Results

Published in the Journal of Ethnopharmacology 175: 518-527

3.3.4 Results

The results of weekly mean body weight (bw) are presented in Figure 3.15. Although fructose feeding for two weeks did not significantly affect the mean bw of the treated animals compared to untreated animals, after STZ injection, the bw of DBC group was decreased for the entire intervention period. On the other hand, AMEF oral treatment ameliorated this reduction when significant (p < 0.05) amelioration was observed after the 2^nd, 3^rd and 4^th week of intervention. Additionally, no significant difference of bw was observed between the AMEF treated groups (DAML and DAMH) and NC and DMF groups (Figure 3.15).

Figure 3.15. Mean body weight change in all animal groups during the entire study period. Data are presented as the mean ± SD of 5-7 animals. ^a-bValues with different letter near the lines for a given week are significantly different from each other group of animals (Tukey's-HSD multiple range post hoc test, p < 0.05). NC, Normal Control; DBC, Diabetic Control; DAML, Diabetic A. melegueta low dose; DAMH, Diabetic A. melegueta high dose; DMF, Diabetic Metformin; NAMH, Normal A.

melegueta high dose.

The results of feed and fluid intake in different group of animals are presented in Figure 3.16.

The results of DBC group suggest that the induction of diabetes significantly (p < 0.05) increased feed

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100 150 200 250 300 350 400

WK-2 WK-1 WK 0 WK 1 WK 2 WK 3 WK 4 WK 5

Mean body weight (g)

Experimental weeks

NC DBC DAML DAMH DMF NAMH

and fluid intake compared to NC group (Figure 3.16). Treatment with various dosages of AMEF for weeks to diabetic animals significantly (p < 0.05) ameliorated the alterations of feed and fluid intake which are comparable to the DMF group (Figure 3.16).

Figure 3.16. Mean food and fluid intake of different animal groups during 4-week intervention period. Data are presented as the mean ± SD of 5-7 animals. ^a–dValues with different letters over the bars for a given parameter are significantly different from each other group of animals (Tukey's-HSD multiple range post hoc test, p < 0.05). NC, Normal Control; DBC, Diabetic Control; DAML, Diabetic A. melegueta low dose; DAMH, Diabetic A. melegueta high dose; DMF, Diabetic Metformin;

NAMH, Normal A. melegueta high dose.

The results of weekly NFBG and FBG (last week only) are presented in Figure 3.17. The data showed that the induction of T2D significantly (p < 0.05) increased NFBG in diabetic animals compared to the normal animals (NC). On the other hand, the oral administration of AMEF significantly (p < 0.05) decreased NFBG in DAML and DAMH groups compared to the DBC group when no significant difference was observed between the AMEF (DAML and DAMH) and metformin (DMF) treated groups. This effect was more pronounced in the DAMH group compared to the DAML group.

Additionally, no significant difference was observed between the last week FBG of DAMH and NC group (Figure 3.17).

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Feed intake (g/day/rat) Fluid intake (ml/day/rat)

Feed and fluid intake

NC DBC DAML

DAMH DMF NAMH

Figure 3.17. Weekly NFBG of all animal groups during the entire experimental period. Data are presented as the mean ± SD of 5-7 animals. ^a–cValues with different letters near the lines for a given week are significantly different from each other group of animals (Tukey's-HSD multiple range post hoc test, p<0.05). NC, Normal Control; DBC, Diabetic Control; DAML, Diabetic A. melegueta low dose; DAMH, Diabetic A. melegueta high dose; DMF, Diabetic Metformin; NAMH, Normal A. melegueta high dose; NFBG, Non-fasting blood glucose; FBG, Fasting blood glucose.

The data of oral glucose tolerance test (OGTT) are presented in Figure 3.18. According to the results, the glucose tolerance ability of the animals in AMEF treated groups was significantly better than the DBC group for the entire period of the test when the results were significantly better in the AMEF treated groups compared to the DMF group from 60-120 min of the test period. No significant difference was observed between the glucose tolerance ability of AMEF treated groups and NC group during this period as well (Figure 3.18). Furthermore, the calculated area under curve (AUC) for DBC group was significantly (p < 0.05) higher compared to NC group when that of DAML and DAMH groups were significantly (p < 0.05) lower compared to DBC groups and were comparable to the DMF group (Table 3.7).

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0 50 100 150 200 250 300 350 400 450 500

WK 0 WK 1 WK 2 WK 3 WK 4

Blood glucose levels (mg/dL)

Week after intervention

NC DBC DAML DAMH DMF NAMH

Figure 3.18. Oral glucose tolerance test (OGTT) in all animal groups in the last week of the 4-week experimental period. Data are presented as the mean ± SD of 5-7 animals. ^a–cValues with different letters near the lines for a given parameter are significantly different from each other group of animals (Tukey's-HSD multiple range post hoc test, p < 0.05). NC, Normal Control; DBC, Diabetic Control; DAML, Diabetic A. melegueta low dose; DAMH, Diabetic A. melegueta high dose; DMF, Diabetic Metformin; NAMH, Normal A. melegueta high dose.

Table 3.7. Area under the curve (AUC) of different animal groups at the end of the experimental period

NC DBC DAML DAMH DMF NAMH

AUC*x10³ 15.75 ± 0.97^a 60.15 ± 7.50 ^c 24.54 ± 3.50 ^b 25.32 ± 5.64 ^b 28.68 ± 7.21 ^b 15.78 ± 0.97 ^a Data are presented as the mean ± SD of 5-7 animals. ^a-cValues with different letter along a row for a given parameter are significantly different from each other group of animals (Tukey's-HSD multiple range post hoc test, p<0.05). NC, Normal Control; DBC, Diabetic Control; DAML, Diabetic A. melegueta low dose; DAMH, Diabetic A. melegueta high dose; DMF, Diabetic Metformin; NAMH, Normal A. melegueta high dose, AUC, Area under curve.

The data for serum insulin, fructosamine and calculated HOMA-IR and HOMA-β scores are presented in Table 3.8. Serum insulin level and the calculated HOMA-β scores were decreased significantly (p < 0.05) whereas serum fructosamine as well as HOMA-IR were increased significantly (p < 0.05) in the DBC group compared to the NC group (Table 3.8). Oral intervention of AMEF for four weeks to diabetic animals elevated serum insulin and HOMA-β scores with subsequent attenuation of serum fructosamine level and HOMA-IR scores in DAML and DAMH groups compared to the DBC group. These effects were found to be more pronounced in the DAMH group compared to the DAML group which is comparable with DMF group (Table 3.8).

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0 100 200 300 400 500 600 700

0 30 60 90 120

Blood glucose (mg/dL)

Time (min)

NC DBC DAML DAMH DMF NAMH

Table 3.8. Serum insulin and fructosamine levels, HOMA-IR and HOMA-β scores of different animal groups at the end of the experimental period

NC DBC DAML DAMH DMF NAMH

Insulin (pmol/L) 76.50 ± 6.26^b 47.05 ± 9.02^a 59.86 ± 11.45^b 64.81 ± 8.77^b 75.88 ± 14.23^b 65.49 ± 11.87^b Fructosamine

(μmol/L) 239.20 ± 47.49^a 365.43 ± 44.94^b 298.00 ± 23.39^a 247.86 ± 28.96^a 283.14 ± 32.46^a 274.00 ± 19.95^a

HOMA-IR٭ 2.50 ± 0.27^a 6.30 ± 1.64^c 3.75 ± 0.53^b 3.11 ± 0.61^b 3.82 ± 0.90^b 2.11 ± 0.44^a

HOMA-β٭٭ 127.66 ± 43.71^d 7.33 ± 1.26^a 26.17 ± 10.18^b 44.32 ± 9.86^b,c 52.57 ± 25.76^c 108.60 ± 19.24^d

Data are presented as the mean ± SD of 5-7 animals. ^a–dValues with different letters along the rows for a given parameter are significantly different from each other group of animals (Tukey's- HSD multiple range post hoc test, p<0.05). NC, Normal Control; DBC, Diabetic Control; DAML, Diabetic A. melegueta low dose; DAMH, Diabetic A. melegueta high dose; DMF, Diabetic Metformin; NAMH, Normal A. melegueta high dose; HOMA-IR, Homeostasis model assessment-insulin resistance; HOMA-β, Homeostasis model assessment-β

٭HOMA-IR = [(Fasting serum insulin in U/l x Fasting blood glucose in mmol/L)/22.5].٭٭HOMA-β = (Fasting serum insulin in U/l x 20/Fasting blood glucose in mmol/L-3.5).

Figure 3.19. Histopathological examination of the pancreatic islets of different animal groups at the end of the intervention period. The NC had a larger islet with high number of β-cells while the DBC had a smaller islet and morphologically deformed β-cells. The DAML, DAMH and DMF groups had relatively larger islets with higher number of β-cells compared to the DBC group. NC, Normal Control; DBC, Diabetic Control; DAML, Diabetic A. melegueta low dose; DAMH, Diabetic A. melegueta high dose; DMF, Diabetic Metformin; NAMH, Normal A. melegueta high dose.

The slides of pancreatic histopathology are presented in Figure 3.19. There was a significant degeneration of pancreatic islets with subsequent reduction on the number of β-cells in the DBC group compared to the NC group. However, treatment with the various dosages of AMEF as well as standard drug attenuated the diabetes induced pancreatic damage and restored the pancreatic morphology to near normal when the number of β-cells were significantly higher in the DAMH group compared to the DAML group (Figure 3.19).

The results of liver weight, relative liver weight and liver glycogen levels are presented in Table 3.9. The induction of T2D did not affect the absolute liver weight but significantly (p < 0.05) increase the relative liver weight and liver glycogen level in the DBC group compared to the NC group. A significant (p < 0.05) reduction of relative liver weight and liver glycogen levels was observed in the DAML and DAMH groups compared to the DBC group when the results were not significantly different compared to the NC and DMF groups (Table 3.9).

The data for serum lipid profile and calculated atherogenic index (AI) and coronary risk index (CRI) are presented in Table 3.10. Elevated serum concentrations of TC, TG and LDL-cholesterol levels as well as calculated AI and CRI with subsequent reduction on serum HDL-cholesterol were observed in the DBC group compared to the NC group (Table 3.10). Treatment with AMEF to diabetic animals significantly (p < 0.05) and dose-dependently reduced TC, TG and LDL-cholesterol, AI and CRI in DAML and DAMH groups compared to the DBC group. Although dose-dependent increase in serum HDL-cholesterol level was observed in the AMEF treated groups compared to the DBC and DMF groups, the data were not significantly different (Table 3.10).

The data for serum ALT, AST, ALP, urea, uric acid, creatinine, LDH and CK-MB are presented in Table 3.11. At the end of the experimental period, the concentrations of all of the above-mentioned serum parameters were increased in the DBC group compared to the NC group, when serum AST level was not affected by the induction of diabetes. On the other hand, oral administration of AMEF to diabetic animals significantly (p < 0.05) ameliorated these alterations in DAML and DAMH groups.

The effects were more pronounced in the DAMH group compared to the DAML group which did not differ significantly (p < 0.05) compared to the DMF group regarding most of the parameters. Similarly, treatment of AMEF to non-diabetic animals demonstrated no significant (p < 0.05) effect on these serum parameters compared to the NC group (Table 3.11).

Table 3.9. Effect of AMEF on liver weights and liver glycogen concentrations in different animal groups as the end of the experimental period.

NC DBC DAML DAMH DMF NAMH

Liver weight(g) 9.25 ± 1.04 10.34 ± 0.31 9.79 ± 0.30 9.75 ± 0.40 9.10 ± 1.33 9.02 ± 0.70 Relative liver weight (%)٭ 2.89 ± 0.29^a 4.55 ± 0.34^b 3.36 ± 0.04^a 3.31 ± 0.40^a 3.10 ± 0.45^a 2.90 ± 0.25^a Liver glycogen (mg/g

tissue)

6.76 ± 0.81^a 8.25 ± 0.83^b 7.03 ± 0.53^a 6.98 ± 0.63^a 6.92 ± 0.56^a 6.80 ± 0.57^a

Data are presented as the mean ± SD of 5-7 animals. ^a-bValues with different letter along a row for a given parameter are significantly different from each other group of animals (Tukey's-HSD multiple range post hoc test, p<0.05). NC, Normal Control; DBC, Diabetic Control; DAML, Diabetic A. melegueta low dose; DAMH, Diabetic A. melegueta high dose; DMF, Diabetic Metformin;

NAMH, Normal A. melegueta high dose.

٭Relative liver weight (%) = [weight of the liver (g)/body weight (g)] x 100%

Table 3.10. Serum lipid profiles atherogenic and coronary risk indices of different animal groups at the end of the experimental period

NC DBC DAML DAMH DMF NAMH

TC (mg/dL) 76.20 ± 7.40^a 116.00 ± 12.92^c 94.86 ± 10.17^b 83.29 ± 8.40^a,b 95.71 ± 7.74^b 78.00 ± 7.91^a TG (mg/dL) 70.20 ± 18.10^a 150.86 ± 24.57^b 107.71 ± 38.13^a 81.14 ± 23.78^a 100.71 ± 32.47^a 68.80 ± 5.50^a LDL-Cholesterol

(mg/dL)*

12.96 ± 4.16^a 57.54 ± 14.45^c 40.03 ± 11.21^b 30.34 ± 7.28^a,b 42.86 ± 8.93^b,c 22.84 ± 10.72^a

HDL-Cholesterol (mg/dL)

49.20 ± 8.35^b 28.29 ± 6.25^a 33.29 ± 6.99^a 36.71 ± 8.52^a,b 32.71 ± 7.43^a 41.40 ± 6.62^a,b

AI 0.57 ± 0.13^a 3.30 ± 1.08^c 2.00 ± 0.91^b 1.36 ± 0.54^a,b 2.06 ± 0.69^b 0.93 ± 0.38^a,b

CRI 1.57 ± 0.13^a 4.30 ± 1.08^c 3.00 ± 0.91^b 2.36 ± 0.54^a,b 3.06 ± 0.69^b 1.93 ± 0.38^a,b

Data are presented as mean ± SD of 5-7 animals. ^a–cValues with different superscript letters along a row for a given parameter are significantly different from each other group of animals (Tukey's- HSD multiple range post hoc test, p<0.05). NC, Normal Control; DBC, Diabetic Control; DAML, Diabetic A. melegueta low dose; DAMH, Diabetic A. melegueta high dose; DMF, Diabetic Metformin; NAMH, Normal A. melegueta high dose; TC, Total cholesterol; TG, Triglyceride; LDL-cholesterol, Low density lipoprotein-cholesterol; HDL-cholesterol; High density lipoprotein- cholesterol; AI, Atherogenic index; CRI, Coronary risk index.

*LDL-Cholesterol (mg/dL)= [TC- HDL- (TG/5)]

Table 3.11. Serum ALT, AST, ALP and other biochemical parameters different animal groups at the end of the experimental period

NC DBC DAML DAMH DMF NAMH

ALT (U/L) 77.40 ± 20.38^a,b 116.14 ± 37.37^c 70.14 ± 10.07^a,b 74.14 ± 8.86^a,b 93.57 ± 6.86^b 58.80 ± 21.67^a AST (U/L) 88.40 ± 11.41 83.29 ± 15.57 80.29 ± 4.19 85.57 ± 10.97 94.71 ± 9.39 82.20 ± 7.46 ALP (U/L) 149.40 ± 11.33^a 451.14 ± 35.10^c 230.14 ± 31.76^b 200.86 ± 26.82^a,b 208.86 ± 27.58^b 158.60 ± 22.55^a Creatinine (mg/dL) 2.52 ± 0.78^b 3.12 ± 0.45^b 1.73 ± 0.52^a 1.53 ± 0.42^a 1.68 ± 0.27^a 2.42 ± 0.77^a,b Urea (mg/dL) 22.00 ± 4.69^a 60.71 ± 15.71^b 21.29 ± 7.80^a 29.29 ± 7.11^a 26.00 ± 5.00^a 27.80 ± 3.90^a Uric acid (mg/dL) 2.06 ± 0.67 2.85 ± 1.18 2.28 ± 0.84 2.77 ± 0.84 2.56 ± 1.36 1.69 ± 1.05 LDH (U/L) 284.00 ± 62.56^a 458.86 ± 68.05^b 419.14 ± 95.60^b 395.14 ± 57.94^a,b 361.57 ± 80.66^a,b 321.20 ± 78.44^a CK-MB (U/L) 374.54 ± 82.56^a 1064.36 ± 537.18^b 692.53 ± 128.69^a,b 368.16 ± 83.06^a 743.97 ± 75.29^a,b 508.68 ± 127.34^a

Data are presented as mean ± SD of 5-7 animals. ^a–cValues with different superscript letters along a row for a given parameter are significantly different from each other group of animals (Tukey's- HSD multiple range post hoc test, p < 0.05). NC, Normal Control; DBC, Diabetic Control; DAML, Diabetic A. melegueta low dose; DAMH, Diabetic A. melegueta high dose; DMF, Diabetic Metformin; NAMH, Normal A. melegueta high dose; ALT, Alanine transaminase; AST, Alanine transaminase; ALP, Alkaline phosphate; LDH, Lactate dehydrogenase; CK-MB, Creatine kinase.