Two antihyperglycaemic compounds from Globimetula braunii (Engl.) Van Tiegh (Loranthaceae)

Large numbers of medicinal plants are constantly being screened for possible pharmacological values, especially for chronic diseases such as diabetes, with the view of discovering new compounds that may serve as templates for synthesis of more active/less toxic drugs. Therefore, the antihyperglycaemic activity of the leaf of Globimetula braunii with antidiabetic ethnomedical usage in Nigeria was investigated is this study to justify this folkloric claim. The median lethal dose, LD50 of the ethanol leaf extract of G. braunii was determined using Lorke’s method and its antihyperglycaemic effect at 100, 200 and 400 mg/kg was evaluated using glucose-induced hyperglycaemic rats while glibenclamide (5 mg/kg) and 1 % Tween 80 in normal saline served as positive and negative controls, respectively. Anti-hyperglycaemic activity-directed purification of the extract of the plant in glucose-loaded rats, led to the isolation and characterisation of phyllanthone and methyl 2, 6-dihydroxy-4-methoxybenzoate from the dichloromethane partitioned fraction. The findings showed that the LD50 of the ethanol leaf extract of G. braunii was greater than 5,000 mg/kg while its 100 mg/kg was the most active dose with comparable activity (p>0.05) to the standard drug, glibenclamide. The dichloromethane and aqueous partitioned fractions of the extract were the most promising fractions. Chromatographic separations of the dichloromethane fraction yielded phyllanthone, and methyl 2, 6dihydroxy-4-methoxybenzoate that elicited comparable activity to glibenclamide (5 mg/kg) at 10 and 20 mg/kg at all time-points. The study justified the antidiabetic folkloric use of G. braunii leaf and confirmed phyllanthone and methyl 2,6-dihydroxy-4-methoxybenzoate as two of its antihyperglycaemic constituents.


Introduction
Medicinal plants are plants which, in one or more of their organs, contain substances that can be used for therapeutic purposes or which are precursors for the synthesis of useful drugs [1]. They have the ability to synthesis a wide variety of chemical compounds that possess pharmacological properties, and defend against attack from predators such as insects, fungi and herbivores [2,3]. These compounds also known as phytochemicals, have been found to exert their effects on the human body through mechanisms that are similar to those already established in conventional drugs revealing that herbal medicines may be as effective as the latter [2,4]. About 25 % of the drugs prescribed worldwide come from medicinal plants and out of the 252 drugs considered as basic and essential by the World Health Organisation, 11 % are exclusively of plant origin and a significant number are synthetic drugs obtained from natural precursors [5]. The plant kingdom is believed to hold many new drug templates; hence, the continued investigation into ethno-medicinal plants [6].

Animals
Male and female healthy Wistar albino rats, average weight of 150 g and bred at 27±3° C, 65 % relative humidity, 12 h day-night and housed in different cages in the animal house, Department of Pharmacology, Faculty of Pharmacy, O.A.U., Ile-Ife, Nigeria, were used for the study. They were fed on a standard pellet diet from 'Bendel Feeds', Benin, Nigeria, with water given as required. Groups of five rats were fasted for 24 h before administration of either glucose, extract, fractions, drugs or vehicle. All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the National Academies Press [18].

Plant material and Extraction
The leaf of Globimetula braunii was collected on Leucena leucocephala (Fabaceae) at Obafemi Awolowo University, Ile-Ife, Osun State and authenticated at the Ife Herbarium, Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria, with the Voucher number IFE 17229. It was then air-dried and milled into powder. A 1.5 kg of the leaf was extracted with 7.5 Litres of 80 % ethanol by maceration at room temperature. It was then filtered and concentrated using rotary evaporator to afford a yield of 105 g of ethanol extract.

Acute toxicity study of the extract
The biological safety of Globimetula braunii was evaluated by determining the LD50 of its ethanol extract, using the 1983 Lorke's method [19]. Animals weighing between 120-150 g were fasted overnight before doses of 10 -5,000 mg/kg of the extract were administered orally. The study was carried out in two phases. Phase 1 of the study involved nine (9) rats that were divided into 3 groups of 3 rats each and were given the extract, with each group receiving 10, 100 and 1000 mg/kg, respectively. These animals were observed for mortality and or toxicity signs within each group over a 24hour period. Due to the results obtained from phase 1, phase 2 study was carried out, using eight (8) rats that were divided into 4 groups of 2 rats each, which were given 1000, 1600, 2900 and 5000 mg/kg of the extract, respectively. The animals were also observed for mortality and/or toxicity signs for 24 hours. The LD50 was calculated as the geometric mean of the dose that resulted in100 % lethality and that which caused no lethality at all [19].

Antihyperglycaemic assay of the extract fractions
The antihyperglycaemic assay of the extract, A and its partition fractions, B1-B4 were carried out in glucose-induced hyperglycaemic normal rats as described by Adebajo, Ayoola and their co-workers [20][21][22][23].

Isolation of Compounds from the Active DCM Fraction
A 20.0 g of active dichloromethane partition fraction (B1) was adsorbed on silica gel and subjected to column chromatography using gradient solvent systems of increasing polarity comprising of n-Hex, DCM, EtOAc and MeOH. A total of four bulked column fractions based on TLC analysis were obtained and coded, C1-C4. Column fractions C1 and C2 formed solid deposits within 12 hrs of standing. Therefore, C1 was washed with 20 % DCM in MeOH, which yielded a whitish amorphous powder, coded, G1 (30 mg). Also, C2 was washed with DCM (100 %) which yielded an ash amorphous powder, coded, G2 (746 mg).

Antihyperglycaemic Assay of Isolated Compounds
The two isolated compounds, G1 and G2 were tested for anti-hyperglycaemic assay as described earlier.

Statistical analysis
The data obtained from the study were expressed as the mean ± SEM for the number (n=5) of animals in the groups. They were analysed with One Way Analysis of Variance (ANOVA), followed by Bonferroni t-test or Student-Newman-Keuls post hoc tests, using GraphPad® Instat, version 5.0 (GraphPad Software Inc., San Diego, USA). P < 0.05 was considered significant.

Safety profile of G. braunii
Acute toxicity studies that were carried out through oral administration of the ethanol leaf extract of G. braunii at 10, 100, 1,000, 1,600, 2,900 and 5,000 mg/kg did not produce death in the animals used. Also, there were no significant changes in their behaviours such as breathing, cutaneous effect, sensory and nervous system responses or on gastrointestinal effects. This showed that the median lethal dose (LD50) of the ethanol leaf extract of G. braunii is above 5,000 mg/kg and that the chosen doses of 100, 200 and 400 mg/kg used in the evaluation of the antihyperglycaemic activity of the extract were therapeutically safe.

Antihyperglycaemic effects of the extract
In glucose-induced antihyperglycaemic experiments of medicinal plants or orthodox drugs involving the use of glibenclamide and other insulinotropic drugs as positive controls, it has been established that results obtained from such experiments can be extrapolated on the Type 2 diabetic state in humans [20,21,24]. Furthermore, the use of glibenclamide in antidiabetic experiments as the standard drug [25] could be used to determine the early extrapancreatic and late insulin stimulating effects in terms of the mechanisms of action of the extract being investigated [22,23,26]. A significant (p < 0.05) time-dependent hyperglycaemia-lowering effects up to the fourth hour was observed in the negative control group of rats that were given normal saline. This activity could be attributed to the normal homeostatic regulatory mechanism in normal animals which confirmed the healthy state of the pancreases of the animals used in the study [20][21][22][23]. Also, the positive control group of rats that were administered with glibenclamide(5 mg/kg) gave a significant time-dependent antihyperglycaemic activity up to fourth hour (Table 1). This observation confirmed the early minor extra pancreatic and late major insulin releasing (insulinotropic) mechanisms of action of glibenclamide [25]. The extract of G. braunii at 100 mg/kg elicited a time-dependent glucose lowering activity up to the fourth hour that was significantly higher (p<0.05) than the 200 and 400 mg/kg doses indicating a non-dose dependent activity of the extract (Table 1). Also, the extract at this dose gave similar profile ofactivity at 0.5-4 h to glibenclamide (5 mg/kg) which suggested that the extract had similar early extra pancreatic and late major insulin stimulating mechanisms ofaction with glibenclamide at this dose [25]. The high blood glucose level reductions of 27, 34 and 38 % elicited by 100 mg/kg of the extract at 0.5-2 h further suggested additional extrapancreatic effect of the extract at this dose ( Table 1). The ethanol leaf extract of G. braunii had previously been reported to show hypoglycaemic effect in alloxan-induced diabetic rats at 250 and 500 mg/kg after 2 and 4 h [27]. This result further supported insulin stimulation as the main mechanism of action of G. braunii leaf extract that was suggested by the results of this present work ( Table 1). The extracts of Carica papaya, Xylopia aethiopica, Parquetina nigrescens, Chrysophylum albidum, Senecio biafrae have similarly been suggested to work through insulin stimulation [28][29][30][31][32]. Data show the mean ± SEM blood glucose levels at the different time points expressed as percentages of levels at 0 h (T0),n = 5. Values in parentheses represent the percentage reductions in blood glucose levels relative to negative control for each time point. Values with different superscripts within columns are significantly different (p < 0.05).GLU: Glucose in < 1 % of Tween 80 in normal saline administered at 10 g/kg (hyperglycaemic negative control); A: Globimetula braunii extract; B1= dichloromethane fraction; B2= Ethylacetate fraction; B3= Butanol fraction; B4= Aqueous fraction; GLI: Glibenclamide (5 mg/kg, positive control).

Antihyperglycaemic Activity of Partition Fractions of G. braunii Extract
Based on the results of the antihyperglycaemic effects of G. braunii ethanol leaf extract (Table 1), the active extract was subjected to solvent partitioning and the resulting partitioned fractions of the extract were tested for antihyperglycaemic activity at the most effective dose of the extract (100 mg/kg). Ethylacetate (B2) and Butanol (B3) fractions were devoid of antihyperglycaemic activity at 0.5-2 h but gave 31 and 11 % blood glucose levels reduction at 4 h, respectively which indicated that they lacked extrapancreatic activity but had insulin stimulating effect with B2 showing higher activity (Table 2). Fractions B1 and B4 gave comparable (p>0.05) and time-dependent activity at alltime points that were similar in profile to glibenclamide which suggested early extrapancreatic and late insulin stimulation mechanism of action [25]. It also indicated that the antihyperglycaemic constituents of the extract were more concentrated in B1 and B4 which were non polar and very polar in nature, respectively. Furthermore, none of the partitioned fractions, B1-B4 was significantly more active than the extract indicating that the antihyperglycaemic constituents of A were working in synergism (Table 2).  Active B1 was subjected to column chromatography as described above and it afforded four bulked column fractions, C1-C4 that were thereafter tested for antihyperglycaemic activity at 100 mg/kg (the most effective dose of the extract). The findings showed that C1-C4 elicited comparable (p>0.05) blood glucose level reduction effect with glibenclamide at 4 h suggesting insulin release as their major mechanism of action ( Table 3). While C1 and C2 gave a non time-dependent antihyperglycaemic effect, the activity of C3 and C4 were time dependent. The additional extrapancreatic effect that was observed in the extract was found in C1 and C2. This showed that chromatographic purification of the extract in this work has successfully separated the extrapancreatic constituents. Furthermore; C1-C4 gave comparable effect both to B1 and the A at 4 h which showed synergism in the antihyperglycaemic constituents of the bulked column fractions (Table 3).

Antihyperglycaemic effects of the isolated compounds from G. braunii
Isolated compound G1, phyllanthone demonstrated a time-dependent and comparable antihyperglycaemic activity at 10 and 20 mg/kg suggesting insulin release as its major mechanism of action and that its activity was not dose dependent (Fig.1). However, the activity of G2,methyl 2, 6-dihydroxy-4-methoxybenzoate was not time-dependent while the 10 mg/kg dose gave a significantly better blood glucose lowering effect than the 20 mg/kg at 4 h. Both phyllanthone, and methyl 2, 6-dihydroxy-4-methoxybenzoate demonstrated comparable and similar profile of antihyperglycaemic effects to glibenclamide which suggested minor extrapancreatic and major insulin stimulating mechanism of action for the compounds [21][22][23][24]. These results thereforeidentified phyllanthone, and methyl 2,6dihydroxy-4-methoxybenzoate as two of the antihyperglycaemic constituents of Globimetula braunni. The spectra data of the isolated compounds, G1 and G2 were compared with those from the literature. Compound G1 has been characterized as a 13, 27-cycloursan-3-one reported from the stem bark of Phyllanthus polyanthus as "phyllanthone" [33]. Also G2 was characterized as methyl 2, 6-dihydroxy-4-methoxybenzoate [34]. The two compounds were isolated for the first time from the leaf of Globimetula braunii and reported in this work.

Conclusion
Lack of death in the animals when administered with high dose of the ethanol leaf extract of G. braunii in acute toxicity study confirmed its safety. Also the antihyperglycaemic activity of the extract at the lowest tested dose, 100 mg/kg indicated its efficacy while phyllanthone and methyl 2, 6-dihydroxy-4-methoxybenzoate with comparable antihyperglycaemic effect to glibenclamide were two of its active constituents. These results justified the antidiabetic ethno-medicinal use of the plant.

Acknowledgments
The authors appreciated Professors Gilbert Arthur and Frank Schweizer of the University of Manitoba, Canada for making their Spectroscopic research laboratory available for the study. The effort of Mr. I.I. Ogunlowo in collecting the plant was also recognized.

Disclosure of conflict of interest
The authors declared that there is no conflict of interest with regard to this work.