Document Type : Research Paper
Authors
1 Research Lab-II, Department of Chemistry Government College University Lahore 54000, Pakistan
2 University College of Pharmacy University of the Punjab, Allama Iqbal Campus Lahore 54000, Pakistan
Abstract
Keywords
1. Introduction
Ruellia tuberosa L. (Acanthaceae) is a tropical perennial plant. It is widely distributed in south East Asia including Thailand and Laos. In folk medicines, R. tuberosa has been used as diuretic, anti-diabetic, antipyretic, analgesic, antihypertensive, thirst quenching and antidotal agent. Recently, it has been introduced as a component of herbal drink in Taiwan [1, 2].
Diabetes mellitus (DM) is a major endocrine disorder that is regarded as hyperglycemia [3]. Asia and Africa are the most viable areas where the disease is feared to raise 2-3 folds [4]. DM is affecting approximately 5% of the world population. Worldwide projections suggest that more than 300 million people will have diabetes by the year 2025 [5]. According to World Ethnobotanical Information of medicinal plants, almost 800 plants are used in the control of DM. However, only few of these plants have been studied [6]. Traditional medicines of plant origin play a vital role in the cure of DM [7]. World Health Organization (WHO) has also recommended the evaluation of traditional plant treatments for diabetes because they are effective, non-toxic, having less or no side effects and are considered good candidates for oral therapy [8]. In this regard, several scientists carried out experimental and clinical trials on medicinal plants and found significant anti-diabetic activity [9-13]. Literature data indicates that some of the flavonoids and triterpenes isolated from medicinal plants have significantly reduced the blood glucose level [14-17].
The objective of this study was to ascertain the scientific basis of the use of R. tuberosa in the treatment of diabetes. The present investigation reports the hypoglycemic activity of methanolic extract, n-hexane, ethyl acetate, and water fractions of the crude methanolic extract of R. tuberosa. To the best of our knowledge this is the first report of the anti-diabetic activity of this plant.
2. Material and methods
2.1. Plant material
The fresh whole plants of R. tuberosa were collected from University of Sargodha, Sargodha campus. The plant was identified at the Department of Botany GC University Lahore where a voucher specimen was deposited (Voucher No. GC.Herb.Bot. 987).
2.2. Preparation and fractionation of the methanolic extract
The plant was cleaned, dried under shade, and the powdered plant material was soaked in methanol for 7 days at room temperature. The methanolic extract (ME) was filtered and evaporated using a rotary evaporator (Laborta 4000-Hedolph). The dried crude extract was dissolved in water and fractionated with n-hexane (HF) and ethyl acetate (EF). The fractions and remaining aqueous layer (WF) were concentrated under reduced pressure. The amounts of solvent fractions for oral administration were calculated from optimum dose (500 mg ME/kg b.w.).
2.3. Preparation of test samples
A weighed portion of each fraction (equivalent to 500 mg ME extract) was suspended in 0.5% aqueous carboxymethyl cellulose (CMC) solution in distilled water prior to oral administration to animals (5 ml/kg b.w.). Animals in control group received only 0.5% CMC suspension in distilled water. Tolbutamide (100 mg/kg b.w.) was used as the reference drug.
2.4. Animals
Adult normal male rabbits (Oryctolagus cuniculus) obtained from the animal house, University College of Pharmacy, University of the Punjab (UCP-PU) Allama Iqbal Campus Lahore, were used for the study. All of the rabbits were kept in individual metal cages located in the UCP-PU. The rabbits were allowed for one week to acclimatize in animal house of Pharmacology Department (UCP-PU). The animal experiments were preceded following the internationally accepted ethical guidelines for the care of laboratory animals. The animal house facility was maintained at standard conditions: temperature (28±2 ˚C), relative humidity (50±5%) and a 12 h light/dark cycle. They were fed with standard diet and water ad libitum. Prior the experiments, they were fasted over night but allowed free access to water.
Table 1. Anti-diabetic effect produced by the oral administration of different doses of R. tuberosa methanol extract in normal and alloxan-induced diabetic rabbits.
|
Study |
Groups |
Samples |
Dose (mg/kg b. w) |
Mean blood glucose concentration±S.E.M. (mg/dL) |
% reduction |
|
|
0 h (in fasting) |
4 h |
|||||
|
|
I-N II-N IV-N V-N VI-N VII-N VIII-N |
Control ME Ext. ME Ext. ME Ext. ME Ext. ME Ext. ME Ext. Tolbutamide |
0.5 % CMC 125 250 500 750 1000 2000 100 |
84.9±1.55 89.5±1.48 86.5±1.37 89.2±0.64 86.6±1.59 86.9±1.48 93.1±1.51 94.5±1.48 |
84.7±1.17 88.2±1.51 83.4*±1.60 73.7***±1.17 73.5±1.52 73.8±0.86 79.9±1.41 76.9±1.18 |
0.07±1.46 1.45±1.86 3.62±0.56 17.33±1.53 15.14±1.44 15.02±0.97 14.10±0.44 18.65±1.73 |
|
|
I-D II-D IV-D V-D VI-D VII-D VIII-D |
Control ME Ext. ME Ext. ME Ext. ME Ext. ME Ext. ME Ext. Tolbutamide |
0.5 % CMC 125 250 500 750 1000 2000 100 |
275.3±1.96 269.4±1.95 268.0±2.33 279.1±1.56 272.9±2.27 276.7±2.15 273.2±1.32 270.0±0.81 |
275.4±2.46 262.6** ±2.21 258.5** ±3.28 226.0*** ±1.36 226.6±0.94 229.0±1.24 230.6±1.09 214.5±1.52 |
0.02±0.94 2.52±0.39 3.56±0.60 19.03±0.68 16.96±0.52 17.21±0.80 15.59±0.29 20.57±0.56 |
ME Ext: Methanol extract; N: normal; D:diabetic;n=6; Values are mean ± S.E.M;.
* p < 0.05 significant from normal control.
** p < 0.025 significant from diabetic control.
*** Optimum dose (p < 0.005).
2.5. Collection and processing of blood for estimation of glucose
Blood glucose concentration (mg/dL) of overnight fasted animals was determined using standard kit (Randox UK) based on glucose oxidase method using a UV-visible spectrophotometer at 546 nm. Blood samples were collected from the marginal ear vein just prior to and 4 h after dosing. Serum was separated by centrifuging the samples at 5000 rpm for 10 min [18].
2.6. Induction of diabetes
Diabetes was induced in rabbits by intraperitonial injection of alloxan monohydrate at a dose of 150 mg/kg b.w. dissolved in normal saline [19]. Seven days after injection, the rabbits were confirmed as diabetic rabbits with blood glucose≥250 mg/dL were selected for the experiments. The results are presented in Table 1.
2.7. Determination of optimum dose
To find out the optimum dose of methanol extract of R. tuberosa, eight groups (I-N to VIII-N; N: normal) comprising of six rabbits each were used. The animals were fasted for 8 h before the experiments. Group I-N animals received 0.5% CMC in distilled water and served as control. After collecting the blood samples at zero h, various doses (125, 250, 500, 750, 1000 and 2000 mg/kg b.w.) of the ME extract of R. tuberosa were administered orally to the animals of group II-N to VII-N, respectively. Blood samples were collected for the estimation of glucose concentration from the marginal ear vein of the rabbits after 4 h of the drug (ME extract) administration [20]. Results were compared with tolbutamide which was administered orally to animals of group VIII-N (reference) at a dose of 100 mg/kg b.w.
Diabetic animals were similarly divided into eight groups (I-D to VIII-D; D: diabetic) of six animals each. Different doses (125, 250, 500, 750, 1000 and 2000 mg/kg b.w.) of ME extract were administered to animals of group II-D to VII-D while group I-D served as control. Tolbutamide was administered to group VIII-D at a dose of 100 mg/kg b.w.
Table 2. Effects of methanolic extract, n-hexane, ethyl acetate and water fractions of R. tuberosa on blood glucose level in normal and alloxan-induced diabetic rabbits.
|
Test Samples |
Dose (mg/kg b. w) |
Mean blood glucose concentration ± S.E.M. (mg/dL) in normal animals |
Mean blood glucose concentration ± S.E.M. (mg/dL) in diabetic animals |
||||
|
0 h |
4 h |
% reduction |
0 h |
4 h |
% reduction |
||
|
Control ME Ext. HF EF WF Tolbutamide |
0.5 % CMC 500 150*** 100*** 275*** 100 |
94.3±0.60 89.2±0.64 88.3±0.88 105.1±0.98 89.9±1.34 100.7±4.12 |
93.8±0.49 73.7±1.17* 81.5±0.38* 87.5±0.84* 90.2±0.34 68.3±1.93* |
0.57±0.53 17.33±1.53 7.58±1.04 16.70±0.73 1.04±0.96 31.70±2.92 |
277.3±1.38 279.1±1.56 274.0±1.71 266.0±1.66 269.8±2.70 289.8±2.20 |
277.2±1.25 226.0±1.36** 232.4±2.08** 189.8±1.80** 266.5±1.63 190.4±1.60** |
0.03±0.53 19.03±1.53 15.17±0.58 28.64±0.28 1.20±0.41 34.31±0.39 |
n = 6; Values are mean±S.E.M; ME Ext: methanol extract; HF: n-hexane fraction, EF: ethyl acetate fraction, WF: water fraction. The doses selected for various fractions are based on their relative amounts present in 500 mg ME extract (500 mg/kg b. w of ME extract is the optimum dose).
* p < 0.005 significant from normal control.
** p < 0.005 significant from diabetic control.
*** ≈500 mg/kg b. w of ME Extract.
2.8. Hypoglycemic effect of fractions of R. tuberosa
Fasted normal rabbits were divided into five groups (IX-N, X-N, XI-N, XII-N and XIII-N; N: normal; six animals in each group). The IX-N group (normal control) received only 0.5% CMC (10 mL/kg b.w.) and served as control. Various fractions (HF, EF and WF) suspended in CMC were administered orally at the dose of 150, 100 and 275 mg/kg b.w. in a volume of 10 mL/kg to the animals of group X-N, XI-N and XII-N, respectively. The doses selected for various fractions are based on their relative amounts present in 500 mg ME; 500 mg/kg b.w. of ME is the optimum dose. Group XIII-N received tolbutamide (100 mg/kg b.w.) as the reference drug suspended in 0.5% CMC.
Similarly, the overnight fasted alloxan-induced diabetic rabbits were divided into five groups (IX-D, X-D, XI-D, XII-D and XII-D; D: diabetic) of six rabbits each. Group IX-D (diabetic control) received vehicle (0.5% CMC). Doses of 150, 100 and 275 mg/kg b.w. of HF, EF and WF, respectively, were given orally to group X-D, XI-D and XII-D. Group XIII-D received tolbutamide as the reference drug (100 mg/kg b.w.) suspended in vehicle (10 mL/kg b.w.).
2.9. Toxicity evaluation in rabbits
The methanol extract was tested for its acute toxicity (if any) in rabbits. To study the acute toxicity, six groups of animals (3 animals in each group) were taken. Different doses (100, 500, 1000, 3000, and 5000 mg/kg b.w.) of the extract were orally administered to the over night starved rabbits of group T1-T5. The control group (T6) was given only CMC. After the administration of extract, the rabbits were observed for gross behavioral, neurological, autonomic and toxic effects continuously for 2 h and then at 6 h interval up to three days and daily up to two weeks.
2.10. Phytochemical studies
Phytochemical study of the various solvent fractions was carried out to determine the presence of alkaloids, flavonoids, triterpenoids, sterols and phenolics.
Table 3. Antioxidant activity and total phenolic content of extract/fractions of R. tuberosa.
|
Samples |
DPPH• scavenging assay |
Total phenolics (mg GAE/g)** |
|
|
% Inhibition (at 100 µg/mL) |
IC50 (μg/mL)* |
||
|
ME ext. HF EF WF BHT |
41.6±1.13 30.9±3.81 64.8±0.53 18.5±4.17 76.2±0.41 |
233.5±3.39 1956.6±26.94 51.0±3.22 513.3±25.04 11.2±0.53 |
544.49±14.28 188.04±11.28 763.46±12.77 385.82±9.64 — |
Values are mean±S.E.M, ME: methanol, HF: n-hexane fraction, EF: ethyl acetate fraction, WF: water fraction and BHT: butylated hydroxytoluene.
* The antioxidant activity was evaluated as the concentration of the test sample required to decrease the absorbance at 517 nm be 50 %in comparison to the control response.
** Total phenolic content is expressed as gallic acid equivalents (GAE; mg gallic acid/g of extract).
2.11. Antioxidant activity
Antioxidant activity of the extract/fractions was analyzed using DPPH assay [1]. 2.0 mL of DPPH (0.2 mg/mL in methanol) was mixed with 1.0 mL sample solution (0.5 mg/mL in methanol). Reduction in the concentration of free radicals was monitored at 517 nm using UV-Visible spectrophotometer. Absorbance of blank sample containing 1.0 mL of methanol and 2.0 mL DPPH solution was also measured. Percent inhibition was calculated by the following formula:
Inhibition%=[(AB–AA)/AB]×100
Where AB=Absorbance of blank sample
AA=Absorbance of fraction (test sample)
Standard antioxidant butylated hydroxytoluene (BHT) was tested and used as reference. The sample solutions having >50 % inhibition were diluted in series to get concentration 0.1-1.0 mg/mL and the percentage of inhibition was calculated. A plot of inhibition% of DPPH against concentration of the various fractions of R. tuberosa was used to determine IC50 values.
2.12. Determination of total phenols
Total phenols were determined by the method of Djeridane [21]. Equal to 0.1 mL of each test sample (0.5 mg/mL) was mixed with 0.1 mL of Folin-Ciocalteu reagent. After 1 min, 2.8 mL of 10% sodium bicarbonate solution was added and the mixture was allowed to stand for 30 min. Absorbance was measured at 725 nm spectrophotometrically. Gallic acid (0.05-0.5 mg/mL) was used to establish standard calibration curve. The mean of three readings was used and the total phenolic contents were expressed as milligrams of gallic acid equivalent/g dry weight of the extract. The coefficient of determination was R2 = 0.9946.
2.13. Statistical analysis
All the data was expressed as mean±SEM for six rabbits in each group. The significance of blood glucose reduction produced by the ME extract and its various fractions was determined by applying student’s unpaired‘t’ test [18].
3. Results
Methodology of Kato [22] was adopted for the hypoglycemic activity of various fractions with slight modification in time pattern for blood glucose level determination. Test samples (ME extract, HF, EF and WF of R. tuberosa and tolbutamide) were given immediately after the collection of initial blood samples. The blood glucose levels of normal and diabetic animals were determined at 0 h and 4 h after the oral administration of various fractions of R. tuberosa [18].
ME extract of R. tuberosa was fractionated to obtain HF, EF and WF in 26.44%, 16.38% and 55.12% yields, respectively. Phytochemical studies of HF and EF indicated the presence of triterpenoids and flavonoids, respectively. WF showed the presence of polyphenols.
3.1. Acute toxicity study
The ME extract of R. tuberosa was subjected to acute toxicity study up to a dose of 5000 mg/kg b.w. which indicated non-toxic nature of the extract. No gross behavioral changes were observed at any of the doses selected until the end of the study period.
3.2. Hypoglycemic study
3.2.1. Determination of optimum dose of R. tuberosa
The effects of ME extract R. tuberosa on blood glucose level of fasting normal and alloxan-induced diabetic rabbits is shown in Table 1. Optimum activity was observed at 500 mg/kg b.w. level in normal animals. Minimum dose (125 mg/kg b.w.) did not show any blood glucose lowering activity. Fasting blood glucose (FBG) decreased to 73.7±1.2 mg/dL (p < 0.05) after 4 h of the drug (500 mg/kg b.w.) administration from an initial value of 89.2±0.64 mg/dL. A dose-dependent effect was observed on FBG upto a dose of 500 mg/kg b.w. Tolbutamide (100 mg/kg b.w.) caused a marked decrease in FBG (glucose concentration from 94.5±1.5 to 76.9±1.2 mg/dL; 18.65±1.73% decrease) in the animals.
A dose-dependent effect was also observed in diabetic animals upto a dose of 500 mg/kg b.w. which decreased the glucose concentration to 226.0±1.4 mg/dL from an initial level of 279.1±1.6 mg/dL (19.03±0.68% reduction; p < 0.005). Tolbutamide showed a significant anti-diabetic activity at a dose of 100 mg/kg b.w. in alloxan-induced diabetic rabbits.
3.2.2. Effect of R. tuberosa fractions on blood glucose level
The effect of R. tuberosa solvent fractions on the blood glucose level of normal and alloxan-induced diabetic rabbits is shown in Table 2.
Normal control group showed no statistically significant hypoglycemic activity (p > 0.2 at 4 h). EF and HF of R. tuberosa produced a significant hypoglycemia in normal rabbits. EF and HF significantly decreased the blood glucose level at 4th h (16.70±0.73% and 7.58±1.04%) respectively in normal animals which is comparable with the standard drug tolbutamide.
The results of the effect of R. tuberosa fractions on blood glucose of alloxan-induced diabetic rabbits are shown in Table 2. The HF showed moderate reduction of blood glucose level to 15.17±0.58 % after 4 h in diabetic rabbits while the effect of EF (100 mg/kg b.w.) is higher with reduction in blood glucose level of 28.64±0.28% after 4 h of drug administration. WF showed no blood glucose lowering activity (p > 0.1) in normal as well as alloxan-induced diabetic animals.
3.3. Antioxidant activity
Table 3 describes the results of antioxidant activity of ME extract and various solvent fractions of R. tuberosa. IC50 values ranged from 51.0±3.2 to 1956.6±26.9 μg/mL. Highest DPPH radical scavenging activity was observed in EF with lowest IC50 51.0 µg/mL followed by ME extract (IC50: 233.5±3.4 μg/mL). Higher antioxidant activity of EF than ME can be attributed to the increase in the concentration of active components through condensation effects during the solvent-solvent partitioning processes [1].
3.4. Total Phenols
Total phenolic contents of the crude ME extract and solvent fractions of R. tuberosa are shown in Table 3. The total phenolic content varied in different fractions and ranged from 188.04±11.28 to 763.46±12.77 mg GAE/g of dry extract. EF showed the highest amount of total phenol (763.46±12.77) followed by ME extract (544.49±14.28) as shown in Table 3.
4. Discussion
Diabetes mellitus (DM) is probably the world’s largest growing metabolic disease which has been identified as a heterogeneous disease [23]. Diabetic complications have been controlled world wide by the use of traditional plant medicines. Drugs which are obtained from natural sources corresponded to 78 % of new drugs approved by Food and Drug Administration (FDA) between 1983 & 1994 [24]. Furthermore, the limitations of currently available pharmacological agents for control of hypoglycemia have stimulated research on novel anti-diabetic agents with different mechanism of action [25]. The present study is a preliminary intent to validate the use of R. tuberosa to control DM. Our results revealed that R. tuberosa has significant blood glucose lowering effect in normal and alloxan-induced diabetic rabbits. Among the extract/fractions of R. tuberosa, ME extract (500 mg/kg b.w.), EF (100 mg/kg b.w.) showed a significant hypoglycemic effect, while HF (150 mg/kg b.w.) exhibited moderate activity after 4 h of the administration. In the present work normal and alloxan-induced diabetic rabbits were used. HF exhibited 7.58±1.04% reduction of blood glucose level in normal, while 15.17±0.58% reduction in diabetic animals. Similarly EF showed 16.70±0.73 and 28.64±0.28% blood glucose reduction in normal and alloxan-induced diabetic rabbits, respectively. The results showed that HF and EF can effectively control diabetic conditions. Alloxan causes hyperglycemia by destroying pancreatic β-cells selectively, mediated by the generation of cytotoxic free radicals [26, 27]. Furthermore, diabetes-induced oxidative stress due to increased reactive oxygen species enhances complications in diabetes [28]. Therefore, it has been suggested that a variety of antioxidants may prevent diabetic complications. Traditional use of R. tuberosa in herbal drink is attributed to its antioxidant activity. The results of antioxidant activity of the extract and solvent fractions of R. tuberosa demonstrated strong antioxidant activity in the EF with IC 50 51.0±3.22 μg/mL, which is in agreement with the literature [1].
Flavonoids constitute one of the well known and wide spread groups of phenolics in higher plants [29]. Several flavonoids; apigenin, luteolin, 3, 5-diglucoside, apigenin 7-O-glucuronoide, apigenin glucoside, apigenin rutinoside, luteolin glucoside, pedalitin, flavone glycoside, cirsimaritin, cirsimarin, cirsiliol 4′-glucoside, sorbifolin and pedalitin are reported in R. tuberosa [2, 30, 31]. Among possible mechanisms to lower blood glucose level, R. tuberosa might act by quenching of free radicals in the body and to improve the metabolic disorder which can be attributed to the presence of flavonoids in the EF. Thus, it can be proposed that the flavonoids of R. tuberosa play a vital role in the reduction of blood glucose level both in normal as well as in diabetic rabbits.
HF of R. tuberosa exhibited reduction in blood glucose level after 4 h of the drug administration. Phytochemical analysis of the n-hexane fraction indicated the presence of sitosterols which has been reported to have anti-diabetic effect and work like sulphonyl urea medicine [32-34]. The hypoglycemic effect of the ME extract could possibly be related to its composition. Thus the significant anti-diabetic effect of R. tuberosa may be due to the presence of more than one hypoglycemic principle and their synergetic properties.
5. Conclusion
In conclusion, we have demonstrated that the methanolic extract and solvent fractions (ethyl acetate and n-hexane) of R. tuberosa possess significant blood glucose lowering effect both in normoglycemic and in alloxan-induced diabetic rabbits. The present results suggest the validity of the clinical use of R. tuberosa in the control of diabetes mellitus. However, further comprehensive chemical and pharmacological investigations are required to isolate and evaluate the hypoglycemic effects of the active components from R. tuberosa.
6. Acknowledgements
The authors wish to thank the Principal, Prof. Dr. Mohammad Jamshaid (University College of Pharmacy, University of the Punjab Lahore) for providing animal house facility along with other necessary laboratory facilities and Dr. Zaheer-ud-Din Khan (Department of Botany, GC University Lahore) for the identification of plant material. We are also very grateful to “Higher Education Commission Islamabad” who financially supported this work.
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