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Effect of Ruzu Herbal Bitters on the Liver Function and Lipid Profile Parameters of Alloxan-Induced Diabetic Rats

Published:September 22, 2021DOI:https://doi.org/10.1016/j.jceh.2021.09.012

      Background

      Ruzu herbal bitters (RHB) is a polyherbal mixture produced in Nigeria indicated for diabetes and other ailments. The consumers of the product testify of its efficacy, but there are not much scientific information on RHB. The study determined the effect of RHB on the liver function and lipid profile parameters of alloxan-induced diabetic rats.

      Method

      Fifty-four adult albino rats were divided into nine groups of six rats each. Group 1 was the normal control, while groups 2–6 were diabetic. Group 2 was not treated, while groups 3–6 were respectively treated with 5 mg/kg b.w of glibenclamide, 0.14, 0.29, and 0.57 ml/kg b.w of RHB. Groups 7–9 were not diabetic but treated as groups 4–6. Diabetes was induced by intraperitoneal injection of freshly prepared alloxan into adult male albino Wister rats with a single dose of 120 mg/kg body weight. The blood sugar level, weight, liver function, and lipid profile of the rats were tested using standard methods.

      Result

      The results showed a significant (P < 0.05) increase in the blood glucose level and decrease in weight in the diabetic-untreated group compared to the normal group. The liver function and lipid profile tests showed significant (P<0.05) increases in the activities of gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST); increases in the levels of total bilirubin, total cholesterol (T.CHOL), triglycerides (TG), very low-density lipoprotein (VLDL) and lowdensity lipoprotein (LDL); decreases in the levels of total protein, albumin and high-density lipoproteins (HDL), in the diabetic-untreated group compared to the normal group. However, treatment of the diabetic rats with different doses of RHB caused the reversal of these effects to near-normal levels in a dose-dependent manner.

      Conclusions

      Our study reveals that RHB has antidiabetic, hepatoprotective, and antihyperlipidemic effects.

      Keywords

      Abbreviations:

      ALB (albumin), ALP (alkaline phosphatase), ALT (alanine aminotransferase), AST (aspartate aminotransferase), CVD (cardiovascular disease), DKA (diabetic ketoacidosis), DM (diabetes mellitus), GC–MS (gas chromatography and mass spectrometry), GGT (gamma-glutamyltransferase), HA (hepatic artery), HDL (high-density lipoprotein cholesterol), HHNK (hyperosmotic hyperglycemic nonketotic state), HHS (hyperosmolar hyperglycemic state/syndrome), HV (hepatic vein), LA (lactic acidosis), LDL (low-density lipoprotein cholesterol), RHB (Ruzu herbal bitters), T. BIL (total bilirubin), T. CHOL/TC (total cholesterol), T. PROT (total protein), T1DM (type 1 diabetes mellitus), T2DM (type 2 diabetes mellitus), TG (triglycerides), VLDL (very-low-density lipoprotein cholesterol)
      Diabetes mellitus (DM) is a heterogeneous metabolic disorder characterized by hyperglycemia due to impaired insulin secretion, defective insulin action, or both.
      • Punthakee Z.
      • Goldenberg R.
      • Katz P.
      Definition, classification and diagnosis of diabetes, prediabetes and metabolic syndrome. Diabetes Canada Clinical Practice Guidelines Expert Committee.
       Impaired insulin secretion or insensitivity leads to poor glucose metabolism resulting in high blood glucose level (hyperglycemia), with inadequacy in the metabolisms of carbohydrates, fats, and proteins. Diabetes mellitus is a global disease, with the prevalence rate differing from country to country.
      • Ivorra M.D.
      • Paya M.
      • Villar A.
      Hypoglycaemic and Insulin release effects of tormenic acid: a new hypoglycaemic natural product.
       The two most common forms of DM are type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), both leading to hyperglycemia, excessive urine production (polyuria), compensatory thirst, increased water intake (polydipsia), blurred vision, sudden weight loss, lethargy, and changes in energy metabolism.
      • Mathur S.
      • Mehta D.K.
      • Kapoor S.
      • Yadav S.
      Liver function in type-2 diabetes mellitus patients.
       Excessive urination leads to the removal of glucose through the urine with a concomitant excessive hunger sensation (polyphagia). The prolonged or chronic hyperglycemia of diabetes is associated with relatively specific long-term microvascular complications affecting the eyes, kidneys, and nerves, as well as an increased risk for cardiovascular disease (CVD). The diagnostic criteria for diabetes are based on thresholds of glycemia that are associated with microvascular disease, especially retinopathy.
      • Punthakee Z.
      • Goldenberg R.
      • Katz P.
      Definition, classification and diagnosis of diabetes, prediabetes and metabolic syndrome. Diabetes Canada Clinical Practice Guidelines Expert Committee.
      The acute metabolic complications of diabetes consist of diabetic ketoacidosis (DKA), hyperglycemic hyperosmolar state/hyperosmolar hyperglycemic syndrome (HHS, also known as hyperosmotic hyperglycemic nonketotic state [HHNK]), lactic acidosis (LA), and hypoglycemia.
      • Fishbein H.
      • Palumbo P.J.
      Acute Metabolic Complications in Diabetes. Chapter 13. Scottsdale A.Z..
      Although it has been centuries since DM was first recognized, it has not been fully understood and managed.
      • Amartey N.A.
      • Nsiah K.
      • Mensah F.O.
      Plasma levels of uric acid, urea and creatinine in diabetics who visit the clinical analysis laboratory (Can-lab) at Kwame Nkrumah University of science and technology, Kumasi, Ghana.
       Despite considerable progress in the treatment of diabetes by oral hypoglycemic agents, search for newer drugs continues because the existing synthetic drugs have many limitations.
      • Ibironke A.A.
      • Olusola O.O.
      Chemical Composition Often Medical Plants Seeds from South-West Nigeria.
       Hence, remedies from medicinal plants, which are natural products are considered to be safe from the side effects when compared to synthetic ones.
      • Ibironke A.A.
      • Olusola O.O.
      Chemical Composition Often Medical Plants Seeds from South-West Nigeria.
       Interests in medicinal plants have also increased due to the higher cost of orthodox drugs in disease management compared to the cheaper cost of herbal medicines in view of the present state of the economy, especially in the rural areas where some poor people die due to their inability to afford medical bills. The importance of plants ranges from food to medicine. Hence, apart from serving as the primary producers in the food chain, plants are the primary sources of therapeutic agents.
      • Obasi D.C.
      • Ogugua V.N.
      • Obasi J.N.
      • Okagu I.U.
      Phytochemical, nutritional and anti-nutritional analyses of Ruzu herbal bitters.
      In some cases, a combination of plants or their extracts is used in the treatment of certain ailments with the belief by the herbalists that the different plants contain different therapeutic agents such that when combined together, will give a better therapeutic efficacy for a particular disease or multiple diseases than a single plant. This indicates that many plants or herbal preparations work in synergy for a better therapeutic effect hence, the need for polyherbal mixtures.
      • Obasi D.C.
      • Ogugua V.N.
      • Obasi J.N.
      • Okagu I.U.
      Phytochemical, nutritional and anti-nutritional analyses of Ruzu herbal bitters.
      One of such polyherbal mixtures is Ruzu herbal bitters.
      The Ruzu herbal bitters used in this study is produced by Ruzu Natural Health Product and Services, Nigeria, with a NAFDAC Registration Number: A7-1102L (Figure 1). It is a mixture of 40% Curculigo pilosa (squirrel groundnut) root, 40% Citrullus colocynthis (bitter apple) bark, and 20% Uvaria chamae (bush banana) stem. The mixture is in liquid form in a well-packaged bottle and commercially available in Nigeria. Ruzu herbal bitters (RHB) is indicated to have the following functions among others: antidiabetic, antihyperlipidemic, antioxidant, anti-inflammatory, analgesic, antibacterial, antifungal, laxative, and hair-growth promoting properties.
      Figure 1
      Figure 1Ruzu herbal bitters - Dosage: Adults – 2–4 tablespoons 1 or 2 times daily. Children – 1 tablespoon (5 ml) once in 3 days or as directed by physician (Producer).
      There are scanty scientific reports on the biochemical effects of RHB, although many consumers of the product have attested to its efficacy. Therefore, in view of these traditional medical claims on RHB, we carried out preliminary studies to ascertain the phytochemical constituents and the possible bioactive compounds present in the polyherbal mixture that could be responsible for its efficacy. The phytochemical screening showed that RHB contains alkaloids, flavonoids, tannins, saponins, phenols, glycosides, and sterols.
      • Obasi D.C.
      • Ogugua V.N.
      • Obasi J.N.
      • Okagu I.U.
      Phytochemical, nutritional and anti-nutritional analyses of Ruzu herbal bitters.
       Furthermore, gas chromatography and mass spectrometry (GC–MS) analyses revealed the presence of 10 bioactive compounds: 2,7-Dioxatricyclodeca-4,9-diene (1.38%), Nonanoic acid (Pelargic/pelargonic acid) (1.68%), Dodecanoic acid (Lauric acid) (7.87%), Tetradecanoic acid (Myristic acid) (16.47%), Methyl-6-methylheptanoate (1.40%), n-Hexadecanoic acid (palmitic acid) (5.64%), 11-Octadecenoic acid, methyl ester (4.05%), 13-Methyloxacyclotetradecane-2-one (4.88%), Cis-Z-α-Bisabolene epoxide (2.66%), and 9-Octadecanamide, (Z) (Crodamide) (3.07%).
      • Obasi D.C.
      • Ogugua V.N.
      GC-MS analysis, pH and antioxidant effect of Ruzu herbal bitters on alloxan-induced diabetic rats.
       Five of these are pharmacologically important bioactive compounds: 11-Octadecenoic acid, Methyl ester (antioxidant, anti-inflammatory, antimicrobial, antiviral, antimutagenic, and anticarcinogenic activities); 2,7-Dioxatricyclodeca-4, 9-diene (antioxidant, cytotoxic, and antimalarial effects); Cis-Z-α- Bisabolene epoxide (antioxidant and antimicrobial agent); Tetradecanoic acid (lauric acid) (antioxidant); and Dodecanoic acid (antibacterial, antifungal, and antiviral agent).
      • Obasi D.C.
      • Ogugua V.N.
      GC-MS analysis, pH and antioxidant effect of Ruzu herbal bitters on alloxan-induced diabetic rats.
      Hence, this study was aimed at investigating the antidiabetic activity and effects of RHB on the liver function and lipid profile parameters of alloxan-induced diabetic rats.

      Materials and methods

      Materials

      Ruzu herbal bitters (RHB) was purchased from the producer and used directly; alloxan monohydrate (Sigma Aldrich, St. Louis, MO, USA); assay kits (Randox Laboratories Limited, BT29 4QY, United Kingdom). All the chemicals and reagents used in this research were of the purest analytical grade commercially available.

      Equipment

      Spectrophotometer (Spectro 21D PEC MEDICALS USA), centrifuge (Techmel and Techmel U.S.A.), electrothermal incubator (Model: DNP), refrigerator (Haier Thermocool; HTF-429H), weighing balance (Golden-Mettler U.S.A.), glucometer (Accu-Check Active, Model-GC, Roche, Germany).

      Methods

      Animal Management

      Adult inbred male Wister albino rats (weighing between 96 and 121 g) were purchased from the animal house of the Department of Zoology, University of Nigeria, Nsukka, and acclimatized for one week before commencement of the experiment. They were kept at room temperature and maintained ad libitum on water and feed; weighed before the commencement of the experiment and weekly till the end of the experiment.

      Induction of Diabetes

      Rats were fasted overnight (24 h), and experimental diabetes was induced by intraperitoneal injection of freshly prepared alloxan (3 g of alloxan monohydrate was dissolved in 30 ml of 0.9% w. v. normal saline to get 0.1 g/ml of alloxan) with a single dose of 120 mg/kg body weight. After three days, rats with fasting blood glucose levels greater than 200 mg/dl were selected for the experiment because most of the rats had blood glucose levels up to 200 mg/dl and above. Only a few rats showed insufficient glycemia, and they were not used for the experiment. The one-touch blood glucose monitoring meter (glucometer) and test strips were used for the assay.
      • Obasi D.C.
      • Ogugua V.N.
      GC-MS analysis, pH and antioxidant effect of Ruzu herbal bitters on alloxan-induced diabetic rats.

      Experimental Design

      Oral administration of 0.14, 0.29, and 0.57 ml/kg body weight of RHB, equivalent to 10, 20, and 40 ml of RHB/70 kg body weight adult daily, as prescribed by the producer, were chosen for the study. Oral administration of RHB was done by using a needle-free 2 ml syringe containing the required dose of RHB and carefully inserting it into the mouth of the rat, held in the vertical position until the drug was swallowed. Fifty-four (54) albino rats were divided into nine groups of six rats each, as follows
      • Obasi D.C.
      • Ogugua V.N.
      GC-MS analysis, pH and antioxidant effect of Ruzu herbal bitters on alloxan-induced diabetic rats.
      :
      • (a)
        Group 1: Normal control rats (nondiabetic).
      • (b)
        Group 2: Positive control rats (diabetic untreated).
      • (c)
        Group 3: Standard control rats (diabetic) treated with 5 mg/kg body weight of glibenclamide.
        • Lav V.K.
        • Gupta P.P.
        • Tripathi P.
        • Pandey A.
        Interaction of aqueous extract of Trigonellafoenum graecum seeds with glibenclamide in streptozotocin induced diabetic rats.
      • (d)
        Group 4: Diabetic rats treated with 0.14 ml/kg body weight of RHB.
      • (e)
        Group 5: Diabetic rats treated with 0.29 ml/kg body weight of RHB.
      • (f)
        Group 6: Diabetic rats treated with 0.57 ml/kg body weight of RHB.
      • (g)
        Group7: Nondiabetic rats treated with 0.14 ml/kg body weight of RHB
      • (h)
        Group 8: Nondiabetic rats treated with 0.29 ml/kg body weight of RHB
      • (i)
        Group 9: Nondiabetic rats treated with 0.57 ml/kg body weight of RHB.
      The baseline fasting blood glucose levels of the rats were determined before the induction of diabetes. The treatment, weight, and blood glucose determinations (7-days intervals) lasted for 21 days. After 21 days, the rats were sacrificed, and blood samples were collected via ocular and cardiac puncture on the first day after treatment for biochemical analyses. The blood samples were allowed to clot in anticoagulantfree bottles and centrifuged at 3,000 rpm for 15 min. The serum collected was used for biochemical assays. Sections of the liver from the rats in the experimental groups were also collected for histological examination.

      Animal Behavior After Induction of Diabetes

      Some of the rats died within 72 h after alloxan administration, while some rats were not hyperglycemic. Few rats showed some signs of weakness, such as sluggish movement and poor feeding habits, and eventually died within the 21 days of the experiment, especially among the diabetic-untreated group 2 (Table 1). Many of the animals survived during the period of the experiment, with some symptoms of diabetes displayed, such as polyurea, polydipsia, and polyphagia. After the experiment and stoppage of the herbal drug intake, it was also observed that some of the rats treated with RHB, which were not sacrificed, recovered without symptoms of diabetes and showed normoglycemia when tested.
      Table 1Number of Dead and Survived Rats After Alloxan Administration.
      GroupsTotal Number of Dead Rats after Day 0Drug/kg b.wTotal Number of Survived Rats with Days
      Day 0Diabetes (Day 0)Day 7Day 14Day 21Day 0Diabetes (Day 0)Day 7Day 14Day 21
      Group 160000066666
      Group 210011201010986
      Group 31021005 mg Glb108777
      Group 41011110.14 ml RHB109876
      Group 51010100.29 ml RHB109988
      Group 61010000.57 ml RHB109999
      Group 7600000.14 ml RHB66666
      Group 8600000.29 ml RHB66666
      Group 9600000.57 ml RHB66666
      Drugs/kg b.w = Drug administered per kilogramme body weight.
      Summary
      ∗ Number of rats administered with alloxan = 50.
      ∗ Number of dead rats within 3 days after alloxan administration = 5.
      ∗ Number of rats with hyperglycemia = 42.
      ∗ Number of rats without hyperglycemia = 3.
      ∗ Number of dead diabetic rats from Day 7 to Day 21 = 9.
      ∗ Number of rats used for study in each group = 6.

      Determination of Blood Glucose Level

      The fasting blood glucose levels of the rats were determined using a glucometer (blood glucose meter) after 12 h postprandial. The Accu-Check strip was inserted into the indicated area of the glucometer and allowed to show light at the point where a drop of blood would be placed. A lancet was used to puncture the tail end of the rat. The tail was pressed to bring out blood that was then placed on the indicated portion of the test strip in the glucometer. The blood glucose level that showed on the screen of the glucometer (in mg/dl) was recorded.
      • Obasi D.C.
      • Ogugua V.N.
      GC-MS analysis, pH and antioxidant effect of Ruzu herbal bitters on alloxan-induced diabetic rats.

      Determination of Liver Function Parameters using Randox Test Kits

      • 1.
        Gamma-glutamyltransferase (GGT) activity was assayed according to Szasz colorimetric method.
        • Szasz G.
        A kinetic photometric method for serum gamma-glutamyltranspeptidase.
      • 2.
        Alkaline phosphatase (ALP) activity was assayed according to Rec. GSCC (DGKC) colorimetric method.
        Rec. GSCC (DGKC)
        Optimised standard colorimetric methods. Serum Alkaline phosphatase (DGKC).
      • 3.
        Alanine aminotransferase (ALT) activity according to Reitman and Frankel method.
        • Reitman S.
        • Frankel S.
        A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminase.
      • 4.
        Aspartate aminotransferase (AST) activity according to Reitman and Frankel method.
        • Reitman S.
        • Frankel S.
        A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminase.
      • 5.
        Total Bilirubin level was determined by the method of Jendrassik and Grof.
        • Jendrassik L.
        • Grof P.
        Colorimetric measurement of bilirubin.
      • 6.
        Serum total protein level was determined according to the method of Tietz.
        • Tietz N.W.
        Clinical Guide to Laboratory Test.
      • 7.
        Serum albumin level was determined according to the method of Doumas et al
        • Doumas B.T.
        • Watson W.A.
        • Biggs H.G.
        Albumin standards and the measurement of serum albumin with bromcresol green.

      Lipid Profile Assay using Randox Test Kits

      • 1.
        Total cholesterol concentration based on the method of NCEP.
        NCEP (National Cholesterol Education Program)
        Expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III). Executive summary of the third report of the National Cholesterol Education Program (NCEP).
      • 2.
        Triacylglycerol (TAG) concentration based on Tietz method.
        • Tietz N.W.
        Clinical Guide to Laboratory Test.
      • 3.
        High-density lipoprotein-cholesterol (HDL-chol.) concentration by the method of NCEP.
        NCEP (National Cholesterol Education Program)
        Expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III). Executive summary of the third report of the National Cholesterol Education Program (NCEP).
      • 4.
        Low-density lipoprotein cholesterol (LDL-chol.) concentration based on Friedewald et al equation.
        • Friedewald W.T.
        • Levy R.I.
        • Fredrickson D.S.
        Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.
      • 5.
        Very low-density lipoprotein (VLDL-chol) concentration based on Friedewald et al equation.
        • Friedewald W.T.
        • Levy R.I.
        • Fredrickson D.S.
        Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.

      Histological Examination of the Liver

      Sections of the liver collected from the animals in the experimental groups were fixed in 10% phosphate-buffered formalin for a minimum of 48 h. The tissues were subsequently trimmed, dehydrated in 4 grades of alcohol (70%, 80%, 90%, and absolute alcohol), cleared in three grades of xylene, and embedded in molten wax. On solidifying, the blocks were sectioned, 5 μm thick with a rotary microtome, floated in a water bath, and incubated at 60 °C for 30 min. The 5 μm thick sectioned tissues were subsequently cleared in three grades of xylene and rehydrated in three grades of alcohol (90%, 80%, and 70%). The sections were then stained with hematoxylin for 15 min. Blueing was done with ammonium chloride. Differentiation was done with 1% acid alcohol before counterstaining with Eosin. Permanent mounts were made on degreased glass slides using a mountant, DPX. The prepared slides were examined with a Motic™ compound light microscope using × 4, × 10, and × 40 objective lenses. The photomicrographs were taken using a Motic™ 5.0 megapixels microscope camera at × 160 & ×400 magnifications.

      Statistical Analysis

      The data obtained from the laboratory tests were subjected to a one-way analysis of variance (ANOVA). Significant differences were obtained at P ≤ 0.05, and the results were expressed as ± standard error of the mean (SEM). The SPSS version 20 and Microsoft excel 2007 software were used.

      Results

      Effect of Ruzu Herbal Bitters on the Blood Glucose Level

      Figure 2 shows the effect of RHB on the blood glucose levels (mg/dl) of the rats. Alloxan administration caused significant (P < 0.05) increases in the blood glucose levels (BGL) of the rat groups 2–6 (DIABETES DAY 0) when compared to their baseline BGL (DAY 0). There was a significant (P < 0.05) increase in the BGL of the diabetic-untreated group from 70.00 mg/dl on day 0–241.25 mg/dl after diabetes induction, and progressively to 514.30 mg/dl on day 21 of the experiment; whereas in the normal control, there was no significant (P < 0.05) increase but a gradual increase in the BGL from 78.50 mg/dl on day 0–88.25 mg/dl on day 21 of the experiment, which could be due to growth and increased food intake. However, treatment of the diabetic groups 4, 5, and 6 with different doses of RHB resulted in significant (P < 0.05) decreases in their BGL from day 0 after diabetes induction to day 21 of the experiment. Also for the nondiabetic groups 7, 8, and 9 treated with 0.14, 0.29, and 0.57 ml/kg b.w of RHB respectively; there was no significant (P < 0.05) decrease in the BGL in group 7 (79.25–75.50 mg/dl) but significant (P < 0.05) decreases in BGL were observed in groups 8 (84.33–70.33 mg/dl) and 9 (81.33–66.33 mg/dl). Therefore, the result showed that RHB has hypoglycemic/antidiabetic property.
      • Obasi D.C.
      • Ogugua V.N.
      GC-MS analysis, pH and antioxidant effect of Ruzu herbal bitters on alloxan-induced diabetic rats.
      Figure 2
      Figure 2Effect of Ruzu herbal bitters (RHB) on the fasting blood glucose levels (BGL) of each rat group. (∗ = indicates no significant (P < 0.05) difference for groups 2 to 6 compared to the control group and/or the baseline BGL (DAY 0) of groups 2 to 6 compared to that of other tested days during the experiment; ∗∗ and ∗∗∗ = indicate increased significant (P < 0.05) differences in the respective BGL of groups 2 to 6 compared to the control group and/or the baseline BGL (DAY 0) with days.) (For groups 7 to 9, ∗ and ∗∗ indicate significant (P < 0.05) differences in the baseline BGL (DAY 0) compared to that of other tested days during the experiment). DAY 0 = baseline blood glucose level (before induction of diabetes); DIABETES (DAY 0) = blood glucose level 3 days after induction of diabetes (alloxan administration). Group 1: normal control; Group 2: diabetic-untreated; Group 3: diabetic treated with 5 mg/kg b.w. of glibenclamide; Group 4: diabetic treated with 0.14 ml/kg b.w. of RHB; Group 5: diabetic treated with 0.29 ml/kg b.w. of RHB; Group 6: diabetic treated with 0.57 ml/kg b.w. of RHB; Group 7: nondiabetic treated with 0.14 ml/kg b.w. of RHB; Group 8: nondiabetic treated with 0.29 ml/kg b.w. of RHB; Group 9: nondiabetic treated with 0.57 ml/kg b.w. of RHB.

      Effect of Ruzu Herbal Bitters on Body Weight

      The effect of Ruzu herbal bitters (RHB) on the body weight (g) of the rats is presented in Figure 3. There was a significant (P < 0.05) increase in the body weight of the normal control group from 117.03 g on day 0–139.27 g on day 21 of the experiment. The body weight of the diabetic-untreated group decreased significantly (P < 0.05) from 104.57 g on day 0 before induction of diabetes to 75.10 g on day 21 of the experiment. On the contrary, there were significant (P < 0.05) increases in the body weights of the diabetic groups treated with different doses of RHB from day 0 to day 21, which are comparable to that observed in the diabetic group 3 treated with 5 mg/kg b.w. of glibenclamide. There were also significant (P < 0.05) increases in the body weights of the nondiabetic groups 7, 8, and 9 treated with different doses of RHB from day 0 to day 21 of treatment, which are comparable to that of the normal control. The result showed that RHB could help in weight gain in diabetics.
      • Obasi D.C.
      • Ogugua V.N.
      GC-MS analysis, pH and antioxidant effect of Ruzu herbal bitters on alloxan-induced diabetic rats.
      Figure 3
      Figure 3Effect of Ruzu herbal bitters (RHB) on the body weight of each rat group. (∗ = indicates no significant (P < 0.05) difference in the initial body weight of each rat group (DAY 0) compared to that of other tested days during the experiment; ∗∗ and ∗∗∗ indicate increased significant (P < 0.05) differences respectively, in the body weight of each rat group compared to the initial body weight (DAY 0) with days.) DAY 0 = initial body weight (before induction of diabetes); DIABETES (DAY 0) = body weight 3 days after induction of diabetes (alloxan administration). Group 1: normal control; Group 2: diabetic—untreated; Group 3: diabetic treated with 5 mg/kg b.w. of glibenclamide; Group 4: diabetic treated with 0.14 ml/kg b.w. of RHB; Group 5: diabetic treated with 0.29 ml/kg b.w. of RHB; Group 6: diabetic treated with 0.57 ml/kg b.w. of RHB; Group 7: nondiabetic treated with 0.14 ml/kg b.w. of RHB; Group 8: nondiabetic treated with 0.29 ml/kg b.w. of RHB; Group 9: nondiabetic treated with 0.57 ml/kg b.w. of RHB.

      Liver Function Parameters

      Table 2 shows the effects of Ruzu herbal bitters (RHB) on some serum liver function parameters of alloxan-induced diabetic and nondiabetic rats.
      Table 2Effects of Ruzu Herbal Bitters (RHB) on Some Serum Liver Function Markers of Alloxan-induced Diabetic Rats.
      Liver function markers expressed in mean ± S.D
      GROUPSGGT (U/L)ALP (U/L)ALT (U/L)AST (U/L)T. PROT (g/dl)ALB (mg/dl)T. BIL (umol/L)
      GROUP 13.26 ± 0.26∗57.65 ± 2.67∗30.30 ± 1.59∗35.83 ± 0.83∗6.41 ± 0.12∗∗∗4.00 ± 0.02∗∗∗0.70 ± 0.02∗
      GROUP 26.08 ± 0.24∗∗∗85.57 ± 1.72∗∗∗43.15 ± 0.50∗∗∗52.82 ± 2.48∗∗∗3.36 ± 0.17∗2.77 ± 0.04∗1.17 ± 0.03∗∗∗
      GROUP 33.32 ± 0.17∗60.56 ± 2.04∗31.33 ± 0.43∗38.17 ± 1.21∗6.140 ± 0.13∗∗∗3.94 ± 0.03∗∗∗0.75 ± 0.02∗
      GROUP 45.43 ± 0.19∗∗∗77.94 ± 1.10∗∗36.55 ± 0.47∗∗45.38 ± 0.68∗∗4.39 ± 0.10∗∗3.00 ± 0.03∗∗1.05 ± 0.02∗∗
      GROUP 54.06 ± 0.36∗∗71.51 ± 1.31∗∗34.03 ± 0.64∗∗41.13 ± 0.45∗∗5.11 ± 0.18∗∗3.59 ± 0.03∗∗0.90 ± 0.05∗∗
      GROUP 63.35 ± 0.12∗60.72 ± l.62∗31.60 ± 0.32∗38.98 ± 0.87∗6.38 ± 0.05∗∗∗3.96 ± 0.03∗∗∗0.77 ± 0.02∗
      GROUP 73.57 ± 0.25∗57.43 ± 1.2∗30.28 ± 1.25∗35.86 ± 0.56∗6.61 ± 0.11∗∗∗4.02 ± 0.01∗∗∗0.69 ± 0.02∗
      GROUP 83.60 ± 0.28∗57.02 ± 0.82∗30.20 ± 0.41∗36.08 ± 0.74∗6.64 ± 0.07∗∗∗4.03 ± 0.02∗∗∗0.69 ± 0.02∗
      GROUP 93.86 ± 0.14∗56.83 ± 0.87∗30.17 ± 0.37∗36.35 ± 0.44∗6.78 ± 0.14∗∗∗4.06 ± 0.02∗∗∗0.68 ± 0.02∗
      (∗ = indicates no significant (P < 0.05) difference in the tested rat groups (groups 2–9) compared to the control group (except for T. PROT and ALB with a reversal trend); ∗∗ and ∗∗∗ = indicate increased significant (P < 0.05) differences, respectively, in the tested rat groups compared to the control group (except for T. PROT and ALB which are compared to diabetes control group 2 with the lowest value)).
      GGT = gamma-glutamyl transferase, ALT = alkaline phosphatase, ALT = alanine aminotransferase, AST = aspartate aminotransferase, T. PROT = total protein, ALB = albumin, T. BIL = total bilirubin.
      Group 1: normal control; Group 2: diabetic - untreated; Group 3: diabetic treated with 5 mg/kg b.w. of glibenclamide.
      Group 4: diabetic treated with 0.14 ml/kg b.w. of RHB; Group 5: diabetic treated with 0.29 ml/kg b.w. of RHB.
      Group 6: diabetic treated with 0.57 ml/kg b.w. of RHB; Group 7: nondiabetic treated with 0.14 ml/kg b.w. of RHB.
      Group 8: nondiabetic treated with 0.29 ml/kg b.w. of RHB; Group 9: nondiabetic treated with 0.57 ml/kg b.w. of RHB.

      Gamma-glutamyl transferase (GGT) activity (U/L)

      There was a significant (P < 0.05) increase in the activity of GGT in the diabetic untreated group (6.08 ± 0.24) when compared with the normal control group (3.26 ± 0.26). There was a significant (P < 0.05) decrease in GGT activity in the diabetic groups 4 (5.43 ± 0.19), 5 (4.06 ± 0.36), and 6 (3.35 ± 0.12) treated with 0.14, 0.29, and 0.57 ml/kg b.w of RHB respectively, in a dose-dependent manner. The decrease in the activity of GGT observed in group 6 treated with 0.57 ml/kg b.w of RHB is comparable with that of diabetic group 3 treated with 5 mg/kg b.w. of glibenclamide (3.32 ± 0.17); both of which are not significantly (P < 0.05) different from that of the normal control group. The nondiabetic groups 7 (3.57 ± 0.25), 8 (3.60 ± 0.28), and 9 (3.86 ± 0.14) treated with 0.14, 0.29, and 0.57 ml/kg b.w of RHB respectively, showed a gradual increase in GGT activity in a dose-dependent manner, with no significant (P < 0.05) difference when compared to that of the normal control.

      Alkaline Phosphatase (ALP) activity (U/L)

      The activity of alkaline phosphatase (ALP) (U/L) increased significantly (P < 0.05) in the diabetic untreated group (85.57 ± 1.72) when compared with the normal control group (57.65 ± 2.67); whereas, there was a gradual decrease (P < 0.05) in ALP activity in the diabetic groups treated with different doses of RHB in a dose-dependent manner. The ALP activity in group 6 treated with 0.57 ml/kg b.w of RHB (60.72 ± 1.62) was comparable with that of diabetic group 3 treated with 5 mg/kg b.w. of glibenclamide (60.56 ± 2.04). There were no significant differences in the ALP activity of the nondiabetic groups treated with the different doses of RHB.

      Alanine Aminotransferase Activity (U/L)

      Alanine aminotransferase (ALT) activity increased significantly (P < 0.05) in the diabetic untreated group (43.15 ± 0.50) compared to that of the normal control group (30.30 ± 1.59). After treatment with different doses of RHB, ALT activity decreased significantly (P < 0.05) in the diabetic groups in a dose-dependent manner. The result of group 6 treated with 0.57 ml/kg b.w of RHB (31.60 ± 0.32) was comparable with that of diabetic group 3 treated with 5 mg/kg b.w. of glibenclamide (31.33 ± 0.43). There was no significant difference in the ALT activity of the RHB-treated nondiabetic groups 7 (30.28 ± 1.25), 8 (30.20 ± 0.41), and 9 (30.17 ± 0.37), which are comparable with that of the normal control group.

      Aspartate Aminotransferase Activity (U/L)

      The aspartate aminotransferase (AST) activity increased significantly (P < 0.05) in the diabetic untreated group (52.82 ± 2.48) compared to that of the normal control group (35.83 ± 0.83). The AST activity decreased significantly (P < 0.05) in the RHB-treated diabetic groups 4, 5, and 6 in a dose-dependent manner. The result of group 6 treated with 0.57 ml/kg b.w of RHB (38.98 ± 0.87) was comparable with that of diabetic group 3 treated with 5 mg/kg b.w. of glibenclamide (38.17 ± 1.21). There were no significant differences in the ALT activities of the RHB-treated nondiabetic groups 7 (35.86 ± 0.56), 8 (36.06 ± 0.74), and 9 (36.35 ± 0.44) when compared with that of the normal control group.

      Total Protein Concentration (g/dl)

      There was a significant (P < 0.05) decrease in the total protein (T. PROT) concentration observed in the diabetic untreated group (3.36 ± 0.17) compared to that of the normal control group (6.41 ± 0.12). However, a significant (P < 0.05) increase in total protein was observed in the RHB-treated diabetic groups in a dose-dependent manner. The total protein of group 6 treated with 0.57 ml/kg b.w of RHB (6.38 ± 0.05) was comparable with that of diabetic group 3 treated with 5 mg/kg b.w. of glibenclamide (6.14 ± 0.13). There was a gradual increase in the total protein concentrations of the RHB-treated nondiabetic groups 7 (6.61 ± 0.11), 8 (6.64 ± 0.07), and 9 (6.78 ± 0.14) in a dose-dependent manner when compared with that of the normal control group.

      Albumin Concentration (mg/dl)

      There was a significant (P < 0.05) decrease in albumin (ALB) concentration observed in the diabetic untreated group (2.77 ± 0.04) compared to that of the normal control group (4.00 ± 0.02). There was a significant (P < 0.05) increase in albumin concentration observed in the RHB-treated diabetic groups 4, 5, and 6 in a dose-dependent manner. The result of group 6 treated with 0.57 ml/kg b.w of RHB (3.96 ± 0.03) was comparable with that of diabetic group 3 treated with 5 mg/kg b.w. of glibenclamide (3.94 ± 0.03). There was a gradual increase in the albumin levels of the RHB-treated nondiabetic groups 7 (4.02 ± 0.01), 8 (4.03 ± 0.02), and 9 (4.03 ± 0.02) in a dose-dependent manner when compared with that of the normal control group.

      Total Bilirubin Concentration (umol/L)

      Total bilirubin (T. BIL) concentration increased significantly (P < 0.05) in the diabetic untreated group (1.17 ± 0.03) compared to that of the normal control group (0.70 ± 0.02). The concentration of bilirubin decreased significantly (P < 0.05) in the RHB-treated diabetic groups in a dose-dependent manner. There was no significant difference between the results of group 6 treated with 0.57 ml/kg b.w of RHB (0.77 ± 0.02) and that of diabetic group 3 treated with 5 mg/kg b.w. of glibenclamide (0.75 ± 0.02). There was also no significant difference in the bilirubin concentrations of the RHB-treated nondiabetic groups 7 (0.69 ± 0.02), 8 (0.69 ± 0.02), and 9 (0.68 ± 0.02), which were comparable with that of the normal control group.

      Histological Examination of the Liver

      The result of the effects of Ruzu herbal bitters (RHB) on the histology of the liver of the different groups of alloxan-induced diabetic and nondiabetic rats is presented in Figure 4 (Plates A – I).
      Figure 4
      Figure 4(a): The histology of liver sections of the normal control Group 1 (Plate A) showing normal histomorphology with normal hepatic lobules containing normal hepatocytes and normal structures of the portal triads—Hepatic vein (HV), Hepatic artery (HA), and Bile duct (BD) (H & E × 160). Central vein (CV). (b): The histology of liver sections of diabetic rats in the positive control Group 2 (Plate B) showing mild, diffuse vacuolar degeneration (black arrows) of hepatocytes and marked aggregations of inflammatory leukocytes around the portal areas (white arrows) (H & E × 400). Portal area (P). (c): The histology of liver sections of diabetic rats in Group 3 treated with 5 mg/kg b.w of glibenclamide (Plate C) showing multifocal individual necrosis of the hepatocytes with infiltration of mononuclear leukocytes (N). Mild to moderate aggregation of inflammatory leukocytes around the portal areas were also observed (H & E × 400). Hepatic vein (HV); Bile duct (BD). (d): The histology of liver sections of diabetic rats in Group 4 treated with 0.14 ml/kg b.w of Ruzu herbal bitters (Plate D) showing normal features of the hepatocytes, with mild widespread infiltrations of inflammatory leukocytes (arrows). (H & E × 160). Central vein (CV), Hepatic vein (HV); Hepatic artery (HA); Bile duct (BD). (e): The histology of liver sections of diabetic rats in Group 5 treated with 0.29 ml/kg b.w. ml/kg b.w. of Ruzu herbal bitters (Plate E) showing normal features of the hepatocytes with mild widespread infiltrations of inflammatory leukocytes (arrows). (H & E × 160). Central vein (CV), Portal area (P). (f): The histology of liver sections of diabetic rats in Group 6 treated with 0.57 ml/kg b.w. ml/kg b.w. of Ruzu herbal bitters (Plate F) showing normal features of the hepatocytes, with mild widespread infiltrations of inflammatory leukocytes (arrows). (H & E × 160). Central vein (CV), Portal area (P). (g): The histology of liver sections of nondiabetic rats in Group 7 treated with 0.14 ml/kg b.w. of Ruzu herbal bitters (Plate G) showing mild to moderate widespread infiltrations of inflammatory leukocytes (arrows). (H & E × 160). Central vein (CV). (h): The histology of liver sections of nondiabetic rats in group 8 treated with 0.14 ml/kg b.w. 0.29 ml/kg b.w. of Ruzu herbal bitters (Plate H) showing mild to moderate widespread infiltrations of inflammatory leukocytes (arrows). (H & E × 160). Central vein (CV), Portal area (P). (i): The histology of liver sections of nondiabetic rats in group 9 treated with 0.57 ml/kg b.w. of Ruzu herbal bitters (Plate I) showing mild to moderate widespread infiltrations of inflammatory leukocytes (arrows). (H & E × 160). Central vein (CV), Portal area (P).
      Figure 4
      Figure 4(a): The histology of liver sections of the normal control Group 1 (Plate A) showing normal histomorphology with normal hepatic lobules containing normal hepatocytes and normal structures of the portal triads—Hepatic vein (HV), Hepatic artery (HA), and Bile duct (BD) (H & E × 160). Central vein (CV). (b): The histology of liver sections of diabetic rats in the positive control Group 2 (Plate B) showing mild, diffuse vacuolar degeneration (black arrows) of hepatocytes and marked aggregations of inflammatory leukocytes around the portal areas (white arrows) (H & E × 400). Portal area (P). (c): The histology of liver sections of diabetic rats in Group 3 treated with 5 mg/kg b.w of glibenclamide (Plate C) showing multifocal individual necrosis of the hepatocytes with infiltration of mononuclear leukocytes (N). Mild to moderate aggregation of inflammatory leukocytes around the portal areas were also observed (H & E × 400). Hepatic vein (HV); Bile duct (BD). (d): The histology of liver sections of diabetic rats in Group 4 treated with 0.14 ml/kg b.w of Ruzu herbal bitters (Plate D) showing normal features of the hepatocytes, with mild widespread infiltrations of inflammatory leukocytes (arrows). (H & E × 160). Central vein (CV), Hepatic vein (HV); Hepatic artery (HA); Bile duct (BD). (e): The histology of liver sections of diabetic rats in Group 5 treated with 0.29 ml/kg b.w. ml/kg b.w. of Ruzu herbal bitters (Plate E) showing normal features of the hepatocytes with mild widespread infiltrations of inflammatory leukocytes (arrows). (H & E × 160). Central vein (CV), Portal area (P). (f): The histology of liver sections of diabetic rats in Group 6 treated with 0.57 ml/kg b.w. ml/kg b.w. of Ruzu herbal bitters (Plate F) showing normal features of the hepatocytes, with mild widespread infiltrations of inflammatory leukocytes (arrows). (H & E × 160). Central vein (CV), Portal area (P). (g): The histology of liver sections of nondiabetic rats in Group 7 treated with 0.14 ml/kg b.w. of Ruzu herbal bitters (Plate G) showing mild to moderate widespread infiltrations of inflammatory leukocytes (arrows). (H & E × 160). Central vein (CV). (h): The histology of liver sections of nondiabetic rats in group 8 treated with 0.14 ml/kg b.w. 0.29 ml/kg b.w. of Ruzu herbal bitters (Plate H) showing mild to moderate widespread infiltrations of inflammatory leukocytes (arrows). (H & E × 160). Central vein (CV), Portal area (P). (i): The histology of liver sections of nondiabetic rats in group 9 treated with 0.57 ml/kg b.w. of Ruzu herbal bitters (Plate I) showing mild to moderate widespread infiltrations of inflammatory leukocytes (arrows). (H & E × 160). Central vein (CV), Portal area (P).
      Group 1: The sections of the liver collected from the normal control group showed the normal hepatic histomorphology for the animals (Plate A). Normal hepatic lobules were also observed. The hepatic lobules contain normal hepatocytes arranged in cords around the central veins (CV), radiating toward the periphery of the lobules where it joins the structures of the portal areas. The hepatic cords are separated by normal-sized sinusoidal spaces. Normal structures of the portal triads [hepatic vein (HV), the hepatic artery (HA), and bile ducts (B)] were also observed.
      Group 2: Sections of the liver collected from the diabetic untreated group (Plate B) showed mild, diffuse vacuolar degeneration of the hepatocytes; the hepatocytes appeared swollen, partially occluding the adjacent hepatic sinusoids and containing tiny variably sized clear vacuoles in their cytoplasm (black arrows). Marked aggregations of inflammatory leukocytes around the portal areas (P, white arrows) were also observed.
      Group 3: In the case of the standard control, treated with 5 mg/kg b.w of glibenclamide (Plate C), multifocal individual necrosis of the hepatocytes with infiltration of mononuclear leukocytes (N) were observed. Mild to moderate aggregation of inflammatory leukocytes around the portal areas were also observed.
      Groups 4–6: The diabetic groups treated with 0.14, 0.29, and 0.57 ml/kg b.w. of Ruzu herbal bitters, respectively (Plates D, E, and F), showed normal features of the hepatocytes. However, there were mild widespread infiltrations of inflammatory leukocytes (arrows). This was suggestive of mild hepatitis.
      Groups 7–9: The nondiabetic groups also treated with 0.14, 0.29, and 0.57 ml/kg b.w. of Ruzu herbal bitters, respectively (Plates G, H, and I) showed mild to moderate widespread of infiltration of inflammatory leukocytes (arrows). This was suggestive of mild hepatitis. Multifocal areas of individual hepatocellular necrosis and apoptosis were also observed.

      Lipid Profile

      The results of the effects of Ruzu herbal bitters (RHB) on the serum lipid profile of alloxan-induced diabetic and nondiabetic rats is presented in Figure 4.

      Total Cholesterol (T. CHOL, TC) Concentration (mg/dl)

      The total cholesterol (T. CHOL, TC) concentration (mg/dl) increased significantly (P < 0.05) in the diabetic untreated group (240.75 ± 2.34) compared to that of the normal control group (158.41 ± 5.03). There was a significant (P < 0.05) decrease in TC concentration in the RHB-treated diabetic groups 4, 5, and 6 in a dose-dependent manner. There was no significant (P < 0.05) difference in the results of TC in group 6 treated with 0.57 ml/kg b.w of RHB (173.72 ± 2.78) and group 3 treated with 5 mg/kg b.w. of glibenclamide (177.38 ± 5.44). Likewise, there was no significant (P < 0.05) difference in the TC concentrations of the RHB-treated nondiabetic groups 7 (155.72 ± 3.30), 8 (153.49 ± 5.39), and 9 (152.96 ± 5.23) compared to that of the normal control group.

      Triglycerides (TG) Concentration (mg/dl)

      There was a significant (P < 0.05) increase in triglycerides (TG) concentration (mg/dl) observed in the diabetic untreated group (191.86 ± 2.80) compared to that of the normal control group (133.92 ± 2.51). However, a significant (P < 0.05) decrease was observed in the RHB-treated diabetic groups 4, 5, and 6 in a dose-dependent manner. The TG concentration of group 6 treated with 0.57 ml/kg b.w of RHB (134.67 ± 2.17) decreased significantly (P < 0.05) compared to that of diabetic group 3 treated with 0.5 mg/kg b.w. of glibenclamide (138.83 ± 2.35). There was a gradual decrease in the TG concentrations of the different doses of RHB-treated nondiabetic groups 7 (131.93 ± 2.90), 8 (131.22 ± 2.75), and 9 (127.70 ± 4.09) in a dose-dependent manner compared to that of the normal control group.

      High-Density Lipoprotein Cholesterol (HDL) Concentration (mg/dl)

      There was a significant (P < 0.05) decrease in the high-density lipoprotein cholesterol (HDL) concentration (mg/dl) in the diabetic untreated group (46.03 ± 2.44) compared to that of the normal control group (69.87 ± 1.63). Significant (P < 0.05) increases in HDL concentrations were observed in the RHB-treated diabetic groups 4, 5, and 6 in a dose-dependent manner. The HDL concentration of group 6 treated with 0.57 ml/kg b.w of RHB (63.03 ± 1.82) showed no significant difference with that of diabetic group 3 treated with 5 mg/kg b.w. of glibenclamide (62.13 ± 1.47). There was a gradual reduction with no significant (P < 0.05) difference in the HDL concentrations of the RHB-treated nondiabetic groups 7 (70.20 ± 1.52), 8 (71.53 ± 1.62), and 9 (71.78 ± 1.73) compared to that of the normal control group.

      Low-Density Lipoprotein Cholesterol (LDL) Concentration (mg/dl)

      The low-density lipoprotein cholesterol (LDL) concentration (mg/dl) increased significantly (P < 0.05) in the diabetic untreated group (156.35 ± 3.97) compared to that of the normal control group (61.76 ± 4.94). There was a significant (P < 0.05) decrease in LDL concentration in the RHB-treated diabetic groups 4, 5, and 6 in a dose-dependent manner. There was no significant (P < 0.05) difference in the LDL concentrations in group 6 treated with 0.57 ml/kg b.w of RHB (83.76 ± 3.19) and that of diabetic group 3 treated with 5 mg/kg b.w. of glibenclamide (87.48 ± 4.18). Likewise, there was no significant (P < 0.05) difference but a gradual decrease in the LDL concentrations of the RHB-treated nondiabetic groups 7 (59.14 ± 4.18), 8 (55.72 ± 5.41), and 9 (55.64 ± 4.47) when compared with that of the normal control group.

      Very Low-Density Lipoprotein Cholesterol (VLDL) Concentration (mg/dl)

      The VLDL concentration increased significantly (P < 0.05) in the diabetic untreated group (38.37 ± 0.56) compared to that of the normal control group (26.79 ± 0.50). There were significant (P < 0.05) decreases in the VLDL concentrations in the RHB-treated diabetic groups 4, 5, and 6 in a dose-dependent manner. The VLDL concentration in group 6 treated with 0.57 ml/kg b.w of RHB (26.94 ± 0.43) is significantly (P < 0.05) lower than that of diabetic group 3 treated with 5 mg/kg b.w. of glibenclamide (27.77 ± 0.46). There was a gradual decrease in the VLDL concentrations of the RHB-treated nondiabetic groups 7 (26.37 ± 0.58), 8 (26.24 ± 0.54), and 9 (25.54 ± 0.82) in a dose-dependent manner.

      Discussions

      Effect of RHB on the Blood Glucose Level

      Alloxan is a beta-cytotoxin shown to induce “chemical diabetes” (alloxan diabetes) in a wide variety of animal species by damaging the insulin-secreting β-cells of the pancreas.
      • Martinez J.A.
      • Milagro F.I.
      Effect of the oral administration of a b3- adrenergic agonist on lipid metabolism in alloxan diabetic rats.
       This causes damages in a large number of beta-cells, resulting in a decrease in endogenous insulin release. Alloxan administered rats, therefore, become hyperglycemic in a short period of time, followed by hepatic glucose overproduction.
      • Isitua C.C.
      • Akinyemi A.J.
      • Akharaiyi F.C.
      • Olubiyi O.O.
      • Anadozie S.O.
      • Olayide I.I.
      Antihyperglycemic and anti-hyperlipidemic effect of Herbamed, a herbal formulation in alloxan-induced diabetic rats.
       In this study, we observed a significant (P < 0.05) increase in the blood glucose levels of the rats after a single dose of 120 mg/kg body weight alloxan injection. The increase in glucose levels was a result of alloxan-induced reactive oxygen species, in addition to a simultaneous massive increase in cytosolic calcium concentrations that led to a rapid destruction of pancreatic islet cells and a concomitant reduction in the synthesis/release of insulin.
      • Etuk E.
      • Muhammed B.
      Evidence-based analysis of chemical method of induction of diabetes mellitus in experimental animals.
      ,
      • Adeyi A.
      • Idowu B.
      • Mafiana C.
      • Oluwalana S.
      • Ajayi O.
      Rat model of food-induced non-obese-type 2 diabetes mellitus: comparative pathophysiology and histopathology.
      The increase in blood glucose level in the alloxan-administered rats observed in our study is in agreement with that of Isitua et al who observed hyperglycemia in rats some period after alloxan administration.
      • Isitua C.C.
      • Akinyemi A.J.
      • Akharaiyi F.C.
      • Olubiyi O.O.
      • Anadozie S.O.
      • Olayide I.I.
      Antihyperglycemic and anti-hyperlipidemic effect of Herbamed, a herbal formulation in alloxan-induced diabetic rats.
       Agarwal et al and Emordi et al also discovered increased blood glucose levels in rats after alloxan administration.
      • Agarwal V.
      • Sharma A.K.
      • Upadhyay A.
      • Singh G.
      • Gupta R.
      Hypoglycemic effects of Citrullus colocynthis roots.
      ,
      • Emordi J.E.
      • Agbaje E.O.
      • Oreagba I.A.
      • Iribhogbe O.I.
      Antidiabetic effects of the ethanolic root extract of Uvaria chamae P. Beauv (Annonaceae) in alloxan-induced diabetic rats: a potential alternative treatment for diabetes mellitus.
      There was a significant (P < 0.05) increase in the blood glucose levels in the diabetic - untreated group after diabetes induction up to the last day of the experiment. However, treatment of the diabetic rats with different doses of Ruzu herbal bitters (RHB) resulted in significant (P < 0.05) reductions in the blood glucose levels in each (Figure 2). The reduction in blood glucose levels in diabetic rats treated with RHB could be due to the ability of the polyherbal mixture to arrest and reverse oxidative stress-induced destruction of pancreatic β-islet cells, thereby causing β-islet cells regeneration, insulin secretion, and consequently enhanced transport of blood glucose to peripheral tissues. Additionally, the hypoglycemic property of RHB may be due to the antioxidant and antibiotic properties of the various bioactive phytocompounds present in the different plant constituents of the mixture that work in synergy since Ruzu herbal bitters have been reported to contain some medicinally important phytochemicals such as flavonoids, alkaloids, steroids, phenols, tannins, and saponins.
      • Obasi D.C.
      • Ogugua V.N.
      • Obasi J.N.
      • Okagu I.U.
      Phytochemical, nutritional and anti-nutritional analyses of Ruzu herbal bitters.
      ,
      • Obasi D.C.
      • Ogugua V.N.
      GC-MS analysis, pH and antioxidant effect of Ruzu herbal bitters on alloxan-induced diabetic rats.
      Our finding agrees with other reports on the antidiabetic activities of polyherbal formulations on alloxan-induced diabetic rats.
      • Otunola G.A.
      • Afolayan A.J.
      Antidiabetic effects of combined spices of Allium sativvum, Zingiber officinale and Capsicum frutescens in alloxan-induced diabetic rats.
      ,
      • Isitua C.C.
      • Akinyemi A.J.
      • Akharaiyi F.C.
      • Olubiyi O.O.
      • Anadozie S.O.
      • Olayide I.I.
      Antihyperglycemic and anti-hyperlipidemic effect of Herbamed, a herbal formulation in alloxan-induced diabetic rats.
       Similarly, reduction in the blood glucose level was observed by Agarwal et al and Lakshmi et al who examined the effects of C. colocynthis root and seeds extracts, respectively, on alloxan-induced diabetic rats.
      • Agarwal V.
      • Sharma A.K.
      • Upadhyay A.
      • Singh G.
      • Gupta R.
      Hypoglycemic effects of Citrullus colocynthis roots.
      ,
      • Lakshmi B.
      • Sendrayaperumal V.
      • Subramanian S.
      Beneficial effects of Citrullus colocynthis seeds extract studied in alloxan-induced diabetic rats.
       It has been reported that U. chamae root extract caused a significant decrease in the concentration of blood glucose in alloxan-induced diabetic rats.
      • Emordi J.E.
      • Agbaje E.O.
      • Oreagba I.A.
      • Iribhogbe O.I.
      Antidiabetic effects of the ethanolic root extract of Uvaria chamae P. Beauv (Annonaceae) in alloxan-induced diabetic rats: a potential alternative treatment for diabetes mellitus.
       C. colocynthis and U. chamae are two of the three constituent plants in Ruzu herbal bitters. However, our result is in variance with another report by Omodamiro et al which concluded that “Ruzu herbal bitter possesses antimicrobial and hyperglycaemic effect, and as such may not be used in the management of diabetes in alloxan-induced diabetic rats but may be used in inhibition of growth of some microorganism”.
      • Omodamiro O.D.
      • Ukpabi-ugo J.C.
      • Obike C.A.
      • F Oledibe O.
      Evaluation of pharmacological potential of Ruzu herbal bitter in experimental animal model.
       The variations in these studies could be that the product used by Omodamiro et al (Ruzu herbal bitter) is a different product from ours (Ruzu herbal bitters). They bought theirs at Umuahia and we bought ours directly from the Head Office of the producer of RHB in Lagos, Nigeria.

      Effect of RHB on the Body Weight

      There was a steady body weight gain observed in the normal control group throughout the 21 days of the experiment; whereas, the diabetic-untreated group showed significant (P < 0.05) weight loss (Figure 3). However, treatment of the diabetic rat groups with different doses of RHB led to a significant (P < 0.05) increase in body weight. The nondiabetic groups treated with RHB also showed a consistent weight gain throughout the experiment. The result showed that diabetes mellitus causes a reduction in body weight, probably as a result of nutrient loss. Therefore, weight gain observed in the RHB-treated groups indicated that RHB contains essential and nonessential nutrients that can boost the body’s nutritional requirements for growth
      • Obasi D.C.
      • Ogugua V.N.
      • Obasi J.N.
      • Okagu I.U.
      Phytochemical, nutritional and anti-nutritional analyses of Ruzu herbal bitters.
      ,
      • Obasi D.C.
      • Ogugua V.N.
      GC-MS analysis, pH and antioxidant effect of Ruzu herbal bitters on alloxan-induced diabetic rats.
      . Increased weight gain after using combined spices for the treatment of diabetes has been reported.
      • Otunola G.A.
      • Afolayan A.J.
      Antidiabetic effects of combined spices of Allium sativvum, Zingiber officinale and Capsicum frutescens in alloxan-induced diabetic rats.
       It was also reported that C. colocynthis seeds extract caused weight gain in alloxan-induced diabetic rats.
      • Lakshmi B.
      • Sendrayaperumal V.
      • Subramanian S.
      Beneficial effects of Citrullus colocynthis seeds extract studied in alloxan-induced diabetic rats.

      Effect of RHB on Liver Function Parameters

      Diabetic untreated rats showed significant increases (P < 0.05) in serum activities of GGT, ALP, ALT, and AST compared to the normal control rats. Whereas a significant increase (P < 0.05) in the level of total bilirubin was observed in the diabetic untreated group (1.17 ± 0.03) compared to the normal control group (0.70 ± 0.02), there were significant decreases (P < 0.05) in the levels of total protein and albumin in the diabetic rats compared to that of the normal control group. Treatment of diabetic groups with different doses of RHB resulted in significant reductions in the serum activities of GGT, ALP, ALT, and AST. Significant reduction in serum total bilirubin level and elevated levels of total protein and albumin in a dose-dependent manner were also observed. There were no significant differences between the results of group 6 treated with 0.57 ml/kg b.w of RHB and that of diabetic group 3 treated with 0.5 mg/kg b.w. of glibenclamide. Hence, 0.57 ml/kg b.w Ruzu herbal bitters was found to be equipotent to 0.5 mg/kg b.w. of glibenclamide in the restoration of the liver biomarkers’ levels to normal, implying the normal functioning of the liver. There was also no significant difference in the results of the RHB-treated nondiabetic groups, which are comparable with that of the normal control group except for GGT and total protein results (Table 2). The findings of this study are in agreement with other findings on the antidiabetic activities of polyherbal formulations on alloxan-induced diabetic rats.
      • Otunola G.A.
      • Afolayan A.J.
      Antidiabetic effects of combined spices of Allium sativvum, Zingiber officinale and Capsicum frutescens in alloxan-induced diabetic rats.
      ,
      • Isitua C.C.
      • Akinyemi A.J.
      • Akharaiyi F.C.
      • Olubiyi O.O.
      • Anadozie S.O.
      • Olayide I.I.
      Antihyperglycemic and anti-hyperlipidemic effect of Herbamed, a herbal formulation in alloxan-induced diabetic rats.
       Similar finding was reported about C. colocynthis root on the liver function parameters of normal and alloxan-induced diabetic rats.
      • Agarwal V.
      • Sharma A.K.
      • Upadhyay A.
      • Singh G.
      • Gupta R.
      Hypoglycemic effects of Citrullus colocynthis roots.
       C. colocynthis seed extract also caused a significant decrease in the activities of serum ALP, ALT, and AST in alloxan-induced diabetic rats.
      • Lakshmi B.
      • Sendrayaperumal V.
      • Subramanian S.
      Beneficial effects of Citrullus colocynthis seeds extract studied in alloxan-induced diabetic rats.
       Another plant used in the formulation of RHB, U. chamae root extract was reported to have caused a significant decrease in the activities of serum ALP, ALT, and AST in alloxan-induced diabetic rats.
      • Emordi J.E.
      • Agbaje E.O.
      • Oreagba I.A.
      • Iribhogbe O.I.
      Antidiabetic effects of the ethanolic root extract of Uvaria chamae P. Beauv (Annonaceae) in alloxan-induced diabetic rats: a potential alternative treatment for diabetes mellitus.
       Furthermore, the hepatoprotective effect of Ruzu herbal bitters on high-fat diet was previously reported.
      • Ogunlana O.O.
      • Ogunlana O.E.
      • Ugochukwu S.K.
      • Adeyemi A.O.
      Assessment of the ameliorative effect of Ruzu herbal bitters on the biochemical and antioxidant abnormalities induced by high fat diet in Wistar rats.
      Alteration in the activity of serum enzymes in diabetic animals is directly linked to changes in the biochemical reactions/metabolism in which the enzymes mediate. The present study found increased activities of serum GGT, ALP, ALT, and AST, which indicated that hepatic dysfunction might be induced due to hyperglycemia in diabetic rats.
      • Sarfraz M.
      • Khaliq T.
      • Khan J.A.
      • Aslam B.
      Effect of aqueous extract of black pepper and ajwa seed on liver enzymes in alloxan-induced diabetic Wister albino rats.
       GGT and AST are sensitive indicators of hepatocellular damage. Their elevation in diabetic rats, as observed in this study, is indicative of severe damage to the liver. The reduction in the activities of these enzymes to near-normal levels in the RHB-treated diabetic rats indicates that the liver function was restored by the polyherbal mixture.
      • Otunola G.A.
      • Afolayan A.J.
      Antidiabetic effects of combined spices of Allium sativvum, Zingiber officinale and Capsicum frutescens in alloxan-induced diabetic rats.
       Elevations of ALT and AST enzyme activities are considered as evidence of hepatic damage. An increase of these enzyme activities is also associated with fatty liver disease and decreased hepatic insulin sensitivity in type 2 diabetes.
      • Schindhelm R.K.
      • Diamant M.
      • Dekker J.M.
      • Tushuizen M.E.
      • Teerlink T.
      • Heine R.J.
      Alanine aminotransferase as a marker of non-alcoholic fatty liver disease in relation to type 2 diabetes mellitus and cardiovascular disease.
       ALP in the liver is found to be associated with cell membrane that adjoins the biliary canaliculus, and so high plasma concentration of the liver isoenzyme indicates cholestasis rather than simply damage to the liver cells. Extremely high levels of ALP are also observed in biliary obstruction.
      • Mathur S.
      • Mehta D.K.
      • Kapoor S.
      • Yadav S.
      Liver function in type-2 diabetes mellitus patients.
      The liver synthesizes all the major blood proteins except the immunoglobulins. The total protein test gives an approximate measure of all plasma proteins (except fibrinogen when testing is on clotted samples).
      • Thompson J.
      Plasma protein tests: how to interpret abnormal results.
      Decreased plasma total protein concentration observed in the diabetic untreated group in our study may be due to the breakdown of proteins by the liver to amino acids and the subsequent utilization of their carbon skeletons in gluconeogenesis or conversion to intermediates of the citric acid cycle for energy generation. Albumin is only synthesized in the liver, and a low concentration of serum albumin occurs when the liver’s synthetic function is significantly impaired, for example, in long-standing liver disease or advanced cirrhosis.
      • Thompson J.
      Plasma protein tests: how to interpret abnormal results.
      High concentration of serum bilirubin causes jaundice, and it occurs in toxic or infectious disease of the liver, such as hepatitis or bile obstruction.
      • Edem D.O.
      • Usoh I.F.
      Biochemical changes in Wistar rats on oral doses of mistletoe (Loranthus micranthus).

      Effect of RHB on the Histology of the Liver

      The result of the liver histology (Figure 4) of the rats showed that alloxan damage of the pancreas induced oxidative stress via hyperglycemia resulting in the liver damage observed in the diabetic untreated rat group with diffuse vacuolar degeneration of the hepatocytes (Plate B). However, treatment of the diabetic rats with different doses of RHB resulted in the gradual restoration of the damaged liver to the normal features of the hepatocytes (Plates D, E, and F) when compared to that of the diabetic untreated group; although there was mild widespread infiltration of inflammatory leukocytes. Similar observations were observed in the RHB-treated nondiabetic groups (Plates G, H, and I). The therapeutic effect of RHB on the histology of the liver was better than that of the glibenclamide (Plate C). Restoration of damages in the liver through the reduction in the congestion, vacuolar degeneration of the hepatocytes, necrosis, and inflammatory cells is an indication that RHB has a hepatoprotective effect. Ruzu herbal bitters also produced significant hypoglycemic and antioxidant effects that restored the liver function and normalized the histopathological and biochemical abnormalities caused by the diabetes in alloxan-induced diabetic rats to the near-normal stage, as shown above by the decrease in the activities of GGT, ALP, AST, and ALT, with decreased bilirubin level, as well as elevation of total protein and albumin levels. However, it should be noted that a small magnitude of change in liver function markers may not have a significant biological effect on liver function. Hence, the liver has the ability to regenerate lost cells and regain normal function, provided that the administration of drugs or toxicants does not take longer than normal or overdosed.
      The incomplete restoration of the normal histology of the liver observed in the treated diabetic (Plates D, E, and F) and nondiabetic (Plates G, H, and I) rat groups, when compared to the normal control group (Plate A), could be as a result of the duration of treatment and probably as a result of dosage and the side effects from the mixture of the phytoconstituents of RHB. The glibenclamide-treated group also showed similar alteration in the liver histology. These histological effects on the liver of the nondiabetic rats treated with RHB could be as a result of stress to the liver on the course of detoxification of the active compounds contained in RHB. Similar results on decreased tissue damage in diabetic rats by apple cider vinegar
      • Omar N.A.A.
      • Allithy A.N.E.
      • El Sayed S.M.
      Hepatoprotective and antidiabetic effects of apple cider vinegar (A Prophetic Medicine Remedy) on the liver of male rats.
      and cinnamon extracts
      • Mhammad H.A.
      • Amad M.
      • Jubrail S.
      • Najeeb M.K.
      Impact of cinnamon extract on liver, kidneys and spleen of diabetic rats.
      have been reported.

      Effect of RHB on Lipid Profiles

      Our study has shown that total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and very-low-density lipoprotein cholesterol (VLDL-C) levels increased significantly (P < 0.05), while high-density lipoprotein cholesterol (HDL-C) was significantly (P < 0.05) lower in the diabetic untreated group compared to that of the normal control group (Figure 5). Treatment of diabetic groups with different doses of RHB resulted in significant (P < 0.05) reductions in the serum TC, TG, LDL-C, and VLDL-C levels, while a significant (P < 0.05) increase in HDL-C levels were observed. A higher hypolipidemic effect was found in group 6 treated with 0.57 ml/kg b.w of RHB than that of group 3 treated with 5 mg/kg b.w. of glibenclamide. Moreover, there were no significant (P < 0.05) differences in the results of the RHB-treated nondiabetic groups, which are comparable with that of the normal control group.
      Figure 5
      Figure 5Effects of Ruzu herbal bitters (RHB) on some serum Lipid Profile Markers of the different rat groups. (∗ = indicates no significant (P < 0.05) difference in the tested rat groups (groups 2–9) compared to the control group (except HDL with a reversal trend); ∗∗ and ∗∗∗ = indicate increased significant (P < 0.05) differences respectively, in the tested rat groups compared to the control group (except HDL, which is compared to diabetes control group 2 with the lowest value).) T. CHOL = total cholesterol, TG = triglycerides, HDL = high-density lipoprotein cholesterol, LDL = low-density lipoprotein cholesterol, VLDL = very-low-density lipoprotein cholesterol. Group 1: normal control; Group 2: diabetic - untreated; Group 3: diabetic treated with 5 mg/kg b.w. of glibenclamide; Group 4: diabetic treated with 0.14 ml/kg b.w. of RHB; Group 5: diabetic treated with 0.29 ml/kg b.w. of RHB; Group 6: diabetic treated with 0.57 ml/kg b.w. of RHB; Group 7: nondiabetic treated with 0.14 ml/kg b.w. of RHB; Group 8: nondiabetic treated with 0.29 ml/kg b.w. of RHB; Group 9: nondiabetic treated with 0.57 ml/kg b.w. of RHB.
      In diabetes, insulin deficiency causes increased blood glucose levels (hyperglycemia) and hypercholesterolemia, as was found in the diabetic control group in our study. This could be due to continuous actions of lipolytic hormones on adipose tissues and hormone-sensitive lipases on fats, leading to increased mobilization of free fatty acids. The excess fatty acids are transported to the liver, where they are converted into phospholipids and cholesterol. Dyslipidemia observed in the diabetic group was restored to near-normal levels in the RHB-treated groups. Our findings are consistent with other reports on dyslipidemia in diabetes mellitus.
      • Omar N.A.A.
      • Allithy A.N.E.
      • El Sayed S.M.
      Hepatoprotective and antidiabetic effects of apple cider vinegar (A Prophetic Medicine Remedy) on the liver of male rats.
      ,
      • Jain H.R.
      • Shetty V.
      • Singh G.S.
      • Shetty S.
      A study of lipid profile in diabetes mellitus.
      • Rai S.
      • Prajna K.
      • Rai T.
      Lipid profile in Type 2 diabetes mellitus and in diabetic nephropathy.
      • Bhowmik B.
      • Siddiquee T.
      • Mujumder A.
      • et al.
      Serum lipid profile and its association with diabetes and prediabetes in a rural Bangladeshi population.
       The antilipidemic effect of Ruzu herbal bitters on high-fat diet was also previously reported.
      • Ogunlana O.O.
      • Ogunlana O.E.
      • Ugochukwu S.K.
      • Adeyemi A.O.
      Assessment of the ameliorative effect of Ruzu herbal bitters on the biochemical and antioxidant abnormalities induced by high fat diet in Wistar rats.
      Lipid abnormalities in patients with diabetes, often termed “diabetic dyslipidemia,” are typically characterized by high total cholesterol (TC), high triglycerides (TG), low high-density lipoprotein cholesterol (HDL-C), and increased levels of small dense low-density lipoprotein (LDL) particles. Low-density lipoprotein cholesterol (LDL-C) levels may be moderately increased or normal.
      • Bhowmik B.
      • Siddiquee T.
      • Mujumder A.
      • et al.
      Serum lipid profile and its association with diabetes and prediabetes in a rural Bangladeshi population.
       Diabetic patients with complications also tend to have higher levels of TG, TC, LDL-C, and VLDL-C, with lower levels of HDL-C. This suggests that there appears to be some relation between the pathogenesis of various vascular complications (microvascular and macrovascular) and the presence of lipid abnormality.
      • Jain H.R.
      • Shetty V.
      • Singh G.S.
      • Shetty S.
      A study of lipid profile in diabetes mellitus.
       It is difficult to point out a particular factor as the cause as multiple mutually interacting factors determine the presence or development of these complications. As good control of diabetes is shown to keep the lipid levels in near-normal range, it appears important to aim at critical control of DM to prevent or at least postpone the onset of various complications.
      • Jain H.R.
      • Shetty V.
      • Singh G.S.
      • Shetty S.
      A study of lipid profile in diabetes mellitus.
      The altered lipid profile in T2DM is due to insulin resistance and defective insulin action on lipoprotein metabolism. Increased lipolysis will increase the synthesis of VLDL and triglyceride-rich LDL-C.
      • Rai S.
      • Prajna K.
      • Rai T.
      Lipid profile in Type 2 diabetes mellitus and in diabetic nephropathy.
       It will also increase triglyceride synthesis and promote the quick breakdown of HDL-C.
      • Trovati M.
      • Cavalot F.
      Optimization of hypolipidemic and antiplatelet treatment in the diabetic patients with renal disease.
       It is recognized that dyslipidemia is an independent risk factor for cardiovascular disease. Elevated blood glucose level combined with dyslipidemia increases atherosclerosis-related inflammation and makes it more extensive.
      • Taskinen M.R.
      • Borén J.
      New insights into the pathophysiology of dyslipidemia in type 2 diabetes.
       A larger extent of coronary artery calcification in asymptomatic patients with newly diagnosed T2DM has been demonstrated.
      • Mrgan M.
      • Funck K.L.
      • Gaur S.
      • Øvrehus K.A.
      • Dey D.
      • Kusk M.W.
      High burden of coronary atherosclerosis in patients with a new diagnosis of type 2 diabetes.
       Dyslipidemia is not only an important risk for macrovascular complications
      • Wu L.
      • Parhofer K.G.
      Diabetic dyslipidemia.
      ; studies have also shown the association of dyslipidemia with microvascular complications related to T2DM, namely diabetic retinopathy, diabetic nephropathy and diabetic neuropathy.
      • Rema M.
      • Srivastava B.K.
      • Anitha B.
      • Deepa R.
      • Mohan V.
      Association of serum lipids with diabetic retinopathy in urban South Indians - the Chennai Urban rural Epidemiology study (CURES) eye study-2.
      • Rutledge J.C.
      • Ng K.F.
      • Aung H.H.
      • Wilson D.W.
      Role of triglyceride-rich lipoproteins in diabetic nephropathy.
      • Al-Ani F.S.
      • Al-Nimer M.S.
      • Ali F.S.
      Dyslipidemia as a contributory factor in etiopathogenesis of diabetic neuropathy.
       Furthermore, our previous study showed that Ruzu herbal bitters possesses nephroprotective effect and reversed hematological disorders in diabetic rats.
      • Obasi D.C.
      • Ogugua V.N.
      Effect of Ruzu herbal bitters on the kidney function and hematological parameters of alloxan-induced diabetic rats.
      Our study showed that Ruzu herbal bitters (RHB) used in the treatment of various ailments has antidiabetic, hepatoprotective, and antihyperlipidemic effects. The reduction of blood glucose levels in diabetic rats treated with RHB could be due to the ability of the polyherbal mixture to arrest and reverse oxidative stress-induced destruction of pancreatic β-islet cells, thereby causing β-islet cells regeneration, insulin secretion, and consequently enhanced transport of blood glucose to peripheral tissues. Additionally, the antidiabetic, hepatoprotective, and antihyperlipidemic effects of RHB may be due to the antioxidant and antibiotic properties of the various bioactive phytocompounds present in the different plant constituents of the mixture that work in synergy. This showed that RHB contains bioactive compounds that could be pharmacologically important. It was also found that 0.57 ml/kg b.w. of RHB (equivalent to 40 ml/70 kg body weight daily) is equipotent with 5 mg/kg b.w of glibenclamide, a standard antidiabetic drug. However, further studies should be carried out to confirm the exact mechanism of actions of Ruzu herbal bitters and its possible side effects.

      CREDIT AUTHORSHIP CONTRIBUTION STATEMENT

      The authors carried out the following functions:
      1. Conception and design of the study: Dr. David C. Obasi and Prof. Victor N. Ogugua.
      2. Analysis and interpretation of data: Dr. David C. Obasi.
      3. Drafting the article: Dr. David C. Obasi.
      4. Revising it critically for important intellectual content: Prof. Victor N. Ogugua.
      5. Final approval of the version to be submitted: Dr. David C. Obasi and Prof. Victor N. Ogugua.

      Conflicts of interest

      The authors have none to declare.

      Acknowledgment

      We appreciate the management of Ruzu Natural Health Product and Services, Nigeria, for permitting us to use their product for this research.

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