Antioxidant potential of theaflavin ameliorates the activities of key enzymes of glucose metabolism in high fat diet and streptozotocin – induced diabetic rats

Figures & data

Figure 1. Structure of theaflavin.

Table

Group I:	Normal control (received 0.5 mL of distilled water).
Group II:	Drug control (normal healthy control rats received intra gastrically theaflavin (100 mg/kg b.wt.) dissolved in 0.5 mL of distilled water for 30 days.
Group III:	Diabetic control.
Group IV:	Diabetic rats received intra gastrically theaflavin (25 mg/kg b.wt.) dissolved in 0.5 mL of distilled water for 30 days.
Group V:	Diabetic rats received intra gastrically theaflavin and metformin (50 mg/kg b.wt.) dissolved in 0.5 mL of distilled water for 30 days.
Group VI:	Diabetic rats received intra gastrically theaflavin (100 mg/kg b.wt.) dissolved in 0.5 mL of distilled water for 30 days.
Group VII:	Diabetic rats received intra gastrically Metformin (500 mg/kg b.wt.) dissolved in 0.5 mL of distilled water for 30 days.

Table 1. Dose dependent effect of theaflavin on blood glucose, insulin levels and HOMA-IR index in control and experimental animals.

Parameters	Control	Normal + Theaflavin (100 mg/kgb.wt)	Diabetes	Diabetes + Theaflavin (25 mg/kg b.wt)	Diabetes + Theaflavin (50 mg/kg b.wt)	Diabetes + Theaflavin 100 mg/kgb.wt	Diabetes + Metformin (500 mg/kg b.wt)
Glucose (mg/dl)	97.65 ± 5.03	95.28 ± 5.15	273.76 ± 16.35^b	239.43 ± 12.54^c	183.98 ± 8.74^d	131.81 ± 7.49^e	129.64 ± 8.12
Insulin (µU/ml)	19.42 ± 1.80	18.99 ± 1.82	9.57 ± 1,18^b	11.80 ± 1.12^c	13.51 ± 1.36^d	16.15 ± 1.35^e	17.66 ± 1.27
HOMA-IR	4.68 ± 0.64	4.38 ± 0.53	6.62 ± 0.70^b	7.20 ± 0.81^c	6.2 ± 0.58^d	5.13 ± 0.37^e	5.57 ± 0.49

Notes: Values are given as mean ± SD for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc LSD test *P < 0.05.

^aControl vs. Drug control (Theaflavin alone treated rats).

^bControl rats vs. Diabetic rats.

^cDiabetic rats vs. Theaflavin 25 mg/kg.

^dDiabetic rats vs. Theaflavin 50 mg/kg.

^eDiabetic rats vs. Theaflavin 100 mg/kg.

^fDiabetic rats treated with Theaflavin 100 mg/kg vs. Diabetic rats treated with metformin (500 mg/kg).

Figure 2. Effect of theaflavin on oral glucose tolerance test. Each value is mean ± S.D. for 6 rats (n = 6) in each group.

Table 2. Effect of theaflavin on body weight, fluid intake and food intake of control and experimental animals.

Groups	Body weight(g)		Food intake (g/rat/day)		Water intake (ml/rat/day)
	Initial	Final	Before	After	Before	After
Control	210.67 ± 6.89	239.67 ± 10.27	15.54 ± 1.21	14.14 ± 1.31	75.58 ± 5.57	73.64 ± 4.36
Normal + Theaflavin (100 mg/kg b.wt)	214.00 ± 7.69	242.83 ± 9.11	16.38 ± 1.19	14.05 ± 1.34	76.52 ± 6.11	78.03 ± 5.47
Diabetes induced	209.17 ± 8.11	150.83 ± 9.45^b	58.50 ± 7.21	78.01 ± 6.72^b	145.11 ± 10.55	155.33 ± 11.84^b
Diabetic + Theaflavin (100 mg/kg b.wt)	208.98 ± 7.12	196.67 ± 9.27^c	42.03 ± 4.19	30.85 ± 2.16^c	135.41 ± 5.27	78.05 ± 6.10^c
Diabetic + Metformin (500 mg/kg b.wt)	207.67 ± 6.86	199.83 ± 7.13	43.77 ± 2.94	29.04 ± 1.88^c	136.12 ± 4.96	76.87 ± 6.37

Notes: Values are given as mean ± SD for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc LSD test *P < 0.05.

^aControl vs. Drug control (Theaflavin alone treated rats).

^bControl rats vs. Diabetic rats.

^cDiabetic rats vs. Theaflavin 100 mg/kg.

^dDiabetic rats treated with Theaflavin 100 mg/kg vs. Metformin (500 mg/kg).

Table 3. Effects of theaflavin on hemoglobin and glycosylated hemoglobin levels in control and experimental animals.

Parameters	Control	Normal+ Theaflavin (100 mg/kg b.wt)	Diabetes	Diabetes + Theaflavin (100 mg/kg b.wt)	Diabetes + Metformin
Haemoglobin(g/dl)	14.62 ± 1.15	13.93 ± 0.87	7.38 ± 1.33^b	11.96 ± 1.30^c	12.87 ± 1.11
HbA1c (%)	4.75 ± 0.41	4.64 ± 0.42	11.15 ± 1.16^b	7.28 ± 0.73^c	6.40 ± 0.78

Notes: Values are given as mean ± SD for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc LSD test *P < 0.05.

^aControl vs. Drug control (Theaflavin alone treated rats).

^bControl rats vs. Diabetic rats.

^cDiabetic rats vs. Theaflavin 100 mg/kg.

^dDiabetic rats treated with Theaflavin 100 mg/kg vs. Metformin (500 mg/kg).

Table 4. Effects of theaflavin on glycogen metabolism in control and experimental animals.

Groups	Glycogen synthase (µ moles of UDP formed/h/mg protein)	Glycogen phosphorylase (µ moles Pi liberated/h/mg protein)	Liver glycogen (mg/g tissue)	Muscle glycogen (mg/g tissue)
Control	820.16 ± 7.13	587.33 ± 7.20	46.83 ± 2.48	17.50 ± 1.64
Normal + Theaflavin 100 mg/kg b.wt	817.66 ± 6.77	586.50 ± 5.20	47.66 ± 2.58	17.83 ± 1.47
Diabetes	514.83 ± 13.22	787.50 ± 11.58^b	21.83 ± 4.40^c	10.66 ± 0.81
Diabetic + Theaflavin 100 mg/kg b.wt	795.66 ± 10.63	660.83 ± 9.17^b	35.50 ± 4.37^c	15.50 ± 1.51
Diabetic + Metformin 100 mg/kg b.wt	798.40 ± 11.20	662.55 ± 8.46	37.67 ± 4.80	17.80 ± 1.42

Notes: Values are given as mean ± SD for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc LSD test *P < 0.05.

^aControl vs. Drug control (Theaflavin alone treated rats).

^bControl rats vs. Diabetic rats.

^cDiabetic rats vs. Theaflavin 100 mg/kg.

^dDiabetic rats treated with Theaflavin 100 mg/kg vs. Metformin (500 mg/kg).

Table 5. Effects of theaflavin on carbohydrate metabolic enzymes in control and experimental.

Parameters	Control	Normal + Theaflavin (100 mg/kg b.wt)	Diabetes	Diabetes + Theaflavin (100 mg/kg b.wt)	Diabetes + Metformin (500 mg/kg b.wt)
Liver
Hexokinase	252.35 ± 14.90	250.97 ± 14.74	153.93 ± 9.65^b	226.04 ± 14.17^c	225.13 ± 13.59
pyruvatekinase	200.96 ± 15.66	202.16 ± 15.7	104.47 ± 10.19^b	181.10 ± 12.51^c	179.24 ± 12.6
LDH	232.28 ± 12.15	230.05 ± 11.74	414.31 ± 23.50^b	249.74 ± 11.42^c	247.57 ± 11.78
Glucose 6 Phosphatase	730.08 ± 47.29	728.00 ± 47.57	1558.39 ± 55.09^b	766.78 ± 49.47^c	765.30 ± 49.42
Fructose 1,6 bisphosphatase	470.65 ± 23.35	467.99 ± 23.41	804.44 ± 55.18^b	500.76 ± 27.29^c	498.82 ± 27.17
glucose 6 phosphate dehydrogenase	510.47 ± 39.41	507.45 ± 38.96	239.24 ± 21.25^b	489.58 ± 35.89^c	487.45 ± 35.90
Kidney
Hexokinase	152.35 ± 14.99	149.56 ± 14.97	82.17 ± 5.59^b	130.87 ± 13.84^c	129.65 ± 13.58
Pyruvatekinase	130.55 ± 11.48	128.12 ± 11.74	70.38 ± 9.57^b	100.02 ± 10.56^c	98.71 ± 10.51
LDH	454.12 ± 24.15	451.09 ± 24.01	750.41 ± 49.73^b	486.35 ± 29.01^c	484.71 ± 29.08
Glucose Phosphatase	381.33 ± 25.63	378.85 ± 25.75	751.33 ± 32.08^b	423.98 ± 29.45^c	422.11 ± 29.53
Fructose 1,6 bisphosphatase	714.58 ± 52.01	711.95 ± 52.19	912.74 ± 81.91^b	794.86 ± 55.40^c	793.15 ± 55.50
glucose 6 phosphate dehydrogenase	657.77 ± 44.71	651.2 ± 40.70	276.50 ± 23.59^b	601.44 ± 40.67^c	600.08 ± 41.18

Notes: Values are given as mean ± SD for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc LSD test *P < 0.05. Units are expressed as: µmoles of glucose-6-phosphate formed/h/mg of protein for hexokinase, µmoles of pyruvate formed/min/mg of protein for pyruvate kinase, µmoles of pyruvate formed/h/mg of protein for lactate dehydrogenase, µmoles of Pi liberated/h/mg of protein for glucose-6- phosphatase and fructose-1, 6-bisphosphatase and µmoles of NADPH/min/mg of protein for glucose-6-phosphate dehydrogenase.

^aControl vs. Drug control (Theaflavin alone treated rats).

^bControl rats vs. Diabetic rats.

^cDiabetic rats vs. Theaflavin 100 mg/kg.

^dDiabetic rats treated with Theaflavin 100 mg/kg vs. Metformin (500 mg/kg).

Table 6. Effect of Theaflavin on the levels of TBARS and HP in the tissues of control and experimental rats.

Groups	TBARS (mM/100 g tissue)		Hydroperoxides (mM/100 g tissue)
	Liver	Kidney	Liver	Kidney
control	0.83 ± 0.05	1.32 ± 0.09	72.03 ± 5.11	41.85 ± 3.55
Normal + Theaflavin (100 mg/kg b.wt)	0.86 ± 0.07	1.26 ± 0.08	70.25 ± 4.95	39.09 ± 3.73
Diabetes Induced	1.88 ± 0.15^b	2.45 ± 0.23^b	137.25 ± 9.95^b	76.73 ± 7.47^b
Diabetic + Theaflavin (100 mg/kg b.wt)	1.19 ± 0.08^c	1.40 ± 0.12^c	97.21 ± 8.21^c	52.04 ± 4.65^c
Diabetic + Metformin (500 mg/kg b.wt)	1.01 ± 0.09	1.20 ± 0.10	92.51 ± 8.28	48.56 ± 4.38

Notes: Values are given as mean ± SD for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc LSD test *P < 0.05.

^aControl vs. Drug control(Theaflavin alone treated control rats).

^bControl rats vs. Diabetic rats.

^cDiabetic rats vs. Theaflavin treated diabetic rats.

^dTheaflavin treated diabetic rats vs. Metformin treated diabetic rats.

Table 7. Effect of theaflavin on the activities SOD, CAT, GPX and GST in control and experimental rats.

Groups	Superoxide dismutase		Catalase		Glutathione peroxidase		Glutathione-S-transferase
	Liver	Kidney	Liver	Kidney	Liver	Kidney	Liver	Kidney
Control	7.19 ± 0.46	8.80 ± 0.50	75.24 ± 4.01	36.95 ± 3.12	8.68 ± 0.63	7.42 ± 0.58	8.53 ± 0.52	5.76 ± 0.46
Normal  +  Theaflavin (100 mg/kg b.wt)	7.52 ± 0.43	9.21 ± 0.51	77.00 ± 3.88	37.31 ± 3.17	9.22 ± 0.71	7.77 ± 0.57	8.92 ± 0.41	6.15 ± 0.38
Diabetes Induced	3.15 ± 0.27^b	3.74 ± 0.35^b	49.57 ± 3.75	24.73 ± 2.35^b	5.19 ± 0.42	4.91 ± 0.44^b	4.06 ± 0.31^b	3.41 ± 0.33^b
Diabetic + Theaflavin (100 mg/kg b.wt)	7.28 ± 0.36^c	7.01 ± 0.40^c	68.11 ± 4.06^c	32.93 ± 2.81^c	7.70 ± 0.71^c	7.00 ± 0.53^c	7.67 ± 0.52^c	4.83 ± 4.63^c
Diabetic + Metformin (500 mg/kg b.wt)	7.57 ± 0.37	7.30 ± 0.39	70.16 ± 4.26	36.33 ± 3.25	7.31 ± 0.77	7.61 ± 0.67	7.20 ± 0.46	5.21 ± 0.37

Notes: Values are given as mean ± SD for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc LSD test *P < 0.05. The activities of enzymes are expressed as follows: SOD – One unit of activity was taken as the enzyme quantity, which gave 50% inhibition of nitroblue tetrazolium reduction in 1 min/mg protein; CAT – µmoles of H₂O₂ consumed/minute; GPx – µg of glutathione consumed/minute/mg protein; GST- µmoles of 1-chloro 2,4-dinitrobenzene-GSH conjugate formed/minute/mg protein.

^aControl vs. Drug control (Theaflavin alone treated control rats).

^bControl rats vs. Diabetic rats.

^cDiabetic rats vs. Theaflavin treated diabetic rats.

^dTheaflavin treated diabetic rats vs. Metformin treated diabetic rats.

Table 8. Effect of theaflavin on the levels of non-enzymatic antioxidants in tissues of control and experimental rats.

Groups	Vitamin C (µM/mg tissue)		Vitamin E (µM/mg tissue)		Reduced Glutathione (GSH) (µM/mg tissue)
	Liver	Kidney	Liver	Kidney	Liver	Kidney
Control	1.35 ± 0.13	1.18 ± 0.07	0.85 ± 0.07	0.58 ± 0.04	44.12 ± 4.67	35.89 ± 3.27
Normal + Theaflavin (100 mg/kg b.wt)	1.48 ± 0.14	1.22 ± 0.09	0.95 ± 0.05	0.72 ± 0.06	45.07 ± 4.48	36.25 ± 3.34
Diabetes Induced	0.88 ± 0.067^b	0.60 ± 0.05^b	0.29 ± 0.02^b	0.35 ± 0.03^b	25.59 ± 2.15^b	21.16 ± 1.98^b
Diabetic + Theaflavin (100 mg/kg b.wt)	1.27 ± 0.10^c	0.94 ± 0.04^c	0.64 ± 0.06^c	0.49 ± 0.04^c	37.04 ± 3.00^c	30.07 ± 2.57^c
Diabetic + Metformin (500 mg/kg b.wt)	1.39 ± 0.12	1.11 ± 0.11	0.69 ± 0.05	0.57 ± 0.04	35.61 ± 3.57	32.40 ± 3.02

Notes: Values are given as mean ± SD for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc LSD test *P < 0.05.

^aControl vs. Drug control(Theaflavin alone treated control rats).

^bControl rats vs. Diabetic rats.

^cDiabetic rats vs. Theaflavin treated diabetic rats.

^dTheaflavin treated diabetic rats vs. Metformin treated diabetic rats.

Figure 3. Histopathological section of pancreas of control and Experimental rats (40X). Control (A), Normal + Theaflavin (B), Diabetes induced (C) Diabetic + Theaflavin (D) Diabetic + Metformin (E). (F) Quantification of granulated cells from pancreas. Values are given as mean ± SD for six animals in each group (n = 6). Values are considered significantly different at P < 0.05 with post hoc LSD test. Comparisons are made as (a) Control vs Drug control (Theaflavin alone treated rat); (b) Control rat vs Diabetic rat; (c) Diabetic rat vs Theaflavin treated diabetic rat; (d) Theaflavin treated diabetic rat vs Metformin.