Potential application of Conyza canadensis ( L ) Cronquist in the management of diabetes : In vitro and in vivo evaluation

Purpose: To investigate the antihyperglycemic activity of Conyza canadensis via α-glucosidase inhibition in alloxan-induced diabetic mice. Methods: In vitro antidiabetic activity was investigated using α-glucosidase inhibition assay with acarbose (62.5, 125, 500 and 1000 μg/ml) as the standard drug. Conyza canadensis crude extract (Cc.Cr) in doses of 10, 30, 100 and 300 mg/kg were administered daily as a single dose to alloxaninduced (200 mg/kg) diabetic mice (Balb/c), and its effect on fasting blood glucose levels and body weight were evaluated for 15 consecutive days; oral glucose tolerance test was conducted. Metformin (500 mg/kg) was used as a standard antidiabetic drug for comparison. Acute toxicity of Cc.Cr was also evaluated at doses of 3 and 5 g/kg. Results: Conyza canadensis crude extract (Cc.Cr) exhibited strong enzyme inhibition at concentrations (μg/ml) of 1000 (74.78 ± 0.92), 500 (65.11 ± 0.07), 250 (57.55 ± 0.41), 125 (51.55 ± 0.67) and 62.5 ( 44.00 ± 0.57), with a median inhibitory concentration (IC50) of 107 μg/ml. Cc.Cr at all test doses (10 300 mg / kg) reduced fasting blood glucose levels in alloxan (200 mg/kg) induced diabetic mice on days 5, 10 and 15 compared to the diabetic control group (p < 0. 001). These effects were similar to those caused by the standard antidiabetic drug, metformin. Cc.Cr at all test doses also increased body weight of treated animals. The extract (300 mg/kg) significantly improved tolerance of oral glucose overload in mice, like metformin. The extract did not cause any mortality up to the maximum dose of 5 g/kg. Conclusion: The results reveal that Conyza canadensis possesses potent secondary metabolites which can cause inhibition of α-glucosidase. Moreover, the plant extract has the ability to reduce blood glucose level in diabetic animals and significantly improves oral glucose overload tolerance.


INTRODUCTION
Diabetes is one of the leading causes of death worldwide and according to World Health Organization (WHO), diabetes will be the 7 th leading cause of death by 2030 [1].It is a metabolic disease with major clinical manifestation of chronic hyperglycemia.The most prominent pathway in the management of diabetes is to reduce the postprandial hyperglycemia by reducing the absorption of glucose from the intestinal tract [2].This reduction in glucose absorption is possible by inhibiting a key enzyme responsible for the process, i.e. α-glucosidase.The inhibitors of αglucosidase will reduce the breakdown of complex carbohydrates to simple sugar units.The reduction in breakdown of complex carbohydrate will lead to the reduction in postprandial glucose level [3].
The present study is designed to investigate the effect of Conyza canadensis on the blood glucose level and oral glucose tolerence in alloxan induced diabetes in mice.

EXPERIMENTAL Plant material and extraction
The whole plant of Conyza canadensis was collected from Islamabad, Pakistan in July 2015 and authenticated by Dr. Mushtaq Ahmad, a taxonomist at Department of Plant Sciences, Quaid-e-Azam University, Islamabad, Pakistan.Plant specimen was deposited at the Department herbarium (voucher no.804).
The plant material was shade dried at room temperature and then grounded to a coarse powder.The powdered material was macerated in 70 % methanol for 10 days with regular shaking at intervals.The crude methanol extract was obtained by concentrating on rotary evaporator under reduced pressure at 40 -50 ° C [14].

Chemicals
The

Preliminary phytochemical screening
Preliminary phytochemical testing was performed for the detection of alkaloids, tannins, triterpenes, proteins, carbohydrates, flavonoids, saponins and steroids, using standard procedures [16].Alkaloids were tested using Mayer's reagent and Dragendroff's reagent.Tannins were considered to be positive when addition of lead acetate to the test material of Cc.Cr gave cream yellow color.Triterpenes were detected positive when addition of sulphuric acid produced a red colouration using Liebermann Burchard test.
Proteins were tested with ninhydrin, appearance of blue colour confirmed the presence of proteins.Benedict's test was used to detect carbohydrates, orange red precipitates were considered positive indication.If plant material treated with chloroform and sulfuric acid produced red colour, confirmed steroids presence [17].Saponins were tested using Froth's test [18].

In-vitro α-glucosidase inhibition assay
The α-glucosidase inhibitory assay for Cc.Cr was performed according to standard procedure [19].The enzyme solution was prepared by dissolving 0.5 unit / mL of α-glucosidase in 0.1M phosphate buffer (pH 6.9).The enzyme solution contains 20 µL of α-glucosidase (0.5 unit / ml) and 120 µl of 0.1 M phosphate buffer.Substrate solution of pnitrophenyl-α-D-glucopyranoside (5 mM) was made in the buffer solution.Test samples were prepared in a concentration range of 62.5-1000 µg/ml, this was then mixed with the enzyme solution and incubated for 15 min at 37 o C. Finally, substrate solution (20 µl) was added to the mixture and this was incubated for another 15 min at 37 o C.
The reaction was completed by the addition of 80 µL of 0.2M sodium carbonate solution.The absorbance were measured at 405 nm using UVspectrophotometer (Thermo Electron Corporation, USA).The system without α-glucosidase was used as blank and acarbose was used as positive control.The experiments were performed in triplicate and inhibitory activity (H) calculated as in Eq 1.
where Ac and As are the absorbance of control and test samples, respectively.

Acute toxicity studies
The acute oral toxicity test was performed as reported by [19].The mice (n = 6) in each group were fasted overnight and administered Cc.Cr orally at doses of 1, 3 and 5 g/kg.Animals were then kept under observation for 48 h and observed for any signs of diarrhea, discomfort, pain, behavioral, neurological changes, convulsions, coma or death.

Induction of diabetes, blood glucose level and body weight measurement
Animals were fasted for 12 h.The solution of alloxan (200 mg/kg) in 0.2 mL saline was injected intra-peritoneally for the induction of diabetes [20].After 48 h, fasting glucose levels of test animals were obtained.Animals with blood glucose levels above 200 mg/dl along with signs of polyuria and polydipsia were considered diabetic and included in the study.The test animals were divided into 7 groups (n = 5).Group I (non-diabetic normal control, given saline), Group II (diabetic control treated with alloxan 200 mg/kg), Group III, IV, V and VI include alloxan treated diabetic animals which received 10, 30, 100 and 300 mg/kg doses of the methanolic extract respectively.The Group VII was diabetic animals, treated with metformin (500 mg/kg), as reference drug.Each group was administered the respective test drug daily for 15 consecutive days with monitoring of blood glucose levels, using EASYGLUCO Auto-coding Glucometer and body weight (g) at regular intervals.

Oral glucose tolerance test
The mice were fasted overnight (14-15 h) and divided into non-diabetic control group (treated with saline 0.2 mL), diabetic control group (administered alloxan 200 mg/kg for induction of diabetes), test group (treated with Cc.Cr 300 mg/kg) and standard drug (treated with metformin 500 mg/kg) group, 30 min prior to oral D-glucose (2 g/kg) challenge.Blood for glucose determination was measured by tail-prick method at different time points: 0 min (before glucose load), then at 30, 60, 90 and 120 min after glucose administration [21].

Statistical analysis
Data was expressed as mean ± standard error of mean (SEM) and were analyzed by one-way analysis of variance (ANOVA) followed by Tukey post-hoc test.P < 0.05 was taken as significantly different [22].The plots were analyzed using the GraphPad software (GraphPad, San Diego, CA, USA).

Phytochemical profile
The methanol extract of Conyza canadensis showed positive results for alkaloids, tannins, triterpenes, proteins, carbohydrates, saponins, flavonoids and steroids.

α-Glucosidase inhibition
The α-glucosidase inhibition of the Cc.Cr was performed at different concentrations and compared with the standard drug acarbose at the same tested concentration (Table 1).Cc.Cr exhibited overwhelming enzyme inhibitory potential at various concentrations.At highest tested concentration (1000 µg/ml), Cc.Cr demonstrate 74.78 ± 0.92 % inhibition which was almost similar to that of the positive control at the same concentration (76.87 ± 0.06 % at 1000 µg/ml).Similarly, the observed inhibitions for Cc.Cr at concentrations of 500, 250, 125 and 62.5 µg/ml were 65.11 ± 0.07, 57.55 ± 0.41, 51.55 ± 0.67 and 44.00 ± 0.57 % respectively.The observed IC 50 s for the Cc.Cr and acarbose were 107 and 23 µg/ml respectively.

Acute toxicity
The crude extract of Conyza canadensis did neither caused any mortality nor any signs of toxicity at the tested doses of 3 and 5 g/kg (n = 5).

DISCUSSION
Several agents have been used to induce diabetes in experimental animals, including alloxan.
All animals treated with 200 mg/kg of alloxan showed marked increase in blood glucose levels up to 200 mg/dl and above after 72 h.The selected test animals were then treated with test drugs for 15 consecutive days.Conyza canadensis in a dose-dependent (10-300 mg/kg) and time-dependent (days 5-15 th ) fashion reduced blood glucose level in diabetic animals, similar to the effect of metformin, the standard antidiabetic drug.Metformin, acts by several pathways including impairing intestinal glucose absorption, enhancing insulin release from beta cells, inhibiting hepatic glucose production and increasing the uptake of glucose into the cells [24].Weight loss is one of the complications of diabetes that occurs due to enhanced lipolysis.Reduction in body weight of diabetics is attributed to inadequacy of insulin and its function in fat metabolism [25].Prevention of weight reduction in animals, treated with Cc.Cr indicates that Conyza canadensis has beneficial outcomes in preventing subsequent complication of weight loss, associated with hyperglycemia.In the glucose-loaded hyperglycemia model, employed to evaluate oral glucose tolerance, Cc.Cr displayed significantly better tolerance of glucose overload at different time intervals (30-120 min), similar to metformin.The excessive amount of glucose in blood impels insulin secretion.This secreted insulin stimulates peripheral glucose utilization and controls processing of glucose through several mechanisms [26].This property of Conyza canadensis in reducing blood glucose levels and simultaneously improving glucose tolerance, suggests its use as antidiabetic.
However, further advanced molecular studies will be needed to elucidate the underlying pharmacodynamics involved.
The observed antihyperglycemic effect of the plant can be attributed to its diverse phytoconstituent profile.Limonene isolated from aerial parts of Conyza canadensis is reported to be effective as an antidiabetic agent by inhibiting protein glycation up to 85.61% and also effectively reduced the blood glucose levels in the in-vivo experiments [27].Other compounds of the plant; β-sitosterol and stigmasterol are also known to possess antidiabetic and antioxidant potentials [28,29].

CONCLUSION
The study reveals that Conyza canadensis exhibits α-glucosidase inhibition and antihyperglycemic action in alloxan-induced diabetic mice and improves tolerance of oral glucose overload.This study thus provides scientific evidence for the use of Conyza canadensis in the treatment of diabetes mellitus in traditional medicine.Future studies will be carried out to identify the active principle(s) of the plant that are responsible for its antidiabetic effect.

Table 1 :
Alpha-glucosidase inhibition assay of crude extract of Conyza Canadensis taken as mean ± SEM (n= 3).The P values less than 0.05 were considered as statistically significant.Values significantly different in comparison to standard drug i.e. ** = P < 0.01 and *** = P < 0.001

Figure 2 :
Figure 2: Blood glucose levels of oral glucose loaded mice of non-diabetic control group diabetic control group , Conyza canadensis and metformin pre-treated groups.Data are expressed as mean ± SEM (n = 5); *** P < 0. 001 compared to alloxan-induced diabetic control group

Table 2 :
Effect of Conyza canadensis crude extract on the body weight of alloxan-treated diabetic mice