Journal of Ethnopharmacology the Freeze-Dried Extracts of Rotheca Myricoides (Hochst.) Steane & Mabb Possess Hypoglycemic, H

Journal of Ethnopharmacology 244 (2019) 112077

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Journal of Ethnopharmacology

journal homepage: www.elsevier.com/locate/jethpharm

The freeze-dried extracts of Rotheca myricoides (Hochst.) Steane & Mabb T possess hypoglycemic, hypolipidemic and hypoinsulinemic on type 2 diabetes rat model Boniface Mwangi Chege*, Mwangi Peter Waweru, Bukachi Frederick, Nelly Murugi Nyaga

Department of Medical Physiology, School of Medicine, University of Nairobi, GPO 30197-00100, Kenya

ARTICLE INFO ABSTRACT

Keywords: Ethnopharmacological relevance: Rotheca myricoides (Hochst.) Steane & Mabb is a plant species used in traditional Type 2 diabetes medicine for the management of diabetes in the lower eastern part of Kenya (Kitui, Machakos and Makueni Antihyperglycaemic Counties, Kenya) that is mainly inhabited by the Kamba community. Antihyperinsulinemic Aim: This study investigated the antihyperglycaemic, antidyslipidemic and antihyperinsulinemic activity of the Streptozocin freeze-dried extracts of Rotheca myricoides (Hochst.) Steane & Mabb (RME) in an animal model of type 2 diabetes Network pharmacology mellitus. Methods: Type 2 diabetes was induced by dietary manipulation for 56 days via (high fat- high fructose diet) and intraperitoneal administration of streptozocin (30 mg/kg). Forty freshly-weaned Sprague Dawley rats were randomly assigned into the negative control (high fat/high fructose diet), low dose test (50mg/kg RME, high dose test (100mg/kg RME and positive control (Pioglitazone, 20mg/kg) groups. Fasting blood glucose and body weight were measured at weekly intervals. Oral glucose tolerance tests were performed on days 28 and 56. Lipid profile, hepatic triglycerides, fasting serum insulin levels and serum uric acid were determined onday56. Results: The RME possessed significant antihyperglycemic [FBG: 6.5 ± 0.11 mmol/l (negative control) vs. 4.62 ± 0.13 mmol/l (low dose test) vs. 5.25 ± 0.15 mmol/l in (high dose test) vs. 4.33 ± 0.09 mmol/l (positive control): p < 0.0001] and antihyperinsulinemic effects [1.84 ± 0.19 (negative control) vs. (0.69 ±0.13 (low dose test) vs. (0.83 ± 0.17 (high dose test) vs. (0.69 ± 0.10 (positive control): F (3, 36) = 0.6421: p < 0.0001. The extracts also possessed significant antidyslipidemic effects [LDL levels: 3.52 ± 0.19 mmol/l (negative control) vs. 0.33 ± 0.14 mmol/l (low dose test) vs. 0.34 ± 0.20 mmol/l (high dose test) vs. 0.33 ± 0.01 mmol/l (positive control): p < 0.0001].RME significantly lowered plasma uric acid levels, as well as hepatic triglycerides and hepatic weights. Network pharmacology analysis indicated that the observed pharmacological effects are mediated via the modulation of Peroxisome proliferator-activated gamma receptor. Conclusions: The freeze dried extracts of Rotheca myricoides possessed significant antihyperglycemic and anti- dyslidemic effects. In addition it lowered serum uric levels, as well as hepatic triglycerides and hepatic weight. These results appear to validate the traditional use of this plant species in the management of diabetes mellitus.

1. Introduction 2004). Approximately 80% of the population in the developing countries routinely utilize traditional herbal medicine often concurrently Type 2 diabetes (T2D) is a complex metabolic disorder character- with allopathic drugs (Wachtel-Galor and Benzie, 2011). It is therefore ized by alterations in lipid metabolism, insulin resistance and pan- imperative to evaluate the efficacy of these traditional remedies. creatic β-cell dysfunction(Podell et al., 2017).The incidence and pre- Rotheca myricoides (Hochst.) Steane & Mabb is a plant species be- valence of diabetes mellitus is rising rapidly(Guthrie and Guthrie, longing to the genus Rotheca the largest genus of the family

List of abbreviations: T2D, Type 2 diabetes; MSG, Monosodium glutamate; FBG, Fasting blood glucose; AUC’S, Area under the curve; HOMA-IR, Homeostasis model of assessment of Insulin Resistance; OD, Optical densities; GLUT, Glucose transporters; OGTT, Oral glucose tolerance test; HDL, High-density lipoprotein; LDL, Low density lipoprotein; RME, Rotheca myricoides (Hochst.) Steane & Mabb extract (RME) * Corresponding author E-mail addresses: [email protected] (B.M. Chege), [email protected] (M.P. Waweru), [email protected] (B. Frederick), [email protected] (N.M. Nyaga). https://doi.org/10.1016/j.jep.2019.112077 Received 28 March 2019; Received in revised form 6 July 2019; Accepted 8 July 2019 Available online 29 July 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved. B.M. Chege, et al. Journal of Ethnopharmacology 244 (2019) 112077

Verbenaceae (Bashwira and Hootele, 1988).It is used for management lyophilized (Free Zone 4.5, USA) to obtain the freeze dried extract of diabetes in the lower eastern part of Kenya (Kitui, Machakos and which was then weighed and placed in amber colored sample con- Makueni Counties, Kenya) that is mainly inhabited by the Kamba tainers and placed in a standard in a laboratory refrigerator. community. A leaf decoction is prepared by boiling and a cup (250 mls) taken daily(Keter and Mutiso, 2012). Other ethnobotanical uses of this species include epilepsy, arthritis, typhoid, cough, eye problems, ton- 2.2. Experimental animals sillitis, rheumatism, gonorrhea(Moshi et al., 2012), cancer (Esubalew et al., 2017), malaria (Mukungu et al., 2016), dysmenorrhea, sterility, Sixty-forty (64), freshly-weaned (four –week old) Sprague Dawley impotence, coughs, furunculosis, inflammation, and snakebites(Richard rats (thirty-two males and thirty-two females), weighing 60–100 g were et al., 2011). It is traditionally brewed and drank as a tea product in obtained from the Department of Zoology, University of Nairobi. They Asian countries to relieve swelling and pain (Herman, 2017). were grouped-housed in the animal within the department of medical This study investigated the antihyperglycaemic, antilipidemic and physiology under the following ambient conditions: room temperature antihyperinsulinemic effects of RME in a diet and low dose streptozocin of 23 ± 2 °C, relative humidity 30–50% and a 12-hr. light/day cycle. animal model of type 2 diabetes. The animals were habituated to both the experimenter and the en- In this study, we report that, the freeze-dried extract of RME not vironmental conditions for seven days prior to the start of the study. only possessed antihyperglycemic, antihyperinsulinemic and anti- This study was carried out in strict accordance with the re- dyslipidemic effects, but also lowered the serum uric levels, hepatic commendations set out in the Guide for care and Use of Laboratory triglycerides and hepatic weight. Animals of the National Institutes of Health (Guillen, 2012). The protocol was approved by the bio-safety, animal use and ethics committee 2. Material and methods Department of Veterinary physiology, University of Nairobi (permit number 161). In addition, the experimental design and procedures were 2.1. Plant material and extract preparation in compliance with the 4Rs (reduction, replacement, refinement and rehabilitation) principles of ethical animal experimental design Rotheca myricoides (Hochst.) Steane & Mabb plant was collected (Guillen, 2012). from its natural habitat in Machakos county, Kenya (Masii Division, Muthetheni Location GPS coordinates: 037 30' 20.27" E 01 29' 46.10" S), its identity verified at the University of Nairobi herbarium anda 2.3. Experimental procedures voucher specimen deposited therein (BMC, 2017/01). The whole plant was used in the study. The plant was air dried for a week and ground The experimental procedures were carried out in two stages. The into a fine powder using a standard laboratory mill. The powder was first stage involved the evaluation of the anti-hyperglycemic, anti-dys- then macerated in distilled water for twenty-five (25) minutes ina lipidemic and anti-hyperinsulinemic activities of the RME while the weight/volume ratio of 1:10 according to the traditional mode of pre- second stage involved experimental procedures aimed at determining paration. The resulting suspension was then filtered in succession using the possible mechanisms of action of the observed pharmacological cotton wool and Whatmans® filter paper. The resulting filtrate was then effects.

2 B.M. Chege, et al. Journal of Ethnopharmacology 244 (2019) 112077

2.3.1. Efficacy experiments grams of solid cooking fat (Frymate® vegetable cooking oil, Pwani oil Forty (40) rats were randomly assigned to the negative control products, Kenya) to 225 g of standard chow pellets (Unga Feeds (normal saline), low dose test (50mg/kg RME), high dose test (100mg/ Limited, Nairobi) which have the following energy content: carbohy- kg RME) and positive control (Pioglitazone 20mg/kg) groups (n = 10 drate:70%, Protein: 20%, Fat: 10%). Monosodium glutamate (MSG) per group). The respective treatments were administered daily by oral (0.8%) (Oshwal Flavours Limited, Nairobi, Kenya) was added to the gavage. high fat diet improve its palatability. The mixture was gently heated Diabetes was induced in all the experimental animals using a over low heat for 20 min with constant mixing. The chow was drained combined dietary and chemical approach. The experimental animals of excess fat, cooled and weighed to ensure achievement of 15% fat were fed on a high fat and high fructose (20% w/v fructose solution) content. It was then stored in air tight containers for later use. diet ad libitum for six (6) weeks after which a low dose of streptozocin The following biochemical parameters were measured in this study. (30 mg/kg) was administered intraperitoneally to all rats at day 42 of the experimental period. Administration of the high fat and high fruc- 2.3.1.1. Fasting blood glucose determination. Fasting blood glucose tose diet continued for a further two weeks after the induction of the (FBG) levels were determined weekly basis using a glucometer diabetes. (StatStrip Xpress® Nova Biomedical, Waltham MA, USA) with the The high fat diet was prepared by the addition of Forty-five (45) respective blood samples being obtained using lateral tail vein blood

3 B.M. Chege, et al. Journal of Ethnopharmacology 244 (2019) 112077 sampling (Topical Lidocaine having been applied ten (10) minutes before the procedure to ease pain/stress associated with the test) (Lee and Goosens, 2015).

2.3.1.2. Oral glucose tolerance tests. Oral glucose tolerance tests were performed on days 28 and 56 of the experimental period according to the protocol described by Barret (Barrett, 2002). Briefly, the rats were fasted for 6–8 h prior to the test. The baseline blood glucose was then determined using the procedure described previously after which a loading dose of glucose (2g/kg) was administered to each rat by oral gavage. Blood glucose levels were then determined at 30, 60, 90 and 120 min after administration of the loading dose of glucose. The Areas under the curve (AUCs) were then calculated.

2.3.1.3. Fasting serum insulin levels. The serum insulin level was determined using the enzyme-linked immunosorbent assay (ELISA) method using a rat insulin kit (Hangzhou Sunlong Biotech co, Ltd, China). Fig. 1. Line graph showing mean fasting blood glucose levels (mmol/l) at The homeostasis model of assessment of Insulin Resistance (HOMA- weekly intervals during the experimental period. (NC-Negative control), (LDT- IR) was calculated using the following equation: Low dose test (50 mg), (HDT-High dose test (100 mg) and (PC-Positive control). Expressed as mean ± SEM. HOMA-IR= Insulin (in mU/L) *glucose (mg/dl)/405

O.D saponified corn oil standard – O.D unsaponified corn oil standard 2.3.1.4. Serum lipid profile. The experimental animals were euthanized after an overnight fast by administration of 20% Phenobarbital (1mg/ And A = volume of aliquot of chloroform extract in ml (1 ml was kg) intraperitoneally on day 56. Blood was then collected by cardiac used in the present study). puncture, allowed to clot then centrifuged at 1500 revolutions per Then triglyceride contents in milligram per gram of tissue minute for ten (10) minutes and the serum obtained transferred into 200 R vacutainers. The sera were then transported to the Department of ×R ×0.05 = 10 Clinical Chemistry, University of Nairobi, where serum uric acid, serum A A triglyceride, total cholesterol, low density lipoprotein and high-density lipoprotein levels were determined. 2.3.2. Mechanism of action experiment Twenty-four rats (24) were randomly assigned to the negative 2.3.1.5. Hepatic triglyceride and hepatic index. After euthanization as control (normal saline), positive control (50mg/kg RME), test group I described above, a midline incision was made on the ventral surface on (50 mg/kg RME plus 23mg/kg indinavir sulfate) 23mg/kg and test the body of each rat to expose the abdominal cavity and the liver group II (Indinavir sulfate 23mg/kg). The inclusion of Indinavir sulfate excised. The liver samples were weighed using a weighing scale (WANT was because it is a known antagonist of GLUT-4 (Hruz et al., 2002). Balance Instrument co.Ltd,China) then deep frozen at −90ᴼ C and the hepatic triglycerides determined using the procedure by Bulter and Mailing (Butler et al., 1961). Briefly, two (2) g portions of the respective 2.4. Network pharmacology analysis of Rotheca myricoides livers were homogenized in eight (8) mls of phosphate Buffer. One (1) ml of the resulting homogenate was then added to four (4) g of Composite compounds of RME were obtained from the LC-MS activated charcoal which had been pre-moistened with two (2) mls of analysis of the RME as well as from the Reaxys® database. A total of 33 chloroform. The resulting paste was then topped up with eighteen (18) compounds were collected and their PubChem ID’s obtained. mls of chloroform and gently shaken for ten (10) minutes after which it The Bioinformatics Analysis Tool for Molecular mechanism of was filtered. The resulting filtrate was then divided into 3 test tubes. Traditional Chinese Medicine (BATMAN-TCM) database was employed One (1) ml of standard oil solution (1%) was pipetted into 3 additional in the target prediction of the composite compounds of RME (Liu et al., test tubes. All the test tubes were placed in a water bath at 80ᴼC and 2016). The BATMAN-TCM tool applies 6 basal scores for the determi- excess chloroform evaporated. 0.5 ml alcoholic potassium hydroxide nation of drug-drug similarity that are based on chemical structure was added to the first & second tube and 0.5 ml of 95% alcohol was (including FP2 fingerprint-based and functional group-based similarity added to the third tube containing the filtrate and the test tube scores), side effects, the Anatomical, Therapeutic and Chemical (ATC) containing the standard corn oil solution. The test tubes were classification system, drug-induced gene expression, and the text maintained in water at 60ᴼC for twenty (20) minutes after which mining score of chemical-chemical associations, and 3 scores to mea- 0.5 ml of 0.2N sulphuric acid were added to each tube and the resulting sure protein-protein similarity respectively based on protein sequence, mixtures heated in a water bath (100ᴼC) for twenty (20) minutes. They closeness in a protein interaction network and Gene Ontology (GO) were then cooled after which 0.1 ml sodium metaperiodate followed by functional annotation. The default parameters were set for the putative 0.1 ml sodium arsenide were added. Five (5) ml of chromotropic acid targets of the composite compounds of RME. were then added to each test tube after ten (10) minutes. The tubes were placed in a water bath (100ᴼC) for half an hour. The optical 2.4.1. Network construction and analysis densities at 540 nm were then determined using spectrophotometer. The RME composite compound-putative target-known therapeutic The optical densities obtained were then used to calculate the hepatic target network was constructed to find the key targets. The target- triglyceride content using the following formula: pathway network was then established to find the relationship between the pathways and the key targets. Cytoscape(“Cytoscape 3.7.1 User Let R = optical density (O.D) saponified unknown – O.D unsaponified Manual — Cytoscape User Manual 3.7.1 documentation,” n.d.) was unknown utilized to directly visualize the networks (Shannon et al., 2003).

4 B.M. Chege, et al. Journal of Ethnopharmacology 244 (2019) 112077

Fig. 2. Fig. 2A and B: Mean area under the curve (mmol/l) during the oral glucose tolerance tests. (NC-Negative control), (LDT-Low dose test (50 mg), (HDT-High dose test (100 mg) and (PC-Positive control).Results are expressed Fig. 4. Fig. 4A and B: Mean hepatic triglycerides content (mg/g) and liver as mean ± SEM. ***- p < 0.001, ****- p < 0.0001. weights (gm) of the experimental groups. (NC-Negative control), (LDT-Low dose test (50 mg), (HDT-High dose test (100 mg) and (PC-Positive control). Results are expressed as mean ± SEM. (****- p < 0.0001).

2.5. Statistical analysis

All the experimental data were expressed as mean ± S.E.M. and statistical analysis was carried using one-way ANOVA followed by Tukey’s test in cases of significance (which was set at p ≤ 0.05) using GraphPad Prism® version 7.0.

3. Results

3.1. Fasting blood glucose

There were no significant differences in the fasting blood glucose between the four experimental groups at the start of the experiment (week 0): [4.42 ± 0.10 mmol/l (negative control) vs. 4.41 ± 0.09 mmol/l (low dose test) vs. 4.43 ± 0.13 mmol/l (high dose test) vs. 4.37 ± 0.10 mmol/l (positive control): F (3,36) = 0.5918: p = 0.6244]. There were no significant differences in the fasting blood glucose between the four experimental groups after week one: [4.25 ± 0.04 mmol/l (negative control) vs. 4.08 ± 0.06 mmol/l (low dose test) vs. 4.15 ± 0.06 mmol/l (high dose test) vs. 4.10 ± 0.04 mmol/l (positive control): F (3, 36) = 0.09901: p = 0.9601]. There were significant differences in fasting blood glucose between the four experimental groups after week two: [4.64 ± 0.80 mmol/l (negative control) vs. 4.14 ± 1.10 mmol/l (low dose test) vs. Fig. 3. Fig. 3A and B: Mean blood glucose response (mmol/L) to an oral glucose 4.36 ± 0.11 mmol/l (high dose test) vs. 4.02 ± 0.15 mmol/l (positive bolus 2 g/kg over a 2-h period. (NC-Negative control), (LDT-Low dose test control): F (3,36) = 0.6251: p = 0.0029]. Post-hoc statistical analysis (50 mg), (HDT-High dose test (100 mg) and (PC-Positive control). using Tukey’s multiple comparisons test revealed significant differences between the negative control and low dose test (50 mg) (p = 0.0198),

5 B.M. Chege, et al. Journal of Ethnopharmacology 244 (2019) 112077 the negative control and positive control (p = 0.0028). Post-hoc statistical analysis using Tukey’s multiple comparisons test There were significant differences in fasting blood glucose between revealed significant differences were between the negative control and the four experimental groups after week three: [5.06 ± 0.08 mmol/l low dose test (50 mg) (p < 0.0001), negative control and high dose (negative control) vs. 4.17 ± 0.10 mmol/l (low dose test) vs. test (100 mg) (p = 0.0002) and negative control and positive control 4.50 ± 0.12 mmol/l (high dose test) vs. 4.12 ± 0.12 mmol/l (positive (p < 0.0001). control): F (3,36) = 0.3253: p < 0.0001]. Post -hoc statistical analysis There were significant differences in AUC between the groups on using Tukey’s multiple comparisons test revealed significant differences week 8: [1626 ± 7.06 mmol/L·min (negative control) vs. between the negative control and low dose test (50 mg) (p < 0.0001), 750.5 ± 24.11 mmol/L·min (low dose test) vs. 861.3 ± 15.07 mmol/ the negative control and high dose test (100 mg) (p = 0.0034) and the l.min (high dose test) vs. 801.8 ± 12.89 mmol/min (positive control): negative control and positive control (p < 0.0001). F (3,36) = 3.395: p = 0.0281]. Post-hoc statistical analysis using There were significant differences in fasting blood glucose between Tukey’s multiple comparisons test showed significant differences were the four experimental groups after week four: [6.14 ± 0.18 mmol/l between the negative control and low dose test (50 mg) (p < 0.0001), (negative control) vs. 4.65 ± 0.14 mmol/l (low dose test) vs. negative control and high dose test (100 mg) (p < 0.0001 and negative 5.17 ± 0.15 mmol/l (high dose test) vs. 4.66 ± 0.11 mmol/l (positive control and positive control (p < 0.0001). control). F (3, 37) = 0.3531: p < 0.0001]. Post -hoc statistical analysis The graphical presentation of the OGTTs on week 4 and 8 shown in using Tukey’s multiple comparisons test revealed significant differences Fig. 2A, B, 3A and 3B. between the negative control and low dose test (50 mg) (p = 0.0026), the negative control and high dose test (100 mg) (p = 0.0142) and the 3.3. Effect on liver weight negative control and positive control (p = 0.0012). There were significant differences in fasting blood glucose between There were significant differences in the mean liver weight between the four experimental groups after week five: [6.5 ± 0.11 mmol/l the experimental groups: [8.55 ± 0.21 g (negative control) vs. (negative control) vs. 4.62 ± 0.13 mmol/l (low dose test) vs. (3.48 ± 0.14 g (low dose test) vs. (3.64 ± 0.10 g (high dose test) vs. 5.25 ± 0.15 mmol/l in (high dose test) vs. 4.33 ± 0.09 mmol/l (po- (3.31 ± 0.09 g (positive control): F (3, 36) = 4.28: p < 0.0001]. Post sitive control). (3, 34) = 0.0.2618: p < 0.0001]. Post -hoc statistical -hoc statistical analysis using Tukey’s multiple comparisons test re- analysis using Tukey’s multiple comparisons test revealed significant vealed significant differences between the negative control andlow differences between the negative control and low dose test (50mg) dose test (50 mg) (p < 0.0001), the negative control and high dose test (p < 0.0001), the negative control and high dose test (100 mg) (100 mg) (p < 0.0001) and the negative control and positive control (p = 0.0008), the negative control and positive control (p < 0.0001) (p < 0.0001) groups. and the high dose test (100 mg) and positive control (p = 0.0012). There were significant differences in fasting blood glucose between 3.4. Effect on hepatic triglyceride the four experimental groups after week six: [7.19 ± 0.1 mmol/l (negative control) vs. 4.02 ± 0.10 mmol/l (low dose test) vs. There were significant differences in the levels of hepatic trigly- 4.43 ± 0.16 mmol/l (high dose test) vs. 4.11 ± 0.13 mmol/l (positive ceride between the experimental groups: [5.09 ± 0.15 mg/g (negative control). F (3, 36) = 0.8722: p < 0.0001]. Post -hoc statistical analysis control) vs. 2.14 ± 0.10 mg/g (low dose test) vs. 2.3 ± 0.12 mg/g using Tukey’s multiple comparisons test revealed significant differences (high dose test) vs. 1.94 ± 0.20 mg/g (positive control): F (3, between the negative control and low dose test (50 mg) (p < 0.0001), 35) = 0.7839: p < 0.0001]. Post -hoc statistical analysis using Tukey’s the negative control and high dose test (100 mg) (p < 0.0001) and the multiple comparisons test revealed significant differences between the negative control and positive control (p < 0.0001). negative control and low dose test (50 mg) (p < 0.0001), the negative There were significant differences in fasting blood glucose between control and high dose test (100 mg) (p < 0.0001) and the negative the four experimental groups after week seven: [7.8 ± 0.24 mmol/l control and positive control (p < 0.0001) groups. (negative control) vs. 3.78 ± 0.09 mmol/l (low dose test) vs. The graphical representation of these results is shown in Fig. 4A and 3.92 ± 0.12 mmol/l (high dose test) vs. 3.86 ± 0.13 mmol/l (positive B. control). F (3, 36) = 0.8722: p < 0.0001]. Post -hoc statistical analysis using Tukey’s multiple comparisons test revealed significant differences 3.5. Effect on lipid profile between the negative control and low dose test (50 mg) (p < 0.0001), the negative control and high dose test (100 mg) (p < 0.0001), the 3.5.1. Total cholesterol negative control and positive control (p < 0.0001). There were significant differences in the levels of total plasma There were significant differences in fasting blood glucose between cholesterol between the experimental groups [5.64 ± 0.11 mmol/l the four experimental groups after week eight: [8.06 ± 0.15 mmol/l (negative control) vs. 1.62 ± 0.14 mmol/l (low dose test) vs. (negative control) vs. 3.54 ± 0.09 mmol/l (low dose test) vs. 1.68 ± 0.11 mmol/l (high dose test) vs. 1.66 ± 0.99 mmol/l (positive 3.73 ± 0.33 (high dose test) vs. 3.68 ± 0.13 mmol/l (positive con- control): F (3, 36) = 0.3513: p < 0.0001]. Post -hoc statistical analysis trol). F (3, 37) = 0.8802: p < 0.0001].Post -hoc statistical analysis using Tukey’s multiple comparisons test revealed significant differences using Tukey’s multiple comparisons test revealed significant differences between the negative control and low dose test (50 mg) (p < 0.0001), between the negative control and low dose test (50 mg) (p < 0.0001), the negative control and high dose test (100 mg) (p < 0.0001) and the the negative control and high dose test (100 mg) (p < 0.0001), the negative control and positive control (p < 0.0001) groups. negative control and positive control (p < 0.0001).The graphical re- presentations of the experimental data are shown in Fig. 1. 3.5.2. Serum triglycerides There were significant differences in the levels of serum triglyceride 3.2. Oral glucose tolerance test between the experimental groups: [3.83 ± 0.22 mmol/l (negative control) vs. 0.60 ± 0.07 mmol/l (low dose test) vs. 0There were significant differences in the area under the curve 0.67 ± 0.10 mmol/l (high dose test) vs. 0.69 ± 0.09 mmol/l (positive (AUC) values between the groups on week four: [1040 ± 32.77 mmol/ control): F (3, 36) = 8.39: p < 0.0001]. Post -hoc statistical analysis l min (negative control) vs. 827.7 ± 18.2 mmol/l min (low dose test) using Tukey’s multiple comparisons test revealed significant differences vs. 880.7 ± 24.11 mmol/l min (high dose test) vs. were between the negative control and low dose test (50 mg) 827.1 ± 15.15 mmol/l min (positive control): F (3,36) = 1.197: (p < 0.0001), the negative control and high dose test (100 mg) p = 0.0424]. (p < 0.0001) and the negative control and positive control

6 B.M. Chege, et al. Journal of Ethnopharmacology 244 (2019) 112077

Fig. 5. Fig. 5A, B, 5C and 5D: Lipid profile (mmol/L) of the experimental groups. (NC-Negative control), (LDT-Low dose test (50 mg), (HDT-High dose test(100mg) and (PC-Positive control) Results are expressed as mean ± SEM. (****- p < 0.0001).

Fig. 6. Mean serum uric acid (mmol/l). (NC-Negative control), (LDT-Low dose test (50 mg), (HDT-High dose test (100 mg) and (PC-Positive control). Results expressed as mean ± SEM. (****- p < 0.0001).

(p < 0.0001) groups.

3.5.3. HDL cholesterol There were significant differences in the levels of HDL cholesterol between the experimental groups: [0.78 ± 0.08 mmol/l (negative control) vs. 1.66 ± 0.12 mmol/l (low dose test) vs. 1.70 ± 0.08 mmol/l (high dose test) vs. 1.69 ± 0.15 mmol/l (positive control): F (3, 36) = 0.8401: p < 0.0001]. Post -hoc statistical analysis using Tukey’s multiple comparisons test revealed significant differences between the negative control and low dose test (50 mg) (p < 0.0001), the negative control and high dose test (100 mg) (p < 0.0001) and the negative control and positive control (p < 0.0001) groups.

3.5.4. LDL cholesterol Fig. 7. Fig. 7A and B: Fasting insulin levels and HOMA-IR. (NC-Negative con- There were significant differences in the levels of LDL cholesterol trol), (LDT-Low dose test (50 mg), (HDT-High dose test (100 mg) and (PC- between the experimental groups: [3.52 ± 0.19 mmol/l (negative Positive control). Results are expressed as mean ± SEM. control) vs. 0.33 ± 0.14 mmol/l (low dose test) vs.

7 B.M. Chege, et al. Journal of Ethnopharmacology 244 (2019) 112077

Table 1 comparisons test revealed significant differences between the negative Quantification (ug analyte/g extract) Rotheca myricoides (Hochst.) Steane & control and low dose (50 mg) (p < 0.0001), the negative control and Mabb. ’high dose (100 mg) (p < 0.0001) and the negative control and posi- # RT Name Ion Mode (M-H)(M + H) Cong (μg/g) tive control (p < 0.0001) groups. The graphical representation of these results is shown in Fig. 6. 1 0.50 Quinic acid Neg 191.0 0.0388 2 0.60 Malic acid Neg 133.1 0.1214 3 0.72 Acotinic acid Neg 172.9 0.3908 3.7. Effects on fasting insulin levels and HOMA-IR 4 0.79 Gallic acid Neg 169.1 0.4734 5 1.02 Chlorogenic acid Neg 353.0 0.1882 6 1.63 Photocetuic acid Neg 153.0 0.5958 There were significant differences in fasting serum insulin levels 7 2.03 Tannic acid Neg 183.0 0.9075 between the experimental groups: [1.84 ± 0.19 (negative control) vs. 8 2.21 Caffeic acid Neg 179.0 1.0076 (0.69 ± 0.13 (low dose test) vs. (0.83 ± 0.17 (high dose test) vs. 9 2.38 Vanillin Neg 151.0 0.4068 (0.69 ± 0.10 (positive control): F (3, 36) = 0.6421: p < 0.0001]. Post 10 2.42 P-Coumaric acid Neg 163.0 1.3437 11 2.73 M-Coumaric acid Neg 164.0 1.4253 -hoc statistical analysis using Tukey’s multiple comparisons test re- 12 2.86 Rutin Neg 609.0 0.7985 vealed significant differences between the negative control andlow 13 2.99 Hesperidin Pos 611.0 0.1324 dose test (50 mg) (p < 0.0001), the negative control and high dose test 14 3.15 Hyperoside Neg 463.1 0.2143 (100 mg) (p < 0.0001) and the negative control and positive control 15 3.32 O-Benzoic acid Neg 137.0 1.3965 (p < 0.0001) groups. 16 3.42 Salicylic acid Neg 138.0 0.6547 17 3.52 Myricertin Neg 317.0 2.8197 There were significant differences in HOMA-IR between theex- 18 3.65 Fisetin Neg 285.0 2.5329 perimental groups: [3.0 ± 0.10 (negative control) vs. (0.76 ± 0.89 19 3.78 Coumarin Pos 147.0 0.3976 (low dose test) vs. (0.89 ± 0.67 (high dose test) vs. (0.62 ± 0.08 20 4.00 Quercetin Neg 300.9 4.4639 (positive control): F (3, 36) = 0.8041: p < 0.0001]. Post -hoc statis- 21 4.22 Naringenin Neg 271.0 2.1987 22 4.44 Hesperidin Neg 301.0 0.9876 tical analysis using Tukey’s multiple comparisons test revealed sig- 23 4.78 Luteolin Neg 285.0 3.9843 nificant differences between the negative control and low dosetest 24 4.99 Kaempferol Neg 285.2 0.5478 (50 mg) (p < 0.0001), the negative control and high dose test 25 5.08 Epigenin Neg 269.0 0.6432 (100 mg) (p < 0.0001) and the negative control and positive control 26 5.23 Rhamnetin Neg 315.3 2.5435 (p < 0.0001) groups. The graphical representation of these results is 27 5.51 Chrysin Neg 253.0 1.5423 shown in Fig. 7A and B.

3.8. Mechanism of action experiment

There were significant differences in area under the curve (AUC) values between the groups [704.2 ± 18.09 mmol/l min (negative control) vs. 427.3 ± 15.97 mmol/l min (positive control) vs. 707 ± 15.5 mmol/l min (test group I) vs. 762 ± 16.92 mmol/l min (test group II): F= (3, 20) = 0.292: p < 0.0001]. Post-hoc statistical analysis using Tukey’s multiple comparisons test revealed significant differences between the positive control andne- gative control (p < 0.0001), positive control and test I (p < 0.0001) as well as between positive control (50 mg) and test II (p < 0.0001) groups. The graphical representation of these results is shown in Fig. 7.

3.9. Interaction network of Rotheca myricoides (Hochst.) Steane & Mabb compounds and putative targets involved in diabetes Fig. 8. Mean area under the curve (mmol/l) (mechanism of action of the extract). (NC-Negative control), (PC-Positive control), (TG 1-Test group I) and A total of 33 composite compounds of Rotheca myricoides (Hochst.) (TG-Test group II). Results are expressed as mean ± SEM. (****- p < 0.0001). Steane & Mabb were retrieved from the Reaxys® database (Supplementary Table S1) and from Liquid chromatography-Mass 0.34 ± 0.20 mmol/l (high dose test) vs. 0.33 ± 0.01 mmol/l (positive spectroscopy: LC-MS (Table 1). The drug target prediction by BATMAN- control): F (3, 36) = 13.63: p < 0.0001]. Post -hoc statistical analysis TCM revealed 130 putative targets of the 33 Rotheca myricoides using Tukey’s multiple comparisons test revealed significant differences (Hochst.) Steane & Mabb compounds. Out of the 130 putative targets as were between the negative control and low dose test (50 mg) shown in Supplementary Tables S2 and 7 were related to diabetes (p < 0.0001), the negative control and high dose test (100 mg) mellitus suggesting their possible role in the observed anti-hypergly- (p < 0.0001) and the negative control and positive control cemic and anti-hyperinsulinemic effects of Rotheca myricoides (Hochst.) (p < 0.0001) groups. Steane & Mabb and are shown in Table 1. A graphical representation of the lipid profile results is shown in A network was constructed based on Rotheca myricoides (Hochst.) Fig. 5A, B, 5C and 5D. Steane & Mabb constitutive compounds-putative targets and known therapeutic targets of the diseases (shown in Table 1) to elucidate the 3.6. Effects on serum uric acid possible pharmacogical mechanisms of action of Rotheca myricoides (Hochst.) Steane & Mabb in type 2 diabetes mellitus and is shown in There were significant differences in the serum uric acid levels be- Fig. 8. PPARG (Peroxisome proliferator-activated receptor gamma) tween the experimental groups: [2.7 ± 0.20 mg/dl (negative control) appears to be a key focal point in the network being targeted by 4 of the vs. 0.33 ± 0.46 mg/dl (low dose) vs. 0.49 ± 0.52 mg/dl (high dose) 7 compounds. vs. 0.48 ± 0.78 mg/dl (positive control): F (3, 36) = 5.64: The graphical representation of these results is shown in Fig. 9 and p < 0.0001]. Post -hoc statistical analysis using Tukey’s multiple Table 1.

8 B.M. Chege, et al. Journal of Ethnopharmacology 244 (2019) 112077

Fig. 9. Interaction network between chemical components of Rotheca myricoides (Hochst.) Steane & Mabb and their putative targets and visualized with Cytoscape. The red dots represent RME compounds and the yellow dots represent the molecular targets. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

4. Discussion Rotheca is genus of flowering plants in the family of Verbenaceae. These plants are widely used as herbal medicine in Africa and Asia to Diabetes mellitus is a metabolic disorder frequently associated with treat diabetes mellitus(Steane and Mabberley, 1998). Rotheca capitalum the development of micro- and macrovascular complications that in- (A. A. Adeneye et al., 2008a), Rotheca phlomidis(Evaluation of im- clude but are not limited to: neuropathy, nephropathy, cardiovascular munomodulatory, 2007) and Rotheca glandulosum(Jadeja et al., 2009) and cerebrovascular disease (Altan, 2003). Diabetes is therefore asso- have all been shown to have hypoglycemic and lipid lowering proper- ciated with a reduced quality of life as well as being a mortality risk ties(Winzell and Ahrén, 2004). factor (Strojek, 2003). The freeze dried extracts of Rotheca myricoides (Hochst.) Steane & Current treatment modalities do not cure or reverse the progression Mabb at the doses tested in this study possessed significant anti- of the condition as end organ damage still develops despite adequate hyperglycemic effects. These antihyperglycemic effects were sig- glycemic control, underscoring the need for newer more efficacious nificantly inhibited when the extract was co-administered with in- drugs for the management of DM (Kasper and Giovannucci, 2006). dinavir which is a known GLUT4 blocker. In addition, they also Additionally; current antidiabetic drugs are often associated with toxic possessed significant effects on insulin sensitivity as shown in theoral adverse effects (Dong et al., 2014). There is therefore need to develop glucose tolerance test. The results obtained in this study are similar to newer safer antidiabetic drugs. The plant Rotheca myricoides (Hochst.) others in published literature. Indeed, extracts from a related species, Steane & Mabb is used in traditional medicine for the management of Rotheca capitatum possessed significant antihyperglycemic effects and diabetes and this study aimed to evaluate its efficacy in an animal improved insulin sensitivity in the oral glucose test in a dose dependent model of type 2 diabetes. manner (A. Adeneye et al., 2008). The observed effects in that study A high fat- high fructose diet was used for the induction of type 2 were attributed to the enhanced glucose uptake by adipose tissue and diabetes(Gan et al., 2015). Such a diet has previously been shown to muscle secondary to the upregulation of GLUT-4 transporters expres- cause the development of obesity, hyperinsulinemia, and insulin re- sion (Shepherd and Kahn, 1999) as well as the inhibition of hepatic sistance (Flanagan et al., 2008). On the other hand, the Initialβ-cell glucose production and increased insulin secretion (Hui et al., dysfunction that mimics that which is observed in type 2 diabetes 2009).These findings were replicated in this study as shown bythe mellitus was achieved by the intraperitoneal administration of low dose blocker experiment involving indinavir. of streptozocin. Streptozocin (STZ) has been observed to be a better The freeze- dried extracts of Rotheca myricoides (Hochst.) Steane & chemical inducer of diabetes than alloxan (Szkudelski, 2001).This Mabb also possessed significant effects on serum insulin levels aswell model best mimics human type 2 diabetes where obesity is associated on the HOMA-IR levels clearly demonstrating potent anti-hyper- with insulin resistance and beta cell dysfunction ensues in the late stage insulinemic effects and/or reductions in insulin resistance. The home- of the disease(Kasper and Giovannucci, 2006). ostasis model of assessment of Insulin Resistance (HOMA-IR) is an index

9 B.M. Chege, et al. Journal of Ethnopharmacology 244 (2019) 112077 that has been used to evaluate both insulin resistance and beta-cell (pioglitazone and rosiglitazone) which function as insulin sensitizers function(Singh and Saxena, 2010). Indeed, HOMA-IR is widely ac- and enhance tissue responses to insulin and consequently improve hy- cepted to be a simple and particularly helpful tool in the assessment of perglycemia in patients with T2DM(Han et al., 2017). The fact that insulin resistance both in pre-clinical and epidemiological studies, in- PPAR-γ is known to contribute to the regulation of adipogenesis as well cluding subjects with both glucose intolerance, mild to moderate dia- as lipid and glucose homeostasis explains the insulin sensitization ef- betes, and in other insulin-resistance conditions(Antunes et al., 2016). fects of the thiazolidinediones(Han et al., 2017). The foregoing discussion therefore indicates that the observed anti- In conclusion, the results of this study validate the traditional use of hyperglycemic effects of the extract are secondary to its insulin sensi- this plant species in the management of Diabetes mellitus and indicate tizing effects. that the main mechanism of action of these antidiabetic effects is via the Insulin resistance is a fundamental aspect of the etiology of type 2 modulation of PPAR-γ. diabetes and plays an important role not only in the development of hyperglycemia of non-insulin dependent diabetes but also in the pa- Conflicts of interest thogenesis of long term complications such as hypertension and hy- perlipidemia (Kahn and Flier, 2000) its amelioration is therefore an The authors declare no competing interests. important therapeutic aim (Huang et al., 2015). Indeed, several groups This manuscript/data, or parts thereof, has not been submitted for of phytochemicals have been shown to possess insulin-sensitizing ac- possible publication to another journal or previously been published tivities including phenolics such as agrimonolide, desmethyla- elsewhere. grimonolide, quercetin (Chen et al., 2016; Huang et al., 2015) triterpenes (Teng et al., 2018), sesquiterpenoids (Chen et al., 2019). Author contributions The freeze-dried extracts of Rotheca myricoides (Hochst.) Steane & Mabb caused significant reductions in hepatic weights as well as inthe Chege Boniface Mwangi, Mwangi Peter Waweru and Bukachi hepatic triglyceride concentrations. These experimental results are si- Frederick designed the study. Chege Boniface Mwangi, Nelly Murugi milar to those in published literature. Indeed, two related plant species Nyaga and Mwangi Peter Waweru did the experimental work. Chege Rotheca infortunatum (Das et al., 2011) and Rotheca capitatum(A. A. Boniface Mwangi, Nelly Murugi Nyaga and Mwangi Peter Waweru Adeneye et al., 2008b) have been reported to have similar effects. The analyzed the experimental data and wrote the paper. All authors re- combination of hepatic steatosis, increased liver weights and increased viewed the manuscript and approved its submission. hepatic index are some of the earliest features of Type 2 diabetes (Kotronen et al., 2008).Hepatic steatosis is often secondary to hepatic Funding insulin resistance (Tolman et al., 2007).It is believed that increases in liver diacylglycerol (DAG) levels lead to protein kinase Cε (PKCε) ac- The project was funded from the authors’ personal resources. tivation and its consequent translocation to the cell membrane, which results in inhibition of hepatic insulin signaling. This then results in the Acknowledgements development of hepatic insulin resistance (Perry et al., 2014). The aforementioned events ultimately result in hepatic accumulation of fat The authors wish to acknowledge the great technical assistance i.e. hepatic steatosis with the consequent increase in hepatic weight and offered by Mr. David Wafula, Mr.Horo Mwaura. The authors wouldalso index. One can therefore argue that compounds in the extract cause a like to acknowledge the help from Mr. Patrick Mutiso Chalo of the reduction in hepatic triglyceride content and consequently prevent University of Nairobi herbarium in the identification and collection of hepatic steatosis by inhibiting one or more of these biochemical path- the plants. ways. The freeze-dried extracts of Rotheca myricoides (Hochst.) Steane & Appendix A. Supplementary data Mabb possessed significant antidyslipidemic effects i.e. decreased total plasma cholesterol, LDL-cholesterol, serum triglyceride and increased Supplementary data to this article can be found online at https:// HDL-cholesterol. The results obtained in this study are similar to those doi.org/10.1016/j.jep.2019.112077. in published literature. Indeed, two plant species from Rotheca genus, Rotheca capitatum(A. A. Adeneye et al., 2008b) and Rotheca phlomidis References (Chidrawa et al., 2011) were reported to possess significant antidyslipidemic effects. These effects were directly attributed to theim- Adeneye, A., Ajagbonna, O., Ayodele, O.W., 2008. Hypoglycemic and antidiabetic ac- provement in insulin signaling, besides other possible lipid-lowering tivities on the stem bark aqueous and ethanol extracts of Musanga cecropioides in normal and alloxan-induced diabetic rats. Fitoterapia 78, 502–505. https://doi.org/ mechanism acting in the cholesterol and/or fatty acids biosynthetic 10.1016/j.fitote.2007.05.001. pathways(Wang et al., 2014). Adeneye, A.A., Adeleke, T.I., Adeneye, A.K., 2008a. Hypoglycemic and hypolipidemic The freeze-dried extracts of Rotheca myricoides (Hochst.) Steane & effects of the aqueous fresh leaves extract of Clerodendrum capitatum in Wistar rats. J. Ethnopharmacol. 116, 7–10. https://doi.org/10.1016/j.jep.2007.10.029. Mabb had significant serum uric acid lowering effects. It is knownthat Adeneye, A.A., Adeleke, T.I., Adeneye, A.K., 2008b. Hypoglycemic and hypolipidemic elevated serum triglyceride is associated with hyperuricemia which effects of the aqueous fresh leaves extract of Clerodendrum capitatum in Wistar rats. results from the increased activity of the pentose phosphate cycle J. Ethnopharmacol. 116, 7–10. https://doi.org/10.1016/j.jep.2007.10.029. leading to increased NADPH requirement for new fatty acid synthesis. Altan, V.M., 2003. The pharmacology of diabetic complications. Curr. Med. Chem. 10, 1317–1327. https://doi.org/10.2174/0929867033457287. The increased NADPH requirements production leads to the enhance- Antunes, L.C., Elkfury, J.L., Jornada, M.N., Foletto, K.C., Bertoluci, M.C., 2016. Validation ment uric acid production (Chen et al., 2007). of HOMA-IR in a model of insulin-resistance induced by a high-fat diet in Wistar rats. Network pharmacology which applies holistic system biology-de- Arch. Endocrinol. Metab. 60, 138–142. https://doi.org/10.1590/2359- 3997000000169. rived concepts to the understanding and remediation of various disease Barrett, C., 2002. The glucose tolerance test: a pitfall in the diagnosis of diabetes mellitus. conditions, has been applied with marked success to understanding the Adv. Intern. Med. 20, 297–323. mechanisms of action of various traditional Chinese Medicine (TCM) Bashwira, S., Hootele, C., 1988. Myricoidine and dihydromyricoidine, two new macro- cyclic spermidine alkaloids from clerodendrum myricoides. Tetrahedron 44, remedies(Zhang et al., 2013)(Zhang et al., 2016).The application of 4521–4526. https://doi.org/10.1016/S0040-4020(01)86153-6. these techniques to Rotheca myricoides (Hochst.) Steane & Mabb re- Butler, W.M., Maling, H.M., Horning, M.G., Brodie, B.B., 1961. The direct determination vealed that modulation of PPAR-γ was the most plausible mechanistic of liver triglycerides. J. Lipid Res. 2, 95–96. Chen, L., Lu, X., El-Seedi, H., Teng, H., 2019. Recent advances in the development of explanation for the observed pharmacological effects of the extract. sesquiterpenoids in the treatment of type 2 diabetes. Trends Food Sci. Technol. 88, PPAR-γ which is the molecular target of the thiazolidine class of drugs

10 B.M. Chege, et al. Journal of Ethnopharmacology 244 (2019) 112077

46–56. https://doi.org/10.1016/j.tifs.2019.02.003. Increased liver fat, impaired insulin clearance, and hepatic and adipose tissue insulin Chen, L., Teng, H., Fang, T., Xiao, J., 2016. Agrimonolide from Agrimonia pilosa sup- resistance in type 2 diabetes. Gastroenterology 135, 122–130. https://doi.org/10. presses inflammatory responses through down-regulation of COX-2/iNOS andin- 1053/j.gastro.2008.03.021. activation of NF-κB in lipopolysaccharide-stimulated macrophages. Phytomedicine Lee, G., Goosens, K.A., 2015. Sampling blood from the lateral tail vein of the rat. J. Vis. Int. J. Phytother. Phytopharm. 23, 846–855. https://doi.org/10.1016/j.phymed. Exp. JoVE.(99), e52766 Basic Protocol. https://doi.org/10.3791/52766. 2016.03.016. Liu, Z., Guo, F., Wang, Y., Li, C., Zhang, X., Li, H., Diao, L., Gu, J., Wang, W., Li, D., He, F., Chen, L., Zhu, W., Chen, Z., Dai, H., Ren, J., Chen, J., Chen, L., Fang, L., 2007. 2016. BATMAN-TCM: a Bioinformatics analysis tool for molecular mechANism of Relationship between hyperuricemia and metabolic syndrome. J. Zhejiang Univ. - Sci. traditional Chinese medicine. Sci. Rep. 6 21146. https://doi.org/10.1038/ B 8, 593–598. https://doi.org/10.1631/jzus.2007.B0593. srep21146. Chidrawa, et al., 2011. Cytoscape 3.7.1 user manual — Cytoscape user manual 3.7.1 Moshi, M.J., Otieno, D.F., Weisheit, A., 2012. Ethnomedicine of the Kagera Region, north documentation [WWW document]. n.d. URL. http://manual.cytoscape.org/en/ western Tanzania. Part 3: plants used in traditional medicine in Kikuku village. stable/ accessed 3.14.19. Muleba District. J. Ethnobiol. Ethnomedicine 8, 14. https://doi.org/10.1186/1746- Das, S., Bhattacharya, S., Prasanna, A., Suresh Kumar, R.B., Pramanik, G., Haldar, P.K., 4269-8-14. 2011. Preclinical evaluation of antihyperglycemic activity of Clerodendron in- Mukungu, N., Abuga, K., Okalebo, F., Ingwela, R., Mwangi, J., 2016. Medicinal plants fortunatum leaf against streptozotocin-induced diabetic rats. Diabetes Ther 2, used for management of malaria among the Luhya community of Kakamega East sub- 92–100. https://doi.org/10.1007/s13300-010-0019-z. County, Kenya. J. Ethnopharmacol. 194, 98–107. https://doi.org/10.1016/j.jep. Dong, Y., Jing, T., Meng, Q., Liu, C., Hu, S., Ma, Y., Liu, Y., Lu, J., Cheng, Y., Wang, D., 2016.08.050. Teng, L., 2014. Studies on the antidiabetic activities of Cordyceps militaris extract in Perry, R.J., Samuel, V.T., Petersen, K.F., Shulman, G.I., 2014. The role of hepatic lipids in diet-streptozotocin-induced diabetic Sprague-Dawley rats. BioMed Res. Int. 2014, hepatic insulin resistance and type 2 diabetes. Nature 510, 84–91. https://doi.org/ 160980. https://doi.org/10.1155/2014/160980. 10.1038/nature13478. Esubalew, S.T., Belete, A., Lulekal, E., Gabriel, T., Engidawor, E., Asres, K., 2017. Review Podell, B.K., Ackart, D.F., Richardson, M.A., DiLisio, J.E., Pulford, B., Basaraba, R.J., of ethnobotanical and ethnopharmacological evidences of some Ethiopian medicinal 2017. A model of type 2 diabetes in the Guinea pig using sequential diet-induced plants traditionally used for the treatment of cancer. Ethiop. J. Health Dev. EJHD 31. glucose intolerance and streptozotocin treatment. Dis. Model. Mech. 10, 151–162. Evaluation of immunomodulatory activity of clerodendrum phlomidis and premna in- https://doi.org/10.1242/dmm.025593. tegrifolia root, Sci. Alert. 2007 3(4), 352-356. [WWW document], n.d. https://doi. Richard, S., Maciuk, A., Banzouzi, J., Champy, P., Figadere, B., Guissou, I.P., Nacoulma, org/10.3923/ijp.2007.352.356. O.G., 2011. Mutagenic effect, antioxidant and anticancer activities of six medicinal Flanagan, A.M., Brown, J.L., Santiago, C.A., Aad, P.Y., Spicer, L.J., Spicer, M.T., 2008. plants from Burkina Faso. https://doi.org/10.1080/14786419.2010.534737. High-fat diets promote insulin resistance through cytokine gene expression in Shannon, P., Markiel, A., Ozier, O., Baliga, N.S., Wang, J.T., Ramage, D., Amin, N., growing female rats. J. Nutr. Biochem. 19, 505–513. https://doi.org/10.1016/j. Schwikowski, B., Ideker, T., 2003. Cytoscape: a software environment for integrated jnutbio.2007.06.005. models of biomolecular interaction networks. Genome Res. 13, 2498–2504. https:// Gan, Y., Yang, C., Tong, X., Sun, H., Cong, Y., Yin, X., Li, L., Cao, S., Dong, X., Gong, Y., doi.org/10.1101/gr.1239303. Shi, O., Deng, J., Bi, H., Lu, Z., 2015. Shift work and diabetes mellitus: a meta-ana- Shepherd, P.R., Kahn, B.B., 1999. Glucose transporters and insulin action–implications for lysis of observational studies. Occup Env. Med. 72, 72–78. https://doi.org/10.1136/ insulin resistance and diabetes mellitus. N. Engl. J. Med. 341, 248–257. https://doi. oemed-2014-102150. org/10.1056/NEJM199907223410406. Guillen, J., 2012. FELASA guidelines and recommendations. J. Am. Assoc. Lab. Anim. Sci. Singh, B., Saxena, A., 2010. Surrogate markers of insulin resistance: a review. World J. JAALAS 51, 311–321. Diabetes 1, 36–47. https://doi.org/10.4239/wjd.v1.i2.36. Guthrie, R.A., Guthrie, D.W., 2004. Pathophysiology of diabetes mellitus. Crit. Care Nurs. Steane, D.A., Mabberley, D.J., 1998. Rotheca (lamiaceae) revived. Novon 8, 204–206. Q. 27, 113. https://doi.org/10.2307/3391997. Han, L., Shen, W.-J., Bittner, S., Kraemer, F.B., Azhar, S., 2017. PPARs: regulators of Strojek, K., 2003. Features of macrovascular complications in type 2 diabetic patients. metabolism and as therapeutic targets in cardiovascular disease. Part II: PPAR-β/δ Acta Diabetol. 40, s334–s337. https://doi.org/10.1007/s00592-003-0115-x. and PPAR-γ. Future Cardiol. 13, 279–296. https://doi.org/10.2217/fca-2017-0019. Szkudelski, T., 2001. The mechanism of alloxan and streptozotocin action in B cells of the Herman, P.P.J., 2017. HERMAN, P.P.J. & CONDY, G. 2017. Rotheca myricoides sensu lato rat pancreas. Physiol. Res. 50, 537–546. (lamiaceae: ajugoideae). Flowering plants of Africa 65: 146–152. Flower. Plants Afr Teng, H., Yuan, B., Gothai, S., Arulselvan, P., Song, X., Chen, L., 2018. Dietary triterpenes 65, 146–152. in the treatment of type 2 diabetes: to date. Trends Food Sci. Technol. 72, 34–44. Hruz, P.W., Murata, H., Qiu, H., Mueckler, M., 2002. Indinavir induces acute and re- https://doi.org/10.1016/j.tifs.2017.11.012. versible peripheral insulin resistance in rats. Diabetes 51, 937–942. Tolman, K.G., Fonseca, V., Dalpiaz, A., Tan, M.H., 2007. Spectrum of liver disease in type Huang, Q., Chen, L., Teng, H., Song, H., Wu, X., Xu, M., 2015. Phenolic compounds 2 diabetes and management of patients with diabetes and liver disease. Diabetes Care ameliorate the glucose uptake in HepG2 cells’ insulin resistance via activating AMPK. 30, 734–743. https://doi.org/10.2337/dc06-1539. J. Funct. Foods 19, 487–494. https://doi.org/10.1016/j.jff.2015.09.020. Wachtel-Galor, S., Benzie, I.F.F., 2011. Herbal medicine: an introduction to its history, Hui, H., Tang, G., Go, V., 2009. Hypoglycemic herbs and their action mechanisms. Chin. usage, regulation, current trends, and research needs. In: Benzie, I.F.F., Wachtel- Med. 4, 11. https://doi.org/10.1186/1749-8546-4-11. Galor, S. (Eds.), Herbal Medicine: Biomolecular and Clinical Aspects. CRC Press/ Jadeja, R.N., Thounaojam, M.C., Ansarullah, A., Devkar, R.V., Ramachandran, A.V., Taylor & Francis, Boca Raton (FL. 2009. A preliminary study on hypolipidemic effect of aqueous leaf extract of Wang, H., Li, H., Jiang, X., Shi, W., Shen, Z., Li, M., 2014. Hepcidin is directly regulated Clerodendron glandulosum. Coleb. Int. J. Green Pharm. IJGP 3. https://doi.org/10. by insulin and plays an important role in iron overload in streptozotocin-induced 22377/ijgp.v3i4.102. diabetic rats. Diabetes 63, 1506–1518. https://doi.org/10.2337/db13-1195. Kahn, B.B., Flier, J.S., 2000. Obesity and insulin resistance. J. Clin. Investig. 106, Winzell, M.S., Ahrén, B., 2004. The high-fat diet–fed mouse: a model for studying me- 473–481. https://doi.org/10.1172/JCI10842. chanisms and treatment of impaired glucose tolerance and type 2 diabetes. Diabetes Kasper, J.S., Giovannucci, E., 2006. A meta-analysis of diabetes mellitus and the risk of 53, S215–S219. https://doi.org/10.2337/diabetes.53.suppl_3.S215. prostate cancer. Cancer Epidemiol. Prev. Biomark. 15, 2056–2062. https://doi.org/ Zhang, G.-B., Li, Q.-Y., Chen, Q.-L., Su, S.-B., 2013. Network pharmacology: a new ap- 10.1158/1055-9965.EPI-06-0410. proach for Chinese herbal medicine research. Evid.-Based Complement. Altern. Med. Keter, L., Mutiso, P., 2012. Ethnobotanical studies of medicinal plants used by traditional ECAM 2013, 621423. https://doi.org/10.1155/2013/621423. Health practitioners in the management of diabetes in lower eastern province, Kenya. Zhang, Y., Mao, X., Guo, Q., Lin, N., Li, S., 2016. Network pharmacology-based ap- J. Ethnopharmacol. 139, 74–80. https://doi.org/10.1016/j.jep.2011.10.014. proaches capture essence of Chinese herbal medicines. Chin. Herb. Med. 8, 107–116. Kotronen, A., Juurinen, L., Tiikkainen, M., Vehkavaara, S., Yki–Järvinen, H., 2008. https://doi.org/10.1016/S1674-6384(16)60018-7.