Synergistic antidiabetic activity of Vernonia amygdalina and Azadirachta indica: Biochemical effects and possible mechanism

Journal of Ethnopharmacology 141 (2012) 878–887

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Synergistic antidiabetic activity of Vernonia amygdalina and Azadirachta indica: Biochemical effects and possible mechanism Item Justin Atangwho a,b,∗ , Patrick Ekong Ebong a , Eyong Ubana Eyong a , Mohd Zaini Asmawi b , Mariam Ahmad b a b

Department of Biochemistry, College of Medical Sciences, University of Calabar, P. M. B. 1115, Calabar, Nigeria School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden 11800, Penang, Malaysia

a r t i c l e

i n f o

Article history: Received 19 October 2011 Received in revised form 6 January 2012 Accepted 17 March 2012 Available online 10 April 2012 Keywords: Synergy Antidiabetic Oxidative stress attenuation Insulin mimetic ␤-Cell regeneration Vernonia amygdalina Azadirachta indica

a b s t r a c t Ethnopharmacological relevance: A decoction from a combination of herbs is commonly used in Traditional African Medicine for the management of chronic ailments. In Nigeria, the leaves of Vernonia amygdalina Del. (VA) and Azadirachta indica A. Juss (AI) are used traditionally as a remedy against diabetes mellitus for which empirical evidence attests to its efficacy. Aim of the study: To evaluate the synergistic antidiabetic action of VA and AI, the biochemical effects and possible mechanism in streptozotocin-induced diabetic rat (SDR) models. Materials and methods: Ethanolic extracts of VA and AI were co-administered (200 mg/kg, 50:50) to nondiabetic rats (NDRs) and SDRs for 28 days. Blood glucose and body weight were monitored during this period, and at end of treatment, serum glucose, insulin, triiodothyronine (T3), tetraiodothyronine (T4) and ␣-amylase activity were studied. Glucose and activities of antioxidant enzymes, e.g., catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), were estimated in hepatocytes, along with the impact on the histology of the liver and pancreas. Medium acting insulin, HU (5 IU/kg, s.c.) was used as a positive control. Results: The study reveals that compared with single extracts, the combined extract (VA/AI) promptly lowered blood glucose and maintained a relatively steady level over the study period, in tandem with HU. During this period, body weight gain successively increased. In SDRs, fasting blood glucose at days 0 and 28 was raised by 4.33 and 3.16 fold, respectively, and the serum glucose was raised by 7.70 fold vs. normal control (P < 0.05). The discrepancies in the individual effects of VA and AI on hepatic glucose and ␣-amylase activity were also restored. In NDRs, VA/AI lowered blood and serum glucose (1.14 and 1.94 fold, respectively), although to a lesser extent when compared with HU. Furthermore, VA/AI was found to lower serum insulin, T3 and T4 by 1.66, 1.57 and 2.16 fold, respectively, in SDR (P < 0.05). This was similar to HU, which demonstrated 1.79 and 1.68 fold reduction of insulin and T3, respectively (P < 0.05), but had no effect on T4. Conversely, in NDRs, VA/AI caused 1.32, 4.93 and 1.04 fold increase in insulin, T3 and T4, respectively, reciprocal to its effect on blood and serum glucose. Oxidative stress in SDR, characterised by decreased GPx and CAT activities, was ameliorated, as the activities of the enzymes and SOD increased following a 28-day treatment with VA/AI (P < 0.05). The features of diabetic pathology, indicated in the histology of the liver and pancreas, were reversed. However, the extent of recovery was partial with VA, better with AI, and distinct and total with VA/AI, compared with a null effect by HU. Conclusion: Taken together, our results contribute towards validation of enhanced antidiabetic efficacy of VA and AI when combined. This synergy may be exerted by oxidative stress attenuation, insulin mimetic action and ␤-cell regeneration. © 2012 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: AI, Azadirachta indica A. Juss; CAT, catalase; DC, diabetic control; FBG, fasting blood glucose; GIT, gastro-intestinal tract; GPx, glutathione peroxidase; HU, humulin (medium acting insulin, NPH); LWH, liver whole homogenate; NC, normal control; NDR, non diabetic rat; SDR, streptozotocin-induced diabetic rat; SOD, superoxide dismutase; STZ, streptozotocin; T3, triiodothyronine; T4, tetraiodothyronine; VA, Vernonia amygdalina Del.; VA/AI, combined extracts of VA and AI; H&E, heamatoxylin and eosin; ANOVA, analysis of variance; DVA, diabetic rat treated with VA extract; DAI, diabetic rat treated with AI extract; DVA/AI, diabetic rat treated with combined extracts of VA and AI; DHU, diabetic rat treated with humulin (insulin); NVA, non diabetic rat treated with VA extract; NAI, non diabetic rat treated with AI extract; NVA/AI, non diabetic rat treated with extracts of VA and AI; NHU, non diabetic rat treated with humulin (insulin); OS, oxidative stress; ROS, reactive oxygen species. ∗ Corresponding author. Tel.: +234 8051684035. E-mail addresses: [email protected], [email protected] (I.J. Atangwho), [email protected] (P.E. Ebong), [email protected] (E.U. Eyong), [email protected] (M.Z. Asmawi), [email protected] (M. Ahmad). 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.03.041

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1. Introduction The ‘silver bullet’ concept, which presupposes that a disease condition can only be corrected by a single agent, is increasingly viewed as inadequate in many clinical situations (Walker, 2007) because in orthodox medicine, a cocktail of drugs is now commonly employed against complex diseases such as HIV infection, cancer, hypertension and diabetes. Cumulative evidence from research with medicinal plants has also revealed that the basic supposition or notion that any plant possessing clinical effectiveness must contain an active principle that can completely replace the plant extract is not necessarily true (Phillipson, 2001). According to Gilbert and Alves (2003), this notion has to be modified in light of the findings that there are, in many cases, adjuvant substances in the plant that enhance the activity of the components that are actually responsible for the effect. Recently, the concept of synergism has become dominant in the pharmacology of medicinal plants, and the use of these plants is gaining reputation as a modern alternative to orthodox medicines or as complementary products to maintain health or treat aspects of diseases, particularly those in which orthodox medication has had only limited success (Houghton, 2009). Strictly speaking, synergism is defined by a combined effect of substances that is greater than would be expected from the individual contributions. In this context, the implication is that secondary metabolites in a plant extract, derived from their diverse chemistry, can act on different targets involved in the pathogenic process, to enhance overall therapeutic efficacy, i.e., pharmacodynamic synergy. Another consideration is the enhancement of biological activity by a combination of active compounds or extracts from different medicinal plants, and/or that the ingredients of the plant extract with little or no direct activity on the pathogenic process may assist the ‘actives’ to reach the target, either by improving bioavailability or decreasing metabolism and excretion of the active principle, i.e., pharmacokinetic synergy (Rasoanaivo et al., 2011). This principle lends credence to the use of a mixture of several herbs in traditional medicine such as in Traditional Chinese Medicine (TCM) to achieve an overall therapeutic biological effect. Synergism has also been largely supported and expounded by a recently identified key feature of drug-target interactions known as polypharmacology, or drug promiscuity, where drugs with multiple target network mechanisms are believed to have enhanced safety and efficacy in disease treatment (Takigawa et al., 2011). It is, however, too simplistic to assume a positive synergy from any two or more herbs combined in traditional practice, as some can be equally harmful. An unexpected decrease in activity, sometimes called ‘negative synergy’ or ‘antagonism’ may also occur, particularly in certain interactions between orthodox medication and herbal products (Barnes et al., 2002). These techniques and approaches are still in their infancy, or according to Mukherjee et al. (2011), we are only at the beginning of an interesting new research field that should shed light on how these remedies work, and ultimately result in reduced side effects and better therapeutic success. Hence, these strategies warrant in-depth research and investigation. Azadirachta indica A. Juss (AI) is a member of the Meliacae family that is predominantly found in the Asian subregion but is also grown in Nigeria and other parts of Africa, where it is functionally used as an antidiabetic medicinal plant. Extracts from Azadirachta indica, locally called “dogonyaro” in Nigeria, are also used to treat gastrointestinal upset, diarrhoea and intestinal infections, skin ulcers and infections and malaria (Anyaehie, 2009). The hypoglycaemic action of its leaves, stem and bark and seed oils as well as other medicinal uses of AI and pharmacologically reported biological actions have been articulated in a review by Biswas et al. (2002). Vernonia amygdalina Del. (VA), commonly known as African

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bitter leaf, is a vegetable of the Compositae family that grows in the African tropics, particularly (but not exclusively) in Nigeria, Cameroon and Zimbabwe and enjoys a very high reputation in traditional medicine as a remedy against several ailments, including bacterial infection, GIT disorders, liver diseases, kidney problems, malaria, infertility, nausea, helmintic diseases, diabetes and hypertension (Abo et al., 2008; Farombi and Owoeye, 2011). Since the maiden report of Akah and Okafor (1992) demonstrating its glucose lowering action, a number of studies have shown that VA possesses antidiabetic properties. However, none of these reports have considered a combined effect with other plants, a common practice in traditional medicine. In fact, in a recent inventory survey of antidiabetic medicinal plants in select districts of Lagos, Nigeria of the 49 plants identified by 100 respondents taken in 50 different recipes, 33 (66%) of the 50 recipes were a combination of herbs. It was also interesting that of the 33 combinations, 14 (42.42%) included VA (Gbolade, 2009). Accordingly, a preliminary investigation of the combined extracts from these two plants suggested a possible synergy in lowering blood glucose of alloxan diabetic rat models treated for 24 days (Ebong et al., 2008). The present study was a 28-day investigation into the reported synergistic antidiabetic action of Vernonia amygdalina Del. and Azadirachta indica A. Juss in streptozotocin diabetic rat models with the aim of elucidating the biochemical effects and possible mechanism by which the synergy is achieved.

2. Materials and methods 2.1. Plant materials Leaves of VA and AI respectively collected from the Endocrine Research Farm and the staff village, University of Calabar, were authenticated by Pastor Frank, a botanist in the Department of Botany, and the voucher specimens (Vernonia amygdalina – ERU/2011/188; Azadirachta indica – ERU/2011/354) were deposited at the same department.

2.2. Preparation of plant extracts One kilogram (1 kg) each of AI and VA leaves was homogenised in 1.95 and 2.25 l of 80% (v/v) ethanol, respectively. The mixtures were incubated for 48 h in the refrigerator at 4 ◦ C and filtered first with a cheesecloth and then with Whatman No. 1 filter paper. The filtrates were concentrated in vacuo (37–40 ◦ C) to 1/10th the original volume using a rotary evaporator. The concentrates were lyophilised to complete dryness, yielding 40.54 g (4.054%) and 34.71 g (3.471%) of greenish brown and brownish substances for VA and AI, respectively, hereinafter referred to as ‘extracts’.

2.3. Experimental animals Sixty male Wistar rats (140–180 g), obtained from the Department of Zoology and Environmental Biology, University of Calabar, were acclimatised in the animal house of the Department of Biochemistry, where the experiment was performed. Adequate environmental conditions were provided (temperature, 25 ± 5 ◦ C; relative humidity, 50 ± 5% and 12 h light/dark cycle). The animals were maintained on palletised Growers feed (Vital Feeds, Jos, Plateau State, Nigeria) and tap water ad libitum. The National Institutes of Health Principles of Laboratory Animal Care (1985) were observed and the protocol of the experiment was duly approved by the College of Medical Sciences’ Animal Ethics Committee, University of Calabar.

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2.4. Diabetes induction Overnight-fasted rats were induced with diabetes by an injection of a single intraperitoneal dose of streptozotocin (65 mg/kg) (Sigma, St. Louis, MO, USA), freshly reconstituted in cold normal saline. Fasting blood glucose (FBG) was measured 7 days after the injection using a One Touch® Glucometer (Lifescan, Inc. 1995 Milpas, California, USA) with blood obtained from a tail vein puncture. Rats with FBG ≥ 200 mg/dl but ≤500 mg/dl were included in the study. 2.5. Experimental design and protocol The 60 rats (30 diabetic and 30 non-diabetic) were divided into 5 parallel groups consisting of a diabetic and non-diabetic pair of 6 animals each (Table 1). The diabetic and non-diabetic animals were accordingly treated with extracts and insulin for 28 days, as shown in Table 1. The dosages of the plant extracts were determined from preliminary dose–response work in our laboratory (data not shown), whereas the insulin dose, HU (5 IU/kg b.w.), was as previously used by Sonia and Srinivasan (1999). The plant extracts were administered via oral gavage, 12 h apart (6:00 a.m. and 6:00 p.m.), and insulin was administered subcutaneously, once per day, postprandially (6:00 p.m.) for the 28 days. Blood glucose and body weight during the study were measured on alternate days, and these measurements were taken 30 min before the administration of the first extract of the day. At end of the study, blood samples, the pancreas, and the liver were collected for biochemical and histological analyses. 2.6. Biochemical analyses Glucose, insulin, triiodothyronine (T3), tetraiodothyronine (T4) and ␣-amylase activities were assayed in the serum. Analytical kits for glucose and ␣-amylase activities were purchased from Randox Laboratories Ltd. (Admore Diamond Road, Crumlin, Co., Antrim, United Kingdom) and rat insulin ELISA kits for hormonal assays were purchased from DialabProduktion und laborinstriementenGessellschaftm.b.H. (A-1160 Wienpanikengasse, Germany). The livers were immediately perfused in heparinised saline (0.9% NaCl), blotted with blotting paper and stored frozen until further use. Exactly 1 g of the liver was homogenised in 10 ml of freshly prepared phosphate buffer (20 mM; pH 7.4) and centrifuged at 3000 × g for 10 min. The supernatant was decanted into clean tubes (liver whole homogenate, LWH) and used for glucose, SOD, GPx and CAT assays. The assay kits for the oxidative stress enzymes were obtained from OxisResearchTM (323 Vintage Park Drive, Suite B Foster City, CA, USA). 2.7. Histopathological studies The pancreas and a portion of the liver were fixed in 10% formalin for 7 days. The tissues were sectioned (5 ␮m thickness) and stained with haematoxylin and eosin (H&E). Sections of the pancreas were also specifically stained for ␤-cells by the aldehyde fuschin procedure (Gomori, 1950), and photomicrographs were taken (400×) as shown below. 2.8. Statistical analysis The results are expressed as the mean ± SEM. The results were analysed for statistical significance by one way ANOVA using the SPSS statistical program and Post Hoc Test (LSD), and differences were considered significant at P < 0.05.

Fig. 1. (a) 28-Day time-course variation in blood glucose of NDRs treated with extracts or HU. Values are the mean ± SEM, n = 5–6. (b) 28-Day time-course variation in blood glucose of SDRs treated with extracts or HU. Values are the mean ± SEM, n = 5–6, *P < 0.05 vs. treated groups.

3. Results 3.1. Time-course blood glucose variation The effects of the treatments on blood glucose in NDRs and SDRs, monitored for 28 days, are shown in Fig. 1a and b respectively. Overall, the 4 treatments lowered blood glucose of SDRs relative to the DCs throughout the study duration (P < 0.05). However, the decrease caused by VA/AI and HU commenced earlier (3 days after), unlike VA and AI alone, which began after 7 days of administration (Fig. 1b). The effects of VA/AI and HU, on both NDRs and SDRs were quite similar and depicted a steady regulation of blood glucose.

3.2. Time-course body weight variation Fig. 2a and b shows the respective effects of treatments on the body weight of NDRs and SDRs measured during the 28-day study. Body weights successively increased over time in NDRs. However, treatment with VA extract successively reduced the gain in body weight over time, compared with the controls, and finally resulted in a decrease by the end of the study (P < 0.05). This effect of VA was not observed when co-administered with AI. Untreated SDRs clearly showed a sustained decrease (P < 0.05) in body weight over the 28-day period, which was the opposite of the observed changes in blood glucose (Fig. 2b). Treatment with extracts of VA, AI, and

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Table 1 Animal grouping and treatment scheme. Group

Treatment (diabetic rats, SDRs)

1 2 3 4

DC DVA DAI DVA/AI

5

DHU

Treatment (non-diabetic rats, NDRs)

Placebo (diabetic control) Vernonia amygdalina extract (200 mg/kg) Azadirachta indica extract (200 mg/kg) Vernonia amygdalina and Azadirachta indica combined extracts (100 mg/kg each; 50:50) Insulin (5 unit/kg)

NC NVA NAI NVA/AI

Placebo (diabetic control) Vernonia amygdalina extract (200 mg/kg) Azadirachta indica extract (200 mg/kg) Vernonia amygdalina and Azadirachta indica combined extracts (100 mg/kg each; 50:50) Insulin (5 unit/kg)

NHU

VA/AI, as well as HU, elicited a gradual increase in body weight, relative to DC, until the end of the study (P < 0.05). 3.3. Glucose in compartments and serum ˛-amylase activity The effect of the treatments on measured glucose and ␣-amylase activity of NDRs and SDRs are shown in Tables 2 and 3, respectively. Seven days after streptozotocin (STZ) injection, a 4.33 fold increase in FBG was observed compared with NC. After a 28-day saline treatment, FBG, serum and hepatic glucose of the DCs were 3.16, 7.70 and 7.75 fold higher than NC values, respectively. ␣-Amylase activity was 1.38 fold lower compared with NCs. Treatment with VA, AI, VA/AI, and HU, however, lowered the fasting glucose (P < 0.05). In NDRs, HU lowered blood and serum glucose levels by 1.42 and 3.86 fold compared with NC values, respectively (P < 0.05). Similarly VA/AI caused a 1.14 and 1.94 fold reduction in the blood and serum glucose levels of NDRs, respectively. The combined extract also modulated the observed discrepancies in the individual effects of VA and AI on LWH glucose. Administration of VA, AI and VA/AI extracts to SDRs caused an observed increase in ␣-amylase activity compared with DCs (P < 0.05), but only a mild increase in the HU-treated rats. However, HU lowered enzyme activity in SDRs compared with NC-and extract-treated groups (P < 0.05). 3.4. The effect on select hormones

Fig. 2. (a) 28-Day time-course body weight variation of NDRs treated with extracts or HU. Values are the mean ± SEM, n = 5–6. (b) 28-Day time-course variation in body weight of SDRs treated with extracts or HU. Values are the mean ± SEM, n = 5–6, *P < 0.05 vs. treated groups.

The results of serum insulin, T3 and T4 levels measured at the end of the experiment are shown in Tables 4 and 5. Compared to DCs, VA/AI lowered serum insulin, T3 and T4 levels by 1.66, 1.57 and 2.16 fold, respectively, in SDRs (P < 0.05). This was similar to HU treated rats that had a 1.79 and 1.68 fold reduction (P < 0.05) in insulin and T3 levels, but no effect on T4 levels. Conversely, in NDRs, VA/AI caused a 1.32, 4.93 and 1.04 fold increase in serum insulin, T3 and T4 levels, respectively, relative to NCs, which was reciprocal to its effect on blood and serum glucose levels. However, a 28-day HU administration in NDRs exerted a null effect on the hormones. Extracts of VA and AI, when co-administered, may mimic HU in its effect on the measured hormones.

Table 2 Effect of treatment on glucose levels in blood, serum, LWH and serum ␣-amylase of non-diabetic rats (NDRs) treated with extracts or HU. Group

Fasting blood glucose (mg/dl) Initial (day-0)

NC NVA NAI NVA/AI NHU

54.60 74.00 78.40 73.00 69.40

Mean ± SEM, n = 6. * P < 0.05. a P < 0.05 vs. initial. b P < 0.05 vs. NC. c P < 0.05 vs. VA/AI. d P < 0.05 vs. HU.

± ± ± ± ±

.29 8.57 8.83 .93 6.19

Serum glucose (mg/dl)

Hepatic glucose (mg/g tissue)

Serum ␣-amylase (U/I)

Final (day-28) 49.60 52.00 43.00 43.40 35.00

± ± ± ± ±

3.49 3.10* , c , d 1.84d 2.32d 0.95a

28.49 26.70 15.28 14.69 7.38

± ± ± ± ±

3.84 10.63d 4.45d 4.18d 3.24b

28.19 82.65 39.14 26.55 28.69

± ± ± ± ±

10.16 20.75b , c , d 13.58 5.94 7.16

452.47 444.60 424.93 437.37 263.60

± ± ± ± ±

27.79 27.66 7.35 17.24 24.43b , c

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Table 3 Effect of treatment on glucose levels in blood, serum, LWH and serum ␣-amylase of STZ diabetic rats (SDRs) treated with extracts or HU. Group

Fasting blood glucose (mg/dl) Initial (day-0)

NC DC DVA DAI DVA/AI DHU

54.60 236.25 260.75 255.00 260.00 268.00

± ± ± ± ± ±

Serum glucose (mg/dl)

Hepatic glucose (mg/g tissue)

Serum ␣-amylase (U/I)

Final (day-28)

2.29 33.94 (4.33 fold↑) 38.38 45.64 56.26 65.01

49.60 206.75 77.75 92.25 101.00 90.00

± ± ± ± ± ±

3.49 34.57 (3.16 fold↑) 13.77b , a 17.44b , a 27.83b , a 27.28b , a

28.49 219.37 77.12 72.32 69.76 84.60

± ± ± ± ± ±

3.84 21.93 (7.70 fold↑) 7.31a 21.90a 11.69a 6.56a

28.19 218.52 205.37 113.33 193.83 153.63

± ± ± ± ± ±

10.16 7.99 (7.75 fold↑) 25.62 53.01* , a 29.70 36.03

452.47 328.13 663.14 520.13 554.96 417.98

± ± ± ± ± ±

27.79 10.65 45.73a 85.84a 56.71a 6.77

Mean ± SEM, n = 6. ↑ = wrt NC. * P < 0.05 vs. VA. a P < 0.05 vs. DC. b P < 0.05 vs. initial. Table 4 Effect of treatments on select hormonal indices in non diabetic rats (NDRs) treated for 28 days. Group/treatment

Insulin (U/ml)

NC NVA NAI NVA/AI NHU

17.62 20.40 20.46 23.25 17.33

± ± ± ± ±

2.76 2.16 2.24 1.93 0.67

T3 (ng/ml) 0.88 3.89 7.95 4.34 0.88

± ± ± ± ±

0.14 1.85 0.27b 2.15 0.11

T4 (ng/ml) 40.00 39.40 33.83 41.54 35.54

± ± ± ± ±

2.78 2.19 3.09c 2.24 1.13

hepatocytes showing distinct single or polynuclei (Fig. 4a1 ). These STZ inflicted lesions were partially reversed by VA extract (indistinct nuclei of the outlined hepatocytes) (Fig. 4b2 ). Extract from AI resulted in better improvement compared with VA extract (distinct hepatocytes together with their nuclei) implying an increase in activity of the cells (Fig. 4c2 ). However, VA/AI restored the

Mean ± SEM, n = 6. b P < 0.05 vs. NC. c P < 0.05 vs. NVA/AI.

3.5. The effect of extracts or HU on oxidative stress in hepatocytes The effect of treatments for 28 days on oxidative stress indices (CAT, GPx and SOD) is shown in Fig. 3a–c. Treatment with VA/AI was found to increase GPx activity relative to both NCs and DCs (P < 0.05), similar to the effect of HU. GPx activity was also increased in NDRs, which received a treatment with the combined extract when compared to the NCs. Furthermore, the study revealed a higher SOD activity in SDRs treated with VA/AI and HU compared with DCs (P < 0.05). The extracts or HU had no obvious effect on SOD activities in NDRs. Catalase activity, which was lower in DC rats compared with NCs (P < 0.05), was increased after 28 days of treatment with VA, AI, VA/AI and HU (P < 0.05); the increase compared well with NCs. Similar to SOD, there were no significant changes in CAT activity in NDRs. 3.6. The effects of extracts or HU on liver histology The cellular integrity of the hepatocytes, as examined in this study, revealed cell sequestration, indistinct cell nuclei, hepatocyte degeneration and reduction in number of nuclei following STZ treatment (Fig. 4a2 ). In comparison, the histology of the NCs showed distinct lobulation, central vein and well stained Table 5 Effect of treatments on select hormonal indices in STZ diabetic rats (SDRs) treated for 28 days. Group/treatment

Insulin (U/ml)

DC NC DVA DAI DVA/AI DHU

16.25 17.62 20.25 14.67 9.63 9.10

Mean ± SEM, n = 6. a P < 0.05 vs. DC. c P < 0.05 vs. DVA/AI. d P < 0.05 vs. DHU.

± ± ± ± ± ±

2.32 2.76 2.72c , d 3.32 2.73 1.93

T3 (ng/ml) 1.38 0.88 1.18 0.65 0.88 0.82

± ± ± ± ± ±

0.55 0.14a 0.16 0.09 0.10a 0.04a

T4 (ng/ml) 36.63 40.00 35.56 36.30 16.98 38.10

± ± ± ± ± ±

2.82 2.78 3.14c 5.50c 7.41a , d 2.56

Fig. 3. (a) Effect of 28-day treatment of extracts or HU on glutathione peroxidase (GPx) activity in hepatocytes of NDRs and SDRs. Values are the mean ± SEM, n = 6, *P < 0.05 vs. DC, a P < 0.05 vs. NC, b P < 0.05 vs. HU. (b) Effect of 28-day treatment of extracts or HU on superoxide dismutase activity in hepatocytes of NDRs and SDRs. Values are the mean ± SEM, n = 6, *P < 0.05 vs. DC. (c) Effect of 28-day treatment of extracts or HU on catalase (CAT) activity in hepatocytes of NDRs and SDRs. Values are the mean ± SEM, n = 6, *P < 0.05 vs. DC.

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Fig. 4. Photomicrograph of histology of liver of NDR (1) and SDR (2) showing effect of 28-day treatment with VA (b), AI (c), VA/AI (d) and HU (e) (400×). CV = central vein, RH = radiating hepatocytes, DCV = degenerated central vein, DS = disoriented sinusoid.

diabetes-induced lesions; hence, the histology is similar to that of the NCs (Fig. 4d2 ). These features were similar to those of insulintreated rats (Fig. 4e2 ). In the NDRs, the VA extract showed features of mild injury (fairly indistinct cell outlines and non-prominent nuclei) (Fig. 4b1 ). This was, however, not the case with AI extract treatment, which presented a better histological architecture than did the NCs (Fig. 4c1 ). Combined extracts of VA and AI restored the histology to a normal phenotype, as no features of injury were observed (Fig. 4d1 ). Overall, the extent of reversal/recovery

was partial with VA, better with AI, and distinct and total with a combination of VA and AI extracts. 3.7. The effects of treatments on the histology of the pancreas The study examined the histology of NDRs and SDRs pancreases, a plausible site of action of antidiabetic agents. Untreated SDRs (Fig. 5a2 ), which presented with damaged islets, were markedly reduced in mass. Less than 20% of the cells survived, and the

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Fig. 5. Photomicrograph of histology of pancreas of NDR (1) and SDR (2) showing effect of 28-day treatment with VA (b), AI (c), VA/AI (d) and HU (e) (400×). IL = islet of Langerhan, DIL = degranulated islet of Langerhan, DAC = degenerated acid cells, H&E = haematoxylin and eosin staining, G = Gomori staining, EA = extra pancreatic acini.

observed infiltration of lymphocytes resembled general fibrosis, unlike the NCs pancreases, which showed preserved (numerous) islets. The cell mass was devoid of fibrosis, and the islets were widely distributed throughout the exocrine pancreas, which demonstrated well stained nuclei (Fig. 5a1 ). Again, the VA extract caused a partial recovery from the damage to islet cells showing

a moderate reduction in islet cell mass and fibrosis (Fig. 5b2 ). A more prominent recovery was produced by the AI extract; islet cell mass was preserved and devoid of islet fibrosis (Fig. 5c2 ). The most prominent islet-cell recovery was from the combined extracts of VA and AI (Fig. 5d2 ). This was not significantly different from the NCs. Treatment with HU could not reverse the damaged islets,

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which was similar to the diabetic controls (Fig. 5e2 ). No significant changes in pancreas histologies were observed in the non-diabetic test animals. As with the liver histology, treatment with the extract reversed the lesions produced by STZ administration, a possible effect of islet cell regeneration. However, the extent of reversal and recovery was partial with VA extract, better with AI and distinct and total with VA/AI. 4. Discussion In diabetes mellitus, the integrated hormonal regulation of blood glucose within physiological limits usually deteriorates to precipitate chronic hyperglycaemia (Becon-McBride, 1992; Champe et al., 2005). Therapeutic efforts have first tried to reestablish this fine control. Accordingly, a 28-day variation in blood glucose was monitored in this study. It was observed that VA/AI promptly reduced blood glucose and maintained a variation curve similar to that maintained by treatment with HU, indicating a tendency towards fine hormonal regulation by the intervention. This effect, not observed with the administration of VA or AI alone, suggests a boost in the content or potency of the insulin-like action of the two extracts when combined, possibly through a synergy. Many such antidiabetic herbs with similar effects to insulin have been reported (Yeh et al., 2003). The prompt blood glucose decrease by only VA/AI and HU may buttress this position because the action of insulin, whether exogenous or endogenous, is usually instantaneous for glucose uptake (Gaw et al., 1995). Insulin mimetics modulate membrane receptors to enhance peripheral glucose entry into tissues or insulin sensitivity (Yeh et al., 2003). It is not likely that functional cellular regeneration of ␤-cells could occur within the first few days, hence excluding the option of endogenous insulin secretion. Untreated animal models of diabetes are characterised by tissue wasting (Ahmed et al., 2005). In our model, DC showed a 28-day successive reduction in body weight, reciprocal in direction to blood glucose, clearly indicating that the deterioration in the glucose control mechanism progresses in stages and would probably climax in the death of the animal if left untreated. The intervention enhanced tissue access to glucose, both to supply energy and to build tissue materials needed for growth, resulting in the observed stepwise appreciation in body weight. STZ injection is known to increase FBG (Sonia and Srinivasan, 1999), serum glucose (Mahdi et al., 2003) and hepatic glucose levels (Ugochukwu and Babady, 2003) by several folds, as was observed in the present study. FBG and serum glucose levels were found to be decreased upon intervention, in accordance with our earlier report (Ebong et al., 2008). This previous study, however, did not measure the effect of the combined extracts on hepatic glucose, hormones, antioxidant enzymes, or the impact on liver and kidney histology because it was not focused on mechanism. In the present study, it was observed for the first time that VA/AI also decreased hepatic glucose levels. Glucose transport across hepatic cell membranes is regulated by the glucokinase and glucose transporter 2 (GLUT 2) systems and the overall glucose tide in the blood (Meyes, 2003). Therefore, the hepatic glucose reducing effect may also suggest a possible glucokinase modulating action of the present treatment. For instance, the Gongronema latifolium an antidiabetic herb is known to reduce hepatic glucose levels, but increase glucokinase activity in SDR (Ugochukwu and Babady, 2003). However, the most striking observation in the present study is the alternate actions of VA and AI on hepatic glucose levels that were modified when combined, to produce a comparable effect with HU and the NCs, suggesting a synergistic action in hepatic glucose regulation. Serum ␣-amylase activity, most commonly utilised for the detection of pancreatic disorders, was reported to increase in

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alloxan-induced diabetic rats (Ebong et al., 2006). The present study showed a contrasting 1.38 fold decreased activity in untreated SDRs. STZ is more selective in attacking ␤-cells than alloxan (Rodrigues et al., 1999), and as such, may spare the exocrine pancreas via minimal cross cell reaction to account for the low serum activity. Moreover, chronic hyperglycaemia is known to overwhelm the regulatory mechanism of ␣-amylase (Cannon, 1992). Therefore, the up-regulation in the enzyme activity observed in this study as a follow-up to the down-regulation of hyperglycaemia by the extracts was necessary. The decrease in ␣-amylase activity caused by HU in NDR attests to the involvement of the enzyme in glycaemic control. Hence, VA/AI may also modulate carbohydrate metabolic enzymes, a subject for further research. Contrary to the individual effects of VA and AI, it was observed that VA/AI could reduce serum insulin levels of SDRs by 1.69 fold, similar to HU (1.79 fold). The implication of this effect is still unclear. However, an in vitro study backed by immunoreactive insulin, glucagon, and c-peptide staining would be useful to clearly elucidate these changes. Diabetogenic hormones, including T3 and T4, are known to increase antagonists to insulin in diabetes mellitus (Karam and Forsham, 1994). The combined extract in our study significantly decreased T3 and T4 levels, an effect not observed with the individual extracts, thus strengthening the reported antidiabetic synergy. Additionally, considering that such a decrease is usually antagonistic to the effects of insulin and that the T3 level was also lowered in HU treated SDRs, our data further support the proposition of insulin mimetic action. VA/AI may, in this regard, have the potential to replace the insulin requirement for the management of type I diabetes, a subject of further study. Antioxidant enzymes (GPx, SOD and CAT) have been used extensively to study peroxidation and oxidation in biological systems. Streptozotocin and hyperglycaemia are pathogenic factors for the oxidative stress-increased imbalance in ROS generation and elimination/neutralisation (Szkudelski, 2001; Goycheva et al., 2006). The resulting excess ROS initially induces antioxidant enzymes, particularly in the liver, the tissue with the most abundant antioxidant defence enzymes (Taksuki et al., 1997). However, as the hyperglycaemia becomes chronic, the enzyme system becomes overwhelmed and exhausted, accounting for the usually reported decrease in activity in untreated diabetes. In this study, the hepatic activities of GPx and CAT were significantly decreased in untreated SDRs, in accordance with previous reports (Ugochukwu et al., 2003; Nwanjo, 2005). Superoxide dismutase activity in DCs was, however, not significantly altered, probably due to the efficient system of the enzyme or decreased production of superoxide radicals within the study period. Twenty-eight-day treatment with VA/AI resulted in an up-regulation of GPx activity, higher than VA and AI. Although, monotherapy with VA extract did not cause significant changes in the activities of GPx and SOD, they were positively modulated by AI when co-administered – a potential positive synergy. This synergy in antioxidant action may be due to a boost in the flavonoid content of the extracts. Quercetin, a flavonoid isolated from AI has demonstrated potent anti-hyperglycaemic action in streptozotocin diabetes (Biswas et al., 2002). Similarly, three flavones with antioxidant activities more potent than the classical antioxidant butylated hydroxy-toluene, at equal concentrations, have also been isolated from VA (Igile et al., 1994). In addition, vitamins A, E and C, and mineral elements including Se, Cu, Zn and Cr were reported to occur in the leaves of VA and AI (Atangwho et al., 2009). These micronutrients with profound antioxidant roles in human nutrition and pathology (Opara and Rockway, 2006) may contribute to the ROS scavenging action and improve enzyme activity. VA/AI is therefore more beneficial and useful, relative to VA or AI, for the amelioration of oxidative stress in diabetes, and hence is a rich repertoire of free radical quenching components.

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Derangements of the gross architecture of the hepatocytes of untreated diabetic rats were reported in an earlier study (Atangwho et al., 2007). In this study, extracts or insulin treatment ameliorated these features, although VA extract could only cause a partial recovery in SDRs and features of mild injury in NDRs. In line with the present observation, Ojiako and Nwanjo (2006) indicated a slight elevation in serum ALT activity of rats given extracts of VA for 14 days. However, the liver histology of VA/AI treated rats showed features of complete recovery, implying a modulation of the potential hepatotoxic effect of VA by AI, an example of positive synergy. This gross tissue protection by VA/AI, reported here for the first time, corroborates and affirms our earlier conclusion drawn from changes in serum liver enzymes (Ebong et al., 2008). The evidence supports a synergy in hepatoprotective action, but cautions abuse for the use of VA extract for the potential risk of hepatotoxicity. Pancreatic lesions induced by STZ diabetes were reversed upon treatment with extracts in this study, possibly due to pancreatic islet cell regeneration. DC pancreas showed markedly reduced islet mass, infiltrated by lymphocytes, indicating general fibrosis, in agreement with other researchers (Bolkent et al., 2000; Noor et al., 2008). As in the cited works, treatment with our extracts ameliorated and/or reversed the lesions. The observed extent of recovery and reversal was partial with VA, better with AI and distinct and total with VA/AI. We had shown in an earlier study that AI has a higher potential to reverse islet cell lesions compared with VA in monotherapy (Ebong et al., 2006). The present investigation indicates, for the first time, complete recovery when the two extracts are co-administered, suggesting that the synergy in antidiabetic action may involve enhanced ␤-cell regeneration. 5. Conclusion In summary, this study further affirms the enhanced antidiabetic efficacy of VA and AI when co-administered, and adduces that the synergy is exerted via fine glucose regulation, oxidative stress attenuation, insulin mimetic action and probable ␤-cell regeneration. Medicinal chemistry of the extracts and immunoreactive insulin studies are on-going in our laboratory to buttress these results. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements The authors sincerely thank the Academy of Science for the Developing World (TWAS) and the Universiti Sains Malaysia (USM) for providing the travel grant and TWAS-USM Fellowship for Item J. Atangwho. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jep.2012.03.041. References Abo, K.A., Fred-Jaiyesimi, A.A., Jaiyesimi, A.E.A., 2008. Ethnobotanical studies of medicinal plants used in the management of diabetes mellitus in South Western Nigeria. Journal of Ethnopharmacology 115, 67–71. Ahmed, S.M., Vrushabendra, S.B.M., Gopkumar, P., Dhanapal, R., Chandrashekara, 2005. Anti-diabetic activity of Terminalia catappa Linn. leaf extracts in alloxaninduced diabetic rats. Iranian Journal of Pharmacology and Therapeutics 4, 38–39. Akah, P., Okafor, C.L., 1992. Blood lowering effects of Vernonia amygdalina Del. in an experimental rabbit model. Phytotherapy Research 6, 111–114.

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