.77) 99.0 ± 9.36(34.73) 122.9 ± 9.90(29.66) 86.94 ± 19.0 (52.64) 192.8 ± 12.96(25.04) 69.48 ± 11.52(59.19) 87.48 ± 14.16(50.29) 85.18 ± 5.04(43.84) 115.2 ± 10.08(34.07) 90.0 ± 8.10(50.98) 293.9 ± 33.24(90.62) 135.0 ± 21.42(20.71) 180.8 ± 55.76(2.72) 283.14 ± 24.48(86.61) 139.68 ± 24.48(20.06) 185.4 ± 44.46(0.98) Data is expressed as MEAN ± SEM, Significant at ap < 0.05, bp < 0.01, cp < 0.001, when compared to control. (n = 6). Values in parenthesis represent% inhibition of elevation of blood glucose calculated relative to 0 min. Table 5 Effect of solvent leaf fractions of Setaria megaphylla on lipid profile of alloxan-induced diabetic rats. Treatment Dose mg/kg Total cholesterol (mMol/L) Triglyceride (mMol/L) HDL-C (mMol/L) LDL-C (mMol/L) VLDL (mMol/L) Control Glibenclamide n-hexane fraction Dichloromethane fraction Ethyl acetate fraction Methanol fraction 10 mL/kg 10 200 200 200 200 2.70 ± 0.05 2.20 ± 0.11 2.50 ± 0.12 2.70 ± 0.05 2.90 ± 0.05 2.66 ± 0.08 1.47 ± 0.08 1.02 ± 0.11a 1.41 ± 0.03 1.45 ± 0.05 1.58 ± 0.10c 1.50 ± 0.09 1.81 ± 0.08 1.39 ± 0.12 1.70 ± 0.25 1.87 ± 0.06 2.17 ± 0.01 1.95 ± 0.24 1.55 ± 1.27 ± 1.46 ± 1.49 ± 1.47 ± 1.38 ± 0.67 ± 0.04 0.46 ± 0.04a 0.63 ± 0.01 0.65 ± 0.02 0.71 ± 0.04 0.67 ± 0.04 0.09 0.03 0.08 0.11 0.03 0.15 Data is expressed as MEAN ± SEM, Significant at ap < 0.05, bp < 0.01, cp < 0.001, when compared to control. (n = 6). oxygen species (ROS) (Okutana et al., 2005; Papachristoforou et al., 2020). Alloxan, which was used to induce diabetes in this study is reported to be biotransformed to dialuric acid with accompanying generation of free radicals (Mathews and Leiter, 1999) and subsequently, partial destruction of pancreatic β-cells of islet of Langerhans (Abdel-Barry et al., 1997). This reduces insulin level resulting in type 2 diabetes mellitus (Ighodaro et al., 2017), with some remnant pancreatic β-cells having insulin producing potentials as observed in a study with hypoglycemic agents like sulphonylureas in alloxan-induced diabetic rats (Subramoniam et al., 1996). The insulin deficiency resultantly stimulates lipolysis in adipose tissues and gives rise to hyperlipidemia (Ahmad et al., 2014). In this study, S. megaphylla leaf fractions demonstrated sustained significant antidiabetic activities during acute and prolonged studies with dichloromethane, ethyl acetate and n-hexane fractions exerting prominent activities. The FBG levels of the treated diabetic rats were significantly reduced when compared to those of untreated diabetic rats (control). The antidiabetic results observed in this study corroborate those of previous reports (Okokon and Antia, 2007; Okokon et al., 2007a). Thus, confirming and validating the antidiabetic potentials of this plant in ethnomedicine. Diabetes mellitus causes body glucose regulatory processes to be compromised leading to chronic hyperglycemia (Champe et al., 2005). The observed reduced FBG of diabetic rats following treatment with the leaf fractions suggests blood glucose lowering potentials probably through insulin secretion stimulation as was observed in this study. The solvents fractions were observed to cause elevation of serum insulin levels of the diabetic rats with n-hexane fraction producing the highest effect followed by DCM fraction. These fractions were observed to offer considerable protection to the pancreas of the diabetic rats and may account for the high insulin levels of the groups. This result suggests insulin secretion stimulatory potential as well as recovery activities of injured β-cells in alloxan-induced diabetic rats. These effects may have resulted from the antioxidative stress activities of the fractions as reported earlier by Okokon et al., al.(2013), and 1-triacontyl cerotate (Snehunsu et al., 2015), as well as pancreatic cell stimulatory effect of stigmasterol which is reported to increase serum insulin levels in diabetic rats (Nualkaew et al., 2015), probably through regeneration of 6 J.E. Okokon et al. Phytomedicine Plus 2 (2022) 100182 Fig. 3. Histological sections of pancreas of alloxan-induced diabetic rats treated with normal saline (control) 10 mL/kg(1), glibenclamide 10 mg/kg bw (2), n-hexane fraction 200 mg/kg bw (3), dichloromethane fraction 200 mg/kg bw (4), ethyl acetate fraction 200 mg/kg bw(5), methanol fraction 200 mg/kg bw(6) at magnification B(x400), stained with H&E method. Keys: Pancreastic acini (PA), Pancreatic Islets (PI), Pancreastic degeneration (PD). pancreatic β-cells. The presence of these compounds and earlier reported compounds (Okokon et al., 2007b, 2013, 2017; Udobang et al., 2017) in the fractions must have offered protection to the pancreas and β-cells against the effect of alloxan as observed in the histopathology which could have been responsible for the high insulin level. Thus, this explains the observed pancreas protection potential. The solvent fractions were found to considerably reduced FBG levels of treated diabetic rats during oral glucose tolerance test (OGTT). Oral glucose tolerance test measures the body’s ability to use glucose, the main source of energy (Gold, 1970). Lowering of BGL effect was observed post administration of the solvent fractions with methanol and hexane fractions exerting the highest inhibition of BGL increases. This may be due to the activities of phytochemicals present in the solvent fraction as earlier reported (Okokon et al., 2006, 2013). Blood glucose level after glucose loading is dependent on insulin secretion, glucose utilization, intestinal absorption and intestinal motility (Peungvicha et al., 1996), alternative mechanism by the respective fractions could have involved insulin secretion stimulation, glucose utilization and inhibition of intestinal absorption and intestinal motility. Naveen and Baskaran (2018) had reported that the possible modes of natural products action in glycemic control include stimulation of glucose uptake by tissues, inhibition of intestinal glucose absorption, increased glycogen synthesis, inhibition of dipeptidyl peptidase-IV (DPP-IV), among others. In this study, the fractions were observed to improved glucose tolerance which could be attributable to potentiation of the insulin effect on the plasma glucose through increased pancreatic insulin secretion from existing β-cells or its release from bound insulin as was observed in this study. This could have also resulted from the inhibitory potentials of the leaf fractions on α-amylase and α-glucosidase activities as reported earlier by Okokon et al. (2021), thereby inhibiting glucose absorption from the intestines, in addition to insulin secretion stimulation in response to glucose load and increased peripheral utilization of glucose which are the probable mechanisms of antidiabetic action (Andrikopoulos et al., 2008; Regginato et al., 2021). In uncontrolled diabetes, high level of glycosylation of some proteins including hemoglobin occurs, contributing to long-term complications of diabetes (Latha and Pari, 2004). Hb1Ac level usually indicates glycemic control (Daisy and Rajathi, 2009) and significant lowering of Hb1Ac levels of diabetic rats by the leaf fractions indicates controlled blood glucose level especially in hexane and dichloromethane fractions-treated groups. However, high Hb1Ac levels with corresponding increased plasma glucose levels were observed in untreated diabetic rats. These results corroborate the findings of previous work (Adeneye and Adeyemi, 2009) and confirm antidiabetic activity of leaf 7 J.E. Okokon et al. Phytomedicine Plus 2 (2022) 100182 Fig. 4. Chemical structures of isolated and identified compounds from S. megaphylla. fractions of S. megaphylla via insulin secretion stimulation, resulting in lowering of BGL and HbA1c levels. Phytochemical screening of the leaf extract revealed the presence of saponins, tannins, flavonoids, alkaloids and terpenes (Okokon et al., 2007). Also, previous GC-MS analysis of the n-hexane fraction of the leaf revealed the presence of (Z,Z,Z)-8,11,14-eicosatrienoic acid, phthalic acid, diisooctyl ester, vitamin E, ᵞ-elemene, urs-12-ene, bicyclogermacrene, α-muurolene, germacrene-A, and guaiol among others (Okokon et al., 2013), while GC-MS analysis of ethyl acetate leaf fraction revealed the presence of (E)-β-ocimene, p-metha-1(7),8-diene, (3a)-D:A-friedooleannan-3-ol,stigmastone-3,6‑dione(5a), bicyclo[2.2.1] heptan-2-ol, 4, 7,7-trimethyl, p-cymene (Okokon et al., 2017). Borneol, astaxanthin, α-terpineol, terpinen-4-ol, β-cis-bergamotene, citronellol, germacrene D among others were reported in dichloromethane fraction and 3-methyl-undecane, hexadecanoic acid, 1,2-benzene dicarboxylic acid, isodecyl octyl ester, carvacrol, linanlool, camphor, borneol, menthofuran, menthone, α-terpineol, and α-eudesmol were found in n-butanol fraction (Udobang et al., 2017). In this study, additional compounds including 1-triacontanal, 1-triacontanol, 1-dotriacontanol, 1-triacontyl cerotate, and stigmasterol were isolated and identified. Some of these phytochemicals in these fractions may in part be responsible for the observed activities of these fractions either singly or in synergy. Several plants rich in terpenes, such as sesquiterpenes and monoterpenes as found in this leaf fractions have been shown to have an effect on blood glucose levels (Alam et al., 2018; Belhadj et al., 2018; Ding et al., 2018) by stimulating insulin secretion and glucose uptake, improving glycogen synthesis and inhibiting α-glucosidase (Zhao et al., 2012; Naveen and Baskaran, 2018). According to Brahmachari (2011), flavonoids also exhibit glycaemic control and also could regulate the rate-determining enzymes vital for metabolic pathways of carbohydrate. Similarly, several studies have shown that ß-sitosterol, stigmasterol, betulin, ergost-8(14)-en-3-ol, n-hexadecanoic acid, and palmitic acid exert hypoglycemic effects by reducing the absorptions of cholesterol from the gut ((Rajasekaran et al., 2006); Gosh et al., 2014) and their presence in these fractions could likely be responsible for the observed antidiabetic activity Hyperglycemia and associated diabetic dyslipidemia lead to several comorbidities including macro-and microvascular damage (Naveen and Baskaran, 2018). Elevated blood glucose levels equally gives rise to low levels of HDL cholesterol and escalation of LDL cholesterol, thus increasing risk of coronary heart diseases (Sudasinghe and Peiris, 2018). Additionally, stimulation of catabolic activity and increased mobilization of free fatty acids from peripheral deposits by diabetic condition as well as lipolytic action of hormones and inhibition of hormone sensitive lipase production by insulin result in elevated lipid levels, thereby, increasing the risk of myocardial dysfunction (Rojop et al., 2012). Therefore, both diabetes and lipid levels must be managed properly to achieve a satisfactory treatment outcome. The leaf fractions were found to have insignificant lowering effect on total cholesterol, TG, VLDL and LDL levels of the diabetic rats and considerably increased the level of HDL –cholesterol in the diabetic rats. The administration of the fractions may have partly increased glucose utiization, thereby suppressing fats mobilization and lipolysis involving plasma lipoprotein lipase, though not significantly (BenKhedher et al., 2018). Plants sterol such as stigmasterol present in the dichloromethane fraction exerts antihyperlipidemic activity through different mechanisms to inhibit the absorption of cholesterol from the gut (Batta et al., 2006). The antihyperlipidemic effects observed in this study may have resulted from the activities of this and other phytochemical compounds. Body weight loss which is due to increased muscle wasting and loss of tissue proteins is common in diabetic rats (Shirwaikar et al., 2005). Treatment with the leaf fractions especially ethyl acetate and hexane fractions, remedied this situation perhaps due to the chemical constituents of these fractions which have the ability to reduce hyperglycaemia by increased glucose metabolism, inhibition of α-amylase and α-glucosidase enzymes, and protein synthesis stimulation thereby suppressing muscle wasting through reversal of gluconeogenesis (Mestry et al., 2017). Alloxan monohydrate has been reported to cause various injuries to the pancreas which results in partial destruction of pancreatic β-cells of islet of Langerhans (Abdel-Barry et al., 1997). The pancreas of untreated diabetic rats were observed to have areas of islets cell degeneration, degranulated islet, degranulated β- and α- cells as well as reduced islet cells among others. These pathologic features were not prominent in the 8 J.E. Okokon et al. Phytomedicine Plus 2 (2022) 100182 leaf fractions treated groups, suggesting regenerative effect of the extract. The phytoconstituents in these leaf fractions such as stigmasterol, hexadecanoic acid and 1-triacontanol cerotate among others, through their antioxidative effect, could have caused the regeneration of the pancreas and increased level of insulin in treated diabetic rats in this study resulting in hypoglycaemic activity. The protection maybe due to the free radical scavenging potentials of the leaf fractions as reported by Okokon et al.,(2013) and recently by Umana (2021) which revealed strong antioxidant potentials of the hexane and ethyl acetate fractions of the leaf extract in the various in vitro models. This activity inherent in the leaf extract/fractions could have counteracted the vast free radicals generated by alloxan and the diabetic condition thereby leading to the observed antidiabetic and pancreas protective effects in this study. However, this study could not determined the quantities of these identified and isolated compounds in the fractions, which is necessary for the standardization of the extract/fractions in order to be used effectively and safely nor established the antioxidant and antidiabetic activities of the isolated compounds. These were the limitations of this study which further study has been recommended to be carried out on the leaf extract. Pharmacognosy and Natural medicine, University of Uyo, Uyo, Nigeria. The authors are grateful to the management of University of Uyo for providing the enabling environment to carry out this work and Mr Nsikan Malachy of Pharmacology and Toxicology Department for providing technical assistance. We thank Dr. Sian Woodfine and Falko Drijfhout at Keele University for recording NMR and obitrap MS spectra, respectively. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phyplu.2021.100182. References Abdel-Barry, J.A., Abdel-Hassan, I.A., Al-Hakiem, M.H., 1997. Hypoglycemic and antihyperglycemic effects of trigonella foenum-graecum leaf in normal and alloxaninduced diabetic rats. J. Ethnopharmacol 58, 149–155. Adeneye, A.A., Adeyemi, O.O., 2009. Further evaluation of antihyperglycaemic activity of Hunteria umbellata (K. Schum) Hallier f. seed extract in experimental diabetes. J. Ethnopharmacol 126 (2), 238–243. 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The use of plants in the traditional management of diabetes in Nigeria: pharmacological and toxicological considerations. J. Ethnopharmacol 155 (2), 857–924. Festing, M.F., 2006. Design and statistical methods in studies using animal models of development. Inst. Laboratory Animal Res. J. 47, 5‑14. Finlay, J.W.A., Dillard, R.F., 2007. Appropriate calibration curve fitting in ligand binding assays. Am. Assoc. Pharm. Scientists Jo. 9 (2), E260–E267. Friedewald, W.T., Levy, R.I., Fredrickson, D.S., 1972. Estimation of the concentration of low density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin. Chem. 18 (6), 499–502. Conclusion The results of this study show that the leaf fractions of S. megaphylla possess antidiabetic, hypolipidemic and pancreas protective potentials, which may be partially attributed to the identified compounds from this plant. Ethics approval and consent to participate Permission and approval for animal studies were obtained from College of Health Sciences Animal Ethics committee, University of Uyo (UU/CHS/AE/21/024). Consent for publication All the authors read and approved the final content of this manuscript for publication. Availability of data and material All data generated in this project were included in this manuscript, supplementary materialsand stored in our computer hard drive and external storage drive which will be available upon request. Authors’ statements Jude E. Okokon designed and supervised the work, Lekara John and Klinton Iwara carried the animal studies, Koofreh Davies did the statistical analysis, Paul S. Thomas carried out the isolation and purification of the compounds, Wen-Wu Li did the GC–MS and NMR analysis and interpretation of the spectra and also edited the work. All data were generated in-house, and no paper mill was used. 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