Demand for herbal medicine is increasing in developing countries and even in USA, where an estimated six billion dollar worth of herbal remedies are being sold yearly and about 80% of the world inhabitants rely mainly on traditional medicine for their health care needs (WHO, 1994; Wild et al, 2004). Of late, emphasis is on the development of drugs for the treatment/management of various diseases, one of which is diabetes mellitus (DM) (WHO, 1980)

DM is a chronic degenerative endocrinologic disorder characterized by high blood glucose levels due to insufficient secretion of insulin by the pancreas, improper utilization of insulin by target cells or both. It is one of the most common non-communicable diseases with prevalence now reaching epidemic proportion (IDF, 2000; Motala et al, 2000; ADA, 2011; Wild et al, 2004). DM has been managed with herbs since antiquity. Recent scientific investigations have confirmed the efficacy of some of these herbal preparations. (Bosni et al, 1995; Dey et al, 2002; James et al 2009; Okokon et al, 2007; Edet et al, 2009; Akinnuga et al, 2011; Teugua et al, 2013). Rothmannia hispida is one of the herds used traditionally for the management of DM. Scientific reports on the antidiabetic potential of this plant medicine have been reported (Antai et al, 2005 and 2010).

Rothmannia hispida (K. Schunn) Fagerl (Syn. Randia Hispida) is a shrub or small tree of about 10 meter tall, with fruits 6-11cm long, the wood turn blue when exposed to air. The leaves of R. hispida are more hairy than the leaves of other species (Jansen, 2005). R. hispida is widely used in Africa for the treatment of various ailments including fever, pain, external lotion, bowel complaints, throat abscesses, tooth decay, leprosy (Jansen, 2005). The leaves are also used against kidney pains and diarrhea and for the treatment of diabetes (Lewis and Elvin-Lewis, 1977, Jansen, 2005). Scientific reports have indicated that R. hipida possesses antidiabetic potential (Antai et al, 2005 and 2010), cytoprotective and anti-gastric ulcer activity (Antai et al, 2008) and anti-malarial effect (Asomaning, 1999). Despite the widespread and age-long use of R. hispida in the management of DM and other ailments, information on the effects of this herb on blood cells is scanty and has not been well established. Thus, the aim of this present study is to evaluate the effect of aqueous extract of R. hispida on glucose tolerance test and on some haematological parameters in normoglycaemic albino rats.

MATERIALS AND METHODS:

Plant materials

Fresh and healthy looking leaves of Rothmannia hispida were collected from the Botanical Garden of the University of Calabar, Calabar, Nigeria during the months of May- August. The collected plant sample was identified at the Department of Botany, University of Calabar, Calabar, Nigeria as Rothmannia hispida (K. Schunn) Fagerl (Syn. Randia hispida

Preparation of plant extract

The collected leaves of Rothmannia hispida were washed, rinsed and blotted. They were then sliced and ground with a manual blender. Wet weights of the paste were obtained using Mettler Weighing Instrument (Ohaus triple beam 700/800, Ohaus, USA). A weighed amount of the paste 1051.3g was mixed with 6.5L of hot water, it was then left to stand for twenty four hours in a thermostatic container. After the set time the mixture was filtered using Whatman’s No. 1 filter paper. The filtrate was evaporated in vacuo and the semi-solid mass (110.8g) obtained was stored in a freezer to prevent bacterial decomposition and possible loss of efficacy prior to experimentation.

Experimental procedure

This study was carried out on healthy albino rats obtained from the animal house of the Department of Pharmacology, University of Calabar, Calabar, Nigeria. They were acclimatized for two weeks in well aerated cages and had free access to standard rat feeds (Vital feeds, Nigeria Limited). The animals were divided into 4 groups of five rats per group. Group 1 (control) were given water and rat feed only, Group 2 (Glucose 2g/kg) were given oral glucose at the concentration of 2g/kg body weight. Group 3 (RHE 125 mg /100g bwt) were given R. Hispida leaf extract at the concentration of 125mg/100g body weight and Group 4 (RHE 250mg/100g bwt) were given R. hipida leaf extract at the concentration of 250mg/100g body weight.

Glucose tolerance test

The animal were fasted overnight and thereafter the fasting blood glucose was determined using automatic glucose analyzer (Fine test Auto-coding^TM Premium, Infopia, Korea). Blood was obtained from tail prick using sterile hypodermic needle. Glucose tolerance test was carried out for groups 1, 2 and 3. Groups 1 and 2 were treated as described above and the blood glucose concentration were then determined from time zero through 180 at 30mins intervals. Group 3 was orally administered aqueous leaf extract of R. hispida at the dose of 125mg/100g body weight 30min before the oral glucose load at the dose of 2g/kg body weight, thereafter blood glucose was determined as for Groups 1and 2 above.

Haematological studies

Extract administration continued for Groups 3 and 4 while groups 1 and 2 were placed on water and rat feed only for 14 days. At the end of the 14 days, the animals in all the groups were weighed and sacrificed using chloroform anesthesia. Blood samples were collected by cardiac puncture into clean heparinized tubes and stored at -4^o for subsequent haematological analysis.

The method of Dacie and Lewis (2001) was employed in red blood cell (RBC) count. The collected blood samples were diluted to 1:200 with Hayem’s fluid and counted with a Neubauer counting chamber using a light microscope. The white blood cell (WBC) count was carried out by the use of the new improved haemocytometer (Hawksley, England) using Turk’s fluid as the diluting fluid in a ratio of 1:20. Hemoglobin (Hb) content was estimated using Sahli’s haemoglobinometer, packed cell volume (PCV) was done by the micro-haematocrit after the method of Dacie and Lewis (2001). Estimation of percentage concentrations of neutrophil (N) and lymphocyte (L) were done using standard method of Dacie and Lewis (2001).

Statistical analysis

Data obtained were expressed as mean ± SEM and the difference between groups compared to each other using the student t-test and One-way ANOVA with Bonferroni’s post-test performed using GraphPad Prism (version 5.00 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com) Values for P<0.05 were considered statistically significant. Graphical representations were designed using GraphPad Prism.

RESULTS:

Food intake

The mean ± SEM daily food intake for Group 1(control) , Group 2 (oral glucose tolerance test-OGTT 2g/kg bwt), Group 3 (Rothmannia hispida extract-RHE 125mg/100g body weight) and Group 4 (Rothmannia hispida extract- RHE 250mg/100g body weight) were 13.2 ± 1.18, 13.9 ± 1.13g, 14.5 ± 1.14g and 14.1 ± 1.15g respectively. This gave the respective percentages of 24%, 25%, 26% and 25% . There was no significant (P>0.05) difference among the groups.

Body weight

The aqueous extract of the leaves of Rothmannia hispida (RHE) produced significant (P<0.05) dose-dependent reduction in mean body weight of the treated groups compared with control and oral glucose treated groups (Table 1). While the mean weight gain for control and oral glucose treated groups were 2.7± 2.4g and 6.7± 3.2g respectively, the mean weight change were -13.7± 2.5g and -28.7± 2.5g for RHE 125mg/kg body weight and RHE 250mg/100g body weight treated groups respectively. However there was no significant (P>0.05) difference in weight change comparing initial weight and final weight within the same animal grouping.

Glucose tolerance test

The result of glucose tolerance test (Figure 1) shows that blood glucose of treated rats significantly (P<0.05) increased at 30 min, with maximal increase at 60min compared with control group. Administration of Rothmannia hispida leaf extract (RHE 125mg/100g bwt) 30min pre-glucose infusion resulted in a significant (P<0.05) reduction in blood glucose level from 30 upwards. The blood glucose level was higher than at time zero but significantly decreased from 30 through 180 min with extract treatment. The blood glucose calculated from areas under the curves for the total periods (0min – 180min) for control, glucose treated and glucose plus extract treated were 11243, 16404 an 14255 respectively.

Effect on haematological indices

The results of the effect of RHE 125mg/100g body weight and RHE 250mg/100g body weight treatment on normoglycaemic albino rats are presented in Table 2. There was no significant (P>0.05) change in RBC, PCV, Hb, N and L among the groups when compared with control except for WBC which was significantly (P<0.05) lower in RHE treated groups when compared with control.

Table 2: Effect of aqueous leaf extract of Rothmannia hispida on haematological indices of normoglycaemic albino rats

Indices	Control	RHE 125mg/100g bwt	RHE 250 mg/100gbwt
RBC (x100⁶)	6.6 ± 0.02	7,76 ± o.17	7.42 ± 0.22
WBC (x10³)	7.14 ± 0.41	4.2 ± 0.66*	4.6 ± 6.0*
PCV (%)	51.6 ± 1.5	45.0 ± 1.58	46.67 ± 1.33
Hb (g/dL)	17.2 ± 0.2	15.1 ± 0.34	15.39 ± 0.37
N (%)	29.6 ± 2.08	30.0 ± 2.4	26.22± 0.56
L (%)	70.2 ± 2.1	70.1 ± 2.98	72.12 ± 0.6

Results show mean ± SEM of five values. *= p<0.05 versus control

RHE 125mg/100g bwt = Rothmannia hispida leaf extract 125mg/100g body weight,

RHE 250mg/100g = Rothmannia hispida leaf extract 250mg/100g body weight.

DISCUSSION:

Diabetes mellitus (DM) has been described as a chronic degenerative endocrinologic disorder characterized by high blood levels of glucose sequel to insufficient secretion of insulin, insulin resistance or both (WHO, 1999; ADA, 2006). DM is associated with reduced life expectancy and morbidity. Diabetic associated morbidity is primarily due to diabetes related microvascular and macrovascular complications. Diabetic macrovascular complications include ischemic heart disease, stroke and peripheral vascular disease (WHO, 2006). Undiagnosed and/or untreated, long term diabetic hyperglycaemia may result to organ damage, dysfunction or failure of many organs to function properly including the eye, kidneys, nerves, heart and blood vessels (Motala et al, 2000).

The present study was designed to evaluate the effect of aqueous extract of the leaves of Rothmannia hispida on glucose tolerance test, body weight and on some haematological indices. Hyperglycaemia in DM has a negative impact on the body’s ability to absorb glucose and other nutrients useful for body building, hence weight loss and glucosuria reported in diabetics (WHO, 1999). The results from this study indicated that R. hispida extract had no significant (P>0.05) effect on food intake but a significant (P<0.05) reduction in mean body weight change in treated groups. It is known that obesity contributes greatly towards insulin resistance, a fore- runner of DM. Insulin sensitivity coupled with improvement in DM management, weight reduction and increased physical exercise have been shown to be beneficial in the treatment of DM (Zimmet, 1992; Wing et al, 1994). The resultant weight reduction in the present study indicates the beneficial role of R. hispida especially in the management of obese diabetic patients.

In oral glucose tolerance test, R. hispida aqueous leaf extract (RHE) showed significant (p<0.05) reduction in blood glucose level in normoglycaemic rats. The rapid onset of action exhibited by R. hispida extract mimics the hypoglycaemic mechanism involved in insulin-like agents, probably acting via increase secretion of insulin and /or increased sensitivity of tissue cells to the stimulatory effect of insulin (Haile, 2000; McNulty, 2000; English, 2000; Soran, 2000; Luna and Feinglos, 2001). Matsuda and DeFrenzo (1999) reported that the oral glucose tolerance test (OGTT) is the most commonly used method in the evaluation of whole body glucose tolerance in vivo. This was confirmed by WHO (2006) who stated that OGTT should be retained as a diagnostic test. In their report, WHO (2006) stated that fasting glucose alone fails to diagnose approximately 30% of cases and that OGTT is needed to confirm or exclude an abnormality of glucose tolerance in asymptomatic people. OGTT may also be used to assess insulin release and insulin sensitivity (Stumvoll et al, 2000; WHO, 2006; Mollar- Puchades et al, 2006). Couple with this is the need to adequately treat DM once diagnosed with effective agents devoid of unwanted side effects associated with some synthetic anti-diabetic agents. R. hispida has been reported to reduce blood glucose levels in diabetic state (Antai et al, 2008; 2009; 2010). The result from the present work indicates that R. hispida can be employed in assessing whole body glucose absorption and probably in the diagnosis of DM, which is essential for the commencement of early treatment and/or prevention.

The results from this study shows that R. hispida extract (125mg/100g bwt and 250mg/100g bwt) had no significant (P>0.05) effect on haematological indices of normoglycaemic albino rats except for WBC that was significantly (P<0.05) reduced. The reduction in WBC count in treated groups was not dose-dependent. There was a non-significant (P>0.05) increase in RBC and non-significant (P>0.05) decrease in PCV and Hb of treated groups compared with control. These differences were within the normal range which could be attributed to stress (Cole, 1986) and not anemia.

The decrease in total WBC count could be due to the presence of some toxic phytochemical components in RHE that are linked to the lyses of blood cell or that could probably be involved in the suppression of WBC synthesis or due to stress (Oguwike et al, 2013). This mechanism of inhibition of WBC synthesis was proposed by Oguwike et al (2013) in their study on the haematological indices of rat. Gongronema latifolium is another medicinal plant scientifically proved to be useful in the management of DM but with reported effects on haematological indices in rats (Edet et al, 2009; Akpaso et al, 2011; Johnkennedy et al, 2012; Obi et al, 2012; Ugochukwu et al, 2013). While Ikpeme et al (2012) indicated that Gongronema latifolium extract enhanced the production of blood cells, Oguwike et al (2013) and Akinnuga et al (2011) reported that Gongronema latifolium extract had a depressant effect on blood cell production, and cautioned against incessant consumption of this medicinal plant. In line with the above, it is advisable to use Rothmannia hispida with caution pending confirmatory report on its effect on haemopoiesis.

CONCLUSION:

It is concluded that Rothmannia hispida leaf extract possess non significant adverse effect on haematological indices in normoglycaemic albino rats. Administration of the leaf extract of R. hispida before oral glucose test resulted in blood glucose lowering effect in the tested animals; this is an indication of its potential not only as an anti-diabetic medicinal herb, but also as a potential investigative tool. Further work is needed to elucidate the active phytochemical components responsible for the haemopoietic and anti-hyperglycaemic effect of this plant drug.

ABBREVIATIONS:

RHE, Rothmannia hispida extract; R. hispida, Rothmannia hispida; bwt, body weight; RBC, red blood cell; WBC, white blood cell; PCV, packed cell volume; Hb, haemoglobin; N, neutrophil, L, lymphocyte; DM, diabetes mellitus; OGTT, oral glucose tolerance test.

REFERENCES:

1. Akinnuga A M, Bamidele O, Ekechi P and Adeniyi O S (). Effects of an Ethanolic Leaf of Gongronema latifolium on Some Haematological Parameters in Rats. African Journal of Biomedical Research, 14 (2); 2011:153-156.

2. Akpaso MI, Atangwo IJ, Akpantah A, Fisher V A, Igiri AO and Ebong PE. Effect of Combined leaf Extracts of Vernonia amygdalina (Bitter leaf) and Gongronema latifolium (Utazi) on the Pancreatic β-Cells of Sreptozotocin-Induced Diabetic Rats. British Journal of Medicine and Medical Research, 1(1); 2011: 24-34.

3. American Diabetes Association (ADA). Diagnosis and Classification of Daibates Mellitus. Diabetes Care, 34 (Suppl. 1); 2011: S62-S69.

4. Antai AB, Anaele BA and Etta KM. Hypoglylaemic actions of a medicinal herb, Rothmannia hispida in diabetic rats. Mary Slessor Journal of Medicine, 5(2); 2005: 21-24.

5. Antai AB, Ofem OE, Nwosu OJ, Okafia SO, Iyadi KC, Nia R and Osim E E. Comparative Effects of Rothmannia hispida Leaves Extract and Protamine-Zinc Insulin on Alloxan Induced Diabetic Rats. African Journal of Biomedical Research, 13; 2010.:47-54.

6. Antai AB, Owu DU and Ofem OE. Effect of aqueous extract of Rothmannia hispida on gastric acid secretion and gastric mucosa protection (Cytoprotection). Mary Slessor Journal of Medicine, 8(1); 2008: 32 - 36.

7. Asomaning WA. Screening of Teclea verdornia and Rothmannia longiflora for antiplasmodial activity. Ghana: Centre for Scientific Research into plant Medicine, University of Ghana Press. Ghana. 1999.

8. Bosni MIK, Osuji PA, Tuah AK and Umunna N. NVernonia amygdalina as a supplement to straw (Eragrasite) fed to Ethiopian menz sheep. Agroforestry System, 2; 1995: 229-241.

9. Cole EH. Veterinary Clinical Pathology, 4^Th Edition, pp457-458. W. b. Saunder Company, Philadelphia. 1896.

10. Dacie, J V and Lewis SM. Practical Haematology, 11th Edition, pp. 11-17. Longman Group Ltd, Hongkong. 2001.

11. Dey L, Attele, SA and Yuan, CS. Alternative therapies for Type 2 diabetes. Alternative Medical Reviews. 7(1); 2002: 45-58.

12. Edet EE, Akpanabiatu MI, Eno AE, Umoh IB and Itam, EA. Effect of Gongronema latifolium leaf extract on some cardiac enzymes of alloxan-induced diabetic rats. African Journal of Biochemistry Research, 3 (11); 2009: 366-369.

13. English P. Oral Agents in the Treatment of Type 2 Diabetes Mellitus. Diabetes International, 10 (3);2000: 70-76.

14. Haile S. Management of diabetic ketoacidosis with lente insulin. Diabetes International, 10(2); 2000: 61.

15. Ikpeme EV, Nta A, Ekaluo UB. and Udensi O. Phytochemical Screening and Haematological Evaluation of Parkia biglobossa and Gongronema latifolium. Journal of Basic and Applied Science Research, 2(3); 2012; 2599-2606.

16. International Diabetes Federation (IDF). Diabetes Atlas (pp. 19-70).

17. Imprimeric L. Vanmelle SA Gent/Mariakerhe. Belgium. 2000.

18. James D B, Elebo N, Hassan N and Odemene L. Glucose tolerance test and biochemical effect of Phyllanthus amarus aqueous extracts on normaglycemic albino rats. African Journal of Biotechnology, 8 (8); 2009: 1637-1642.

19. Jansen PCM. Rothmannia longiflora Salib. In Jansen, P.C.M. & Cardon, D. (Eds.). PROTA 3: Dyes and tannins/ colorants et tannins. [CD.Rom] PROTA, Washington, Netherlands. 2005.

20. Johnkennedy N, Adamma E, Augustin I and Ifeoma UH. Influence of Gongronema latifolium on Some Biochemical Parameters in Alloxan Induced Diabetes. International Journal of Analytical, Pharmaceutical and Biomedical Sciences, 1(1); 2012: 13-17.

21. Lewis W and Elvin –Lewis MPF. Medical Botany (pp. 140-250). New York: John Wiley and Sons, New York. 1977.

22. Luna B and Feinglos MN. Oral agents in the management of Type 2 diabetes mellitus. American Family Physicians, 67; 2001: 1741-1780.

23. Matsuda M, and DeFrenzo R A. Insulin Sensitivity Indices Obtained From Oral Glucose Tolerance Testing – Comparing With Euglycemic Insulin Clamps. Diabetes Care, 22(9); 1999: 1462-1470.

24. McNulty SJ. Modern insulin therapies. Diabetes International, 10(2);2000: 38-41.

25. Mollar-Puchades MA, Pallare's-Carratala' V, Navas de Solis MS and PinoSelle's F. Fasting Glucose versus Oral Glucose Tolerance testing in the Diagnosis of Diabetes Mellitus. Revista Espaňola Cardiologia, 59(12); 2006: 1345-1350.

26. Motala AA, Omar MAK and Pirie FJ. Type 1 Diabetes in Africa: Epidemiology and pathogenesis. Diabetes International, 10(2); 2000: 44-47.

27. Obi HI, Ilodigwe EE, Ajaghaku DE and Okonta JM. Antidiabetic Activity of Combined aqueous Extracts of Gongronema latifolium (Benth) and Allium cepa. Journal of Pharmaceutical and Biomedical Sciences, 19 (19); 2012: 1-5.

28. Oguwike FN, Okpala CN and Ofor CC. Haemostatic and Haematological Indices of Aqueous of gongronema latifolium on Female Albino Rats. IOSR Journal of Dental and Medical Sciences, 8(1); 2013: 61-63.

29. Okokon JE, Antia BS, Osuji LC and Udia PM. Antidiabetic and Hypolipidemic Effects of Marmea Africana (Guttiferae) in Streptozotocin Induced Diabetic Rats. Journal of Pharmacology and Toxicology, 2 (3); 2007: 278-283.

30. Soran H. Oral hypoglycaemic agents: Past, present and future. Diabetes International, 10(3); 2000: 77-80.

31. Stumvoll M, Mitrakou A, Pimenta W and Jensen T. Use of Oral Glucose Tolerance Test to Assess Insulin Release and Insulin Sensitivity. Diabetes Care, 23(3); 2000: 295-301.

32. Subramanian SS. (-) – Epicatechin as an antidiabetic drug. Indian drugs, 18; 1981: 259-263.

33. Teugua CM, Boudjeko T, Tchinda BT and Mejiato PC. Anti-hyperglyvemic globulins from selected Cucurbitaceae seeds used as antidiabetic medicinal plants in Africa. BMC Complimentary and Alternative Medicine, 13; 2013: 63.

34. Ugochukwu NH, Babady NE, Cobourne M and Gasset S. The Effect of Gongronema latifolium extracts on serum profile and oxidative stress in hepatocytes of diabetic rats. Journal of Bioscience, 28(10); 3002: 1-5.

35. Wild S, Roglic G, Green A, Sicree R and King H. Global Prevalence of Diabetes. Diabetes care, 27(5); 2004: 1047-1053.

36. Wing RR, Blair EH, Bononi P, Marcus MD, Watanabe R, Bergman R.. Caloric restriction per se is a significant factor in improvements in glycemic control and insulin sensitivity during weight loss in obese NIDDM patients. Diabetes Care, 17; 1994: 30-36.

37. World Health Organization (WHO). Definition, Diagnosis and Classification of Diabetes Mellitus and its Complications. Part 1: Diagnosis and Classification of Diabetes, WHO (pp. 1-26). Geneva. 1999.

38. World Health Organization (WHO).Expert Committee on diabetes Mellitus. Tech. Ref. Series No. 646. Geneva. 1980.

39. World Health Organization (WHO) Prevention of diabetes mellitus. Technical Report Series, 844 (pp. 3-16). Geneva. 1994

40. World Health Organization (WHO). Definition and Diagnosis of Diabetes Mellitus and Intermediate Hyperglycaemia. Report of a WHO/IDF Consultation, (pp. 1-26). WHO, Geneva. 2006

41. Zimmet PZ. Kelly West Lecture 1991: Challenges in diabetes epidemiology: from West to the rest. Diabetes Care, 15; 1992: 232 – 236.

Received on 19.09.2013

Modified on 27.10.2013

Accepted on 02.11.2013

Research Journal of Pharmacognosy and Phytochemistry. 5(6): November –December 2013, 300-305

Treatment	Initial body weight (g)	Final body weight (g)	Weight change (g)
Control	193.7 ± 11.6	196.4± 13.0	2.7 ± 2.4
Glu 2g/kg bwt	222.0 ± 17.3	228.7± 14.5	6.7 ± 3.2
RHE 125mg/100g bwt	200.6 ± 13.7	186.9 ± 10.9	-13.7 ± 2.5*
RHE 250mg/100g bwt	220.3 ± 13.1	191.3 ± 6.5	-28.7 ± 2.0*